Bifidobacteria as indicators of faecal contamination along a sheep meat production chain.

AIMS: The potential use of bifidobacteria as indicators for faecal contamination was studied along a sheep meat production and processing chain. The levels of bifidobacteria were compared with those of Escherichia coli. Total viable counts were followed along the chain (244 samples). METHODS AND RESULTS: Forty-three per cent of the samples contained bifidobacteria, of which 15% were solely detected using a PCR method based on the hsp60 gene and not by a culture-based method. Bifidobacteria were detected in only three of nine sheep faeces samples using one or the other method. However, carcasses (types C and E) were highly contaminated. These sample types (30% and 28%, respectively) were positive for bifidobacteria and negative for E. coli. The species Bifidobacterium pseudolongum and Bif. thermophilum, isolated from faecal samples, were predominant. Bifidobacterium choerinum were found in C, D, E and F sample types. CONCLUSIONS: Bifidobacteria were shown more efficient than E. coli in carcasses samples. The presence of Bif. choerinum suggested a faecal pork contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: Detection and identification of bifidobacteria, in correlation with E. coli counting, should improve hygiene quality of mutton processing chains.

Description of a new species, Bifidobacterium crudilactis sp. nov., isolated from raw milk and raw milk cheeses.

A new Bifidobacterium species is described based on the study of ten Gram-positive strains with fructose-6-phosphate phosphoketolase activity. They are part of a phenotypic group comprising 141 strains isolated from raw milk and raw milk cheeses in French raw milk cheese factories. This group was separated by a numerical analysis based on API 50CH, API 32A tests and growth at 46 degrees C. A strong similarity of 16S rRNA sequences (99.8%) was shown between strain FR62/b/3(T) and Bifidobacterium psychraerophilum LMG 21775(T). However, low DNA-DNA relatedness was observed between their DNAs (31%). The new isolates are able to grow at low temperatures (all ten strains up to 5 degrees C) and strain FR62/b/3(T) grows under aerobic conditions, as does B. psychraerophilum. However, contrary to B. psychraerophilum, they do not ferment L-arabinose, D-xylose, arbutin or melezitose, but they do acidify lactose. The DNA G+C content of FR62/b/3(T) is 56.4mol%. Therefore, the name Bifidobacterium crudilactis sp. nov. is proposed, with its type strain being FR62/b/3(T) (=LMG 23609(T)=CNCM I-3342(T)).

Bifidobacterium species isolated from animal feces and from beef and pork meat.

Bifidobacteria were isolated from 122 of 145 samples of animal feces (from cattle, swine, sheep, goats, horses, rabbits, chickens, geese, and pigeons) from farms in France and Austria and from 92 of 955 production and processing chain samples of beef and pork (obtained at slaughter, cutting, and retail). Bacterial strains were identified to species by phenotypic numerical classification based on API 50CH and ID 32A tests and DNA-DNA hybridization. Bifidobacterium pseudolongum was present in 81% (99 of 122 samples) of all Bifidobacterium-positive fecal samples and predominated in samples from all animal species except those from swine from Austria. In these Austrian swine samples, the majority of strains were identified as Bifidobacterium thermophilum (78%), followed by B. pseudolongum (48%). The distribution of B. thermophilum and B. pseudolongum differed significantly between Austrian swine and cattle samples such as those collected along beef and pork production and processing chains. Bifidobacterium animalis was isolated from swine feces, and Bifidobacterium ruminantium was isolated from cow dung. Six fecal isolates (from cattle, swine, rabbits, goats, and horses) were identified as belonging to Bifidobacterium species of predominantly human origin: B. adolescentis, B. bifidum, and B. catenulatum. Only one other species, Bifidobacterium choerinum, was detected with low frequency in a pork processing chain. B. pseudolongum subsp. pseudolongum was predominant in pig feces, whereas B. pseudolongum subsp. globosum was predominant in feces from other animal species. Four strains closely related to both subspecies (58 to 61% DNA reassociation) formed a distinct genomic group. PCR techniques, which are more rapid and sensitive than culture-based methods, could be used to detect directly B. pseudolongum and B. thermophilum as indicators of fecal contamination along the meat processing chain.

A PCR method for detection of bifidobacteria in raw milk and raw milk cheese: comparison with culture-based methods.

