# Plasmid-Mediated Ciprofloxacin Resistance Imparts a Selective Advantage on Escherichia coli ST131

Minh-Duy Phan, Kate M. Peters, Laura Alvarez Fraga, Steven C. Wallis,

Steven J Hancock, Nguyen Thi Khanh Nhu, Brian M Forde, Michelle J.

Bauer, David L Paterson, Scott A Beatson, et al.

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## To cite this version:

Minh-Duy Phan, Kate M. Peters, Laura Alvarez Fraga, Steven C. Wallis, Steven J Hancock, et al. "Plasmid-Mediated Ciprofloxacin Resistance Imparts a Selective Advantage on Escherichia coli ST131." Antimicrobial Agents and Chemotherapy, 2022, 66 (1), pp.e02146-21. 10.1128/AAC.02146-21. hal-04958943

HAL Id: hal-04958943

https://hal.inrae.fr/hal-04958943v1

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Distributed under a Creative Commons Attribution 4.0 International License
1 Plasmid-mediated ciprofloxacin resistance imparts a

2 selective advantage on *Escherichia coli* ST131

3 Minh-Duy Phan¹,², Kate M. Peters¹,², Laura Alvarez Fraga¹,²,⁵, Steven C. Wallis³,

4 Steven Hancock¹,², Nguyen Thi Khanh Nhu¹,², Brian Forde³, Michelle J. Bauer³,

5 David L. Paterson³, Scott A Beatson¹,², Jeffrey Lipman³,⁴, Mark A. Schembri¹,²*

6

7 ¹ School of Chemistry and Molecular Biosciences, The University of Queensland,
Brisbane, Queensland, Australia

8

9 ² Australian Infectious Diseases Research Centre, The University of Queensland,
Brisbane, Queensland, Australia

10

11 ³ University of Queensland Centre for Clinical Research, The University of
Queensland, Brisbane, Australia

12

13 ⁴ Jamison Trauma Institute and Department of Intensive Care Medicine, Royal
Brisbane and Women's Hospital, Brisbane, Queensland, Australia

14

15 ⁵ University Hospital Complex of A Coruña (CHUAC), Biomedical Research Institute
of A Coruña (INIBIC), A Coruña, Spain

16

17

18

19 *Corresponding author

20 ² Mark Schembri: School of Chemistry and Molecular Biosciences, Building 76, The
University of Queensland, Brisbane, Queensland 4072, Australia. Telephone:

21 +6173365 3306; E-mail: m.schembri@uq.edu.au

22

23
30

**Abstract**

Escherichia coli ST131 is a recently emerged antibiotic resistant clone responsible for high rates of urinary tract and bloodstream infections. Despite its global dominance, the precise mechanisms that have driven the rapid dissemination of ST131 remain unknown. Here, we show that the plasmid-associated resistance gene encoding the AAC(6')-Ib-cr enzyme that inactivates the fluoroquinolone antibiotic ciprofloxacin is present in >70% of strains from the most rapidly expanding subgroup of multidrug resistant ST131. Using a series of genome-edited and plasmid-cured isogenic strains, we demonstrate that the *aac(6')-Ib-cr* gene confers a selective advantage on ST131 in the presence of ciprofloxacin, even in strains containing chromosomal GyrA and ParC FQ-resistance mutations. Further, we identify a pattern of emerging carbapenem resistance in other common *E. coli* clones carrying both *aac(6')-Ib-cr* and chromosomal FQ-resistance mutations, suggesting this dual resistance combination may also impart a selective advantage on these non-ST131 antibiotic resistant lineages.

45
46 **Introduction**

47 The *Escherichia coli* ST131 clone is of major clinical importance due to its multidrug
48 resistance phenotype and high prevalence worldwide. Alarmingly, just over a decade
49 since its first identification, ST131 has become the overwhelmingly predominant *E*.
50 *coli* clone responsible for urinary tract and bloodstream infections. Genomic analyses
51 have classified ST131 into three major clades (A, B and C), with all strains from the
52 globally dominant clade C (*H30*) sublineage containing target-modifying mutations in
53 the DNA gyrase (GyrA; S83L, D87N) and topoisomerase IV (ParC; S80I)
54 chromosome replication enzymes that together confer high-level resistance to
55 fluoroquinolones (FQs) ¹⁻⁴. Clade C strains can be further subdivided into clade C1
56 (*H30* non-Rx) and C2 (*H30Rx*), with clade C2 strains largely positive for the CTX-M-
57 15 extended-spectrum β-lactamase (ESBL) ¹,², the most common type of ESBL
58 worldwide ⁵.

59

60 FQs are one of the most commonly prescribed antibiotics globally, with resistance to
61 FQs considered to be a strong driver of ST131 dissemination. While chromosomal
62 GyrA and ParC modifications form a ubiquitous mechanism of FQ resistance in clade
63 C ST131, other mechanisms of FQ resistance have also been characterised, including
64 enhanced drug efflux and target modification by the plasmid-encoded AAC(6')-Ib-cr
65 enzyme 6. AAC(6')-Ib-cr is a variant of the AAC(6')-Ib aminoglycoside
66 acetyltransferase that has been modified by the acquisition of two mutations (W102R
67 and D179Y) necessary for its acetyltransferase function 7. Among the FQ antibiotics,
68 ciprofloxacin and norfloxacin are subjected to N-acetylation (deactivation) by
69 AAC(6')-Ib-cr due to the availability of an unsubstituted piperazinyl group 8.
70 Levofloxacin and moxifloxacin lack this unsubstituted piperazinyl group and
therefore are not subjected to acetylation. AAC(6')-Ib-cr activity confers low-level
reduced susceptibility to ciprofloxacin and norfloxacin, however it can act additively
with other mechanisms to achieve clinically significant resistance<sup>7, 9</sup>.

