Clinical Methicillin-resistant Staphylococcus Aureus Research Articles

Evaluation of in vitro activity of ceftaroline on methicillin resistant Staphylococcus aureus blood isolates from Iran.

Background and Objectives:Ceftaroline (CPT) is a novel cephalosporin with potent activity against methicillin-resistant Staphylococcus aureus (MRSA). Despite its recent introduction, CPT resistance in MRSA has been described worldwide. We aimed in the current study to evaluate the in vitro activity of CPT against 91 clinical MRSA and 3 MSSA isolates.Materials and Methods:Susceptibility of isolates to CPT was tested using E-test and disk diffusion (DD) method. The nucleotide sequence of the mecA gene and molecular types of isolates with reduced susceptibility to CPT were further studied to identify resistance conferring mutations in PBP2a and the genetic relatedness of the isolates respectively.Results:Overall, 92.5% of isolates were found to be CPT susceptible (MICs≤1mg/l) and 7 MRSA isolates were characterized with MIC=2mg/l and categorized as susceptible dose dependent. Compared to E-test, DD revealed a categorical agreement rate of 93.6% and the obtained rates for minor, major /very major error were found to be 6.3% and 0% respectively. The MRSA isolates with increased CPT MICs (n=7), belonged to spa types t030 (n=6) and t13927 (n=1) and all carried N146K substitution in PBP2a allosteric domain, except for one isolate which harbored a wild-type PBP2a.Conclusion:While resistance to CPT was not detected we found increased CPT MICs in 7.69% of MRSA isolates. Reduced susceptibility to CPT in the absence of mecA mutations is indicative of contribution of secondary chromosomal mutations in resistance development.

Biofilm Formation of Multidrug-Resistant MRSA Strains Isolated from Different Types of Human Infections.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main pathogens causing chronic infections, mainly due to its capacity to form biofilms. However, the mechanisms underlying the biofilm formation of MRSA strains from different types of human infections are not fully understood. MRSA strains isolated from distinct human infections were characterized aiming to determine their biofilm-forming capacity, the biofilm resistance to conventional antibiotics and the prevalence of biofilm-related genes, including, icaA, icaB, icaC, icaD, fnbA, fnbB, clfA, clfB, cna, eno, ebpS, fib and bbp. Eighty-three clinical MRSA strains recovered from bacteremia episodes, osteomyelitis and diabetic foot ulcers were used. The biofilm-forming capacity was evaluated by the microtiter biofilm assay and the biofilm structure was analyzed via confocal scanning laser microscopy. The antimicrobial susceptibility of 24-h-old biofilms was assessed against three antibiotics and the biomass reduction was measured. The metabolic activity of biofilms was evaluated by the XTT assay. The presence of biofilm-related genes was investigated by whole-genome sequencing and by PCR. Despite different intensities, all strains showed the capacity to form biofilms. Most strains had also a large number of biofilm-related genes. However, strains isolated from osteomyelitis showed a lower capacity to form biofilms and also a lower prevalence of biofilm-associated genes. There was a significant reduction in the biofilm biomass of some strains tested against antibiotics. Our results provide important information on the biofilm-forming capacity of clinical MRSA strains, which may be essential to understand the influence of different types of infections on biofilm production and chronic infections.

Molecular Characterization and Antimicrobial Susceptibility of Methicillin-Resistant Staphylococcus Aureus Isolates from Clinical Samples and Asymptomatic Nasal Carriers in Istanbul (Turkey)

