Evaluation of the chromogenic agar chromID C. difficile.

Three selective media (chromID C. difficile agar, taurocholate cycloserine cefoxitin agar [TCCA; homemade], and CLO medium) were compared from 406 stool samples of patients suspected of having Clostridium difficile infection. The sensitivities of chromID C. difficile agar at 24 h and 48 h, CLO medium, and TCCA were 74.1%, 87%, 85.2%, and 70.4%, respectively.

Clinical features of Clostridium difficile-associated infections and molecular characterization of strains: results of a retrospective study, 2000-2004.

BACKGROUND: Recent outbreaks of severe cases of Clostridium difficile-associated diarrhea (CDAD) reported in North America, the United Kingdom, and The Netherlands have emphasized the importance of an ongoing epidemiological surveillance of CDAD. OBJECTIVE: To determine the epidemiology of CDAD over the years 2000-2004 and the rate of nosocomial transmission of C. difficile. DESIGN: Retrospective survey of inpatients with CDAD and molecular characterization of the strains isolated. SETTING: A 760-bed teaching hospital. METHODS: All CDAD cases diagnosed from January 1, 2000, to December 31, 2004, were reviewed. A CDAD case was defined as diarrhea in a hospitalized patient who had a stool specimen that tested positive for C. difficile cytotoxin or had a positive toxigenic culture result. CDAD was considered to be severe if a patient fulfilled at least 1 of the following 3 criteria: (1) presence of a fever (defined as temperature higher than 38.5 degrees C), abdominal pain, and leukocyte count greater than 10,000 cells/mm(3); (2) endoscopically or histologically proven pseudomembranous colitis; or (3) complications (defined as death with C. difficile infection as the primary or a contributing cause, toxic megacolon, perforation, toxic shock, and/or colectomy). CDAD was considered community-acquired if the diarrhea occurred in the patient within 72 hours after admission and if the patient had no history of hospitalization in the previous month; otherwise, CDAD was considered healthcare-associated. All the strains isolated were serogrouped and were characterized by toxinotyping and PCR ribotyping. Detection of toxin A, toxin B, and binary toxin was performed by PCR. RESULTS: One hundred fifty-one cases of CDAD were diagnosed; 147 clinical records could be reviewed, and 131 strains were studied. The overall incidence of CDAD was 1.1 cases per 1,000 patients admitted, but incidence rates were higher in 2003-2004, compared with 2000-2002 (P=.017). Diarrhea was community acquired in 28 patients (19%). For patients with healthcare-associated CDAD, transmission of the strain from patient to patient (ie, infection with a strain of the same serogroup and PCR ribotype as the strain isolated from another patient hospitalized in the same ward or in a linked ward in the previous 2 months) was demonstrated in 12 cases (10.1%). Eleven percent of strains were positive for binary toxin. Binary toxin-positive strains were associated with more-severe diarrhea (P=.01) and with a higher case-fatality rate (P=.03). A specific clone of C. difficile (serogroup H, PCR ribotype sa026) accounted for 35 (26.7%) of all the strains isolated, but this clone was found both in healthcare-associated and community-acquired cases. Three strains belonged to toxinotype III, but only 1 was related to the hypervirulent clone involved in recent outbreaks. CONCLUSION: The incidence of CDAD is low in our hospital, and cross-infection is limited. These results also suggest that strains with binary toxin might be more virulent.

Clinical features of Clostridium difficile-associated diarrhoea due to binary toxin (actin-specific ADP-ribosyltransferase)-producing strains.

