# Antimicrobial resistance and virulence common mechanisms (ebook)

Etienne Giraud, Ivan Rychlik, Axel Cloeckaert

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Etienne Giraud, Ivan Rychlik, Axel Cloeckaert (Dir.). Antimicrobial resistance and virulence common mechanisms (ebook). Frontiers Media, 138 p., 2017, Frontiers in Microbiology, 9782889451814. 10.3389/978-2-88945-181-4. hal-01605380

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Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
ANTIMICROBIAL RESISTANCE
AND VIRULENCE COMMON
MECHANISMS

EDITED BY: Etienne Giraud, Ivan Rychlik and Axel Cloeckaert

PUBLISHED IN: Frontiers in Microbiology
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ISSN 1664-8714

ISBN 978-2-88945-181-4

DOI 10.3389/978-2-88945-181-4

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ANTIMICROBIAL RESISTANCE AND
VIRULENCE COMMON MECHANISMS

Topic Editors:

**Etienne Giraud**, National Institute for Agricultural Research, France

**Ivan Rychlik**, Veterinary Research Institute, Czechia

**Axel Cloeckaert**, National Institute for Agricultural Research, France

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Scanning electron microscopy image of a *Streptococcus suis* biofilm. Certain physiological adaptations like biofilm formation may affect both virulence by increasing resistance to host defense response and resistance to antibiotics by decreasing their access to biofilm embedded bacteria.

Image taken from: Wang S, Yang Y, Zhao Y, Zhao H, Bai J, Chen J, Zhou Y, Wang C and Li Y (2016) Sub-MIC Tylosin Inhibits *Streptococcus suis* Biofilm Formation and Results in Differential Protein Expression. Front. Microbiol. 7:384. doi: 10.3389/fmicb.2016.00384

Multiple relationships exist between antimicrobial resistance and bacterial virulence, and the spread of clones combining multiple antibiotic resistance and a high virulence level is an increasing problem. It was previously described how mutation-driven or horizontally acquired resistance mechanisms can also have effects on virulence. It was also reported that mobile genetic elements often carry both resistance determinants and virulence-modulating genes, which favors the co-selection of both traits. In the present volume, we present a collection of articles which document additional aspects of the interactions between antimicrobial resistance and virulence in bacteria, and describe their potential therapeutic consequences.

**Citation:** Giraud, E., Rychlik, I., Cloeckaert, A., eds. (2017). Antimicrobial Resistance and Virulence Common Mechanisms. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-181-4
Table of Contents

**05 Editorial: Antimicrobial Resistance and Virulence Common Mechanisms**
Etienne Giraud, Ivan Rychlik and Axel Cloeckaert

**08 Multidrug Efflux Pumps at the Crossroad between Antibiotic Resistance and Bacterial Virulence**
Manuel Alcalde-Rico, Sara Hernando-Amado, Paula Blanco and José L. Martínez

**22 PSM-Mec—A Virulence Determinant that Connects Transcriptional Regulation, Virulence, and Antibiotic Resistance in Staphylococci**
Li Qin, Joshua W. McCausland, Gordon Y. C. Cheung and Michael Otto

**30 Sub-MIC Tylosin Inhibits Streptococcus suis Biofilm Formation and Results in Differential Protein Expression**
Shuai Wang, Yanbei Yang, Yulin Zhao, Honghai Zhao, Jingwen Bai, Jianqing Chen, Yonghui Zhou, Chang Wang and Yanhua Li

**39 Absence of Protoheme IX Farnesyltransferase CtaB Causes Virulence Attenuation but Enhances Pigment Production and Persister Survival in MRSA**
Tao Xu, Jian Han, Jia Zhang, Jiazhen Chen, Nan Wu, Wenhong Zhang and Ying Zhang

**53 Polyphosphate Kinase Mediates Antibiotic Tolerance in Extraintestinal Pathogenic Escherichia coli PCN033**
Jing Chen, Lijie Su, Xiangru Wang, Tao Zhang, Feng Liu, Huanchun Chen and Chen Tan

**63 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

**74 Carriage of Extended-Spectrum Beta-Lactamase-Plasmids Does Not Reduce Fitness but Enhances Virulence in Some Strains of Pandemic E. coli Lineages**
Katharina Schaufler, Torsten Semmler, Derek J. Pickard, María de Toro, Fernando de la Cruz, Lothar H. Wieler, Christa Ewers and Sebastian Guenther

**86 Fitness Cost of Fluoroquinolone Resistance in Clinical Isolates of Pseudomonas aeruginosa Differs by Type III Secretion Genotype**
Melissa Agnello, Steven E. Finkel and Annie Wong-Beringer

**98 Virulence and Genomic Feature of Multidrug Resistant Campylobacter jejuni Isolated from Broiler Chicken**
Haihong Hao, Ni Ren, Jing Han, Steven L. Foley, Zahid Iqbal, Guyue Cheng, Xiuhua Kuang, Jie Liu, Zhenli Liu, Menghong Dai, Yulian Wang and Zonghui Yuan

**112 Is Quorum Sensing Interference a Viable Alternative to Treat Pseudomonas aeruginosa Infections?**
Rodolfo García-Contreras
**119 PA3297 Counteracts Antimicrobial Effects of Azithromycin in Pseudomonas aeruginosa**

Hao Tan, Lu Zhang, Yuding Weng, Ronghao Chen, Feng Zhu, Yongxin Jin, Zhihui Cheng, Shouguang Jin and Weihui Wu

**131 Efflux Pump Blockers in Gram-Negative Bacteria: The New Generation of Hydantoin Based-Modulators to Improve Antibiotic Activity**

Ewa Otrębska-Machaj, Jacqueline Chevalier, Jadwiga Handzlik, Ewa Szymańska, Jakub Schabikowski, Gérard Boyer, Jean-Michel Bolla, Katarzyna Kieć-Kononowicz, Jean-Marie Pagès and Sandrine Alibert
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updates

# Editorial: Antimicrobial Resistance and Virulence Common Mechanisms

Etienne Giraud<fnref n="1" />,<fnref n="2" />*, Ivan Rychlik³ and Axel Cloeckaert<fnref n="1" />

**Keywords:** antimicrobial resistance mechanisms, virulence, co-evolution, biofilms, fitness costs

## Editorial on the Research Topic

### Antimicrobial Resistance and Virulence Common Mechanisms

Multiple relationships exist between antimicrobial resistance and bacterial virulence, and the spread of clones combining multiple antibiotic resistance and a high virulence level is an increasing problem. These relationships have been previously reviewed, notably by Beceiro et al. (2013), who described how mutation-driven or horizontally acquired resistance mechanisms can also have effects on virulence. It was also reported that mobile genetic elements often carry both resistance determinants and virulence-modulating genes, which favors the co-selection of both traits. In the present volume, we present a collection of articles which document additional aspects of the interactions between antimicrobial resistance and virulence in bacteria, and describe their potential therapeutic consequences.

In an excellent review article, Alcade-Rico et al. established that multidrug efflux pumps are “at the cross-road between resistance and virulence of bacterial pathogens.” They describe how multidrug efflux pumps are involved, besides antibiotic resistance, in many physiological and virulence-related processes controlled by complex regulatory networks. A special focus is made on the role of these efflux pumps in bacterial cell-to-cell communication and in bacterial-host interactions.

Another example of link between resistance and virulence in *Staphylococci* is given in a mini review by Qin et al. The authors summarized how the *psm-mec* locus present in some methicillin-resistant *Staphylococcus aureus* (MRSA) and coagulase-negative *Staphylococci* influences methicillin resistance and virulence-related phenotypes, including the ability to form biofilms.

In another paper also dealing with biofilms, Wang et al. used an up-to-date iTRAQ protein labeling technology to identify proteins that are differencially expressed in *Streptococcus suis* treated with sub-MIC concentrations of tylosin. These concentrations were indeed capable of inhibition of biofilm formation by this zoonotic pathogen, which is of interest, since biofilms are known to display an increased tolerance to antibiotics.

This antibiotic tolerance of bacteria present in biofilms is thought to be at least partially due to the presence of higher numbers of persisters than in planktonic bacterial populations. Persisters are phenotypic variants of bacteria which are able to survive antibiotic treatments in a dormancy state and to resume growth when the (antibiotic) stress is withdrawn. Like antibiotic-resistant bacteria, antibiotic-tolerant persisters are also often responsible for failures of antibiotic therapies. However, the mechanisms underlying their production are still not well-understood. An article by Xu et al. lifts a corner of the veil on this subject in *Staphylococcus aureus* by suggesting that heme biosynthesis, in addition to being necessary to a full expression of virulence, is also important in the formation of persisters in *S. aureus*.

**OPEN ACCESS**

**Edited by:**

Rustam Aminov,
University of Aberdeen, UK

**Reviewed by:**

Rustam Aminov,
University of Aberdeen, UK
Jose L. Martinez,
Consejo Superior de Investigaciones
Científicas, Spain
Kunihiko Nishino,
Osaka University, Japan

**\*Correspondence:**

Etienne Giraud
etienne.giraud@inra.fr

**Specialty section:**

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

**Received:** 30 January 2017

**Accepted:** 15 February 2017

**Published:** 03 March 2017

**Citation:**

Giraud E, Rychlik I and Cloeckaert A
(2017) Editorial: Antimicrobial
Resistance and Virulence Common
Mechanisms. Front. Microbiol. 8:310.
doi: 10.3389/fmicb.2017.00310

<footnote n="1">¹ ISP, Institut National de la Recherche Agronomique, Université François Rabelais de Tours, UMR 1282, Nouzilly, France,</footnote>

<footnote n="2">² TOXALIM (Research Centre in Food Toxicology), ENVT, INP-Purpan, Institut National de la Recherche Agronomique, UPS, Université de Toulouse, Toulouse, France, ³ Department of Immunology, Veterinary Research Institute, Brno, Czechia</footnote>
Another study by Chen et al. addressed the problem of antibiotic tolerance in extraintestinal pathogenic *Escherichia coli* (ExPEC). Authors studied the role of polyphosphate kinase (PPK), an enzyme already known for its implication in motility, quorum sensing or virulence, in antibiotic tolerance. Their results indicate that PPK is important for the antibiotic stress response and may therefore be a valuable antibacterial target, especially since it is absent in mammals.

Small Colony Variants (SCV) represent another state in which bacteria are more tolerant to antibiotics. The clinical importance of SCV has been well-documented in cases of chronic infections by *S. aureus* and *Pseudomonas aeruginosa* but their existence was only recently reported in *Listeria monocytogenes*, a food pathogen producing recurrent infections despite being rarely resistant to antibiotics. In an original research article, Curtis et al. described a heme mutant displaying a SCV phenotype mutant with an increased tolerance toward most of the relevant antibiotics. They suggest that the SCV phenotype of *L. monocytogenes* should be screened in clinical laboratories, as it might be associated to complicated antibiotic treatments.

Genetic changes leading to antimicrobial resistance are expected to result in higher fitness costs in the absence of antibiotic selection pressure. On the other hand, increased resistance is sometimes associated with increased virulence or to a better adaptation to stress conditions. This topic is developed in a study by Schaufler et al. who reported that the carriage of extended spectrum beta-lactamase (ESBL)-plasmids by strains of pandemic *E. coli* lineages did not lead to a fitness cost. Instead, these plasmids could enhance virulence or adaptation to specific habitats by influencing the expression of chromosomal genes.

In another article dealing with bacterial fitness, Agnello et al. presented results that possibly explain why highly virulent *P. aeruginosa* strains, which often possess the *exoU* toxin gene, are more frequently resistant to fluoroquinolones than the generally less virulent strains harboring the *exoS* toxin gene. Their results support the hypothesis that fitness cost imposed by fluoroquinolone resistance may be lower and "more easily" compensable by *exoU* strains than by *exoS* strains.

In a whole genome sequence-based study, Hao et al. analyze a multidrug resistant and virulent *Campylobacter jejuni* strain isolated from a broiler chicken. By comparison to reference strains, they revealed that the phenotype of this isolate was associated with large differences in the genome structure and content. This study provides an example of how a stepwise accumulation of mutations in antibiotic target genes, acquisition of exogenous resistance genes and virulence associated genes can result in the emergence of isolates that are both multidrug-resistant and virulent.

Since quorum sensing is a major player in the control of virulence factors in *P. aeruginosa*, therapies based on quorum sensing interference (QSI) have been envisaged as possible alternatives to conventional antibiotic therapies

against this pathogen. In a thoughtful perspective article, García-Contreras, however, compiles many recent experimental evidence which actually indicate that early optimistic expectations about QSI should be put in perspective. Indeed, he estimates that a far better understanding of *P. aeruginosa* virulence and behavior during infection is needed before efficient QSI-based clinical applications can be designed.

In addition to some immunomodulating activities, azithromycin (AZM) has various inhibiting effects on *P. aeruginosa* such as bacterial killing or the repression of multiple virulence factors. This antibiotic is therefore currently used in the treatment of cystic fibrosis patients, whose lungs are commonly colonized by this pathogen. However, *P. aeruginosa* responds to AZM by yet unknown mechanisms and is able to counteract its killing and virulence-inhibitory effects. An interesting work by Tan et al. revealed that PA3297, a *P. aeruginosa* gene encoding a DEAH-box helicase, is involved in this response. Indeed, deficiency of this gene renders *P. aeruginosa* more susceptible to the killing and to virulence suppression by AZM. Authors therefore suggested that targeting the regulatory pathway of PA3297 or its function might increase the beneficial effect of AZM in chronic *P. aeruginosa* infections such as those occuring in cystic fibrosis patients.

At last, in a pharmacological perspective, Obtreska-Machaj et al. report the development of hydantoin derivatives that have increased activity as inhibitors of the AcrAB-TolC efflux pump of *Enterobacter aerogenes*. Close homologs of this multidrug pump are also present in other pathogenic enterobacteria and involved in their virulence. The authors defined important pharmacophoric groups whose modulation may allow to develop still more potent inhibitors capable of restoring antibiotic activity in AcrAB producing bacteria.

It is likely that both resistant and virulent high risk clones will continue to emerge and spread in the next years. Several factors supports this hypothesis, among which the facts that highly pathogenic bacterial strains are more likely to be subjected to the selective pressure of antibiotics treatments, that virulence and resistance determinants are sometimes genetically associated and that fitness and virulence impairments that often accompany acquisition of resistance can be compensated in the long term by suppressor mutations (Beceiro et al., 2013; Mathers et al., 2015). In spite of these facts, virulence and antimicrobial resistance are studied usually separately rather than jointly. But now, as exemplified in this research topic, time has come for more integrative studies linking both traits and their co-evolution, so that we may be better armed against future bacterial threats.

## AUTHOR CONTRIBUTIONS

All authors have substantially contributed to this work and approved it for publication.
REFERENCES

Beceiro, A., Tomás, M., and Bou, G. (2013). Antimicrobial resistance and virulence: a successful or deleterious association in the bacterial world? *Clin. Microbiol. Rev.* 26, 185–230. doi: 10.1128/CMR.0059-12

Mathers, A. J., Peirano, G., and Pitout, J. D. D. (2015). The role of epidemic resistance plasmids and international high-risk clones in the spread of multidrug-resistant enterobacteriaceae. *Clin. Microbiol. Rev.* 28, 565–591. doi: 10.1128/CMR.00116-14

**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 © 2017 Giraud, Rychlik and Cloeckaert. 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.
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# Multidrug Efflux Pumps at the Crossroad between Antibiotic Resistance and Bacterial Virulence

Manuel Alcalde-Rico, Sara Hernando-Amado, Paula Blanco and José L. Martínez*

Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Madrid, Spain

Multidrug efflux pumps can be involved in bacterial resistance to antibiotics at different levels. Some efflux pumps are constitutively expressed at low levels and contribute to intrinsic resistance. In addition, their overexpression may allow higher levels of resistance. This overexpression can be transient, in the presence of an effector (phenotypic resistance), or constitutive when mutants in the regulatory elements of the expression of efflux pumps are selected (acquired resistance). Efflux pumps are present in all cells, from human to bacteria and are highly conserved, which indicates that they are ancient elements in the evolution of different organisms. Consequently, it has been suggested that, besides antibiotic resistance, bacterial multidrug efflux pumps would likely contribute to other relevant processes of the microbial physiology. In the current article, we discuss some specific examples of the role that efflux pumps may have in the bacterial virulence of animals' and plants' pathogens, including the processes of intercellular communication. Based in these evidences, we propose that efflux pumps are at the crossroad between resistance and virulence of bacterial pathogens. Consequently, the comprehensive study of multidrug efflux pumps requires addressing these functions, which are of relevance for the bacterial-host interactions during infection.

