Surveillance of antimicrobial susceptibilities reveals high proportions of multidrug resistance in toxigenic Clostridium difficile strains in different areas of Poland

Journal Pre-proof Surveillance of antimicrobial susceptibilities reveals high proportions of multidrug resistance in toxigenic Clostridium difficile strains in different areas of Poland

D. Lachowicz, H. Pituch, D. Wultańska, E. Kuijper, P. Obuch-Woszczatyński PII:

S1075-9964(20)30023-8

DOI:

https://doi.org/10.1016/j.anaerobe.2020.102167

Reference:

YANAE 102167

To appear in:

Anaerobe

Received Date:

26 September 2019

Accepted Date:

25 January 2020

Please cite this article as: D. Lachowicz, H. Pituch, D. Wultańska, E. Kuijper, P. ObuchWoszczatyński, Surveillance of antimicrobial susceptibilities reveals high proportions of multidrug resistance in toxigenic Clostridium difficile strains in different areas of Poland, Anaerobe (2020), https://doi.org/10.1016/j.anaerobe.2020.102167

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Journal Pre-proof Surveillance of antimicrobial susceptibilities reveals high proportions of multidrug resistance in toxigenic Clostridium difficile strains in different areas of Poland D. Lachowicz1,2, H. Pituch1, D. Wultańska1, E. Kuijper3, P. Obuch-Woszczatyński1 1. Department of Medical Microbiology, Medical University of Warsaw, Poland 2. Department of Medical Microbiology, The Infant Jesus Teaching Hospital, Warsaw, Poland. 3. Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, Netherlands

Corresponding author Hanna Pituch Department of Medical Microbiology Medical University of Warsaw Address 5 Chalubinski Street 02-004 Warsaw, Poland ([email protected])

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Abstract Two hundred and fifty-three non-duplicate toxigenic Clostridium difficile isolates, collected from February 2012 to December 2014, were evaluated for phenotypic resistance to ten antimicrobial drugs with the E-test gradient diffusion method. All strains of C. difficile were susceptible to metronidazole, vancomycin, and tigecycline. The metronidazole MIC values of the hyperepidemic PCR-ribotypes RT027 and RT176 were higher than those of non-epidemic PCR-ribotypes (p<0.05, as evidenced by Mann-Whitney U test). In contrast, vancomycin susceptibility did not differ between hyperepidemic and non-epidemic strains, although the difference was almost significant (p=0.065). Clostridium difficile RT027 and RT176 isolates could be assessed to five and four different susceptibility patterns, respectively, representing various combinations of resistance to different antimicrobial classes. A single point mutation (Thr82Ile) in the gyrA gene was detected in 11 (78.6%) of 14 isolates with high level of resistance to ciprofloxacin and moxifloxacin and four different types of single point mutations (Arg447Lys, Ser416Ala, Asp426Val, Asp426Asn) in the gyrB gene were detected in 4 strains, also with high level of resistance to ciprofloxacin and moxifloxacin. Four different point mutations were detected in the rpoB gene in 21 rifampicin-resistant strains of which one has not been reported previously, Gln489Leu. This study demonstrates the presence of multidrugresistant C. difficile strains in Polish hospitals over the study period, irrespective of geographical location or reference level of the hospital.

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Keywords: Clostridium difficile, epidemiology, Poland, multi-center study, PCR-ribotypes, multidrug resistance

Introduction

Clostridium difficile infection (CDI) is one of the most common healthcare-associated infections worldwide [1, 2, 3].

The main virulence factors of Clostridium difficile (C. difficile) are toxins, such as toxin A (TcdA; enterotoxin), toxin B (TcdB; cytotoxin), and, in some strains, a binary toxin CDT. The C. difficile strains exhibit genetic variation, with a number of known genotypes, which can be classified as North American pulsed-field (NAP) genotypes (by using pulsed-field gel electrophoresis, PFGE), restriction endonuclease analysis (REA) genotypes, polymerase chain reaction ribotypes (PCR-ribotypes), and toxinotypes (identified by polymerase chain reaction restriction fragment length polymorphism, PCR-RFLP). The increase in CDI incidence is predominantly

due

to

hyperepidemic

strains

of

the

genotype

known

as

NAP1/BI/RT027/toxinotype III, which emerged in North America and Europe at the beginning of the new millennium [4, 5, 6]. The strains were highly resistant to multiple antibiotics (especially fluoroquinolones), which contributed to their survival and expansion in the hospital environment. Strains of NAP1/BI/RT027/toxinotype III posed a serious epidemiological problem in Poland in 2010–2014 [7, 8, 9]. 3

