Characterization, Antioxidant and Antibacterial Potentials of Tamarindus indica L. Fruit Pulp Extract Loaded O/W Nanoemulsions

Abstract The main purposes of the current study were to formulate o/w nanoemulsions as a carrier for Tamarindus indica (tamarind) fruit pulp extract and to study the antioxidant and antibacterial potentials of nanoemulsions containing tamarind extract, focusing on cosmetic/hygiene applications. The o/w nanoemulsions using a mixture of Tween 80 and Span 80 as an emulsifier (5%w/w) were prepared by a high pressure homogenization process. Two concentrations of sweet tamarind extract, 3.3 and 6.6%w/w, based on the bioactivity study, were incorporated into the blank nanoemulsions to produce loaded nanoemulsions, F1-3.3TE (3.3%) and F1- 6.6TE (6.6%). As compared with the unloaded nanoemulsion, both tamarind extract loaded nanoemulsions showed reduced pH and significantly increased viscosity. Overall, the loaded nanoemulsions had droplet sizes of approximately 130 nm, zeta potential around -38 mV and polydispersity index (PDI) values less than 0.2. The nanoemulsion F1-3.3TE had better stability (e.g. significantly greater % tartaric acid content and lesser PDI value) than the nanoemulsion F1-6.6TE did. The antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl assay revealed that the nanoemulsions F1-3.3TE and F1-6.6TE had scavenging activities of 81.66 ± 0.77% and 63.80 ± 0.79%, respectively. However, antioxidant activity of these two formulations decreased under stress conditions (heating-cooling cycles). Such incidence did not occur for their antibacterial properties investigated by agar well diffusion technique. The two formulations exhibited inhibition zones of approximately 24.0-27.7 mm against Staphylococcus aureus and Staphylococcus epidermidis, responsible for malodor of underarms. The results suggest the potential of using sweet tamarind pulp extract loaded nanoemulsions as hygiene products.

Molecular epidemiology of methicillin resistant Staphylococcus species in healthcare workers of a blood bank in the Brazilian Amazon.

BACKGROUND: Healthcare workers are susceptible to colonization by multiresistant bacteria, which can increase the risk of outbreaks. METHODS: Samples were collected from the nasopharynx, hands, and lab coats of healthcare workers. The phenotypic identification was carried out using a VITEK®2 rapid test system. PCR tests for the mecA gene and the sequencing of the amplicons were performed. Staphylococcus epidermidis and Staphylococcus aureus phylogenies were reconstructed using the Bayesian inference. RESULTS: A total of 225 healthcare workers participated in this study. Of these, 21.3% were male and 78.7% female. S. epidermidis and S.aureus showed high levels of resistance to penicillin, ampicillin, erythromycin, tetracycline and cefoxitin. The prevalence of methicillin resistant S. aureus was 3.16% and methicillin resistant S. epidermidis was 100%. Multilocus sequence typing identified 23 new S. epidermidis sequence types, and one new allele and sequence type for S. aureus. The frequency of methicillin-resistant S. epidermidis in nursing and hemotherapy technicians as a percentage of the total number of healthcare workers was 5.8-3.1%, while the frequency of methicillin resistant S. aureus in hemotherapy technicians and biomedics, as a percentage of the total number of healthcare workers was 4.2-8.9%%. CONCLUSIONS: The healthcare workers at the city's blood bank, even when taking the necessary care with their hands, body and clothes, harbour methicillin-resistant S. aureus and S. epidermidis sequence types, which, as a potential source of multidrug resistant bacteria, can contribute to nosocomial infections among hematological patients.

Distinct clonal lineages and within-host diversification shape invasive Staphylococcus epidermidis populations.

