Antimicrobial susceptibility of polymerase chain reaction ribotypes of Clostridium difficile commonly isolated from symptomatic hospital patients in the UK

Journal of Hospital Infection (2005) 61, 11–14

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Antimicrobial susceptibility of polymerase chain reaction ribotypes of Clostridium difficile commonly isolated from symptomatic hospital patients in the UK R. John1, J.S. Brazier* Anaerobe Reference Laboratory, NPHS Microbiology Cardiff, University Hospital of Wales, Heath Park, Cardiff CF14 4XW, UK Received 12 November 2004; accepted 21 January 2005 Available online 5 July 2005

KEYWORDS Clostridium difficile; Antibiotics; PCR ribotyping

Summary Two hundred and seventy-one clinical isolates of Clostridium difficile, including the six most common polymerase chain reaction (PCR) ribotypes isolated from symptomatic patients in UK hospitals, were tested against nine antibiotics (imipenem, erythromycin, levofloxacin, piperacillin/tazobactam, ciprofloxacin, co-amoxiclav, cefotaxime, amoxicillin and clindamycin). All 271 strains were susceptible to co-amoxiclav, piperacillin/tazobactam and amoxicillin, and resistant to cefotaxime and ciprofloxacin. Variable degrees of resistance were found to imipenem, erythromycin, levofloxacin and clindamycin. Significantly greater resistance to erythromycin, levofloxacin and imipenem was found in virtually all members of the two most common PCR ribotypes, 001 and 106. Resistance to these agents may have played a part in their selection as the most common strains of C. difficile found in UK hospitals. Q 2005 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.

Introduction Clostridium difficile is recognized as a major cause * Corresponding author. Tel.: C44 02920 742378; fax: C44 02920 746403. E-mail address: [email protected] 1 Present address: Department of Microbiology, Southmead Hospital, Westbury-on-Trym, Bristol BS10 5NB, UK.

of nosocomial antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in UK hospitals, and the major risk factor in development of C. difficile diarrhoeal disease is antibiotic treatment. The epidemiology of nosocomial C. difficile AAD in the UK has been monitored using the isolates referred for typing to the Anaerobe Reference Laboratory (ARL) in Cardiff since 1998. Of over 3000 UK hospital isolates examined, the majority

0195-6701/$ - see front matter Q 2005 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jhin.2005.01.020

12 (55%) have been identified as polymerase chain reaction (PCR) ribotype 001. Of O116 other PCR ribotypes currently recognized by this typing scheme,1 PCR ribotype 106 is the next most common, with types 014, 015, 005 and 002 following in order of rank. Although resistance of C. difficile to the agents commonly used for treatment in the UK (metronidazole and vancomycin) is not a problem,2 very little is known about the relative susceptibilities of the common PCR ribotypes, some of which may be implicated in initiating C. difficile disease. The aim of this study was to determine the susceptibilities of representatives of common PCR ribotypes against nine commonly used antibiotics, using the E-test method (AB Biodisk Ltd, Sweden), and to determine if any relationships exist between type and antibiotic susceptibility.

Materials and methods Isolates Two hundred and seventy-one isolates of C. difficile referred from symptomatic UK hospital patients to the ARL for PCR ribotyping method were selected at random from our database, ensuring that too many strains did not all come from one centre. They included the six most commonly isolated types, namely PCR ribotypes 001 (NZ49), 106 (NZ50), 015 (NZ47), 014 (NZ44), 005 (NZ41) and 002 (NZ42). Prior to testing, each isolate was recovered from storage on beads at K80 8C on fastidious anaerobe agar (FAA, Lab M Ltd; Bury, UK) incubated anaerobically overnight at 37 8C. A control strain of Clostridium perfringens NCTC 11229 was used for susceptibility testing.

