Evaluation of 16S rRNA gene PCR sensitivity and specificity for diagnosis of prosthetic joint infection: a prospective multicenter cross-sectional study.

There is no standard method for the diagnosis of prosthetic joint infection (PJI). The contribution of 16S rRNA gene PCR sequencing on a routine basis remains to be defined. We performed a prospective multicenter study to assess the contributions of 16S rRNA gene assays in PJI diagnosis. Over a 2-year period, all patients suspected to have PJIs and a few uninfected patients undergoing primary arthroplasty (control group) were included. Five perioperative samples per patient were collected for culture and 16S rRNA gene PCR sequencing and one for histological examination. Three multicenter quality control assays were performed with both DNA extracts and crushed samples. The diagnosis of PJI was based on clinical, bacteriological, and histological criteria, according to Infectious Diseases Society of America guidelines. A molecular diagnosis was modeled on the bacteriological criterion (≥ 1 positive sample for strict pathogens and ≥ 2 for commensal skin flora). Molecular data were analyzed according to the diagnosis of PJI. Between December 2010 and March 2012, 264 suspected cases of PJI and 35 control cases were included. PJI was confirmed in 215/264 suspected cases, 192 (89%) with a bacteriological criterion. The PJIs were monomicrobial (163 cases [85%]; staphylococci, n = 108; streptococci, n = 22; Gram-negative bacilli, n = 16; anaerobes, n = 13; others, n = 4) or polymicrobial (29 cases [15%]). The molecular diagnosis was positive in 151/215 confirmed cases of PJI (143 cases with bacteriological PJI documentation and 8 treated cases without bacteriological documentation) and in 2/49 cases without confirmed PJI (sensitivity, 73.3%; specificity, 95.5%). The 16S rRNA gene PCR assay showed a lack of sensitivity in the diagnosis of PJI on a multicenter routine basis.

CC9 livestock-associated Staphylococcus aureus emerges in bloodstream infections in French patients unconnected with animal farming.

We report 4 bloodstream infections associated with CC9 agr type II Staphylococcus aureus in individuals without animal exposure. We demonstrate, by microarray analysis, the presence of egc cluster, fnbA, cap operon, lukS, set2, set12, splE, splD, sak, epiD, and can, genomic features associated with a high virulence potential in humans.

Variability of neuD transcription levels and capsular sialic acid expression among serotype III group B Streptococcus strains.

Serotype III group B Streptococcus (GBS) is the major cause of neonatal meningitis, but the risk of infection in the colonized neonates is variable. Capsular sialic acid (Sia), whose synthesis is encoded by neu genes, appears to be a major virulence factor in several bacterial species able to reach the cerebrospinal fluid. Therefore, variations of Sia expression related to the genetic diversity of strains may have an impact on the risk of meningitis in colonized neonates. We characterized by MLST the phylogenetic diversity of 64 serotype III GBS strains isolated from vaginal flora and randomly selected. These strains mostly belonged to three major sequence types (STs): ST1 (11%), ST17 (39%) and ST19 (31%). The genetic diversity of strains of these lineages, characterized by PFGE, allowed the selection of 17 representative strains, three ST1, six ST17 and eight ST19, with NEM316 as reference, in order to evaluate (i) by quantitative RT-PCR, the level of transcription of the neuD gene as a marker for the transcription of neu genes and (ii) by enzymological analysis, the expression of Sia. The mean transcription level of neuD was higher for ST17 strains than for ST1 and ST19 strains in the early, mid- and late exponential growth phases, and was maximum in the early exponential growth phase for ST17 strains and in the mid-exponential growth phase for ST1 and ST19 strains. Mean Sia concentration was higher for ST17 than for ST1 and ST9 strains in all three growth phases. For the total population, Sia concentration varied notably in the stationary phase, from 0.38 to 9.30 nmol per 10(8) viable bacteria, with a median value of 2.99 nmol per 10(8) bacteria. All ST17 strains, only one-third of the ST19 strains and none of the ST1 strains had Sia concentrations higher than the median Sia concentration. Therefore, differences in the level of expression of Sia by strains of the major serotype III GBS phylogenetic lineages might be one of the factors that explain the leading role of ST17 strains in neonatal meningitis.

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.

12/111phiA Prophage Domestication Is Associated with Autoaggregation and Increased Ability to Produce Biofilm in Streptococcus agalactiae.

CC17 Streptococcus agalactiae carrying group-A prophages is increasingly responsible for neonatal infections. To investigate the impact of the genetic features of a group-A prophage, we first conducted an in silico analysis of the genome of 12/111phiA, a group-A prophage carried by a strain responsible for a bloodstream infection in a parturient. This revealed a Restriction Modification system, suggesting a prophage maintenance strategy and five ORFs of interest for the host and encoding a type II toxin antitoxin system RelB/YafQ, an endonuclease, an S-adenosylmethionine synthetase MetK, and an StrP-like adhesin. Using the WT strain cured from 12/111phiA and constructing deleted mutants for the ORFs of interest, and their complemented mutants, we demonstrated an impact of prophage features on growth characteristics, cell morphology and biofilm formation. Our findings argue in favor of 12/111phiA domestication by the host and a role of prophage features in cell autoaggregation, glycocalyx and biofilm formation. We suggest that lysogeny may promote GBS adaptation to the acid environment of the vagina, consequently colonizing and infecting neonates.

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.

Infected breast milk associated with late-onset and recurrent group B streptococcal infection in neonatal twins: a genetic analysis.

