Clinical Isolates Of Clostridium Difficile Research Articles

Antimicrobial susceptibility and molecular typing of toxigenic clinical isolates of Clostridium difficile causing infections in the south of Spain

Antimicrobial susceptibility to 6 antimicrobial agents, PCR-ribotyping and molecular genetics of fluoroquinolone resistance was assessed in 70 toxigenic clinical isolates of C. difficile recovered from patients attended in a hospital in southern Spain with suspected Clostridium difficile infection. Moxifloxacin was the least active drug, mainly driven by the aminoacid substitution Thr82Ile in GyrA, while PCR-ribotype 078 was the most prevalent lineage identified and grouped several of the fluoroquinolone resistant isolates.

Antimicrobial susceptibility and mechanisms of resistance of Greek Clostridium difficile clinical isolates.

This study examined the antimicrobial susceptibility and resistance mechanisms of Clostridium difficile recovered in Greek hospitals during 2012-2015. C. difficile isolates (n=88) were collected from clinically-confirmed C. difficile infection from symptomatic patients in 10 Greek hospitals. Minimum inhibitory concentrations (MICs) of various antimicrobial agents were determined by Etest. Isolates were typed by multilocus sequence typing (MLST). Toxin and resistance genes were detected by PCR. Chromosomal mutations in gyrA, gyrB and rpoB were identified by PCR and sequencing. The genetic environment of resistance genes was characterised by Illumina sequencing. The 88 C. difficile isolates comprised 27 sequence types (STs), with ST37 (n=26) and ST11 (n=21) being the most prevalent. All isolates were susceptible to vancomycin and metronidazole, with variable resistance rates to other antimicrobials. Of the 88 isolates, 45.5% were multidrug-resistant and the majority belonged to ST11 and ST37. The presence of chromosomal mutations in gyrA, gyrB and rpoB was mainly observed in high-risk clones such as ST11 and ST37. The antimicrobial resistance genes ermB, mefA, msrA and tetM were identified at different prevalences and combinations. Additionally, cfrB and cfrC were identified for the first time in Greece and were carried by a Tn6218 transposon and a novel plasmid, respectively. To our knowledge, this is the first study examining the resistance profiles and respective mechanisms of C. difficile recovered in Greek hospitals. Gut commensals such as C. difficile may serve as hubs for further transfer of antimicrobial resistance genes.

Characterization of Bacteriophages Infecting Clinical Isolates of Clostridium difficile.

Clostridium difficile is recognized as a problematic pathogen, causing severe enteric diseases including antibiotic-associated diarrhea and pseudomembranous colitis. The emergence of antibiotic resistant C. difficile has driven a search for alternative anti-infection modalities. A promising strategy for controlling bacterial infection includes the use of bacteriophages and their gene products. Currently, knowledge of phages active against C. difficile is still relatively limited by the fact that the isolation of phages for this organism is a technically demanding method since bacterial host themselves are difficult to culture. To isolate and characterize phages specific to C. difficile, a genotoxic agent, mitomycin C, was used to induce temperate phages from 12 clinical isolates of C. difficile. Five temperate phages consisting of ΦHR24, ΦHN10, ΦHN16-1, ΦHN16-2, and ΦHN50 were successfully induced and isolated. Spotting assays were performed against a panel of 92 C. difficile isolates to screen for susceptible bacterial hosts. The results revealed that all the C. difficile phages obtained in this work displayed a relatively narrow host range of 0–6.5% of the tested isolates. Electron microscopic characterization revealed that all isolated phages contained an icosahedral head connected to a long contractile tail, suggesting that they belonged to the Myoviridae family. Restriction enzyme analysis indicated that these phages possess unique double-stranded DNA genome. Further electron microscopic characterization revealed that the ΦHN10 absorbed to the bacterial surface via attachment to cell wall, potentially interacting with S-layer protein. Bacteriophages isolated from this study could lead to development of novel therapeutic agents and detection strategies for C. difficile.