Methicillin-resistant Staphylococcus aureus (MRSA) has been a widespread problem in Turkish hospitals. The aim of this study was to investigate the staphylococcal toxin genes of the clinical and nasal MRSA isolates, and their antibiotic resistance profiles. Isolation of nasal and clinical bacteria was done following standard microbiological methods. The presence of antimicrobial resistance genes (mec A, pvl, tsst-1, and SEs genes) was determined using the real-time polymerase chain reaction (PCR) assay. Among nasal MRSA isolates, 66.7% were toxigenic. The distribution of genes was as follows: pvl 26.7%, tsst-1 3.3%, and SEs 36.7%. Therefore, the nasal MRSA isolates had a rate of 23.3% multidrug resistance (MDR) pattern to the non-beta-lactams antibiotics. All (100%) clinical MRSA isolates were found to be toxigenic. The distribution of genes was as follows; pvl 10%, tsst-1 6.7%, and SEs 100%. The clinical MRSA isolates had a rate of 60% MDR. Following detection of pvl, tsst-1, and SEs among nasal and clinical MRSA isolates, and the presence of high antimicrobial resistance, the spread of these strains may be an additional factor contributing to the emergence of community-acquired (CA)-MRSA and hospital-acquired (HA)-MRSA. This study is the first to determine the resistance to linezolid and tigecycline in both nasal and clinical MRSA isolates, for the first time in Turkey. All nasal and clinical MRSA isolates were uniformly susceptible to vancomycin and quinupristin-dalfopristin. Our findings show that MRSA infections in Turkey can be empirically treated with vancomycin and quinupristin-dalfopristin based on the lack of demonstrable resistance to these drugs.

The Link between Occurrence of Class I Integron and Acquired Aminoglycoside Resistance in Clinical MRSA Isolates.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial infections because of its high resistance. Here, we study the antibiotic resistance in MRSA clinical isolates and their relation to integron I occurrence. A total of 88 clinical Staphylococcus aureus isolates were collected. MRSA were identified by the disk diffusion method (DDM) and confirmed by PCR, and antibiogram was determined by DDM. Integron I, II and the aacA4 gene were investigated by PCR. Integrase-positive strains were analyzed for the presence of resistance gene cassettes by sequencing. All isolates were identified as MRSA by DDM and confirmed by PCR. All isolates were resistant to ampicillin and cefoxitin. Concerning aminoglycosides, the frequency of resistance was reported for streptomycin (60.7%), tobramycin (37.1%) gentamicin (36%), and for amikacin (15.9%). Integron I was detected in 41 isolates (46.6%), while integron II was detected in three isolates (3.4%). Sequencing of the integron I-cassette indicated the exclusive prevalence of addA gene variants mediating aminoglycoside resistance. The aacA4 gene was found in DNA of 31 isolates (35.22%). This study revealed the high existence of MRSA. Furthermore, the AacA4 gene and class I integron harboring aadA gene were predominant in MRSA isolates.

The First Report of a Methicillin-Resistant Staphylococcus aureus Isolate Harboring Type IV SCCmec in Thailand.

Methicillin-resistant Staphylococcus aureus (MRSA) is mostly found in Thailand in the hospital as a nosocomial pathogen. This study aimed to report the genetic characterization of a clinical community-acquired MRSA (CA-MRSA) isolate collected from hospitalized patients in Thailand. Among 26 MRSA isolates, S. aureus no. S17 preliminarily displayed the presence of a staphylococcal cassette chromosome mec (SCCmec) type IV pattern. The bacterial genomic DNA was subjected to whole-genome sequencing. Panton–Valentine leukocidin (PVL) production, virulence toxins, and antibiotic resistance genes were identified, and multi-locus sequence typing (MLST) and spa typing were performed. The strain was matched by sequence to MLST type 2885 and spa type t13880. This strain carried type IV SCCmec with no PVL production. Five acquired antimicrobial resistance genes, namely blaZ, mecA, Inu(A), tet(K), and dfrG conferring resistance to β-lactams, lincosamides, tetracycline, and trimethoprim, were identified. The detected toxins were exfoliative toxin A, gamma-hemolysin, leukocidin D, and leukocidin E. Moreover, there were differences in seven regions in CR-MRSA no. S17 compared to CA-MRSA type 300. In summary, we have reported the ST2885-SCCmec IV CA-MRSA clinical strain in Thailand for the first time, highlighting the problem of methicillin resistance in community settings and the consideration in choosing appropriate antibiotic therapy.

