Antimicrobial resistance in Clostridium difficile

International Journal of Antimicrobial Agents 34 (2009) 516–522

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Review

Antimicrobial resistance in Clostridium difficile Haihui Huang a,b , Andrej Weintraub b , Hong Fang b , Carl Erik Nord b,∗ a b

Institute of Antibiotics, Huashan Hospital, Fudan University, 12 Wulumuqi Zhong Road, Shanghai 200040, China Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, SE-141 86 Stockholm, Sweden

a r t i c l e

i n f o

Article history: Received 21 September 2009 Accepted 22 September 2009 Keywords: Clostridium difficile Antimicrobial susceptibility Resistance mechanisms

a b s t r a c t Clostridium difficile is the leading cause of hospital-acquired diarrhoea and the number of outbreaks has risen markedly since 2003. The emergence and spread of resistance in C. difficile is complicating treatment and prevention. Most isolates are still susceptible to vancomycin and metronidazole (MTZ), however transient and heteroresistance to MTZ have been reported. The prevalence of resistance to other antimicrobial agents is highly variable in different populations and in different countries, ranging from 0% to 100%. Isolates of common polymerase chain reaction (PCR) ribotypes are more resistant than uncommon ribotypes. Most of the resistance mechanisms that have been identified in C. difficile are similar to those in other Gram-positive bacteria, including mutation, selection and acquisition of the genetic information that encodes resistance. Better antibiotic stewardship and infection control are needed to prevent further spread of resistance in C. difficile. © 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

1. Introduction Clostridium difficile, a Gram-positive, spore-forming bacillus, was first described in 1935 as a component of the intestinal flora in healthy neonates [1]. The role of C. difficile as a cause of diarrhoea was described in 1978 [2]. Today, C. difficile is a leading cause of hospital-acquired diarrhoea, ranging from mild cases to severe pseudomembranous colitis, collectively known as C. difficile infection (CDI). The number of outbreaks has risen markedly since 2003. These outbreaks are thought to be due in part to the emergence of a hypervirulent C. difficile strain 027 [3]. Another new hypervirulent strain 078, whose incidence increased from 3% to 13% during 2005–2008 in The Netherlands, has also been described [4]. New risk groups of patients are peripartum women and children [5]. Antimicrobial therapy plays a central role in the development of CDI. The risk is increased if C. difficile is resistant to the antimicrobial agents used [6]. One of the proposed theories behind the major reported outbreaks was that the fluoroquinolone-resistant strain 027 was circulating at the same time as the use of fluoroquinolones was common in Canadian hospitals [7]. The standard antimicrobial therapy for CDI has changed little since the disease was first described. Metronidazole (MTZ) and vancomycin (VAN) are used as primary therapeutics. However, decreased susceptibility and increased refractoriness to MTZ have been reported [8].

∗ Corresponding author. Present address: Division of Clinical Microbiology F68, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden. Tel.: +46 8 585 878 38; fax: +46 8 585 879 33. E-mail address: [email protected] (C.E. Nord).

This article will review the antimicrobial susceptibility patterns and resistance mechanisms of C. difficile. PubMed, Medline and Google Scholar were searched for C. difficile during the period January 1980 to June 2009. The search terms included ‘Clostridium difficile’, ‘susceptibility’, ‘resistance’, ‘resistant’, ‘metronidazole’, ‘vancomycin’, ‘erythromycin’, ‘clindamycin’, ‘tetracycline’, ‘fluoroquinolones’, ‘moxifloxacin’, ‘rifampin’, ‘rifaximin’ and ‘fusidic acid’. 2. Antimicrobial susceptibility and antimicrobial resistance mechanisms 2.1. Resistance to antimicrobials varies widely between countries Although all isolates are usually susceptible to MTZ and VAN, resistance to other antimicrobials varies widely from one country to another (Table 1). A 2-month prospective study of CDI was conducted in 38 hospitals from 14 different European countries during 2005 [22]. The resistance rates to erythromycin (ERY) and moxifloxacin (MFX) were 87.5% for both antibiotics among UK isolates. However, among isolates from Switzerland, no strain was resistant to ERY and only 7.1% of isolates were resistant to MFX. Another study conducted in Québec, Canada [12] showed that all isolates were susceptible to rifampicin (RIF) and meropenem (MER) but were resistant to bacitracin, cefotaxime and levofloxacin (LFX). More than 80% of isolates were resistant to ceftriaxone, clarithromycin, gatifloxacin and MFX. In a study conducted in the USA [16], several new agents were also tested. The most active agent was rifaximin (RFX), with minimal inhibitory concentrations for 50% and 90% of the organisms (MIC50 and MIC90 values)

