Comparative Genomics between Two Xenorhabdus bovienii Strains Highlights Differential Evolutionary Scenarios within an Entomopathogenic Bacterial Species.

Bacteria of the genus Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema. This symbiotic association constitutes an insecticidal complex active against a wide range of insect pests. Within Xenorhabdus bovienii species, the X. bovienii CS03 strain (Xb CS03) is nonvirulent when directly injected into lepidopteran insects, and displays a low virulence when associated with its Steinernema symbiont. The genome of Xb CS03 was sequenced and compared with the genome of a virulent strain, X. bovienii SS-2004 (Xb SS-2004). The genome size and content widely differed between the two strains. Indeed, Xb CS03 had a large genome containing several specific loci involved in the inhibition of competitors, including a few NRPS-PKS loci (nonribosomal peptide synthetases and polyketide synthases) producing antimicrobial molecules. Consistently, Xb CS03 had a greater antimicrobial activity than Xb SS-2004. The Xb CS03 strain contained more pseudogenes than Xb SS-2004. Decay of genes involved in the host invasion and exploitation (toxins, invasins, or extracellular enzymes) was particularly important in Xb CS03. This may provide an explanation for the nonvirulence of the strain when injected into an insect host. We suggest that Xb CS03 and Xb SS-2004 followed divergent evolutionary scenarios to cope with their peculiar life cycle. The fitness strategy of Xb CS03 would involve competitor inhibition, whereas Xb SS-2004 would quickly and efficiently kill the insect host. Hence, Xenorhabdus strains would have widely divergent host exploitation strategies, which impact their genome structure.

Attenuated virulence and genomic reductive evolution in the entomopathogenic bacterial symbiont species, Xenorhabdus poinarii.

Bacteria of the genus Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema. This symbiotic association constitutes an insecticidal complex active against a wide range of insect pests. Unlike other Xenorhabdus species, Xenorhabdus poinarii is avirulent when injected into insects in the absence of its nematode host. We sequenced the genome of the X. poinarii strain G6 and the closely related but virulent X. doucetiae strain FRM16. G6 had a smaller genome (500-700 kb smaller) than virulent Xenorhabdus strains and lacked genes encoding potential virulence factors (hemolysins, type 5 secretion systems, enzymes involved in the synthesis of secondary metabolites, and toxin-antitoxin systems). The genomes of all the X. poinarii strains analyzed here had a similar small size. We did not observe the accumulation of pseudogenes, insertion sequences or decrease in coding density usually seen as a sign of genomic erosion driven by genetic drift in host-adapted bacteria. Instead, genome reduction of X. poinarii seems to have been mediated by the excision of genomic blocks from the flexible genome, as reported for the genomes of attenuated free pathogenic bacteria and some facultative mutualistic bacteria growing exclusively within hosts. This evolutionary pathway probably reflects the adaptation of X. poinarii to specific host.

Photorhabdus heterorhabditis sp. nov., a symbiont of the entomopathogenic nematode Heterorhabditis zealandica.

The bacterial symbionts SF41T and SF783 were isolated from populations of the insect pathogenic nematode Heterorhabditis zealandica collected in South Africa. Both strains were closely related to strain Q614 isolated from a population of Heterorhabditis sp. collected from soil in Australia in the 1980s. Sequence analysis based on a multigene approach, DNA-DNA hybridization data and phenotypic traits showed that strains SF41T, SF783 and Q614 belong to the same species of the genus Photorhabdus with Photorhabdus temperata subsp. cinerea as the most closely related taxon (DNA-DNA hybridization value of 68%). Moreover, the phylogenetic position of Photorhabdus temperata subsp. cinerea DSM 19724T initially determined using the gyrB sequences, was reconsidered in the light of the data obtained by our multigene approach and DNA-DNA hybridization experiments. Strains SF41T, SF783 and Q614 represent a novel species of the genus Photorhabdus, for which the name Photorhabdus heterorhabditis sp. nov. is proposed (type strain SF41T=ATCC BAA-2479T=DSM 25263T).

