Genomic portraits of methicillin-resistant staphylococci (MRS) from food fish unveiled the genes associated with staphylococcal food poisoning (SFP), virulence and antimicrobial resistance.

BACKGROUND: Characteristics of non-clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) especially from fishery environment are poorly understood. This research, in addition to comprehensive characterisation, sought to delineate the genetic relatedness between the MRSA strains originating from clinical as well as non-clinical settings. Out of 39 methicillin-resistant staphylococcal isolates from 197 fish samples, 6 (Three each of methicillin-resistant S. haemolyticus (MRSH) and MRSA) with distinct resistance profiles were selected for whole-genome sequencing. Using respective bioinformatics tools, MRSA genomes were comprehensively characterized for resistome, virulomes, molecular epidemiology and phylogenetic analysis. Simultaneously, MRSH genomes were specifically examined to characterize antimicrobial resistance genes (ARGs), owing to the fact that MRSH is often recognized as a reservoir for resistance determinants. RESULTS: Three MRSA clones identified in this study include ST672-IVd/t13599 (sequence type-SCCmec type/spa type), ST88-V/t2526, and ST672-IVa/t1309. Though, the isolates were phenotypically vancomycin-sensitive, five of the six genomes carried vancomycin resistance genes including the VanT (VanG cluster) or VanY (VanM cluster). Among the three MRSA, only one harbored the gene encoding Panton-Valentine Leukocidin (PVL) toxin, while staphylococcal enterotoxin (SEs) genes such as sea and seb, associated with staphylococcal food poisoning were identified in two other MRSA. Genomes of MRSH carried a composite of type V staphylococcal cassette chromosome mec (SCCmec) elements (5C2 & 5). This finding may be explained by the inversion and recombination events that may facilitate the integration of type V elements to the SCC elements of S. aureus with a methicillin-susceptible phenotype. Phylogenetically, MRSA from a non-clinical setting displayed a considerable relatedness to that from clinical settings. CONCLUSION: This study highlights the genetic diversity and resistance profiles of MRSA and MRSH, with non-clinical MRSA showing notable relatedness to clinical strains. Future research should explore resistance gene transfer mechanisms and environmental reservoirs to better manage MRSA spread.

Characterization of staphylococcal cassette chromosome mec in Staphylococcus haemolyticus ST3 and ST42 isolates from bovine mastitis milk: identification of novel variant in class C1 mec complex.

BACKGROUND: Methicillin-resistant Staphylococcus haemolyticus (MRSH) is an important pathogenic agent of bovine mastitis. Among the prominent clone lineages in dairy cows are MRSH sequence types ST3 and ST42. Little information is available on the complete characterization of SCCmec elements in MRSH. OBJECTIVE: In this study, two clinical isolates of MRSH ST3 and ST42 from bovine mastitis milk were selected, and their nontypable SCCmec structures were compared. METHODS: Two MRSH strains, MRSH-ST3 strain M62.3 and MRSH-ST42 strain M81.1, were identified from bovine mastitis milk in Thailand in 2022. Minimum inhibitory concentration was used to screen for antimicrobial resistance susceptibility. Oxford Nanopore Technologies and Illumina sequencing were performed in combination to complete the genome. Their gene organization and structure of SCCmec types were analysed and compared with the whole sequences of other strains in the same sequence types. RESULTS: Both MRSH-ST3 strain M62.3 and MRSH-ST42 strain M81.1 possessed the class C1 mec complex but lacked the ccr gene complex. Notably, MRSH-ST42 strain M81.1 contained a novel variant of C1 mec complex, which consisted of IS431-mecA-ISSha1-paaZ-upgQ-IS431, with IS431 organized in the same orientation. Apart from class C1 mec and the heavy metal-resistant cluster, the gene composition and order of the SCCmec element varied. In ST3, variations in the SCCmec type, gene content and organization were observed. CONCLUSIONS: The distinct evolution of the MRSH lineage was indicated by the various SCCmec elements. The insertion of ISSha1 resulted in a unique variant of class C1 mec complex that demonstrated the important role of the insertion sequence in SCCmec diversification.

Molecular characteristics and antibiotic resistance of Staphylococcus aureus and Staphylococcus haemolyticus isolated from bovine mastitis.

