Distribution of Clostridium difficile strains from a North American,European and Australian trial of treatment for C. difficile infections: 2005–2007

Clostridium difficile is a widely distributed pathogen with multiple strain types as determined by restriction endonuclease analysis (REA) and by PCR ribotyping, two well-characterized typing systems. In this study, REA typing was performed on 894 C. difficile isolates from patients enrolled from 16 countries on three continents in two large, recently conducted clinical treatment trials of C. difficile infection. REA group BI (Ribotype 027) isolates were the most common strains identified and were widely distributed throughout North America, but restricted to three of thirteen countries in Europe. REA group J (Ribotype 001) isolates were the most common strains identified in Europe and non-specific REA groups (historically less frequent) were the most common strains identified in Australia. REA groups BI, J, G and CF correlated with specific PCR ribotypes whereas more than one ribotype was found within REA groups Y, BK, and K. International surveillance of C. difficile strains is important to document the changing epidemiology of this enteric pathogen that continues to cause healthcare facility outbreaks and sporadic infections in other settings.     

Comparative genomics analysis of Clostridium difficile epidemic strain DH/NAP11/106

Clostridium difficile PCR ribotype 106 (also identified as restriction endonuclease analysis [REA] group DH) recently emerged as the most common strain causing C. difficile infection (CDI) among US adults. We previously identified this strain predominating our pediatric cohort. Pediatric clinical CDI isolates previously characterized by REA underwent antibiotic resistance testing and whole genome sequencing. Of 134 isolates collected from children, 31 (23%) were REA group DH. We performed a comparative genomics analysis to identify DH-associated accessory genes. We identified five DH-associated genes that are associated with virulence in other bacterial species but not previously known to contribute to CDI. These genes are associated with intestinal mucosal adhesion (collagen-binding surface protein), sporulation (sporulation integral membrane protein YtvI), and protection from oxidative stress and foreign DNA (DNA phosphorothioation-dependent restriction proteins, sulfurtransferase, and DNA sulfur modification proteins). The association of these genes was validated in a cohort of 623 publicly available C. difficile sequences, 10 (1.6%) of which were monophyletic to REA group DH through in silico multilocus sequence typing and core genome phylogenetic analysis. Further investigation is required to determine the contribution of these genes to the emergence and virulence of this epidemic strain.     

Typing and susceptibility of bacterial isolates from the fidaxomicin (OPT-80) phase II study for C. difficile infection

BackgroundClostridium difficile infection (CDI) has been increasing in incidence and severity in recent years, coincident with the spread of a “hypervirulent” strain, REA type BI (ribotype 027, PFGE NAP 1). Exacerbating the problem has been the observation that metronidazole may be showing decreased effectiveness, particularly in the more severe cases. Fidaxomicin is an 18-membered macrocycle currently in phase 3 trials for the treatment of C. difficile infection (CDI). An open-label, phase II study in CDI patients has been completed and the clinical results published. C. difficile organisms were isolated from patient stool specimens and typed by restriction endonuclease analysis (REA) in order to determine the frequency and susceptibility of the C. difficile isolates and their response to treatment.MethodsFecal samples were plated on CCFA agar for isolation of C. difficile. These isolates were tested for susceptibility to fidaxomicin, vancomycin, and metronidazole using CLSI agar dilution methods and were typed by REA.ResultsC. difficile was isolated from 38 of 49 subjects and 16 (42%) were the epidemic C. difficile BI group. The BI strain was distributed approximately equally in the three dosing groups. Overall antibiotic susceptibilities were consistent with the previously reported MIC₉₀ values for the three antibiotics tested, but the MIC₉₀ of BI strains was two dilutions higher than non-BI strains for metronidazole and vancomycin (for both antibiotics, MIC₉₀ was 2 μg/mL vs. 0.5 μg/mL, P < 0.01 for metronidazole, P = NS for vancomycin). Clinical cure for BI isolates (11/14, 79%) was not significantly different from non-BI isolates (21/22, 95%).ConclusionThese results underscore the high prevalence of the BI epidemic strain and demonstrate that mild to moderate CDI infection as well as severe disease can be caused by these strains. Fidaxomicin cure rates for subjects with BI and with non-BI strains are similar, although the small numbers of subjects preclude a robust statistical comparison.     

