Epidermidis Strains Research Articles

Antimicrobial resistance and genetic characterization of coagulase-negative staphylococci from bovine mastitis milk samples in Korea

Coagulase-negative staphylococci (CNS) are one of the most common bovine mastitis pathogens found worldwide. In this study, we investigated the prevalence and distribution of CNS species in mastitis milk samples and further characterized the methicillin-resistant (MR) CNS. A total of 311 CNS were isolated from 3,692 quarter milk samples from 1,373 dairy cattle at 81 farms between 2013 and 2017. Further evaluation of the CNS isolates revealed 14 CNS species among the samples and 3 predominant species-namely, Staphylococcus chromogenes, Staphylococcus simulans, and Staphylococcus epidermidis. Resistance was higher in S. epidermidis than in other CNS species except for resistance against oxacillin in Staphylococcus sciuri. Resistance to β-lactams was the most common in all CNS species (8.4% in ampicillin, 21.2% in oxacillin, and 13.5% in penicillin). Conversely, only minimal resistance to cephalothin, ceftiofur, and pirlimycin/novobiocin was found. Twenty-one isolates from 4 species were mecA-carrying MRCNS strains, including 18 S. epidermidis and 1 each of S. sciuri, Staphylococcus equorum, and Staphylococcus hominis. The majority of the mecA-carrying MRCNS isolates were produced in the biofilm. Furthermore, multidrug-resistant sequence type 179 isolate produced the strongest biofilm. Seven genotypes were detected in the 18 MR S. epidermidis strains, the most predominant of which persisted on a farm for 2 yr. Our findings for the antimicrobial susceptibility profiles and genotypic characterization of the MRCNS isolates could provide valuable information for controlling the spread of resistance and the selection of appropriate antimicrobial therapies for mastitis in the future. Further, strategic antibiotic use for mastitis treatment and hygienic management practices aimed at the prevention of the growth of resistant bacteria are urgently needed on dairy farms.

Proteomic Analysis Reveals a Biofilm-Like Behavior of Planktonic Aggregates of Staphylococcus epidermidis Grown Under Environmental Pressure/Stress.

Prosthetic joint replacement failure has a huge impact on quality of life and hospitalization costs. A leading cause of prosthetic joint infection is bacteria-forming biofilm on the surface of orthopedic devices. Staphylococcus epidermidis is an emergent, low-virulence pathogen implicated in chronic infections, barely indistinguishable from aseptic loosening when embedded in a mature matrix. The literature on the behavior of quiescent S. epidermidis in mature biofilms is scarce. To fill this gap, we performed comparative analysis of the whole proteomic profiles of two methicillin-resistant S. epidermidis strains growing in planktonic and in sessile form to investigate the molecular mechanisms underlying biofilm stability. After 72-h culture of biofilm-forming S. epidermidis, overexpression of proteins involved in the synthesis of nucleoside triphosphate and polysaccharides was observed, whereas planktonic bacteria expressed proteins linked to stress and anaerobic growth. Cytological analysis was performed to determine why planktonic bacteria unexpectedly expressed proteins typical of sessile culture. Images evidenced that prolonged culture under vigorous agitation can create a stressful growing environment that triggers microorganism aggregation in a biofilm-like matrix as a mechanism to survive harsh conditions. The choice of a unique late time point provided an important clue for future investigations into the biofilm-like behavior of planktonic cells. Our preliminary results may inform comparative proteomic strategies in the study of mature bacterial biofilm. Finally, there is an increasing number of studies on the aggregation of free-floating bacteria embedded in an extracellular matrix, prompting the need to gain further insight into this mode of bacterial growth.

Total alkaloids of Sophora alopecuroides and matrine inhibit auto-inducer 2 in the biofilms of Staphylococcus epidermidis

Quorum sensing (QS) mediates the coordination of population-based behavior in bacteria, which is highly involved in the formation of bacterial biofilms and virulence of bacteria in vivo. Therefore, an inhibition of QS and biofilm growth is of therapeutic interest. This study exhibited the an auto-inducer molecule (AI-2) activity as the most important component of the QS system was positively correlated with the growth and biofilm formation of S. epidermidis strains. In addition, TASA and matrine have a capacity to inhibit AI-2 in three S. epidermidis strains compared to the control (p < 0.01). This result indicated TASA and matrine can also decrease AI-2 activity in the biofilm of S. epidermidis (p < 0.05). By comparison, TASA was more effective than ceftazidime and matrine to inhibit the AI-2 activity in biofilm S.epidermidis reference strain ATCC35984 (p < 0.05). This result indicated potentials of TCM compounds TASA and matrine in prevention of biofilm formation in Staphylococcus epidermidis infections.

