Metronidazole resistance in Prevotella spp. and description of a new nim gene in Prevotella baroniae.

Nonduplicate clinical isolates of Prevotella spp. recovered from patients hospitalized between 2003 and 2006 in two French tertiary-care teaching hospitals were investigated for their susceptibility to metronidazole and the presence of nim genes. Of the 188 strains tested, 3 isolates displayed reduced susceptibility to metronidazole after 48 h of incubation, while 27 additional isolates exhibited heterogeneous resistance after prolonged incubation; all 30 of the isolates were nim negative. Among the remaining 158 isolates, 7 nim-positive isolates were detected. All of these strains were identified as Prevotella baroniae by 16S rRNA gene sequence analysis and contained a new nim gene, named nimI, as determined by DNA sequence analysis. Chromosomal localization of this single-copy gene was demonstrated in all clinical isolates as well as in type strain P. baroniae DSM 16972 by using Southern hybridization. No known associated insertion sequence elements were detected upstream of the nimI gene in any of the nim-positive strains by PCR mapping. After prolonged exposure to metronidazole, stable resistant subpopulations could be selected in nimI-positive Prevotella isolates (n = 6) as well as in nim-negative Prevotella isolates (n = 6), irrespective of their initial susceptibility to this antibiotic. This study is the first description of a new nitroimidazole resistance gene in P. baroniae which seems to be silent and which might be intrinsic in this species. Moreover, our findings highlight the fact that high-level resistance to metronidazole may be easily induced in both nim-positive and nim-negative Prevotella sp. strains.

Production of an antibacterial substance in the digestive tract involved in colonization-resistance against Clostridium perfringens.

Ruminococcus gnavus E1, Bacteroides thetaiotaomicron LEMF4, Clostridium hathewayi LEMC7, and Clostridium orbiscindens LEMH9 were isolated from ex germ-free mice inoculated with a human faecal microbiota. When initially germ-free mice who were previously inoculated with the strain E1 alone, or a four-strain consortium [E1, LEMF4, LEMC7, and LEMH9], were then challenged with 108 counts of Clostridium perfringens; the target strain was rapidly eliminated from the digestive tract of the animals (<10² cfu g⁻¹ of faeces). R. gnavus E1 was able to produce a diffusible anti-C. perfringens substance that accumulated in the faeces of monoassociated animals, but failed to be detected in the faeces of mice associated with the four-strain consortium. The capability to produce the antibacterial substance was transferred in the digestive tract of gnotobiotic mice to a Dorea longicatena strain. Further experiments realized with the D. longicatena wild type strain and the transconjugant support the assumption that the diffusible antibacterial substance was necessary for obtaining the antagonistic effect against C. perfringens, but that it acted as a precursor in the mechanism of interaction of the four-strain consortia.

Botulism in patients who inhale cocaine: the first cases in France.

We describe 2 cases of mild botulism in patients who inhaled cocaine. Botulism, though rare, is increasing in incidence among illicit drug users. To our knowledge, these are the first cases of botulism in illicit drug users in France. Clinicians should be aware of this phenomenon; botulism should be considered in illicit drug users with neurological symptoms.

Identification of a Clostridium cocleatum strain involved in an anti-Clostridium difficile barrier effect and determination of its mucin-degrading enzymes.

We isolated Gram-positive circular bacterium HB1 from intestinal microflora showing resistance to colonization by Clostridium difficile in mice (Su et. al., 1986a,b). We studied its enzymatic capacity to degrade mucin the first potential barrier to implantation of strains in the intestine. Its biochemical characteristics, terminal metabolites and the electrophoretic profiles of proteins and DNA-DNA homology indicated that it was a strain of Clostridium cocleatum. This strain displayed numerous glucosidase activities which were assumed to play a role in the degradation of mucin oligosaccharide chains in the digestive tract. These enzymes included alpha- and beta-galactosidases, beta-glucosidase, beta-N-acetylglucosaminidase, sialidase and alpha-N-acetylgalactosaminidase.

Phenotypic diversity of anaerobic glycerol dissimilation shown by seven enterobacterial species.

