Comparison between the biofilm initiation of Campylobacter jejuni and Campylobacter coli strains to an inert surface using BioFilm Ring Test.

AIMS: The adhesion to an inert surface (the first step of biofilm formation) of the two main pathogenic Campylobacter species, Campylobacter jejuni and Campylobacter coli, isolated from diverse origins, was compared. METHODS AND RESULTS: Adhesion assays were conducted in 96-well, polystyrene microtiter plates using the BioFilm Ring Test method. This new technique, based on magnetic bead entrapment, was shown to be suitable for analysing the adhesion of Campylobacter sp. strains by comparing the adhesion of four C. jejuni strains as revealed by the BioFilm Ring Test and immunodetection. Among the 46 strains tested, C. jejuni and C. coli displayed different adhesion capabilities ranging from no adhesion to strong adhesion. However, no strain of C. coli was strongly adherent, and statistically, C. coli adhered less to an inert surface than C. jejuni. In addition, strains isolated from animals or carcasses were less adherent than those isolated from food-processing and clinical cases. CONCLUSIONS: These observations suggest that the food environment and the human body could have selected strains with greater adhesion. SIGNIFICANCE AND IMPACT OF THE STUDY: The adhesion capability of strains could partly explain the cross-contamination or re-contamination of food products by Campylobacter. This property could provide a mode of survival for Campylobacter in the food chain.

Sodium chloride affects Listeria monocytogenes adhesion to polystyrene and stainless steel by regulating flagella expression.

AIM: To study the adhesion capability of seven strains of Listeria monocytogenes to polystyrene and stainless steel surfaces after cultivation at various NaCl concentrations. METHODS AND RESULTS: Determination of growth limits indicated that all seven strains were able to grow in up to 11% NaCl in rain heart infusion and 3 g l(-1) yeast extract-glucose at 20 degrees C, but no growth was detected at 15% NaCl. Adhesion of L. monocytogenes was estimated after 4-h incubation at 20 degrees C in 96-well microtitre plates. Statistical results revealed no significant difference between adhesion to polystyrene and stainless steel although surface properties were different. Adhesion between 0% and 6% NaCl was not different, whereas adhesion at 11% NaCl was significantly lower. This discrepancy in adhesion was correlated with the down-regulation of flagella at 11% NaCl. CONCLUSIONS: Only high salinity levels, close to nongrowth conditions, repressed the expression of flagella, and consequently, decreased the adhesion capability of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Adhesion of L. monocytogenes to inert surfaces depends on environmental conditions that affect flagellum expression. High salinity concentrations would delay biofilm formation.

Impacts of treatment parameters on the inactivation of Campylobacter jejuni by high pressure: a statistical study of main effects and interactions.

AIM: The influence of environmental (temperature and pH) and biological (strain) parameters on the inactivation of Campylobacter jejuni by high hydrostatic pressure (HHP) was investigated. METHODS AND RESULTS: Two clinical strains harvested in stationary phase were pressurized at 20 degrees C and 37 degrees C within a range of 50-400 MPa, in a phosphate (pH 7.0) or a citrate phosphate buffer (pH 5.6), for 10 min. Treatment efficiencies were determined by logarithmic comparisons of culturable cells on blood agar before and after treatment. Results were statistically compared using an anova of culturable cells after treatment to evaluate the effect of all factors. At least a 7-log reduction in cell numbers was observed for both strains. The pH and the strains had no effect on HHP treatment at 20 degrees C while at 37 degrees C, both pH and strain influenced significantly the HHP treatment on C. jejuni. CONCLUSIONS: The pressure efficacy on C. jejuni eradication was affected by both environmental and biological factors. SIGNIFICANCE AND IMPACT OF THE STUDY: Depending on the treatment conditions, C. jejuni sensitivity to HHP can significantly vary. The determination of the inactivation treatment by HPP has to be normalized considering the interaction of environmental and biological factors.

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)).

Comparative evaluation of adhesion, surface properties, and surface protein composition of Listeria monocytogenes strains after cultivation at constant pH of 5 and 7.

AIMS: To analyse the cellular mechanisms that influence Listeria monocytogenes adhesion onto inert surfaces under acidic growth conditions. METHODS AND RESULTS: The adhesion capability of all the strains was significantly reduced after cultivation at constant pH 5 than at constant pH 7 and the cell surface was significantly less hydrophobic at pH 5 than at 7. At pH 5, the analyses of surface protein composition revealed that the flagellin was downregulated for all strains, which was confirmed by the absence of flagella and the P60 protein was upregulated for L. monocytogenes EGD-e, X-Li-mo 500 and 111. The use of L. monocytogenes EGD mutants revealed that flagellin could be involved in the adhesion process, but not P60 protein. It was also observed that the hydrophobic character was not linked to the presence or the absence of flagellin or P60 protein at the cell surface of L. monocytogenes. CONCLUSIONS: The decrease of L. monocytogenes adhesion at pH 5 could be attributed to the downregulation of the flagellin synthesis under the acidic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Conservation of food product at pH 5 will delay bacterial adhesion and biofilm formation during food processing on inert surfaces when the product is contaminated with L. monocytogenes.