Journal Pre-proof Metronidazole and vancomycin are the first drugs of choice for CDI treatment [10]. Although C. difficile has been reported to be susceptible to metronidazole and vancomycin in most studies, resistance and reduced sensitivity of C. difficile to metronidazole was reported recently [11]. Since 2011, a network of 13 hospital-based laboratories in Poland surveyed the epidemiology of CDI and characterized the C. difficile strains. Within the network, the mean annual hospital CDI incidence rates increased with time; 6.1, 8.6 and 9.6 CDI per 10,000 patient-days in 2011, 2012, and 2013, respectively [8]. We conducted this study to investigate the antimicrobial susceptibility patterns of clinical C. difficile isolates collected from 2012 to 2014. Additionally, the mechanism of resistance of randomly selected strains to fluoroquinolones and rifampicin, and macrolide-clindamycin-and streptogramin B (ermB) gene were determined.

Material and methods

Study Design

The original design of this study has been described previously [8]. Definitions of CDI were used according to the proposal by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) [12]. Among the 16 participating hospitals (designated H1 to H16), 14 provided secondary (n=7) or tertiary care (n=7), and two specialized in pulmonology/thoracic surgery (H1) and oncology (H8). During the first period of the study in 2012, 10 from the 13 randomly selected regional laboratories sent the C. difficile isolates to the Anaerobe Laboratory (AL). In total, 88 C. difficile strains from patients with CDI were submitted for PCR-ribotyping and antimicrobial drug susceptibility testing. The 88 strains

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Journal Pre-proof were isolated from in-patients admitted to the following hospitals: none (H1), 9 (H2), none (H3), 17 (H4), none (H5), 4 (H6), 3 (H7), 6 (H8), 3 (H9), 6 (H10), 10 (H11), 19 (H12), and 11 (H13). Of the 88 C. difficile isolates, 76 had been isolated between February and March 2012, and 12 were isolated in January, or in April. Between February and March 2013, the second surveillance program for CDI was conducted in Poland. During this period of the study, 91 C. difficile strains, consecutively isolated from patients with CDI hospitalized in different wards, were sent to the AL by 13 (10 from the first program and three new hospitals) randomly selected regional laboratories. Of the original 91 strains that were isolated from in-patients admitted to the 13 hospitals (H1-H13), 1 originated from H1, 5 (H2), 10 (H3), 9 (H4), 5 (H5), 7 (H6), 6 (H7), 10 (H8), 8 (H9), 10 (H10), 10 (H11), and 10 (H12). In the third part of this survey, organized in 2014, 82 C. difficile strains were sent for PCR-ribotyping and antimicrobial drug susceptibility testing. Of the original 82 strains that were isolated from the in-patients admitted to 16 hospitals (H1-H16), only six hospitals sent the strains to the AL; 16 (H3), 2 (H5), 12 (H12), 35 (H14), 9 (H15), and 8 (H16).

Clostridium difficile culture, identification, and toxigenicity A total of 261 strains were collected; 88 in 2012, 91 in 2013, and 82 strains in 2014, respectively. The C. difficile strains or fecal samples were inoculated anaerobically on selective media for 48 h (CLO, bioMérieux, SA; Marcy l’Etoile, France) or 24 h (CHROM ID bioMérieux, SA; Marcy l’Etoile, France), and C. difficile colonies were sub-cultured on blood agar and identified using standard methods, as described previously [7]. Presumptive C. difficile isolates were cultured on C. difficile selective medium, identified by Gram stain, and characterized odor and typical colony morphology. All C. difficile strains were included in the genetic study and tested in AL for genetic markers of toxigenicity using the Xpert CD assay (Cepheid; Sunnyvale, CA, USA). The Xpert CD assay can be used to detect toxigenic C.

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Journal Pre-proof difficile strains and identify the presumptive C. difficile NAP1/BI/027 strain or the closelyrelated RT176. Real-time multiplex PCR (RT-PCR) was performed to detect the tcdB, cdtA, and cdtB genes (binary toxin genes), and a deletion in the tcdC gene (encoding a negative regulator of toxin A/B production). Toxin production of the isolates was detected by the immunochromatographic test that detects the A and B toxin (C. DIFF QUIK CHEK COMPLETE, TECHLAB, US). Only toxigenic strains were used for further susceptibility testing. Eight isolates were non-toxigenic and were excluded from the study, leaving 253 toxigenic isolates for further characterization of susceptibility.

