Prospective evaluation of a real-time PCR assay for detection of group B streptococci in vaginal swabs from pregnant women.
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Bacteriological Techniques↗
Biomedical subjects
P Trieu-Cuot
Publications and source records attributed to P Trieu-Cuot.
Use of genotypic identification by sodA sequencing in a prospective study to examine the distribution of coagulase-negative Staphylococcus species among strains recovered during septic orthopedic surgery and evaluate their significance.
A total of 212 coagulase-negative Staphylococcus strains recovered prospectively during 119 surgeries for proven or suspected bone and joint infection (BJI) were identified by sodA sequencing. These strains were identified as 151 Staphylococcus epidermidis isolates, 15 S. warneri isolates, 14 S. capitis isolates, 9 S. hominis isolates, 6 S. lugdunensis isolates, 5 S. haemolyticus isolates, 4 S. caprae isolates, 4 S. pasteuri isolates, 3 S. simulans isolates, and 1 S. cohnii isolate. Only S. epidermidis, S. lugdunensis, S. capitis, and S. caprae were found to be infecting organisms and were involved, respectively, in 35 (81.4%), 3 (7.0%), 3 (7.0%), and 2 (4.6%) cases of BJI.
Adult↗
Use of sodA sequencing for the identification of clinical isolates of coagulase-negative staphylococci.
This study evaluated the possible advantages provided by a genotypic method over commercially available biochemical systems for the identification of clinical isolates of coagulase-negative staphylococci (CNS). Partial sequencing of the sodA gene was performed for 168 coagulase-negative clinical isolates of staphylococci identified previously with the ID32 STAPH system. Of these, 101 (60.1%) were identified to the species level with ID32 STAPH, while 67 (39.9%) were misidentified or not identified with certainty. Sequencing of sodA proved useful for resolving all ambiguities or inconclusive identifications generated by the commercially available biochemical identification system.
Bacterial Proteins↗
Minimizing potential resistance: the molecular view.
The major contribution of molecular biology to the study of antibiotic resistance has been the elucidation of nearly all biochemical mechanisms of resistance and the routes for dissemination of genetic information among bacteria. In this review, we consider the potential contribution of molecular biology to counteracting the evolution of resistant bacteria. In particular, we emphasize the fact that fundamental approaches have had direct practical effects on minimizing potential resistance: by improving interpretation of resistance phenotypes, by providing more adequate human therapy, by fostering more prudent use of antibiotics, and by allowing the rational design of new drugs that evade existing resistance mechanisms or address unexploited targets.
Animals↗
Identification of new genes involved in the virulence of Listeria monocytogenes by signature-tagged transposon mutagenesis.
Listeria monocytogenes is a gram-positive, facultative intracellular pathogen that can cause severe food-born infections in humans and animals. We have adapted signature-tagged transposon mutagenesis to L. monocytogenes to identify new genes involved in virulence in the murine model of infection. We used transposon Tn1545 carried on the integrative vector pAT113. Forty-eight tagged transposons were constructed and used to generate banks of L. monocytogenes mutants. Pools of 48 mutants were assembled, taking one mutant from each bank, injected into mice, and screened for those affected in their multiplication in the brains of infected animals. From 2,000 mutants tested, 18 were attenuated in vivo. The insertions harbored by these mutants led to the identification of 10 distinct loci, 7 of which corresponded to previously unknown genes. The properties of four loci involving putative cell wall components were further studied in vitro and in vivo. The data suggested that these components are involved in bacterial invasion and multiplication in the brain.
Amino Acid Sequence↗
Contribution of Mn-cofactored superoxide dismutase (SodA) to the virulence of Streptococcus agalactiae.
