*Lactobacillus reuteri* in the murine gut. *Appl. Environ. Microbiol.* 71, 979-986.
doi: 10.1128/AEM.71.2.979-986.2005

Wang, K., and Lu, C. (2007). Adhesion activity of glyceraldehyde-3-phosphate dehydrogenase in a Chinese *Streptococcus suis* type 2 strain. *Berl. Munch. Tierarztl. Wochenschr.* 120, 207-209. doi: 10.2376/0005-9366-120-207

Wang, Y., Yi, L., Wu, Z., Shao, J., Liu, G., Fan, H., et al. (2012). Comparative proteomic analysis of *Streptococcus suis* biofilms and planktonic cells that identified biofilm infection-related immunogenic proteins. *PLoS ONE* 7:e33371. doi: 10.1371/journal.pone.0033371

Wang, Y., Zhang, W., Wu, Z. F., and Lu, C. P. (2011). Reduced virulence is an important characteristic of biofilm infection of *Streptococcus suis*. *FEMS Microbiol. Lett.* 316, 36-43. doi: 10.1111/j.1574-6968.2010.02189.x

Wisniewski, J. R., Zougman, A., Nagaraj, N., and Mann, M. (2009). Universal sample preparation method for proteome analysis. *Nat. Methods* 6, 359-362. doi: 10.1038/nmeth.1322

Yang, K., Meng, J., Huang, Y.-C., Ye, L.-H., Li, G.-J., Huang, J., et al. (2014). The role of the QseC quorum-sensing sensor kinase in epinephrine-enhanced motility and biofilm formation by *Escherichia coli*. *Cell Biochem. Biophys.* 70, 391-398. doi: 10.1007/s12013-014-9924-5

Yang, W.-Z., Yu, H.-J., Jing, H.-Q., Xu, J.-G., Chen, Z.-H., Zhu, X.-P., et al. (2006). An outbreak of human *Streptococcus suis* serotype 2 infections presenting with toxic shock syndrome in Sichuan, China. *Zhonghua Liu Xing Bing Xue za Zhi* 27, 185-191.

Yang, Y. B., Wang, S., Wang, C., Huang, Q. Y., Bai, J. W., Chen, J. Q., et al. (2015). Emodin affects biofilm formation and expression of virulence factors in *Streptococcus suis* ATCC700794. *Arch. Microbiol.* 197, 1173-1180. doi: 10.1007/s00203-015-1158-4

Yu, J., Bryant, A. P., Marra, A., Lonetto, M. A., Ingraham, K. A., Chalker, A. F., et al. (2001). Characterization of the *Streptococcus pneumoniae* NADH oxidase that is required for infection. *Microbiology* 147(Pt 2), 431-438. doi: 10.1099/00221287-147-2-431

Zhang, A., Xie, C., Chen, H., and Jin, M. (2008). Identification of immunogenic cell wall-associated proteins of *Streptococcus suis* serotype 2. *Proteomics* 8, 3506-3515. doi: 10.1002/pmic.200800007

Zhang, W., and Lu, C. P. (2007a). Immunoproteomic assay of membrane-associated proteins of *Streptococcus suis* type 2 china vaccine strain HA9801. *Zoonoses Public Health* 54, 253-259. doi: 10.1111/j.1863-2378.2007.01056.x

Zhang, W., and Lu, C. P. (2007b). Immunoproteomics of extracellular proteins of Chinese virulent strains of *Streptococcus suis* type 2. *Proteomics* 7, 4468-4476. doi: 10.1002/pmic.200700294

Zhao, Y. L., Zhou, Y. H., Chen, J. Q., Huang, Q. Y., Han, Q., Liu, B., et al. (2015). Quantitative proteomic analysis of sub-MIC erythromycin inhibiting biofilm formation of S-suis *in vitro*. *J. Proteomics* 116, 1-14. doi: 10.1016/j.jprot.2014.12.019

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Wang, Yang, Zhao, Bai, Chen, Zhou, Wang and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
<figure><img src="image_15.png" /></figure>

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# Absence of Protoheme IX Farnesyltransferase CtaB Causes Virulence Attenuation but Enhances Pigment Production and Persister Survival in MRSA

Tao Xu<fnref n="1" />, Jian Han², Jia Zhang<fnref n="1" />, Jiazhen Chen<fnref n="1" />, Nan Wu<fnref n="1" />, Wenhong Zhang<fnref n="1" />* and Ying Zhang<fnref n="1" />,<fnref n="3" />*

OPEN ACCESS

**Edited by:**

Ivan Rychlik,
Veterinary Research Institute,
Czechia

**Reviewed by:**

Atte Von Wright,
University of Eastern Finland, Finland
Dinesh Sriramulu,
Shres Consultancy (Life Sciences),
India

*Correspondence:

Wenhong Zhang
zhangwenhong@fudan.edu.cn
Ying Zhang
yzhang@jhsph.edu

**Specialty section:**

This article was submitted to
Antimicrobials, Resistance and
Chemotherapy,
a section of the journal
Frontiers in Microbiology

Received: 14 July 2016

Accepted: 29 September 2016

Published: 24 October 2016

**Citation:**

Xu T, Han J, Zhang J, Chen J, Wu N, Zhang W and Zhang Y (2016) Absence of Protoheme IX Farnesyltransferase CtaB Causes Virulence Attenuation but Enhances Pigment Production and Persister Survival in MRSA. *Front. Microbiol.* 7:1625. doi: 10.3389/fmicb.2016.01625

The membrane protein CtaB in S. aureus is a protoheme IX farnesyltransferase involved in the synthesis of the heme containing terminal oxidases of bacterial respiratory chain. In this study, to assess the role of CtaB in S. aureus virulence, pigment production, and persister formation, we constructed a ctaB mutant in the methicillin-resistant Staphylococcus aureus (MRSA) strain USA500. We found that deletion of ctaB attenuated growth and virulence in mice but enhanced pigment production and formation of quinolone tolerant persister cells in stationary phase. RNA-seq analysis showed that deletion of ctaB caused decreased transcription of several virulence genes including RNAIII which is consistent with its virulence attenuation. In addition, transcription of 20 ribosomal genes and 24 genes involved in amino acid biosynthesis was significantly down-regulated in the ctaB knockout mutant compared with the parent strain. These findings suggest the importance of heme biosynthesis in virulence and persister formation of S. aureus.

**Keywords:** Staphylococcus aureus, heme, antibiotics, pigment, virulence, persister formation

## INTRODUCTION

Staphylococcus aureus, named according to production of golden pigment, is an important human pathogen causing a variety of infection types including rampant skin and soft tissue infections, pneumonia, septicaemia, endocarditis, and central nervous system (CNS) infections. Methicillin-resistant S. aureus (MRSA) is notorious for its development of antibiotic resistance and expression of multiple virulence factors. (Li et al., 2012; Carrel et al., 2015).

S. aureus virulence factors are multifactorial and previous studies have been mainly focused on toxins (α-toxin, γ-toxin, Panton-Valentine leucocidin, exfoliative toxin and phenol-soluble modulins, etc.), surface proteins (FnbP, Bap, SasX, etc.) that help bind to host cells, facilitate

**Abbreviations:** MRSA, methicillin resistant Staphylococcus aureus; FPP, farnesyl diphosphate; SCV, small colony variants; MIC, minimum inhibitory concentration.

<footnote n="1">¹ Key Laboratory of Medical Molecular Virology, Huashan Hospital, Shanghai Medical College of Fudan University, Shanghai, China, ² Department of Pathogenic Biology, School of Basic Medical Sciences, Lanzhou University, Lanzhou, China,</footnote>

<footnote n="3">³ Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA</footnote>
internalization and immune evasion. Staphyloxanthin, synthesized from farnesyl diphosphate (FPP) by CrtM and CrtN, is the main component of *S. aureus* golden pigment (Liu et al., 2005). Staphyloxanthin not only plays a protecting role in bacterial fitness, but enhances virulence and survive attack by neutrophils (Clauditz et al., 2006). In addition, global regulatory systems (Agr, SaeRS, SarA, etc.) govern different aspects of physiology and expression of virulence traits, maintaining a balance between fitness and virulence.

It was in *staphylococcus* that persisters were first described in Bigger (1944). Persisters represent a certain portion of a bacterial culture that is genetically identical but phenotypically resistant or tolerant to antibiotics and stresses. In the model organism *Escherichia coli*, much research reveals the mechanisms of persister formation, including involvement of toxin-antitoxin, protein degradation, energy production and DNA repair (Zhang, 2014). However, less understood are the mechanisms of *S. aureus* persister formation. The portion of persisters in *S. aureus* is so high that a hypothesis was proposed that unlike *E. coli*, all *S. aureus* cells in stationary phase are persisters (Keren et al., 2004). Subsequently, however, Lechner et al. proved that stationary phase cultures of *S. aureus* are also a mixture of regular and persister cells (Lechner et al., 2012).

Though the key mechanisms of *S. aureus* persister formation are poorly understood, progress has been made recently. It has been reported that biofilm formation (Lewis, 2001; Resch et al., 2006) and small colony variants (SCV; Lechner et al., 2012) are two key features involving *S. aureus* persister formation, probably because the cells in biofilms and SCV cells have a different profile of gene expression, which makes them more readily to form persisters. Glycerol uptake has been reported to play a role in persister formation. Mutation in the glycerol transporter encoding gene *glpF* caused defective survival of *S. aureus* to ampicillin and norfloxacin (Han et al., 2014). A point mutation of the inorganic phosphate transporter gene *pitA* enhanced tolerance to daptomycin (Mechler et al., 2015). Mutations in purine biosynthesis genes (*purB*, *purF*, *purH*, *purM*,) amino acid, lipid, carbohydrate metabolism, and energy production genes efflux etc. were found to cause decreased persister formation in recent transposon mutant library screens (Yee et al., 2015; Wang et al., 2015).

Heme synthesis is an important pathway in Gram positive bacteria and provide substrate to production of terminal oxidases (Mogi et al., 1994). Within vertebrates *S. aureus* fulfills its requirement of iron by uptaking heme-iron from transferrin or heme or hemoglobin with its several transporters including StrA, StrB, IsdA, and IsdE, etc. (Drabkin, 1951; Mazmanian et al., 2003; Liu et al., 2008; Mason and Skaar, 2009). However, in an environment without heme-iron, *S. aureus* has to synthesize heme A with a complex pathway starting from glutamate (Hammer et al., 2016). CtaB and CtaA catalyzes the last two steps of the process. CtaB is a heme O synthase (protoheme IX farnesyltransferase) and while CtaA is an integral membrane protein that converts heme O to heme A (Svensson et al., 1993; Svensson and Hederstedt, 1994; Clements et al., 1999). Heme A is essential for functional expression of the terminal oxidases. Among terminal oxidases synthesized with heme A, cytochrome

aa 3 are quinol oxidases (QoxA, QoxB, etc.) and cytochrome c aa 3 is a cytochrome c oxidase.

Though heme synthesis mainly contributes to the pathway of synthesis of terminal oxidases that mediate bacterial respiration, it has also been reported to participate in fitness and virulence of *S. aureus*. For example, CtaA was found to be required for starvation survival and recovery from glucose starvation (Clements et al., 1999). A correlation between heme production and pigment production was reported by Lan et al., as depletion of CtaA and QoxB both enhanced pigment production, while attenuating hemolytic activity and virulence (Lan et al., 2010). However, no study has been done to address the specific effects of *ctaB* mutation on the heme-to-respiratory chain pathway and associated phenotypic changes. In this study, we created a CtaB deletion mutant of *S. aureus* and found associations of CtaB with heme synthesis, pigment production as well as persister cell formation. In addition, we performed a transcriptome analysis to provide new insights into the basis of the above associations.

# MATERIAL AND METHODS

## Bacterial Strains, Growth, and Chemical Reagents

*S. aureus* USA500 (Diep et al., 2006) was used for construction of gene knockout and complementation strains. *E. coli* DC10B (Monk et al., 2012) was used for shuttle plasmid construction. Luria Broth medium was composed of 1% tryptone (Oxoid), 0.5% yeast extract (Oxoid) and 0.5% NaCl; BM (B-Medium) was composed of 1% tryptone, 0.5% yeast extract, 0.5% glucose, 0.1% K₂HPO₄ and 0.5% NaCl; BM and TSB (Tryptic soy broth, Oxoid) were used for *S. aureus* cultivation. Bacterial strains were inoculated in BM, and their growth rate at 37°C was monitored by measuring the OD values at 600 nm. Anhydrotetracycline (ATC) was used for induction of *secY* antisense RNA during gene knockout. Antibiotics were added to medium at the following concentrations: chloramphenicol, 10 µg/ml; ampicillin, 100 µg/ml; levofloxacin, 50 µg/ml.

## Construction of Plasmids for Homologous Recombination and Complementary Strains

We constructed plasmid pMX10 by replacing *ccdB* element with multiple cloning sites in pKOR1 (Bae and Schneewind, 2006) and used it for construction of gene knock out strains. Primers pMX10-f and pMX10-r were mixed equally to a final concentration of 100 uM, incubated at 72°C for 20 min and slowly cooled to 4°C. The resulting dimers were digested with BamHI and KpnI and ligated to pKOR1 backbone digested with the same restriction enzymes. To construct Δ*ctaB* in USA500, the upstream (us) fragment (about 1000 bp) at the upstream of *ctaB* gene of USA500 strain was amplified with primer *ctaB-uf* and *ctaB-ur*, while the downstream (ds) fragment with primers *ctaB-df* and *ctaB-dr*. The two fragments were then used as templates for fusion PCR with primer *ctaB-uf* and *ctaB-dr*. The final PCR product was digested with KpnI and MluI and then ligated into pMX10. The recombinant plasmids was transformed into
USA500 by electroporation and mutants were selected according to the method reported by Bae et al. (Bae and Schneewind, 2006). To construct the complementation strain Δ*ctaB*:pRBctaB, a fragment containing the promoter region and coding sequence of *ctaB* gene was amplified with primers cp-*ctaB*-f and cp-*ctaB*-r. The PCR product was digested with EcoRI and BamHI and then ligated into plasmid PRB473. The resulting plasmid was transformed into the Δ*ctaB* mutant via electroporation. The sequences of primers are listed in Table 1.

## Detection of Pigment Production and Hemolytic Activity

To compare pigment production, USA500 and USA500Δ*ctaB* were dropped onto TSA plates and USA500Δ*ctaB* with pRB473 or pRBctaB on TSA plates with 10 µg/ml chloramphenicol. The plates were incubated at 37°C for 24 h and pictured. For quantitate assay of pigment production, the same strains were cultured in TSB at 37°C for 24 h. For each sample, pigment was extracted with methanol and detected with a parameter (GeneSpec III, Hitachi, Japan), following a previously reported protocol (Morikawa et al., 2001). For hemolytic activity determination, the strains were analyzed by growing the strains on 5% sheep blood agar at 37°C for 48 h. The result represents three independent experiments.

## Mouse Infection

The mouse virulence test was performed on Balb/C mice. USA500 and the Δ*ctaB* mutant strains were cultured for 18 h and 1 ml of the each culture was mixed with 2% Cytodex-1 beads by 1:1. The mice were randomized into two groups (5 mice/group). Each mouse was challenged with 200 µl bacterial mixture (each containing approximately 2 × 10⁵ bacterial cells) via injection under skins on the back. After 48 h, the mice were sacrificed and the abscess under skin was homogenized in 2 ml PBS. The samples were diluted and plated on TSA plates at 37°C for 18 h. CFU counting was performed and a Student's *t*-test was used for statistical analysis using Microsoft Excel.

Animal studies on mice were performed according to relevant national and international guidelines (the Regulations for the Administration of Affairs Concerning Experimental Animals, China) and were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Medical College, Fudan University (IACUC Animal Project Number: 20110630). Standard operation procedures were followed to carry out animal experiments in bio-safety level 2 labs.

## Susceptibility Testing

The MIC of each antimicrobial compound was determined in triplicate by a conventional broth microdilution technique in TSB medium, following the protocol previously published (Andrews, 2001) and the CLSI guidelines. The MIC was defined as the lowest antibiotic concentration that inhibited visible bacterial growth (also according to OD600 measurements) after 24 h of incubation at 37°C.

<table><caption>TABLE 1 | Primers used in this study.</caption><thead><tr><th>Primers</th><th>Sequence 5'-3'</th><th>Purpose</th></tr></thead><tbody><tr><td>pMX10-f</td><td>GGGGTACCGCTAGCCGGCCGG</td><td>Construction of pMX10</td></tr><tr><td>pMX10-r</td><td>GGCCACGCGTGAATTCCG</td><td>Construction of pMX10</td></tr><tr><td>ctaB-uf</td><td>CGGAATTCACGCGTGGGCCCC</td><td>Construction of ΔctaB</td></tr><tr><td>ctaB-ur</td><td>GGGGTACCGCTGTATAACCAT</td><td>Construction of ΔctaB</td></tr><tr><td>ctaB-df</td><td>AATTTAAGGAGGGGGATTATTAAT</td><td>Construction of ΔctaB::pRBctaB</td></tr><tr><td>ctaB-dr</td><td>TAAGTTAGGATGAAAAAATATGGG</td><td>Construction of ΔctaB::pRBctaB</td></tr><tr><td>cp-ctaB-f</td><td>CGGAATTCAAAAAAGAACTTA</td><td>Construction of ΔctaB::pRBctaB</td></tr><tr><td>cp-ctaB-r</td><td>ATCGTAATGATTTTTTTATTG</td><td>Construction of ΔctaB::pRBctaB</td></tr><tr><td>RThld-f</td><td>CGGGATCCCTTAATTAATCT</td><td>Real-time PCR</td></tr><tr><td>RThld-r</td><td>AGATCAAAGTAAGTAATGAAAC</td><td>Real-time PCR</td></tr><tr><td>RTesaB-f</td><td>CACTGTGTCGATAATCCATT</td><td>Real-time PCR</td></tr><tr><td>RTesaB-r</td><td>ATTAAGGAAGGAGTGATTTCAAT</td><td>Real-time PCR</td></tr><tr><td>RTset18-f</td><td>ACTTAGCAGTACCAGCATAT</td><td>Real-time PCR</td></tr><tr><td>RTset18-r</td><td>AATATCTCCATCAGCGATTTG</td><td>Real-time PCR</td></tr><tr><td>RTset18-f</td><td>CAGAGCGATTAGCAATGATAA</td><td>Real-time PCR</td></tr><tr><td>RTset18-r</td><td>GCGTTCTTGTCTTTGTGTTA</td><td>Real-time PCR</td></tr><tr><td>RThtrA-f</td><td>TGTGCTATTGAACGATAACG</td><td>Real-time PCR</td></tr><tr><td>RThtrA-r</td><td>CTTGCTCTGCTTGATAACTC</td><td>Real-time PCR</td></tr><tr><td>RTarcB2-f</td><td>TGAACCTGATGAAGTATGGA</td><td>Real-time PCR</td></tr><tr><td>RTarcB2-r</td><td>TGGAAAGATGGTAAGCAATG</td><td>Real-time PCR</td></tr><tr><td>RTsdhA2-f</td><td>CAGCAGATTTAGCATTAGCA</td><td>Real-time PCR</td></tr><tr><td>RTsdhA2-r</td><td>TACGACCAACCTTATCCATT</td><td>Real-time PCR</td></tr><tr><td>RTnrdE-f</td><td>CGATGGTATGGCTATTCCTA</td><td>Real-time PCR</td></tr><tr><td>RTnrdE-r</td><td>CGATTGGCATTACAGAACTT</td><td>Real-time PCR</td></tr><tr><td>RTpyrF-f</td><td>TAGATGGCGTTGTTTGTCATT</td><td>Real-time PCR</td></tr><tr><td>RTpyrF-r</td><td>GTAATACGGTGTTGGTCATT</td><td>Real-time PCR</td></tr><tr><td>RTrpmC-f</td><td>TTAGAGACTTAACCACTTCAGA</td><td>Real-time PCR</td></tr><tr><td>RTrpmC-r</td><td>CTTTCACGAGCAACAGTTT</td><td>Real-time PCR</td></tr><tr><td>RTagrD-f</td><td>AACATTGGTAACATCGCAG</td><td>Real-time PCR</td></tr><tr><td>RTagrD-r</td><td>GTGTTAATTCTTTTGGTACTTCA</td><td>Real-time PCR</td></tr><tr><td>RTdltA-f</td><td>TGGTTCATTCAAGGTCGTA</td><td>Real-time PCR</td></tr><tr><td>RTdltA-r</td><td>GCATTGTCCGTAACTTCAG</td><td>Real-time PCR</td></tr><tr><td>RTrrs1-f</td><td>GTGCTACAATGGACAATACAA</td><td>Real-time PCR</td></tr><tr><td>RTrrs1-r</td><td>ACTACAATCCGAACTGAGAA</td><td>Real-time PCR</td></tr></tbody></table>

## Persister Assay

To determine the number of persister cells in exponential phase, cells were grown overnight in 4 ml and were inoculated to 10 ml of fresh medium to an initial OD600 of 0.05. Cultures were shaken for 1.5–2 h (for normally growing cells), until an OD600 of approximately 0.5 was reached. To determine the number of persister cells in stationary phase, overnight cultures (16 or 24 h) were used without dilution.

For heat stress assay, stationary phase cultures were incubated at 57°C for up to 3 h. For oxidative stress assay, stationary phase cultures were diluted by 1:100 in TSB that contained 50 mM hydrogen peroxide (H₂O₂) for 4 h.
For starvation stress assay, stationary phase cultures were centrifuged, washed and resuspended in 3% NaCl. The survival of bacteria was determined by CFU counting at each hour. All stress assays were conducted using at least three biological replicates.

For antibiotic exposure, 2 ml of the overnight or the exponential phase cultures was transferred to 14 ml culture tubes (Greiner), antimicrobials were added at 100-fold MIC as indicated and the cultures were shaken for 12 h, or for 7 days during long-term experiments. For CFU determination, 100 µl was taken before and during antimicrobial challenge on an hourly basis during the first 8 h and after 24 h, or after 1, 2, 3, 5, 6 days during long-term experiments. Cells were washed in PBS and spotted as 10 µl aliquots of serial dilutions onto TSA plates. Colonies were counted after incubation for 24 h at 37°C. The lower limit of quantification was 100 CFU/ ml. All time-kill experiments were conducted using at least three biological replicates.

## RNA Isolation, mRNA Enrichment and Sequencing

S. aureus USA500 parent strain and Δ*ctaB* mutant were cultured for 6 or 24 h as log phase and stationary phase cultures. The cultures were divided into 3 aliquots and treated with RNAprotect (Qiagen) and frozen at -80°C. Total RNA was extracted from bacterial cells using the RNeasy Mini kit (Qiagen) as described (Atshan et al., 2012). The quality of RNA samples was examined with Bioanalyzer 2100 RNA-6000 Nano Kit. To remove 16S and 23S rRNAs, 10 µg of high-quality total RNA was processed using the Ribo-Zero™ Gold Kit before precipitating with ethanol and resuspending into 25 µL of nuclease-free water. The cDNA libraries with 150- to 250-bp multiplexed cDNA were generated from the enriched mRNA samples using the TruSeq Illumina kit (Illumina, San Diego, CA), following instructions from the manufacturer.

Sequencing was performed with HiSeq2500 (Illumina). The Cufflinks suite of tools were used to assess and quantify the total number of reads. With the program Cuffdiff as part of the suite, transcripts were quantified by assessing the total number of reads for the entire transcript. Briefly, reads were mapped to annotated coding sequences (CDSs) from genome of S. aureus USA300 TCH1516 strain since USA500 is the progenitor of USA300. The samples to be compared were evaluated for variance and tested for differential expression. Reads' P-values were determined, and significance was assessed by conducting Benjamini-Hochberg correction for multiple testing. The transcript sequencing data were submitted to the NCBI Sequence Read Archive, available for access under a RUN number RSS3919726.

## Quantitative Real-Time PCR

For quantitative Real-time PCR, the same RNA samples were taken from that used for RNA-seq. After reverse transcription with cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA), qRT-PCR was performed using SYBR Green PCR reagents (Takara Biotechnology) to determine the relative expression levels of the target genes with gene-specific primers listed in Table 1. The housekeeping gene *rrs1* (16s RNA) was used as an

endogenous control. All qRT-PCR experiments were carried out in triplicate with independent RNA samples and the 2⁻⁰ΔΔCT method was performed for analysis of relative gene expression data (Livak and Schmittgen, 2001).

## Statistics

The significance of experimental differences in pigment production, hemolytic activity, survival *in vivo* and persister assay was evaluated by unpaired Student's t-test.

## RESULTS

### Construction and Properties of the S. aureus *ctaB* Deletion mutant Δ*ctaB*

To investigate the functions of CtaB, we constructed a *ctaB* deletion mutant, Δ*ctaB*, via homologous recombination, as well as made a complemented strain Δ*ctaB*::pRBctaB by inserting *ctaB* with its own promoter into plasmid pRB473. When grown in TSB, the Δ*ctaB* mutant showed a slight growth defect, compared with the parent strain USA500 (Figure 1A). The Δ*ctaB* mutant displayed enhanced golden pigment production when grown on TSA for 24 h compared with the control strain (Figure 1B), and complementation of the Δ*ctaB* mutant reduced pigment production to normal levels (Figure 1B). Quantification of pigment production by extracting carotenoid products confirmed that CtaB depletion afforded enhanced pigmentation than USA500 strain (Figure 1C).

