Dalbavancin reduces biofilms of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> (MRSA) and methicillin-resistant <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> (MRSE)

Activity of dalbavancin against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) in biofilm was investigated and the microbicidal biofilm concentrations (MBC) were determined. Biofilms obtained from ten MRSA and ten MRSE bloodstream isolates, collected from patients in the General Hospital of Vienna between 2012 and 2015, were incubated with dalbavancin in trypticase soy broth (TSB) in serial dilution from 0.0625 mg/l to 256 mg/l using a microtiter plate biofilm model. The plates were incubated for 24 h at 37 ° C and 50% humidity. Biofilms were fixed with 2.5% glutaraldehyde and stained with crystal violet. Subsequently the optical density (OD₆₂₀) was used to measure the MBC, defined as the concentration of dalbavancin leading to a 50% reduction of biofilm. MBC for MRSA was 1 mg/l–4 mg/l (minimal inhibitory concentrations (MIC) 0.0312 mg/l–0.064 mg/l). MBC for MRSE was 2 mg/l–16 mg/l (MIC 0.023 mg/l–0.0625 mg/l). Dalbavancin successfully reduced MRSA and MRSE in biofilms, and therefore provides a promising option for the treatment of biofilm-associated infections.     

Recurrent necrotizing fasciitis caused by methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Reported here is a rare case of recurrent necrotizing fasciitis due to methicillin-resistant Staphylococcus aureus (MRSA). A 46-year-old female with poorly controlled diabetes and chronic ingestion of steroid-containing medications was admitted for treatment of necrotizing fasciitis of the right thigh. Three months following hospital discharge she was readmitted with necrotizing fasciitis of the left hand. On both occasions, MRSA was isolated from tissue cultures obtained during surgical debridement. Patients who develop necrotizing fasciitis are predisposed to severe soft tissue infections due to associated comorbid conditions such as diabetes mellitus. Recurrent soft tissue infection in a patient with previous MRSA-related necrotizing fasciitis should therefore be treated with a high index of suspicion.     

Rapid diagnosis of<Emphasis Type="Italic"> Staphylococcus aureus</Emphasis> bacteremia using <Emphasis Type="Italic">S. aureus</Emphasis> PNA FISH

In the study presented here, the performance of the S. aureus PNA FISH assay was evaluated using 285 blood cultures (from 104 patients) that had gram-positive cocci resembling staphylococci on Gram stain. The new molecular test is based on a fluorescence in situ hybridization assay using peptide nucleic acid probes targeting Staphylococcus aureus 16S rRNA and is designed for the rapid identification of Staphylococcus aureus directly from positive blood cultures. The sensitivity, specificity, and positive and negative predictive values of the S. aureus PNA FISH for the rapid identification of Staphylococcus aureus directly from positive blood culture bottles were 100, 99.4, 99.2 and 100%, respectively.     

Development of a vaccine against <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

A vaccine to prevent infections caused by Staphylococcus aureus would have a tremendously beneficial impact on public health. In contrast to typical encapsulated bacterial pathogens, such as Streptococcus pneumoniae, H. influenzae, and Neisseria meningitides, the capsule of S. aureus is not clearly linked to strain virulence in vivo. Furthermore, it is not clear that natural infection caused by S. aureus induces a protective humoral immune response, as does infection caused by typical encapsulated bacteria. Finally, pure B cell or antibody deficiency, in either animal models or in patients, does not predispose to more frequent or more severe S. aureus infections, as it does for infections caused by typical encapsulated bacteria. Rather, primary immune mechanisms necessary for protection against S. aureus infections include professional phagocytes and T lymphocytes (Th17 cells, in particular) which upregulate phagocytic activity. Thus, it is not clear whether an antibody-mediated neutralization of S. aureus virulence factors should be the goal of vaccination. Rather, the selection of antigenic targets which induce potent T cell immune responses that react to the broadest possible array of S. aureus strains should be the focus of antigen selection. Of particular promise is the potential to select antigens which induce both humoral and T cell-mediated immunity in order to generate immune synergy against S. aureus infections. A single-antigen vaccine may achieve this immune synergy. However, multivalent antigens may be more likely to induce both humoral and T cell immunity and to induce protection against a broader array of S. aureus isolates. A number of candidate vaccines are in development, raising the promise that effective vaccines against S. aureus will become available in the not-so-distant future. Possible development programs for such vaccines are discussed.     