S. epidermidis is a substantial component of the human skin microbiota, but also one of the major causes of nosocomial infection in the context of implanted medical devices. We here aimed to advance the understanding of S. epidermidis genotypes and phenotypes conducive to infection establishment. Furthermore, we investigate the adaptation of individual clonal lines to the infection lifestyle based on the detailed analysis of individual S. epidermidis populations of 23 patients suffering from prosthetic joint infection. Analysis of invasive and colonizing S. epidermidis provided evidence that invasive S. epidermidis are characterized by infection-supporting phenotypes (e.g. increased biofilm formation, growth in nutrient poor media and antibiotic resistance), as well as specific genetic traits. The discriminating gene loci were almost exclusively assigned to the mobilome. Here, in addition to IS256 and SCCmec, chromosomally integrated phages was identified for the first time. These phenotypic and genotypic features were more likely present in isolates belonging to sequence type (ST) 2. By comparing seven patient-matched nasal and invasive S. epidermidis isolates belonging to identical genetic lineages, infection-associated phenotypic and genotypic changes were documented. Besides increased biofilm production, the invasive isolates were characterized by better growth in nutrient-poor media and reduced hemolysis. By examining several colonies grown in parallel from each infection, evidence for genetic within-host population heterogeneity was obtained. Importantly, subpopulations carrying IS insertions in agrC, mutations in the acetate kinase (AckA) and deletions in the SCCmec element emerged in several infections. In summary, these results shed light on the multifactorial processes of infection adaptation and demonstrate how S. epidermidis is able to flexibly repurpose and edit factors important for colonization to facilitate survival in hostile infection environments.

Comparative genomics of Staphylococcus epidermidis from prosthetic-joint infections and nares highlights genetic traits associated with antimicrobial resistance, not virulence.

There is increased awareness of the worldwide spread of specific epidemic multidrug-resistant (MDR) lineages of the human commensal Staphylococcus epidermidis. Here, using bioinformatic analyses accounting for population structure, we determined genomic traits (genes, SNPs and k-mers) that distinguish S. epidermidis causing prosthetic-joint infections (PJIs) from commensal isolates from nares, by analysing whole-genome sequencing data from S. epidermidis from PJIs prospectively collected over 10 years in Sweden, and contemporary S. epidermidis from the nares of patients scheduled for arthroplasty surgery. Previously suggested virulence determinants and the presence of genes and mutations linked to antimicrobial resistance (AMR) were also investigated. Publicly available S. epidermidis sequences were used for international extrapolation and validation of findings. Our data show that S. epidermidis causing PJIs differed from nasal isolates not by virulence but by traits associated with resistance to compounds used in prevention of PJIs: ß-lactams, aminoglycosides and chlorhexidine. Almost a quarter of the PJI isolates did not belong to any of the previously described major nosocomial lineages, but the AMR-related traits were also over-represented in these isolates, as well as in international S. epidermidis isolates originating from PJIs. Genes previously associated with virulence in S. epidermidis were over-represented in individual lineages, but failed to reach statistical significance when adjusted for population structure. Our findings suggest that the current strategies for prevention of PJIs select for nosocomial MDR S. epidermidis lineages that have arisen from horizontal gene transfer of AMR-related traits into multiple genetic backgrounds.

The Epidome - a species-specific approach to assess the population structure and heterogeneity of Staphylococcus epidermidis colonization and infection.

BACKGROUND: Although generally known as a human commensal, Staphylococcus epidermidis is also an opportunistic pathogen that can cause nosocomial infections related to foreign body materials and immunocompromized patients. Infections are often caused by multidrug-resistant (MDR) lineages that are difficult and costly to treat, and can have a major adverse impact on patients' quality of life. Heterogeneity is a common phenomenon in both carriage and infection, but present methodology for detection of this is laborious or expensive. In this study, we present a culture-independent method, labelled Epidome, based on an amplicon sequencing-approach to deliver information beyond species level on primary samples and to elucidate clonality, population structure and temporal stability or niche selection of S. epidermidis communities. RESULTS: Based on an assessment of > 800 genes from the S. epidermidis core genome, we identified genes with variable regions, which in combination facilitated the differentiation of phylogenetic clusters observed in silico, and allowed classification down to lineage level. A duplex PCR, combined with an amplicon sequencing protocol, and a downstream analysis pipeline were designed to provide subspecies information from primary samples. Additionally, a probe-based qPCR was designed to provide valuable absolute abundance quantification of S. epidermidis. The approach was validated on isolates representing skin commensals and on genomic mock communities with a sensitivity of < 10 copies/µL. The method was furthermore applied to a sample set of primary skin and nasal samples, revealing a high degree of heterogeneity in the S. epidermidis populations. Additionally, the qPCR showed a high degree of variation in absolute abundance of S. epidermidis. CONCLUSIONS: The Epidome method is designed for use on primary samples to obtain important information on S. epidermidis abundance and diversity beyond species-level to answer questions regarding the emergence and dissemination of nosocomial lineages, investigating clonality of S. epidermidis communities, population dynamics, and niche selection. Our targeted-sequencing method allows rapid differentiation and identification of clinically important nosocomial lineages in low-biomass samples such as skin samples.

