# Temocillin Resistance in the Enterobacter cloacae Complex Is Conferred by a Single Point Mutation in BaeS, Leading to Overexpression of the AcrD Efflux Pump

François Guérin, François Gravey, Sophie Reissier, Malo Penven, Charlotte

Michaux, Simon Le Hello, Vincent Cattoir

► To cite this version:

François Guérin, François Gravey, Sophie Reissier, Malo Penven, Charlotte Michaux, et al. Temocillin Resistance in the Enterobacter cloacae Complex Is Conferred by a Single Point Mutation in BaeS, Leading to Overexpression of the AcrD Efflux Pump. Antimicrobial Agents and Chemotherapy, 2023, 67 (6), pp.e00358-23. 10.1128/aac.00358-23. hal-04117033

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1 Temocillin resistance in *Enterobacter cloacae* complex is conferred by a single point

2 mutation in BaeS leading to an overexpression of AcrD efflux pump

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4 Running title: Temocillin resistance in ECC

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6 François Guérina,b*, François Graveyc,d, Sophie Reissiera,b, Malo Penvena,b, Charlotte Michauxb,

7 Simon Le Helloc,d, Vincent Cattoira,b*

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9 aDepartment of Clinical Microbiology, Rennes University Hospital, 35033 Rennes, France

10 bUniversity of Rennes, Inserm UMR 1230 BRM, 35043 Rennes, France

11 cNormandie Univ, UNICAEN, UNIROUEN, Inserm UMR 1311 DYNAMICURE, 14000 Caen, France

12 dMicrobiology Department, CHU Caen, 14000 Caen, France

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14 *Correspondence: Dr François Guérin or Prof. Vincent Cattoir, CHU de Rennes, Service de
15 Bactériologie-Hygiène Hospitalière, 2 rue Henri Le Guilloux, 35033 Rennes Cedex 9, France.

16 +33-2-99-28-98-28, Fax: +33-2-99-28-41-59, Email: [francois.guerin@chu-rennes.fr](mailto:francois.guerin@chu-rennes.fr) or
17 vincent.cattoir@chu-rennes.fr.

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19 Keywords: ECC; *E. asburiae*; BaeSR, AcrD, MdtABC.

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21 Word count: abstract = 249 words; text = 2,872 words; 3 Tables; 1 Figure; 64 References; 2
22 Supplemental materials.

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ABSTRACT

Enterobacter cloacae complex (ECC) has become a major opportunistic pathogen with antimicrobial resistance issues. Temocillin, an ‘old’ carboxypenicillin remarkably stable towards β-lactamases, has been used as an alternative for the treatment of multidrug-resistant ECC infections. Here, we aimed at deciphering the never-investigated mechanisms of temocillin resistance acquisition in Enterobacterales. By comparative genomic analysis of two clonally-related ECC clinical isolates, one susceptible (Temo_S; MIC of 4 mg/L), and the other resistant (Temo_R; MIC of 32mg/L), we found that they only differed by 14 SNPs including one non-synonymous mutation (Thr175Pro) in the two-component system (TCS) sensor histidine kinase BaeS. By site-directed mutagenesis in E. coli CFT073, we demonstrated that this unique change in BaeS was responsible for a significant (16-fold) increase in temocillin MIC. Since the BaeSR TCS regulates the expression of two RND-type efflux pumps (namely AcrD and MdtABCD) in E. coli and Salmonella, we evidenced by qRT-PCR that *mdtB*, *baeS*, and *acrD* genes were significantly overexpressed (15-, 11-, and 3-fold, respectively) in Temo_R. To confirm the role of each efflux pump in this mechanism, multi-copy plasmids harboring *mdtABCD* or *acrD* were introduced into either Temo_S or the reference strain E. cloacae subsp. cloacae ATCC 13047. Interestingly, only the overexpression of *acrD* conferred a significant increase (from 8- to 16-fold) of the temocillin MIC. Altogether, we have shown that temocillin resistance in ECC can result from a single BaeS alteration, likely resulting in the permanent phosphorylation of BaeR leading to AcrD overexpression and temocillin resistance by enhanced active efflux.