### CtaB Affects Hemolytic Activity and Survival *In vivo*

Hemolytic ability is an important aspect of S. aureus virulence (Wang and Muir, 2016). We analyzed the level of bacterial growth and hemolysis of the Δ*ctaB* mutant on sheep blood agar plates. While all strains showed similar sized colonies, deletion of *ctaB* generated a strain with reduced hemolytic activity, which could be restored by complementation of the Δ*ctaB* mutant with the wild type *ctaB* gene (Figure 2A).

Having observed that the Δ*ctaB* mutant enhanced pigment production but reduced hemolytic activity, we wondered whether Δ*ctaB* mutant would affect virulence *in vivo*. Skin is one of the most frequently targeted sites for S. aureus infection (Liu, 2009). To determine whether CtaB is associated with virulence during S. aureus infection, we compared the Δ*ctaB* mutant and the parent strain in a mouse model of skin abscess. Colony counting of bacteria from mouse skin lesions showed that inactivation of *ctaB* attenuated bacterial survival *in vivo*. After 24 h of infection, the CFU counting of USA500 increased from 6.18 ± 0.46E + 6 to 6.79 ± 1.02E + 6. Within contrast, the Δ*ctaB* mutant survived less well with a decrease of CFU, from 4.74 ± 0.57E + 6 to 2.96 ± 1.3E + 6. (Figure 2B).

### CtaB is Involved in Persister Cell Formation under Stress and Antibiotic Treatment

To determine if CtaB is involved in persister formation or survival, we subjected stationary cultures of USA500, Δ*ctaB* and Δ*ctaB*::pRBctaB under stress conditions including heat, oxidative stress, and starvation. The CtaB mutation attenuated the ability of
<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

WT

<figure><img src="image_18.png" /></figure>

ctaB

<figure><img src="image_19.png" /></figure>

ctaB::pRBctaB

<figure><img src="image_20.png" /></figure>

**FIGURE 1** | (A) Comparison of the growth rate of USA500 and the *ΔctaB* mutant. A saturated overnight culture of each strain was inoculated in a 12 ml tube and cultured at 37°C. Cultures were monitored by measuring absorption at OD600 each hour. (B) Pigmentation display of S. aureus strains grown on TSA plates at 37°C for 24 h. (C) Measurement of the golden pigment of different strains by methanol extraction. The relative optical density units were detected at 465 nm and normalized to the USA500 strain, which was set at 100. Results are means with standard error (error bars) of three independent experiments.

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

**FIGURE 2** | (A) Hemolytic activity assay. Overnight cultures of USA500, *ΔctaB* mutant and complemented strains were spotted (20 µl) on sheep blood agar plates and grown at 37°C for 24 h. The result is representative of triplicate experiments. (B) Survival of S. aureus strains in a Balb/C challenged by subcutaneous injection. Comparison of CFU counts was performed using the Student's *t*-test. Results are means with standard error (errors bars).

S. aureus to survive starvation in 3% NaCl, which provides similar osmotic pressure as TSB, and the impact was reversed by gene complementation (Figure 3A). However, CtaB knockout did not affect survival of S. aureus under treatment with heat or H₂O₂ (data not shown).

Before persister assay, we measured the antibiotic sensitivity of *ctaB* mutant and found no difference in MIC tests for multiple antibiotics (data not shown). Challenging the stationary phase cultures of USA500, *ΔctaB* and *ΔctaB::pRBctaB* with 100 X MIC ciprofloxacin or levofloxacin yielded disparate killing curves. As shown in Figures (3B,C), the surviving ratios of *ΔctaB* were similar with that of USA500 in the first 3 days, but became higher than the control strain in day four and day five. Meanwhile, complementation with plasmid pRBctaB but not pRB473 partially reversed the augmentation of persister formation caused by deletion of *ctaB* in the last 2 days of treatment. We also tested other antibiotics such as vancomycin,

rifampicin, streptomycin, tobramycin, and gentamycin at 100X MIC concentration but found no significant difference in persister formation between the *ΔctaB* mutant and the parent strain from either exponential phase or stationary phase (data not shown).

## RNA-seq Analysis of the *ΔctaB* Mutant Compared with its Parent Strain USA500

The above results indicate an intriguing and paradoxical role of CtaB in persistence and virulence of S. aureus, as its deletion attenuated virulence and survival in 3% NaCl while increasing persister numbers for quinolone antibiotics. To gain insights into the role of CtaB in altered S. aureus virulence and persistence, we performed RNA-seq analysis of USA500 and *ΔctaB* mutant grown for 6 h (log phase) or 24 h (stationary phase) in TSB medium. Based on the results of read counts of all annotated
<figure><img src="image_23.png" /><figcaption>**FIGURE 3** | Time dependent killing of S. aureus stationary phase bacteria. (A) Effects of starvation (3% NaCl) on survival kinetics of S. aureus USA500, Δ*ctaB* mutant and complemented strain. Results are representative of three independent experiments. (B, C) Persister assay with antibiotics. Strains were treated with (B) 20 µg/ml ciprofloxacin or (C) 50 µg/ml levofloxacin for 6 days. The limit of detection was 100 CFU/ml throughout all killing experiments. Results are representative of three independent experiments.</figcaption></figure>

genes, a total of 4 RNA-seq samples were clustered without supervision (Figure 4). The results indicated that the effect of CtaB knockout on the bacterial transcriptome at 24 h were more apparent than that at 6 h (Figures 5A,B).

In log phase cultures, we found 18 genes with significant changes in transcription (cutoff > 2-fold) and *p*-values less than 0.05 between Δ*ctaB* mutant and the parent strain (Table 2). Most strikingly, the virulence gene *hld* was significantly down regulated (0.35, *p* = 8.47E-05) in the Δ*ctaB* mutant. In S. aureus, gene *hld* is located inside the coding sequence for small regulatory RNA RNAIII which regulates the expression of many S. aureus genes encoding exoproteins and cell-wall-associated

<figure><img src="image_24.png" /><figcaption>**FIGURE 4** | Clustering of 4 RNA-seq samples. Sample_W6, USA500 grown to 6 h; Sample_B6, USA500Δ*ctaB* grown to 6 h; Sample_W24, USA500 grown to 24 h; Sample_B24, USA500Δ*ctaB* grown to 24 h. Heatmap shows the Euclidean distances between the samples as calculated from the variance-stabilizing transformation of the count data.</figcaption></figure>

proteins. The data indicated that deletion of CtaB could attenuate expression of various virulence genes regulated by RNAIII in log phase. Interestingly, CtaB deletion did not affect expression of the Agr system (encoded by *agrB*, *agrD*, *agrC*, and *agrA*), which is the well-known upstream regulator of RNAIII and a virulence factor. The virulence gene *esaB* (Burts et al., 2008; Anderson et al., 2011) was also down regulated. Meanwhile, the virulence genes *set18* and *set19* were up regulated in the Δ*ctaB* mutant. Two hemin ABC transporter super family genes *htrB* and *htrA* were significantly up regulated, as a consequence of lack of heme caused by deletion of CtaB (Table 2).

For the transcripts at 24 h, 119 genes showed significant changes between Δ*ctaB* mutant and the parent strain (Table 3), indicating that CtaB has a major impact on stationary phase S. aureus. The proposed pathways that these genes participate in were analyzed (Figure 6). Nineteen genes encoding ribosome proteins were strongly down regulated, as well as nine genes involved in biosynthesis of Aminoacyl-tRNA. Genes involved in arginine, proline, cysteine, methionine, glycine, serine, threonine, lysine, phenylalanine, tyrosine, tryptophan, valine, leucine, and isoleucine were down regulated in the CtaB mutant. Expression of several ABC transporters was also up regulated, but other transporters such as OppA1, OppB3, OppC1, OppD1, and OppF1, were down regulated. Twenty two genes that encode factors for amino acid metabolism showed difference in expression. Expression of two genes (*arcB2* and *arcC1*) involved in arginine metabolism was up regulated while the others were down regulated. The deletion of CtaB also down regulated genes from pathways involved in purine
<figure><img src="image_25.png" /><figcaption>**FIGURE 5** | Scatter plot of the expression levels of pairs of adjacent genes. The expression levels of two genes located within the same transcript of log phase bacteria (A) and stationary phase bacteria (B) are plotted in log₁₀ scale.</figcaption></figure>

metabolism, pyrimidine metabolism, and fatty acid biosynthesis. Though out of 17 genes associated with *S. aureus* infection 12 were up regulated, genes in the *dlt* operon (*dltA*, *dltB*, *dltC*, and *dltD*) were significantly down regulated (Collins et al., 2002). The CtaB deletion also induced expression of genes of five two-component systems, including PhoPR, LgrAB, SaeRS, and LytSR, indicating that these systems might play a role when *S. aureus* is confronted with lack of heme biosynthesis (Table 3).

Quantitative Real-time PCR was performed to validate the RNA-seq results. Genes were chosen from the list of genes with significant changes of transcription, favoring those associated

with virulence and protein production but had a *p*-value < 0.05. All showed similar fold change with those from the RNA-seq results (Figure 7).

DISCUSSION

The aim of this study is to address the role of CtaB in pigment production, virulence and persister formation in *S. aureus*. We found that deletion of *ctaB* attenuated survival under starvation and virulence in mice but had enhanced pigment production and formation of quinolone tolerant persister cells. Our study is the first to report the complex relationship
<table><caption>TABLE 2 | List of genes differentially expressed in USA500 and ΔctaB grown to 6 h.</caption><thead><tr><th>Gene</th><th>Gene symbol</th><th>Log<sub>2</sub> fold change</th><th>p-value</th><th>Description</th></tr></thead><tbody><tr><td>USA300HOU_RS14135</td><td></td><td>-2.04</td><td>4.4E-05</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS09600</td><td></td><td>-1.96</td><td>1.1E-03</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS10850</td><td></td><td>-1.83</td><td>1.2E-02</td><td>Hypothetical bacteriophage protein</td></tr><tr><td>USA300HOU_RS 10955</td><td>hld</td><td>-1.53</td><td>8.5E-05</td><td>Delta-hemolysin</td></tr><tr><td>USA300HOU_RS 03725</td><td></td><td>-1.42</td><td>9.0E-04</td><td>Hypothetical membrane protein</td></tr><tr><td>USA300HOU_RS 05170</td><td></td><td>-1.24</td><td>1.9E-03</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS 04240</td><td></td><td>-1.07</td><td>3.7E-02</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS 10770</td><td></td><td>1.02</td><td>1.8E-02</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS 09400</td><td>ribD</td><td>1.06</td><td>9.6E-07</td><td>Diaminohydroxyphosphoribosylaminopyrimidine deaminase</td></tr><tr><td>USA300HOU_RS 12385</td><td>ureB</td><td>1.09</td><td>1.3E-02</td><td>Urease beta subunit</td></tr><tr><td>USA300HOU_RS 09390</td><td>ribA</td><td>1.13</td><td>2.0E-07</td><td>Bifunctional 3,4-dihydroxy-2-butanone-4-phosphate synthase/GTP cyclohydrolase II</td></tr><tr><td>USA300HOU_RS 10800</td><td></td><td>1.17</td><td>2.8E-03</td><td>Hypothetical bacteriophage protein</td></tr><tr><td>USA300HOU_RS 09395</td><td>ribB</td><td>1.32</td><td>3.5E-08</td><td>Riboflavin synthase alpha subunit</td></tr><tr><td>USA300HOU_RS 13155</td><td></td><td>1.32</td><td>1.6E-02</td><td>ABC superfamily ATP binding cassette transporter, ABC/membrane protein</td></tr><tr><td>USA300HOU_RS 06710</td><td></td><td>1.32</td><td>2.3E-02</td><td>ABC superfamily ATP binding cassette transporter, membrane protein</td></tr><tr><td>USA300HOU_RS 03970</td><td></td><td>1.39</td><td>4.5E-07</td><td>Iron (Fe+3) ABC superfamily ATP binding cassette transporter, membrane protein</td></tr><tr><td>USA300HOU_RS 12770</td><td>htrB</td><td>4.14</td><td>1.2E-51</td><td>Hemin ABC superfamily ATP binding cassette transporter, ABC protein</td></tr><tr><td>USA300HOU_RS 12775</td><td>htrA</td><td>4.34</td><td>1.6E-55</td><td>Hemin ABC superfamily ATP binding cassette transporter, membrane protein</td></tr></tbody></table>

between heme production, persister formation, and virulence in *S. aureus*.

We have shown that CtaB depletion barely affected growth in rich medium (TSB), but caused faster death under starvation stress (Figure 3A). The result echoes the finding by Clements et al., as CtaA mutation caused growth defect in glucose-limiting chemically defined medium (Clements et al., 1999). Heme production is a key step for cellular aerobic respiration and energy conversion, providing resources for synthesis of heme A-containing terminal oxidases (Svensson and Hederstedt, 1994; Hederstedt et al., 2005). The changes in the respiratory chain by mutation of CtaB could account for the defects. Meanwhile, it is more difficult to explain the enhanced pigment production caused by CtaB depletion. The production of staphyloxanthin, the main pigment of Staphylococci, is mediated by factors encoded by *crtOPQMN*, using FPP as the substrate (Wieland et al., 1994; Pelz et al., 2005). Regulators such as *rsbUVW-sigB* are known to regulate expression of pigment genes in *S. aureus* (Kullik et al., 1998; Giachino et al., 2001). In previous reports, suppression of genes from metabolic pathways (purine biosynthesis, the TCA cycle and oxidative phosphorylation) has also been found to affect pigment production (Lan et al., 2010). We detected the expression of pigment associated genes and found that CtaB deletion did not affect expression of *rsbUVW-sigB*, *fliA* (*sigB*) or *crtOPQMN*, while expression of *citZ* in *ΔctaB* mutant was down regulated (0.45) while *qoxB* was induced (2.09), and the other metabolic genes were not affected (Table 4). FPP is a key intermediate in mevalonate pathway that serves as a substrate of several pathways including synthesis of heme A and staphyloxanthin (Szkopinska and Plochocka, 2005). Since CtaB deletion did not affect pigment production by altering expression of the currently known genes of pigment production

pathway, the possibility is worth considering that the absence of competition by heme A production pathway leaves more FPP to staphyloxanthin synthesis pathway, thus enhancing pigment production provided.

From the RNA-seq data, we show the down regulation of multiple virulence genes was caused by CtaB depletion. Despite the depression of global regulatory RNA RNAIII and several classic virulence factors (EsaB, EsaC, EsXB, etc.), DltA-D and most of the amino acid ABC transporters were down regulated. The four proteins (DltA-D) incorporate D-alanine into cell wall polymers during teichoic acid synthesis (Reichmann et al., 2013) and their inactivation has been shown to impact the defense of *S. aureus* against antimicrobial agents (Peschel et al., 1999). Expression of many amino acid transporter genes (*oppA1*, *oppC1*, *oppD1*, and *oppF1*) were found down regulated in the CtaB knockout strain. These ABC transporters not only function by obtaining nutrients, but play important roles in adherence and processing of secreting toxins (Podbielski et al., 1996). They also showed up frequently in screening of virulence genes of *S. aureus* with transposon libraries in different animal models (Mei et al., 1997; Coulter et al., 1998; Bae et al., 2004).

Pigment production has been found to enhance fitness and virulence and help the bacteria cope with oxidative stress (Clauditz et al., 2006). However, our results seem to contradict this finding of association of pigment production and virulence as we see enhanced pigment production of CtaB mutant but less virulence. Nevertheless, CtaB deletion had multiple effects on *S. aureus*, despite enhanced pigment production, it caused attenuated hemolytic activity and survival in animal model. It is likely the virulence attenuation of CtaB mutant is combined effect of more important attenuated hemolytic activity over the increased pigment production such that the net outcome is
<table><caption>TABLE 3 | List of genes differentially expressed in USA500 and ΔctaB grown to 24 h.</caption><thead><tr><th>Gene</th><th>Gene symbol</th><th>log<sub>2</sub> fold change</th><th>p-value</th><th>Description</th></tr></thead><tbody><tr><td>USA300HOU_RS1190</td><td></td><td>-3.37</td><td>3.36E-06</td><td>Acetyl-CoA C-acetyltransferase</td></tr><tr><td>USA300HOU_RS1195</td><td></td><td>-3.30</td><td>7.44E-06</td><td>3-hydroxyacyl-CoA dehydrogenase</td></tr><tr><td>USA300HOU_RS1200</td><td></td><td>-3.19</td><td>9.02E-06</td><td>Acyl-CoA dehydrogenase</td></tr><tr><td>USA300HOU_RS1205</td><td></td><td>-3.16</td><td>1.08E-05</td><td>Long-chain-fatty-acid-CoA ligase</td></tr><tr><td>USA300HOU_RS02265</td><td>cobW1</td><td>-2.78</td><td>6.77E-04</td><td>Cobalamin (vitamin B12) biosynthesis protein</td></tr><tr><td>USA300HOU_RS01210</td><td></td><td>-2.76</td><td>1.58E-04</td><td>3-oxoacid CoA-transferase</td></tr><tr><td>USA300HOU_RS11065</td><td>ilvB1</td><td>-2.69</td><td>1.10E-03</td><td>Acetolactate synthase large subunit</td></tr><tr><td>USA300HOU_RS02055</td><td>xprT</td><td>-2.66</td><td>1.47E-03</td><td>Xanthine phosphoribosyltransferase</td></tr><tr><td>USA300HOU_RS12265</td><td></td><td>-2.57</td><td>2.28E-03</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS14500</td><td>lip</td><td>-2.16</td><td>2.42E-03</td><td>Triacylglycerol lipase</td></tr><tr><td>USA300HOU_RS02060</td><td>pbuX</td><td>-2.16</td><td>3.25E-03</td><td>NCS2 family nucleobase:cation symporter-2</td></tr><tr><td>USA300HOU_RS05895</td><td></td><td>-2.14</td><td>1.26E-02</td><td>Antibacterial protein</td></tr><tr><td>USA300HOU_RS04630</td><td>dltC</td><td>-2.11</td><td>7.32E-03</td><td>D-alanine-poly(phosphoribitol) ligase</td></tr><tr><td>USA300HOU_RS08560</td><td></td><td>-2.07</td><td>1.27E-02</td><td>Acetyl-CoA carboxylase biotin carboxyl carrier subunit</td></tr><tr><td>USA300HOU_RS12135</td><td>rpmC</td><td>-2.04</td><td>3.26E-03</td><td>Ribosomal protein L29</td></tr><tr><td>USA300HOU_RS11075</td><td>ilvC</td><td>-2.03</td><td>1.12E-02</td><td>Ketol-acid reductoisomerase</td></tr><tr><td>USA300HOU_RS00655</td><td></td><td>-2.03</td><td>3.65E-02</td><td>Hypothetical membrane protein</td></tr><tr><td>USA300HOU_RS01875</td><td></td><td>-2.01</td><td>4.74E-02</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS04875</td><td>fabH1</td><td>-1.95</td><td>4.11E-03</td><td>3-oxoacyl-[acyl-carrier-protein] synthase</td></tr><tr><td>USA300HOU_RS02840</td><td>rplL1</td><td>-1.94</td><td>5.40E-03</td><td>Ribosomal protein L7/L12</td></tr><tr><td>USA300HOU_RS12130</td><td>rpsQ</td><td>-1.93</td><td>5.02E-03</td><td>Ribosomal protein S17</td></tr><tr><td>USA300HOU_RS04900</td><td>oppD1</td><td>-1.91</td><td>4.92E-03</td><td>Oligopeptide ABC superfamily ATP binding cassette transporter, ABC protein</td></tr><tr><td>USA300HOU_RS11060</td><td>ilvD</td><td>-1.89</td><td>1.79E-02</td><td>Dihydroxy-acid dehydratase</td></tr><tr><td>USA300HOU_RS07110</td><td>dapB</td><td>-1.84</td><td>1.91E-02</td><td>Dihydrodipicolinate reductase</td></tr><tr><td>USA300HOU_RS13735</td><td></td><td>-1.82</td><td>1.46E-02</td><td>Transcriptional regulator</td></tr><tr><td>USA300HOU_RS08760</td><td>rplU</td><td>-1.79</td><td>1.10E-02</td><td>Ribosomal protein L21</td></tr><tr><td>USA300HOU_RS07115</td><td>dapD</td><td>-1.75</td><td>2.17E-02</td><td>2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase</td></tr><tr><td>USA300HOU_RS07100</td><td>asd</td><td>-1.74</td><td>2.52E-02</td><td>Aspartate-semialdehyde dehydrogenase</td></tr><tr><td>USA300HOU_RS06985</td><td>trpB</td><td>-1.72</td><td>4.42E-02</td><td>Tryptophan synthase beta subunit</td></tr><tr><td>USA300HOU_RS06055</td><td>pyrE</td><td>-1.71</td><td>2.74E-02</td><td>Orotate phosphoribosyltransferase</td></tr><tr><td>USA300HOU_RS06045</td><td>carB</td><td>-1.68</td><td>1.67E-02</td><td>Carbamoyl-phosphate synthase (glutamine-hydrolyzing), large subunit</td></tr><tr><td>USA300HOU_RS01940</td><td>ssb1</td><td>-1.67</td><td>1.64E-02</td><td>Single-stranded DNA-binding protein</td></tr><tr><td>USA300HOU_RS01120</td><td>rplF</td><td>-1.66</td><td>1.69E-02</td><td>Ribosomal protein L6</td></tr><tr><td>USA300HOU_RS02835</td><td>rplJ</td><td>-1.65</td><td>1.75E-02</td><td>Ribosomal protein L10</td></tr><tr><td>USA300HOU_RS02375</td><td>gltB1</td><td>-1.63</td><td>2.23E-02</td><td>Glutamate synthase (NADPH), large subunit</td></tr><tr><td>USA300HOU_RS06050</td><td>pyrF</td><td>-1.62</td><td>3.08E-02</td><td>Orotidine-5'-phosphate decarboxylase</td></tr><tr><td>USA300HOU_RS04905</td><td>oppF1</td><td>-1.58</td><td>1.92E-02</td><td>Oligopeptide ABC superfamily ATP binding cassette transporter, ABC protein</td></tr><tr><td>USA300HOU_RS01935</td><td>rpsF</td><td>-1.56</td><td>2.29E-02</td><td>Ribosomal protein S6</td></tr><tr><td>USA300HOU_RS04620</td><td>dltA</td><td>-1.55</td><td>2.45E-02</td><td>Long-chain-fatty-acid-CoA ligase</td></tr><tr><td>USA300HOU_RS07105</td><td>dapA</td><td>-1.52</td><td>4.99E-02</td><td>dihydrodipicolinate synthase</td></tr><tr><td>USA300HOU_RS04635</td><td>dltD</td><td>-1.51</td><td>3.00E-02</td><td>D-alanine transfer protein DltD</td></tr><tr><td>USA300HOU_RS07190</td><td></td><td>-1.51</td><td>4.86E-02</td><td>Nitric-oxide reductase</td></tr><tr><td>USA300HOU_RS12610</td><td>hutI</td><td>-1.50</td><td>2.99E-02</td><td>Imidazolonepropionase</td></tr><tr><td>USA300HOU_RS02380</td><td>gltD</td><td>-1.50</td><td>4.97E-02</td><td>Glutamate synthase (NADPH) small subunit</td></tr><tr><td>USA300HOU_RS05945</td><td>murD</td><td>-1.50</td><td>2.66E-02</td><td>UDP-N-acetylmuramoylalanine-D-glutamate ligase</td></tr><tr><td>USA300HOU_RS08555</td><td></td><td>-1.49</td><td>2.77E-02</td><td>Biotin carboxylase</td></tr><tr><td>USA300HOU_RS12615</td><td>hutU</td><td>-1.49</td><td>3.30E-02</td><td>Urocanate hydratase</td></tr><tr><td>USA300HOU_RS13410</td><td></td><td>-1.47</td><td>3.09E-02</td><td>Possible decarboxylase</td></tr><tr><td>USA300HOU_RS12085</td><td>rpmD</td><td>-1.46</td><td>2.95E-02</td><td>Ribosomal protein L30</td></tr><tr><td>USA300HOU_RS11770</td><td></td><td>-1.46</td><td>3.46E-02</td><td>Hypothetical membrane protein</td></tr><tr><td>USA300HOU_RS06545</td><td>glpK</td><td>-1.45</td><td>3.79E-02</td><td>Glycerol kinase</td></tr></tbody></table>

