Clinical Methicillin-resistant S. Aureus Research Articles

Inhibition of Clinical MRSA Isolates by Coagulase Negative Staphylococci of Human Origin.

Staphylococcus aureus is frequently highlighted as a priority for novel drug research due to its pathogenicity and ability to develop antibiotic resistance. Coagulase-negative staphylococci (CoNS) are resident flora of the skin and nares. Previous studies have confirmed their ability to kill and prevent colonization by S. aureus through the production of bioactive substances. This study screened a bank of 37 CoNS for their ability to inhibit the growth of methicillin-resistant S. aureus (MRSA). Deferred antagonism assays, growth curves, and antibiofilm testing performed with the cell-free supernatant derived from overnight CoNS cultures indicated antimicrobial and antibiofilm effects against MRSA indicators. Whole genome sequencing and BAGEL4 analysis of 11 CoNS isolates shortlisted for the inhibitory effects they displayed against MRSA led to the identification of two strains possessing complete putative bacteriocin operons. The operons were predicted to encode a nukacin variant and a novel epilancin variant. From this point, strains Staphylococcus hominis C14 and Staphylococcus epidermidis C33 became the focus of the investigation. Through HPLC, a peptide identical to previously characterized nukacin KQU-131 and a novel epilancin variant were isolated from cultures of C14 and C33, respectively. Mass spectrometry confirmed the presence of each peptide in the active fractions. Spot-on-lawn assays demonstrated both bacteriocins could inhibit the growth of an MRSA indicator. The identification of natural products with clinically relevant activity is important in today's climate of escalating antimicrobial resistance and a depleting antibiotic pipeline. These findings also highlight the prospective role CoNS may play as a source of bioactive substances with activity against critical pathogens.

Effect of Bioactive Peptide, KR-12-a5, on Growth of Clinical Methicillin-resistant Staphylococcus aureus Isolates

Staphylococcus aureus is one of the most frequent infectious agents causing hospitals and community associated infections. Developing the treatment for methicillin resistant S. aureus (MRSA) is a challenging arena for effective therapeutics. Application of antimicrobial peptides is an alternative option to control MRSA infection. The potential of the bioactive peptide against MRSA was previously revealed; however, the study on the effect of KR-12-a5 on the various clinical MRSA growth was not elucidated. This study aimed to investigate the effect of bioactive peptide KR-12-a5 on the growth of MRSA isolated from patients. Evaluation of KR-12-a5 peptide using antimicrobial susceptibility test was performed. Antimicrobial activity assay showed that the KR-12-a5 at 2-64 μM could inhibit the growth of S. aureus ATCC 29213 in the range of 55.0±0.8%-81.0±6.2%. In case of KR-12-a5 treatment at 2, 4, and 8 μM, growth of all twenty clinical MRSA isolates could be inhibited less than 90% in the range of 0.0-75.9±1.6%, 0.0-73.0±4.2%, and 0.0-77.1±1.7%, respectively. Whereas the treatment of those at 16, 32, and 64 μM, growth of clinical MRSA could be significantly inhibited more than 90% (p < 0.05) in the range of 94.3±6.7%-100.4±0.4% in 3 isolates (15%), 97.3±0.3%-99.5±0.2% in 4 isolates (20%), and 91.6±0.8%-99.0±0.1% in 9 isolates (45%), respectively. These findings indicated that the KR-12-a5 effectively controls growth of both S. aureus ATCC 29213 and clinical MRSA isolates

Synthesis, characterization, and antibacterial activities of a heteroscorpionate derivative platinum complex against methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus is one of the species with the greatest clinical importance and greatest impact on public health. In fact, methicillin-resistant S. aureus (MRSA) is considered a pandemic pathogen, being essential to develop effective medicines and combat its rapid spread. This study aimed to foster the translation of clinical research outcomes based on metallodrugs into clinical practice for the treatment of MRSA. Bearing in mind the promising anti-Gram-positive effect of the heteroscorpionate ligand 1,1'-(2-(4-isopropylphenyl)ethane-1,1-diyl)bis(3,5-dimethyl-1H-pyrazole) (2P), we propose the coordination of this compound to platinum as a clinical strategy with the ultimate aim of overcoming resistance in the treatment of MRSA. Therefore, the novel metallodrug 2P-Pt were synthetized, fully characterized and its antibacterial effect against the planktonic and biofilm state of S. aureus evaluated. In this sense, three different strains of S. aureus were studied, one collection strain of S. aureus sensitive to methicillin and two clinical MRSA strains. To appraise the antibacterial activity, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC), and minimum biofilm eradication concentration (MBEC) were determined. Moreover, successful outcomes on the development of biofilm in a wound-like medium were obtained. The mechanism of action for 2P-Pt was proposed by measuring the MIC and MBC with EDTA (cation mediated mechanism) and DMSO (exogenous oxidative stress mechanism). Moreover, to shed light on the plausible antistaphylococcal mechanism of this novel platinum agent, additional experiments using transmission electron microscopy were carried out. 2P-Pt inhibited the growth and eradicated the three strains evaluated in the planktonic state. Another point worth stressing is the inhibition in the growth of MRSA biofilm even in a wounded medium. The results of this work support this novel agent as a promising therapeutic alternative for preventing infections caused by MRSA.

