Surveillance of antibiotic resistance among common Clostridium difficile ribotypes in Hong Kong

Incidence of Clostridium difficile infection (CDI) is rapidly increasing and it poses a major health burden globally. However, data regarding the epidemiology of CDI in Asia are limited. We aimed to characterize the antimicrobial susceptibility patterns of common ribotypes of toxigenic C. difficile in Hong Kong. Fifty-three PCR ribotypes were identified among 284 toxigenic C. difficile clinical isolates. The five most prevalent ribotypes were 002 (13%), 017 (12%), 014 (10%), 012 (9.2%), and 020 (9.5%). All tested C. difficile strains remained susceptible to metronidazole, vancomycin, meropenem and piperacillin/tazobactam, but highly resistant to cephalosporins. Of the fluoroquinolones, highest resistance to ciprofloxacin was observed (99%), followed by levofloxacin (43%) and moxifloxacin (23%). The two newly emerged PCR ribotypes, 017 and 002, demonstrated high levels of co-resistance towards clindamycin, tetracycline, erythromycin and moxifloxacin. PCR ribotypes 017 and 002 with multi-drug resistance are rapidly emerging and continuous surveillance is important to monitor the epidemiology of C. difficile to prevent outbreaks of CDI.

PCR ribotypes. Fifty-three PCR ribotypes were identified among the 284 toxigenic C. difficile clinical isolates and their respective distribution frequencies are shown in Fig. 1 number of isolates included in this study. The less-common ribotypes included 001, 046, 159, 220 and 265 with frequencies between 2.1% and 4.5% (Fig. 1). The remaining 31% isolates (i.e. 89 out of 284) were composed of 43 different ribotypes, with frequencies of ≤1.8% for each ribotype (Fig. 1). In our C. difficile collection, only 2 isolates of the collection were identified as PCR ribotype 027, whilst ribotype 078 remained unobserved.
Relationship of resistance profile to prevalent ribotypes. All C. difficile isolates were susceptible to metronidazole, meropenem and piperacillin-tazobactam, while all isolates were resistant to cefotaxime. The pattern of co-resistance to clindamycin, tetracycline, erythromycin, moxifloxacin, and rifampicin by the five most prevalent PCR ribotypes were investigated (Fig. 2). These five antibiotics were chosen as they represented common standalone antibiotics prescribed clinically, and showed intermediate levels of resistance. Co-resistance between clindamycin and tetracycline was most notable for ribotype 017 (97%) and ribotype 012 (96%). In addition to clindamycin and tetracycline, nearly 50% of the ribotype 017 isolates were multi-drug resistant (i.e. also resistant to moxifloxacin and rifampicin). A similar multi-drug resistance profile was observed for ribotype 020; however, the multi-drug resistance rate was much lower at only 3.7% in contrast to ribotype 017. Different patterns of co-resistance were observed for other ribotypes -isolates of both 002 and 014 ribotypes were susceptible to rifampicin, whereas all ribotype 012 stains were susceptible to moxifloxacin (Fig. 2).

Discussion
Metronidazole has long been used as the first-line drug for treatment of CDI while vancomycin is reserved for patients with complicated infections, severe or recurrent diseases of CDI 27 . In vitro susceptibility testing against C. difficile is not routinely performed and therefore the susceptibility profiles of clinical isolates remains largely unknown. A previous study that examined 100 C. difficile isolates in Hong Kong identified one strain that exhibited an unexpectedly high MIC towards metronidazole (64 mg/L by E-test) 28 . Although metronidazole resistance in C. difficile have been described in previous reports 29,30 , our present study revealed that all C. difficile isolates were susceptible to metronidazole with MICs ranging from ≤0.125 to 1 mg/L (Table 1), which was significantly lower than the CLSI described breakpoint of 32 mg/L 24 . Thus, metronidazole should remain effective as a first-line therapy for CDI in Hong Kong. According to the EUCAST guideline 25 , four vancomycin-resistant isolates with MIC of 4 mg/L (1.4%) were identified. Isolates with reduced susceptibility to vancomycin had been described in China and Taiwan 31,32 . Nevertheless, it should be noted that the EUCAST-defined vancomycin breakpoint of >2 mg/L is only a reference point to distinguish strains with reduced susceptibility to vancomycin. In clinical settings, the faecal level of vancomycin can reach a value of ≥2000 mg/L with standard oral vancomycin treatment 33 . Therefore, despite four isolates showing reduced susceptibility to vancomycin, standard oral administration of vancomycin should remain effective for treating CDI in Hong Kong.
