A multi locus variable number of tandem repeat analysis (MLVA) scheme for Streptococcus agalactiae genotyping.

BACKGROUND: Multilocus sequence typing (MLST) is currently the reference method for genotyping Streptococcus agalactiae strains, the leading cause of infectious disease in newborns and a major cause of disease in immunocompromised children and adults. We describe here a genotyping method based on multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) applied to a population of S. agalactiae strains of various origins characterized by MLST and serotyping. RESULTS: We studied a collection of 186 strains isolated from humans and cattle and three reference strains (A909, NEM316 and 2603 V/R). Among 34 VNTRs, 6 polymorphic VNTRs loci were selected for use in genotyping of the bacterial population. The MLVA profile consists of a series of allele numbers, corresponding to the number of repeats at each VNTR locus. 98 MLVA genotypes were obtained compared to 51 sequences types generated by MLST. The MLVA scheme generated clusters which corresponded well to the main clonal complexes obtained by MLST. However it provided a higher discriminatory power. The diversity index obtained with MLVA was 0.960 compared to 0.881 with MLST for this population of strains. CONCLUSIONS: The MLVA scheme proposed here is a rapid, cheap and easy genotyping method generating results suitable for exchange and comparison between different laboratories and for the epidemiologic surveillance of S. agalactiae and analyses of outbreaks.

Molecular characterization and prophage DNA contents of Streptococcus agalactiae strains isolated from adult skin and osteoarticular infections.

Skin and osteoarticular infections (SKI and OAI, respectively) account for almost one-third of Streptococcus agalactiae infections in nonpregnant adults. We evaluated the genetic diversity and phylogeny of 58 S. agalactiae strains responsible for adult SKI or OAI and of 61 S. agalactiae strains from cases of adult human colonization (HCol) by serotyping and multilocus sequence typing (MLST). We also assessed the prophage DNA content of the genomes of these strains by a PCR-based method. We found that 63% of SKI and 56% of OAI occurred in people aged 55 years and over. Overall, 71% of SKI strains were of serotype Ia or V, and 91% of OAI strains were of serotype Ia, III, or V. Strains of clonal complexes 1 and 23 (CC1 and CC23) were associated with 79% of SKI cases and 62% of OAI cases. Seven groups of strains, groups A, B, C, D, E, F, and G, were obtained by performing a hierarchical analysis on the basis of prophage DNA-PCR data. We found that 85% of CC1 strains clustered in DNA prophage group D, the group with the highest prophage DNA content (average, 4.4; average of absolute deviations [AVEDEV], 0.9). The CC23 strains displayed the greatest diversity in prophage DNA fragment content, but 47% of CC23 strains clustered in group B, which also had a high average prophage DNA content per strain (average, 2.3; AVEDEV, 0.6). Many (65%) of the OAI strains were in prophage DNA group D, whereas 83% of the SKI strains were in prophage DNA groups B and D. These data suggest that S. agalactiae strains from CC1 and CC23 may be subject to particular transduction mechanisms in gene recombination, rendering them particularly capable of invading the skin, bone, or joints in adults.

Molecular characterization and lytic activities of Streptococcus agalactiae bacteriophages and determination of lysogenic-strain features.

The application of mitomycin C induction to 114 genetically diverse Streptococcus agalactiae strains generated 36 phage suspensions. On electron microscopy of the phage suspensions, it was possible to assign the phages to the Siphoviridae family, with three different morphotypes (A, B, and C). Phage genetic diversity was evaluated by a PCR-based multilocus typing method targeting key modules located in the packaging, structural, host lysis, lysogeny, replication, and transcriptional regulation clusters and in the integrase genes and by DNA digestion with EcoRI, HindIII, and ClaI. Thirty-three phages clustering in six distantly related molecular phage groups (I to VI) were identified. Each molecular group was morphotype specific except for morphotype A phages, which were found in five of the six phage groups. The various phage groups defined on the basis of molecular group and morphotype had specific lytic activities, suggesting that each recognized particular host cell targets and had particular lytic mechanisms. Comparison of the characteristics of lysogenic and propagating strains showed no difference in the serotype or clonal complex (CC) identified by multilocus sequence typing. However, all the lysogenic CC17 and CC19 strains presented catabolic losses due to a lack of catabolic decay of dl-alpha-glycerol-phosphate substrates (CC17) and of alpha-d-glucose-1-phosphate (CC19). Moreover, the phages from CC17 lysogenic strains displayed lytic replication in bacterial hosts from all S. agalactiae phylogenetic lineages other than CC23, whereas phages obtained from non-CC17 lysogenic strains lysed bacteria of similar evolutionary origin. Our findings suggest that the adaptive evolution of S. agalactiae exposed the bacteria of this species to various phage-mediated horizontal gene transfers, which may have affected the fitness of the more virulent clones.