Comparison of the in vitro antibacterial activity of Ramizol, fidaxomicin, vancomycin, and metronidazole against 100 clinical isolates of Clostridium difficile by broth microdilution

Antibiotic drug development remains a major challenge with few candidates in clinical development. Ramizol, a first-in-class styrylbenzene antibiotic, is under development for the treatment of Clostridium difficile associated disease. Here, we investigate the in vitro antibacterial activity of Ramizol in comparison to fidaxomicin, vancomycin and metronidazole against 100 clinical isolates of C. difficile by the broth microdilution method. We show there is no apparent impact of ribotype, toxin-production, or resistance to fidaxomicin, vancomycin or metronidazole on the activity of Ramizol. Moreover, we show Ramizol has a narrower MIC range translating to potentially better control over the therapeutic dose. Together, these results support the further development of Ramizol for the treatment of C. difficile associated disease.

Molecular characterization, toxin detection and resistance testing of human clinical Clostridium difficile isolates from Lebanon

Clostridium (Clostridioides) difficile is the main cause for nosocomial diarrhoea in industrialised nations. Epidemiologic data on the pathogen’s occurrence in other world regions are still scarce. In this context we characterized with phenotypic and molecular genetic methods C. difficile isolates stemming from hospitalised patients with diarrhoea in Lebanon.From 129 stool samples of symptomatic patients at a tertiary care University hospital in Lebanon, a total of 107 C. difficile strains were cultivated and underwent ribotyping, toxin gene detection and antibiotic resistance testing.Ribotype 014 (RT014, 16.8%) predominated, followed by RT002 (9.3%), RT106 (8.4%) and RT070 (6.5%). Binary toxin gene-positive isolates (RT023, RT078 and RT126) were rarely detected and RT027 was absent. Interestingly, within one isolate only the toxin A gene (tcdA) was detected. Multiple-locus variable-number tandem repeat analysis (MLVA) revealed strong strain diversity in most RTs. The isolates were sensitive to metronidazole and vancomycin, and only a small proportion of strains displayed resistance against moxifloxacin, rifampicin, and clarithromycin (5.6%, 1.9%, and 2.8%), respectively.The data indicate that the genetic strain composition of Lebanese strains differs markedly from the situation seen in Europe and North America. Especially the epidemic RTs seen in the latter regions were almost absent in Lebanon. Interestingly, most strains showed almost no resistance to commonly used antibiotics that are suspected to play a major role in the development of C. difficile infection, despite frequent use of these antibiotics in Lebanon. Thus, the role of antimicrobial resistance as a major driving force for infection development remains uncertain in this area.

Antimicrobial activity of fidaxomicin against Clostridium difficile clinical isolates in Aichi area in Japan.

We evaluated the susceptibility of 100 Japanese Clostridium difficile isolates to fidaxomicin, a new macrocyclic antibiotic. The minimum inhibitory concentration (MIC) range of fidaxomicin was 0.03-0.5μg/mL, with a MIC for inhibition of 50% (MIC50) of 0.12μg/mL, and for inhibition of 90% (MIC90) of 0.25μg/mL. We also evaluated the susceptibilities of the same 100 C.difficile isolates to vancomycin, metronidazole, moxifloxacin, clindamycin, meropenem, and ampicillin. Of all the antibiotics tested, fidaxomicin showed the most potent antimicrobial activity against this group of C.difficile isolates. MIC levels against C.difficile isolates, including those producing binary toxin, did not substantially differ from those previously reported in Europe, North America and Taiwan.

Toxin profiles, PCR ribotypes and resistance patterns of Clostridium difficile: a multicentre study in China, 2012–2013

A total of 178 clinical isolates of Clostridium difficile were collected from five major teaching hospitals representing northern, eastern and southern China from August 2012 to July 2013. Among the 178 isolates, 162 (91.0%) were toxigenic, including 66 (40.7%) toxin A-negative, toxin B-positive, 95 (58.6%) toxin A-positive, toxin B-positive and only 1 (0.6%) toxin A-, toxin B- and binary toxin-positive. Twenty-nine different PCR ribotypes were identified, of which 017 (21.0%), 012 (17.3%) and novel type H (16.7%) were the most prevalent. PCR ribotypes 027 and 078 were not found. All toxigenic strains were susceptible to metronidazole, vancomycin and piperacillin/tazobactam. Resistance to moxifloxacin, clindamycin, erythromycin, tetracycline, chloramphenicol, fusidic acid, imipenem, linezolid and rifampicin was observed in 45.1%, 79.6%, 75.3%, 46.9%, 3.7%, 29.6%, 4.9%, 2.5% and 12.3% of the isolates, respectively. Ribotype 017 showed resistance to more antimicrobial agents tested than other ribotype strains.

