Antibiotic Resistances of Clostridioides difficile.

The rapid evolution of antibiotic resistance in Clostridioides difficile and the consequent effects on prevention and treatment of C. difficile infections (CDIs) are a matter of concern for public health. Antibiotic resistance plays an important role in driving C. difficile epidemiology. Emergence of new types is often associated with the emergence of new resistances, and most of the epidemic C. difficile clinical isolates is currently resistant to multiple antibiotics. In particular, it is to worth to note the recent identification of strains with reduced susceptibility to the first-line antibiotics for CDI treatment and/or for relapsing infections. Antibiotic resistance in C. difficile has a multifactorial nature. Acquisition of genetic elements and alterations of the antibiotic target sites, as well as other factors, such as variations in the metabolic pathways or biofilm production, contribute to the survival of this pathogen in the presence of antibiotics. Different transfer mechanisms facilitate the spread of mobile elements among C. difficile strains and between C. difficile and other species. Furthermore, data indicate that both genetic elements and alterations in the antibiotic targets can be maintained in C. difficile regardless of the burden imposed on fitness, and therefore resistances may persist in C. difficile population in absence of antibiotic selective pressure.

Comparison of microbiological profile of enterotoxigenic Bacteroides fragilis (ETBF) isolates from subjects with colorectal cancer (CRC) or intestinal pre-cancerous lesions versus healthy individuals and evaluation of environmental factors involved in intestinal dysbiosis.

OBJECTIVE: The aim of this study was to analyze enterotoxigenic Bacteroides fragilis (ETBF) isolates from colorectal biopsies of subjects with a histological analysis positive for colorectal cancer (CRC), pre-cancerous lesions (pre-CRC) or with a healthy intestinal tissue and to evaluate the environmental factors that may not only concur to CRC development but may also affect gut microbiota composition. METHODS: ETBF isolates were typed using the ERIC-PCR method, while PCR assays were performed to investigate the bft alleles, the B. fragilis pathogenicity island (BFPAI) region and the cepA, cfiA and cfxA genes. Susceptibility to antibiotics was tested using the agar dilution method. Environmental factors that could play a role in promoting intestinal dysbiosis were evaluated throughout a questionnaire administered to the subjects enrolled. RESULTS: Six different ERIC-PCR types were identified. The type denominated C in this study was the most prevalent, in particular among the biopsies of subjects with pre-CRC, while an isolate belonging to a different type, denominated F, was detected in a biopsy from a subject with CRC. All the ETBF isolates from pre-CRC or CRC subjects had a B. fragilis pathogenicity island (BFPAI) region pattern I, while those from healthy individuals showed also different patterns. Furthermore, 71% of isolates from subjects with pre-CRC or CRC were resistant to two or more classes of antibiotics vs 43% of isolates from healthy individuals. The B. fragilis toxin BFT1 was the most frequently detected in this study, confirming the constant circulation of this isoform strains in Italy. Interestingly, BFT1 was found in 86% of the ETBF isolates from patients with CRC or pre-CRC, while the BFT2 was prevalent among the ETBF isolates from healthy subjects. No substantial differences based on sex, age, tobacco and alcohol consumption were observed between healthy and non-healthy individuals included in this study, while most of the subjects with CRC or pre-CRC lesions were subjected to pharmacological therapy (71%) and showed a body mass index (BMI) that falls within the overweight range (86%). CONCLUSIONS: Our data suggest that some types of ETBF seem to better adapt and colonize the human gut and that the selective pressure exerted by factors related to lifestyle, such as pharmacological therapy and weight, could facilitate their persistence in the gut and their possible involvement in CRC development.

Clostridioides difficile infection (CDI) during the COVID-19 pandemic.