The precise molecular mechanisms that have led to the dominance of ST131 over
other FQ-resistant *E. coli* clones remain elusive. Several studies have reported an
association between the *aac(6')-Ib-cr* gene and clade C2 FQ-resistant ST131<sup>4, 6, 10, 11</sup>,
supporting the assertion that expression of the plasmid-encoded AAC(6')-Ib-cr
enzyme amplifies the level of FQ resistance mediated by chromosomal GyrA and
ParC modifications<sup>6</sup>. Here, we curated a large ST131 genome dataset to explore the
prevalence of the *aac(6')-Ib-cr* gene in different ST131 sublineages. We also
generated a series of genome-edited and plasmid modified isogenic strains to
experimentally define the precise contribution of the *aac(6')-Ib-cr* gene to FQ
resistance in ST131 backgrounds comprising different GyrA and ParC modifications.
We show that the *aac(6')-Ib-cr* gene provides a selective advantage in the presence of
ciprofloxacin, and extend this observation to reveal an emerging link between
*aac(6')-Ib-cr* carriage, chromosomal FQ-resistance mutations and resistance to last-
line carbapenems. Overall, our data identifies the acquisition of plasmids carrying the
*aac(6')-Ib-cr* gene as one element of a complex multifactorial genetic landscape
linked to the global dominance of clade C2 ST131, and reveals that acquisition of the
*aac(6')-Ib-cr* gene can be linked to emerging carbapenem resistance in other FQ-
resistant *E. coli* clones.

93
94 **Material and Methods**

95 **Ethics statement**

96 Approval for the collection of human urine was obtained from the University of

97 Queensland Institutional Human Research Ethics Committee (2015000347).

98 Participation was voluntary and all individuals provided informed consent prior to
99 participation in the study.

100 **Bacterial strains and growth conditions**

101 The *E. coli* strain EC958 was isolated from the urine of a patient with community-
102 acquired UTI in the UK <sup>12</sup>. *E. coli* strain S101EC was from a global collection of *E.
103 *coli* ST131 <sup>1</sup>. The EC958Δ*lac* mutant has been described previously <sup>13</sup>. Bacterial
104 strains were routinely cultured on or in lysogeny broth (LB) medium supplemented
105 with appropriate antibiotics. Alternatively, bacterial strains were cultured in Mueller-
106 Hinton (MH) broth or pooled human urine, or on MacConkey lactose agar,
107 supplemented with appropriate antibiotics as required.

108 **Plasmid curing**

109 The IncF plasmids harbouring the *aac(6′)-Ib-cr* gene were cured from EC958 and
110 S101EC using the curing vector pMDP5-cureEC958 designed based on the pCURE2
111 plasmid <sup>14</sup>. The pMDP5-cureEC958 vector contained the FIA and FIIA replicons plus
112 the *ccdA*, *sok*, *pemI*, and *vagC* anti-toxin genes from pEC958 (synthesized by Epoch
113 Life Science). The curing procedure has been described previously <sup>14</sup>.

114 **Molecular methods**

115 Chromosomal DNA purification, PCR and DNA Sanger sequencing of PCR products
116 were performed as previously described <sup>15</sup>. The full list of primers used are outlined in
Table S1. Mutation of the *aac(6')-Ib-cr* gene was performed using the λ-Red recombinase method with some modifications as previously described ¹³, ¹⁶. The chromosomal mutations on *gyrA* (GyrA L(TTG)-83-S(TCG), N(AAC)-87-D(GAC)) and *parC* (ParC I(ATT)-80-S(TCC)) were generated using the pORTMAGE method ¹⁷. Genome sequencing of these mutants was performed using Illumina methodology (by Australian Centre for Ecogenomics) to confirm the mutations and to examine for off-target mutations (Table S2).

## Bacterial growth kinetics

Bacterial growth kinetics were measured as changes in optical density (OD₆₀₀) over time using the FLUOstar OPTIMA Microplate Reader (BMG Labtech). Briefly, overnight cultures were standardised to OD₆₀₀ = 0.05 in cation adjusted MH broth (BD, USA) or MH supplemented with ciprofloxacin at specified concentrations, and loaded in triplicate onto a flat bottom 96-well plate at 200 µl per well. The plate was then sealed using the Breathe-Easy sealing membrane (Diversified Biotech) and incubated at 37°C with shaking inside the microplate reader. Optical measurements were recorded every 15 minutes for 12 hours; all experiments were performed in triplicate.

## Mixed competition assays

Competition assays were performed using similar conditions as described for bacterial growth kinetics. A 50:50 mixture of wildtype (EC958Δ*lac* ) and mutant strains was used. Viable counts were determined at t = 0h and t = 18h by plating on MacConkey lactose agar, which allowed the differentiation of EC958Δ*lac* (non-lactose fermenter) and the mutant strains. Human urine, when used as culture media for competition
140 assays, was pooled from at least three healthy female donors with no recent history of
141 antibiotic use.

142 **Antimicrobial susceptibility testing**

143 Minimal inhibitory concentrations (MICs) were determined by broth microdilution
144 method ¹⁸. The interpretation of MIC was performed using EUCAST breakpoints
145 (http://www.eucast.org/clinical breakpoints/). All MIC assays were performed in
146 triplicate.