Staphylococci are classified as one of the pathogens causing bovine mastitis that can pose not only an economic loss to the dairy farms, but a serious public-health threat based on their zoonotic potential. We focused to monitor phenotypes of the isolated strains of Staphylococcus aureus (S. aureus) and Staphylococcus haemolyticus (S. haemolyticus) from milk of cows with clinical mastitis, including antibiotic resistance, biofilm forming ability and the presence of biofilm- and toxin- related genes. From a total of 191 milk samples were identified as S. aureus - 12% (22 isolates) and S. haemolyticus - 6% (12 isolates). Automatic interpreted reading of the antibiogram evaluated potentially 12 isolates as methicillin-resistant S. aureus and methicillin-resistant coagulase-negative Staphylococci. Genotypically, the isolates were positive for blaZ and negative for mecA and mecC. Others important mechanisms were inducible macrolide-lincosamide-streptogramin B (iMLSB) resistance with presence of msrA, ermC, vgaA. The most detected biofilm-associated and toxins genes were clfA, sdrD, sdrE, fnbpB, bbp, isdA, isdB, hla and see. S. aureus isolates were subjected to spa typing. It turned out that despite the strains coming from different farms, they were either resistant or sensitive to antibiotic, were all of the same spa-type t 10035. Our findings revealed the presence iMLSB, which, to our best knowledge, were described in Slovakian bovine staphylococci rarely. The majority of isolates were multidrug-resistant and carried multiple virulence genes, posing a potential public-health risk.

Emergence of multidrug-resistant Staphylococcus haemolyticus in neonatal intensive care unit in Southern France, a genomic study.

An emergence of multidrug-resistant (MDR) Staphylococcus haemolyticus has been observed in the neonatal intensive care unit (NICU) of Nîmes University Hospital in southern France. A case-control analysis was conducted on 96 neonates, to identify risk factors associated with S. haemolyticus infection, focusing on clinical outcomes. Forty-eight MDR S. haemolyticus strains, isolated from neonates between October 2019 and July 2022, were investigated using routine in vitro procedures and whole-genome sequencing. Additionally, five S. haemolyticus isolates from adult patients were sequenced to identify clusters circulating within the hospital environment. The incidence of neonatal S. haemolyticus was significantly associated with low birth weight, lower gestational age, and central catheter use (p < 0.001). Sepsis was the most frequent clinical manifestation in this series (20/46, 43.5%) and was associated with five deaths. Based on whole-genome analysis, three S. haemolyticus genotypes were predicted: ST1 (6/53, 11%), ST25 (3/53, 5.7%), and ST29 (44/53, 83%), which included the subcluster II-A, predominantly emerging in the neonatal department. All strains were profiled in silico to be resistant to methicillin, erythromycin, aminoglycosides, and fluoroquinolones, consistent with in vitro antibiotic susceptibility tests. Moreover, in silico prediction of biofilm formation and virulence-encoding genes supported the association of ST29 with severe clinical outcomes, while the persistence in the NICU could be explained by the presence of antiseptic and heavy metal resistance-encoding genes. The clonality of S. haemolyticus ST29 subcluster II-A isolates confirms healthcare transmission causing severe infections. Based on these results, reinforced hygiene measures are necessary to eradicate the nosocomial transmission of MDR strains.

Novel insights into the role of the mobilome in ecological diversification and success of Staphylococcus haemolyticus as an opportunistic pathogen.

Staphylococcus haemolyticus is a species of coagulase-negative staphylococci that has primarily been studied as a human skin microbiome member and an emerging nosocomial pathogen. Here, we present the first complete genome of S. haemolyticus strains SE3.9, SE3.8 and SE2.14 reported as an endophyte of rice seed. Detailed investigation of the genome dynamics of strains from diverse origins revealed an expanded genome size in clinical isolates, and a role of many insertion sequence (IS) elements in strain diversification. Interestingly, several of the IS elements are also unique or enriched in a particular habitat. Comparative studies also revealed the potential movement of mobile elements from rice endophytic S. haemolyticus to strains from other pathogenic species such as Staphylococcus aureus. The study highlights the importance of ecological studies in the systematic understanding of genome plasticity and management of medically important Staphylococcus species.

Identification of surface proteins in a clinical Staphylococcus haemolyticus isolate by bacterial surface shaving.