(Continued)
<table><caption>TABLE 3 | Continued</caption><thead><tr><th>Gene</th><th>Gene symbol</th><th>log<sub>2</sub> fold change</th><th>p-value</th><th>Description</th></tr></thead><tbody><tr><td>USA300HOU_RS03955</td><td>nrdF</td><td>-1.45</td><td>3.80E-02</td><td>Ribonucleoside-diphosphate reductase subunit beta</td></tr><tr><td>USA300HOU_RS12120</td><td>rplX</td><td>-1.41</td><td>4.01E-02</td><td>Ribosomal protein L24</td></tr><tr><td>USA300HOU_RS01785</td><td>glpT</td><td>-1.41</td><td>3.96E-02</td><td>MOP superfamily multidrug/oligosaccharidyl-lipid/polysaccharide flippase transporter</td></tr><tr><td>USA300HOU_RS11480</td><td>pyrG</td><td>-1.38</td><td>4.72E-02</td><td>CTP synthase</td></tr><tr><td>USA300HOU_RS06895</td><td>parC</td><td>-1.35</td><td>4.42E-02</td><td>DNA topoisomerase (ATP-hydrolyzing) ParC</td></tr><tr><td>USA300HOU_RS01150</td><td>pfl</td><td>1.33</td><td>4.65E-02</td><td>Formate C-acetyltransferase</td></tr><tr><td>USA300HOU_RS12815</td><td></td><td>1.37</td><td>4.46E-02</td><td>Hypothetical lipoprotein</td></tr><tr><td>USA300HOU_RS09730</td><td></td><td>1.39</td><td>4.50E-02</td><td>Hypothetical bacteriophage protein</td></tr><tr><td>USA300HOU_RS14200</td><td>nrdD</td><td>1.42</td><td>3.47E-02</td><td>Anaerobic ribonucleotide reductase large subunit</td></tr><tr><td>USA300HOU_RS03820</td><td></td><td>1.46</td><td>3.56E-02</td><td>Hypothetical membrane protein</td></tr><tr><td>USA300HOU_RS05800</td><td></td><td>1.47</td><td>4.78E-02</td><td>Fibrinogen-binding protein</td></tr><tr><td>USA300HOU_RS03270</td><td></td><td>1.49</td><td>3.19E-02</td><td>Hydrolase</td></tr><tr><td>USA300HOU_RS12795</td><td></td><td>1.51</td><td>2.88E-02</td><td>Hypothetical membrane protein</td></tr><tr><td>USA300HOU_RS03030</td><td></td><td>1.51</td><td>2.58E-02</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS01345</td><td>scdA</td><td>1.52</td><td>2.94E-02</td><td>Cell division and morphogenesis protein ScdA</td></tr><tr><td>USA300HOU_RS11555</td><td></td><td>1.54</td><td>3.52E-02</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS12775</td><td>htrA</td><td>1.55</td><td>4.66E-02</td><td>Hemin ABC superfamily ATP binding cassette transporter, membrane protein</td></tr><tr><td>USA300HOU_RS12700</td><td></td><td>1.56</td><td>3.28E-02</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS03830</td><td></td><td>1.61</td><td>4.27E-02</td><td>Hypothetical membrane protein</td></tr><tr><td>USA300HOU_RS13375</td><td></td><td>1.62</td><td>2.50E-02</td><td>Oligopeptide ABC superfamily ATP binding cassette transporter, binding protein</td></tr><tr><td>USA300HOU_RS13655</td><td></td><td>1.62</td><td>3.16E-02</td><td>Possible hydrolase</td></tr><tr><td>USA300HOU_RS07060</td><td>pstA</td><td>1.63</td><td>3.71E-02</td><td>Phosphate ABC superfamily ATP binding cassette transporter, membrane protein</td></tr><tr><td>USA300HOU_RS08965</td><td></td><td>1.66</td><td>1.63E-02</td><td>Sensor histidine kinase</td></tr><tr><td>USA300HOU_RS05785</td><td></td><td>1.69</td><td>4.13E-02</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS13875</td><td></td><td>1.71</td><td>1.13E-02</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS11550</td><td>dps</td><td>1.71</td><td>1.41E-02</td><td>Dps family stress protein</td></tr><tr><td>USA300HOU_RS14195</td><td>nrdG</td><td>1.72</td><td>1.43E-02</td><td>Anaerobic ribonucleotide reductase small subunit</td></tr><tr><td>USA300HOU_RS01630</td><td></td><td>1.72</td><td>3.52E-02</td><td>Acid phosphatase</td></tr><tr><td>USA300HOU_RS13840</td><td></td><td>1.77</td><td>2.44E-02</td><td>FeoB family ferrous iron (Fe2+) uptake protein</td></tr><tr><td>USA300HOU_RS13100</td><td>hlgC</td><td>1.80</td><td>1.30E-02</td><td>Gamma hemolysin component C</td></tr><tr><td>USA300HOU_RS06060</td><td></td><td>1.83</td><td>4.16E-02</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS01155</td><td>pflA</td><td>1.85</td><td>6.42E-03</td><td>[Formate-C-acetyltransferase]-activating enzyme</td></tr><tr><td>USA300HOU_RS12770</td><td>htrB</td><td>1.86</td><td>2.23E-02</td><td>Hemin ABC superfamily ATP binding cassette transporter, ABC protein</td></tr><tr><td>USA300HOU_RS14125</td><td>EDA</td><td>1.87</td><td>6.17E-03</td><td>Fructose-bisphosphate aldolase</td></tr><tr><td>USA300HOU_RS13320</td><td></td><td>1.89</td><td>2.63E-02</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS01220</td><td></td><td>1.89</td><td>1.78E-02</td><td>ABC superfamily ATP binding cassette transporter, binding protein</td></tr><tr><td>USA300HOU_RS02105</td><td>set3</td><td>1.91</td><td>1.38E-02</td><td>Staphylococcal exotoxin</td></tr><tr><td>USA300HOU_RS01390</td><td></td><td>1.93</td><td>2.02E-02</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS13085</td><td></td><td>1.96</td><td>4.09E-03</td><td>Immunoglobulin G-binding protein SBI</td></tr><tr><td>USA300HOU_RS00580</td><td>spa</td><td>1.99</td><td>3.51E-03</td><td>Immunoglobulin G binding protein A</td></tr><tr><td>USA300HOU_RS00555</td><td></td><td>2.09</td><td>3.39E-03</td><td>Myosin-cross-reactive antigen</td></tr><tr><td>USA300HOU_RS14135</td><td></td><td>2.14</td><td>3.07E-02</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS10525</td><td>mapW2</td><td>2.14</td><td>4.61E-03</td><td>Cell surface protein MapW2</td></tr><tr><td>USA300HOU_RS12790</td><td></td><td>2.18</td><td>5.84E-03</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS07065</td><td>pstC</td><td>2.23</td><td>4.71E-03</td><td>Phosphate ABC superfamily ATP binding cassette transporter, membrane protein</td></tr><tr><td>USA300HOU_RS01225</td><td></td><td>2.26</td><td>9.38E-03</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS01665</td><td></td><td>2.29</td><td>2.74E-03</td><td>Possible CNT family concentrative nucleoside transporter</td></tr><tr><td>USA300HOU_RS13665</td><td></td><td>2.29</td><td>3.12E-03</td><td>MarR family transcriptional regulator</td></tr><tr><td>USA300HOU_RS13095</td><td>hlgA</td><td>2.30</td><td>8.97E-04</td><td>Gamma-hemolysin component A</td></tr><tr><td>USA300HOU_RS01655</td><td></td><td>2.35</td><td>2.51E-03</td><td>PfkB family carbohydrate kinase</td></tr></tbody></table>

(Continued)
<table><caption>TABLE 3 | Continued</caption><thead><tr><th>Gene</th><th>Gene symbol</th><th>log<sub>2</sub> fold change</th><th>p-value</th><th>Description</th></tr></thead><tbody><tr><td>USA300HOU_RS03725</td><td></td><td>2.41</td><td>1.29E-02</td><td>Hypothetical membrane protein</td></tr><tr><td>USA300HOU_RS04290</td><td>nuc</td><td>2.48</td><td>3.60E-03</td><td>Micrococcal nuclease</td></tr><tr><td>USA300HOU_RS13635</td><td></td><td>2.50</td><td>2.35E-03</td><td>ABC superfamily ATP binding cassette transporter, membrane protein</td></tr><tr><td>USA300HOU_RS13830</td><td>clp</td><td>2.51</td><td>2.93E-04</td><td>S14 family endopeptidase Clp</td></tr><tr><td>USA300HOU_RS13660</td><td></td><td>2.53</td><td>1.74E-03</td><td>Possible lactoylglutatione lyase</td></tr><tr><td>USA300HOU_RS01660</td><td></td><td>2.62</td><td>9.10E-04</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS05775</td><td></td><td>2.62</td><td>2.08E-04</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS05855</td><td></td><td>2.80</td><td>1.39E-02</td><td>Exotoxin</td></tr><tr><td>USA300HOU_RS10450</td><td></td><td>2.80</td><td>8.14E-04</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS07070</td><td>pstS</td><td>2.85</td><td>5.80E-05</td><td>Phosphate ABC superfamily ATP binding cassette transporter, binding protein</td></tr><tr><td>USA300HOU_RS05795</td><td></td><td>2.90</td><td>8.96E-05</td><td>Fibrinogen-binding protein</td></tr><tr><td>USA300HOU_RS01235</td><td>hmp</td><td>2.96</td><td>5.40E-05</td><td>Possible nitric oxide dioxygenase</td></tr><tr><td>USA300HOU_RS10965</td><td>agrD</td><td>3.45</td><td>6.02E-04</td><td>Accessory gene regulator protein D</td></tr><tr><td>USA300HOU_RS04615</td><td></td><td>4.03</td><td>5.89E-03</td><td>Hypothetical protein</td></tr><tr><td>USA300HOU_RS13630</td><td></td><td>4.07</td><td>6.62E-06</td><td>ABC superfamily ATP binding cassette transporter, ABC protein</td></tr><tr><td>USA300HOU_RS01365</td><td>lrgB</td><td>4.08</td><td>9.13E-08</td><td>Murein hydrolase regulator LrgB</td></tr><tr><td>USA300HOU_RS01360</td><td>lrgA</td><td>4.36</td><td>1.25E-08</td><td>Murein hydrolase regulator LrgA</td></tr><tr><td>USA300HOU_RS01230</td><td></td><td>6.07</td><td>1.16E-06</td><td>Hypothetical protein</td></tr></tbody></table>

<figure><img src="image_26.png" /></figure>

still attenuated virulence despite increased pigment production which is often associated with virulence.

Persister formation is a phenomenon with highly complex mechanisms. Energy production and protein translation are two vital pathways for bacterial survival and reproduction, and it is generally believed that an overall suppression of

metabolism and replication is a universal cause for bacterial persister formation (Lewis, 2012; Kwan et al., 2013). In *E. coli*, deficiency of energy production genes such as *sucB* and *ubiF* has been found to decrease persister survival (Ma et al., 2010). It has also been shown in *E. coli* that bacteriostatic antibiotic treatment enhances persister formation via suppression of
<figure><img src="image_27.png" /><figcaption>**FIGURE 7 | Validation of RNA-seq by quantitative real-time PCR.**</figcaption></figure>

Relative mRNA levels of transcripts corresponding to USA500 and Δ*ctaB* mutant grown to 6 or 24 h were determined. RNA was obtained from the same samples for RNA-seq and experiments were performed in triplicate. Bars show the fold change of Δ*ctaB* mutant vs. USA500 and error bars indicate standard deviations calculated with the 2⁻ΔΔCT method based on three independent experiments.

<table><caption>**TABLE 4 | Transcription change of pigment production associated genes.**</caption><thead><tr><th>Genes</th><th>Fold change (ΔctaB vs. USA500)</th><th>SD</th></tr></thead><tbody><tr><td>rsbU</td><td>0.90</td><td>0.122</td></tr><tr><td>crtM</td><td>1.18</td><td>0.143</td></tr><tr><td>qoxB</td><td>2.09</td><td>0.276</td></tr><tr><td>citZ</td><td>0.45</td><td>0.089</td></tr><tr><td>flaA (sigB)</td><td>0.91</td><td>0.129</td></tr><tr><td>purA</td><td>0.56</td><td>0.113</td></tr><tr><td>USA300HOU_0726 (NWMN_0672)</td><td>1.38</td><td>0.127</td></tr><tr><td>USA300HOU_ (NWMN_1144)</td><td>3.54</td><td>0.323</td></tr></tbody></table>

cellular respiration (Lobritz et al., 2015). In *S. aureus*, a recent study correlated the drop of ATP level to enhanced persister formation in stationary phase (Pontes et al., 2015). CtaB is a key factor in *S. aureus* respiratory chain and energy production. In stationary phase when most glucose is consumed, *S. aureus* turns to utilize amino acids such as arginine and histidine for energy production (Makhlin et al., 2007). We also found that in stationary phase, multiple genes involved in amino acid metabolism (*argG*, *hutI*, *hisD*, etc.) were inhibited in Δ*ctaB* strain (Table 3). Based on these findings, CtaB depletion might account for augmented persister formation. However, the correlation between respiration and persister formation is far from unveiled. A counter-example has been provided by Mehmet et al. who reported that inhibition of respiration during stationary phase with KCN reduced persister levels in *E. coli* (Orman and Brynildsen, 2015).

While more work needs to be done to investigate the role of respiratory chain in persister formation, it is also important to

further investigate how repression of protein production affects persister formation. The well-understood mechanism of HipAB Toxin-antitoxin system affecting persister formation in *E. coli*, relies on (p)ppGpp to trigger a regulatory cascade involving inorganic polyphosphate (polyP) and Lon, which eventually results in accumulation of (p)ppGpp and persister formation (Rodionov and Ishiguro, 1995; Korch et al., 2003; Germain et al., 2013; Maisonneuve et al., 2013). In our study, CtaB depletion caused strong inhibition of translation by repressing genes involved in multiple aspects of protein production, including amino acid transport (*oppA1*, *oppC1*, *oppD1*, etc.), amino acid synthesis (trpC, aroB), aminoacyl-tRNA biosynthesis (*aspS*, *alaS*, *ileS*, etc.) and ribosome proteins (*rpmC*, *rplF*, *rpsE*, etc.) (Table 3; Figure 6).

It is generally assumed that elevated persistence is associated with better survival and therefore higher virulence in animal models. However, our observation that mutation of CtaB caused attenuated virulence but elevated persister formation, seems paradoxical. Indeed, many infectious diseases are difficult to be treated with antibiotics due to persisters but not resistance (Mulcahy et al., 2010; Welsh et al., 2011). However, we propose that in most cases with MRSA infection the role of virulence is greater than persister formation because: the proportion of persisters is generally small; after antibiotics kill the majority of infecting population of bacteria, the host immune system generally eliminates persisters non-selectively. Nevertheless, the importance of persister formation by MRSA should not be neglected. Future studies are needed to better understand the roles virulence and persistence play in *S. aureus* infection.

In our study, the CtaB mutant only showed elevated persister formation with levofloxacin and ciprofloxacin but no other antibiotics. This may be attributed to the multi-drug resistance of the MRSA strain, which may have masked the defect in persistence to other antibiotics. The difference in persister formation between MRSA and methicillin-sensitive *S. aureus* (MSSA) is worth further investigation.

Our study suggests the importance of heme synthesis in virulence and persister formation of *S. aureus* and provide new insights into the role of CtaB in bacterial respiration in *S. aureus* virulence and persistence. However, one limitation of the study is that we have not dealt specifically with the metabolic aspects of CtaB mutation, such as the efficiency of the respiratory chain in the mutant and possible changes in components of the TCA cycle as well as comparing the phenotypes of *S. aureus* and the *ctaB* mutant in anaerobic conditions. Future studies are needed to address these issues and better understand the relationship between *S. aureus* respiration and virulence and persistence.

## AUTHOR CONTRIBUTIONS

YZ, TX, and WZ designed the work and revised the manuscript; TX, JH, JZ, JC, and NW completed all the experiments; TX and JH performed the statistically analysis and made the figures; TX and YZ wrote the manuscript.
ACKNOWLEDGMENTS

We thank Timothy J. Foster, Moyne Institute of Preventive
Medicine, Department of Microbiology, Trinity College,
Dublin, Ireland, for providing the *E. coli* strain DC10B.
We also thank Ralph Bertram, Klinikum Nürnberg Medical
School GmbH, Research Department, Paracelsus Medical

University, Nuremberg, Germany for helpful discussions about S. aureus gene manipulation and persister assay. This work was supported by the National Natural Science Foundation of China (81572046 and 81471987), and the Key Technologies Research and Development Program for Infectious Diseases of China (2013ZX10003008-003). YZ was supported in part by NIH grants AI99512 and AI108535.

REFERENCES

Anderson, M., Chen, Y. H., Butler, E. K., and Missiakas, D. M. (2011). EsaD, a secretion factor for the Ess pathway in *Staphylococcus aureus*. *J. Bacteriol.* **193**, 1583–1589. doi: **10.1128/JB.01096-10**

Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. *J. Antimicrob. Chemother.* **48**(Suppl. 1), 5–16. doi: **10.1093/jac/48.suppl_1.5**

Atshan, S. S., Shamsudin, M. N., Lung, L. T., Ling, K. H., Sekawi, Z., Pei, C. P., et al. (2012). Improved method for the isolation of RNA from bacteria refractory to disruption, including S. aureus producing biofilm. *Gene* **494**, 219–224. doi: **10.1016/j.gene.2011.12.010**

Bae, T., Banger, A. K., Wallace, A., Glass, E. M., Aslund, F., Schneewind, O., et al. (2004). *Staphylococcus aureus* virulence genes identified by bursa aurealis mutagenesis and nematode killing. *Proc. Natl. Acad. Sci. U.S.A.* **101**, 12312–12317. doi: **10.1073/pnas.0404728101**

Bae, T., and Schneewind, O. (2006). Allelic replacement in *Staphylococcus aureus* with inducible counter-selection. *Plasmid* **55**, 58–63. doi: **10.1016/j.plasmid.2005.05.005**

Bigger, J. (1944). Treatment of staphylococcal infections with penicillin by intermittent sterilisation. *Lancet* **244**, 497–500. doi: **10.1016/S0140-6736(00)74210-3**

Burts, M. L., DeDent, A. C., and Missiakas, D. M. (2008). EsaC substrate for the ESAT-6 secretion pathway and its role in persistent infections of *Staphylococcus aureus*. *Mol. Microbiol.* **69**, 736–746. doi: **10.1111/j.1365-2958.2008.06324.x**

Carrel, M., Perencevich, E. N., and David, M. Z. (2015). USA300 methicillin-
resistant *Staphylococcus aureus*, United States, 2000–2013. *Emer. Infect. Dis.*
**21**, 1973–1980. doi: **10.3201/eid2111.150452**

Clauditz, A., Resch, A., Wieland, K. P., Peschel, A., and Götz, F. (2006). Staphyloxanthin plays a role in the fitness of *Staphylococcus aureus* and its ability to cope with oxidative stress. *Infect. Immun.* **74**, 4950–4953. doi: **10.1128/IAI.00204-06**

Clements, M. O., Watson, S. P., Poole, R. K., and Foster, S. J. (1999). CtaA of *Staphylococcus aureus* is required for starvation survival, recovery, and cytochrome biosynthesis. *J. Bacteriol.* **181**, 501–507.

Collins, L. V., Kristian, S. A., Weidenmaier, C., Faigle, M., Van Kessel, K. P., Van Strijp, J. A., et al. (2002). *Staphylococcus aureus* strains lacking D-alanine modifications of teichoic acids are highly susceptible to human neutrophil killing and are virulence attenuated in mice. *J. Infect. Dis.* **186**, 214–219. doi: **10.1086/341454**

Coulter, S. N., Schwan, W. R., Ng, E. Y., Langhorne, M. H., Ritchie, H. D., Westbrock-Wadman, S., et al. (1998). *Staphylococcus aureus* genetic loci impacting growth and survival in multiple infection environments. *Mol. Microbiol.* **30**, 393–404.

Diep, B. A., Carleton, H. A., Chang, R. F., Sensabaugh, G. F., and Perdreau-
Remington, F. (2006). Roles of 34 virulence genes in the evolution of hospital-
and community-associated strains of methicillin-resistant *Staphylococcus
aureus*. *J. Infect. Dis.* **193**, 1495–1503. doi: **10.1086/503777**

Drabkin, D. L. (1951). Metabolism of the hemin chromoproteins. *Physiol. Rev.* **31**, 345–431.

Germain, E., Castro-Roa, D., Zenkin, N., and Gerdes, K. (2013). Molecular mechanism of bacterial persistence by HipA. *Mol. Cell* **52**, 248–254. doi: **10.1016/j.molcel.2013.08.045**

Giachino, P., Engelmann, S., and Bischoff, M. (2001). Sigma(B) activity depends on RsbU in *Staphylococcus aureus*. *J. Bacteriol.* **183**, 1843–1852. doi: **10.1128/JB.183.6.1843-1852.2001**

Hammer, N. D., Schurig-Briccio, L. A., Gerdes, S. Y., Gennis, R. B., and Skaar, E. P. (2016). CtaM is required for menaquinol oxidase aa3 function in *Staphylococcus aureus*. *mBio* **7:e00823-16. doi: 10.1128/mBio.00823-16**

Han, J., He, L., Shi, W., Xu, X., Wang, S., Zhang, S., et al. (2014). Glycerol uptake is important for L-form formation and persistence in *Staphylococcus aureus*. *PLoS ONE* **9:e108325. doi: 10.1371/journal.pone.0108325**

Hederstedt, L., Lewin, A., and Throne-Holst, M. (2005). Heme A synthase enzyme functions dissected by mutagenesis of *Bacillus subtilis* CtaA. *J. Bacteriol.* **187**, 8361–8369. doi: **10.1128/JB.187.24.8361-8369.2005**

Keren, I., Kaldalu, N., Spoering, A., Wang, Y., and Lewis, K. (2004). Persister cells and tolerance to antimicrobials. *FEMS Microbiol. Lett.* **230**, 13–18. doi: **10.1016/S0378-1097(03)00856-5**

Korch, S. B., Henderson, T. A., and Hill, T. M. (2003). Characterization of the hipA7 allele of *Escherichia coli* and evidence that high persistence is governed by (p)ppGpp synthesis. *Mol. Microbiol.* **50**, 1199–1213. doi: **10.1046/j.1365-2958.2003.03779.x**

Kullik, I., Giachino, P., and Fuchs, T. (1998). Deletion of the alternative sigma factor sigmaB in *Staphylococcus aureus* reveals its function as a global regulator of virulence genes. *J. Bacteriol.* **180**, 4814–4820.

Kwan, G. W., Valenta, J. A., Benedik, M. J., and Wood, T. K. (2013). Arrested protein synthesis increases persister-like cell formation. *Antimicrob. Agents Chemother.* **57**, 1468–1473. doi: **10.1128/AAC.02135-12**

Lan, L., Cheng, A., Dunman, P. M., Missiakas, D., and He, C. (2010). Golden pigment production and virulence gene expression are affected by metabolisms in *Staphylococcus aureus*. *J. Bacteriol.* **192**, 3068–3077. doi: **10.1128/JB.00928-09**

Lechner, S., Lewis, K., and Bertram, R. (2012). *Staphylococcus aureus* persisters tolerant to bactericidal antibiotics. *J. Mol. Microbiol. Biotechnol.* **22**, 235–244. doi: **10.1159/000342449**

Lewis, K. (2001). Riddle of biofilm resistance. *Antimicrob. Agents Chemother.* **45**, 999–1007. doi: **10.1128/AAC.45.4.999-1007.2001**

Lewis, K. (2012). “Persister cells: molecular mechanisms related to antibiotic tolerance,” in *Antibiotic Resistance*, ed A. R. M. Coate (Heidelberg: Springer), 121–133.

Li, M., Du, X., Villaruz, A. E., Diep, B. A., Wang, D., Song, Y., et al. (2012). MRSA epidemic linked to a quickly spreading colonization and virulence determinant. *Nat. Med.* **18**, 816–819. doi: **10.1038/nm.2692**

Liu, G. Y. (2009). Molecular pathogenesis of *Staphylococcus aureus* infection. *Pediatr Res* **65**(5 Pt 2), 71R–77R. doi: **10.1203/PDR.0b013e31819dc44d**

Liu, G. Y., Essex, A., Buchanan, J. T., Datta, V., Hoffman, H. M., Bastian, J. F., et al. (2005). *Staphylococcus aureus* golden pigment impairs neutrophil killing and promotes virulence through its antioxidant activity. *J. Exp. Med.* **202**, 209–215. doi: **10.1084/jem.20050846**

Liu, M., Tanaka, W. N., Zhu, H., Xie, G., Dooley, D. M., and Lei, B. (2008). Direct hemin transfer from IsdA to IsdC in the iron-regulated surface determinant (Isd) heme acquisition system of *Staphylococcus aureus*. *J. Biol. Chem.* **283**, 6668–6676. doi: **10.1074/jbc.M708372200**

Livak, K. J., and Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. *Methods* **25**, 402–408. doi: **10.1006/meth.2001.1262**

Lobritz, M. A., Belenky, P., Porter, C. B., Gutierrez, A., Yang, J. H., Schwarz, E. G., et al. (2015). Antibiotic efficacy is linked to bacterial cellular respiration. *Proc. Natl. Acad. Sci. U.S.A.* **112**, 8173–8180. doi: **10.1073/pnas.1509743112**

Ma, C., Sim, S., Shi, W., Du, L., Xing, D., and Zhang, Y. (2010). Energy production genes sucB and ubiF are involved in persister survival and tolerance to multiple antibiotics and stresses in *Escherichia coli*. *FEMS Microbiol. Lett.* **303**, 33–40. doi: **10.1111/j.1574-6968.2009.01857.x**
Maisonneuve, E., Castro-Camargo, M., and Gerdes, K. (2013). (p)ppGpp controls bacterial persistence by stochastic induction of toxin-antitoxin activity. *Cell* 154, 1140–1150. doi: 10.1016/j.cell.2013.07.048

Makhlin, J., Kofman, T., Borovok, I., Kohler, C., Engelmann, S., Cohen, G., et al. (2007). *Staphylococcus aureus* ArcR controls expression of the arginine deiminase operon. *J. Bacteriol.* 189, 5976–5986. doi: 10.1128/JB.00592-07

Mason, W. J., and Skaar, E. P. (2009). Assessing the contribution of heme-iron acquisition to *Staphylococcus aureus* pneumonia using computed tomography. *PLoS ONE* 4:e6668. doi: 10.1371/journal.pone.006668

Mazmanian, S. K., Skaar, E. P., Gaspar, A. H., Humayun, M., Gornicki, P., Jelenska, J., et al. (2003). Passage of heme-iron across the envelope of *Staphylococcus aureus*. *Science* 299, 906–909. doi: 10.1126/science.1081147

Mechler, L., Herbig, A., Paprotka, K., Fraunholz, M., Nieselt, K., and Bertram, R. (2015). A novel point mutation promotes growth phase-dependent daptomycin tolerance in *Staphylococcus aureus*. *Antimicrob. Agents Chemother.* 59, 5366–5376. doi: 10.1128/AAC.00643-15

Mei, J. M., Nourbakhsh, F., Ford, C. W., and Holden, D. W. (1997). Identification of *Staphylococcus aureus* virulence genes in a murine model of bacteraemia using signature-tagged mutagenesis. *Mol. Microbiol.* 26, 399–407.