The anaerobic glycerol pathway was studied in seven enterobacterial species selected as representative of different behaviours in terms of anaerobic glycerol dissimilation. The presence of oxidative and reductive pathways of the dha regulon in Klebsiella pneumoniae enabled the cells to grow fermentatively on glycerol. The first two enzymes of the dha regulon (glycerol dehydrogenase type I and dihydroxyacetone kinase) represent the oxidative branch, while the latter two (glycerol dehydratase and 1,3-propanediol dehydrogenase) represent the reductive branch of glycerol fermentation. The slower utilization of glycerol by K. oxytoca was attributed to low production of 1,3-propanediol. K. oxytoca lacked glycerol dehydratase and demonstrated low 1,3-propanediol dehydrogenase activity. K. planticola and K. ozaenae differed from K. pneumoniae and K. oxytoca in lacking the ability to grow on glycerol. K. planticola lacked both enzymes of the reductive branch of glycerol fermentation, and K. ozaenae possessed glycerol dehydrogenase only. K. rhinoscleromatis and Hafnia alvei, like Escherichia coli, did not possess a dha regulon. The glycerol dehydrogenase type II of H. alvei was distinct from that of E. coli. The phenotypic diversity of anaerobic glycerol dissimilation may have taxonomic applications.

Prevotella nanceiensis sp. nov., isolated from human clinical samples.

Three strains of anaerobic, non-pigmented, Gram-negative bacilli isolated from various human clinical samples were characterized in terms of phenotypic and genotypic tests, including sequence analysis of 16S rRNA and rpoB genes. The strains were most closely related to the type strains of Prevotella marshii and Prevotella shahii on the basis of both 16S rRNA (89.8 and 89.0 % identity, respectively) and rpoB gene sequences (83.1 and 82.8 % identity, respectively). Phylogenetic analysis showed that the isolates constituted a robust homogeneous group distinct from known species in the genus Prevotella. The rrn skeleton (as determined by PFGE) and the DNA G+C content, determined to be 39.4 mol% for strain LBN 293(T), distinguished the novel isolates from the type strains of P. marshii and P. shahii. The three strains were saccharolytic and produced acetic, lactic and succinic acids as major metabolic end products. Polyphasic investigations supported the proposal of a novel species, Prevotella nanceiensis sp. nov., with LBN 293(T) (=AIP 261.03(T) =CIP 108993(T) =CCUG 54409(T)) as the type strain.

[Gas-liquid chromatography of non-volatile fatty acid produced by "Yersinia enterocolitica" (author's transl)]. / Chromatographie en phase gazeuse des acides gras fixes produits par Yersinia enterocolitica dans divers milieux liquides.

The metabolic products (non-volatile fatty acids) of 24 strains belonging to four Yersinia enterocolitica groups (typical, sucrose negative, rhamnose positive, melibiose and rhamnose positive) were examined by gas liquid chromatography. All strains produced lactic, pyruvic and succinic acids. This technique did not allow any classification for taxonomic purpose. The evolution of lactic and succinic acid production was monitored for 72 h in shaked and non-shaked cultures. The concentration of both acids increased in the first 24 h, then decreased and increased again after 38 h. Shaked culture produced a lesser amount of lactic acid than did non-shaked culture. Only traces of succinic acid were detected in shaked culture. The quantities of metabolic products of the four strains grown in five media were significantly different. These observations suggest that the composition of the medium, the bacterial growth, inoculum size, incubation technique and the incubation time are important factors in metabolic production by aeroanerobic bacteria. Only constant results in the face of such varying factors could be of taxonomic value.

Gas chromatographic-mass spectral studies after methylation of metabolites produced by some anaerobic bacteria in spent media.

