In vitro susceptibility to 17 antimicrobials of clinical Clostridium difficile isolates collected in in Sweden

Transcription

1 ORIGINAL ARTICLE BACTERIOLOGY In vitro susceptibility to 17 antimicrobials of clinical Clostridium difficile isolates collected in in Sweden T. Norén 1,2, I. Alriksson 2,T.Åkerlund 3, L. G. Burman 3 and M. Unemo 2 1) Department of Infectious Diseases and 2) Department of Laboratory Medicine, Clinical Microbiology, Örebro University Hospital, Örebro and 3) Swedish Institute for Infectious Disease Control, Solna, Sweden Abstract This study investigated the MICs of 17 antimicrobials, for 606 toxigenic clinical isolates of Clostridium difficile collected between 1993 and 2007 in Sweden. Low MIC 90 values were found for metronidazole (0.5 mg/l), vancomycin (1.0 mg/l), teicoplanin (0.125 mg/l), fusidic acid (1.0 mg/l), linezolid (2.0 mg/l), daptomycin (2.0 mg/l) and tigecycline (0.064 mg/l). Three isolates (0.5%) had elevated MICs for vancomycin (4 8 mg/l); however, these isolates originated from the same patient, who was receiving long-term intravenous vancomycin treatment. High-level clindamycin resistant isolates (MIC >256 mg/l) peaked in 1997 with 39 of 95 (41%) and out of these, 36% were also highly resistant to erythromycin. b-lactams such as penicillin V and piperacillin displayed MIC 90 s of 8 and 32 mg/l, respectively, whereas MICs of cefuroxime were >256 mg/l for all isolates. Universal resistance to ciprofloxacin and levofloxacin was found, and resistance to moxifloxacin increased from 4% of isolates in 2004 to 23% in Notably, these moxifloxacin-resistant isolates did not belong to the recent epidemic PCR ribotype 027, but to the pre-existing epidemic type 012 (82%), and these isolates accounted for the majority of isolates that were resistant to clindamycin (70%), tetracycline (84%) and rifampicin (92%) as well. This investigation of susceptibility data on clinical C. difficile isolates showed variations of multiresistance to be due to a specific PCR ribotype 012, emphasizing the importance of genotyping when evaluating emerging resistance over time. Keywords: 027/NAP1, antimicrobial resistance, Clostridium difficile, Clostridium difficile infection, Etest, Sweden Original Submission: 15 June 2009; Revised Submission: 17 August 2009; Accepted: 17 August 2009 Editor: F. Allerberger Article published online: 3 September 2009 Clin Microbiol Infect 2010; 16: /j x Corresponding author and reprint requests: T. Norén, Department of Infectious Diseases, Örebro University Hospital, SE Örebro, Sweden torbjorn.noren@orebroll.se Introduction PCR ribotype 001 in the UK renews this concern [11]. The main purpose of the present study was to obtain a comprehensive overview, including trends in antimicrobial susceptibility or resistance in toxigenic C. difficile isolates collected from primary CDI patients in a well-defined area of central Sweden over 15 years ( ). Clostridium difficile infection (CDI) is increasing in incidence and morbidity, partly owing to the appearance of the C. difficile 027/NAP1 strain, exhibiting moxifloxacin resistance [1,2], and high levels of toxin and spore production [3,4]. Both the suggested epidemic impact of widespread fluoroquinolone use [5] and the decline in therapeutic efficacy of metronidazole [6] have brought more attention to active antimicrobial susceptibility surveillance of clinical C. difficile isolates [7 9]. When recommended therapies are used, in vitro resistance of C. difficile isolates has not yet been associated with treatment failure [10], although the recent increase in the resistant Materials and Methods C. difficile isolates A total of 606 clinical toxigenic C. difficile isolates from primary CDI patients were recovered by sampling the first consecutive 105 toxin-positive isolates obtained during the years 1993, 1997, 1999, 2002, 2004 and 2007 at Örebro University Hospital in Örebro County (which has approximately inhabitants) in Sweden. Both hospital and community isolates were included, and only primary CDI isolates were selected, avoiding recurrences by excluding Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases

2 CMI Norén et al. Antimicrobial resistance of C. difficile in Sweden 1105 individuals with positive laboratory reports 6 months prior to the actual request date. Toxin testing, culture and preservation of isolates were performed as described elsewhere [12]. The 630 primarily frozen toxin-positive faecal samples yielded a total recovery rate of 96% (606 isolates culturepositive for C. difficile). Accordingly, 24 samples five (1993), ten (1997), one (1999), three (2002), three (2004) and two (2007) were either culture-negative (n = 5) or did not yield isolates identified as C. difficile (n = 19) when analysed by colony appearance and agglutination (Microgen Bioproduct Ltd, Camberley, UK). MIC determination Preserved C. difficile isolates from all years were thawed and cultured anaerobically on pre-reduced fastidious anaerobic agar (FAA; Lab M Ltd, Bury, UK) at 37 C for 48 h. Colonies were subsequently suspended in nutrient broth (Oxoid Ltd, Basingstoke, UK) to a turbidity of 4.0, using the McFarland scale, and seeded on IsoSensitest Agar (Oxoid) supplemented with defibrinated horse blood (5%) and 20 mg/l b- nicotinamide adenine dinucleotide (Sigma-Aldrich, Saint Louis, MO, USA). Initially, from 1993 to 1999, PDM agar (AB Biodisk, Solna, Sweden) was used, as recommended by the manufacturer, but parallel use of both agar media in 1999 showed no significant discrepancies, and the new proposed IsoSensitest medium was used from 2002 onwards (data not shown). Etest strips (AB Biodisk) were applied, and this was followed by anaerobic incubation at 37 C for 48 h. C. difficile ATCC 13124, Bacteroides fragilis ATCC and Enterococcus faecalis ATCC were included as quality control strains. Pharmacological breakpoints were used when available, as recommended by EUCAST (European Committee on Antimicrobial Susceptibility Testing, and the Swedish Reference Group for Antibiotics (SRGA, Antimicrobial agents The following 17 antibiotics were tested: metronidazole, vancomycin, teicoplanin, fusidic acid, clindamycin, erythromycin, rifampicin, piperacillin, penicillin V, cefuroxime, ciprofloxacin, levofloxacin, moxifloxacin, daptomycin, linezolid, tigecycline and tetracycline. The more recently introduced antibiotics, i.e. moxifloxacin, levofloxacin, daptomycin, linezolid and tigecycline (and the older but similar tetracycline), were only included in 2004 and Teicoplanin, cefuroxime and penicillin V were not tested in 2004 and (1997), 102 (1999), 37 (2002), 103 (2004) and 102 (2007)) were subjected to additional PCR ribotyping as well as MIC determination. Statistical analysis The Mann Whitney U-test was used when appropriate. Results The MIC distributions for all antimicrobials specified for each year investigated are provided in Tables 1 3, and MIC 50, MIC 90 and geometric means of MICs are given in Table 4. All C. difficile isolates were inhibited by a concentration of 2 mg/l of metronidazole, and all but three isolates (0.5%) also displayed vancomycin MICs of 2 mg/l (Table 1). Of the other therapeutic alternatives, teicoplanin was the most active agent, displaying MICs 0.5 mg/l for all isolates. Fusidic acid also had favourable MICs ( 4 mg/l), except in the case of four isolates (0.7%) identified in 2002 and 2007 (Table 1). For erythromycin, clindamycin and rifampicin, the prevalence of isolates with high-level resistance (MICs >256 mg/l) peaked in 1997 (36%, 41% and 38% of isolates, respectively, Table 2). For rifampicin, the C. difficile isolates were either highly susceptible (MIC <0.016 mg/l, 80% of isolates) or highly resistant (MIC >256 mg/l, the remaining 20% of isolates) (Table 2). The b-lactams penicillin V and piperacillin displayed MIC 90 s of 8 and 32 mg/l, respectively (Table 4). As expected, all C. difficile isolates were resistant to the cephalosporin cefuroxime (>256 mg/l, Table 2). The activity of tetracycline was generally high, with 84% of isolates