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.

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.

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.

Updated French guidelines for diagnosis and management of pelvic inflammatory disease.

BACKGROUND: Pelvic inflammatory disease (PID) is commonly encountered in clinical practice. OBJECTIVES: To provide up-to-date guidelines on management of PID. SEARCH STRATEGY: An initial search of the Cochrane database, PubMed, and Embase was performed using keywords related to PID to identify reports in any language published between January 1990 and January 2012, with an update in May 2015. SELECTION CRITERIA: All identified reports relevant to the areas of focus were included. DATA COLLECTION AND ANALYSIS: A level of evidence based on the quality of the data available was applied for each area of focus and used for the guidelines. MAIN RESULTS: PID must be suspected when spontaneous pelvic pain is associated with induced adnexal or uterine pain (grade C). Pelvic ultrasonography is necessary to exclude tubo-ovarian abscess (grade B). Microbiological diagnosis requires vaginal and endocervical sampling for molecular and bacteriological analysis (grade B). First-line treatment for uncomplicated PID combines ofloxacin and metronidazole for 14days (grade B). Treatment of tubo-ovarian abscess is based on drainage if the collection measures more than 3cm (grade B), with combined ceftriaxone, metronidazole, and doxycycline for 14-21days. CONCLUSIONS: Current management of PID requires easily reproducible investigations and treatment, and thus can be applied worldwide.

Current Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in Elderly French People: Troublesome Clones on the Horizon.

OBJECTIVE: In 2015, we conducted at 44 healthcare facilities (HCFs) and 21 nursing homes (NHs) a 3-month bloodstream infection (BSI) survey, and a 1-day prevalence study to determine the rate of carriage of methicillin-resistant Staphylococcus aureus (MRSA) in 891 patients and 470 residents. We investigated the molecular characteristics of the BSI-associated and colonizing MRSA isolates, and assessed cross-transmission using double-locus sequence typing and pulsed-field gel electrophoresis protocol. RESULTS: The incidence of MRSA-BSI was 0.040/1000 patient-days (19 cases). The prevalence of MRSA carriage was 4.2% in patients (n = 39) and 8.7% in residents (n = 41) (p < 0.001). BSI-associated and colonizing isolates were similar: none were PVL-positive; 86.9% belonged to clonal complexes 5 and 8; 93.9% were resistant to fluoroquinolones. The qacA/B gene was carried by 15.8% of the BSI-associated isolates [3/3 BSI cases in intensive care units (ICUs)], and 7.7% of the colonizing isolates in HCFs. Probable resident-to-resident transmission was identified in four NHs. CONCLUSION: Despite generally reassuring results, we identified two key concerns. First, a worryingly high prevalence of the qacA/B gene in MRSA isolates. Antisepsis measures being crucial to prevent healthcare-associated infections, our findings raise questions about the potential risk associated with chlorhexidine use in qacA/B(+) MRSA carriers, particularly in ICUs. Second, NHs are a weak link in MRSA control. MRSA spread was not controlled at several NHs; because of their frequent contact with the community, conditions are favorable for these NHs to serve as reservoirs of USA300 clone for local HCFs.

Marked increase in incidence for bloodstream infections due to Escherichia coli, a side effect of previous antibiotic therapy in the elderly.

We conducted a survey including 3334 bloodstream infections (BSIs) due to E. coli diagnosed in 2005-2014 at a stable cohort of hospitals. Marked increases in incidence were observed for community-acquired (CA) BSIs in patients aged >75 years, CA-BSIs of digestive origin in patients aged 60-74 years, healthcare-associated BSIs, and BSIs associated with ESBL (extended-spectrum B-lactamase)-producing E. coli (ESBLEc). Using MLST, we studied the genetic diversity of 412 BSI isolates recovered during the 2014 survey: 7 major sequence type complexes (STCs) were revealed in phylogenetic group B2, 3 in group A/B1 and 2 in group D. Among the 31 ESBLEc isolates, 1/3 belonged to STC 131. We searched for possible associations between clonal groups, clinical determinants and characteristics of BSIs: isolates from groups B2 (except STC 131) and D were susceptible to antibiotics and associated with BSIs of urinary origin in patients <60 years. STC 131 and group A/B1 isolates were multi-drug resistant and associated with CA-BSIs of digestive origin in patients aged 60-74 with a recent history of antibiotic treatment. STC 131 isolates were associated with HCA-BSIs in patients with recent/present hospitalization in a long-stay unit. We provide a unique population-based picture of the epidemiology of E. coli BSI. The aging nature of the population led to an increase in the number of cases caused by the B2 and D isolates generally implicated in BSIs. In addition, the association of a trend toward increasing rates of gut colonization with multi drug-resistant isolates revealed by the rise in the incidence of BSIs of digestive origin caused by STC 131 and A/B1 (STCs 10, 23, and 155) isolates, and a significant increase in the frequency of BSIs in elderly patients with recent antibiotic treatment suggested that antibiotic use may have contributed to the growing incidence of BSI.

