Large scale preparation of Rhizobium meliloti bacteriophages by fermenter culture.

A simple method for preparation of Rhizobium meliloti bacteriophages was established employing fermenter culture. This technique allowed phage production to be checked by dissolved oxygen measure. Phage suspensions ranging from 5.10(12) to 1.2.10(13) PFU/ml were found after polyethylene glycol precipitation and centrifugation in CsCl.

Occurrence and Multiple Antibiotic Resistance Profiles of Non-fermentative Gram-Negative Microflora in Five Brands of Non-carbonated French Bottled Spring Water.

Five brands of French bottled mineral water were analyzed by heterotrophic plate counts (HPC) and for the presence of multiple antibiotic resistant bacteria. HPC at 22 degrees C were around 10(4) colony forming units ml(-1) on R2A medium. Enumeration on PCA/10, MH, and especially PCA and King B media was less efficient. At 37 degrees C, HPC were two to three orders of magnitude less than at 22 degrees C. Moreover, phenotypic diversity (7 to 15 phenotypes) was optimal on R2A incubated at 22 degrees C. All isolates were identified as non-fermentative Gram-negative rods and 75% were non-identifiable with the API 20NE system. Stenotrophomonas maltophilia and fluorescent Pseudomonas were isolated on VIA and CFC selective agar media, respectively. Burkholderia cepacia strains were not isolated on BCSA medium. The species S. maltophilia was found in 33%, 28%, and 11% of sample from springs A, D, and E, respectively. Independent of brand, isolates from HPC media were less efficient to achieve confluent growth in 18 h on MH at 30 or 37 degrees C (0 to 40%) than isolates from selective media (28 to 63%). Seventy percent of the total isolates from dominant microflora (1-5 x 10(3) CFU ml(-1) on HPC media) were resistant against two or four antibiotics. The antibiotics concerned were principally aztreonam, ampicillin, and nalidixic acid. The remaining dominant bacteria showed a 6-9 multiple antibiotic resistant (MAR) pattern. All isolates were susceptible to newer antimicrobial agents. Owing to their low nutrient and temperature requirements, these isolates are unlikely to cause concern to public heath. Fifty percent of strains isolated from selective media (non-dominant microflora, 4-40 CFU l(-1)) showed a 10-18 MAR pattern and 33%, identified as S. maltophilia, a 20-27 MAR pattern. However, minocycline was effective against all isolates. Owing to its low concentration, colonization of human intestine by MAR S. maltophilia is unlikely.

Growth and survival of clinical vs. environmental species of Aeromonas in tap water.

The ability of four species of Aeromonas (two of clinical and two of environmental origin) to survive and/or grow in tap water microcosms supplemented with sodium thiosulphate was tested. After bottling, the autochthonous microflora reached 6 x 10(5) cfu ml(-1) after a 5-day incubation period in tap water unfiltered and which was non-autoclaved. In filtered tap water, "ultramicrocells" were detected and final populations of ca. 10(6) cfu ml(-1) after 7 days were obtained. Aeromonas was inoculated at an initial cell concentration of ca. 10(4) cfu ml(-1). All strains were able to grow in tap water samples, which were filtered and autoclaved, and a final concentration of 10(5)-10(6) cfu ml(-1) was observed. Any inherent capability of Aeromonas to grow in tap water was eliminated by the presence of autochthonous microflora and "ultramicrocells" bacteria. Survival rates were strain- and microcosm-dependent. In unfiltered-non-autoclaved water, viable counts declined to below the detection limit (i.e. 1 log cfu ml(-1)) in 1.5 to 20 days. The declines in viable counts were even more pronounced in the filtered microcosm. Although inoculation ratios (100/1 in unfiltered-non-autoclaved and 1,000/1 in filtered microcosms) were favourable for aeromonads, at least for I to 3 days, the organisms disappeared in these microcosms. Thus, competition for nutrients was an unlikely cause of the limitation of aeromonads. The bacteriolytic effect of enzymes released by membrane vesicles from the autochthonous microflora and of "tail phage-like particles" bacteriocins were suggested as an in situ control of aeromonad populations. The present study showed that environmental strains of Aeromonas had no ecological advantage over clinical isolates. Thus, waterborne infections and contaminations of foods by pathogenic Aeromonas species could not be discounted.

Roles of Exopolysaccharides and Lipopolysaccharides in the Adsorption of the Siphovirus Phage NM8 to Rhizobium meliloti M11S Cells

The exopolysaccharides produced by Rhizobium meliloti M11S inhibited nonspecifically the adsorption of phage NM8 by coating the cells. But lipopolysaccharides (LPS) had a specific inhibitory effect. Only the polysaccharide moiety of LPS, composed of glucose, glucosamine, galactose, 3-deoxy-D-manno-octulosonic acid (KDO), and large amounts of sialic acid, inhibited phage adsorption; neither the lipid A moiety nor a cellular glucan was involved. Rhizobium strains lacking sialic acids did not bind phage NM8. Inhibition of phage binding by lectin specific for N-acetylneuraminic acid demonstrated that phage NM8 bound to sialic acids. Preincubation of the phage with monosaccharides showed that inactivation of phage was very stereospecific for N-acetylneuraminic acid. Phage adsorption was also strongly inhibited by N-acetylglucosamine, which is not present in the LPS. Therefore, the receptor for phage NM8 appears to be a saccharide site, probably involving the acetyl groups of sialic acids.

