A negative role for MyD88 in the resistance to starvation as revealed in an intestinal infection of Drosophila melanogaster with the Gram-positive bacterium Staphylococcus xylosus.

Drosophila melanogaster is a useful model to investigate mucosal immunity. The immune response to intestinal infections is mediated partly by the Immune deficiency (IMD) pathway, which only gets activated by a type of peptidoglycan lacking in several medically important Gram-positive bacterial species such as Staphylococcus. Thus, the intestinal host defense against such bacterial strains remains poorly known. Here, we have used Staphylococcus xylosus to develop a model of intestinal infections by Gram-positive bacteria. S. xylosus behaves as an opportunistic pathogen in a septic injury model, being able to kill only flies immunodeficient either for the Toll pathway or the cellular response. When ingested, it is controlled by IMD-independent host intestinal defenses, yet flies eventually die. Having excluded an overreaction of the immune response and the action of toxins, we find that flies actually succumb to starvation, likely as a result of a competition for sucrose between the bacteria and the flies. Fat stores of wild-type flies are severely reduced within a day, a period when sucrose is not yet exhausted in the feeding solution. Interestingly, the Toll pathway mutant MyD88 is more resistant to the ingestion of S. xylosus and to starvation than wild-type flies. MyD88 flies do not rapidly deplete their fat stores when starved, in contrast to wild-type flies. Thus, we have uncovered a novel function of MyD88 in the regulation of metabolism that appears to be independent of its known roles in immunity and development.

Fly culture collapse disorder: detection, prophylaxis and eradication of the microsporidian parasite Tubulinosema ratisbonensis infecting Drosophila melanogaster.

Drosophila melanogaster is a robust model to investigate many biological problems. It is however prone to some infections, which may endanger fly stocks if left unchecked for. One such infection is caused by an obligate fungal intracellular parasite, Tubulinosema ratisbonensis, which can be found in laboratory stocks. Here, we identify and briefly characterize a T. ratisbonensis strain that was infesting our Drosophila cultures and that required intensive measures to contain and eradicate the infection. We describe the phenotypes of infested stocks. We also report PCR-based techniques that allow the detection of infested stocks with a high sensitivity. We have developed a high-throughput qPCR assay that allows the efficient parallel screening of a large number of potentially-infested stocks. We also have investigated several prophylactic measures to prevent the further contamination of stocks, namely UV-exposure, ethanol treatment, bleaching, and desiccation. Bleaching was found to kill all spores. Other treatments were less effective but were found to be sufficient to prevent further contamination of noninfested stocks. Two treatments were efficacious in curing infested stocks (1) bleaching of eggs and subsequent raising of the larvae in clean vials; (2) fumagillin treatment. These cures only work on stocks that have not become too weak to withstand the procedures.

Molecular typing of Mycoplasma agalactiae: tracing European-wide genetic diversity and an endemic clonal population.

Mycoplasma agalactiae causes chronic infections in small ruminants and remains endemic in many regions of the world, despite intensive and costly eradication programs. In this study, the innate genomic plasticity of M. agalactiae was exploited to design and assess a combination of molecular epidemiological tools to trace the pathogen in different geographic locations and to understand its emergence or re-emergence after eradication campaigns. For this purpose, two collections of M. agalactiae isolates, representing European outbreaks or localized endemic disease in a single region of France, were subjected to RFLP (Restriction Fragment Length Polymorphism) analyses using two sets of DNA probes (distributed across the genome and specific for the vpma gene locus), and a previously described VNTR (Variable Number Tandem Repeats) analysis. A combination of four genome-specific DNA probes and two VNTRs gave the highest discriminative power. Molecular typing revealed that, while isolates from diverse geographical origins fell into clearly different groups, the endemic disease repeatedly observed in the Western Pyrenees region over the past 30 years has been caused by a unique subtype of M. agalactiae. This indicates that the re-emergence of the pathogen after seemingly successful eradication programs is not due to the importation of exotic strains, but to the persistence of local reservoirs of infection.