**Keywords:** multidrug efflux pumps, quorum sensing, antibiotic resistance mechanisms, virulence, global regulation

**OPEN ACCESS**

**Edited by:**
Axel Cloeckaert,
French National Institute
for Agricultural Research, France

**Reviewed by:**
Jessica M. A. Blair,
University of Birmingham, UK
Kunihiko Nishino,
Osaka University, Japan

*Correspondence:
José L. Martínez
jlmtnez@cnb.csic.es

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

**Received:** 23 June 2016
**Accepted:** 06 September 2016
**Published:** 21 September 2016

**Citation:**
Alcalde-Rico M, Hernando-Amado S, Blanco P and Martínez JL (2016) *Multidrug Efflux Pumps at the Crossroad between Antibiotic Resistance and Bacterial Virulence*. *Front. Microbiol.* 7:1483.
doi: 10.3389/fmicb.2016.01483

## INTRODUCTION

Multidrug resistance (MDR) efflux pumps are relevant elements belonging to the microbial repertoire that bacteria harbor for resisting the action of antimicrobial drugs (Piddock, 2006a; Vila and Martínez, 2008; Li et al., 2015; Jang, 2016). Indeed, several works have shown that these elements are involved in resistance of *in vitro* selected mutants as well as in the reduced susceptibility to antimicrobials of clinical isolates of different bacterial pathogens. The expression of efflux pumps is usually down regulated; only some of them present a substantial level of expression under regular growing conditions in the laboratory (Grkovic et al., 2001, 2002). However, constitutive high-level expression of these elements can be achieved by means of mutations in the elements regulating their expression. Transient high-level expression of efflux pumps can also be triggered in the presence of their effectors or under some specific growing conditions. In agreement with this situation, efflux pumps contribute to antibiotic resistance at three different levels: they can be involved in intrinsic resistance when presenting a basal level of expression under any condition.
They can contribute to acquired resistance when mutants achieving high-level of expression of the efflux pumps are selected. Finally, they can contribute to transient, non-inheritable, phenotypic resistance when bacteria are growing in the presence of an effector of the efflux pump or under growing conditions that trigger their overexpression. As reviewed in Hernando-Amado et al. (2016), efflux pumps are grouped in five structural families, namely the resistance-nodulation-division (RND), the small multidrug resistance (SMR), the multi antimicrobial extrusion (MATE), the major facilitator superfamily (MFS), and the ATP-binding cassette (ABC) superfamilies. Whereas some efflux pumps can work independently of any other protein, mainly in the case of Gram-positive organisms, in the case of Gram-negative organisms, they form tripartite complexes capable to traverse both bacterial membranes. These complexes include the inner-membrane efflux pump, a membrane fusion protein and an outer membrane protein.

When compared with other classical resistance genes, MDR efflux pumps present some specific features that support they should have other roles in the bacterial physiology besides their well-known involvement in antibiotic resistance. First, MDR efflux pumps are ubiquitous; they are present in all living cells, from humans to bacteria (Alonso et al., 1999; Alonso and Martinez, 2001; Gould et al., 2004; Sanchez et al., 2004). Second, the genes encoding them belong to the bacterial core genome in the sense that all (or most) members of a given species harbor the same efflux pumps (Alonso et al., 1999). Third, they are redundant; a single bacterial cell usually contains more than 10 different efflux pumps (Crossman et al., 2008). Fourth, they are rather unspecific; each efflux pump is able to extrude a variety of different substrates, including synthetic antibiotics as quinolones (Hernandez et al., 2011; Redgrave et al., 2014). Fifth, as above mentioned the expression of efflux pumps is tightly regulated; this regulation includes local regulators usually encoded upstream the structural genes of the operon encoding the efflux pump, as well as global regulators (Randall and Woodward, 2002; Luong et al., 2003; Nikaido et al., 2008; De Majumdar et al., 2013), frequently controlling the expression of a set of genes involved in the adaptation to a given ecosystem, as is the infected host. Sixth, at least in occasions, antibiotics are not good effectors of the expression of efflux pumps, whereas host-produced compounds as bile salts or plant-produced signals may induce the expression of MDR pumps (Rosenberg et al., 2003; Prouty et al., 2004; García-León et al., 2014). Altogether, these characteristics support that MDR efflux pumps are ancient elements (present in all organisms), important for the bacterial physiology (all members of a given species present the same, conserved efflux pumps), likely displaying different functions besides antibiotic resistance (a single microorganism contains a large number of different efflux pumps, with overlapping substrate ranges, including synthetic antibiotics not present in nature) and frequently integrated in complex response networks (they form part of global regulons and their expression is triggered by host produced compounds). In the present article we discuss some examples of the potential functions, besides antibiotic resistance, of MDR efflux pumps with a particular focus

on the role that they may have in bacterial-host interactions in animals (humans) and plants as well as in intercellular signaling (Piddock, 2006b; Martinez et al., 2009; Alvarez-Ortega et al., 2013).

## EFFLUX PUMPS AND CELL-TO-CELL COMMUNICATION

The capability to sense the environment and the organisms that are living in the same niche is critical to allow the microorganisms for choosing the best strategy to survive and colonize such niche. Along evolution, a battery of different mechanisms to sense the continuously changing environment has been selected in different microbial species. One of these mechanisms consists on the cell-to-cell communication systems. These inter-cellular signaling systems are based on the production of one or more low-molecular weight compounds which are sensed by molecular receptors of other cells, promoting a specific response in the target organism. In the bacterial world, this phenomenon is known as quorum sensing (QS) because it was initially described as a mechanism to sense the density of the bacterial population belonging to the same species present in a given habitat. The QS system allows the establishment of a cooperative genetic program of the whole population that increases the microbial efficacy for colonizing a given environment, including the infected host (Williams et al., 2007). The QS signal molecules (QSSMs) are constitutively produced at low quantities by all cells in the population. Their release outside the cells allows a progressive accumulation of QS signals in the intercellular space. The consequence is an increasingly production and accumulation of QSSMs while the population size increases, activating the QS response when the concentration of the signal reaches a threshold level. Further, these molecules (known as autoinducers) are able to induce their own production when they bind to their cognate transcriptional regulators, which produce a feed-forward regulation circuit. The regulator-QSSM complex is the main responsible of triggering the QS response by increasing the expression of a large number of genes, including those encoding the autoinducer synthase enzymes, which further increases the QSSM production. This response coordinates a number of physiological changes at a population-scale level, which allows among other issues improving bacterial competition for nutrients with other species, forming morphological-resistance structures to overcome environmental threats or triggering the expression of virulence factors (Swift and Downie, 2001; Williams, 2007). Further, it has been shown that several QSSMs can be sensed by other species promoting responses mediated by inter-species communication processes (Williams et al., 2007; Shimada et al., 2013; Lee and Zhang, 2015), including eukaryotic cells (interkingdom signaling; Martinez, 2014). As above stated these QSSMs have to cross the cells membranes for their extra-cellular accumulation, and it has been reported that some MDR efflux pumps might be involved in their transport outside the cell. In this section, we will discuss the role of MDR efflux pumps in the modulation of intercellular signaling and how the acquisition of antibiotic resistance in mutants overexpressing efflux pumps
<figure><img src="image_6.png" /></figure>

may challenge bacterial virulence through alterations in the
diffusion of QS signals.

Pseudomonas aeruginosa is one of the most important opportunistic pathogens causing infections at hospitals as well as in cystic fibrosis patients (Buhl et al., 2015; Kaye and Pogue, 2015; Oliver et al., 2015; Talwalkar and Murray, 2016). This pathogen is able to produce different virulence factors, many of them being regulated by a hierarchically organized QS signaling system (Figure 1), which consists in three different and interconnected regulatory networks each one respectively governed by the transcriptional regulators LasR, RhlR, and PqsR (Lee and Zhang, 2015). These three QS regulators recognize respectively one of different QSSMs produced by P. aeruginosa: N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL), N-butanoyl-L-homoserine lactone (C4-HSL),

and *Pseudomonas* quinolone signal (PQS). The presence of an additional regulatory QS network has been recently described (Lee et al., 2013; Lee and Zhang, 2015). Despite being tightly regulated by the *las* system in standard conditions of growth, this new system is able to trigger the PQS and C4-HSL production in absence of the *las* system or under phosphate stress conditions. However, the mechanisms responsible of the regulation mediated by this QS system are not fully understood and consequently will not be discussed along the present review.

Some works have shown that the expression of different efflux pumps encoded in the *P. aeruginosa* chromosome may have an impact in the QS-regulation networks of this microorganism. For instance, the RND efflux pump MexAB-OprM is highly integrated within the *las* and *rhl* QS regulons, since it has
been shown that its expression can be induced by C4-HSL (Maseda et al., 2004; Sawada et al., 2004). In addition, this efflux pump is able to extrude the 3-oxo-C12-HSL QS signal (Evans et al., 1998; Pearson et al., 1999; Minagawa et al., 2012). In such a way, the constitutive high-level expression of MexAB-OprM entails the extrusion of 3-oxo-C12-HSL, increasing the concentration of this QS signal around the cell, but reducing its intracellular accumulation. Indeed, antibiotic resistant mutants overexpressing MexAB-OprM present defects in the production of several virulence factors and are impaired in their QS response (Evans et al., 1998).

It is important to highlight that the 3-oxo-C12-HSL autoinducer is not just an intra-specific signal compound but may also entail inter-kingdom signaling. For instance, it has been reported that this QS signal may inhibit the filamentous differentiation, which is linked to a virulent state of *Candida albicans*, a fungal pathogen commonly found in patients with *P. aeruginosa* infections (Hogan et al., 2004). Conversely, it has been also shown that the *C. albicans* QS compound farnesol in turn can inhibit the production of QSSMs and the production of virulence factors by *P. aeruginosa* (Méar et al., 2013), evidencing the relevance of these cell-to-cell communication “weapons” in the establishment of a competitive interaction between these two opportunistic pathogens. In addition to its role in inter-microbial interactions, a role as activator of the human immune system has been attributed to 3-oxo-C12-HSL. This autoinducer signal can act as a chemoattractant for polymorphonuclear neutrophils and is able to induce the expression of adhesion proteins and immunoglobulin receptors implicated in the recognition and localization of microbial infections (Smith et al., 2001; Zimmermann et al., 2006; Wagner et al., 2007). On the other hand, it has been shown that 3-oxo-C12-HSL can induce the apoptosis of neutrophils and macrophages (Tateda et al., 2003). The fact that 3-oxo-C12-HSL has been detected directly in the sputum of cystic fibrosis patients with *P. aeruginosa* infections indicates that this QS signal might be involved in *in vivo* interkingdom signaling (Erickson et al., 2002; Middleton et al., 2002).

*Pseudomonas aeruginosa* MDR strains overexpressing MexAB-OprM produce lower amounts of virulence factors as well as of 3-oxo-C12-HSL (Evans et al., 1998). In addition, * mexAB-OprM * defective mutants are avirulent in a mouse model of infection as well as in MDCK cells (Hirakata et al., 2002). These results support the notion that this efflux system is directly involved in the appropriate and coordinate *las* response needed for a successful host infection. Further, it has been proposed that this system is used by *P. aeruginosa* to optimize indirectly the specific binding of the LasR regulator to 3-oxo-C12-HSL by extruding other 3-oxo-Cn-HSL, which can affect the *las* network activation because they are able of competing with 3-oxo-C12-HSL for the same binding site in LasR (Minagawa et al., 2012). All these intra- and interspecific responses mediated by 3-oxo-C12-HSL suggest that MexAB-OprM is an important element modulating cell-to-cell communication and host-pathogen interactions. As a consequence, the acquisition of antimicrobial resistance through the overexpression of this system could have an impact, at several levels, on the virulence of *P. aeruginosa*.

Besides MexAB-OprM, other *P. aeruginosa* efflux pumps might be involved in the regulation of the expression of QS-dependent virulence factors. One of them is MexEF-OprN, an efflux pump able to extrude both the QS signal HHQ (Lamarche and Deziel, 2011) and its precursor, kynurenine (Olivares et al., 2012), making it an important element in the QS response (Köhler et al., 2001). As we have mentioned above, the virulence and pathogenicity of *P. aeruginosa* is partially controlled by the PQS communication system. Therefore, it is not surprising that, as it happens in the case of MexAB-OprM, mutants overexpressing MexEF-OprN are affected in the production of QS-regulated virulence factors and, in consequence, they are impaired in the host infection process (Olivares et al., 2012). It is worth mentioning that the extrusion of kynurenine by this system could have an additional role in *P. aeruginosa*-host interaction. It has been shown that the expression of the MexEF-OprN system can be induced upon contact with human airway epithelial cells (Frisk et al., 2004). On the other hand, it has been suggested that *P. aeruginosa* production of kynurenine may have a role in the bacterial resistance to the toxic reactive oxygen species (ROS) produced by neutrophils, a key cell component in the innate immune system and in the inflammatory response in lung infections (Genestet et al., 2014). Based on this situation, it is possible that the increased expression of MexEF-OprN upon contact with the lung epithelial cells may allow *P. aeruginosa* to secrete high levels of kynurenine, thus promoting resistance against the neutrophil ROS production. This possibility, which has not been explored yet, would provide MexEF-OprN a new role in the infected lungs beyond the modulation of the QS response.

Another *P. aeruginosa* efflux pump with potential relevance in the virulence of this pathogen is MexGHI-OprD (Aendekerk et al., 2005; Dietrich et al., 2006). It has been shown that this efflux pump is able of extruding 5-methylphenazine-1-carboxylate (5-Me-PCA), a precursor of the phenazine pyocyanin, and anthranilate, the immediate precursor of PQS (Aendekerk et al., 2005). Further, the *mexGHI-oprD* expression could be induced by 5-Me-PCA and, in consequence, is under the transcriptional control of the QS response. It is worth mentioning that the phenazine extruded by this efflux pump is required for biofilm development by *P. aeruginosa* (Sakhtah et al., 2016). If we take into consideration that biofilms are the regular way of growing of *P. aeruginosa* in the lungs of chronically infected patients (Martinez-Solano et al., 2008; Wagner and Iglewski, 2008), this indicates that MexGHI might have a relevant role in the adaptation of *P. aeruginosa* for colonizing that habitat.

