Exploring extra-cellular proteins in methicillin susceptible and methicillin resistant Staphylococcus aureus by liquid chromatography–tandem mass spectrometry

Staphylococcus aureus (S. aureus) strains cause several diseases in humans from minor skin infections to severe lethal infections. To explore the virulence determinants of this important microorganism, two clinical isolates of methicillin susceptible S. aureus (MSSA) and methicillin resistant S. aureus (MRSA) were subjected to proteomic analysis of their extracellular products using liquid chromatography–tandem mass spectrometry. The numbers of proteins identified in MSSA and MRSA extracellular products were 168 and 261; respectively, from them 117 were shared, while 144 proteins were unique to MRSA. The shared proteins, having a higher protein score with increased number of peptide matches in MRSA over MSSA, reflect the relatively active secretory state of MRSA rather than biased analytical variances. Characteristic determinants for MRSA were identified; mostly found to play a role in the virulence. We conclude that MRSA produces distinct proteins considered as its virulence determinants and we found that the shared extracellular products are more abundant in MRSA than MSSA that supporting the high invasiveness of MRSA over MSSA in pathogenesis.     

β-Lapachone activity in synergy with conventional antimicrobials against methicillin resistant Staphylococcus aureus strains

The aim of this study was to evaluate the antimicrobial activity of lapachol, α-lapachone, β-lapachone and six antimicrobials (ampicillin, amoxicillin/clavulanic acid, cefoxitin, gentamicin, ciprofloxacin and meropenem) against twelve strains of Staphylococcus aureus from which resistance phenotypes were previously determined by the disk diffusion method. Five S. aureus strains (LFBM 01, LFBM 26, LFBM 28, LFBM 31 and LFBM 33) showed resistance to all antimicrobial agents tested and were selected for the study of the interaction between β-lapachone and antimicrobial agents, busing checkerboard method. The criteria used to evaluate the synergistic activity were defined by the Fractional Inhibitory Concentration Index (FICI). Among the naphthoquinones, β-lapachone was the most effective against S. aureus strains. FICI values ranged from 0.07 to 0.5, suggesting a synergistic interaction against multidrug resistant S. aureus (MRSA) strains. An additive effect was observed with the combination β-lapachone/ciprofloxacin against the LFBM 33 strain. The combination of β-lapachone with cefoxitin showed no added benefit against LFBM 31 and LFBM 33 strains. This study demonstrated that, in general, β-lapachone combined with beta lactams antimicrobials, fluoroquinolones and carbapenems acts synergistically inhibiting MRSA strains.     

Synergistic effects between aminoethyl-chitosans and β-lactams against methicillin-resistant Staphylococcus aureus (MRSA)

Two kinds of aminoethyl-chitosans (AEC), AEC90 and AEC50, which had degrees of deacetylation of 90% and 50%, respectively, were prepared and their synergistic effects in combination with β-lactams including ampicillin, penicillin, and oxacillin against two standard methicillin-resistant Staphylococcus aureus (MRSA) strains and twelve clinical isolated MRSA strains were investigated. When AECs and β-lactams were combined, synergistic effects were observed with fractional inhibitory concentration (FIC) indices of 0.252–0.508, and the MICs of β-lactams in the presence of AECs were dramatically reduced.     

Characterisation of virulence genes in methicillin susceptible and resistant Staphylococcus aureus isolates from a paediatric population in a university hospital of Medellín, Colombia

Virulence and antibiotic resistance are significant determinants of the types of infections caused by Staphylococcus aureus and paediatric groups remain among the most commonly affected populations. The goal of this study was to characterise virulence genes of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains isolated from a paediatric population of a Colombian University Hospital during 2009. Sixty MSSA and MRSA isolates were obtained from paediatric patients between zero-14 years. We identified the genes encoding virulence factors, which included Panton-Valentine leucocidine (PVL), staphylococcal enterotoxins A-E, exfoliative toxins A and B and toxic shock syndrome toxin 1. Typing of the staphylococcal chromosome cassette mec (SCCmec) was performed in MRSA strains. The virulence genes were more diverse and frequent in MSSA than in MRSA isolates (83% vs. 73%). MRSA strains harboured SCCmec types IVc (60%), I (30%), IVa (7%) and V (3%). SCCmec type IVc isolates frequently carried the PVL encoding genes and harboured virulence determinants resembling susceptible strains while SCCmec type I isolates were often negative. PVL was not exclusive to skin and soft tissue infections. As previously suggested, these differences in the distribution of virulence factor genes may be due to the fitness cost associated with methicillin resistance.     

