Survival and spread of Shiga toxin-producing Escherichia coli in alpine pasture grasslands.

AIMS: To determine the fate of Shiga toxin-producing Escherichia coli (STEC) strains defecated onto alpine grassland soils. METHODS AND RESULTS: During the summers of 2005 and 2006, the field survival of STEC was monitored in cowpats and underlying soils in four different alpine pasture units. A most probable number (MPN)-PCR stx assay was used to enumerate STEC populations. STEC levels ranged between 3.9 and 5.4 log(10) CFU g(-1) in fresh cowpats and slowly decreased until their complete decay (inactivation rates k < 0.04 day(-1)). PFGE typing of STEC strains isolated from faecal and soil samples assessed the persistence of various clonal types for at least 2 months in cowpats and their vertical dispersal down through the soil at a depth up to at least 20 cm. STEC cells counts in soil were always below 2 log(10) CFU g(-1), regardless of the pasture unit investigated. The soil became rapidly free of detectable STEC once the cowpat had decomposed. The eight STEC strains isolated during this study belonged to six distinct serotypes and tested positive for the gene(s) stx2, including the stx2g and stx2 NV206 variants. CONCLUSIONS: STEC were able to persist in cowpats and disseminate down through the soil but were unable to establish. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides useful information concerning the ecology of STEC in alpine pasture grasslands and may have implications for land and cattle management.

Assessment of SCAR markers to design real-time PCR primers for rhizosphere quantification of Azospirillum brasilense phytostimulatory inoculants of maize.

AIMS: To assess the applicability of sequence characterized amplified region (SCAR) markers obtained from BOX, ERIC and RAPD fragments to design primers for real-time PCR quantification of the phytostimulatory maize inoculants Azospirillum brasilense UAP-154 and CFN-535 in the rhizosphere. METHODS AND RESULTS: Primers were designed based on strain-specific SCAR markers and were screened for successful amplification of target strain and absence of cross-reaction with other Azospirillum strains. The specificity of primers thus selected was verified under real-time PCR conditions using genomic DNA from strain collection and DNA from rhizosphere samples. The detection limit was 60 fg DNA with pure cultures and 4 x 10(3) (for UAP-154) and 4 x 10(4) CFU g(-1) (for CFN-535) in the maize rhizosphere. Inoculant quantification was effective from 10(4) to 10(8) CFU g(-1) soil. CONCLUSION: BOX-based SCAR markers were useful to find primers for strain-specific real-time PCR quantification of each A. brasilense inoculant in the maize rhizosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: Effective root colonization is a prerequisite for successful Azospirillum phytostimulation, but cultivation-independent monitoring methods were lacking. The real-time PCR methods developed here will help understand the effect of environmental conditions on root colonization and phytostimulation by A. brasilense UAP-154 and CFN-535.

Pseudomonas fluorescens and closely-related fluorescent pseudomonads as biocontrol agents of soil-borne phytopathogens.

Many strains of Pseudomonas fluorescens show potential for biological control of phytopathogens especially root pathogens. In taxonomic terms, several of them are indeed P. fluorescens sensu stricto, while others belong in fact to neighbouring species of the 'P. fluorescens' complex or to ill-defined related species within the fluorescent Pseudomonas spp. These bacteria have become prominent models for rhizosphere ecological studies and analysis of bacterial secondary metabolism, and in recent years knowledge on their plant-beneficial traits has been considerably enhanced by widening the focus beyond the case of phytopathogen-directed antagonism. Current genomic analyses of rhizosphere competence and biocontrol traits will likely lead to the development of novel tools for effective management of indigenous and inoculated P. fluorescens biocontrol agents and a better exploitation of their plant-beneficial properties for sustainable agriculture.

Long-term survival of Shiga toxin-producing Escherichia coli in cattle effluents and environment: an updated review.

