Molecular epidemiology of Clostridioides (previously Clostridium) difficile isolates from a university hospital in Minas Gerais, Brazil.

The molecular epidemiology of 38 non-duplicate toxigenic Clostridioides (previously Clostridium) difficile isolates from inpatients from a hospital in Brazil during a 6-year period (2012-2017) were investigated by multilocus sequence typing (MLST) and ribotyping. These isolates were classified into 20 sequence types (ST), six (30%) of which were novel, revealing a high diversity in a single hospital. Classic hypervirulent strains ST1/RT027 and ST11/RT078 were not identified, while ST42 (almost all RT106) was the most common type, being detected in 11 (28.9%) strains. Noteworthy, six (15.8%) isolates were classified into five STs from clade 2, four of which were new ST and RT. Our study suggests that possible hypervirulent strains other than ST1/RT027 might be inadvertently circulating in Brazilian hospitals and highlights the importance of permanent surveillance on circulating strains in a national scale.

Two Distinct Patterns of Clostridium difficile Diversity Across Europe Indicating Contrasting Routes of Spread.

Background: Rates of Clostridium difficile infection vary widely across Europe, as do prevalent ribotypes. The extent of Europe-wide diversity within each ribotype, however, is unknown. Methods: Inpatient diarrheal fecal samples submitted on a single day in summer and winter (2012-2013) to laboratories in 482 European hospitals were cultured for C. difficile, and isolates the 10 most prevalent ribotypes were whole-genome sequenced. Within each ribotype, country-based sequence clustering was assessed using the ratio of the median number of single-nucleotide polymorphisms between isolates within versus across different countries, using permutation tests. Time-scaled Bayesian phylogenies were used to reconstruct the historical location of each lineage. Results: Sequenced isolates (n = 624) were from 19 countries. Five ribotypes had within-country clustering: ribotype 356, only in Italy; ribotype 018, predominantly in Italy; ribotype 176, with distinct Czech and German clades; ribotype 001/072, including distinct German, Slovakian, and Spanish clades; and ribotype 027, with multiple predominantly country-specific clades including in Hungary, Italy, Germany, Romania, and Poland. By contrast, we found no within-country clustering for ribotypes 078, 015, 002, 014, and 020, consistent with a Europe-wide distribution. Fluoroquinolone resistance was significantly more common in within-country clustered ribotypes (P = .009). Fluoroquinolone-resistant isolates were also more tightly clustered geographically with a median (interquartile range) of 43 (0-213) miles between each isolate and the most closely genetically related isolate, versus 421 (204-680) miles in nonresistant pairs (P < .001). Conclusions: Two distinct patterns of C. difficile ribotype spread were observed, consistent with either predominantly healthcare-associated acquisition or Europe-wide dissemination via other routes/sources, for example, the food chain.

Patient and Strain Characteristics Associated With Clostridium difficile Transmission and Adverse Outcomes.

Background: No study has used whole-genome sequencing (WGS) to investigate risk factors for Clostridium difficile (CD) transmission between cases, or assessed the impact of recent acquisition on patient outcome. Methods: This 20 month retrospective cohort study included consecutive cytotoxin-positive diarrheal samples, which underwent culture, ribotyping, and WGS (Illumina). Sequenced isolates were compared using single nucleotide variants (SNVs). Independent predictors of acquisition from another case, onward transmission, 120-day recurrence, and 30-day mortality were identified using logistic regression with backwards elimination. Results: Of 660 CD cases, 640 (97%) were sequenced, of which 567 (89%) shared a ribotype with a prior case, but only 227 (35%) were ≤2 SNVs from a prior case, supporting recent acquisition. Plausible (<2 SNVs) recent ward-based acquisition from a symptomatic case was more frequent in certain ribotypes; 64% (67/105) for ribotype-027 cases, compared with 11% (6/57) for ribotype-078. Independent risk factors (adjusted P < .05) for CD acquisition included older age, longer inpatient duration, and ribotype; these factors, and male sex, increased onward transmission. Patients with a plausible donor had a greater risk of recurrence (adjusted P = .001) and trended towards greater 30-day mortality (adjusted P = .06). Ribotype had no additional mortality or recurrence impact after adjusting for acquisition (P > .1). Conclusions: Greater transmission of certain lineages suggests CD may have different reservoirs and modes of transmission. Acquiring CD from a recent case is associated with poorer clinical outcomes. Clinical characteristics associated with increased healthcare-associated CD transmission could be used to target preventative interventions.

