Epidermidis Strains Research Articles

English

Plants have been an important source of secondary metabolites known for their diverse biological activities; some have been shown to inhibit the development of certain pathogenic microorganisms. Herein, the antimicrobial activity of the carbon tetrachloride, hexane, ethanol, and aqueous extracts of leaves of Stevia rebaudiana Bertoni against Staphylococcus aureus (strain 921), Staphylococcus epidermidis (strains 965, 982, and 735), and Pseudomonas aeruginosa (strains RO3 and RO4) was presented. The antibacterial activity was evaluated using the disk diffusion method, and the minimal inhibitory concentration (MIC) was determined. The results show that, even at the lowest evaluated concentration (1.06 mg/mL), the hexane extract had an inhibitory effect for all the studied microorganisms. The aqueous extract exhibited high inhibition values (84.4%), on S. epidermidis (strain 965). These results indicate that compounds contained in non-polar extracts of S. rebaudiana could be potential candidates as conventional pharmaceutical drugs against bacteria, resistant to conventional antibiotics. Key words: Stevia rebaudiana, antimicrobial properties, plant extracts.

The methicillin-resistant S. epidermidis strain RP62A genome mining for potential novel drug targets identification

In unique biofilm-forming methicillin-resistant (MRSE) strains of S. epidermidis have become a serious medical problem due to the emergence of antibiotic resistance strains. There is an essential need to develop novel drug targets to address the new intervention challenge for such multidrug-resistant bacteria. Here we utilized the available genomes and proteome datasets resources of MRSE S. epidermidis RP62A for comparative and subtractive genome analyses to identify novel drug targets for future drug discovery approaches. The comparative genome based molecular biology database resources scanning identified 255 proteins as essential human non-homologs targets for epidermidis RP62A that involve in essential metabolic pathways. Among these 12 targets were found to be involved in pathogen unique metabolic pathways. Druggability potential of each of these targets by Drug Bank database further identified 5 proteins as druggable essential proteins. As antimicrobial agents inflict beneficial gut microbiome and the antibiotics causes less agitation in gut flora are considered as best candidates. We pursued additional analysis and screen our lead targets proteins against metagenomes databases holding whole genome sequences of human gut microbiome. The negligible biological similarity with gut microbiota genomes datasets predicting the future implementation of our lead identified proteins as druggable targets for S. epidermidis RP62A that may not affect the essential humans gut microbiota. Based on these evidences, we are speculating that the putative druggable proteins addressed in this study are potent enough for their future evaluation as therapeutic targets to combat the S. epidermidis RP62A infections.

Ica-status of clinical Staphylococcus epidermidis strains affects adhesion and aggregation: a thermodynamic analysis.

Staphylococcus epidermidis is a major nosocomial pathogen associated with infections of indwelling medical devices. One important virulence factor of these organisms is their ability to adhere to devices and form biofilms. In this study, we evaluated the effect of the ica operon on cell surface hydrophobicity, thermodynamics of adhesion, and biofilm formation for seven S. epidermidis strains. The surface free energy parameters of the bacterial cell surface and the substratum were determined by contact angle measurement. Biofilm formation was assayed using crystal violet staining. Results showed that ica-positive strains demonstrated a higher hydrophobic characteristic than ica-negative strains, suggesting that the ica-operon seems to determine the cell surface hydrophobicity of S. epidermidis. Interaction of ica-positive strains with a tissue-culture treated polystyrene surface was energetically favourable (ΔGTot<0), in contrast to ica-negative strains (ΔGTot>0). The interfacial free energy of aggregation of S. epidermidis was lower for ica-positive than for ica-negative strains. Our study suggests that, in addition to biofilm formation, adhesion and aggregation of clinical S. epidermidis is stimulated in ica-positive strains by influencing the thermodynamics of interaction.

Prevalence, clonality, and pathogenicity of Staphylococcus epidermidis isolates in newborn feces.