Shiga toxin-producing Escherichia coli (STEC) are one of the most important emergent foodborne pathogens. STEC are common as colonizers in the intestine of healthy cattle and are spread into the environment by fecal shedding or following the surface application of farm effluent on soil. The bacteria can be transmitted to humans through food, such as inadequately cooked ground beef or unpasteurized milk. During the last decade, a wide variety of environmentally related exposures have emerged as new routes of transmission. Major outbreaks due to the consumption of raw fruits and vegetables or accidental ingestion of soil or water contaminated by STEC have been increasingly reported. STEC survival in cattle effluents, soil, plants and water is discussed in the light of new knowledge regarding both biotic and abiotic factors which may affect their survival or enhance their dissemination in the environment. The ability to persist in cattle production environments contributes to the contamination and recontamination of cattle, as well as for human infection. Consequently, effective control strategies must be considered on cattle farms, in order to limit entry of STEC cells into the environment.

Persistence of Shiga toxin-producing Escherichia coli O26 in various manure-amended soil types.

AIMS: To evaluate the behaviour of Shiga toxin-producing Escherichia coli (STEC) O26 strains inoculated in manure-amended soils under in vitro conditions. METHODS AND RESULTS: Four green fluorescent protein (GFP)-labelled STEC O26 strains were inoculated in duplicate (at 10(6) CFU g(-1)) in three different manure-amended soil types, including two loam soils (A and B) and one clay loam soil (C), and two incubation temperatures (4 and 20 degrees C) were tested. STEC counts and soil physical parameters were periodically monitored. STEC O26 cells were able to persist during extended periods in soil even in the presence of low moisture levels, i.e. less than 0 x 08 g H2O g(-1) dry soil. At 4 and 20 degrees C, STEC could be detected in soil A for 288 and 196 days, respectively, and in soils B and C for at least 365 days postinoculation at both temperatures. The ambient temperature (i.e. 20 degrees C) was significantly associated with the highest STEC count decline in all soils tested. CONCLUSIONS: The temperature and soil properties appear to be contributory factors affecting the long-term survival of STEC O26 in manure-amended soils. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides useful information regarding the ecology of STEC O26 in manure-amended soils and may have implications for land and waste management.

Involvement of the Cpx signal transduction pathway of E. coli in biofilm formation.

In a genetic screening directed to identify genes involved in biofilm formation, mutations in the cpxA gene were found to reduce biofilm formation by affecting microbial adherence to solid surfaces. This effect was detected in Escherichia coli K12 as well as in E. coli strains isolated from patients with catheter-related bacteremia. We show that the negative effect of the cpxA mutation on biofilm formation results from a decreased transcription of the curlin encoding csgA gene. The effect of the cpxA mutation could not be observed in cpxR- mutants, suggesting that they affect the same regulatory pathway. The cpxA101 mutation abolishes cpxA phosphatase activity and results in the accumulation of phosphorylated CpxR. Features of the strain carrying the cpxA101 mutation are a reduced ability to form biofilm and low levels of csgA transcription. Our results indicate that the cpxA gene increases the levels of csgA transcription by dephosphorylation of CpxR, which acts as a negative regulator at csgA. Thus, we propose the existence of a new signal transduction pathway involved in the adherence process in addition to the EnvZ-OmpR two-component system.

Growth and survival of non-O157:H7 Shiga-toxin-producing Escherichia coli in cow manure.

AIMS: The main objective of this study was to evaluate the behaviour of non-O157:H7 Shiga-toxin-producing Escherichia coli (STEC) strains in cow manure. METHODS AND RESULTS: A mixture of eight green-fluorescent-protein-labelled STEC strains was inoculated around 10(6)-10(7) CFU g(-1) into four manure heaps. Two heaps were regularly turned and the two others remained unturned. STEC counts and physical parameters (temperature, pH, moisture content and oxido-reduction potential) were monitored for 1000 manure samples. The highest mean pH values were obtained near the surface at the base of all manure heaps. At the surface, the moisture content decreased from 76.5% to 42% in turned heaps. Temperatures reached 65 degrees C near the main body of all manure heaps, and only 35 degrees C near the superficial parts located at the base of them. These two sites (the centre and the base) were associated with D values for the STEC counts of 0.48 and 2.39 days, respectively. We were able to detect STEC strains during 42 days in turned manure heaps and during at least 90 days in unturned ones. CONCLUSIONS: These results emphasize the long-term survival of non-O157:H7 STEC in cow manure. SIGNIFICANCE AND IMPACT OF THE STUDY: Good management practices (e.g. turning) should be respected in order to minimize the risk of environmental contamination by STEC.