Characterization of Biocontrol Traits in Heterorhabditis floridensis: A Species with Broad Temperature Tolerance.

Biological characteristics of two strains of the entomopathogenic nematode, Heterorhabditis floridensis (332 isolated in Florida and K22 isolated in Georgia) were described. The identity of the nematode's symbiotic bacteria was elucidated and found to be Photorhabdus luminescens subsp. luminescens. Beneficial traits pertinent to biocontrol (environmental tolerance and virulence) were characterized. The range of temperature tolerance in the H. floridensis strains was broad and showed a high level of heat tolerance. The H. floridensis strains caused higher mortality or infection in G. mellonella at 30°C and 35°C compared with S. riobrave (355), a strain widely known to be heat tolerant, and the H. floridensis strains were also capable of infecting at 17°C whereas S. riobrave (355) was not. However, at higher temperatures (37°C and 39°C), though H. floridensis readily infected G. mellonella, S. riobrave strains caused higher levels of mortality. Desiccation tolerance in H. floridensis was similar to Heterorhabditis indica (Hom1) and S. riobrave (355) and superior to S. feltiae (SN). H. bacteriophora (Oswego) and S. carpocapsae (All) exhibited higher desiccation tolerance than the H. floridensis strains. The virulence of H. floridensis to four insect pests (Aethina tumida, Conotrachelus nenuphar, Diaprepes abbreviatus, and Tenebrio molitor) was determined relative to seven other nematodes: H. bacteriophora (Oswego), H. indica (Hom1), S. carpocapsae (All), S. feltiae (SN), S. glaseri (4-8 and Vs strains), and S. riobrave (355). Virulence to A. tumida was similar among the H. floridensis strains and other nematodes except S. glaseri (Vs), S. feltiae, and S. riobrave failed to cause higher mortality than the control. Only H. bacteriophora, H. indica, S. feltiae, S. riobrave, and S. glaseri (4-8) caused higher mortality than the control in C. nenuphar. All nematodes were pathogenic to D. abbreviatus though S. glaseri (4-8) and S. riobrave (355) were the most virulent. S. carpocapsae was the most virulent to T. molitor. In summary, the H. floridensis strains possess a wide niche breadth in temperature tolerance and have virulence and desiccation levels that are similar to a number of other entomopathogenic nematodes. The strains may be useful for biocontrol purposes in environments where temperature extremes occur within short durations.

Photorhabdus luminescens subsp. noenieputensis subsp. nov., a symbiotic bacterium associated with a novel Heterorhabditis species related to Heterorhabditis indica.

The bacterial symbiont AM7(T), isolated from a novel entomopathogenic nematode species of the genus Heterorhabditis, displays the main phenotypic traits of the genus Photorhabdus and is highly pathogenic to Galleria mellonella. Phylogenetic analysis based on a multigene approach (16S rRNA, recA, gyrB, dnaN, gltX and infB) confirmed the classification of isolate AM7(T) within the species Photorhabdus luminescens and revealed its close relatedness to Photorhabdus luminescens subsp. caribbeanensis, P. luminescens subsp. akhurstii and P. luminescens subsp. hainanensis. The five concatenated protein-encoding sequences (4197 nt) of strain AM7(T) revealed 95.8, 95.4 and 94.9 % nucleotide identity to sequences of P. luminescens subsp. caribbeanensis HG29(T), P. luminescens subsp. akhurstii FRG04(T) and P. luminescens subsp. hainanensis C8404(T), respectively. These identity values are less than the threshold of 97 % proposed for classification within one of the existing subspecies of P. luminescens. Unlike other strains described for P. luminescens, strain AM7(T) produces acid from adonitol, sorbitol and xylitol, assimilates xylitol and has no lipase activity on medium containing Tween 20 or 60. Strain AM7(T) is differentiated from P. luminescens subsp. caribbeanensis by the assimilation of N-acetylglucosamine and the absence of haemolytic activity. Unlike P. luminescens subsp. akhurstii, strain AM7(T) does not assimilate mannitol, and it is distinguished from P. luminescens subsp. hainanensis by the assimilation of trehalose and citrate, the inability to produce indole from tryptophan and the presence of acetoin production and urease activity. Strain AM7(T) ( = ATCC BAA-2407(T)  = DSM 25462(T)) belongs to a novel subspecies, and is proposed as the type strain of Photorhabdus luminescens subsp. noenieputensis sp. nov.