Surveillance of Antibiotic Resistance among Hospital- and Community-Acquired Toxigenic Clostridium difficile Isolates over 5-Year Period in Kuwait.

Clostridium difficile infection (CDI) is a leading and an important cause of diarrhea in a healthcare setting especially in industrialized countries. Community-associated CDI appears to add to the burden on healthcare setting problems. The aim of the study was to investigate the antimicrobial resistance of healthcare-associated and community-acquired C. difficile infection over 5 years (2008–2012) in Kuwait. A total of 111 hospital-acquired (HA-CD) and 35 community-acquired Clostridium difficile (CA-CD) clinical isolates from stool of patients with diarrhoea were studied. Antimicrobial susceptibility testing of 15 antimicrobial agents against these pathogens was performed using E test method. There was no evidence of resistance to amoxicillin-clavulanic acid, daptomycin, linezolid, piperacillin-tazobactam, teicoplanin and vancomycin by both HA-CD and CA-CD isolates. Metronidazole had excellent activity against CA-CD but there was a 2.9% resistance rate against HA-CD isolates. Ampicillin, clindamycin, levofloxacin and imipenem resistance rates among the HC-CD vs. CA-CD isolates were 100 vs. 47.4%; 43 vs. 47.4%; 100 vs. 100% and 100 vs. 89%, respectively. An unexpected high rifampicin resistance rate of 15.7% emerged amongst the HA-CD isolates. In conclusion, vancomycin resistance amongst the HA-CD and CA-CD isolates was not encountered in this series but few metronidazole resistant hospital isolates were isolated. High resistance rates of ampicillin, clindamycin, levofloxacin, and imipenem resistance were evident among both CA-CD and HA-CD isolates. Rifampicin resistance is emerging among the HA-CD isolates.

Molecular characterization and antimicrobial susceptibility of tcdA-negative Clostridium difficile isolates from Guangzhou, China

This study aimed to investigate the molecular characteristics and antimicrobial susceptibility of Clostridium difficile clinical isolates in Guangzhou, China. One hundred twenty isolates were collected from Guangzhou General Hospital at the Guangzhou Military Command in China from March 2014 to April 2015, and 9 isolates were identified as tcdA-negative/tcdB-positive (A−B+) strains. Results showed that all of the strains were confirmed to be ST37 and 0 single nucleotide variants (SNVs) were found in the PaLoc region, and >60 SNVs were identified throughout the whole genome sequence. The results show the diversity of the antibiotic and gene mutations present in these strains. All of the A−B+ isolates were highly resistant to clindamycin and erythromycin; showed an average sensitivity to fluoroquinolones; and maintained a high susceptibility to metronidazole, vancomycin, and tigecycline.

Molecular characterization and antimicrobial susceptibilities of Clostridium difficile clinical isolates from Victoria, Australia

Some Australian strain types of Clostridium difficile appear unique, highlighting the global diversity of this bacterium. We examined recent and historic local isolates, finding predominantly toxinotype 0 strains, but also toxinotypes V and VIII. All isolates tested were susceptible to vancomycin and metronidazole, while moxifloxacin resistance was only detected in recent strains.

Variation in germination of Clostridium difficile clinical isolates correlates to disease severity

Over the past two decades, Clostridium difficile infections have been increasing in both number and severity throughout the world. As with other spore forming bacteria, germination is a vital step in the life cycle of this pathogen. Studies have examined differences in sporulation and toxin production among a number of C. difficile clinical isolates; however, few have examined differences in germination and the relationship between this phenotype and disease severity. Here, over 100 C. difficile isolates from the University of Michigan Health System were examined for overall germination in response to various combinations of known germinants (taurocholate) and co-germinants (glycine and histidine). Significant variation was observed among isolates under all conditions tested. Isolates representing ribotype 014-020, which was the most frequently isolated ribotype at our hospital, exhibited increased germination in the presence of taurocholate and glycine when compared to isolates representing other ribotypes. Interestingly, isolates that caused severe disease exhibited significantly lower germination in response to minimal germination conditions (taurocholate only), indicating increased control over germination in these isolates. These data provide a broad picture of C. difficile isolate germination and indicate a role for precise control of germination in disease severity.