147 **Time-kill kinetics assays**

148 The four strains EC958 wild-type, EC958cured, EC958 *gyrA<sup>S</sup>parC<sup>S</sup>*, and EC958cured
149 *gyrA<sup>S</sup>parC<sup>S</sup>* were used to investigate the killing effects of ciprofloxacin at various
150 concentrations. Overnight cultures were standardised to ~10<sup>8</sup> CFU/ml in MH or MH
151 supplemented with ciprofloxacin at specified concentrations and loaded in triplicate
152 onto a flat bottom 96-well plate at 200 µl per well, sealed with the Breathe-Easy
153 membrane (Diversified Biotech) and incubated at 37°C with shaking. At each time
154 point, a 10 µl sample was taken for serial dilution and enumeration of viable counts.
155 All experiments were performed in triplicate.

156 **Ultra-high-performance liquid chromatographic-photodiode array**

157 Total concentrations of ciprofloxacin and N-acetylciprofloxacin in cation adjusted
158 Mueller Hinton broth were measured by an ultra-high performance liquid
159 chromatography-photo diode array (UHPLC-PDA) method on a Nexera2 liquid
160 chromatograph connected to an SPD-M30 photo diode array detector (Shimadzu,
161 Kyoto, Japan). The filtered sample (1 µL) was injected onto the UHPLC-PDA. The
162 stationary phase was Pinnacle DB IBD, 50 x 2.1 mm (1.9 µm) column (Restek,
163 Bellefonte, USA) preceded by a C18 UHPLC analytical guard column (Phenomenex,
Torrence, USA). The mobile Phase A was 30 mM sodium phosphate at pH3, and the Mobile Phase B was a mixture of 50% 30 mM sodium phosphate at pH3 and 50% acetonitrile (v/v). Separation of ciprofloxacin and N-acetylcyprofloxacin was effected by a gradient from 65% to 95% mobile phase B at a flow 0.3 mL/min, producing a backpressure of approximately 2600 psi. Peaks for the analytes were detected at 315 nm.

## 170 Bioinformatic analysis

A total of 3,391 *E. coli* ST131 genomes were downloaded from EnteroBase (25/05/2017). The genome assemblies of 100 randomly selected isolates from each of the 100 most common STs in Enterobase were downloaded on 18/12/2020. The phylogenetic tree was built from a core genome alignment generated by parsnp with recombination filtering <sup>19</sup>, using FastTree2 <sup>20</sup>. The clade structure of ST131 was inferred from the tree. The presence of *aac(6')-Ib-cr* and other resistance genes was detected by blastn (template sequences downloaded from the Comprehensive Antibiotic Resistance Database <sup>21</sup>, August 2020 release) using a stringent threshold of 100% identity and 100% coverage. The resistance mutations in *gyrA* and *parC* were detected by pointfinder <sup>22</sup>. The association of the *aac(6')-Ib-cr* gene and clade C2 was tested using Pearson's Chi-squared test followed by post-hoc pairwise Chi-squared tests (R v4.0).

## 184 Results

The plasmid-encoded *aac(6')-Ib-cr* gene is highly prevalent in clade C2 ST131
To examine the prevalence of the *aac(6')-Ib-cr* gene in the context of the ST131 phylogeny, we downloaded a large dataset of 3,391 ST131 genomes from EnteroBase and built a core SNP maximum likelihood tree that reproduced its established clade A-B-C1/C2 structure. We then used a blast search to detect the presence of *aac(6')-Ib-cr* in each genome. Among a total of 2,737 clade C strains, the *aac(6')-Ib-cr* gene was present in significantly more clade C2 strains (1,378/1,902; 72%) compared to clade C1 strains (31/835; 3.7%) (Figure 1A). Only 1% of clade A strains and 0.6% of clade B strains contained the *aac(6')-Ib-cr* gene. We also used the same dataset to probe the prevalence of the *bla*<sub>CTX-M-15</sub> gene, and revealed a very similar distribution to that observed for *aac(6')-Ib-cr*, with 1,582/1,902 (83%) clade C2 strains harbouring the *bla*<sub>CTX-M-15</sub> gene compared to 45/835 (5.4%) clade C1 strains (Figure 1B). Taken together, this demonstrates the *aac(6')-Ib-cr* gene is largely contained within clade C2 ST131, and for the majority of these strains the *aac(6')-Ib-cr* gene is likely to be co-localised on a plasmid with the *bla*<sub>CTX-M-15</sub> gene.

200

The *aac(6')-Ib-cr* gene imparts a growth advantage on ST131 in the presence of sub-MIC ciprofloxacin

We hypothesised that the *aac(6')-Ib-cr* gene confers a growth advantage in the presence of ciprofloxacin, and tested this by examining the growth kinetics of two clade C2 strains, EC958 and S101EC, as well as their plasmid-cured and *aac(6')-Ib-cr* deletion-derivative mutants. Both EC958 and S101EC displayed an identical growth profile in the presence of sub-MIC ciprofloxacin (128 µg/mL; Figure 2), consistent with their resistance phenotype (MIC ≥256 µg/mL). However, when the plasmid harbouring the *aac(6')-Ib-cr* gene was cured from both strains they exhibited reduced growth in the presence of 128 µg/mL ciprofloxacin (Figure 2). To confirm this growth
211 defect was specifically due to loss of *aac(6')-Ib-cr*, we deleted the gene in both strains
212 and re-examined their growth profile. Both EC958 and S101EC *aac(6')-Ib-cr*
213 deletion-derivative mutants displayed the same growth-impaired phenotype as the
214 plasmid-cured strains in the presence of 128 µg/mL ciprofloxacin (Figure 2).