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Widely encountered in natural environments, members of the *Enterobacter cloacae* complex (ECC) are also commensals of the gut microbiota of both animals and humans (1). The ECC presents a high genetic diversity and comprises 12 different genetic clusters: C-I (*Enterobacter asburiae*), C-II (*Enterobacter kobei*), C-III (*E. cloacae* subsp. *hoffmannii*), C-IV (*E. roggenkampii*), C-V (*Enterobacter ludwigii*), C-VI (*Enterobacter hormaechei* subsp. *oharae* or *E. hormaechei* susbp. *xiangfangensis*), C-VII (*E. hormaechei* subsp. *hormaechei*), C-VIII (*E. hormaechei* subsp. *steigerwaldii*), C-IX (*E. bugandensis*), C-X (*Lelliota nimipressuralis*), C-XI (*E. cloacae* subsp. *cloacae*), and C-XII (*E. cloacae* subsp. *dissolvens*) (2-4). From a clinical point of view, ECC has recently become a major opportunistic pathogen associated with many hospital-acquired infections and outbreaks. Often multidrug-resistant, ECC belongs to the ESPAKE group (*Enterococcus faecium*, *Staphylococcus aureus*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, *Pseudomonas aeruginosa*, and *Enterobacter* spp.), which comprises nosocomial pathogens with limited antimicrobial treatment options (5). All ECC members harbor a chromosomal *bla*<sub>AmpC</sub> gene coding for an AmpC-type cephalosporinase conferring an intrinsic resistance to aminopenicillins and first- and second-generation cephalosporins (6). Due to its high genomic plasticity, ECC is also able to easily develop acquired resistances. For instance, there is a large proportion of ECC clinical isolates resistant to third-generation cephalosporins (3GC) due to AmpC overproduction and/or acquisition of plasmid-mediated extended-spectrum β-lactamases (ESBLs) (7). Therefore, only a limited number of carbapenem-sparing therapeutic options remain for the treatment of 3GC-resistant ECC infections, including neglected and disused antibiotics such as colistin, fosfomycin, and temocillin (8).

Temocillin, a 6-α-methoxy derivative of ticarcillin, is an antibiotic commercialized in the early 1980s and only used in a few European countries (Belgium, France, Luxembourg, and the UK)
mainly due to the concomitant marketing of 3GCs (8,9). The use of this antibiotic in clinics has been based on its multiple advantageous characteristics such as (i) a narrow spectrum almost restricted to *Enterobacterales*, (ii) a remarkable stability towards hydrolysis by numerous β-lactamases including ESBLs and AmpC-type cephalosporinases, and (iii) a minimal risk of *Clostridioides difficile* infection by the preservation of intestinal microbiota (10). In addition, the frequency of resistant mutants *in vitro* appears low (ranging from $1 \times 10^{-8}$ to $1 \times 10^{-10}$) (11-13) whereas no *in vivo* development of temocillin resistance has been detected after treatment so far (13,14). Therefore, it has recently become a useful alternative to carbapenems for the treatment of infections caused by ESBL-producing *Enterobacterales* (9,10). Surprisingly, mechanisms of temocillin resistance have been very poorly studied with the exception of intrinsic resistance in *Pseudomonas aeruginosa*, mainly driven by active efflux (15,16). Whereas hydrolysis of temocillin by class B- and D-type β-lactamases is well known, mechanisms of acquired chromosomal-encoded resistance have never been reported in *Enterobacterales*.

Two-component systems (TCS) are universal signal transduction systems in bacteria and participate in various important cellular processes such as pathogenicity, antibiotic resistance, environmental stresses, quorum sensing, and biofilm production (17,18). These environmental response systems are typically composed of a sensor histidine kinase (HK) and a cognate response regulator (RR) (19). HKs are usually transmembrane proteins, which are able to perceive environmental signals. When stimulated, HK triggers its autophosphorylation. The phosphate group is then transferred to an aspartate residue on the cognate RR located in the cytoplasm (20). The phosphorylated RR can therefore bind to promoter regions of target genes leading to expression regulation (20). Among TCS identified in *Enterobacterales*, several of them are known to be involved in antibiotic resistance such as BaeSR, CpxAR, EnvZ/OmpR,
EvgAS, PhoPQ, PmrAB, and RcsBCD (21). Nonetheless, only two TCSs have been characterized so far in ECC, namely PhoPQ and CrrAB, which are both involved in colistin heteroresistance (22-24).

Here, by comparing a temocillin-susceptible and -resistant ECC clinical isolates, we identified a mutation in the HK BaeS of the TCS BaeSR. By using knockout mutants and overexpressing strains of genes known to be regulated by BaeSR, we identified the genetic basis of acquired temocillin resistance in ECC, which will pave the way for a better understanding of mechanisms of antimicrobial resistance deployed by this worrisome pathogen.
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# RESULTS

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## *E. asburiae* isolates have different antibiotic susceptibility profiles

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Two clinical isolates, first identified as belonging to the *E. cloacae* complex by MALDI-TOF mass spectrometry, were recovered from the same urinary specimen (Table 1). By *hsp60* sequencing, both isolates were shown to belong to the cluster C-I (namely *E. asburiae*) and were presumably clonally related. A comparison of antibiotic susceptibility profiles showed that these isolates only differed by their susceptibility to three β-lactams: ticarcillin, temocillin, and aztreonam (Table 2). Indeed, *E. asburiae* Temo_R exhibited 32-, 8-, and 4-fold higher MICs of ticarcillin, temocillin, and aztreonam, respectively, compared to those determined for the *E. asburiae* Temo_S strain (Table 2). Note that no change in MICs was observed in the presence of Phe-Arg-β-naphthylamide (PAβN) for these three antibiotics (data not shown).