A fusidic acid-resistant (PVL+) clone is associated with the increase in methicillin-resistant Staphylococcus aureus in New Caledonia

ObjectivesSince 2014, Staphylococcus aureus methicillin resistance has been rapidly increasing in New Caledonia and is associated with potential serious clinical repercussions. In the present study, we investigated the epidemiology of methicillin-resistant S. aureus (MRSA) in New Caledonia and the possible emergence of a particular clonal strain. MethodsAn overview of the distribution of MRSA in New Caledonia in 2019 is presented. We collected and analysed 171 clinical MRSA isolates from New Caledonia medical laboratories during August and September 2019. Among this collection, 49 representative isolates were analyzed by the French National Reference Center for Staphylococci using the StaphyType DNA microarray, allowing genetic characterization of the isolates. ResultsAmong the 1144 S. aureus isolated over the year 2019, 442 isolates (39%) were resistant to methicillin, and 62% of these isolates were resistant to fusidic acid (FA). During the inclusion period, FA resistance rate was similar (60%). Genetic characterization evidenced CC6 as the predominant clonal complex (70%) with 26 isolates (53%) identified as CC6-MRSA-[IV+fus] (PVL+). ConclusionsThese findings demonstrated a low diversity of MRSA in New Caledonia, with the dominance of a clonal complex not reported previously. The frequent fusidic acid (FA) resistance in MRSA was associated with a high prevalence of fusC gene, suggesting that FA misuse contributed to driving the selection of this clone. Our findings suggest the recommendation to stop the topical use of FA to control the emergence of this severe MRSA clone and decrease the rate of MRSA in New Caledonia.

Comparison of peaks in the matrix-assisted laser desorption ionization time-of-flight mass spectrometry spectra of Staphylococcus aureus grown on various blood agar plates

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is routinely used for bacterial identification in clinical laboratories. Bacterial protein expression may differ according to their growth conditions, especially the culture medium composition. We aimed to study the peak variations of Staphylococcus aureus grown on various blood agar plates (BAP), especially phenol-soluble modulin-mec (PSM-mec) peak (m/z 2409) associated with mecA gene conferring methicillin resistance. Methicillin-resistant S. aureus (MRSA) ATCC 43300 and eight clinical MRSA isolates were cultured on various commercial BAPs including tryptic soy agar-based BAPs, Columbia agar-based BAP and in-house BAPs with the addition of yeast extract. Analysis of the MALDI-TOF peaks of S. aureus, cultured on various BAPs, revealed the peak intensities of low-molecular weight proteins to vary depending on the composition of BAPs, especially the presence or absence of yeast extract. Especially, the PSM-mec and delta-toxin peaks showed low intensity for S. aureus ATCC 43300 and clinical isolates. No significant differences were found in the number of peaks, but some peaks had lower intensity, corresponding to the medium containing yeast extract, in low-mass region (<m/z 4000). BAPs based on tryptic soy agar rather than Columbia agar seems to be appropriate for the detection of PSM-mec, a methicillin resistance marker of S. aureus and delta-toxin, an agr function indicator.

Ultrastructural changes in methicillin-resistant Staphylococcus aureus (MRSA) induced by metabolites of thermophilous fungi Acrophialophora levis.

