Plasmid-mediated metronidazole resistance in Clostridioides difficile

Metronidazole was until recently used as a first-line treatment for potentially life-threatening Clostridioides difficile (CD) infection. Although cases of metronidazole resistance have been documented, no clear mechanism for metronidazole resistance or a role for plasmids in antimicrobial resistance has been described for CD. Here, we report genome sequences of seven susceptible and sixteen resistant CD isolates from human and animal sources, including isolates from a patient with recurrent CD infection by a PCR ribotype (RT) 020 strain, which developed resistance to metronidazole over the course of treatment (minimal inhibitory concentration [MIC] = 8 mg L−1). Metronidazole resistance correlates with the presence of a 7-kb plasmid, pCD-METRO. pCD-METRO is present in toxigenic and non-toxigenic resistant (n = 23), but not susceptible (n = 563), isolates from multiple countries. Introduction of a pCD-METRO-derived vector into a susceptible strain increases the MIC 25-fold. Our finding of plasmid-mediated resistance can impact diagnostics and treatment of CD infections.


Introduction 72
Clostridioides difficile (Clostridium difficile) is a gram-positive obligate anaerobe capable of causing 73 Clostridium difficile Infection (CDI) upon disruption of the normal intestinal flora. 1 Although it is one 74 of the major causes of nosocomial infectious diarrhea, community-acquired CDI is becoming more 75 frequent. 2,3 CDI infection poses a significant economic burden with an estimated cost at €3 billion 76 per year in the European Union and impairs the quality of life in infected individuals. 4,5 The incidence 77 of CDI has increased over the last two decades with outbreaks caused by epidemic types such as PCR 78 ribotype (RT) 027 (NAP1/BI). 6 CDI is not restricted to this type, however, as infections caused by 79 RT001, RT002, RT014/020 and RT078 are frequently reported in both Europe and the United 80 States. 7,8 Metronidazole is frequently used for the treatment of mild-to-moderate infections and 81 vancomycin for severe infections, though vancomycin is increasingly indicated as a general first-line 82 treatment 9,10 Fidaxomicin has recently also been approved for CDI treatment, but its use is limited 83 by high costs. 11 Fecal Microbiota Transplantation (FMT) is effective at treating recurrent CDI (rCDI) 84 that is refractory to antimicrobial therapy. 12 Reduced susceptibility and resistance to clinically used 85 antimicrobials, including metronidazole, has been reported and this, combined with the intrinsic 86 multiple drug-resistant nature of C. difficile, stresses the importance for the development of new 87 effective treatment modalities. 8 88 Routine antimicrobial susceptibility testing is generally not performed for C. difficile and 89 consequently, reports of resistance to metronidazole are rare. [13][14][15] Longitudinal surveillance in 90 Europe found that 0•2% of clinical isolates investigated were resistant to metronidazole, 14 but 91 reported rates from other studies vary from 0-18•3%. [16][17][18][19] These differences may reflect geographic 92 distributions in resistant strains, or differences in testing methodology and breakpoints used. 20,21 93 Moreover, metronidazole resistance can be unstable, inducible and heterogeneous. 22 Finally, 94 metronidazole resistance appears to be more frequent in non-toxigenic strains such as those 95 belonging to RT010, which have a 7-9 fold increase in Minimal Inhibitory Concentration (MIC) values 96 compared to RT001, RT027 and RT078. 16,21 Metronidazole is a 5-nitroimidazole drug that upon intracellular reductive activation induces 98 cellular damage through nitrosoradicals. 22 Mechanisms associated with metronidazole resistance 99 described in other organisms include the presence of 5-nitroimidazole reductases (nim genes), 100 altered pyruvate-ferredoxin oxidoreductase (PFOR) activity and adaptations to (oxidative) stress. 22 101 The knowledge on resistance mechanisms in C. difficile is very limited, but may involve modulation 102 of core metabolic and stress pathways as well. 23 103 Here, we present a case of a patient with rCDI due to an initially metronidazole susceptible 104 (MTZ S ) RT020 strain which developed resistance to metronidazole over time. We analyzed the 105 genome sequences of these toxigenic MTZ S and metronidazole resistant (MTZ R ) strains, together 106 with 4 MTZ S and 8 MTZ R non-toxigenic RT010 strains. We identified pCD-METRO, a 7-kb plasmid 107 conferring metronidazole resistance. This plasmid is internationally disseminated and also occurs in 108 epidemic types. This is the first report of a clinically relevant phenotype associated with plasmid-109 carriage in C. difficile. 110