Emergence of a novel subpopulation of CC398 Staphylococcus aureus infecting animals is a serious hazard for humans.

Until recently, Staphylococcus aureus from clonal complex (CC)398 were mostly described as colonizing asymptomatic raised pigs and pig-farmers. Currently, the epidemiology of the CC398 lineage is becoming more complex. CC398 human-adapted isolates are increasingly being identified in bloodstream infections in humans living in animal-free environments. In addition, CC398 isolates are increasingly responsible for invasive infections in various animals. CC398 isolates that colonize asymptomatic pigs and the isolates that infect humans living in animal-free environments (human-adapted isolates) both lack several clinically important S. aureus-associated virulence factors but differ on the basis of their prophage content. Recent findings have provided insight into the influence of a φMR11-like helper prophage on the ability of CC398 isolates to infect humans. To assess the recent spread of the CC398 lineage to various animal species and to investigate the links between the φMR11-like prophage and the emergence of CC398 isolates infecting animals, we studied 277 isolates causing infections in unrelated animals. The prevalence of CC398 isolates increased significantly between 2007 and 2013 (p < 0.001); 31.8% of the animal isolates harbored the φMR11-like prophage. High-density DNA microarray experiments with 37 representative infected-animal isolates positive for φMR11-like DNA established that most infected-animal isolates carried many genetic elements related to antimicrobial resistance and virulence genes, and a φ3 prophage encoding immune-modulating proteins and associated with animal-to-human jumps. Our findings suggest recent clonal expansion and dissemination of a new subpopulation of CC398 isolates, responsible for invasive infections in various animals, with a considerable potential to colonize and infect humans, probably greater than that of human-adapted CC398 isolates, justifying active surveillance.

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.

Extended-spectrum ß-lactamase-producing Enterobacteriaceae in French nursing homes: an association between high carriage rate among residents, environmental contamination, poor conformity with good hygiene practice, and putative resident-to-resident transmission.

OBJECTIVE: We evaluated the spread of multidrug-resistant Enterobacteriaceae in 38 nursing homes (NHs) in the Centre region of France. METHODS: We conducted a multicenter prevalence study and evaluated extended-spectrum ß-lactamase- and carbapenemase-producing Enterobacteriaceae (ESBLE and CPE, respectively) colonization of 1,155 residents. The colonizing isolates were studied by randomly amplified polymorphic DNA typing. We observed hygiene practices and studied the contamination of the environment in 8 NHs. RESULTS: A total of 114 residents were ESBLE carriers (9.9%); none were CPE carriers. A total of 82.6% of the ESBLE were Escherichia coli. ESBLE colonization was associated with poor health status (P = .002), malignancy (P = .006), urinary incontinence (P = .007), fecal incontinence (P = .002), previous hospitalization (P = .033), and carbapenem treatment (P = .040). The clonal relationship between isolates within NHs suggested resident-to-resident ESBLE transmission in 15 NHs. ESBLE isolates were recovered from 6 of 232 bedrooms studied. A total of 1,533 observations revealed low rates of conformity for hand hygiene (25.7%), the use of gloves (45.9%) and protective clothing (13.3%), and waste management (46.7%). Conformity rates correlated inversely with ESBLE carriage rates. CONCLUSIONS: In most of the participating NHs, improved application of standard precautions during incontinence care is needed, and greater efforts to clean the environment of residents are required.

The transcription of the neuD gene is stronger in serotype III group B streptococci strains isolated from cerebrospinal fluid than in strains isolated from vagina.

Group B streptococci (GBS) are a major cause of neonatal meningitis, and sialic acid is a determinant of the development of meningitis. The transcription level of the neuD gene, used as a marker of neu gene expression and capsular production, was significantly higher in serotype III GBS strains isolated from meningitis than from vaginal carriage. This was irrespective both of the phylogenetic position of strains and of the presence of a thymine at position 264 in the neuD gene. Differences in neuD gene transcription may explain in part why particular isolates among the GBS strains colonizing the vagina can cause meningitis.

Whole-Genome Sequences of Staphylococcus aureus ST398 Strains of Animal Origin.

Staphylococcus aureus sequence type 398 (ST398) was originally associated with animal infections. We announce the complete genome sequences of two ST398 methicillin-susceptible S. aureus strains from the livestock environment. These genome sequences assist in the characterization of interesting ST398 features relying on host tropism and epidemiological settings.

Whole-Genome Sequences of Two Staphylococcus aureus ST398 Strains of Human Origin, S94 and S100.

Sequence type 398 (ST398) Staphylococcus aureus was originally associated with animal infection. We announce the complete genome sequences of two ST398 methicillin-susceptible S. aureus strains of human origin, S94 and S100. The genome sequences assist in the characterization of interesting ST398 features related to host specificities.

Whole-Genome Sequence of the Ancestral Animal-Borne ST398 Staphylococcus aureus Strain S123.