The ongoing coronavirus disease (COVID-19) pandemic has dramatically tested healthcare systems around the world, with serious repercussions on the measures of prevention and control of hospital-acquired infections (HAIs). Among HAIs, Clostridioides difficile infection (CDI) represents one of the most important global public health threats. Although the full impact of the COVID-19 pandemic on CDI remains undetermined, depending on the development of the pandemic in the coming months, in this review literature studies of the last three years have been considered in order to depict the current situation, and make some considerations about possible future developments. If on the one hand, a general reduction in CDI incidence has been reported in several settings, mainly due to the extraordinary reinforcement of infection prevention measures, on the other hand, the critical circumstances experienced in many hospitals have limited the effectiveness of these measures, particularly in the intensive care units (ICUs), increasing the possibility of the occurrence of hospital-acquired CDI (HA-CDI). New concerns have arisen from the decrease in C. difficile testing and the increased use of broad-spectrum antibiotics reported during the pandemic. In particular, overuse of antibiotics and disinfectants may lead to a selection of resistant C. difficile strains not only in hospitals but also in the community. Furthermore, patients infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and patients that have survived COVID-19 may represent a new group of frail patients potentially at a higher risk of CDI, a group that could potentially increase in size due to SARS-CoV-2 evolution. In the dramatic COVID-19 era, the multifactorial nature of CDI has emerged more clearly than before, highlighting the necessity of a strong refocus on efforts to improve prevention strategies and to integrate CDI surveillance in a One Health prospective in order to curtail the public health threat posed by this infection in the next future.

Mechanisms of antibiotic resistance of Clostridioides difficile.

Clostridioides difficile (CD) is one of the top five urgent antibiotic resistance threats in USA. There is a worldwide increase in MDR of CD, with emergence of novel strains which are often more virulent and MDR. Antibiotic resistance in CD is constantly evolving with acquisition of novel resistance mechanisms, which can be transferred between different species of bacteria and among different CD strains present in the clinical setting, community, and environment. Therefore, understanding the antibiotic resistance mechanisms of CD is important to guide optimal antibiotic stewardship policies and to identify novel therapeutic targets to combat CD as well as other bacteria. Epidemiology of CD is driven by the evolution of antibiotic resistance. Prevalence of different CD strains and their characteristic resistomes show distinct global geographical patterns. Understanding epidemiologically driven and strain-specific characteristics of antibiotic resistance is important for effective epidemiological surveillance of antibiotic resistance and to curb the inter-strain and -species spread of the CD resistome. CD has developed resistance to antibiotics with diverse mechanisms such as drug alteration, modification of the antibiotic target site and extrusion of drugs via efflux pumps. In this review, we summarized the most recent advancements in the understanding of mechanisms of antibiotic resistance in CD and analysed the antibiotic resistance factors present in genomes of a few representative well known, epidemic and MDR CD strains found predominantly in different regions of the world.

COVID-19 and Clostridioides difficile infection (CDI): Possible implications for elderly patients.

COVID-19 dramatically affects the elderly. Due to the large usage of antibiotics during the current pandemic and the gastrointestinal manifestations of COVID-19, the elderly population, hospitalized patients, residents in LTCFs and persons that survived the COVID-19 might be more prone to Clostridioides difficile infections (CDI). A renewed attention to CDI is necessary during the ongoing COVID-19 pandemic.

Microbiological characteristics of human and animal isolates of Clostridioides difficile in Italy: Results of the Istituto Superiore di Sanità in the years 2006-2016.

The increased incidence of Clostridioides difficile infection (CDI) and the emergence of highly virulent types highlight the need of microbiological characterization to gain insight CDI epidemiological changes. This paper, reporting data obtained by the Istituto Superiore di Sanità Central Laboratory Service for C. difficile (ISS-CLSCD) in 2006-2016, provides a first long-term microbiological analysis of human and animal C. difficile strains circulating in Italy. The number of human isolates analyzed by ISS-CLSCD significantly increased over the time (170 in 2006-2011 vs 661 in 2012-2016). Independently from the year of isolation, 42% of the clinical isolates belonged to the PCR-ribotype (RT) 018-lineage (RT 018, RT 607, RT 541, PR07661 and PR14328), with RT 018 and RT 607 grouping the majority of isolates. This lineage was significantly associated to CDIs occurred in the General Medicine Units, Clinic Units or Long-Term Care Facilities, while it was rarely found in pediatric patients. Although the percentage of isolates positive for the binary toxin (CDT) was stable during the study (20%), several CDT-positive RTs emerged in 2012-2016, including RT 027. In total, 32 RTs overlapped between animals and humans and six of these RTs were non-toxigenic. The two lineages prevalent in animals, the RT 078-lineage and the RT 569-lineage (RT 569, RT 049, RT 056 and RT 727), were also found in humans, while the RT 018-lineage was rarely detected in animals, suggesting that it is prevalently associated to human infections. Sixty-two percent of clinical isolates showed a multidrug-resistance (MDR) phenotype, with resistance to rifampicin characterizing successful RTs. A MDR phenotype was also observed in 18% of animal isolates, in particular from dogs, supporting animals as potential reservoirs of resistant C. difficile strains. Interestingly, multiple resistances were observed in both human and animal non-toxigenic isolates suggesting their contribution to antibiotic resistance spread among C. difficile population. All these data indicate that CDI is an issue of growing concern in Italy, highlighting the need for a standardized surveillance in our Country and an interdisciplinary approach to deal successfully with this infection.