A hemocyte gene expression signature correlated with predictive capacity of oysters to survive Vibrio infections.

BACKGROUND: The complex balance between environmental and host factors is an important determinant of susceptibility to infection. Disturbances of this equilibrium may result in multifactorial diseases as illustrated by the summer mortality syndrome, a worldwide and complex phenomenon that affects the oysters, Crassostrea gigas. The summer mortality syndrome reveals a physiological intolerance making this oyster species susceptible to diseases. Exploration of genetic basis governing the oyster resistance or susceptibility to infections is thus a major goal for understanding field mortality events. In this context, we used high-throughput genomic approaches to identify genetic traits that may characterize inherent survival capacities in C. gigas. RESULTS: Using digital gene expression (DGE), we analyzed the transcriptomes of hemocytes (immunocompetent cells) of oysters able or not able to survive infections by Vibrio species shown to be involved in summer mortalities. Hemocytes were nonlethally collected from oysters before Vibrio experimental infection, and two DGE libraries were generated from individuals that survived or did not survive. Exploration of DGE data and microfluidic qPCR analyses at individual level showed an extraordinary polymorphism in gene expressions, but also a set of hemocyte-expressed genes whose basal mRNA levels discriminate oyster capacity to survive infections by the pathogenic V. splendidus LGP32. Finally, we identified a signature of 14 genes that predicted oyster survival capacity. Their expressions are likely driven by distinct transcriptional regulation processes associated or not associated to gene copy number variation (CNV). CONCLUSIONS: We provide here for the first time in oyster a gene expression survival signature that represents a useful tool for understanding mortality events and for assessing genetic traits of interest for disease resistance selection programs.

Description of Xenorhabdus magdalenensis sp. nov., the symbiotic bacterium associated with Steinernema australe.

A symbiotic bacterium, strain IMI 397775(T), was isolated from the insect-pathogenic nematode Steinernema australe. On the basis of 16S rRNA gene sequence similarity, this bacterial isolate was shown to belong to the genus Xenorhabdus, in agreement with the genus of its nematode host. The accurate phylogenetic position of this new isolate was defined using a multigene approach and showed that isolate IMI 397775(T) shares a common ancestor with Xenorhabdus doucetiae FRM16(T) and Xenorhabdus romanii PR06-A(T), the symbiotic bacteria associated with Steinernema diaprepesi and Steinernema puertoricense, respectively. The nucleotide identity (less than 97%) between isolate IMI 397775(T), X. doucetiae FRM16(T) and X. romanii PR06-A(T) calculated for the concatenated sequences of five gene fragments encompassing 4275 nt, several phenotypic traits and the difference between the upper temperatures that limit growth of these three bacteria allowed genetic and phenotypic differentiation of isolate IMI 397775(T) from the two closely related species. Strain IMI 397775(T) therefore represents a novel species, for which the name Xenorhabdus magdalenensis sp. nov. is proposed, with the type strain IMI 397775(T) ( = DSM 24915(T)).

The first record of entomopathogenic nematodes (Rhabiditiae: Steinernematidae and Heterorhabditidae) in natural ecosystems in Lebanon: A biogeographic approach in the Mediterranean region.