Coagulase-negative staphylococci (CoNS) are the most prevalent pathogens causing late-onset sepsis in neonates. The question is whether neonates acquire endemic hospital-adapted clones or incidentally occurring CoNS strains after birth during their hospital stay. Therefore, a prospective study was performed on the prevalence of CoNS in the stool of babies (born vaginally or by cesarean section) during their first days of life. Their clonal relatedness and potential to induce invasive disease were characterized. CoNS were analyzed from the stool samples of newborns with a load of CoNS above 103 colony-forming units (CFU)/mL. The identification of CoNS was performed phenotypically and genotypically. For typing, repetitive polymerase chain reaction (PCR), pulsed-field gel electrophoresis, and multilocus sequence typing were used. Resistance profiles, biofilm production, the presence of icaAD and of IS256 were determined as well. From a total of 207 stool samples (56 newborns), CoNS were detected in 41% of the newborns, mostly on day 3 for the first time (62.5%). Staphylococcus epidermidis was isolated in 85.7% of cases, harbored no IS256 element, and mostly expressed no biofilm. The isolates were separated into four main clusters by repetitive sequence-based PCR. 24% of the strains showed no antimicrobial resistance. 20% were resistant against four antibiotics of two different antibiotic classes. The remaining strains were resistant only against one antimicrobial substance class. Thus, it can be concluded that newborns do not acquire hospital-adapted endemic, multidrug-resistant S. epidermidis isolates during their first days of life. Yet, the results support the thesis that, during hospital stay, environmental parameters may convert sensible/noninvasive S. epidermidis strains into multidrug-resistant strains with characteristics of invasiveness.

Antibacterial effect of hydrogen peroxide-titanium dioxide suspensions in the decontamination of rough titanium surfaces

The chemical decontamination of infected dental implants is essential for the successful treatment of peri-implantitis. The aim of this study was to assess the antibacterial effect of a hydrogen peroxide-titanium dioxide (H2O2–TiO2) suspension against Staphylococcus epidermidis biofilms. Titanium (Ti) coins were inoculated with a bioluminescent S. epidermidis strain for 8 h and subsequently exposed to H2O2 with and without TiO2 nanoparticles or chlorhexidine (CHX). Bacterial regrowth, bacterial load and viability after decontamination were analyzed by continuous luminescence monitoring, live/dead staining and scanning electron microscopy. Bacterial regrowth was delayed on surfaces treated with H2O2–TiO2 compared to H2O2. H2O2-based treatments resulted in a lower bacterial load compared to CHX. Few viable bacteria were found on surfaces treated with H2O2 and H2O2–TiO2, which contrasted with a uniform layer of dead bacteria for surfaces treated with CHX. H2O2–TiO2 suspensions could therefore be considered an alternative approach in the decontamination of dental implants.

Role of extracellular polymeric substances in polymicrobial biofilm infections of Staphylococcus epidermidis and Candida albicans modelled in the nematode Caenorhabditis elegans.

Biofilms are formed by communities of microorganisms living in a self-produced extracellular polymeric matrix attached to a surface. When living in a biofilm microorganisms change phenotype and thus are less susceptible to antibiotic treatment and biofilm infections can become severe. The aim of this study was to determine if the presence of multikingdom microorganisms alters the virulence of a biofilm infection in a host organism. The coexistence of Candida albicans and Staphylococcus epidermidis in biofilm was examined in the nematode model Caenorhabditis elegans. It was evaluated if the hyphal form of C. albicans and extracellular polymeric substances (EPS) formed by S. epidermidis increases biofilm virulence. Survival assays were performed, where C. elegans nematodes were exposed to S. epidermidis and C. albicans. Single inoculation assays showed a decreased survival rate after 2 days following exposure, while dual inoculation assays showed that a clinical S. epidermidis strain together with C. albicans significantly increased the virulence and decreased nematode survival. EPS seem to interfere with the bacterial attachment to hyphae, since the EPS overproducing S. epidermidis strain was most virulent. The clinical S. epidermidis paired with C. albicans led to a severe infection in the nematodes resulting in reduced survival.