Comparison of the microbial population dynamics and phylogenetic characterization of a CANOXIS reactor and a UASB reactor degrading trichloroethene.

AIMS: To understand the microbial ecology underlying trichloethene (TCE) degradation in a coupled anaerobic/aerobic single stage (CANOXIS) reactor oxygenated with hydrogen peroxide (H2O2) and in an upflow anaerobic sludge bed (UASB) reactor. METHODS AND RESULTS: The molecular study of the microbial population dynamics and a phylogenetic characterization were conducted using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). In both reactors, TCE had a toxic effect on two uncultured bacterial populations whereas oxygen favoured the growth of aerobic species belonging to Rhizobiaceae and Dechloromonas. No methanotrophic bacteria were detected when targeting 16S rRNA gene with universal primers. Alternatively, pmo gene encoding the particulate methane monooxygenase of Methylomonas sp. LW21 could be detected in the coupled reactor when H2O2 was supplied at 0.7 g O2 l day(-1). CONCLUSIONS: Methylomonas sp. LW21 that could be responsible for the aerobic degradation of the TCE by-products is not among the predominant bacterial populations in the coupled reactor. It seems to have been outcompeted by heterotrophic bacteria (Rhizobiaceae and Dechloromonas sp.) for oxygen. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained show the limitations of the coupled reactor examined in this study. Further investigations should focus on the operating conditions of this reactor in order to favour the growth of the methanotrophs.

Dynamics of living and dead bacterial cells within a mixed-species biofilm during toluene degradation in a biotrickling filter.

AIMS: To study the accumulation of the bacterial living cells (LC) and dead cells (DC) in a mixed-species biofilm developed in a 3 l biotrickling filter (BTF) challenged with toluene. METHODS AND RESULTS: The bacterial LC and DC within the biofilm developed on polypropylene Pall rings in a toluene-degrading BTF were enumerated as fluoro-microscopic counts during a 62-operating day period using nucleic acid staining and the direct epifluorescence filter technique. The biofilm development could be separated into three distinct phases: (i) cell attachment, (ii) biofilm establishment and (iii) biofilm maturation. The LC were always dominant (>/=72%) in the biofilm during the establishment phase whereas the average LC fraction decreased to 51% of the total cells in the maturation phase. The concentration of LC and DC was observed to level off after 41 days at 1010 cells per ring. The biofilm thickness and the dry weight increased independently of the cell number during the maturation phase. CONCLUSIONS: After the LC reached a maximum concentration in the biofilm, the biofilm proliferation was only characterized by the accumulation of DC and organic matter. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained in the present study are of particular relevance for biofilm mathematical modelling and numerical simulations. They will also be useful to estimate the contribution of the living bacteria within the biofilm in bioprocesses.

Population dynamics of free-floating and attached bacteria in a styrene-degrading biotrickling filter analyzed by denaturing gradient gel electrophoresis.

Population dynamics was studied in a 52-l biotrickling filter (BTF) operated for 182 days and used to clean air contaminated with styrene vapors. In the BTF, biomass grew either as free-floating (planktonic) or attached (sessile) microorganisms. PCR-amplified 16S rDNA fragments from planktonic and sessile cells within the bioreactor were analyzed using denaturing gradient gel electrophoresis (DGGE). The results indicated that the complexity of biofilm community was always more pronounced than the complexity of the planktonic cell community. Notably, Rhodococcus erythropolis was identified, based on DNA sequence analysis, as one of the biofilm-specific strains. It was also shown that the inoculum, even when enriched with styrene-degrading bacteria, was not adapted to the growth conditions imposed by the BTF. After a 35-day microbial acclimation period, the DGGE analysis also showed less variation in the banding pattern representing the microbial complexity of the biofilm. In addition, the phylogenic fingerprinting method used demonstrated similar banding profiles in the biofilm along the filter bed.

Bacterial survival and mineralization of p-nitrophenol in soil by green fluorescent protein-marked Moraxella sp. G21 encapsulated cells.

Moraxella sp. G21 cells marked with the green fluorescent protein (gfp) survived in kappa-carrageenan beads and as free cells for a month after inoculation into autoclaved soil and non-sterile soil contaminated with p-nitrophenol (PNP). Similar [U-(14)C]PNP mineralization values were produced by encapsulated Moraxella sp. G21 cells and as free cells (53 and 60% mineralization). There was no significant difference between cell survival and [U-(14)C]PNP mineralization activity in soil by the rifampicin-resistant Moraxella sp. mental strain and Moraxella sp. G21. The ability of encapsulated Moraxella sp. G21 cells to survive, retain their green fluorescence and mineralize [U-(14)C]PNP suggests that the GFP-marked strain encapsulated in kappa-carrageenan may be useful for bioremediation of toxic chemicals in soil.

Green fluorescent protein as a visual marker in a p-nitrophenol degrading Moraxella sp.