Toxins A and B are known to be the primary virulence factors of Clostridium difficile. Other potential virulence factors have been identified such as binary toxin (actin-specific ADP-ribosyltransferase toxin, or CDT). A retrospective case-control study was performed in order to identify clinical features and risk factors of C. difficile-associated diarrhoea due to binary toxin-producing strains. Each case (a patient with diarrhoea due to binary toxin-producing strain) was compared with two controls (patients with diarrhoea due to a C. difficile strain that did not produce binary toxin) matched for ward and date of hospitalization. cdtA and cdtB genes were screened by PCR. Production of CDT was studied by Western blotting using an antiserum against Ia and Ib from the Clostridium perfringens iota toxin, and the activity of the binary toxin was assessed using an ADP-ribosyltransferase assay. Twenty-six cases (14 males and 12 females) were identified in 1999 and 2000. Cases and controls did not differ significantly for sex, age, previous administration of antibiotics or frequency of endoscopic examination. Diarrhoea was community-acquired more often in cases than in controls (65.4 vs 35.7 %, P = 0.017) and more often represented the cause of hospitalization (61.5 vs 26.2 %, P = 0.003). Moreover, diarrhoea in cases was more frequently associated with abdominal pain (63.6 vs 39.4 %, P = 0.07) and with liquid stools (76.9 vs 59.5 %, P = 0.14) than in controls. These results suggest that there could be a correlation between the production of binary toxin and the severity of diarrhoea.

Contamination of ready-to-eat raw vegetables with Clostridium difficile in France.

The presence of Clostridium difficile in food like shellfish, vegetables and meat has been reported in several publications during the past few years. The objective of this study was to assess the prevalence of ready-to-eat raw vegetables contaminated with C. difficile in France. One hundred and four ready-to-eat salads and vegetables were studied. Toxigenic C. difficile strains were isolated in three samples (2.9 %): two ready-to-eat salads (one heart of lettuce and one lamb's lettuce salad) and one portion of pea sprouts. The strains belonged to three different PCR ribotypes: 001, 014/020/077 and 015. The detection thresholds for vegetative cells and spores cells varied between 1 and 3 c.f.u. in 20 g salad and between 6 and 15 c.f.u. in 20 g salad, respectively, for the method employed.

First outbreak of multidrug-resistant Klebsiella pneumoniae carrying blaVIM-1 and blaSHV-5 in a French university hospital.

OBJECTIVES: We studied eight imipenem-resistant isolates of Klebsiella pneumoniae involved in an outbreak in a French teaching hospital. METHODS: The eight isolates were recovered from clinical specimens or rectal swabs. Antibiotic susceptibilities were determined using standard agar diffusion and dilution methods including synergy tests. PFGE was used to study the relatedness of isolates. Genes encoding beta-lactamases were characterized by transfer assays, specific amplification and cloning. RESULTS: The eight isolates were closely related by PFGE analysis and highly related to a K. pneumoniae strain from Greece. They were highly resistant to beta-lactams, including aztreonam and imipenem (MIC > or =32 mg/L), and were positive by the imipenem-EDTA disc synergy test. Isolates were also resistant to aminoglycosides, newer quinolones and sulfamethoxazole, and showed an intermediate level of resistance to tetracycline. VIM-1 and SHV-5 beta-lactamases were revealed in all isolates by PCR. The analysis of plasmid contents of Escherichia coli DH10B electroporants expressing the VIM-1 beta-lactamase or the SHV-5 beta-lactamase confirmed that the two enzymes were coded by two different plasmids. The bla(VIM-1) gene was part of a class 1 integron that also included aac6, dhfrI and aadA genes and was similar to those reported from strains isolated in Greece. CONCLUSIONS: This study confirms the potential risk of spread of multiresistant bacteria with international transfer of patients.

In vivo transfer of plasmid-encoded ACC-1 AmpC from Klebsiella pneumoniae to Escherichia coli in an infant and selection of impermeability to imipenem in K. pneumoniae.

We describe in vivo selection of a Klebsiella pneumoniae strain with diminished imipenem susceptibility attributable to plasmid-encoded ACC-1 beta-lactamase production and loss of a 36-kDa major outer membrane protein, together with transfer of this plasmid from K. pneumoniae to Escherichia coli in a Tunisian infant.