Bacterial Colonization of the Hospitalized Newborn: Competition Between Staphylococcus aureus and Staphylococcus epidermidis.

In adults, Staphylococcus epidermidis and Staphylococcus aureus compete for colonization of the nasal mucosa and S. epidermidis strains that produce the Esp serine protease eradicate S. aureus nasal colonization. Whether similar phenomena are seen in newborn infants is unknown. Nasal swabs were obtained on admission and discharge from newborn infants (n = 90 and 83, respectively) in the neonatal intensive care unit at UC Davis Children's Hospital. Swabs were cultured for S. aureus and S. epidermidis. S. epidermidis isolates were tested for Esp expression, overall secreted protease activity and biofilm inhibition. No infant had S. aureus on admission. S. epidermidis colonization was rare on admission in inborn infants (2.5%), but common in infants transferred from referring hospitals (50%). At discharge, most infants (96%) were colonized by staphylococci. S. aureus colonization was less common in infants with S. epidermidis colonization (9%) and more common in infants without S. epidermidis (77%) (relative risk of S. aureus colonization in infants colonized with S. epidermidis 0.18, 95% confidence interval: 0.089-0.34, P < 0.0001). Compared with S. epidermidis strains from infants without S. aureus, S. epidermidis from infants co-colonized with S. aureus had lower total proteolytic enzyme activity and decreased biofilm inhibition capacity, but did not have lower frequency of Esp positivity. In hospitalized neonates, S. epidermidis colonization has a protective effect against S. aureus colonization. Secretion of proteases by S. epidermidis is a possible mechanism of inhibition of S. aureus colonization; however, in this cohort of neonates, the source of major protease activity is likely other than Esp.

An underestimated pathogen: Staphylococcus epidermidis induces pro-inflammatory responses in human alveolar epithelial cells

ObjectivesConventionally regarded as a harmless skin commensal, Staphylococcus epidermidis accounts for the majority of neonatal late-onset sepsis and is shown to be associated with neonatal inflammatory morbidities, especially bronchopulmonary dysplasia. This study addressed the pro-inflammatory capacity of different S. epidermidis strains on human alveolar epithelial cells. MethodsA549 cell monolayers were stimulated by live bacteria of S. epidermidis RP62A strain (biofilm-positive) and ATCC 12228 strain (biofilm-negative) at a multiplicity of infection ratio of 10 for 24 h. LPS (100 ng/ml) and Pam3CSK4 (1 µg/ml) were used for comparisons. Cell viability was measured by MTT method. The mRNA and protein expression of inflammatory mediators and toll-like receptor (TLR)-2 were assessed using RT-PCR, immunoassays and immunofluorescence. ResultsBoth S. epidermidis strains induced expression of tumor necrosis factor (TNF)-α, IL-1β, interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP)-1, interferon γ-induced protein 10 (IP-10) and intercellular adhesion molecule (ICAM)-1, but not IL-10. The stimulatory effect of RP62A exceeded that of LPS (p < 0.05). RP62A strain showed a trend towards higher induction of pro-inflammatory mediators than ATCC 12228 strain. The co-stimulation with RP62A strain decreased cell viability compared to control and TLR agonists (p < 0.05). RP62A but not ATCC 12228 stimulated mRNA and protein expression of TLR2. ConclusionsS. epidermidis drives pro-inflammatory responses in lung epithelial cells in vitro. The pro-inflammatory capacity of S. epidermidis may differ between strains. Biofilm-positive S. epidermidis strain seems to induce more potent pulmonary pro-inflammation than biofilm-negative S. epidermidis strain.