Antibiotic Resistances of Clostridium difficile.

The rapid evolution of antibiotic resistance in Clostridium difficile and the consequent effects on prevention and treatment of C. difficile infections (CDIs) are matter of concern for public health. Antibiotic resistance plays an important role in driving C. difficile epidemiology. Emergence of new types is often associated with the emergence of new resistances and most of epidemic C. difficile clinical isolates is currently resistant to multiple antibiotics. In particular, it is to worth to note the recent identification of strains with reduced susceptibility to the first-line antibiotics for CDI treatment and/or for relapsing infections. Antibiotic resistance in C. difficile has a multifactorial nature. Acquisition of genetic elements and alterations of the antibiotic target sites, as well as other factors, such as variations in the metabolic pathways and biofilm production, contribute to the survival of this pathogen in the presence of antibiotics. Different transfer mechanisms facilitate the spread of mobile elements among C. difficile strains and between C. difficile and other species. Furthermore, recent data indicate that both genetic elements and alterations in the antibiotic targets can be maintained in C. difficile regardless of the burden imposed on fitness, and therefore resistances may persist in C. difficile population in absence of antibiotic selective pressure.

Clostridium difficile causing pediatric infections: New findings from a hospital-based study in Italy.

Recent studies support a change of Clostridium difficile infections (CDIs) epidemiology in pediatric patients. Since limited information is available about C. difficile in this population, we investigated the epidemiology of CDI in a large pediatric hospital that acts as reference centre in Italy and analyzed C. difficile isolates to identify the prevalent PCR-ribotypes (RTs), the binary toxin (CDT)-positive strains and the antibiotic susceptibility patterns. The CDI incidence was 6.6 cases/1000 admissions and the majority (92%) of CDI were healthcare-associated (47% occurred in the Hematology-Oncology and in the Gastroenterology units). Most of symptomatic children <3 years with a positive culture for C. difficile were negative for other gastrointestinal pathogens, supporting C. difficile as cause of disease in these patients, including those showing recurrences. Strains RT020 (16%) and RT014 (14%) were identified as the main cause of infection, while RT356/607 and RT018, predominant in Italian adult patients, were absent (RT356/607) or rarely found (RT018) among children. CDT-positive strains represented the 20% of the total number of isolates analyzed. In particular, two emerging types, RT033 and RT442, were recognized as Toxin A-/Toxin B-/CDT+. Resistance to antibiotics characterized almost 50% of the toxigenic isolates analyzed in this study and, in particular, 20% of them were multidrug resistant (MDR). The emergence and circulation of strains with peculiar toxins profiles and/or MDR strongly highlight the necessity of a rapid CDI diagnosis, a careful monitoring of C. difficile in pediatric patients and a more strict control of antibiotics usage in the Italian pediatric hospitals.

Epidemiology and outcome of Clostridium difficile infections in patients hospitalized in Internal Medicine: findings from the nationwide FADOI-PRACTICE study.

BACKGROUND: Clostridium difficile (CD) is a leading cause of diarrhoea among hospitalized patients. The objective of this study was to evaluate the rate, the optimal diagnostic work-up, and outcome of CD infections (CDI) in Internal Medicine (IM) wards in Italy. METHODS: PRACTICE is an observational prospective study, involving 40 IM Units and evaluating all consecutive patients hospitalized during a 4-month period. CDI were defined in case of diarrhoea when both enzyme immunoassay for GDH, and test for A/B toxin were positive. Patients with CDI were followed-up for recurrences for 4 weeks after the end of therapy. RESULTS: Among the 10,780 patients observed, 103 (0.96 %) showed CDI, at admission or during hospitalization. A positive history for CD, antibiotics in the previous 4 weeks, recent hospitalization, female gender and age were significantly associated with CDI (multivariable analysis). In-hospital mortality was 16.5 % in CD group vs 6.7 % in No-CD group (p < 0.001), whereas median length of hospital stay was 16 (IQR = 13) vs 8 (IQR = 8) days (p < 0.001) among patients with or without CDI, respectively. Rate of CD recurrences was 14.6 %. As a post-hoc evaluation, 23 out of 34 GDH+/Tox- samples were toxin positive, when analysed by molecular method (a real-time PCR assay). The overall CD incidence rate was 5.3/10,000 patient-days. CONCLUSIONS: Our results confirm the severity of CDI in medical wards, showing high in-hospital mortality, prolonged hospitalization and frequent short-term recurrences. Further, our survey supports a 2-3 step algorithm for CD diagnosis: EIA for detecting GDH, A and B toxin, followed by a molecular method in case of toxin-negative samples.