Correlation between restriction endonuclease analysis and PCR ribotyping for the identification of Clostridioides (Clostridium) difficile clinical strains.

Restriction endonuclease analysis (REA) and PCR ribotyping are two typing systems that have been frequently utilized for molecular epidemiologic characterization of Clostridioides (Clostridium) difficile. To correlate typing data obtained from each method, we performed both REA and PCR ribotyping on a large and diverse set of historical and contemporary C. difficile infection clinical isolates. Eighty isolates were selected from each reference laboratory in the United States (Microbiology Reference Laboratory, Hines VA Medical Center) and United Kingdom (Clostridium difficile Network for England and Northern Ireland laboratory, University of Leeds). The 160 isolates were assigned to 82 unique ribotypes and 51 unique REA groups (116 unique REA types). In general, concordance between typing methods was good. Dendrogram analysis of PCR ribotype band patterns demonstrated close genetic relationships among strain types with discordant REA and ribotype assignments. While REA typing was more discriminatory, several REA types in this study were further discriminated by PCR ribotyping, indicating that discriminatory value of these typing methods may be strain dependent. These data will assist with molecular epidemiologic surveillance of strains identified by these two commonly used C. difficile typing systems.

Contribution to Clostridium Difficile Transmission of Symptomatic Patients With Toxigenic Strains Who Are Fecal Toxin Negative.

Background: The role of symptomatic patients who are toxigenic strain positive (TS+) but fecal toxin negative (FT-) in transmission of Clostridium difficile is currently unknown. Methods: We investigated the contribution of symptomatic TS+/FT- and TS+/FT+ patients in C. difficile transmission in 2 UK regions. From 2-step testing, all glutamate dehydrogenase (GDH)-positive specimens, regardless of fecal toxin result, from Oxford (April 2012 through April 2013) and Leeds (July 2012 through April 2013) microbiology laboratories underwent culture and whole-genome sequencing (WGS), using WGS to identify toxigenic strains. Plausible sources for each TS+/FT+ case, including TS+/FT- and TS+/FT+ patients, were determined using WGS, with and without hospital admission data. Results: A total of 1447 of 12772 (11%) fecal samples were GDH positive, 866 of 1447 (60%) contained toxigenic C. difficile, and fecal toxin was detected in 511 of 866 (59%), representing 235 Leeds and 191 Oxford TS+/FT+ cases. TS+/FT+ cases were 3 times more likely to be plausibly acquired from a previous TS+/FT+ case than a TS+/FT- patient. Fifty-one of 265 (19%) TS+/FT+ cases diagnosed >3 months into the study were genetically related (≤2 single-nucleotide polymorphisms) to ≥1 previous TS+/FT+ case or TS+/FT- patient: 27 (10%) to only TS+/FT+ cases, 9 (3%) to only TS+/FT- patients, and 15 (6%) to both. Only 10 of 265 (4%) were genetically related to a previous TS+/FT+ or TS+/FT- patient and shared the same ward simultaneously or within 28 days. Conclusions: Symptomatic TS+/FT- patients were a source of C. difficile transmission, although they accounted for less onward transmission than TS+/FT+ cases. Although transmission from symptomatic patients with either fecal toxin status accounted for a low overall proportion of new cases, both groups should be infection control targets.

Comparison of Control of Clostridium difficile Infection in Six English Hospitals Using Whole-Genome Sequencing.