Differences in distribution of MLS antibiotics resistance genes in clinical isolates of staphylococci belonging to species: S. epidermidis, S. hominis, S. haemolyticus, S. simulans and S. warneri

BackgroundMacrolides and lincosamides are two leading types of antibiotics commonly used in therapies. The study examines the differences in resistance to these antibiotics and their molecular bases in S. epidermidis as well as in rarely isolated species of coagulase-negative staphylococci such as S. hominis, S. haemolyticus, S. warneri and S. simulans. The isolates were tested for the presence of the erm(A), erm(B), erm(C), lnu(A), msr(A), msr(B), mph(C), ere(A) and ere(B) genes. Phenotypic resistance to methicillin and mecA presence were also determined.ResultsThe MLSB resistance mechanism was phenotypically found in isolates of species included in the study. The most prevalent MLSB resistance mechanism was observed in S. hominis, S. haemolyticus and S. epidermidis isolates mainly of the MLSB resistance constitutive type. Macrolide, lincosamide and streptogramin B resistance genes were rarely detected in isolates individually. The erm(B), ere(A) and ere(B) genes were not found in any of the strains. The erm(A) gene was determined only in four strains of S. epidermidis and S. hominis while lnu(A) was seen in eight strains (mainly in S. hominis). The erm(C) gene was present in most of S. epidermidis strains and predominant in S. hominis and S. simulans isolates. The examined species clearly differed between one another in the repertoire of accumulated genes.ConclusionsThe presence of genes encoding the MLSB resistance among CoNS strains demonstrates these genes’ widespread prevalence and accumulation in opportunistic pathogens that might become gene reservoir for bacteria with superior pathogenic potential.

Association between biofilm formation, structure and antibiotic resistance in Staphylococcus epidermidis isolated from neonatal septicemia in southwest Iran.

Background: Staphylococcus epidermidis has emerged as the pathogen from neonatal septicemia. Antibiotic resistance and the capability of biofilm formation make these infections much harder to treat. Hence, the aim of this study was to investigate the association between biofilm formation, structure and antibiotic resistance in S. epidermidis isolated from neonatal septicemia.Methods: Overall, 65 S. epidermidis isolates were recovered from blood cultures of neonatal septicemia. Antibiotic resistance pattern and the biofilm production were determined using phenotypic methods. The presence of ica operon, the bhp, the aap genes and SCCmec types were screened using PCR.Results: Most S.epidermidis isolates were resistant to erythromycin, while all isolates were sensitive to linezolid and vancomycin. Fifty-three percent of S.epidermidis isolates were resistant to methicillin. SCCmec types II was found commonly among methicillin-resistant S. epidermidis (MRSE) strains. The biofilm formation was observed in 65% of S.epidermidis isolates and the majority have polysaccharide matrix. icaA and icaD genes were found in 40% and 19% of isolates. Twenty-three isolates (62%) produced dissolvable polysaccharide intercellular adhesion (PIA)-dependent biofilms in SM after growth in TSB with NaCl and 14 (37%) isolates produced dissolvable protein-dependent biofilms in PK after growth in TSB with glucose. Three isolates (62%) produced dissolvable polysaccharide intercellular adhesion.Conclusion: Our data indicate the high rates of antibiotic resistance and the capability of biofilm formation among S. epidermidis isolates. Hence, the transmission of these strains can cause an increased risk of serious nosocomial infections.

Nasal commensal Staphylococcus epidermidis enhances interferon-\u03bb-dependent immunity against influenza virus

BackgroundStaphylococcus epidermidis is one of the most abundant colonizers of healthy human mucosa including that in the respiratory tract. As the respiratory microbiome has been linked to host immune responses, this study sought to determine the role of nasal mucosa-associated S. epidermidis in innate immune responses against the influenza A virus (IAV). S. epidermidis strains were isolated from nasal mucus samples of healthy individuals. The effects of these mucosa-derived commensal strains on interferon (IFN)-dependent innate immunity and IAV infection dynamics were tested in vitro using normal human nasal epithelial (NHNE) cells and human turbinate mucosa. The effects of S. epidermidis on antiviral immunity were also tested in vivo using an acute IAV infection mouse model.ResultsExposure of NHNE cells to nasal mucosa-derived S. epidermidis increased IFN-λ mRNA and secreted protein levels in the absence of viral stimulation. In the context of IAV infection, NHNE exposure to S. epidermidis prevented an increase in the viral burden, as revealed by IAV PA mRNA abundance, IAV nucleoprotein levels, and viral titers. S. epidermidis also enhanced transcription of IFN-stimulated genes independently of Toll-like receptor 2 and further induced IFN-λ production in IAV-infected cells by promoting phosphorylation of interferon regulatory factor 7. In a murine infection model, S. epidermidis prevented the spread of IAV to the lungs by stimulating IFN-λ innate immunity and suppressing IAV replication in the nasal mucosa.ConclusionThe human nasal commensal S. epidermidis mediates front-line antiviral protection against IAV infection through modulation of IFN-λ-dependent innate immune mechanisms in the nasal mucosa, thereby demonstrating the role of host-bacterial commensalism in shaping human antiviral responses.