Commensal bacterial modulation of the host immune response to ameliorate pain in a murine model of chronic prostatitis.

The human commensal microflora plays an essential role in modulating the immune response to control homeostasis. Staphylococcus epidermidis, a commensal bacterium most commonly associated with the skin exerts such effects locally, modulating local immune responses during inflammation and preventing superinfection by pathogens such as Staphylococcus aureus. Although the prostate is considered by many to be sterile, multiple investigations have shown that small numbers of gram-positive bacterial species such as S. epidermidis can be isolated from the expressed prostatic secretions of both healthy and diseased men. Chronic pelvic pain syndrome is a complex syndrome with symptoms including pain and lower urinary tract dysfunction. It has an unknown etiology and limited effective treatments but is associated with modulation of prostate immune responses. Chronic pelvic pain syndrome can be modeled using murine experimental prostatitis (EAP), where CD4+ve IL17A+ve T cells have been shown to play a critical role in disease orchestration and development of pelvic tactile allodynia. Here, we report that intraurethral instillation of a specific S. epidermidis strain (designated NPI [non-pain inducing]), isolated from the expressed prostatic secretion of a healthy human male, into EAP-treated mice reduced the pelvic tactile allodynia responses and increased CD4+ve IL17A+ve T-cell numbers associated with EAP. Furthermore, a cell wall constituent of NPI, lipoteichoic acid, specifically recapitulates these effects and mediates increased expression of CTLA4-like ligands PDL1 and PDL2 on prostatic CD11b+ve antigen-presenting cells. These results identify a new potential therapeutic role for commensal S. epidermidis NPI lipoteichoic acid in the treatment of prostatitis-associated pain.

612 Specific strains of S. epidermidis suppress UV-induced skin tumor formation by production of 6-N-hydroxyaminopurine, a DNA synthesis inhibitor

Evidence continues to accumulate that specific members of the skin microbiome impart important elements to cutaneous defense. In this study, we sought to understand the mechanism of action of specific strains of Staphylococcus epidermidis that we previously screened from healthy human skin based on capability to kill Staphylococcus aureus. Remarkably, these strains were shown to abundantly produce 6-N-hydroxyaminopurine (6-HAP), a molecule not previously known to be produced in nature. Given the structural similarity of this small molecule to adenine, we tested if it could inhibit DNA synthesis in vitro and noted that 6-HAP was a potent competitive inhibitor of Klenow DNA polymerase by competing with adenosine for DNA extension. 6-HAP also potently inhibited BrdU incorporation and the growth of several tumor lines, such as Pam212 SCC, L5178 lymphoma and B16F10 melanoma. In contrast, 6-HAP did not affect growth of normal keratinocytes (NHEK), indicating selective anti-proliferative activity. The selective resistance of NHEKs to 6-HAP was due to expression of Mitochondrial Amidoxime Reducing Components (mARCs), an enzyme that catalyzes reduction of N-hydroxylated compounds, since NHEKs treated with siRNA for mARC1 lost resistance to 6-HAP. We next explored activity of 6-HAP in several mouse tumor models and observed that I.V. injection with 6-HAP was non-toxic but suppressed tumor size of B16F10 melanoma by 60% (P < 0.01) compared to vehicle control. Furthermore, the skin of SHK-1 hairless mice was treated with DMBA and low-dose UV-B (180mJ/cm2) and then colonized by a S. epidermidis strain producing 6-HAP. These mice had 54% lesser tumor incidence at 10 wks and 26% of the total tumor number at 12 wks (P < 0.001) compared to mice colonized by a similar abundance of control strain. These findings show that some skin commensal bacteria may provide selective resistance against neoplasia.

Characterization of Staphylococcus epidermidis strains isolated from industrial cleanrooms under regular routine disinfection.