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## *E. asburiae* isolates are clonally related

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By genome sequencing, we determined that the two strains belonged to the sequence type ST24 and confirmed that they were isogenic with a difference of only 14 single-nucleotide polymorphisms (SNPs). Detailed analysis revealed that around one third of the SNPs (5/14) occurred within noncoding regions. On the seven SNPs appearing in coding regions, six led to synonymous mutations, and three were responsible for an amino acid change (Table 3). The three non-synonymous mutations were found in three different genes coding for a hypothetical protein (Leu118Phe), the TCS sensor HK BaeS (Thr175Pro), and a beta-1,3-glucosyltransferase (Ile38Val) (Table 3).

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## Unique Thr175Pro change in BaeS confers temocillin resistance

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TCS systems, including BaeRS, have been associated with antibiotic resistance. Therefore, the consequence of the amino-acid change found in the BaeS sequence was of high interest to investigate in the context of temocillin resistance. In order to confirm the role of the
Thr175Pro mutation in BaeS, we introduced it in the *baeS* gene of *E. coli* CFT073 by site-directed mutagenesis. As expected, we demonstrated experimentally that this unique point mutation was alone responsible for a significant (16-fold) increase in MICs of temocillin, ticarcillin, and aztreonam (Table 2). Note that the mutation was located in a transmembrane domain of BaeS, which is not in the vicinity of the histidine residue (*His*<sub>250</sub>) within the cytoplasmic catalytic domain (Figure S1).

## 130 **Temocillin resistance is due to overexpression of AcrD efflux pump system**

The BaeSR TCS regulates the expression of two efflux pump systems (namely AcrAD-TolC and MdtABCD) belonging to the RND (Resistance-Nodulation-cell Division) superfamily in *E. coli* and *Salmonella* (25,26). In addition, *baeS* is part of the *mdtABCD-baeSR* operon (27). Then, we compared by qRT-PCR the expression of *mdtB* and *acrD* genes as well as *baeS* in both *E. asburiae* *Temo_R* and *Temo_S* clinical isolates. We evidenced that these three genes were significantly overexpressed in *E. asburiae* *Temo_R* as compared to *E. asburiae* *Temo_S* with a fold change of 15, 11, and 3 for *mdtB*, *baeS*, and *acrD*, respectively. These results strongly suggested that decreased susceptibility to ticarcillin, temocillin, and aztreonam in *E. asburiae* *Temo_R* was likely related to an increase of the efflux mediated by MdtABCD and/or AcrD RND-type efflux pump systems.

To confirm the role of these two RND-type efflux pump systems, both *mdtABCD* and *acrD* genes (together with their own promoters) were cloned into the pBAD202/D-TOPO vector and expressed in *E. asburiae* *Temo_S* strain. We observed that the overexpression of *acrD*, but not *mdtABCD*, was able to confer temocillin resistance (64 mg/L) with a 16-fold increase in MIC (Table 2). The overexpression of *acrD* also led to an increase in MICs of ticarcillin (32-fold) and aztreonam (4-fold) while overexpression of *mdtABCD* only conferred a four-fold increase in ticarcillin MIC (Table 2). The same mutants and *trans*-complemented strains were also
obtained in the reference strain *E. cloacae* subsp. *cloacae* ATCC 13047 (C-XI). Although we
showed that the deletion of *baeS* (ECL_03405), *mdtB* (ECL_03403) or *acrD* (ECL_03767) did
not modify susceptibility profiles to β-lactams, we confirmed that the sole overexpression of
*acrD* conferred significant increase (8 fold) in MICs of temocillin (Table 2).

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DISCUSSION

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This study deciphers for the first time the genetic basis of temocillin resistance in *Enterobacterales*, which is a cross-resistance to carboxypenicillins (ticarcillin and temocillin) and aztreonam. Indeed, mechanisms of temocillin intrinsic resistance/acquired susceptibility have only been studied in *P. aeruginosa* (15,16). In this species, it was demonstrated that temocillin was a substrate of the RND-type efflux system MexAB-OprM while its uptake marginally requires anion-specific porins OpdK and OpdF (15,16).

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Here, we showed that temocillin resistance was due to a unique point non-synonymous mutation in the *baeS* gene (leading to the Thr175Pro change) encoding the HK of the TCS BaeSR. In *E. coli*, BaeSR is a well-known TCS involved in the regulation of the envelope stress response (28,29). It is worth notifying that this TCS can be induced by a pleiotropic number of compounds including indole, copper, zinc, ethanol, sodium tungstate tannin, and flavonoid (26,30-37). BaeSR is also one of the most studied TCS associated with multidrug resistance in *E. coli*, *Salmonella*, and *A. baumannii*. However, it has never been linked to temocillin resistance and intriguingly enough, has never been studied in ECC, species in which only 2 TCS have been described so far to be linked with antibiotic resistance. Indeed, PhoPQ and CrrAB have been both associated with colistin hetero-resistance (22-24).