BACKGROUND: The skin commensal Staphylococcus haemolyticus is an emerging nosocomial pathogen. Despite its clinical relevance, published information about S. haemolyticus virulence factors is scarce. In this study, the adhesive and biofilm forming properties of ten clinical and ten commensal S. haemolyticus strains were examined using standard adhesion and biofilm assays. One of the clinical strains was used to identify expressed surface proteins using bacterial surface shaving. Protein abundance was examined by a comparative analysis between bacterial protein expression after human keratinocyte (HaCaT) colonization and growth in cell culture media supplemented with serum. Relative protein quantification was performed by labeling peptides with tandem mass tags (TMT) prior to Mass Spectrometry analysis. Surface proteins can be used as novel targets for antimicrobial treatment and in diagnostics. RESULTS: Adherence to fibronectin, collagen and plastic was low in all tested strains, but with significantly higher adhesion to fibronectin (p = 0.041) and collagen (p = 0.001) in the commensal strains. There was a trend towards higher degree of biofilm formation in the clinical strains (p = 0.059). By using surface shaving, 325 proteins were detected, of which 65 were classified as surface proteins. Analyses showed that the abundance of nineteen (5.8%) proteins were significantly changed following HaCaT colonization. The bacterial Toll/interleukin-1 like (TIRs) domain containing protein (p = 0.04), the transglycosylase SceD (p = 0.01), and the bifunctional autolysin Atl (p = 0.04) showed a 1.4, 1.6- and 1.5-fold increased abundance. The staphylococcal secretory antigen (SsaA) (p = 0.04) was significantly downregulated (- 1.5 fold change) following HaCaT colonization. Among the 65 surface proteins the elastin binding protein (Ebps), LPXAG and LPXSG domain containing proteins and five LPXTG domain containing proteins were identified; three Sdr-like proteins, the extracellular matrix binding protein Embp and a SasH-like protein. CONCLUSIONS: This study has provided novel knowledge about expression of S. haemolyticus surface proteins after direct contact with eukaryotic cells and in media supplemented with serum. We have identified surface proteins and immune evasive proteins previously only functionally described in other staphylococcal species. The identification of expressed proteins after host-microbe interaction offers a tool for the discovery and design of novel targets for antimicrobial treatment.

Detection of sasX Gene and Distribution of SCCmec Types in Invasive and Non-invasive Coagulase-negative Staphylococci

Background: Coagulase-negative staphylococci, which belong to the normal microbiota of the skin and mucous membranes, are opportunistic pathogens. sasX, a newly described protein, is thought to play an important role in nasal colonization and methicillin-resistant Staphylococcus aureus virulence, and it may be acquired from coagulase-negative staphylococci by horizontal gene transfer. It has been considered that understanding the function of sasX gene may help clarify the relevance of the different adhesion mechanisms in the pathogenesis of infections associated with biofilm. Aims: To investigate the sasX gene presence, staphylococcal cassette chromosome mec types, and antimicrobial resistance patterns of invasive and noninvasive coagulase-negative staphylococci isolates. Study Design: Cross-sectional study. Methods: The study included a total of 180 coagulase-negative staphylococci strains. Non-invasive isolates (n=91) were obtained from the hands of healthy volunteers who do not work at the hospital (n=30), the nasal vestibule of healthy volunteer hospital workers (n=26), and central venous catheter (n=35). Invasive isolates (n=89) were isolated from peripheral blood cultures of inpatients who do not have catheters. All isolates were identified by conventional microbiological methods, automated systems, and, if needed, with matrix-assisted laser desorption/ionization-time of flight. Staphylococcal cassette chromosome mec typing, sasX and mec gene detection, antibiotic susceptibility, and sasX gene sequence analysis were performed. Results: Peripheral blood, central venous catheter colonization, and nasal vestibule isolates were positive for the sasX gene, whereas hand isolates were negative. sasX gene was present in 17 isolates, and no statistical significance was found between invasive and noninvasive isolates (p=0.173). Sequence analysis of the sasX genes showed high homology to related proteins of Staphylococcus phage SPbeta-like and Staphylococcus epidermidis RP62A. staphylococcal cassette chromosome mec type V was the most prevalent regardless of species. staphylococcal cassette chromosome mec type II was more frequent in invasive isolates and found to be statistically important for invasive and noninvasive S. epidermidis isolates (p=0.029). Staphylococcus haemolyticus isolates had the overall highest resistance rates. Resistance to ciprofloxacin, trimethoprim-sulfamethoxazole, and erythromycin was found to be higher in isolates from catheter and blood culture. Staphylococcus hominis isolates had the highest rate for inducible clindamycin resistance. None of the isolates were resistant to vancomycin, teicoplanin, and linezolid. Conclusion: The sasX gene is detected in 9.44% of the isolates. There is no statistical difference between the sasX-positive and -negative isolates in terms of antibacterial resistance and the presence of sasX and SCCmec types. Further studies about the role of sasX at virulence in coagulase-negative staphylococci, especially from clinical samples such as tracheal aspirate and abscess isolates, and distribution of staphylococcal cassette chromosome mec types are needed.