displaying MICs of 0.25 mg/l, although a doubling of the numbers of highly resistant isolates was observed between 2004 (9.8%) and 2007 (21%). The related antibiotic tigecycline had a very low MIC 90 (0.064 mg/l), and no resistant isolate was found. Similarly, daptomycin and linezolid had relatively low MIC 90 s (Table 4). All isolates tested were highly resistant (MIC >32 mg/l) to ciprofloxacin. This was also the case for levofloxacin; that is, all isolates tested in 2004 and 2007, (Table 3) as well as 49 random isolates collected during , displayed an MIC of >32 mg/l (data not shown). In contrast, only 4% (4/102) of the isolates were moxifloxacin-resistant in 2004, as compared with 23% (24/103) in Discussion PCR ribotyping PCR ribotyping was performed as previously described [12] and, altogether, a subset of 307 of 606 isolates (16 (1993), This study provides a comprehensive description of the susceptibility to 17 antimicrobials, including their MIC distributions, of clinical C. difficile isolates (n = 606) from

3 1106 Clinical Microbiology and Infection, Volume 16 Number 8, August 2010 CMI TABLE 1. Susceptibility to metronidazole, vancomycin, teicoplanin and fusidic acid of Clostridium difficile isolates from primary C. difficile infection (CDI) patients in in Örebro County, Sweden Boxed numbers 1993 to These C. difficile isolates were sampled consecutively every second to fourth year from primary CDI patients in Örebro County, Sweden, and this made possible identification of any imported or local emergence of resistance. All 606 isolates were susceptible ( 4 mg/l, to metronidazole, as has been found in previous studies [7,13], although the highest MICs (2 mg/l) observed could cause some therapeutic concern, considering the low gut concentrations of metronidazole [14]. Both intermediate and/or high-level resistance to metronidazole and vancomycin have been described in vitro [15], but the clinical relevance in vivo remains unclear [10]. All but three of our isolates displayed vancomycin MICs of 2 mg/l. These three isolates were all from CDI episodes in the same individual subjected to haemodialysis and long-term intravenous vancomycin therapy for recurrent Staphylococcus aureus septicaemia. Treatment of additional pneumonia and urinary tract infection precipitated subsequent multiple CDI episodes. Interestingly, all three episodes were caused by PCR ribotype 002 (data not shown). The other glycopeptide tested, teicoplanin, had high activity, with an MIC 90 of mg/l. Notably, the two isolates from 1999 with the highest teicoplanin MIC (0.5 mg/l; Table 1) were of the same PCR ribotype (002) as isolates that displayed elevated MICs of vancomycin. Fusidic acid has been proven to be comparable to metronidazole in clinical efficacy in a few clinically controlled CDI trials [16,17]. C. difficile isolates are mostly susceptible to fusidic acid [18], as was also confirmed in the present study. Only 0.7% of the isolates showed high-level resistance, and all of these were from patients treated with fusidic acid prior to the onset of CDI (data not shown). Accordingly, like other bacteria, such as the staphylococci, C. difficile may rapidly develop high-level fusidic acid resistance after treatment [19]. Cephalosporins are known to have a high propensity to precipitate CDI [20] and, as expected, cefuroxime had no activity against the C. difficile isolates tested. However, penicillin V, which is known to affect Gram-positive anaerobes, showed an MIC 90 of 8 mg/l, similar to the 4 mg/l found by Tyrell et al. [21]. Fifteen isolates from 1993 to 1999 displayed MICs of >8 mg/l; four of these were highly resistant (MIC 128 mg/l), which could suggest the presence of a b- lactamase. The MIC 90 was higher (32 mg/l) in the case of the b-lactam piperacillin, but a general trend of fewer resistant isolates in later years (2% of isolates with MICs of