Molecular genetic characterization of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates recovered from Moscow clinics

Methicillin-resistant Staphylococcus aureus (MRSA) is the causative agent of nosocomial infections observed worldwide. The goal of this work was to study the genetic variations in MRSA isolates recovered from Moscow clinics and compare various methods of molecular typing (multiplex PCR, SNP genotyping based on the determination of single-nucleotide polymorphisms). A total of 62 epidemiologically unrelated hospital-acquired MRSA isolates were studied. A previously described multiplex PCR assay was utilized for the molecular typing of staphylococcal cassette chromosome mec (SCCmec). SNP genotyping that targets the seven sequences in five housekeeping genes (arcC162, arcC210, aroE132, gmk123, tpi241, tpi243, and yqiL333) was employed. Primer extension was used to screen single nucleotide variants followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Multilocus sequence typing (MLST) was used as a reference assay. Most MRSA isolates (93.6%) were assigned to the clonal complex (CC) 8 disseminated worldwide. Three MRSA isolates (4.8%) proved to belong to CC1. The following correlation was established in this study between SCCmec cassette types and sequence types (STs): ST8-MRSA carried SCCmec type IV and ST239-MRSA carried type-III a SCCmec. Four SNP groups associated with certain SCCmec types were also identified. The developed SNP genotyping assay aided by MALDI-MS enables the rapid genotyping of S. aureus isolates according to their clonal types.     

Risk factors for long-term mortality of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> bacteremia

Staphylococcus aureus bacteremia (SAB) is a fatal disease. We aimed to describe risk factors for long-term mortality with SAB. We analyzed data from a retrospectively collected database including 1,692 patients with SAB. We considered variables of infection and background conditions for the analysis of long-term survival. The Kaplan–Meier procedure was used for analysis of long-term survival. Variables significantly associated with mortality were analyzed using a Cox regression model. We included 1,692 patients in the analysis. Patients were followed for up to 22 years. Within one year, 62% of patients died and within 5 years 72% died. A total of 82% of patients aged 65 years and older died within 5 years. Independent predictors of long-term mortality were older age (Hazard ratio 1.029, 95% confidence interval 1.022–1.036), female gender (HR 1.302, 95% CI 1.118–1.517), pneumonia or primary/ unknown source of infection (HR 1.441, 95% CI 1.230–1.689), dementia (HR 1.234, 95% CI 1.004–1.516), higher Charlson score (HR 1.155, 95% CI 1.115–1.196), shock at onset (HR 1.776, 95% CI 1.430–2.207) and arrival to hospitalization from an institution (HR 1.319, 95% CI 1.095–1.563). Long-term survival of patients older than 65 years and of women with SAB is severely curtailed.     

CC398 <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> subpopulations in Belgian patients

Studies based on genome-wide single nucleotide polymorphisms (SNPs) supported the existence of two subpopulations in clonal complex (CC) 398 Staphylococcus aureus: an ancestral human-adapted clade (HC) and an animal-associated clade (AC). In this study, we have investigated the occurrence of genetic markers that allow discrimination of these subpopulations among CC398 isolates collected during 2014 to 2016 from human patients in Belgium. A collection of isolates was investigated by means of spa-typing and 16S-mecA-nuc PCR. CC398 isolates were classified as belonging to the human or the animal clade by using a canonical SNPs PCR and further studied by antimicrobial susceptibility and the presence of toxins, immune evasion cluster (IEC), and resistance genes. A total of 124 (7.8%) human isolates belonged to CC398. They were grouped into HC (n?=?58) or AC (n?=?66). The genes erm(T), pvl, chp, and scn were predominantly found in HC-CC398, while AC-CC398 isolates carried more frequently than the mecA, erm(C), tet(K), tet(M), and tet(L) genes. Different combinations of gene profiles were observed according to the clade. CC398 isolates from Belgian patients belonged to different subpopulations including typical HC and AC-isolates. Few HC-strains with mecA and AC-isolates harboring IEC were found. CC398 isolates from Belgian patients belonged to different subpopulations including typical HC and AC-isolates, as well as new emerging subpopulations that underline the ability of this lineage to acquire resistance and virulence genes. Further research is needed to evaluate the emergence of these subpopulations in the clinical setting.     

Cassette chromosome mec typing of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates from patients in Tehran

Background

Methicillin-resistant Staphylococcus aureus is the cause of nosocomial infections leading to high mortality. Since these strains have become prevalent in the world, it is necessary to identify and type them.