Triphenyl-sesquineolignan analogues derived from Illicium simonsii Maxim exhibit potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) by disrupting bacterial membranes

Infections caused by clinical methicillin-resistant Staphylococcus aureus (MRSA) are a serious public problem. Triphenyl-sesquineolignans from Illicium genus possess antibacterial activity, but few researches have reported their antibacterial spectrums, structure–activity relationships (SARs) and antibacterial mechanism. In this study, three triphenyl-sesquineolignans, dunnianol (1), macranthol (2) and isodunnianol (3) were isolated from the stems and leaves of I. simonsii Maxim, and seven dunnianol derivatives were prepared through esterification, etherification and halogenation reactions. Among all triphenyl-sesquineolignan analogues, compound 6 showed the best antibacterial activity against four Gram-positive bacteria (MICs = 1–2 µg/mL) and ten clinical MRSA strains (MICs = 2–8 µg/mL), and also exhibited characteristics of killing MRSA more rapidly than tigecycline. Meanwhile, compound 6 did not only show a low probability of drug resistance development, but also exhibited relatively low hemolysis, and good stability in 50% plasma. Further mechanism studies revealed that 6 could kill bacterial strains by disrupting bacterial membranes. These results suggested that 6 may be developed into a new antibacterial candidate for combating MRSA infections.

Single nucleotide polymorphism leads to daptomycin resistance causing amino acid substitution-T345I in MprF of clinically isolated MRSA strains.

Daptomycin (DAP) is one of the most potent antibiotics used for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. Due to an increase in its administration for combating MRSA infections, DAP non-susceptible (DAP-NS) MRSA strains have recently been reported in clinical settings. The presence of single nucleotide polymorphisms (SNPs) in the multiple peptide resistance factor (mprF) gene is the most frequently reported cause for the evolution of DAP-NS MRSA strains; however, there are some variations of SNPs that could lead to DAP-NS. In this study, we used two clinical MRSA strains, including DAP susceptible (DAP-S) and DAP-NS, isolated from the same patient at different time points. We introduced T345I SNP to mprF of the DAP-S MRSA strain using the gene exchange method with pIMAY vector. Further, we investigated the phenotype of the mutant strain, including drug susceptibility, cell surface positive charge, and growth speed. The mutant strain exhibited (i) resistance to DAP, (ii) up-regulation of positive surface charge, (iii) slower growth speed, and (iv) thickened cell walls. Hence, the SNP in mprF may have caused an up-regulation in MprF function, with a subsequent increase in positive surface charge. Cumulatively, these results demonstrated that the T345I amino acid substitution in mprF represents one of the primary causes of DAP-NS in MRSA strains.

Reduced Daptomycin Susceptibility in Clinical MRSA Isolates Showing Vancomycin MIC Creep Phenomenon

Introduction: Infections caused by Methicillin Resistant Staphylococcus aureus (MRSA) are associated with increased morbidity, longer antimicrobial therapy, etc. First option for treating invasive MRSA infections is glycopeptide vancomycin. Daptomycin, a lipopeptide rapidly bactericidal invitro against MRSA, is an acceptable alternative. Aim: To identify MRSA isolates from clinical specimens and assess their vancomycin and daptomycin susceptibility pattern. Materials and Methods: The cross-sectional study was conducted over a period of six months (January 2019 to June 2019) on 90 clinical samples in a rural teaching hospital in Pune, Maharashtra, India, including all samples except sputum received in the Microbiology laboratory. MRSA isolates were tested for vancomycin and daptomycin susceptibility by Epsilometer (E) test Minimum Inhibitory Concentration (MIC) method. Results: Among 90 MRSA isolates, most were from pus (51) followed by Urine (23), Blood (9), followed by Miscellaneous samples (7). Data analysis was done using Statistical Package for the Social Sciences (SPSS) version 16.0 software. All MRSA isolates in this study were susceptible to daptomycin with MIC in the range of 0.25-1 μg/mL with maximum isolates (39) with MIC of 0.38 μg/mL. Vancomycin MIC creep phenomenon was observed in 68 isolates. All these isolates also showed reduced susceptibility to daptomycin. Conclusion: MRSA in hospital set up mandates strict infection control practices in place. Daptomycin can be a good therapeutic alternative to treat infections caused by MRSA keeping in mind its therapeutic limitations and prior vancomycin usage in the same patient. Empirical therapy should always be based on antibiogram pattern. Adherence to hospital antibiotic policy and constant surveillance of antimicrobial resistance is the need of the hour.

Efficacy of vancomycin in combination with various antimicrobial agents against clinical methicillin resistant Staphylococcus aureus strains.