Characterisation and carriage ratio of Clostridium difficile strains isolated from a community-dwelling elderly population in the United Kingdom

Background

Community-associated Clostridium difficile infection (CDI) appears to be an increasing problem. Reported carriage rates by C.difficile are debatable with suggestions that primary asymptomatic carriage is associated with decreased risk of subsequent diarrhoea. However, knowledge of potential reservoirs and intestinal carriage rates in the community, particularly in the elderly, the most susceptible group, is limited. We have determined the presence of C.difficile in the faeces of a healthy elderly cohort living outside of long-term care facilities (LCFs) in the United Kingdom.

Methods

Faecal samples from 149 community-based healthy elderly volunteers (median age 81 years) were screened for C.difficile using direct (Brazier''s CCEY) and enrichment (Cooked Meat broth) culture methods and a glutamate dehydrogenase (GDH) immunoassay. Isolates were PCR-ribotyped and analysed for toxin production and the presence of toxin genes.

Results

Of 149 faecal samples submitted, six (4%) were found to contain C.difficile. One particular sample was positive by both the GDH immunoassay and direct culture, and concurrently produced two distinct strain types: one toxigenic and the other non-toxigenic. The other five samples were only positive by enrichment culture method. Overall, four C.difficile isolates were non-toxigenic (PCR-ribotypes 009, 026 (n = 2) and 039), while three were toxigenic (PCR-ribotypes 003, 005 and 106). All individuals who had a positive culture were symptom-free and none of them had a history of CDI and/or antibiotics use in the 3 month period preceding recruitment.

Conclusions

To our knowledge, this is the first study of the presence of C.difficile in healthy elderly community-dwelling individuals residing outside of LCFs. The observed carriage rate is lower than that reported for individuals in LCFs and interestingly no individual carried the common epidemic strain PCR-ribotype 027 (NAP1/BI). Further follow-up of asymptomatic carriers in the community, is required to evaluate host susceptibility to CDI and identify dynamic changes in the host and microbial environment that are associated with pathogenicity.     

Bactericidal activity of telavancin,vancomycin and metronidazole against Clostridium difficile

We compared the time-kill activities of telavancin, vancomycin and metronidazole at concentrations of 2 times, 4 times, and 8 times their respective MICs against five Clostridium difficile strains including REA type BI, using inocula that contained predominantly either vegetative cells or spores. Telavancin MICs (0.125–0.25 μg/ml) were 2–8-fold lower than those of vancomycin. Telavancin was bacteriostatic, reducing the inoculum by 1–2.5 log₁₀ after 24 h. No major differences occurred with the different inoculum forms or the REA types. Although telavancin MICs were 2–8 times lower than vancomycin, both demonstrated similar bacteriostatic activity against C. difficile while metronidazole was bactericidal.     

New PCR ribotypes of Clostridium difficile detected in children in Brazil

A total of 35 Brazilian isolates of Clostridium difficile from faecal stools and four isolates from hospital environments were analyzed by PCR ribotyping. A whole cell protein profile (as an alternative for serogrouping), in vitro toxin production and susceptibility to vancomycin, metronidazole and clindamycin were also investigated. All strains were typeable by both phenotypic and genotypic methods, and a total of 13 different PCR ribotypes were identified, of which seven (132, 133, 134, 135, 136, 142 and 143) were considered new types and accounted for 78.5% of all samples evaluated (including hospital environments). A non-toxigenic C. difficile PCR ribotype 133 was detected in all children groups examined (inpatients, outpatients and healthy children), whilst toxigenic PCR ribotypes 015, 131, 134 and 135 were associated mostly with symptomatic children. Serogroups G and D were disseminated both in patients from the community and from the pediatric hospital, with group G prevalent among outpatient children. All strains were susceptible to vancomycin and metronidazole but high levels of resistance to clindamycin were found, especially among serogroups G and D. Co-existence of different ribotypes and serogroups in the same individual was observed. The new seven ribotypes found in this investigation may represent strains characteristic of this region of Brazil.     