Materials and Methods

This cross-sectional study was conducted to study a total of 1475 clinical specimens from Iranian patients in 2012–2013. Using phenotypic tests such as Gram stain, catalase, coagulase, DNase and mannitol fermentation 169 isolates of Staphylococcus aureus and by utilizing methicillin-resistance test 100 MRSA isolates were identified. SCCmec typing was performed by multiplex PCR method and the results were analyzed using chi-square tests by SPSS-18 software.

Results

Disk agar diffusion test using cefoxitin disk (30 μg) showed methicillin resistance in 59% of our isolates. mecA and femB genes were identified in all of the MRSA isolates using PCR method. Frequency of SCCmec types and sub-types were as follow; SCCmecIII (77%), SCCmecI (5%), SCCmecIVa (1%), SCC-mecIVc (1%), Mixed isolates SCCmecIVc-III (1%) and Non typeable isolates (15%). Non typeable isolates recovered in two groups (10% without any band and 5% of multi-bands III-I). In this study, 82% of isolates were HA-MRSA, 3% were CA-MRSA and 15% were Non typeable.

Conclusion

In our S. aureus isolates methicillin resistance was 59%. The most frequent SCCmec type was SCCmecIII (77%). Our results demonstrated the spread of HA-MRSA isolates in the community and propagating CA-MRSA isolates in the studied hospitals.

     

Intra-strain variability of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> strains ST228-MRSA-I and ST5-MRSA-II

Isolates belonging to two major epidemic strains of methicillin-resistant Staphylococcus aureus (MRSA) from clonal complex 5 were characterised using diagnostic microarrays in order to detect and analyse intra-strain variability. Isolates were sampled from hospitals scattered all over Germany. The study included 56 isolates of ST228-MRSA-I, which is also known as the South German Epidemic Strain, and 40 isolates of ST5-MRSA-II (UK-EMRSA-3, Rhine-Hesse Epidemic Strain, New York/Japan Clone), as well as, for comparison, some control strains and overseas isolates of ST5-MRSA-II. Both strains showed a remarkable variability. This affected plasmid-borne resistance genes (tetK, blaZ/R/I, aacA–aphD, qacA), genes from SCCmec elements (aadD, ermA, merA/B/R/T), toxin gene clusters on pathogenicity islands (sec/l, tst1) or, probably, on plasmids, (sed/j/r), the presence or absence of beta-haemolysin-converting phages (sea, sea-N315, sak, chp, scn), deletions of single chromosomal genes (bbp, clfA) or, occasionally, of rather large clusters of neighbouring genes (seg, sei, sem, sen, seo, seu, lukD/E). Both strains could be split into four major clusters each, based on the presence of a mercury resistance operon (merA/B/R/T) and lukD/E in ST228-MRSA-I or of tst1 and enterotoxin genes seD/J/R in ST5-MRSA-II. The use of this variability for typing purposes as well as its phylogenetic significance are discussed.     

The remarkably multifunctional fibronectin binding proteins of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Staphylococcus aureus expresses two distinct but closely related multifunctional cell wall-anchored (CWA) proteins that bind to the host glycoprotein fibronectin. The fibronectin binding proteins FnBPA and FnBPB comprise two distinct domains. The C-terminal domain comprises a tandem array of repeats that bind to the N-terminal type I modules of fibronectin by the tandem β-zipper mechanism. This causes allosteric activation of a cryptic integrin binding domain, allowing fibronectin to act as a bridge between bacterial cells and the α₅β₁ integrin on host cells, triggering bacterial uptake by endocytosis. Variants of FnBPA with polymorphisms in fibronectin binding repeats (FnBRs) that increase affinity for the ligand are associated with strains that infect cardiac devices and cause endocarditis, suggesting that binding affinity is particularly important in intravascular infections. The N-terminal A domains of FnBPA and FnBPB have diverged into seven antigenically distinct isoforms. Each binds fibrinogen by the ‘dock, lock and latch’ mechanism characteristic of clumping factor A. However, FnBPs can also bind to elastin, which is probably important in adhesion to connective tissue in vivo. In addition, they can capture plasminogen from plasma, which can be activated to plasmin by host and bacterial plasminogen activators. The bacterial cells become armed with a host protease which destroys opsonins, contributing to immune evasion and promotes spreading during skin infection. Finally, some methicillin-resistant S. aureus (MRSA) strains form biofilm that depends on the elaboration of FnBPs rather than polysaccharide. The A domains of the FnBPs can interact homophilically, allowing cells to bind together as the biofilm accumulates.     

