Meticillin-resistant S. Aureus Isolates Research Articles

Does biofilm formation have different pathways in Staphylococcus aureus?

Objective(s):Biofilm formation is one of the most important factors in the development of infections caused by Staphylococcus aureus. In this study, the expression levels of genes responsible for biofilm formation were studied in methicillin sensitive and methicillin resistant S. aureus.Materials and Methods:A total of 100 meticillin-resistant s.aureus (MRSA) and meticillin-sensetive s.aureus (MSSA) isolates were studied. Bacterial biofilm formation was evaluated phenotypically using microtiter plate method. Real-time PCR tests were conducted to determine the expression levels of genes involved in biofilm formation.Results:Quantitative biofilm formation test was repeated three times for each specimen. The prevalence of weak, medium, and strong biofilm producers were 16%, 49%, and 35%, respectively. In MSSA isolates, expression levels of ica genes increased compared to the fnbA, fnbB, clfA and clfB genes. These results were different in MRSA isolates, and ica genes showed a decreased gene expression levels compared to the aforementioned genes. Conclusion:Considering the results of this study, clf genes probably contribute to the same extent in both MRSA and MSSA isolates, and there is probably no significant difference in the role of these genes in these isolates. In addition, the results of this study indicated that MRSA may not use the conventional route for biofilm formation and may use independent pathways through Polysaccharide intercellular adhesion (PIA).

Virulence determinants, biofilm production and antimicrobial susceptibility in Staphylococcus aureus causing device-associated infections in a Tunisian hospital

Staphylococcus aureus is a clinically relevant pathogen that causes device-related infections (DRI) driven by several virulence factors. This study characterized S. aureus isolates involved in DRI in Tunisian patients. Forty consecutive S. aureus strains causing DRI and 47 randomly selected S. aureus strains causing non-device-related infections (NDRI) were collected. All strains were screened phenotypically for antibiotic susceptibility and biofilm forming ability. They were investigated for accessory gene regulator (agr) types, biofilm encoding genes (icaADBC), adhesins, leukotoxins, toxic shock toxin, enterotoxins and exotoxins encoding genes by polymerase chain reaction. Meticillin-resistant S. aureus (MRSA) strains were further characterized by staphylococcal cassette chromosome mec (SCCmec) typing. MRSA rates among DRI and NDRI isolates were 23% and 49% (P=0.02), respectively. The DRI isolates formed biofilm more frequently (n=32) than the NDRI isolates (n=28) (P=0.04), with predominance of the moderate biofilm producer category (P=0.027). All biofilm-positive isolates except four harboured icaADBC genes. A significant difference was observed between DRI and NDRI isolates for fnbA (53-77%), spa (45-26%), sdrD (80-55%) and sen (33-11%) genes. DRI strains were agrI (48%) and agrII (30%) types, whereas NDRI strains were agrI (36%) and agrIII (43%) types. SCCmec type IV was carried by 50% of MRSA isolates. This study highlights the virulence potential displayed by S. aureus isolated from DRI in comparison with NDRI.

Preliminary investigations into developing all-D Omiganan for treating Mupirocin-resistant MRSA skin infections.

Staphylococcus aureus is the primary pathogen responsible for the majority of human skin infections, and meticillin-resistant S.aureus (MRSA) currently presents a major clinical concern. The overuse of Mupirocin, the first-line topical antibacterial drug over 30years, has led to the emergence of Mupirocin-resistant MRSA, creating a clinical concern. The antimicrobial peptide Omiganan was touted to be a promising antibacterial drug candidate due to its rapid membrane-disrupting bactericidal mode of action, entering clinical trials in 2005 as a topical gel to prevent catheter site infections. However, drug development ceased in 2009 due to a lack of efficacy. We postulate this to be due to proteolytic degradation caused by endogenous human skin proteases. Herein, we tested our hypothesis using Omiganan and its all-D enantiomer in a human skin protease stability assay, followed by anti-MRSA activity assay against of a panel of clinical MRSA isolates, a bactericidal/static determination and a time-kill assay to gauge all-D Omiganan's potential for further topical antibacterial drug development.