Methods 111
Strains 112 The 18 strains sequenced as part of this study were isolated from a single patient at the Leiden 113 University Medical Center (LUMC) or derived from the collection of the Dutch National Reference 114 Laboratory (NRL) for C. difficile, which is hosted at the LUMC. Informed consent was given for the 115 use of the patient samples for research purposes. Other clinical isolates (n=567)  Real time quantitative PCR (qPCR) experiments were performed essentially as described. 34 In short, 162 total DNA was isolated after 17h of growth using a phenol-chloroform extraction protocol and 163 diluted to a concentration of 10 ng/µl. 4 µl of the diluted DNA sample was added to 6 µl of a mixture 164 containing SYBR Green Supermix (Bio-Rad) and gene-specific primers (0•4 µM total) for a total 165 volume of 10 µl per well. Gene specific primers used were targeting rpoB (chromosome) and catR 166 (plasmid) and copy number was calculated using the ΔCT method. Statistical significance was 167 calculated using two-way analysis of variance (ANOVA) and Tukey's test for multiple comparisons 168 (Prism 8,GraphPad). 169 170

Role of the funding source 171
The funders had no role in the study design, data collection, data analysis, data interpretation, or 172 writing of the report. The corresponding author had full access to all the data in the study and had 173 final responsibility for the decision to submit for publication. 174 175

In-patient development of a metronidazole resistant toxigenic strain 178
A 54 year old kidney-pancreas transplant patient with a medical history of Type I diabetes mellitus, 179 vascular disease and a double lower leg amputation was on hemodialysis when developing diarrhea. 180 The patient was subsequently diagnosed with CDI and a toxigenic metronidazole sensitive (MIC=0•25 181 mg/L) RT020 strain was isolated from the fecal material of the patient. Treatment with 182 metronidazole was started, leading to initial resolution of the symptoms (figure 1). Two more 183 episodes of CDI occurred during which the patient was treated primarily with vancomycin prior to an 184 FMT provided by the Netherlands Donor Faeces Bank. At the start of the second episode a MTZ S 185 RT020 strain was once more isolated. 186 Three months after the first FMT, the patient once again developed bloody diarrhea and two 187 more episodes of rCDI were diagnosed which were treated with a vancomycin and a fidaxomicin 188 regime. At two instances, RT020 strains were again isolated from the fecal material of the patient. 189 Strikingly, these two clinical isolates were now phenotypically resistant to metronidazole (MIC=8 190 mg/L as determined by agar dilution). Ultimately the patient was cured by a second FMT. 191 We hypothesized that the rCDI episodes were due to clonal RT020 strains that persisted 192 despite antimicrobial therapy and a FMT. Clonal MTZ S and MTZ R strains would allow us to determine 193 the underlying genetic changes that resulted in metronidazole resistance. To determine the 194 relatedness between these RT020 isolates whole genome sequencing (WGS) was performed (table  195 1). We also included a non-related RT078 strain isolated from the same patient and 4 MTZ S and 8 196 MTZ R RT010 strains from our laboratory collection (supplemental table 1 Assembly of the MTZ R RT020 strain IB136 (see appendix) resulted in a genome of 4166362 202 bp with 57 contigs, and an average G+C-content of 28•5% (N50= 263391 bp, mapping rate 98•97%). A 203 BLAST comparison between this genome and the NCBI nt database showed that the genome is 204 closest to the genome of strain LEM1. 35 As expected, 5/6 strains isolated from the patient (all RT020) 205 showed 100% identity over the majority of all contigs, suggesting they are highly similar. The sixth 206 strain, IB137 (RT078), was a clear outlier and was identified as being closest to strain M120, the 207 RT078 reference strain, consistent with the different ribotype assignment. 36 208 209