Bifidobacteria are well known for their beneficial effects on health and are used as probiotics in food and pharmaceutical products. As they form one of the most important groups in both human and animal feces, their use as fecal indicator organisms in raw milk products has recently been proposed. Bifidobacteria species isolated in humans are different from those isolated in animals. It should therefore be possible to determine contamination origin (human or animal). A method of detecting the Bifidobacterium genus was developed by PCR targeting the hsp60 gene. The genus Bifidobacterium was identified by PCR amplification of a 217-bp hsp60 gene fragment. The degenerated primer pair specific to the Bifidobacterium genus used was tested for it specificity on 127 strains. Sensitivity was measured on artificially contaminated samples. Food can however be a difficult matrix for PCR testing since it contains PCR inhibitors. So an internal PCR control was used. An artificially created DNA fragment of 315 bp was constructed. The PCR detection method was tested on raw milk and cheese samples and compared with three culture-based methods, which comprised enrichment and isolation steps. The enrichment step used Brain Heart Infusion medium with propionic acid, iron citrate, yeast extract, supplemented with mupirocin (BHMup) or not (BH) and the isolation step used Columbia blood agar medium, supplemented with mupirocin (CMup) or not (C). The method using mupirocin at both enrichment and isolation steps and the PCR method performed from the culture in BHMup enrichment medium were shown to be the most efficient. No significant difference was observed in raw milk samples between PCR from BHMup and the culture-based method BHMup/CMup, while a significant difference was noticed between the same methods in raw milk cheese samples, which would favor using PCR. The results suggested that PCR on the hsp60 gene was convenient for a rapid detection of bifidobacteria in raw milk and raw milk cheese samples and that bifidobacteria always present throughout raw milk cheese production could be efficiently used as fecal indicators.

Antimicrobial susceptibility of bifidobacteria.

OBJECTIVES: The aim of our study was to analyse the antibiotic susceptibility of various strains of Bifidobacterium spp. to a wide range of antimicrobial agents. METHODS: Fifty strains belonging to eight species of bifidobacteria, isolated from humans, animals or probiotic products, were tested for susceptibility to 30 antibiotics by disc diffusion on Brucella agar supplemented with 5% laked sheep blood and vitamin K1 (1 mg/L). MICs of nine anti-anaerobe agents, including three new molecules (telithromycin, linezolid and gatifloxacin), were determined using the reference agar-dilution method. RESULTS: All strains of bifidobacteria, whatever the species, were sensitive to penicillins: penicillin G, amoxicillin (MIC(50) 0.06 mg/L), piperacillin, ticarcillin, imipenem and usually anti-Gram-positive antibiotics (macrolides, clindamycin, pristinamycin, vancomycin and teicoplanin). Susceptibility to cefalothin and cefotetan was variable. Most isolates (70%) were resistant to fusidic acid. As expected, high resistance rates were observed for aminoglycosides. Metronidazole, an agent known for its anti-anaerobe activity, was ineffective against 38% of the strains. The newly commercialized molecules, telithromycin, linezolid and gatifloxacin, were active with MIC(50)S of 1 mg/L. The only variation in susceptibility observed among the different species concerned Bifidobacterium breve, which appeared to be generally more resistant. Potentially acquired resistance was only observed against tetracycline and minocycline, in 14% of the strains. CONCLUSIONS: With regard to a general concern about the safety of probiotics, such as potential transferability of resistance determinants, bifidobacteria, with their low natural and acquired resistance to 30 antibiotics, appear risk-free.

Distribution of genes encoding the trypsin-dependent lantibiotic ruminococcin A among bacteria isolated from human fecal microbiota.

Fourteen bacterial strains capable of producing a trypsin-dependent antimicrobial substance active against Clostridium perfringens were isolated from human fecal samples of various origins (from healthy adults and children, as well as from adults with chronic pouchitis). Identification of these strains showed that they belonged to Ruminococcus gnavus, Clostridium nexile, and Ruminococcus hansenii species or to new operational taxonomic units, all from the Clostridium coccoides phylogenetic group. In hybridization experiments with a probe specific for the structural gene encoding the trypsin-dependent lantibiotic ruminococcin A (RumA) produced by R. gnavus, seven strains gave a positive response. All of them harbored three highly conserved copies of rumA-like genes. The deduced peptide sequence was identical to or showed one amino acid difference from the hypothetical precursor of RumA. Our results indicate that the rumA-like genes have been disseminated among R. gnavus and phylogenetically related strains that can make up a significant part of the human fecal microbiota.