Sensitive and Rapid Detection of ermB Gene in Clostridium difficile by Loop-Mediated Isothermal Amplification

Introduction: Macrolide-lincosamide-streptogramin B resistance in Clostridium difficile is mostly due to the ermB resistance determinant. This paper describes a sensitive and rapid molecular method to detect ermB in clinical isolates to guide therapeutic treatment. Methods: Five sets of loop-mediated isothermal amplification (LAMP) primers were designed to target 6 or 8 distinct sequences on erm B gene by Primer Explorer V4 soft ware. The optimal primers were selected for rapid detection and chosen to determine the specificity and sensitivity of the LAMP reactions for erm B by using DNA extracted from reference strain API10463. Twenty-six enteric pathogenic bacteria strains were selected for specificity assays. Both the real-time monitoring of turbidity and the chromogenic reaction using calcein/Mn2+ complex were included to determine negative and positive results. The results demonstrated that target DNA was amplified and visualized by the 2 detection methods within 60 minutes at an isothermal temperature of 62°C. Besides, erm B gene in Clostridium difficile from 62 clinical isolates were tested by the developed LAMP assay followed by conventional PCR. Simultaneously, the tcdA, tcdB, cdtA, cdtB, and tetM genes were detected by PCR. Results: A total of 26 bacteria strains were negative for erm B, which indicated the high-specificity of the primers. The detection limit of LAMP was 36.1 pg/μl DNA, and its sensitivity was 10-fold greater than that of conventional PCR. The positive rate of erm B obtained from 62 clinical isolates of Clostridium difficile were 98.4% (61/62) by LAMP, which was completely consistent with PCR. Besides, among the 62 isolates analyzed in this study, 60 were toxin A-positive and toxin B-positive (A+B+), 1 was toxin Anegative but toxin B-positive (A−B+), 1 was toxin A, toxin B and binary toxin-positives (A+B+CDT+). Both the A−B+ isolate and 2 A+B+ isolates were negative for tet M, while the rest of isolates were positive (95.2%,59/62). Conclusion: This study is the first report regarding the application of the LAMP assay for detection of erm B gene in Clostridium difficile. The developed LAMP method is exhibited to be a potentially valuable, sensitive, specific, simple, and inexpensive assay for the rapid detection of erm B.

Effect of Lactobacillus plantarum Strains on Clinical Isolates of Clostridium difficile in vitro

Probiotic bacteria are proposed for prevention of Clostridium difficile associated diarrhea. The aim of this in vitro study was to evaluate the influence of five Lactobacillus plantarum strains to the survival of C. difficile reference strains (M13042 and VPI 10463) and clinical isolates (n=12) using co-culturing and micro-titre plate assay. Changes in bacterial growth were assessed over the time period of 48 h. Quantitative analysis of C. difficile population revealed that there was a significant decrease of C. difficile in co-culture compared to the control (p=0.01). Susceptibility against L. plantarum was C. difficile strain specific, while L. plantarum was not affected by the presence of C. difficile. Reference strains were more sensitive to inhibition than most of the clinical strains (M13042 strain vs eight clinical strains, p=0.03; VPI vs six clinical strains, p=0.04). Fluoroquinolone resistant C. difficile strains were less inhibited by L. plantarum than sensitive strains (p<0.05). In the micro-titre plate assay experiment the inhibition of C. difficile was not related to any particular C. difficile strains however, inhibitory activity was affected by treatment of supernatants. Supernatants of tested lactobacilli inhibited the C. difficile growth from 72% to 82% if nonneutralized (p=0.001); 43% to 68% if neutralized (p=0.003) and 92% to 99% (p=0.001) if supernatant was neutralized and heated as compared to controls.

Multilocus sequence typing of 33 clinical Clostridium difficile isolates

Objective To investigate the multilocus sequence typing (MLST) for clinical Clostridium difficile isolates in Shijiazhuang of China. Methods A laboratory-based study was carried out retrospectively to characterize 33 C.difficile strains isolated from diarrhea patients during July 2010 to December 2011 according to toxin types,PCR ribotyping and MLST.START2 system was used to analyze the results. Results Ten ST types (ST2/3/4/35/37/53/54/55/102/172) and 12 PCR ribotyping were identified in 33 C.difficile strains.The most prevalent ST types and PCR ribotyping were ST37/SⅠ (9/33,27.3%) and ST54/SⅢ (8/33,24.2%).ST172 was a new ST type reported firstly in the world,and has been included in the MLST database.Phylogenetic analysis also revealed that 6 STs (ST2,ST3,ST4,ST53,ST54,ST102) were placed in one large group.Genotypes ST35,ST37,ST55,ST172 were considered as singletons. Conclusions The most common ST types for 33 clinical C.difficile strains isolated from Shijiazhuang were ST37 and ST54.No significant homology was found between ST types and hospitals.(Chin J Lab Med,2013,36:920-925) Key words: Clostridium difficile; Molecular typing; PCR ribotyping; Multilocus sequence typing