Metronidazole resistance does not correlate with a SNP across multiple isolates 210
Previous studies analysing the mechanism behind metronidazole resistance in C. difficile only studied 211 one single isolate. 23,37 We performed a core genome SNP analysis on all WGS obtained for this study 212 (n=18; 6 MTZ S , 12 MTZ R ; see table 1), comparing MTZ S and MTZ R strains within and between the 213 different PCR ribotypes (RT010, RT020 and RT078). 214 The evolutionary rate of C. difficile has been estimated at 0-2 SNPs/genome/year but might 215 vary based on intrinsic (strain type) and extrinsic (selective pressure) factors. 38 Our analysis 216 identified a single SNP in MTZ R RT020, compared to the MTZ S RT020 strains derived from the same 217 patient, conclusively demonstrating that these strains are clonal. This implies the MTZ S RT020 strain 218 acquired metronidazole resistance. In contrast, between the MTZ S -and MTZ R RT010 isolates (which 219 come from diverse human and animal sources) 457 SNPs were detected. Moreover, RT010 and 220 RT020 were separated by >25.000 SNPs. 221 The SNP identified in the RT020 strains discriminating the MTZ S from the MTZ R isolates is 222 located in a conserved putative cobalt transporter (CbiN, IPR003705). However, the SNP is not 223 observed in the MTZ R RT010 strains. Thus, metronidazole resistance is either multifactorial or not 224 contained within the core genome. We did not investigate the contribution of this SNP to 225 metronidazole resistance further. 226 227 MTZ R C. difficile strains contain a 7-kb plasmid 228 Next, we investigated extrachromosomal elements (ECEs), which can include plasmids. Although 229 plasmids containing antimicrobial resistance determinants have been described in gram-positive 230 organisms, they appear to be more common in gram-negatives. 39 Plasmids in C. difficile are known 231 to exist, but no phenotypic consequences of plasmid carriage have been described to date. 40 The 232 investigation of the pan-genome of all sequenced strains, including a prediction of ECEs predicted by 233 an in-house pipeline similar to PLACNET (appendix), 41 showed a single contig that was present in all 234 MTZ R strains (4•6% -19•27% of reads mapped, with a minimum of 479.497), but absent from MTZ S 235 strains, of both RT010 and RT020 (0% of reads mapped with a maximum 327 reads). Circularization 236 based on terminal repeats yielded a putative plasmid of 7056bp with a G+C-content of 41•6% (figure 237 2a). Correct assembly was confirmed by PCR (figure 2b) and Sanger sequencing (data not shown). 238 To confirm the circular nature of the contig, total DNA isolated from the MTZ R RT010 strain 239 IB138 before and after PlasmidSafe DNase (PSD, Epicentre) 40 treatment was analyzed by PCR using 240 primers specific for chromosomal DNA (gluD) and the putative plasmid (figure 2c). A positive signal 241 for gluD was only observed in samples that had not been treated with PSD, demonstrating that PSD 242 treatment degrades chromosomal DNA to below the detection limit of the PCR. By contrast, a signal 243 specific for the putative plasmid was visible both before and after PSD treatment. Consequently, we 244 conclude that our whole genome sequence identified a legitimate 7-kb plasmid. ORF6 is a small ORF that is likely a pseudogene, and the remaining ORFs encode a 250 metallohydrolase/oxidoreductase protein (ORF7; IPR001279) and a Tn5-like transposase gene (ORF8; 251 PF13701). Intriguingly, ORF6 showed homology on the protein level to the 5-nitroimidazole 252 reductase (nim) gene nimB (33% identity, 54% positives over 61 amino acids) described in 253 Bacteroides fragilis (CAA50578.1) and found in both metronidazole resistant-and susceptible 254 isolates of anaerobic gram-positive cocci. 42,43 The ORF lacks the region encoding the N-terminal part 255 of the Nim protein, and the Phyre2-predicted protein structure shows it lacks the catalytic site 256 residues (data not shown). Of note, the plasmid sequences from all strains are highly similar. 257 Compared to the plasmid of strain IB136, only strains IB143, IB144 and IB145 contained a single SNP 258 resulting in a Y314S mutation within the Tn5-like transposase ORF. 259 Together, these results show that all of the MTZ R strains, but none of the MTZ S strains, 260 sequenced in this study contain a plasmid hereafter referred to as pCD-METRO (for plasmid from C.