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H. Huang et al. / International Journal of Antimicrobial Agents 34 (2009) 516–522

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Table 1 Comparison of minimal inhibitory concentrations (MICs) and resistance rates of Clostridium difficile isolates from different countries. Country, year [Reference]

Sample size

Australia, 2002 [9] Germany, 2003 [10] UK, 2003 [11] Canada, 2006 [12] Sweden, 2006 [13] Germany, 2007 [14] Scotland, 2007 [15] USA, 2007 [16] UK, 2008 [17] Canada, 2008 [18] China, 2009 [19] Hungary, 2009 [20] Sweden, 2009 [21]

80 192 186 258 238 317 116 110 677 1080 136 80 80

MIC90 (mg/L) (% resistance rate) MTZ

VAN

0.38 (0)

1.5 (0)

2 (0) 0.5 (0) 0.25 (0) 1 (0) 2 (0) 0.25 (0) 1 (0) 1 (1.6) 0.5 (0) 1 (0) 0.25 (0)

CLI

ERY

>256 (36) 16 (66.7) >128 (14.7) >256 (43.7) ≥256 (65) 16 (62.9)

2 (0) 1 (0) 1 (0) 2 (0) 4 (0) 1 (0) 1 (0) 1 (0) 2 (0)

TET

>256 (27)

64 (82.2) ≥256 (49) ≥32 (94.8)

1 (0)

128 (71.4) 256 (25) 64 (13.8)

RIF

FA

4 (6.3) >32 (12)

2 (4.3)

>256 >256 (90.9) >128 (71.4) 256 (27.5) 128 (65)

MFX

64 (35.7) 0.25 (7.5)

32 (40) ≥32 (87.1) 16 (13.6) >32 64 (46.4) 32 (25) 16 (15)

2 (17.5)

≥0.06 (0)

2 (1.2) 0.5 (1.3)

0.015 (2.7)a

32 (25) 0.5 (6.3) 0.06 (3.8)

1 (17.9) 0.5 (2.5)

MTZ, metronidazole; VAN, vancomycin; CLI, clindamycin; ERY, erythromycin; TET, tetracycline; MFX, moxifloxacin; RIF, rifampicin; FA, fusidic acid. a Rifaximin was tested instead of RIF in this study.

of 0.0075 mg/L and 0.015 mg/L, respectively, followed by fidaxomicin (OPT-80), with MIC50 and MIC90 values of 0.125 mg/L. In Asia [19], a 1-year survey was conducted in Shanghai, China, in 2007. All isolates were susceptible to piperacillin/tazobactam and MER. Resistance to MFX, ERY, clindamycin (CLI), tetracycline (TET) and RIF was found in 46.4%, 71.4%, 71.4%, 35.7% and 25.0% of the isolates, respectively.