Genetic analysis of Tn916-like elements conferring tetracycline resistance in clinical isolates of Clostridium difficile

As an important clinically relevant pathogen, Clostridium difficile has a high multidrug resistance rate. Conjugative transposons play a vital role in its resistance phenotype. In the present study, 34 tetracycline-resistant clinical isolates of C. difficile were studied to detect tetracycline resistance genes and the presence of transposons. Thirty-two isolates were found to harbour Tn916-like elements carrying the tet(M) resistance gene, of which only one copy existed in the genome by Southern blot analysis. To characterise the genetic organisation of the Tn916-like elements, overlap PCR assays were performed with nine primer pairs, revealing three types of elements designated T1 to T3. The prevalent element T1 lacking PCRA (ORF23 to ORF21) and PCRB (ORF21 to ORF20) products, present in the epidemic ST37 clone, was further analysed by genome walking PCR in the left and right end sequences of the novel Tn916-like element. A gene coding for an FtsK/SpoIIIE family protein was found to replace the ORF24 to ORF21 region in Tn916. Moreover, the element could hardly conjugate between cells by filter mating experiments. These findings suggest that the dissemination of Tn916-like elements in epidemic ST37 strains in China was likely to have been conferred by clonal spread, signifying the importance of future surveillance and characterisation of conjugative transposons.

Diversity of cwp loci in clinical isolates of Clostridium difficile

An increased incidence of Clostridium difficile infection (CDI) is associated with the emergence of epidemic strains characterized by high genetic diversity. Among the factors that may have a role in CDI is a family of 29 paralogues, the cell-wall proteins (CWPs), which compose the outer layer of the bacterial cell and are likely to be involved in colonization. Previous studies have shown that 12 of the 29 cwp genes are clustered in the same region, named after slpA (cwp1), the slpA locus, whereas the remaining 17 paralogues are distributed throughout the genome. The variability of 14 of these 17 cwp paralogues was determined in 40 C. difficile clinical isolates belonging to six of the currently prevailing PCR ribotypes. Based on sequence conservation, these cwp genes were divided into two groups, one comprising nine cwp loci having highly conserved sequences in all isolates, and the other five loci showing low genetic conservation among isolates of the same PCR ribotype, as well as between different PCR ribotypes. Three conserved CWPs, Cwp16, Cwp18 and Cwp25, and two variable ones, Cwp26 and Cwp27, were characterized further by Western blot analysis of total cell extracts or surface-layer preparations of the C. difficile clinical isolates. Expression of genetically invariable CWPs was well conserved in all isolates, whilst genetically variable CWPs were not always expressed at comparable levels, even in strains containing identical sequences but belonging to different PCR ribotypes. This is the first report on the distribution and variability of a number of genes encoding CWPs in C. difficile.

Clostridium difficile clinical isolates exhibit variable susceptibility and proteome alterations upon exposure to mammalian cationic antimicrobial peptides

Clostridium difficile is a leading cause of hospital-acquired bacterial infections in the United States, and the increased incidence of recurrent C. difficile infections is particularly problematic. The molecular mechanisms of C. difficile colonization, including its ability to evade host innate immune responses, is poorly understood. We hypothesized that epidemic-associated C. difficile clinical isolates would exhibit increased resistance to mammalian, gut-associated, cationic antimicrobial peptides such as the cathelicidin LL-37. Standardized susceptibility tests as well as comparative proteomic analyses revealed that C. difficile strains varied in their responses to LL-37, with epidemic-associated 027 ribotype isolates displaying greater resistance. Further, exposure of C. difficile strains to sub-lethal concentrations of LL-37 resulted in increased resistance to subsequent peptide challenge, suggesting the presence of inducible resistance mechanisms. Correspondingly, LL-37 exposure altered the C. difficile proteome, with marked changes in abundance of cell wall biosynthesis proteins, surface layer proteins, ABC transporters and lysine metabolism pathway components. Taken together, these results suggest that innate immune avoidance mechanisms could facilitate robust colonization by C. difficile.

