Véronique Avesani

Distribution of nocardia species in clinical samples and their routine rapid identification in the laboratory.

ABSTRACT Eighty-six Nocardia strains isolated from clinical samples in Belgium were identified by 16S rRNA gene sequencing. Eighty-three (96%) strains belonged to only six Nocardia species: N. farcinica (38 [44%]), N. nova (19 [22%]), N. cyriacigeorgica (13 [15%]), N. brasiliensis (6 [6.9%]), N. abscessus (5 [5.8%]), and N. paucivorans (2 [2.3%]). A gallery of nine conventional and enzymatic tests was developed for the rapid identification of the most common species isolated during this survey. Pyrrolidonyl aminopeptidase, γ-glutamyl aminopeptidase, α-mannosidase, and α-glucosidase were found to be highly discriminating and could be used to develop an identification scheme.

Serogroup-f Strains of Clostridium-difficile Produce Toxin-b But Not Toxin-a

Most toxigenic strains of Clostridium difficile produce two toxins: an enterotoxin (toxin A) and a cytotoxin (toxin B). Only one strain (strain 8864) has been reported to produce toxin B but no toxin A. Serogroup F strains (44) of C. difficile, often isolated from asymptomatic infants, have been examined for toxin production. These strains, which were from distinct geographical and clinical sources, did not produce any detectable toxin A in vitro when examined in three distinct immunoassays. Nevertheless, all the strain produced a cytotoxin. Immunological differences between the cytotoxin of the serogroup F strains and that produced by C. difficile strain VPI 10463 (serogroup G) were demonstrated with monoclonal antibodies specific for either the toxin B produced by C. difficile strain VPI 10463 or C. sordellii lethal toxin (LT). Polymerase chain reaction amplification with primers derived from C. difficile strain VPI 10463 toxin A and B genes showed that serogroup F strains seem to possess a toxin B gene homologous with that of strain VPI 10463 and at least fragments of the toxin A gene. When axenic mice were inoculated with serogroup F strains, the animals survived; they did not develop diarrhoea and no toxin A could be detected in their faeces. However, cytotoxin was detected. Furthermore, these mice were protected against subsequent challenge with the otherwise lethally toxigenic C. difficile strain VPI 10463. The serogroup F strains appeared to be homogeneous and distinct from other C. difficile strains with regard to toxin production.

International Typing Study of Toxin A-Negative, Toxin B-Positive Clostridium difficile Variants

ABSTRACT Clinically important strains of Clostridium difficile that do not produce toxin A but produce toxin B and are cytotoxic (A−/B+) have been reported from multiple countries. In order to compare the relatedness of these strains, we typed 23 A−/B+C. difficile isolates from the United Kingdom (6 isolates), Belgium (11 isolates), and the United States (6 isolates) by three well-described typing methods. Restriction endonuclease analysis (REA), PCR ribotyping, and serogrouping differentiated 11, 4, and 3 different strain types, respectively. Twenty-one of the 23 A−/B+ variants had a 1.8-kb truncation of the toxin A gene characteristic of toxinotype VIII strains; 20 of the 21 toxinotype VIII-like strains were PCR type 17. PCR type 17 isolates could be differentiated into two separate strain groups by serogrouping and by REA. REA further discriminated these isolates into eight subgroups (REA types). PCR type 17-serogroup F-REA group CF isolates were recovered from all three countries, and one specific REA type, CF4, was recovered from patients with C. difficile disease in the United Kingdom and the United States. C. difficile A−/B+ variants of apparent clonal origin are widely distributed in Europe and North America.

Virulence of ten serogroups of Clostridium difficile in hamsters.

A slide agglutination technique identifying 10 serogroups of Clostridium difficile (A,B,C,D,F,G,H,I,K and X) has been described previously. In this study, we have used the hamster to compare the ability of the 10 serogroup reference strains to colonise and produce disease. Groups of four hamsters were each given a single intraperitoneal injection of either clindamycin or cefoxitin, and an oral challenge dose of C. difficile. The time taken to establish faecal colonisation and the length of survival after colonisation were monitored. All hamsters treated with cefoxitin became colonised by day 3 and those challenged with the cytotoxigenic strains of serogroups A,C,H and K developed colitis and died. Among those challenged with the non-toxigenic strains of groups B,D,I and X and the toxigenic strains of groups F and G, faecal colonisation was established without signs of disease. This demonstrates that there are differences in virulence even among toxigenic strains of C. difficile. The same phenomenon was observed after treatment with clindamycin but the pattern of colonisation was quite different with some strains. In the hamsters challenged with toxigenic strains of groups C and K and non-toxigenic strains of groups D and I, which are highly resistant to clindamycin, the response was the same as with cefoxitin. The results were different with strains which were susceptible to clindamycin. Some animals became colonised much later than those treated with cefoxitin but the mortality was similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidemiology and prevention of Clostridium difficile infections in a leukemia unit.