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BaeSR system has been linked to antibiotic resistance in different bacterial species throughout the regulation of genes encoding for efflux pump systems. In *E. coli* and *Salmonella*, the overexpression of BaeR has been shown to regulate expression of *acrD* and *mdtA*, both part of the AcrAD-ToIC and MdtABC efflux pumps, conferring resistance to different classes of antibiotics such as novobiocin, some β-lactams or deoxycholate (25-27,38,39). In addition, BaeR overexpression slightly reduced susceptibility to cephalosporins by decreasing the expression of outer membrane proteins in *E. coli* (40), whereas in *Salmonella*, overexpression

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177 of BaeR also increased polymyxin susceptibility by altering the expression of polymyxin resistance-associated genes (41). In *A. baumannii*, BaeSR affects polymyxin susceptibility by regulating the expression of efflux pump systems such as MacAB and AdelJK while it is also involved in tigecycline resistance by activating the expression of AdeABC, AdelJK, and MacAB-TolC (42-44).

182 As opposed to other antibiotics, very few studies have linked temocillin with RND-type efflux pumps with only the AcrAD-TolC system being involved in its export. The AcrD RND-type efflux pump functions in cooperation with AcrA and TolC, and the AcrAD-TolC system and effluxes structurally-diverse compounds including SDS, novobiocin, and deoxycholate as well as aminoglycosides and dianionic β-lactams such as carbenicillin, oxacillin, nafcillin, and aztreonam (25,45-47). Very recently, it was also demonstrated in S. Typhimurium that AcrD expelled unreported substrates such as temocillin, dicloxacillin, cefazolin, and fusidic acid (48).

189 Interestingly, the overexpression of *acrD* in an *ΔacrBΔacrD E. coli* strain increased MICs of both temocillin and aztreonam by 8-fold (48).

191 In ECC, temocillin resistant mutant carried a Thr175Pro point mutation in the BaeS-encoding gene, known to regulate together with BaeR, efflux pump systems in other bacterial species.

193 Recently in *E. coli*, five different gain-of-function mutations in *baeS* were identified as constitutively activating the BaeSR TCS with increased expression of MdtABC efflux pump (49).

195 These modifications likely alter the BaeS structure with constitutive elevation of kinase activity and/or reduction of phosphatase activity, which in turn cause permanent phosphorylation of BaeR. Phosphorylated BaeR will then be able to bind *mdtABCD-baeSR* and *acrD* promoter regions and increase corresponded genes expression (49). In this study, DGE analysis through RNAseq revealed that the mutation C310T in the *baeS51* mutant (leading to Arg104Cys amino acid change) was responsible for a 37- and 6-fold change in expression of *mdtABCD-baeSR* and
201 *acrD*, respectively, as compared to the parental strain (49). This difference of change in
202 expression is similar to what we observed here between *E. asburiae* Temo_R and Temo_S
203 strains, possibly due to the expression of the *mdtABCD-baeSR* operon, which is directly
204 controlled by BaeR whereas *acrD* is indirectly regulated (50).

205 In conclusion, we describe for the first the genetic basis of temocillin resistance in
206 *Enterobacterales* through a single mutation (Thr175Pro) in BaeS and as a consequence, an
207 increase of AcrD-mediated efflux (Figure 1).
208 **MATERIALS AND METHODS**

209 **Bacterial strains and growth conditions.** Bacterial strains used in this study are listed in Table 210
1. The two clinical isolates (Temo_S and Temo_R) were recovered from a single urine sample 211
from the same patient. The *E. cloacae* subsp. *cloacae* ATCC 13047 reference strain (51) was 212
used to construct deletion and *trans*-complemented strains while *E. coli* CFT073 (52) was used 213
to construction the *baeS*<sup>Thr175Pro</sup> point-mutation strain by site-directed mutagenesis. All strains 214
were cultured with constant agitation (200 rpm) at 35°C in Luria-Bertani (LB) medium.

215 **Antimicrobial susceptibility testing.** MICs were determined using the broth microdilution 216
reference method according to the guidelines recommended by the European Committee on 217
Antimicrobial Susceptibility (EUCAST; http://www.eucast.org/). The following antibiotics were 218
tested: amoxicillin, ticarcillin, temocillin, piperacillin, cefoxitin, cefotaxime, cefepime, 219
aztreonam, ertapenem, gentamicin, ciprofloxacin, tigecycline, colistin, and chloramphenicol.
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All experiments were performed in biological triplicate. MICs of ticarcillin, temocillin, and 221
aztreonam were also determined by broth microdilution in the absence or presence of the 222
efflux pump inhibitor PAβN at the concentration of 40 mg/L.