The purpose of this study was the genotypic and phenotypic characterization of 57 strains of Staphylococcus epidermidis isolated from cleanroom environments, based on their biofilm formation and antimicrobial resistance profiles. Biofilm formation was investigated using real-time PCR (icaA, aap, bhp genes), the Congo red agar method and the crystal violet assay. The majority of the strains (59·7%; 34/57) did not form biofilms according to the crystal violet assay, although the biofilm-associated genes were present in 94·7% (54/57) of the strains. Of the biofilm formers (40·4%; 23/57), 39·1% (9/23) have been identified as strong biofilm formers (>4× crystal violet absorbance cut-off). Resistance to a commercial disinfectant and its quaternary ammonium active component, didecyl-dimethyl-ammonium chloride (DDAC), was determined according to minimum inhibitory concentrations (MICs) and the presence of the qac (quaternary ammonium compound) genes. More than 95% (55/57) of the Staph. epidermidis strains had the qacA/B and qacC genes, but not the other qac genes. The MICs for the disinfectant and DDAC varied among the Staph. epidermidis strains, although none were resistant. Although 59·6% of the Staph. epidermidis strains did not form biofilms and none were resistant to DDAC, more than 94% had the genetic basis for development of resistance to quaternary ammonium compounds, and among them at least 14·0% (8/57) might represent a high risk to cleanroom hygiene as strong biofim formers with qacA/B and qacC genes. To assure controlled cleanroom environments, bacterial strains isolated from cleanroom environments need to be characterized regularly using several investigative methods.

Molecular Characterization of Staphylococcus Epidermidis Isolates Recovered from Patients Admitted to a Referral Hospital in Iran

Background: Coagulase-negative Staphylococci (CoNS), especially Staphylococcus epidermidis, are considered as commensal bacteria of human skin and oral-nasal mucosa. Because of having many various virulence factors as well as the emergence of the multidrug-resistant (MDR) strains, this microorganism is regarded as a major cause of hospital-acquired bacteremia and invasive nosocomial infections. Objectives: Due to the significance of S. epidermidisassociated infections in different health care units, the aim of this study was to determine the molecular epidemiology of S. epidermidis in true infection-associated isolates. Methods: In our cross sectional study, a total of 183 S. epidermidis strains was collected during 8 months. Only 40 strains, which were identified as “true” infection-associated strains, were assessed via antibiotic susceptibility testing and pulsed field gel electrophoresis (PFGE) typing. Finally, strains with specific assigned pulsotypes were also analyzed by multilocus sequence typing (MLST). Results: Specimens were most commonly obtained from the bloodstream, wound, and catheter. More than half (75%) of the tested strains were found to be resistant to cefoxitin and ciprofloxacin (60%), while 45% of strains showed resistance to tetracycline. Using PFGE, 3 clones (A, B and C) were identified. According to MLST, the frequency of ST2 and ST5 was more prominent than the other STs. Conclusions: In accordance with the life cycle of S. epidermidis, molecular characterization of invasive isolates is essential for controlling the epidemic strains in health care units.

Genetic and biochemical characterization of staphylococci occurring in Novosibirsk, Russia