In the present study, most C. difficile isolates tested were resistant to cefotaxime, cefoperazone and ceftazidime. For quinolones, 281 of the 284 (99%) clinical isolates were resistant to ciprofloxacin, followed by levofloxacin (121/284, 43%) and moxifloxacin (65/284, 23%) ( Table 1). Resistance to fluoroquinolone in C. difficile is mediated through changes in gyrA or gyrB, in which single mutations may raise the MIC and produced a level of resistance above peak drug concentrations achievable in serum 34 . Given the different potencies of fluoroquinolones in targeting DNA gyrase in C. difficile, mutations affecting the enzyme targets may confer resistance to different extents. Other factors affecting drug resistance to fluoroquinolones may include drug permeation and presence of an efflux system 35 , although the latter has not yet been demonstrated for C. difficile.
High resistance rate to clindamycin was also observed in our collection (239/284, 84%; Table 1). Resistance to erythromycin, tetracycline and fusidic acid were observed at 46%, 30% and 40%, respectively (Table 1). Mutations at the erythromycin ribosomal methylases gene class B (ermB) is a predominant mechanism of resistance to the macrolide-lincosamide-streptogramin B (MLS B ) family of antibiotics. Nevertheless, several ermB-negative strains resistant to both erythromycin and clindamycin, or only to erythromycin have been identified [36][37][38][39] , suggesting presence of other resistance mechanisms. Alterations in the 23 S rDNA or ribosomal proteins (L4 or L22) have been found in some of these strains 39 , whereas the multidrug resistance (MDR) gene cfr have also been suggested to cause resistance to the MLS B family of antibiotics 40 . Furthermore, the antibiotics may also induce ermB differentially, resulting in the heterogeneity of antibiotic resistance 41 . These may have accounted for the different resistance rates to clindamycin and erythromycin as observed in some studies 36,42 . Given the high levels of antibiotic resistance, prescription of antibiotics must be justified to minimize the risk of secondary infections such as CDI. Rifampicin is an anti-tuberculosis drug and the reported resistance rates of C. difficile were 3.8% in Sweden, 7.9% in North America, and 25% in Shanghai 2,26,43 . The resistance rate in our study was 10% (Table 1). Nevertheless, out of the 26 rifampicin-resistant isolates, 15 of them (58%) belonged to ribotype 017. Given the large number of Chinese population being affected by tuberculosis, the high resistance rate to rifampicin in China could be a result of selective pressure exerted by the widespread use of this drug [44][45][46] . Since ribotype 017 was identified as the dominant clone in Shanghai 43 , the subsequent emergence of ribotype 017 in Hong Kong suggested that there might have been a clonal spread of this ribotype across the region.
Different C. difficile PCR ribotypes were found circulating in Hong Kong during the study period, as a total of 53 PCR ribotypes was identified. Previous study had shown that the PCR ribotype 002, with a frequency of 10%, was the dominant strain in Hong Kong in 2009, and the frequencies for ribotypes 012, 014, 017 and 020 were 2.3%, 1.2%, 0.6%, and 0%, respectively 22 . Our study confirmed that ribotype 002 remained as the predominant clone in Hong Kong (13%). However, PCR ribotype 017, with a distribution frequency of 12%, has become the second most prevalent ribotype. Other PCR ribotypes including 012, 014, and 020 were also identified as major clones at frequencies of 9.2%, 10% and 9.5%, respectively (Fig. 1). The differences between our findings and those of Cheng et al. 23 suggested that the epidemiology of C. difficile in Hong Kong has constantly been changing. The increased sporulation rate of ribotype 002 might render this ribotype with a better survival, which might be contributing factor for their increasing prevalence 22,47 . This may also be related to the local antibiotic usage, as antibiotic prescriptions were observed to correlate highly with incidence of C difficile infections 48,49 .
Consider the heavy flow of international trading and long survival of C. difficile spores, it has been speculated that PCR ribotypes 012, 014 and 020 might have spread from European countries to Hong Kong, as these ribotypes has been described to cause major epidemics in Europe 50,51 . Ribotype 027 was identified as a hypervirulent strain, responsible for severe outbreaks in North America and Europe 52,53 , while ribotype 078 has emerged as another hypervirulent strain in the Netherlands 54 . Ribotype 027 arrived in Hong Kong in 2008 22 but ribotype 078 has not yet been identified. Nonetheless, repeated outbreaks associated with the PCR ribotype 027 has not been reported in Hong Kong and its incidence rate remained low. In North America, multi-drug resistance (i.e. to clindamycin, moxifloxacin, and rifampicin) was frequently associated with ribotype 027 and was also observed among several isolates of ribotype 017 2 . Interestingly, the two ribotype 027 isolates identified in this study did not show multi-drug resistance. Among the five major ribotypes identified in this study, the rates of concurrent resistance to clindamycin, tetracycline, erythromycin, moxifloxacin and rifampicin were the highest for ribotype 017 (Fig. 2). An association between multi-drug resistance and ribotype 017 has also been reported in Poland, Korea and Shanghai 26,55,56 . A high -level of clindamycin and erythromycin co-resistance was displayed by ribotype 012 (Fig. 2). However, in contrast to ribotype 012 strains isolated in Sweden that had high resistance rates to moxifloxacin and rifampicin 57 , the ribotype 012 isolates from Hong Kong were all susceptible to moxifloxacin and largely (96%) susceptible to rifampicin. A recent surveillance report showed that ribotype 002 remained as the most prevalent strain in Hong Kong, despite the lower multi-drug resistance rate (Fig. 2) 8 . All together, these results indicated that even the same ribotype from different regions could display significantly different levels of virulence and patterns of antibiotic resistance. This may imply that environmental factors can pose a strong evolutionary pressure for their survival, and further shape their genomes in their resistance to antibiotics.