223 **Whole-genome sequencing and bioinformatic analysis.** The genome sequences of both *E. 224 asburiae* Temo_S and Temo_R clinical isolates were determined by WGS at the "Plateforme 225 de microbiologie mutualisée" (P2M) of the Pasteur International Bioresources network 226
(Institut Pasteur, Paris, France). DNA was extracted using the MagNAPure 96 system (Roche 227 Diagnostics GmbH, Mannheim, Germany). The libraries were prepared using the Nextera XT 228
kit (Illumina, San Diego, CA), and the sequencing was performed using an Illumina NextSeq 229
500 system generating 150-bp paired-end reads.

230 FastQ files were trimmed using the `fqCleanER` tool 231
(https://gitlab.pasteur.fr/GIPhy/fqCleanER), and the qualities of the filtered FastQ files were
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evaluated

using

the

FastQC

software

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(https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Genomes were de novo

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assembled using the SPAdes v 3.12 (53) while the quality of assemblies was assessed using the

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Quast software (54).

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The identification to the species level was performed using the rMLST algorithm (55). The

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sequence type (ST) was resolved using the appropriate MLST schema (56). The cluster

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attribution based on the partial sequence of the *hsp60* gene was determined using blastn

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algorithm (2). The identification to the species level was also confirmed by average nucleotide

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identity (ANI) analysis using all reference ECC genomes (3) using the mash tool. Acquired

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resistance genes and point mutations were detected using Resfinder and PointFinder

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databases, respectively (https://cge.food.dtu.dk/services/ResFinder/; 57). The genome of the

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Temo_S strain was annotated using the RAST server and then used as the reference genome

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to perform variant calling between the two Temo_R and Temo_S strains using Freebayes after

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a mapping of the reads using Burrows-Wheeler transform (58).

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The genomic sequences of both *E. asburiae* Temo_S and Temo_R clinical isolates were

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deposited in GenBank as BioProject no. PRJNA902787.

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**Construction of the knockout mutants and trans-complemented strains in *E. cloacae*.** The

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deletion of the genes encoding the histidine kinase BaeS (ECL_03405), the response effector

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BaeR (ECL_03406), and the two RND pumps MdtB (ECL_03403) and AcrD (ECL_03767) were

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performed in ATCC 13047 using the Red helper plasmid pKOBEG as previously described (59-

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63) (Tables 1 and S1). The RND pump-encoding genes (MdtABCD or ECL_03403-ECL_03407;

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and AcrD or ECL_03767) including their own promoters were amplified by PCR (Table S1) and

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obtained amplicons were TA-cloned into the overexpression plasmid, pBAD202 (low-copy

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number plasmid: ~20 copies/cell) Directional TOPO (Invitrogen, Villebon-sur-Yvette, France).
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E. coli TOP10 cells (Invitrogen) carrying pBAD202 with correctly oriented inserts were then selected on LB plates with 40 mg/L of kanamycin. After extraction, plasmids carrying the RND efflux pump-encoding genes were transformed in E. cloacae ATCC 13047 strain or E. asburiae Temo_S clinical isolate (Table 1).

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## Site-Directed Mutagenesis

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To confirm the role of the Thr175Pro mutation in *baeS*, single-nucleotide allelic replacement was carried out using the suicide vector pDS132 in *E. coli* CFT073 (52,64). The cloning steps were performed in *E. coli* DH5α λpir strain to allow replication of the plasmid (Table S1). The recombinant plasmids were then purified and introduced in *E. coli* CFT073 by electro-transformation. Clones that integrated the plasmid were selected by plating cells on LB plates containing 30 mg/L of chloramphenicol. After overnight growth at 35°C, one colony was picked, diluted in 10 mM MgSO₄ solution, and serial dilutions were plated on LB agar plates with 5% sucrose and without NaCl to force plasmid excision. After overnight incubation at 35°C, 100 clones were streaked on chloramphenicol-containing LB agar plates and on LB agar with 5% sucrose and without NaCl. Several clones were tested by PCR-sequencing to identify those carrying the mutation (Table S1).

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Quantification of gene expression by RT-qPCR. Total RNAs were extracted from E. asburiae Temo_S and Temo_R strains grown to the late-exponential phase by using the Direct-Zol RNA mini-prep kit (Zymo Research, Irvine, CA). Residual chromosomal DNA was removed by treating samples with the TURBO DNA-free kit (Life Technologies, Saint-Aubin, France). Samples were quantified using the NanoDrop One spectrophotometer (Thermo Fisher Scientific, Courtabœuf, France). cDNA was synthesized from total RNAs (~1 µg) using the QuantiTect reverse transcription kit (Qiagen) according to the manufacturer's instructions. Transcript levels were determined by the ΔΔCt method using *rpoB* as a housekeeping control
280 gene. The expression, and the fold change (FCs) of *mdtB*, *baeS*, and *acrD* genes were calculated

281 by Ct comparison between Temo_S and Tem_R clinical isolates. Experiments were performed

282 at least in three biological replicates. Student t-test was employed and *p*-values less than 0.05

283 were considered as statistically significant.
284 ACKNOWLEDGMENTS

285 A part of this work was performed using bioinformatical resources provided by CRIANN

286 (Normandy, France). The three-year doctoral contract of François Gravey was funded by a grant

287 from the Normandy Region.