Disrupted tongue microbiota and detection of nonindigenous bacteria on the day of allogeneic hematopoietic stem cell transplantation.

Disruption of the intestinal microbiota caused by intensive chemotherapy, irradiation and antibiotics can result in development of severe gut graft-versus-host disease and infectious complications, leading to poorer outcomes among allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. Although the oral cavity is also densely colonized by indigenous microorganisms, the bacterial composition in allo-HSCT recipients remains unclear. We determined the tongue microbiota composition of 45 patients with hematological disorders on the day of transplantation and compared them to 164 community-dwelling adults. The V1-V2 regions of the 16S rRNA gene sequences demonstrated that the allo-HSCT recipients had less diverse and distinct microbiota from that of community-dwelling adults. The full-length 16S rRNA gene sequences identified 146 bacterial taxa in the microbiota of allo-HSCT recipients, of which 34 bacterial taxa did not correspond to bacteria primarily inhabiting the oral cavity deposited in the expanded Human Oral Microbiome Database. Notably, the detection of Staphylococcus haemolyticus and/or Ralstonia pickettii was significantly associated with a higher risk of mortality during the follow-up period. These results demonstrate that the oral cavity of allo-HSCT recipients is colonized by a disrupted microbiota on the day of transplantation and suggest that detection of specific nonindigenous taxa could be a predictor of transplant outcome.

Linezolid Resistance in Staphylococcus haemolyticus - Case Series and Review of Literature.

BACKGROUND: Coagulase negative Staphylococci (CoNS) are important. The common antibiotics used for the treatment of the infections caused by CoNS are penicillin, oxacillin, ciprofloxacin, clindamycin, erythromycin, gentamicin, and vancomycin. Linezolid is an oxazolidinone group of antibiotic with activity against Gram-positive bacteria. It is used for the treatment of serious infections caused by Gram-positive bacteria resistant to other antibiotics, including streptococci, vancomycin-resistant enterococci (VRE). AIMS AND OBJECTIVE: This study emphasizes on the judicious use of newer antibiotics to contain the spread of resistance. METHOD: We are discussing five cases of Linezolid resistant Staphylococcus Haemolyticus which were reported in our laboratory during one year from patients with device related infections and also review of literature is being presented for an update. RESULT: In our study, the isolates were resistant to other groups of antimicrobials but susceptible to glycopeptides. All the isolates were methicillin-resistant. CONCLUSION: Linezolid is approved as an alternative drug to be given for catheter-related bloodstream infections. In earlier studies, linezolid-resistant staphylococci have been reported increasingly all over the world. This study is to create awareness amongst clinicians that improper and excessive use of linezolid will make this antibiotic-resistant and thus will be of no help in future, so judicious and relevant use of antibiotics needs to be emphasized.

Clinical efficacy of teicoplanin in the treatment of bloodstream infection caused by methicillin-resistant coagulase-negative staphylococci.

