Jean-Philippe Carlier

Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis

ABSTRACT A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of Clostridium spp. was confirmed by sequencing of excised bands. C. tyrobutyricum and C. beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C. butyricum and C. sporogenes were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species, C. tyrobutyricum and C. sporogenes, could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C. tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses. The sensitivity of the method was estimated to be 100 CFU/g.

Anaeroglobus geminatus gen. nov., sp. nov., a novel member of the family Veillonellaceae.

A hitherto unknown anaerobic coccus isolated from a post-operative fluid collection was characterized by phenotypic and phylogenetic methods. 16S rDNA sequence analysis revealed an affiliation of this isolate to the family Veillonellaceae. Also, a high level of sequence similarity was observed to some oral clone sequences of Megasphaera spp. contained in the GenBank database under designations BB166, CS025 and BS073. These clones and the unknown bacterium form a well-separated phylogenetic branch that may represent a novel lineage within the family Veillonellaceae. Based on phenotypic and phylogenetic evidence, a new genus, Anaeroglobus gen. nov., is proposed for the unknown bacterium, with one species, Anaeroglobus geminatus gen. nov., sp. nov. The type strain of Anaeroglobus geminatus is strain AIP 313.00T (= CIP 106856T = CCUG 44773T). It is also suggested that the oral clones BB166, CS025 and BS073 belong to the genus Anaeroglobus.

Proposal to unify Clostridium orbiscindens Winter et al. 1991 and Eubacterium plautii (Séguin 1928) Hofstad and Aasjord 1982, with description of Flavonifractor plautii gen. nov., comb. nov., and reassignment of Bacteroides capillosus to Pseudoflavonifractor capillosus gen. nov., comb. nov.

We isolated several strains from various clinical samples (five samples of blood, four of intra-abdominal pus and one of infected soft tissue) that were anaerobic, motile or non-motile and Gram-positive rods. Some of the strains formed spores. Phylogenetic analysis of the 16S rRNA gene sequence showed that these organisms could be placed within clostridial cluster IV as defined by Collins et al. [(1994). Int J Syst Bacteriol 44, 812-826] and shared more than 99 % sequence similarity with Clostridium orbiscindens DSM 6740(T) and Eubacterium plautii DSM 4000(T). Together, they formed a distinct cluster, with Bacteroides capillosus ATCC 29799(T) branching off from this line of descent with sequence similarities of 97.1-97.4 %. The next nearest neighbours of these organisms were Clostridium viride, Oscillibacter valericigenes, Papillibacter cinnamivorans and Sporobacter termitidis, with sequence similarities to the respective type strains of 93.1-93.4, 91.2-91.4, 89.8-90 and 88.7-89.3 %. On the basis of biochemical properties, phylogenetic position, DNA G+C content and DNA-DNA hybridization, it is proposed to unify Clostridium orbiscindens and Eubacterium plautii in a new genus as Flavonifractor plautii gen. nov., comb. nov., with the type strain Prévot S1(T) (=ATCC 29863(T) =VPI 0310(T) =DSM 4000(T)), and to reassign Bacteroides capillosus to Pseudoflavonifractor capillosus gen. nov., comb. nov., with the type strain CCUG 15402A(T) (=ATCC 29799(T) =VPI R2-29-1(T)).

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.

Bacteremia Caused by a Metronidazole-Resistant Prevotella sp. Strain

ABSTRACT Metronidazole resistance among Prevotella spp. is rare. We report here the first case of bacteremia due to a high-level metronidazole-resistant Prevotella sp. responsible for treatment failure.

First Isolation of Desulfovibrio Species as Part of a Polymicrobial Infection from a Brain Abscess

9. Condoluci C, Chessa M, Butera G, Cipriani A, Pelargonio S: Pericarditis caused by Gemella morbillorum. Description of a case. Minerva Pediatrica (1995) 47 :545–547 10. DaCosta CT, Porter C, Parry K, Morris A, Quoraishi AH: Empyema thoracis and lung abscess due to Gemella morbillorum. European Journal of Clinical Microbiology & Infectious Diseases (1996) 15 :75–77 11. Martin MJ, Wright DA, Jones ARR: A case of Gemella morbillorum endocarditis. Lancet (1993) 342 :188

Brain abscess due to Actinobacillus actinomycetemcomitans Case report

Actinobacillus actinomycetemcomitans, a constituent of the oral flora, is a rare cause of brain abscesses. We report the case of a 47‐year‐old male who presented with multiple brain abscesses due to this organism, presumably originating from his poor dentition. Problems met in isolating and identifying A. actinomycetemcomitans suggest that its true rate of isolation from non‐oral samples may have been underestimated.

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.

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.

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.