Seasonal and circadian evaluation of a citral-chemotype from Lippia alba essential oil displaying antibacterial activity

In Brazil, the infusion from leaves of Lippia alba (Verbenaceae), popularly known as Erva-cidreira, is used for the treatment of infections, digestive disorders, cough, asthma, colds and anxiolytic, among others. It has been used in phytotherapy practices of communities of Santarém, Pará State, Brazil, cultivated in a public program of medicinal plants and phytotherapy. In this work, the local influence of seasonality and planting time on essential oil yield and composition were evaluated, as well as in the development of a topical formulation to determine the oil efficacy in inhibition the Staphylococcus epidermidis strain. The plant was sampled during the dry and rainy seasons, three times a day, the oils extracted and its constituents identified by GC and GC-MS. The formulation of topical use was prepared with the incorporation of the oil (10%) to an anionic cream, followed by analysis of its organoleptic characteristics. The antibacterial activity of the topical formulation was evaluated by the Well Diffusion Method, using the S. epidermidis strain. The oil yield in the plant was from 2.2 to 4.3%, and its main constituents were geranial, neral, elemol, p-cymene, limonene, carvone, and geraniol, with the quantitative variation depending on the seasonality and the collection time. The results for the seasonal and circadian study were compatible with the data variance obtained by Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA), with the formation of two distinct groups of primary constituents for the rainy and dry seasons. The formulated product maintained about 45% of the primary constituents of the oil (neral and geranial) for various days. It was homogeneous after centrifugation test, having shown excellent stability at 5 °C and 25 °C. Storage of the formulated product at higher temperatures appears to be a limiting factor for the antimicrobial action of the oil.

Antimicrobial Agent Susceptibility and Typing of Staphylococcal Isolates from Subclinical Mastitis in Ewes.

Objective was to study susceptibility to antimicrobial agents of 142 staphylococcal isolates from subclinical mastitis in ewes. In total, 41.5% of these were resistant and 5.6% multidrug resistant. More coagulase-negative staphylococci (47.0%) were resistant than Staphylococcus aureus (18.5%) isolates. Resistance was greater to penicillin (22.5%), tetracycline, or ampicillin (18.3%). More biofilm-forming (20.6%) isolates were resistant to tetracycline than nonbiofilm-forming (0.0%) ones. Presence of tetK was associated with presence of icaA in the same strains. Further, 76.6% of resistant isolates versus 57.7% of susceptible ones were recovered immediately postpartum and 23.4% of resistant isolates versus 9.9% of susceptible ones were recovered in farms that practiced routine administration of antimicrobial agents at the end of a lactation period. Most S. aureus (59.3%) were classified in ST133 and most Staphylococcus epidermidis were classified in ST100, ST142, or ST152 (19.0% each). There was no association of sequence types with resistance. Whole genome sequencing showed that, in a Staphylococcus lentus strain, the ermB gene was part of transposon Tn917 integrated into the chromosome; also, a small plasmid was observed in an ermC-carrying Staphylococcus hominis strain and, finally, in an S. aureus and an S. epidermidis strains, small tetK-carrying plasmids (pSau-2716Lar, pSau-3893Lar) of 4.439 kb were found.

Synthesis of conjugated PIA-rSesC and immunological evaluation against biofilm-forming Staphylococcus epidermidis.