Empirical combination therapy with ß-lactams and glycopeptides is recommended for patients with presumed staphylococcal bloodstream infection (BSI). While coagulase-negative staphylococci (CNS) remain susceptible to vancomycin, such isolates have become less susceptible to teicoplanin. The aim of this retrospective study was to evaluate the clinical efficacy of teicoplanin in the treatment of BSI caused by methicillin-resistant CNS according to teicoplanin susceptibility. Inclusion criteria were patients with intravascular-catheter related BSIs caused by methicillin-resistant CNS (positive for two or more specimens); teicoplanin therapy; and at least one of the signs or symptoms caused by BSI. Antimicrobial resistance was defined as minimum inhibitory concentration (MIC) ≥8 µg/mL. The primary efficacy endpoint was clinical success evaluated 2 weeks after the completion of teicoplanin therapy [test of cure (TOC)]. Resistant rate of CNS was 0% for vancomycin and 22.9% for teicoplanin, and geometric mean MICs were 1.31 µg/mL and 3.41 µg/mL, respectively (p < 0.001). The catheter was removed in all patients except one, and high early clinical response at 72 h after starting therapy was obtained irrespective of teicoplanin susceptibility. The clinical success rate at TOC was 60% in patients with BSIs caused by teicoplanin-resistant strains, while 90% in patients with BSIs caused by susceptible strains (p = 0.052). In multivariate analyses, teicoplanin resistance was significant factor for decreased clinical success at TOC (adjusted odds ratio 0.138, 95% confidence interval 0.020-0.961, p = 0.045). Because of the poor clinical efficacy of teicoplanin against teicoplanin-resistant CNS, combination therapy comprising vancomycin and ß-lactam antibiotics should be considered in presumed staphylococci BSI.

Antimicrobial Resistance Profiles and Molecular Characteristics of Coagulase-Negative Staphylococci Isolated from Two Tertiary Hospitals Before and 15 Years After Implementation of the Separation of Drug Prescribing and Dispensing Policy of Korea.

This study compared changes in antimicrobial susceptibilities and molecular characteristics of coagulase-negative staphylococci (CNS) between the year 2000 and the year 2014-2015 to evaluate the policy of separating drug prescribing and dispensing in Korea. We obtained 68 CNS clinical isolates from two tertiary general hospitals before (the year 2000; n = 25) and after (the year 2014 - 2015; n = 43) implementation of the separation. Isolates were identified as Staphylococcus capitis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, and Staphylococcus warneri. When minimal inhibitory concentrations of 14 antimicrobials were applied to isolates, resistance rates to gentamicin and oxacillin in 2000 were significantly higher than in 2014-2015 (p < 0.05). Fifty-seven isolates were methicillin-resistant CNS (MR-CNS), 42 of which were also multidrug resistant; overall, multidrug resistance decreased from 72% in the year 2000 to 55.8% in 2014-2015. Staphylococcal cassette chromosome mec (SCCmec) type III was the dominant type of MR-CNS in the year 2000, while SCCmec type IV was the dominant type in 2014-2015. Twenty-five sequence types (STs) were identified; ST2 appeared most frequently in both periods. After 15 years of implementation of this policy, multidrug resistance as well as methicillin and gentamicin resistance in CNS decreased, but not resistance to other antibiotics. Long-term surveillance at both genotypic and phenotypic levels of various species is necessary for further evaluation of this policy.

Non-aureus staphylococci in fecal samples of dairy cows: First report and phenotypic and genotypic characterization.

The aims of this study were to determine whether non-aureus staphylococci (NAS) are present in rectal feces of healthy dairy cows, and if so, to delineate species to which they belong and to study several phenotypic and genotypic traits as a first step toward determining the potential impact of fecal shedding of NAS on bovine udder health. Fecal samples were aseptically collected from the rectum of 25 randomly selected clinically healthy dairy cows in a commercial dairy herd using an automated milking system. Fecal NAS were isolated and then identified at the species level using transfer RNA-intergenic spacer PCR and sequencing of the 16S rRNA housekeeping gene. Strain typing was performed using random amplification of polymorphic DNA (RAPD)-PCR. The antimicrobial resistance profiles, biofilm formation, and growth and inhibitory characteristics of all NAS isolates were evaluated. Half of the cows were shedding NAS, resulting in 31 NAS isolates belonging to 11 different species. The most prevalent species were Staphylococcus rostri (23%, n = 7), Staphylococcus cohnii (16%, n = 5), and Staphylococcus haemolyticus (13%, n = 4) with all Staphylococcus agnetis, Staphylococcus chromogenes, and Staph. rostri isolates belonging to the same strain according to RAPD banding patterns. Acquired antimicrobial resistance was observed in 28 of the 31 NAS isolates, mainly due to ß-lactamase production. Most of the isolates (84%, n = 27) had a weak biofilm-forming potential, but only 2 contained the bap gene. The ica and aap genes were not detected in any of the isolates. In vitro growth of Staphylococcus aureus and Streptococcus dysgalactiae was inhibited by Staph. agnetis isolates, and Staph. chromogenes isolates were able to inhibit the growth of Strep. dysgalactiae and Streptococcus uberis. All fecal isolates were able to grow when oxygen and iron were limitedly available, mimicking the growth conditions in the mammary gland.