The gas chromatographic analysis of metabolites such as volatile fatty acids (VFAs) and non-volatile fatty acids (NVFAs) for identification of anaerobic bacteria is now widely performed. Cultures of anaerobes tested for NVFAs as methyl esters were found to contain several unidentified compounds not previously detected and/or reported with methylation procedures. Gas chromatographic-mass spectrometric studies demonstrated that these compounds correspond to the methyl esters of both saturated and unsaturated short-chain fatty acids, and also of 2-hydroxy and 2-oxo acids. The distribution of these acids among different species of anaerobes was determined and their amounts were measured. The effects of supplementing the culture medium with either glucose or amino acids on the production of these acids are described. The use of very polar stationary phases is suggested for a better separation of all NVFAs.

A simple technique useful for clinical laboratory testing for invasive potential applied to Bacteroides fragilis.

A simple technique using fluorescent microscopy examines the association between Bacteroides fragilis and different types of tissue cells (epithelial, fibroblast, osteoblast). Through this stepwise intracellular staining and extracellular quenching some latent signs of invasive quality may be exhibited. The bacteria are incubated with monolayers of tissue cells, and the reactions immediately visualized after staining with acridine orange and masking by crystal violet. Intact bacterial rods exhibit a greenish hue which will reveal their location within the tissue cells but varies depending on tissue cell type. This direct means of testing for invasive potential applied to B. fragilis relies on the use of a readily available clinical tool.

Demonstration by confocal laserscanning microscopy of invasive potential with Bacteroides fragilis.

The confocal laserscanning microscopy (CLSM) system provides a stereoscopic view of an object. By this system the penetration of B. fragilis into HeLa cells was observed. The intensity of contact is highlighted with time. The CLSM system consolidates the recently described fluorescence technique to test invasive potential. This work purports that certain gram-negative anaerobes should be considered for invasiveness.

Taxonomic position of lecithinase-negative strains of Clostridium sordellii.

Eleven out of 43 strains of Clostridium sordellii from clinical sources did not produce lecithinase activity and were not toxic to mice. However, these strains did belong to the C. sordellii group and could readily be differentiated from C. bifermentans and C. difficile on the basis of DNA-DNA homologies, carbohydrate fermentation patterns, enzyme activities, GLC analysis of fatty acid fermentation products and the electrophoretic analysis of whole cell protein extracts.

Identification of Clostridium butyricum and Clostridium beijerinckii by gas-liquid chromatography and sugar fermentation: correlation with DNA homologies and electrophoretic patterns.

Sixty-five strains of clostridia of the butyricum group were studied by DNA-DNA hybridization, electrophoresis of cell proteins, gas-liquid chromatography, and fermentation of glycerol, inositol and ribose. The DNA--DNA hybridization results confirmed that strains of this group belong to two main species, Clostridium butyricum and C. beijerinckii. Five strains did not hybridize with the reference strains of these two species. Most of the strains could be identified by quantitative gas-liquid chromatographic analysis combined with fermentation patterns. The other strains could be identified by their protein electrophoretic patterns.

Effect of growth of Bacteroides fragilis at different redox levels on potential pathogenicity in a HeLa cell system: demonstration by confocal laser scanning microscopy.

During trauma, the intestinal anaerobe, Bacteroides fragilis, may enter into a pathogenic state. The process coincides with changing environmental conditions particularly the redox level in situ. To gain insight into this phenomenon B. fragilis was grown at different redox levels, and the invasive potential was examined using an in vitro model consisting of HeLa cell monolayers. The clinical strain AIP 5-86 was taken from a small collection of B. fragilis strains able to penetrate into tissue cell monolayers when selected by an acridine orange-crystal violet fluorescent staining technique. Following preliminary investigation by confocal laser scanning microscopy (CLSM), this particular strain was regarded as representative for examining the invasive potential. After growth in a defined medium under oxidizing, reducing or intermediate Eh7 conditions, the washed mid-log phase bacteria were allowed to interact with HeLa cell monolayers for 45 min at 37 degrees C. The results were extensively monitored by CLSM to follow the reactions in a stereoscopic dimension. In addition, the bacteria were examined by transmission and scanning electron microscopy before interaction to distinguish characteristics in surface configuration. The growth of the bacteria at particular redox levels seemed to influence their potential for pathogenicity. After growth at relatively high Eh, the bacteria easily penetrated into the HeLa cells, but not at low Eh, as determined by the laser scanning studies. Examination of the bacteria alone by transmission and scanning electron microscopy revealed small vesicles and a tendency to aggregate after growth at the low redox level while there were rather few vesicles and an implied dispersion at the high redox level. This leaves it open whether the invasiveness was based on the alterations found during growth of the bacteria. Different redox levels as well as the respective changes of the bacterial surface may help to discern the commensal from the pathogenic state of B. fragilis.