2.2. Strains of common polymerase chain reaction (PCR) ribotypes are more resistant In a UK surveillance programme [17], 667 isolates were characterised by PCR ribotyping. PCR ribotype 027 was the most common, accounting for >40%, followed by types 106 and 001. Antimicrobial susceptibility testing revealed significantly lower

Table 2 Antimicrobial resistance patterns of major polymerase chain reaction (PCR) ribotypes of Clostridium difficile. Country, year [Reference]/ribotype

Sample size

Resistance rate (%) ERY

CLI

TET

MFX

0 3.4 0 0 27.8 0

82.9 6.9 100 0 100 15.8

Europe (2005) [22] 001 014 027 002 017 020

41 31 20 19 19 19

82.9 3.4 100 10.5 100 26.3

85.4 27.6 20 52.6 100 52.6

UK (2005) [24] 001 106

49 50

98 100

40.8

Scotland (2007) [15] 001 106

87 10

100 100

62.1 70

Canada (2008) [25] 027 001

141 72

UK (2008) [17] 001 027 106 002 015

53 280 137 27 25

88a 100a 1.2 0

98.9 90 MIC90 > 32b MIC90 = 32b

MIC90 = 8 MIC90 > 64 MIC90 > 256 MIC90 > 256 MIC90 > 256 MIC90 = 2 MIC90 = 2

MIC90 > 32 MIC90 > 32 MIC90 > 32 MIC90 = 2 MIC90 = 2

The Netherlands (2008) [4] 078

49

78

43c

China (2009) [21] 017 012 014

14 13 4

92.9 92.3 0

92.9 100 75

85.7 7.7 0

78.6 0 25

0 0

77.8 37.5

0 0

11.1 0

Sweden (2009) [21] 005 014

9 8

12c

ERY, erythromycin; CLI, clindamycin; TET, tetracycline; MFX, moxifloxacin; MIC90 , minimal inhibitory concentration for 90% of the organisms. a Levofloxacin was tested instead of MFX in this study. b Gatifloxacin was tested instead of MFX in this study. c Intermediate and complete resistance.

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susceptibility to MTZ in the more common strains (mean MIC range 0.5–0.61 mg/L) compared with the less common ribotypes (mean MIC range 0.09–0.2 mg/L), although no strain was classified as clinically resistant. Similarly, strains of common PCR ribotypes were more resistant to MFX and ERY than the less common strains (MIC90 for MFX >32 mg/L vs. 2 mg/L and for ERY >256 mg/L vs. 2 mg/L). In another study conducted in Poland [23], type 017 was the dominant strain and 94% of macrolide–lincosamide–streptogramin B (MLSB ) type resistance belonged to this type. The studies conducted by Barbut et al. [22] and John and Brazier [24] showed similar results (Table 2). 2.3. Metronidazole MTZ demonstrates high activity against C. difficile strains and resistance is still rare. In 2001, Brazier et al. [26] found one MTZresistant strain among 1000 C. difficile isolates tested by the UK Public Health Laboratory Service Anaerobe Reference Laboratory. However, microbiology laboratories should not consider C. difficile as a pathogen not developing resistance to MTZ. Several reports have emphasised the problem with MTZ resistance. In 1994 and 2002, Peláez et al. [27,28] reported two series (7.7% and 6.3% resistance rates) of MTZ-resistant C. difficile isolates in Spain. In 2008, the same group described that the resistance (12%) to MTZ in toxigenic C. difficile isolates was heterogeneous and inducible [29]. The strain presented slow-growing subpopulations within the inhibition zones around both the disk and the Etest strip. However, all initially resistant isolates became susceptible following serial passages. In addition, the emergence of reduced susceptibility to MTZ has recently been reported in C. difficile isolates in the UK. MICs for the historical C. difficile ribotype 001 were 1.03 mg/L (range 0.25–2 mg/L) compared with 5.94 mg/L (range 4–8 mg/L) (P < 0.001) for recent isolates with reduced MTZ susceptibility (24.4% of isolates) [30]. The exact mechanism of reduced susceptibility to MTZ remains to be determined. MTZ resistance studies in Helicobacter pylori and Bacteroides fragilis identified nitroimidazole genes (nimA, B, C, D and E) associated with resistance [31]. Homologous genes have been identified in Clostridium tetani and Clostridium bifermentans [32,33]. However, resistance due to nim genes in C. difficile has not yet been described. Besides, alternative MTZ resistance mechanisms have been suggested, such as reduced uptake of MTZ and reduced nitroreductase activity. 2.4. Vancomycin To our knowledge, VAN resistance in C. difficile was only reported once in Poland in 1991 using the disk diffusion method [34]. However, decreased susceptibility to VAN has been reported in several studies. Peláez et al. [28] reported a total of 13 isolates with high-level MICs (8–16 mg/L) for VAN in Spain during 1999–2000. The first strain was isolated in 1996 and since then a low but persistent number of resistant isolates was found every year. Mutlu et al. [15] reported that the number of isolates with MICs of 4 mg/L for VAN increased from 2.7% in 1999–2000 to 21.6% in 2005 in Scotland.