Superoxide dismutases convert superoxide anions to molecular oxygen and hydrogen peroxide, which, in turn, is metabolized by catalases and/or peroxidases. These enzymes constitute one of the major defense mechanisms of cells against oxidative stress and hence play a role in the pathogenesis of certain bacteria. We previously demonstrated that group B streptococci (GBS) possess a single Mn-cofactored superoxide dismutase (SodA). To analyze the role of this enzyme in the pathogenicity of GBS, we constructed a sodA-disrupted mutant of Streptococcus agalactiae NEM316 by allelic exchange. This mutant was subsequently cis complemented by integration into the chromosome of pAT113/Sp harboring the wild-type sodA gene. The SOD specific activity detected by gel analysis in cell extracts confirmed that active SODs were present in the parental and complemented strains but absent in the sodA mutant. The growth rates of these strains in standing cultures were comparable, but the sodA mutant was extremely susceptible to the oxidative stress generated by addition of paraquat or hydrogen peroxide to the culture medium and exhibited a higher mutation frequency in the presence of rifampin. In mouse bone marrow-derived macrophages, the sodA mutant showed an increased susceptibility to bacterial killing by macrophages. In a mouse infection model, after intravenous injection the survival of the sodA mutant in the blood and the brain was markedly reduced in comparison to that of the parental and complemented strains whereas only minor effects on survival in the liver and the spleen were observed. These results suggest that SodA plays a role in GBS pathogenesis.
Animals↗
Regulation of D-alanyl-lipoteichoic acid biosynthesis in Streptococcus agalactiae involves a novel two-component regulatory system.
The dlt operon of gram-positive bacteria comprises four genes (dltA, dltB, dltC, and dltD) that catalyze the incorporation of D-alanine residues into the lipoteichoic acids (LTAs). In this work, we characterized the dlt operon of Streptococcus agalactiae, which, in addition to the dltA to dltD genes, included two regulatory genes, designated dltR and dltS, located upstream of dltA. The dltR gene encodes a 224-amino-acid putative response regulator belonging to the OmpR family of regulatory proteins. The dltS gene codes for a 395-amino-acid putative histidine kinase thought to be involved in the sensing of environmental signals. The dlt operon of S. agalactiae is mainly transcribed from the P(dltR) promoter, which directs synthesis of a 6.5-kb transcript encompassing dltR, dltS, dltA, dltB, dltC, and dltD, and from a weaker promoter, P(dltA), which is located in the 3' extremity of dltS. We demonstrate that P(dltR), but not P(dlA), is activated by DltR in the presence of DltS in D-Ala-deficient LTA mutants resulting from insertional inactivation of the dltA gene, which encodes the cytoplasmic D-alanine-D-alanyl carrier ligase DltA. Expression of the dlt operon does not require DltR and DltS, since the basal activity of P(dltR) is high, being 20-fold that of the constitutive promoter P(aphA-3) which directs synthesis of the kanamycin resistance gene aphA-3 in various gram-positive bacteria. We hypothesize that the role of DltR and DltS in the control of expression of the dlt operon is to maintain the level of D-Ala esters in LTAs at a constant and appropriate value whatever the environmental conditions. The DltA(-) mutant displayed the ability to form clumps in standing culture and exhibited an increased susceptibility to the cationic antimicrobial polypeptide colistin.
Bacterial Proteins↗
Rapid and accurate species-level identification of coagulase-negative staphylococci by using the sodA gene as a target.
Simple PCR and sequencing assays that utilize a single pair of degenerate primers were used to characterize a 429-bp-long DNA fragment internal (sodA(int)) to the sodA gene encoding the manganese-dependent superoxide dismutase in 40 coagulase-negative staphylococcal (CNS) type strains. The topology of the phylogenetic tree obtained was in general agreement with that which was inferred from an analysis of their 16S rRNA or hsp60 gene sequences. Sequence analysis revealed that the staphylococcal sodA genes exhibit a higher divergence than does the corresponding 16S ribosomal DNA. These results confirm that the sodA gene constitutes a highly discriminative target sequence for differentiating closely related bacterial species. Clinical isolates that could not be identified at the species level by phenotypical tests were identified by use of this database. These results demonstrate the usefulness of this method for rapid and accurate species identification of CNS isolates, although it does not allow discrimination of subspecies. The sodA sequence polymorphisms observed with staphylococcal species offer good opportunities for the development of assays based on DNA chip technologies.
Bacterial Proteins↗
Optimization of green fluorescent protein expression vectors for in vitro and in vivo detection of Listeria monocytogenes.