In vitro activity of dalbavancin against multidrug-resistant Staphylococcus aureus and streptococci from patients with documented infections in Europe and surrounding regions (2011–2013)

The in vitro activity of dalbavancin was evaluated against 9303 Staphylococcus aureus and 2670 streptococci, including multidrug-resistant (MDR) isolates, collected from hospitalised patients in Europe and surrounding regions from 2011 to 2013. Dalbavancin recently received approval for the treatment of acute bacterial skin and skin-structure infections by the US Food and Drug Administration (FDA) and the European Medicines Agency. Bacterial identification was confirmed by standard microbiological methods (including MALDI-TOF), and susceptibility testing was performed by reference broth microdilution methods. Dalbavancin susceptibility interpretations followed FDA/EUCAST criteria. Meticillin-resistant S. aureus (MRSA) and streptococci exhibiting resistance to at least three other drug classes were considered as MDR. Dalbavancin was highly active (MIC50/90, 0.06/0.06 mg/L; ≥99.9% susceptible) against MDR and non-MDR MRSA isolates. Vancomycin, daptomycin and linezolid were also active (99.6–100.0% susceptible) against MDR MRSA, however MIC90 values for these drugs were 8- to 16-fold higher than dalbavancin (MIC90 values of 1, 0.5 and 1 mg/L, respectively). All viridans group streptococci (VGS) and β-haemolytic streptococci were susceptible to dalbavancin regardless of resistance phenotype (MIC50/90 values of ≤0.03 mg/L and 0.06 mg/L, respectively). Dalbavancin MIC50/90 results (MIC50/90, ≤0.03/0.06 mg/L) against MDR VGS were at least eight-fold lower than those of vancomycin (MIC50/90, 0.5/1 mg/L), daptomycin (MIC50/90, 0.5/1 mg/L) and linezolid (MIC50/90, 0.5/1 mg/L). Overall, dalbavancin exhibited potent in vitro antibacterial activity against S. aureus and streptococci, including MDR phenotypes. Dalbavancin had the lowest MIC50/90 results against the isolates tested, relative to comparator agents, regardless of resistance phenotypes.

Frequency of MRSA in diabetic foot infections

Staphylococcus aureus is one of the most common bacterial pathogens isolated from diabetic foot infections (DFIs). The increasing prevalence of meticillin-resistant S. aureus (MRSA) in patients with diabetes is associated with complications. The aim of this study was to determine the prevalence of S. aureus in DFIs and antibiotic susceptibility patterns of MRSA and non-MRSA isolates. Identification of S. aureus and MRSA was performed by the phenotypic and molecular methods. The Kirby-Bauer and agar dilution methods were performed for determination of antibiotic susceptibility patterns. Thirty-four isolates of S. aureus were isolated from March 2014 to February 2015. The rate of MRSA was 38.23 % according to the disk cefoxitin and oxacillin agar dilution methods, and as by PCR method 12) 35.29 %), isolates were found to have the mecA gene. All MRSA and non-MRSA isolates were susceptible to linezolid and vancomycin. The resistance rate to ceftriaxone was high followed by amoxicillin-clavulanic acid, tetracycline, gentamicin, and erythromycin. The most common bacterial pathogen isolated from DFIs was S. aureus. To ensure effective treatment, accurate detection of MRSA is critical. Our findings showed that MRSA isolates had high-level resistance to antimicrobial agents and that appropriate antibiotic therapy, based on the antibiotic susceptibility pattern, is essential to ensure a good result.

Healthcare-associated Staphylococcus aureus Bacteremia in Children: Evidence for Reverse Vancomycin Creep and Impact of Vancomycin Trough Values on Outcome.