Growth, morphology and surface properties of Listeria monocytogenes Scott A and LO28 under saline and acid environments.

AIMS: The effect of salt and acid on the growth and surface properties of two strains of Listeria monocytogenes was investigated. METHODS AND RESULTS: Medium acidification and NaCl supplementation induced a marked increase in the lag and growth times (up to fivefold higher) and a decrease in the maximal optical density. Due to a strong synergic effect of pH and NaCl, growth was only detected after 280 h incubation for Scott A and not detected after 600 h for LO28 at pH 5.0 and 10% NaCl. Furthermore, the addition of NaCl in acidic conditions gave rise to cell filamentation and cell surfaces became strongly hydrophilic. CONCLUSIONS: Some L. monocytogenes strains subjected to high NaCl concentrations in acidic conditions are able to grow but may present altered adhesion properties due to modification of their surface properties. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted that L. monocytogenes do represent a hazard in acid and salted foods, such as soft cheese.

A colony immunoblotting method for quantitative detection of a Bifidobacterium animalis probiotic strain in human faeces.

A colony immunoblotting method has been developed to allow detection of the probiotic Bifidobacterium animalis strain DN-173 010 in human faecal samples. Rabbits were immunized with heat-killed DN-173 010 bacteria resulting in the production of an antiserum highly specific for bacteria belonging to Bif. animalis species. Of the 89 strains representative of 29 different bifidobacterial species tested, only the 15 strains of the Bif. animalis species could be detected with the antiserum. In Western immunoblotting the serum reacts with a protein of 45-kDa apparent molecular weight. None of the bacteria classically encountered in human faecal samples and able to grow on non-selective Columbia blood agar (enterobacteria, Bacteroides or Lactobacillus for instance) reacted with the antiserum. Taking advantage of the high specificity of the antiserum and of the absence of Bif. animalis bacteria in faeces samples of five human volunteers, we demonstrated that strain DN-173 010 survives the intestinal transit. Being based on a combination of semiselective cultivation and colony immunoblotting techniques, the method allowed detection of the Bif. animalis strain even when it represented only one thousandth of the total bifidobacterial population.

Evaluation of the hygienic quality of raw milk based on the presence of bifidobacteria: the cow as a source of faecal contamination.

Fifty-eight samples of raw milk from three different farms were examined for the presence of bifidobacteria. Isolates were identified and compared with bifidobacteria isolated from dung of the cows that provided the milk. Of the raw milk samples, 88% harboured Bifidobacterium pseudolongum subsp. globosum, as did 95% of the dung samples.

Origin and identification of bifidobacteria strains isolated from meat and meat products.

Forty-seven strains of bifidobacteria isolated from meat and meat products have been identified following phenotypic numerical analysis and DNA-DNA hybridization. Twenty-three strains were identified to the species B. thermophilum and 14 to B. pseudolongum subsp. pseudolongum. All others were also of animal origin, except for two strains -- B. longum and B. pseudocatenulatum -- that were of human origin. These strains were isolated from artificially contaminated meat by manual handling.

Two epidemiologically related cases of Rahnella aquatilis bacteremia.

Rahnella aquatilis was isolated from the blood cultures of two patients who were in different units of the same hospital. Both isolates were susceptible to aminoglycosides, fluoroquinolones, cotrimoxazole, piperacillin, third generation cephalosporins and amoxicillin-clavulanate, but resistant to amoxicillin, ticarcillin, and first generation cephalosporins. The synergistic activity of amoxicillin and clavulanic acid suggested the presence of a beta-lactamase, confirmed by a positive nitrocefin test and by analytical isoelectric focusing. Pulsed-field gel electrophoresis and ribotyping with the pKK3535 probe showed that the isolates shared the same banding pattern. The results of an epidemiological study suggested that an in-house total parenteral nutrition solution might be the source of this unusual gram-negative rod.