215

216 To better understand the impact of *aac(6')-Ib-cr*, we performed mixed competition
217 assays using the EC958 *aac(6')-Ib-cr* deletion strain and wild-type EC958. The
218 competition was performed in both MH broth and human urine, with and without the
219 presence of 64 µg/mL ciprofloxacin (Figure 3). The *aac(6')-Ib-cr* deletion strain was
220 out-competed by wild-type EC958 in the presence of ciprofloxacin in both MH broth
221 and human urine. However, no selective advantage was observed in the absence of
222 ciprofloxacin. Together, these data demonstrate a selective advantage conferred by the
223 *aac(6')-Ib-cr* gene in the presence of ciprofloxacin in a strain already harbouring the
224 *gyrA* and *parC* FQ-resistance alleles.

225

226 Consistent with these results, both the plasmid-cured and *aac(6')-Ib-cr* deletion
227 mutants also exhibited a 4-fold reduction in MIC against ciprofloxacin (Table 1)
228 despite the presence of FQ resistance-defining chromosomal GyrA and ParC
229 modifications. AAC(6')-Ib-cr is a dual function enzyme with aminoglycoside
230 acetyltransferase activity, and its loss also caused a reduction in the MIC to amikacin
231 (4-fold) and kanamycin (32-fold) (Table 1).

232
233 Stepwise acquisition of *aac(6')-Ib-cr* and chromosomal GyrA/ParC

234 modifications shape resistance to ciprofloxacin

235 All clade C ST131 strains possess chromosomal GyrA (S83L, D87N) and ParC (S80I)

236 modifications, henceforth referred to as *gyrA*<sup>R</sup>*parC*<sup>R</sup>, that confer FQ resistance. To

237 better understand the combinatory effect of the *aac(6')-Ib-cr* gene and *gyrA*<sup>R</sup>*parC*<sup>R</sup>

238 chromosomal modifications on ciprofloxacin susceptibility, we generated a unique set

239 of genome-edited isogenic EC958 strains where the *gyrA*<sup>R</sup> and *parC*<sup>R</sup> alleles were

240 reverted to the ‘sensitive’ allele type. Three isogenic mutants were constructed,

241 namely *parC*<sup>S</sup> (EC958 ParC I80S), *gyrA*<sup>S</sup> (EC958 GyrA L83S, N87D), *gyrA*<sup>S</sup>*parC*<sup>S</sup>

242 (EC958 GyrA L83S, N87D, and ParC I80S), and confirmed by genome sequencing

243 (Table S2). These three mutants were also cured of the plasmid pEC958 (which

244 harbours the *aac(6')-Ib-cr* gene), yielding a set of eight isogenic strains containing

245 different combinations of chromosomal and plasmid-encoded FQ susceptibility

246 profiles. Analysis of the MIC against ciprofloxacin for all of these strains revealed the

247 precise contributions of modifications to GyrA and ParC to FQ susceptibility

248 (GyrA<sup>R</sup>ParC<sup>R</sup> >256 µg/ml; GyrA<sup>R</sup>ParC<sup>S</sup> = 8 µg/ml; GyrA<sup>S</sup>ParC<sup>R</sup> = 0.125 µg/ml;

249 GyrA<sup>S</sup>ParC<sup>S</sup> <0.125 µg/ml) (Figure 4). Notably, loss of plasmid pEC958, and hence

250 the *aac(6')-Ib-cr* gene, resulted in a ~4-fold reduction in MIC to ciprofloxacin in all

251 of the different FQ resistant/susceptible chromosome backgrounds.

252

253 Bacterial regrowth after ciprofloxacin treatment is mediated by *aac(6')-Ib-cr*

254 gene

255 To further characterise the role of the *aac(6')-Ib-cr* gene in response to ciprofloxacin

256 treatment, we performed time-kill kinetic assays using the EC958 *gyrA*<sup>R</sup>*parC*<sup>R</sup> and
257 EC958 *gyrA<sup>S</sup>parC<sup>S</sup>* strains in the presence and absence of *aac(6')-Ib-cr*. Survival of
258 the EC958 *gyrA<sup>R</sup>parC<sup>R</sup>* strain containing *aac(6')-Ib-cr* was only minimally impacted
259 in the presence of ciprofloxacin, and growth occurred even at a concentration of 1024
260 µg/mL (4x MIC). In contrast, the absence of *aac(6')-Ib-cr* in EC958 *gyrA<sup>R</sup>parC<sup>R</sup>* led
261 to a loss in viability after 24 hours in the presence of 256 µg/mL (4x MIC)
262 ciprofloxacin (Figure 5A and B). Time-kill assays for the EC958 *gyrA<sup>S</sup>parC<sup>S</sup>* strains ±
263 *aac(6')-Ib-cr* were performed at lower concentrations of ciprofloxacin, in line with
264 their increased FQ susceptibility. However, an identical pattern was observed,
265 whereby compared to EC958 *gyrA<sup>S</sup>parC<sup>S</sup>* harbouring *aac(6')-Ib-cr*, loss of the
266 *aac(6')-Ib-cr* gene in EC958 *gyrA<sup>S</sup>parC<sup>S</sup>* resulted in complete killing after 24 hours in
267 the presence of 0.25 µg/mL (4x MIC) ciprofloxacin (Figure 5C and D). Taken
268 together, these data demonstrate that the AAC(6')-Ib-cr enzyme enhances survival
269 and growth against ciprofloxacin administered at concentrations above its MIC.
270