Elevated vancomycin minimum inhibitory concentrations (MICs) in Staphylococcus aureus have been associated with worse clinical outcomes in adults. For invasive meticillin-resistant S. aureus (MRSA) infections in adults, the Infectious Diseases Society of America recommends targeting vancomycin serum trough concentrations between 15 and 20 μg/mL. We evaluated trends in vancomycin MICs from healthcare-associated (HCA) S. aureus bacteremia isolates in children in addition to correlating vancomycin serum trough levels with clinical outcomes. Patients and isolates were identified from a prospective S. aureus surveillance study at Texas Children's Hospital (TCH). HCA S. aureus bacteremia isolates from 2003 to 2013 were selected. Vancomycin MICs by E-test were determined and medical records were reviewed. Acute kidney injury (AKI) was defined as doubling of the baseline serum creatinine. Three hundred forty-one isolates met inclusion criteria. We observed a reverse vancomycin creep among MRSA isolates in the study period with a decline in the proportion of isolates with vancomycin MIC ≥ 2 μg/mL (from 32.7% to 5.6%; P < 0.001). However, the proportion of MSSA isolates with MIC ≥ 2 μg/mL increased (from 2.9% to 9%; P = 0.04). Among patients who had vancomycin troughs performed, there was no difference in duration of bacteremia or fever with vancomycin trough >15 versus <15 μg/mL. A vancomycin trough >15 μg/mL was, however, an independent risk factor for AKI. Vancomycin MICs are shifting among HCA S. aureus bacteremia isolates with significant differences between MRSA and MSSA at TCH. Higher vancomycin troughs did not improve outcomes in pediatric HCA S. aureus bacteremia but were associated with increased nephrotoxicity. Further studies are needed to better understand optimal management of children with S. aureus bacteremia.

Genotype-specific prevalence of heterogeneous vancomycin-intermediate Staphylococcus aureus in Asian countries

Although heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) has been increasingly reported, the true prevalence of hVISA is unclear, especially in Asia. In this study, the genotype-specific prevalence of hVISA among meticillin-resistant S. aureus (MRSA) clinical isolates collected from Asian countries was determined. Among MRSA collections from South Korea, Taiwan, Hong Kong, Thailand, the Philippines, Vietnam, India and Sri Lanka in the ANSORP surveillance study during 2004 and 2006, isolates with a vancomycin minimum inhibitory concentration of ≥0.25mg/L were randomly selected. After screening by macro Etest, hVISA was confirmed using the modified population analysis profile method. MRSA isolates were typed by spa tying and multilocus sequence typing (MLST). Among 462 MRSA isolates, 16 (3.5%) were confirmed as hVISA. The proportion of hVISA was highest in South Korea and Vietnam (both 7.0%), followed by Thailand (3.2%) and Taiwan (1.9%). spa type t601 belonging to clonal complex (CC) 5 showed the highest proportion of hVISA (33.3%), and hVISA accounted for 6.9% among isolates of t002 belonging to CC5. Among CC239 isolates, only those of t037 were hVISA (1.6%). Among isolates of community-associated MRSA genotypes, hVISA was found only in those of t437 (4.8%) belonging to CC59, and no hVISA was found among those of CC30 or CC72. The prevalence of hVISA in the Asian region differed by country and was dependent upon the genotype of MRSA strains. It suggests that differences in hVISA prevalence between countries can be affected by the genotype distribution of MRSA strains.

The distribution of pathogenic and toxigenic genes among MRSA and MSSA clinical isolates