Staphylococcus aureus and Methicillin-resistant S. aureus (MRSA) remains one of the major concerns of healthcare associated and community-onset infections worldwide. The number of cases of treatment failure for infections associated with resistant bacteria is on the rise, due to the decreasing efficacy of current antibiotics. Notably, Acrophialophora levis, a thermophilous fungus species, showed antibacterial activity, namely against S. aureus and clinical MRSA strains. The ethyl acetate extract of culture filtrate was found to display significant activity against S. aureus and MRSA with a minimum inhibitory concentration (MIC) of 1 μg/mL and 4 μg/mL, respectively. Scanning electron micrographs demonstrated drastic changes in the cellular architecture of metabolite treated cells of S. aureus and an MRSA clinical isolate. Cell wall disruption, membrane lysis and probable leakage of cytoplasmic are hallmarks of the antibacterial effect of fungal metabolites against MRSA. The ethyl acetate extract also showed strong antioxidant activity using two different complementary free radicals scavenging methods, DPPH and ABTS with efficiency of 55% and 47% at 1 mg/mL, respectively. The total phenolic and flavonoid content was found to be 50 mg/GAE and 20 mg/CAE, respectively. More than ten metabolites from different classes were identified: phenolic acids, phenylpropanoids, sesquiterpenes, tannins, lignans and flavonoids. In conclusion, the significant antibacterial activity renders this fungal strain as a bioresource for natural compounds an interesting alternative against resistant bacteria.

Functional Characterization of Type III-A CRISPR-Cas in a Clinical Human Methicillin-R Staphylococcus aureus Strain.

CRISPR with its cas genes is an adaptive immune system that protects prokaryotes against foreign genetic elements. The type III-A CRISPR-Cas system is rarely found in Staphylococcus aureus, and little is known about its function in S. aureus. Here, we describe the genome characteristics of the clinical methicillin-resistant S. aureus (MRSA) strain TZ0912, carrying a type III-A CRISPR-Cas system. Phylogenetic analysis of 35 reported CRISPR-Cas-positive S. aureus strains revealed that the CRISPR-Cas system is prevalent in CC8 clones (10/35) and is located in the staphylococcal cassette chromosome mec (SCCmec) V, which confers methicillin resistance. Plasmid transformation and phage infection assays reveal that the type III-A CRISPR-Cas system protects TZ0912 against foreign DNA with sequence homology to the spacers located in the CRISPR array. We observed that the CRISPR-Cas immune system could effectively protect MRSA against phage attacks in both liquid culture and solid medium. In accordance with previous reports, using RNA-seq analysis and plasmid transformation assays, we find that the crRNAs close to the leading sequence of the CRISPR array are more highly expressed and are more effective at directing plasmid elimination compared to the distant spacers. This study established a model for evaluating the efficiency of naive CRISPR-Cas system in MRSA against phage, which could contribute to future research on the function of CRISPR-Cas in clinical MRSA isolates and improve phage therapy against MRSA infections.

The Role of Subinhibitory Concentrations of Daptomycin and Tigecycline in Modulating Virulence in Staphylococcus aureus.

Staphylococcus aureus (S. aureus) infections are notoriously complicated by the ability of the organism to grow in biofilms and are difficult to eradicate with antimicrobial therapy. The purpose of the current study was to clarify the influence of sub-inhibitory concentrations (sub-MICs) of daptomycin and tigecycline antibiotics on biofilm adhesion factors and exoproteins expressions by S. aureus clinical isolates. Six clinical isolates representing positive biofilm S. aureus clones (3 methicillin-sensitive S. aureus (MSSA) and 3 methicillin-resistant S. aureus (MRSA)) were grown with sub-MICs (0.5 MIC) of two antibiotics (daptomycin and tigecycline) for 12 h of incubation. RNA extracted from culture pellets was used via relative quantitative real-time-PCR (qRT-PCR) to determine expression of specific adhesion (fnbA, fnbB, clfA, clfB, fib, ebps, cna, eno) and biofilm (icaADBC) genes. To examine the effect of sub-MIC of these antibiotics on the expression of extracellular proteins, samples from the culture supernatants of six isolates were collected after 12 h of treatment with or without tigecycline in order to profile protein production via 2D gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D gel-SDS-PAGE). Sub-MIC treatment of all clinical MRSA and MSSA strains with daptomycin or tigecycline dramatically induced or suppressed fnbA, fnbB, clfA, clfB, fib, ebps, cna, eno, and icaADBC gene expression. Furthermore, sub-MIC use of tigecycline significantly reduced the total number of separated protein spots across all the isolates, as well as decreasing production of certain individual proteins. Collectively, this study showed very different responses in terms of both gene expression and protein secretion across the various isolates. In addition, our results suggest that sub-MIC usage of daptomycin and tigecycline could signal virulence induction by S. aureus via the regulation of biofilm adhesion factor genes and exoproteins. If translating findings to the clinical treatment of S. aureus, the therapeutic regimen should be adapted depending on antibiotic, the virulence factor and strain type.