Conversion of DL-threonine, D-threonine and 2-oxobutyrate into propionate and 2-hydroxybutyrate by Fusobacterium species.

The present investigation examined DL-threonine, D-threonine and 2-oxobutyrate conversion into propionate and 2-hydroxybutyrate by various type strains and clinical isolates of Fusobacterium. Except for Fus. naviforme, the type strains were able to produce varying degrees of propionate and/or 2-hydroxybutyrate from DL-threonine. Additionally, D-threonine was converted into an equimolar amount of propionate by Fus. necrophorum subsp. necrophorum, Fus. nucleatum subsp. nucleatum and Fus. varium, and to a lower but significant amount by Fus. mortiferum and Fus. perfoetens. However, the level of propionate remained unchanged for Fus. nucleatum subsp. fusiforme, Fus. nucleatum subsp. vincentii, Fus. naviforme, Fus. necrophorum subsp. funduliforme, Fus. gonidiaforme and Fus. russii. 2-Oxobutyrate was fermented to propionate by all type strains, although Fus. russii reduced it mainly to 2-hydroxybutyrate. Thus, an attempt was made to make use of these features in order to identify clinical isolates.

Metabolism of a 5-nitroimidazole in susceptible and resistant isogenic strains of Bacteroides fragilis.

We investigated the metabolism of dimetridazole (1,2-dimethyl-5-nitroimidazole) (DMZ) by the resting cell method in a susceptible strain of Bacteroides fragilis and in the same strain containing the nimA gene, which conferred resistance to 5-nitroimidazole drugs. In both cases, under strict anaerobic conditions DMZ was metabolized without major ring cleavage or nitrate formation. However, one of two distinct metabolic pathways is involved, depending on the susceptibility of the strain. In the susceptible strain, the classical reduction pathway of nitroaromatic compounds is followed at least as far as the nitroso-radical anion, with further formation of the azo-dimer: 5,5'-azobis-(1,2-dimethylimidazole). In the resistant strain, DMZ is reduced to the amine derivative, namely, 5-amino-1,2-dimethylimidazole, preventing the formation of the toxic form of the drug. The specificity of the six-electron reduction of the nitro group, which is restricted to 4- and 5-nitroimidazole, suggests an enzymatic reaction. We thus conclude that nimA and related genes may encode a 5-nitroimidazole reductase.

[Hydrolysis of tributyrin by species of "Neisseria" and "Branhamella" (author's transl)]. / Hydrolyse de la tributyrine par les Neisseria et les Branhamella.

A comparative study of 60 strains of Neisseriaceae was done using 4 different substrates: tween 20, tween 80, tributyrin and naphthylmyristate. Production of butyric acid from tributyrin was measured by gas chromatography. Strains of the genus Branhamella produced large amounts of butyric acid, whereas strains of the genus Neisseria produced little amounts. Evidence of lipolytic activity can be used to separate Branhamella from Neisseria. A biochemical method is described using a buffered medium containing tributyrin and phenol red which can best separate lipolytic and non-lipolytic strains of Neisseriaceae. All B. catarrhalis gave positive results as well as related species B. caviae, B. ovis and B. cuniculi. All Neisseria strains gave negative results.

Low-level vancomycin resistance in Clostridium innocuum.

Low-level vancomycin resistance was observed for 28 clinical Clostridium innocuum isolates and C. innocuum NCIB 10674, whereas teicoplanin was active. DNA from three clinical isolates and the type strain could not be amplified by PCR with primers specific for the genes vanA, vanB, and vanC, suggesting that C. innocuum is intrinsically resistant to vancomycin.