288
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453

LEGEND OF THE FIGURE

454

**FIG 1** Mechanism of temocillin resistance in ECC through the two-component system BaeSR.

455

The alteration of BaeS by a single mutation (Thr175Pro; red star), responsible for a constitutive elevation of kinase activity and/or reduction of phosphatase activity, is responsible for permanent phosphorylation of BaeR. Phosphorylated BaeR can then activate directly (solid line arrow) *mdtABCD-baeSR* and indirectly (dotted line arrow) *acrD* expression. As a consequence, the expression of those genes contribute to increase the active efflux of ticarcillin (yellow rectangle) and temocillin (blue round)/ticarcillin/aztreonam (green triangle) through MdtABC-TolC and AcrAD-TolC RND-type efflux pump systems, respectively.

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<table><caption>TABLE 1 Strains and plasmid used in the study</caption><thead><tr><th>Strain or plasmid</th><th>Genotype/relevant characteristic(s)</th><th>Reference</th></tr></thead><tbody><tr><td><strong>Strains</strong></td><td></td><td></td></tr><tr><td>E. asburiae Temo_S</td><td>Human clinical isolate Ecc20200302 (urine, 2020/02/28), Tem<sup>S</sup></td><td>This study</td></tr><tr><td>E. asburiae Temo_R</td><td>Human clinical isolate Ecc20200303 (urine, 2020/02/28), Tem<sup>R</sup></td><td>This study</td></tr><tr><td>Tem_S/pBAD</td><td>E. asburiae Temo_S trans-complemented strain carrying pBAD202/D-TOPO</td><td>This study</td></tr><tr><td>Tem_S/pBADΩmdtABCD</td><td>E. asburiae Temo_S trans-complemented strain carrying pBAD202/D-TOPOΩmdtABCD (ECL_03401-ECL_03404)</td><td>This study</td></tr><tr><td>Tem_S/pBADΩacrD</td><td>E. asburiae Temo_S trans-complemented strain carrying pBAD202/D-TOPOΩacrD (ECL_03767)</td><td>This study</td></tr><tr><td>E. cloacae subsp. cloacae ATCC 13047</td><td>Reference strain ATCC 13047 (cluster XI)</td><td>51</td></tr><tr><td>ATCC13047ΔbaeS</td><td>ATCC 13047 deleted for baeS (ECL_03405)</td><td>This study</td></tr><tr><td>ATCC13047ΔmdtB</td><td>ATCC13047 deleted for mdtB (ECL_03403)</td><td>63</td></tr><tr><td>ATCC13047ΔacrD</td><td>ATCC13047 deleted for acrD (ECL_03767)</td><td>63</td></tr><tr><td>ATCC13047/pBAD</td><td>ATCC 13047 trans-complemented strain carrying pBAD202/D-TOPO</td><td>63</td></tr><tr><td>ATCC13047/pBADΩmdtABCD</td><td>ATCC 13047 trans-complemented strain carrying pBAD202/D-TOPOΩmdtABCD (ECL_03401-ECL_03404)</td><td>63</td></tr><tr><td>ATCC13047/pBADΩacrD</td><td>ATCC 13047 trans-complemented strain carrying pBAD202/D-TOPOΩacrD (ECL_03767)</td><td>63</td></tr><tr><td>E. coli CFT073</td><td>Wild-type susceptible clinical isolate (phylogenetic group B2)</td><td>52</td></tr><tr><td>CFT073_baeS<sup>Thr175Pro</sup></td><td>CFT073 derivative with allelic replacement of baeS by baeS<sup>Thr175Pro</sup></td><td>This study</td></tr><tr><td>E. coli DH5α λpir</td><td>Reference strain (supE4 ΔlacU169 (Φ80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 λpir)</td><td>Laboratory collection</td></tr><tr><td><strong>Plasmids</strong></td><td></td><td></td></tr><tr><td>pBAD202/D-TOPO</td><td>General expression vector with arabinose-inducible promoter, Kan<sup>R</sup></td><td>Life Technologies</td></tr><tr><td>pKOBEG</td><td>Recombination vector, phage λ recγβα operon under the control of the pBAD promoter, Cm<sup>R</sup></td><td>60</td></tr><tr><td>pKD4</td><td>Plasmid containing an FRT-flanked kanamycin, Kan<sup>R</sup></td><td>59</td></tr><tr><td>pCP20_Gm</td><td>FLP-mediated recombination vector, Gm<sup>R</sup></td><td>61</td></tr><tr><td>pDS132</td><td>Conditional replication vector; R6K origin, mobRP4 transfer origin, sucrose-inducible sacB, Cm<sup>R</sup></td><td>64</td></tr></tbody></table>