<Emphasis Type="Italic">Staphylococcus aureus</Emphasis> colonization related to severity of hand eczema

Knowledge on Staphylococcus aureus colonization rates and epidemiology in hand eczema is limited. The aim of this study was to clarify some of these issues. Samples were collected by the “glove juice” method from the hands of 59 patients with chronic hand eczema and 24 healthy individuals. Swab samples were taken from anterior nares and throat from 43 of the 59 patients and all healthy individuals. S. aureus were spa typed and analysed by DNA-microarray-based genotyping. The extent of the eczema was evaluated by the hand eczema extent score (HEES). The colonization rate was higher on the hands of hand eczema patients (69 %) compared to healthy individuals (21 %, p?<?0.001). This was also seen for bacterial density (p?=?0.002). Patients with severe hand eczema (HEES?≥?13) had a significantly higher S. aureus density on their hands compared to those with milder eczema (HEES?=?1 to 12, p?=?0.004). There was no difference between patients and healthy individuals regarding colonization rates in anterior nares or throat. spa typing and DNA-microarray-based genotyping indicated certain types more prone to colonize eczematous skin. Simultaneous colonization, in one individual, with S. aureus of different types, was identified in 60–85 % of the study subjects. The colonization rate and density indicate a need for effective treatment of eczema and may have an impact on infection control in healthcare.     

In vitro activity of beta-lactam antibiotics to community-associated methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> (CA-MRSA)

Community-associated (CA) MRSA often display low MIC values against oxacillin. The in vitro activity of various beta-lactam antibiotics against heterogeneous CA-MRSA (n = 98) isolated in a low endemic area was determined by Etest, and Mueller–Hinton agar (MUHAP) was compared with Mueller–Hinton agar supplemented with 2% NaCl (MUHSP). In general, the CA-MRSA isolates showed higher MIC values for the various beta-lactam antibiotics on MUHSP compared with MUHAP. MIC values for oxacillin ranged from 1 to >256 mg/L on MUHSP. Cephalothin, representing the first generation of cephalosporins, showed MICs from 0.75 to 96 mg/L and the MIC₅₀ and MIC₉₀ for cefuroxime, cefotaxime and cefepime, representing the second, third and fourth generations, respectively, were rather high. However, the MIC₅₀ and MIC₉₀ for ceftobiprole (fifth generation) were 1.5 and 2 mg/L, respectively, on MUHSP. The MIC₅₀ and MIC₉₀ for imipenem were 0.75 and 2 mg/L, respectively, on MUHSP. Only 3/98 (3%) CA-MRSA isolates showed a MIC >4 mg/L. Consequently, low MIC values for imipenem, lower than those of the newly developed fifth generation cephalosporins, were found among CA-MRSA. These findings may be considered for further studies including clinical trials in order to evaluate carbapenems as a potential treatment option for infections caused by CA-MRSA.     

Methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> with <Emphasis Type="Italic">mecC</Emphasis>: a description of 45 human cases in southern Sweden

In 2011, a novel mecA gene homologue, mecC, was reported in isolates from both humans and dairy cattle. The epidemiology of mecC methicillin-resistant Staphylococcus aureus (MRSA) in humans is not yet well known. In this retrospective study, we present the epidemiology of human clinical cases with mecC MRSA detected in the southern part of Sweden during the period 2005–2014. A total of 45 patients with an isolate positive for mecC MRSA were included in the study. Twenty-six isolates were found before 2012 and were retrospectively tested for mecC. Nineteen isolates were detected in 2012–2014 through routine testing. Culture results, resistance patterns, Panton–Valentine leukocidin (PVL) genes, and spa types were collected from the Clinical Microbiology Laboratory. Epidemiological data were received from the database at the Regional Centre for Communicable Disease Control and the patient’s medical files. The majority of the patients with mecC MRSA were of Swedish origin, had underlying diseases, and lived in rural areas. The median age was 60 years. Of the mecC MRSA, 76 % belonged to spa types t373 and t843. The median minimum inhibitory concentration (MIC) value for oxacillin was 16 mg/L (1–64 mg/L) and only one isolate was resistant to other classes of antibiotics. The most common type of infection was skin and soft tissue infections, most often in an existing skin lesion. The patients with mecC MRSA were colonized for a short time and gave rise to few secondary cases. mecC MRSA in our region appears to have a domestic origin and mainly affects patients with underlying diseases or patients with an existing skin lesion. Our data indicate that it could be a poor colonizer.     