Evolving MRSA: High-level β-lactam resistance in Staphylococcus aureusis associated with RNA Polymerase alterations and fine tuning of gene expression.

Most clinical MRSA (methicillin-resistant S. aureus) isolates exhibit low-level β-lactam resistance (oxacillin MIC 2-4 μg/ml) due to the acquisition of a novel penicillin binding protein (PBP2A), encoded by mecA. However, strains can evolve high-level resistance (oxacillin MIC ≥256 μg/ml) by an unknown mechanism. Here we have developed a robust system to explore the basis of the evolution of high-level resistance by inserting mecA into the chromosome of the methicillin-sensitive S. aureus SH1000. Low-level mecA-dependent oxacillin resistance was associated with increased expression of anaerobic respiratory and fermentative genes. High-level resistant derivatives had acquired mutations in either rpoB (RNA polymerase subunit β) or rpoC (RNA polymerase subunit β') and these mutations were shown to be responsible for the observed resistance phenotype. Analysis of rpoB and rpoC mutants revealed decreased growth rates in the absence of antibiotic, and alterations to, transcription elongation. The rpoB and rpoC mutations resulted in decreased expression to parental levels, of anaerobic respiratory and fermentative genes and specific upregulation of 11 genes including mecA. There was however no direct correlation between resistance and the amount of PBP2A. A mutational analysis of the differentially expressed genes revealed that a member of the S. aureus Type VII secretion system is required for high level resistance. Interestingly, the genomes of two of the high level resistant evolved strains also contained missense mutations in this same locus. Finally, the set of genetically matched strains revealed that high level antibiotic resistance does not incur a significant fitness cost during pathogenesis. Our analysis demonstrates the complex interplay between antibiotic resistance mechanisms and core cell physiology, providing new insight into how such important resistance properties evolve.

First report of mecC gene in clinical methicillin resistant S. aureus (MRSA) from tertiary care hospital Islamabad, Pakistan

BackgroundStaphylococcus aureus (S. aureus) is one of the leading causes of community and hospital acquired infections globally. The objective of the study was to assess the prevalence, study the carriage of antibiotic resistance genes and evaluate the molecular typing of S. aureus isolates from a tertiary care hospital in Islamabad. MethodsA total of 1528 staphylococci isolates were included in this study. Standard microbiological procedures were applied to identify S. aureus. Antimicrobial susceptability was evaluated using the disk diffusion method and Minimum Inhibitory Concentrations (MICs) were determined using microbroth dilution method following Clinical and Laboratory Standards Institute (CLSI) guidelines. Multiplex PCR was used to detect antibiotic resistance genes, and molecular typing was performed using agr, SCCmec, spa, and Multi-Locus Sequence Typing (MLST) and clonal relatedness by Pulse Field Gel Electrophoresis (PFGE) methods. ResultsOverall 65% were MRSA and 35% were methicillin sensitive Staphylococcus aureus (MSSA). Among MRSA isolates, 83% were multi-drug resistant and mecA was found in 54% isolates, mecC was in 3% while 1 MRSA carried both mecA and mecC genes. agrI (22%) was most prevalent group in MRSA, while agrIII (16%) was observed in MSSA. SCCmec types I, II, III, IV, and VI were detected, with high prevalence of type III while type V was absent. The prevelant spa type in MRSA was t657 with SCCmecIII elelments while in MSSA it was t021. One NEW spa type identified in MSSA isolates. In a subset of isolates, ST772 with SCCmecIV, ST1 carrying PVL marker, and ST1535 was reported first time from Pakistan. ConclusionsThe study presents a comprehensive analysis of prevalent S. aureus types and their antibiotic resistance profiles. It also reports for the first time SCCmec type VI and clinical MRSA isolates with mecC alone and in combination with mecA from Islamabad, Pakistan. This calls for further detailed investigations in other hospital settings in the region.