The resistance mechanism of C. difficile to VAN has not yet been reported. Acquired VAN resistance in Enterococcus spp. is due to the acquisition of vanA, vanB, vanD, vanE and vanG genes, resulting in the production of peptidoglycan precursors with reduced affinity for glycopeptide antibiotics [35]. However, these genes have not been described in C. difficile strains. 2.5. Erythromycin and clindamycin ERY and CLI are members of the MLSB group of protein synthesis inhibitors. Resistance to CLI and/or ERY is the most common phenotype in C. difficile, with the resistant rate usually >50%. Spigaglia et al. [36] have described five phenotypic classes (EC-a to EC-e) with characteristic susceptibility/resistance patterns to ERY and CLI. CLI has been considered as a ‘protective’ antibiotic with regard to the development of CDI due to type 027. However, in a surveillance programme in Europe in 2008 [37] it was reported that outbreaks due to CLI-resistant type 027 strains have occurred in France, Ireland and Switzerland. A similar outbreak occurred in Denmark in March 2009 [38]. The multiplicity of mechanisms of resistance, which include ribosomal modification, efflux of the antibiotic and drug inactivation, results in a variety of resistance phenotypes. Ribosomal methylation is the most widespread mechanism of resistance and is mediated by more than 20 known classes of erm (erythromycin ribosomal methylase) genes [39]. Numerous erm genes have been characterised and divided into distinct classes based on their sequence similarity. The most prevalent class of Erm determinants is the Erm B-AM class, which has recently been renamed as the ErmB class [40]. High-level resistance against MLS antimicrobials usually requires an erm gene, whereas efflux-mediated resistance confers low-level resistance [39]. MLSB resistance in C. difficile is encoded by the ermB gene located on a mobilisable conjugative transposon called Tn5398 (Table 3) [41,42]. Two studies have reported detection of two copies of the ermB gene in C. difficile [56,57]. In addition, 23S rDNA mutation in high-level ERY-resistant and low-level CLI-resistant (8 mg/L < MIC < 24 mg/L) but erm-negative isolates was recently identified. All isolates harboured a nucleotide substitution (C → T) at position 656 within the 23S rDNA copy on the genome [14]. However, erm genes have not been identified in all clinical isolates expressing high-level resistance to ERY. Other mechanisms, probably other erm genes, confer MLS resistance in C. difficile that were not detectable with the primers used in those studies. It is also possible that efflux mechanisms, encoded by msr genes in staphylococci and mef genes in streptococci, could have caused resistance to MLS [39]. 2.6. Tetracyclines TET resistance varies widely between countries, from 0% (UK and The Netherlands) to 38.9% (Greece) [22]. However, in most studies it is <10% [14,15,22]. Resistance is primarily due to either energy-dependent efflux of TET or protection of the ribosomes from the action of TET. Most TET resistance genes (tet) are carried on

Table 3 Major mechanisms of antimicrobial resistance in Clostridium difficile. Antimicrobial agent

