Virulence and cord blood mononuclear cells cytokine production induced by perinatal listeria monocytogenes strains from different phylogenetic lineages.

Some of the phylogenetic lineages of Listeria monocytogenes are more likely to cause invasive disease in humans than are strains from other phylogenetic lineages. This suggests that strains belonging to these lineages display different levels of pathogenicity. To investigate this, we carried out a plaque-forming assay with HT-29 cells to evaluate the virulence of six perinatal strains from the three lineages that compose the species. All of the strains were largely over the 3.34 cutoff (between 4.29 and 5.97 mean log) with the HT-29 model and can therefore be considered to be equally virulent. We also explored part of the immune response of cord blood mononuclear cells by measuring cytokine production. All strains induced the production of similar amounts of TNF-alpha and IL-1beta. High concentrations of IL-6 and IL-8 were produced (between 6,000 and 17,000 pg/ml), whereas little or no IFN-gamma or IL-12 was produced. Thus, there is no difference between the strains from the three genetic lineages in terms of virulence or cytokine response. Given the epidemiological distribution of the serotypes responsible for human listeriosis and the genetic structure of the L. monocytogenes species, our results suggest: (i) that all strains from lineage I (serotypes 1/2b and 4b), a genetically homogeneous subpopulation, have a similar level of pathogenicity, and (ii) that lineage II (serotypes 1/2a and 1/2c), which is genetically more heterogeneous, is composed of strains with different levels of pathogenicity. The ones responsible for invasive diseases, particularly perinatal infections, display a similar level of pathogenicity to lineage-I strains and are not virulence-attenuated strains that can only infect the most immunocompromised hosts, whereas the other lineage-II strains are probably less pathogenic for humans.

Genetic features of Pseudomonas aeruginosa isolates from cystic fibrosis patients compared with those of isolates from other origins.

In order to improve our understanding of the colonization of the pulmonary tract of cystic fibrosis (CF) patients by Pseudomonas aeruginosa, 162 isolates from five different ecological origins were studied. The genetic features of each isolate were determined by random amplification of polymorphic DNA (RAPD) and by searching for eight virulence genes (six known virulence genes, algD, lasB, toxA, plcH, plcN and exoS, and two genes encoding putative neuraminidases, nan1 and nan2). Five RAPD groups were identified. Most of the CF isolates were distributed equally in three of these groups (RA, RB and RC). The CF isolates in RB were related to isolates from a wide variety of origins. The CF isolates in RA were related to a population composed of 65 % of the non-CF isolates from pulmonary tract infections. RC was mainly composed of CF isolates that were related to 30 % of isolates from plants. All genes except exoS and nan1 were present in all isolates. The exoS and nan1 virulence factor genes were most prevalent in CF isolates. exoS, which encodes exoenzyme S, was present in 94 % of CF isolates but also in 80 % of non-CF isolates from pulmonary tract infections. nan1, which encodes a putative neuraminidase, was found in 82.5 % of the isolates from group RC, which was composed largely of CF isolates. In conclusion, three major genogroups of P. aeruginosa isolates, each of which exhibits peculiar genetic features, are able to colonize CF patients. This may have different consequences on the outcome of pulmonary disease.

Typing of nonencapsulated haemophilus strains by repetitive-element sequence-based PCR using intergenic dyad sequences.

Intergenic dyad sequences (IDS) are short repeated elements that have been described for several Haemophilus genomes and for only two other bacterial genera. We developed a repetitive-element sequence-based PCR using an IDS-specific primer as a typing method (IDS-PCR) for nonencapsulated Haemophilus strains and compared this technique with pulsed-field gel electrophoresis (PFGE) of DNA restricted with SmaI. IDS-PCR was rapid, easy to perform, and reproducible, with a high discriminatory capacity for nontypeable Haemophilus influenzae (NTHI) strains. The 69 NTHI strains tested generated 65 different banding patterns. Epidemiologically related strains gave similar or identical fingerprints, and all of the unrelated strains except two showed different patterns. These results were in agreement with those obtained by PFGE. For 20 genital strains usually identified as being biotype IV NTHI and belonging to a cryptic genospecies of Haemophilus with remarkable genetic homogeneity, four bands were significantly present and six bands were significantly absent from the fingerprints. The 20 strains were gathered in 11 closely related profiles, whereas PFGE provided no band when DNA was treated with SmaI. IDS-PCR improved the differentiation previously obtained within this species by ribotyping and multilocus enzyme electrophoresis. Our findings suggest that IDS-PCR is a rapid, reliable, and discriminatory method for typing NTHI strains and is currently the most efficient method for distinguishing strains within the cryptic genospecies of HAEMOPHILUS:

Escherichia coli strains from pregnant women and neonates: intraspecies genetic distribution and prevalence of virulence factors.

To determine the extent to which the vagina, endocervix, and amniotic fluid screen the Escherichia coli strains responsible for neonatal infections, we studied the genetic relationships among 105 E. coli strains isolated from all of the ecosystems involved in this infectious process. Twenty-four strains were isolated from the intestinal flora, and 25 strains were isolated from the vaginas of pregnant women. Twenty-seven strains were isolated from the amniotic fluid, blood, and cerebrospinal fluid (CSF) of infected neonates. The intraspecies genetic characteristics of all of the isolates were determined by random amplified polymorphic DNA (RAPD) analysis, PCR ECOR (E. coli reference) grouping, and PCR virulence genotyping. A correlation was found between the intraspecies distributions of the strains in the A, B1, B2, and D ECOR groups and in the two major RAPD groups (I and II). Nevertheless, the distribution of the E. coli strains in the RAPD groups according to their anatomical origins was more significant than their distribution in the ECOR groups. This may be explained by the existence of an E. coli subpopulation, defined by the RAPD I group, within the ECOR B2 group. This RAPD I group presents a major risk for neonates: 75% of the strains isolated from patients with meningitis and 100% of the strains isolated from patients with bacteremia were in this group. The vagina and the amniotic fluid are two barriers that favor colonization by highly infectious strains. Indeed, only 17% of fecal strains belonged to the RAPD I group, whereas 52% of vaginal strains and 67% of amniotic fluid strains belonged to this subpopulation. The ibeA and iucC genes were significantly associated with CSF strains, whereas the hly and sfa/foc genes were more frequent in blood strains. These findings could serve as a basis for developing tools to recognize vaginal strains, which present a high risk for neonates, for use in prophylaxis programs.

tRNA gene clusters at the 3' end of rRNA operons are specific to virulent subgroups of Streptococcus agalactiae strains, as demonstrated by molecular differential analysis at the population level.

The aim of this work was to characterize a 2.4 kb randomly amplified polymorphic DNA (RAPD) fragment described as a marker for a phylogenetic group of Streptococcus agalactiae strains significantly associated with neonatal meningitis. This fragment was analysed by cloning and sequencing, and showed that two types of tRNA gene cluster flank the 3' end of the rRNA operons in S. agalactiae strains. Both types of tRNA gene cluster act as markers for phylogenetic subgroups of strains within the species. One type could be used to distinguish two of the three virulent intraspecies subgroups to which most of the S. agalactiae strains able to invade the central nervous system of neonates belong. This raises the possibility that there is a link between these tRNA genes and the virulence of the bacterium. Based on this analysis, PCR primers were designed to determine whether S. agalactiae strains are likely to belong to lineages of organisms in which most of the highly virulent strains isolated from cerebrospinal fluid cluster. In addition, this work demonstrated that RAPD can be used to detect novel particularities within intraspecies variants of pathogens.