PCR-ribotyping analysis

All isolates of C. difficile were frozen (-70°C) and stored in Microbank™ (PROLAB DIAGNOSTICS) until the tests were carried out in the AL. PCR-ribotyping was performed on all C. difficile isolates using standard methods previously described [13]. Using the support of the Reference Laboratory in Leiden, 22 different PCR-ribotypes were identified, of which four were not present in the database. The Cardiff-ECDC collection of reference isolates of C. difficile was used as a reference.

Antimicrobial susceptibility testing

Minimal inhibitory concentrations (MICs) for the 253 toxigenic C. difficile strains were determined for 10 antimicrobial agents by using Etest strips (bioMérieux SA; Marcy l’Etoile, France) that contained gradients of each agent tested. The Etest strips were pre-reduced in an anaerobic atmosphere. The following antimicrobials were tested: metronidazole (MZ), vancomycin (VA), clindamycin (CM), erythromycin (EM), tetracycline (TC), and tigecycline

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Journal Pre-proof (TG), which had Etest strips ranging from 0.016 to 256 mg/L; and ciprofloxacin (CI), moxifloxacin (MX), imipenem (IP), and rifampicin (RI), which had Etest strips ranging from 0.002 to 32 mg/L. EUCAST clinical breakpoints for C. difficile were applied to the antimicrobial drugs MZ, VA, MX, RI, TG, ME, and ER (http://www.eucast.org) [14]. For EM, CM, CI, IP, and TC, the Clinical & Laboratory Standards Institute (CLSI, 2007) clinical breakpoints were assessed [15]. Standard reference strains Bacteroides fragilis ATCC 25285, and Bacteroides thetaiotaomicron ATCC 29741 were included as controls. The next step in our study was to describe the susceptibility patterns, representing various combinations of susceptibility and resistance to epidemiologically important antimicrobial agents, i.e., erythromycin (EM), clindamycin (CM), moxifloxacin (MX), and rifampicin (RI), with “R” indicating resistance and “S” indicating susceptibility.

Genetics of resistance and gene sequencing The PCR technique was used to detect the ermB gene fragment, most commonly responsible for high-level, simultaneous resistance of C. difficile strains to both erythromycin and clindamycin, as described earlier [16]. The mechanism of resistance to fluoroquinolones (FQ) and rifampicin was assessed in strains selected based on their high levels of phenotypic resistance to these drugs. The PCR-amplified gyrA, gyrB, and rpoB gene fragments were sequenced to search for the mutations responsible for resistance to fluoroquinolones (genes gyrA and gyrB) and rifampicin (gene rpoB) [17]. The gyrA and gyrB quinolone resistance determining regions (QRDRs) of FQ-resistant strains were amplified using the primer couple gyrA1 and gyrA2 , amplifying 390 bp of gyrA, and the primer couple gyrB1 and gyrB2, amplifying 390 bp of gyrB, as already described [18]. DNA extraction was performed by Genomic Mini (A&A BIOTECHNOLOGY, Poland).

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The rpoB gene of rifampicin resistant strains was amplified using the primer couple rpoB2F and rpoB2R, amplifying 200 bp, as already described [19].

DNA products were purified and sequenced on BigDye® Terminator v3.1 (Applied Biosystems, Life Technologies). The products of the sequencing reaction were separated in a 3730xl DNA capillary sequencer. Mutations in the resistance genes were aligned with the reference sequence of C. difficile 630 (GenBank accession NC 009089.1) using Mutation Surveyor V4.0.9.

Statistical analysis

Differences in the MIC values of the 10 antimicrobial drugs between the hyperepidemic group of C. difficile strains (RT027 and RT176) and non-epidemic RTs were performed using Mann-Whitney U test. This difference was considered statistically significant when the P value was <0.05. The study was approved by the local Institutional Review Board. The written informed consents were waived because of the retrospective studies of clinical isolates without interventions from the patients.

Results

PCR-ribotyping

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Journal Pre-proof A total of 253 toxigenic C. difficile strains were included in this study. The 253 strains evaluated for antimicrobial susceptibility belonged to 24 different PCR-ribotypes. In three years (2012-2014), 261 isolates of C. difficile from 16 hospitals were submitted to the Anaerobe Laboratory for testing. Of these, 96.9% (n=253) were toxin-positive with the highest proportions of the following ribotypes: RT027 (65.2%; n=165), RT176 (13.0%; n=33), RT014 and the genetically similar RT020, i.e. RT014/RT020 (constituting 5.9%; n=15), and RT023 (2.4%; n=6). The other PCR-ribotypes, RT001 (n=1), RT002 (n=4), RT003 (n=3), RT005 (n=3), RT012 (n=1), RT017 (n=2), RT018 (n=3), RT045 (n=1), RT046 (n=2), RT053 (n=1), RT056 (n=2), RT081 (n=3), RT087 (n=1), RT112 (n=1), RT152 (n=1), RT231 (n=1), and four unknown PCR-ribotypes constituted 13.5%.