Resistance mechanism

Locus

Example (gene/mutation)

Reference(s)

Erythromycin and clindamycin Tetracycline Moxifloxacin Rifampicin

Altered target (ribosomal methylation) Altered target (ribosomal protection) Altered target (mutation in QRDR) Altered target (mutation leading to reduced binding to RNA polymerase) Altered target (mutation)

Transposon Transposon Chromosome Chromosome

ermB genes tetM Thr82 → Ile substitution in gyrA Arg505 → Lys substitution in rpoB

[14,41,42] [43–47] [48–52] [53,54]

Chromosome

Ala374 → Val substitution in fusA

[55]

Fusidic acid

QRDR, quinolone-resistance-determining region.

H. Huang et al. / International Journal of Antimicrobial Agents 34 (2009) 516–522

autonomously transferable mobile elements and this is one of the reasons for their wide distribution among bacterial genera [58]. Among the genes coding for ribosomal protection proteins, tetM is the most widespread gene class and is usually found on conjugative elements of the Tn916 family, whereas tetW has the second largest host range and is associated with conjugative or non-conjugative elements that may vary among different bacterial species [43,44]. Previous studies showed that the majority of resistant C. difficile strains have the tetM gene carried by the conjugative transposon Tn5397. This element is related to Tn916 but differs in that it contains a Group II intron and has different integration/excision modules. In particular, Tn5397 has a large resolvase gene, tndX, instead of the int and xis genes of Tn916 [45]. Interestingly, Tn916like elements have only been found in some C. difficile clinical isolates, whereas the elements are predominating in other Grampositive bacteria [46]. Recently, the tetW gene region of a human clinical isolate of C. difficile resistant to TET was characterised. This gene was a new allele showing 99% sequence identity to the gene found in the human Bifidobacterium longum F8 strain [47]. 2.7. Fluoroquinolones Fluoroquinolones are a family of broad-spectrum antibiotics and are strongly associated with CDI [7,59]. The in vitro activity of the older fluoroquinolones, such as ciprofloxacin, has been reported to be moderate or poor against anaerobes, including C. difficile. On the other hand, the third- and fourth-generation fluoroquinolones are characterised by improved activity against anaerobic bacteria. However, recent studies indicate that the resistance rate to MFX in C. difficile has increased dramatically compared with the rate of 7% reported in France in 1991 and 1997 [60] and the rate of 12% reported in Germany between 1986 and 2001 [10]. Barbut et al. [22] reported that 37.5% of European isolates were resistant to MFX. Bourgault et al. [12] found that >80% strains isolated from a multiinstitutional outbreak in Québec, Canada, were resistant to MFX. Huang et al. [19] reported a resistance rate of 46.4% among Shanghai isolates. Furthermore, strains resistant to MFX are also resistant to other fluoroquinolones, in particular to LFX, with high-level MICs [48]. Resistance to fluoroquinolones is generally caused by two main mechanisms: (i) alteration of target enzymes caused by chromosomal mutations in encoding genes, leading to decreased affinity for the drug; and (ii) reduced intracellular accumulation due to increased efflux of the drug or reduced permeability. The first mechanism of resistance is widespread in many bacterial species and is due to amino acid substitutions in the quinolone resistance-determining region (QRDR) of the target enzymes [49]. The principal mechanism of quinolone resistance in C. difficile is also determined by alterations in the QRDR of either DNA gyrase subunit GyrA or GyrB [50,51]. Until now, five different amino acid substitutions in gyrA (Thr82 → Ile, Thr82 → Val, Asp71 → Val, Asp81 → Asn and Ala118 → Thr) and six substitutions in gyrB (Asp426 → Val, Asp426 → Asn, Arg447 → Leu, Arg447 → Lys, Ser366 → Ala and Ser416 → Ala) have been identified in clinical isolates [19,48–52]. Thr82 → Ile in gyrA is the most frequent amino acid change, which also characterises the hypervirulent epidemic clone 027. Asp426 → Val has been described in toxin A-negative/toxin B-positive C. difficile epidemic strains [51]. Two substitutions, Ser366 → Ala and Ser416 → Ala, do not appear to have a key role in resistance since they are also detected in susceptible strains. As in other bacteria, gyrA mutations in C. difficile occur more commonly than gyrB mutations. In a European surveillance study [48], sequence analysis of both gyrA and gyrB indicated that 83% of MFX-resistant C. difficile isolates had a substitution in gyrA, 10% had an amino acid substitution in both gyrA and gyrB and 7% had a single amino acid change in gyrB. Furthermore, double