Variable-number tandem repeat analysis and multilocus sequence typing data confirm the epidemiological changes observed with Staphylococcus aureus strains isolated from bloodstream infections.

Since 2000, our geographical region in France systematically surveys bloodstream infections (BSI) due to Staphylococcus aureus. This survey involves 39 health care institutions (HCIs) encompassing 6,888 short-stay beds and was performed during two 3-month periods during 2007 and 2008. The study periods of this survey identified 292 S. aureus isolates causing BSI. Extensive molecular characterization, including genotyping as well as toxin, agr, and staphylococcal cassette chromosome content determinations, allowed us to describe epidemiological evolution in comparison to that discussed in our previous study. Our main epidemiological observation shows that the incidence of BSI remained constant but that methicillin (meticillin)-resistant S. aureus strains with a wider variety of genetic backgrounds now harbor pyl, as has already been reported in different European countries. We noticed stable numbers of BSI episodes involving community-acquired methicillin-sensitive S. aureus (MSSA), whereas a drastic increase in the number of strains harboring the tst gene was recorded. The increase in the number of tst gene-harboring strains is related to known hospital-acquired MSSA isolates and appears related to epidemic episodes in specific HCIs. Monitoring the increase in prevalence of specific strains helps us understand where the standard precautions are not satisfactorily applied or do not efficiently prevent the spread of epidemic MSSA strains in these HCIs. The recent increases in incidence of these strains call for particular vigilance to avoid the spread of potentially virulent MSSA strains harboring the tst gene and for continuance of this strategy of BSI surveillance.

Identification of Streptococcus agalactiae isolates from various phylogenetic lineages by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Variations in proteins related to bacterial diversity may affect species identification performed using matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry. Using this method, we identified 110 Streptococcus agalactiae isolates characterized by serotyping and multilocus sequence typing. Serotype III and sequence type 23 strains expressed the widest variation in molecular weight of putative "species-identifying" biomarker ions. Recognition of the diversity of MALDI patterns observed in strains that represent all major intraspecies lineages assists in the constitution of an optimal reference database.

Genetic diversity of Streptococcus agalactiae strains and density of vaginal carriage.

We screened 500 pregnant women who had no risk factors for Streptococcus agalactiae vaginal carriage, and isolated 39 S. agalactiae strains (8 %). The density of carriage was low in 16 cases (41 %), intermediate in 16 cases (41 %) and heavy in seven cases (18 %). Strains were mostly of serotype III (41 %), Ia (26 %) and V (18 %). Thirty-five strains had at least one of five genetic markers that have been associated with virulent phylogenetic subgroups of strains. Using PCR, nine strains (23 %) were identified as belonging to CC17. The 39 vaginal strains that were studied exhibited a substantial genetic diversity; there were 39 PFGE profiles and 13 variants defined on the basis of the five genetic markers studied. The prevalence of the studied genetic characteristics was similar for strains associated with all three classes of density of carriage. These data suggest that genetic features that are markers of S. agalactiae strains able to invade the central nervous system of neonates are not determinants for vaginal adaptation.

Molecular characterization of temporally and geographically matched Streptococcus agalactiae strains isolated from food products and bloodstream infections.

In a defined geographic area, during a 3-month period, 914 food products were screened for Streptococcus agalactiae, and S. agalactiae strains isolated from bloodstream infections (BSI) in nonpregnant adults were collected. Eleven S. agalactiae strains were isolated from 1.2% of food products, with high rates in pastries (7.0%) and seafood products (11.8%). These findings indicate that S. agalactiae is a food product contaminant. Seven S. agalactiae BSI were observed in nonpregnant adults representing an incidence of 0.015/100 admissions. The distribution of strains in serotypes did not differ according to origin of the strains; food products and clinical strains were of serotypes Ia (22%), Ib (11%), II (5%), III (22%), IV (5%), and V (33%). The strains isolated from seafoods were of serotypes Ia and Ib. The distribution of strains in Sequence Types differed according to their origin; food strains were equally distributed between the major clonal complex (CC), CC1 (27%), CC9 (18%), CC17 (18%), and CC23 (27%), whereas a high proportion of BSI strains belonged to CC1 (57%). DNA macrorestriction using SmaI revealed diversity; nine different patterns were found for the 11 food strains and seven for the 7 BSI strains. One pattern was similar for two food strains and one BSI strain. On account of the molecular characteristics previously described for S. agalactiae strains of human carriage and fish and mice infections, the serotype characteristics of seafood strains suggest contamination by aquatic S. agalactiae, whereas the molecular characteristics of strains from pastries suggest human contamination, but may also originate from rodents. Indeed, serotype V CC1 strains, found in food and responsible for a high percentage of BSI in nonpregnant adults, belong to a known clone spreading worldwide, and have also been described in mice.