pCD-METRO is found in metronidazole resistant strains from different countries 264
Very few clinical isolates with stable metronidazole resistance have been described and we 265 evaluated the presence of pCD-METRO in the assembled genome sequences from these strains using 266 BLAST. 23,37 We failed to identify pCD-METRO in the draft genome of a toxigenic NAP1 isolate that 267 acquired stable metronidazole resistance through serial passaging under selection. 37 We did identify 268 pCD-METRO (fragmented over multiple contigs) in the draft genome a non-toxigenic Spanish RT010 269 strain with stable metronidazole resistance (strain 7032989), whereas neither the reduced-270 susceptible strain nor the susceptible strain from the same study contained the plasmid. 23 We 271 confirmed these results using PCR, as described for strain IB138 (figure 3A; lanes SP), demonstrating 272 pCD-METRO is indeed present in strain 7032989. These data show that the presence of pCD-METRO 273 may explain at least part of the cases of metronidazole resistance described in literature. We did not 274 detect pCD-METRO in the sequence read archive in entries labelled as C. difficile, or otherwise. 275 Our observations above raise the question how prevalent pCD-METRO is in MTZ R C. difficile 276 isolates and if there is a bias towards specific types or geographic origins. As metronidazole 277 resistance in C. difficile is rare, we expanded our collection of clinical isolates through our network 278 (including the ECDC) and with selected strains from the Tolevamer and MODIFY clinical trials. [24][25][26] To 279 correct for interlaboratory differences in typing and antimicrobial susceptibility testing, all strains 280 were retyped by capillary ribotyping and tested for metronidazole resistance using agar dilution 281 according to CLSI guidelines in our laboratory with inclusion of appropriate control strains. 28,29 282 Although these strains, with the exception of the Tolevamer strains, were characterized as having 283 altered metronidazole susceptibility by the senders (n=122), agar dilution performed in our own 284 laboratory classified nearly all of these strains as metronidazole susceptible (MIC <2 mg/L). We 285 expected pCD-METRO to be present in MTZ R strains, but not in MTZ S strains. 286 We identified three additional metronidazole resistant strains: a RT027 isolate from Poland 287 Taken together, our results shows that pCD-METRO is internationally disseminated and can 298 explain metronidazole resistance in both non-toxigenic-and toxigenic isolates of C. difficile, including 299 those belonging to epidemic ribotypes such as RT027. 300 301

pCD-METRO is acquired through horizontal gene transfer 302
Our whole genome sequence analysis revealed the acquisition of pCD-METRO by a toxigenic RT020 303 strain during treatment of rCDI. We made use of longitudinal fecal samples that were stored during 304 treatment to investigate the presence of pCD-METRO in total fecal DNA at various timepoints. Total 305 DNA derived from the fecal sample harboring the MTZ S RT020 was positive for the presence of pCD-306

METRO (figure 4). This indicates that pCD-METRO was present in the gut reservoir of the patient. 307
Post-FMT, pCD-METRO was no longer detected in total fecal DNA, suggesting that the fecal 308 transplant reduced levels of pCD-METRO containing C. difficile and/or the donor organism to below 309 the limit of detection of the assay. Fecal samples were stored in the absence of cryoprotectant and 310 as a result we were unable to reculture the possible donor organism. 