Glucose and galactose transport in Bifidobacterium bifidum DSM 20082.

Sugar uptake was measured with 3H-galactose and 14C-glucose. Galactose transport system was not modified by inhibitors of known translocases and did not present a saturation kinetic with high concentration of galactose. Glucose incorporation was inhibited by lasalocid (cation symport inhibitor) and increased by KCl. The kinetic parameters KM and Vmax were respectively 9.16 mM and 26.56 nmol/min/mg cell protein. On the basis of this study, galactose crossed through the membrane by diffusion, and glucose was incorporated by a cation symport which is regulated by K+ ions.

Polyphasic approach to the classification and identification of Gardnerella vaginalis and unidentified Gardnerella vaginalis-like coryneforms present in bacterial vaginosis.

A taxonomic study of Gardnerella vaginalis and G. vaginalis-like coryneforms was performed in order to clarify the phylogenetic affiliation of these organisms and to improve future identification. We examined 50 strains by performing whole-cell protein and fatty acid analyses, a 16S rRNA sequence analysis, and an extensive phenotypic characterization analysis. The results of both chemotaxonomic techniques which we used divided the organisms into two main clusters, and the 16S rRNA sequence analysis revealed that the clusters represent different genera, which were easily distinguished by the results of classical phenotypic tests. The cluster I strains were identified as G. vaginalis, which was shown to be a close relative of the genus Bifidobacterium. An improved description of G. vaginalis is presented. The cluster II strains belong to or are closely related to Actinomyces turicensis.

Characterization of the lactose transport system in the strain Bifidobacterium bifidum DSM 20082.

Lactose was fermented but not assimilated by the strain Bifidobacterium bifidum DSM 20082. The sugar uptake was measured with lactose 14C. Km and V(max) values were respectively 2.6 mM and 12.11 nmol/min/mg of cell protein. The lactose transport system and the beta-D-galactosidase were stimulated when the cells were grown with lactose, but isopropyl-beta-D-thiogalactopyranoside had no effect. Lactose uptake was inhibited by compounds which interfered with proton and metal ionophore. Na+, Li+, or K+ did not affect incorporation of lactose. Furthermore, the lactose uptake decreased when an inhibitor of ATP synthesis was used. From the results of this study, the stain contained an active lactose transport system, probably a proton symport as described for Escherichia coli but with a different regulation system.

Phenotypic and genomic analyses of human strains belonging or related to Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium breve.

A numerical analysis based on phenotypic characteristics (89 enzymatic tests and 49 carbohydrate acidification tests), in which experimental strips from Biomerieux-API, La Balme les Grottes, France, were used, was performed to characterize 82 new isolates belonging or related to Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium breve. A total of 72 strains were isolated from child or adult feces, and the other strains were obtained from human vaginas and bronchi. In this study we also included 38 type and reference strains that were representative of all species of the genus Bifidobacterium and 6 strains belonging to the genus Lactobacillus. DNA-DNA relationships between B. longum and B. infantis were determined by using 19 strains related to these species, as determined by the numerical analysis. The degree of DNA binding was determined by the S1 nuclease method. The phenotypic study revealed that there were six main clusters, which were subdivided into nine subclusters. Subcluster Va contained the type strains of B. longum and B. infantis. The DNA-DNA relatedness values of some of the new isolates were very similar to the DNA-DNA relatedness values of the type strain of B. longum. On the basis of these data, it was difficult to isolate B. infantis strains and then to define B. infantis as a single species separated from B. longum. Subclusters IVb to IVf comprised reference strains of B. breve. Cluster III and subcluster Ia were not identified.

Phenotypic differentiation of bifidobacteria of human and animal origins.