The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is a useful reporter molecule for monitoring in vivo gene expression in eukaryotic and prokaryotic cells. We constructed a series of GFP vectors for in situ detection of the intracellular pathogen Listeria monocytogenes. The gfp-mutl gene, which encodes a red-shifted GFP, was transcriptionally fused to a strong L. monocytogenes promoter and inserted into various Escherichia coli-Listeria shuttle vectors: i) the integrative monocopy plasmid pAT113; ii) the low copy number plasmid pTCV-Exl; iii) the high copy number plasmid pAT18. Listeria cells harboring pNF6 and pNF7, constructed from pAT113 and pTCV-Exl, respectively, gave low fluorescence intensities, and were optically detected in cultured macrophages, but not in tissue sections. The fluorescence of Listeria with the pAT18 derivative pNF8 was about 40 times greater than that with pNF6 and 15 times greater than that with pNF7. Listeria cells harboring pNF8 were readily detected in both cultured macrophages and tissue sections. Constructed GFP vectors did not affect the virulence of L. monocytogenes in a murine model of infection.
Animals↗
Characterization of the Tn916-like transposon Tn3872 in a strain of abiotrophia defectiva (Streptococcus defectivus) causing sequential episodes of endocarditis in a child.
Clinical blood isolates from three sequential episodes of endocarditis occurring over a 6-month period in a child with a malformative cardiopathy were investigated. All isolates identified as Abiotrophia defectiva were resistant to erythromycin-clindamycin and to tetracycline-minocycline, due to the presence of sequences homologous to the erythromycin resistance gene ermB and to the tetracycline resistance gene tet(M), respectively. These resistance genes were located on a chromosomally borne composite Tn916-related transposon. These results demonstrate the involvement of conjugative transposons in the dissemination of antibiotic resistance in the genus Abiotrophia.
Anti-Bacterial Agents↗
Sequencing the gene encoding manganese-dependent superoxide dismutase for rapid species identification of enterococci.
Simple PCR and sequencing assays that utilize a single pair of degenerate primers were used to characterize a 438-bp-long DNA fragment internal (sodA(int)) to the sodA gene encoding the manganese-dependent superoxide dismutase in 19 enterococcal type strains (Enterococcus avium, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus columbae, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus flavescens, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, Enterococcus raffinosus, Enterococcus saccharolyticus, Enterococcus seriolicida, Enterococcus solitarius, and Enterococcus sulfureus). Sequence analysis of the sodA(int) fragments enabled reliable identification of 18 enterococcal species, including E. casseliflavus-E. flavescens and E. gallinarum. The sodA(int) fragments of E. casseliflavus and E. flavescens were almost identical (99.5% sequence identity), which suggests that they should be associated in a single species. Our results confirm that the sodA gene constitutes a more discriminative target sequence than 16S rRNA gene in differentiating closely related bacterial species.
Bacterial Proteins↗
Characterization of IS1541-like elements in Yersinia enterocolitica and Yersinia pseudotuberculosis.
We characterized Yersinia enterocolitica and Yersinia pseudotuberculosis insertion sequences related to insertion sequence 1541, recently identified in Yersinia pestis. For each of the two species, two insertion sequence copies were cloned and sequenced. Genetic elements from Y. pseudotuberculosis were almost identical to insertion sequence 1541, whereas these from Y. enterocolitica were less related. Phylogenetic analysis of the putative transposases encoded by insertion sequences from the three pathogenic members of the genus Yersinia showed that they clustered with those encoded by Escherichia coli and Salmonella enterica elements belonging to the insertion sequence 200/insertion sequence 605 group. Insertion sequences originating from Y. pestis and Y. pseudotuberculosis constitute a monophyletic lineage distinct from that of Y. enterocolitica.
Base Sequence↗
Circularization of Tn916 is required for expression of the transposon-encoded transfer functions: characterization of long tetracycline-inducible transcripts reading through the attachment site.