Staphylococcus epidermidis is an opportunistic pathogen and a leading cause of morbidity and premature mortality in patients with medical device-related infections, which is a concern in hospitalized patients. Developing a strategy to raise opsonic antibodies against polysaccharide intracellular adhesin (PIA) could be promising for the elimination of colonizing and biofilm-forming S. epidermidis. Following the purification of truncated rSesC protein and PIA, for the first time, PIA was conjugated to rSesC as a carrier to increase the immunogenicity of PIA and its efficacy in mice was evaluated. The structure of the conjugate was analysed using the Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance spectroscopy (H1- NMR) methods. Afterwards, the immune response was evaluated by measuring the total IgG, IgG2a and IgG2b titres. The immunization of mice with the PIA-rSesC conjugate raised the levels of opsonic antibodies, and the vaccinated mice were protected when challenged intravenously by wild-type S. epidermidis strain 1457. Further studies indicated that the conjugated vaccine was able to eliminate S. epidermidis biofilm formation in in vitro or in vivo assays. This study confirms the proposal that the immunization of mice with PIA-rSesC conjugate vaccine could protect against S. epidermidis infection.

Deposition of Host Matrix Proteins on Breast Implant Surfaces Facilitates Staphylococcus Epidermidis Biofilm Formation: In Vitro Analysis.

Staphylococcus epidermidis is a primary cause of breast implant-associated infection. S epidermidis possesses several virulence factors that enable it to bind both abiotic surfaces and host factors to form a biofilm. In addition S epidermidis colocalizes with matrix proteins coating explanted human breast implants. The authors sought to identify matrix proteins that S epidermidis may exploit to infect various breast implant surfaces in vitro. A combination of in vitro assays was used to characterize S epidermidis strains isolated from human breast implants to gain a better understanding of how these bacteria colonize breast implant surfaces. These included determining the (1) minimum inhibitory and bactericidal concentrations for irrigation solutions commonly used to prevent breast implant contamination; (2) expression and carriage of polysaccharide intercellular adhesin and serine-aspartate repeat proteins, which bind fibrinogen (SdrG) and collagen (SdrF), respectively; and (3) biofilm formation on varying implant surface characteristics, in different growth media, and supplemented with fibrinogen and Types I and III collagen. Scanning electron microscopy and immunofluorescence staining analyses were performed to corroborate findings from these assays. Textured breast implant surfaces support greater bacterial biofilm formation at baseline, and the addition of collagen significantly increases biomass on all surfaces tested. We found that S epidermidis isolated from breast implants all encoded SdrF. Consistent with this finding, these strains had a clear affinity for Type I collagen, forming dense, highly structured biofilms in its presence. The authors found that S epidermidis may utilize SdrF to interact with Type I collagen to form biofilm on breast implant surfaces.

Identification of ability to form biofilm in Candida parapsilosis and Staphylococcus epidermidis by Raman spectroscopy.

Aim: Finding rapid, reliable diagnostic methods is a big challenge in clinical microbiology. Raman spectroscopy is an optical method used for multiple applications in scientific fields including microbiology. This work reports its potential in identifying biofilm positive strains of Candida parapsilosis and Staphylococcus epidermidis. Materials & methods: We tested 54S. epidermidis strains (23 biofilm positive, 31 negative) and 51C. parapsilosis strains (27 biofilm positive, 24 negative) from colonies on Mueller-Hinton agar plates, using Raman spectroscopy. Results: The accuracy was 98.9% forC. parapsilosis and 96.1% forS. epidermidis. Conclusion: The method showed great potential for identifying biofilm positive bacterial and yeast strains. We suggest that Raman spectroscopy might become a useful aid in clinical diagnostics.

Tokiinshi, a traditional Japanese medicine (Kampo), suppresses Panton-Valentine leukocidin production in the methicillin-resistant Staphylococcus aureus USA300 clone.

It is necessary to develop agents other than antimicrobials for the treatment of Staphylococcus aureus infections to prevent the emergence of antimicrobial-resistant strains. Particularly, anti-virulence agents against the Panton-Valentine leukocidin (PVL)-positive methicillin-resistant S. aureus (MRSA), USA300 clone, is desired due to its high pathogenicity. Here, we investigated the potential anti-virulence effect of Tokiinshi, which is a traditional Japanese medicine (Kampo) used for skin diseases, against the USA300 clone. A growth inhibition assay showed that a conventional dose (20 mg/ml) of Tokiinshi has bactericidal effects against the clinical USA300 clones. Notably, the growth inhibition effects of Tokiinshi against S. epidermidis strains, which are the major constituents of the skin microbiome, was a bacteriostatic effect. The data suggested that Tokiinshi is unlikely to affect skin flora of S. epidermidis. Furthermore, PVL production and the expression of its gene were significantly suppressed in the USA300 clone by a lower concentration (5 mg/ml) of Tokiinshi. This did not affect the number of viable bacteria. Moreover, Tokiinshi significantly suppressed the expression of the agrA gene, which regulates PVL gene expression. For the first time, our findings strongly suggest that Tokiinshi has the potential to attenuate the virulence of the USA300 clone by suppressing PVL production via agrA gene suppression.