271 **Deactivation of ciprofloxacin by the AAC(6')-Ib-cr enzyme enhances the**
272 **survival of FQ resistant and FQ susceptible strains**

273 The AAC(6')-Ib-cr enzyme converts ciprofloxacin to an inactive N-acetyl-
274 ciprofloxacin form, and thus we hypothesized it would reduce the local concentration
275 of ciprofloxacin during bacterial growth, leading to increased survival of both
276 resistant and sensitive cells. To test this, we grew EC958 and the EC958 *aac(6')-Ib-cr*
277 deletion mutant in MH broth containing 64 µg/mL ciprofloxacin (Figure 6A). At
278 periodic intervals, samples were collected from both cultures, filter-sterilised, and the
279 concentration of ciprofloxacin and N-acetyl-ciprofloxacin was measured directly by
280 ultra-high-performance liquid chromatographic-photodiode array (UHPLC-PDA).
281 These analyses demonstrated rapid inactivation of ciprofloxacin during growth, with
282 the concentrations of ciprofloxacin and inactive N-acetyl-ciprofloxacin displaying an
283 inverse relationship over time (Figure 6B). To measure the impact of ciprofloxacin
deactivation, we employed a disc diffusion assay using the ciprofloxacin sensitive E.
284 coli strain ATCC 25922 in combination with filter sterilized broth collected during the
285 growth assay. Strain ATCC 25922 was seeded onto a MH agar plate, a filter paper
286 disc loaded with filtered broth was placed on the agar, and the plates were incubated
287 overnight, with the size of the zone of inhibition used as an indicator of the amount of
288 active ciprofloxacin in the filtered broth. We observed that the zone of inhibition from
289 EC958 broth decreased from 35 mm at 0h (corresponding to 64 µg/mL ciprofloxacin)
290 to 12 mm at 8h. In contrast, the zone of inhibition from the EC958 *aac(6')-Ib-cr*
291 deletion mutant was the same at both time points. Thus, the AAC(6')-Ib-cr enzyme
292 rapidly deactivates ciprofloxacin, thereby generating a local environment that
293 enhances the survival of susceptible cells.
294

295

296 **Emergence of carbapenem resistant E. coli carrying both aac(6')-Ib-cr and**
297 **chromosomal FQ-resistance modifications**

298 Given the selective advantage of carrying both chromosomal FQ-resistance
299 modifications and the plasmid-associated *aac(6')-Ib-cr* gene, we hypothesized that *E.
*coli* isolates carrying this dual FQ-resistance combination would be more common
300 than isolates that carry *aac(6')-Ib-cr* alone. To investigate this, we randomly selected
301 100 isolates from the top 100 most common *E. coli* sequence types (STs) from
302 EnteroBase and calculated the percentage of isolates carrying both FQ-resistance
303 mechanisms compared to *aac(6')-Ib-cr* alone. Isolates carrying both FQ-resistance
304 mechanisms were identified in 22 STs, ranging from 1% to 58% (Figure 7A). In
305 contrast, isolates from 10 STs carried the *aac(6')-Ib-cr* gene alone, ranging from 1%
306
307 to 11%. These combinations were largely mutually exclusive, with carriage of the
308 dual FQ-resistance combination excluding carriage of the plasmid-encoded *aac(6')*-Ib-cr gene alone within an individual ST.

310
311 Five STs contained isolates that carried the FQ-resistance combination at levels approximately equal to ST131 (ST448 = 31% vs ST131 = 33%) or greater than ST131 (ST405 = 41%; ST648 = 42%; ST167 = 43%; ST410 = 46%; ST617 = 58%). Given the FQ-resistance combination in ST131 is strongly associated with the *bla*<sub>CTX-M-15</sub> gene, hence promoting additional resistance to 3<sup>rd</sup>-generation cephalosporins, we also examined the carriage of *bla*<sub>CTX-M</sub> ESBL genes and genes encoding resistance to last-line carbapenems in these five STs. Carriage of a *bla*<sub>CTX-M</sub> ESBL gene was identified in >50% of isolates from all STs except ST448, with ST617, ST410 and ST167 displaying high co-carriage rates of the *aac(6')-Ib-cr* and *bla*<sub>CTX-M</sub> genes as we observed in clade C2 ST131 (Figure 7B-F). In the case of carbapenem resistance genes, the *bla*<sub>NDM</sub> gene was most common. We identified at least one cluster in each ST that was enriched for the combined carriage of the FQ-resistance combination as well as at least one type of carbapenem resistance gene (Figure 7B-F). Interestingly, the possession of carbapenem resistance genes was less frequent in a similarly generated set of 100 randomly selected ST131 genomes (Figure S1). Taken together, this highlights the existence of multiple global *E. coli* clones armed with critical resistance combinations that threaten to cause increased rates of infection associated with fewer treatment options.
330 **Discussion**

331 FQ resistance in *E. coli* generally develops following a stepwise acquisition of
332 resistance mutations in which alterations in the primary target, the DNA gyrase
333 (*gyrA*), precede those in the secondary target, the topoisomerase IV (*parC*); mutations
334 in both DNA gyrase and topoisomerase IV confer a higher level of resistance<sup>24</sup>.