BACKGROUND: Variation in Clostridium difficile infection (CDI) rates between healthcare institutions suggests overall incidence could be reduced if the lowest rates could be achieved more widely. METHODS: We used whole-genome sequencing (WGS) of consecutive C. difficile isolates from 6 English hospitals over 1 year (2013-14) to compare infection control performance. Fecal samples with a positive initial screen for C. difficile were sequenced. Within each hospital, we estimated the proportion of cases plausibly acquired from previous cases. RESULTS: Overall, 851/971 (87.6%) sequenced samples contained toxin genes, and 451 (46.4%) were fecal-toxin-positive. Of 652 potentially toxigenic isolates >90-days after the study started, 128 (20%, 95% confidence interval [CI] 17-23%) were genetically linked (within ≤2 single nucleotide polymorphisms) to a prior patient's isolate from the previous 90 days. Hospital 2 had the fewest linked isolates, 7/105 (7%, 3-13%), hospital 1, 9/70 (13%, 6-23%), and hospitals 3-6 had similar proportions of linked isolates (22-26%) (P ≤ .002 comparing hospital-2 vs 3-6). Results were similar adjusting for locally circulating ribotypes. Adjusting for hospital, ribotype-027 had the highest proportion of linked isolates (57%, 95% CI 29-81%). Fecal-toxin-positive and toxin-negative patients were similarly likely to be a potential transmission donor, OR = 1.01 (0.68-1.49). There was no association between the estimated proportion of linked cases and testing rates. CONCLUSIONS: WGS can be used as a novel surveillance tool to identify varying rates of C. difficile transmission between institutions and therefore to allow targeted efforts to reduce CDI incidence.

Enhanced surveillance of Clostridium difficile infection occurring outside hospital, England, 2011 to 2013.

There are limited national epidemiological data for community-associated (CA)-Clostridium difficile infections (CDIs). Between March 2011 and March 2013, laboratories in England submitted to the Clostridium difficile Ribotyping Network (CDRN) up to 10 diarrhoeal faecal samples from successive patients with CA-CDI, defined here as C. difficile toxin-positive diarrhoea commencing outside hospital (or less than 48 hours after hospital admission), including those cases associated with community-based residential care, with no discharge from hospital within the previous 12 weeks. Patient demographics and C. difficile PCR ribotypes were compared for CA-CDIs in our study and presumed healthcare-associated (HA) CDIs via CDRN. Ribotype diversity indices, ranking and relative prevalences were very similar in CA- vs HA-CDIs, although ribotypes 002 (p ≤ 0.0001),020 (p = 0.009) and 056 (p < 0.0001) predominated in CA-CDIs; ribotype 027 (p = 0.01) predominated in HA-CDIs. Epidemic ribotypes 027 and 078 predominated in institutional residents with CDI (including care/nursing homes) compared with people with CDI living at home. Ribotype diversity decreased with increasing age in HA-CDIs, but not in CA-CDIs. Ribotype 078 CA-CDIs were significantly more common in elderly people (3.4% (6/174) vs 8.7% (45/519) in those aged < 65 and ≥ 65 years, respectively; p = 0.019). No antibiotics were prescribed in the previous four weeks in about twofold more CA-CDI vs HAs (38.6% (129/334) vs 20.3% (1,226/6,028); p < 0.0001). We found very similar ribotype distributions in CA- and HA-CDIs, although a few ribotypes significantly predominated in one setting. These national data emphasise the close interplay between, and likely common reservoirs for, CDIs, particularly when epidemic strains are not dominant.

Outcome of relapsing Clostridium difficile infections do not correlate with virulence-, spore- and vegetative cell-associated phenotypes.