Low occurrence of the new species <Emphasis Type="Italic">Staphylococcus argenteus</Emphasis> in a <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> collection of human isolates from Belgium

Staphylococcus argenteus is a novel Staphylococcus species closely related to Staphylococcus aureus that has been recently described. In this study, we investigated the proportion and the characteristics of S. argenteus recovered from humans in Belgium. S. aureus. human isolates collected in Belgium from 2006 to 2015 (n?=?1,903) were retrospectively characterised via the presence of non-pigmented colonies on chocolate agar, spa typing and rpoB sequencing to determine if some of them were in fact S. argenteus. Out of 73 strains non-pigmented on chocolate plates, 3 isolates (0.16 %) showed rpoB sequences, in addition to spa and sequence types (ST2250/t5787, ST2250/t6675, ST3240/t6675), related to S. argenteus. Two of them were methicillin-resistant, harbouring a SCCmec type IV. The three S. argenteus isolates carried genes (sak, scn) of the immune evasion cluster. This first Belgian nationwide analysis showed a low occurrence of S. argenteus. Further studies should be conducted to identify the distribution range and the clinical impact of this new species.     

Genetic diversity of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in a tertiary hospital in The Netherlands between 2002 and 2006

The aim of this study was to investigate the methicillin-resistant Staphylococcus aureus (MRSA) clones isolated in a Dutch university hospital, situated near the borders of Belgium and Germany, between 2002 and 2006. MRSA strains (n = 175) were characterized using spa and SCCmec typing. The presence of Panton Valentine leukocidin (PVL) was determined. Between 2002 and 2005, ST5-MRSA-IV was predominant, and the spa type of ST5-MRSA-IV changed from t002 to t447. ST5-MRSA-I, ST5-MRSA-II, ST228-MRSA-I, and ST247-MRSA-I were also observed in this period. From 2004, the MRSA genetic background became more diverse, and in 2006, ST5-MRSA-IV was only sporadically observed. From 2005, ST5-MRSA-II, ST8-MRSA-IV, ST22-MRSA-IV, and ST45-MRSA-IV were increasingly observed. Several other MRSA clones, such as ST239-MRSA-III, were found sporadically. Four PVL-positive MRSA isolates were observed, associated with ST80-MRSA-IV and ST8-MRSA-IV. ST5-MRSA-I, ST5-MRSA-II, ST5-MRSA-IV, and ST228-MRSA-I have not been described previously in The Netherlands.     

Phenotypic changes of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> during vancomycin therapy for persistent bacteraemia and related clinical outcome

Persistent bacteraemia (PB) due to methicillin-resistant Staphylococcus aureus (MRSA) that fails to respond to glycopeptide therapy is a well-documented clinical problem. There are limited data on changes in agr functionality, vancomycin susceptibility and heteroresistance during MRSA PB. Thus, the frequency of these changes and their clinical significance remain unclear. Only patients with MRSA PB (≥7 days) from a prospective cohort of S. aureus bacteraemia were included. We collected isogenic paired strains and compared vancomycin MIC, vancomycin heteroresistance, and agr functionality between initial and final blood isolates. We also assessed the clinical outcome. A total of 49 patients had MRSA PB over 22 months. Bacteraemia persisted for a median of 13 days and most patients (98%) received glycopeptide as initial therapy. Among 49 isogenic pairs, only one pair showed a vancomycin MIC increase ≥2-fold by broth microdilution method, and only seven (14%) by E-test. Significant portions of initial isolates had vancomycin heteroresistance (49%) and agr dysfunction (76%). Development of vancomycin heteroresistance during PB occurred in four (16%) among 25 initial vancomycin-susceptible isolates, and acquisition of agr dysfunction occurred in two (16%) among 12 initial agr-functional isolates. Changes in the opposite direction occasionally occurred. These phenotypic changes during PB were not associated with mortality, whereas agr dysfunction of the initial isolates was significantly associated with mortality. During MRSA PB, phenotypic changes of MRSA isolates occurred occasionally under prolonged vancomycin exposure but were not significantly associated with clinical outcome. In contrast, initial agr dysfunction could be a predictor for mortality in MRSA PB.