Sensitivity to antibiotics of Clostridium difficile toxigenic nosocomial strains

Clostridium difficile is the etiological agent of diarrhoea and colitis, especially in elderly patients. The incidence of these diseases has increased during the last 10 years. Emergence of so-called hypervirulent strains is considered as one of the main factors responsible for the more severe disease and changed profile of sensitivity to antimicrobial agents. The aim of this work was to determine the sensitivity profile of toxigenic strains of C. difficile in the Czech Republic in 2011–2012 to selected antibiotics. The antibiotics clindamycin, metronidazole, vancomycin and amoxicillin with clavulanic acid were used for this purpose. Isolates cultured on Brazier's C. difficile selective agar were analysed for the presence of toxin genes using Xpert detection system. Xpert analysis revealed that 33 strains carried the genes for toxins tcdB, cdt and tcdCΔ117, thus showing characteristics typical for the hypervirulent ribotype 027/PFGE type NAP1/REA type B1. The remaining 29 strains carried only the gene for toxin B (tcdB) and not cdt and tcdCΔ117. Our results indicate the higher susceptibility of C. difficile hypertoxigenic strains to three out of four tested antibiotics (except vancomycin) than it is for the other toxigenic strains. We found that only 10.34 % of other toxigenic strains were resistant to clindamycin, and no resistance was found in all other cases. All the isolates were sensitive to amoxicillin/clavulanic acid in vitro. However, its use is not recommended for therapy of infections caused by C. difficile.     

Biodiversity of Clostridium botulinum Type E Strains Isolated from Fish and Fishery Products

The genetic biodiversity of Clostridium botulinum type E strains was studied by pulsed-field gel electrophoresis (PFGE) with two macrorestriction enzymes (SmaI-XmaI and XhoI) and by randomly amplified polymorphic DNA (RAPD) analysis with two primers (OPJ 6 and OPJ 13) to characterize 67 Finnish isolates from fresh fish and fishery products, 15 German isolates from farmed fish, and 10 isolates of North American or North Atlantic origin derived mainly from different types of seafood. The effects of fish species, processing, and geographical origin on the epidemiology of the isolates were evaluated. Cluster analysis based on macrorestriction profiles was performed to study the genetic relationships of the isolates. PFGE and RAPD analyses were combined and resulted in the identification of 62 different subtypes among the 92 type E isolates analyzed. High genetic biodiversity among the isolates was observed regardless of their source. Finnish and North American or North Atlantic isolates did not form distinctly discernible clusters, in contrast with the genetically homogeneous group of German isolates. On the other hand, indistinguishable or closely related genetic profiles among epidemiologically unrelated samples were detected. It was concluded that the high genetic variation was probably a result of a lack of strong selection factors that would influence the evolution of type E. The wide genetic biodiversity observed among type E isolates indicates the value of DNA-based typing methods as a tool in contamination studies in the food industry and in investigations of botulism outbreaks.     

Molecular typing and antimicrobial susceptibility testing to six antimicrobials of <Emphasis Type="Italic">Clostridium difficile</Emphasis> isolates from three Czech hospitals in Eastern Bohemia in 2011–2012