Influence of antibiotics on biofilm formation by different clones of nosocomial Staphylococcus haemolyticus.

Staphylococcus haemolyticus is the most common organism among clinical isolatesof methicillin-resistant staphylococci. Aim: This study evaluated the ability to produce biofilm with the presence of the antibiotics (1/4 minimum inhibitory concentrations) of S. haemolyticus strains isolated from blood culture. Methods: Clonal distribution was assessed in pulsed-field gel electrophoresis. PCR assays were performed to detect mecA, icaA, aap, atlE, atl, fbp genes. S. haemolyticus strains grown in the presence of the antibiotics were investigated for biofilm formation on glass, polystyrene and catheter surfaces. Results: Biofilm formation was independent of the presence of the icaA and mecA genes, pulsed-field gel electrophoresis type. Vancomycin, oxacillin, moxifloxacin, rifampicin, teicoplanin, tigecycline and linezolid did not inhibit biofilm formation on abiotic surfaces. Conclusion: This study demonstrated that the biofilm formation process is complex and may not be related to ica gene carriage. Furthermore, in this study the biofilm formation was increased in the presence of antimicrobial agents.

Molecular characterization of multi-drug resistant coagulase negative cocci in non-hospital environment.

Antibiotic resistance crisis occasioned by sporadic appearance of multi-drug resistance (MDR) in human pathogens to clinically applied antimicrobials is a serious threat to global health. In this study, we investigated the drug resistant phenotype of Gram-positive cocci isolates from environment. Staphylococcus capitis and Staphylococcus haemolyticus colonies were isolated on mannitol-salt agar plates supplemented with tetracycline. Antibiotic susceptibility profile of the isolates via minimum inhibitory concentration (MIC) determination was examined. Isolates showed decreased sensitivity to clinically applied antimicrobial agents: tetracycline, kanamycin, erythromycin, norfloxacin, teicoplanin, and ampicillin. Genomic analysis demonstrated the presence of multiple antibiotic resistant genes in these bacteria, suggesting the origin of the multiple antimicrobials resistant phenotype. Tetracycline resistance of these isolates was transduced to Staphylococcus aureus-RN4220 strain. These findings indicate the presence of multiple antimicrobials resistant S. capitis and S. haemolyticus strain in a non-hospital setting. Moreover, the presence of plethora of genes responsible for MDR suggest that these strains could present potential threat to human health by serving as reservoir for lateral transference of antimicrobial resistance conferring foreign genetic elements to other clinically relevant pathogens.

Catheter related recurrent blood stream infection caused by linezolid-resistant, methicillin resistant Staphylococcus haemolyticus; an emerging super bug.

A 61 year male, admitted in Combined Military Hospital Rawlpindi on 12th March 2017, operated for diverticulitis became colonized with Staphylococcus haemolyticus. Patient suffered repeated septic episodes caused by the same organism during his stay in hospital. The strain was identified as methicillin resistant Staphylococcus haemolyticus (MRSH) also resistant to Linezolid by analytical profile index for Staphylococcus (API Staph) and VITEK 2 Gram positive cocci panel. The isolate was cultured from blood cultures, Central Venous Catheter (CVC) tip and skin swabs. Patient was successfully treated with injectable vancomycin and skin decolonization was acheived with chlorhexidine bath after which no episode of MRSH infection occurred. Patient had an uneventful recovery and was discharged on 21st June. His follow up visit showed clinical improvement.

Comparative exoproteome profiling of an invasive and a commensal Staphylococcus haemolyticus isolate.