Methicillin-Resistant Staphylococcus aureus (MRSA) Is Increasing in Norway: A Time Series Analysis of Reported MRSA and Methicillin-Sensitive S. aureus Cases, 1997–2010

Background

Accurate estimates of the incidence and prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infections are needed to inform public health policies. In Norway, where both MRSA infection and carriage are notifiable conditions, the reported incidence of MRSA is slowly increasing. However, the proportion of MRSA in relation to all S. aureus isolates is unknown, making it difficult to determine if the rising incidence is real or an artifact of an increasing number of tests performed.

Aim

To characterize recent trends in MRSA infections and obtain a more complete understanding of the MRSA level in Norway.

Methods

All reported cases of MRSA and methicillin-sensitive S. aureus (MSSA) from Oslo County (1997–2010) and Health Region East (2008–2008), representing approximately 11% and 36% of the Norwegian population, respectively, were analyzed using a stochastic time series analysis to characterize trends.

Results

In Oslo County, the proportion of methicillin-resistant cases increased from 0.73% to 3.78% during the study period and was well modeled by an exponential growth with a doubling constant of 5.7 years (95% CI 4.5–7.4 years). In Health Region East, the proportion of MRSA cases increased from 0.4% to 2.1% from 2002 to 2008, with a best-fitting linear increase of 0.26% (95% CI 0.21–0.30%) per year. In both cases, the choice of a linear or exponential model for the time trend produced only marginally different model fits. We found no significant changes due to revised national MRSA guidelines published in June 2009. Significant variations in the increasing time trend were observed in the five hospitals within the region. The yearly reported incidence of MSSA was relatively stable in both study areas although we found seasonal patterns with peaks in August.

Conclusion

The level of MRSA is increasing in Norway, and the proportion of methicillin resistance in all S. aureus isolates are higher than the reported proportion of MRSA in invasive infections.     

Comparison of ESBL – And AmpC Producing Enterobacteriaceae and Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated from Migratory and Resident Population of Rooks (Corvus frugilegus) in Austria

In order to test whether rooks (Corvus frugilegus) represent good indicators for the potential circulation of antibiotics in their native habitat, two populations with different migratory behavior were tested for the presence of beta-lactamase producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA). In all, 54 and 102 samples of fresh feces of a migratory and a resident population were investigated. A total of 24 and 3 cefotaxime-resistant enterobacterial isolates were obtained from the migratory and resident population, respectively. In these isolates CTX-M-1 (n = 15), CTX-M-3 (n = 3), and CTX-M-15 (n = 3) genes were detected. TEM-1 and OXA-1 were associated with CTX-M in 3 and 2 isolates, respectively. In two E. coli isolates CMY-2 could be detected, where from one isolate displayed an overexpression of chromosomal AmpC as well. Among E. coli isolates the most common phylogenetic group was A (n = 11) and ST1683 (n = 5). In one E. coli of B2-ST131 the rfbO25b locus was detected. Three Enterobacter isolates were stably derepressed AmpC-producers. In five samples of the migratory population, PVL positive MRSA could be isolated. Two isolates were typed SCCmec IVa, spa type t127, and ST1. Three isolates carried a SCCmec type IVc, with spa type t852 and ST22. The highly significant difference of the occurrence of antibiotic resistance between the migratory population from eastern Europe compared to resident population in our study indicates that rooks may be good indicator species for the evaluation of environmental contamination with antibiotic resistant bacteria, especially due to their ecology, foraging behavior and differing migratory behavior.     

Alarming Proportions of Methicillin-Resistant Staphylococcus aureus (MRSA) in Wound Samples from Companion Animals,Germany 2010–2012