A survey of entomopathogenic nematodes in Lebanon was conducted for the first time during 2008-2009. Samples were collected on the coastal strip and in nine vegetation types extending from the coastal line to 3088m above sea level. Wooded and herbaceous ecosystems were considered for sampling purposes. A total of 570 samples were taken, out of which 1% were positive for entomopathogenic nematodes. Approximately, 15.8% out of the 19 sites sampled revealed entomopathogenic nematodes presence (representing three samples). Two entomopathogenic nematodes species Heterorhabditis bacteriophora and Steinernema feltiae were recovered, and identification of their symbiotic bacteria revealed the presence of a Xenorhabdus bovienii, Photorhabdus temperata subsp. thracensis, Photorhabdus luminescens subsp. kayaii and Photorhabdus luminescens subsp. Laumondii.

Units of plasticity in bacterial genomes: new insight from the comparative genomics of two bacteria interacting with invertebrates, Photorhabdus and Xenorhabdus.

BACKGROUND: Flexible genomes facilitate bacterial evolution and are classically organized into polymorphic strain-specific segments called regions of genomic plasticity (RGPs). Using a new web tool, RGPFinder, we investigated plasticity units in bacterial genomes, by exhaustive description of the RGPs in two Photorhabdus and two Xenorhabdus strains, belonging to the Enterobacteriaceae and interacting with invertebrates (insects and nematodes). RESULTS: RGPs account for about 60% of the genome in each of the four genomes studied. We classified RGPs into genomic islands (GIs), prophages and two new classes of RGP without the features of classical mobile genetic elements (MGEs) but harboring genes encoding enzymes catalyzing DNA recombination (RGPmob), or with no remarkable feature (RGPnone). These new classes accounted for most of the RGPs and are probably hypervariable regions, ancient MGEs with degraded mobilization machinery or non canonical MGEs for which the mobility mechanism has yet to be described. We provide evidence that not only the GIs and the prophages, but also RGPmob and RGPnone, have a mosaic structure consisting of modules. A module is a block of genes, 0.5 to 60 kb in length, displaying a conserved genomic organization among the different Enterobacteriaceae. Modules are functional units involved in host/environment interactions (22-31%), metabolism (22-27%), intracellular or intercellular DNA mobility (13-30%), drug resistance (4-5%) and antibiotic synthesis (3-6%). Finally, in silico comparisons and PCR multiplex analysis indicated that these modules served as plasticity units within the bacterial genome during genome speciation and as deletion units in clonal variants of Photorhabdus. CONCLUSIONS: This led us to consider the modules, rather than the entire RGP, as the true unit of plasticity in bacterial genomes, during both short-term and long-term genome evolution.

Phylogeny of Photorhabdus and Xenorhabdus based on universally conserved protein-coding sequences and implications for the taxonomy of these two genera. Proposal of new taxa: X. vietnamensis sp. nov., P. luminescens subsp. caribbeanensis subsp. nov., P. luminescens subsp. hainanensis subsp. nov., P. temperata subsp. khanii subsp. nov., P. temperata subsp. tasmaniensis subsp. nov., and the reclassification of P. luminescens subsp. thracensis as P. temperata subsp. thracensis comb. nov.

We used the information from a set of concatenated sequences from four genes (recA, gyrB, dnaN and gltX) to investigate the phylogeny of the genera Photorhabdus and Xenorhabdus (entomopathogenic bacteria associated with nematodes of the genera Heterorhabditis and Steinernema, respectively). The robustness of the phylogenetic tree obtained by this multigene approach was significantly better than that of the tree obtained by a single gene approach. The comparison of the topologies of single gene phylogenetic trees highlighted discrepancies which have implications for the classification of strains and new isolates; in particular, we propose the transfer of Photorhabdus luminescens subsp. thracensis to Photorhabdus temperata subsp. thracensis comb. nov. (type strain CIP 108426T =DSM 15199T). We found that, within the genus Xenorhabdus, strains or isolates that shared less than 97 % nucleotide identity (NI), calculated on the concatenated sequences of the four gene fragments (recA, gyrB, dnaN and gltX) encompassing 3395 nucleotides, did not belong to the same species. Thus, at the 97% NI cutoff, we confirm the current 20 species of the genus Xenorhabdus and propose the description of a novel species, Xenorhabdus vietnamensis sp. nov. (type strain VN01T =CIP 109945T =DSM 22392T). Within each of the three current species of the genus Photorhabdus, P. asymbiotica, P. luminescens and P. temperata, strains or isolates which shared less than 97% NI did not belong to the same subspecies. Comparisons of the four gene fragments plus the rplB gene fragment analysed separately led us to propose four novel subspecies: Photorhabdus luminescens subsp. caribbeanensis subsp. nov. (type strain HG29T =CIP 109949T =DSM 22391T), P. luminescens subsp. hainanensis subsp. nov. (type strain C8404T = CIP 109946T =DSM 22397T), P. temperata subsp. khanii subsp. nov. (type strain C1T =NC19(T) =CIP 109947T =DSM 3369T), and P. temperata subsp. tasmaniensis subsp. nov. (type strain T327T =CIP 109948T =DSM 22387T).