Genotypic and phenotypic analysis of biofilm formation Staphylococcus epidermidis isolates from clinical specimens.

OBJECTIVES: Staphylococcus epidermidis is the primary causative agent of infections associated with indwelling biomaterials. Antibiotic susceptibility patterns, Biofilm formation capability, and screening of responsible genes in biofilm formation procedure in clinical isolates (icaA, icaB, icaC, icaD, sdrG, and atlE) were assigned as the main objectives in this study. The clinical samples were analyzed via standard biochemical assays for identifying different bacteria which were confirmed using the multiplex colony PCR method. Subsequently, biofilm-formation capability, antibiotic susceptibility testing, and the frequency of genes responsible for biofilm formation in the confirmed strains were checked. RESULTS: Out of 183 clinical specimens 54 S. epidermidis isolates were detected by targeting a housekeeping gene (sesc) taking advantage of the PCR procedure. All of the strains were Biofilm forming producers. The in vitro biofilm formation assays determined that 45 (83.33%), 5 (9.26%), 4 (7.41%) were strong, moderate, and weak biofilm former strains respectively. Among the isolated strains, the specific frequencies of the biofilm-forming genes were specified to be (98%) for sdrG, (84%) for atlE, (80%) for icaC, and (70%) for icaD. Cefamandole and Amikacin are the most effective antibiotics in isolated strains. All strains were ascertained to be methicillin and amoxicillin/clavulanic acid resistant.

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.

Whole-genome sequencing of Staphylococcus epidermidis bloodstream isolates from a prospective clinical trial reveals that complicated bacteraemia is caused by a limited number of closely related sequence types.

OBJECTIVES: The significance of isolating Staphylococus epidermidis from a blood culture is highly heterogeneous, ranging from contamination to an indication of a serious infection. Herein we sought to determine whether there is a relationship between S. epidermidis genotype and clinical severity of bacteraemia. METHODS: S. epidermidis bacteraemias from a prospective, multicentre trial at 15 centres in the United States and one in Spain were classified as simple (including possible contamination), uncomplicated, and complicated. Whole-genome sequencing (WGS) was performed on 161 S. epidermidis isolates, and clinical outcomes were correlated with genotypic information. RESULTS: A total of 49 S. epidermidis sequence types (STs) were identified. Although strains of all 49 STs were isolated from patients with either simple or uncomplicated infection, all strains causing complicated infections were derived from five STs: ST2, ST5, ST7, ST16, and ST32. ST2 and ST5 isolates were significantly more likely to cause uncomplicated and complicated bloodstream infections compared to simple bacteraemia (odds ratio 2.0, 95%CI 1.1-3.9, p 0.04). By multivariate regression analysis, having an ST2 or ST5 S. epidermidis bacteraemia was an independent predictor of complicated bloodstream infection (odds ratio 3.7, 95%CI 1.2-11.0, p 0.02). ST2/ST5 strains carried larger numbers of antimicrobial resistance determinants compared to non-ST2/ST5 isolates (6.34 ± 1.5 versus 4.4 ± 2.5, p < 0.001). CONCLUSION: S. epidermidis bacteraemia was caused by a genetically heterogeneous group of organisms, but only a limited number of STs-particularly multidrug-resistant ST2 and ST5 strains-caused complicated infections.

Genotypic and phenotypic changes of Staphylococcus epidermidis during relapse episodes in prosthetic joint infections.

Staphylococcus epidermidis is a coagulase-negative bacterium capable of causing recurrent relapses in prosthetic joint infection (PJI). The aim of this study was to determine if Staphylococcus epidermidis isolates from patients with recurrent relapses of prosthetic joint infection (PJI) changed genotypically (pulsed-field gel electrophoresis (PFGE) pattern analysis and genes involved in biofilm formation) and phenotypically (antimicrobial resistance, biofilm formation) during the different episodes. Four patients with PJI recurrent relapses were evaluated clinically and microbiologically. Genotypic and phenotypic characteristics of 31 S. epidermidis isolates were determined. In all cases, PJI was treated with antimicrobial therapy and resection of the prosthesis without reimplantation. Months later, all patients had a relapse episode and treated with rifampin plus vancomycin and surgical debridement. Changes in the antibiotics resistance profile in isolates from patients 1 and 2 were observed in the two episodes. Patient 1 had four clones A, B, C, and D that were distributed differentially in the two episodes. Similarly, patients 2 and 3 had two clones and subclones (E-E1 and F-F1, respectively), and patient 4 had only the clone G in both episodes. The clone F formed small-colony variants (SCVs). High level of biofilm formation was found in all clones, except for clones D and G. Clones/subclones showed a genotypic variation in icaA, sdrF, bap, sesI, and embp genes. The principal coordinate analysis showed that all clones/subclones were different. These results showed that the initial infective clone of S. epidermidis from PJI, changed genotypically and phenotypically after a second relapse as a response to the treatment.