Mogi, T., Saiki, K., and Anraku, Y. (1994). Biosynthesis and functional role of haem O and haem A. *Mol. Microbiol.* 14, 391–398. doi: 10.1111/j.1365-2958.1994.tb02174.x

Monk, I. R., Shah, I. M., Xu, M., Tan, M. W., and Foster, T. J. (2012). Transforming the untransformable: application of direct transformation to manipulate genetically *Staphylococcus aureus* and *Staphylococcus epidermidis*. *MBio* 3:e00277–11. doi: 10.1128/mBio.00277-11

Morikawa, K., Maruyama, A., Inose, Y., Higashide, M., Hayashi, H., and Ohta, T. (2001). Overexpression of Sigma Factor, ζB, urges *Staphylococcus aureus* to thicken the cell wall and to resist β-Lactams. *Biochem. Biophys. Res. Commun.* 288, 385–389. doi: 10.1006/bbrc.2001.5774

Mulcahy, L. R., Burns, J. L., Lory, S., and Lewis, K. (2010). Emergence of *Pseudomonas aeruginosa* strains producing high levels of persister cells in patients with cystic fibrosis. *J. Bacteriol.* 192, 6191–6199. doi: 10.1128/JB.01651-09

Orman, M. A., and Brynildsen, M. P. (2015). Inhibition of stationary phase respiration impairs persister formation in *E. coli*. *Nat. Commun.* 6:7983. doi: 10.1038/ncomms8983

Pelz, A., Wieland, K. P., Putzbach, K., Hentschel, P., Albert, K., and Götz, F. (2005). Structure and biosynthesis of staphyloxanthin from *Staphylococcus aureus*. *J. Biol. Chem.* 280, 32493–32498. doi: 10.1074/jbc.M505070200

Peschel, A., Otto, M., Jack, R. W., Kalbacher, H., Jung, G., and Götz, F. (1999). Inactivation of the dlt operon in *Staphylococcus aureus* confers sensitivity to defensins, protegrins, and other antimicrobial peptides. *J. Biol. Chem.* 274, 8405–8410.

Podbielski, A., Pohl, B., Woischnik, M., Körner, C., Schmidt, K. H., Rozdzinski, E., et al. (1996). Molecular characterization of group A streptococcal (GAS) oligopeptide permease (opp) and its effect on cysteine protease production. *Mol. Microbiol.* 21, 1087–1099.

Pontes, M. H., Sevostyanova, A., and Groisman, E. A. (2015). When too much ATP is bad for protein synthesis. *J. Mol. Biol.* 427, 2586–2594. doi: 10.1016/j.jmb.2015.06.021

Reichmann, N. T., Cassona, C. P., and Gründling, A. (2013). Revised mechanism of D-alanine incorporation into cell wall polymers in Gram-positive bacteria. *Microbiology* 159(Pt 9), 1868–1877. doi: 10.1099/mic.0.069898-0

Resch, A., Leicht, S., Saric, M., Päsztor, L., Jakob, A., Götz, F., et al. (2006). Comparative proteome analysis of *Staphylococcus aureus* biofilm and planktonic cells and correlation with transcriptome profiling. *Proteomics* 6, 1867–1877. doi: 10.1002/pmic.200500531

Rodionov, D. G., and Ishiguro, E. E. (1995). Direct correlation between overproduction of guanosine 3',5'-bispyrophosphate (ppGpp) and penicillin tolerance in *Escherichia coli*. *J. Bacteriol.* 177, 4224–4229.

Svensson, B., and Hederstedt, L. (1994). *Bacillus subtilis* CtaA is a heme-containing membrane protein involved in heme A biosynthesis. *J. Bacteriol.* 176, 6663–6671.

Svensson, B., Lübben, M., and Hederstedt, L. (1993). *Bacillus subtilis* CtaA and CtaB function in haem A biosynthesis. *Mol. Microbiol.* 10, 193–201. doi: 10.1111/j.1365-2958.1993.tb00915.x

Szkopinska, A., and Plochocka, D. (2005). Farnesyl diphosphate synthase; regulation of product specificity. *Acta Biochim. Pol.* 52, 45–55. doi: 055201045

Wang, B., and Muir, T. W. (2016). Regulation of virulence in *Staphylococcus aureus*: molecular mechanisms and remaining puzzles. *Cell Chem. Biol.* 23, 214–224. doi: 10.1016/j.chembiol.2016.01.004

Wang, W., Chen, J., Chen, G., Du, X., Cui, P., Wu, J., et al. (2015). Transposon mutagenesis identifies novel genes associated with *Staphylococcus aureus* persister formation. *Front. Microbiol.* 6:1437. doi: 10.3389/fmicb.2015.01437

Welsh, K. J., Skrobarcek, K. A., Abbott, A. N., Lewis, C. T., Kruzel, M. C., Lewis, E. M., et al. (2011). Predictors of relapse of methicillin-resistant *Staphylococcus aureus* bacteremia after treatment with vancomycin. *J. Clin. Microbiol.* 49, 3669–3672. doi: 10.1128/JCM.05287-11

Wieland, B., Feil, C., Gloria-Maercker, E., Thumm, G., Lechner, M., Bravo, J. M., et al. (1994). Genetic and biochemical analyses of the biosynthesis of the yellow carotenoid 4,4'-diaponeurosporene of *Staphylococcus aureus*. *J. Bacteriol.* 176, 7719–7726.

Yee, R., Cui, P., Shi, W., Feng, J., and Zhang, Y. (2015). Genetic screen reveals the role of purine metabolism in *Staphylococcus aureus* persistence to rifampicin. *Antibiotics* 4, 627–642. doi: 10.3390/antibiotics4040627

Zhang, Y. (2014). Persisters, persistent infections and the Yin–Yang model. *Emerg. Microbes Infect.* 3:e3. doi: 10.1038/emi.2014.3

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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<figure><img src="image_28.png" /></figure>

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# Polyphosphate Kinase Mediates Antibiotic Tolerance in Extraintestinal Pathogenic *Escherichia coli* PCN033

Jing Chen¹, Lijie Su², Xiangru Wang¹, Tao Zhang¹, Feng Liu¹, Huanchun Chen¹,³ and Chen Tan¹,³*

¹ State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China, ² School of Public Health, Guangzhou Medical University, Guangzhou, China, ³ Key Laboratory for the Development of Veterinary Diagnostic Products, The Cooperative Innovation Center for Sustainable Pig Production, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, China

Extraintestinal pathogenic *Escherichia coli* (ExPEC) causes a variety of acute infections in its hosts, and multidrug-resistant strains present significant challenges to public health and animal husbandry. Therefore, it is necessary to explore new drug targets to control *E. coli* epidemics. Previous studies have reported that *ppk* mutants of *Burkholderia pseudomallei* and *Mycobacterium tuberculosis* are more susceptible than the wild types (WTs) to stress. Therefore, we investigated the stress response to antibiotics mediated by polyphosphate kinase (PPK) in ExPEC strain PCN033. We observed that planktonic cells of a *ppk* knockout strain (Δ*ppk*) were more susceptible to antibiotics than was WT. However, biofilm-grown Δ*ppk* cells showed similar susceptibility to that of the WT and were more tolerant than the planktonic cells. During the planktonic lifestyle, the expression of genes involved in antibiotic tolerance (including resistance-conferring genes, and antibiotic influx, and efflux genes) did not change in the Δ*ppk* mutant without antibiotic treatment. However, the resistance-conferring gene *bla* and efflux genes were upregulated more in the WT than in the Δ*ppk* mutant by treatment with tazobactam. After treatment with gentamycin, the efflux genes and influx genes were upregulated and downregulated, respectively, more in the WT than in the Δ*ppk* mutant. The expression of genes involved in biofilm regulation also changed after treatment with tazobactam or gentamycin, and which is consistent with the results of the biofilm formation. Together, these observations indicate that PPK is important for the antibiotic stress response during the planktonic growth of ExPEC and might be a potential drug target in bacteria.

**Keywords:** ExPEC, antibiotic resistance, PPK, gene expression, RNA-seq, qPCR

## INTRODUCTION

Extraintestinal pathogenic *Escherichia coli* (ExPEC) is a major cause of urinary tract infections in women, abdominal sepsis, and septicemia in elderly or immunocompromised individuals, and meningitis in newborns, with high morbidity and mortality (Gaschignard et al., 2011; Weston et al., 2011; Mellata, 2013). ExPEC strains commonly colonize domestic animals, such as pigs, chickens, and cattle, causing significant losses in animal husbandry and threatening human health (Girardeau et al., 2003; Johnson et al., 2005; Bergeron et al., 2012). We previously investigated the prevalence

## OPEN ACCESS

**Edited by:**
Etienne Giraud,
Institut National de la Recherche
Agronomique, France

**Reviewed by:**
César De La Fuente-Núñez,
Massachusetts Institute of
Technology, USA
Herman Sintim,
Purdue University, USA

*Correspondence:
Chen Tan
tanchen@mail.hzau.edu.cn

**Specialty section:**
This article was submitted to
Antimicrobials, Resistance and
Chemotherapy,
a section of the journal
Frontiers in Microbiology

Received: 01 December 2015

Accepted: 02 May 2016

Published: 19 May 2016

**Citation:**
Chen J, Su L, Wang X, Zhang T, Liu F, Chen H and Tan C (2016) Polyphosphate Kinase Mediates Antibiotic Tolerance in Extraintestinal Pathogenic Escherichia coli PCN033. *Front. Microbiol.* 7:724. doi: 10.3389/fmicb.2016.00724
of ExPEC in swine across China and detected ExPEC in 10.1% of porcine samples. The frequency of ExPEC isolated from pigs increased between 2004 and 2007 from 3.1 to 14.6% (Tan et al., 2012). The emergence of multidrug-resistant strains has significantly hindered the prevention and control of ExPEC epidemics (Sedláková et al., 2014; Sidrach-Cardona et al., 2014). Therefore, it is urgent that we identify new drug targets to control these increasing E. coli outbreaks.

Polyphosphate kinase (PPK) is an essential enzyme in polyphosphate (polyP) synthesis and has been implicated in many intracellular biological processes. *Pseudomonas aeruginosa* in which *ppk* was deleted showed impairments in motility, quorum sensing, and virulence (Rashid et al., 2000), compacted nucleoids, membrane distortion, extracellular polymer production, and a susceptibility to desiccation (Fraley et al., 2007). PPK also plays a prominent role in the stress response, and a *Burkholderia pseudomallei* *ppk* mutant was susceptible to hydrogen peroxide under oxidative stress conditions (Tunpiboonsak et al., 2010). A *ppk1* mutant strain of *Mycobacterium tuberculosis* displayed a survival defect in response to nitrosative stress, and the negligible levels of polyP were associated with its increased susceptibility to certain tuberculosis drugs (Singh et al., 2013). PPK is highly conserved in bacteria, but is absent in higher mammals (Brown and Kornberg, 2004), indicating that PPK has potential utility as an antibacterial drug target.

As an opportunistic pathogen, E. coli mainly causes acute infections in immunocompromised individuals (Chaudhuri and Henderson, 2012; Mellata, 2013); further, acute infections are associated with its planktonic growth mode (Li et al., 2014). Therefore, we explored the role of PPK in antibiotic resistance in the planktonic cells of ExPEC strain PCN033. Biofilm formation contributes to chronic bacterial infections, such as the recurrent pyelonephritis caused by uropathogenic E. coli in children (Tapiainen et al., 2014). Therefore, we also studied the role of PPK in antibiotic tolerance in biofilm-grown cells.

# MATERIALS AND METHODS

## Bacterial Strains and Culture Conditions

The wild-type (WT) strain used in this study, PCN033, was isolated from a diseased swine in Hubei Province, Central China (Liu et al., 2015). The Δ*ppk* mutant was obtained by in-frame deletion with the suicide plasmid pRE112 (He et al., 2012). Details of both these strains and the primers used in this study are listed in Table S1 (available as Supplementary Data). The antibiotic susceptibility and biofilm formation assays were performed in MOPS broth (an inorganic phosphorus [P₁]-limited medium) at 28°C (Neidhardt et al., 1974). The antibiotics used in the biofilm inhibition assay were added below the minimum inhibitory concentrations (MICs), and had no bactericidal effect on the planktonic cells.

## Growth Characteristics

Fresh colonies of both strains were taken from Luria-Bertani (LB) agar plates, used to inoculate LB broth, and then cultured in MOPS in a shaker incubator for 12 h. The growth

characteristics were monitored turbidimetrically at 600 nm on a spectrophotometer (Eppendorf, Hamburg, Germany) and the colony-forming units (cfu) were counted at 1 h intervals. The generation times were calculated with the formula (Penfold and Norris, 1912):

$$G = \frac{T}{\text{Log}_2^a b}$$

where G is the generation time; T is the length of the logarithmic phase; a is the initial number of bacteria; and b is the final number of bacteria.

## Susceptibility Assay

Each MIC was determined with a series of two-fold dilutions of the antibiotic in MOPS broth, according to the Clinical Laboratory and Standards Institute guidelines. A pre-grown inoculum of each strain was diluted in MOPS to a final concentration of 10⁷ cfu/mL, and the concentration of antibiotic added varied from 0.25 to 512 mg/L. The plates were incubated for 24 h, and the MICs were determined as the lowest antibiotic concentrations that produced no visible growth.

The susceptibility assay of the biofilms was performed as described previously (Benthall et al., 2015), with some modifications. The MIC on the biofilm was determined by allowing a biofilm to form in a 96-well-plate for 24 h. The unattached cells were washed off three times with 0.9% saline. The biofilm was incubated for 24 h with a range of antibiotic concentrations from 512 to 0.25 mg/L. The MIC was defined as the lowest antibiotic concentration at which no bacterial growth was detected. To determine the effect of the biofilm on the bacterial susceptibility to antibiotics, the viability of planktonic and biofilm-grown cells was calculated after the antibiotic treatments. About 10⁷ cfu were incubated with antibiotic concentrations of 2 × MIC for 3 h, and the cfu were then counted.

To quantify the bactericidal activity of these antibiotics on the biofilm, their activity percentage was assessed according to a previous report (Sánchez-Gómez et al., 2015), with some modifications. The ability of these antibiotics to remove the biofilm attached to the microplate was determined with crystal violet (CV) staining. For this purpose, the treated biofilm was stained with CV for 30 min at room temperature. The excess stain was then rinsed off with saline and the CV remaining on the biofilm was dissolved in 33% acetic acid. The absorbance was measured at 595 nm with a Synergy HT microplate reader (BioTek, USA). The activity percentage was calculated according to Sánchez-Gómez et al., with the formula:

$$\text{Activity Percentage} = \frac{(C - B) - (T - B)}{C - B} \times 100$$

where C is the absorbance of the control well-containing untreated biofilm; T is the absorbance of the well-containing treated biofilm; and B is the absorbance of the blank well (i.e., no biofilm).
## Biofilm Formation Assay

Static cultures of biofilm grown at 28°C were analyzed in flat-bottom 96-well-microtiter plates (Corning, USA) using CV. Briefly, approximately $10^7$ cfu were inoculated with sub-MIC antibiotics and cultured for 24 h. The unattached cells were then washed off as described above, fixed with absolute ethanol for 30 min, dried, and stained with 0.1% CV solution for 1 h. The microplates were then washed three times to remove any unattached CV. The CV in the stained biofilm was then dissolved in 33% acetic acid solution and the absorbance read at 595 nm. Each experiment was repeated twice with three technical replicates.

## RNA-seq Assay and Quantitative Real-Time (qRT)-PCR Validation

RNA samples from each strain were prepared for RNA sequencing. Sequencing was performed on an Illumina HiSeq 2500 sequencer (Illumine Inc.) by Shanghai Hanyu Biotechnology Co., Ltd (Shanghai, China). The RNA-seq results were confirmed with qRT-PCR. Before qRT-PCR, the RNA of both strains was extracted with RNAiso Plus reagent (Takara, China). Any genomic DNA contamination was eliminated, and the RNA was reverse transcribed to cDNA with the PrimeScript™RT reagent Kit with gDNA Eraser (Takara). Quantitative real-time PCR was performed in triplicate in optical 96-well-reaction plates (Life Technologies, China) using Power SYBR Green PCR Master Mix (Life Technologies). The primers are listed in Table S1. The mRNA levels of the target genes were normalized to the internal 16S rRNA control with the $ΔΔCt$ method (Kubista et al., 2006). The planktonic cells of the WT and $Δppk$ mutant were cultured to an optical density at a wavelength of 600 nm ($OD_{600}$) of about 0.5. Tazobactam or gentamycin was added at a concentration of 2 × MIC and the cells were incubated for 2 h. The cells were then collected for RNA extraction and qRT-PCR.

## Statistical Analysis

Statistical analysis was performed with the SPSS software (SPSS, Inc., Chicago, IL, USA) on a Windows XP system. Biofilm formation, differential expression, and generation times were compared with one-way analysis of variance (ANOVA). Values are expressed as means ± SD, and statistically significant differences are marked with asterisks. To identify the levels of gene expression, the RNA-seq data were analyzed with an MA-plot-based method with the random sampling model (MARS) in the DEGseq software (http://www.bioconductor.org/packages/release/bioc/html/DESeq.html). Genes showing two-fold changes in expression, a false discovery rate <0.001, and reads per kilobase per million >20 in at least one sample were considered to be differentially expressed under the conditions used.

# RESULTS

## Growth Characteristics

In MOPS minimal medium, the density of the $Δppk$ cultures was slightly higher than that of the WT at each time point tested

(Figure 1). However, the generation times, calculated from both the optical density and cfu, for $Δppk$ ($G_{OD} = 56.5 \pm 4.73$, $G_{CFU} = 42.0 \pm 1.88$) were not significantly different ($P > 0.05$) from those for WT ($G_{OD} = 56.3 \pm 3.64$, $G_{cfu} = 45.9 \pm 4.35$).

## Planktonic Δppk Cells are More Susceptible to Antibiotics than WT Cells

As shown in Table 1, the 17 antibiotics screened in this study were categorized based on their targets: cell wall biosynthesis (type A), protein biosynthesis (type B), nucleotide metabolism (type C), and cell membrane (type D). There were no significant differences in the MIC values for the $Δppk$ strain and WT in LB broth. However, in MOPS broth, $Δppk$ was more susceptible to antibiotics than WT. Specifically, $Δppk$ was much more susceptible to type B antibiotics than was WT, followed by type A antibiotics. Because the WT strain can accumulate more polyP in MOPS broth than in other medi (Ault-Riché et al., 1998), we performed all further assays in MOPS.

## Biofilm-Grown Δppk and WT Cells Are Similarly Tolerant and More Tolerant than Planktonic Cells

To clarify the role of biofilms in antibiotic resistance, an MIC assay using biofilm-grown cells was performed as described previously (Benthall et al., 2015). As shown in Table 2, biofilm-grown $Δppk$ cells showed almost no difference from WT cells in their antibiotic susceptibility, and biofilm-grown cells of both strains were more tolerant than the corresponding planktonic cells. Consistent with this, there was no significant difference in the ability of antimicrobial compounds to kill biofilm-grown cells of the WT and $Δppk$ strains (Figure S1). However, more planktonic cells of the $Δppk$ strain were killed than WT cells (Figure 2). In a future study, we will investigate the role of PPK in antibiotic tolerance within the planktonic growth mode.

## Expression of Antibiotic-Resistance Genes without Antibiotic Treatment

RNA-seq data for the $Δppk$ and WT strains regarding the expression of genes involved in antibiotic resistance, including resistance-conferring genes and antibiotic efflux and influx genes, are presented in Table S2. Of 53 genes known to be involved in antibiotic resistance or multidrug resistance, the expression of one resistance-conferring gene (*tetB*), two efflux genes (*mdtE* and *mdtG*), and one influx gene (*ompC*) was upregulated, and the expression of three efflux genes (*marA*, *marB*, and *mdtA*) was downregulated in the mutant compared with their expression in WT (Figure 3A). The expression of some of these genes was confirmed with qRT-PCR (Figure 3B).

## Expression of Resistance-Conferring Genes after Antibiotic Treatment

Five resistance-conferring genes were selected for analysis when WT was treated with tazobactam, as shown in Figure 4. The expression of beta-lactamase (*bla*) was upregulated, as was that of aminoglycoside 3′N-acetyltransferase III (*aac*), fused UDP-L-Ara4N formyltransferase (*arnA*), and nitroreductase A
<figure><img src="image_29.png" /><figcaption>FIGURE 1 | Growth characteristics of *E. coli* strains PCN033 (wild type) and Δ*ppk* in MOPS broth: (A) optical density, and (B) colony-forming units.</figcaption></figure>

<table><caption>TABLE 1 | Assay of the susceptibility of planktonic cells to antibiotics (mg/L).</caption><thead><tr><th rowspan="2">Target (type)</th><th rowspan="2">antibiotic category</th><th rowspan="2">compound</th><th colspan="2">MIC in LB</th><th rowspan="2">MIC change (fold)</th><th colspan="2">MIC in MOPS</th><th rowspan="2">MIC fold (fold)</th><th rowspan="2">References</th></tr><tr><th>PCN033</th><th>Δppk</th><th>PCN033</th><th>Δppk</th></tr></thead><tbody><tr><td rowspan="6">Cell wall biosynthesis(A)</td><td rowspan="6">β-lactams</td><td>cefotaxime</td><td>2</td><td>1</td><td>2</td><td>16</td><td>4</td><td>4</td><td rowspan="6">Bush, 2012</td></tr><tr><td>ceftazidime</td><td>256</td><td>256</td><td>1</td><td>512</td><td>512</td><td>1</td></tr><tr><td>ampicillin</td><td>>512</td><td>>512</td><td>1</td><td>>512</td><td>>512</td><td>1</td></tr><tr><td>cefazolin</td><td>>512</td><td>>512</td><td>1</td><td>>512</td><td>>512</td><td>1</td></tr><tr><td>tazobactam</td><td>>512</td><td>>512</td><td>1</td><td>>512</td><td>256</td><td>>2</td></tr><tr><td>ticarcillin</td><td>>512</td><td>>512</td><td>1</td><td>>512</td><td>>512</td><td>1</td></tr><tr><td rowspan="4">Protein biosynthesis (B)</td><td rowspan="4">Glycopeptide</td><td>vancomycin</td><td>>512</td><td>>512</td><td>1</td><td>128</td><td>64</td><td>2</td><td rowspan="4">Davis, 1987</td></tr><tr><td rowspan="3">Aminoglycosides</td><td>gentamicin</td><td>>512</td><td>>512</td><td>1</td><td>256</td><td>32</td><td>8</td></tr><tr><td>gentamicin sulfate</td><td>>512</td><td>>512</td><td>1</td><td>256</td><td>64</td><td>4</td></tr><tr><td>amikacin</td><td>>512</td><td>>512</td><td>1</td><td>>512</td><td>64</td><td>≥4</td></tr><tr><td rowspan="4">Nucleotide metabolism (C)</td><td rowspan="2">Macrolide</td><td>Erythromycin</td><td>512</td><td>512</td><td>1</td><td>512</td><td>128</td><td>4</td><td rowspan="2">Brisson-Noël et al., 1988</td></tr><tr><td>norfloxacin</td><td>>512</td><td>512</td><td>>1</td><td>512</td><td>512</td><td>1</td><td rowspan="2">Aldred et al., 2014</td></tr><tr><td rowspan="2">Quinolones</td><td>levofloxacin</td><td>128</td><td>64</td><td>2</td><td>64</td><td>16</td><td>4</td><td rowspan="2">Pérez-Trallero and Iglesias, 2003</td></tr><tr><td>Trimethoprim</td><td>128</td><td>128</td><td>1</td><td>256</td><td>128</td><td>2</td><td rowspan="2">Hof, 1988</td></tr><tr><td rowspan="3">Membrane (D)</td><td rowspan="3">Nitrofurans</td><td>sulfadiazine</td><td>>512</td><td>>512</td><td>>1</td><td>>512</td><td>>512</td><td>1</td><td rowspan="3">Grau-Campistany et al., 2015</td></tr><tr><td>macrodantin</td><td>512</td><td>256</td><td>2</td><td>128</td><td>128</td><td>1</td></tr><tr><td>polymyxin B</td><td>0.5</td><td>≤0.25</td><td>≥2</td><td>2</td><td>1</td><td>2</td></tr></tbody></table>

(nfsA). The bla, arnA, and sulfate adenylyltransferase (cysN) genes were also upregulated in the Δ*ppk* mutant after tazobactam treatment, but their expression was higher in WT than in the Δ*ppk* mutant. The expression of resistance-conferring gene was not significantly altered by gentamycin treatment, except for cysN.

## Expression of Genes Involved in Antibiotic Transport Was Altered More Strongly in WT than in the Δ*ppk* Mutant

The expression of six genes involved in antibiotic efflux was determined, as shown in Figure 5A. The efflux genes were upregulated in both the WT and Δ*ppk* mutant strains by tazobactam and gentamycin. However, the expression of acrA, cusC, and marA was higher in WT than in the Δ*ppk* mutant after

treatment with tazobactam. After treatment with gentamycin, the expression of acrA and marA was also higher in WT than in the Δ*ppk* mutant. The expression of three genes involved in antibiotic influx was determined, as shown in Figure 5B. All the genes tested were downregulated by antibiotic treatment. However, *ompF* expression decreased more strongly in WT than in the Δ*ppk* mutant when treated with tazobactam. The expression of *ompF*, *ompC*, and *phoE* also decreased more strongly in WT than in the Δ*ppk* mutant after treatment with gentamycin.