Staphylococcus (S.) aureus is an important cause of wound infections in companion animals, and infections with methicillin-resistant S. aureus (MRSA) are of particular concern due to limited treatment options and their zoonotic potential. However, comparable epidemiological data on MRSA infections in dogs, cats and horses is scarce, also limiting the knowledge about possible links to MRSA isolates from human populations. To gain more knowledge about the occurrence and genotypic variation of MRSA among wound swabs of companion animal origin in Germany we performed a survey (2010–2012) including 5,229 samples from 1,170 veterinary practices. S. aureus was identified in 201 (5.8%) canine, 140 (12.2%) feline and 138 (22.8%) equine swabs from a total of 3,479 canine, 1,146 feline and 604 equine wounds, respectively. High MRSA rates were identified with 62.7%, 46.4% and 41.3% in S. aureus of canine, feline and equine origin, respectively. Further genotyping including spa typing and multilocus sequence typing (MLST) revealed a comparable distribution of spa types among canine and feline MRSA with CC22 (47.6%; 49.2%) and CC5 (30.2%; 29.2%) as predominant lineages followed by CC398 (13.5%; 7.7%) and CC8 (4.0%; 9.2%). In contrast, the majority of equine MRSA belonged to CC398 (87.7%). Our data highlight the importance of S. aureus and MRSA as a cause of wound infections, particularly in cats and horses in Germany. While “human-associated” MRSA lineages were most common in dogs and cats, a remarkable number of CC398-MRSA was detected in horses, indicating a replacement of CC8-MRSA as the predominant lineage within horses in Germany. These data enforce further longitudinal epidemiological approaches to examine the diversity and temporal relatedness of MRSA populations in humans and animals to assess probable sources of MRSA infections. This would enable a sound risk assessment and establishment of intervention strategies to limit the additional spread of MRSA.     

Molecular Typing of MRSA and of Clinical Staphylococcus aureus Isolates from Ia?i,Romania

Romania is one of the countries with the highest prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in the world. To obtain data on affiliation of MRSA to strains and clonal complexes and on the population of methicillin susceptible S. aureus (MSSA), clinical isolates from bloodstream infections, skin and soft tissue infections as well as from screening swabs were collected at hospitals in Ia?i, a city in the North-Eastern part of Romania. Isolates were characterised by microarray hybridisation. Nearly half of all isolates (47%), and about one third (34%) of bloodstream isolates were MRSA. The prevalence of the Panton-Valentine leukocidin (PVL) was also high (31% among MRSA, 14% among MSSA). The most common MRSA strain was a PVL-negative CC1-MRSA-IV that might have emerged locally, as a related MSSA was also common. PVL-positive CC8-MRSA-IV (“USA300”) and PVL-negative ST239-like MRSA-III were also frequently found while other MRSA strains were only sporadically detected. Among MSSA, PVL-positive CC121 as well as PVL-negative CC1, CC22 and CC45 predominated. Although this study provides only a snapshot of S. aureus/MRSA epidemiology in Romania, it confirms the high burden of MRSA and PVL on Romanian healthcare settings.     

Methicillin-resistant Staphylococcus aureus (MRSA) with high resistance to mupirocin in hospitals of the Gdańsk region

Occurrence of high-level mupirocin resistance in methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from 18 hospitals in Gdańsk area was determined. The study was carried out on 190 MRSA isolated in 1997-2000 from various clinical samples. The strains were tested for high-level mupirocin resistance by 200 micrograms mupirocin disc. The minimum inhibitory concentrations (MIC) for methicillin were estimated by agar dilution. Sensitivity to other antibiotics was determined in disc-diffusion method and to vancomycin in agar dilution method additionally. The strains were typed by set of 10 experimental phages and compared by the method of PCR-RFLP analysis of coagulase gen restriction fragment length polymorphism. There were low frequency of high-level mupirocin resistance in MRSA strains (4.7%) that were found only in 3 hospitals, in 6 patients. All of them were high-resistant also to methicillin and resistant to doxycyclin, gentamycin, erytromycin, klindamycin, ciprofloksacin, rifampicin, resistant or intermediate sensitive to fusidic acid but sensitive to vancomycin, teikoplanin and bacitracin. The origin all of the MRSA strains high-resistant to mupirocin probably was the same, except one strain, because they were belonged to one genetic type and possessed the same phage pattern.     

Sepicanin A—A new geranyl flavanone from Artocarpus sepicanus with activity against methicillin-resistant Staphylococcus aureus (MRSA)

Bioassay-guided fractionation of the EtOH extract of Artocarpus sepicanus Diels leaves has led to the isolation of a new geranyl flavanone (1), along with the known compounds, afzelechin-3-O-α-l-rhamnopyranoside and β-sitosterol glucoside. The structure of the new compound was established by UV, IR, HRESIMS, 1D and 2D NMR experiments. Antimicrobial testing of the three compounds indicated that 1 displayed a significant selective antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) with IC₅₀ and MIC values of 1.4 and 2.9 μM, respectively.     