In 2011–2012, a survey was performed in three regional hospitals in the Czech Republic to determine the incidence of Clostridium difficile infections (CDIs) and to characterize bacterial isolates. C. difficile isolates were characterized by PCR ribotyping, toxin genes detection, multiple-locus variable-number tandem-repeat analysis (MLVA), and antimicrobial susceptibility testing to fidaxomicin, vancomycin, metronidazole, clindamycin, LFF571, and moxifloxacin using agar dilution method. The incidence of CDI in three studied hospitals was 145, 146, and 24 cases per 100,000 inhabitants in 2011 and 177, 258, and 67 cases per 100,000 inhabitants in 2012. A total of 64 isolates of C. difficile was available for molecular typing and antimicrobial susceptibility testing. 60.9% of the isolates were classified as ribotype 176. All 41 isolates of ribotypes 176 and 078 were positive for the presence of binary toxin genes. Ribotype 176 also carried 18-bp deletion in the regulatory gene tcdC. Tested isolates of C. difficile were fully susceptible to vancomycin and metronidazole, whereas 65.1% of the isolates were resistant to moxifloxacin. MLVA results indicated that isolates from three different hospitals were genetically related, suggesting transmission between healthcare facilities.     

Molecular Characterization of Clostridium difficile Isolates from Human Subjects and the Environment

Clostridium difficile is a spore-forming, gram-positive, anaerobic bacillus that can cause C. difficile infection (CDI). However, only a few studies on the prevalence and antibiotic resistance of C. difficile in healthy individuals in China have been reported. We employed a spore enrichment culture to screen for C. difficile in the stool samples of 3699 healthy Chinese individuals who were divided into 4 groups: infants younger than 2 years of age and living at home with their parents; children aged 1 to 8 years of age and attending three different kindergarten schools; community-dwelling healthy adult aged 23–60 years old; and healthcare workers aged 28–80 years old. The C. difficile isolates were analyzed for the presence of toxin genes and typed by PCR ribotyping and multilocus sequence typing (MLST). The minimum inhibitory concentration of 8 antimicrobial agents was determined for all of the isolates using the agar dilution method. The intestinal carriage rate in the healthy children was 13.6% and ranged from 0% to 21% depending on age. The carriage rates in the 1654 community-dwelling healthy adults and 348 healthcare workers were 5.5% and 6.3%, respectively. Among the isolates, 226 were toxigenic (225 tcdA+/tcdB+ and 1 tcdA+/tcdB+ ctdA+/ctdB+). Twenty-four ribotypes were found, with the dominant type accounting for 29.7% of the isolates. The toxigenic isolates were typed into 27 MLST genotypes. All of the strains were susceptible to vancomycin, metronidazole, fidaxomicin, and rifaximin. High resistance to levofloxacin and ciprofloxacin at rates of 39.8% and 98.3%, respectively, were observed. ST37 isolates were more resistant to levofloxacin than the other STs. The PCR ribotypes and sequence types from the healthy populations were similar to those from the adult patients.     

Sequence Similarity of Clostridium difficile Strains by Analysis of Conserved Genes and Genome Content Is Reflected by Their Ribotype Affiliation

PCR-ribotyping is a broadly used method for the classification of isolates of Clostridium difficile, an emerging intestinal pathogen, causing infections with increased disease severity and incidence in several European and North American countries. We have now carried out clustering analysis with selected genes of numerous C. difficile strains as well as gene content comparisons of their genomes in order to broaden our view of the relatedness of strains assigned to different ribotypes. We analyzed the genomic content of 48 C. difficile strains representing 21 different ribotypes. The calculation of distance matrix-based dendrograms using the neighbor joining method for 14 conserved genes (standard phylogenetic marker genes) from the genomes of the C. difficile strains demonstrated that the genes from strains with the same ribotype generally clustered together. Further, certain ribotypes always clustered together and formed ribotype groups, i.e. ribotypes 078, 033 and 126, as well as ribotypes 002 and 017, indicating their relatedness. Comparisons of the gene contents of the genomes of ribotypes that clustered according to the conserved gene analysis revealed that the number of common genes of the ribotypes belonging to each of these three ribotype groups were very similar for the 078/033/126 group (at most 69 specific genes between the different strains with the same ribotype) but less similar for the 002/017 group (86 genes difference). It appears that the ribotype is indicative not only of a specific pattern of the amplified 16S–23S rRNA intergenic spacer but also reflects specific differences in the nucleotide sequences of the conserved genes studied here. It can be anticipated that the sequence deviations of more genes of C. difficile strains are correlated with their PCR-ribotype. In conclusion, the results of this study corroborate and extend the concept of clonal C. difficile lineages, which correlate with ribotypes affiliation.     