Clonally Diverse Methicillin and Multidrug Resistant Coagulase Negative Staphylococci Are Ubiquitous and Pose Transfer Ability Between Pets and Their Owners.

Sixty-eight owners and 66 pets, from 43 unrelated pet-owning households were screened for methicillin-resistant coagulase negative staphylococci (MRCoNS), potential cases of MRCoNS interspecies transmission (IT), and persistence. MRCoNS isolates were identified by microbiological and molecular tests. MLST-based phylogenetic analysis was performed in Staphylococcus epidermidis isolates. Antimicrobial susceptibility was evaluated using phenotypic and molecular methods. SCCmec type and the presence of biofilm-related ica locus was PCR-tested. Isolates suspected for MRCoNS IT cases were subjected to SmaI-PFGE analysis and individuals from positive households were followed-up for 1 year for carriage dynamics (every 3 months, T0–T4). Nineteen MRCoNS isolates from owners (27.9%) and 12 from pets (16.7%) were detected, coming from 20 households (46.5%). S. epidermidis was predominant (90 and 67% of human and animal strains, respectively), showing high phylogenetic diversity (16 STs among 24 strains). Methicillin-resistant S. epidermidis (MRSE) strains belonged to CC5 (75%), CC11 (12.5%), singleton S556 (8.3%), and S560 (4.17%). Significant host-associated differences were observed for resistance to aminoglycosides, co-trimoxazole, chloramphenicol (higher in animal isolates) and tetracycline (higher among human strains). Multidrug resistance (MDR) was common (68.4%) and associated with human strains. Great diversity of ccr and mec complexes were detected, most strains being non-typeable, followed by SCCmecIV and V. Over one third of isolates (most from owners), carried the ica locus, all MRSE CC5. Two sporadic IT cases (T0) were identified in owners and dogs from two households (4.7%), with diverse interspecies-exchanged clones detected along the sampling year, especially in dogs. A comparative analysis of all MRCoNS, with all nasal coagulase positive staphylococci (CoPS) recovered from the same individuals at T0, revealed that CoPS alone was predominant in owners and pets, followed by co-carriage of CoPS and MRCoNS in owners but single MRCoNS in pets. Statistical analyses revealed that owners are more prone to co-carriage and that co-existence of IT cases and co-carriage are positively interrelated. MRCoNS from healthy owners and their pets are genetically heterogeneous MDR strains that are spread in the community. Therefore, pets also contribute to the dissemination of successful human clones. Owner-pet inhabitancy increases the risk for staphylococcal temporal concomitance with its subsequent risk for bacterial infection and genetic exchange.

Biofilm production, quorum sensing system and analysis of virulence factors of Staphylococcus epidermidis collected from sheep milk samples

Intramammary infections are a serious problem in dairy sheep farming, and Staphylococcus epidermidis is one of the main etiological agents of ovine mastitis. In this work, 199 S. epidermidis isolates collected in Sardinia (Italy) from sheep milk samples and used for the preparation of inactivated autogenous vaccines were characterized with phenotypic and genotypic methods. The potential for biofilm production was evaluated phenotypically by assessing colony characteristics on Congo Red Agar (CRA), and genotypically by PCR for i) the icaA/D genes coding the polysaccharide intercellular adhesin (PIA) and ii) for the bhp, aap and embp genes coding biofilm accumulation proteins. Isolates were also investigated by PCR for the presence of autolysins (atlE and aae genes), microbial surface components recognizing adhesive matrix molecules (MSCRAMMs, sdrG and sdrF genes), enterotoxins (sea, seb, sec, sed and see genes), and the toxic shock syndrome toxin (tsst genes). In addition, the staphylococcal accessory gene regulator (agr), the most important locus responsible for the regulation of virulence factors, was characterized for the first time. As a result, only two isolates showed biofilm production on CRA and harbored the icaA/D genes. Four isolates, phenotypically classified as non-biofilm producers, possessed the icaA/D genes (n = 3) or only the icaA gene (n = 1). All isolates harbored the atlE, aae, embp and sdrG genes, but none harbored the genes encoding enterotoxins and tsst. Most isolates belonged to the agr-3se group (56.3%), followed by agr-1se (41.2%) and agr-2se (2.5%). All six isolates that harbored ica genes belonged to agr-1se.In conclusion, this study provides a detailed characterization of the putative virulence genes present in S. epidermidis strains circulating in Sardinian sheep. Because of their role in ovine mastitis pathogenesis, these might represent valuable candidates for vaccine development.