A 29-month prospective study was carried out in a leukemia unit with the aim of investigating the epidemiology ofClostridium difficileinfections and limiting their spread. Systematic cultures of stools and assays for cytotoxin were performed on patient admission and at weekly intervals, yielding 1,355 cultures and assays. The study period was divided in period A, before total unit renovation, and period B, afterwards. During period B all patient carriers ofClostridium difficilereceived vancomycin. A comparison of the two periods showed that the percentage of positive cultures fell from 16.6 % to 3.6 % and the positive toxin assays from 9.9% to 1.2%. It was concluded that colonization byClostridium difficilecan be prevented in hospital wards with generally high rates of infection by a combination of decontamination of the environment, introduction of preventive measures and treatment ofClostridium difficilecarriage with vancomycin.

Bacteremia Due to a Novel Microbacterium Species in a Patient with Leukemia and Description of Microbacterium paraoxydans sp. nov.

ABSTRACT A yellow-pigmented coryneform rod was isolated from the blood of a child with acute lymphoblastic leukemia who was perfused with a central venous catheter. The culture bottles were positive twice, at a 2-month interval. The isolate was identified as a Microbacterium sp. and studied along with five other similar strains. Phenotypic, chemotaxonomic, and genetic characteristics indicated that they are closely related to Microbacteriumoxydans but that they belong to a distinct species, for which the name Microbacteriumparaoxydans sp. nov. is proposed. The type strain of M. paraoxydans is CF36T = DSM 15019T. The G+C content of its DNA is 69.9 mol%.

Clostridium difficile-associated diarrhea in HIV-infected patients: Epidemiology and risk factors

A retrospective analysis of all the cases of Clostridium difficile-associated diarrhea (CDAD) in hospitalized patients infected with HIV was performed over a 52-month period to assess the incidence, epidemiology, and risk factors of CDAD. A case of CDAD was defined as a patient with diarrhea and a positive stool cytotoxin B assay. Sixty-seven cases of CDAD were recorded in HIV-infected patients between January 1991 and April 1995. The annual incidence of CDAD ranged from 1.7 to 6.4 per 100 HIV-infected patients discharged from hospital. The 67 CDAD cases included 48 (72%) first episodes and 19 (28%) relapses. Serogroup C accounted for 69% of strains from initial episodes of CDAD. To identify risk factors for CDAD, 34 HIV-infected patients with a first episode were compared with 66 HIV-infected controls matched for the length of hospital stay. Three independent factors remained significantly associated with CDAD among HIV-infected patients: CD4+ cell counts <50/mm3 (OR = 5.2; 95% CI = 1.4-19.3; p = 0.01), clindamycin use (OR = 5.0; 95% CI = 1.3-18.3; p = 0.02) and penicillin use (OR = 4.6; 95% CI = 1.1-18.8; p = 0.03). C. difficile is a common enteric pathogen responsible for nosocomial diarrhea in HIV-infected patients. Clinicians should keep this pathogen in mind when searching for the cause of diarrhea in these patients, especially those who are severely immunocompromised or have received clindamycin or penicillin.

Description of Chryseobacterium anthropi sp. nov. to accommodate clinical isolates biochemically similar to Kaistella koreensis and Chryseobacterium haifense, proposal to reclassify Kaistella koreensis as Chryseobacterium koreense comb. nov. and emended description of the genus Chryseobacterium.

A collection of eight strains, NF 1366(T), NF 450, NF 1101, NF 1107, NF 1123, NF 1413, CCUG 15260 and CCUG 15624, from various clinical origins, were characterized biochemically as similar to Kaistella koreensis and Chryseobacterium haifense. They differed from K. koreensis, which is unable to alkalinize acetate, and from C. haifense, which is ONPG-positive (beta-galactosidase) and acidifies sucrose, fructose and lactose. Based on 16S rRNA gene sequence comparisons, this collection of strains was most closely related to the type strains of K. koreensis (97.3-97.5 %) and C. haifense (99.1 %). Representative strain NF 1366(T) showed only 41.8 % DNA-DNA relatedness with K. koreensis DSM 12107(T) and only 51.9 % with C. haifense DSM 19056(T). DNA-DNA hybridization of strains NF 450 and CCUG 15624 to strain NF 1366(T) was 41.7 and 74.6 %, respectively, and relatedness of these strains with C. haifense DSM 19056(T) was 72.6 and 70.2 %. With the present information, these two strains must be classified as intermediate between C. haifense and strain NF 1366(T). The fatty acid composition and polar lipid profile of strain NF 1366(T) were similar to those reported for other Chryseobacterium species. Like other chryseobacteria, strain NF 1366(T) exhibited a polyamine pattern with the predominant compound sym-homospermidine and a quinone system consisting of menaquinone MK-6 only. For this collection of clinical strains, the name Chryseobacterium anthropi sp. nov. is proposed, with NF 1366(T) (=CCUG 52764(T) =CIP 109762(T)) as the type strain. K. koreensis was shown to be very similar genotypically and phenotypically to Chryseobacterium. Its polar lipid profile exhibited the major characteristics shown for recently described Chryseobacterium species and the fatty acid profile of K. koreensis was also very similar to those of the Chryseobacterium species. Hence, no striking genotypic or phenotypic differences could be found that could justify the classification of this species into a separate genus, and we therefore propose to reclassify Kaistella koreensis in the genus Chryseobacterium as Chryseobacterium koreense comb. nov. (type strain Chj707(T) =IAM 15050(T) =JCM 21512(T) =KCTC 12107(T) =NBRC 103027(T)). An emended description of the genus Chryseobacterium is also proposed.

Clostridium difficile in neonates: serogrouping and epidemiology.

A typing scheme for clostridium difficile based on serogrouping, toxigenicity and sorbitol fermentation was applied to 270 strains isolated in one neonatal ward during a 6-month prospecitive study. Two hundred and twenty-three strains were isolated from 377 faecal samples of 114 neonates and 47 from 92 environmental specimens. The isolates were distributed among five different types; 87% of the faecal and 85% of the environmental isolates belonged to two of these types (toxigenic, sorbitol negative, serogroup F and nontoxigenic, sorbitol positive, serogroup A). Nosocomial spread was clearly demonstrated and the environment appeared to be the main source of contamination: most of the neonates were colonized after admission by strains found in their environment; clusters of colonization with unusual isolates were observed following referral of patients from the intensive care unit or from other hospitals. No relation was found between the acquisition or the carriage of C. difficile and any intestinal symptoms. All the strains belonged to types different from those usually found in cases of antibiotic associated colitis (AAC) suggesting differences of pathogenicity among the different types.

Molecular Characterization of fliD Gene Encoding Flagellar Cap and Its Expression among Clostridium difficile Isolates from Different Serogroups

ABSTRACT The fliD gene encoding the flagellar cap protein (FliD) of Clostridium difficile was studied in 46 isolates belonging to serogroups A, B, C, D, F, G, H, I, K, X, and S3, including 30 flagellated strains and 16 nonflagellated strains. In all but three isolates, amplification by PCR and reverse transcription-PCR demonstrated that the fliD gene is present and transcribed in both flagellated and nonflagellated strains. PCR-restriction fragment length polymorphism (RFLP) analysis of amplifiedfliD gene products revealed interstrain homogeneity, with one of two major patterns (a and b) found in all but one of the strains, which had pattern c. A polyclonal monospecific antiserum raised to the recombinant FliD protein reacted in immunoblots with crude flagellar preparations from 28 of 30 flagellated strains but did not recognize FliD from nonflagellated strains. The fliDgenes from five strains representative of the three different RFLP groups were sequenced, and sequencing revealed 100% identity between the strains with the same pattern and 88% identity among strains with different patterns. Our results show that even though FliD is a structure exposed to the outer environment, the flagellar cap protein is very well conserved, and this high degree of conservation suggests that it has a very specific function in attachment to cell or mucus receptors.