Identification of a novel phage targeting methicillin-resistant Staphylococcus aureus In vitro and In vivo

IntroductionStaphylococcus aureus is a common human pathogen that causes various diseases including infections on the skin, in the bloodstream and the lower respiratory tracts. The emergence of methicillin-resistant S. aureus (MRSA) made the treatment of the bacterial infection more difficult, calling for development of new therapeutics. Compared with conventional antibiotic therapy, phage therapy offers a promising alternative to combat infections caused by MRSA. ResultsHere we showed that phage VB_SauS_SH-St 15644 isolated from sewage inhibited MRSA isolates in vitro and in the murine skin infection model. Phage VB_SauS_SH-St 15644 belongs to Siphoviridae. The genome of the phage is a linear, 45,111 bp double-stranded DNA with GC content of 33.35%. Among the 37 clinical MRSA isolates tested, 12 (32%) were lysed by the phage in vitro. The phage was relatively stable at temperatures up to 40 °C or between pH 6 and 9. However, the phage was sensitive to UV light. 80% of the phage was approximately adsorbed to the host MRSA isolate in 4 min. The one-step growth curve showed that the latent period was about 12 min followed by the growth period (about 9 min). The burst size was estimated at 13 PFU per infected cell. Furthermore, in a murine skin infection model, the phage significantly inhibited MRSA infection. ConclusionsOur study suggested that phage VB_SauS_SH-St 15644 has a potential to inhibit MRSA skin infection.

Staphylococcus aureus biofilm organization modulated by YycFG two-component regulatory pathway

BackgroundStaphylococcus aureus (S. aureus) infection accounts for more than 50% of the osteomyelitis cases. Currently, methicillin-resistant S. aureus (MRSA) strains present an urgent medical problem. The YycFG two-component regulatory system (TCS) can allow bacteria to rapidly adapt to physical, chemical, and biological stresses. To define the role of YycFG in modulation virulence of S. aureus in osteomyelitis, we isolated clinical MRSA strains and compared these with ATCC29213 methicillin-sensitive S. aureus (MSSA).MethodsIn the present study, 13 MRSA strains from chronic osteomyelitis tissues were isolated. The in-depth sequencing of 16S rRNA amplicons of the samples was conducted. Bacterial growth was monitored, and biofilm biomass was determined by crystal violet microtiter assay. Furthermore, quantitative RT-PCR analysis was adopted to identify the expression of yycF/G/H and icaA/D in MRSA and MSSA strains. Analysis of variance with one-way ANOVA was used for statistical analysis.ResultsThe in-depth sequencing of 16S rRNA amplicons of the clinical samples indicated a polymicrobial infection, with the phylum Firmicutes made up 13% of the microbial population. The MRSA strains showed an accelerated growth rate compared to the MSSA strains. Of note, MRSA biofilms showed an accumulation of an intercellular polysaccharides matrix and enhanced biomass upon microscopic examination. Furthermore, MRSA strains had a higher expression of the yycF/G/H and icaA/D genes and adhesion force.ConclusionsThese data suggested the roles of intercellular polysaccharide in S. aureus pathogenesis, indicating a possible association between YycFG pathways and MRSA strain virulence.

Sub-Inhibitory Concentrations of Mupirocin Strongly Inhibit Alpha-Toxin Production in High-Level Mupirocin-Resistant MRSA by Down-Regulating agr, saeRS, and sarA.

Mupirocin, a topical antibiotic, has been utilized for decades to treat Staphylococcus aureus skin infections, as well as to decolonize patients at risk of methicillin-resistant S. aureus (MRSA) infection. The aims of this study were to investigate the expression of α-toxin (encoded by the hla gene) in ten clinical MRSA strains (MIC = 1024 μg/ml) in response to a sub-inhibitory concentration of mupirocin (1/32 minimum inhibitory concentration [MIC]) (32 μg/ml) by using α-toxin activity determination and enzyme-linked immune sorbent assay (ELISA). Subsequently, real-time polymerase chain reaction (RT-PCR) was used to examine the expression of saeR, agrA, RNAIII, and sarA genes under sub-inhibitory concentration of mupirocin in order to investigate the mechanism of action of this treatment regarding its strong inhibition of α-toxin expression. For all the strains tested, mupirocin dramatically reduced mRNA levels of α-toxin. The results indicated that α-toxin activity in mupirocin-treated groups was significantly lower than that in untreated groups. The results show that the levels of agrA, RNAIII, saeR, and sarA expression significantly decrease by 11.82- to 2.23-fold (P < 0.01). Moreover, we speculate that mupirocin-induced inhibition of α-toxin expression may be related to the inhibition of regulatory loci, such as agr, sarA and saeRS. More specifically, we found that the mechanism involves inhibiting the expression of agrA and RNAIII. In conclusion, sub-inhibitory concentrations of mupirocin strongly inhibit alpha-toxin production in high-level mupirocin-resistant MRSA by down-regulating agr, saeRS and sarA, which could potentially be developed as a supplemental treatment to control high-level mupirocin-resistant MRSA infection and reduce the risk of infection and colonization.