Another relevant pathogen whose virulence has been associated to the extrusion of QS signals by efflux pumps is *Burkholderia pseudomallei*, the causal agent of melioidosis. Different works have suggested that the virulence of this pathogen is modulated by the stationary phase sigma factor RpoS in addition to the regulatory activity of a QS system based on both acyl-homoserine lactones (AHLs) and 2-alkyl-4-quinolones (Ulrich et al., 2004; Wongtrakoongate et al., 2012; Butt et al., 2016). *B. pseudomallei* produces up to six different homoserine lactones: N-octanoyl-homoserine lactone (C8-HSL), N-decanoyl-homoserine lactone
(C10-HSL), *N*--(3-hydroxy)-octanoyl-homoserine lactone (3-OH-C8-HSL), *N*--(3-hydroxy)-decanoyl-homo-serine lactone (3-OH-C10-HSL), *N*--(3-oxo)-decanoyl-homoserine lactone (3-oxo-C10-HSL), and *N*--(3-oxo)-tetradecanoyl-homoserine lactone (3-oxo-C14-HSL). It has been reported that in the clinical strain KHW, the BpeAB-OprB efflux pump is strictly needed for the synthesis and full extrusion of these six AHLs and it has been suggested that another efflux pump, AmrAB-OprA could also be involved in the extrusion of 3-oxo-C10-HSL (Chan et al., 2007). In agreement with a potential role of these efflux pumps in QS communication, it has been shown that *bpeAB-oprB* expression is induced in presence of exogenous C8-HSL and C10-HSL (Chan and Chua, 2005). Moreover, a *bpeAB-oprB* defective mutant is affected in the expression of QS-dependent virulence factors as well as in biofilm formation. In addition, the mutant presents impaired invasiveness and cytotoxicity in both human macrophages and lung epithelial cells. Even though the role of BpeAB-OprB in virulence might be strain-dependent (Mima and Schweizer, 2010), all of the aforementioned results support the hypothesis that some efflux systems could be involved in the modulation of *B. pseudomallei* virulence and in the host-pathogen interactions through the extrusion of QS communication signals or by responding to their presence.

Other signaling networks involved in intra- and inter-specific communication are based in the use of indole and its derived compounds as signal molecules (Bommarius et al., 2013; Shimada et al., 2013; Lee J.H. et al., 2015). Indole is synthesized by plants and by many different bacteria and has different roles depending on the target species. Even though it was proposed that the efflux pump AcrEF could be implicated in indole export in *Escherichia coli* (Kawamura-Sato et al., 1999), other works have shown that indole is able to diffuse easily across the bacterial envelope (Gaede et al., 2005; Pinero-Fernandez et al., 2011) so that a clear role of efflux pumps in indole trafficking remains controversial. However, a crosstalk between virulence and efflux pumps-linked antibiotic resistance mediated through indole signaling is possible. Indeed, indole may affect the expression of efflux pumps, the resistance to antibiotics and the behavior of *Salmonella enterica* serovar Typhimurium when growing inside the host (Nikaido et al., 2012). This bacterial pathogen does not produce indole. However, it usually lives together with indole-producing species in the host and is able to respond to this signal (Nikaido et al., 2011). It is proposed that indole blocks the activity of the RamR transcriptional regulator through the interaction with its C-terminal domain, leading to the overexpression of RamA activator (Nikaido et al., 2011, 2012). This regulatory protein is able to induce the expression of *acrAB*, *acrEF*, and *tolC*, in addition of repressing the expression of some virulence determinants (Bailey et al., 2010; Nikaido et al., 2012). This entails the development of a low-virulence phenotype, which presents, however, increased resistance to antibiotics as well as to host-produced toxic compounds as bile salts and fatty acids, a phenotype closer to a commensal behavior than to an infective one. This is another example of the relationship between the communication signals, with inter-specific function in this case and the expression of efflux pumps, a situation with clear implications in the virulence potential of bacterial pathogens.

Indeed, the role of AcrAB-TolC in the virulence of *Salmonella* goes beyond the acquired phenotype upon induction by indole, since, as described below, it has been shown that *acrAB-tolC* deficient mutants have reduced invasiveness in animal models (Buckley et al., 2006; Webber et al., 2009).

MULTIDRUG EFFLUX PUMPS AND
THEIR ROLE IN THE VIRULENCE OF
HUMAN PATHOGENS

During the course of an infection, a bacterial pathogen has to be capable of surviving from the anti-infective defense mechanisms of the host. These mechanisms include, among others, the production of a diverse set of antimicrobial compounds as fatty acids, peptides or even detergents as bile salts, which function is in food uptake, but also present antimicrobial activity (Fernando and Kumar, 2013). In addition of extruding antibiotics regularly used for treating bacterial infections, different MDR efflux pumps have the ability to extrude a wide variety of compounds, including those antimicrobials produced by the host as well as QS signals involved in the regulation of the expression of virulence determinants (see above). Consequently, these efflux pumps are relevant payers on both antibiotic resistance and virulence of bacterial populations (Piddock, 2006b; Martinez et al., 2009; Alvarez-Ortega et al., 2013).

In the case of enteric bacteria, it has been shown that the efflux of several host-derived antimicrobial compounds, such as bile salts, allows the colonization and promotes the bacterial adaptation to the animal intestinal tract. The best-studied system able to confer resistance to bile salts is the *E. coli* RND efflux pump AcrAB-TolC, which is also a major contributor to intrinsic resistance to antibiotics in this organism (Thanassi et al., 1997). Similar roles have been reported for different AcrAB homologs from other Enterobacteriaceae species, such as *S. enterica* serovar Typhimurium. In this case, it has been described that mutants lacking *acrB* and *tolC* are less proficient for adhering, invading and surviving in mouse monocyte macrophages. The same study reported that an *acrB* mutant was able to colonize chicks. However, it was unable to survive gastrointestinally, which suggests that AcrB is not particularly relevant for the early steps of the gut colonization but it is needed for gastrointestinal persistence. On the other hand, the *tolC* mutant was able to colonize and persist in the chicken intestinal tract, but with a much lower efficiency than the wild-type strain, which could be due to the bile hypersensitivity displayed by this mutant (Buckley et al., 2006). A more recent study, using total genome transcriptional analysis, showed that the inactivation of *acrA*, *acrB*, and *tolC* rendered changes in the level of expression of several genes involved in bacterial pathogenicity, further supporting a crosstalk between resistance and virulence (Webber et al., 2009). For instance, the disruption of *acrB* or *tolC* led to a general repression of the SPI-1 pathogenicity island, which promotes the invasion of non-phagocytic intestinal epithelial cells, as well as bacterial survival and persistence within the host, while inactivation of *acrA* is associated with a repression of SPI-2, which promotes the survival and multiplication in
phagocytic cells (Dieye et al., 2009). Therefore, inactivation of *acrB* led to the inability to grow anaerobically, which would negatively impact in the capacity to survive in the host gut; as well as a reduced bacterial motility (Webber et al., 2009), which is also an important factor in the *S. enterica* serovar Typhimurium pathogenicity (Khoramian-Falsafi et al., 1990). As above stated, RND efflux systems are tripartite protein complexes formed by an inner membrane protein (the pump itself), an outer membrane protein and a linker, membrane fusion protein (Yamaguchi et al., 2015; Daury et al., 2016). The inner membrane and the membrane fusion proteins are always encoded in the same operon and are supposed to be specifically associated in each efflux pump, while the outer membrane protein can frequently form part of different efflux pumps. Consequently, while a differential response to the inactivation of the outer membrane protein of the complex (TolC in this case) as compared with the other members of the complex is conceivable, the observed differences between the *acrA* and *acrB* are more difficult to explain and suggests that a certain degree of trans-complementation between the components of different efflux pumps may happen. Although some information on this possibility has been published (Smith and Blair, 2014), this is a topic that remains to be studied in detail, although it merits to be addresses if we wish to understand in full the role that efflux pumps may play in the bacterial physiology besides antibiotic resistance.

An homologous of AcrAB is also present in the respiratory tract pathogen *Moraxella catarrhalis*. In addition of contributing to antibiotic resistance, this efflux pump is also involved in the efficient invasion of nasopharyngeal epithelial cells, since mutants lacking *acrA*, *acrB*, or *tolC* present decreased invasion levels as compared to the wild-type strain (Spaniol et al., 2015). This study also showed that exposure to cold shock (26°C) led to an increase in the expression of the efflux pump genes (Spaniol et al., 2015). Since temperature is an important factor for the adaptation and survival in the respiratory tract, as well as for the colonization properties and the virulence of *M. catarrhalis* (Heiniger et al., 2005; Spaniol et al., 2013), these results indicate that the levels of expression of this efflux pump are controlled by cues with relevance for the infectious success of *M. catarrhalis*. This further supports that efflux pumps may form part of global regulatory networks that include resistance and virulence determinants among other elements (Randall and Woodward, 2002; Luong et al., 2003; Nikaido et al., 2008; De Majumdar et al., 2013).

The ability to persist and to replicate in bile-rich environments is also critical for the pathogenesis of the food-borne pathogen *Listeria monocytogenes*. Among the elements that contribute to its survival in bile salts are the MDR efflux pumps MdrM and MdrT, belonging to the MFS family (Quillin et al., 2011). Expression of both MDR efflux pumps is strongly induced by cholic acid, a bile component, but only the MdrT efflux pump is able to extrude this compound, which is toxic for mutants lacking this efflux pump. Besides, MdrT is an important virulence factor involved in the colonization of the gallbladder *in vivo*, since mutants lacking *mdrT* are 100-fold attenuated. This study also suggests that MdrM has a synergistic role with MdrT in *L. monocytogenes* liver

colonization, although MdrM substrates have not been identified yet (Quillin et al., 2011).

RND efflux systems also play a role in the pathogenesis of *Vibrio cholerae*. It has been reported that VexAB, VexCD, VexIJK, and VexGH contribute not only to antimicrobial resistance, but also to the colonization of the infant mouse small intestine; deletion of these systems impair the colonization of the mice intestine by *V. cholerae* (Bina et al., 2008; Taylor et al., 2012). Besides being relevant factors for colonization, these efflux pumps are required for the expression of the genes that encode two of the most important virulence factors in *V. cholerae*: the cholera toxin (CT) and the toxin-coregulated pilus (TCP). In a mutant lacking *vexB*, *vexD*, *vexH*, and *vexK*, the production of CT and TcpA (the pilin subunit of the TCP) is reduced by 45%, while a six RND-null strain showed a 70% reduction in the expression of these virulence determinants, suggesting that the remaining efflux pumps, VexF and VexM, also contribute to virulence in *V. cholerae* (Taylor et al., 2012).

Multidrug efflux systems are also relevant elements in the defense against oxidative stress produced in the host during phagocytosis. For example, the ABC family efflux pump MacAB is required for the survival of *S. enterica* serovar Typhimurium inside macrophages, where they are exposed to ROS. It has been observed that mutants lacking *macAB* showed an impaired intracellular replication in macrophages as compared with the wild-type parental strain and also failed in growing in the inflamed intestine, where neutrophils release ROS. Further, the same mutants were able to grow inside macrophages that do not produce ROS, which implies that MacAB is needed for *S. enterica* serovar Typhimurium replication inside macrophages and for the survival under oxidative stress conditions. Besides, the *macAB* deletion mutant had a defect in liver colonization in BALB/c mice after intraperitoneal infection, which indicates another function of this system in the infective program of *S. enterica* serovar Typhimurium (Bogomolnaya et al., 2013).

In addition to the role of efflux pumps in the interaction of bacterial pathogens with the compounds present in the host, they also may play a direct role in virulence. This is the case for the *Mycobacterium tuberculosis* RND proteins designated as MmpL (*Mycobacterial membrane protein Large*; Cole et al., 1998). The genome of *M. tuberculosis* encodes 13 of these proteins, which role seems to be transporting lipids for their incorporation on the cell envelope, providing protection against host-derived compounds and contributing to the bacterial virulence (Neyrolles and Guilhot, 2011). Domenech et al. (2005) examined the contribution of these proteins to the bacterium virulence by using a murine model of infection and mutants for each MmpL protein. Among these proteins, they found that MmpL4, MmpL7, MmpL8, and MmpL11 were required for the virulence maintenance, since there is an increase in the survival time when the host is infected with these mutants as compared with the wild-type strain. The attenuation of the *mmpL7* mutant might be due to the lack of phthiocerol dimycocerosate (PDIM), which is an abundant wax of the outer cell matrix involved in the cell permeability (Camacho et al., 2001); and the *mmpL8* mutant is deficient in SL-1 because MmpL8 is involved in the transport of SL-N, a precursor of
SL-1 (Domenech et al., 2004). It has been shown that SL-N stimulates human CD1b-restricted T cells (Gilleron et al., 2004), a feature that might explain the attenuation of the *mmpL8* mutant if this molecule has a similar effect on murine CD1d-restricted T cells. The mechanisms by which *mmpL4* and *mmpL11* mutants were more attenuated have not been fully elucidated; however, it has been recently described a mutation in *mmpL4a* (Tyr842His) in *Mycobacterium bolletii*, which is responsible for the smooth-to-rough morphotype change, since MmpL4 is involved in the transport of glycopeptidolipid. This variant also contributes to the bacterial virulence in a zebrafish model (Bernut et al., 2016). The Tyr842 residue is conserved in all other mycobacterial MmpL4 orthologs and in all 13 MmpL RND proteins in *M. tuberculosis*, which indicates the functional relevance of this residue (Szekely and Cole, 2016).

Biofilms are complex microbial associations attached to a variety of surfaces. Bacteria that grow forming biofilms are more resistant to antibiotics than planktonic cells, being also important elements in the bacterial virulence and pathogenesis. The link between antimicrobial tolerance on biofilms and efflux pumps has been reported in several microorganisms (Soto, 2013). For instance, in the opportunistic pathogen *P. aeruginosa*, the MerR-like regulator BrlR plays a role in the high-level tolerance to antimicrobials in biofilms because it is able to activate under these growing conditions the expression of the multidrug efflux pumps MexAB-OprM and MexEF-OprN (Liao et al., 2013). A novel efflux pump in *P. aeruginosa* involved in biofilm tolerance, named as PA1874-1877, has been identified. The expression of this efflux pump was 10-fold increased in biofilms when compared with planktonic cells. Besides, deletion of the genes encoding this efflux pump resulted in an increased susceptibility to ciprofloxacin, gentamicin, and tobramycin (Zhang and Mah, 2008). Efflux pump expression can also impact the flagellar motility, which plays a relevant role in biofilm formation (Houry et al., 2012) and enhances pathogenicity by improving bacterial motility (Duan et al., 2013). In *Stenotrophomonas maltophilia*, another opportunistic pathogen, it has been observed that deletion of the RND efflux pump SmeYZ resulted in a reduced ability to form biofilm and the abolition of flagella formation. Besides, this deletion mutant was more susceptible to redox compounds, human serum and neutrophils, which indicates that this efflux pump is also involved in the protection against ROS (Lin et al., 2015).

Expression of efflux pumps not always enhance virulence; constitutive overexpression of these systems in antibiotic resistant mutants, can compromise the bacterial fitness and the virulence as well (Sanchez et al., 2002), indicating that the expression of these elements is finely regulated and deviations on this regulation, altering their expression below or above the physiological levels, may impair bacterial physiology and virulence. This is the case of the overexpression of the MDR efflux pumps MexCD-OprJ and MexEF-OprN in *P. aeruginosa*, which negatively affect the expression of the type III secretion system (T3SS; Linares et al., 2005). The T3SS is an important virulence mechanism, since bacteria are able to inject effector proteins manipulating the host cell function (Coburn et al., 2007). The effect of over-expression of efflux pumps on T3S was due to

the lack of expression of *exsA* gene, which is the transcriptional activator of the T3SS in *P. aeruginosa* (Linares et al., 2005). More recently, it was found that MexT, the positive regulator of MexEF, was able to repress the expression of the T3SS through the regulators MexS and PtrC (Jin et al., 2011), in such a way linking resistance and virulence within a single regulon.