4 CMI Norén et al. Antimicrobial resistance of C. difficile in Sweden 1107 TABLE 2. Susceptibility to erythromycin, clindamycin, rifampicin, penicillin V, piperacillin and cefuroxime in Clostridium difficile isolates from primary C. difficile infection patients in in Örebro County, Sweden Boxed numbers >16 mg/l in vs. 33% for ) was noted (Table 2). This observation remains unexplained, since the usage of this antibiotic has not decreased accordingly. In 2004 and 2007, however, 81% (22/27) of the piperacillinresistant (MIC 16 mg/l) C. difficile isolates were of PCR ribotype 012 (p )(data not shown), representing the epidemic genotype in the geographical area studied. Significant outbreaks caused by C. difficile isolates that are highly resistant to clindamycin (MIC >256 mg/l) are well known [22], but only 10% of Swedish isolates are highly resistant under endemic conditions [14]. Considering only highly resistant isolates, we identified a peak proportion of clindamycin resistance of 41% in 1997, and these clindamycin-resistant isolates represented 32 of 36 of the erythromycin-resistant isolates (36%) and all of the rifampicin-resistant isolates (38%). This association of clindamycin, erythromycin and rifampicin resistance was found consistently over the years examined, and such isolates were found in 1999, 2004 and 2007 to be of PCR ribotype 012 in 89%, 69% and 79% of cases, respectively, indicating a clonal relationship (data not shown). Furthermore, we found this clindamycin erythromycin rifampicin-resistant PCR ribotype 012 strain to be also resistant to piperacillin, tetracycline and moxifloxacin (Table 3). Similar multiresistance has been described in Germany [23], although the relationship with PCR ribotype 012 is not known. In the search for new CDI treatment regimens, rifampicinlike agents such as rifalazil have been tested, but recent

5 1108 Clinical Microbiology and Infection, Volume 16 Number 8, August 2010 CMI TABLE 3. Susceptibility to tetracycline, tigecycline, daptomycin, linezolid, moxifloxacin, levofloxacin and ciprofloxacin in Clostridium difficile isolates from primary C. difficile infection patients in in Örebro County, Sweden Boxed numbers reports of highly resistant isolates (MIC >256 mg/l) are discouraging [24]. In the present study, the C. difficile isolates were either highly susceptible or, in the case of as many as 38%, highly resistant to rifampicin (Table 2). This is similar to the findings in some more epidemic settings, e.g. in Bristol, UK [25], but different from the situations encountered in other places [26]. Perhaps resistance may explain the poor results in the past studies that have used rifampicin for CDI treatment [27]. Fluoroquinolones have selective activity on the microflora of the colon, and both selection of pre-existing and new resistant mutants and activity against other anaerobic bacteria will increase the risk of CDI. The risk thought to be associated with moxifloxacin usage has been attributed to its extended anti-anaerobe spectrum and, thus, its propensity to disrupt this major part of the colonic flora [28]. Perhaps decades of ciprofloxacin use have contributed to the selection of fluoroquinolone resistance mutations in colonizing C. difficile strains [29]. The possibility is supported by our findings of 2% of moxifloxacin-resistant isolates as early as 1993, before the drug was marketed (data not shown), alongside 100% ciprofloxacin and levofloxacin resistance. Moreover, the 4 23% increase in moxifloxacin resistance among isolates from 2004 to 2007 is explained mainly by the spread of the multiresistant PCR ribotype 012 (Table 3). The mechanism of moxifloxacin resistance in these PCR ribotype 012 isolates and the cause of their increase in later years is unknown. As moxifloxacin is rarely used in Sweden, the increase in this ribotype could instead be related to co-selection by other antibiotics or increased virulence of the actual epidemic strain. Notably, none of the moxafloxacin-resistant isolates belonged to the current epidemic PCR ribotype 027, which has so far been rarely found in Sweden [3]. In conclusion, the present study confirms the reassuring susceptibility data for metronidazole regarding clinical C. difficile isolates, and supports its further use as a first-line therapeutic agent in treating CDI. The unique 15-year timespan of consecutive sampling of isolates from a well-defined geographical area enabled us to detect variations of multiresistance in C. difficile and unsuspected elevations of MICs of common b-lactams agents such as penicillin V and piperacillin. Finally, monitoring of fluctuating susceptibility data over