Staphylococci are capable of penetrating many human tissues and organs, causing superficial and deep purulent infections, respiratory and urinary tract infections, food poisoning and intoxication. Last years, coagulase­negative staphylococci were the cause of infection in many cases. Infectious agents, namely Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis, were detected more often as nosocomial infections. A particular danger of these infections is a high virulence and pathogenicity of bacterial strains and their resistance to various anti ­ biotics. Methicillin­resistant staphylococci are especially difficult to treat. The correct identification of staphylococci and their sensitivity to antibiotics are important for clinical diagnosis and appointment of adequate drug therapy. Rapid and accurate identification of Staphylococcus species and detection of their sensitivity to antibiotics is quite important. The aim of this study was to study staphylococci isolated in Novosibirsk from human, animal and environmental samples. A collection of 100 staphylococcus strains was analyzed. Staphylococcus species were identified by sequencing the 16S rRNA gene. Eleven staphylococcus species were identified. Among the strains obtained from hospitalized patients, Staphylococcus aure us dominated (79.1 %), Staphylococcus epidermidis amounted to about 12.5 %. However, S. aureus and S. epi dermidis strains were isolated in an approximately equal proportion from community­associated samples. Identification of coagulase positive strains was performed using a standard biochemical method and by real­time PCR of the coa gene. 100 % coincidence between the presence of the gene and coagulase activity for S. aureus strains was recorded, which suggests that detection of the coa gene can be used as a correct method for S. aure us identification. A high coincidence rate (99 %) was reveal ed between the phenotypic resistance to oxacillin and the presence of the staphylococcal mecA gene. The study of staphylococci for the presence of the mecA gene can be considered as an alternative to the phenotypical method for identification of methicillin­resistant strains of staphylococci.

Hastanelerde sık kullanılan bazı antiseptiklerin biyofilm oluşturan ve oluşturmayan Staphylococcus epidermidis suşlarına karşı antibakteriyel etkinliğinin araştırılması

Staphylococcus epidermidis, a commensal bacterium, commonly found on the surfaces of the human body especially on skin. It is a member of human normal flora. Nowadays, S. epidermidis is regarded as the most frequent cause of nosocomial infections especially in immunocompromised patients. Biofilm formation is essential for its pathogenicity in these infections. The aim of this study was to investigate the antibacterial efficacy of five commonly used antiseptics (7.5% povidone-iodine, 10% povidone-iodine, 70% ethanol-0.5% chlorhexidine digluconate, 70% 2-propanol- chlorhexidine digluconate, 70% 2-propanol) against biofilm forming (S. epidermidis ATCC 35984) and non-biofilm forming (S. epidermidis ATCC 12228) S. epidermidis strains. Susceptibilities of the strains to antiseptics were investigated by quantitative suspension test at contact times of 30 seconds and one minute. According to the results of this in vitro study, there is no difference found between the antibacterial efficacies of tested antiseptics against biofilm forming and non-biofilm forming S. epidermidis.

Overview of Genetic Background Beyond Polysaccharide Intercellular Adhesion Production in Staphylococcus epidermidis

Background: An important observation during quantification experiments of Staphylococcus epidermidis biofilm is that there is a great difference in the biofilm biomass of different strains despite the same experimental conditions. Objectives: This study aimed to study the genotypic background beyond differential rates of Polysaccharide Intercellular Adhesion (PIA) production in S. epidermidis biofilm forming strains. Methods: A number of 126 strains were isolated from blood cultures (n = 40), catheter cultures (n = 50), and other specimens (n = 36). The strains were obtained from patients hospitalized at the bone marrow transplant center of Tunis. Biofilm micro-plate assay, hemagglutination, and susceptibility to proteinase K methods were used to assess biofilm characteristics in the studied strains. Conventional and real time PCR were used to assess genotypic background of biofilm formation. Results: Using PCR method, we demonstrated that there is a significant difference in ica genes (P < 0.01) and not in adhesion rsb and sar genes distribution between biofilm forming and non-biofilm forming strains. Almost all strains harbored agr type I. None of studied strains harbored IS256 inside ica operon. Ica-independent biofilm formation was detected in 11 strains that were confirmed to have proteinaceous matrix. Using Kernel density estimation, we established that biofilm biomass was higher in ica-dependent than ica-independent biofilm forming population. Using qRT-PCR, we found a significant correlation between biofilm biomass and RNAIII expression level (r2 = 0.95); but no correlation was found for biofilm biomass neither with icaA nor with ccpA genes. Conclusions: Data reported here indicated that there is no specific genetic combination beyond the quantity of biofilm biomass in S. epidermidis. Biofilm biomass seemed to be controlled by RNAIII expression level. Further interest should be directed to biofilm dispersal since it seems that the key difference in biofilm biomass ability of S. epidermidis strains relates to factors regulating this stage.