Background:Multi-drug resistant methicillin resistant Staphylococcus aureus (MRSA) strains that have been isolated frequently worldwide have difficulties in the treatment and therefore alternative choices for the treatment of the infections are required. The aim of the study was to evaluate the interaction of various antimicrobials in combination with vancomycin against MRSA.Methods:Twenty five clinical MRSA strains isolated in 2016 were included in the study. The interaction between vancomycin and new generation/conventional antimicrobials against MRSA strains was analyzed by E-test.Results:All of the strains tested was found to be susceptible to vancomycin, telavancin, dalbavancin, ceptobiprole, daptomycin, linezolid, quinupristin-dalfopristin, trimethoprim-sulfamethoxazole, rifampicin and tigecycline. The susceptibility rates of the isolates were found to be high, with the lowest rate (48%) against azithromycin. According to the fractional inhibitory concentration index results, synergistic interaction with vancomycin was determined with trimethoprim-sulfamethoxazole, azithromycin, linezolid, minocycline, dalbavancin, clindamycin in five, three, two, two, one, one and one strain(s), respectively. Additionally, all combinations studied showed additive interaction at high rates.Conclusions:The results of the study indicate that the use of vancomycin in combination with conventional and new generation antibiotics is promising.

Antimicrobial Action and Reversal of Resistance in MRSA by Difluorobenzamide Derivatives Targeted at FtsZ.

The bacterial cell division protein, FtsZ, has been identified as a target for antimicrobial development. Derivatives of 3-methoxybenzamide have shown promising activities as FtsZ inhibitors in Gram-positive bacteria. We sought to characterise the activity of five difluorobenzamide derivatives with non-heterocyclic substituents attached through the 3-oxygen. These compounds exhibited antimicrobial activity against methicillin resistant Staphylococcus aureus (MRSA), with an isopentyloxy-substituted compound showing modest activity against vancomycin resistant Enterococcus faecium (VRE). The compounds were able to reverse resistance to oxacillin in highly resistant clinical MRSA strains at concentrations far below their MICs. Three of the compounds inhibited an Escherichia coli strain lacking the AcrAB components of a drug efflux pump, which suggests the lack of Gram-negative activity can partly be attributed to efflux. The compounds inhibited cell division by targeting S. aureus FtsZ, producing a dose-dependent increase in GTPase rate which increased the rate of FtsZ polymerization and stabilized the FtsZ polymers. These compounds did not affect the polymerization of mammalian tubulin and did not display haemolytic activity or cytotoxicity. These derivatives are therefore promising compounds for further development as antimicrobial agents or as resistance breakers to re-sensitive MRSA to beta-lactam antibiotics.

Housefly Phormicin inhibits Staphylococcus aureus and MRSA by disrupting biofilm formation and altering gene expression in vitro and in vivo

The increasing drug resistance of pathogenic bacteria is a crisis that threatens public health. Antimicrobial peptides (AMPs) have been suggested to be potentially effective alternatives to solve this problem. Here, we tested housefly Phormicin-derived peptides for effects on Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) infections in vitro and in vivo. A decreased bacterial load of MRSA was observed in the mouse scald model after treatment with Phormicin and in the positive control group (vancomycin). A mouse scrape model indicated that Phormicin helps the host fight drug-resistant MRSA infections. The protective effect of Phormicin on MRSA was confirmed in the Hermetia illucens larvae model. Phormicin also disrupted the formation of S. aureus and MRSA biofilms. Furthermore, this effect coincided with the downregulation of biofilm formation-related gene expression (agrC, sigB, RNAIII, altA, rbf, hla, hld, geh and psmɑ). Notably, virulence genes and several regulatory factors were also altered by Phormicin treatment. Based on these findings, housefly Phormicin helps the host inhibit MRSA infection through effects on biofilm formation and related gene networks. Therefore, housefly Phormicin potential represents a candidate agent for clinical MRSA chemotherapy.

Novel Antibiotic Combinations of Diverse Subclasses for Effective Suppression of Extensively Drug-Resistant Methicillin-Resistant Staphylococcus aureus (MRSA).