Prevalence and genetic characterization of toxin A variant strains of Clostridium difficile among adults and children with diarrhea in France.

Toxin A variant strains (toxin A-negative, toxin B-positive strains) of Clostridium difficile have been reported to be responsible for diarrhea or pseudomembranous colitis in humans. These strains lack parts of the repeating sequences of the toxin A gene (tcdA) and are toxin A negative by commercial enzyme immunoassays (EIA). Here, we report the prevalence of the toxin A variant strains in 334 patients with C. difficile-associated diarrhea in France. The repeating segment of the tcdA gene (1,200 bp) was amplified by PCR using the primers NK9 and NK11 (H. Kato et al., J. Clin. Microbiol. 36:2178-2182, 1998). In the case of amplified fragments of unexpected size, the entire tcdA gene was studied by PCRs A1, A2, and A3 (Rupnik et al., J. Clin. Microbiol. 36:2240-2247, 1998), and strains were characterized by serotyping, pulsed-field gel electrophoresis and PCR ribotyping. By PCR with primers NK9 and NK11, C. difficile variant strains were detected in 2.7% of patients. Several variant types were found. A deletion of approximately 1,700 bp was observed in six strains from five patients. These strains belonged to serotype F and were characterized by the same pulsotype and the same PCR ribotype. They were toxin A negative by EIA and exhibited an atypical cytopathic effect on MRC-5 cells. Two other tcdA variant types that exhibited a positive result for toxin A by EIA were identified: one from serotype H with a longer amplified fragment (insertion of 200 bp) and one with a deletion of 600 bp. Diagnosis of C. difficile-associated diseases would have been missed in five patients (1.5%) by laboratories that screen the stools only for the presence of toxin A. This result underlines the need for testing stool by the cytotoxicity assay in patients with a high suspicion of C. difficile-associated diarrhea but a negative immunoassay for toxin A.

Prevalence and characterization of a binary toxin (actin-specific ADP-ribosyltransferase) from Clostridium difficile.

In addition to the two large clostridial cytotoxins (TcdA and TcdB), some strains of Clostridium difficile also produce an actin-specific ADP-ribosyltransferase, called binary toxin CDT. We used a PCR method and Southern blotting for the detection of genes encoding the enzymatic (CDTa) and binding (CDTb) components of the binary toxin in 369 strains isolated from patients with suspected C. difficile-associated diarrhea or colitis. Twenty-two strains (a prevalence of 6%) harbored both genes. When binary toxin production was assessed by Western blotting, 19 of the 22 strains reacted with antisera against the iota toxin of C. perfringens (anti-Ia and anti-Ib). Additionally, binary toxin activity, detected by the ADP-ribosyltransferase assay, was present in only 17 of the 22 strains. Subsequently, all 22 binary toxin-positive strains were tested for the production of toxins TcdA and TcdB, toxinotyped, and characterized by serogrouping, PCR ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis. All binary toxin-positive strains also produced TcdB and/or TcdA. However, they had significant changes in the tcdA and tcdB genes and belonged to variant toxinotypes III, IV, V, VII, IX, and XIII. We could differentiate 16 profiles by using typing methods, indicating that most of the binary toxin-positive strains were unrelated.

Outbreak of multi-resistant Klebsiella oxytoca involving strains with extended-spectrum beta-lactamases and strains with extended-spectrum activity of the chromosomal beta-lactamase.