335 Plasmid-mediated FQ resistance mechanisms only confer a small decrease in
336 susceptibility and may prime the development of the first mutation in the DNA gyrase
337 <sup>25</sup>. All isolates from clade C of the *E. coli* ST131 clone harbour two mutations in
338 DNA gyrase (S83L and D87N) and one in topoisomerase IV (S80I) and therefore the
339 presence of the plasmid-encoded *acc(6')*-Ib-cr gene in these isolates attracts little
340 attention because its phenotype is dominated by the higher level of resistance
341 provided by the chromosomal *gyrA* and *parC* mutations. Here, we show that the
342 *acc(6')*-Ib-cr gene is found in the overwhelming majority of multidrug resistant
343 ST131 strains from clade C2, leading to a selective advantage in the presence of
344 ciprofloxacin at a much higher concentrations than previously associated with the
345 *acc(6')*-Ib-cr gene.

346

347 AAC(6')-Ib-cr is a dual function enzyme with activity against aminoglycosides and
348 the FQ antibiotics ciprofloxacin and norfloxacin<sup>7</sup>. Our data showed that loss of the
349 *aac(6')*-Ib-cr gene in ST131 clade C2 strains (EC958 and S101EC) caused a growth
350 defect in the presence of sub-MIC ciprofloxacin compared to the corresponding WT
351 strain, which translated into a 4-fold reduction in MIC. In contrast to a previous study
352 that started with the sensitive ATCC 25922 strain and introduced isogenic FQ
353 resistance mutations in *gyrA* and *parC*<sup>26</sup>, we assessed the dynamics of FQ
354 susceptibility by converting the *gyrA* and *parC* mutations in EC958, a clade C2
355 ST131 strain with clinically significant FQ resistance, into the sensitive allele type.
356 These allelic reversion mutants were generated using the pORTMAGE vector
357 exchange system, which uses transient suppression of the mismatch repair MutL
358 enzyme to introduce oligonucleotide-directed mutations on the chromosome ¹⁷. The
359 EC958 isogenic strains converted to the sensitive allele of *parC*⁵ (EC958 ParC I80S),
360 *gyrA*⁵ (EC958 GyrA L83S, N87D), and *gyrA*⁵*parC*⁵ (EC958 GyrA L83S, N87D, and
361 ParC I80S) all displayed a ~4-fold reduction in MIC against ciprofloxacin in the
362 absence of *aac(6')-Ib-cr* (compared to the corresponding parent strain containing
363 *aac(6')-Ib-cr*). Thus, we were able to show that the increased resistance conferred by
364 AAC(6')-Ib-cr is consistent across a very wide range of MICs (0.064 to >256 µg/mL),
365 including a clinically significant range that could influence therapeutic outcomes.
366

367 The presence of *aac(6')-Ib-cr* also led to a time-dependent reduction in ciprofloxacin-
368 mediated killing. Strains carrying the *gyrA/parC* sensitive allele type were susceptible
369 to the bactericidal effect of ciprofloxacin in the first 5 hours, with subsequent bacterial
370 regrowth driven by the presence of the *aac(6')-Ib-cr* gene. This phenomenon has also
371 been observed previously, but at lower ciprofloxacin concentrations and employing
372 strains engineered to possess ciprofloxacin resistance ²⁶, ²⁷. It is worth noting that
373 growth of the wild-type EC958 strain was only inhibited in the first 3h at a
374 ciprofloxacin concentration of 1056 µg/mL. This concentration is the maximum
375 human urine concentration achieved from a single treatment dose of 500mg
376 ciprofloxacin (ranging from 23 – 1000 µg/mL, with the median ranging from 237 –
377 407 µg/mL in the first 6h ²⁸⁻³⁰). The ability of EC958 to grow in high concentrations
378 of ciprofloxacin was also linked to the acetylation of ciprofloxacin by AAC(6')-Ib-cr.
379 During growth of EC958 in broth containing ciprofloxacin, AAC(6')-Ib-cr activity
caused the rapid conversion of active ciprofloxacin to inactive N-acetyl-ciprofloxacin, consistent with previous studies examining environmental *E. coli* isolates and other clinical *Enterobacteriaceae* isolates harbouring the *aac(6')-Ib-cr* gene<sup>31, 32</sup>. In addition to priming the development of clinically relevant resistance, AAC(6')-Ib-cr mediated reduction of local ciprofloxacin concentrations would lead to the enhanced survival of sensitive strains, thereby driving population-wide resistance. Indeed, this may contribute to the diversification of plasmid composition and resistance genes carried by ST131 that persist in the gut of patients that suffer recurrent UTI despite extensive antibiotic treatment<sup>33</sup>.

The strong association between *gyrA*<sup>R</sup>*parC*<sup>R</sup> chromosomal mutations and the plasmid-encoded *aac(6')-Ib-cr* gene in ST131 suggests this dual resistance combination may be a driver of its clonal expansion. Thus, we also explored the prevalence of this resistance combination in other common *E. coli* STs linked to increasing infection rates. Among the top 100 *E. coli* STs in Enterobase, 22 STs comprised strains harbouring both FQ resistance mechanisms, some at an incidence as high as 58%.

Closer examination of the five STs with the highest prevalence of the *gyrA*<sup>R</sup>*parC*<sup>R</sup> chromosomal mutations and the *aac(6')-Ib-cr* gene revealed a strong association with carbapenem resistance genes. Indeed, three STs strongly linked to carriage of the *bla*<sub>NDM</sub> gene, ST167, ST617 and ST410<sup>34</sup>, were among the top 5 STs in our analysis.