The emergence of multidrug-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), the chief etiological agent for a range of refractory infections, has rendered all β-lactams ineffective against it. The treatment process is further complicated with the development of resistance to glycopeptides, primary antibiotics for treatment of MRSA. Antibiotic combination therapy with existing antimicrobial agents may provide an immediate treatment option. Minimum inhibitory concentrations (MICs) of 18 different commercially available antibiotics were determined along with their 90 possible pairwise combinations and 64 triple combinations to filter out 5 best combinations. Time-Kill kinetics of these combinations were then analyzed to find collateral bactericidal combinations which were then tested on other randomly selected MRSA isolates. Among the top 5 combinations including levofloxacin-ceftazidime; amoxicillin/clavulanic acid-tobramycin; amoxicillin/clavulanic acid-cephradine; amoxicillin/clavulanic acid-ofloxacin; and piperacillin/tazobactam-tobramycin, three combinations were found to be collaterally effective. Levofloxacin-ceftazidime acted synergistically in 80% of the tested clinical MRSA isolates. First-line β-lactams of lower generations can be used effectively against MRSA infection when used in combination. Antibiotics other than glycopeptides may still work in combination.

Combination Therapy Using Low-Concentration Oxacillin with Palmitic Acid and Span85 to Control Clinical Methicillin-Resistant Staphylococcus aureus.

The overuse of antibiotics has led to the emergence of multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). MRSA is difficult to kill with a single antibiotic because it has evolved to be resistant to various antibiotics by increasing the PBP2a (mecA) expression level, building up biofilm, introducing SCCmec for multidrug resistance, and changing its membrane properties. Therefore, to overcome antibiotic resistance and decrease possible genetic mutations that can lead to the acquisition of higher antibiotic resistance, drug combination therapy was applied based on previous results indicating that MRSA shows increased susceptibility to free fatty acids and surfactants. The optimal ratio of three components and the synergistic effects of possible combinations were investigated. The combinations were directly applied to clinically isolated strains, and the combination containing 15 μg/mL of oxacillin was able to control SCCmec type III and IV isolates having an oxacillin minimum inhibitory concentration (MIC) up to 1024 μg/mL; moreover, the combination with a slightly increased oxacillin concentration was able to kill SCCmec type II. Phospholipid analysis revealed that clinical strains with higher resistance contained a high portion of 12-methyltetradecanoic acid (anteiso-C15:0) and 14-methylhexadecanoic acid (anteiso-C17:0), although individual strains showed different patterns. In summary, we showed that combinatorial therapy with a low concentration of oxacillin controlled different laboratory and highly diversified clinical MRSA strains.

Antimicrobial Nonsusceptibility Among Invasive MRSA USA300 Strains by Healthcare Exposure, Three Sites, 2005–2016

Background: Incidence of community-associated (CA) and healthcare-associated, community-onset (HACO) USA300 methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections has remained unchanged in recent years. Traditionally considered a CA strain, USA300 is increasingly associated with healthcare settings. We examined whether antimicrobial nonsusceptibility among USA300 strains could distinguish epidemiologic class (community vs hospital), and whether divergences in susceptibility were occurring over time. Methods: We used data on invasive MRSA infections from active, population, and laboratory-based surveillance during 2005–2016 from 11 counties in 3 states. Invasive cases were defined as MRSA isolation from a normally sterile site in a surveillance area resident. Cases were considered hospital-onset (HO) if the culture was obtained >3 days after hospitalization and HACO if ≥1 of the following risk factors was present: hospitalization, surgery, dialysis, or residence in a long-term care facility in the past year; or central vascular catheter ≤2 days before culture. Otherwise, cases were considered CA. Sites submitted a convenience sample of clinical MRSA isolates for molecular typing and antimicrobial susceptibility testing. Molecular typing was performed by pulsed-field gel electrophoresis until 2008, when typing was inferred using a validated algorithm based on molecular characteristics. Reference broth microdilution was performed for 8 antimicrobials and interpreted based on CLSI interpretive criteria. We compared USA300 nonsusceptibility for HO and CA isolates. For antimicrobials with >5% nonsusceptibility and for which HO isolates had greater nonsusceptibility than CA isolates, we compared nonsusceptibility for HACO and CA and analyzed annual trends in nonsusceptibility within each epidemiologic class (ie, CA, HACO, and HO) using linear regression. Results: Of 17,947 MRSA cases during 2005–2016, isolates were available for 6,685 (37%), and 2,120 were USA300 (34% CA, 52% HACO, 14% HO). HO isolates had more nonsusceptibility than CA isolates to gentamicin (2.2% vs 0.6%; P = .03), levofloxacin (47.8% vs 39.7%; P = .02), rifampin (3.7 vs 1.1%; P = .01), and trimethoprim-sulfamethoxazole (3.4% vs 0.6%; P = .04). HACO isolates also had more nonsusceptibility than CA isolates to levofloxacin (50.9% vs 39.7%; P < .01). Levofloxacin nonsusceptibility increased during 2005–2016 for HACO and CA isolates (P < .01), but not among HO isolates (P = .36) (Fig. 1). Conclusions: Overall, nonsusceptibility across drugs cannot distinguish USA300 isolates causing HO versus CA disease. Although HO isolates had higher levofloxacin nonsusceptibility than CA and HACO isolates early on, USA300 MRSA HACO isolates now have levofloxacin nonsusceptibility most similar to that of HO isolates. Further study could help to explore whether increases in fluoroquinolone nonsusceptibility among CA and HACO cases may be contributing to the persistence of USA300 strains.Disclosures: NoneFunding: None