6 CMI Norén et al. Antimicrobial resistance of C. difficile in Sweden 1109 TABLE 4. Range, MIC 50, MIC 90 and geometric mean of MIC of 17 antimicrobial agents for toxigenic Clostridium difficile isolates from primary C. difficile infection patients in in Örebro County, Sweden MIC (mg/l) Antimicrobial agent Isolates tested Range MIC 50 MIC 90 mean a Geometric Metronidazole b 606 < Vancomycin Teicoplanin 401 < Fusidic acid to > Erythromycin to > > Clindamycin to > > Rifampicin 606 <0.016 to >256 <0.016 > Penicillin V to > Piperacillin b to > Cefuroxime 401 >256 >256 >256 >256 Tetracycline to > > Tigecycline 205 < < Daptomycin Linezolid Moxifloxacin to > > Levofloxacin 205 >32 >32 >32 >32 Ciprofloxacin 606 >32 >32 >32 >32 a MIC values <0.016, >32 and >256 mg/l were approximated to 0.016, 32 and 256 mg/l. b All isolates were susceptible to metronidazole (MIC <4.0 mg/l), and 106 of 606 (17%) of isolates were resistant to piperacillin (MIC >16.0 mg/l) according to the only established breakpoints by the European Committee on Antimicrobial Susceptibility Testing (EUCAST; time identified a high prevalence of the local multiresistant epidemic strain, PCR ribotype 012, which was responsible for the 2007 emergence of resistance to moxifloxacin. This again emphasizes the importance of monitoring antimicrobial susceptibility, and also of genotyping C. difficile isolates, when evaluating any change in antimicrobial susceptibility [30]. Transparency Declaration This study was supported by grants from the Örebro County Council Research Committee, Örebro University Hospital, Örebro, Sweden. No conflicts of interest to declare among authors. References 1. Kuijper EJ, Coignard B, Tull P. Emergence of Clostridium difficile-associated disease in North America and Europe. Clin Microbiol Infect 2006; 12 (suppl 6): McDonald LC, Killgore GE, Thompson A et al. An epidemic, toxin gene-variant strain of Clostridium difficile. N Engl J Med 2005; 353: Åkerlund T, Persson I, Unemo M et al. Increased sporulation rate of epidemic Clostridium difficile Type 027/NAP1. J Clin Microbiol 2008; 46: Warny M, Pepin J, Fang A et al. Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe. Lancet 2005; 366: Pepin J, Saheb N, Coulombe MA et al. Emergence of fluoroquinolones as the predominant risk factor for Clostridium difficile-associated diarrhea: a cohort study during an epidemic in Quebec. Clin Infect Dis 2005; 41: Musher DM, Aslam S, Logan N et al. Relatively poor outcome after treatment of Clostridium difficile colitis with metronidazole. Clin Infect Dis 2005; 40: Hecht DW, Galang MA, Sambol SP, Osmolski JR, Johnson S, Gerding DN. In vitro activities of 15 antimicrobial agents against 110 toxigenic Clostridium difficile clinical isolates collected from 1983 to Antimicrob Agents Chemother 2007; 51: John R, Brazier JS. Antimicrobial susceptibility of polymerase chain reaction ribotypes of Clostridium difficile commonly isolated from symptomatic hospital patients in the UK. J Hosp Infect 2005; 61: Drummond LJ, McCoubrey J, Smith DG, Starr JM, Poxton IR. Changes in sensitivity patterns to selected antibiotics in Clostridium difficile in geriatric in-patients over an 18-month period. J Med Microbiol 2003; 52: Johnson S, Sanchez JL, Gerding DN. Metronidazole resistance in Clostridium difficile. Clin Infect Dis 2000; 31: Baines SD, O Connor R, Freeman J et al. Emergence of reduced susceptibility to metronidazole in Clostridium difficile. J Antimicrob Chemother 2008; 62: Norén T, Åkerlund T, Bäck E et al. Molecular epidemiology of