## Effects of Tazobactam and Gentamycin in Biofilm Formation

The RNA-seq data showed that the transcription levels of some genes associated with biofilm formation were altered
<table><caption>TABLE 2 | Assay of the susceptibility of biofilm-grown cells to antibiotics (mg/L).</caption><thead><tr><th rowspan="2">Target (type)</th><th rowspan="2">antibiotic category</th><th rowspan="2">compound</th><th colspan="2">MIC for biofilm</th><th rowspan="2">MIC change (fold)</th></tr><tr><th>PCN033</th><th>Δppk</th></tr></thead><tbody><tr><td rowspan="7">Cell wall biosynthesis (A)</td><td rowspan="7">β-lactams</td><td>cefotaxime</td><td>128</td><td>256</td><td>0.5</td></tr><tr><td>ceftazidime</td><td>512</td><td>512</td><td>1</td></tr><tr><td>ampicillin</td><td>>512</td><td>>512</td><td>1</td></tr><tr><td>cefazolin</td><td>>512</td><td>>512</td><td>1</td></tr><tr><td>tazobactam</td><td>512</td><td>>512</td><td>≤0.5</td></tr><tr><td>ticarcillin</td><td>>512</td><td>>512</td><td>1</td></tr><tr><td rowspan="7">Protein biosynthesis (B)</td><td rowspan="7">Aminoglycosides</td><td>gentamicin</td><td>512</td><td>512</td><td>1</td></tr><tr><td>gentamicin sulfate</td><td>512</td><td>256</td><td>2</td></tr><tr><td>amikacin</td><td>512</td><td>512</td><td>1</td></tr><tr><td>erythromycin</td><td>512</td><td>512</td><td>1</td></tr><tr><td>norfloxacin</td><td>512</td><td>512</td><td>1</td></tr><tr><td>levofloxacin</td><td>32</td><td>32</td><td>1</td></tr><tr><td rowspan="3">Nucleotide metabolism (C)</td><td rowspan="3">Sulfonamides</td><td>Trimethoprim</td><td>512</td><td>512</td><td>1</td></tr><tr><td>sulfadiazine</td><td>>512</td><td>>512</td><td>1</td></tr><tr><td>macrodantin</td><td>512</td><td>512</td><td>1</td></tr><tr><td>Membrane (D)</td><td>Lipopeptde</td><td>polymyxin B</td><td>4</td><td>4</td><td>1</td></tr></tbody></table>

<figure><img src="image_30.png" /></figure>

in the Δppk strain (Tables S3, S4), especially those encoding the flagella cluster, which simultaneously promotes biofilm generation and impedes biofilm maturation (Laverty et al., 2014). The expression of fimbrial and curli genes was also reduced in the Δppk mutant. The expression levels of some genes were confirmed with qRT-PCR (Figure 6A). The expression of four genes (yddV, mcbR, bolA, and csgD) involved in biofilm regulation was determined during antibiotic treatment, as shown

in Figure 6B (Laverty et al., 2014; Lord et al., 2014; Dressaire et al., 2015; Wu et al., 2015). The expression of yddV, mcbR, and bolA was upregulated in WT but downregulated in the Δppk mutant when treated with tazobactam, and all four genes were upregulated in WT but downregulated in the Δppk mutant when treated with gentamycin. Biofilm formation was also evaluated in the presence of tazobactam or gentamycin, as shown Figure 6C. Biofilm formation increased in WT planktonic
<figure><img src="image_31.png" /><figcaption>**FIGURE 6** | Biofilm formation assay. (A) Validation of the expression of genes involved in biofilm formation. (B) Expression levels of genes involved in biofilm regulation during tazobactam and gentamycin treatment. (C) Biofilm formation during tazobactam or gentamycin treatment. Expression levels of genes were compared with those in WT (PCN033). 33-TZP/PCN033 indicates the expression levels in PCN033 during tazobactam treatment compared with those in untreated PCN033; K-TZP/PCN033 indicates the expression levels in the *Δppk* mutant during tazobactam treatment compared with those in untreated PCN033; 33-GEN/PCN033 indicates the expression levels in PCN033 during gentamycin treatment compared with those in untreated PCN033; K-GEN/PCN033 indicates the expression levels in the *Δppk* mutant during gentamycin treatment compared with those in untreated PCN033. WT indicates PCN033 without antibiotic treatment; WT treated indicates PCN033 with antibiotic treatment; PPK-KO indicates the *Δppk* mutant without antibiotic treatment; PPK-KO treated indicates the *Δppk* mutant with antibiotic treatment. \*\*\*p < 0.000, \*p < 0.01, \*p < 0.05.</figcaption></figure>

cells but decreased in Δ*ppk* planktonic cells when treated with antibiotics.

DISCUSSION

Multidrug-resistant strains of ExPEC present significant challenges to public health and animal husbandry (Girardeau et al., 2003; Johnson et al., 2005; Bergeron et al., 2012). Because pathogenic *E. coli* mainly causes acute infections in its planktonic growth mode (Li et al., 2014), we initially investigated the role of PPK in the antibiotic resistance of ExPEC in the planktonic growth mode. We investigated in detail its susceptibility to different types of antibiotics, mediated by PPK, in *E. coli*. We

found that PPK is very important in aminoglycoside tolerance, regulating the expression levels of antibiotic efflux and influx genes in the planktonic growth mode. Our findings indicate that PPK could have utility as a novel antimicrobial drug target.

As reported previously, resistance-conferring proteins and antibiotic efflux and influx porins play important roles in multidrug-resistance. Efflux proteins contribute to antibiotic tolerance by transporting compounds to the extracellular environment, whereas influx proteins have the opposite effect (Wilson, 2014). The expression levels of these genes were determined with RNA-seq, and showed that without antibiotic treatment, they did not differ significantly between WT and the Δ*ppk* mutant. Because PPK is reported to play prominent
roles in the stress responses elicited by other stimuli (Alcántara
et al., 2014), we investigated the role of PPK in the antibiotic
stress response. Gentamycin and tazobactam were selected to
treat the planktonic cells of WT and the Δppk mutant. With
gentamycin treatment, the expression of the efflux genes acrA
and marA increased more strongly in WT than that in the Δppk
mutant, and the influx porin genes ompC and ompF decreased
more strongly in WT than in the Δppk mutant. Gentamycin
binds the 30S ribosomal subunit and interrupts protein synthesis,
thus inhibiting bacterial multiplication (Wargo and Edwards,
2014). According to Gray et al., compounds that interrupt protein
metabolism cause intracellular polyP accumulation (Gray et al.,
2014). Because it is a high-energy phosphate compound, polyP
can be used to phosphorylate the response regulators of two-
component systems to regulate gene expression (Sureka et al.,
2007). As reported previously, the two-component systems CpxR
and BaeR are implicated in antibiotic resistance by regulating
the efflux genes of the acr operon and mar operon (Hu et al.,
2011; Weatherspoon-Griffin et al., 2014; Pletzer et al., 2015).
We speculated that phosphorylation of BaeR or CpxR using
polyP as phosphate donor to modulate expression of acrA and
marA during gentamycin treatment. The expression of porin
genes ompF and ompC is upregulated by cAMP (Dalhoff, 1983),
and the level of cAMP is negatively regulated by polyP, which
potently inhibits the activity of the class III adenylate cyclases
(Guo et al., 2009). Therefore, we speculated that the expression of
ompC and ompF was downregulated by polyP during gentamycin
treatment. Therefore, polyP may influence gentamycin tolerance
by regulating the expression of antibiotic efflux and influx
genes.

With tazobactam treatment, the expression of the resistance gene *bla* and efflux genes *acrA*, *cusC*, and *marA* was upregulated. Tazobactam binds to the periplasmic β-lactamase, and the efflux pump is implicated in resistance to beta-lactams and beta-lactamase inhibitors (Zhanel et al., 2014). However, there are insufficient data to clarify the role of PPK in regulating the expression of efflux pump genes induced by β-lactams. It will be interesting to explore the role of PPK in this process.

Biofilms contribute to antibiotic tolerance and chronic
infection; thus, we also investigated the role of PPK in the
antibiotic resistance of biofilm-grown cells. We observed that
biofilm formation was impaired in the Δppk mutant when
treated with antibiotics. The genes involved in biofilm formation
(such as those encoding the fimbriae cluster, flagella cluster,
and biofilm regulators BoA and McbR), were downregulated
in the Δppk mutant by both antibiotic treatments. PolyP
acts as a “chemical chaperone,” stabilizing cytoplasmic proteins
intracellularly, similarly to heat shock proteins (Gray et al., 2014),
and chaperones are known to be involved in biofilm formation.
For example, the chaperone CsgE directs the intracellular
localization of CsgA, the major subunit of the extracellular
amyloid protein known as curli, which is essential for biofilm

formation (Andersson et al., 2013). The FliS protein acts as a
chaperone for FliC, a flagellar structural protein that promotes
biofilm generation (Xu et al., 2014). The universal heat shock
protein chaperones are also implicated in biofilm formation by
fungi, such as *Candida albicans* (Robbins et al., 2011; Becherelli
et al., 2013), and by Gram-negative bacteria, such as *E. coli*
(Grudniak et al., 2013). Ultimately, these different effects reduce
biofilm impairment during antibiotic treatment. Interestingly,
PPK did not affect the antibiotic tolerance of ExPEC in the
biofilm growth mode. Biofilms manifest antibiotic tolerance
through many different mechanisms, including preventing the
passage of antimicrobial compounds into the cytoplasm and
possessing densely adherent growth (Qu et al., 2010). As reported
previously, the planktonic and biofilm modes of growth are
two distinct bacterial “lifestyles” (Chua et al., 2014), so it will
be interesting to explore the roles of PPK in these distinct
contexts.

AUTHOR CONTRIBUTIONS

JC and CT designed and supervised the research project.
JC wrote the paper, and LS, XW, HC, and TZ revised the
manuscript. JC, TZ, and FL performed the experiments.
JC and LS processed the data and performed the statistical
analysis. All the authors have read and approved the
manuscript.

FUNDING

This work was supported by grants from the National Natural Science Foundation of China (grant numbers 31472201, 31030065, and 31421064) and the Programme for Introducing Talents of Discipline to Universities (grant number B12005).

ACKNOWLEDGMENTS

The authors wish to thank Dr. Janine Miller from the University
of Tasmania for writing improvement.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found
online at: http://journal.frontiersin.org/article/10.3389/fmicb.
2016.00724

<caption>**Figure S1 | Percentage activity of different antibiotics in removing biofilm.**
(A) Targeting the cell wall, (B) targeting protein biosynthesis, and (C) targeting
nucleotide metabolism.</caption>

<caption>Table S1 | Bacteria strains and primers.</caption>

<caption>Table S2 | Transcription level of genes involved in antibiotic and multi-drug resistance.</caption>

<caption>Table S3 | Expression of genes implicated in biofilm.</caption>

<caption>Table S4 | Genes involved in biofilm formation (part results of RNA-seq).</caption>
REFERENCES

Alcántara, C., Blasco, A., Zúñiga, M., and Monedero, V. (2014). Accumulation of polyphosphate in *Lactobacillus* spp. and its involvement in stress resistance. *Appl. Environ. Microbiol.* 80, 1650–1659. doi: 10.1128/AEM.03997-13

Aldred, K. J., Kerns, R. J., and Osheroff, N. (2014). Mechanism of quinolone action and resistance. *Biochemistry* 53, 1565–1574. doi: 10.1021/bi000564

Andersson, E. K., Bengtsson, C., Evans, M. L., Chorell, E., Sellstedt, M., and Lindgren, A. E. (2013). Modulation of curli assembly and pellicle biofilm formation by chemical and protein chaperones. *Chem. Biol.* 20, 1245–1254. doi: 10.1016/j.chembiol.2013.07.017

Ault-Riché, D., Fraley, C. D., Tzeng, C. M., and Kornberg, A. (1998). Novel assay reveals multiple pathways regulating stress-induced accumulations of inorganic polyphosphate in *Escherichia coli*. *J. Bacteriol.* 180, 1841–1847.

Becherelli, M., Tao, J., and Ryder, N. S. (2013). Involvement of heat shock proteins in *Candida albicans* biofilm formation. *J. Mol. Microbiol. Biotechnol.* 23, 396–400. doi: 10.1159/000351619

Benthall, G., Touzel, R. E., Hind, C. K., Titball, R. W., Sutton, J. M., and Thomas, R. J. (2015). Evaluation of antibiotic efficacy against infections caused by planktonic or biofilm cultures of *Pseudomonas aeruginosa* and *Klebsiella pneumoniae* in *Galleria mellonella*. *Int. J. Antimicrob. Agents* 46, 538–545. doi: 10.1016/j.ijantimicag.2015.07.014

Bergeron, C. R., Prussing, C., Boerlin, P., Daignault, D., Dutil, L., and Reid-Smith, R. J. (2012). Chicken as reservoir for extraintestinal pathogenic *Escherichia coli* in humans, Canada. *Emerg. Infect. Dis.* 18, 415–421. doi: 10.3201/eid1803.111099

Brisson-Noël, A., Trieu-Cuot, P., and Courvalin, P. (1988). Mechanism of action of spiramycin and other macrolides. *J. Antimicrob. Chemother.* 22 (Suppl B), 1–23.

Brown, M. R., and Kornberg, A. (2004). Inorganic polyphosphate in the origin and survival of species. *Proc. Natl. Acad. Sci. U.S.A.* 101, 16085–16087. doi: 10.1073/pnas.0406909101

Bush, K. (2012). Antimicrobial agents targeting bacterial cell walls and cell membranes. *Rev. Sci. Tech.* 31, 43–56. doi: 10.20506/rst.31.1.2096

Chaudhuri, R. R., and Henderson, I. R. (2012). The evolution of the *Escherichia coli* phylogeny. *Infect. Genet. Evol.* 12, 214–226. doi: 10.1016/j.meegid.2012.01.005

Chua, S. L., Liu, Y., Yam, J. K., Chen, Y., Vejborg, R. M., and Tan, B. G. (2014). Dispersed cells represent a distinct stage in the transition from bacterial biofilm to planktonic lifestyles. *Nat. Commun.* 21, 4462. doi: 10.1038/ncomms5462

Dalhoff, A. (1983). Transport of aminoglycosides in *Escherichia coli*. *Z. Bakteriol. Mikrobiol. Hyg. A.* 254, 379–387.

Davis, B. D. (1987). Mechanism of bactericidal action of aminoglycosides. *Microbiol. Rev.* 51, 341–350.

Dressaire, C., Moreira, R. N., Barahona, S., Alves de Matos, A. P., and Arraiano, C. M. (2015). BolA is a transcriptional switch that turns off motility and turns on biofilm development. *MBio* 6, e02352–e02314. doi: 10.1128/mBio.02352-14

Fraley, C. D., Rashid, M. H., Lee, S. S., Gottschalk, R., Harrison, J., and Wood, P. J. (2007). A polyphosphate kinase 1 (ppk1) mutant of *Pseudomonas aeruginosa* exhibits multiple ultrastructural and functional defects. *Proc. Natl. Acad. Sci. U.S.A.* 104, 3526–3531. doi: 10.1073/pnas.0609733104

Gaschignard, J., Levy, C., Romain, O., Cohen, R., Bingen, E., and Aujard, Y. (2011). Neonatal bacterial meningitis: 444 cases in 7 years. *Pediatr. Infect. Dis. J.* 30, 212–217. doi: 10.1097/INF.0b013e3181fa1e7

Girardeau, J. P., Lalioui, L., Said, A. M., De Champs, C., and Le Bouguénec, C. (2003). Extended virulence genotype of pathogenic *Escherichia coli* isolates carrying the afa-8 operon: evidence of similarities between isolates from humans and animals with extraintestinal infections. *J. Clin. Microbial.* 41, 218–226. doi: 10.1128/JCM.41.1.218-226.2003

Grau-Campistany, A., Manresa, Á., Pujol, M., Rabanal, F., and Cajal, Y. (2015). Tryptophan-containing lipopeptide antibiotics derived from polymyxin B with activity against Gram positive and Gram negative bacteria. *Biochim. Biophys. Acta* 1858, 333–343. doi: 10.1016/j.bbamem.2015.11.011

Gray, M. J., Wholey, W. Y., Wagner, N. O., Cremers, C. M., Mueller-Schickert, A., and Hock, N. T. (2014). Polyphosphate is a primordial chaperone. *Mol. Cell.* 53, 689–699. doi: 10.1016/j.molcel.2014.01.012

Grudniak, A. M., Pawlak, K., Bartosik, K., and Wolska, I. K. (2013). Physiological consequences of mutations in the htpG heat shock gene of *Escherichia coli*. *Mutat. Res.* 745–746, 1–5. doi: 10.1016/j.mrfmmm.2013.04.003

Guo, Y. L., Mayer, H., Vollmer, W., Dittrich, D., Sander, P., and Schultz, A. (2009). Polyphosphates from *Mycobacterium bovis*-potent inhibitors of class III adenylate cyclases. *FEBS J.* 276, 1094–1103. doi: 10.1111/j.1742-4658.2008.06852.x

He, Y., Xu, T., Fossheim, L. E., and Zhang, X. H. (2012). FliC, a flagellin protein, is essential for the growth and virulence of fish pathogen *Edwardsiella tarda*. *PLoS ONE.* 7:e45070. doi: 10.1371/journal.pone.0045070

Hof, H. (1988). Antimicrobial therapy with nitroheterocyclic compounds, for example, metronidazole and nitrofurantoin. *Immun. Infekt.* 16, 220–225.

Hu, W. S., Chen, H. W., Zhang, R. Y., Huang, C. Y., and Shen, C. F. (2011). The expression levels of outer membrane proteins STM1530 and OmpD, which are influenced by the CpxAR and BaeSR two-component systems, play important roles in the ceftriaxone resistance of *Salmonella enterica* serovar Typhimurium. *Antimicrob. Agents Chemother.* 55, 3829–3837. doi: 10.1128/AAC.00216-11

Johnson, J. R., Kuskowski, M. A., Smith, K., O'Bryan, T. T., and Tatini, S. (2005). Antimicrobial-resistant and extraintestinal pathogenic *Escherichia coli* in retail foods. *J. Infect. Dis.* 191, 1040–1049. doi: 10.1086/428451

Kubista, M., Andrade, J. M., Bengtsson, M., Forootan, A., Jonák, J., and Lind, K. (2006). The real-time polymerase chain reaction. *Mol. Aspects Med.* 27, 195–125. doi: 10.1016/j.mam.2005.12.007

Laverty, G., Gorman, S. P., and Gilmore, B. F. (2014). Biomolecular mechanisms of *Pseudomonas aeruginosa* and *Escherichia coli* biofilm formation. *Pathogens* 3, 596–632. doi: 10.3390/pathogens3030596

Li, Y., Petrova, O. E., Su, S., Lau, G. W., Panmanee, W., and Na, R. (2014). BdLA, DipA and induced dispersion contribute to acute virulence and chronic persistence of *Pseudomonas aeruginosa*. *PLoS Pathog.* 10:e1004168. doi: 10.1371/journal.ppat.1004168

Liu, C., Zheng, H., Yang, M., Xu, Z., Wang, X., and Wei, L. (2015). Genome analysis and *in vivo* virulence of porcine extraintestinal pathogenic *Escherichia coli* strain PCN033. *BMC Genomics.* 16:717. doi: 10.1186/s12864-015-1890-9

Lord, D. M., Uzgoren Baran, A., Soo, V. W., Wood, T. K., Peti, W., and Page, R. (2014). McbR/YncC: implications for the mechanism of ligand and DNA binding by a bacterial GntR transcriptional regulator involved in biofilm formation. *Biochemistry* 53, 7223–7231. doi: 10.1021/bi500871a

Mellата, M. (2013). Human and avian extraintestinal pathogenic *Escherichia coli*: infections, zoonotic risks, and antibiotic resistance trends. *Foodborne Pathog. Dis.* 10, 916–932. doi: 10.1089/fpd.2013.1533

Neidhardt, F. C., Bloch, P. L., and Smith, D. F. (1974). Culture medium for enterobacteria. *J. Bacteriol.* 119, 736–747.

Penfold, W. J., and Norris, D. (1912). The relation of concentration of food supply to the generation-time of bacteria. *J. Hyg.* 12, 527–531. doi: 10.1017/S0022172400005180

Pérez-Trallero, E., and Iglesias, L. (2003). Tetracyclines, sulfonamides and metronidazole. *Enferm. Infecc. Microbiol. Clin.* 21, 520–528. doi: 10.1016/S0213-005X(03)72999-1

Pletzer, D., Stahl, A., Oja, A. E., and Weingart, H. (2015). Role of the cell envelope stress regulators BaeR and CpxR in control of RND-type multidrug efflux pumps and transcriptional cross talk with exopolysaccharide synthesis in *Erwinia amylovora*. *Arch. Microbiol.* 197, 761–772. doi: 10.1007/s00203-015-1109-0

Qu, Y., Daley, A. J., Istivan, T. S., Rouch, D. A., and Deighton, M. A. (2010). Densely adherent growth mode, rather than extracellular polymer substance matrix build-up ability, contributes to high resistance of *Staphylococcus epidermidis* biofilms to antibiotics. *J. Antimicrob. Chemother.* 65, 1405–1411. doi: 10.1093/jac/dkq119

Rashid, M. H., Rumbaugh, K., Passador, L., Davies, D. G., Hamood, A. N., and Iglewski, B. H. (2000). Polyphosphate kinase is essential for biofilm development, quorum sensing, and virulence of *Pseudomonas aeruginosa*. *Proc. Natl. Acad. Sci. U.S.A.* 97, 9636–9641. doi: 10.1073/pnas.170283399

Robbins, N., Uppuluri, P., Nett, J., Rajendran, R., Ramage, G., and Lopez-Ribot, J. L. (2011). Hsp90 governs dispersion and drug resistance of fungal biofilms. *PLoS Pathog.* 7:e1002257. doi: 10.1371/journal.ppat.1002257

Sánchez-Gómez, S., Ferrer-Espada, R., Stewart, P. S., Pitts, B., Lohner, K., and Martínez de Tejada, G. (2015). Antimicrobial activity of synthetic cationic peptides and lipopeptides derived from human lactoferricin against *Pseudomonas aeruginosa* planktonic cultures and biofilms. *BMC Microbiol.* 15:137. doi: 10.1186/s12866-015-0473-x
Sedláková, M. H., Urbánek, K., Vojtová, V., Suchánková, H., Imwensi, P., and Kolář, M. (2014). Antibiotic consumption and its influence on the resistance in *Enterobacteriaceae*. *BMC Res. Notes*. 7:454. doi: 10.1186/1756-0500-7-454

Sidrach-Cardona, R., Hijosa-Valsero, M., Marti, E., Balcázar, J. L., and Becares, E. (2014). Prevalence of antibiotic-resistant fecal bacteria in a river impacted by both an antibiotic production plant and urban treated discharges. *Sci. Total Environ.* 488, 220–227. doi: 10.1016/j.scitotenv.2014.04.100

Singh, R., Singh, M., Arora, G., Kumar, S., Tiwari, P., and Kidwai, S. (2013). Polyphosphate deficiency in Mycobacterium tuberculosis is associated with enhanced drug susceptibility and impaired growth in guinea pigs. *J. Bacteriol.* 195, 2839–2851. doi: 10.1128/JB.00038-13

Sureka, K., Dey, S., Datta, P., Singh, A. K., Dasgupta, A., and Rodrigue, S. (2007). Polyphosphate kinase is involved in stress-induced mprAB-sigE-rel signalling in mycobacteria. *Mol. Microbiol.* 65, 261–276. doi: 10.1111/j.1365-2958.2007.05814.x

Tan, C., Tang, X., Zhang, X., Ding, Y., Zhao, Z., and Wu, B. (2012). Serotypes and virulence genes of extraintestinal pathogenic *Escherichia coli* isolates from diseased pigs in China. *Vet. J.* 192, 483–488. doi: 10.1016/j.tvjl.2011.06.038

Tapiainen, T., Hanni, A. M., Salo, J., Ikäheimo, I., and Uhari, M. (2014). *Escherichia coli* biofilm formation and recurrences of urinary tract infections in children. *Eur. J. Clin. Microbiol. Infect. Dis.* 33, 111–115. doi: 10.1007/s10096-013-1935-4

Tunpiboonsak, S., Mongkolrob, R., Kitudomsub, K., Thanwatanaying, P., Kiettipirodom, W., and Tungboontina, Y. (2010). Role of a *Burkholderia pseudomallei* polyphosphate kinase in an oxidative stress response, motilities, and biofilm formation. *J. Microbiol.* 48, 63–70. doi: 10.1007/s12275-010-9138-5

Wargo, K. A., and Edwards, J. D. (2014). Aminoglycoside-induced nephrotoxicity. *J. Pharm. Pract.* 27, 573–577. doi: 10.1177/0897190014546836

Weatherspoon-Griffin, N., Yang, D., Kong, W., Hua, Z., and Shi, Y. (2014). The CpxR/CpxA two-component regulatory system up-regulates the multidrug resistance cascade to facilitate *Escherichia coli* resistance to a model antimicrobial peptide. *J. Biol. Chem.* 289, 32571–32582. doi: 10.1074/jbc.M114.565762

Weston, E. J., Pondo, T., Lewis, M. M., Martell-Cleary, P., Morin, C., and Jewell, B. (2011). The burden of invasive early-onset neonatal sepsis in the United States, 2005-2008. *Pediatr. Infect. Dis. J.* 30, 937–941. doi: 10.1097/INF.0b013e318223bad2

Wilson, D. N. (2014). Ribosome-targeting antibiotics and mechanisms of bacterial resistance. *Nat. Rev. Microbiol.* 12, 35–48. doi: 10.1038/nr micro3155

Wu, Y., Ding, Y., Cohen, Y., and Cao, B. (2015). Elevated level of the second messenger c-di-GMP in *Comamonas testosteroni* enhances biofilm formation and biofilm-based biodegradation of 3-chloroaniline. *Appl. Microbiol. Biotechnol.* 99, 1967–1976. doi: 10.1007/s00253-01 4-6107-7

Xu, S., Peng, Z., Cui, B., Wang, T., Song, Y., and Zhang, L. (2014). FliS modulates FlgM activity by acting as a non-canonical chaperone to control late flagellar gene expression, motility and biofilm formation in *Yersinia pseudotuberculosis*. *Environ. Microbiol.* 16, 1090–1104. doi: 10.1111/1462-2920. 12222

Zhanel, G. G., Chung, P., Adam, H., Zelenitsky, S., Denisuik, A., and Schweizer, F. (2014). Ceftolozane/tazobactam: a novel cephalosporin/β-lactamase inhibitor combination with activity against multidrug-resistant gram-negative bacilli. *Drugs* 74, 31–51. doi: 10.1007/s40265-013-0168-2

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Chen, Su, Wang, Zhang, Liu, Chen and Tan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
# The Small Colony Variant of *Listeria monocytogenes* Is More Tolerant to Antibiotics and Has Altered Survival in RAW 264.7 Murine Macrophages

Thomas D. Curtis, Lone Gram* and Gitte M. Knudsen

Gram Lab, Department of Systems Biology, Technical University of Denmark, Kongens Lyngby, Denmark

Small Colony Variant (SCV) cells of bacteria are a slow-growing phenotype that result from specific defects in the electron transport chain. They form pinpoint colonies on agar plates and have a variety of phenotypic characteristics, such as altered carbon metabolism, decreased toxin and lytic enzyme production, aminoglycoside resistance, and increased intracellular persistence. They are clinically relevant in *Staphylococcus aureus* and *Pseudomonas aeruginosa*, serving as a reservoir for recurrent or prolonged infections. Here, we found that a SCV mutant in the foodborne pathogen *Listeria monocytogenes* (strain SCV E18), similar to the high persister mutant phenotype, survived significantly better than the wild type when exposed over a 48-h period to concentrations above Minimal Inhibitory Concentration for most tested antibiotics. SCV E18 survived more poorly than the wildtype in unactivated RAW264.7 macrophage cells, presumably because of its reduced listeriolysin O expression, however, it survived better in reactive oxygen species producing, phorbol 12-myristate 13-acetate-activated macrophages. Although SCV E18 was sensitive to oxygen as it entered the stationary phase, it was significantly more tolerant to H₂O₂ than the wild type, which may result from a shift in metabolism, however, further investigation is needed to resolve this. SCV E18 is a spontaneous mutant with a point mutation in the *hemA* gene. A wild type copy of *hemA* was complemented on plasmid pSOG30222, which restored the wild type phenotype. The results reported here suggest that the SCV of *L. monocytogenes* could be of clinical importance and highlight a need for adequate clinical screening for this phenotype, as it could affect antibiotic treatment outcomes.