Molecular characterization of human-colonizing Streptococcus agalactiae strains isolated from throat, skin, anal margin, and genital body sites.

Streptococcus agalactiae carriage was evaluated by sampling four body sites in a group of 249 healthy individuals including both sexes and a wide range of ages; the aims were to study the population structure of colonizing strains by multilocus sequence typing (MLST) and to evaluate their diversity by serotyping, SmaI macrorestriction analysis, and PCR screening for genetic markers of highly virulent clones for neonates. The prevalences of carriage were 27% in women and 32% in men. The major positive body site was the genital tract (23% in women and 21% in men); skin, throats, and anal margins were also positive in 2%, 4%, and 14%, respectively. These human-colonizing strains belonged mostly to serotypes III (24%), Ia (21%), V (18%), and Ib (17%). Twenty-three sequence types (STs) were identified. The MLST characteristics of the strains isolated from a single anatomic site-genital (vagina [women] or from a sample of the first urination after arising from a night's sleep [men]), throat, skin, or anal margin-suggest a body site colonization specificity for particular STs: strains of STs 2, 10, 19, and 196 were isolated only from genital sites; strains of STs 1, 8, and 23 were isolated more frequently from throat florae; and strains recovered only from anal margin samples were more closely related to strains isolated from throats than to those from genital sites. Most strains of STs 1, 8, and 23-STs that are increasingly described as being responsible for adult infections-did not carry any markers of strains virulent for neonates, suggesting that the virulence of these strains is probably associated with other genetic determinants. In addition, the genetic diversities of the strains varied between STs: STs 2, 8, 10, 23, and 196 were the most diverse; STs 1 and 19 were more homogeneous; and ST 17 strains formed three distant groups.

Molecular characterization of erythromycin-resistant Streptococcus agalactiae strains.

OBJECTIVES: The aim of this study is to identify the molecular characteristics of erythromycin-resistant (Erm(r)) Streptococcus agalactiae strains and to correlate with the clinical origin of strains. METHODS: From 711 S. agalactiae strains, 119 Erm(r) strains (17%) were collected, serotyped and screened for macrolide resistance genes. The genetic relationship between strains was established by the PFGE analysis. Strains were tested for the group II intron GBSi1 downstream of the scpB gene, IS1548 in the hylB gene, four prophage DNA fragments and a lineage defined by multilocus sequence typing as ST-17. RESULTS: Erythromycin resistance involved 8% of serotype Ia, 15% of serotype Ib, 9% of serotype II, 16% of serotype III, 31% of serotype IV and 35% of serotype V. The prevalence of Erm(r) strains was higher among strains isolated from the gastric fluid of neonates (33%) than in those isolated from bacteraemia and meningitis during early-onset disease (EOD) or late-onset disease (7% and 11%) (P = 0.001). In serotype III, Erm(r) strains were more frequent in vaginal carriage (22%) and colonized neonates (18%) than in EOD (0%) (P = 0.03). The mef(A) gene was the most common in serotype Ia (55%), the erm(A) gene in serotype Ib (75%) and the erm(B) gene in the other serotypes (56% to 75%). All resistant strains with IS1548 also had the erm(B) gene. Erm(r) strains were not randomly distributed in the different PFGE genogroups, and 11% had the GBSi1 intron, 37% had at least one prophage DNA fragment and 7% belonged to ST-17. CONCLUSIONS: Erythromycin resistance varied according to the clinical origin, serotype and molecular characteristics of S. agalactiae strains.

Rapid detection of "highly virulent" Group B Streptococcus ST-17 and emerging ST-1 clones by MALDI-TOF mass spectrometry.

MALDI-TOF MS identified a 6250-Da protein specific to Sequence Type-1 (ST-1) strains and a 7625-Da protein specific to ST-17 strains when used for identification of Group B streptococci. The strains of these STs are major causes of meningitis and late-onset-disease in neonates. This rapid method of identification could thus be valuable in the evaluation of risk of neonatal diseases.

Diversity of prophage DNA regions of Streptococcus agalactiae clonal lineages from adults and neonates with invasive infectious disease.