Profile of Biofilm-Producing Staphylococcus epidermidis from Intravenous Catheter Colonisation at Prof. Dr. Margono Soekarjo Hospital Purwokerto

Biofilm- producing Staphylococcus epidermidis has evolved to be a significant human pathogen, particularly in the use of medical devices such as an intravenous catheter. Furthermore, biofilm-producing bacteria 10-1000 fold less susceptible to several antimicrobial agents than free-bacteria. This simple survey aimed to describe the profile of biofilm-producing S. epidermidis from intravenous catheter colonization of some patients in surgical and internal medicine wards at the hospital Margono Soekarjo, Purwokerto, and the antibiotics resistance pattern. A vitek® 2 compact (Enseval Medika Prima) was performed to identify the bacterial species and to examine the 73 antibiotics for understanding the resistance pattern automatically. Microtiter plate biofilm assay with crystal violet staining was performed to measure biofilm optical density (OD) for analyzing the biofilm production capabilities. A scanning electron microscopy (SEM) was done to compare the thickness of ultrastructure of biofilm-producing S. epidermidis visually. The present study found that 2 of 8 Gram-positive bacteria (25%) were biofilm-producing S. epidermidis . One of S. epidermidis was moderate whereas the other was high biofilm-producing bacteria. Images of SEM showed that a high biofilm-producing S. epidermidis has a thicker ultrastructure of biofilm than the moderate biofilm-producing, whereas a control, the weak biofilm-producing S. epidermidis ATCC 12228 has the least biofilm. Both of S. epidermidis strains were sensitive to Gentamicin, Moxifloxacin, Quinupristin/Dalfopristin, Linezolid, Vancomycin, Doxycycline, Minocycline, Tetracycline, Tigecycline, and Nitrofurantoin. Furthermore, both S. epidermidis strains were resistant to the other (63) antibiotics. In conclusion, two strains of S. epidermidis in this study have different capabilities to form the biofilm which were showed that high biofilm-producing strain was thicker than moderate biofilm-producing strain by scanning electron microscopy. However, both of them were resistant to the same number of antibiotics.

A Comprehensive In Vitro Evaluation of Medihoney as an Anti-Biofilm Agent in Preventing Ventricular Assist Device Driveline Infections

Purpose Medihoney is routinely applied to the exit site of Ventricular Assist Device (VAD) drivelines in patients in many Australian hospitals to prevent driveline infections. Its effectiveness as a prophylactic measure remains unclear. We performed a comprehensive in vitro study using microbiological assays that closely mimic the in vivo environment of the driveline exit-site to assess the effectiveness of medihoney in preventing biofilm-related driveline infections. Methods Antimicrobial efficacy testing of medihoney was performed against 24 clinically-relevant bacterial and fungal strains grown as planktonic and biofilm cells. The minimum inhibitory concentration (MIC), minimum biofilm inhibitory concentration, minimum eradication concentration of medihoney, and the activity of individual components of medihoney were assessed. A colony biofilm assay and a drip-flow biofilm reactor assay that mimic chronic wounds and the driveline exit site were used to evaluate medihoney activity in inhibiting early adherent organism monolayers and mature biofilms. Results Medihoney demonstrated activity against planktonic organism cells in 5/6 S. aureus strains, 6/6 of S. epidermidis strains, 1/6 of P. aeruginosa strains and 4/6 of Candida strains using a qualitative disk diffusion susceptibility assay and a quantitative broth microdilution assay [MICs, 10% weight/volume (W/V) medihoney concentrations]. Higher concentrations of medihoney (30-50%, W/V) were required to inhibit the growth of biofilm cells. Eradication of biofilms could not be achieved by medihoney, even at the highest concentration studied. The antibiofilm properties of medihoney were multi-faceted, including sugar content, pH, osmolality and the proposed active ingredient, methylglyoxal (MGO). MGO actually played a less important role than other medihoney characteristics. The colony biofilm assay and the drip flow biofilm reactor showed that medihoney was unable to prevent the maturation of biofilms after early organism adherence, and only had a weak activity against established biofilms. Conclusion Our work suggests a suboptimal effectiveness of medihoney in preventing driveline exit site infections due to biofilm development, warranting further clinical trials before its widespread use can be justified.