Characterization of Clostridium difficile strains isolated from immunosuppressed inpatients in a hospital in Rio de Janeiro,Brazil

The aim of this work was to identify and characterize Clostridium difficile strains from fecal and hospital environmental samples. C. difficile toxins were detected by ELISA in 28.5% of the analyzed samples. Four strains were isolated from immunosuppresssed inpatients presenting antibiotic-associated diarrhea. All strains possessed tcdA and tcdB genes and did not present neither the cdtA and cdtB genes nor any significant deletions in the tcdC gene. PFGE and PCR-ribotyping analysis showed that two strains belonged to the same clonal type (ribotype 014) and the other two were grouped into ribotype 106, in spite of presenting a similar, but not identical genetic fingerprint. This report shows that for the first time ribotype 106 was found outside the United Kingdom. All isolates were equally sensitive to metronidazole. The ribotype 014 isolates were highly resistant to clindamycin, while the ribotype 106 isolates were resistant to all fluoroquinolones tested. This work reveals the spread of C. difficile in the hospital unit studied and the presence of three genetically related types, two of them presenting resistance to fluoroquinolones.     

Porcine and bovine <Emphasis Type="Italic">Clostridium difficile</Emphasis> ribotype 078 isolates demonstrate similar growth and toxigenic properties

Clostridioides (C.) difficile are found in cows, pigs and poultry suggesting that this pathogen is present and more importantly animals could act as a reservoir, via food or environment, of human C. difficile infection. Molecular methods together with phenotypical characterisation bring integrated and important tools to describe diversity and nature of bacteria including C. difficile. Moreover, similar or identical C. difficile types are found in different farm animals. This study aimed to phenotypically characterise C. difficile isolates belonging to ribotype 078 and to identify differences such as growth and toxicity between porcine and bovine isolates. C. difficile isolates were assessed for the growth behaviour (turbidimetry), metabolic potential (Biolog AN) and toxin production (ELISA method) in vitro. The concentration of released either toxin A (TcdA) or toxin B (TcdB) varied greatly between the isolates tested; however, it did not differ between the porcine and bovine ribotypes. Also, the TcdA/TcdB ratio of the isolates did not show a difference either. The most common metabolised substrates were pyruvic acid followed by α-ketobutyric acid. The results show that both porcine and bovine C. difficile RT 078 share similar phenotypical characteristics including growth and production of toxins. The findings may help understand the virulence of C. difficile RT 078 in porcine and bovine species.     

Surface-Layer Protein A (SlpA) Is a Major Contributor to Host-Cell Adherence of Clostridium difficile

Clostridium difficile is a leading cause of antibiotic-associated diarrhea, and a significant etiologic agent of healthcare-associated infections. The mechanisms of attachment and host colonization of C. difficile are not well defined. We hypothesize that non-toxin bacterial factors, especially those facilitating the interaction of C. difficile with the host gut, contribute to the initiation of C. difficile infection. In this work, we optimized a completely anaerobic, quantitative, epithelial-cell adherence assay for vegetative C. difficile cells, determined adherence proficiency under multiple conditions, and investigated C. difficile surface protein variation via immunological and DNA sequencing approaches focused on Surface-Layer Protein A (SlpA). In total, thirty-six epidemic-associated and non-epidemic associated C. difficile clinical isolates were tested in this study, and displayed intra- and inter-clade differences in attachment that were unrelated to toxin production. SlpA was a major contributor to bacterial adherence, and individual subunits of the protein (varying in sequence between strains) mediated host-cell attachment to different extents. Pre-treatment of host cells with crude or purified SlpA subunits, or incubation of vegetative bacteria with anti-SlpA antisera significantly reduced C. difficile attachment. SlpA-mediated adherence-interference correlated with the attachment efficiency of the strain from which the protein was derived, with maximal blockage observed when SlpA was derived from highly adherent strains. In addition, SlpA-containing preparations from a non-toxigenic strain effectively blocked adherence of a phylogenetically distant, epidemic-associated strain, and vice-versa. Taken together, these results suggest that SlpA plays a major role in C. difficile infection, and that it may represent an attractive target for interventions aimed at abrogating gut colonization by this pathogen.     