Biodiversity of indigenous staphylococci of naturally fermented dry sausages and manufacturing environments of small-scale processing units.

The staphylococcal community of the environments of nine French small-scale processing units and their naturally fermented meat products was identified by analyzing 676 isolates. Fifteen species were accurately identified using validated molecular methods. The three prevalent species were Staphylococcus equorum (58.4%), Staphylococcus saprophyticus (15.7%) and Staphylococcus xylosus (9.3%). S. equorum was isolated in all the processing units in similar proportion in meat and environmental samples. S. saprophyticus was also isolated in all the processing units with a higher percentage in environmental samples. S. xylosus was present sporadically in the processing units and its prevalence was higher in meat samples. The genetic diversity of the strains within the three species isolated from one processing unit was studied by PFGE and revealed a high diversity for S. equorum and S. saprophyticus both in the environment and the meat isolates. The genetic diversity remained high through the manufacturing steps. A small percentage of the strains of the two species share the two ecological niches. These results highlight that some strains, probably introduced by the meat, will persist in the manufacturing environment, while other strains are more adapted to the meat products.

Safety improvement and preservation of typical sensory qualities of traditional dry fermented sausages using autochthonous starter cultures.

Traditional dry fermented sausages are manufactured without addition of starter cultures in small-scale processing units, their fermentation relying on indigenous microflora. Characterisation and control of these specific bacteria are essential for the sensory quality and the safety of the sausages. The aim of this study was to develop an autochthonous starter culture that improves safety while preserving the typical sensory characteristics of traditional sausages. An autochthonous starter composed of Lactobacillus sakei, Staphylococcus equorum and Staphylococcus succinus isolated from a traditional fermented sausage was developed. These strains were tested for their susceptibility to antibiotics and their production of biogenic amines. This starter was evaluated in situ at the French traditional processing unit where the strains had been isolated. Effects of the autochthonous starter were assessed by analysing the microbial, physico-chemical, biochemical and sensory characteristics of the sausages. Inoculation with the chosen species was confirmed using known species-specific PCR assays for L. sakei and S. equorum and a species-specific PCR assay developed in this study for S. succinus. Strains were monitored by pulse-field gel electrophoresis typing. Addition of autochthonous microbial starter cultures improved safety compared with the traditional natural fermentation of sausages, by inhibiting the pathogen Listeria monocytogenes, decreasing the level of biogenic amines and by limiting fatty acid and cholesterol oxidation. Moreover, autochthonous starter did not affect the typical sensory quality of the traditional sausages. This is the first time to our knowledge that selection, development and validation in situ of autochthonous starter cultures have been carried out, and also the first time that S. equorum together with S. succinus have been used as starter cultures for meat fermentation. Use of autochthonous starter cultures is an effective tool for limiting the formation of unsafe compounds in traditional sausage while preserving their original and specific sensory quality.

Evaluation of the Vitek 2 system with a variety of Staphylococcus species.

The Vitek 2 gram-positive (GP) card was compared with an oligonucleotide array approach for the identification of 190 Staphylococcus strains, including 35 species, isolated from clinical and environmental specimens. The GP card provided a rapid and reliable identification of most species, whatever their origin.

A model of bacterial intestinal infections in Drosophila melanogaster.