The antimicrobial potential of a new derivative of cathelicidin from Bungarus fasciatus against methicillin-resistant Staphylococcus aureus.

Cathelicidins are a family of antimicrobial peptides which exhibit broad antimicrobial activities against antibiotic-resistant bacteria. Considering the progressive antibiotic resistance, cathelicidin is a candidate for use as an alternative approach to treat and overcome the challenge of antimicrobial resistance. Cathelicidin-BF (Cath-BF) is a short antimicrobial peptide, which was originally extracted from the venom of Bungarus fasciatus. Recent studies have reported that Cath-BF and some related derivatives exert strong antimicrobial and weak hemolytic properties. This study investigates the bactericidal and cytotoxic effects of Cath-BF and its analogs (Cath-A and Cath-B). Cath-A and Cath-B were designed to increase their net positive charge, to have more activity against methicillin resistant S. aureus (MRSA). The results of this study show that Cath-A, with a +17-net charge, has the most noteworthy antimicrobial activity against MRSA strains, with minimum inhibitory concentration (MIC) ranging between 32-128 μg/ml. The bacterial kinetic analysis by 1 × MIC concentration of each peptide shows that Cath-A neutralizes the clinical MRSA isolate for 60 min. The present data support the notion that increasing the positive net charge of antimicrobial peptides can increase their potential antimicrobial activity. Cath-A also displayed the weakest cytotoxicity effect against human umbilical vein endothelial and H9c2 rat cardiomyoblast cell lines. Analysis of the hemolytic activity reveals that all three peptides exhibit minor hemolytic activity against human erythrocytes at concentrations up to 250 μg/ml. Altogether, these results suggest that Cath-A and Cath-B are competent candidates as novel antimicrobial compounds against MRSA and possibly other multidrug resistant bacteria.

Cephalosporin-Glycopeptide Combinations for Use against Clinical Methicillin-Resistant Staphylococcus aureus Isolates: Enhanced In vitro Antibacterial Activity.

The empirical combination of both a beta-lactam and glycopeptide to counter potential staphylococcal pathogens may improve the clinical outcomes for cases of Staphylococcus aureus bacteremia. We reported comparative in vitro studies of combination effects of different cephalosporins (i.e., cefazolin, cefmetazole, cefotaxime, and cefepime) combined with glycopeptides for 34 randomly selected methicillin-resistant S. aureus (MRSA) isolates by three methods, including the checkerboard, time-killing, and combination MIC measurement methods. Thirteen SCCmec type III isolates with a cefazolin MIC of ≥ 128 μg/mL were classified as the high-cefazolin MIC (HCM) group, whereas 13 SCCmec type IV and 8 SCCmec type V isolates were classified as the low-cefazolin MIC (LCM) group. With the checkerboard method, synergism was present for vancomycin-based combinations at 30.8–69.2 and 13.6–66.7%, as well as teicoplanin-based combinations of 38.5–84.6 and 0–47.6%, of the HCM and LCM isolates, respectively. No antagonism was noted. The in vitro inhibitory activity was evident even at a low concentration of 1/512x MIC of cephalosporin combined with sub-inhibitory concentrations (1/2x MIC) of a glycopeptide. With time-killing assays, synergism was noted at 1/2x or 1x susceptible breakpoint concentrations (SBCs) of a cephalosporin combined with 1/4 or 1/2 MIC of a glycopeptide. In the presence of 1/2 SBC of a cephalosporin, vancomycin or teicoplanin MICs decreased an average of 2.0- to 6.6- or 1.6- to 5.5-fold, respectively. With 8 μg/mL cephalosporin, the decline of glycopeptide MICs was most obvious in the presence of cefmetazole. In conclusion, cephalosporin-glycopeptide combinations at clinically achievable concentrations can exhibit in vitro synergistic antibacterial activity against clinical MRSA isolates. Such combinations require more clinical data to support their application for use in human MRSA infections.