Identification of Cyst Nematodes of Agronomic and Regulatory Concern with PCR-RFLP of ITS1

The first internally transcribed spacer region (ITS1) from cyst nematode species (Heteroderidae) was compared by nucleotide sequencing and PCR-RFLP. European, Asian, and North American isolates of five heterodefid species were examined to assess intraspecific variation. PCR-RFLP patterns of amplified ITS1 DNA from pea cyst nematode, Heterodera goettingiana, from Northern Ireland were identical with patterns from Washington State. Sequencing demonstrated that ITS1 heterogeneity existed within individuals and between isolates, but did not result in different restriction patterns. Three Indian and two U.S. isolates of the corn cyst nematode, Heterodera zeae, were compared. Sequencing detected variation among ITS1 clones from the same individual, between individuals, and between isolates. PCR-RFLP detected several restriction site differences between Indian and U.S. isolates. The basis for the restriction site differences between isolates from India and the U.S. appeared to be the result of additional, variant ITS1 regions amplified from the U.S. isolates, which were not found in the three India isolates. PCR-RFLP from individuals of the U.S. isolates created a composite pattern derived from several ITS1 types. A second primer set was specifically designed to permit discrimination between soybean (H. glycines) and sugar beet (H. schachtii) cyst nematodes. Fok I digestion of amplified product from soybean cyst nematode isolates displayed a uniform pattern, readily discernible from the pattern of sugar beet and clover cyst nematode (H. trifolii).     

Clinical Clostridium difficile: clonality and pathogenicity locus diversity

Clostridium difficile infection (CDI) is an important cause of mortality and morbidity in healthcare settings. The major virulence determinants are large clostridial toxins, toxin A (tcdA) and toxin B (tcdB), encoded within the pathogenicity locus (PaLoc). Isolates vary in pathogenicity from hypervirulent PCR-ribotypes 027 and 078 with high mortality, to benign non-toxigenic strains carried asymptomatically. The relative pathogenicity of most toxigenic genotypes is still unclear, but may be influenced by PaLoc genetic variant. This is the largest study of C. difficile molecular epidemiology performed to date, in which a representative collection of recent isolates (n = 1290) from patients with CDI in Oxfordshire, UK, was genotyped by multilocus sequence typing. The population structure was described using NeighborNet and ClonalFrame. Sequence variation within toxin B (tcdB) and its negative regulator (tcdC), was mapped onto the population structure. The 69 Sequence Types (ST) showed evidence for homologous recombination with an effect on genetic diversification four times lower than mutation. Five previously recognised genetic groups or clades persisted, designated 1 to 5, each having a strikingly congruent association with tcdB and tcdC variants. Hypervirulent ST-11 (078) was the only member of clade 5, which was divergent from the other four clades within the MLST loci. However, it was closely related to the other clades within the tcdB and tcdC loci. ST-11 (078) may represent a divergent formerly non-toxigenic strain that acquired the PaLoc (at least) by genetic recombination. This study focused on human clinical isolates collected from a single geographic location, to achieve a uniquely high density of sampling. It sets a baseline of MLST data for future comparative studies investigating genotype virulence potential (using clinical severity data for these isolates), possible reservoirs of human CDI, and the evolutionary origins of hypervirulent strains.     

Molecular analysis of isolates of Streptococcus suis capsular type 2 by restriction-endonuclease-digested DNA separated on SDS-PAGE and by hybridization with an rDNA probe.