Serratia marcescens is an entomopathogenic bacterium that opportunistically infects a wide range of hosts, including humans. In a model of septic injury, if directly introduced into the body cavity of Drosophila, this pathogen is insensitive to the host's systemic immune response and kills flies in a day. We find that S. marcescens resistance to the Drosophila immune deficiency (imd)-mediated humoral response requires the bacterial lipopolysaccharide O-antigen. If ingested by Drosophila, bacteria cross the gut and penetrate the body cavity. During this passage, the bacteria can be observed within the cells of the intestinal epithelium. In such an oral infection model, the flies succumb to infection only after 6 days. We demonstrate that two complementary host defense mechanisms act together against such food-borne infection: an antimicrobial response in the intestine that is regulated by the imd pathway and phagocytosis by hemocytes of bacteria that have escaped into the hemolymph. Interestingly, bacteria present in the hemolymph elicit a systemic immune response only when phagocytosis is blocked. Our observations support a model wherein peptidoglycan fragments released during bacterial growth activate the imd pathway and do not back a proposed role for phagocytosis in the immune activation of the fat body. Thanks to the genetic tools available in both host and pathogen, the molecular dissection of the interactions between S. marcescens and Drosophila will provide a useful paradigm for deciphering intestinal pathogenesis.

Development of a new oligonucleotide array to identify staphylococcal strains at species level.

The genus Staphylococcus is made up of 36 validated species which contain strains that are pathogenic, saprophytic, or used as starter cultures for the food industry. An oligonucleotide array targeting the manganese-dependent superoxide dismutase (sodA) gene was developed to overcome the drawbacks of the conventional methods of identification. Divergences of the sodA gene were used to design oligonucleotide probes, and we showed that each of the 36 species had a characteristic pattern of hybridization. To evaluate the array, we analyzed 38 clinical and 38 food or food plant Staphylococcus isolates identified by the phenotype-based system VITEK 2 (bioMérieux). This commercial kit failed to identify 8 (21%) of the clinical isolates and 32 (84%) of the food and food plant isolates. In contrast, the oligonucleotide array we designed provided an accurate and rapid method for the identification of staphylococcal strains, isolated from clinical, environmental, or food samples, at species level.

Role of RegM, a homologue of the catabolite repressor protein CcpA, in the virulence of Streptococcus pneumoniae.

As part of a study of virulence gene regulation in Streptococcus pneumoniae, we have identified a gene encoding a homologue of the staphylococcal catabolite control protein CcpA in the pneumococcal genome sequence. The pneumococcal protein, designated RegM, has significant similarity to members of the LacI/GalR family of bacterial regulatory proteins. S. pneumoniae D39 derivatives with insertion-duplication or deletion mutations in regM were significantly attenuated in virulence with respect to the wild-type strain. In defined media containing either sucrose or lactose as sole carbon sources, the in vitro growth rates of D39 and the regM mutants were essentially the same. However, in the presence of galactose the regM mutants grew significantly faster than the wild-type strain, whereas growth rates were significantly lower in the presence of glucose or maltose. These data are consistent with the involvement of regM in the catabolism of carbohydrates in S. pneumoniae. RegM was a repressor of both alpha-glucosidase and beta-galactosidase activities in S. pneumoniae, but unlike the situation in certain other bacteria, it does not mediate the repression of these enzymes by glucose. The observed attenuation in virulence was not attributable to poorer growth of the regM mutants in mouse blood ex vivo, but nevertheless, the mutants were rapidly cleared from the blood of infected mice in vivo. The regM mutation had no apparent impact on expression of several confirmed pneumococcal virulence proteins, but studies employing a lacZ transcriptional fusion construct indicated that mutation of regM resulted in a significant reduction in transcription of the capsular polysaccharide biosynthesis locus (cps). Thus, regM is the first gene outside of the cps locus to be implicated in regulation of capsular gene expression.

The genes encoding virulence-associated proteins and the capsule of Streptococcus pneumoniae are upregulated and differentially expressed in vivo.