Survey of diagnostic and typing capacity for Clostridium difficile infection in Europe, 2011 and 2014.

Suboptimal laboratory diagnostics for Clostridium difficile infection (CDI) impedes its surveillance and control across Europe. We evaluated changes in local laboratory CDI diagnostics and changes in national diagnostic and typing capacity for CDI during the European C. difficile Infection Surveillance Network (ECDIS-Net) project, through cross-sectional surveys in 33 European countries in 2011 and 2014. In 2011, 126 (61%) of a convenience sample of 206 laboratories in 31 countries completed a survey on local diagnostics. In 2014, 84 (67%) of these 126 laboratories in 26 countries completed a follow-up survey. Among laboratories that participated in both surveys, use of CDI diagnostics deemed 'optimal' or 'acceptable' increased from 19% to 46% and from 10% to 15%, respectively (p < 0.001). The survey of national capacity was completed by national coordinators of 31 and 32 countries in 2011 and 2014, respectively. Capacity for any C. difficile typing method increased from 22/31 countries in 2011 to 26/32 countries in 2014; for PCR ribotyping from 20/31 countries to 23/32 countries, and specifically for capillary PCR ribotyping from 7/31 countries to 16/32 countries. While our study indicates improved diagnostic capability and national capacity for capillary PCR ribotyping across European laboratories between 2011 and 2014, increased use of 'optimal' diagnostics should be promoted.

Clostridium difficile ribotype 033 colitis in a patient following broad-spectrum antibiotic treatment for KPCproducing Klebsiella pneumoniae infection, Italy.

This report describes a case of Clostridium difficile ribotype 033 colitis in a patient treated with multiple antibiotics for KPC-producing Klebsiella pneumoniae pancreatitis. Diagnostic, clinical and therapeutic features are discussed. To the best of our knowledge, this is the first case of C. difficile ribotype 033 clinical infection reported from Italy.

Characterization of Clostridium difficile PCR-ribotype 018: A problematic emerging type.

Recent surveys indicate that the majority of toxigenic Clostridium difficile strains isolated in European hospitals belonged to PCR-ribotypes (RTs) different from RT 027 or RT 078. Among these types, RT 018 has been reported in Italy and, more recently, in Korea and Japan. In Italy, strains RT 018 have become predominant in the early 2000s, whereas the majority of strains isolated before were RT 126, a type belonging to the same lineage as the RT 078. In this study, we have found that Italian strains RT 018 are resistant to erythromycin, clindamycin, moxifloxacin and rifampicin. Rifampicin resistance is rarely observed in strains RT 018 from other countries and in Italian strains RT 078 and RT 126, therefore the decennial use of rifamycin antibiotics in Italy may be one of the driving factors for the spread of RT 018 in our country. The strains RT 018 examined showed a significant higher adhesion to Caco-2 cells compared to strains RT 078 and RT 126. Furthermore, strains RT 018 became predominant in in vitro competition assays with strains RT 078 or RT 126. If maintained in vivo, these characteristics could lead to a rapid colonization of the intestine by strains RT 018. Under the conditions used, isolates RT 018 produced significantly higher toxins levels compared to strains RT 078 and RT 126, while heat-resistant CFUs production seems to be strain-dependent. Robust toxin production and enhanced sporulation could in part explain the high diffusion and interpatient transmissibility observed for strains RT 018 in the hospital environment. In conclusion, the characteristics observed in the Italian isolates RT 018 seem to contribute in conferring an adaptive advantage to these strains, allowing their successful spread in our country.

Diagnostic testing for Clostridium difficile in Italian microbiological laboratories.