Metal Susceptibility of a Hetero-Vancomycin-Intermediate Methicillin-Resistant Staphylococcus aureus Isolate

The Gram-positive bacterium Staphylococcus aureus is well known for its ability to develop resistance to various antimicrobial substances. Methicillin-resistant S. aureus (MRSA), a cause of nosocomial infections worldwide, is becoming increasingly resistant to the glycopeptide antibiotic vancomycin, one of few antibiotics used to treat serious multiple-antibiotic-resistant staphylococcal infections. With the increase in the number of MRSA strains showing reduced sensitivity to vancomycin, it has become important to investigate alternative treatment options. In this study, we examined the effects of five metals: silver, copper, arsenate, zinc, and cadmium on the growth of a clinical MRSA strain MM66 demonstrating heterogeneous intermediatelevel resistance to vancomycin. Disc diffusion and gradient plate experiments were used to compare the metal susceptibility levels of strain MM66 to that of the methicillin-resistant S. aureus laboratory control strain. MM66 grew less successfully when exposed to metals, and showed an overall increased level of susceptibility to metals compared to the laboratory control strain. Of the metals tested, silver exerted the highest inhibitory effect on the growth of MM66.

Vancomycin (VAN) Combinations with Β-Lactams (BLs) against Methicillin-Resistant Staphylococcus aureus (MRSA), Heterogeneous Intermediate-Level Resistance to Vancomycin (hVISA) and Vancomycin-Intermediate Staphylococcus aureus (VISA)

Background Staphylococcus aureus (S. aureus), especially Methicillin-resistant S. aureus (MRSA) remains a major cause of serious infection and is associated with increased morbidity and mortality. Vancomycin (VAN) has been the mainstay of therapy for MRSA infections. However, decades of selective pressure have led to increasing concerns regarding the efficacy of VAN against MRSA. In vitro data suggest the potential for potent synergy between several Β-lactams (BLs) and VAN. The objective of this study is to further explore the synergistic effect between BLs and VAN against MRSA strains with varying susceptibility to VAN.MethodsFifty randomly selected clinical MRSA strains from the Anti-Infective Research Laboratory library with varying susceptibility to VAN were evaluated for VAN alone and VAN in combination with Cefazolin (CFZ), Cefepime (FEP), Ceftaroline (CPT), and Nafcillin (NAF) minimum inhibitory concentration (MIC) by microdilution in duplicate. The potential for synergy was assessed by 24 hours time-kills (TK). Synergy was defined as >2 log10 CFU/mL difference between combination and the most active single agent at 24 hours.ResultsBLs reduced VAN MIC values against all strains (4–16 fold reduction). In TK studies against MRSA, all BLs demonstrated a similar extent of killing at 24 hours and showed synergy with VAN against all strains. Each combination was superior to any single agent alone, and each was bactericidal (3.42 ± 0.26 log10 CFU/cm2 reduction, P < 0.001 for all comparisons). All single agent exposures demonstrated no activity at 24 hours.Phenotype (Number of Strains)VAN MIC Range (µg/mL)VAN MIC Range with CFZVAN MIC Range with FEPVAN MIC Range with CPTVAN MIC Range with NAFMRSA (15)0.5 – 20.0625 – 0.250.0625 – 10.0625 – 0.250.125 – 0.5hVISA (20)0.5 – 20.125 – 0.50.0625 – 0.50.0625 – 0.50.125 – 1VISA (15)4 (all)0.0625 – 0.50.5 – 20.0625 – 0.250.125 – 1ConclusionThe combination of VAN and BLs significantly improved antibacterial activity against MRSA, hVISA, and VISA compared with any agent alone, supporting the potential use of Vancomycin/BL combination therapy in infections caused by MRSA. Further clinical research is warranted to investigate the synergistic activity of vancomycin against these Staphylococcus strains.Disclosures M. J. Rybak, Allergen: Scientific Advisor, Consulting fee

Activity of novel inhibitors of Staphylococcus aureus biofilms.