[Cryoresistance during sporulation in Bacillus (author's transl)]. / Apparition de la cryorésistance au cours de la sporulation chez Bacillus.

The maximal resistance at -- 20 degrees C is obtained as soon as stage V of sporulation in Bacillus cereus and for the spore of the mutant Marburg strain of B. subtilis bloked at stage V of sporulation.

[Survival of microorganisms frozen on solid medium (author's transl)]. / Survie de microorganismes congelés sur milieu solide.

Different freezing methods have been studied for 12 microorganisms. This study has been carried out on solid support in the conditions near the freezing of foods. The survival rates are on relation with species and freezing methods. The survival of some microorganisms during a storage at -20 degrees C has been studied.

Enumeration and characterization of bacteria in mineral water by improved direct viable count method.

Fifteen strains from two emergent mineral waters were isolated and tentatively identified with API 20NE and BIOLOG GN systems. These strains were screened for their sensitivities to seven replication-inhibiting antibiotics of the (fluoro)quinolone group (nalidixic and pipemidic acid, flumequine, norfloxacin, ofloxacin, pefloxacin and ciprofloxacin). It was shown that the direct viable count (DVC) procedure could be improved by using certain antibiotic cocktails, which were active against the isolates. Geometric bacterial features were successfully determined with image analysis and adapted software (ICONIX, Perfect Image). Elongations were significant and allowed rapid discrimination of antibiotic inhibited and non-inhibited strains. Particular isolates in a mixed culture were characterized and enumerated after only 14 h exposure with the appropriate antibiotic cocktail. This method can also be applied to other communities, such as mixed cultures in bio-fermentors or in food with known microflora.

Starvation survival and viable but nonculturable states in Aeromonas hydrophila.

The behavior of Aeromonas hydrophila stored at 4 degrees C and 25 degrees C in nutrient-poor filtered sterilized distilled water was investigated. At 4 degrees C, the A. hydrophila population declined below the detection level (0.1 cell mL(-1)) after 7 weeks, whereas the number of cells with intact membrane as determined by the LIVE/DEAD method decreased only by 1 log unit. Although, this response is reminiscent of the so-called VBNC state, the cells could not be resuscitated by an upshift to 25 degrees C. A mixture of rods with normal size and elongated cells was observed in this state. At 25 degrees C, viable cells and cells with intact membrane declined only by 0.8 log unit over the 10-week storage period, and thus A. hydrophila entered the classical starvation survival state. During this state, a mixture of rods and cocci was observed. Prestarvation at 25 degrees C for 24 h and especially 49 days delayed significantly the rate of entry into the VBNC state. However, stationary phase cells were not significantly more tolerant than exponential phase cells. No significant improvements in recovery yield were obtained on LB agar plates amended with catalase or sodium pyruvate. During cold incubation, high variability in responses was observed. Intermittent cryptic regrowth might be responsible for this variability in responses.

Total counts, culturable and viable, and non-culturable microflora of a French mineral water: a case study.

The changes in bacterial counts during the storage of a natural mineral water from a French spring were studied. Samples were taken from the spring and the bottling line. Viable cultivable (VC) bacteria were counted on R2A medium. Total counts, viable and dead bacteria were counted using the LIVE/DEAD Bac Light VIABILITY kit and epifluorescence microscopy. Viable but non-cultivable (VNC) bacteria were estimated by difference between viable and VC counts. Isolates were clustered by phenotype. The microflora in the spring water increased from < 10-3 x 10(5) bacteria ml-1 after 6 d in storage and then stabilized. Mechanical bottling increased the allochthonous bacteria in the water that stabilized at 10(5) bacteria ml-1. Maximal growth is controlled by the low concentration of nutrients in the mineral water and the lysis of dead cells. The allochthonous bacteria came from the aquifer and colonized the filling line. The changes in the VC and VNC populations showed that the bacteria used starvation-survival and entry into the VNC state to adapt to the bottling stress and the enclosed oligotrophic environment.

Preparative electroelution of specific protein antigens from Mycoplasma pneumoniae: use in an enzyme-linked immunosorbent assay (ELISA).

A rapid, simple and preparative method is described for the recovery of the seven highest molecular weight proteins (HMWP) from Mycoplasma pneumoniae membrane. The yield of proteins obtained was approximately 90%. The method involved the separation of M. pneumoniae proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), followed by electroelution of HMWP. These eluted antigens were used in an ELISA to measure IgG antibodies in sera from 9 blood donors and 9 patients with M. pneumoniae infection. The specificity of M. pneumoniae HMWP was examined by competition ELISA and immunoblotting with different mycoplasma species encountered in the respiratory tract.