Machine Learning Analyses on Data including Essential Oil Chemical Composition and In Vitro Experimental Antibiofilm Activities against Staphylococcus Species.

Biofilm resistance to antimicrobials is a complex phenomenon, driven not only by genetic mutation induced resistance, but also by means of increased microbial cell density that supports horizontal gene transfer across cells. The prevention of biofilm formation and the treatment of existing biofilms is currently a difficult challenge; therefore, the discovery of new multi-targeted or combinatorial therapies is growing. The development of anti-biofilm agents is considered of major interest and represents a key strategy as non-biocidal molecules are highly valuable to avoid the rapid appearance of escape mutants. Among bacteria, staphylococci are predominant causes of biofilm-associated infections. Staphylococci, especially Staphylococcus aureus (S. aureus) is an extraordinarily versatile pathogen that can survive in hostile environmental conditions, colonize mucous membranes and skin, and can cause severe, non-purulent, toxin-mediated diseases or invasive pyogenic infections in humans. Staphylococcus epidermidis (S. epidermidis) has also emerged as an important opportunistic pathogen in infections associated with medical devices (such as urinary and intravascular catheters, orthopaedic implants, etc.), causing approximately from 30% to 43% of joint prosthesis infections. The scientific community is continuously looking for new agents endowed of anti-biofilm capabilities to fight S. aureus and S epidermidis infections. Interestingly, several reports indicated in vitro efficacy of non-biocidal essential oils (EOs) as promising treatment to reduce bacterial biofilm production and prevent the inducing of drug resistance. In this report were analyzed 89 EOs with the objective of investigating their ability to modulate bacterial biofilm production of different S. aureus and S. epidermidis strains. Results showed the assayed EOs to modulated the biofilm production with unpredictable results for each strain. In particular, many EOs acted mainly as biofilm inhibitors in the case of S. epidermidis strains, while for S. aureus strains, EOs induced either no effect or stimulate biofilm production. In order to elucidate the obtained experimental results, machine learning (ML) algorithms were applied to the EOs’ chemical compositions and the determined associated anti-biofilm potencies. Statistically robust ML models were developed, and their analysis in term of feature importance and partial dependence plots led to indicating those chemical components mainly responsible for biofilm production, inhibition or stimulation for each studied strain, respectively.

Identification and characterization of bioengineered nisin derivatives that inhibit the opportunistic pathogen Staphylococcus epidermidis

Staphylococcus epidermidis is a major cause of hospital-acquired infections particularly on indwelling medical devices and implants. Infections caused by this pathogen are difficult to treat with standard antimicrobial agents mainly as the bacterium can establish biofilms on artificial surfaces. Novel control methods are needed and one such alternative may be bacteriocins; these are antimicrobial peptides produced by some bacteria that inhibit other specific bacteria. They are potent, safe, and stable and are easy to produce using biotechnological based strategies. Additionally, as they are gene encoded they can be easily modified or engineered to enhance their activity. In the present study, we demonstrate the ability of the bacteriocin nisin to inhibit a collection of clinical S. epidermidis strains under standard laboratory conditions. In addition, a bank of bioengineered nisin derivatives was screened using agar-based deferred antagonism assays and derivatives with enhanced antimicrobial activity compared to the wild-type nisin were identified. These derivative peptides are currently being purified using a combination of chromatography-based approaches and their potency and stability are being examined. Future experiments will focus on examining the ability of the nisin derivatives in inhibiting S. epidermidis biofilms on medical device materials (e.g. stainless steel and polyvinyl chloride) both alone and in combination with conventional antibiotics. It is hoped that one of the nisin derivatives analysed in this study may ultimately be used to control or prevent infections caused by S. epidermidis.