A detailed transcriptional analysis of the conjugative transposon Tn916 was carried out, which revealed that transcription of the transfer functions requires excision of the element and dramatically increases in the presence of tetracycline. The key components of this regulatory system are two contiguous transposon-borne genes, orf7 and orf8, located downstream from and having the same polarity of transcription as the tetracycline resistance determinant tetM. The gene orf7 encodes a 140-amino-acid (aa) protein exhibiting limited homology with sigmaF of Bacillus subtilis, whereas orf8 encodes a 76-aa peptide that does not share any sequence homology with any cognate proteins. In the presence of tetracycline, an attenuation mechanism enables the transcription of orf7 and orf8 from the tetM promoter. The resulting increased synthesis of ORF7 and ORF8 activates the promoter Porf7 located upstream from orf7, which then directs the expression of the transfer functions in the transposon circular intermediate through long transcripts encompassing the attachment site. The apparently non-regulated promoter Pxis located upstream of the excisionase encoding gene xis could also participate in the expression of the tra genes. We also demonstrate that Tn916 carries another regulated promoter, Porf9, which directs transcription of a single gene, orf9, located downstream from and transcribed counterclockwise to tetM. This gene encodes a 117-aa putative transcriptional repressor, but the exact role of this protein in the mobility of Tn916, as well as the regulation of its expression, remains to be elucidated. Our results constitute the molecular basis for the observation that tetracycline increased the transfer frequency of this type of element.
Bacillus subtilis↗
A novel extended-spectrum TEM-type beta-lactamase (TEM-52) associated with decreased susceptibility to moxalactam in Klebsiella pneumoniae.
Klebsiella pneumoniae NEM865 was isolated from the culture of a stool sample from a patient previously treated with ceftazidime (CAZ). Analysis of this strain by the disk diffusion test revealed synergies between amoxicillin-clavulanate (AMX-CA) and CAZ, AMX-CA and cefotaxime (CTX), AMX-CA and aztreonam (ATM), and more surprisingly, AMX-CA and moxalactam (MOX). Clavulanic acid (CA) decreased the MICs of CAZ, CTX, and MOX, which suggested that NEM865 produced a novel extended-spectrum beta-lactamase. Genetic, restriction endonuclease, and Southern blot analyses revealed that the resistance phenotype was due to the presence in NEM865 of a 13.5-kb mobilizable plasmid, designated pNEC865, harboring a Tn3-like element. Sequence analysis revealed that the blaT gene of pNEC865 differed from blaTEM-1 by three mutations leading to the following amino acid substitutions: Glu104-->Lys, Met182-->Thr, and Gly238-->Ser (Ambler numbering). The association of these three mutations has thus far never been described, and the blaT gene carried by pNEC865 was therefore designated blaTEM-52. The enzymatic parameters of TEM-52 and TEM-3 were found to be very similar except for those for MOX, for which the affinity of TEM-52 (Ki, 0.16 microM) was 10-fold higher than that of TEM-3 (Ki, 1.9 microM). Allelic replacement analysis revealed that the combination of Lys104, Thr182, and Ser238 was responsible for the increase in the MICs of MOX for the TEM-52 producers.
Amino Acid Sequence↗
Molecular characterization of IS1541 insertions in the genome of Yersinia pestis.
The genome of Yersinia pestis, the causative agent of plague, contains at least 30 copies of an element, designated IS1541, which is structurally related to IS200 (85% identity). One such element is inserted within the chromosomal inv gene (M. Simonet, B. Riot, N. Fortineau, and P. Berche, Infect. Immun. 64:375-379, 1996). We characterized other IS1541 insertions by cloning 14 different Y. pestis 6/69M loci carrying a single copy of this insertion sequence (IS) into Escherichia coli and, for each element, sequencing 250 bp of both flanking regions. In no case was this IS element inserted into large open reading frames; however, in eight cases, it was detected downstream (17 to 139 bp) of genes thought to be transcribed monocistronically or which constituted the last gene of an operon, and in only one case was it detected upstream (37 bp) of the first gene of an operon. Sequence analysis revealed stem-loop structures (deltaG, < -10 kcal) resembling rho-independent transcription terminators in 8 of the 14 insertion sites. These motifs might constitute hot spots for insertion of this IS1541 element within the Y. pestis genome.