Staphylococcus aureus (S. aureus) is considered as a notorious nosocomial pathogen among hospitalized patients and community-dwelling subjects. Its increasing morbidity and mortality is believed to be due to antibiotic resistance. However, the data concerning molecular properties of infecting strains are few.In this study, a total of 192 S. aureus strains, including 88 (45.8%) meticillin-sensitive S. aureus (MSSA) and 104 (54.2%) meticillin-resistant S. aureus (MRSA) were recovered from clinical samples. The prevalence of subtypes containing staphylococcal cassette chromosome mec (SSCmec), staphylococcal enterotoxins (SEs), toxic shock syndrome toxin (TSST) and exfoliative toxin was assessed by PCR. Antibiotic susceptibility pattern and vancomycin resistance of each isolate were evaluated by disk diffusion method and micro-dilution method, respectively.9 (2.3%) strains required MIC > 2 mg/l of vancomycin, which significantly increased among multi drug resistant (MDR), MRSA and SCCmec type III strains (p < 0.05). 171 (89%), 140 (72.91%), 7 (3.6), 78 (48.6%), 5 (2.6%), 151 (78.64%), 129 (67.18%), 178 (92.7%) and 15 (7.8%) of 192 isolates harbored mecA, entA, entB, entC, entD, entE, eta, etb and tsst-1 genes, respectively. 31 (16.14%), 5 (2.6%), 95 (49.48%) and 7 (3.64%) of 192 isolates carried SCCmec type I, II, III and IV, respectively. We found a significantly higher rate of MRSA and resistance to all tested antibiotics, except to penicillin G, kanamycin and linezolide among the SCCmec type III class (p < 0.05).According to our findings, MSSA isolates should be taken as seriously as MRSA strains due to the potential presence of broad spectrum virulence factor genes.

Selection and characterisation of Staphylococcus aureus mutants with reduced susceptibility to the investigational oxazolidinone MRX-I

MRX-I is a new oxazolidinone antimicrobial under development. In this study, the potential for development of resistance to MRX-I in Staphylococcus aureus was investigated and key mutations were characterised. Determination of spontaneous resistance frequency and the mutant selection window (MSW) were performed with meticillin-susceptible S. aureus (MSSA) ATCC 29213, meticillin-resistant S. aureus (MRSA) ATCC 33591 and two clinical MRSA isolates SA 0016 and SA 0017. Selected resistant mutants were sequenced for 23S rRNA as well as genes encoding the ribosomal proteins L3, L4 and L22. Resistance frequencies for the aforementioned strains were <8.25×10−12, <6.33×10−12, <2.96×10−13 and <4.52×10−13, respectively, and the MSW of MRX-I was 2–4, 1–4, 1–2 and 1–4mg/L, respectively. After 30 serial passages, MRX-I minimum inhibitory concentrations (MICs) increased up to 8- to 16-fold both against MSSA and MRSA, whilst linezolid MICs increased 128-fold against MSSA and 16- to 32-fold against MRSA. MRX-I resistance mutations were clustered mainly in 23S rRNA and L3 protein regions. The U2504A transversion in 23S rRNA dominated in MRX-I-resistant mutants. No mutations in L4 and L22 proteins were observed. MRX-I exhibits a low potential to develop resistance in S. aureus, with a reduced resistance propensity compared with linezolid.

High vancomycin minimum inhibitory concentrations with heteroresistant vancomycin-intermediate Staphylococcus aureus in meticillin-resistant S. aureus bacteraemia patients

Patients with high vancomycin minimum inhibitory concentrations (MICs) and heteroresistant vancomycin-intermediate Staphylococcus aureus (hVISA) infection are associated with treatment failure and poor outcomes. The main purpose of this study was to investigate the effect of hVISA on patient outcome, considering both the high vancomycin MIC and the existence of heteroresistant phenotypes. From January 2005 to December 2009, consecutive meticillin-resistant S. aureus (MRSA) isolates from 284 cases of MRSA bacteraemia receiving glycopeptide therapy were collected for further MIC and hVISA testing. The demographic distribution, clinical features and outcomes in bacteraemia patients with different vancomycin MICs and hVISA status in MRSA isolates were subsequently compared. Subjects were divided into three groups: low vancomycin MIC (<1.5mg/L) with vancomycin-sensitive S. aureus (VSSA) (n=50); high vancomycin MIC (≥1.5mg/L) with VSSA (n=218); and high vancomycin MIC with hVISA (n=16). Cox regression analysis demonstrated that the high MIC with VSSA group exhibited significantly higher 30-day mortality than the low MIC with VSSA group [odds ratio (OR)=2.349, 95% confidence interval (CI) 1.078–5.118]. The high MIC with hVISA phenotype was not associated with higher mortality but was independently associated with persistent MRSA bacteraemia (OR=5.996, 95% CI 1.438–25.005). To summarise, although hVISA is correlated with persistent bacteraemia, higher mortality in high vancomycin MIC infections could not be explained by the existing hVISA phenotype. Facing persistent bacteraemia under glycopeptide therapy for 7 days, clinicians should consider shifting to an alternative class of antibiotics to treat hVISA infection.