Persistence of Shiga toxin-producing Escherichia coli O26 in cow slurry.

AIMS: The main objective of this study was to evaluate the growth and survival of Shiga toxin-producing Escherichia coli (STEC) O26 in cow slurry; this serogroup is regarded as an important cause of STEC-associated diseases. METHODS AND RESULTS: Four STEC were examined by polymerase chain reaction (PCR) to determine whether they harbour key virulence determinants and also by pulsed-field gel electrophoresis (PFGE) to obtain overview fingerprints of their genomes. They were transformed with the pGFPuv plasmid and were separately inoculated at a level of 10(6) CFU ml(-1) in 15 l of cow slurry. All STEC O26 strains could be detected for at least 3 months in cow slurry without any genetic changes. The moisture content of the slurry decreased over time to reach a final value of 75% while the pH increased from 8.5 to 9.5 units during the last 50 days. CONCLUSION: STEC O26 strains were able to survive in cow slurry for an extended period. SIGNIFICANCE AND IMPACT OF THE STUDY: Long-term storage of waste slurry should be required to reduce the pathogen load and to limit environmental contamination by STEC O26.

Freshwater selenium-methylating bacterial thiopurine methyltransferases: diversity and molecular phylogeny.

The diversity of bacterial thiopurine methyltransferases (bTPMT) among five natural Se-methylating freshwaters was investigated by polymerase chain reaction (PCR) screenings and sequencings. DNA sequence analyses confirmed the cloned products' identity and revealed a broad diversity of freshwater TPMTs. Neighbour-joining (NJ) phylogenetic analyses combining these sequences, all GenBank entries closely related to these sequences and deduced TPMTs obtained in this work from selected gamma-proteobacteria showed TPMTs to form a distinct radiation, closely related to UbiG methyltransferases. Inside the TPMT phylogenetic cluster, eukaryote sequences diverged early from the bacterial ones, and all the bacterial database entries belonged to a subgroup of gamma-proteobacteria, with an apparent lateral transfer of a particular allele to beta-proteobacteria of Bordetella. The NJ phylogenetic tree revealed 22 bTPMT lineages, 10 of which harboured freshwater sequences. All lineages showed deep and long branches indicative of major genetic drifts outside regions encoding highly conserved domains. Selected residues among these highly variable domains could reflect adaptations for particular ecological niches. PCR lineage-specific primers differentiated Se-methylating freshwaters according to their 'tpm lineage' signatures. Most freshwater tpm alleles were found to be distinct from those available in the databases, but a group of tpm was found encoding TPMTs identical to an Aeromonas veronii TPMT characterized in this work.

Abiotic surface sensing and biofilm-dependent regulation of gene expression in Escherichia coli.

To get further information on bacterial surface sensing and biofilm-dependent regulation of gene expression in Escherichia coli K-12, random insertion mutagenesis with Mu dX, a mini-Mu carrying the promoterless lacZ gene, was performed with an ompR234 adherent strain, and a simple screen was developed to assess changes in gene expression in biofilm cells versus planktonic cells. This screen revealed that major changes in the pattern of gene expression occur during biofilm development: the transcription of 38% of the genes was affected within biofilms. Different cell functions were more expressed in sessile bacteria: the OmpC porin, the high-affinity transport system of glycine betaine (encoded by the proU operon), the colanic acid exopolysaccharide (wca locus, formerly called cps), tripeptidase T (pepT), and the nickel high-affinity transport system (nikA). On the other hand, the syntheses of flagellin (fliC) and of a putative protein of 92 amino acids (f92) were both reduced in biofilms. Such a genetic reprogramming of gene expression in biofilms seems to result from changes in multiple environmental physicochemical conditions. In this work, we show that bacteria within biofilms encounter higher-osmolarity conditions, greater oxygen limitation, and higher cell density than in the liquid phase.

Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression.

Classical laboratory strains of Escherichia coli do not spontaneously colonize inert surfaces. However, when maintained in continuous culture for evolution studies or industrial processes, these strains usually generate adherent mutants which form a thick biofilm, visible with the naked eye, on the wall of the culture apparatus. Such a mutant was isolated to identify the genes and morphological structures involved in biofilm formation in the very well characterized E. coli K-12 context. This mutant acquired the ability to colonize hydrophilic (glass) and hydrophobic (polystyrene) surfaces and to form aggregation clumps. A single point mutation, resulting in the replacement of a leucine by an arginine residue at position 43 in the regulatory protein OmpR, was responsible for this phenotype. Observations by electron microscopy revealed the presence at the surfaces of the mutant bacteria of fibrillar structures looking like the particular fimbriae described by the Olsén group and designated curli (A. Olsén, A. Jonsson, and S. Normark, Nature 338:652-655, 1989). The production of curli (visualized by Congo red binding) and the expression of the csgA gene encoding curlin synthesis (monitored by coupling a reporter gene to its promoter) were significantly increased in the presence of the ompR allele described in this work. Transduction of knockout mutations in either csgA or ompR caused the loss of the adherence properties of several biofilm-forming E. coli strains, including all those which were isolated in this work from the wall of a continuous culture apparatus and two clinical strains isolated from patients with catheter-related infections. These results indicate that curli are morphological structures of major importance for inert surface colonization and biofilm formation and demonstrate that their synthesis is under the control of the EnvZ-OmpR two-component regulatory system.

Developmental pathway for biofilm formation in curli-producing Escherichia coli strains: role of flagella, curli and colanic acid.

This work was performed to establish a model describing bacterial surface structures involved in biofilm development, in curli-overproducing Escherichia coli K-12 strains, at 30 degrees C, and in minimal growth medium. Using a genetic approach, in association with observations of sessile communities by light and electron microscopic techniques, the role of protein surface structures, such as flagella and curli, and saccharidic surface components, such as the E. coli exopolysaccharide, colanic acid, was determined. We show that, in the context of adherent ompR234 strains, (i) flagellar motility is not required for initial adhesion and biofilm development; (ii) both primary adhesion to inert surfaces and development of multilayered cell clusters require curli synthesis; (iii) curli display direct interactions with the substratum and form interbacterial bundles, allowing a cohesive and stable association of cells; and (iv) colanic acid does not appear critical for bacterial adhesion and further biofilm development but contributes to the biofilm architecture and allows for the formation of voluminous biofilms.

Complex regulatory network controls initial adhesion and biofilm formation in Escherichia coli via regulation of the csgD gene.

The Escherichia coli OmpR/EnvZ two-component regulatory system, which senses environmental osmolarity, also regulates biofilm formation. Up mutations in the ompR gene, such as the ompR234 mutation, stimulate laboratory strains of E. coli to grow as a biofilm community rather than in a planktonic state. In this report, we show that the OmpR234 protein promotes biofilm formation by binding the csgD promoter region and stimulating its transcription. The csgD gene encodes the transcription regulator CsgD, which in turn activates transcription of the csgBA operon encoding curli, extracellular structures involved in bacterial adhesion. Consistent with the role of the ompR gene as part of an osmolarity-sensing regulatory system, we also show that the formation of biofilm by E. coli is inhibited by increasing osmolarity in the growth medium. The ompR234 mutation counteracts adhesion inhibition by high medium osmolarity; we provide evidence that the ompR234 mutation promotes biofilm formation by strongly increasing the initial adhesion of bacteria to an abiotic surface. This increase in initial adhesion is stationary phase dependent, but it is negatively regulated by the stationary-phase-specific sigma factor RpoS. We propose that this negative regulation takes place via rpoS-dependent transcription of the transcription regulator cpxR; cpxR-mediated repression of csgB and csgD promoters is also triggered by osmolarity and by curli overproduction, in a feedback regulation loop.