519

substitution in gyrA or gyrB has been reported and is associated with an increase in fluoroquinolone resistance, as observed in bacteria showing multiple substitutions [19,52]. Neither the sequence of topoisomerase IV, the second target enzyme for fluoroquinolone antimicrobials, nor efflux-mediated resistance mechanisms of C. difficile have been described in the literature. However, it is reported that although Thr82 → Ile is usually associated with high-level resistance (MIC ≥ 32 mg/L), sometimes it could be related to an intermediate MIC level. Similar results are found for the substitution of Asp426 → Asn. Further analysis should be performed to verify whether the different phenotypes associated with the same amino acid substitution may be due to the presence of other mechanisms of resistance and/or amino acid changes outside of gyrA and gyrB. 2.8. Rifamycins Two members of the rifamycin class, RIF and RFX, have been used extensively for the treatment of CDI on the basis of in vitro susceptibility data [61]. Hecht et al. [16] reported RFX resistance in 3 (2.7%) of 110 clinical isolates from the USA. Bourgault et al. [12] found that all isolates from an outbreak in Canada were susceptible to RIF. However, Curry et al. [53] reported that 81.5% of the epidemic clone (BI/NAP1) in a large teaching hospital in the USA were resistant to RIF. In most studies, RFX susceptibility data were in agreement with RIF susceptibility; the MICs of both antimicrobials for C. difficile isolates were either very low or very high [53,54]. However, in one study 7% of 163 isolates were resistant to RIF and only 1% were resistant to RFX [62]. Studies of a variety of bacterial genera have shown that exposure to rifamycins in vitro and in vivo can lead to selection of resistant organisms. The major resistance mechanism of rifamycins is mutations leading to reduced binding to RpoB, the ␤ subunit of RNA polymerase. In three-dimensional space, the RpoB amino acids that confer rifamycin resistance either directly interact with RIF or are in close proximity to those that are involved in RIF interactions [63]. In C. difficile, RIF and RFX resistance are also associated with point mutations in rpoB as mentioned above. No other rifamycin resistance mechanisms have been described in C. difficile. To date, eight different RpoB amino acid substitutions have been identified. All of the identified amino acid substitutions reside between amino acids 488 and 548. The Arg505 → Lys substitution is the most frequent single substitution, which usually results in high-level resistance (MIC > 32 mg/L). In contrast, RIFintermediate isolates contain only a His502 → Asn substitution (MIC range 0.003–32 mg/L). Double substitution has also been described [53,54]. 2.9. Fusidic acid (FA) FA is a steroid with a narrow spectrum, with activity against Gram-positive bacteria but no activity against Gram-negative agents. It has been used in Europe for the treatment of staphylococcal infections since the early 1960s and has occasionally been evaluated in CDI with a clinical efficacy comparable with that of MTZ and VAN [64]. FA shows good activity against C. difficile. Leroi et al. [9] reported that the MIC values of FA ranged from 0.125 mg/L to 4 mg/L, with a MIC50 value of 0.75 mg/L and a MIC90 value of 2 mg/L among 80 Australian isolates. Similarly, Aspevall et al. [13] reported that >98% of Swedish isolates were susceptible to FA (MIC range 0.032–1 mg/L). However, FA monotherapy appeared to rapidly select conserved resistant mutants. It was reported by Norén et al. [65] that development of resistance in C. difficile was frequent in patients given FA. Resistance to FA was found in 1 of 88 pre-therapy isolates available, plus in at least one subsequent isolate from 11/20 patients (55%) who remained culture-positive after