The phylogenetic position and prophage DNA content of the genomes of 142 S. agalactiae (group-B streptococcus, GBS) isolates responsible for bacteremia and meningitis in adults and neonates were studied and compared. The distribution of the invasive isolates between the various serotypes, sequence types (STs) and clonal complexes (CCs) differed significantly between adult and neonatal isolates. Use of the neighbor-net algorithm with the PHI test revealed evidence for recombination in the population studied (PHI, P = 2.01 × 10(-6)), and the recombination-mutation ratio (R/M) was 6:7. Nevertheless, the estimated R/M ratio differed between CCs. Analysis of the prophage DNA regions of the genomes of the isolates assigned 90% of the isolates to five major prophage DNA groups: A to E. The mean number of prophage DNA fragments amplified per isolate varied from 2.6 for the isolates of prophage DNA group E to 4.0 for the isolates of prophage DNA group C. The isolates from adults and neonates with invasive diseases were distributed differently between the various prophage DNA groups (P < 0.00001). Group C prophage DNA fragments were found in 52% of adult invasive isolates, whereas 74% of neonatal invasive isolates had prophage DNA fragments of groups A and B. Differences in prophage DNA content were also found between serotypes, STs and CCs (P < 0.00001). All the ST-1 and CC1 isolates, mostly of serotype V, belonged to the prophage DNA group C, whereas 84% of the ST-17 and CC17 isolates, all of serotype III, belonged to prophage DNA groups A and B. These data indicate that the transduction mechanisms, i.e., gene transfer from one bacterium to another by a bacteriophage, underlying genetic recombination in S. agalactiae species, are specific to each intraspecies lineage and population of strains responsible for invasive diseases in adults and neonates.

Methicillin-susceptible ST398 Staphylococcus aureus responsible for bloodstream infections: an emerging human-adapted subclone?

In the course of an annual 3-month bloodstream infections (BSI) survey conducted during a four-year period in 31 healthcare institutions located in three noncontiguous French regions, we report 18 ST398 Staphylococcus aureus BSI. ST398 BSI incidence showed a seven-fold increase during the study period (0.002 per 1,000 patient days in 2007 vs. 0.014 in 2010). ST398 BSI isolates differed from the pig-borne multiresistant clone: 17/18 BSI isolates were methicillin susceptible and none was of t011, t034 or t108 pig-borne spa-types. ST398 BSI isolates had homogenous resistance patterns (15/18 with only Ery(r)) and prophagic content (all harboured the hlb-converting Sau3int phage). The clustering of BSI and pig-borne isolates by spa-typing and MLVA, the occurrence of Sau3int phage in BSI isolates and the lack of this phage in pig-borne isolates suggest that the emergence of BSI isolates could have arisen from horizontal transfer, at least of the Sau3int phage, in genetically diverse MSSA ST398 isolates. The acquisition of the phage likely plays a role in the increasing ability of the lysogenic ST398 isolates to colonize human. The mode of acquisition of the non pig-borne ST398 isolates by our 18 patients remains unclear. ST398 BSI were diagnosed in patients lacking livestock exposure and were significantly associated with digestive portals of entry (3/18 [16.7%] for ST398 vs. 19/767 [2.5%] for non ST398 BSI; p = .012). This raises the question of possible foodborne human infections. We suggest the need for active surveillance to study and control the spread of this human-adapted subclone increasingly isolated in the hospital setting.

Staphylococcus aureus strains isolated from bloodstream infections changed significantly in 2006.

We studied 358 Staphylococcus aureus strains isolated from bloodstream infections (BSI) observed during an epidemiological study covering 2,007,681 days of hospitalization in 32 healthcare institutions (HCIs) between 2004 and 2006. The strains were tested for antibiotic susceptibility and characterized genetically. The incidence of S. aureus BSI declined regularly through 2004 and 2005 and then significantly increased in 2006 (+80%). This was largely due to an increase in BSI involving methicillin-sensitive S. aureus (MSSA) strains and nonmultiresistant methicillin-resistant S. aureus (NORSA) strains. Ninety-six percent of the NORSA strains were resistant only to methicillin and fluoroquinolones. Most of the MSSA strains belonged to a small number of pulsed-field gel electrophoresis (PFGE) divisions and were associated with epidemic phenomena in HCIs. The NORSA strains also clustered into a limited number of PFGE divisions but could not be related to any local outbreak in HCIs. In 2006, there was a significant increase in the incidence of BSI associated with tst gene-positive MSSA strains (+275%) and the first three BSI associated with tst gene-positive MRSA were observed. PFGE data revealed a limited heterogeneity among the tst gene-positive strains without any outbreak in the HCIs. Our study underlines the need for infection control teams to focus efforts on preventing both MRSA and MSSA BSI. As recently demonstrated in vitro, fluoroquinolones may enhance horizontal transfer of virulence and antibiotic resistance genes. These antibiotics are widely used in France, so our findings raise the issue of whether their use has contributed to the acquisition of mecA and tst genes by S. aureus strains.

