Group B streptococcus neonatal invasive infections, France 2007-2012.

Streptococcus agalactiae (group B streptococcus (GBS)) is the leading cause of invasive infections among newborns in industrialized countries, with two described syndromes: early-onset disease (EOD) and late-onset disease (LOD). Since the introduction in many countries of intrapartum antibioprophylaxis (IAP), the incidence of EOD has dramatically decreased, whereas that of LOD remains unchanged. We describe the clinical and bacteriological characteristics of 438 GBS neonatal invasive infections notified to the French National Reference Centre for Streptococci in France from 2007 to 2012. Clinical data were retrieved from hospitalization reports or questionnaires. Capsular type, assignment to the hypervirulent clonal complex (CC)17 and antibiotic susceptibility profiles were determined. One hundred and seventy-four (39.7%) and 264 (60.3%) isolates were responsible for EOD, including death in utero, and LOD, respectively. EOD was associated with bacteraemia (n = 103, 61%) and LOD with meningitis (n = 145, 55%). EOD was mainly due to capsular polysaccharide (CPS) III isolates (n = 99, 57%) and CPS Ia isolates (n = 40, 23%), and CPS III isolates were responsible for 80% (n = 211) of LOD cases. CC17 accounted for 80% (n = 121) of CPS III isolates responsible for meningitis (n = 151; total cases of meningitis, 188). Bad outcome risk factors were low gestational age and low birthweight. LOD represents almost 60% of cases of neonatal GBS disease in France and other countries in which IAP has been implemented. This observation reinforces the need to develop new prevention strategies targeting CC17, which is predominant in GBS neonatal infections.

Invasive group A streptococcal infections in adults, France (2006-2010).

Severe invasive group A streptococcal diseases have re-emerged during the past 10-20 years. In order to provide a better insight into the current epidemiological situation in France, we analysed the questionnaires regarding all invasive strains received at the National Reference Center for Streptococci (CNR-Strep) between 2006 and 2010 from patients aged ≥ 18 and characterized them by emm typing, spe gene detection and antibiotic resistance. Among the 1542 invasive GAS strains studied, 78% (n=1206) were from blood cultures, and a streptococcal toxic shock syndrome (STSS) was described in 22% (n=340) of cases, mainly associated with necrotizing fasciitis (NF) and pleuro-pulmonary infections (p<0.001). The in-hospital fatality rate was 15%. A total of 83 different emm types were recovered but the three predominant emm types, representing almost 60% of the isolates, were emm1 (24%), emm28 (17%) and emm89 (15%). The preponderance of each emm type varied according to the year, with a significant constant increase of emm28 strains, whereas emm1 strains, representing approximately 32% of GAS invasive isolates in 2007 and 2008, dropped to <15% in 2010 (p<0.001). The distribution of phage-associated superantigen genes (speA, speC and ssa) was linked to certain emm types. Between 2006 and 2010, the percentage that was macrolide-resistant decreased from 11% to 5%, confirming the trend observed in 2007. Fortunately, emm1 strains associated with the most life-threatening clinical manifestations remain susceptible to all anti-streptococcal antibiotics.

Epidemiology of invasive Streptococcus pyogenes infections in France in 2007.

Invasive group A streptococcal (GAS) infections cause significant morbidity and mortality. A national survey was initiated to assess the burden of invasive GAS infections in France, describe their clinical characteristics, and assess the molecular characteristics of GAS strains responsible for these infections. The survey was conducted in 194 hospitals, accounting for 51% of acute care hospital admissions in France. Clinical data, predisposing factors, and demographic data were obtained, and all GAS isolates were emm sequence typed. We identified 664 cases of invasive GAS infections, with an annual incidence of 3.1 per 100,000 population. The case-fatality ratio was 14% and rose to 43% in the case of streptococcal toxic shock syndrome. Bacteremia without identified focus (22%) and skin/soft tissue infections (30%) were the most frequent clinical presentations. Necrotizing fasciitis was frequent in adults (18%) and uncommon in children (3%). The 3 predominant emm types were emm1, emm89, and emm28, accounting for 33%, 16%, and 10% of GAS isolates, respectively. The emm1 type was associated with fatal outcomes and was more frequent in children than in adults. Six clusters of cases were identified, with each cluster involving 2 invasive cases due to GAS strains which shared identical GAS emm sequence types. Four clusters of cases involved eight postpartum infections, one family cluster involved a mother and child, and one cluster involved two patients in a nursing home. Invasive GAS infection is one of the most severe bacterial diseases in France, particularly in persons aged ≥ 50 years or when associated with toxic shock syndrome.

Invasive group B streptococcal infections in adults, France (2007-2010).

Group B streptococcus (GBS) has emerged as an important cause of invasive infection in adults. Here, we report the clinical and microbiological characteristics of 401 non-redundant GBS strains causing adult invasive infections collected during a 4-year period (2007-2010). Bacteraemia without focus (43.4%) and bone and joint infections (18.7%) were the main clinical manifestations. The distribution of capsular polysaccharide (CPS) type showed that types Ia, III, and V accounted for 71.8% of all strains. Resistance to erythromycin increased from 20.2% in 2007 to 35.3% in 2010, and was mainly associated with CPS type V harbouring the erm(B) resistant determinant.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.