One of the main clinical challenges of Clostridium difficile infections (CDI) is the high rate of relapse episodes. The main determinants involved in relapse of CDI include the presence of antibiotic-resistant C. difficile spores in the colonic environment and a permanent state of dysbiosis of the microbiota caused by antibiotic therapy. A possible scenario is that phenotypes related to the persistence of C. difficile spores might contribute to relapsing infections. In this study, 8 C. difficile isolates recovered from 4 cases with relapsing infection, and 9 isolates recovered from single infection cases were analyzed for PCR ribotyping and the presence of tcdA, tcdB and cdtAB genes. Factors associated to spore persistence, sporulation, spore adherence and biofilm formation and sporulation during biofilm formation were characterized. We also evaluated motility and cytotoxicity. However, we observed no significant difference in the analyzed phenotypes among the different clinical outcomes, most likely due to the high variability observed among strains within clinical backgrounds in each phenotype and the small sample size. It is noteworthy that C. difficile spores adhered to similar extents to undifferentiated and differentiated Caco-2 cells. By contrast, spores of all clinical isolates tested had increased germination efficiency in presence of taurocholate, while decreased sporulation rate during biofilm development in the presence of glucose. In conclusion, these results show that, at least in this cohort of patients, the described phenotypes are not detrimental in the clinical outcome of the disease.

Clostridium difficile ribotype 126 in southern Taiwan: a cluster of three symptomatic cases.

INTRODUCTION: Several virulent Clostridium difficile clones, designated as polymerase chain reaction (PCR) ribotypes 017, 027, or 078, are well recognized in western countries. However, the ribotype distribution of clinical C. difficile isolates in Taiwan remains unclear. METHOD: Between 2010 and 2012, we identified three patients with C. difficile-associated diarrhea (CDAD) at a hospital in southern Taiwan. The C. difficile strains isolated from these patients were further characterized by PCR detection of tcdA, tcdB, tcdC, cdtA, and cdtB, toxinotyping, multilocus sequence typing, ribotyping and repetitive-based PCR. RESULTS: Three C. difficile strains harbored tcdCΔ39 and belonged to multilocus sequence typing 11 (ST11), toxinotype V, and ribotype 126 (a ribotype 078-like clone). Notably, one patient developed pseudomembranous colitis and recurrent CDAD. These three isolates were noted between January 2012 and June 2012 and were identical, as evidenced by repetitive sequence-based PCR, suggestive of case clustering. CONCLUSION: A hypervirulent C. difficile clone, ribotype 126, causing pseudomembranous colitis and recurrent CDAD, is present in southern Taiwan.

Recombinational switching of the Clostridium difficile S-layer and a novel glycosylation gene cluster revealed by large-scale whole-genome sequencing.

BACKGROUND: Clostridium difficile is a major cause of nosocomial diarrhea, with 30-day mortality reaching 30%. The cell surface comprises a paracrystalline proteinaceous S-layer encoded by the slpA gene within the cell wall protein (cwp) gene cluster. Our purpose was to understand the diversity and evolution of slpA and nearby genes also encoding immunodominant cell surface antigens. METHODS: Whole-genome sequences were determined for 57 C. difficile isolates representative of the population structure and different clinical phenotypes. Phylogenetic analyses were performed on their genomic region (>63 kb) spanning the cwp cluster. RESULTS: Genetic diversity across the cwp cluster peaked within slpA, cwp66 (adhesin), and secA2 (secretory translocase). These genes formed a 10-kb cassette, of which 12 divergent variants were found. Homologous recombination involving this cassette caused it to associate randomly with genotype. One cassette contained a novel insertion (length, approximately 24 kb) that resembled S-layer glycosylation gene clusters. CONCLUSIONS: Genetic exchange of S-layer cassettes parallels polysaccharide capsular switching in other species. Both cause major antigenic shifts, while the remainder of the genome is unchanged. C. difficile genotype is therefore not predictive of antigenic type. S-layer switching and immune escape could help explain temporal and geographic variation in C. difficile epidemiology and may inform genotyping and vaccination strategies.

Mixed infection by Clostridium difficile in an in vitro model of the human gut.