Mutation in the L3 Ribosomal Protein Could Be Associated with Risk of Selection of High-Level Linezolid-Resistant Staphylococcus epidermidis Strains.

Linezolid (LZD) has arisen as an alternative treatment in diabetic foot osteitis due to staphylococci. LZD resistance selection is difficult and involved various molecular mechanisms. As a better knowledge of those mechanisms could be beneficial for pathogenic strains' screening, we simulated in vitro the spontaneous mutagenesis process that leads to LZD-resistant strains from two Staphylococcus epidermidis strains responsible for monomicrobial diabetic foot osteitis. LZD high resistance was selected for both strains, with the same timeline of mutation appearance. Mutation in L3 protein (G152D) occurred first and quickly, but did not cause phenotypically detectable resistance or fitness cost. It was later followed by different 23S rRNA mutations (G2505A, G2447T), leading this time to detectable resistance (minimum inhibitory concentration [MIC] ≥8 mg/L). This phenomenon underlies the difficulty of resistance selection in coagulase-negative staphylococci (CoNS). This study is the first description of G2505A mutation in CoNS. Various phenotypical impacts were observed depending on strain and mutation: (i) fitness cost of G2505A and G2447T mutations; (ii) loss of erythromycin resistance concomitantly with L3 mutation selection; (iii) correlation between number of mutated rrl copies and LZD resistance level for G2447T. In conclusion, the risk of selection of high-level LZD-resistant S. epidermidis strains is weak, but does exist. It could probably appear in case of long-term treatment and be favored in the case of a pre-existing mutation in L3 ribosomal protein. Thus, broad screening conditions for pathogenic strains should probably be considered.

Staphylococcus epidermidis ΔSortase A strain elicits protective immunity against Staphylococcus aureus infection.

Staphylococcus aureus and Staphylococcus epidermidis are two of the most significant opportunistic human pathogens, causing medical implant and nosocomial infections worldwide. These bacteria contain surface proteins that play crucial roles in multiple biological processes. It has become apparent that they have evolved a number of unique mechanisms by which they can immobilise proteins on their surface. Notably, a conserved cell membrane-anchored enzyme, sortase A (SrtA), can catalyse the covalent attachment of precursor bacterial cell wall-attached proteins to peptidoglycan. Considering its indispensable role in anchoring substrates to the cell wall and its effects on virulence, SrtA has attracted great attention. In this study, a 549-bp gene was cloned from a pathogenic S. epidermidis strain, YC-1, which shared high identity with srtA from other Staphylococcus spp. A mutant strain, YC-1ΔsrtA, was then constructed by allelic exchange mutagenesis. The direct survival rate assay suggested that YC-1ΔsrtA had a lower survival capacity in healthy mice blood compare with the wild-type strain, indicating that the deletion of srtA affects the virulence and infectious capacity of S. epidermidis YC-1. YC-1ΔsrtA was then administered via intraperitoneal injection and it provided a relative percent survival value of 72.7% in mice against S. aureus TC-1 challenge. These findings demonstrate the possbility that YC-1ΔsrtA might be used as a live attenuated vaccine to produce cross-protection against S. aureus.

IL-10 plays an important role in the control of inflammation but not in the bacterial burden in S. epidermidis CNS catheter infection.