Sequence type 398 (ST398) Staphylococcus aureus was originally reported in livestock. We announce the complete genome sequence of an ST398 methicillin-susceptible S. aureus strain of animal origin, S123. The genome sequence reveals a wild-type genome that probably corresponds to an ancestral clone.

Analysis of prophages harbored by the human-adapted subpopulation of Staphylococcus aureus CC398.

Staphylococcus aureus clonal complex 398 is a livestock-associated pathogen that poses a worldwide threat because of its ability to colonize and infect both humans and animals. We used high-resolution whole-genome microarrays, prophage profiling, immune evasion cluster characterization and whole-genome sequencing to investigate the roles of prophages in the emerging human-adapted subpopulation of CC398 that has been associated with invasive infections in humans living in animal-free environments. We characterized one phage and two prophages specifically harbored by CC398 isolates belonging to the emerging subpopulation. We introduced the phage into permissive prophage-free isolates. We investigated the effects of lysogeny on the host ability to resist further phage infection and transformation, to acquire the capacity to invade human cells, and to express virulence factors encoded by prophages. We report evidence of a defective ϕMR11-like helper prophage, named StauST398-5pro, specifically associated with the emerging non-LA CC398 subpopulation. StauST398-5pro confers substantial protection against horizontal genetic transfer to its host. It interacts with a human-associated ß-converting prophage encoding immune-modulating proteins such that virulence genes are expressed during stress situations. Our findings provide insight into the role of phages in the expression of virulence and in the spread of genetic information among new host-adapted S. aureus isolates. We demonstrate that functional prophage elements can condition host specificity and confer new virulence traits on emerging intra-species clones of bacteria.

Difficult-to-detect carbapenem-resistant IMP13-producing P. aeruginosa: experience feedback concerning a cluster of urinary tract infections at a surgical clinic in France.

BACKGROUND: We report a carbapenem-resistant P. aeruginosa clone responsible for a cluster of urinary tract infections in elderly surgery patients, diagnosed during a three-month period in a 59-bed surgical clinic. FINDINGS: The clonal nature of the cluster was established by molecular study of the P. aeruginosa isolates (PFGE and MLST). Despite an MIC of imipenem in the susceptibility range for two isolates, all were metallo-ß-lactamase-producers (IMP13-type, clone ST621). We conducted a review of the medical and surgical procedures. We tested water delivered into the clinic and urological devices for the presence of the epidemic strain. The hygiene nurse observed hygiene practices. A week after the implementation of barrier precautions around the fourth infected patient, we studied the extent to which the patients hospitalised were colonised to assess whether the spread of the epidemic strain had been controlled. CONCLUSIONS: 1/ Our findings indicate the difficulties in the detection of the metallo-ß-lactamase in this clone, that resulted in the alert being delayed. 2/ Unlike most investigations of UTI outbreaks described in urology wards, we did not detect any contaminated urological devices or water colonisation. 3/ Consistent with outbreaks involving the IMP-13 clone in critical care units, the observation of inadequate application of standard precautions argued for patient-to-patient transmission during urinary management of the urology patients. 4/ The implementation of barrier precautions around infected patients resulted in control of the spread of the epidemic clone. This report serves as an alert concerning a difficult-to-detect multidrug-resistant P. aeruginosa clone in elderly urology patients.

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.

Biocidal activity of metalloacid-coated surfaces against multidrug-resistant microorganisms.

BACKGROUND: The antimicrobial effects of a coating of molybdenum trioxide (MoO3) has been recently described. The metalloacid material produces oxonium ions (H3O+), which creates an acidic pH that is an effective, non specific antimicrobial. We determined the in vitro antimicrobial activity of molybdenum trioxide metalloacid-coated surfaces. METHODS: Metalloacid-coated and non-coated (control) surfaces were contaminated by exposing them for 15 minutes to microbial suspensions containing 105 cfu/mL. Eleven microorganisms responsible for nosocomial infections were tested: two Staphylococcus aureus strains (the hetero-vancomycin intermediate MRSA Mu50 strain and a ST80-PVL-producing MRSA strain); a vancomycin-resistant vanA Enterococcus faecium strain; three extended-spectrum beta-lactamase-producing Enterobacteriaceae strains; a MBL-producing Pseudomonas aeruginosa strain; a multidrug-resistant Acinetobacter baumannii strain; a toxin-producing Clostridium difficile strain; and two fungi (Candida albicans and Aspergillus fumigatus). The assay tested the ability of the coated surfaces to kill microorganisms. RESULTS: Against all non-sporulating microorganisms tested, metalloacid-coated surfaces exhibited significant antimicrobial activity relative to that of the control surfaces within two to six hours after contact with the microorganisms (p < 0.001). Microorganism survival on the coated surfaces was greatly impaired, whereas microorganism survival on control surfaces remained substantial. CONCLUSIONS: We suggest that, facing the continuing shedding of microorganisms in the vicinity of colonized or infected patients, the continuous biocidal effect of hydroxonium oxides against multidrug-resistant microorganisms may help limit environmental contamination between consecutive cleaning procedures.

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.