Screening Anti-MRSA Activities of Indigenous Microbes and Prediction of The Biosynthetic Gene Clusters

Methicillin-Resistant Staphylococcus aureus (MRSA) is a species of S. Aureus, which shows resistance to the methicillin class of antibiotics. MRSA infection poses significant health problems because it causes severe disease in a vulnerable population and also because effective antibiotics are limited. Therefore, the research to find the new anti-MRSA compound is a necessity. The genome mining approach through Biosynthetic Gene Clusters (BGC) analysis can detect the ability of microbes to produce antibiotics. Thus, the study aimed to screen potential anti-MRSA microbes and predict their BGC. This study used ten microorganisms isolated from mangrove areas in Segara Anakan Cilacap and clinical MRSA 2983 from the Faculty of Medicine, UNSOED. All samples microorganisms were cultivated in starch casein nitrate agar. DNA isolation used Quick-DNA Fungal/Bacterial Miniprep Kit, 16S rRNA gene amplification used 27F and 1492R primers, and DNA amplicons were sequenced by the Sanger sequencing method. BCG prediction of the associated genes used antiSMASH 5.1.2. The results showed that W-5A, P-6B, and W-5B isolates have anti-MRSA activities with inhibition indexes of 0.58; 0.53; and 0.47, respectively. Species identification revealed similarity of W-5A with Streptomyces longisporoflavus strain Moghannam M1, P-6B with Ochrobactrum intermedium LMG 3301, and W-5B with Streptomyces cellulosae strain NBRC 13027. The results of BGC analysis showed that Streptomyces longisporoflavus has one region involved in the synthesis of tetronasin. Ochrobactrum intermedium has one region involved in the synthesis ambactin. Streptomyces cellulosae has eight gene clusters for tiacumicin B, actinorhodin, ulleungmycin, albaflavenone, desferrioxamine B/E, stenothricin, auricin, and prejadomycin productions. These results indicate that the microbes isolated from Segara Anakan Cilacap serve as promising antibacterial producers.

Use of Varying Single-Nucleotide Polymorphism Thresholds to Identify Strong Epidemiologic Links Among Patients with Methicillin-Resistant Staphylococcus aureus (MRSA)