P106 Comparing susceptibility profiles of antibiotics by contour interval analysis: Ceftaroline and Belgian Streptococcus pneumoniae (SP) and MRSA isolates

Ceftaroline, the active metabolite of the prodrug ceftaroline fosamil, is a novel cephalosporin exhibiting bactericidal activity in vitro against Gram-positive organisms, including meticillin-susceptible Staphylococcus aureus (MSSA), meticillin-resistant S. aureus (MRSA), β-haemolytic streptococci, viridans group streptococci (VGS) and Streptococcus pneumoniae, as well as against many common Gram-negative organisms. The objective of this study was to determine the spectrum and potency of ceftaroline against recent (2010) leading pathogens causing complicated skin and soft-tissue infections (cSSTIs) isolated in Europe. A total of 2438 isolates from the Assessing Worldwide Antimicrobial Resistance and Evaluation (AWARE) programme were identified as cSSTI pathogens. Isolates were collected from patients in 52 medical centres in 19 European countries. Reference susceptibility testing for ceftaroline and commonly used antimicrobials was performed by broth microdilution methodologies. Ceftaroline was very active overall against S. aureus (MIC50/90 = 0.25/1 mg/L) and inhibited 100.0% of isolates at a MIC of ≤2 mg/L. Activity against MRSA was documented (MIC50/90 = 1/2 mg/L) but was lower than that observed against MSSA (MIC50/90 = 0.25/0.25 mg/L). Ceftaroline was also very active against 460 β-haemolytic streptococci and 93 VGS (MIC90 = 0.015 mg/L and 0.06 mg/L, respectively). Ceftaroline was active against Escherichia coli (MIC50 = 0.12 mg/L) and Klebsiella pneumoniae (MIC50 = 0.06 mg/L) not expressing extended-spectrum β-lactamases, with activity similar to ceftriaxone and ceftazidime. In summary, this study documents the potent in vitro activity of ceftaroline when tested against recent pathogens isolated from patients with cSSTI in Europe.

Phagocytosis and intracellular killing of heterogeneous vancomycin-intermediate Staphylococcus aureus strains

Risk factors for invasive infections by heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) may involve resistance to opsonophagocytosis and bacterial killing. hVISA strains typically have a thickened cell wall with altered peptidoglycan cross-linking. To determine whether hVISA may be endowed with an increased resistance to phagocytosis, this study assessed the characteristics of uptake and killing by granulocytes of three hVISA strains. All isolates were analysed by multilocus sequence typing and staphylococcal chromosome cassette mec typing. One of the strains belonged to the Hungarian meticillin-resistant S. aureus (MRSA) clone ST239-MRSA-III and the other two to the New York/Japan MRSA clone ST5-MRSA-II. In the presence of 10 % normal serum, the extent of phagocytosis and killing by blood granulocytes was equivalent for hVISA, MRSA and meticillin-sensitive S. aureus (MSSA) strains. Using granulocytes and serum from one patient who survived hVISA infection, the rate of phagocytosis and killing was also found to be comparable to that by control cells in the presence of 10 % serum. However, phagocytosis and killing of hVISA and MRSA (ATCC 25923) strains by normal granulocytes was markedly decreased in the presence of low concentrations (1 and 2.5 %) of serum from the patient who survived hVISA infection compared with that found with normal human serum. These data suggest that hVISA and MRSA isolates may be more resistant to opsonophagocytosis and bacterial killing than MSSA isolates, at least in some cases.