OBJECTIVES: Clostridium difficile infection (CDI) is still a major clinical challenge. Previous studies have demonstrated multiple distinct C. difficile strains in the faeces of patients with CDI; yet whether true mixed CDI occurs in vivo is unclear. In this study we evaluated whether two distinct C. difficile strains could co-germinate and co-proliferate in an in vitro human gut model. METHODS: An in vitro triple-stage chemostat was used to study the responses of two PCR ribotype 001 C. difficile strains following exposure to ceftriaxone at concentrations observed in vivo (7 days). C. difficile viable counts (vegetative and spore forms), cytotoxin titres and indigenous microflora viable counts were monitored throughout the experiment. RESULTS: Both C. difficile strains germinated and proliferated following exposure to ceftriaxone. Cytotoxin production was detected in the gut model following C. difficile spore germination and proliferation. Ceftriaxone elicited reduced viable counts of Bifidobacterium spp. and elevated viable counts of Enterococcus spp. CONCLUSIONS: These data suggest that multiple C. difficile strains are able to proliferate concurrently in an in vitro model reflective of the human colon. Previous studies in the gut model have reflected clinical observations so clinicians should be mindful of the possibility that multiple C. difficile strains may infect patients. These observations augment recent human epidemiological studies in this area.

Renal failure and leukocytosis are predictors of a complicated course of Clostridium difficile infection if measured on day of diagnosis.

Nonsevere Clostridium difficile infection (CDI) and severe CDI, which carries a higher risk than nonsevere CDI for treatment failure and CDI recurrence, are difficult to distinguish at the time of diagnosis. To investigate the prognostic value of 3 markers of severe CDI suggested by recent guidelines (fever, leukocytosis, and renal failure), we used the database of 2 randomized controlled trials, which contained information for 1105 patients with CDI. Leukocytosis (risk ratio [RR], 2.29; 95% confidence interval [CI], 1.63-3.21) and renal failure (RR, 2.52; 95% CI, 1.82-3.50) were associated with treatment failure. Fever, although associated with treatment failure (RR, 2.45; 95% CI, 1.07-5.61), was rare. Renal failure was the only significant predictor of recurrence (RR, 1.45; 95% CI, 1.05-2.02). Different timing of measurements of leukocyte count and serum creatinine level around the CDI diagnosis led to a different severity classification in many cases. In conclusion, both leukocytosis and renal failure are useful predictors, although timing of measurement is important.

Oritavancin does not induce Clostridium difficile germination and toxin production in hamsters or a human gut model.

OBJECTIVES: To evaluate the relative propensities of oritavancin and vancomycin to induce Clostridium difficile infection (CDI) in hamster and in vitro human gut models. METHODS: Hamsters received clindamycin (100 mg/kg orally or subcutaneously), oritavancin (50 mg/kg orally) or vancomycin (50 mg/kg orally). C. difficile spores were administered orally the next day. Control hamsters received vehicle only (polyethylene glycol 400) plus spores or clindamycin but no spores. Hamsters were monitored for clinical signs for 20 days. Caecal contents were analysed for C. difficile cells, spores and the presence of (cyto)toxin. Oritavancin and vancomycin were instilled over 7 days into separate in vitro gut models primed with pooled human faeces and inoculated with C. difficile ribotype 027 spores. Gut flora, C. difficile total viable and spore counts, toxin titres and antimicrobial concentrations were determined. RESULTS: All hamsters treated with oritavancin survived up to 20 days, with no evidence of C. difficile spores, vegetative cells or toxin in their caeca. No hamsters treated with clindamycin or vancomycin survived >6 days after spore administration. Death was associated with high C. difficile counts and toxin in caecal contents. In the gut model, oritavancin dosing elicited a rapid, marked decrease in total viable C. difficile and spore counts to below the limit of detection. Vancomycin did not elicit germination or toxin production in the gut model, but C. difficile remained present as spores throughout. CONCLUSIONS: Oritavancin exposure, unlike exposure to vancomycin or clindamycin, did not lead to CDI in hamsters. In both models, oritavancin reduced C. difficile total counts and spores to below detectable limits. The data indicate the potential of oritavancin for CDI treatment, since exposure did not induce C. difficile germination and toxin production, which are known to exacerbate the disease state.