The phenotypes of 153 strains belonging or related to the genus Bifidobacterium were studied. These organisms included 38 collection strains and 115 wild strains (41 strains of human origin, 56 strains of animal origin, and 18 strains obtained from rivers or sewage). Our phenotypic analysis revealed seven main groups that were subdivided into 20 subgroups. Seven subgroups contained no type or collection strain. Among the human strains, the type strains of Bifidobacterium pseudocatenulatum and B. catenulatum fell into group I, which contained the type strains of B. adolescentis (subgroup Ib), B. dentium (subgroup Ic), and B. angulatum (ungrouped). The type strain of B. breve belonged to subgroup IIIa1, and the type strains of B. infantis and B. longum fell into subgroup IIIb1. Group VII comprised only wild strains that were isolated from human infant feces. Among the animal strains, group II consisted mainly of bifidobacteria that were isolated from pig feces and contained the type strains of B. suis (subgroup IIb), B. thermophilum (subgroup IIf), B. choerinum, and B. boum (ungrouped). Wild strains belonging to group V were isolated from pig, calf, cow, and chicken feces; this included the type strains of B. animalis (subgroup Va), B. magnum (subgroup Vb), B. pseudolongum, and B. globosum (subgroup Vc). The strains of human origin (groups I, III, and VII) were well separated from the animal strains (groups II, IV, and V). It was not surprising that the wild strains isolated from surface water or sewage were distributed in the animal groups as well as the human groups. Thus, bifidobacteria can be considered to be successful indicators of human or animal fecal pollution when they are correctly classified. The acidification patterns were not adequate to differentiate Bifidobacterium species, as determined previously by Mitsuoka (Bifidobacteria Microflora 3:11-28, 1984) and Scardovi (p. 1418-1434, in P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt, ed., Bergey's Manual of Systematic Bacteriology, vol. 2, 1986). However, enzymatic tests furnished new taxonomic criteria for the genus.

Rahnella aquatilis, a potential contaminant in lager beer breweries.

The species Rahnella aquatilis has been isolated mostly from water, soil, and, in a few cases, from human clinical specimens; little is known about its ecological role. The application of polyacrylamide gel electrophoresis of soluble proteins, DNA-DNA hybridizations and API 20 E systems has shown that Rahnella aquatilis might also be encountered as a contaminant in lager beer breweries.

Development of an expert system for bacterial identification: study of a prototype for identifying beta-galactosidase positive enterobacteria.

A prototype of an expert system for the identification of beta-galactosidase positive Enterobacteriaceae has been developed, for use with the API 20 EC kit. The system is implemented in Prolog on an IBM PC AT with 640 K of central memory and 20 megabytes of secondary memory. Its objectives are to highlight errors that can occur when the kit is in use. It can indicate the presence of new groups or species and give advice or suggest additional tests for the differentiation of the new species from those included in the kit.

[Comparison of two systems for identifying coliform enterobacteria newly described or rarely found in clinical practice]. / Comparaison de deux systèmes d'identification d'entérobactéries coliformes nouvellement décrites ou rarement rencontrées en clinique.

The API 20 EC and ATB 32 GN identification systems were compared for their ability to identify 231 coliform bacteria strains. Agreement with the identification given by conventional methods was achieved for 96.1 p. cent of strains by the API 20 EC gallery and for 95.9 p. cent by the ATB 32 GN system. Complementary tests were needed to identify 9.5 p. cent of strains using the API 20 EC system but 30.3 per cent using the ATB 32 GN system.

Isolation and characterization of monoclonal antibodies against alkaline phosphatase of Pseudomonas aeruginosa.

Monoclonal antibodies against the alkaline phosphatase of Pseudomonas aeruginosa were produced from spleen cells of BALB/c mice primed with purified alkaline phosphatase of P. aeruginosa ATCC 10145 and SP20/Ag-14 myeloma cells. The eight stable clones established produced antibodies that reacted by enzyme-linked immunosorbent and indirect immunofluorescence assays with all bacterial strains of P. aeruginosa, including the 17 serotypes and two nontypable strains. Three of the clones cross-reacted only with some Pseudomonas species of the rRNA homology group I defined by N. J. Palleroni (in N. R. Krieg and J. G. Holt, ed., Bergey's Manual of Systematic Bacteriology, 8th ed., p. 140-218, 1984). The other clones also interacted with other species, including Pseudomonas acidovorans and Xanthomonas maltophilia. Because other species of the genera Aeromonas and Acinetobacter and species of the family Enterobacteriaceae were not detected by these monoclonal antibodies, the antibodies could be used as reagents for routine detection of P. aeruginosa in clinical specimens. Interactions of the antibodies with other Pseudomonas species such as P. fluorescens and P. stutzeri are not important, since these species are susceptible to the same antipseudomonal agents.