hospital-associated and community-acquired Clostridium difficile infection in a Swedish county. J Clin Microbiol 2004; 42: Aspevall O, Lundberg A, Burman LG, Åkerlund T, Svenungsson B. Antimicrobial susceptibility pattern of Clostridium difficile and its relation to PCR ribotypes in a Swedish university hospital. Antimicrob Agents Chemother 2006; 50: Bolton RP, Culshaw MA. Faecal metronidazole concentrations during oral and intravenous therapy for antibiotic associated colitis due to Clostridium difficile. Gut 1986; 27: Pelaez T, Alcala L, Alonso R, Rodriguez-Creixems M, Garcia-Lechuz JM, Bouza E. Reassessment of Clostridium difficile susceptibility to metronidazole and vancomycin. Antimicrob Agents Chemother 2002; 46: Wullt M, Odenholt I. A double-blind randomized controlled trial of fusidic acid and metronidazole for treatment of an initial episode of Clostridium difficile-associated diarrhoea. J Antimicrob Chemother 2004; 54: Wenisch C, Parschalk B, Hasenhundl M, Hirschl AM, Graninger W. Comparison of vancomycin, teicoplanin, metronidazole, and fusidic

7 1110 Clinical Microbiology and Infection, Volume 16 Number 8, August 2010 CMI acid for the treatment of Clostridium difficile-associated diarrhea. Clin Infect Dis 1996; 22: Leroi MJ, Siarakas S, Gottlieb T. E test susceptibility testing of nosocomial Clostridium difficile isolates against metronidazole, vancomycin, fusidic acid and the novel agents moxifloxacin, gatifloxacin, and linezolid. Eur J Clin Microbiol Infect Dis 2002; 21: Norén T, Wullt M, Åkerlund T, Bäck E, Odenholt I, Burman LG. Frequent emergence of resistance in Clostridium difficile during treatment of C. difficile-associated diarrhea with fusidic acid. Antimicrob Agents Chemother 2006; 50: Thomas C, Riley TV. Restriction of third generation cephalosporin use reduces the incidence of Clostridium difficile-associated diarrhoea in hospitalised patients. Commun Dis Intell 2003; 27 (suppl): S28 S Tyrrell KL, Citron DM, Warren YA, Fernandez HT, Merriam CV, Goldstein EJ. In vitro activities of daptomycin, vancomycin, and penicillin against Clostridium difficile, C. perfringens, Finegoldia magna, and Propionibacterium acnes. Antimicrob Agents Chemother 2006; 50: Johnson S, Samore MH, Farrow KA et al. Epidemics of diarrhea caused by a clindamycin-resistant strain of Clostridium difficile in four hospitals. N Engl J Med 1999; 341: Ackermann G, Degner A, Cohen SH, Silva J, Jr., Rodloff AC. Prevalence and association of macrolide-lincosamide-streptogramin B (MLS (B)) resistance with resistance to Moxifloxacin in Clostridium difficile. J Antimicrob Chemother 2003; 51: Curry SR, Marsh JW, Shutt KA et al. High frequency of rifampin resistance identified in an epidemic Clostridium difficile clone from a large teaching hospital. Clin Infect Dis 2009; 48: Bendle JS, James PA, Bennett PM, Avison MB, Macgowan AP, Al-Shafi KM. Resistance determinants in strains of Clostridium difficile from two geographically distinct populations. Int J Antimicrob Agents 2004; 24: Bishara J, Bloch Y, Garty M, Behor J, Samra Z. Antimicrobial resistance of Clostridium difficile isolates in a tertiary medical center, Israel. Diagn Microbiol Infect Dis 2006; 54: Lagrotteria D, Holmes S, Smieja M, Smaill F, Lee C. Prospective, randomized inpatient study of oral metronidazole versus oral metronidazole and rifampin for treatment of primary episode of Clostridium difficile-associated diarrhea. Clin Infect Dis 2006; 43: Deshpande A, Pant C, Jain A, Fraser TG, Rolston DD. Do fluoroquinolones predispose patients to Clostridium difficile associated disease? A review of the evidence. Curr Med Res Opin 2008; 24: Samore MH, Venkataraman L, DeGirolami PC et al. Genotypic and phenotypic analysis of Clostridium difficile correlated with previous antibiotic exposure. Microb Drug Resist 2006; 12: Wilcox MH, Fawley W, Freeman J, Brayson J. In vitro activity of new generation fluoroquinolones against genotypically distinct and indistinguishable Clostridium difficile isolates. J Antimicrob Chemother 2000; 46: