Longitudinal survey of Clostridium difficile presence and gut microbiota composition in a Belgian nursing home.

BACKGROUND: Increasing age, several co-morbidities, environmental contamination, antibiotic exposure and other intestinal perturbations appear to be the greatest risk factors for C. difficile infection (CDI). Therefore, elderly care home residents are considered particularly vulnerable to the infection. The main objective of this study was to evaluate and follow the prevalence of C. difficile in 23 elderly care home residents weekly during a 4-month period. A C. difficile microbiological detection scheme was performed along with an overall microbial biodiversity study of the faeces content by 16S rRNA gene analysis. RESULTS: Seven out of 23 (30.4 %) residents were (at least one week) positive for C. difficile. C. difficile was detected in 14 out of 30 diarrhoeal samples (43.7 %). The most common PCR-ribotype identified was 027. MLVA showed that there was a clonal dissemination of C. difficile strains within the nursing home residents. 16S-profiling analyses revealed that each resident has his own bacterial imprint, which was stable during the entire study. Significant changes were observed in C. difficile positive individuals in the relative abundance of a few bacterial populations, including Lachnospiraceae and Verrucomicrobiaceae. A decrease of Akkermansia in positive subjects to the bacterium was repeatedly found. CONCLUSIONS: A high C. difficile colonisation in nursing home residents was found, with a predominance of the hypervirulent PCR-ribotype 027. Positive C. difficile status is not associated with microbiota richness or biodiversity reduction in this study. The link between Akkermansia, gut inflammation and C. difficile colonisation merits further investigations.

Faecal microbiota characterisation of horses using 16 rdna barcoded pyrosequencing, and carriage rate of clostridium difficile at hospital admission.

BACKGROUND: The equine faecal microbiota is very complex and remains largely unknown, while interspecies interactions have an important contribution to animal health. Clostridium difficile has been identified as an important cause of diarrhoea in horses. This study provides further information on the nature of the bacterial communities present in horses developing an episode of diarrhoea. The prevalence of C. difficile in hospitalised horses at the time of admission is also reported. RESULTS: Bacterial diversity of the gut microbiota in diarrhoea is lower than that in non-diarrhoeic horses in terms of species richness (p-value <0.002) and in population evenness (p-value: 0.02). Statistical differences for Actinobacillus, Porphyromonas, RC9 group, Roseburia and Ruminococcaceae were revealed. Fusobacteria was found in horses with diarrhoea but not in any of the horses with non-diarrheic faeces. In contrast, Akkermansia was among the three predominant taxa in all of the horses studied. The overall prevalence of C. difficile in the total samples of hospitalised horses at admission was 3.7 % (5/134), with five different PCR-ribotypes identified, including PCR-ribotype 014. Two colonised horses displayed a decreased bacterial species richness compared to the remaining subjects studied, which shared the same Bacteroides genus. However, none of the positive animals had diarrhoea at the moment of sampling. CONCLUSIONS: The abundance of some taxa in the faecal microbiota of diarrhoeic horses can be a result of microbiome dysbiosis, and therefore a cause of intestinal disease, or some of these taxa may act as equine enteric pathogens. Clostridium difficile colonisation seems to be transient in all of the horses studied, without overgrowth to trigger infection. A large proportion of the sequences were unclassified, showing the complexity of horses' faecal microbiota.

Description of Acidovorax wautersii sp. nov. to accommodate clinical isolates and an environmental isolate, most closely related to Acidovorax avenae.

Three Gram-negative strains, NF 1078(T), NF 1598 and NF 1715, were isolated from clinical (two) and environmental (one) samples, respectively. Sequence analysis of the 16S rRNA genes revealed similarity of 100% among the three strains and next highest similarity to the type strain of Acidovorax avenae (98.16%). The three strains were able to acidify lactose and rhamnose on low peptone phenol red agar and to alkalinize citrate on Simmons' agar and were negative for nitrate reduction. The DNA G+C content of strain NF 1078(T) was 67.1 mol%. The level of DNA-DNA relatedness between this strain and the type strains of A. avenae (ATCC 19860(T), LMG 2117(T)) was 29%. Based on these phylogenetic, phenotypic and genotypic analyses, the three strains could be distinguished clearly from all other recognized Acidovorax species and should be classified as representatives of a novel species of the genus Acidovorax, for which the name Acidovorax wautersii sp. nov. is proposed. The type strain is NF 1078(T) (=LMG 26971(T)=CCUG 62584(T)).

Laboratory diagnosis of Clostridium difficile-associated diarrhoea: a plea for culture.

A routine protocol for diagnosing Clostridium difficile-associated diarrhoea (CDAD) based on both faecal-cytotoxin detection and toxigenic culture was adopted by the microbiology laboratory of the St Luc-UCL University Hospital in Brussels in 1997. A toxigenic culture is a faecal culture followed, in the case of positivity, by a direct immunoassay on colonies to detect toxin A production. The results obtained over the past 7 years in the hospital are reviewed here. A total of 10,552 diarrhoeal stools from 7042 patients were analysed, of which 9494 were negative for all tests. A total of 1058 samples (10 %) from 794 patients were culture-positive, of which 460 (4.4 %) were positive for a faecal cytotoxin. The remaining 598 cultures were tested for toxin A on colonies; 355 of them were positive, which is 3.4 % of the total, and the remaining 243 (2.3 %) were negative. The positivity of the faecal-cytotoxin assay was statistically linked to the number of colonies observed on the culture plate. In conclusion, over a 7 year period, toxigenic culture allowed the diagnosis of 355 cases of CDAD that would have been missed by a protocol using a faecal-cytotoxin assay alone. In terms of both patient care, prevention of environmental contamination and prevention of risk of a hospital outbreak, it is proposed that these results justify the recommendation to perform both faecal-toxin assay and culture in routine medical practice.

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.

Chryseobacterium hominis sp. nov., to accommodate clinical isolates biochemically similar to CDC groups II-h and II-c.

A collection of eight clinical strains from Belgian hospitals and three clinical strains of the CCUG collection were characterized biochemically as being similar to CDC groups II-h and II-c; the latter differs from group II-h only by positivity for sucrose acidification. These 11 strains were found to cluster according to 16S rRNA gene sequence similarity at a level of >or=99.5%, and on the basis of their tDNA-PCR profile. Based on 16S rRNA gene sequence analysis, this collection of strains was related most closely to Chryseobacterium hispanicum (97.2%), but they differed from the type strain of this species by the following phenotypic characteristics: growth at 37 degrees C, negativity for xylose acidification, positivity for acetate assimilation-alkalinization on Simmons' agar base and absence of flexirubin pigments, and by their tDNA-PCR profile. Strain NF802T showed only 57.8% DNA-DNA relatedness to the type strain of C. hispanicum. Fatty acid composition did not enable differentiation from C. hispanicum. The DNA G+C content of strain NF802T is 36.5 mol%. The name Chryseobacterium hominis sp. nov. is proposed for this taxon, with type strain NF802T (=CCUG 52711T=CIP 109415T).

Description of Wautersiella falsenii gen. nov., sp. nov., to accommodate clinical isolates phenotypically resembling members of the genera Chryseobacterium and Empedobacter.

A total of 26 isolates of non-fermenting, Gram-negative rods, obtained between 1980 and 2004 by various clinical laboratories in Belgium, with phenotypic characteristics resembling those of members of the genera Chryseobacterium and Empedobacter (indole-positive) and a biochemical profile resembling that of CDC group II-h, but urease-positive, were collected at the Université Catholique de Louvain Microbiology Laboratory, Belgium. The 16S rRNA gene sequences were determined for most of the isolates and showed 94-95 % similarity with the type strain of Empedobacter brevis as the closest relative, indicating that these isolates might belong to a separate genus. Furthermore, the 16S rRNA gene sequences of the isolates were similar, but two clusters (genomovars) could be distinguished. The sequence similarities were 99.5-100 % for the 14 isolates of genomovar 1 and 99.4-100 % for the 12 isolates of genomovar 2. The similarity between the two clusters was 98.3-99.5 %. The presence of two clearly different groups was corroborated by using tRNA intergenic length polymorphism analysis, which also enabled differentiation of the novel species from all other species studied thus far using this technique. DNA-DNA hybridization results excluded a close relatedness to Empedobacter brevis. The DNA G+C contents of the reference strains of genomovars 1 and 2 were 33.8+/-0.4 and 34.4+/-0.2 mol%, respectively. The name Wautersiella falsenii gen. nov., sp. nov., is proposed for this group, comprising two closely related genomovars. The type strain of the species and reference strain for genomovar 1 is NF 993(T) (=CCUG 51536(T)=CIP 108861(T)), which was isolated from a surgical wound. The reference strain for genomovar 2 is NF 770 (=CCUG 51537=CIP 108860), which was isolated from blood.

Mycobacterium mucogenicum sepsis in an immunocompetent patient.

We report a case of Mycobacterium mucogenicum sepsis in an immunocompetent woman with Münchausen syndrome presenting with multiple abscesses.

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

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, gamma-glutamyl aminopeptidase, alpha-mannosidase, and alpha-glucosidase were found to be highly discriminating and could be used to develop an identification scheme.

Toxin A detection on Clostridium difficile colonies from 24-h cultures.

OBJECTIVE: Performance of a combined approach for the detection of toxigenic strains in patients suspected of having Clostridium difficile-associated disease was evaluated. METHODS: In this approach, stools were cultured for 24 h on a selective medium supplemented with sodium taurocholate (TCCFA), in anaerobic conditions created with the Martreg Anoxomat system, and toxin A detection was performed directly on C. difficile colonies, by enzyme immunoassay (EIA). This method was compared with three others: cytotoxigenic culture consisting of a 48-h culture on selective medium followed by detection of in vitro cytotoxin production on cell monolayers, fecal cytotoxin detection and fecal toxin A detection by EIA. RESULTS: From 548 stools, 108 yielded a positive culture by at least one of the methods, and 81 isolates were cytotoxin producers. Cultures for 24 h on TCCFA were positive in 106 cases and EIA performed on colonies gave 73 positive results, giving a sensitivity of 90.1% and a specificity of 100%. By comparison, the sensitivity and specificity of cytotoxigenic culture, stool cytotoxin and stool toxin A were respectively 96.2% and 100%, 61.7% and 100%, and 66.7% and 95.9%. CONCLUSIONS: Performing EIA on colonies recovered after 24 h culture allows us to improve the detection of toxigenic strains in patients suspected of having C. difficile-associated disease.

Biochemical and susceptibility tests useful for identification of nonfermenting gram-negative rods.

Six hundred nineteen strains of nonfermenting gram-negative rods were tested for alkaline phosphatase, benzyl-arginine arylamidase, pyrrolidonyl arylamidase, ethylene glycol acidification, and susceptibility to desferrioxamine and colistin. The results were highly discriminant. Therefore, the proposed tests may be helpful for the identification of this group of organisms.

Bacteremia due to a novel Microbacterium species in a patient with leukemia and description of Microbacterium paraoxydans sp. nov.

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 Microbacterium oxydans but that they belong to a distinct species, for which the name Microbacterium paraoxydans sp. nov. is proposed. The type strain of M. paraoxydans is CF36(T) = DSM 15019(T). The G+C content of its DNA is 69.9 mol%.

Evaluation of Oxoid combination discs for detection of extended-spectrum beta-lactamases.

OBJECTIVES: We evaluated the reliability of cefpirome/clavulanate (CD04) compared with ceftazidime/clavulanate (CD02) and cefotaxime/clavulanate (CD03) Oxoid combination discs for the detection of extended-spectrum beta-lactamases (ESBL) in several Enterobacteriaceae isolates, including Enterobacter spp. METHODS: Overall, a total of 105 ESBL-positive [positive double-disc synergy test (DDST)] and 94 ESBL-negative (negative DDST) Gram-negative isolates were evaluated. Ninety-eight isolates were confirmed as ESBL-positive on the basis of the sequence alignments of the blaTEM and/or blaSHV gene amplification products, which matched with previously identified ESBLs. The phenotypic detection of ESBLs was performed by the three combination discs according to the NCCLS and BSAC methods. The CD04 disc was evaluated with the manufacturer's recommended zone size difference breakpoint of > or =4 mm. RESULTS: In Escherichia coli and Klebsiella spp., the sensitivities (%)/specificities (%) of CD02, CD03 and CD04 discs, and the combination of CD02 or CD04 discs, were, respectively, 88/92, 90/92, 95/84 and 100/82, while the corresponding figures were 94/100, 4/100, 94/100 and 100/100 in Enterobacter aerogenes. NCCLS and BSAC methods yielded concordant results in 99% of the isolates. CONCLUSIONS: CD04 and CD02 discs were the best combination for detection of ESBLs in our collection of Enterobacteriaceae isolates, including E. aerogenes.

Histidine decarboxylase in Enterobacteriaceae revisited.

With a modification of Taylor's decarboxylation broth, histidine decarboxylase was detected in Enterobacter aerogenes, Morganella morganii, Raoultella ornithinolytica, and some strains of Citrobacter youngae and Raoultella planticola. This method provides a useful confirmatory test for identification of E. aerogenes strains.

Study of genotypes and virB4 secretion gene of Bartonella henselae strains from patients with clinically defined cat scratch disease.

Bartonella henselae is the causative agent of cat scratch disease (CSD), which usually presents as a self-limiting lymphadenopathy. Occasionally, the bacteria will spread and be responsible for tissue and visceral involvement. Two B. henselae genotypes (genotypes I and II) have been described to be responsible for uncomplicated CSD on the basis of 16S rRNA sequence analysis. A type IV secretion system (T4SS) similar to the virulence-associated VirB system of Agrobacterium tumefaciens was recently identified in the B. henselae Houston-1 genotype I strain. We studied the correlations of the B. henselae genotypes with the clinical presentations and with the presence of T4SS. Isolates originated from CSD patients whose lymph nodes were prospectively analyzed. B. henselae genotype I was identified in 13 of 42 patients (30%). Among these, two teenage twins presented with hepatosplenic CSD and one immunocompetent adult presented with osteomyelitis. Genotype II was detected in 28 of 42 patients (67%), all of whom presented with uncomplicated CSD. The last patient was infected with both genotypes. T4SS was studied by PCR amplification of the virB4 gene. Amplification of virB4 codons 146 to 256, 273 to 357, and 480 to 537 enabled us to detect 66, 90, and 100% of the B. henselae isolates, respectively. Sequence analysis revealed sequence variations that correlated with genotype distribution. Our studies suggest that B. henselae genotype I strains harbor virB4 genes that are different from those harbored by genotype II strains and that genotype I strains might be more pathogenic.

Brevibacterium lutescens sp. nov., from human and environmental samples.

Three strains of coryneform rods isolated from clinical samples and one of environmental origin exhibited phenotypic and chemotaxonomic properties characteristic of the genus Brevibacterium and their 16S rRNA gene sequences were closely related (98.5-99.0 %) to that of Brevibacterium otitidis. However, DNA-DNA hybridization of one strain (CF87(T)) showed only 59.6 % relatedness to the type strain of B. otitidis, DSM 10718(T), and 75-82 % relatedness to the three other strains. The four strains could be differentiated from B. otitidis by cellular fatty acid composition and some phenotypic characteristics. These findings suggest that the four strains belong to a novel species, for which the name Brevibacterium lutescens sp. nov. is proposed. The type strain of B. lutescens is CF87(T) (=DSM 15022(T)=CCUG 46604(T)).

Identification of a novel Brevibacterium species isolated from humans and description of Brevibacterium sanguinis sp. nov.

Six coryneforms isolated from blood and dialysate fluid were phenotypically similar to Brevibacterium casei, but 16S rRNA gene sequencing and DNA-DNA hybridization indicate that they belong to a new species for which the name Brevibacterium sanguinis is proposed.