Despite increasing antibiotic stewardship, ciprofloxacin remains one of the most widely used antibiotics worldwide, with its consumption (including both human and animal) reported by the WHO as high as 9.8% of total defined daily doses in the Regions of the Americas, 7% in the European Region, and 22.9% in the Western
404 Pacific Region (WHO Report on Surveillance of Antibiotic consumption ³⁵),
405 emphasizing the importance of monitoring epidemiological co-resistance patterns.

406
407 In conclusion, we have shown that in *E. coli* ST131, the combination of *aac(6')-Ib-cr* and chromosomal mutations in *gyrA* and *parC* represents an important dual resistance mechanism that has contributed to its global dissemination. Although the precise timeline of *aac(6')-Ib-cr* acquisition remains to be determined, we hypothesize that the *aac(6')-Ib-cr* gene was acquired after the gain of FQ resistance via *gyrA* and *parC* mutations (i.e. this occurred after the emergence of clade C), and that the *aac(6')-Ib-cr* gene acts in concert with the chromosomal mutations to increase the selective advantage of clade C2 strains in the presence of ciprofloxacin. Furthermore, this combination has emerged in multiple other STs, including those that also harbour carbapenem resistance genes. As our dependence on last-line carbapenems grows, this presents the alarming scenario that multiple clones are poised to survive and spread, highlighting the need for ongoing surveillance of *aac(6')-Ib-cr* carriage and improved control of ciprofloxacin usage.

420

421

422

423 Acknowledgement

424
425 We thank Prof. Mark Achtman and the EnteroBase team for facilitating our data analysis using large numbers of genome assemblies from EnteroBase.

427
428 **Funding**

429

430 This work was supported by grants APP1181958 and APP2001431 from the National

431 Health and Medical Research Council of Australia (NHMRC). L.A.F is supported by

432 the Galician Ministry of Culture, Education and University Planning and Galician

433 Ministry of Economy, Employment and Industry (Regional Government of Galicia-

434 Spain) (IN606B-2018/011). The funders had no role in study design, data collection

435 and interpretation, or the decision to submit this work for publication.

436 **Author contributions**

437

438 MDP and KMP performed experiments with assistance from SCW, SH, NTKN, and

439 MJB. MDP and MAS designed the study. MDP, LAF, SCW, SH, NTKN, BF and

440 MAS analysed the data. DLP, SAB, JL and MAS supervised aspects of the project

441 and provided essential expert analysis. All authors contributed to the interpretation of

442 the results. MDP and MAS wrote the manuscript. All authors read and approved the

443 final manuscript.

444 **Transparency declarations**

445

446 None to declare.

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## Tables

<table><caption>Table 1: MIC of ST131 strains with and without the *aac(6')-Ib-cr* gene</caption><thead><tr><th></th><th>Ciprofloxacin</th><th>Amikacin</th><th>Kanamycin</th></tr></thead><tbody><tr><td>EC958</td><td>&gt;256</td><td>16</td><td>96</td></tr><tr><td>EC958cured</td><td>64</td><td>4</td><td>3</td></tr><tr><td>EC958Δaac(6')-Ib-cr</td><td>64</td><td>3</td><td>3</td></tr><tr><td>S101EC</td><td>256</td><td>16</td><td>128</td></tr><tr><td>S101ECcured</td><td>64</td><td>4</td><td>4</td></tr><tr><td>S101ECΔaac(6')-Ib-cr</td><td>64</td><td>3</td><td>3</td></tr></tbody></table>

564

Note: MIC for ciprofloxacin was performed using broth microdilution method;

amikacin and kanamycin were determined using the E-test.

567

568
570

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

571

572 **Figure 1.** Prevalence of the *aac(6')-Ib-cr* and *bla*<sub>CTX-M-15</sub> genes in ST131. **A** (i)

573 Barplot showing the prevalence of *aac(6')-Ib-cr* in each ST131 based on clade designation, and (ii) core SNP ML tree of 3,391 ST131 genomes with the presence of the *aac(6')-Ib-cr* gene indicated by pink dots. **B** (i) Barplot showing the prevalence of *bla*<sub>CTX-M-15</sub> in each ST131 clade and (ii) core SNP ML tree of 3,391 ST131 genomes with the presence of the *bla*<sub>CTX-M-15</sub> gene indicated by blue dots. Statistical analyses based on Pearson's Chi-squared test; *P* < 0.001 for pairs of C2-A, C2-B, C2-C1 and C2-Other).

580
<figure><img src="image_5.png" /><figcaption>Figure 2. Growth kinetics of wild-type ST131 strains and their *aac(6′)-Ib-cr* deletion derivatives in sub-MIC of ciprofloxacin. (A) Growth curves comparing growth of EC958 (WT), EC958cured (cured), and EC958Δ*aac(6′)-Ib-cr* (Δ*aac*). (B) Growth curves comparing growth of S101EC (WT), S101ECcured (cured) and S101ECΔ*aac(6′)-Ib-cr* (Δ*aac*). All experiments were performed in triplicate in MH supplemented with 128 µg/mL ciprofloxacin at 37°C. The grey shades indicate SE range.</figcaption></figure>
<figure><img src="image_6.png" /></figure>

592

593 Figure 3. Competition assays showing the selective advantage of EC958 compared to EC958Δ*aac(6′)-Ib-cr* in human urine and MH broth in the absence and presence of sub-MIC of ciprofloxacin (64 µg/mL). The data represents biological replicates with individual data points shown; the group median is indicated by a horizontal line.