BackgroundShunt infection is a frequent and serious complication in the surgical treatment in hydrocephalus. Previous studies have shown an attenuated immune response to these biofilm-mediated infections. We proposed that IL-10 reduces the inflammatory response to Staphylococcus epidermidis (S. epidermidis) CNS catheter infection.MethodsIn this study, a murine model of catheter-associated S. epidermidis biofilm infection in the CNS was generated based on a well-established similar model for S. aureus. The catheters were pre-coated with a clinically derived biofilm-forming strain of S. epidermidis (strain 1457) which were then stereotactically implanted into the lateral left ventricle of 8-week-old C57BL/6 and IL-10 knockout (IL-10 knockout) mice. Bacterial titers as well as cytokine and chemokine levels were measured at days 3, 5, 7, and 10 in mice implanted with sterile and S. epidermidis-coated catheters.ResultsCultures demonstrated a catheter-associated and parenchymal infection that persisted through 10 days following infection. Cytokine analysis of the tissue surrounding the catheters revealed greater levels of IL-10, an anti-inflammatory cytokine, in the infected group compared to the sterile. In IL-10 KO mice, we noted no change in bacterial burdens, showing that IL-10 is not needed to control the infection in a CNS catheter infection model. However, IL-10 KO mice had increased levels of pro-inflammatory mediators in the tissues immediately adjacent to the infected catheter, as well as an increase in weight loss.ConclusionsTogether our results indicate that IL-10 plays a key role in regulating the inflammatory response to CNS catheter infection but not in control of bacterial burdens. Therefore, IL-10 may be a useful therapeutic target for immune modulation in CNS catheter infection but this should be used in conjunction with antibiotic therapy for bacterial eradication.

Bactericidal effect of γ-radiation with 137Cesium in platelet concentrates

Introductionγ-radiation is a method that was originally designed for inactivation of T lymphocytes in blood and blood components in order to prevent transfusion associated–graft versus host disease (TA–GVHD). Klebsiella pneumoniae, Escherichia coli, Enterococcus faecium and Staphylococcus epidermidis strains are important pathogens in blood banks since they have been related to post-transfusional sepsis. This study was conducted to demonstrate that γ-radiation is effective in reducing the viability of bacteria in platelet concentrates (PC). Material and MethodsKlebsiella pneumoniae, E. coli, E. faecium and S. epidermidis strains were adjusted at 101 to 106 CFU/ml and used in artificial contamination assays in PC. Contaminated platelet concentrates were subjected to γ–radiation with doses of 2500 cGy in a 137Cesium irradiator. The average of surviving bacteria at different bacterial concentrations, logarithmic reduction values (LRV) and bacterial death after γ–radiation percentage was calculated. ResultsEscherichia coli and K. pneumoniae were eliminated in 101 to 103 CFU; in contrast with 104 to 106 CFU, the LRV were 2.4, 2.6 and 2.6 for E. coli and 3.3, 2.7 and 3.0 for K. pneumoniae strains at 104, 105 and 106 CFU respectively. For Gram-positive strains, 101 CFU in PC, the inactivation post γ-radiation was not completed. Logarithmic reduction values post γ-radiation were 0.8 to 1.2 for E. faecium and S. epidermidis strains respectively. Conclusionγ-radiation cannot be an alternative for the inactivation of pathogens in PC, because of the bacterial concentration and pathogen nature – being resistant to γ-radiation, the Gram-positive bacteria.

Characterization of CRISPR-Cas system in clinical Staphylococcus epidermidis strains revealed its potential association with bacterial infection sites

Staphylococcus epidermidis is considered as a major cause of nosocomial infections, bringing an immense burden to healthcare systems. Virulent phages have been confirmed to be efficient in combating the pathogen, but the prensence of CRISPR-Cas system, which is a bacterial immune system eliminating phages was reported in few S. epidermidis strains. In this study, the CRISPR-Cas system was detected in 12 from almost 300 published genomes in GenBank and by PCR of cas6 gene in 18 strains out of 130 clinical isolates obtained in Copenhagen. Four strains isolated in 1965–1966 harboured CRISPR elements confirming that this immunity system was not recently acquired by S. epidermidis. In these CRISPR-positive strains, 44 and 12 spacers were found to belong to CRISPR1 and CRISPR2 elements, respectively. However, only 15 spacers displayed homology to reported phages and plasmids DNA. Interestingly, 5 different spacers located in the CRISPR1 locus with homolgy to virulent phage 6ec DNA sequences, and 19 strains each carrying 2 or 3 different spacers recognizing this phage, implied that the CRISPR-Cas immunity could be abrogated by nucleotide mismatch between the spacer and its target phage sequence, while new spacers obtained from the evolved phage could recover the CRISPR interference. In addition, phylogenetic analysis of the 29 CRISPR-positive isolates divided them into four lineages, with 81% human blood isolates as a distinct sub-lineage, suggesting that the CRISPR difference is closely related to diverse habitats. Knowledge of CRISPR and its prevalence may ultimately be applied in the understanding of origin and evolution of CRISPR-positive S. epidermidis strains.