311 Though we cannot exclude the possibility that the MTZ R RT020 strain was already present at 312 the moment the MTZ S RT020 strain was isolated, our results indicate that pCD-METRO was most 313 likely acquired through horizontal gene transfer between the MTZ S C. difficile strain and an as-of-yet 314 uncharacterized donor organism in the gut of the patient. to 38 (pCD-METRO shuttle , in IB125) (figure 6). In line with these results, a strain harboring a catP-349 containing plasmid with the pCD-METRO replicon demonstrates a growth advantage over a strain 350 harboring a similar plasmid with the pCD6 replicon when exposed to high levels (256 μg/mL) of 351 thiamphenicol (supplemental figure 2, appendix). As pIB90 containing strains are not MTZ R (figure 5), 352 resistance to metronidazole is not mediated by a higher copy number plasmid per se, but is 353 dependent on a determinant specific to pCD- METRO. 354 A difference between the read-depth estimate and the qPCR can be explained by technical 355 bias or differences in strain background. Nevertheless, our experiments clearly demonstrate that the 356 pCD-METRO replicon sustains plasmid levels that are approximately 10-fold greater than that of 357 currently used replicons. 358 Together, these results demonstrate that pCD-METRO encodes a functional replicon that is 359 responsible for a high copy number in C. difficile. 360 361 Discussion 362 In this study we describe the first plasmid linked to clinically relevant antimicrobial resistance in C. 363 difficile. We show that the high-copy number plasmid pCD-METRO is internationally disseminated, 364 present in diverse PCR ribotypes -including those known to cause outbreaks -and we provide 365 evidence for the horizontal transmission of the plasmid. 366 Though the presence of plasmids in C. difficile has been known for many years, no 367 phenotypes associated with plasmid carriage have been described. 40,45 We show that introduction of 368 pCD-METRO in susceptible strains leads to stable metronidazole resistance. Plasmids may play a 369 broader role in antimicrobial resistance of C. difficile. A putative plasmid containing the 370 aminoglycoside/linezolid resistance gene cfrC was recently identified in silico, but in contrast to our 371 work no experiments were presented to verify the contig was in fact a plasmid conferring 372 resistance. 46 The presence of an antimicrobial resistance gene does not always result in resistance, 373 and DNA-based identification of putative resistance genes without phenotypic confirmation may 374 lead to an overestimation of the resistance frequencies. 14,47,48 375 At present, it is unknown which gene(s) on pCD-METRO are responsible for metronidazole 376 resistance. Nitroimidazole reductase (nim) genes have been implicated in resistance to 377 nitroimidazole type antibiotics. 22 Though the presence of a truncated nim gene on pCD-METRO is 378 intriguing, we do not believe this gene to be responsible for the phenotype for several reasons. 379 Structural modelling of the predicted protein shows that it lacks the catalytic domain, and 380 introduction of the ORF under the control of an inducible promoter did not confer resistance in our 381 laboratory strain (data not shown). Moreover, the RT027 strain R20291 encodes a putative 5-382 nitroimidazole reductase (R20291_1308) and is not resistant to metronidazole, implying the 383 presence of a nim gene is not causally related to metronidazole resistance in C. difficile. Further 384 research is necessary to determine the mechanism for metronidazole resistance in C. difficile 385 conferred by pCD-METRO, and to investigate the contribution of the high copy number (figure 6) to 386 the resistance phenotype. 387 Our work, combined with that of others, suggests that metronidazole resistance is 388 multifactorial and other factors than pCD-METRO can cause or contribute to metronidazole 389 resistance in C. difficile. For instance, pCD-METRO may not explain low level resistance, 390 heterogeneous resistance, or stable resistance resulting from serial passaging of isolated strains 391 under metronidazole selection. 