Isolation and identification of entomopathogenic nematodes and their symbiotic bacteria from Hérault and Gard (Southern France).

Isolation and identification of native nematode-bacterial associations in the field are necessary for successful control of endemic pests in a particular location. No study has yet been undertaken to recover and identify EPN in metropolitan France. In the present paper, we provide results of a survey of EPN and their symbiotic bacteria conducted in Hérault and Gard regions in Southern France. Molecular characterization of isolated nematodes depicted three different Steinernema species and one Heterorhabditis species, H. bacteriophora. Steinernema species recovered were identified as: S. feltiae and S. affine and an undescribed species. Xenorhabdus symbionts were identified as X. bovienii for both S. feltiae and S. affine. Phylogenetic analysis placed the new undescribed Steinernema sp. as closely related to S. arenarium but divergent enough to postulate that it belongs to a new species within the "glaseri-group". The Xenorhabdus symbiont from this Steinernema sp. was identified as X. kozodoii. All Heterorhabditis isolates recovered were diagnosed as H. bacteriophora and their bacterial symbionts were identified as Photorhabdus luminescens. Molecular characterization of these nematodes enabled the distinction of two different H. bacteriophora strains. Bacterial symbiontic strains of these two H. bacteriophora strains were identified as P. luminescens ssp. kayaii and P. luminescens ssp. laumondii.

Diversity of lactic acid bacteria associated with fish and the fish farm environment, established by amplified rRNA gene restriction analysis.

Lactic acid bacteria have become a major source of concern for aquaculture in recent decades. In addition to true pathogenic species of worldwide significance, such as Streptococcus iniae and Lactococcus garvieae, several species have been reported to produce occasional fish mortalities in limited geographic areas, and many unidentifiable or ill-defined isolates are regularly isolated from fish or fish products. To clarify the nature and prevalence of different fish-associated bacteria belonging to the lactic acid bacterium group, a collection of 57 isolates of different origins was studied and compared with a set of 22 type strains, using amplified rRNA gene restriction analysis (ARDRA). Twelve distinct clusters were delineated on the basis of ARDRA profiles and were confirmed by sequencing of sodA and 16S rRNA genes. These clusters included the following: Lactococcus raffinolactis, L. garvieae, Lactococcus l., S. iniae, S. dysgalactiae, S. parauberis, S. agalactiae, Carnobacterium spp., the Enterococcus "faecium" group, a heterogeneous Enterococcus-like cluster comprising indiscernible representatives of Vagococcus fluvialis or the recently recognized V. carniphilus, V. salmoninarum, and Aerococcus spp. Interestingly, the L. lactis and L. raffinolactis clusters appeared to include many commensals of fish, so opportunistic infections caused by these species cannot be disregarded. The significance for fish populations and fish food processing of three or four genetic clusters of uncertain or complex definition, namely, Aerococcus and Enterococcus clusters, should be established more accurately.

New insight into diversity in the genus Xenorhabdus, including the description of ten novel species.