Rising incidence of Staphylococcus aureus with reduced susceptibility to vancomycin and susceptibility to antibiotics: a global analysis 2004–2009

During 2004–2009, 20004 isolates of Staphylococcus aureus were collected from the Tigecycline Evaluation and Surveillance Trial (T.E.S.T.). Of these isolates, 8249 (41.2%) were meticillin-resistant S. aureus (MRSA) and 11755 (58.8%) were meticillin-susceptible S. aureus (MSSA). A total of 4.0%, 5.3% and 3.0% of all S. aureus, MRSA and MSSA isolates, respectively, exhibited vancomycin minimum inhibitory concentrations (MICs) ≥2μg/mL. Whilst no vancomycin-resistant S. aureus were encountered in this study and the majority of these isolates remained susceptible to vancomycin at the Clinical and Laboratory Standards (CLSI) breakpoint of 2μg/mL, the total number of isolates with MICs creeping up to 2μg/mL and above increased in all S. aureus from 4.0% in 2004 to 7.7% in 2009 (P<0.001). Moreover, in MRSA this phenotype increased from 5.6% in 2004 to 11.1% in 2009 (P<0.001). The increase was also notable for MSSA, which rose from 2.6% in 2004 to 5.6% in 2009 (P<0.001). Of the 12 antibiotics tested, linezolid, minocycline, tigecycline and vancomycin were the most active agents by susceptibility against all S. aureus, all MRSA and all MSSA isolates. Against MRSA isolates with vancomycin MICs≥2μg/mL, susceptibility to vancomycin decreased from 100% in 2004 to 95.77% in 2009 (P>0.05). Similarly, in MSSA isolates susceptibility to vancomycin decreased from 100% in 2004 to 91.07% in 2009 (P>0.05). These data suggest that although the number of isolates of S. aureus with reduced susceptibility to vancomycin has increased significantly from 2004 to 2009, this upward creep of MICs has not yet impacted significantly on the overall susceptibility of vancomycin against either MRSA or MSSA.

Activity of telavancin and comparator antimicrobial agents tested against Staphylococcus spp. isolated from hospitalised patients in Europe (2007–2008)

The activity of telavancin was evaluated against Staphylococcus spp. collected from European hospitals as part of an international surveillance study (2007–2008). A total of 7534 staphylococcal clinical isolates [5726 Staphylococcus aureus and 1808 coagulase-negative staphylococci (CoNS)] were included. Isolates were tested for susceptibility according to reference methods and minimum inhibitory concentration (MIC) values were interpreted based on Clinical and Laboratory Standards Institute (CLSI) 2010 and European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2009 criteria. Telavancin breakpoints approved by the US Food and Drug Administration (FDA) were applied. Telavancin activity was evaluated against meticillin-resistant S. aureus (MRSA) displaying several antibiogram resistance patterns, including multidrug-resistant isolates. Telavancin was active against S. aureus [MIC 50/90 values (MICs for 50% and 90% of the isolates, respectively) = 0.12/0.25 mg/L; 100.0% susceptible] and CoNS (MIC 50/90 = 0.12/0.25 mg/L), inhibiting all isolates at ≤0.5 mg/L. Similar results were observed when S. aureus were stratified by year or country of origin (MIC 50/90 = 0.12/0.25 mg/L). When MRSA isolates were clustered according to 48 different resistance patterns, telavancin showed consistent MIC 90 values (0.25 mg/L) regardless of multidrug resistance. Amongst CoNS, telavancin was slightly more active against Staphylococcus capitis, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus lugdunensis and Staphylococcus xylosus (MIC 50 = 0.12 mg/L) compared with Staphylococcus haemolyticus, Staphylococcus saprophyticus and Staphylococcus warneri (MIC 50 = 0.25 mg/L). Overall, telavancin exhibited MIC 90 results two- to eight-fold lower than comparators (daptomycin, quinupristin/dalfopristin, vancomycin and linezolid). Based upon MIC 90 values, telavancin demonstrated potent in vitro activity against a contemporary (2007–2008) collection of Staphylococcus spp. recovered from nearly 30 European medical centres.