A laboratory diagnosis survey of Clostridium difficile infection (CDI) was performed in Italy in 2012-2013. Questionnaires from 278 healthcare settings from 15 regions of Italy were collected and analysed. Eighty seven percent of the laboratories declared to routinely perform CDI diagnosis, 99% of them only after the clinician's request. Among the 216 laboratories providing information on the size of the hospitals in which they were located, 65 had more than 500 beds (large hospitals), while 151 had less than 500 beds (small hospitals). The average percentage of positive tests for C. difficile toxins was 12.2%. Almost half of the laboratories (42%) used immunoenzymatic assay (EIA) for Tox A/B as a stand-alone method, while only 34% used an algorithm for CDI as indicated by the European guidelines. A low percentage of laboratories performed molecular assays or C. difficile culture, 25% and 29%, respectively. Most laboratories (161/278) declared to type C. difficile strains, the majority in collaboration with a reference laboratory. Among the 103 C. difficile clinical isolates collected during the study, 31 different PCR-ribotypes were identified. PCR-ribotype 356/607 (27%) was predominant, followed by 018 (12%). These two PCR-ribotypes show 87.5% of similarity in ribotyping profile. PCR-ribotypes 027 and 078 represented 8% and 4% of the strains, respectively. Four PCR-ribotypes (027, 033, 078 and 126) were positive for the binary toxin CDT. In particular, PCR-ribotype 033 produces only CDT, and it has recently been associated with symptomatic cases. The majority of strains were multidrug resistant. In particular, all strains PCR-ribotypes 356/607 and 018 were resistant to moxifloxacin, rifampicin, erythromycin and clindamycin. The results obtained highlight the need to raise awareness to the microbiological diagnosis of CDI among clinicians and to implement and harmonize diagnostic methods for CDI in Italian laboratories in the perspective of a future national surveillance.

Fluoroquinolone resistance does not impose a cost on the fitness of Clostridium difficile in vitro.

Point mutations conferring resistance to fluoroquinolones were introduced in the gyr genes of the reference strain Clostridium difficile 630. Only mutants with the substitution Thr-82→Ile in GyrA, which characterizes the hypervirulent epidemic clone III/027/NAP1, were resistant to all fluoroquinolones tested. The absence of a fitness cost in vitro for the most frequent mutations detected in resistant clinical isolates suggests that resistance will be maintained even in the absence of antibiotic pressure.

Clostridium difficile infection (CDI) in children due to hypervirulent strains PCR-ribotype 027: An emblematic report of two cases.

In this report, the first two cases of pediatric Clostridium difficile infection (CDI) due to the hypervirulent PCR-ribotype 027 in Italy are described as emblematic of the role of both the infecting C. difficile strain and patient status in the occurrence and clinical manifestation of CDI in children.

Antibiotic resistance patterns and PCR-ribotyping of Clostridium difficile strains isolated from swine and dogs in Italy.

Recent studies suggest animals, in particular farm and companion animals, as possible reservoir for Clostridium difficile human pathogenic strains. The aim of this study was to give a first characterization of C. difficile isolates from Italian swine and dogs. In total, 10 different PCR-ribotypes were identified among porcine strains and six among canine strains. The predominant type found among porcine strains was 078 (50%), whereas the most frequently detected among canine strains was the non-toxinogenic 010 (64%). Considering the CLSI breakpoints, 60% of porcine isolates was resistant to ERY, 35% to MXF, 15% to CLI, 5% to RIF, and none to MTZ or VAN. Among dogs, 51% of strains was resistant to CLI, 46% to ERY, 21% to MTZ and 5% to MXF or RIF, and none to VAN. Five porcine strains (10%) and 9 canine isolates (41%) were MDR. Interestingly, 8 MDR canine strains were highly resistant to MTZ, with MICs ≥32 mg/L. Considering the EUCAST cut-off for MTZ (MIC >2 mg/L), 13 canine isolates and one porcine strain were found with reduced susceptibility to MTZ (MICs ranging from 3 to ≥256 mg/L). Swine and canine strains showing resistance or reduced susceptibility to MTZ belonged to PCR-ribotype 010 and 078. These PCR-ribotypes have been associated to reduced susceptibility to MTZ also in human, suggesting a potential risk for the emergence of C. difficile strains resistant to the current first-line antibiotic for CDI treatment. The agar incorporation method (AIM) was confirmed as the best method to detect C. difficile strains with this phenotype also after strains manipulations. The results obtained add further evidences about the possible role of animals as source of MDR C. difficile strains and reservoir of antibiotic resistance determinants.

Multidisciplinary analysis of a nontoxigenic Clostridium difficile strain with stable resistance to metronidazole.