Genetic and phenotypic traits of staphylococcus epidermidis strains causing post‐cataract endophthalmitis compared to commensal conjunctival flora

PurposeThe aim of this study was to evaluate the virulence and antibiotic resistance traits of S. epidermidis strains causing post‐cataract endophthalmitis compared to those of the normal conjunctival flora in non‐infected control patients.MethodsThis prospective study included 22 patients (22 eyes) with acute post‐cataract endophthalmitis and 72 uninfected control patients (72 eyes). Antibiotic susceptibilities were evaluated using the Vitek II® automated system. These strains were then tested for the presence of eight virulence genes (icaA, icaB, icaC, icaD, atlE, aap, and capA), the insertion sequence IS256, and the mecA gene coding for methicillin‐resistance.ResultsCulture of 72 conjunctival samples collected from 41 patients allowed isolation of 43 S. epidermidis strains. The strains from the endophthalmitis group patients displayed higher prevalence rates for aap, atlE and mecA genes carriage compared to those of the control group (77% vs. 42%, p = 0.007; 100% vs. 79%, p = 0.02; and 54% vs. 11%, p &lt; 0.001, respectively) and more frequently harbored simultaneously the mecA and icaA genes (13%) compared to the control group (2.3%, p = 0.01). They were also significantly more resistant than control strains to methicillin, fluoroquinolones, and the aminoglycosides.ConclusionsA higher capacity of adhesion to intraocular lens and formation of biofilms, and a higher resistance to antibiotics were found in S. epidermidis strains causing post‐cataract endophthalmitis compared to those of the normal conjunctival flora. The selection of such strains in endophthalmitis patients could be related to their specific virulence traits and/or their resistance to antibiotics used for prophylaxis of endophthalmitis.

Methicillin resistance and biofilm production of Staphylococcus epidermidis isolates from infectious and normal flora conjunctiva.

Coagulase-negative staphylococci have been reported to be the most frequent cause of bacterial postoperative endophthalmitis. Biofilm formation is the major virulence factor of Staphylococcus epidermidis and is often associated with methicillin resistance. This study aims at evaluating the presence of biofilm-producing and methicillin resistance genes (mecA) in S. epidermidis. S. epidermidis isolated from clinically infected sites (group 1) and from normal human conjunctiva (group 2) were studied. All the isolates were tested for their ability to produce biofilm by the conventional Christensen´s method and the presence of mecA by PCR using the 22-mer oligonucleotides as primers. In total 20 isolates from group 1 and 22 from group 2 were studied. Biofilm and mecA were detected in 15 (75%) and in 14 (70%) in group 1 as compared to 8 (36.3%) and 4 (18.2%) in group 2 (p=0.016). Simultaneously, biofilm production and presence of mecA genes were observed in 13/20 (65.0%) in group 1, and 4/22 (18.2%) in group 2 (p=0.002). Multi-resistance was observed in 55% in group 1 and 9% in group 2 (p=0.002); 57% of the biofilm-producing strains was multi-resistant in contrast to none of the non-producing strains. In all multi-resistant strains, biofilm production was seen. Biofilm formation capacity was widely distributed, particularly among mecA (+) S. epidermidis strains, which also displayed a high diversity of antibiotic resistance profiles.