Association between PCR ribotypes and antimicrobial susceptibility among Clostridium difficile isolates from healthcare-associated infections in South Korea

In this study, the association between antimicrobial susceptibility, PCR ribotype and presence of the ermB gene in clinical isolates of Clostridium difficile was investigated. PCR ribotyping and ermB gene PCR were performed on 131 C. difficile isolates. The susceptibility of these isolates to metronidazole, vancomycin, piperacillin/tazobactam (TZP), clindamycin, moxifloxacin and rifaximin was also determined. Use of antibiotics within the previous 2 months was documented. Resistance rates to clindamycin, moxifloxacin and rifaximin were 67.9%, 62.6% and 19.1%, respectively. No metronidazole, vancomycin or TZP resistance was detected. Previous exposure to moxifloxacin was significantly correlated with resistance to this antibiotic, but prior use of clindamycin was not significantly correlated with clindamycin resistance. Sixty-four strains (48.9%) carried the ermB gene, of which all but one (98.5%) were resistant to clindamycin. The clindamycin resistance rates of the common PCR ribotypes (018, 017 and 001) were 91.4%, 100% and 84.2%, respectively, and their moxifloxacin resistance rates were 91.4%, 95.0% and 78.9%, respectively. Resistance rates to rifaximin were 5.7% and 95.0% in ribotype 018 and 017 strains, whilst none of the 001 strains were resistant to rifaximin. In conclusion, the common ribotypes 018, 017 and 001 of C. difficile have high rates of resistance to clindamycin and moxifloxacin, but differ greatly in the frequency of rifaximin resistance.

Characterizations of Clinical Isolates of Clostridium difficile by Toxin Genotypes and by Susceptibility to 12 Antimicrobial Agents, Including Fidaxomicin (OPT-80) and Rifaximin: a Multicenter Study in Taiwan

A total of 403 nonduplicate isolates of Clostridium difficile were collected at three major teaching hospitals representing northern, central, and southern Taiwan from January 2005 to December 2010. Of these 403 isolates, 170 (42.2%) were presumed to be nontoxigenic due to the absence of genes for toxins A or B or binary toxin. The remaining 233 (57.8%) isolates carried toxin A and B genes, and 39 (16.7%) of these also had binary toxin genes. The MIC(90) of all isolates for fidaxomicin and rifaximin was 0.5 μg/ml (range, ≤ 0.015 to 0.5 μg/ml) and >128 μg/ml (range, ≤ 0.015 to >128 μg/ml), respectively. All isolates were susceptible to metronidazole (MIC(90) of 0.5 μg/ml; range, ≤ 0.03 to 4 μg/ml). Two isolates had reduced susceptibility to vancomycin (MICs, 4 μg/ml). Only 13.6% of isolates were susceptible to clindamycin (MIC of ≤ 2 μg/ml). Nonsusceptibility to moxifloxacin (n = 81, 20.1%) was accompanied by single or multiple mutations in gyrA and gyrB genes in all but eight moxifloxacin-nonsusceptible isolates. Two previously unreported gyrB mutations might independently confer resistance (MIC, 16 μg/ml), Ser416 to Ala and Glu466 to Lys. Moxifloxacin-resistant isolates were cross-resistant to ciprofloxacin and levofloxacin, but some moxifloxacin-nonsusceptible isolates remained susceptible to gemifloxacin or nemonoxacin at 0.5 μg/ml. This study found the diversity of toxigenic and nontoxigenic strains of C. difficile in the health care setting in Taiwan. All isolates tested were susceptible to metronidazole and vancomycin. Fidaxomicin exhibited potent in vitro activity against all isolates tested, while the more than 10% of Taiwanese isolates with rifaximin MICs of ≥ 128 μg/ml raises concerns.

Reproducibility of broth microdilution and comparison to agar dilution for testing CB-183,315 against clinical isolates of Clostridium difficile

This study evaluated the reproducibility and agreement of broth microdilution to agar dilution (AD) for testing CB-183,315, a novel lipopeptide antibiotic for Clostridium difficile. Reproducibility was 100% within ± one 2-fold dilution for 10 strains tested; agreement was 90–95% within one 2-fold dilution with AD for 103 clinical isolates.