We investigated the diversity of a collection of 76 Xenorhabdus strains, isolated from at least 27 species of Steinernema nematodes and collected in 32 countries, using three complementary approaches: 16S rRNA gene sequencing, molecular typing and phenotypic characterization. The 16S rRNA gene sequences of the Xenorhabdus strains were highly conserved (similarity coefficient >95 %), suggesting that the common ancestor of the genus probably emerged between 250 and 500 million years ago. Based on comparisons of the 16S rRNA gene sequences, we identified 13 groups and seven unique sequences. This classification was confirmed by analysis of molecular typing profiles of the strains, leading to the classification of new isolates into the Xenorhabdus species described previously and the description of ten novel Xenorhabdus species: Xenorhabdus cabanillasii sp. nov. (type strain USTX62(T)=CIP 109066(T)=DSM 17905(T)), Xenorhabdus doucetiae sp. nov. (type strain FRM16(T)=CIP 109074(T)=DSM 17909(T)), Xenorhabdus griffiniae sp. nov. (type strain ID10(T)=CIP 109073(T)=DSM 17911(T)), Xenorhabdus hominickii sp. nov. (type strain KE01(T)=CIP 109072(T)=DSM 17903(T)), Xenorhabdus koppenhoeferi sp. nov. (type strain USNJ01(T)=CIP 109199(T)=DSM 18168(T)), Xenorhabdus kozodoii sp. nov. (type strain SaV(T)=CIP 109068(T)=DSM 17907(T)), Xenorhabdus mauleonii sp. nov. (type strain VC01(T)=CIP 109075(T)=DSM 17908(T)), Xenorhabdus miraniensis sp. nov. (type strain Q1(T)=CIP 109069(T)=DSM 17902(T)), Xenorhabdus romanii sp. nov. (type strain PR06-A(T)=CIP 109070(T)=DSM 17910(T)) and Xenorhabdus stockiae sp. nov. (type strain TH01(T)=CIP 109067(T)=DSM 17904(T)). The Xenorhabdus strains studied here had very similar phenotypic patterns, but phenotypic features nonetheless differentiated the following species: X. bovienii, X. cabanillasii, X. hominickii, X. kozodoii, X. nematophila, X. poinarii and X. szentirmaii. Based on phenotypic analysis, we identified two major groups of strains. Phenotypic group G(A) comprised strains able to grow at temperatures of 35-42 degrees C, whereas phenotypic group G(B) comprised strains that grew at temperatures below 35 degrees C, suggesting that some Xenorhabdus species may be adapted to tropical or temperate regions and/or influenced by the growth and development temperature of their nematode host.

Common virulence factors and genetic relationships between O18:K1:H7 Escherichia coli isolates of human and avian origin.

Extraintestinal pathogenic (ExPEC) Escherichia coli strains of serotype O18:K1:H7 are mainly responsible for neonatal meningitis and sepsis in humans and belong to a limited number of closely related clones. The same serotype is also frequently isolated from the extraintestinal lesions of colibacillosis in poultry, but it is not well known to what extent human and avian strains of this particular serotype are related. Twenty-two ExPEC isolates of human origin and 33 isolates of avian origin were compared on the basis of their virulence determinants, lethality for chicks, pulsed-field gel electrophoresis (PFGE) patterns, and classification in the main phylogenetic groups. Both avian and human isolates were lethal for chicks and harbored similar virulence genotypes. A major virulence pattern, identified in 75% of the isolates, was characterized by the presence of F1 variant fimbriae; S fimbriae; IbeA; the aerobactin system; and genomic fragments A9, A12, D1, D7, D10, and D11 and by the absence of P fimbriae, F1C fimbriae, Afa adhesin, and CNF1. All but one of the avian and human isolates also belonged to major phylogenetic group B2. However, various subclonal populations could be distinguished by PFGE in relation to animal species and geographical origin. These results demonstrate that very closely related clones can be recovered from extraintestinal infections in humans and chickens and suggest that avian pathogenic E. coli isolates of serotype O18:K1:H7 are potential human pathogens.