Mining the Methylome Reveals Extensive Diversity in Staphylococcus epidermidis Restriction Modification.

Staphylococcus epidermidis is a significant opportunistic pathogen of humans. Molecular studies in this species have been hampered by the presence of restriction-modification (RM) systems that limit introduction of foreign DNA. Here, we establish the complete genomes and methylomes for seven clinically significant, genetically diverse S. epidermidis isolates and perform the first systematic genomic analyses of the type I RM systems within both S. epidermidis and Staphylococcus aureus Our analyses revealed marked differences in the gene arrangement, chromosomal location, and movement of type I RM systems between the two species. Unlike S. aureus, S. epidermidis type I RM systems demonstrate extensive diversity even within a single genetic lineage. This is contrary to current assumptions and has important implications for approaching the genetic manipulation of S. epidermidis Using Escherichia coli plasmid artificial modification (PAM) to express S. epidermidishsdMS, we readily overcame restriction barriers in S. epidermidis and achieved electroporation efficiencies equivalent to those of modification-deficient mutants. With these functional experiments, we demonstrated how genomic data can be used to predict both the functionality of type I RM systems and the potential for a strain to be electroporation proficient. We outline an efficient approach for the genetic manipulation of S. epidermidis strains from diverse genetic backgrounds, including those that have hitherto been intractable. Additionally, we identified S. epidermidis BPH0736, a naturally restriction-defective, clinically significant, multidrug-resistant ST2 isolate, as an ideal candidate for molecular studies.IMPORTANCEStaphylococcus epidermidis is a major cause of hospital-acquired infections, especially those related to implanted medical devices. Understanding how S. epidermidis causes disease and devising ways to combat these infections have been hindered by an inability to genetically manipulate clinically significant hospital-adapted strains. Here, we provide the first comprehensive analyses of the barriers to the uptake of foreign DNA in S. epidermidis and demonstrate that these are distinct from those described for S. aureus Using these insights, we demonstrate an efficient approach for the genetic manipulation of S. epidermidis to enable the study of clinical isolates for the first time.

Genetic Diversity of Composite Enterotoxigenic Staphylococcus epidermidis Pathogenicity Islands.

The only known elements encoding enterotoxins in coagulase-negative staphylococci are composite Staphylococcus epidermidis pathogenicity islands (SePIs), including SePI and S. epidermidis composite insertion (SeCI) regions. We investigated 1545 Staphylococcus spp. genomes using whole-genome MLST, and queried them for genes of staphylococcal enterotoxin family and for 29 ORFs identified in prototype SePI from S. epidermidis FRI909. Enterotoxin-encoding genes were identified in 97% of Staphylococcus aureus genomes, in one Staphylococcus argenteus genome and in nine S. epidermidis genomes. All enterotoxigenic S. epidermidis strains carried composite SePI, encoding sec and sel enterotoxin genes, and were assigned to a discrete wgMLST cluster also containing genomes with incomplete islands located in the same region as complete SePI in enterotoxigenic strains. Staphylococcus epidermidis strains without SeCI and SePI genes, and strains with complete SeCI and no SePI genes were identified but no strains were found to carry only SePI and not SeCI genes. The systematic differences between SePI and SeCI regions imply a lineage-specific pattern of inheritance and support independent acquisition of the two elements in S. epidermidis. We provided evidence of reticulate evolution of mobile elements that contain elements with different putative ancestry, including composite SePI that contains genes found in other coagulase-negative staphylococci (SeCI), as well as in S. aureus (SePI-like elements). We conclude that SePI-associated elements present in nonenterotoxigenic S. epidermidis represent a scaffold associated with acquisition of virulence-associated genes. Gene exchange between S. aureus and S. epidermidis may promote emergence of new pathogenic S. epidermidis clones.