OBJECTIVES: This study was conducted to analyse broad-spectrum cephalosporin-resistant Klebsiella oxytoca strains. METHODS: The 57 isolates studied were recovered from clinical specimens (n=23) or from rectal swabs (n=34) during a 26-month period. Antibiotic susceptibility patterns were determined using standard agar diffusion and dilution methods including the synergy test between extended-spectrum cephalosporins and clavulanic acid. ERIC-2 PCR and pulsed-field gel electrophoresis (PFGE) methods were used to study the clonal relatedness of the strains. Plasmid-mediated and chromosomal beta-lactamases were characterized by mating and specific bla gene amplification and sequencing. RESULTS: Four different antibiotic resistance patterns were identified whereas ERIC-2 PCR and PFGE revealed six main profiles. Extended-spectrum beta-lactamases (ESBLs) were found in 32 strains: TEM-7 (n=26), TEM-129 (n=1), TEM-3 (n=4), SHV-2 (n=1). The new TEM-type beta-lactamase, TEM-129, differed from TEM-7 by one mutation (Glu-104-->Lys). All TEM-7 or TEM-129 producers were genetically related. Twenty-five other strains with identical ERIC-2 PCR and PFGE profiles harboured a bla(OXY-2) gene different from the reference gene: 24 strains displayed one substitution (Ala-237-->Ser) in the KTG motif and one strain, highly resistant to ceftazidime, showed an additional substitution (Pro-167-->Ser). CONCLUSIONS: The study demonstrated that the majority of strains (n=52) harbouring the OXY-2-type beta-lactamase corresponded to two clones. The first clone (n=27) corresponded to ESBL-producing strains. The second clone (n=25) displayed extended-spectrum activity of the chromosomal beta-lactamase.

gyrA and gyrB mutations are implicated in cross-resistance to Ciprofloxacin and moxifloxacin in Clostridium difficile.

A total of 198 nonrepetitive clinical strains of Clostridium difficile isolated from different French hospitals in 1991 (n = 100) and 1997 (n = 98) were screened for decreased susceptibility to fluoroquinolones by plating onto Wilkins-Chalgren agar containing 16 micro g of ciprofloxacin per ml. The frequency of decreased susceptibility was 7% (14 of 198) and was identical for the years 1991 and 1997. Serogroups C, H, D, A9, and K accounted for five, four, two, one, and one of the resistant strains, respectively, one strain being nontypeable. Arbitrarily primed PCR typing showed that all resistant strains had unique patterns except two serotype C strains, which could not be clearly distinguished. All isolates with decreased susceptibility carried a mutation either in gyrA (eight mutations, amino acid changes Asp71-->Val in one, Thr82-->Ile in six, and Ala118-->Thr in one) or in gyrB (six mutations, amino acid changes Asp426-->Asn in five and Arg447-->Leu in one). These changes are similar to those already described in other species except for Asp71-->Val, which is novel, and Ala118-->Thr, which is exceptional. Attempts to detect the topoisomerase IV parC gene by PCR amplification with universal parC primers or DNA-DNA hybridization under low-stringency conditions were unsuccessful. The susceptibilities of all resistant strains to ciprofloxacin and ethidium bromide were not affected by the addition of reserpine at 20 micro g/ml. In conclusion, decreased susceptibility to fluoroquinolones in C. difficile is rare in France and is associated with the occurrence of a gyrA or gyrB mutation.

Characterization of CMY-type beta-lactamases in clinical strains of Proteus mirabilis and Klebsiella pneumoniae isolated in four hospitals in the Paris area.

We isolated five clinical strains (three Proteus mirabilis and two Klebsiella pneumoniae) with beta-lactam resistance phenotypes consistent with production of an AmpC-type beta-lactamase. The predicted amino acid sequences of the enzymes were typical of class C beta-lactamases. The enzymes were identified as CMY-2, CMY-4 and a new CMY-variant beta-lactamase, CMY-12. The AmpC beta-lactamases from the two K. pneumoniae isolates were found to be encoded on self-transferable plasmids. The genes encoding the AmpC-type beta-lactamase produced by the three P. mirabilis isolates were chromosomal. Four of the five clinical isolates were from patients transferred from Greece, Algeria and Egypt; one of the K. pneumoniae strains was recovered from a French patient. PFGE analysis and rep-PCR fingerprinting showed that the two P. mirabilis isolates from Greek patients were closely related.