This study was undertaken to assess the discriminatory value of restriction endonuclease (RE) digestion patterns of Streptococcus suis chromosomal DNA using polyacrylamide gel electrophoresis (SDS-PAGE) and DNA-rDNA hybridization. For the RE digestion patterns, DNAs were digested separately with the enzymes BamHI and BglII and the resultant fragments were separated by SDS-PAGE. An Escherichia coli rDNA probe derived from pKK3535 was used for the hybridization. Twenty-three S. suis capsular type 2 isolates recovered from diseased and clinically healthy pigs, from a human case, and from a cow were compared in this study. The majority of isolates associated with septicaemia belonged to one restriction endonuclease analysis (REA) profile group. Isolates associated with pneumonia belonged either to the REA profile group of isolates associated with septicaemia or to a second REA profile group. The REA profiles of isolates from clinically healthy animals were more heterogeneous. The REA profile of the type 2 reference strain, S735, which was originally isolated from a pig, was very different from those of the porcine and bovine isolates but similar to the profile of the human isolate. The profiles obtained after rDNA hybridization were more homogeneous. Although different patterns were detected in the 23 isolates, there was no correlation between the source of the isolate and the patterns observed with this technique.     

Molecular Epidemiology of Clostridium difficile at a Medical Center in Taiwan: Persistence of Genetically Clustering of A?B+ Isolates and Increase of A+B+ Isolates

Introduction

We investigated the changing trend of various toxigenic Clostridium difficile isolates at a 3 500-bed hospital in Taiwan. Genetic relatedness and antimicrobial susceptibility of toxigenic C. difficile isolates were also examined.

Methods

A total of 110 non-repeat toxigenic C. difficile isolates from different patients were collected between 2002 and 2007. Characterization of the 110 toxigenic isolates was performed using agar dilution method, multilocus variable-number tandem-repeat analysis (MLVA) genotyping, tcdC genotyping, and toxinotyping.

Results

Among the 110 toxigenic isolates studied, 70 isolates harbored tcdA and tcdB (A⁺B⁺) and 40 isolates harbored tcdB only (A⁻B⁺). The annual number of A⁺B⁺ isolates considerably increased over the 6-year study (P = 0.055). A total of 109 different MLVA genotypes were identified, in which A⁺B⁺ isolates and A⁻B⁺ isolates were differentiated into two genetic clusters with similarity of 17.6%. Twenty-four (60%) of the 40 A⁻B⁺ isolates formed a major cluster, MLVA-group 1, with a similarity of 85%. Seven (6.4%) resistant isolates were identified, including two metronidazole-resistant and five vancomycin-resistant isolates.

Conclusions

This study indicated a persistence of a MLVA group 1 A⁻B⁺ isolates and an increase of A⁺B⁺ isolates with diverse MLVA types. Moreover, C. difficile isolates with antimicrobial resistance to metronidazole or vancomycin were found to have emerged. Continuous surveillance is warranted to understand the recent situation and control the further spread of the toxigenic C. difficile isolates, especially among hospitalized patients.     

Molecular epidemiology of Clostridium difficile strains from nosocomial-acquired infections

The purpose of this study is to analyze isolates of Clostridium difficile from patients with nosocomial acquired infection in respect to their molecular type and antimicrobial susceptibility. Fifty-nine randomly selected clinical isolates were characterized. Molecular typing was performed by rep-PCR (DiversiLab). Isolates were tested by disk diffusion towards 11 different antibiotics. All isolates were susceptible to metronidazole and vancomycin. Fifty five (93 %) isolates were resistant to erythromycin and fifty six (95 %) exhibited resistance to both clindamycin and moxifloxacin. Twenty rep-PCR types were identified, but most clinical isolates formed four major rep-PCR clusters (A₁ 24/59, 40 %; A₂ 20/59, 33 %; A₃ 5/59, 8 %; A₄ 3/59, 5 %). These results show high genetic variability, which demonstrate clearly the complexity of the strains of C. difficile and also show an increasing rate of resistance to fluoroquinolones in our region emphasizing the importance of implementing surveillance programs in order to prevent further spread of resistance in C. difficile.