Pathogen Analysis of Central Nervous System Infections in a Chinese Teaching Hospital from 2012-2018: A Laboratory-based Retrospective Study.

Central nervous system (CNS) infections are associated with high mortality rates. The clinical presentation of many CNS infections by different pathogens is difficult to distinguish, but the definite diagnosis of the etiology is critical for effective therapy and prognosis. The aim of this study was to explore the etiology of CNS infections with definite diagnoses based on data from a clinical microbiology laboratory in Tongji Hospital, a teaching hospital in China, obtained over a six-year period. We conducted a retrospective study on all cerebrospinal fluid (CSF) specimens submitted to our clinical microbiology laboratory from September, 2012 to December, 2018. The etiology of CNS infections caused by Cryptococcus neoformans, Mycobacterium tuberculosis and common bacteria was analyzed. Antimicrobial susceptibility testing was conducted on all isolates. The results showed that 1972 cases of CNS infections were identified from 18 300 CSF specimens. Common bacterial meningitis (BM), cryptococcal meningitis (CM) and tuberculous meningitis (TM) accounted for 86.3% (677/785), 9.4% (74/785) and 4.3% (34/785) respectively of cases over the six-year period. BM was the most common among the different age groups, followed by CM. Of the TM cases, 44.1% (15/34) were distributed within the age group of 15-34 years, whereas for CM cases, 52.7% (39/74) occurred within the 35-54-year age group, and the age distribution of BM cases was fairly even. Among the bacterial pathogens isolated, Staphylococcus epidermidis was the most common, accounting for 12.5% (98/785), followed by Acinetobacter baumannii (ABA) and Staphylococcus aureus (SAU), accounting for 11.8% (93/785) and 7.6% (60/785) respectively. The resistance rates to antibiotics were >75%, with the exception of the resistance rate of ABA to tegafycline, which was <3%. More than 60% of SAU strains displayed resistance to penicillin, oxacillin, ampicillin/sulbactam, cefazolin, cefuroxime, gentamycin, tobramycin, erythromycin and levofloxacin, whereas more than 90% of SAU strains showed susceptibility to trimethoprim/sulfamethoxazole, tegafycline, vancomycin, teicoplanin and linezolid. For C. neoformans, the susceptibility rates to amphotericin B, 5-fluorocytosine, fluconazol and voriconazole were >95%. Analysis of samples from patients with CNS infection in a clinical microbiology laboratory at a teaching hospital in China over a six-year period indicated that the most common etiological agents were the bacteria ABA and SAU. The antibiotic resistance levels of ABA were found to be high and of concern, whereas isolates of C. neoformans were found to be sensitive to antifungal antibiotics.

Draft genome sequences of three clinical isolates of teicoplanin-resistant Staphylococcus epidermidis from patients without prior exposure to glycopeptide antibiotics.

OBJECTIVES: The aim of this study was to analyse the DNA sequences of three teicoplanin-resistant Staphylococcus epidermidis isolates collected from patients not previously treated with glycopeptide antibiotics. METHODS: The minimum inhibitory concentrations (MICs) of 12 antibiotics, including teicoplanin and vancomycin, were determined by the broth microdilution method. Genomic DNA was isolated, was sequenced by HiSeqX paired-end sequencing and was assembled into draft genome sequences using MyPro pipeline. RESULTS: Analysis of the draft genome sequences demonstrated that the teicoplanin-resistant S. epidermidis isolates belonged to multilocus sequence typing (MLST) sequence types ST5 and ST87 and encoded multiple antimicrobial resistance genes, including the methicillin resistance gene mecA. CONCLUSIONS: This report highlights the risk of dissemination of S. epidermidis strains resistant to a wide range of clinically important antibiotics.

Understanding the microbial basis of body odor in pre-pubescent children and teenagers.