Stable resistance to metronidazole in a nontoxigenic Clostridium difficile strain was investigated at both the genomic and proteomic levels. Alterations in the metabolic pathway involving the pyruvate-ferredoxin oxidoreductase were found, suggesting that reduction of metronidazole, required for its activity, may be less efficient in this strain. Proteomic studies also showed a cellular response to oxidative stress.

First clinical and microbiological characterization of Clostridium difficile infection in a Croatian University Hospital.

Clinical background and molecular epidemiology of Clostridium difficile infection (CDI) in the University Hospital Centre Split were investigated from January 2010 to December 2011. In total, 54 patients with first episode of CDI were consecutively included in the study based on the positive EIA test specific for A and B toxins. Demographic and clinical data were prospectively analyzed from medical records. CDI incidence rate was 0.6 per 10,000 patient-days. Thirty six cases (70.6%) were healthcare-associated, twelve cases (23.5%) were community-associated and three (5.9%) were indeterminate. Six patients (11.7%) had suffered one or more recurrences and 37 patients (72.5%) showed severe CDI. Prior therapy with third generation cephalosporin was significantly associated with severe CDI (P<0.021). Fifty four toxigenic C. difficile strains were isolated and 50 of them were available for PCR-ribotyping. Sixteen different PCR-ribotypes were identified. The most prevalent were PCR-ribotype 001 (27.8%) and 014/020 (24.1%). Twenty three strains were resistant to at least one of the antibiotics tested. Among resistant strains, three (13.0%)--all PCR-ribotype 001--were multi-resistant. Resistance to fluoroquinolones was significantly higher in strains that caused infection after previous use of fluoroquinolones (P=0.04).

Clostridioides difficile and Gut Microbiota: From Colonization to Infection and Treatment.

Clostridioides difficile is the main causative agent of antibiotic-associated diarrhea (AAD) in hospitals in the developed world. Both infected patients and asymptomatic colonized individuals represent important transmission sources of C. difficile. C. difficile infection (CDI) shows a large range of symptoms, from mild diarrhea to severe manifestations such as pseudomembranous colitis. Epidemiological changes in CDIs have been observed in the last two decades, with the emergence of highly virulent types and more numerous and severe CDI cases in the community. C. difficile interacts with the gut microbiota throughout its entire life cycle, and the C. difficile's role as colonizer or invader largely depends on alterations in the gut microbiota, which C. difficile itself can promote and maintain. The restoration of the gut microbiota to a healthy state is considered potentially effective for the prevention and treatment of CDI. Besides a fecal microbiota transplantation (FMT), many other approaches to re-establishing intestinal eubiosis are currently under investigation. This review aims to explore current data on C. difficile and gut microbiota changes in colonized individuals and infected patients with a consideration of the recent emergence of highly virulent C. difficile types, with an overview of the microbial interventions used to restore the human gut microbiota.

Analysis of metronidazole susceptibility in different Clostridium difficile PCR ribotypes.

OBJECTIVES: Susceptibility to metronidazole was investigated in 81 Clostridium difficile strains, belonging to nine different PCR ribotypes, by three different laboratory methods. METHODS: MICs for 81 C. difficile clinical isolates were determined by Etest, the agar dilution method (ADM) and the agar incorporation method (AIM). Twenty selected strains were also subjected to subinhibitory concentrations of metronidazole and the MIC heterogeneity was analysed in colonies from each strain that showed increased values before and after exposure to the antibiotic, using ADM and AIM. RESULTS: Overall, the MICs obtained by Etest were lower compared with those obtained by ADM and AIM, causing discrepancies in the categorization (as susceptible or having reduced susceptibility) of some strains. Reduced susceptibility to metronidazole was observed using both ADM and AIM, with higher MIC values by AIM in isolates belonging to PCR ribotypes 001 and 010. An increase in MICs after exposure to metronidazole was observed for strains belonging to these PCR ribotypes (by Etest and ADM, but not by AIM). In particular, MICs for colonies from strains belonging to either PCR ribotype 001 or 010 were less heterogeneous by AIM compared with by ADM, suggesting a better ability of AIM to detect strains with reduced susceptibility. CONCLUSIONS: These results suggest that the presence of C. difficile subpopulations with reduced susceptibility to metronidazole in the human intestine may be one of the factors responsible for reduced antibiotic efficacy in vivo. The possibility that higher MICs may have often gone unnoticed underlines the importance of choosing the best method for MIC determination and the necessity to monitor C. difficile susceptibility to metronidazole.