Staphylococcus haemolyticus is a skin commensal emerging as an opportunistic pathogen. Nosocomial isolates of S. haemolyticus are the most antibiotic resistant members of the coagulase negative staphylococci (CoNS), but information about other S. haemolyticus virulence factors is scarce. Bacterial membrane vesicles (MVs) are one mediator of virulence by enabling secretion and long distance delivery of bacterial effector molecules while protecting the cargo from proteolytic degradation from the environment. We wanted to determine if the MV protein cargo of S. haemolyticus is strain specific and enriched in certain MV associated proteins compared to the totalsecretome. The present study shows that both clinical and commensal S. haemolyticus isolates produce membrane vesicles. The MV cargo of both strains was enriched in proteins involved in adhesion and acquisition of iron. The MV cargo of the clinical strain was further enriched in antimicrobial resistance proteins. Data are available via ProteomeXchange with identifier PXD010389. BIOLOGICAL SIGNIFICANCE: Clinical isolates of Staphylococcus haemolyticus are usually multidrug resistant, their main virulence factor is formation of biofilms, both factors leading to infections that are difficult to treat. We show that both clinical and commensal S. haemolyticus isolates produce membrane vesicles. Identification of staphylococcal membrane vesicles can potentially be used in novel approaches to combat staphylococcal infections, such as development of vaccines.

In vitro activity of tedizolid against clinical isolates of Staphylococcus lugdunensis and Staphylococcus haemolyticus from Europe and the United States.

Staphylococcus lugdunensis and Staphylococcus haemolyticus are unique among CoNS in that the former often causes aggressive disease, while the latter consistently exhibits high rates of multidrug resistance. We evaluated the in vitro susceptibility of contemporary (2012-2013) isolates from both pathogens to tedizolid and comparators, using standard methodology. Results were interpreted using CLSI and EUCAST breakpoints. Overall, 106 S. lugdunensis and 103 S. haemolyticus isolates were collected from 51 medical centers in the United States and 30 centers in 18 European countries. Tedizolid showed good activity against S. lugdunensis (MIC50/MIC90: 0.12/0.12 mg/L) and S. haemolyticus (MIC50/MIC90: 0.12/0.12 mg/L), inhibiting all isolates at MIC ≤0.25 mg/L. Based on the EUCAST breakpoint for staphylococci and when substituting the CLSI breakpoint for Staphylococcus aureus, all isolates were tedizolid susceptible. All isolates were also susceptible to linezolid, but the in vitro potency of tedizolid was 4-fold greater than that of linezolid against both S. lugdunensis and S. haemolyticus, based on MIC90 values. S. lugdunensis exhibited ≥99% susceptibility to vancomycin, teicoplanin, gentamicin, levofloxacin, and trimethoprim-sulfamethoxazole; 7% of isolates were resistant to tetracycline, 11% to clindamycin, and 2% were methicillin-resistant. S. haemolyticus exhibited high rates of resistance to commonly used anti-staphylococcal agents: 71% of isolates were resistant to methicillin, 36%-37% to clindamycin, and 30%-50% to gentamicin. These in vitro findings suggest that tedizolid could be an alternative treatment option for infections due to these medically important CoNS pathogens. Additional clinical evaluation and continued surveillance of tedizolid in vitro activity against S. lugdunensis and S. haemolyticus are warranted.

Alarming and Threatening Signals from Health Centers About Multi Drug Resistance Staphylococcus haemolyticus.

OBJECTIVES: Nowadays, due to the biofilm formation among coagulase-negative staphylococci (CoNS), acquisition of antibiotic resistance and virulence genes could be noted. These organisms resemble other staphylococcus that harbor mecA gene, which encode methicillin resistance but the diversity in CoNS is higher than other species. Based on increasing antibiotic resistance in Staphylococcus haemolyticus, analyzing of phenotypic and genotypic biofilm formation, antibiotic resistance and genes involved in this process, SCCmec and ACME typing were the aims of the current study. METHODS: 256 clinical CoNS isolates were collected that 49 isolates were identified as S. haemolyticus. For evaluation the antibiotic resistance patterns, disk agar diffusion method was applied, and also biofilm assay carried out among methicillin resistant S. haemolyticus and SCCmec and ACME typing. RESULTS: The results of antibiotic susceptibility indicated that the highest resistance was found for cotrimoxazole and erythromycin (86.6%) and the frequency of blaA (96.5%) and msrA (79.3%) genes was much higher than others. Among MRSH isolates 58.6 % showed a weak biofilm phenotype and 41.4% demonstrated a moderate biofilm density. Also, among the biofilm correlated genes, IS 256 (79.31%) was the most frequent. The SCCmec typing of MRSH isolates indicated that the type V was dominant. CONCLUSION: Our findings indicated that the correlation between high rates of existence Is 256 gene and high prevalence of weak biofilm phenotype was among MRSH isolates. The current study revealed that multiple antibiotic resistance existed in S. haemolyticus isolates that is a warning for public health.