An enhanced DNA fingerprinting service to investigate potential Clostridium difficile infection case clusters sharing the same PCR ribotype.

Of 53 potential Clostridium difficile infection (CDI) case clusters/outbreaks, affecting 2 to 41 patients in 27 institutions, 19% comprised unrelated isolates and 34% had highly related and distinct isolates as shown by multilocus variable-number tandem-repeat analysis, despite sharing a common ribotype. These findings emphasize the value of enhanced fingerprinting to confirm or refute suspected CDI case clusters.

The potential for airborne dispersal of Clostridium difficile from symptomatic patients.

BACKGROUND. The high transmissibility and widespread environmental contamination by Clostridium difficile suggests the possibility of airborne dissemination of spores. We measured airborne and environmental C. difficile adjacent to patients with symptomatic C. difficile infection (CDI). METHODS. We conducted air sampling adjacent to 63 patients with CDI for 180 h in total and for 101 h in control settings. Environmental samples were obtained from surfaces adjacent to the patient and from communal areas of the ward. C. difficile isolates were characterized by ribotyping and multilocus variable-number tandem-repeat analysis to determine relatedness. RESULTS. Of the first 50 patients examined (each for 1 h), only 12% had positive air samples, most frequently those with active symptoms of CDI (10%, vs 2% for those with no symptoms). We intensively sampled the air around 10 patients with CDI symptoms, each for 10 h over 2 days, as well as a total of 346 surface sites. C. difficile was isolated from the air in the majority of these cases (7 of 10 patients tested) and from the surfaces around 9 of the patients; 60% of patients had both air and surface environments that were positive for C. difficile. Molecular characterization confirmed an epidemiological link between airborne dispersal, environmental contamination, and CDI cases. CONCLUSIONS. Aerosolization of C. difficile occurs commonly but sporadically in patients with symptomatic CDI. This may explain the widespread dissemination of epidemic strains. Our results emphasize the importance of single-room isolation as soon as possible after the onset of diarrhea to limit the dissemination of C. difficile.

Clostridioides difficile ribotypes 001 and 126 were predominant in Tehran healthcare settings from 2004 to 2018: a 14-year-long cross-sectional study.

Clostridioides difficile infection (CDI) remains a major healthcare problem worldwide, however, little is known about CDI epidemiology in Iran. Between December 2004 and November 2018, 3649 stool samples were collected from patients in 69 hospitals and medical centres in Tehran and were cultured for the presence of C. difficile; isolates were characterized by PCR ribotyping and toxin genes detection. A total of 582 C. difficile isolates were obtained and the overall CDI prevalence was 15.9%; 290 (49.8%) cases were healthcare-associated (HA) and 292 (50.2%) cases were community-associated (CA). Of these, DNA of 513 isolates submitted for ribotyping. The ribotype and/or WEBRIBO type could be assessed in 366 (62.9%) isolates. The most frequent RTs were 001 (n = 75, 12.9%), 126 (n = 65, 11.2%) and 084 (n = 19, 3.3%); the toxin gene profile tcdA + B + /cdtA + B + (n = 112, 19.2%) was the most common. Fifteen C. difficile isolates (2.6%) did not carry any toxin genes. There was no difference between frequently found RTs in HA-CDI and CA-CDI, except for RT 029 which was more likely to be associated with healthcare origin (12/15, p-value = 0.02). No isolate of RTs 027 or 078 was identified. Importantly, RTs 031, 038, 039, 084, 085 reported previously as RTs with an absence of toxin genes, revealed the presence of toxin genes in our study. Using Simpson's reciprocal index of diversity, we found that RT diversity decreased as the prevalence of the RT 084 increased (R = -0.78, p-value = 0.041). Different patterns in CDI epidemiology underscore the importance of local surveillance and infection control measures in Tehran healthcare settings.

A Role for Tetracycline Selection in Recent Evolution of Agriculture-Associated Clostridium difficile PCR Ribotype 078.

