Antimicrobial susceptibilities of Clostridium difficile.

The antimicrobial susceptibilities of 78 strains of Clostridium difficile isolated from patients with and without gastrointestinal symptoms were determined and compared. Strains from patients with symptoms were more likely to show resistance to antibiotics. The antimicrobial susceptibilities of toxigenic and non-toxigenic strains were found to be similar.     

Antimicrobial resistance, toxinotype, and genotypic profiling of Clostridium difficile isolates of swine origin

The occurrence of Clostridium difficile infections in patients that do not fulfill the classical risk factors prompted us to investigate new risk factors of disease. The goal of this study was to characterize strains and associated antimicrobial resistance determinants of C. difficile isolated from swine raised in Ohio and North Carolina. Genotypic approaches used include PCR detection, toxinotyping, DNA sequencing, and pulsed-field gel electrophoresis (PFGE) DNA fingerprinting. Thirty-one percent (37/119) of isolates carried both tetM and tetW genes. The ermB gene was found in 91% of isolates that were resistant to erythromycin (68/75). Eighty-five percent (521/609) of isolates were toxin gene tcdB and tcdA positive. A total of 81% (494/609) of isolates were positive for cdtB and carry a tcdC gene (a toxin gene negative regulator) with a 39-bp deletion. Overall, 88% (196/223) of pigs carry a single C. difficile strain, while 12% (27/223) of pigs carried multiple strains. To the best of our knowledge, this is the first report of individual pigs found to carry more than one strain type of C. difficile. A significant difference in toxinotype profiles in the two geographic locations was noted, with a significantly (P < 0.001) higher prevalence of toxinotype V found in North Carolina (84%; 189/224) than in Ohio (55%; 99/181). Overall, the study findings indicate that significant proportions of C. difficile in swine are toxigenic and often are associated with antimicrobial resistance genes, although they are not resistant to drugs that are used to treat C. difficile infections.     

Antimicrobial susceptibility testing of Clostridium difficile using EUCAST epidemiological cut-off values and disk diffusion correlates

With the emergence of reduced susceptibility of Clostridium difficile to metronidazole and vancomycin the value of antimicrobial susceptibility testing has increased. The aim of our study was to evaluate disk diffusion for susceptibility testing of C. difficile by comparing disk diffusion results with MICs from gradient tests and to propose zone diameter breakpoint correlates for the EUCAST epidemiological cut-off values (ECOFFs) recently published. We tested 211 clinical isolates of C. difficile, from patients with diarrhoea hospitalized at Aarhus and Odense University Hospitals, Denmark. Furthermore, ten clinical isolates of C. difficile from the Anaerobe Reference Laboratory, University Hospital of Wales, with known reduced susceptibility to either metronidazole or vancomycin, were included. Isolates were tested with Etest gradient strips and disk diffusion towards metronidazole, vancomycin and moxifloxacin on Brucella Blood Agar supplemented with hemin and vitamin K. We found an excellent agreement between inhibition zone diameter and MICs. For each MIC value, the inhibition zones varied from 0 to 8 mm, with 93% of values within 6 mm for metronidazole, 95% of values within 4 mm for vancomycin, and 98% of values within 4 mm for moxifloxacin. With proposed zone diameter breakpoints for metronidazole, vancomycin and moxifloxacin of WT ≥ 23 mm, WT ≥ 19 and WT ≥ 20 mm, respectively, we found no very major errors and only major errors below 2%. In conclusion, we suggest that disk diffusion is an option for antimicrobial susceptibility testing of C. difficile.     