Antimicrobial susceptibility All the 253 strains of C. difficile strains isolated during 2012–2014 were evaluated with a preselected drug susceptibility testing (E-test method). The toxigenic C. difficile isolates belonging to the different RTs showed resistance to 7 out of the 10 antimicrobial agents tested. All strains were susceptible to metronidazole (MIC90=0.5 mg/L) and vancomycin (MIC90=0.75 mg/L). Only one strain with reduced sensitivity (MIC=2 mg /L) to metronidazole was isolated in 2014. The overall proportion of C. difficile strains resistant to erythromycin was 81.0% (n=205); MIC90=256 mg/L. The proportion of strains resistant to clindamycin was 17.4% (n=44); MIC90=256 mg/L. Among the erythromycin-resistant strains, those simultaneously resistant to clindamycin constituted 20.5% (42 out of 205) (MIC≥256 mg/L). The strains resistant simultaneously to erythromycin and clindamycin constituted 16.6% (n=42). Only two strains (both isolated in 2014) were resistant to clindamycin (MIC=12 mg/L) while exhibiting susceptibility to erythromycin. All 253 strains (100%) were resistant to ciprofloxacin (MIC90=32 mg/L), with 80.6% of those (n=204) simultaneously

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Journal Pre-proof resistant to moxifloxacin (MIC90=32 mg/L). Only 1.6% (n=4) out of 253 C. difficile strains proved to be resistant to tetracycline. No tigecycline-resistant (MIC90=0.125 mg/L) strains were detected (Table 1). All RT027 strains (100%) showed high-level resistance to moxifloxacin (MIC≥32 mg/L). Among RT027 strains, those resistant to erythromycin constituted 99.4% (MIC≥256 mg/L), with only a single RT027 strain exhibiting susceptibility to erythromycin (MIC=1 mg/L). All RT176 strains were resistant to moxifloxacin and erythromycin. In the case of hyperepidemic strains (RT027 and RT176) 5.4% (n=9) of RT027 strains and 84.8% (n=28) of RT176 strains were simultaneously resistant to clindamycin and erythromycin. Simultaneous resistance to erythromycin, moxifloxacin, and rifampicin was detected in 30.8% (n=78) of the evaluated strains, 74 of which were RT027. The non-RT027 and RT176 C. difficile strains resistant to erythromycin and clindamycin were 14.5% and 12.7%, respectively. We observed a higher percentage of resistance to tetracycline among the non-epidemic strains compared to the epidemic strains (RT027), which was 5.4% and 0.6%, respectively. We did not observe resistance to tetracycline among the non-epidemic strains isolated in 2014. Susceptibility to carbapenems varied, 93.3% (n=236) out of 253 strains were resistant to imipenem (MIC90=32 mg/L), 59.7% (n=46) out of 77 randomly selected toxigenic strains were resistant to ertapenem, and 100% (n=77) exhibited susceptibility to meropenem (MIC90=2 mg/L) (data not shown). The percentage and numbers of C. difficile strains resistant to antimicrobial agents in the 16 Polish hospitals are presented in Fig 1 and Fig 2.

Analysis of the drug resistance of C. difficile strains belonging to PCR-epidemic RT027 and RT176 and other PCR-ribotypes

The metronidazole MIC values of the hyperepidemic RT027 and RT176 were higher than those of non-epidemic PCR-ribotypes. This difference was statistically significant (p<0.05),

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Journal Pre-proof as evidenced by the Mann-Whitney U test. However, in the case of vancomycin, the difference in the MIC values of hyperepidemic and non-epidemic strains was not statistically significant (p=0.065). Statistically significant differences in the median values of MICs were also demonstrated for erythromycin (p<0.05), moxifloxacin (p<0.05), and rifampicin (p<0.05). For these three drugs, the MIC values for the RT027 and RT176 epidemic strains were higher than for the strains belonging to other PCR-ribotypes. A statistically significant (p<0.05) difference in the median value was also demonstrated for the tigecycline MIC. In this case, MIC values were higher for strains belonging to other RT-PCR-ribotypes than RT027 and RT176. There were no statistically significant differences in median values for MIC of the other drugs tested, i.e., clindamycin (p=0.216), imipenem (p=0.175), and tetracycline (p=0.431). The MIC values for ciprofloxacin were constant; all strains were resistant, and the MICs were 32 mg/L.

Patterns of susceptibility of C. difficile strains The evaluated clinical C. difficile strains demonstrated nine different antimicrobial susceptibility patterns. Each individual Polish clinical hyperepidemic strain of C. difficile RT027 was found to exhibit one out of five (I, II, III, IV, or V) susceptibility patterns, representing various combinations of susceptibility and resistance to epidemiologically important antimicrobial agents, i.e., erythromycin (EM), clindamycin (CM), moxifloxacin (MX), and rifampicin (RI), with “R” indicating resistance and “S” indicating susceptibility. The PCR-ribotype 176 (RT176) exhibited I, II, III, or IV susceptibility/resistant patterns. Conversely, strains of other PCR-ribotypes were characterized by the highest proportion, i.e., 78.2% (n=43) of susceptibility pattern IX, indicating susceptibility to all evaluated antibiotics. However, this susceptibility/resistance patterns were present in this group and accounted for 21.8% of strains. Among the strains not belonging to the hyperepidemic PCR-group, only one

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Journal Pre-proof strain (1.8%) belonging to an undetermined PCR-ribotype, similar to RT027, was resistant to these four drugs. Similarly, 84.8% of strains belonging to RT176 were resistant to clindamycin, erythromycin, moxifloxacin, and imipenem. However, only two strains (3.6%) belonging to other PCR-ribotypes (RT045 and RT003) were resistant to these drugs. The susceptibility/resistant patterns of the C. difficile strains are shown in Table 2.

Analysis of the mechanisms of resistance to antimicrobial drugs All 14 evaluated C. difficile strains showed high levels of resistance for both ciprofloxacin and moxifloxacin (MIC≥32 mg/L). The QRDRs of GyrA and GyrB were individually sequenced from the nucleotide codons 48 to 152 and 371 to 479, respectively. The results indicated that a single amino acid substitution (Thr82Ile) in GyrA was detected in 11 out of the 14 evaluated strains, while the remaining 3 strains were negative for this mutation (Table 3). Four different types of amino acid substitutions (Arg447Lys, Ser416Ala, Asp426Val, and Asp426Asn) in the GyrB were detected in 4 out of 14 of the evaluated strains, while the remaining 10 strains did not exhibit any substitutions in the evaluated part of GyrB. No point substitution was detected in one strain. The most frequent mutation among the tested isolates was the point mutation within the gyrA gene at position 82, conditioning the conversion of threonine (Thr) to isoleucine (Ile). This mutation was detected in 11 of the tested strains. Two strains having Thr82Ile mutations also had a point mutation within the gyrB gene. A strain belonging to RT027 (no.7) had a mutation at position 447, conditioning the change of arginine (Arg) to lysine (Lys), R447K, and strain RT045 (no.11) had a mutation at position 416, conditioning the change of serine (Ser) to alanine (Ala), S416. Two different single point mutations in the gyrB gene at position 426 were detected in two strains. In strains RT053 and RT231, a mutation conditioning the alteration of aspartic acid (Asp) to valine (Val), D426V and aspartic acid (Asp) to asparagine (Asn), D426N, respectively, were detected. In the strain

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Journal Pre-proof belonging to RT003, no point mutation was detected within the examined fragments of gyrA and gyrB genes; thus, this strain requires further analysis. Sequencing to detect mutations in the rpoB gene conferring resistance to rifamycins (RI) was performed for a total of 21 selected C. difficile isolates. Twenty strains were highly resistant to RI (MIC≥32 mg/L), while one strain, with non-typable PCR-ribotype, showed lower resistance, MIC=0.5 mg/L. Most of the tested strains belonged to RT027 (n=14). The remaining isolates belonged to RT176 (n=2) and RT046 (n=3). One of the tested strains, no. 20, belonged to a previously non-isolated RT027-like PCR-ribotype. In all tested strains, at least one point mutation was detected within the rpoB gene. The most frequent point mutation among the tested strains was at position 505, conditioning the replacement of arginine (Arg) with lysine (Lys), R505K. The presence of this point mutation was detected in 19 isolates. In addition, 9 of these isolates had a second point mutation, 8 of them at position 502, conditioning the conversion of histidine (His) to asparagine (Asn), H502N, and one isolate at position 488, which conditions the exchange of serine (Ser) to threonine (Thr), S488T. Two other point mutations were also detected in two strains; in one strain belonging to RT027, a point mutation at position 489 was found, conditioning the conversion of glutamine (Gln) to leucine (Leu), Q489L. This is a newly detected mutation, not previously described in the literature. However, in one strain for which the rifampicin MIC was 0.5 mg/L, there was a point mutation at position 550, which determines the conversion of serine (Ser) to phenylalanine (Phe), S550F. The presence of the ermB gene was confirmed in 29 out of 44 strains phenotypically resistant to clindamycin, while in 15 strains the ermB gene was not detected. Discussion Clostridium difficile is recognized as a major agent of nosocomial diarrhea. In recent years, the incidence of CDI has increased in the United States, Canada, and Europe, and a recent 13

Journal Pre-proof study in Poland found c.a. 8.3 CDI per 10,000 patient-days in 2012-2013 [5, 9]. It was higher compared to Europe, where the incidence was c.a 7.0 per 10,000 patients-days in the same period [20]. Metronidazole and vancomycin are regarded as the primary therapeutic options for CDI, depending on the severity of the disease [10]. Recent guidelines favor vancomycin and consider metronidazole as inferior [21]. Though all 253 clinical C. difficile isolates in our study were susceptible to metronidazole, significantly higher MIC values were found against RT027 and RT176 strains, similar to the study by Debast et al [22]. This could be one of the likely causes for the lack of efficacy of metronidazole in the treatment of patients infected with strain RT027 or RT176. All Polish isolates were susceptible to tigecycline. Resistance to tigecycline is rare; among n=1112 strains isolated in Taiwan, resistance was found in 0.6%– 3.2% of the isolates in 5 hospitals and was more common among RT078 lineage than nonRT078 (20.7%, 12/58 vs 6.5%, 51/784; p<0.001) [23]. Multidrug resistance (MDR) is defined as resistance to at least three classes of antimicrobial drugs. Among all strains belonging to RT027, 43.0% of the strains were characterized by multi-drug resistance. These strains were simultaneously resistant to drugs belonging to five different groups (fluoroquinolones, macrolide, lincosamide, rifamycin, and carbapenems). The study showed that 84.8% of strains belonging to RT176 are multi-drug resistant strains. The susceptibility analysis of Polish clinical strains of C. difficile, conducted for epidemiological purposes, to the antimicrobials, such as erythromycin, clindamycin, moxifloxacin, and rifampicin revealed the presence of nine susceptibility patterns representing various combinations of susceptibility and resistance to these antibiotics. This finding demonstrates highly diversified susceptibility patterns of hospital C. difficile strains in Poland. This study showed that hyperepidemic PCR-ribotypes of C. difficile (RT027 and the closelyrelated RT176) exhibit the highest antimicrobial resistance, indicating a relationship between

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Journal Pre-proof this genotype and acquired antimicrobial resistance. However, definitive confirmation of such a relationship requires further studies. This study demonstrated the presence of multidrug-resistant C. difficile strains in Polish hospitals over the consecutive years of the study period, irrespective of either geographical location or reference level of the hospital. This high prevalence may be a result of overuse of individual types of antibiotics. The presence of C. difficile strains with a high resistance potential in some Polish hospitals calls for the development of effective means for limiting the uncontrolled spread of these strains as well as monitoring the future rates of antimicrobial resistance; particularly, resistance to the drugs used to combat C. difficile infections. The analysis of the genetic determinants of resistance to selected drugs demonstrated the presence of similar mechanisms of resistance to those observed in international C. difficile strains [24]. The resistance mechanism to fluoroquinolones in bacteria is caused by two mechanisms: a mutation in the encoding genes, leading to a reduced affinity for the drug; and either an increase in the active efflux of the drug or a decrease in permeability [25]. C. difficile resistance to quinolone is due to changes in the DNA gyrase subunit GyrA and/or GyrB [26], and these mutations can be found in the quinolone-resistance-determining region (QRDR) [18]. The T82I is the most common site of mutation in GyrA, whereas D71V, T82V, D81N, A83V, A118V, and A118T are the most common mutation sites in GyrB [26, 27]. The substitution Thr-82→Ile in GyrA is prevalent in the moxifloxacin resistant epidemic clone NAP1/027 [27]. Although rare, the substitution Thr-82→Val has also been detected. In GyrB substitutions, Asp-426→Asn and Asp-426→Val are some of the most common changes found in resistant isolates [28]. Recent studies have demonstrated that C. difficile resistance to tetracycline varies among different countries from 2.4% to 62.7% [11, 29, 30]. In previous studies conducted in Poland, including 330 clinical C. difficile strains isolated in two Polish hospitals H1 and H2 in 2004-

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Journal Pre-proof 2006, significantly higher resistance to tetracycline (23% and 22.6%, respectively) were observed, while the isolates in the years 2012-2014 showed only 1.6% resistance [31]. Two members of rifamycin drugs are used for the treatment of CDI, namely rifampicin and rifaximin. A study conducted in 2009 in Spain reported that rifamycin (RI) resistance was ca. 24% [32]. A Hungarian study demonstrated that the resistance rate of C. difficile was ca. 11.5%–14.9% from 2008 to 2010 [33,34]. In 2010, a study in Poland reported a high rate of resistance to RI of the C. difficile clone, PCR-ribotype 046 in tuberculosis patients [35]. Interestingly, in Asia (China and South Korea), the resistance rate of C. difficile to RI is quite low compared with that in Europe countries (ca. 19.8%), and strains resistant to RI (MIC≥32 µg/ml) have also been shown to be resistant to rifaximine (RFX) [36,37]. In the present study, higher percentage of RI resistant strains was observed, especially among RT027: 31.2% (2012), 49.1% (2013), and 51.6% (2014). Among RT176 C. difficile strains, the observed RI resistance was 0% (n=0/21; 2012), 50% (n=3/6; 2013), and 0% (n=0/6; 2014). In Chinese hospitals, H502N and/or R505K mutations were identified in the rpoB gene in all of the rifaximin-resistant and intermediate strains [24]. The most frequent point mutation among Polish strains was at position 505, conditioning the replacement of arginine (Arg) with lysine (Lys), R505K. In previous studies conducted in Poland (2004-2006), a higher percentage of clindamycin resistant strains were found compared to the present study. It was 53.6% and 47.5%, respectively, in two hospitals (H1 and H2) in Warsaw [31]. In the present study the percentage of clindamycin-resistant strains was lower: 27.7% (2012); 17.0% (2013), and 7.3% (2014). Significant changes in the susceptibility of Polish clinical isolates of C. difficile have occurred in 10 years. In addition to the differences in the resistance to clindamycin and tetracycline, differences in resistance to other drugs, such as erythromycin, imipenem, and moxifloxacin,

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Journal Pre-proof are also noticeable. Interestingly, the antibiotic resistance of the Polish C. difficile strains isolated in 2012-2014 for these three drugs is much higher compared to 2004-2006.

Conclusions Occurrence of C. difficile strains resistant to various antimicrobial drugs in Polish hospitals with a high incidence rate indicates the abuse of individual groups of drugs, warranting the development of effective methods to control the spread of these strains, as well as prevent them from acquiring drug resistance, particularly against the drugs used to control C. difficile. Acknowledgements We wish to thank the Polish clinical microbiologist for their help with C. difficile strains collection. Conflicts of interest We have no competing interests to declare Funding This work was supported by the National Centre of Science, Poland (Grant number: UMO2011/01/B/NZ7/02720).

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22

Journal Pre-proof Conflicts of interest We have no competing interests to declare

Journal Pre-proof

Fig. 2 Resistance of Polish C. difficile strains in different areas of Poland

Journal Pre-proof Fig. 1 Percentage of C. difficile strains resistant to selected antibiotics / chemotherapeutics depending on PCR-ribotype, in different years

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

RT 027 (2013) RT 027 (2014) RT 176 (2012) RT 176 (2013) RT 176 (2014) other ribotypes (2012) other ribotypes (2013)

nc

id a

zo le om Er y yt hr cin om yc Cl in da in m Ci pr ycin of lo x M ox acin ifl ox Ri acin fa m pi Im cyn ip e Te ne tra m cy cli Ti ge ne cy cli ne

other ribotypes (2014)

Va

ro n et M

percent of resistant isolates

RT 027 (2012)

Journal Pre-proof Highlights 

We demonstrates highly diversified susceptibility patterns of Polish C. difficile



We clarified that MDR C. difficile strains dominated in hospital setting



Higher percentage of RI resistant strains was observed

Journal Pre-proof Tab. 1 In vitro susceptibilities of toxigenic C. difficile strains to ten antimicrobial agents

Antimicrobial agent

All toxigenic C. difficile strains MIC mg/L 50%* 90%*

No. (%) of resistant strains

C. difficile strains belonging to PCR-RT 027 MIC mg/L 50%

90%

No. (%) of resistant strains

C. difficile strains belonging to PCR-RT 176 MIC mg/L 50%

90%

No. (%) of resistant strains

2012 Metronidazol Vancomycin Erythromycin Clindamycin Ciprofloxacin Moxifloxacin Rifampicyn Imipenem Tetracycline Tigecycline

0.125 0.5 256 3 32 32 0.002 32 0.19 0.094

n**=83 0.75 0 (0) 0.75 0 (0) 256 71 (85.5) 256 23 (27.7) 32 83 (100) 32 69 (83.1) 32 15 (18.1) 32 73 (87.9) 0.38 2 (2.4) 0.19 0 (0)

n=48 1 0.75 256 4 32 32 32 32 0.38 0.19

0 (0) 0 (0) 48 (100) 2 (4.2) 48 (100) 48 (100) 15 (31.2) 46 (95.8) 1 (2.1) 0 (0)

0.094 0.75 256 256 32 32 0.002 32 0.19 0.064

n=21 0.5 1 256 256 32 32 0.003 32 0.38 0.19

n=55 0.19 0.75 256 256 32 32 32 32 0.19 0.094

0 (0) 0 (0) 54 (98.2) 7 (12.7) 55 (100) 55 (100) 27 (49.1) 52 (94.5) 0 (0) 0 (0)

0.064 0.25 256 256 32 32 0.002 32 0.064 0.023

n=6 0.094 0 (0) 0.38 0 (0) 256 6 (100) 256 4 (66.7) 32 6 (100) 32 6 (100) 32 3 (50) 32 6 (100) 0.125 0 (0) 0.094 0 (0)

0 (0) 0 (0) 62 (100) 0 (0) 62 (100) 62 (100) 32 (51,6) 61 (98,4) 0 (0) 0 (0)

0.094 0.38 256 256 32 32 0.002 32 0.094 0.047

n=6 0.125 0 (0) 0.5 0 (0) 256 6 (100) 256 4 (66.7) 32 6 (100) 32 6 (100) 0.002 0 (0) 32 6 (100) 0.19 0 (0) 0.047 0 (0)

0.19 0.5 256 2 32 32 0.002 32 0.25 0.125

0 (0) 0 (0) 21 (100) 20 (95.2) 21 (100) 21 (100) 0 (0) 21 (100) 0 (0) 0 (0)

2013 Metronidazol Vancomycin Erythromycin Clindamycin Ciprofloxacin Moxifloxacin Rifampicyn Imipenem Tetracycline Tigecycline

0.094 0.38 256 3 32 32 0.002 32 0.125 0.047

n=88 0.19 0.75 256 256 32 32 32 32 0.25 0.125

0 (0) 0 (0) 65 (73.9) 15 (17.0) 88 (100) 65 (73.9) 30 (34.1) 85 (96.6) 2 (2.3) 0 (0)

0.125 0.38 256 2 32 32 0.002 32 0.125 0.047

2014 Metronidazol Vancomycin Erythromycin Clindamycin Ciprofloxacin Moxifloxacin Rifampicyn Imipenem Tetracycline Tigecycline

0.19 0.38 256 3 32 32 0.002 32 0.125 0.047

n=82 0.5 0,75 256 8 32 32 32 32 0,25 0.094

0 (0) 0 (0) 69 (84.1) 6 (7.3) 82 (100) 70 (85.4) 34 (41.5) 79 (96.3) 0 (0) 0 (0)

0.19 0.38 256 3 32 32 32 32 0.125 0.032

n=62 0.5 0.75 256 6 32 32 32 32 0.25 0.064

Journal Pre-proof Legend: *MIC 50% - MIC50; MIC 90% - MIC90 ; ** n-number of strains

Journal Pre-proof Tab. 2 Patterns of susceptibility of C. difficile depending on PCR-ribotype

Pattern of susceptibility

No. (%) of C. difficile strains PCR-RT 027 n=165

PCR-RT 176 n=33

PCR-RT non 027 non 176 n=55

I

EM-R CM-R MX-R RI-R

5 (3.0)

2 (6.1)

0 (0.0)

II

EM-R CM-S MX-R RI-R

67 (40.0)

1 (3.0)

1 (1.8)

III

EM-R CM-R MX-R RI-S

4 (2.4)

26 (78.0)

2 (3.6)

IV

EM-R CM-S MX-R RI-S

88 (53.4)

4 (12.1)

1 (1.8)

V

EM-R CM-R MX-S RI-S

0 (0.0)

0 (0.0)

3 (5.6)

VI

EM-S CM-S MX-R RI-S

1 (0.6)

0 (0.0)

2 (3.6)

VII

EM-S CM-R MX-S RI-S

0 (0.0)

0 (0.0)

2 (3.6)

VIII

EM-R CM-S MX-S RI-S

0 (0.0)

0 (0.0)

1 (1.8)

IX

EM-S CM-S MX-S RI-S

0 (0.0)

0 (0.0)

43 (78.2)

Journal Pre-proof

Legend: EM – erythromycin, CM – clindamycin, MX – moxifloxacin, RI – rifampicin, S – susceptible, R – resistance.