Antimicrobial susceptibility profiles of meticillin-susceptible and -resistant Staphylococcus aureus: focus on daptomycin minimum inhibitory concentrations at a tertiary care centre in Mumbai, India

In this study, daptomycin minimum inhibitory concentrations (MICs) for Staphylococcus aureus isolates were determined using Etest strips and were correlated with staphylococcal cassette chromosome mec (SCC mec) types and Panton–Valentine leukocidin (PVL) gene positivity. In total, 60 meticillin-resistant S. aureus (MRSA) and 60 meticillin-susceptible S. aureus (MSSA) isolates from clinical samples were subjected to antimicrobial susceptibility testing by disk diffusion according to Clinical and Laboratory Standards Institute guidelines, polymerase chain reaction (PCR) detection of PVL and mecA genes, and SCC mec typing. Daptomycin MICs were determined using Etest strips. The mecA gene was present in all MRSA isolates and was absent in 59 MSSA isolates. The PVL gene was present in 41 MRSA isolates and 25 MSSA isolates. Amongst the MRSA isolates, 10 were SCC mec type III, 27 were SCC mec IV and 23 were SCC mec V. Moreover, 26 SCC mec IV and 15 SCC mec V isolates were PVL-positive. All SCC mec III isolates were multidrug-resistant and PVL-negative. Daptomycin MICs ranged from 0.047 μg/mL to 1 μg/mL for MRSA and from 0.19 μg/mL to 1 μg/mL for MSSA. All MRSA and MSSA isolates were susceptible to daptomycin. Although SCC mec III MRSA and PVL-positive MSSA were resistant to more antimicrobial classes than SCC mec IV and V MRSA and PVL-negative MSSA, there does not appear to be a significant correlation between SCC mec types, PVL positivity and daptomycin MICs. MICs were not as low as expected for a newly introduced drug.

Comparative molecular analysis of meticillin-resistantStaphylococcus aureusisolates from children with atopic dermatitis and healthy subjects in Taiwan

Children with atopic dermatitis (AD) are more frequently colonized by Staphylococcus aureus than healthy children. To assess whether any relationship exists between nasal meticillin-resistant S. aureus (MRSA) colonization and subsequent skin and soft-tissue infections (SSTI). From 2005 through 2006, comparative molecular analyses of 23 MRSA-colonizing isolates from 133 children with AD, 44 MRSA-colonizing isolates from 490 healthy controls, and 12 MRSA-infecting isolates from 20 children with AD and concurrent SSTI were conducted. Nasal MRSA colonization in children with AD was significantly higher compared with normal individuals (17.3% vs. 9.0%; P = 0.01). The molecular characteristics differed significantly between the MRSA isolates from children with AD and the MRSA-colonizing isolates from healthy controls. The clone characterized as sequence type (ST)59 (338)/pulsotype B/staphylococcal cassette chromosome mec (SCCmec) V(T)/Panton-Valentine leucocidin (PVL)-positive/staphylococcal enterotoxin B (SEB)-positive accounted for half of the MRSA isolates from children with AD, and another clone, characterized as ST59/pulsotype A/SCCmec IV/PVL-negative/SEB-positive accounted for 61% of the MRSA-colonizing isolates from healthy controls. We found MRSA colonizing the anterior nares of a large number of Taiwanese children, especially among those with AD. Analysis of our data provides evidence that links MRSA-colonizing isolates to MRSA-infecting isolates from concurrent SSTI in children with AD.

Change in environmental bacterial flora in a new hospital building

Microbial surveillance of environmental bacteria was performed in order to study the microbial changes in a newly established hospital building. Airborne bacteria and surface-associated bacteria on floors and sinks were systematically collected between 2002 and 2005. The number of isolates obtained from frequently used floors was significantly higher than that obtained from those floors used less often. A significant increase in Staphylococcus aureus, the appearance of Pseudomonas aeruginosa, and changes among species of Gram-negative bacilli were observed 8-11 months after the new building had been opened. Furthermore, pulsed-field gel electrophoresis (PFGE) typing of meticillin-resistant S. aureus (MRSA) and P. aeruginosa showed that strains of the same PFGE groups were isolated from different sinks, floors and the adjoining old buildings. The number of MRSA isolates obtained from the new building increased as time passed. The sinks from which P. aeruginosa strains of the same PFGE type were isolated are connected by the same drainage pipe. Human movement has considerable effects on bacterial flora and their subsequent spread.