The green fluorescent protein gene (gfp) was introduced into a p-nitrophenol-metabolizing strain of Moraxella sp. by chromosomal integration. The gfp-marked transformants, designated Moraxella sp. strains G21 and G25, exhibited green fluorescence under UV light. Molecular characterization by PCR and Southern hybridization showed the presence of gfp in both transformants. Both transformants and the parent strain degraded 720 microM of p-nitrophenol with nitrite release within 4 h after inoculation in minimal medium supplemented with yeast extract. Transformants degraded up to 1440 microM p-nitrophenol and mineralized about 60% of 720 microM p-nitrophenol, both in broth and in soil, to the same extent as the parent strain. Insertion of gfp did not adversely affect the expression of p-nitrophenol-degrading genes in the transformants. Survival studies indicated that individual green fluorescent colonies of transformants can be detected up to 2 weeks after inoculation in soil. These marked strains could be of value in studies on microbial survival in the environment.

Antibacterial efficacy of tobramycin against anaerobic Escherichia coli cultures in the presence of electron acceptors.

The antimicrobial activity of tobramycin against anaerobic cultures of Escherichia coli was tested in the presence of various electron carriers. The presence of 2,6-dichlorophenol 4-indophenol (DCIP) significantly enhanced the killing efficacy of tobramycin. Only 0.003% of the initial cell population (i.e. 10(6) cfu/mL) remained viable after exposure for 10 h to the mixture of antibiotic (20 x MIC, i.e. 40 mg/L) and electron acceptor (10(-3) M), as compared with 9% of surviving organisms in the presence of tobramycin alone. Less synergy was obtained with p-benzoquinone and 1,2-naphthoquinone. Fumarate did not affect the efficiency of the antibiotic. The mixture of tobramycin and DCIP was ineffective against agar-entrapped bacteria which, like biofilm organisms, are subject to oxygen limitation.

Underexpression of porin protein OmpF in agar-entrapped, sessile-like Escherichia coli.

The electrophoretic patterns of the outer membrane proteins of agar-entrapped Escherichia coli cells were found to be different from those of free organisms. In particular, the porin protein OmpF was underexpressed in immobilized bacteria, that displayed enhanced resistance to latamoxef.

The role of oxygen limitation in the resistance of agar-entrapped, sessile-like Escherichia coli to aminoglycoside and beta-lactam antibiotics.

Viable cells of Escherichia coli were entrapped in agar gel layers to form artificial biofilm-like structures. Killing assays of immobilized organisms by latamoxef and tobramycin were performed under different oxygenation conditions of the culture medium and compared with free-cell experiments. Under moderate aeration, agar-entrapped bacteria displayed higher resistance to the two antibiotics than suspended cells. Slow-growing free-cell cultures were resistant to latamoxef but not to tobramycin. In anaerobic incubation conditions, suspended organisms were highly resistant to the two antibiotics. Sustained oxygenation enhanced tobramycin efficacy against free and immobilized cells. These results show that oxygen deficiency in the gel layer contributes to the enhanced antibiotic resistance of sessile-like cells.

Susceptibility of agar-entrapped Escherichia coli cultures to cefotaxime as assessed by the potentiometric measurement of lipoic acid reduction.

We used an original potentiometric procedure to monitor the metabolic activity of biofilm-like structures and to assess their susceptibility to betalactam antibiotics. The reduction of exogeneous alpha-lipoic acid to dihydrolipoic acid by planktonic and agar-entrapped bacteria, metabolizing glucose, was followed by potential-time measurements using gold versus reference electrodes. Immobilized cultures displayed a lower susceptibility to cefotaxime than their free counterparts. The redox potential-time courses showed that the gel matrix was more rapidly oxygen depleted than the free-cell culture medium. The depletion of both glucose and dissolved O2 inside the gel induced a breakdown in the reducing activity of agar-entrapped organisms.

Agar-entrapped bacteria as an in vitro model of biofilms and their susceptibility to antibiotics.

A simple in vitro system was developed as a model structure of biofilms and to evaluate their susceptibility to antibiotics. Viable Escherichia coli cells were entrapped in agar gel layers and incubated for 2 days in a minimal salt medium supplemented with glucose. After subsequent culture for 3 weeks under metal ion depletion, the biomass distribution inside the gel layer was highly heterogeneous. The cell concentration reached 10(11) cfu/g gel in the outer regions of the agar structure whereas the inner gel areas were less colonized (10(9) cfu/g gel). Immobilized cells displayed enhanced resistance to latamoxef as compared with free microorganisms. Moreover, a 3-week-old immobilized-cell membrane was less susceptible to the antibiotic than a younger (2 days old) one. The exposure for 11 h to 64 micrograms/cm3 latamoxef killed about 90% of the bacteria entrapped in the older agar layer, whereas the number of killed cells was 100-fold higher in the younger structure. Effective diffusivity measurements showed that the diffusion of latamoxef in the biofilm-like agar structures was moderately restricted as compared to that in water, and independent of the immobilized-cell content.