Altogether, these works show that, in addition of being involved in antibiotic resistance, efflux pumps can participate in bacterial virulence as well. In some cases, a direct effect can be foreseen; this is the situation of efflux pumps able to extrude host-produced antimicrobial compounds. However, in other occasions the reasons behind the effect of efflux pumps on virulence are not so straightforward. In some cases, the effect on virulence is derived from the integration of the efflux pumps in a regulon that also includes virulence determinants. This could be the case of MexEF, a part of the MexT regulon that also includes the *P. aeruginosa* T3SS. Mutations in this global regulator will simultaneously alter antibiotic resistance and virulence, although the efflux pump itself is not directly involved in T3S. Other situation, also described for MexEF, is the capability of some efflux pumps for extruding intercellular signal molecules (see above) or their precursors (Olivares et al., 2012). If this efflux pump is abnormally expressed, the levels of expression of the genes belonging to the regulatory network (frequently including virulence genes) triggered by such signals will be also altered.

## THE FUNCTIONAL ROLE OF MULTIDRUG EFFLUX PUMPS IN PLANT-BACTERIA INTERACTIONS

Multidrug efflux pumps, in addition of being relevant antibiotic resistance determinants, are relevant key players for the behavior of microorganisms in their natural (non-clinical) habitats. Indeed, whereas most studies in human pathogens have concentrated in the role on antibiotic resistance of these elements, the analyses of efflux pumps from plants pathogens or epiphytes, has mainly focused on their role in plant-bacteria or bacteria-bacteria interactions. The rhizosphere is a natural ecosystem that includes a complex microbiome formed by microorganisms that live in contact with plants' roots. Roots' and other plants' exudates contain a large array of natural products, such as flavonoids, which confer them protection against microbial attack. However, different microorganisms have developed mechanisms to deal with the activity of those compounds. One of them is the flavonoid-responsive RND family of efflux pumps, which includes several members as MexAB-OprM from *Pseudomonas syringae*, AcrAB from *Erwinia amylovora*, AcrD from *Erwinia chrysanthemi*, IfeAB from *Agrobacterium tumefaciens*, XagID2689 from *Xanthomonas axonopodis*, SmeDEF from *S. maltophilia*, EmrAB from *Sinorhizobium meliloti* and BjG30 from *Bradyrhizobium japonicum* (Palumbo et al., 1998; Burse et al., 2004b; Vargas et al., 2011; Takeshima et al., 2013; García-León et al., 2014; Pletzer and Weingart, 2014; Rossbach et al., 2014; Chatnaparat et al., 2016). Some of these efflux pumps are implicated in plant colonization, whereas
some others are involved in bacteria/plant symbiosis processes. As stated before MexAB-OprM is described as a significant determinant of multidrug resistance in *P. aeruginosa* (Poole, 2001) having a basal expression level enough to contribute to the intrinsic antimicrobial resistance of these bacteria (Li et al., 1995) and it also has a role in virulence. Indeed, as above described, mutant strains overexpressing this efflux pump are less virulent because of the extrusion of the QS homoserine lactone 3-oxo-C₁₂-HSL (Minagawa et al., 2012). In addition of extruding antibiotics or QS signaling molecules, MexAB-OprM is able to pump out plant antimicrobial compounds from leaves of *Melaleuca alternifolia*, supporting that, in addition of its role in clinical settings, this efflux pump (and several others, see below) may allow bacterial survival in a vegetal environment (Papadopoulos et al., 2008). Indeed, this efflux pump is required for the efficient colonization of tomato plants by *P. syringae*, since the inactivation MexAB-OprM lead to a defective colonization capacity of the plant by this bacterial species (Vargas et al., 2011). In line with the role of efflux pumps in a general bacterial response to the plant antimicrobial defense, it has been shown that flavonoids are inducers and substrates of this transporter (Vargas et al., 2011). In addition, the same flavonoids are able to inhibit the GacS/GacA two component system (TCS) of *P. syringae*, which is implicated, among other regulatory elements, in the activation of the motility and the T3SS in this species (Chatterjee et al., 2003). In fact, the absence of MexAB-OprM and the accumulation of flavonoids inside bacteria lead to a reduction of swarming and swimming motility and a significant impairment in the production of flagella and T3S (Vargas et al., 2013). Therefore, when the amount of plant flavonoids is enough to reduce bacterial virulence by inhibiting GacS/GacA, *P. syringae* may extrude such flavonoids using the MexAB efflux pump (which expression is now induced), shrewdly regulating the intracellular level of flavonoids and consequently, ensuring the viability of the bacteria by the de-repression of this TCS. This is an elegant example of the adaptive co-evolution of plant resistance and pathogen virulence in which the role of this efflux pump goes beyond being a mere detoxification system (Figure 2).

Another plant-pathogenic bacteria is *E. amylovora*, an Enterobacteria that causes fire blight on apple and pear trees. The flavonoid inducible AcrAB-TolC efflux pump from *E. amylovora* confers resistance, in addition to antibiotics, to plant compounds as phytoalexins and flavonoids (phloretin, naringenin, and quercetin) leading to the successful colonization of plants (Burse et al., 2004b; Maggiorani Valecillos et al., 2006; Al-Karablieh et al., 2009). Moreover, an AcrAB deletion mutant has a strong reduction of *E. amylovora* virulence in apple plants (Burse et al., 2004b). A close homolog of AcrB is AcrD, an efflux pump that is also induced by plant flavonoids, such as luteolin, but whose deletion mutant exhibits full virulence on apple and pear fruits (Pletzer and Weingart, 2014). *E. chrysanthemi* constitutes another example in which efflux pumps are required to infect plants. A tolC mutant of *E. chrysanthemi* is unable to extrude the plant antimicrobial compound berberine, being then unable to cause plant tissue maceration. Moreover, TolC plays a role also

modulating the fitness of the bacteria growing in the microbial community (Barabote et al., 2003), indicating that efflux pumps might also be involved in inter-microbial interactions (see below). In addition of plant-derived antimicrobials, the AcrAB efflux pump of *E. chrysanthemi* is inducible by salicylic acid (SA), an important plant hormone implicated in local and systemic plant resistance (Raviralra et al., 2007). SA and others plant phenolic acids are able of reducing the expression of the T3SS, by inhibiting the GacS/A pathway, in *P. syringae* (Lee J.S. et al., 2015), suggesting that a role of efflux pumps in non-clinical ecosystems might be to adjust the intracellular level of plant signals, as a mechanism to deal with plant defense systems. Other efflux pump that has a role in plant colonization is IfeAB from *A. tumefaciens*, which extrudes coumestrol bestowing measurable ecological benefits to this bacterium in flavonoids rich environments. *X. axonopodis* pv. *glycines* is another plant pathogen, causative of bacterial pustule of *Glycine max*, one of the most important diseases in soybean. It contains another flavonoid inducible RND efflux pump, called XagID2689 (Chatnaparat et al., 2016), which in addition of being induced by flavonoids contributes as well to flavonoids' resistance; its deletion strongly reduces bacterial virulence in soybean. Moreover, this deletion mutant shows a higher susceptibility than the wild-type parental counterpart to the isoflavonoids phloretin, naringenin and berberine, as well as to the antibiotics acriflavine and tetracycline, suggesting a role in the intracellular reduction of the levels of several compounds produced by soybean and involved in its antimicrobial response program. In *S. maltophilia*, the SmeDEF efflux pump, which is the most important quinolone resistance determinant of this microorganism (Alonso and Martinez, 2000, 2001; Zhang et al., 2001; Garcia-Leon et al., 2014), is induced by flavonoids, that are able to bind to its SmeT repressor (García-León et al., 2014); in addition, a mutant lacking smeE is unable to colonize the roots of *Arabidopsis* plants (García-León et al., 2014), further supporting that MDR efflux pumps, with a relevant role for antibiotic resistance at clinical settings, might have been selected in nature for different ecological purposes.

In addition of easening the bacterial plants' colonization and infection, efflux pumps have also a role in the interactions between plants and their symbiotic bacteria. For example, EmrAB from *S. meliloti* is an inducible flavonoid efflux pump with a role in symbiosis with *Medicago sativa*; the symbiotic process is impaired when the regulator of this efflux system, the TetR repressor EmrR, is deleted (Rossbach et al., 2014; Santos et al., 2014). Other examples of efflux pumps with a role in symbiotic nitrogen-fixation activity processes in *G. max* are BdeAB and BjG30 from *B. japonicum* (Lindemann et al., 2010; Takeshima et al., 2013). BdeAB deficient mutants, in addition of presenting symbiotic defects, are more susceptible to aminoglycosides, highlighting the multifunctional role of efflux pumps (Lindemann et al., 2010).

Efflux pumps have also an important role in the inter-
microbial interactions in the host plant and in its rhizosphere,
where each bacteria have to compete for space and nutrients
to survive. As with antibiotic resistance mechanisms, that
can be considered as a colonization factor in the treated
<figure><img src="image_7.png" /></figure>

**FIGURE 2** | Role of the MexAB-OprM efflux pump in the adaptive co-evolution of plant defense and *P. syringae* virulence. The intracellular amount of the plant flavonoids is regulated by MexAB-OprM efflux pump. (A) Expression of *mexAB-oprM* is controlled by the transcriptional repressor PmeR; in the absence of flavonoids the expression of this efflux pump is repressed and expression of virulence determinants as the T3SS or bacterial motility is activated through the GacS/A TCS (Vargas et al., 2011). (B) The presence of flavonoids inhibits GacS/A, hence leading a transient low virulence phenotype, in which the expression of the T3SS and the motility are low (Vargas et al., 2013). (C) The accumulation of flavonoids inside the cell leads to over-expression of *mexAB-oprM* through the release of its transcriptional regulator PmeR from its operator. Since flavonoids are substrates of MexAB-OprM, under these conditions they are efficiently extruded and their intracellular concentration falls down below the threshold required for inactivating GacS/GacA. As a consequence of this situation, the virulent state of *P. syringae* is restored.

patient, where antibiotics are present (Martinez and Baquero, 2002), resistance to antimicrobials produced by epiphytes may be considered as a colonization tool of phytopathogenic bacteria and *vice versa*. If the epiphytes can inhibit the phytopathogens, the plant will be protected from infection, whereas if pathogens can inhibit epiphytes, this situation will provide a colonization advantage and hence better possibilities for infecting the plant. Resistance to compounds produced by bacterial competitors will then increase the chances for colonization of the epiphyte or the pathogen, with important consequence in terms of crops protection. This is the case of

the efflux pump NorM from *E. amylovora* that, in contrast to the previously mentioned efflux pump AcrAB from *E. amylovora* (Burse et al., 2004b), is not able to extrude the high amount of isoflavonoids that produce the members of the Rosaceae family (Burse et al., 2004a). Moreover, a *norM*-deficient mutant causes comparable symptoms as the wild-type parental counterpart in plant tissues, indicating that this efflux pump does not contribute to virulence of *E. amylovora* against apple plants. Nevertheless, while the NorM efflux pump does not directly contribute to virulence *E. amylovora*, it has an important role extruding toxic molecules produced by *Pantoea*
*agglomerans* (Pusey et al., 2011), an epiphytic bacteria that is an excellent colonizer of stigmas of apple and pear and it may effectively inhibit the multiplication of *E. amylovora* when both microorganisms co-colonize rosaceous (Burse et al., 2004a; Piddock, 2006b), hence constituting a potential biocontrol agent for fire blight (Kim et al., 2012). It is worth mentioning that NorM is involved in the capability *E. amylovora* to reach high-density populations at low temperatures. Indeed, at 18°C the growth of an *E. amylovora* norM mutant is significantly impaired as compared with the wild-type strain. In addition, the level of expression of *norM* is twofold greater at 18°C gene than at 28°C (Burse et al., 2004a). Altogether these results suggest that the intrinsic resistance of *E. amylovora* to *P. agglomerans* competition at 18°C may be due to the increased expression level of *norM*, which might allow the colonization by *E. amylovora* of the stigma surface of the blossom when it coexists with *P. agglomerans*. These evidences lead to hypothesize that the utilization of *P. agglomerans* as a biocontrol mechanism for fire blight would be compromised at low temperatures or by mutations causing of overexpression of *norM* in *E. amylovora*.

## AUTHOR CONTRIBUTIONS

All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.

## FUNDING

Work in our laboratory is supported by grants from the Spanish Ministry of Economy and Competitiveness (BIO2014-54507-R and JPI Water StARE JPIW2013-089-C02-01); from Madrid Autonomous Community [S2010/BMD2414 (PROMPT)]; and from the Instituto de Salud Carlos III [Spanish Network for Research on Infectious Diseases (REIPI RD12/0015)]. MA-R and PB are recipients of FPI fellowships from MINECO.

## ACKNOWLEDGMENT

Thanks are given to Mary Higgins for grammar corrections.

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**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 Alcalde-Rico, Hernando-Amado, Blanco and Martínez. 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_8.png" /></figure>

CrossMark

# PSM-Mec—A Virulence Determinant that Connects Transcriptional Regulation, Virulence, and Antibiotic Resistance in Staphylococci

Li Qin¹,², Joshua W. McCausland¹, Gordon Y. C. Cheung¹ and Michael Otto¹*

¹Pathogen Molecular Genetics Section, Laboratory of Bacteriology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA, ²Department of Dermatology, Wuhan No. 1 Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

PSM-mec is a secreted virulence factor that belongs to the phenol-soluble modulin (PSM) family of amphipathic, alpha-helical peptide toxins produced by *Staphylococcus* species. All known PSMs are core genome-encoded with the exception of PSM-mec, whose gene is found in specific sub-types of SCC*mec* methicillin resistance mobile genetic elements present in methicillin-resistant *Staphylococcus aureus* and coagulase-negative staphylococci. In addition to the cytolytic translational product, PSM-mec, the *psm-mec* locus encodes a regulatory RNA. In *S. aureus*, the *psm-mec* locus influences cytolytic capacity, methicillin resistance, biofilm formation, cell spreading, and the expression of other virulence factors, such as other PSMs, which results in a significant impact on immune evasion and disease. However, these effects are highly strain-dependent, which is possibly due to differences in PSM-mec peptide vs. *psm-mec* RNA-controlled effects. Here, we summarize the functional properties of PSM-mec and the *psm-mec* RNA molecule and their roles in staphylococcal pathogenesis and physiology.

**Keywords:** *Staphylococcus aureus*, *Staphylococcus epidermidis*, phenol-soluble modulin, PSM-mec, regulatory RNA, SCC*mec*, virulence

## OPEN ACCESS

**Edited by:**
Axel Cloeckaert,
French National Institute for
Agricultural Research (INRA), France

**Reviewed by:**
Jan Maarten Van Dijl,
University Medical Center Groningen,
Netherlands
Dinesh Sriramulu,
Shres Consultancy (Life Sciences),
India

*Correspondence:
Michael Otto
motto@niaid.nih.gov

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

**Received:** 07 June 2016
**Accepted:** 05 August 2016
**Published:** 22 August 2016

**Citation:** Qin L, McCausland JW, Cheung GYC and Otto M (2016) PSM-Mec—A Virulence Determinant that Connects Transcriptional Regulation, Virulence, and Antibiotic Resistance in *Staphylococci*. *Front. Microbiol.* 7:1293. doi: 10.3389/fmicb.2016.01293

## INTRODUCTION

*Staphylococcus aureus* is a serious human pathogen responsible for a multitude of human diseases, which range from acute skin and soft tissue infections to more severe illnesses such as catheter-associated bacteremia, necrotizing pneumonia, and osteomyelitis. Since the emergence of methicillin-resistant *S. aureus* (MRSA) in the early 1960s, *S. aureus* has continued to cause significant morbidity, mortality, and a considerable financial burden for public health systems. For *S. aureus* and its closely related cousin, *Staphylococcus epidermidis*, the acquisition of antibiotic resistance, coupled with the ability to attach to surfaces and form sticky, multicellular agglomerations (biofilms; Costerton, 1999) on indwelling catheters, are two defining hallmarks that account for the ongoing difficulty in treating immuno-compromised individuals (Otto, 2008) with infections caused by Hospital-Associated MRSA [HA-MRSA] (Otto, 2012) and *S. epidermidis* (Otto, 2009).