The increasing clinical importance of human infections (frequently severe) caused by Clostridium difficile PCR ribotype 078 (RT078) was first reported in 2008. The severity of symptoms (mortality of ≤30%) and the higher proportion of infections among community and younger patients raised concerns. Farm animals, especially pigs, have been identified as RT078 reservoirs. We aimed to understand the recent changes in RT078 epidemiology by investigating a possible role for antimicrobial selection in its recent evolutionary history. Phylogenetic analysis of international RT078 genomes (isolates from 2006 to 2014, n = 400), using time-scaled, recombination-corrected, maximum likelihood phylogenies, revealed several recent clonal expansions. A common ancestor of each expansion had independently acquired a different allele of the tetracycline resistance gene tetM Consequently, an unusually high proportion (76.5%) of RT078 genomes were tetM positive. Multiple additional tetracycline resistance determinants were also identified (including efflux pump tet40), frequently sharing a high level of nucleotide sequence identity (up to 100%) with sequences found in the pig pathogen Streptococcus suis and in other zoonotic pathogens such as Campylobacter jejuni and Campylobacter coli Each RT078 tetM clonal expansion lacked geographic structure, indicating rapid, recent international spread. Resistance determinants for C. difficile infection-triggering antimicrobials, including fluoroquinolones and clindamycin, were comparatively rare in RT078. Tetracyclines are used intensively in agriculture; this selective pressure, plus rapid, international spread via the food chain, may explain the increased RT078 prevalence in humans. Our work indicates that the use of antimicrobials outside the health care environment has selected for resistant organisms, and in the case of RT078, has contributed to the emergence of a human pathogen.IMPORTANCEClostridium difficile PCR ribotype 078 (RT078) has multiple reservoirs; many are agricultural. Since 2005, this genotype has been increasingly associated with human infections in both clinical settings and the community. Investigations of RT078 whole-genome sequences revealed that tetracycline resistance had been acquired on multiple independent occasions. Phylogenetic analysis revealed a rapid, recent increase in numbers of closely related tetracycline-resistant RT078 (clonal expansions), suggesting that tetracycline selection has strongly influenced its recent evolutionary history. We demonstrate recent international spread of emergent, tetracycline-resistant RT078. A similar tetracycline-positive clonal expansion was also identified in unrelated nontoxigenic C. difficile, suggesting that this process may be widespread and may be independent of disease-causing ability. Resistance to typical C. difficile infection-associated antimicrobials (e.g., fluoroquinolones, clindamycin) occurred only sporadically within RT078. Selective pressure from tetracycline appears to be a key factor in the emergence of this human pathogen and the rapid international dissemination that followed, plausibly via the food chain.

Emergence of reduced susceptibility to metronidazole in Clostridium difficile.

OBJECTIVES: Antimicrobial treatment for Clostridium difficile infection (CDI) has typically been metronidazole, although reports have questioned the efficacy of this option. We screened recently isolated C. difficile (2005-06) for susceptibility to metronidazole and compared results for historic isolates (1995-2001). METHODS: C. difficile ribotypes 001 (n = 86), 106 (n = 81) and 027 (n = 48) and isolates from the 10 other most prevalent ribotypes in Leeds (n = 57) were screened using spiral gradient endpoint analysis (SGE). C. difficile with metronidazole SGE MICs > or = 6 mg/L were analysed further by agar incorporation and Etest. Multiple-locus variable-number tandem-repeat analysis (MLVA) typing was performed for 28 C. difficile isolates. RESULTS: No reduced metronidazole susceptibility was observed in C. difficile ribotypes 106 and 027 (geometric mean SGE MICs 1.11 and 0.90 mg/L, respectively). In contrast, 21 (24.4%) C. difficile ribotype 001 demonstrated reduced susceptibility to metronidazole (geometric mean SGE MICs 3.51 mg/L, P < 0.001). Variations in susceptibility were observed relating to the method and media, but increased metronidazole MICs were confirmed by an agar incorporation method. Geometric mean agar incorporation MICs for historic C. difficile ribotype 001 (n = 72) were 1.03 (range 0.25-2) mg/L compared with 5.94 (4-8) mg/L (P < 0.001) for recent isolates displaying reduced metronidazole susceptibility. MLVA typing revealed two clonal complexes of C. difficile with reduced susceptibility to metronidazole. CONCLUSIONS: We have demonstrated the emergence of reduced susceptibility to metronidazole in 24.4% of the recent C. difficile ribotype 001 isolates from our institution. Our observations could have implications in the clinical setting due to the poor penetration of metronidazole into the colon.