Optimization and structure–activity relationships of a series of potent inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase as novel antimicrobial agents

A novel series of hydrazones were synthesized and evaluated as inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). PK has been identified as one of the most highly connected ‘hub proteins’ in MRSA. PK has been shown to be critical for bacterial survival which makes it a potential target for development of novel antibiotics and the high degree of connectivity implies it should be very sensitive to mutations and thus less able to develop resistance. PK is not unique to bacteria and thus a critical requirement for such a PK inhibitor would be that it does not inhibit the homologous human enzyme(s) at therapeutic concentrations. Several MRSA PK inhibitors (including 8d) were identified using in silico screening combined with enzyme assays and were found to be selective for bacterial enzyme compared to four human PK isoforms (M1, M2, R and L). However these lead compounds did not show significant inhibitory activity for MRSA growth presumably due to poor bacterial cell penetration. Structure–activity relationship (SAR) studies were carried out on 8d and led us to discover more potent compounds with enzyme inhibiting activities in the low nanomolar range and some were found to effectively inhibit bacteria growth in culture with minimum inhibitory concentrations (MIC) as low as 1 μg/mL. These inhibitors bind in two elongated flat clefts found at the minor interfaces in the homo-tetrameric enzyme complex and the observed SAR is in keeping with the size and electronic constraints of these binding sites. Access to the corresponding sites in the human enzyme is blocked.     

Crystal Structures of Penicillin-Binding Protein 3 (PBP3) from Methicillin-Resistant Staphylococcus aureus in the Apo and Cefotaxime‐Bound Forms

Staphylococcus aureus is a widespread Gram‐positive opportunistic pathogen, and a methicillin‐resistant form (MRSA) is particularly difficult to treat clinically. We have solved two crystal structures of penicillin‐binding protein (PBP) 3 (PBP3) from MRSA, the apo form and a complex with the β-lactam antibiotic cefotaxime, and used electrospray mass spectrometry to measure its sensitivity to a variety of penicillin derivatives. PBP3 is a class B PBP, possessing an N-terminal non-penicillin‐binding domain, sometimes called a dimerization domain, and a C-terminal transpeptidase domain. The model shows a different orientation of its two domains compared to earlier models of other class B PBPs and a novel, larger N-domain. Consistent with the nomenclature of “dimerization domain”, the N-terminal region forms an apparently tight interaction with a neighboring molecule related by a 2-fold symmetry axis in the crystal structure. This dimer form is predicted to be highly stable in solution by the PISA server, but mass spectrometry and analytical ultracentrifugation provide unequivocal evidence that the protein is a monomer in solution.     

Successful eradication of methicillin-resistantStaphylococcus aureus (MRSA) intestinal carrier status in a healthcare worker — Case report

We describe bacteriophage therapy in the case of a healthcare worker whose gastrointestinal tract was colonized by methicillin-resistant Staphylococcus aureus (MRSA) with subsequent urinary tract infection caused by the same pathogen. Oral treatment with anti-MRSA phages resulted in eradication of the carrier status.     

Methicillin-Resistant Staphylococcus aureus USA300 Latin American Variant in Patients Undergoing Hemodialysis and HIV Infected in a Hospital in Bogotá, Colombia

We aimed to determine the prevalence of MRSA colonization and examine the molecular characteristics of colonizing isolates in patients receiving hemodialysis and HIV-infected in a Colombian hospital. Patients on hemodialysis and HIV-infected were prospectively followed between July 2011 and June 2012 in Bogota, Colombia. Nasal and axillary swabs were obtained and cultured. Colonizing S. aureus isolates were identified by standard and molecular techniques. Molecular typing was performed by using pulse-field gel electrophoresis and evaluating the presence of lukF-PV/lukS-PV by PCR. A total of 29% (n = 82) of HIV-infected and 45.5% (n = 15) of patients on hemodialysis exhibited S. aureus colonization. MSSA/MRSA colonization was observed in 28% and 3.6% of the HIV patients, respectively and in 42.4% and 13.3% of the hemodialysis patients, respectively. Staphylococcal cassette chromosome mec typing showed that four MRSA isolates harbored the type IV cassette, and one type I. In the hemodialysis group, two MRSA isolates were classified as belonging to the USA300-LV genetic lineage. Conversely, in the HIV infected group, no colonizing isolates belonging to the USA300-Latin American Variant (UDA300-LV) lineage were identified. Colonizing isolates recovered from the HIV-infected group belonged to the prevalent hospital-associated clones circulating in Latin America (Chilean [n = 1] and Pediatric [n = 2]). The prevalence of MRSA colonization in the study groups was 3.6% (HIV) and 13.3% (hemodialysis). Surveillance programs should be implemented in this group of patients in order to understand the dynamics of colonization and infection in high-risk patients.