Genetic mutations and/or the acquisition of mobile genetic elements (MGEs) that harbor drug resistance genes are two common mechanisms that are responsible for antibiotic resistance in microorganisms. Arguably the most important staphylococcal MGE is *Staphylococcus* cassette
chromosome *mec* (SCC*mec*), which codes for methicillin
resistance. Recently, a unique locus was discovered in specific
types of SSC*mec* elements in *S. aureus* and *S. epidermidis*, which
comprises a small regulatory (sr)RNA and an embedded gene
encoding a cytolytic peptide called PSM-mec (Queck et al.,
2009). PSM-mec belongs to the phenol-soluble modulin (PSM)
family of amphipathic, alpha-helical peptide toxins produced by
*Staphylococcus* species (Wang et al., 2007, 2011; Diep and Otto,
2008; Otto, 2014; Cheung et al., 2014a), which collectively play
an important role as virulence determinants in many facets of
*S. aureus* and *S. epidermidis* pathogenesis (Wang et al., 2011;
Periasamy et al., 2012; Cassat et al., 2013; Cheung et al., 2014a).
Here, we will discuss the current knowledge on the PSM-mec and
the *psm-mec* srRNA molecule, with a focus on their roles in *S.
aureus* pathogenesis.

THE DISTRIBUTION OF THE *psm-mec*
LOCUS IN SCC*mec*

SCC*mec* elements, which range in size from 21 to 67 kb, comprise as characteristic components, the *mecA* gene that codes for a penicillin-binding protein, a *ccr* gene complex for site-specific recombination, and flanking repeat sequences for integration into the genome (International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements, 2009). SCC*mec* elements are classified into 11 different “types,” as defined by the presence of different *ccr* and *mec* resistance genes (International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements, 2009; Li et al., 2011; Shore et al., 2011) and further divided into “subtypes” based on the presence of other resistance genes and transposons in the non-essential J regions (Ito et al., 2003). The *mec* gene complex has four core elements; IS431, *mecA*, *mecR1*, and *mecI*, which represent the genes coding for insertion sequence IS431, a modified penicillin binding protein 2a (PBP2a), the signal transducer protein, and the methicillin resistance regulatory protein (International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements, 2009; Shore and Coleman, 2013; **Figure 1A**).

The *psm-mec* locus is found in the J2 region adjacent to
*mecI* of the class A *mec* gene complex in several widespread
HA-MRSA lineages, but most prominently in Sequence Type
(ST) 5, ST36, ST45, ST225, and ST239 strains (Deurenberg and
Stobberingh, 2009; Nübel et al., 2010; Castillo-Ramirez et al.,
2012; Schulte et al., 2013), which all have either SCC*mec* types
II or III (Chatterjee et al., 2011; Monecke et al., 2012; Josten et al.,
2014). However, more detailed analysis revealed that the *psm-mec*
locus is found in SCC*mec* types II, IIA, IIB, IID, III, and VIII
of many *Staphylococcus* species (Monecke et al., 2012; Shore and
Coleman, 2013).

Antibiotic resistance in bacteria is often associated with a
reduced fitness cost to the bacterial host (Otto, 2013a). We
recently reported that the expression of the PSM-mec peptide
impacted oxacillin resistance in *S. aureus* (Cheung et al., 2014b),
suggesting that a delicate balance may exist between virulence

gene expression and methicillin resistance in MRSA strains
carrying the *psm-mec* locus.

EXPRESSION AND REGULATION OF THE
PSM-mec PEPTIDE

The production of the PSM-mec peptide, which can be
identified in bacterial culture filtrates by high-pressure liquid
chromatography/mass spectrometry (HPLC/MS) (Queck et al.,
2009) or Matrix-assisted laser desorption/ionization Time of
Flight (MALDI-TOF) analysis (Josten et al., 2014), is a reliable
indicator of methicillin resistance. However, the restricted
distribution of the *psm-mec* locus severely limits the potential
use of HPLC-based detection of PSM-mec for routine clinical
screening of methicillin resistance.

The expression of all PSMs including the MGE-encoded PSM-mec is positively regulated by the accessory gene regulatory (*agr*) quorum sensing system (Novick, 2003; Queck et al., 2008; Chatterjee et al., 2011). This up-regulation is dependent on the binding of activated AgrA, the response regulator of *agr*, to promoter sequences upstream of the *psm* genes (Queck et al., 2008), but this has not directly been demonstrated for the *psm-mec* promoter. Interestingly, PSM expression is also enhanced in the presence of calf serum (Oogai et al., 2011) and up-regulated within intracellular environments after the activation of the stringent response (Geiger et al., 2012). Whether this is also the case for PSM-mec is not known.

PSM-mec production was shown to be especially frequent
among clinical isolates of S. epidermidis compared to isolates
from the skin of healthy individuals (Queck et al., 2009), which is
a direct consequence of the genetic connection of *psm-mec* with
methicillin resistance, which occurs much more frequently in
clinical isolates. However, expression levels of PSM-mec can vary
strongly among isolates (Queck et al., 2009). The mechanistic
reasons for varying levels of PSM-mec expression between
isolates cannot be fully explained but a documented $-7T > C$
mutation in the *psm-mec* promoter is known to diminish PSM-
mec expression in a subset of HA-MRSA strains (Kaito et al.,
2011, 2013; Aoyagi et al., 2014).

CHARACTERIZATION OF PSM-mec
PEPTIDE

Like all members of the PSM family, PSM-mec is expressed with
an N-terminal formyl-methionine and secreted without a signal
peptide (Wang et al., 2007; Queck et al., 2009). Although this has
not yet been shown directly for PSM-mec, it is likely that it is
also exported by one or both of the two dedicated ATP-Binding
Cassette (ABC) transporters that secrete PSMs (Chatterjee et al.,
2013; Yoshikai et al., 2016). The 22 amino acid PSM-mec fits into
the class of shorter α-type PSMs, which are anywhere between~
20–25 amino acids in length, as opposed to the β-type, which are
approximately twice as long (43–45 amino acids) (Cheung et al.,
2014a).

PSM-mec has a unique cysteine residue at position 17, while
cysteine residues are not present in the sequences of any other
<figure><img src="image_9.png" /></figure>

known PSM (Figure 1B). The notion of a hypothetical dimeric
form of PSM-mec, due to the formation of disulfide bridges
created from oxidized cysteine residues, was formally discounted
after size exclusion chromatography experiments showed no
difference in extracted ion chromatograms of PSM-mec mutants
compared to that of the unaltered PSM-mec peptide (Queck
et al., 2009). However, the cysteine residue was crucial for
pro-inflammatory and cytolytic activity, probably because it is
essential for the PSM-mec secondary structure (Queck et al.,
2009).

THE PRO-INFLAMMATORY AND
CYTOLYTIC PROPERTIES OF PSM-mec

At nanomolar concentrations, the PSMs are powerful
pro-inflammatory agents and have an extraordinary capacity
for activating and stimulating neutrophils (Wang et al., 2007),
which is exerted through their interactions with human formyl
peptide receptor 2 (FPR2; Kretschmer et al., 2010), a G protein-
coupled receptor expressed on multiple immune cell types
(Migeotte et al., 2006). Although differences in activity are
observed between PSM members, PSM-mec readily up-regulates both CD11b and gp91phox expression, induces calcium flux, chemotaxis, and IL-8 release in neutrophils (Queck et al., 2009).

On the other hand, several PSM members of the α-type possess potent cytolytic activities, and an ever-increasing number of immune and non-immune cells are being discovered that are subject to PSM-mediated cytolysis (Cheung et al., 2014a). The lysis of eukaryotic cells by PSMs, which occurs at micromolar concentrations, is likely mediated in a receptor-independent manner (Kretschmer et al., 2010), While the involvement of receptors cannot be formally excluded, the facts that PSMs are surfactant molecules, lyse artificial phospholipid vesicles (Duong et al., 2012; Laabei et al., 2014), and FPR2 is not involved in lysis (Kretschmer et al., 2010), speak for such a receptor-independent mechanism. Lytic activity is, however, heavily influenced by membrane composition (Laabei et al., 2014). It is believed to be crucial for PSM-mediated immune evasion and progression of staphylococcal pathogenesis. PSM-mec follows the characteristic cytolytic profile of some α-type PSMs and can lyse human erythrocytes and neutrophils at a comparatively moderate level that is similar to that exerted by δ-toxin (Queck et al., 2009).

## THE *psm-mec* srRNA

The first clue for the presence of an srRNA within the *psm-mec* locus stemmed from experiments with MRSA strains transformed with a plasmid carrying 575 nucleotides amplified from SCC*mec* type II, termed the “F region” (Kaito et al., 2008). The authors reported that an open reading frame (ORF) transcribed in the opposite direction of the *psm-mec* gene, coined “fudoh,” encoded a protein that controlled colony spreading (Kaito et al., 2008). It was later discovered from experiments using S. aureus isogenic *fudoh* point-deletion mutants that the phenotypic differences were in fact influenced by an srRNA (Kaito et al., 2011), which contains the *psm-mec* gene (Kaito et al., 2011, 2013), in a way similar to the genetic layout of δ-toxin, whose gene is found within the regulatory RNA of the *agr* system, RNAIII (Novick, 2003). Interestingly, our group reported that the *psm-mec* srRNA transcript length was 143 bp, 14 nucleotides shorter than that described by Kaito et al. (2013) (Figure 1C). Notably, in any case, the *psm-mec* srRNA only barely exceeds the ORF coding for PSM-mec. This contrasts with RNAIII, which is considerably longer than the δ-toxin gene, *hld*. Additionally, the *psm-mec* srRNA is estimated to have a half-life of approximately 20 min (Kaito et al., 2013), which is significantly shorter than the ~45 min half-life described for RNAIII (Huntzinger et al., 2005). The only reported mechanism by which the *psm-mec* srRNA exerts it gene regulatory activity is through its interaction with the *agrA* transcript resulting in the overall decrease of AgrA activity (Kaito et al., 2013), which is entirely dependent on the first 60 nucleotides of the transcript (Kaito et al., 2013), where key anti-sense base-pair interactions occur (Cheung et al., 2014b). Because AgrA strictly regulates activity of the *psma* and *psmβ* promoters (Queck et al., 2008), many of the *psm-mec* srRNA-mediated phenotypes likely are mostly due to a repression of the production of the strongly cytolytic and pro-inflammatory PSMα peptides (Table 1). Kaito et al. initially observed up to eight-fold reduction in PSM expression when the *psm-mec* locus

was over-expressed on a plasmid in methicillin-sensitive S. aureus strains or strains with SCC*mec* type IV (Kaito et al., 2011). However, the differences in PSM reduction were less dramatic, and often inconsistent, in isogenic *psm-mec* mutants naturally harboring SCC*mec* types II and III (Chatterjee et al., 2011), indicating a high degree of strain dependence regarding the role of the *psm-mec* srRNA in S. aureus.

## THE IMPACT OF THE *psm-mec* srRNA ON BIOFILM FORMATION, COLONY SPREADING, AND EXPRESSION OF S. aureus VIRULENCE FACTORS

PSM peptides have a considerable impact on biofilm formation, which is based on their detergent-like properties (Vuong et al., 2000; Kong et al., 2006). PSMs cause the structuring and maturation of biofilms, which includes the formation of nutrient-transporting channels (Wang et al., 2011; Periasamy et al., 2012). PSMs also facilitate the detachment of biofilm clusters *in vitro* and *in vivo* (Wang et al., 2011; Periasamy et al., 2012; Otto, 2013b). It was shown that isogenic *agr* mutants (Vuong et al., 2000, 2004), total *psm* deletion mutants in all *psm* genes, and even single mutants in either the *psma*, *psmβ*, or *hld* loci (Wang et al., 2011; Periasamy et al., 2012) formed thicker biofilms compared to their wild type counterparts. Therefore, it was surprising to find that several isogenic *psm-mec* S. aureus mutants showed a reverse phenotype, albeit the change in biofilm formation was overall minor (Queck et al., 2009; Kaito et al., 2013). A phenotype of increased aggregation similar to that conferred by other PSMs (Dastgheyb et al., 2015) was only found in a strain in which the PSM-mec peptide was expressed in high relative amounts as compared to other PSMs (Queck et al., 2009). In most *psm-mec*-positive S. aureus strains, the regulatory effect of the *psm-mec* srRNA on the expression of other biofilm-dispersing

<table><caption>TABLE 1 | Activities of the PSM-mec peptide and the *psm-mec* srRNA.</caption><thead><tr><th>Virulence determinant within the <i>psm-mec</i> locus</th><th>Known in vitro activities in S. aureus</th></tr></thead><tbody><tr><td>PSM-mec peptide</td><td>FPR2-mediated pro-inflammatory activity at nM concentrations:<ul><li>Increased CD11b expression</li><li>Increased gp91phox expression</li><li>Increased IL-8 expression</li><li>Increased calcium flux</li><li>Increased chemotaxis</li></ul>Receptor-independent cytolytic activity at µM concentrations toward:<ul><li>Erythrocytes</li><li>Neutrophils</li></ul>Peptide expression decreases oxacillin resistance<sup>a</sup></td></tr><tr><td>*psm-mec* srRNA</td><td>Represses AgrA activity through direct interaction<ul><li>Decreased PSM production<sup>b</sup></li><li>Enhanced biofilm formation and aggregation<sup>b</sup></li><li>Suppresses colony spreading</li><li>Up-regulates SpA expression<sup>b</sup></li></ul></td></tr></tbody></table>

<footnote><sup>a</sup>Only tested in one background strain.</footnote>

<footnote><sup>b</sup>Strain-specific phenotype.</footnote>
PSMs (Periasamy et al., 2012) appears to over-shadow the lack of the direct dispersion effect of the PSM-mec peptide in *psm-mec* mutants (Kaito et al., 2013). Thus, in most strains, the impact of the *psm-mec* locus on biofilm formation is opposite to that of other *psm* loci, although that effect is generally not very pronounced.

For many pathogenic bacteria, motility plays an important role in colonization and virulence (Cossart, 2002; Josenhans and Suerbaum, 2002; Krukonis and DiRita, 2003). Motility is commonly dependent on the expression of flagella and type IV pili (Kearns, 2010). Even though *S. aureus* has always been historically regarded as non-motile, as it does not have the genes for flagella or pili, *S. aureus* has repeatedly been shown to spread efficiently across the surface of soft agar (Kaito et al., 2011; Tsompanidou et al., 2012) and more recently, on fresh pork meat (Tsompanidou et al., 2013), through a PSM-dependent mechanism (Lin et al., 2016), an effect now known as "colony-spreading" (Kaito and Sekimizu, 2007).