Asymptomatic excretion of group B streptococcus (GBS) in breast milk may be an underrecognized cause of neonatal and recurrent infection. We report the case of late-onset and recurrent infection in newborn twins resulting from ingestion of maternal breast milk infected with GBS. Genetic analysis of isolates is equally presented.

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.

phiD12-Like Livestock-Associated Prophages Are Associated With Novel Subpopulations of Streptococcus agalactiae Infecting Neonates.

Group B Streptococcus (GBS) is a major cause of invasive disease in neonates worldwide. Monitoring data have revealed a continuing trend toward an increase in neonatal GBS infections, despite the introduction of preventive measures. We investigated this trend, by performing the first ever characterization of the prophage content for 106 GBS strains causing neonatal infections between 2002 and 2018. We determined whether the genome of each strain harbored prophages, and identified the insertion site of each of the prophages identified. We found that 71.7% of the strains carried at least one prophage, and that prophages genetically similar to livestock-associated phiD12, carrying genes potentially involved in GBS pathogenesis (e.g., genes encoding putative virulence factors and factors involved in biofilm formation, bacterial persistence, or adaptation to challenging environments) predominated. The phiD12-like prophages were (1) associated with CC17 and 1 strains (p = 0.002), (2) more frequent among strains recovered during the 2011-2018 period than among those from 2002-2010 (p < 0.001), and (3) located at two major insertion sites close to bacterial genes involved in host adaptation and colonization. Our data provide evidence for a recent increase in lysogeny in GBS, characterized by the acquisition, within the genome, of genetic features typical of animal-associated mobile genetic elements by GBS strains causing neonatal infection. We suggest that lysogeny and phiD12-like prophage genetic elements may have conferred an advantage on GBS strains for adaptation to or colonization of the maternal vaginal tract, or for pathogenicity, and that these factors are currently playing a key role in the increasing ability of GBS strains to infect neonates.

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.

Effect of hydroxyapatite coating and polymethylmethacrylate on stainless steel implant-site infection with Staphylococcus epidermidis in a sheep model.

We aimed to study the influence of hydroxyapatite (HA) coating and polymethylmethacrylate (PMMA) cement on the risk of development of stainless steel implant-site infection with Staphylococcus epidermidis in a sheep model. Uncoated, HA-coated, and PMMA-cemented stainless steel implants were inserted in the left femur of 30 sheep. For each type of implant, sheep were inoculated with S. epidermidis in the intramedullary canal and one non-inoculated group was used as control. After 6 weeks, infection was evaluated using clinical, radiological, bacteriological, and histological criteria. Radiological and clinical results were normal. Cultures were negative in the control sheep. In the inoculated sheep, interposition tissue and bone cultures were positive in 2 of 6 uncoated, 6 of 6 HA, and 6 of 6 PMMA implants with a mean bacteria count of 5.2 +/- 1.17, 3.5 +/- 0.7, and 3.9 +/- 0.9 log10 cfu/g, respectively (NS), for interposition tissue, and 4 +/- 0.01, 2.9 +/- 0.6, and 2.5 +/- 1.3 log10 cfu/g, respectively (NS) for bone. The polymorphonuclear leukocyte (PMN) score (mean number of PMN per 10 different microscopic high-power fields >or=5) in interposition tissue was >or=3 in 6 of 6 HA, significantly different from uncoated (3 of 6) and PMMA (2 of 6) groups (p = 0.04). The HA and PMMA inoculated groups had a higher infection rate than the uncoated inoculated group (p = 0.06). In this experimental sheep model of S. epidermidis infection at the bone-biomaterial interface, HA seems to be at higher risk of infection compared with uncoated or PMMA-cemented stainless steel, when inoculation is intramedullary and contemporary with implantation.

Multiresistant Enterobacter cloacae outbreak in an intensive care unit associated with therapeutic beds.

We report a multiresistant Enterobacter cloacae outbreak in an intensive care unit, associated with mattresses and with antibacterial-treated and vapour-permeable polyurethane synthetic mattress covers of therapeutic beds.

Regulatory RNAs in the Less Studied Streptococcal Species: From Nomenclature to Identification.

Streptococcal species are Gram-positive bacteria involved in severe and invasive diseases in humans and animals. Although, this group includes different pathogenic species involved in life-threatening infections for humans, it also includes beneficial species, such as Streptococcus thermophilus, which is used in yogurt production. In bacteria virulence factors are controlled by various regulatory networks including regulatory RNAs. For clearness and to develop logical thinking, we start this review with a revision of regulatory RNAs nomenclature. Previous reviews are mostly dealing with Streptococcus pyogenes and Streptococcus pneumoniae regulatory RNAs. We especially focused our analysis on regulatory RNAs in Streptococcus agalactiae, Streptococcus mutans, Streptococcus thermophilus and other less studied Streptococcus species. Although, S. agalactiae RNome remains largely unknown, sRNAs (small RNAs) are supposed to mediate regulation during environmental adaptation and host infection. In the case of S. mutans, sRNAs are suggested to be involved in competence regulation, carbohydrate metabolism, and Toxin-Antitoxin systems. A new category of miRNA-size small RNAs (msRNAs) was also identified for the first time in this species. The analysis of S. thermophilus sRNome shows that many sRNAs are associated to the bacterial immune system known as CRISPR-Cas system. Only few of the other different Streptococcus species have been the subject of studies pointed toward the characterization of regulatory RNAs. Finally, understanding bacterial sRNome can constitute one step forward to the elaboration of new strategies in therapy such as substitution of antibiotics in the management of S. agalactiae neonatal infections, prevention of S. mutans dental caries or use of S. thermophilus CRISPR-Cas system in genome editing applications.