Steinernema sichuanense n. sp. (Rhabditida, Steinernematidae), a new species of entomopathogenic nematode from the province of Sichuan, east Tibetan Mts., China.

Steinernema sichuanense n. sp. is characterized by male, female and IJ. For male, the spicules are robust with prominent rostrum; gubernaculum has blunt anterior end; cuneus is arrow-shaped, pointed posteriorly. Second-generation male has a prominent mucron. For female, tail usually has one to four papillae-like projections on tail tip; post anal swelling is absent. For IJ, body length is about 710 microm; lateral field has six ridges; the formula of lateral field is 2, 5, 6, 4, 2 with two prominent submarginal ridges; tail usually has a dorsal depression. In Steinernema affine/intermedium group, the IJ of S. sichuanense n. sp. differs from S. affine by its absence of the internal tail spine; differs from Steinernema beddingi by its six ridges in lateral field compared to 4 for S. beddingi. For male mucron is absent in both generations of S. affine, S. intermedium and S. beddingi, whereas it is present in the second-generation of S. sichuanense sp. n. Morphology and morphometrics of spicules and gubernacula of the four species in S. affine/intermedium group are quite different based on SEM photographs. For female, the postanal swelling is absent in the first-generation of S. sichuanense n. sp. whereas S. affine and S. intermedium have slight swelling and S. beddingi has conspicuous swelling. The new species is further recognized by characterization of sequences of ITS and D2/D3 regions of the ribosomal DNA. The symbiotic bacterium associated to S. sichuanense belongs to the species Xenorhabdus bovienii.

Effects of orally administered Lactobacillus casei DN-114 001 on the composition or activities of the dominant faecal microbiota in healthy humans.

The composition and activities of the faecal microbiota in twelve healthy subjects analysed in a single open study were monitored before (1-week baseline step), during (10 d supplementation step) and after (10 d follow-up step) the ingestion of a fermented milk containing Lactobacillus casei DN-114 001. Fluorescent in situ hybridisation with group-specific DNA probes, real-time PCR using L. paracasei group-specific primers and temporal temperature gradient gel electrophoresis (TTGE) using group-specific primers were carried out, together with bacterial enzyme activity and metabolite analyses to monitor the structure and activities of the faecal microbiota. L. casei DNA was detected in the faeces of all of the subjects by TTGE after 10 d supplementation. Its quantification by real-time PCR showed a 1000-fold increase during the test step compared with initial levels. No major modification in either the dominant members of the faecal microbiota or their activities was observed during the trial. In conclusion, the short-term consumption of a milk product containing L. casei DN-114 001 was accompanied by a high, transient increase in the quantity of this strain in the faeces of all of the subjects without markedly affecting biochemical or bacteriological factors.

Genetic characterization of the beta-glucuronidase enzyme from a human intestinal bacterium, Ruminococcus gnavus.

beta-Glucuronidase activity (encoded by the gus gene) has been characterized for the first time from Ruminococcus gnavus E1, an anaerobic bacterium belonging to the dominant human gut microbiota. beta-Glucuronidase activity plays a major role in the generation of toxic and carcinogenic metabolites in the large intestine, as well as in the absorption and enterohepatic circulation of many aglycone residues with protective effects, such as lignans, flavonoids, ceramide and glycyrrhetinic acid, that are liberated by the hydrolysis of the corresponding glucuronides. The complete nucleotide sequence of a 4537 bp DNA fragment containing the beta-glucuronidase locus from R. gnavus E1 was determined. Five ORFs were detected on this fragment: three complete ORFs (ORF2, gus and ORF3) and two partial ORFs (ORF4 and ORF5). The products of ORF2 and ORF3 show strong similarities with many beta-glucoside permeases of the phosphoenolpyruvate : beta-glucoside phosphotransferase systems (PTSs), such as Escherichia coli BglC, Bacillus subtilis BglP and Bacillus halodurans PTS Enzyme II. The product of ORF5 presents strong similarities with the amino-terminal domain of Clostridium acetobutylicum beta-glucosidase (bglA). The gus gene product presents similarities with several known beta-glucuronidase enzymes, including those of Lactobacillus gasseri (69%), E. coli (61%), Clostridium perfringens (59%) and Staphylococcus aureus (58%). By complementing an E. coli strain in which the uidA gene encoding the enzyme was deleted, it was confirmed that the R. gnavus gus gene encodes the beta-glucuronidase enzyme. Moreover, it was found that the gus gene was transcribed as part of an operon that includes ORF2, ORF3 and ORF5.