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FA therapy. The majority of resistant follow-up isolate(s) belonged to the same PCR ribotype as the susceptible Day 1 isolate, confirming frequent emergence of resistance to FA during treatment. Mechanisms of FA resistance have only been extensively studied in Staphylococcus aureus. It is mainly due to mutations in the chromosomal gene, fusA, which codes for elongation factor G (EFG), a ribosomal translocation enzyme. The mutation alters EF-G so that FA is no longer able to bind to it. Similar mechanisms are characterised in C. difficile. The FA resistance-associated mutations in C. difficile are mainly located in clusters and, as described for S. aureus, distinct locations in fusA appear to be especially important for resistance, such as amino acid residues 432–459. Twelve distinct mutations have been identified. Five of the resistanceassociated mutations identified, i.e. Ala374 → Val, Asp432 → Asn, Thr434 → Ile, His455 → Asn and His455 → Gln, have been described at corresponding positions of the S. aureus fusA. The remaining are novel mutations associated with FA resistance and comprised either a variant substitution at a classical position, i.e. Ser449 → Pro, His455 → Arg and Ile459 → Arg substitutions, or mutations not previously identified in any species, i.e. Met16 → Ile, Ser116 → Leu and Val119 → Ala mutations and the deletion of Ala375. Except the deletion of one amino acid (Ala375) that results in a moderately increased MIC (4 mg/L), the amino acid alterations in EF-G yield MICs of 64–256 mg/L [55]. 3. Antimicrobial susceptibility testing 3.1. Methods for antimicrobial susceptibility testing The value of antibiotic sensitivity testing of anaerobes has been questioned in the past owing to the predictable sensitivity to most anti-anaerobe antibiotics and lack of a simple method for testing. However, with increasing resistance to agents with excellent activity against anaerobes such as MTZ, antibiotic susceptibility testing of anaerobic bacteria therefore becomes important. The methods for antibiotic susceptibility testing of C. difficile have mainly been extensively investigated for MTZ and VAN. This fact clearly affects the magnitude of MICs for C. difficile and changes in susceptibility may be missed by some methods. The correlation coefficient is low for MTZ and VAN by the disk diffusion test with MICs as determined by the Etest; hence the zone of inhibition by disk diffusion test cannot predict the MIC satisfactorily [66]. Poilane et al. [67] reported that although no discrepancies between MICs measured by agar incorporation and Etest were found, MICs determined by the latter are slight lower. However, Baines et al. [30] reported that despite the consistent demonstration of increased MTZ MICs in C. difficile PCR ribotype 001 by both spiral gradient endpoint and agar dilution methods, the results obtained by Etest and the Clinical and Laboratory Standards Institute (CLSI) agar dilution methods failed to correlate. They also found that agar base composition and broth inocula may also affect the MICs. It is described that more reproducible growth of C. difficile occurred on Wilkins–Chalgren agar in comparison with other agar bases including supplemented Brucella agar. Since MTZ heteroresistance of C. difficile isolates is an unstable mechanism that goes undetected if MICs are determined by the CLSI standard method, Peláez et al. [29] recommended that the disk diffusion method (5 ␮g MTZ disk) or Etest with primary fresh C. difficile isolates should be used. 3.2. In vitro susceptibility profiles and clinical outcomes Various organisations have issued guidelines for susceptibility testing and/or recommended different breakpoints for MICs, e.g. CLSI, European Committee on Antimicrobial Susceptibility Testing (EUCAST), British Society for Antimicrobial Chemotherapy (BSAC),