This study showed that metronidazole and vancomycin remain effective for the treatment of infection caused by toxigenic strains of C. difficile in Hong Kong. Ribotype 002 was identified as the most prevalent ribotype, with a high rate of co-resistance between clindamycin and erythromycin. Ribotype 017 was the second major clone in our study and is associated with multi-drug resistance. Although metronidazole and vancomycin remain effective for CDI treatment, PCR ribotypes 002 and 017 with multi-drug resistant patterns are rapidly emerging. These data inform the susceptibility patterns of these regionally prevalent ribotypes, and emphasize the need for continual surveillance on the disease. Growth conditions and cytotoxicity assay. Vero cell line (ATCC CCL-81) was maintained in minimum essential medium (MEM) containing 10% fetal calf serum (Gibco ® ) and gentamicin (Rotexmedica) at a final concentration of 24 mg/L. C. difficile isolates were maintained on anaerobic blood agar plate, supplemented with vitamin K1 (Oxoid), and grown in pre-reduced brain heart infusion (BHI) broth (Oxoid) anaerobically at 37 °C. All C. difficile isolates were confirmed as toxigenic by testing for the presence of toxin B in culture supernatant with the C. difficile Toxin/Antitoxin Kit (Techlab) by following the manufacturer's instructions. In brief, each well of a 96-well plate (Greiner) was seeded with 200 μL of Vero cell culture and incubated for 24 h at 37 °C with 5% CO 2 to achieve a confluent homogenous monolayer. Grown C. difficile broth culture was centrifuged at 6000 × g for 5 min and the resulting supernatant was filtered through a membrane filter with a pore size of 0.45 μm (Millipore). Each filtered supernatant was serially diluted and added to the grown Vero cells. A positive cytotoxic reaction was noted by rounding of the Vero cells observed by light microscopy after 48 h of incubation at 37 °C with 5% CO 2 . Neutralization of cytotoxic effect by the C. difficile antitoxin confirmed the presence of toxin B in the supernatant.

Methods
Antimicrobial susceptibility testing. The susceptibilities of the 284 toxigenic C. difficile clinical isolates to 15 antimicrobial agents were determined by the agar dilution method described by the Clinical and Laboratory Standards Institute (CLSI) 24 . The antimicrobial agents tested include cefotaxime, cefoperazone, ceftazidime (GlaxoSmithKline), ciprofloxacin, clindamycin, erythromycin, fusidic acid, levofloxacin, metronidazole (B. Braun Medical Industries), meropenem (Astra Zeneca), moxifloxacin (Bayer), piperacillin-tazobactam, rifampicin, tetracycline and vancomycin, all reagents were purchased from Sigma unless otherwise stated. C. difficile ATCC 700057 and Bacteroides fragilis ATCC 25285 were used as control strains for each run of agar dilution testing. The minimal inhibitory concentration (MIC) was defined as the lowest concentration of the drug that inhibits bacterial growth. The breakpoints for metronidazole, clindamycin, tetracycline, moxifloxacin, meropenem, piperacillin-tazobactam, cefotaxime and cefoperazone were determined with MIC criteria described by CLSI guidelines 24 . For vancomycin and fusidic acid, breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were used 25 . For erythromycin, ciprofloxacin, levofloxacin, and rifampicin, we adapted the breakpoints from Huang et al. 26 . No breakpoint for ceftazidime was available at the time of this study. PCR ribotyping. PCR ribotyping was performed as previously described 58 . In brief, crude template nucleic acid was prepared by resuspending C. difficile cells, which were grown on Anaerobe Agar (LabM) supplemented with 6% horse blood, in a 5% (wt/vol) solution of Chelex-100 (Bio-Rad) and boiling. After removal of cellular debris by centrifugation, the resulting supernatant (10% vol/vol) was added to PCR mixture containing 50 pmol of each primer, 5′-CTGGGGTGAAGTCGTAACAAGG-3′ (positions 1445 to 1466 of the 16 S rRNA gene) and 5′-GCGCCCTTTGTAGCTTGACC-3′ (positions 20 to 1 of the 23 S rRNA gene). Reaction mixtures were subjected to 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min. Amplification products were separated by electrophoresis in 3% Metaphor agarose. Amplified products were visualized by ethidium bromide staining and the ribotype patterns were analyzed with image analysis software.