BACKGROUND: Even though human sweat is odorless, bacterial growth and decomposition of specific odor precursors in it is believed to give rise to body odor in humans. While mechanisms of odor generation have been widely studied in adults, little is known for teenagers and pre-pubescent children who have distinct sweat composition from immature apocrine and sebaceous glands, but are arguably more susceptible to the social and psychological impact of malodor. RESULTS: We integrated information from whole microbiome analysis of multiple skin sites (underarm, neck, and head) and multiple time points (1 h and 8 h after bath), analyzing 180 samples in total to perform the largest metagenome-wide association study to date on malodor. Significant positive correlations were observed between odor intensity and the relative abundance of Staphylococcus hominis, Staphylococcus epidermidis, and Cutibacterium avidum, as well as negative correlation with Acinetobacter schindleri and Cutibacterium species. Metabolic pathway analysis highlighted the association of isovaleric and acetic acid production (sour odor) from enriched S. epidermidis (teen underarm) and S. hominis (child neck) enzymes and sulfur production from Staphylococcus species (teen underarm) with odor intensity, in good agreement with observed odor characteristics in pre-pubescent children and teenagers. Experiments with cultures on human and artificial sweat confirmed the ability of S. hominis and S. epidermidis to independently produce malodor with distinct odor characteristics. CONCLUSIONS: These results showcase the power of skin metagenomics to study host-microbial co-metabolic interactions, identifying distinct pathways for odor generation from sweat in pre-pubescent children and teenagers and highlighting key enzymatic targets for intervention.

Global spread of three multidrug-resistant lineages of Staphylococcus epidermidis.

Staphylococcus epidermidis is a conspicuous member of the human microbiome, widely present on healthy skin. Here we show that S. epidermidis has also evolved to become a formidable nosocomial pathogen. Using genomics, we reveal that three multidrug-resistant, hospital-adapted lineages of S. epidermidis (two ST2 and one ST23) have emerged in recent decades and spread globally. These lineages are resistant to rifampicin through acquisition of specific rpoB mutations that have become fixed in the populations. Analysis of isolates from 96 institutions in 24 countries identified dual D471E and I527M RpoB substitutions to be the most common cause of rifampicin resistance in S. epidermidis, accounting for 86.6% of mutations. Furthermore, we reveal that the D471E and I527M combination occurs almost exclusively in isolates from the ST2 and ST23 lineages. By breaching lineage-specific DNA methylation restriction modification barriers and then performing site-specific mutagenesis, we show that these rpoB mutations not only confer rifampicin resistance, but also reduce susceptibility to the last-line glycopeptide antibiotics, vancomycin and teicoplanin. Our study has uncovered the previously unrecognized international spread of a near pan-drug-resistant opportunistic pathogen, identifiable by a rifampicin-resistant phenotype. It is possible that hospital practices, such as antibiotic monotherapy utilizing rifampicin-impregnated medical devices, have driven the evolution of this organism, once trivialized as a contaminant, towards potentially incurable infections.

Use of MALDI-TOF MS to Discriminate between Biofilm-Producer and Non-Producer Strains of Staphylococcus epidermidis.

For the management of Staphylococci coagulase-negative infection, often related to biofilm formation, rapid and accurate identification is necessary in choosing a correct antibiotic therapy. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is becoming increasingly important for bacterial identification over traditional methods. Our aim was to validate the use of MALDI to discriminate Staphylococcus epidermidis biofilm-producing strains. Clinical strains coming from suture wires were identified and their protein profiles were compared to that obtained from two ATCC reference strains (biofilm producer and non-producer). MALDI identified the eighteen isolates as S. epidermidis, combining sixteen profiles with the biofilm producer and two with the non-producer, confirming the results of crystal violet assay. Our data highlight that MALDI can be considered a good tool to discriminate between biofilm-producer and non-producer strains of S. epidermidis, thus helping to establish an effective antibiotic therapy.

The prevalence, antibiotic resistance and mecA characterization of coagulase negative staphylococci recovered from non-healthcare settings in London, UK.