Interest of the disk diffusion method for screening Clostridium difficile isolates with decreased susceptibility to antibiotics

AimIn vitro determination of Clostridium difficile susceptibility to antibiotics is not routinely performed. The aim of this study was to evaluate the performance of antibiotic susceptibility determination with the disk diffusion method for screening C. difficile isolates with decreased susceptibility to antibiotics.MethodsThirty-six C. difficile isolates (toxigenic or not) isolated in 2005 and 2006 from three hospitals Assistance publique–Hôpitaux de Paris (Jean-Verdier, René-Muret, Beaujon) were studied by disk diffusion method with 14 antibiotics. Mueller-Hinton agar supplemented with sheep blood (Bio-Rad*) were swabbed with a C. difficile suspension at 1 McFarland. To check the results obtained with the disk diffusion method, Minimal Inhibitory Concentration (MIC) were performed respectively with E-test for glycopeptides and metronidazole and with the agar dilution reference method and E-test for new molecules with a potential activity against anaerobes: imipenem, ertapenem, linezolid and moxifloxacin.ResultsThe decreased susceptibility (resistant and intermediate) observed was 40% for amoxicillin–clavulanate, 60% for piperacillin–tazobactam, 100% for ceftriaxone, 81% for imipenem, 61% for ertapenem, 2% for chloramphenicol, 34% for erythromycin, 90% for lincomycin, 2% for linezolid, 98% for levofloxacin, 17% for moxifloxacin and 0% for vancomycin, teicoplanin and metronidazole. The results obtained with the disk diffusion method were compared to MICs obtained with E-test and reference method.ConclusionThe disk diffusion method seems to be a good method to detect isolates suspected to have a decreased susceptibility and consequently to reduce MIC determinations.     

Antimicrobial susceptibility of equine isolates of Actinobacillus spp. and identification of beta-lactamases in some strains

A total number of 149 Actinobacillus strains isolated from clinical samples (73 strains) and from the oral cavity of healthy horses (76 strains) were tested for their susceptibility to 17 antimicrobial substances. The antibiograms were generally very similar between the various strains and no differences could be clearly correlated to either phenotype or source of isolates. However, when tested against penicillin, ampicillin, trimethoprim-sulfa, and streptomycin, small groups of strains with what appeared to be acquired resistance could be identified. Eight of the penicillin-resistant strains were found to produce beta-lactamase. The beta-lactamases appeared to be bound tightly to the cell wall, thereby frustrating further characterization by isoelectric focusing. Plasmids of approximately 3 kb were found in four out of seven beta-lactamase-producing strains submitted to plasmid analysis.     

Comparison of typing methods for Clostridium difficile isolates.

A simple discriminative typing method for Clostridium difficile has been developed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and restriction enzyme analysis are relatively simple techniques but are difficult to evaluate, especially the restriction enzyme analysis. Immunoblotting and restriction fragment length polymorphism typing facilitate simple discrimination of patterns.     

Restriction endonuclease analysis of nosocomial isolates of Clostridium difficile.

A total of 110 clinical isolates of Clostridium difficile were analyzed by agarose gel electrophoresis by using both bacterial restriction endonuclease analysis (REA) and plasmid profiles. A total of 72 isolates were divided into 12 groups according to their REA patterns. Some 38 isolates exhibited unique patterns. Pattern A occurred in 20% of isolates. Isolates with patterns B, E, and G were cytotoxin negative. The remaining groups were cytotoxin positive. Multiple isolates obtained from two stool specimens were studied to examine the variation in REA profiles found in single specimens. In these specimens no variation in REA profiles was found. The stability of C. difficile was studied by examining sequential in vitro subcultures of a single isolate and strains isolated over a 4-month period from two long-term carriers. REA patterns were stable over time, both in vitro and in vivo. Because plasmid DNA was observed in 53% of isolates, plasmid profiles alone could not be used to study the spread of C. difficile; however, they were necessary for the interpretation of REA patterns in some instances.     

The recognition and characterisation of Finnish Clostridium difficile isolates resembling PCR-ribotype 027

Purpose

To characterise and compare twenty-eight Finnish Clostridium difficile RT027-like isolates, selected based on the presence of 18 bp deletion in the tcdC gene and toxin gene profile (A, B, binary), with eleven RT027 isolates from different Finnish geographical areas and time periods.