Staphylococcus aureus is one of the most important pathogens causing chronic biofilm infections. These are becoming more difficult to treat owing to drug resistance, particularly because S. aureus biofilms limit the efficacy of antimicrobial agents, leading to high morbidity and mortality. In the present study, we screened for inhibitors of S. aureus biofilm formation using a natural product library from the Korea Chemical Bank (KCB). Screening by crystal violet-based biomass staining assay identified hit compounds. Further examination of antibiofilm properties of these compounds was conducted and led to the identification of celastrol and telithromycin. In vitro, both celastrol and telithromycin were toxic to planktonic S. aureus and also active against a clinical methicillin-resistant S. aureus (MRSA) isolate. The effect of the compounds on preformed biofilms of clinical MRSA isolates was evaluated by confocal laser scanning microscopy (CLSM), which revealed the absence of typical biofilm architecture. In addition, celastrol and telithromycin inhibited the production of extracellular protein at selected sub-MIC concentrations, which revealed the reduced extracellular polymeric substance (EPS) secretion. Celastrol exhibited greater cytotoxicity than telithromycin. These data suggest that the hit compounds, especially telithromycin, could be considered novel inhibitors of S. aureus biofilm. Although the mechanisms of the effects on S. aureus biofilms are not fully understood, our data suggest that telithromycin could be a useful adjuvant therapeutic agent for S. aureus biofilm-related infections.

Molecular characterization of Staphylococcus aureus isolates from various healthcare institutions in Nairobi, Kenya: a cross sectional study.

BackgroundStaphylococcus aureus (S. aureus) has established itself over the years as a major cause of morbidity and mortality both within the community and in healthcare settings. Methicillin resistant S. aureus (MRSA) in particular has been a major cause of nosocomial infections resulting in significant increase in healthcare costs. In Africa, the MRSA prevalence has been shown to vary across different countries. In order to better understand the epidemiology of MRSA in a setting, it is important to define its population structure using molecular tools as different clones have been found to predominate in certain geographical locations.MethodsWe carried out PFGE, MLST, SCCmec and spa typing of selected S. aureus isolates from a private and public referral hospital in Nairobi, Kenya.ResultsA total of 93 S. aureus isolates were grouped into 19 PFGE clonal complexes (A–S) and 12 singletons. From these, 55 (32 MRSA and 23 MSSA) representative isolates from each PFGE clonal complex and all singletons were spa typed. There were 18 different MRSA spa types and 22 MSSA spa types. The predominant MRSA spa type was t037 comprising 40.6 % (13/32) of all MRSA. In contrast, the MSSA were quite heterogeneous, only 2 out of 23 MSSA shared the same spa type. Two new MRSA spa types (t13149 and t13150) and 3 new MSSA spa types (t13182, t13193 and t13194) were identified. The predominant clonal complex was CC 5 which included multi-locus sequence types 1, 8 and 241.ConclusionIn contrast to previous studies published from Kenya, there’s marked genetic diversity amongst clinical MRSA isolates in Nairobi including the presence of well-known epidemic MRSA clones. Given that these clones are resident within our referral hospitals, adherence to strict infection control measures needs to be ensured to reduce morbidity and mortality associated with hospital acquired MRSA infections.

Effects of Subinhibitory Concentrations of Ceftaroline on Methicillin-Resistant Staphylococcus aureus (MRSA) Biofilms.

Ceftaroline (CPT) is a novel cephalosporin with in vitro activity against Staphylococcus aureus. Ceftaroline exhibits a level of binding affinity for PBPs in S. aureus including PBP2a of methicillin-resistant S. aureus (MRSA). The aims of this study were to investigate the morphological, physiological and molecular responses of MRSA clinical strains and MRSA biofilms to sub-MICs (1/4 and 1/16 MIC) of ceftaroline by using transmission, scanning and confocal microscopy. We have also used quantitative Real-Time PCR to study the effect of sub-MICs of ceftaroline on the expression of the staphylococcal icaA, agrA, sarA and sasF genes in MRSA biofilms. In one set of experiments, ceftaroline was able to inhibit biofilm formation in all strains tested at MIC, however, a strain dependent behavior in presence of sub-MICs of ceftaroline was shown. In a second set of experiments, destruction of preformed biofilms by addition of ceftaroline was evaluated. Ceftaroline was able to inhibit biofilm formation at MIC in all strains tested but not at the sub-MICs. Destruction of preformed biofilms was strain dependent because the biofilm formed by a matrix-producing strain was resistant to a challenge with ceftaroline at MIC, whereas in other strains the biofilm was sensitive. At sub-MICs, the impact of ceftaroline on expression of virulence genes was strain-dependent at 1/4 MIC and no correlation between ceftaroline-enhanced biofilm formation and gene regulation was established at 1/16 MIC. Our findings suggest that sub-MICs of ceftaroline enhance bacterial attachment and biofilm formation by some, but not all, MRSA strains and, therefore, stress the importance of maintaining effective bactericidal concentrations of ceftaroline to fight biofilm-MRSA related infections.