Determination of minimum inhibitory concentration A few colonies of an overnight culture of each isolate under test were streaked on to a dried FAA plate using a sterile swab to form a lawn growth and left to dry on the bench for 10 min. One E-test strip for each antibiotic [imipenem, erythromycin, levofloxacin, piperacillin/tazobactam, ciprofloxacin, co-amoxiclav, cefotaxime, amoxicillin and clindamycin (Biostat Diagnostic Systems Ltd, UK)] was aseptically placed diametrically on each plate and the plates were incubated in an anaerobic chamber at 37 8C for 48 h. After removal from the chamber, the minimum inhibitory concentrations (MICs) were read where the zone of inhibition intersected with the E-test strip. The control strain of C. perfringens

R. John, J.S. Brazier was tested with each batch against all the drugs on the same batch of agar media. Breakpoints of susceptibility for each drug were chosen at the levels listed by the National Committee for Clinical Laboratory Standards (NCCLS).3

Statistical analysis Fisher’s exact test (SPSS version 9) was used to determine whether there was any correlation between a strain’s PCR ribotype and its susceptibility to a given antibiotic. Statistical significance required a P value !0.05.

Results All 271 isolates were sensitive to piperacillin/tazobactam (MIC%8 mg/L), amoxicillin (MIC%1.5 mg/L) and co-amoxiclav (MIC%1 mg/L), and resistant to cefotaxime (MICR64 mg/L) and ciprofloxacin (MICR6 mg/L). Antibiotic susceptibilities expressed as the means and ranges of MICs are shown in Tables I and II. Significantly greater numbers of PCR ribotypes 001 and 106 were resistant to erythromycin, levofloxacin, imipenem and clindamycin than the other PCR ribotypes (Figure 1). The MICs of the C. perfringens control only varied by a maximum of plus or minus one dilution to each drug. PCR ribotypes 015, 014, 005 and 002 had very low numbers of resistant isolates to both antibiotics, with the average MIC being !1.67 mg/L (Table II). Clindamycin resistance occurred at a lower Table I In vitro susceptibility [minimum inhibitory concentration (MIC) in mg/L] to imipenem (IP), erythromycin (EM), levofloxacin (LE), piperacillin/ tazobactam (PTc), ciprofloxacin (CI), co-amoxiclav (XL), cefotaxime (CT), amoxicillin (AC) and clindamycin (CM) for polymerase chain reaction (PCR) ribotypes 001 and 106 Clostridium difficile PCR ribotype 001 (NZ49) 106 (NZ50)

IP EM LE PTc CI XL CT AC CM

Mean MIC

MIC range

Mean MIC

MIC range

5.3 O256 O32 2.77 O32 0.42 O256 0.54 6.18

3R32 O256 4R32 1.5–3 8R32 0.19–0.75 128R256 0.19–0.75 2–6

5.68 O256 O32 6.45 O32 0.59 O256 0.76 5.65

2R32 O256 16R32 4–12 O32 0.25–1 64R256 0.19–1.5 1.5–24

Antibiotic resistance of C. difficile PCR ribotypes

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Table II In vitro susceptibility [minimum inhibitory concentration (MIC) in mg/L] to imipenem (IP), erythromycin (EM), levofloxacin (LE), piperacillin/tazobactam (PTc), ciprofloxacin (CI), co-amoxiclav (XL), cefotaxime (CT), amoxicillin (AC) and clindamycin (CM) for polymerase chain reaction (PCR) ribotypes 015, 014, 005 and 002 Clostridium difficile PCR ribotype 015 (nZ47) Mean MIC IP EM LE PTc CI XL CT AC CM

2.96 0.81 3.47 4.04 12.27 0.45 106.4 0.57 5.57

MIC range 1.5–8 0.25R256 2R32 2–6 6R32 0.19–0.75 64R256 0.25–1.5 3R256

014 (nZ44)

005 (nZ41)

002 (nZ40)