Swedish Reference Group for Antimicrobials (SRGA) or the German Institute for Standardization (DIN). However, in CDI little correlation between clinical treatment outcomes with antibiotic susceptibility results has been reported. The reason might be that all the breakpoints are based on serum drug concentrations but that effective antimicrobial therapy of CDI requires bactericidal intracolonic concentrations. For example, a decreasing effectiveness of MTZ in CDI treatment has been documented recently, however no MTZ-resistant isolates were found in these studies. The suboptimal response of some patients may be related to the wide variability in MTZ levels that have been reported in watery stools during acute CDI (mean 9.3 ± 7.5 ␮g/g, range 0.8–24.2 ␮g/g). Even a modest increase in the MIC of MTZ for C. difficile may result in insufficient faecal antibiotic concentrations to inhibit (vegetative) bacteria [68,69]. Therefore, clinical breakpoints are subject to revision in order to help in predicting outcome and detecting resistance mechanisms. Clinical data, mechanisms of resistance and pharmacokinetics/pharmacodynamics are the major factors that drive the change [70]. 4. Conclusion Resistance to antimicrobials in C. difficile varies widely between countries. Most isolates are susceptible to MTZ and VAN, however decreased sensitivity is emerging. Strains of common PCR ribotypes are more resistant to MFX and ERY than those of the less common ribotypes. Most of the resistance mechanisms that have been identified in C. difficile are similar to those in other Gram-positive bacteria. Further investigations are required to monitor the emergence of specific highly virulent clones and resistance as well as to understand the resistance mechanisms. Better antibiotic stewardship and infection control are also needed to prevent further spread of resistance in C. difficile. Funding: This work was supported by grants from the Scandinavian Society for Antimicrobial Chemotherapy and from the Stockholm County Council. Competing interests: None declared. Ethical approval: Not required. References [1] Hall IC, O’Toole E. Intestinal flora in new-born infants: with a description of a new pathogenic anaerobe, Bacillus difficilis. Am J Dis Child 1935;49:390–402. [2] Bartlett JG, Chang TW, Gurwith M. Antibiotic-associated pseudomembranous colitis due to toxin-producing clostridia. N Engl J Med 1978;298:531–4. [3] O’Connor JR, Johnson S, Gerding DN. Clostridium difficile infection caused by the epidemic BI/NAP1/027 strain. Gastroenterology 2009;136:1913–24. [4] Goorhuis A, Bakker D, Corver J, Debast SB, Harmanus C, Notermans DW, et al. Emergence of Clostridium difficile infection due to a new hypervirulent strain, polymerase chain reaction ribotype 078. Clin Infect Dis 2008;47:1162–70. [5] Rouphael NG, O’Donnell JA, Bhatnagar J, Lewis F, Polgreen PM, Beekmann S, et al. Clostridium difficile-associated diarrhea: an emerging threat to pregnant women. Am J Obstet Gynecol 2008;198:635.e1–6. [6] Owens Jr RC, Donskey CJ, Gaynes RP, Loo VG, Muto CA. Antimicrobial-associated risk factors for Clostridium difficile infection. Clin Infect Dis 2008;46(Suppl. 1):S19–31. [7] Muto CA, Pokrywka M, Shutt K, Mendelsohn AB, Nouri K, Posey K, et al. A large outbreak of Clostridium difficile-associated disease with an unexpected proportion of deaths and colectomies at a teaching hospital following increased fluoroquinolone use. Infect Control Hosp Epidemiol 2005;26:273–80. [8] Kelly CP, LaMont JT. Clostridium difficile—more difficult than ever. N Engl J Med 2008;359:1932–40. [9] 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;21:72–4. [10] Ackermann G, Degner A, Cohen SH, Silva J, Rodloff AC. Prevalence and association of macrolide–lincosamide–streptogramin B (MLSB ) resistance with resistance to moxifloxacin in Clostridium difficile. J Antimicrob Chemother 2003;51:599–603. [11] 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;52:259–63.

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