Biofilm formation by Scottish clinical isolates of Staphylococcus aureus

The biofilm-forming capacity of 972 clinical isolates of Staphylococcus aureus was tested using a high-throughput polystyrene 96-peg plate format. Isolates of S. aureus were collected from patients in hospitals throughout Scotland from 2004 to 2006; 763 of these were meticillin-resistant S. aureus (MRSA) and 209 were meticillin-sensitive S. aureus (MSSA). The biomass of each biofilm was quantified using a crystal violet staining technique. Isolates were divided into those that formed fully established biofilms, moderately attached biofilms and weakly adherent biofilms by comparison with a known biofilm-forming strain. The majority of MRSA (53.8 %) and MSSA (43.5 %) isolates formed moderately attached biofilms. Fully established biofilms were formed by 20.5 % of MRSA isolates and 28.0 % of MSSA isolates, whilst 25.7 % of MRSA isolates and 28.5 % of MSSA isolates formed negligible biofilms. There was no significant correlation between susceptibility to meticillin and biofilm formation (P=0.77). MRSA isolates were divided into clonal types (EMRSA-15, EMRSA-16 and sporadic isolates) based on PFGE genotyping results. EMRSA-15 isolates formed significantly more moderately and fully established biofilms than EMRSA-16 isolates (P<0.001). S. aureus strains isolated from the skin of patients had a significantly greater capacity to form biofilms than isolates from other body sites, including the blood. Microscopic examination of biofilms by scanning electron microscopy (SEM) revealed that poorly adherent biofilm formers failed to colonize the entire surface of the peg, whilst moderately adherent biofilm formers grew in uniform monolayers but failed to develop a mature three-dimensional structure. SEM analysis of an isolate representative of the group that formed fully established biofilms confirmed that this isolate developed a dense biofilm with a textured, multi-layered, three-dimensional structure.

Prevalence of genes encoding extracellular virulence factors among meticillin-resistant Staphylococcus aureus isolates from the University Hospital, Olomouc, Czech Republic

A rather fast and complicated progression of an infection caused by some strains of Staphylococcus aureus could be associated with the expression and co-action of virulence factor complexes in these strains. This study screened the antibiotic susceptibility and prevalence of virulence markers in isolates of meticillin-resistant S. aureus (MRSA) obtained from patients hospitalized at the University Hospital in Olomouc, Czech Republic. A total of 100 isolates was screened for 13 genes encoding extracellular virulence determinants (tst, pvl, eta, etb, sea, seb, sec, sed, see, seg, seh, sei and sej) and for their distribution in sample types. Eighty-nine isolates were positive for at least one of the genes. Genes for etb, pvl, see and seh were not detected in any of the MRSA isolates. No statistically significant differences in the occurrence of the determinants studied among sample types were found.

Rapid determination of hospital-acquired meticillin-resistant Staphylococcus aureus lineages

Multilocus sequence typing (MLST) and multi-strain microarray analysis have shown that most human Staphylococcus aureus strains belong to ten dominant clonal complexes (CCs) or lineages, each with unique surface architecture. Meticillin-resistant S. aureus (MRSA) strains currently belong to six of these lineages (CC1, CC5, CC8, CC22, CC30 and CC45), each of which has independently acquired mobile genetic elements (MGEs) carrying antibiotic resistance genes. MLST and microarrays are expensive and time consuming methods for routine determination of S. aureus lineage. A restriction-modification (RM) test has now been developed that is rapid, simple, inexpensive and accurately determines lineage of hospital-acquired MRSA. The RM test is based on three PCRs for hsdS gene variants, as hsdS genes likely control the independent evolution of S. aureus lineages. The RM test correctly identified 102 MRSA isolates as belonging to one of the six lineages/CCs. Real-time MRSA typing can be used to identify and track changes in local MRSA outbreaks, and provide support for targeting infection control strategies. Simple and accurate typing methods will also support large scale epidemiological studies, and could lead to greater understanding of the carriage, spread and virulence of different MRSA lineages.