Mean MIC

MIC range

Mean MIC

MIC range

Mean MIC

MIC range

2.28 0.91 2.63 3.14 7.67 0.34 103.79 0.32 4.96

1.5–6 0.38R256 1.5R32 1.5–8 4R32 0.125–0.75 48R256 0.125–0.5 1.5R256

6.24 1.67 4.56 5.74 10.56 0.59 152.61 0.71 4.45

1.5R32 0.25R256 3R32 3–12 6R32 0.25–1 96R256 0.25–1 2R256

2.85 1.46 5.13 4.6 12.05 0.49 109.68 0.57 4.48

2R32 0.75R256 2R32 3–6 6R32 0.25–0.75 64R256 0.38–0.75 3–6

frequency in PCR ribotypes 001 and 106 than erythromycin resistance, but PCR ribotypes 015, 014, 005 and 002 all had a higher frequency of clindamycin resistance than erythromycin resistance. Resistance to both antibiotics was rare (less than three isolates for each PCR ribotype) (Figure 2). Only 38% (19/49) and 20% (10/50) of PCR ribotypes 001 and 106, respectively, were resistant to both clindamycin and erythromycin. Isolates belonging to PCR ribotypes 001 and 106 were significantly more likely to be resistant to erythromycin than other PCR ribotypes (PZ!0.01) with 98% (48/49) and 100% (50/50) resistance, respectively. Imipenem resistance in PCR ribotype 001 was also significantly higher (10/49 isolates, 20.4%, P%0.01), as was clindamycin resistance (20/49 isolates, 40.8%, P%0.039). Eighty-eight percent of PCR ribotype 001 (43/49) strains were resistant to levofloxacin, as were all strains of PCR ribotype 106 (50/50) (PZ!0.01).

Figure 1 Distribution of resistant isolates throughout the six polymerase chain reaction ribotypes for imipenem (IP), erythromycin (EM), levofloxacin (LE) and clindamycin (CM).

Discussion Of the six ribotypes studied, PCR ribotypes 001 and 106 ranked one and two in order of occurrence in symptomatic hospitalized patients, and showed similar antibiotic resistance profiles, with more resistant isolates than PCR ribotypes 015, 014, 005 and 002. Erythromycin, levofloxacin and imipenem resistance may give a survival advantage within the clinical environment, and could have contributed to the widespread proliferation, particularly of PCR ribotype 001. The common use of erythromycin derivatives such as clarithromycin and azithromycin, particularly in treatment of communityacquired pneumonia, may well have encouraged the development of erythromycin resistance within C. difficile. PCR ribotype 106 was also highly resistant to erythromycin, which could have contributed to its

Figure 2 Comparisons of clindamycin and erythromycin resistance for all six polymerase chain reaction ribotypes CMr, clindamycin resistant; EMr, erythromycin resistant; CMs, clindamycin susceptible; EMs, erythromycin susceptible.

14 nationwide dissemination. This strain was originally detected in the Midlands and has since been identified from symptomatic patients in hospitals throughout the UK. It is possible, however, that this is due to increased numbers of strains submitted to the ARL for PCR ribotyping rather than a true increase in the spread of this PCR ribotype. However, if erythromycin resistance was an important factor in the nosocomial spread of C. difficile, the high susceptibility of the other four PCR ribotypes would explain why they are much less common in the hospital environment. Clindamycin-resistant PCR ribotype 001 strains have been responsible for outbreaks of diarrhoeal disease in four American hospitals,4 and erythromycin resistance has been documented in a clonal PCR ribotype 001 strain5 compared with lower susceptibility in genotypically distinct isolates. Erythromycin resistance in C. difficile has been well documented, with a high level of resistance being found in PCR ribotype 001 isolates endemic to Leeds General Hospital.3 The higher levels of single resistance to erythromycin and clindamycin illustrate that both resistance factors are not always jointly inherited. The mechanism of erythromycin and clindamycin inheritance should be considered for future study. The high level of ciprofloxacin resistance was expected since it has relatively low activity against anaerobes, although PCR ribotypes 015, 014, 005 and 002 were generally susceptible to the newer fluoroquinolone, levofloxacin. The NCCLS breakpoints are based on systemic levels of antibiotics; however, gut levels of ciprofloxacin and other quinolones are much greater than the NCCLS breakpoints. Interestingly, PCR ribotypes 001 and 106 both displayed significantly higher degrees of resistance to levofloxacin. This antibiotic is sometimes used to treat community-acquired

R. John, J.S. Brazier pneumonia, and resistance to it might also confer a selective advantage to these two PCR ribotypes. In summary, there is an apparently significant correlation between the two most commonly isolated PCR ribotypes of C. difficile, and increased resistance to erythromycin, levofloxacin and imipenem compared with other less common types. This suggests that resistance to one or more of these agents may have played a part in their selection. More work is needed to compare these profiles with those of PCR ribotypes found within the community, such as PCR ribotypes 010 and 020.

Acknowledgements This work was supported by a grant from the Hospital Infection Society (Grant 98/04).

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