Loss of catabolic function in Streptococcus agalactiae strains and its association with neonatal meningitis.

The abilities of 151 Streptococcus agalactiae strains to oxidize 95 carbon sources were studied using the Biolog system. Two populations were constituted: one with a high risk of causing meningitis (HR group; 63 strains), and the other with a lower risk of causing meningitis (LR group; 46 strains). Strains belonging to the HR group were significantly less able to use four carbon sources, i.e., alpha-D-glucose-1-phosphate, D-ribose, beta-methyl-D-glucoside, and D,L-alpha-glycerol phosphate, than strains from the LR group (P

Legionella anisa, a possible indicator of water contamination by Legionella pneumophila.

Legionella anisa is one of the most frequent species of Legionella other than Legionella pneumophila in the environment and may be hospital acquired in rare cases. We found that L. anisa may mask water contamination by L. pneumophila, suggesting that there is a risk of L. pneumophila infection in immunocompromised patients if water is found to be contaminated with Legionella species other than L. pneumophila.

Prophagic DNA fragments in Streptococcus agalactiae strains and association with neonatal meningitis.

We identified-by randomly amplified polymorphic DNA (RAPD) analysis at the population level followed by DNA differential display, cloning, and sequencing-three prophage DNA fragments (F5, F7, and F10) in Streptococcus agalactiae that displayed significant sequence similarity to the DNA of S. agalactiae and Streptococcus pyogenes. The F5 sequence aligned with a prophagic gene encoding the large subunit of a terminase, F7 aligned with a phage-associated cell wall hydrolase and a phage-associated lysin, and F10 aligned with a transcriptional regulator (ArpU family) and a phage-associated endonuclease. We first determined the prevalence of F5, F7, and F10 by PCR in a collection of 109 strains isolated in the 1980s and divided into two populations: one with a high risk of causing meningitis (HR group) and the other with a lower risk of causing meningitis (LR group). These fragments were significantly more prevalent in the HR group than in the LR group (P < 0.001). Our findings suggest that lysogeny has increased the ability of some S. agalactiae strains to invade the neonatal brain endothelium. We then determined the prevalence of F5, F7, and F10 by PCR in a collection of 40 strains recently isolated from neonatal meningitis cases for comparison with the cerebrospinal fluid (CSF) strains isolated in the 1980s. The prevalence of the three prophage DNA fragments was similar in these two populations isolated 15 years apart. We suggest that the prophage DNA fragments identified have remained stable in many CSF S. agalactiae strains, possibly due to their importance in virulence or fitness.

Epidemiology and typing of Staphylococcus aureus strains isolated from bloodstream infections.

We carried out an epidemiological study covering 2,365,067 patient days of hospitalization between 2000 and 2003. During this time, 413 Staphylococcus aureus bloodstream infections occurred. This corresponds to 15% of the 2,676 bloodstream infections observed during this period in the 31 hospitals in our region of France, which has 2.5 million inhabitants. The incidence of nosocomial S. aureus bloodstream infections was 0.11 per 1,000 days of hospitalization. The prevalence of methicillin-resistant S. aureus (MRSA) strains, of which 13% were nonmultiresistant MRSA (NORSA), was 33%, and this percentage was stable over the 4 years. In contrast, the prevalence of S. aureus strains susceptible to methicillin but resistant to quinolones or susceptible to methicillin but multiresistant to antibiotics (EMSSA strains) increased from 4% in 2000 to 23% in 2003. As previously reported, MRSA strains were mostly recovered from nosocomial bloodstream infections, whereas NORSA strains-generally considered to be responsible for community-acquired infections-were always isolated from nosocomial bloodstream infections. Pulsed-field gel electrophoresis (PFGE) analysis of 109 MRSA strains and 15 EMSSA strains demonstrated clonal diffusion of the three major French MRSA clones and revealed considerable genetic heterogeneity among EMSSA strains. Although no epidemiologically related NORSA strains clustered in particular PFGE groups, the distribution of MRSA strains isolated from bloodstream infections according to the portal of entry (vascular devices, pulmonary, and urinary) was not random for the major PFGE clones, suggesting that each MRSA lineage displays particular virulence features.