Molecular quantification of lactic acid bacteria in fermented milk products using real-time quantitative PCR.

Real-time quantitative PCR assays were developed for the absolute quantification of lactic acid bacteria (LAB) (Streptococcus thermophilus, Lactobacillus delbrueckii, L. casei, L. paracasei, L. rhamnosus, L. acidophilus and L. johnsonii) in fermented milk products. The results of molecular quantification and classic bacterial enumeration did not differ significantly with respect to S. thermophilus and the species of the L. casei group which were detected in the six commercial fermented products tested, thus showing that DNA extraction was efficient and that genomic DNA solutions were free of PCR inhibitors. For L. delbrueckii, the results of bacterial enumeration were generally lower by a factor 10 to 100 than those of PCR quantification, suggesting a loss of viability during storage of the dairy products at 1-8 degrees C for most of the strains in this species. Real-time quantitative assays enabled identification of the species of lactic acid bacterial strains initially present in commercial fermented milk products and their accurate quantification with a detection threshold of 10(3) cells per ml of product.

Molecular diversity of new Thermococcales isolates from a single area of hydrothermal deep-sea vents as revealed by randomly amplified polymorphic DNA fingerprinting and 16S rRNA gene sequence analysis.

Members of the Thermococcales are anaerobic Archaea belonging to the kingdom Euryarchaea that are studied in many laboratories as model organisms for hyperthermophiles. We describe here a molecular analysis of 86 new Thermococcales isolates collected from six different chimneys of a single hydrothermal field located in the 13 degrees N 104 degrees W segment of the East Pacific ridge at a depth of 2,330 m. These isolates were sorted by randomly amplified polymorphic DNA (RAPD) fingerprinting into nine groups, and nine unique RAPD profiles were obtained. One RAPD group corresponds to new isolates of Thermococcus hydrothermalis, whereas all other groups and isolates with unique profiles are different from the 22 reference strains included in this study. Analysis of 16S rRNA gene sequences of representatives of each RAPD group and unique profiles showed that one group corresponds to Pyrococcus strains, whereas all the other isolates are Thermococcus strains. We estimated that our collection may contain at least 11 new species. These putative species, isolated from a single area of hydrothermal deep-sea vents, are dispersed in the 16S rRNA tree among the reference strains previously isolated from diverse hot environments (terrestrial, shallow water, hydrothermal vents) located around the world, suggesting that there is a high degree of dispersal of Thermococcales: About one-half of our isolates contain extrachromosomal elements that could be used to search for novel replication proteins and to develop genetic tools for hyperthermophiles.

Use of predictive modeling for Propionibacterium strain classification.

Computed data analysis of biochemical or molecular profiles is currently used in studies of microbial taxonomy, epidemiology, and microbial diversity. We assessed the use of Partial Least Squares (PLS) regression for multivariate data analysis in bacteriology. We identified clear relationships between RAPD profiles of propionibacteria strains and their species classification, autolytic capacities, and their origins. The PLS regression also predicted species identity of some strains with RAPD profiles partially related to those of reference strains. The PLS analysis also allowed us to identify key characteristics to use to classify strains. PLS regression is particularly well adapted to i) describing a collection of bacterial isolates, ii) justifying bacterial groupings using several sets of data, and iii) predicting phenotypic characters of strains that have been classified by routine typing methods.