Cm<sup>R</sup>, chloramphenicol resistant; Gm<sup>R</sup>, gentamicin resistant; Kan<sup>R</sup>, kanamycin resistant; Tem<sup>R</sup>, temocillin resistant; Tem<sup>S</sup>, temocillin susceptible.
<table><caption>TABLE 2 MIC values of antibiotics for *E. cloacae* clinical isolates, *E. cloacae* ATCC 13047, and *E. coli* CFT073 (and their derivative strains)</caption><thead><tr><th rowspan="2">Strain</th><th colspan="12">MIC (mg/L)<sup>a,b</sup></th></tr><tr><th>AMX</th><th>TEM</th><th>TIC</th><th>PIP</th><th>FOX</th><th>CTX</th><th>FEP</th><th>ATM</th><th>ERT</th><th>GEN</th><th>CIP</th><th>TGC</th><th>COL</th><th>CHL</th></tr></thead><tbody><tr><td><strong>E. asburiae</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Temo_S</td><td>&gt;256</td><td>4</td><td>2</td><td>4</td><td>&gt;256</td><td>0.5</td><td>0.03</td><td>0.03</td><td>0.25</td><td>0.12</td><td>&lt;0.01</td><td>0.5</td><td>32</td><td>4</td></tr><tr><td>Temo_R</td><td>&gt;256</td><td><strong>32 (8)</strong></td><td><strong>64 (32)</strong></td><td>4</td><td>&gt;256</td><td>0.5</td><td>0.03</td><td><strong>0.12 (4)</strong></td><td>0.25</td><td>0.12</td><td>&lt;0.01</td><td>0.5</td><td>64</td><td>4</td></tr><tr><td>Temo_S/pBAD</td><td>&gt;256</td><td>4</td><td>2</td><td>2</td><td>&gt;256</td><td>0.5</td><td>0.03</td><td>0.03</td><td>0.25</td><td>0.12</td><td>&lt;0.01</td><td>0.5</td><td>32</td><td>4</td></tr><tr><td>Temo_S/pBADΩmdtABCD</td><td>&gt;256</td><td>4</td><td><strong>8 (4)</strong></td><td>2</td><td>&gt;256</td><td>0.5</td><td>0.03</td><td>0.03</td><td>0.25</td><td>0.12</td><td>&lt;0.01</td><td>0.5</td><td>16</td><td>4</td></tr><tr><td>Temo_S/pBADΩacrD</td><td>&gt;256</td><td><strong>64 (16)</strong></td><td><strong>64 (32)</strong></td><td>2</td><td>&gt;256</td><td>0.5</td><td>0.03</td><td><strong>0.12 (4)</strong></td><td>0.25</td><td>0.12</td><td>&lt;0.01</td><td>0.5</td><td>32</td><td>4</td></tr><tr><td><strong>E. cloacae subsp. cloacae</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ATCC 13047</td><td>&gt;256</td><td>8</td><td>4</td><td>8</td><td>&gt;256</td><td>2</td><td>0.03</td><td>0.5</td><td>0.25</td><td>0.25</td><td>0.03</td><td>0.5</td><td>&gt;256</td><td>4</td></tr><tr><td>ATCC13047ΔbaeS</td><td>&gt;256</td><td>4</td><td>4</td><td>8</td><td>&gt;256</td><td>2</td><td>0.03</td><td>0.5</td><td>0.25</td><td>0.25</td><td>0.03</td><td>0.5</td><td>256</td><td>4</td></tr><tr><td>ATCC13047ΔmdtB</td><td>&gt;256</td><td>8</td><td>4</td><td>8</td><td>&gt;256</td><td>2</td><td>0.03</td><td>0.5</td><td>0.25</td><td>0.25</td><td>0.03</td><td>0.5</td><td>256</td><td>4</td></tr><tr><td>ATCC13047ΔacrD</td><td>&gt;256</td><td>8</td><td>4</td><td>8</td><td>&gt;256</td><td>2</td><td>0.03</td><td>0.25</td><td>0.25</td><td>0.25</td><td>0.03</td><td>0.5</td><td>&gt;256</td><td>4</td></tr><tr><td>ATCC13047/pBAD</td><td>&gt;256</td><td>8</td><td>4</td><td>8</td><td>&gt;256</td><td>2</td><td>0.03</td><td>0.5</td><td>0.25</td><td>0.25</td><td>0.03</td><td>0.5</td><td>&gt;256</td><td>4</td></tr><tr><td>ATCC13047/pBADΩmdtABCD</td><td>&gt;256</td><td>8</td><td><strong>16 (4)</strong></td><td>8</td><td>&gt;256</td><td>2</td><td>0.03</td><td>0.5</td><td>0.5</td><td>0.25</td><td>0.03</td><td>0.5</td><td>&gt;256</td><td>4</td></tr><tr><td>ATCC13047/pBADΩacrD</td><td>&gt;256</td><td><strong>64 (8)</strong></td><td><strong>128 (16)</strong></td><td>8</td><td>&gt;256</td><td>2</td><td>0.03</td><td><strong>2 (4)</strong></td><td>0.25</td><td>0.25</td><td>0.03</td><td>0.5</td><td>&gt;256</td><td>4</td></tr><tr><td><strong>E. coli</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>CFT073</td><td>4</td><td>4</td><td>4</td><td>2</td><td>2</td><td>0.12</td><td>0.06</td><td>0.06</td><td>0.06</td><td>0.12</td><td>&lt;0.01</td><td>0.12</td><td>0.12</td><td>4</td></tr><tr><td>CFT073_baeS<sup>Thr175Pro</sup></td><td>4</td><td><strong>64 (16)</strong></td><td><strong>64 (16)</strong></td><td>2</td><td>2</td><td>0.12</td><td>0.06</td><td><strong>1 (16)</strong></td><td>0.06</td><td>0.12</td><td>&lt;0.01</td><td>0.12</td><td>0.12</td><td>4</td></tr></tbody></table>