**Keywords:** *Listeria monocytogenes*, small colony variants, antibiotic tolerance, persister cells, cell invasion, oxidative stress

## INTRODUCTION

*Listeria monocytogenes* is a Gram-positive, foodborne pathogen that can cause the rare, but often lethal infection listeriosis. This typically affects pregnant women, neonates, the elderly and the immunocompromised, and can cause mortality rates of up to 34% (Farber and Peterkin, 1991). Upon entry into the gastrointestinal tract, *L. monocytogenes* infects host epithelial cells

## OPEN ACCESS

**Edited by:**
Etienne Giraud,
Institut National de la Recherche
Agronomique, France

**Reviewed by:**
Stephen Peter Kidd,
University of Adelaide, Australia
Barbara C. Kahl,
University Hospital Münster, Germany

*Correspondence:
Lone Gram
gram@bio.dtu.dk

**Specialty section:**
This article was submitted to *Antimicrobials, Resistance and Chemotherapy*, a section of the journal *Frontiers in Microbiology*

Received: 01 March 2016

Accepted: 23 June 2016

Published: 05 July 2016

**Citation:**
Curtis TD, Gram L and Knudsen GM (2016) The Small Colony Variant of *Listeria monocytogenes* Is More Tolerant to Antibiotics and Has Altered Survival in RAW 264.7 Murine Macrophages. *Front. Microbiol.* 7:1056. doi: 10.3389/fmicb.2016.01056
and immediately escapes the phagosome where it is free to replicate within the cytosol. Using actin-mediated mobility it spreads to neighboring cells and eventually enters the bloodstream, causing systemic listeriosis if unchecked by the innate immune system (Portnoy et al., 2002). Macrophages are a key part of the innate immune response and control the initial infection by engulfing and killing the bacteria with of a variety of antimicrobial compounds, such as reactive oxygen and nitrogen species (Shaughnessy and Swanson, 2007).

Treatment options for listeriosis are limited as many antibiotics are only bacteriostatic against *L. monocytogenes*. Ampicillin is the treatment of choice, and is sometimes used in combination with gentamicin, although the necessity of this supplemental treatment has been debated (Temple and Nahata, 2000; Mitjà et al., 2009). For patients who cannot tolerate ampicillin, a combination of trimethoprim and sulfamethoxazole, known as co-trimoxazole (SXT), or macrolides are substituted. (Temple and Nahata, 2000). While resistance in *L. monocytogenes* is uncommon, particularly for the clinically relevant antibiotics (Morvan et al., 2010), we recently demonstrated that this bacterium can form multi-antibiotic tolerant persister cells (Knudsen et al., 2013). However, it is not known if such cells are formed *in vivo* during infection, which could further complicate antibiotic treatment.

Persister cells are a very small subpopulation of bacteria that enter a dormant-like state making them refractory to most antibiotics. Their tolerance to antibiotics is not genetically inherited, and can be operationally defined by a biphasic killing curve when treated with bactericidal antibiotics and no observed increase in the Minimum Inhibitory Concentration (MIC) (Lewis, 2010). Persister cells are thought to exist in all bacteria and have been linked to recurrent infections with a number of diseases (Maisonneuve and Gerdes, 2014). Their formation can be either stochastic and the result of phenotypic switching from normally growing cells to those with reduced growth rates (Balaban et al., 2004), or they can be induced by certain conditions, such as biofilms (Lewis, 2010) or internalization by macrophages (Helaine et al., 2014). One of the primary genetic mechanisms of persister formation identified so far are toxin/antitoxin (TA) modules, which consist of a stable toxin that slows bacterial growth or metabolism and an unstable antitoxin that neutralizes the activity of the toxin under growth conditions. By repeatedly exposing *Escherichia coli* to high concentrations of ampicillin, Fridman et al. (2014) found that bacteria can also tolerate antibiotics by extending the lag-phase by mutations in a variety of pathways, essentially rendering the cells dormant until the transient antibiotic pressure is removed.

Another potential reservoir of persistent and recurrent bacterial infections is the so-called Small Colony Variant (SCV) (Kahl et al., 2016). The SCV is a slow growing phenotypic variant that forms pinpoint colonies when plated on agar, and is the result of either thymidine auxotrophy caused by mutations in the thymidylate synthase gene (Besier et al., 2007; Chatterjee et al., 2008) or an interruption in the electron transport chain (ETC), specifically resulting from an absence of menadione or hemin biosynthesis and metabolism. These variants can either be

fixed, which is the result of a mutation in one of the biosynthesis or metabolism genes for the aforementioned compounds, or transient, alternating between the wild type and SCV phenotypes during replication (Edwards, 2012). Additional characteristics include altered carbon metabolism, reduced toxin and lytic enzyme production, and resistance to aminoglycosides (Proctor et al., 2014). Aminoglycosides have also been shown to select for the SCV phenotype, as the reduction in the membrane potential resulting from the disrupted ETC will lower the active transport necessary for aminoglycosides to cross the membrane (Balwit et al., 1994). The combination of trimethoprim/sulfamethoxazole (co-trimoxazole) has been found to select specifically for thymidine auxotrophic SCVs due to the anti-folate action of the drug (Garcia et al., 2013). While SCVs have been primarily studied in *Staphylococcus aureus*, they have been described in a number of bacteria including *Pseudomonas aeruginosa*, *Echerichia coli*, *Vibrio cholera*, *Salmonella*, and *Lactobacillus acidophilus* (Proctor et al., 2006). Furthermore, they have been linked to a number of recurrent infections including those caused by *S. aureus* (Proctor et al., 1995), *P. aeruginosa* (Haussler et al., 1999) and *E. coli* (Roggenkamp et al., 1998; for a comprehensive review see: Kahl et al., 2016). Although typically less virulent due to their slower growth and reduced expression of virulence factors, such as toxins and lytic enzymes, their ability to adhere, invade and persist within the host cell increases, which is thought to be a crucial factor in the ability of SCVs to cause recurrent or persistent infections (Sendi and Proctor, 2009).

We have previously found that exposure to sub-lethal concentrations of triclosan with subsequent selection on gentamicin could generate stable *L. monocytogenes* SCV cells (Christensen et al., 2011), which all had mutations in one of the heme biosynthesis or metabolism genes and exhibited the same traits observed in SCVs of other species (e.g., colony size, decreased haemolytic activity, aminoglycoside resistance and altered carbon utilization). Our SCV E18 strain also showed reduced growth typical of SCVs from other species, taking roughly four more hours to reach the same maximal cell density as the wild type strain (~$10^9$ CFU/ml), however, we also observed a sensitivity to aerated growth conditions (Kastbjerg et al., 2014), which to our knowledge has not been shown in SCVs from other species.

The phenotypic switching of the SCV and the persister cell are thought to be part of a bacterial bet-hedging strategy, allowing a small percentage of the population to survive and repopulate following stress exposure (Sousa et al., 2012). These two phenotypes share a number of other characteristics including slowed growth, intracellular persistence and a link to chronic infections. Thus, given these similarities, we speculate that a stable *L. monocytogenes* heme deficient SCV would show a similar tolerance toward a broad range of antibiotics that persister cells do. Furthermore, we sought to determine if *L. monocytogenes* SCVs are better at invading and surviving intracellularly, as they have been shown to in *S. aureus* (Sendi and Proctor, 2009). Taken together, these results will help to determine the clinical significance of Small Colony Variants in *L. monocytogenes*.
# MATERIALS AND METHODS

## Bacterial Strains and Growth Conditions

*Listeria monocytogenes* strain N53-1 and a mutant thereof (E18) were included in the present study. N53-1 represents a food-processing plant persistent molecular subtype and was isolated from a fish smokehouse (Wulff et al., 2006). Strain E18 [denoted as strain (1) 1-1 in Christensen et al. (2011)] is a stable SCV of N53-1, with a Single Nucleotide Polymorphism (SNP) in the glutamyl tRNA reductase (*hemA*) gene (Kastbjerg et al., 2014), generated through an adaptive evolution experiment where N53-1 was exposed to sub-lethal levels of triclosan and subsequently selected on gentamicin (Christensen et al., 2011). Strains were stored at -80°C and grown on Brain Heart Infusion (BHI; Oxoid CM 1135) agar at 37°C for 24 h. Overnight cultures were achieved by inoculating single colonies in 10 ml of BHI broth (Oxoid CM 1135) and incubating at 37°C with shaking at 250 rpm.

## Preparation of Antibacterial Agents

Fresh antibiotic solutions were prepared for each experiment: ampicillin (dissolved in sterile MilliQ water; Sigma-Aldrich A9518), erythromycin (dissolved in 96% ethanol; Sigma-Aldrich E6376), gentamicin (dissolved in sterile MilliQ water; Sigma-Aldrich G3632), norfloxacin (dissolved in sterile MilliQ with 1% glacial acetic acid; Fluka N9890) vancomycin (dissolved in sterile MilliQ; Sigma-Aldrich V2002) and co-trimoxazole, which is comprised of one part trimethoprim (dissolved in sterile MilliQ with 1% glacial acetic acid; Sigma-Aldrich 92131) and five parts sulphamethoxazole (dissolved in acetone; Fluka S7507). H₂O₂ (30% in water; Sigma-Aldrich 216763) was diluted to 20 mM in sterile MilliQ water.

## Minimal Inhibitory Concentration (MIC) Assay

Antibiotic MIC values were determined in BHI broth as previously described (Cockerill et al., 2012). In brief, stationary phase cultures of N53-1 and SCV E18 were adjusted to OD₆₀₀ = 0.2 and further diluted 1000-fold, corresponding to a CFU/ml of 10⁵. The adjusted cultures were tested against norfloxacin, ampicillin, gentamicin, and co-trimoxazole (trimethoprim/sulfamethoxazole) using a twofold dilution series of concentrations ranging from 100 to 0.10 µg/ml in 96-well plates (Thermo Scientific 163320) and incubated at 37°C. MIC for N53-1 and SCV E18 was determined by visual inspection after 24 and 48 h of incubation at 37°C, respectively. Two biological replicates were performed.

## Killing Kinetic Assays

To compare the antibiotic and H₂O₂ sensitivity of SCV E18 and N53-1, killing kinetics were performed according to Knudsen et al. (2013). Overnight cultures were diluted 10⁶-fold in 10 ml BHI broth and incubated into early stationary phase for 16 h at 37°C with shaking at 250 rpm. Each culture was then adjusted to an optical density at 600 nm (OD₆₀₀) of 0.2 in 2 ml BHI broth and challenged with antibiotic (30X MIC except nitrofurantoin, which was 10X MIC) at 37°C. Bacterial counts

were performed at 0, 4, 10, 24, 48, and 72 h. The antibiotics and concentrations used were norfloxacin (100 µg/ml), ampicillin (185 µg/ml), erythromycin (6 µg/ml), vancomycin (47 µg/ml), and co-trimoxazole (95 µg/ml). Cultures treated with H₂O₂ were grown and adjusted as described above, then treated with 20 mM and incubated under stagnant conditions at 37°C for 2 h. Three independent biological replicates were performed for each experiment and the limit of detection was 10² CFU/ml.

## Anaerobic Culturing Conditions

To verify that E18 was sensitive to oxygen, it, along with N53-1, were cultured in anaerobic conditions. Oxygen was removed from the media by autoclaving, followed by incubation in a HP0011 anaerobic jar (Oxoid) with an anaerobe gas generation bag (Sigma-Aldrich 68061) for 24 h. Ten microliters of an overnight culture was added to the anaerobic BHI, along with a new anaerobe gas generation bag and incubated on the lab bench for 3 h to allow for an anaerobic atmosphere to be generated before incubation with shaking. The cultures were grown for 72 h at 37°C with shaking at 200 rpm. Two biological replicates were performed.

## hemA Complementation

E18 carries several single nucleotide polymorphisms (SNPs) of which one is in the *hemA* gene (Kastbjerg et al., 2014). To verify the role of the SNP in *hemA* in the SCV E18 strain, a *hemA* complementation strain was constructed. The *hemA* gene with a 128 bp upstream region including the native promoter was amplified from genomic DNA of N53-1 with the forward primer 5'-AAACTCGAGTCATCCGTTAACTCCTCG and the reverse primer 5'-AAAGAATTCATAGAAGGAGTTGGAATG GA, which contained terminal XhoI and EcoRI sites, respectively, using Phusion High Fidelity DNA Polymerase (NEB, M0530S). The pSOG30222 plasmid (Hain et al., 2008) and *hemA* amplicon were digested with XhoI and EcoRI, and ligated overnight using a 3:1 insert to vector ratio, then electroporated into 60 µl of electrocompetent DH5α cells using a MicroPulser (BioRad 165-2100). Transformants were selected on Luria-Bertani plates with 100 µg/ml erythromycin and verified by colony PCR and sequencing (GATC Biotech). Plasmids containing the correct pSOG30222::*hemA* construct, along with an unmodified pSOG30222 control plasmid, were transformed into the SCV E18 strain. Transformants were selected on BHI agar with 5 µg/ml erythromycin and confirmed with colony PCR and sequencing. With the exception of the killing kinetic assay, all complementation experiments were carried out in the presence of 5 µg/ml erythromycin. Colony pictures were taken of each strain with an Olympus BX51 microscope at 40× magnification.

## Macrophage Internalization and Survival Assay

A murine macrophage cell line, RAW 264.7 (Sigma-Aldrich 91062702), was used to test bacterial internalization and intracellular survival using a protocol adapted from Bateman and Seed (Bateman and Seed, 2012). RAW 264.7 macrophages were grown in Dulbecco's Modified Eagle's Medium - high glucose
(Sigma-Aldrich D6546), 10% heat inactivated fetal bovine serum (Sigma-Aldrich F9665) and L-glutamine (Sigma-Aldrich G7513) in 150cc tissue culture flasks, for 10-15 passages prior to use. A cell scraper was used to disjoin the adherent cells from the surface. RAW 264.7 cells were seeded into 24-well plates at a density of 2 × 10⁶ cells/ml in RAW 264.7 culture media and then incubated for a total of 48 h at 37°C with 5% CO₂. During the 48 h, one group of RAW 264.7 cells was stimulated for 18 h with 1 ng/ml IFNγ, required for macrophage activation, and a second group was stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA), a known inducer of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity (Walloschke et al., 2010) for 2 h, while a third control group was left unstimulated. Prior to infection, IFNγ and PMA were removed from the RAW 264.7 containing wells. Overnight cultures of N53-1 and SCV E18 were diluted 10⁶-fold in 10 ml of BHI media and grown at 37°C for 16 h. *L. monocytogenes* cultures were adjusted to a density of 2 × 10⁷ CFU/ml in PBS and added to the confluent RAW 264.7 containing wells, achieving a multiplicity of infection (MOI) of 10. Plates were centrifuged for 5 min at 1,000 rpm to synchronize the infection and incubated at 37°C for 1 h. The plates were then incubated for an additional 2 h in media containing 100 µg/ml gentamicin, which was reduced to 50 µg/ml gentamicin for the remainder of the incubation. In order to verify that gentamicin did not favor the resistant SCV E18 during the 21 h incubation step, a control experiment was performed using the same methods as the unactivated macrophage experiment, with the exception that the media with 50 µg/ml gentamicin was substituted with PBS. To enumerate the surviving *L. monocytogenes* per well, RAW 264.7 cells were washed twice with PBS, followed by lysis with 0.1% Triton-X 100 (Sigma-Aldrich T8787) and then serial dilutions of the intracellular bacteria were plated on BHI agar plates. Internalization was measured following 3 h of incubation, while survival was measured after 24 h.

## Statistical Analysis

CFU/ml values for each biological replicate were log₁₀ transformed prior to statistical analysis. For the killing kinetic and O₂ sensitivity assays, significant differences between the N53-1 and SCV E18 were determined using a paired *t*-test, where significance is equal to *P* < 0.05. Significance between N53-1 and SCV E18 for the RAW 264.7 internalization and survival assays

was determined using a paired *t*-test (*P* < 0.05) with the log₁₀ transformed CFU/ml values. Significance for each strain between the RAW264.7 treatment groups was calculated using a one-way ANOVA test for the log₁₀ transformed CFU/ml values, where *P* < 0.05 determined significance. Significance between the two strains within each RAW264.7 treatment group was calculated using a paired *t*-test (*P* < 0.05). The limit of detection for all CFU/ml values was 10². All data analysis was performed in Excel. Replicates falling below the detection limit were set to 99 for statistical analysis.

## RESULTS

### SCV E18 Shows Increased Tolerance to Antibiotics

The *L. monocytogenes* SCVs, selected on gentamicin, were resistant to other aminoglycosides (Kastbjerg et al., 2014). To determine if the SCV phenotype conveys resistance to other antibiotics, MIC values of selected antibiotics were tested for both N53-1 and SCV E18, which were found to be the same for the two strains (Table 1).

Because *L. monocytogenes* SCV grow slower than the wild type (Kastbjerg et al., 2014), and because of the lowered oxidative phosphorylation observed in other SCV organisms, such as *S. aureus* (Proctor et al., 1998), we hypothesized that *L. monocytogenes* SCVs would have an increased tolerance to other classes of antibiotics, which was evaluated using time dependent killing experiments. With the exception of erythromycin, the SCV E18 strain survived significantly better for each antibiotic challenge up to 48 h (Figure 1; ampicillin, *P* = 0.0007; co-trimoxazole, *P* = 0.02; vancomycin, *P* = 0.02 and norfloxacin, *P* = 0.03), with between one and threefold higher log₁₀ CFU/ml as compared to the N53-1. When treated with norfloxacin, both N53-1 and SCV E18 exhibited a biphasic killing curve with a rapid decline to approximately three and five log₁₀ CFU/ml, respectively, which remained stable throughout the 72 h experiment (Figure 1A). Ampicillin has a delayed bactericidal effect on *L. monocytogenes* (Winslow et al., 1983), which was also observed with SCV E18, as is evidenced by the roughly three-fold log₁₀ reduction in CFU/ml over the 48-72 h time points (Figure 1B). However, this delayed bactericidal effect of ampicillin was not observed for N53-1 over the course of the

<table><caption>TABLE 1 | Minimum Inhibitory Concentration (MIC) values for *Listeria monocytogenes* wild type strain N53-1 strain and SCV E18 strain.</caption><thead><tr><th rowspan="2">Strain</th><th colspan="6">MIC µg/ml</th></tr><tr><th>Ampicillin</th><th>Co-trimoxazole</th><th>Norfloxacin</th><th>Erythromycin</th><th>Vancomycin</th><th>Gentamicin</th></tr></thead><tbody><tr><td>N53-1</td><td>3.13</td><td>0.78</td><td>3.13</td><td>0.20</td><td>1.56</td><td>0.40</td></tr><tr><td>E18</td><td>3.13</td><td>0.78</td><td>3.13</td><td>0.20</td><td>1.56</td><td>12.50</td></tr><tr><td>E18 pSOG30222::hemA</td><td>1.56</td><td>0.78</td><td>3.13</td><td>-</td><td>1.56</td><td>0.40</td></tr><tr><td>E18 pSOG30222</td><td>1.56</td><td>0.78</td><td>3.13</td><td>-</td><td>1.56</td><td>12.50</td></tr></tbody></table>

The MIC assay was performed using BHI broth and incubated at 37°C for 24 and 48 h for N53-1 and SCV E18, respectively. All experiments were done with two independent replicates. -, not tested.
<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

**FIGURE 1** | Killing of *Listeria monocytogenes* N53-1 (■) and SCV E18 (○) over the course of 72 h under shaking conditions with (A) norfloxacin, (B) ampicillin, (C) co-trimoxazole, (D) vancomycin, and (E) erythromycin. Error bars represent standard deviation of the mean for three biological replicates. Significance at the 48-h time point was determined using a paired *t*-test (*p* < 0.05 and **p** < 0.005).

72 h killing kinetic (Figure 1B). By the end of the 72 h treatment with co-trimoxazole, SCV E18 decreased in a linear manner to a comparable level with N53-1 (2.3 × 10⁵ and 5.6 × 10⁴ CFU/ml, respectively; *P* = 0.45; Figure 1C). Vancomycin had an initial bacteriostatic effect on both strains, followed by linear killing, however, the bactericidal effect began earlier for N53-1 (*T* = 10), while the SCV E18 strain resisted killing until the 24 h time point (Figure 1D). Erythromycin was bacteriostatic on both strains up to the 48 h time point, however, bacterial counts for SCV E18 decreased after 48 h, while the N53-1 remained stable (Figure 1E).

While the SCV phenotype in *L. monocytogenes* does not confer notable resistance to antibiotics besides the aminoglycosides, they survived significantly better under high antibiotic concentrations for up to 48 h under ampicillin or co-trimoxazole, and up to 72 h when exposed to norfloxacin or vancomycin. In contrast, by 72 h of exposure to erythromycin or ampicillin, the SCV has fewer CFU/ml than the wild type.

## Oxygen Sensitivity, But H₂O₂ Resistance, of the SCV E18

When grown under shaking conditions, SCV E18 grows to a maximum density of approximately 1 × 10⁹ CFU/ml after 12 h, where after the CFU/ml decreases rapidly, eventually dropping below the limit of detection after 24 h (Kastbjerg et al., 2014). Here, we grew cultures of the SCV E18, along with the N53-1, anaerobically to test if the observed decrease in CFU/ml is caused by exposure to oxygen. When grown anaerobically, cultures of the SCV E18 grow to higher densities (8.1 × 10⁸ CFU/ml) after 72 h, than the N53-1 strain (2.5 × 10⁸ CFU/ml; *P* = 0.002) (Table 2), suggesting that oxygen is toxic to the SCV E18 strain. Under anaerobic growth conditions the SCV E18 strain retained the pin point colony morphology when plated, whereas the N53-1 strain exhibited variable colony size.