Originally, the observations of colony spreading and several other phenotypes were made using plasmid-based expression of the *psm-mec* locus in MRSA strains that did not carry the *psm-mec* locus, such as *S. aureus* isolates that were either methicillin- sensitive or MRSA strains that have recently emerged in the community (Community-Associated MRSA, CA-MRSA (Lowy, 1998; DeLeo et al., 2010). CA-MRSA strains are genetically distinct and notably more aggressive than HA-MRSA strains, show enhanced production of virulence factors, and are capable of causing disease in otherwise healthy individuals outside public healthcare settings (Tacconelli et al., 2004; Diep and Otto, 2008; Li et al., 2009; DeLeo et al., 2010). Furthermore, CA-MRSA strains characteristically possess SCC*mec* types IV or V (Diep and Otto, 2008; DeLeo et al., 2010) and therefore lack the *psm-mec* locus. Interestingly, CA-MRSA strains were reported to have a greater capacity to spread on soft agar compared to HA-MRSA strains (Kaito et al., 2008) and the absence of the *psm-mec* locus from CA-MRSA strains was speculated to cause that phenotype. Indeed, CA-MRSA strains transformed with plasmids containing the *psm-mec* locus were impaired in colony spreading. Conversely, isogenic HA-MRSA *psm-mec* mutants showed enhanced colony spreading (Kaito et al., 2013). Moreover, the inability of synthetic PSM-mec to rescue the colony spreading phenotype in an *agr* deletion strain of *S. aureus* (Tsompanidou et al., 2013) indicated that this regulation is predominantly mediated by the *psm-mec* srRNA. However, other studies showed that colony spreading is positively regulated by *agr* (Tsompanidou et al., 2011) and mainly controlled by mechanisms independent of the *psm-mec* srRNA, which include the production of several staphylococcal virulence factors, such as PSMs (Omae et al., 2012; Tsompanidou et al., 2013) and teichoic acids (Kaito and Sekimizu, 2007). In contrast, colony spreading is suppressed in the presence of multiple staphylococcal cell surface proteins (Tsompanidou et al., 2012). Taken together, the molecular mechanisms behind colony spreading in *S. aureus* reveal a somewhat complex network involving different, and often opposing, signals that affect the final phenotypic outcome. Lastly, it remains unknown whether colony spreading on agar surface is a mechanism universally adopted by all staphylococci

and to what extent it matters for the colonization of epithelial surfaces *in vivo*.

In an effort to investigate *psm-mec*-dependent regulation in the natural strain background without artificial plasmid expression-induced effects, our group used microarray profiling to compare the transcriptomes of wild-type strains compared with those of isogenic *psm-mec* mutants (Cheung et al., 2014b). Most strikingly, the primary conserved target of *psm-mec*-dependent gene regulation was *spa*, the gene coding for the immune evasion protein, surface protein A (SpA), which was independently confirmed (Kaito et al., 2011). We also found that the strength of *psm-mec* srRNA transcription affected SpA expression (Cheung et al., 2014b). The up-regulation of SpA by the *psm-mec* srRNA is especially interesting because it adds to a number of existing transcriptional regulators known to control *spa* (Gao and Stewart, 2004). The up-regulated expression of SpA may play an important but yet unidentified role in the manifestation of staphylococcal disease (Gomez et al., 2004; Cheng et al., 2009; Merino et al., 2009; Kim et al., 2011) in some *psm-mec*-harboring *S. aureus* strains.

## THE IMPACT OF THE *psm-mec* LOCUS ON *S. aureus* VIRULENCE

Our group demonstrated that the ability of an isogenic *psm-mec* *S. aureus* mutant to form skin abscesses and cause sepsis in mouse models of infection was severely attenuated compared to the parent strain (Queck et al., 2009), indicating that the PSM-mec peptide contributed significantly to *S. aureus* pathogenesis. However, this phenotype could only be produced using an isogenic *psm-mec* *S. aureus* mutant whose parent strain produced more PSM-mec relative to other PSMs as compared to most *psm-mec*-harboring strains. Kaito et al. reported that the *psm-mec* srRNA contributes to virulence, but in a fashion opposite to that mediated by the PSM-mec peptide (Kaito et al., 2011, 2013). Furthermore, those authors proposed that the *psm-mec* srRNA was generally responsible for the differences in virulence between HA- and CA-MRSA strains (Kaito et al., 2013). They also speculated that point mutations in the *psm-mec* locus underlie increased virulence of a certain lineage, ST764, but this was not directly shown using isogenic mutants (Suzuki et al., 2016). However, a phenotype of increased virulence is not always observed in isogenic *psm-mec* HA-MRSA strains (Chatterjee et al., 2011), suggesting that the contribution of *psm-mec* srRNA toward *S. aureus* pathogenesis is strongly strain-dependent and cannot serve as a generally applicable explanation for differences in virulence between CA- and HA-MRSA strains.

## THE ROLES OF THE PSM-mec PEPTIDE AND THE *psm-mec* REGULATORY RNA IN OTHER *Staphylococcus* SPECIES

In addition to *S. aureus*, the *psm-mec* locus is detected in many different methicillin-resistant *Staphylococcus* species (Monecke et al., 2012). However, PSM-mec expression levels have only been rigorously studied in *S. epidermidis* (Queck et al., 2009). Our
group showed that PSM-mec expression was only detected in methicillin-resistant S. epidermidis infection isolates, but never in methicillin-sensitive S. epidermidis strains (Queck et al., 2009). There is no amino acid or nucleotide sequence difference (100% identity) between the S. aureus and S. epidermidis PSM-mec peptide or srRNA, respectively, based on a comparison of strains S. aureus MRSA252 and S. epidermidis RP62A, and differences are also absent or very minor in other strains. This is in accordance with the notion of a relatively recent move of SCC*mec* from coagulase-negative species to S. aureus.

To date, only one study has studied the impact of the *psm-mec* locus on the phenotypes in S. epidermidis and S. haemolyticus (Ikuo et al., 2014). The results, which were based on S. epidermidis and S. haemolyticus strains without SCC*mec* transformed with plasmids harboring the *psm-mec* locus, showed a decrease in PSM production, as demonstrated in similar experiments with S. aureus (Kaito et al., 2011). Intriguingly, an increase of biofilm formation was only observed in experiments with S. epidermidis, as reported previously (Kaito et al., 2011), but not S. haemolyticus, suggesting that the impact of the *psm-mec* locus on virulence phenotypes among staphylococci is also highly strain-dependent. The role of PSM-mec in coagulase-negative staphylococci has not yet been directly investigated using isogenic deletion mutants.

CONCLUSION

It has become clear that the effects exerted by the *psm-mec*
locus in S. *aureus* on virulence and virulence phenotypes

such as biofilm formation are highly strain-dependent. They
are also commonly rather moderate in extent compared
to those conferred by other members of the PSM family.
While great strides have been made to better understand the
molecular underpinnings of gene regulatory *psm-mec*-dependent
mechanisms in S. *aureus* pathogenesis, in the future the focus
should be shifted toward the investigation of how the *psm-mec*
locus affects the pathogenesis in other *Staphylococcus* species,
especially S. *epidermidis* and S. *haemolyticus*, which, alongside S.
*aureus*, are the most frequent and pathogenic agents of hospital-
associated infections around the world. As it is a commonly
accepted notion that SCC*mec* elements originate from coagulase-
negative staphylococci (Otto, 2013c), such investigation is
bound to shed light on the “original” role of the *psm-mec*
locus in staphylococcal physiology, with the potential to also
explain the contrasting roles of *psm-mec* in the S. *aureus*
background.

AUTHOR CONTRIBUTIONS

LQ, JM, GC, and MO contributed to the drafting of the
manuscript and approved the final version.

FUNDING

This work was supported by the Intramural Research Program
of the National Institute of Allergy and Infectious Diseases, U.S.
National Institutes of Health (grant ZIA AI000904-16).

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**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 Qin, McCausland, Cheung and Otto. 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_10.png" /></figure>

# Sub-MIC Tylosin Inhibits *Streptococcus suis* Biofilm Formation and Results in Differential Protein Expression

Shuai Wang<fnref n="1" />, Yanbei Yang<fnref n="1" />, Yulin Zhao<fnref n="1" />, Honghai Zhao<fnref n="2" />, Jingwen Bai<fnref n="1" />, Jianqing Chen<fnref n="1" />, Yonghui Zhou<fnref n="1" />, Chang Wang<fnref n="1" /> and Yanhua Li<fnref n="1" />*

*Streptococcus suis* (S.suis) is an important zoonotic pathogen that causes severe diseases in humans and pigs. Biofilms of S. suis can induce persistent infections that are difficult to treat. In this study, the effect of tylosin on biofilm formation of S. suis was investigated. 1/2 minimal inhibitory concentration (MIC) and 1/4 MIC of tylosin were shown to inhibit S. suis biofilm formation in vitro. By using the iTRAQ strategy, we compared the protein expression profiles of S. suis grown with sub-MIC tylosin treatment and with no treatment. A total of 1501 proteins were identified by iTRAQ. Ninety-six differentially expressed proteins were identified (Ratio > ±1.5, *p* < 0.05). Several metabolism proteins (such as phosphoglycerate kinase) and surface proteins (such as ABC transporter proteins) were found to be involved in biofilm formation. Our results indicated that S. suis metabolic regulation, cell surface proteins, and virulence proteins appear to be of importance in biofilm growth with sub-MIC tylosin treatment. Thus, our data revealed the rough regulation of biofilm formation that may provide a foundation for future research into mechanisms and targets.

**Keywords:** proteomics, S. suis, biofilms, tylosin, iTRAQ

**OPEN ACCESS**

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

**Reviewed by:**
Rui Vitorino,
University of Aveiro, Portugal
Raymond Allan,
University of Southampton, UK

*Correspondence:
Yanhua Li
liyanhua1970@163.com

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

**Received:** 19 November 2015
**Accepted:** 11 March 2016
**Published:** 30 March 2016

**Citation:**
Wang S, Yang Y, Zhao Y, Zhao H, Bai J, Chen J, Zhou Y, Wang C and Li Y (2016) Sub-MIC Tylosin Inhibits Streptococcus suis Biofilm Formation and Results in Differential Protein Expression. *Front. Microbiol.* 7:384. doi: 10.3389/fmicb.2016.00384

## INTRODUCTION

*Streptococcus suis* (S. suis) is an important zoonotic pathogen that causes a wide range of diseases in pigs, including meningitis, septicaemia, pneumonia, endocarditis, and arthritis (Gottschalk et al., 2010). In addition, this pathogen is an emerging zoonotic agent and an important public health issue in East and Southeast Asia (Sriskandan and Slater, 2006; Gottschalk et al., 2007). During a single outbreak in China in 2005, more than 200 human cases of S. suis were reported, with a death total of 39 (Yang et al., 2006). Current studies show that S. suis can cause persistent infections by forming biofilms *in vivo* (Wang et al., 2011).

Biofilms are assemblages of microorganisms characterized by cells that are irreversibly attached to a substratum and embedded in a matrix of self-produced extracellular polymeric substances such as exopolysaccharides (EPS), proteins, nucleic acids and other substances; this type of sessile community-based existence is a critical characteristic for bacterial persistence (Davey and O'Toole, 2000). The physical and biological properties of the biofilm, such as slow growth and mechanical barrier, have a substantial role in the development of increased antimicrobial tolerance. Because the bacteria in chronic infections are aggregated and are in close proximity, genes coding for resistance

<footnote n="1">¹Department of Veterinary Pharmacy, College of Veterinary Medicine, Northeast Agricultural University, Harbin, China,</footnote>

<footnote n="2">²Department of Biotechnology, Heilongjiang Vocational College for Nationalities, Harbin, China</footnote>
to antimicrobials can be passed horizontally from one bacterium to the another (Bjarnsholt et al., 2013). The bacteria in biofilms could be 1000-times more difficult to kill by antibiotics than the same organism growing planktonically (Gilbert et al., 1997). Therefore, the control of biofilms is understood to be crucial.

Apart from surgical intervention (when applicable), antibiotics are the main option for the treatment of biofilm infections (Bjarnsholt et al., 2013). Previous studies showed that macrolides successfully inhibited *Staphylococcus aureus* biofilm formation and reduced its virulence when used at sub-inhibitory concentrations (Fujimura et al., 2008). In addition, a sub-minimal inhibitory concentration of erythromycin can inhibit S. suis biofilm formation (Zhao et al., 2015). Tylosin, a macrolide antibiotic produced by *Streptomyces fradiae*, is widely used as a veterinary medicine. However, there is still not much effective research of sub-MIC tylosin inhibiting biofilm formation of S. suis in vitro.

At present, most of the available information regarding biofilm formation by drug intervention is based on transcriptomic analyses. However, a limitation of transcriptomic analysis for identifying biofilm-regulated gene network raised concerns among investigators. The method of proteomics is thought to be an essential complement to transcriptomic analysis for discovering key regulators of biofilm (Sauer, 2003). Different immunogenic components of planktonically grown S. suis proteins such as secreted or cell wall-associated proteins had been studied by using immunoproteomic assays (Zhang and Lu, 2007a,b; Geng et al., 2008; Zhang et al., 2008). Additionally, our lab found that quorum-sensing played a crucial role leading to biofilm formation through quantitative proteomic analysis of S. suis biofilm inhibited by sub-MIC erythromycin treatment in vitro (Zhao et al., 2015). However, there are no reports regarding the proteomic analysis of sub-MIC tylosin inhibiting biofilm formation of S. suis in vitro.

We identified several proteins in sub-MIC tylosin inhibiting biofilm formation of S. suis by using iTRAQ technology in this study. The findings from the present study may provide a theoretical foundation for therapy of S. suis biofilm infection and provide references for finding new potential therapeutic targets.

# MATERIALS AND METHODS

## Growth of S. suis Planktonic Cells

S. suis (ATCC 700794) was grown in Todd-Hewitt yeast Broth (THB; Summus Ltd., Harbin, Heilongjiang, China) for 16–18 h at 37°C with constant shaking for biofilm assays (Wang et al., 2011).

## Observation by Scanning Electron Microscopy (SEM)

Mid-exponential growth phase cultures of S. suis ATCC 700794 were adjusted to an optical density of 0.1 at 600 nm (OD600). Then, 2 mL cultures were transferred to the wells of a 6-well microplate containing an 11 × 11 mm sterilized rough glass slide (Mosutech Co., Ltd., Shanghai, China) on the bottom. After culturing for 72 h at 37°C without shaking, the glass slide was removed with tweezers, and the biofilms on the rough glass slide were washed with sterile PBS. The remaining biofilms were fixed with fixative solution [4% (w/v) paraformaldehyde, 2.5% (w/v) glutaraldehyde, 2 mM CaCl₂ in 0.2 M cacodylate buffer, pH 7.2] for 6 h and washed three times with 0.1 M PBS 10 min each, then fixed in 2% osmium tetroxide containing 2 mM potassium ferrocyanide and 6% (w/v) sucrose in cacodylate buffer. The samples were dried, gold sputtered with an ion sputtering instrument (current 15 mA, 2 min) and observed using SEM (FEI Quanta, Netherland).

## Effect of Tylosin on Biofilm Formation Determined by the TCP Assay

Mid-exponential growth phase cultures of S. suis were adjusted to 0.2 of OD600. Then, 100 µL of cultures were added to each wells of a 96-well microplate with equal volume of tylosin solution with the final concentrations of 1/2 MIC (0.25 µg/mL), 1/4 MIC (0.125 µg/mL), 1/8 MIC (0.0625 µg/mL), and 1/16 MIC (0.03125 µg/mL), respectively. In addition, a negative control (with THB alone) and a positive control (with bacteria alone) were also included. After incubation at 37°C for 72 h without shaking, the medium was removed by aspiration and the wells were washed three times with sterile physiological saline. The remaining attached bacteria were fixed with 200 µL of 99% methanol (Guoyao Ltd., China) per well, and the plates were emptied after 15 min and left to dry. Then, the plates were stained for 5 min with 200 µL of 2% crystal violet (Guoyao Ltd., China) per well. The excess stain was rinsed off by placing the plate under running tap water. After the plates were air dried, the dye bound to the adherent cells was resolubilized with 200 µL of 33% (v/v) glacial acetic acid (Guoyao Ltd., China) per well. The amount of released stain was quantified by measuring the absorbance at 570 nm with a microplate reader (DG5033A, Huadong Ltd., Nanjing, Jiangsu, China). The reported values are the means of three measurements. The experiments were performed in triplicate.