Amino Acid Sequence↗
Structural and functional characterization of IS1358 from Vibrio cholerae.
The new epidemic serovar O139 of Vibrio cholerae has emerged from the pandemic serovar O1 biotype El Tor through the replacement of a 22-kbp DNA region by a 40-kbp O139-specific DNA fragment. This O139-specific DNA fragment contains an insertion sequence that was described previously (U. H. Stroeher, K. E. Jedani, B. K. Dredge, R. Morona, M. H. Brown, L. E. Karageorgos, J. M. Albert, and P. A. Manning, Proc. Natl. Acad. Sci. USA 92:10374-10378, 1995) and designated IS1358O139. We studied the distribution of the IS1358 element in strains from various serovars by Southern analysis. Its presence was detected in strains from serovars O1, O2, O22, O139, and O155 but not in strains from serovars O15, O39, and O141. Furthermore, IS1358 was present in multiple copies in strains from serovars O2, O22, and O155. We cloned and sequenced four copies of IS1358 from V. cholerae O22 and one copy from V. cholerae O155. A comparison of their nucleotide sequences with those of O1 and O139 showed that they were almost identical. We constructed a transposon consisting of a kanamycin resistance gene flanked by two directly oriented copies of IS1358 to study the functionality of this element. Transposition of this element from a nonmobilizable plasmid onto the conjugative plasmid pOX38-Gen was detected in an Escherichia coli recA donor at a frequency of 1.2 x 10(-8). Sequence analysis revealed that IS1358 duplicates 10 bp at its insertion site.
Base Sequence↗
Identification of streptococci to species level by sequencing the gene encoding the manganese-dependent superoxide dismutase.
We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (sodA(int)) representing approximately 85% of the genes encoding the manganese-dependent superoxide dismutase in various streptococcal type strains (S. acidominimus, S. agalactiae, S. alactolyticus, S. anginosus, S. bovis, S. constellatus, S. canis, S. cricetus, S. downei, S. dysgalactiae, S. equi subsp. equi, S. equi subsp. zooepidemicus, S. equinus, S. gordonii, S. iniae, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguis, S. pneumoniae, S. porcinus, S. pyogenes, S. salivarius, S. sanguis, S. sobrinus, S. suis, S. thermophilus, and S. vestibularis). Phylogenetic analysis of these sodA(int) fragments yields an evolutionary tree having a topology similar to that of the tree constructed with the 16S rRNA sequences. We have shown that clinical isolates could be identified by determining the positions of their sodA(int) fragments on the phylogenetic tree of the sodA(int) fragments of the type species. We propose this method for the characterization of strains that cannot be assigned to a species on the basis of their conventional phenotypic reactions.
Base Sequence↗
Molecular characterization and expression analysis of the superoxide dismutase gene from Streptococcus agalactiae.
We have cloned and sequenced a 3103-bp DNA fragment carrying the gene encoding the Mn-SOD from Streptococcus agalactiae NEM318 serotype III. This DNA fragment contained four orfs that have the same polarity of transcription. Orf1 was truncated by molecular cloning and the corresponding 228-aa-long polypeptide did not exhibit any significant homology with other cognate proteins. Orf2 encodes a protein of 345 aa that displays some similarity (29% identity) with the YqeN peptide of Bacillus subtilis, the function of which is unknown. Orf3 encodes the 202-aa-long Mn-SOD which was functionally expressed in Escherichia coli. Orf4 was also truncated by molecular cloning and encodes 99 aa of the N-terminal moiety of a protein that displays significant homology (40% f identity) with the antiterminator LicT of B. subtilis. Transcriptional analysis revealed that the sodA gene of S. agalactiae NEM318 was transcribed monocistronically from a promoter, the activity of which is neither regulated by pH, O2, nor CO2 concentrations of the culture medium. Analysis by high resolution agarose gel electrophoresis of the AluI DNA polymorphism of the sodA locus in wild-type strains of S. agalactiae belonging to serogroups I, II, or III revealed no detectable difference.
Bacterial Proteins↗