Characterization of surface layer proteins from different Clostridium difficile clinical isolates

In a previous study we suggested that two surface proteins of a Clostridium difficile strain were involved in the formation of a regularly assembled surface layer (S-layer) external to the cell wall. In the present paper six C. difficile strains isolated from cases and healthy carriers were studied. By using freeze-etching and negative staining techniques two superimposed structurally different lattices were detected on the cell surface of the different C. difficile strains. In each strain, the outer S-layer lattice was arranged in a square symmetry and the inner S-layer lattice in hexagonal symmetry. The S-layer proteins from the different strains were isolated and characterized. Each strain showed two distinct S-layer glycoproteins ranging in molecular mass 36-56 kDa. Antigenic cross-reactivity among the S-layer proteins of higher molecular masses extracted from each strain was demonstrated whereas no antigenic relationship was observed among the different S-layer proteins of lower molecular masses. N-terminal sequence analysis showed the presence of common structural motifs conserved among the high S-layer proteins as well as among the low S-layer proteins. These data indicate that the presence of S-layer on C. difficile strains is common and that its glycoprotein subunits show a certain degree of heterogeneity.     

Typing and subtyping of Clostridium difficile isolates by using multiple-locus variable-number tandem-repeat analysis

Using the genomic sequence of Clostridium difficile strain 630, we developed multiple-locus variable-number tandem-repeat analysis (MLVA) with automated fragment analysis and multicolored capillary electrophoresis as a typing method for C. difficile. All reference strains, representing 31 serogroups, 25 toxinotypes, and 7 known subtypes of PCR ribotype 001, could be discriminated from each other. Application of MLVA to 28 isolates from 7 outbreaks due to the emerging hypervirulent PCR ribotype 027-pulsed-field gel electrophoresis type NAP1 resulted in recognition of 13 clusters. Additionally, 29 toxin A-negative, toxin B-positive isolates belonging to PCR ribotype 017 from eight different countries revealed eight country-specific clusters. MLVA is a highly discriminatory genotyping method and a new tool for subtyping of newly emerging variants of C. difficile.     

Antimicrobial susceptibility pattern of clinical isolates of non-fermentative bacteria

152 nonfermentative bacteria were isolated from a total number of 965 clinical samples processed routinely in the laboratory of Microbiology Department, M.K.C.G Medical College in South Orissa accounting to a prevalence rate of 15.75%. Pseudomonas spp. (both pigmented and non-pigmented strains) were isolated in maximum percentage (73.6%) followed by Acinetobacter spp. (19.7%) and Alkaligenes faecalis (4.6%). Rarely encountered species were Eikenella corrodens (1.3%) and Stenotrophomonas maltophila (0.6%). Pus from various sites was the major source (116; 76%). 81% of all isolates were sensitive to amikacin and 74% to ofloxacin. Sensitivity to cefotaxime, ciprofloxacin, tobramycin, gentamicin and netlimycin ranged from 53% to 68%. Least effective drugs were carbenicillin and ceftriaxone (48% each).     

Antimicrobial susceptibility of sixty human fecal isolates ofAeromonas species

The MICs of 21 antimicrobial agents were determined for 60 strains ofAeromonas spp. isolated from human feces. All isolates tested were susceptible to aztreonam, tetracycline, imipenem, moxalactam, pipemidic acid, gentamicin, trimethoprim-sulfamethoxazole, pefloxacin and ciprofloxacin. Resistance to erythromycin and streptomycin was observed in all 60 strains.Aeromonas caviae was less susceptible to cefamandole, cefotaxime, norfloxacin, chloramphenicol, tetracycline, sulfamethoxazole and trimethoprim than was eitherAeromonas hydrophila orAeromonas sobria. It was concluded that cotrimoxazole or one of the newer quinolones can be considered for treatment of aeromonas-associated diarrhea.     