## Colony Forming Unit (CFU) Enumeration

Overnight cultures of S. suis were adjusted to an OD 600 of 0.2. Then, the bacteria were inoculated into 96-well microtiter plate wells containing 200 µL of THB alone (untreated wells) or adding the tylosin solution with the final concentrations of 1/4 MIC (0.125 µg/mL). In addition, a negative control (with THB alone) and a positive control (with bacteria alone) were also included. After incubation at 37°C for 72 h without shaking, the medium was removed by aspiration, and the wells were washed three times with sterile physiological saline. Biofilm cells were removed from wells by sonication for 5 min in 200 µL of THB. The cell suspensions (n = 3) underwent 10-fold dilutions in THB, and 100 µL of each dilution was spot plated onto THB soft-agar plates and incubated at 37°C for 24 h. All the experiments were performed in triplicate.

## Preparation of Protein Extracts

For biofilm cultures, S. suis was grown in THB in 100 mm polystyrene petri dishes at 37°C for 24 h. Then, the supernatant was removed and the dishes were washed twice with Tris-HCl buffer (50 mM, pH 7.5). The biofilms were detached by scraping. After being sonicated for 5 min (Branson 220; Branson Consolidated Ultrasonic Pvt. Ltd., Australia), the cells were centrifuged at 12,000×g for 10 min at 4°C. Then, the cell
pellets were washed twice with Tris-HCl buffer (Wang et al., 2012).

## Protein Digestion and iTRAQ Labeling

Protein digestion was performed according to the reported FASP procedure (Wisniewski et al., 2009). In brief, 200 µg of proteins at two different conditions (1/4 MIC of tylosin treated cells and nontreated cells) were added into 30 µL STD buffer (4% SDS, 100 mM DTT, 150 mM Tris-HCl pH 8.0) and ultrafiltered (Microcon units, 30 kD) with UA buffer (8 M Urea, 150 mM Tris-HCl pH 8.0). To block reduced cysteine residues, 100 µL 0.05 M iodoacetamide was added into UA buffer and incubated for 20 min in the dark. The filters were washed three times with 100 µL UA buffer and twice with 100 µL DS buffer (50 mM triethylammoniumbicarbonate at pH 8.5). Finally, the proteins were digested with 2 µg trypsin (Promega) in 40 µL DS buffer at 37°C for 16–18 h. Then, the resulting peptides were collected as a filtrate. The peptide content was estimated by UV light spectral density at 280 nm using an extinctions coefficient of 1.1 of 0.1% (g/l) solution calculated on the basis of the frequency of tryptophan and tyrosine in vertebrate proteins.

For the iTRAQ labeling, the peptides were labeled with the 8-plex iTRAQ reagent by following the manufacturer's instructions (Applied Biosystems). Each iTRAQ reagent was dissolved in 70 µL of ethanol and added to the respective peptide mixture. The peptides from the *S. suis* biofilms treated by tylosin were labeled with 115 isobaric reagent, and the peptides from the nontreated *S. suis* biofilms were labeled with 116 isobaric reagent. Then, the samples were multiplexed and vacuum dried. Three independent biological experiments were performed.

## Peptide Fractionation with Strong Cation Exchange (SCX) Chromatography

SCX chromatography using the AKTA Purifier system (GE Healthcare) was used to fractionate the iTRAQ labeled peptides. After being reconstituted and acidified with 2 mL buffer A (10 mM KH₂PO₄ in 25% of ACN, pH 2.7), the peptides were loaded onto a PolySULFOETHYL 4.6 × 100 mm column (5 µm, 200 Å, PolyLC Inc., Maryland, U.S.A.). Then, the peptides were eluted at 1 ml/min with a gradient of 0–10% buffer B (500 mM KCl, 10 mM KH₂PO₄ in 25% of ACN, pH 2.7) for 2 min, 10–20% buffer B for 25 min, 20–45% buffer B for 5 min, and 50–100% buffer B for 5 min. The elution was monitored by absorbance at 214 nm, and the fractions were collected after every 1 min. The collected fractions (~30 fractions) were combined into 10 pools and desalted on C18 Cartridges [Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 ml, Sigma]. Each pooled fraction was concentrated by vacuum centrifugation and reconstituted in 40 µl of 0.1% (v/v) trifluoroacetic acid and stored at –80°C for LC-MS/MS analysis.

## Liquid Chromatography (LC) Electrospray Ionization (ESI) Tandem Ms (MS/MS) Analysis by Q Exactive

Experiments were performed on a Q Exactive mass spectrometer that was coupled to Easy nLC (Thermo Fisher Scientific). A

sample (10 µL) of each fraction was injected for the nano LC-MS/MS analysis. The peptide mixture (5 µg) was loaded onto a C18-reversed phase column (Thermo Scientific Easy Column, 10 cm long, 75 µm inner diameter, 3 µm resin) in buffer A (0.1% formic acid) and separated with a linear gradient of buffer B (80% acetonitrile and 0.1% formic acid) at 250 nl/min controlled by IntelliFlow technology for 140 min. MS data were acquired using a data-dependent top10 method dynamically choosing the most abundant precursor ions from the survey scan (300–1800 m/z) for HCD fragmentation. Determination of the target value was based on predictive Automatic Gain Control (pAGC). The dynamic exclusion duration was 60 s. Survey scans were acquired at a resolution of 70,000 at m/z 200, and the resolution for HCD spectra was set to 17,500 at m/z 200. The normalized collision energy was 30 eV, and the underfill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as 0.1%. The instrument was run with the peptide recognition mode enabled.

## Sequence Database Searching and Data Analysis

The MS/MS spectra were searched using the MASCOT engine (Matrix Science, London, UK; version 2.2) in the Proteome Discoverer 1.3 (Thermo Electron, San Jose, USA.) against the Uniprot *S. suis* fasta database (38,369 sequences, downloaded March 4th, 2013) and the decoy database. False discovery rates (FDR) were calculated by running all spectra against a decoy database using the MASCOT software. To identify proteins, the following options were used: Peptide mass tolerance = 20 ppm, MS/MS tolerance = 0.1 Da, Enzyme = Trypsin, Missed cleavage = 2, Fixed modification: Carbamidomethyl (C), iTRAQ 8plex (K), iTRAQ 8plex (N-term), Variable modification: Oxidation (M). The quantification was performed based on the peak intensities of the reporter ions in the MS/MS spectra. The ratio of label 115 and 116 represents the expression of proteins with the protein identification confidence of a 1% FDR (Unwin et al., 2010). The proteins were considered over expressed when the iTRAQ ratio was above 1.5 and under expressed when the iTRAQ ratio was lower than 0.67. The Proteome Discoverer tool was used to categorize the proteins detected by Gene Ontology (GO) annotation according to the cellular component, biological process and molecular function.

## RESULTS

### Effect of Tylosin against Biofilm Formation
*In vitro* by the TCP Assay

We evaluated the action of tylosin on biofilm growth *in vitro*. The MIC against *S. suis* was 0.5 µg·mL⁻¹. Tylosin at 1/2 MIC and 1/4 MIC caused a significantly higher reduction in the biofilm-forming ability of *S. suis* compared with positive control (p < 0.05). However, there was no pronounced effect for 1/16 MIC and 1/8 MIC of tylosin on biofilm formation of *S. suis* (p > 0.05; **Figure 1**).
## Direct Observation of Biofilm Formation *In vitro* by Sem

SEM analysis was performed to observe the 1/4 MIC of tylosin treated cells and nontreated cells biofilm formation by *S. suis* under same growth conditions. As shown in **Figure 2A**, the surface of the glass slide is almost entirely covered by the aggregates and microcolonies of *S. suis* when growth was carried out in the culture medium without tylosin. However, when the culture medium was added to 1/4 MIC of tylosin, the biofilms were characterized by the presence of small clusters of cells interspersed amongst individual cells (**Figure 2B**). This result showed that the biofilm formation of *S. suis* was inhibited by the tylosin of 1/4 MIC *in vitro*.

## Colony Forming Unit (CFU) Enumeration

To better assess action of tylosin on biofilm quantitatively, the CFUs of *S. suis* were counted. The viability of *S. suis* treated with 1/4 MIC of tylosin was different from the viability of untreated *S. suis*. The number of CFUs/mL in treated biofilms (5.3 log₁₀ CFUs/mL) was significantly fewer than in nontreated biofilms (6.5 log₁₀ CFUs/mL; *p* < 0.05). The number of CFUs/mL in treated biofilms (5.3 log₁₀ CFUs/mL) was significantly fewer than in nontreated biofilms (6.5 log₁₀ CFUs/mL; *p* < 0.05) (**Figure 3**). The findings demonstrated that 1/4 MIC of tylosin remained effective in decreasing the viability of *S. suis*.

Tylosin

<figure><img src="image_11.png" /><figcaption>**FIGURE 1** | Effect of tylosin at different concentrations on *S. suis* ATCC 700794 biofilm formation. The data are expressed as the means ± standard deviations. Significant decrease (\*p* < 0.05) compared with control biofilm formation of *S. suis* *in vitro*.</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**FIGURE 2** | (A) Biofilm formation of *S. suis* without tylosin. (B) Biofilm formation of *S. suis* with 1/4 MIC tylosin treatment.</figcaption></figure>

## Sub-MIC Tylosin Inhibits Biofilm Formation and Differentially Expressed Proteins by iTRAQ

A total of 1501 proteins were identified by iTRAQ. Detailed information is shown in the Supplementary File. A ratio of proteins with >1.5 or <0.67 (*p* < 0.05) was considered to be differentially expressed. Based on this criterion, 96 differentially expressed proteins were identified in 1/4 MIC tylosin treated cells and nontreated cells. Detailed information could be shown **Table 1**. These proteins were detected by GO annotation. Thirty-five proteins were deleted from the database. The remaining 61 proteins were classified into biological process, molecular function and cellular component (**Figure 4**). The results regarding the biological process were as follows: single-organism process (8, 13%), response to stimulus (4, 7%), localization (4, 7%), biological adhesion (1, 2%), cellular component organization or biogenesis (6, 10%), cellular process (25, 41%), biological regulation (3, 5%), metabolic process (30, 49%). The results regarding molecular function were as follows: binding (29, 48%), nucleic acid binding transcription factor activity (1, 2%), transporter activity (3, 5%), structural molecule activity (5, 8%), catalytic activity (25, 41%). The results regarding cellular component were as follows: organelle (6, 10%), virion (1, 2%), cell (13, 21%), extracellular region (1, 2%), membrane (6, 10%), macromolecular complex (8, 13%).

## DISCUSSION

A TCP assay is based on the ability of bacteria to form biofilms on the bottom of tissue culture plates and is mainly used to identify the formation of bacterial biofilms (Mathur et al., 2006; Okajima et al., 2006; Presterl et al., 2007). Bacteria are grown in cell culture plates, and the quantity of a biofilm is detected by staining the wells with crystal violet according to the correlation between OD values and biofilm formation (Mathur et al., 2006).

To further confirm *S. suis* biofilm formation, the structure of the *S. suis* biofilm was identified by scanning electron microscopy

Tylosin

<figure><img src="image_13.png" /><figcaption>**FIGURE 3** | Effect of 1/4 MIC of tylosin on *S. suis* ATCC 700794 biofilm formation by colony forming unit (CFU) enumeration. The data are expressed as the means ± standard deviations. Significant decrease (\*p* < 0.05) compared with control biofilm formation of *S. suis* *in vitro*.</figcaption></figure>
<table><caption>TABLE 1 | List of differentially expressed proteins in tylosin treated cells.</caption><thead><tr><th>Accession</th><th>Description</th><th>Fold change<sup>a</sup></th></tr></thead><tbody><tr><td>G7SDH9</td><td>Putative uncharacterized protein</td><td>0.555777</td></tr><tr><td>A4W1G9</td><td>Phosphotransferase system cellobiose-specific component IIC</td><td>0.521547</td></tr><tr><td>G7SA24</td><td>Cold-shock DNA-binding domain protein</td><td>0.394546</td></tr><tr><td>F4EDP5</td><td>Putative uncharacterized protein</td><td>0.568016</td></tr><tr><td>R4NL31</td><td>MF3-like protein</td><td>0.63094</td></tr><tr><td>E9NQ29</td><td>CPS16V</td><td>0.452748</td></tr><tr><td>G5KZ73</td><td>NADH:flavin oxidoreductase/NADH oxidase family protein</td><td>0.459622</td></tr><tr><td>G7S4C2</td><td>Major membrane immunogen, membrane-anchored lipoprotein</td><td>0.643938</td></tr><tr><td>A8CUL3</td><td>Integrase</td><td>0.46773</td></tr><tr><td>G7SM99</td><td>Type I site-specific restriction-modification system, R (Restriction) subunit and related helicase</td><td>0.506277</td></tr><tr><td>G7SLJ0</td><td>Putative competence-damage inducible protein</td><td>0.564703</td></tr><tr><td>A4W1W3</td><td>FOF1-type ATP synthase, subunit a</td><td>0.586873</td></tr><tr><td>G7RZW0</td><td>Sugar ABC transporter permease</td><td>1.508325</td></tr><tr><td>G7S2T8</td><td>Alcohol dehydrogenase</td><td>0.610195</td></tr><tr><td>G7SDG9</td><td>Laminin binding protein</td><td>1.524593</td></tr><tr><td>R4NLJ6</td><td>Oligopeptide ABC transporter, periplasmic oligopeptide-binding protein OppA</td><td>0.644177</td></tr><tr><td>G7S371</td><td>Fructose-bisphosphate aldolase</td><td>0.623705</td></tr><tr><td>M1UGE2</td><td>Uncharacterized protein</td><td>0.647168</td></tr><tr><td>A4VZM2</td><td>Cell cycle protein GpsB</td><td>0.631018</td></tr><tr><td>A4W2D2</td><td>Cation transport ATPase</td><td>1.676356</td></tr><tr><td>G5L1V6</td><td>50S ribosomal protein L9</td><td>0.6638821</td></tr><tr><td>G7SEP2</td><td>Putative uncharacterized protein</td><td>1.502566</td></tr><tr><td>A4VZV3</td><td>tRNA dimethylallyltransferase</td><td>1.510883</td></tr><tr><td>G7S5B8</td><td>3-isopropylmalate dehydrogenase</td><td>1.513117</td></tr><tr><td>G7SA68</td><td>UDP-N-acetylmuramate-alanine ligase</td><td>0.656503</td></tr><tr><td>G5L098</td><td>Transcriptional regulator Spx</td><td>1.514636</td></tr><tr><td>E8UKC3</td><td>Uncharacterized protein</td><td>1.507837</td></tr><tr><td>G7SFU4</td><td>Putative uncharacterized protein</td><td>1.543591</td></tr><tr><td>A4VV99</td><td>DNA repair protein</td><td>1.545594</td></tr><tr><td>A4W2Z7</td><td>FMN-dependent NADH-azoreductase</td><td>1.527496</td></tr><tr><td>F4EC05</td><td>Putative uncharacterized protein</td><td>1.57743</td></tr><tr><td>C6GNL8</td><td>30S ribosomal protein S21</td><td>1.545471</td></tr><tr><td>K7ZNG8</td><td>DNA recombination/repair protein (Fragment)</td><td>1.558023</td></tr><tr><td>G7S3K5</td><td>ABC-type cobalt transport system, ATPase component</td><td>1.583854</td></tr><tr><td>G7SM56</td><td>Ribosomal RNA small subunit methyltransferase H</td><td>1.594936</td></tr><tr><td>A2VC24</td><td>Muramidase-released protein</td><td>1.579304</td></tr><tr><td>A4W3C1</td><td>Putative phosphotyrosine protein phosphatase</td><td>1.60975</td></tr><tr><td>G7S4U0</td><td>Putative uncharacterized protein</td><td>1.596783</td></tr><tr><td>G7SP92</td><td>Putative uncharacterized protein</td><td>1.658448</td></tr><tr><td>G7S7E3</td><td>Helicase</td><td>1.516925</td></tr><tr><td>M1VK55</td><td>Glycosyltransferase</td><td>1.662795</td></tr><tr><td>G7S3P9</td><td>Putative uncharacterized protein</td><td>1.673475</td></tr><tr><td>A4W106</td><td>Signal transduction histidine kinase</td><td>1.62492</td></tr><tr><td>B0M0G7</td><td>Phosphomethylpyrimidine kinase (Fragment)</td><td>1.633436</td></tr><tr><td>A5JSJ7</td><td>Putative uncharacterized protein</td><td>1.681661</td></tr><tr><td>G5L226</td><td>Type III restriction-modification system, restriction endonuclease subunit</td><td>1.697991</td></tr></tbody></table>