Subtyping of Clostridium difficile PCR ribotypes 591, 106 and 002, the dominant strain types circulating in Medellin, Colombia.

We aimed to achieve a higher typing resolution within the three dominant Clostridium difficile ribotypes (591,106 and 002) circulating in Colombia. A total of 50 C. difficile isolates we had previously typed by PCR-ribotyping, representing the major three ribotypes circulating in Colombia, were analyzed. Twenty-seven isolates of ribotype 591, 12 of ribotype 106 and 11 of ribotype 002 were subtyped by multiple locus variable-number tandem-repeat analysis (MLVA). The presence of the PaLoc genes (tcdA/tcdB), toxin production in culture and antimicrobial susceptibility were also determined. From the total C. difficile ribotypes analyzed, 20 isolates (74%) of ribotype 591, nine (75%) of ribotype 106 and five (45.5%) of ribotype 002 were recovered from patients with Clostridium difficile infection (CDI). MLVA allowed us to recognize four and two different clonal complexes for ribotypes 591 and 002, respectively, having a summed tandem-repeat difference (STRD) <2, whereas none of the ribotype 106 isolates were grouped in a cluster or clonal complex having a STRD >10. Six ribotype 591 and three ribotype 002 isolates belonging to a defined clonal complex were isolated on the same week in two different hospitals. All ribotypes harbored either tcdA+/tcdB+ or tcdA-/tcdB+ PaLoc genes. Moreover, 94% of the isolates were positive for toxin in culture. All isolates were susceptible to vancomycin and metronidazole, while 75% to 100% of the isolates were resistant to clindamycin, and less than 14.8% of ribotype 591 isolates were resistant to moxifloxacina. No significant differences were found among ribotypes with respect to demographic and clinical patients' data; however, our results demonstrated a high molecular heterogeneity of C. difficile strains circulating in Colombia.

Efficacy of hospital cleaning agents and germicides against epidemic Clostridium difficile strains.

OBJECTIVE: To compare the effects of hospital cleaning agents and germicides on the survival of epidemic Clostridium difficile strains. METHODS: We compared the activity of and effects of exposure to 5 cleaning agents and/or germicides (3 containing chlorine, 1 containing only detergent, and 1 containing hydrogen peroxide) on vegetative and spore forms of epidemic and non-epidemic C. difficile strains (3 of each). We carried out in vitro exposure experiments using a human fecal emulsion to mimic conditions found in situ. RESULTS: Cleaning agent and germicide exposure experiments yielded very different results for C. difficile vegetative cells, compared with those for spores. Working-strength concentrations of all of the agents inhibited the growth of C. difficile in culture. However, when used at recommended working concentrations, only chlorine-based germicides were able to inactivate C. difficile spores. C. difficile epidemic strains had a greater sporulation rate than nonepidemic strains. The mean sporulation rate, expressed as the proportion of a cell population that is in spore form, was 13% for all strains not exposed to any cleaning agent or germicide, and it was significantly increased by exposure to cleaning agents or germicides containing detergent alone (34%), a combination of detergent and hypochlorite (24%), or hydrogen peroxide (33%). By contrast, the mean sporulation rate did not change substantially after exposure to germicides containing either a combination of detergent and dichloroisocyanurate (9%) or dichloroisocyanurate alone (15%). CONCLUSIONS: These results highlight differences in the activity of cleaning agents and germicides against C. difficile spores and the potential for some of these products to promote sporulation.