Antimicrobial susceptibility of Bacteroides fragilis group isolates in Europe

Objective  To evaluate the activity of old and newer antianaerobic drugs against clinical isolates of Bacteroides fragilis group strains from different parts of Europe.
Methods  Bacteroides fragilis group isolates from 37 laboratories in 19 countries were biochemically characterized. The MICs of seven antimicrobial agents were determined by the agar dilution method as recommended by the NCCLS. Production of beta-lactamase was detected by nitrocefin.
Results  There were 1284 B. fragilis group isolates included in the study. Abdominal infections and wounds were the most common sources of isolation and B. fragilis was the dominating species. Ninety-nine percent of the strains were resistant to ampicillin (breakpoint 2 mg/L), 6% to cefoxitin (64 mg/L), 15% to clindamycin (8 mg/L) and 9% to moxifloxacin (8 mg/L). Less than 1% were resistant to imipenem (16 mg/L), piperacillin-tazobactam (128 mg/L) and metronidazole (32 mg/L). Ninety-six percent of the isolates were beta-lactamase producers.
Conclusions  Antimicrobial resistance among the B. fragilis group is increasing.     

Comparison of strain typing results for Clostridium difficile isolates from North America

Accurate strain typing is critical for understanding the changing epidemiology of Clostridium difficile infections. We typed 350 isolates of toxigenic C. difficile from 2008 to 2009 from seven laboratories in the United States and Canada. Typing was performed by PCR-ribotyping, pulsed-field gel electrophoresis (PFGE), and restriction endonuclease analysis (REA) of whole-cell DNA. The Cepheid Xpert C. difficile test for presumptive identification of 027/NAP1/BI isolates was also tested directly on original stool samples. Of 350 isolates, 244 (70%) were known PCR ribotypes, 224 (68%) were 1 of 8 common REA groups, and 187 (54%) were known PFGE types. Eighty-four isolates typed as 027, NAP1, and BI, and 83 of these were identified as presumptive 027/NAP1/BI by Xpert C. difficile. Eight additional isolates were called presumptive 027/NAP1/BI by Xpert C. difficile, of which three were ribotype 027. Five PCR ribotypes contained multiple REA groups, and three North American pulsed-field (NAP) profiles contained both multiple REA groups and PCR ribotypes. There was modest concordance of results among the three methods for C. difficile strains, including the J strain (ribotype 001 and PFGE NAP2), the toxin A-negative 017 strain (PFGE NAP9 and REA type CF), the 078 animal strain (PFGE NAP7 and REA type BK), and type 106 (PFGE NAP11 and REA type DH). PCR-ribotyping, REA, and PFGE provide different but overlapping patterns of strain clustering. Unlike the other methods, the Xpert C. difficile 027/NAP1/BI assay gave results directly from stool specimens, required only 45 min to complete, but was limited to detection of a single strain type.     

Genotyping of outbreak-related and sporadic isolates of Clostridium difficile belonging to serogroup C.

Serogroup C of Clostridium difficile is the serogroup most frequently related to outbreaks. Fifty-six toxigenic serogroup C isolates of C. difficile were genotyped by ribotyping PCR (ribo-PCR), random amplified polymorphic DNA (RAPD) assay, and pulsed-field gel electrophoresis (PFGE). Thirty-five of the 56 isolates were recovered from four unrelated outbreaks (Belgium, 1987, 1992, and 1995; France, 1992 to 1993) 7 derived from a spatiotemporal cluster in Cotonou, Benin (1992), and 14 were sporadic isolates. The serogroup C reference strain, also isolated during an outbreak (Belgium, 1983), was genotyped too. Ribo-PCR, the RAPD assay, and PFGE generated 2, 5, and 11 major genotypes, respectively. Combination of the three methods finally yielded 13 general types, although ribo-PCR did not play any role in enhancing resolution. Three general types were recovered from all the isolates from the five outbreaks and the cluster, with two types being predominant. The 14 sporadic serogroup C isolates were divided into 11 overall genotypes. These results indicate that genotyping methods, and more particularly the combination of the RAPD assay and PFGE, can resolve genetic diversity within toxigenic, serogroup C C. difficile strains. Also, this study suggests that outbreak-related serogroup C strains are limited to a few genetically stable and apparently very widely (internationally and intercontinentally) distributed genotypes.