(Continued)

<table><caption>TABLE 1 | Continued.</caption><thead><tr><th>Accession</th><th>Description</th><th>Fold change<sup>a</sup></th></tr></thead><tbody><tr><td>G7S7K4</td><td>Putative uncharacterized protein</td><td>1.645719</td></tr><tr><td>B9WY95</td><td>Peptidase M20</td><td>1.647536</td></tr><tr><td>G7S4T2</td><td>Branched-chain alpha-keto acid dehydrogenase subunit E2</td><td>1.673032</td></tr><tr><td>G5KX48</td><td>ABC-type uncharacterized transport system, permease component</td><td>1.705109</td></tr><tr><td>G7S178</td><td>Putative uncharacterized protein</td><td>1.774886</td></tr><tr><td>M1VDL7</td><td>ABC transporter permease protein</td><td>1.942183</td></tr><tr><td>G7S6Z3</td><td>Putative uncharacterized protein</td><td>1.956584</td></tr><tr><td>J7KIA5</td><td>Abortive infection bacteriophage resistance related protein</td><td>1.999106</td></tr><tr><td>G7SJZ7</td><td>Primosomal protein N'</td><td>1.720079</td></tr><tr><td>G7SHW7</td><td>Putative uncharacterized protein</td><td>2.001559</td></tr><tr><td>Q9EZW2</td><td>Elongation factor Tu (Fragment)</td><td>1.739971</td></tr><tr><td>A4VVJ5</td><td>Uncharacterized protein</td><td>2.141695</td></tr><tr><td>M1VJZ8</td><td>Zeta toxin</td><td>1.74309</td></tr><tr><td>G5L2V0</td><td>NADPH:quinone reductase and related Zn-dependent oxidoreductase</td><td>1.79706</td></tr><tr><td>R4NU43</td><td>Aromatic amino acid aminotransferase gamma @ N-acetyl-L,L-diaminopimelate aminotransferase</td><td>2.144605</td></tr><tr><td>A4W296</td><td>Uncharacterized protein</td><td>1.837367</td></tr><tr><td>G7SKQ0</td><td>CHAP domain containing protein</td><td>2.179882</td></tr><tr><td>B9WVD6</td><td>Putative uncharacterized protein (Fragment)</td><td>2.226792</td></tr><tr><td>G5L2N5</td><td>L-fucose isomerase</td><td>1.859887</td></tr><tr><td>B9WTA1</td><td>Putative uncharacterized protein</td><td>2.290848</td></tr><tr><td>B9WXU6</td><td>ABC transporter related protein</td><td>1.873566</td></tr><tr><td>G8DU82</td><td>Transposase</td><td>2.376768</td></tr><tr><td>G7S242</td><td>Putative ABC transporter</td><td>1.883471</td></tr><tr><td>A4VZ91</td><td>50S ribosomal protein L32</td><td>1.915877</td></tr><tr><td>G7SDX6</td><td>50S ribosomal protein L7/L12</td><td>1.918196</td></tr><tr><td>G5KZ86</td><td>Phosphatidylserine/phosphatidylglycerophosphate/ cardiolipin synthase-like protein</td><td>1.965488</td></tr><tr><td>G7SIQ5</td><td>Putative uncharacterized protein</td><td>2.451703</td></tr><tr><td>G7S7J2</td><td>Putative uncharacterized protein</td><td>2.481527</td></tr><tr><td>G7SPA1</td><td>Putative scaffolding protein</td><td>2.584249</td></tr><tr><td>G7S7A9</td><td>FAD-dependent pyridine nucleotide-disulfide oxidoreductase</td><td>1.970356</td></tr><tr><td>G7S8Q2</td><td>Peptidase M22 glycoprotease</td><td>1.978772</td></tr><tr><td>M1TIM7</td><td>Methylated DNA-protein cysteine methyltransferase</td><td>2.161583</td></tr><tr><td>G7SBK2</td><td>Elongation factor Ts</td><td>2.266517</td></tr><tr><td>M1VE47</td><td>Fic/DOC family protein</td><td>2.606274</td></tr><tr><td>M1UFP9</td><td>Thiamine-phosphate synthase</td><td>2.298028</td></tr><tr><td>G7S535</td><td>Fructose-6-phosphate aldolase</td><td>2.553516</td></tr><tr><td>G5L351</td><td>NsuB</td><td>2.694988</td></tr><tr><td>B9WUV5</td><td>Transcriptional regulator, DeoR family</td><td>2.65073</td></tr><tr><td>R4NW55</td><td>Plasmid replication protein Rep and AAA-class ATPase domain protein</td><td>2.779765</td></tr><tr><td>G7RZ18</td><td>Sortase-like protein</td><td>2.791504</td></tr><tr><td>S6B433</td><td>RecN protein (Fragment)</td><td>3.007243</td></tr><tr><td>G5KZR3</td><td>Glutathione S-transferase</td><td>3.262393</td></tr><tr><td>R4NLK5</td><td>SSU ribosomal protein S1p</td><td>3.46851</td></tr><tr><td>R4NWB6</td><td>Uncharacterized protein</td><td>8.306942</td></tr></tbody></table>

(Continued)
<table><caption>TABLE 1 | Continued.</caption><thead><tr><th>Accession</th><th>Description</th><th>Fold change<sup>a</sup></th></tr></thead><tbody><tr><td>G5L259</td><td>NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, putative</td><td>2.781098</td></tr><tr><td>D5AFN4</td><td>Xaa-Pro dipeptidyl-peptidase</td><td>3.095053</td></tr><tr><td>G7SMG3</td><td>Elongation factor G</td><td>3.667679</td></tr><tr><td>G7SD52</td><td>ABC superfamily ATP binding cassette transporter, membrane protein</td><td>3.201581</td></tr><tr><td>G5L1N9</td><td>Nucleoid DNA-binding protein</td><td>3.796473</td></tr><tr><td>G7S8P5</td><td>Phosphoglycerate kinase</td><td>11.34908</td></tr></tbody></table>

Fold change<sup>a</sup>, the ratio of different expression level between Sub-MIC tylosin treated cells and nontreated cells.

(SEM). The biofilm examination relied heavily on SEM. Because of its high magnification, the biofilm microstructure is observed clearly. These findings further confirm that S. suis can form biofilms *in vitro*. Although these procedures have their advantages, the TCP procedure lacks specificity and sensitivity because crystal violet stains all of the components of the biofilm and could potentially stain non-biofilm material. More specific and sensitive techniques can be performed to assess each of the individual components. This method is good for screening purposes.

Tylosin is a frequently used drug for the treatment of S. suis infection. Upon treatment with this antimicrobial agent, S. suis is inevitably exposed to a sub-inhibitory level of the agent. Therefore, we studied its effect in sub-inhibitory concentrations on S. suis biofilm formation. Tylosin can inhibit S. suis biofilm formation at sub-inhibitory concentrations in a dose-dependent manner. In addition, the inhibition of biofilm formation varied among the antimicrobial agents. Numerous reports have showed biofilm formation in the presence of sub-inhibitory concentrations of antimicrobial agents (Dunne, 1990; Carsenti-Etesse et al., 1993; Rupp and Hamer, 1998; Rachid et al., 2000; Yang et al., 2015). Our results are in agreement with these previous findings. Our results showed a better activity of tylosin against S. suis biofilm formation. However, further studies should be conducted to confirm and clarify the relationship between the relative adherence-inhibiting properties of tylosin and their mechanisms. These results may provide information regarding the clinical use of antimicrobial agents against biofilm-forming bacteria.

Our data identified proteins related to biofilm growth that have previously been uncharacterized. iTRAQ analyses showed that the regulation of metabolism plays a key role during S. suis biofilm growth. First, the iTRAQ quantitative data revealed that carbohydrate metabolism may be particularly important during S. suis biofilm growth. It was reported that fructose bisphosphate aldolase and glyceraldehyde-3-phosphate dehydrogenase were significantly decreased in *Streptococcus pneumoniae* biofilms (Allan et al., 2014). In our study, there was a >two-fold increase of glyceraldehyde-3-phosphate dehydrogenase in the tylosin inhibiting biofilm formation. In addition, the levels of fructose bisphosphate aldolase (fold change: 0.62) were down-regulated. It was reported that phosphoglycerate kinase was up-regulated in S. suis biofilms compared with planktonic cells by comparative proteomic analysis (Wang et al., 2012).

Similarly, biofilms of *Pseudomonas aeruginosa* (Sauer et al., 2002) and *Staphylococcus xylosus* (Planchon et al., 2009) exhibit up-regulated phosphoglycerate kinase. There was a >11-fold increase in the level of phosphoglycerate kinase in the tylosin inhibiting biofilm formation. Phosphoglycerate kinase is a glycolytic enzyme that functions in the conversion of glyceraldehyde 3-phosphate into 1, 3-diphosphoglycerate. Glycolysis may play a pivotal role during S. suis biofilm growth. Furthermore, histidine metabolism may play an important role in biofilm formation. Histidine kinase (fold change: 1.62) was up-regulated in the tylosin-inhibited biofilm formation. Compared with other amino acids, L-His had the strongest effect on biofilm induction (Cabral et al., 2011). Histidine metabolism is also found to be involved in biofilm formation and is confirmed by gene disruption (Cabral et al., 2011). Histidine kinase is an important signaling molecule in biofilm formation in gram-positive and negative bacteria (McLoon et al., 2011; Shemesh and Chai, 2013; Yang et al., 2014; Grau et al., 2015). In addition, several ABC transporter system proteins were significantly up-regulated in the tylosin inhibiting biofilm formation. This finding is consistent with the previous results of our laboratory showing that sub-MIC erythromycin inhibits S. suis biofilm formation (Zhao et al., 2015). The carbohydrate substrate selection and fermentation determine ABC transporter proteins were significantly up-regulated in tylosin-treated cells, suggesting that S. suis had the capability of metabolizing a wide range of carbohydrates during biofilm development (Hardy et al., 2001; Marion et al., 2011; Bidossi et al., 2012; Haertel et al., 2012). Moreover, ABC transporters are important because they regulate respiration and biofilm formation, which in turn affect the rate of electricity production and bioremediation (Selvaraj et al., 2014). Furthermore, in the mutants of a *pneumococcal* biofilm screen, ABC transporters were shown to be defective in colonization (Munoz-Elias et al., 2008).

Cell surface proteins play a crucial role in biofilms. Within the biofilm, bacterial cells are embedded in a self-produced extracellular matrix. This matrix protects bacteria against a number of environmental insults. However, the matrix also confines bacterial access to fresh nutrients. Thus, the verified increase in the expression of transmembrane channels appears to be an essential requirement for the entrance of important nutrient-containing fluid (Costerton et al., 1994). In addition to acting, as channels, porins may act as potential targets for adhesion to other cells and may mediate cell attachment through binding to the proteins released for biofilm formation (Dallo et al., 2010). The outer membrane proteins may mediate cell attachment through binding to the released proteins for biofilm formation. Membrane proteins such as OmpA mediate cell adhesion in *Acinetobacter baumannii* (Dallo et al., 2010). The key developmental stages of biofilm development have been reported to be adhesion to surfaces, aggregation of micro colonies, and further expansion of the microbial community. The putative involvement of many genes encoding large cell surface proteins is adhesion to the epithelium and biofilm formation (Pridmore et al., 2004; Walter et al., 2005; Frese et al., 2011). Pham (Pham et al., 2010) found increased expression of outer membrane proteins in the *Tannerella forsythia* biofilm cells by using quantitative non-gel-based proteomic techniques. In
<figure><img src="image_14.png" /><figcaption>FIGURE 4 | Annotation of differentially regulated protein (ratio >1.5 or <0.67) functions by Gene Ontology (GO).</figcaption></figure>

our study, the expression of membrane-anchored lipoprotein, phosphoglycerate kinase, sugar ABC transporter permease, ABC superfamily ATP binding cassette transporter membrane protein and L-fucose isomerase changed; these proteins belong to membrane proteins and cell-surface proteins and might be involved in some molecular functions including catalytic activity, motor activity, nucleotide binding, protein binding and transporter activity. For example, phosphoglycerate kinase is a *S. suis* surface protein that promotes cell adhesion and plays a key role in bacterial infection and invasion (Brassard et al., 2004; Wang and Lu, 2007). We predicted that these membrane proteins might affect the bacterial cell-cell interaction. Thus, membrane proteins might play a significant role in biofilm formation.

*S. suis* virulence proteins related to infection, persistence and competitive fitness were mostly down regulated when sub-MIC tylosin was used to inhibit biofilm formation. There was a >1.5-fold decrease in the level of NADH oxidase. NADH oxidase regulates competence, virulence, and pneumococcal persistence by its actions as an oxygen sensor, in detoxifying oxygen, and in increasing the efficiency of glucose breakdown (Auzat et al., 1999) and plays an important role in *pneumococcal* infection in animal models of pneumonia (Yu et al., 2001). NADH oxidase is encoded by *nox*. The virulence and persistence in mice of a blood isolate was attenuated by a *nox* insertion mutation (Auzat et al., 1999). Thus, this protein appears to be of major importance in the growth of biofilms.

## CONCLUSIONS

Our study suggested that sub-MICs of tylosin could inhibit *S. suis* biofilm formation in vitro. We used a robust and reliable comparative proteomic technique (iTRAQ) to compare the abundances of proteins from 1/4 MIC of tylosin treated cells

and nontreated cells. Finally, in sub-MIC tylosin inhibiting cells, we identified 96 differentially expressed proteins when the protein had a fold-change of more than a ratio >1.5 or <0.67 (p < 0.05). Our proteomic data suggested general changes in metabolism (such as phosphoglycerate kinase) and surface proteins (such as ABC transporter proteins) involved in biofilm formation. Overall, our results indicated that *S. suis* metabolic regulation, cell-surface proteins, and virulence proteins appear to be of importance in biofilm growth by sub-MIC tylosin treatment. Thus, our data revealed the rough regulation of biofilm formation that might potentially be utilized to manage biofilm infections of *S. suis*.

## AUTHOR CONTRIBUTIONS

SW the design whole experiment. YL directed the completion of the experiment. YY, YZhao, HZ, JB, JC, YZhou, CW provided help during the experiment.

## ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (No. 31472231) and Guangdong Technology Research Center for Traditional Chinese Veterinary Medicine and Natural Medicine (No.20141020002). We thank Shanghai Applied Protein Technology Co. Ltd. for the help with iTRAQ.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb.2016.00384
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