23,37,49 We also observed that MIC values in agar dilution experiments 392 differed between MTZ R isolates of different PCR ribotypes despite carriage of pCD-METRO, 393 suggesting a contribution of chromosomal or other extrachromosomal loci to absolute resistance 394 levels. Though the SNP we identified in the MTZ R RT020 strain was not found in the MTZ R RT010, we 395 cannot exclude that it contributes to the resistance in the patient strain. Notably, all natural isolates 396 of C. difficile with a MIC ≥8mg/L tested positive for pCD-METRO, whereas a plasmid-negative MTZ R 397 strain showed MICs below these levels (MIC=4 mg/L). 398 The pCD-METRO plasmid appears to be internationally disseminated (table 1), although 399 further research is necessary to determine how prevalent the plasmid is in metronidazole resistant 400 C. difficile isolates. This study attempted to enrich for metronidazole resistant strains as this 401 resistance is scarce in C. difficile. We received strains which were reported to be metronidazole 402 resistant by the senders. However, when performing antimicrobial susceptibility testing for these 403 strains with agar dilution in our own laboratory, virtually all strains had MIC values below the 404 epidemiological cut-off value from EUCAST for metronidazole and were considered susceptible. It is 405 not entirely clear how these differences came into existence. Depending on handling of the sample 406 material and freeze-thawing cycles, it is possible that inducible metronidazole resistance, unrelated 407 to pCD-METRO, was initially measured and that this was lost after storage and lack of selection. For 408 this reason we ended up having very few metronidazole resistant isolates of other PCR ribotypes 409 than RT010 (RT020 and RT027). 410 The pCD-METRO plasmid appears to be transmissible (figures 1, 2 and 4). Horizontal gene 411 transfer is consistent with the observed high level of sequence conservation between the RT010 and 412 RT020 pCD-METRO plasmids sequenced in this study. Nevertheless, we failed to demonstrate 413 intraspecies transfer with different donor and recipient strains of C. difficile under laboratory 414 conditions (appendix), suggesting that the strains tested (or possible the species) lack a determinant 415 required for transfer. Together with its size and the presence of mobilization genes (figure 2A), we 416 therefore hypothesize that pCD-METRO is mobilizable from an uncharacterized donor organism. 50 417 We screened the complete sequence read archive of the NCBI (paired-end Illumina data) for 418 potential sources of the plasmid, but failed to identify any entries with reliable mapping (>1% of 419 data) to pCD-METRO (data not shown). 420 As more reports are published associating metronidazole with higher disease recurrence and 421 treatment failure, a shift in consensus for using metronidazole as first line treatment for mild to 422 moderate CDI is occurring. 51 The reason for treatment failure is currently unknown, but no 423 correlation between MTZ R C. difficile isolates and treatment failure seems to exist. 47 We also 424 observed that clinical isolates from subjects in which metronidazole treatment failed, were 425 metronidazole susceptible and pCD-METRO negative (supplemental table 1   RT012 laboratory strain. IB30: 630Δerm + pIB20 (contains pCD6 replicon); IB90: 630Δerm + pIB80 505 (contains pCD-METRO replicon); IB125: 630Δerm + pCD-METRO shuttle (pIB86, contains pCD-METRO 506 replicon). *** p<0,001, **** p<0,0001. Copy number is determined as the ratio of a plasmid locus 507 (catP) relative to a chromosomal locus (rpoB) as determined by qPCR on total DNA. Data from strains 508 containing a plasmid with the pCD6-replicon are indicated in blue, data from strains containing a 509 plasmid with the pCD-METRO replicon are indicated in red. Experiments were performed in triplicate 510 on three different technical replicates. 511