To test if the oxygen sensitivity was related to a general deficiency in the oxidative stress response, such as the absence of catalase in the SCV E18, we treated with 20 mM H₂O₂
<table><caption>TABLE 2 | Oxidative stress sensitivity of *L. monocytogenes* N53-1 and SCV E18 as measured by aerobic and anaerobic growth, and exposure to 20 mM H₂O₂ for 2 h.</caption><thead><tr><th rowspan="3"></th><th colspan="4">Bacterial count (Log<sub>10</sub> CFU/ml)</th></tr><tr><th colspan="2">Growth in BHI for 72 h</th><th colspan="2">Survival in 20 mM H<sub>2</sub>O<sub>2</sub></th></tr><tr><th>Aerobic</th><th>Anaerobic</th><th>0 h</th><th>2 h</th></tr></thead><tbody><tr><td>N53-1</td><td>9.4 ± .01</td><td>8.4 ± 0.03</td><td>7.2 ± 0.04</td><td>3.7 ± 0.09</td></tr><tr><td>E18</td><td>BD</td><td>8.9 ± 0.02</td><td>7.2 ± 0.13</td><td>6.3 ± 1.02</td></tr><tr><td>E18 pSOG30222::<i>hemA</i></td><td>8.1 ± 0.09</td><td>-</td><td>8.0 ± 0.22</td><td>5.2 ± 0.17</td></tr><tr><td>E18 pSOG30222</td><td>BD</td><td>-</td><td>8.0 ± 0.06</td><td>6.5 ± 0.41</td></tr></tbody></table>

All experiments are log₁₀ CFU/ml mean values of three biological replicates ± standard deviation. BD, below the limit of detection; -, not tested.

for 2 h. Surprisingly, the SCV E18 survived significantly better (P = 0.008) than the wild type, with the number of colony forming bacteria following treatment being 33% of the inoculum, whereas the N53-1 strain survived at a rate of 0.03% (Table 2). Thus, similar to the increased tolerance toward antibiotics, and regardless of the oxygen sensitivity and lack of catalase (Kastbjerg et al., 2014), the SCV E18 strain exhibits a greater ability to resist killing by H₂O₂ over the wild type.

## hemA Complementation Restores Wild Type Phenotype of SCV E18

By genome sequencing and analyses, we previously demonstrated that all of the *L. monocytogenes* SCVs had several mutations, including a mutation in the *hemA* gene (Kastbjerg et al., 2014). To determine if the tested aspects of the SCV phenotype were due to the SNP in the *hemA* gene, a wild type *hemA* complemented strain of E18 was constructed. This complemented strain regained the large colony phenotype (Figure 2A), had restored gentamicin sensitivity identical to the N53-1 strain (Table 1), regained sensitivity to H₂O₂ and lost its sensitivity to oxygen, as demonstrated by the presence of viable colonies after culturing for 72 h (Table 2). Furthermore, the level of antibiotic tolerance was evaluated with a time dependent killing experiment using norfloxacin. The killing curve of the complemented *hemA* strain was significantly different from the empty vector control (P = 0.0000046), and was similar to the killing of N53-1 strain, while the empty vector control showed a very similar curve to its non-transformed counterpart SCV E18 (Figure 2B).

## SCV E18 Is Less Affected by Macrophage Status Than Wild Type N53-1

We investigated the survival ability of the wild type and the SCV E18 in the IFNγ activated and un-stimulated macrophage model. Additionally, due to the increased tolerance of SCV E18 toward H₂O₂, we used macrophages stimulated with PMA, a known activator of the NADPH oxidase pathway. Three hours following infection of the un-stimulated macrophage there was a high degree of internalization and intracellular growth of N53-1 and SCV E18, with 7.6 log₁₀ CFU/ml and 7.5 log₁₀ CFU/ml recovered, respectively, a difference that was not statistically significant (P = 0.77). These internalization findings were similar for the other two treatment groups (Figure 3A). By 24 h of incubation in

the un-stimulated macrophage, bacterial counts were drastically reduced for both strains, although N53-1 survived significantly better (P = 0.006) with 3.6 log₁₀ CFU/ml of the N53-1 and 2.3 log₁₀ CFU/ml for SCV E18 (Figure 3B). When macrophages

<figure><img src="image_37.png" /><figcaption>FIGURE 2 | *hemA* complementation experiments showing (A) colony morphology at 40x magnification of I: N53-1, II: SCV E18, III: E18 pSOG30222, and IV: E18 pSOG30222::*hemA* after 24 h of incubation at 37°C on BHI plates and (B) Killing of *L. monocytogenes* N53-1 (■) and SCV E18 (○) strains, as well as the *hemA* complemented (□) and empty vector control E18 (●) strains exposed to 100 µg/ml norfloxacin for 72 h. The data represents three biological replicates with standard deviation of the mean. Significance was determined using a paired t-test (***p < 0.000005).</figcaption></figure>
<figure><img src="image_38.png" /><figcaption>**FIGURE 3** | Intracellular (A) internalization and (B) survival of *L. monocytogenes* N53-1 and SCV E18 in RAW264.7 cells treated with IFNγ, PMA and no treatment using a multiplicity of infection (MOI) of 10. Bacterial counts for the internalization assay were determined after 2 h of 100 µg/ml gentamicin exposure. Survival was determined after 21 additional hours of incubation with 50 µg/ml gentamicin. Values are displayed as the log₁₀ transformed mean of three biological replicates with standard deviation. Significance was determined using a paired *t*-test (*p* < 0.05).</figcaption></figure>

were activated with IFNγ, the survival difference between strains vanished, as the number of recovered N53-1 bacteria at 24 h was reduced to 2.06 log₁₀ CFU/ml, while the SCV E18 burden was almost unchanged by IFNγ activation (2.15 log₁₀ CFU/ml) (Figure 3B). Increased reactive oxygen species (ROS) production, via PMA activation of the NADPH oxidase pathway, resulted in

a slight, but significantly higher bacterial burden of macrophages infected with SCV E18 (2.7 log₁₀ CFU/ml), than N53-1 (2.1 log₁₀ CFU/ml; *P* = 0.02) (Figure 3B). While activation of macrophages with IFNγ or stimulation with PMA significantly reduced the intracellular bacterial load of N53-1 (*P* = 0.01), the SCV E18 was not sensitive to either treatment and remained unchanged across groups (*P* = 0.17).

To summarize, macrophages were equally poor at controlling the bacterial burden of both N53-1 and SCV E18 in the short term (*T* = 3 h), however, by 24 h the CFU/ml for all intracellular bacteria had been severely reduced. While survival in the un-activated macrophage was attenuated for SCV E18, as compared to N53-1, it was unaffected by the status of the macrophage unlike N53-1, which was significantly reduced in CFU/ml upon macrophage activation with IFNγ or stimulation with PMA.

DISCUSSION

The Small Colony Variant (SCV) has been described for decades in several bacteria such as *S. aureus*, *E. coli* and *P. aeruginosa* (Proctor et al., 2006). However, they were only recently identified in *L. monocytogenes* and, with the exception of aminoglycoside resistance (Kastbjerg et al., 2014), their importance with respect to the treatment of listeriosis has not been investigated. This study is the first to demonstrate that the SCV phenotype of *L. monocytogenes* caused by a single point mutation in the *hemA* gene has the potential to complicate treatment by causing an increase in tolerance toward most of the clinically relevant antibiotics.

As shown previously (Kastbjerg et al., 2014), SCV E18 exhibited resistance to the aminoglycoside gentamicin, a defining trait of the electron-transport deficient SCV (Proctor et al., 2006). This is particularly relevant for listeriosis, as gentamicin is the most common secondary antibiotic used for treatment (Temple and Nahata, 2000). While the SCV E18 showed no increase in MIC for the other antibiotics tested, indicating a lack of acquired resistance, it was nonetheless able to survive lethal concentrations of norfloxacin, ampicillin (up to 48 h), co-trimoxazole, and vancomycin significantly better than the wild type, which taken together, are the definition of antibiotic tolerance (Dimitrijovski et al., 2015). The increase in tolerance toward vancomycin, second generation fluoroquinolones (e.g., norfloxacin) and beta-lactams (e.g., ampicillin) has been observed with *S. aureus* SCVs and is presumably the result of their slow growing SCV phenotype (Chuard et al., 1997; Garcia et al., 2012; Lechner et al., 2012). Antibiotic tolerance poses a unique clinical dilemma, as any testing on isolates relying solely on MIC values could obscure the true antimicrobial susceptibility of the SCV and lead to sub-optimal treatment strategies.

In contrast to the other antibiotics, SCV E18 did not survive better under erythromycin pressure at any time point during the experiment and, as with ampicillin, became more sensitive after 48 h of killing. This could indicate that whatever protective effect the SCV phenotype contributes toward other antibiotics does not apply to the macrolide class, which target protein synthesis. Interestingly, *S. aureus* SCVs were not isolated from cystic fibrosis
patients treated exclusively with the macrolide azithromycin, whereas when treated with other antibiotics, SCVs were observed in 46% of patients colonized with *S. aureus* (Kahl et al., 2003; Green et al., 2011). Although the SCV *L. monocytogenes* E18 tolerates ampicillin better than the wild type N53-1 in the short term, the sensitivity toward extended exposure (as evidenced by the pronounced decrease in bacterial count following 48 h of treatment) and the fact that long-term (between 2 and 8 weeks) ampicillin therapy is the primary treatment of choice for listeriosis (Temple and Nahata, 2000), could explain why the SCV phenotype has, to our knowledge, not been observed in clinical cases. However, it is also possible that current screening procedures in the clinical laboratory are inadequate, leading the SCV sub-population to go unnoticed (Proctor et al., 2006). The slower growth of SCV E18 would likely cause it to be overshadowed in a mixed culture containing the wild type. Furthermore, the altered sugar metabolism and lack of catalase observed in SCV E18 (Kastbjerg et al., 2014), as well as the fact that some of the media used to grow *L. monocytogenes* contain the compounds SCVs are auxotrophic for, would complicate proper identification of any SCV *L. monocytogenes* clinical isolates.

We were expecting the SCV E18 to be more sensitive to H₂O₂ because it does not produce catalase, however, it was able to better withstand H₂O₂ than the wild type. As H₂O₂ reacts with the heme cofactor of proteins (Nagababu and Rifkind, 1998), our observation may simply be the result of fewer targets in the heme deficient SCV E18. Alternatively, tolerance toward H₂O₂ may be due to a change in the metabolism of the SCV, as suggested by Painter et al. (2015). In addition to showing that heme auxotrophic SCVs of *S. aureus* were more tolerant of H₂O₂, they added 4-hydroxy-2-heptylquinoline-*N*-oxide (HQNO), which can induce the SCV phenotype by blocking the electron transport chain of Gram-positive bacteria, to wild type *S. aureus* either hours or minutes before exposure to lethal levels of H₂O₂. Those bacteria grown for hours in the presence of HQNO survived the H₂O₂ exposure significantly better than the wild type, whereas those that received the HQNO only minutes before did not. This demonstrates that the tolerance is not simply the result of reduced electron transport, but as the authors suggest, a global physiological change caused by the loss of the electron transport chain (e.g., a switch to anaerobic metabolism), which could also explain our similar observation in *L. monocytogenes*.

In addition to the typical characteristics of the SCV, *L. monocytogenes* SCVs exhibit a unique sensitivity to growth under shaking conditions as they enter the stationary phase (Kastbjerg et al., 2014). Here, we demonstrate that this results from the presence of oxygen in the media. The addition of catalase, which converts H₂O₂ to water and oxygen, or heme (necessary for the biosynthesis of catalase) to the growth media abolished this effect, suggesting that the oxygen sensitivity is due to the inability of the heme-deficient SCV mutant to produce catalase (Kastbjerg et al., 2014). However, to our knowledge, this oxygen sensitivity has not been observed in heme-deficient SCVs of other catalase-dependent species (Roggenkamp et al., 1998; Zahra et al., 2013), and this fails to explain why the associated toxicity only manifests in the stationary phase. Few explanations seem to coincide with the observed increased tolerance toward

H₂O₂ and extended viability of SCV E18 cells when treated with antibiotics, however, we believe this is an important observation that warrants further research.

When the wild type and SCV E18 were inoculated with RAW264.7 murine macrophages over the course of 3 h, we observed no difference between the internalization of the two strains. However, prolonged incubation of the infected macrophages revealed differences between the fates of each strain depending on the activation status of the macrophages. N53-1 survived better than SCV E18 in the naïve macrophages, however, this difference disappeared once macrophages were activated with IFNγ where the wild type was reduced to the same CFU/ml as the mutant. The former observation is likely explained by the reduced expression of the pore-forming toxin Listeriolysin O (LLO) (Kastbjerg et al., 2014) in SCV E18, which has been shown to be produced in murine macrophages (Moors et al., 1999) and is necessary for *L. monocytogenes* to escape from the phagosome (Bielecki et al., 1990). The latter observation corresponds with studies showing that IFNγ-activated macrophages are able to block the escape of *L. monocytogenes* (Portnoy et al., 1989) by a process thought to be mediated by ROS inactivation of the LLO toxin (Myers et al., 2003). This suggests that macrophages need not first be activated by IFNγ in order to control *L. monocytogenes* SCVs, which contrasts to SCVs from other species, and could explain the lack of observed clinical *L. monocytogenes* SCVs. On the other hand, LLO is the most immunogenic antigen for the T cell response, which mediates the ultimate removal and adaptive immunity to *L. monocytogenes* (Vijh and Pamer, 1997), thus a reduction in LLO expression may lead to a weaker adaptive immune response to SCV E18 and could explain the observation that mutants incapable of producing LLO persist for significantly longer in the bone marrow of infected mice (Hardy et al., 2009). Furthermore, the PMA stimulated macrophage showed that SCV E18 is significantly better at surviving the bactericidal effects of ROS within the phagosome, which is consistent with its observed increase in H₂O₂ tolerance, and could have implications on the fate of SCV *L. monocytogenes* in neutrophils that rely predominantly on NADPH oxidase for their bactericidal activity (Segal, 2005).

Our complementation experiment showed that this phenotype can arise through a single base-pair substitution in the *hemA* gene, and given the prevalence of verified SCV selection pressures *L. monocytogenes* faces both in the environment and during treatment (e.g., triclosan, gentamicin and co-trimoxazole), the probability of exposure to SCV *L. monocytogenes* seems high. Therefore, it is surprising that no clinical cases of SCV associated listeriosis have been reported. Perhaps the long treatment with ampicillin is enough to eradicate the SCV, or the immune system is better able to control SCV *L. monocytogenes*, as indicated by our findings with non-IFNγ activated RAW264.7 murine macrophages. However, it seems equally likely that this elusive phenotype has gone unnoticed due to a lack of adequate screening procedures in clinical laboratories.

Much like persister cells, SCVs have also been shown to revert to the wildtype phenotype. When clinical and laboratory strains of *S. aureus* were cyclically grown with and without gentamicin, an identical proportion of SCVs upon each
exposure to the antibiotic was observed (Massey et al., 2001). Drenkard and Ausubel (2002) found the same phenotypic switching in *P. aeruginosa*, and also identified a protein that modulates the switch between the wild type and SCV phenotypes. This phenotypic heterogeneity, often referred to as a bet-hedging strategy, allows a portion of the bacterial population to survive a stress (e.g., antibiotic exposure or the phagosome), without giving up any fitness advantages associated with mutation. It is plausible that the majority of SCVs *in vivo* are the result of phenotypic switching, and although, we observed no reversion with SCV E18, this mutant and others like it could represent a small subset of SCVs locked in the on position. This would add another treatment challenge, as the bacterial population as a whole would be protected from antibiotics, while simultaneously capable of full virulence (Tuchscherr et al., 2011). Reports of SCVs from a variety of bacterial species being linked to persistent and chronic infections are numerous (Proctor et al., 1995; von Eiff et al., 1997; Roggenkamp et al., 1998; Seifert et al., 1999; Häussler et al., 2003), and while uncommon, cases of recurrent listeriosis have been reported and the cause of these treatment failures were not determined (McLauchlin et al., 1991; Sauders et al., 2001; Kleemann et al., 2009).

Although *L. monocytogenes* SCVs may be unique in that macrophages are able to control them without first becoming activated by IFNγ, they may yet be able to better survive other aspects of the immune system, and the presence of an easily generated, multi-antibiotic tolerant sub-population

is nonetheless a cause for concern. Awareness and adequate screening for SCVs in clinical isolates, such as extended incubation times and inclusion of isolates with non-standard sugar metabolism or those lacking catalase activity, would also help to determine the role (if any) this phenotype plays in human infection with *L. monocytogenes*, and we believe the evidence presented here warrants such measures being employed in clinical laboratories.

## AUTHOR CONTRIBUTIONS

Conceived and designed the experiments: TC, LG, and GK. Performed the experiments: TC. Analyzed the data: TC, LG, and GK. Wrote the manuscript: TC, LG, and GK.

## FUNDING

This work was supported by a Ph.D. grant from the Department of Systems Biology, Technical University of Denmark.

## ACKNOWLEDGMENT

The authors would like to thank all members of the Gram Lab for their support and feedback.

## REFERENCES

* Balaban, N. Q., Merrin, J., Chait, R., Kowalik, L., and Leibler, S. (2004). Bacterial persistence as a phenotypic switch. *Science* 305, 1622-1625. doi: 10.1126/science.1099390

* Balwit, J. M., van Langevelde, P., Vann, J. M., and Proctor, R. A. (1994). Gentamicin-resistant menadione and hemin auxotrophic *Staphylococcus aureus* persist within cultured endothelial cells. *J. Infect. Dis.* 170, 1033-1037. doi: 10.1093/infdis/170.4.1033

* Bateman, S. L., and Seed, P. C. (2012). Epigenetic regulation of the nitrosative stress response and intracellular macrophage survival by extraintestinal pathogenic *Escherichia coli*. *Mol. Microbiol.* 83, 908-925. doi: 10.1111/j.1365-2958.2012.07977.x

* Besier, S., Ludwig, A., Ohlsen, K., Brade, V., and Wichelhaus, T. A. (2007). Molecular analysis of the thymidine-auxotrophic small colony variant phenotype of *Staphylococcus aureus*. *Int. J. Med. Microbiol.* 297, 217-225. doi: 10.1016/j.ijmm.2007.02.003

* Bielecki, J., Youngman, P., Connelly, P., and Portnoy, D. A. (1990). *Bacillus subtilis* expressing a haemolysin gene from *Listeria monocytogenes* can grow in mammalian cells. *Nature* 345, 175-176. doi: 10.1038/345175a0

* Chatterjee, I., Kriegeskorte, A., Fischer, A., Deiwick, S., Theimann, N., Proctor, R. A., et al. (2008). In vivo mutations of thymidylate synthase (encoded by thyA) are responsible for thymidine dependency in clinical small-colony variants of *Staphylococcus aureus*. *J. Bacteriol.* 190, 834-842. doi: 10.1128/JB.00912-07

* Christensen, E. G., Gram, L., and Kastbjerg, V. G. (2011). Sublethal triclosan exposure decreases susceptibility to gentamicin and other aminoglycosides in *Listeria monocytogenes*. *Antimicrob. Agents Chemother.* 55, 4064-4071. doi: 10.1128/AAC.00460-11

* Chuard, C., Vaudaux, P. E., Proctor, R. A., and Lew, D. P. (1997). Decreased susceptibility to antibiotic killing of a stable small colony variant of *Staphylococcus aureus* in fluid phase and on fibronectin-coated surfaces. *J. Antimicrob. Chemother.* 39, 603-608. doi: 10.1093/jac/39.5.603

* Cockerill, F. R., Wiker, M. A., Alder, J., Dudley, M. N., Eliopoulos, G. M., Ferraro, M. J., et al. (2012). *Methods for Dilution Antimicrobial Susceptibility Testing for Bacteria that Grow Aerobically*. Approved Standard — 9th Edn, Vol. 32. Wayne, PA: Clinical and Laboratory Standards Institute, 18-20.

* Dimitrijovski, B., Jensen, S. O., Espido, B. A., and van Hal, S. J. (2015). “Tolerance” of Misused Terminology? Enforcing Standardized Phenotypic Definitions. *MBio* 6:e00446-15. doi: 10.1128/mBio.00446-15

* Drenkard, E., and Ausubel, F. M. (2002). *Pseudomonas* biofilm formation and antibiotic resistance are linked to phenotypic variation. *Nature* 416, 740-743. doi: 10.1038/416740a

* Edwards, A. M. (2012). Phenotype switching is a natural consequence of *Staphylococcus aureus* replication. *J. Bacteriol.* 194, 5404-5412. doi: 10.1128/JB.00948-12

* Farber, J. M., and Peterkin, P. I. (1991). *Listeria monocytogenes*, a food-borne pathogen. *Microbiol. Rev.* 55, 476-511.

* Fridman, O., Goldberg, A., Ronin, I., Shores, N., and Balaban, N. Q. (2014). Optimization of lag time underlies antibiotic tolerance in evolved bacterial populations. *Nature* 513, 418-421. doi: 10.1038/nature13469

* Garcia, L. G., Lemaire, S., Kahl, B. C., Becker, K., Proctor, R. A., Denis, O., et al. (2012). Pharmacodynamic evaluation of the activity of antibiotics against hemin- and menadione-dependent small-colony variants of *Staphylococcus aureus* in models of extracellular (broth) and intracellular (THP-1 monocytes) infections. *Antimicrob. Agents Chemother.* 56, 3700-3711. doi: 10.1128/AAC.00285-12

* Garcia, L. G., Lemaire, S., Kahl, B. C., Becker, K., Proctor, R. A., Denis, O., et al. (2013). Antibiotic activity against small-colony variants of *Staphylococcus aureus*: review of in vitro, animal and clinical data. *J. Antimicrob. Chemother.* 68, 1455-1464. doi: 10.1093/jac/dkt072

* Green, N., Burns, J. L., Mayer-Hamblett, N., Kloster, M., Lands, L. C., Anstead, M., et al. (2011). Lack of association of small-colony-variant *Staphylococcus aureus* strains with long-term use of azithromycin in patients with cystic fibrosis. *J. Clin. Microbiol.* 49, 2772-2773. doi: 10.1128/JCM.00835-11
Hain, T., Hossain, H., Chatterjee, S. S., Machata, S., Volk, U., Wagner, S., et al. (2008). Temporal transcriptomic analysis of the *Listeria monocytogenes* EGD-e sigmaB regulon. *BMC Microbiol.* 8:20. doi: 10.1186/1471-2180-8-20

Hardy, J., Chu, P., and Contag, C. H. (2009). Foci of *Listeria monocytogenes* persist in the bone marrow. *Dis. Mode. Mech.* 2, 39-46. doi: 10.1242/dmm.000836

Häussler, S., Lehmann, C., Breselge, C., Rohde, M., Classen, M., Tümmler, B., et al. (2003). Fatal outcome of lung transplantation in cystic fibrosis patients due to small-colony variants of the *Burkholderia cepacia* complex. *Eur. J. Clin. Microbiol. Infect. Dis.* 22, 249-253.

Haussler, S., Tummler, B., Weissbrodt, H., Rohde, M., and Steinmetz, I. (1999). Small-colony variants of *Pseudomonas aeruginosa* in cystic fibrosis. *Clin. Infect. Dis.* 29, 621-625. doi: 10.1086/598644

Helaine, S., Cheverton, A. M., Watson, K. G., Faure, L. M., Matthews, S. A., and Holden, D. W. (2014). Internalization of *Salmonella* by macrophages induces formation of nonreplicating persisters. *Science* 343, 204-208. doi: 10.1126/science.1244705

Kahl, B. C., Becker, K., and Löffler, B. (2016). Clinical significance and pathogenesis of *Staphylococcal* small colony variants in persistent infections. *Clin. Microbiol. Rev.* 29, 401-427. doi: 10.1128/CMR.00069-15

Kahl, B. C., Duebbers, A., Lubritz, G., Haeberle, J., Koch, H. G., Ritzerfeld, B., et al. (2003). Population dynamics of persistent *Staphylococcus aureus* isolated from the airways of cystic fibrosis patients during a 6-year prospective study. *J. Clin. Microbiol.* 41, 4424-4427. doi: 10.1128/JCM.41.9.4424-4427.2003

Kastbjerg, V. G., Hein-Kristensen, L., and Gram, L. (2014). Triclosan-induced aminoglycoside-tolerant *Listeria monocytogenes* isolates can appear as small-colony variants. *Antimicrob. Agents Chemother.* 58, 3124-3132. doi: 10.1128/AAC.02266-13

Kleemann, P., Domann, E., Chakraborty, T., Bernstein, I., and Lohoff, M. (2009). Chronic prosthetic joint infection caused by *Listeria monocytogenes*. *J. Med. Microbiol.* 58, 138-141. doi: 10.1099/jmm.0.004234-0

Knudsen, G. M., Ng, Y., and Gram, L. (2013). Survival of bactericidal antibiotic treatment by a persister subpopulation of *Listeria monocytogenes*. *Appl. Environ. Microbiol.* 79, 7390-7397. doi: 10.1128/AEM.02184-13

Lechner, S., Lewis, K., and Bertram, R. (2012). *Staphylococcus aureus* persisters tolerant to bactericidal antibiotics. *J. Mol. Microbiol. Biotechnol.* 22, 235-244. doi: 10.1159/000342449

Lewis, K. (2010). Persister cells. *Annu. Rev. Microbiol.* 64, 357-372. doi: 10.1146/annurev.micro.112408.134306

Maisonneuve, E., and Gerdes, K. (2014). Molecular mechanisms underlying bacterial persisters. *Cell* 157, 539-548. doi: 10.1016/j.cell.2014.02.050

Massey, R. C., Buckling, A., and Peacock, S. J. (2001). Phenotypic switching of antibiotic resistance circumvents permanent costs in *Staphylococcus aureus*. *Curr. Biol.* 11, 1810-1814. doi: 10.1016/S0960-9822(01)00507-3

McLauchlin, J., Audurier, A., and Taylor, A. G. (1991). Treatment failure and recurrent human listeriosis. *J. Antimicrob. Chemother.* 27, 851-857. doi: 10.1093/jac/27.6.851

Mitjä, O., Pigrau, C., Ruiz, I., Vidal, X., Almirante, B., Planes, A.-M., et al. (2009). Predictors of mortality and impact of aminoglycosides on outcome in listeriosis in a retrospective cohort study. *J. Antimicrob. Chemother.* 64, 416-423. doi: 10.1093/jac/dkp180

Moors, M. A., Levitt, B., Youngman, P., and Portnoy, D. A. (1999). Expression of listeriolysin O and ActA by intracellular and extracellular *Listeria monocytogenes*. *Infect. Immun.* 67, 131-139.