<footnote>a AMX, amoxicillin; ATM, aztreonam; CHL, chloramphenicol; CIP, ciprofloxacin; COL, colistin; CTX, cefotaxime; ERT, ertapenem; FEP, cefepime; FOX, cefoxitin; GEN, gentamicin; PIP, piperacillin; TEM, temocillin; TIC, ticarcillin; TGC, tigecycline.</footnote>

<footnote>b Numbers in boldface are MICs that are significantly different (fold changes of ≥4 are indicated in parentheses).</footnote>
<table><caption>TABLE 3 Nucleotide changes identified between *E. asburiae* Temo_S and Temo_R isogenic isolates</caption><thead><tr><th>Gene<sup>a</sup></th><th>Predicted product</th><th>Nucleotide change<sup>a</sup></th><th>Amino acid change</th><th>Homolog in <i>E. cloacae</i> subsp. <i>cloacae</i> ATCC 13047<sup>b</sup></th></tr></thead><tbody><tr><td>OSB85_02280</td><td>General stress protein YmdF</td><td>24A&gt;C<br>36A&gt;T</td><td>None<br>None</td><td>ECL_02630</td></tr><tr><td>OSB85_09375</td><td>Hypothetical protein</td><td>354G&gt;T<br>390T&gt;C</td><td>Leu118Phe<br>None</td><td>Not found</td></tr><tr><td>OSB85_12740</td><td>IS3 family transposase</td><td>120C&gt;T<br>132C&gt;T</td><td>None<br>None</td><td>Not found</td></tr><tr><td>OSB85_20490</td><td>Two-component system sensor histidine kinase BaeS</td><td>523A&gt;C</td><td>Thr175Pro</td><td>ECL_03405</td></tr><tr><td>OSB85_21180</td><td>Beta-1,3-glucosyltransferase</td><td>112A&gt;G<br>117C&gt;T</td><td>Ile38Val<br>None</td><td>Not found</td></tr></tbody></table>

<footnote><sup>a</sup> Gene names and positions from the annotated sequence of the *E. asburiae* Temo_S clinical isolate (GenBank accession no. SAMN31775694).</footnote>

<footnote><sup>b</sup> Gene names are from the annotated sequence of the *E. cloacae* subsp. *cloacae* ATCC 13047 (GenBank accession no. NC_014121).</footnote>
<table><caption>TABLE 3 Nucleotide changes identified between *E. asburiae* Temo_S and Temo_R isogenic isolates</caption><thead><tr><th>Gene<sup>a</sup></th><th>Predicted product</th><th>Nucleotide change<sup>a</sup></th><th>Amino acid change</th><th>Homolog in E. cloacae subsp. cloacae ATCC 13047<sup>b</sup></th></tr></thead><tbody><tr><td>OSB85_02280</td><td>General stress protein YmdF</td><td>24A&gt;C<br>36A&gt;T</td><td>None<br>None</td><td>ECL_02630</td></tr><tr><td>OSB85_09375</td><td>Hypothetical protein</td><td>354G&gt;T<br>390T&gt;C</td><td>Leu118Phe<br>None</td><td>Not found</td></tr><tr><td>OSB85_20490</td><td>Two-component system sensor histidine kinase BaeS</td><td>523A&gt;C</td><td>Thr175Pro</td><td>ECL_03405</td></tr><tr><td>OSB85_21180</td><td>Beta-1,3-glucosyltransferase</td><td>112A&gt;G<br>117C&gt;T</td><td>Ile38Val<br>None</td><td>Not found</td></tr></tbody></table>

<footnote><sup>a</sup> Gene names and positions from the annotated sequence of the *E. asburiae* Temo_S clinical isolate (GenBank accession no. SAMN31775694).</footnote>

<footnote><sup>b</sup> Gene names are from the annotated sequence of the *E. cloacae* subsp. cloacae ATCC 13047 (GenBank accession no. NC_014121).</footnote>