Background: Coagulase negative staphylococci (CoNS) are important reservoirs of antibiotic resistance genes and associated mobile genetic elements and are believed to contribute to the emergence of successful methicillin resistant Staphylococcus aureus (MRSA) clones. Although, these bacteria have been linked to various ecological niches, little is known about the dissemination and genetic diversity of antibiotic resistant CoNS in general public settings. Methods: Four hundred seventy-nine samples were collected from different non-healthcare/general public settings in various locations (n = 355) and from the hands of volunteers (n = 124) in London UK between April 2013 and Nov 2014. Results: Six hundred forty-three staphylococcal isolates belonging to 19 staphylococcal species were identified. Five hundred seventy-two (94%) isolates were resistant to at least one antibiotic, and only 34 isolates were fully susceptible. Sixty-eight (11%) mecA positive staphylococcal isolates were determined in this study. SCCmec types were fully determined for forty-six isolates. Thirteen staphylococci (19%) carried SCCmec V, followed by 8 isolates carrying SCCmec type I (2%), 5 SCCmec type IV (7%), 4 SCCmec type II (6%), 1 SCCmec type III (2%), 1 SCCmec type VI (2%), and 1 SCCmec type VIII (2%). In addition, three isolates harboured a new SCCmec type 1A, which carried combination of class A mec complex and ccr type 1.MLST typing revealed that all S. epidermidis strains possess new MLST types and were assigned the following new sequence types: ST599, ST600, ST600, ST600, ST601, ST602, ST602, ST603, ST604, ST605, ST606, ST607 and ST608. Conclusions: The prevalence of antibiotic resistant staphylococci in general public settings demonstrates that antibiotics in the natural environments contribute to the selection of antibiotic resistant microorganisms. The finding of various SCCmec types in non-healthcare associated environments indicates the complexity of SCCmec. We also report on new MLST types that were assigned for all S. epidermidis isolates, which demonstrates the genetic variability of these isolates.

Whole Genome Sequence and Comparative Genomics Analysis of Multi-drug Resistant Environmental Staphylococcus epidermidis ST59.

Staphylococcus epidermidis is a major opportunistic pathogen primarily recovered from device-associated healthcare associated infections (DA-HAIs). Although S. epidermidis and other coagulase-negative staphylococci (CoNS) are less virulent than Staphylococcus aureus, these bacteria are an important reservoir of antimicrobial resistance genes and resistance-associated mobile genetic elements that can be transferred between staphylococcal species. We report a whole genome sequence of a multidrug resistant S. epidermidis (strain G6_2) representing multilocus sequence type (ST) 59 and isolated from an environmental sampling of a hotel room in London, UK. The genome of S. epidermidis G6_2 comprises of a 2408357 bp chromosome and six plasmids, with an average G+C content of 32%. The strain displayed a multi-drug resistance phenotype which was associated with carriage of 7 antibiotic resistance genes (blaZ, mecA, msrA, mphC, fosB, aacA-aphD, tetK) as well as resistance-conferring mutations in fusA and ileS Antibiotic resistance genes were located on plasmids and chromosome. Comparative genomic analysis revealed that antibiotic resistance gene composition found in G6_2 was partly preserved across the ST59 lineage.

Simple and economical method for identification and speciation of Staphylococcus epidermidis and other coagulase negative Staphylococci and its validation by molecular methods.

Coagulase-negative staphylococci (CoNS) have been increasingly recognized as a clinically important group of species that can cause several opportunistic nosocomial infections. There are at least 47 known species of Staphylococci and to differentiate all these species >40 biochemical tests need to be performed. The present study was able to refine the CoNS identification process by using only five tests to identify S. epidermidis from the rest and used six other tests to identify eleven other clinically significant CoNS species. A total of 242 CoNS isolates were collected from tertiary care hospitals and included in the study. The five-biochemical test scheme devised based on mathematical probability derived from a computer algorithm included fermentation of mannitol, maltose, mannose, trehalose and novobiocin susceptibility to differentiate S. epidermidis from other CoNS species. The remaining CoNS isolates other than S. epidermidis were further characterized with the help of six additional tests, which identified another eleven species. Species-specific PCR and 16SrDNA sequencing were used to confirm and validate the identification scheme. Species-specific PCR and 16SrDNA sequencing showed 100% agreement with non-divergent phenotypic test results, indicating that the five selected assays are highly specific for identifying S. epidermidis. In conclusion, this study used only 11 tests to identify most of the clinically significant CoNS that can reduce cost and time. This scheme is easy to perform in any laboratory with basic resources, the results of this study were validated using more accurate molecular methods such as PCR and 16S rDNA typing to confirm the utility of the proposed scheme.

Identification of Staphylococcus epidermidis with transferrable mupirocin resistance from canine skin.

Resistance to mupirocin was analysed in Staphylococcus spp. isolated from healthy dogs (n=21) and dogs with pyoderma (n=47) or otitis externa (n=52). Isolates were identified to species level by MALDI-TOF and PCR-RFLP of the groEL gene. One isolate of Staphylococcus epidermidis from the skin of a healthy dog, which harboured a plasmid carrying the mupA gene, was resistant to mupirocin.