The polysaccharide capsule of Streptococcus pneumoniae and several well-characterized virulence proteins are known to contribute to the pathogenesis of pneumococcal disease. However, there is a paucity of data on the expression of their respective genes in vivo. In this study, the relative abundance of the mRNA transcripts of the genes encoding pneumolysin (ply), pneumococcal surface protein A (pspA), pneumococcal surface antigen A (psaA) and choline-binding protein A (cbpA), and of the first gene of the capsular polysaccharide biosynthesis locus (cps2A), was measured in virulent type 2 pneumococci harvested from the blood of BALB/c mice at 12 h and 24 h following intraperitoneal infection. The mRNA levels were then compared, using relative quantitative RT-PCR, with those present in organisms grown in serum broth. The expression of ply was upregulated threefold at 12 h, and 10-fold at 24 h post-infection; the expression of pspA and psaA was upregulated threefold and fivefold, respectively, at 12 h post-infection. Interestingly, the expression of pspA was 36-fold higher at 24 h post-infection whereas the expression of cps2A was upregulated approximately fourfold at 12 and 24 h post-infection. However, cbpA mRNA levels remained comparable in vivo and in vitro. When organisms were grown in whole blood or THY broth, the relative expression of these genes in the two growth media also differed markedly. This work provides direct molecular evidence that known virulence-associated genes of S. pneumoniae are differentially expressed in vivo. Data on the relative expression of these genes in different growth media also suggests that the regulation of expression of these genes is highly complex and multifactorial.

Genetic and physiological studies of the CiaH-CiaR two-component signal-transducing system involved in cefotaxime resistance and competence of Streptococcus pneumoniae.

A mutation in the ciaH gene of Streptococcus pneumoniae induces cefotaxime resistance and transformation deficiency. ciaH encodes a putative sensor protein that belongs to the family of signal-transducing histidine kinases. This gene is adjacent to ciaR, which encodes a DNA-binding protein involved in the regulation of genes responding to environmental signals sensed by the histidine kinase. The authors have characterized a mutation that induces reversion of both cefotaxime resistance and transformation deficiency. It is a T/A deletion in the ciaR gene resulting in the synthesis of a truncated protein containing only 125 amino acids instead of 224. The ciaH mutation requires a functional CiaR protein for expression. Northern blot analysis, using ciaR-ciaH as a probe, revealed one mRNA from the wild-type strain, indicating that the two genes constitute an operon. Comparisons of Northern blots show that the operon is constitutively activated in the strain carrying only the ciaH mutation. In the wild-type strain the activation occurs when the Ca2+ concentration is very low, demonstrating that Ca2+ is the environmental signal. The pleiotropic effects caused by the ciaH mutation include sensitivity to antibiotics and toxins, the ability to form protoplasts and the susceptibility to lysis with deoxycholate. Null-mutants were constructed in both genes and the particular features of the ciaR null mutant determined. It is able to grow in choline-deprived medium, and competence development occurs in a phosphate-deprived competence medium (CH-maleate), suggesting that the CiaH-CiaR system regulates several pathways, including teiochoic acid synthesis.

Organization around the dnaA gene of Streptococcus pneumoniae.

The dnaA gene region of Streptococcus pneumoniae was cloned and sequenced. A tRNA gene, seven ORFs and three DnaA box clusters were identified. The order of the genes and intergene regions found was tRNA(Arg)-orf1-DnaA box cluster 3-htrA-spoOJ-DnaA box cluster 2-dnaA-DnaA box cluster 1-dnaN-orfX-orfY. Five ORFs are homologous to known bacterial genes. The tRNA(Arg) gene and orf1, also called orfL, have already been described in pneumococci and have been reported to be preceded by the competence regulation locus comCDE. In Escherichia coli, htrA encodes a serine protease. In Bacillus subtilis, spoOJ plays a role in sporulation and partition. dnaA encodes an initiator replication protein, very well conserved in several bacteria and dnaN encodes the beta subunit of DNA polymerase III in E. coli. The function of orfX is unknown. The N-terminal part of another reading frame, orfY, revealed high homology with a GTP-binding protein, DnaA box clusters were found upstream and downstream from dnaA. The presence of two such clusters suggests that the chromosomal origin of S. pneumoniae is located within this region. The position of dnaA, and therefore the putative origin of replication, were localized on the physical map of S. pneumoniae.