Morvan, A., Moubareck, C., Leclercq, A., Hervé-Bazin, M., Bremont, S., Lecuit, M., et al. (2010). Antimicrobial resistance of *Listeria monocytogenes* strains isolated from humans in France. *Antimicrob. Agents Chemother.* 54, 2728-2731. doi: 10.1128/AAC.01557-09

Myers, J. T., Tsang, A. W., and Swanson, J. A. (2003). Localized reactive oxygen and nitrogen intermediates inhibit escape of *Listeria monocytogenes* from vacuoles in activated macrophages. *J. Immunol.* 171, 5447-5453. doi: 10.4049/jimmunol.171.10.5447

Nagababu, E., and Rifkind, J. M. (1998). Formation of fluorescent heme degradation products during the oxidation of hemoglobin by hydrogen peroxide. *Biochem. Biophys. Res. Commun.* 247, 592-596. doi: 10.1006/bbrc.1998.8846

Painter, K. L., Strange, E., Parkhill, J., Bamford, K. B., Armstrong-James, D., and Edwards, A. M. (2015). *Staphylococcus aureus* adapts to oxidative stress by producing H2O2-resistant small-colony variants via the SOS response. *Infect. Immun.* 83, 1830-1844. doi: 10.1128/IAI.03016-14

Portnoy, D. A., Auerbuch, V., and Glomski, I. J. (2002). The cell biology of *Listeria monocytogenes* infection: the intersection of bacterial pathogenesis and cell-mediated immunity. *J. Cell Biol.* 158, 409-414. doi: 10.1083/jcb.200 205009

Portnoy, D. A., Schreiber, R. D., Connelly, P., and Tilney, L. G. (1989). Gamma interferon limits access of *Listeria monocytogenes* to the macrophage cytoplasm. *J. Exp. Med.* 170, 2141-2146. doi: 10.1084/jem.170.6.2141

Proctor, R. A., Kahl, B., Von Eiff, C., Vaudaux, P. E., Lew, D. P., and Peters, G. (1998). Staphylococcal small colony variants have novel mechanisms for antibiotic resistance. *Clin. Infect. Dis.* 27, 68-74. doi: 10.1086/ 514906

Proctor, R. A., Kriegeskorte, A., Kahl, B. C., Becker, K., Löffler, B., and Peters, G. (2014). *Staphylococcus aureus* Small Colony Variants (SCVs): a road map for the metabolic pathways involved in persistent infections. *Front. Cell. Infect. Microbiol.* 4:99. doi: 10.3389/fcimb.2014.00099

Proctor, R. A., van Langevelde, P., Kristjansson, M., Maslow, J. N., and Arbeit, R. D. (1995). Persistent and relapsing infections associated with small-colony variants of *Staphylococcus aureus*. *Clin. Infect. Dis.* 20, 95-102. doi: 10.1093/clinids/20.1.95

Proctor, R. A., von Eiff, C., Kahl, B. C., Becker, K., McNamara, P., Herrmann, M., et al. (2006). Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. *Nat. Rev. Microbiol.* 4, 295-305. doi: 10.1038/nrmicro1384

Roggenkamp, A., Sing, A., Hornef, M., Brunner, U., Autenrieth, I. B., and Heesemann, J. (1998). Chronic prosthetic hip infection caused by a small-colony variant of *Escherichia coli*. *J. Clin. Microbiol.* 36, 2530-2534.

Sauders, B. D., Wiedmann, M., Desjardins, M., Fenlon, C., Davenport, N., Hibbs, J. R., et al. (2001). Recurrent *Listeria monocytogenes* infection: relapse or reinfection with a unique strain confirmed by molecular subtyping. *Clin. Infect. Dis.* 33, 257-259. doi: 10.1086/321821

Segal, A. W. (2005). How neutrophils kill microbes. *Annu. Rev. Immunol.* 23, 197-223. doi: 10.1146/annurev.immunol.23.021704.115653

Seifert, H., Von Eiff, C., and Fätkenheuer, G. (1999). Fatal case due to methicillin-resistant *Staphylococcus aureus* small colony variants in an AIDS patient. *Emerg. Infect. Dis.* 5, 450-453. doi: 10.3201/eid0503.990319

Sendi, P., and Proctor, R. A. (2009). *Staphylococcus aureus* as an intracellular pathogen: the role of small colony variants. *Trends Microbiol.* 17, 54-58. doi: 10.1016/j.tim.2008.11.004

Shaughnessy, L. M., and Swanson, J. A. (2007). The role of the activated macrophage in clearing *Listeria monocytogenes* infection. *Front. Biosci.* 12:2683-2692. doi: 10.2741/2364

Sousa, A. M., Machado, I., and Pereira, M. O. (2012). "Phenotypic switching: an opportunity to bacteria thrive," in *Science against Microbial Pathogens: Communicating Current Research and Technological Advances*, ed. A. Méndez-Vilas (Berlin: Springer), 252-262.

Temple, M. E., and Nahata, M. C. (2000). Treatment of listeriosis. *Ann. Pharmacother.* 34, 656-661. doi: 10.1345/aph.19315

Tuchscherr, L., Medina, E., Hussain, M., Völker, W., Heitmann, V., Niemann, S., et al. (2011). *Staphylococcus aureus* phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection. *EMBO Mol. Med.* 3, 129-141. doi: 10.1002/emmm.201 000115

Vijh, S., and Pamer, E. G. (1997). Immunodominant and subdominant CTL responses to *Listeria monocytogenes* infection. *J. Immunol.* 158, 3366-3371.

von Eiff, C., Bettin, D., Proctor, R. A., Rolauffs, B., Lindner, N., Winkelmann, W., et al. (1997). Recovery of small colony variants of *Staphylococcus aureus* following gentamicin bead placement for osteomyelitis. *Clin. Infect. Dis.* 25, 1250-1251. doi: 10.1086/516962

Walloschke, B., Fuhrmann, H., and Schumann, J. (2010). Macrophage cell line RAW264.7 but not P-388D1 is an appropriate in vitro-model for studying oxidative burst as well as cytokine production in context of fatty acid enrichment. *Cell. Immunol.* 262, 58-61. doi: 10.1016/j.cellimm.2009.12.008

Winslow, D., Damme, J., and Dieckman, E. (1983). Delayed bactericidal activity of beta-lactam antibiotics against *Listeria monocytogenes*: antagonism of
chloramphenicol and rifampin. *Antimicrob. Agents Chemother.* 23, 555–558.
doi: 10.1128/AAC.23.4.555

Wulff, G., Gram, L., Ahrens, P., and Vogel, B. F. (2006). One group of genetically similar *Listeria monocytogenes* strains frequently dominates and persists in several fish slaughter- and smokehouses. *Appl. Environ. Microbiol.* 72, 4313–4322. doi: 10.1128/AEM.02288-05

Zahra, M., Ferreri, M., Alkasir, R., Yin, J., Han, B., and Su, J. (2013). Isolation and characterization of small-colony variants of ornithobacterium rhinotracheale. *J. Clin. Microbiol.* 51, 3228–3236. doi: 10.1128/JCM.01337-13

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Curtis, Gram and Knudsen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
# Carriage of Extended-Spectrum Beta-Lactamase-Plasmids Does Not Reduce Fitness but Enhances Virulence in Some Strains of Pandemic *E. coli* Lineages

Katharina Schaufler¹*, Torsten Semmler¹,², Derek J. Pickard³, María de Toro⁴, Fernando de la Cruz⁴, Lothar H. Wieler¹,², Christa Ewers⁵ and Sebastian Guenther¹

**OPEN ACCESS**

**Edited by:**
Ivan Rychlik,
Veterinary Research Institute,
Czech Republic

**Reviewed by:**
Vishvanath Tiwari,
Central University of Rajasthan, India
Dinesh Sriramulu,
Shres Consultancy (Life Sciences),
India

**\*Correspondence:**
Katharina Schaufler
katharina.schaufler@fu-berlin.de

**Specialty section:**
This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

**Received:** 15 December 2015

**Accepted:** 03 March 2016

**Published:** 17 March 2016

**Citation:**
Schaufler K, Semmler T, Pickard DJ, de Toro M, de la Cruz F, Wieler LH, Ewers C and Guenther S (2016) Carriage of Extended-Spectrum Beta-Lactamase-Plasmids Does Not Reduce Fitness but Enhances Virulence in Some Strains of Pandemic *E. coli* Lineages. *Front. Microbiol.* 7:336.
doi: 10.3389/fmicb.2016.00336

¹ Veterinary Faculty, Institute of Microbiology and Epizootics, Freie Universität Berlin, Berlin, Germany, ² Robert Koch Institute, Berlin, Germany, ³ Wellcome Trust Sanger Institute, Cambridge, UK, ⁴ Departamento de Biologia Molecular, Instituto de Biomedicina y Biotecnología de Cantabria (UC-SODERCAN-CSIC), Universidad de Cantabria, Santander, Spain, ⁵ Veterinary Faculty, Institute of Hygiene and Infectious Diseases of Animals, Justus-Liebig-Universität Giessen, Giessen, Germany

Pathogenic ESBL-producing *E. coli* lineages occur frequently worldwide, not only in a human health context but in animals and the environment, also in settings with low antimicrobial pressures. This study investigated the fitness costs of ESBL-plasmids and their influence on chromosomally encoded features associated with virulence, such as those involved in the planktonic and sessile behaviors of ST131 and ST648 *E. coli*. ESBL-plasmid-carrying wild-type *E. coli* strains, their corresponding ESBL-plasmid-"cured" variants (PCV), and complementary ESBL-carrying transformants were comparatively analyzed using growth curves, Omnilog® phenotype microarray (PM) assays, macrocolony and biofilm formation, swimming motility, and RNA sequence analysis. Growth curves and PM results pointed toward similar growth and metabolic behaviors among the strains. Phenotypic differences in some strains were detected, including enhanced curli fimbriae and/or cellulose production as well as a reduced swimming capacity of some ESBL-carrying strains, as compared to their respective PCVs. RNA sequencing mostly confirmed the phenotypic results, suggesting that the chromosomally encoded csgD pathway is a key factor involved. These results contradict the hypothesis that ESBL-plasmid-carriage leads to a fitness loss in ESBL-carrying strains. Instead, the results indicate an influence of some ESBL-plasmids on chromosomally encoded features associated with virulence in some *E. coli* strains. In conclusion, apart from antibiotic resistance selective advantages, ESBL-plasmid-carriage may also lead to enhanced virulence or adaption to specific habitats in some strains of pandemic ESBL-producing *E. coli* lineages.

**Keywords:** ESBL-producing *E. coli*, ESBL-plasmids, fitness costs, enhanced virulence, biofilm formation, plasmid and host interaction
# INTRODUCTION

The global emergence of antimicrobial resistance, including extended-spectrum beta-lactamases (ESBL), is driven not only by plasmids encoding for these factors, but is also crucially influenced by pandemic bacterial clonal lineages (Naseer and Sundsfjord, 2011). The success of the pathogenic ESBL-producing *E. coli* clonal lineage of sequence type ST131 and virulence-associated phylogenetic group B2 is particularly noteworthy (Nicolas-Chanoine et al., 2014). B2-ST131 is found worldwide in environments with high antimicrobial selection pressures, including human and veterinary clinics and communities (Nicolas-Chanoine et al., 2008; Ewers et al., 2010). ST131 is also found in remote areas and wildlife (Bonnedahl et al., 2014), where antimicrobial influence is thought to be of lower importance. Despite the recognition of the human clinical reservoir as most abundant, several studies have demonstrated increasing prevalence of ST131 in animals and extra-clinical settings (Ewers et al., 2012). The acquisition of ESBL-genes happened over time, and the initial spread of ST131 most likely evolved from an emergence of chromosomally encoded fluoroquinolone resistance (Nicolas-Chanoine et al., 2014). Besides ST131, several STs, including ST648, ST617, ST167, ST410, ST224, and ST117, appear to be associated with ESBL-production, which demonstrates that ESBL-producing isolates are not equally distributed over all phylogenetic backgrounds (Ewers et al., 2012). This is expected in cases of solely plasmid-driven spread (Ewers et al., 2012). Regarding the success of pathogenic clonal lineages of B2-ST131, similar scenarios might also apply to CTX-M-producing lineages of ST648 belonging to phylogenetic group D, also known for harboring virulent isolates (Pitout, 2012; Ewers et al., 2014). Many strains of ST131 and ST648 carry plasmids encoding ESBL-enzymes, often of the CTX-M-15 type, and have become problematic due to limitations in antimicrobial therapies (Johnson et al., 2010). Besides ESBL-encoding genes, plasmids of these *E. coli* isolates contain antibiotic resistance determinants affecting various antimicrobial classes, which often results in multi-drug resistant phenotypes (Woodford et al., 2011). Prior studies suggest trade-offs between antibiotic-resistance and fitness in such strains (Dasilva and Bailey, 1986; Lenski, 1997; Andersson and Hughes, 2010). However, this does not necessarily apply to ST131 and ST648. In contrast, the combination of multi-resistance, virulence and phylogenetic background is hypothesized to be a recipe for their successful pandemic spread (Johnson et al., 2010; Pitout, 2012; Calhau et al., 2013). In addition to antibiotic resistance genes, ESBL-plasmids harbor non-resistance factors, which are partly unexplored. These include fertility and virulence factors, genes for plasmid maintenance including toxin-antitoxin systems, resistances against heavy metals (Seiler and Berendonk, 2012; Schaufler et al., 2013), and putative protein-coding genes (Smet et al., 2010b).

Why do ESBL-associated STs exist, and why are certain clonal lineages so successful not only in environments with high and moderate antibiotic pressures, but also in antimicrobially isolated areas? It might be due to their ubiquity and frequent detection, whereas rare lineages are found less often, or for reasons beyond

antibiotic resistance such as virulence-associated factors. One possibility has rarely been studied: the interaction between ESBL-plasmids and the chromosomal content of particular clonal lineages. The influence of non-resistance genes on the chromosome may be of particular importance. For the closely related species *Klebsiella pneumoniae*, it was shown that acquisition of ESBL-plasmids lead to expression changes of chromosomally encoded fimbriae genes, subsequently affecting the overall invasion ability of tested strains (Sahy et al., 2008). Another main bacterial virulence factor is the chromosomally encoded ability to form biofilms. The subtle interactions between biofilm formation and its counterpart, motility, are mostly regulated by the transcriptional regulator csgD (curlin subunit gene D; Hammar et al., 1995; Dudin et al., 2014). Biofilm formation has previously been linked to antimicrobial resistance (Ito et al., 2009); however, the influence of ESBL-plasmids on csgD-associated virulence features remains to be investigated.

This study addressed two hypotheses: (i) ESBL-plasmid carriage does not negatively influence the host's growth/metabolic fitness; and (ii) ESBL-plasmid carriage supports the host through the ESBL-plasmid's influence on chromosomally encoded virulence-associated features.

# MATERIALS AND METHODS

## Strains

Seven wild-type (WT) ESBL-producing *E. coli* strains, their ESBL-"plasmid" cured variants (PCV) (Schaufler et al., 2013), and transformants (T) with the reintroduced large ESBL-plasmid constructed from PCVs (Green and Sambrook, 2012) were analyzed in this study; in total, 21 strains were studied. The WT B2-ST131 and D-ST648 strains originated from different hosts including humans, companion animals, and wild birds (Supplementary Table S1).

## ESBL-Plasmid-"Curing" and Transformations

As previously described by Schaufler et al. (2013), large ESBL-plasmids were extracted from seven WT ESBL-carrying *E. coli* strains using a heat technique (Dale and Park, 2004). Loss of the large ESBL-plasmid as well as the clonal character of WT and its corresponding PCV strain were tested via plasmid-profile analysis and XbaI-pulsed-field gel electrophoresis (PFGE) (Schierack et al., 2009). To assure genetic identity and to exclude chromosomal changes in the PCV, the whole genome sequences of WT and PCV strains were analyzed using bioinformatics. This included a pairwise comparison of the number of orthologous genes using the OrthoMCL pipeline (Chen et al., 2006) and computation of the phylogenetic distances of all strains based on their Maximum Common Genome (MCG) (Von Mentzer et al., 2014) as previously described (Schaufler et al., 2013). Transformants with the reintroduced large ESBL-plasmid were constructed to verify the observed phenotypic differences (Supplementary Table S1). PCVs functioned as electrocompetent acceptor strains; thus, the respective large ESBL-plasmid was transformed via electroporation (Green and Sambrook, 2012). Transformed
strains were screened for ESBL-production on cefotaxime (4 µg/mL; Sigma-Aldrich, Taufkirchen, Germany) containing CHROMagar™ (MAST Diagnostica, Reinfeld, Germany) plates for ESBL-enzyme-production. Plasmid profile analysis was used to evaluate the success of electroporation.

## ESBL-Plasmid Characteristics

Whole genomes of all WT and their corresponding PCV strains were sequenced using an Illumina HiSeq2000 sequencer in collaboration with the Wellcome Trust Sanger Institute (Cambridge, United Kingdom). The resulting reads were used for a *de novo* assembly (CLC Genomics Workbench 6.5, CLC Bio, Denmark). PLACNET analysis (Lanza et al., 2014) was then performed for all genomes to extract putative plasmid contiguous sequences (contigs) and to assign them to the large ESBL-plasmid and to smaller, additional non-ESBL-plasmids. These were confirmed via BLAST analysis, in which they were compared to reference sequences of known plasmids. All previously defined plasmid contigs showed a high similarity to parts of plasmids that have already been described. Annotation of the contigs was performed using the annotation feature of the program Geneious version 7.1.2 (Kearse et al., 2012) with 100% similarity to an in-house plasmid reference data base (data not shown). ESBL types, relaxase (REL) and plasmid replication initiator (RIP) proteins, incompatibility (Inc) groups, approximate ESBL-plasmid sizes, the number of additional plasmids, and virulence factors were detected based on PLACNET ESBL-plasmid sequences and using VirulenceFinder 1.4 (Joensen et al., 2014) and ResFinder 2.1 (Zankari et al., 2012; **Table 1**). BLAST ring image generator (BRIG) (Alikhan et al., 2011) was used to visualize the annotated PLACNET ESBL-plasmid sequences (pIMT17433, pIMT19205, pIMT27685, pIMT16316, pIMT17887, pIMT21183, and pIMT23463) of the seven WT strains with pEC_L8 as a reference ESBL-plasmid (Smet et al., 2010b; **Figure 1**).

## Growth Curves

Growth curves in LB medium were performed in triplicate using standard protocols.

## Omnilog® Phenotype Microarray (PM)

Using 96-well microtiter plates spotted with different substrates, growth/metabolic activity in 379 single substrates and sensitivity to 48 antimicrobial and chemical compounds was analyzed for all strains [Omnilog® (Biolog, Hayward, USA), Supplementary Table S2]. Three biological replicates were tested on six PM plates (PM1 and PM2: carbon sources; PM3: nitrogen sources; PM4: phosphorus and sulfur sources [http://www.biolog.com/pdf/pm_lit/PM1-PM10.pdf]; PM13 and PM14: chemical sensitivity [http://www.biolog.com/pdf/pm_lit/PM11-PM20.pdf]). Plates were inoculated with the bacterial suspension according to Omnilog® PM protocols. Plates were incubated for 48 h at 37°C. Respiration was measured by dye (tetrazolium violet) reduction every 15 min. The calculated longitudinal respiration kinetics were analyzed in R (Vaas et al., 2013). In confidence interval plots computed with extract defined by enlisted metadata for the parameter “area under the curve,” only wells in which the normalized mean point estimates of WT and T groups showed

<table><caption>TABLE 1 | Characteristics of large ESBL-plasmids of the seven ESBL-producing wild-type strains.</caption><thead><tr><th>Strain</th><th>ESBL type</th><th>ESBL-plasmid REL/RIP proteins and Inc group</th><th>ESBL-plasmid size (bp)</th><th>Number of additional plasmids (sizes in bp)</th><th>ESBL-plasmid additional resistance genes</th><th>ESBL-plasmid virulence genes</th></tr></thead><tbody><tr><td>IMT17433</td><td>CTX-M-15</td><td>MOB-F12, RepFIA/FII, IncF</td><td>122,823</td><td>1 (1600)</td><td>blaTEM-1, blaOXA-1, tet(A), tet(R), aadA, aac(6')-lb-cr, catB4</td><td>finO, traT</td></tr><tr><td>IMT19205</td><td>CTX-M-27</td><td>MOB-F12, RepFIA/FIB/FII, IncF</td><td>166,151</td><td>2 (1500, 5200)</td><td>blaTEM-1, tet(A), sul2, strA, aadA, aac(3')-IId, aac(3')-IV, aac(6')-lb-cr</td><td>finO, traT, senB</td></tr><tr><td>IMT27685</td><td>CTX-M-15</td><td>MOB-F12, RepFIA/FII, IncF</td><td>133,611</td><td>1 (1600)</td><td>blaOXA-1, tet(A), sul1, strA, strB, aadA, aac(6')-lb-cr, dfrA17</td><td>traT</td></tr><tr><td>IMT16316</td><td>CTX-M-15</td><td>MOB-F12, RepFIA/FIB, IncF</td><td>136,508</td><td>4 (2500, 3200, 4100, 7000)</td><td>blaTEM-1, tet(A), tet(R), sul1, sul2, strA, strB, aadA, aac(3')-I, dfrVII, dfrA17</td><td>finO, traT</td></tr><tr><td>IMT17887</td><td>CTX-M-15</td><td>MOB-F12, RepFIA/FIB, IncF</td><td>143,494</td><td>2 (2600, 7100)</td><td>blaTEM-1, tet(A), tet(R), sul1, sul2, strA, strB, aadA, aac(3')-IId, aph(3')-Ia, dfrVII, dfrA17</td><td>finO, traT</td></tr><tr><td>IMT21183</td><td>CTX-M-15</td><td>delta-Tal, RepFIA, IncF</td><td>103,420</td><td>1 (7100)</td><td>blaTEM-1, tet(A), tet(R), sul1, sul2, strA, strB, aadA, aac(3')-I, aph(3')-Ia, dfrVII, dfrA17</td><td>traT</td></tr><tr><td>IMT23463</td><td>CTX-M-14</td><td>MOB-F12, RepFIB/FII, IncF</td><td>143,193</td><td>1 (4100)</td><td>blaTEM-1, blaOXA-1, tet(A), sul2, strA, strB, aadA, aac(3')-IId, aac(6')-lb-cr, dfrA17</td><td>finO, traT, sitABCD, cma</td></tr></tbody></table>

REL, relaxase; RIP, replication initiator protein; Inc, incompatibility; bp, base pairs.
<figure><img src="image_39.png" /><figcaption>FIGURE 1 | Circular visualization of the seven wild-type ESBL-plasmid sequences as compared to *E. coli* ESBL-plasmid pEC_L8 using BRIG (Alikhan et al., 2011).</figcaption></figure>

no overlapping 95% confidence intervals as compared to those of the PCV group, were considered to be significantly different. The group means of these wells underwent further statistical analysis using Tukey's method for multiple comparisons.

## Macrocolonies

Three microliters of overnight culture (from a single colony grown in 5 mL BHI broth) from all strains were dropped on span agar plates (H. Carroux, Germany) with or without sodium chloride (5%) and congo red solution [0.5% congo red (Sigma-Aldrich, Taufkirchen, Germany) and 0.25% coomassie-brilliant-blue (Carl Roth, Karlsruhe, Germany) diluted in ethanol]. Plates were incubated for 5 days at 28°C (Romling, 2005; Richter, 2011). Following an initial comprehensive screening to detect differences between WT and PCV strains regarding their cellulose and/or curli fimbriae production, follow-up runs were performed on plates containing and lacking sodium chloride for all strains (Table 2). These follow-up runs were repeated six times. Reference strains (AAEC189, Blomfield et al., 1991, IMT26949, and W3110 Hayashi et al., 2006) were included in all runs for all plates and at all temperatures.

<table><caption>TABLE 2 | Cellulose and curli fimbriae expression by 17433 and 16316 strains on span agar plates with congo red, with or without sodium chloride, incubated at 28°C for 5 days.</caption><thead><tr><th rowspan="2">Strain</th><th colspan="2">With sodium chloride</th><th colspan="2">Without sodium chloride</th></tr><tr><th>Cellulose</th><th>Curli</th><th>Cellulose</th><th>Curli</th></tr></thead><tbody><tr><td>IMT17433</td><td>*****</td><td>*****</td><td>*****</td><td>*****</td></tr><tr><td>PCV17433</td><td></td><td></td><td></td><td></td></tr><tr><td>T17433</td><td>*****</td><td>*****</td><td>*****</td><td>*****</td></tr><tr><td>IMT16316</td><td>*****</td><td>*****</td><td></td><td></td></tr><tr><td>PCV16316</td><td></td><td>*****</td><td></td><td></td></tr><tr><td>T16316</td><td>*****</td><td>*****</td><td></td><td></td></tr></tbody></table>

Six repetition runs followed a comprehensive initial screening. *, positive cellulose and/or curli fimbriae phenotype per run; 16316 strains were not tested on plates lacking sodium chloride as they did not show any differences during the initial screening.

## Biofilm Formation Assays

Overnight culture (from a single colony grown in 5 mL LB and minimal medium M63 overnight, Pardee et al., 1959) was set to optical density OD$_{600}$ = 0.05. Technical triplicates of