595
596
597 Statistical analyses were performed using the Wilcoxon signed rank test comparing
598
each group against theoretical median of 0. * Indicates P-value < 0.05.
<figure><img src="image_7.png" /><figcaption>Figure 4. Comparative effects of chromosomal mutations and the *aac(6′)-Ib-cr* gene EC958 susceptibility to ciprofloxacin. The four chromosomal backgrounds were WT (EC958), *parC*<sup>S</sup> (EC958 ParC I80S), *gyrA*<sup>S</sup> (EC958 GyrA L83S, N87D), *gyrA*<sup>S</sup>*parC*<sup>S</sup> (EC958 GyrA L83S, N87D, and ParC I80S) as shown in the lower panel. For each background, a strain with and a strain without plasmid pEC958 was tested. MIC determinations were performed using the broth microdilution method in triplicate. The data are presented as the mid-point and range of the three MIC values. The EUCAST breakpoint of 0.5 µg/mL is shown as a red dashed line.</figcaption></figure>
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

Figure 5. Time-kill kinetic assays showing regrowth during ciprofloxacin treatment in the presence and absence of the *aac(6′)-Ib-cr* gene. The assays were performed on (A) EC958 wild-type, (B) EC958cured, (C) EC958 *gyrA<sup>S</sup>parC<sup>S</sup>*, and (D) EC958cured *gyrA<sup>S</sup>parC<sup>S</sup>*. The line type and symbols denote CFU counts following exposure to different ciprofloxacin concentrations, with concentrations relative to MIC shown in brackets as fold difference (i.e., 1x, 2x and 4x). All experiments were performed in triplicate and the data are shown as mean ± SE.
625

<figure><img src="image_12.png" /></figure>

626

Figure 6. Kinetics of ciprofloxacin conversion to N-acetyl-ciprofloxacin by AAC(6')-Ib-cr during growth. (A) Growth kinetics of EC958 and EC958aac (EC958Δ*aac(6')*-Ib-cr) in MH broth supplemented with 64µg/mL ciprofloxacin. (B) Measurements of ciprofloxacin or N-acetyl-ciprofloxacin concentrations performed by ultra-high-performance liquid chromatographic-photodiode array, demonstrating a decrease of ciprofloxacin and increase of N-acetyl-ciprofloxacin in EC958 broth cultures compared to EC958acc broth cultures, where the concentration of ciprofloxacin was unchanged. (C) Zones of inhibition from disc diffusion assays using 5µl of filter-sterilised broth from each time point in (A) loaded onto filter paper and placed on MH agar seeded with the ciprofloxacin susceptible *E. coli* ATCC 25922 strain. In each case, the zone of inhibition is proportional to the amount of active ciprofloxacin in the respective broth culture.

639
<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

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

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

640
641

<caption>Figure 7. ST-based phylogenetic clustering of strains carrying *gyrA*^R^*parC*^R, *aac*(6')-*Ib*-cr gene and carbapenem resistance gene combinations. (A) Barplot demonstrating the strong association between the *aac*(6')-*Ib*-cr gene and chromosomal *gyrA*^R^*parC*^R modifications compared to carriage of *aac*(6')-*Ib*-cr gene alone in different *E. coli* STs. (B) to (F) Phylogenetic trees of each ST built from 100 randomly selected genomes using *parsnps*. The tips with red dots indicate genomes that possess the</caption>
gyrA<sup>R</sup> parC<sup>R</sup> FQ resistance modifications, the *aac(6')-Ib-cr* gene and any of the indicated carbapenem resistance genes. The clustering of genomes that carry this resistance combination is indicated by pink shading. The side columns depict the absence (grey) or presence (blue) of *gyrA<sup>R</sup> parC<sup>R</sup>* modifications (GyrA S83L D87N and ParC S80I), the *aac(6')-Ib-cr* gene, *bla*<sub>CTX-M</sub> genes (CTX-M), *bla*<sub>KPC</sub> genes (KPC), *bla*<sub>VIM</sub> genes (VIM), *bla*<sub>IMP</sub> genes (IMP), *bla*<sub>NDM</sub> genes (NDM), *bla*<sub>OXA</sub> genes (OXA, including *bla*<sub>OXA-48</sub> and *bla*<sub>OXA-181</sub>).

655
<figure><img src="image_19.png" /></figure>

aac(6')-Ib-cr

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

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

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

bla<sub>CTX-M-15</sub>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>
<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>
<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>
<figure><img src="image_31.png" /></figure>

<table><tr><td rowspan="2">GyrA</td><td>83</td><td>L</td><td>L</td><td>S</td><td>S</td></tr><tr><td>87</td><td>N</td><td>N</td><td>D</td><td>D</td></tr><tr><td>ParC</td><td>80</td><td>I</td><td>S</td><td>I</td><td>S</td></tr></table>
A

<figure><img src="image_32.png" /></figure>

C

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

B

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

D

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

C

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

D

<figure><img src="image_37.png" /></figure>

B

<figure><img src="image_38.png" /></figure>

D

<figure><img src="image_39.png" /></figure>

C

<figure><img src="image_40.png" /></figure>

D

<figure><img src="image_41.png" /></figure>

B

<figure><img src="image_42.png" /></figure>

D

<figure><img src="image_43.png" /></figure>
<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /></figure>
<figure><img src="image_47.png" /></figure>