Background: Whole-genome sequencing (WGS) has a high discriminatory power in confirming outbreaks. Outbreak investigation models that categorize the possibility of an outbreak based on the degree of genetic relatedness of isolates are highly dependent on the single-nucleotide polymorphism (SNP) threshold used. Methods: NYU Langone Medical center is a 725-bed academic center that has implemented WGS of methicillin-resistant Staphylococcus aureus (MRSA) isolates since 2016. Patients admitted to a medical or intensive care unit were screened on admission and transfer. The first surveillance and clinical MRSA isolate during each hospitalization was sequenced. We conducted a retrospective analysis to identify strong epidemiologic links among patients involved in genetically related clusters. We used different SNP thresholds to define genetic relatedness to identify the optimal threshold that should prompt an outbreak investigation. We considered strong hospital epidemiologic links sharing the same room or unit or having resided in the same room or unit within 7 days. A pairwise analysis was conducted to compare the epidemiologic links among patients involved in genetically related clusters. Results: Among 1,070 isolates, our analysis focused on 777 belonging to USA100 and USA300 clones. For USA100 isolates, we identified 8, 14, and 20 clusters comprising of 16, 29, and 42 patients when the threshold for genetic relatedness was set at 20, 40, and 60 SNP differences, respectively. Patients identified in a cluster yielded a strong hospital epidemiologic link in 62.5%, 87.5%, and 91.7% of cases (Fig. 1). For USA300 isolates, SNP differences of 10, 20, and 30 were used, identifying 20, 34, and 40 clusters of 43, 79, and 127 patients. The expansion of the threshold from 10 to 30 resulted in a decrease of the percentage of pairwise analyses with a strong hospital epidemiologic link from 57.7% to 13.6% by increasing 13-fold the number of analyses that were conducted to identify only 3 times more cases with strong epidemiologic links (Fig. 2). Conclusions: The results of our study indicate that SNPs thresholds determined by intrapatient variability of MRSA isolates might need to be tailored to the individual setting to guide infection control interventions because optimal thresholds might vary depending on characteristics of the population, MRSA isolates, and screening practices. Establishing conservative thresholds might allow the identification and quantification over time of the locations (eg, rooms or units) where transmission is occurring as well as the investigation of the clusters without strong epidemiologic links that might be valuable in elucidating unrecognized routes of transmission.Funding: NoneDisclosures: None

Transcriptomic analysis of stress response to novel antimicrobial coatings in a clinical MRSA strain

Multi-drug resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) cause nosocomial infections that can have deleterious effects on human health. Thus, it is imperative to find solutions to treat these detrimental infections as well as to control their spread. We tested the effect of two different antimicrobial materials, functionalised graphene oxide (GOX), and AGXX® coated on cellulose fibres, on the growth and transcriptome of the clinical MRSA strain S. aureus 04-02981. In addition, we investigated the effect of a third material as a combination of GOX and AGXX® fibres on S. aureus 04-02981. Standard plate count assay revealed that the combination of fibres, GOX-AGXX® inhibited the growth of S. aureus 04-02981 by 99.98%. To assess the effect of these antimicrobials on the transcriptome of our strain, cultures of S. aureus 04-02981 were incubated with GOX, AGXX®, or GOX-AGXX® fibres for different time periods and then subjected to RNA-sequencing. Uncoated cellulose fibres were used as a negative control. The antimicrobial fibres had a huge impact on the transcriptome of S. aureus 04-02981 affecting the expression of 2650 genes. Primarily genes related to biofilm formation and virulence (such as agr, sarA, and those of the two-component system SaeRS), and genes crucial for survival in biofilms (like arginine metabolism arc genes) were repressed. In contrast, the expression of siderophore biosynthesis genes (sbn) was induced, a probable response to stress imposed by the antimicrobials and the conditions of iron-deficiency. Genes associated with potassium transport, intracellular survival and pathogenesis (kdp) were also differentially expressed. Our data suggest that the combination of GOX and AGXX® acts as an efficient antimicrobial against S. aureus 04-02981. Thus, these materials are potential candidates for applications in antimicrobial surface coatings.

A Fluorinated Analogue of Marine Bisindole Alkaloid 2,2-Bis(6-bromo-1H-indol-3-yl)ethanamine as Potential Anti-Biofilm Agent and Antibiotic Adjuvant Against Staphylococcus aureus.

Methicillin resistant Staphylococcus aureus (MRSA) infections represent a major global healthcare problem. Therapeutic options are often limited by the ability of MRSA strains to grow as biofilms on medical devices, where antibiotic persistence and resistance is positively selected, leading to recurrent and chronic implant-associated infections. One strategy to circumvent these problems is the co-administration of adjuvants, which may prolong the efficacy of antibiotic treatments, by broadening their spectrum and lowering the required dosage. The marine bisindole alkaloid 2,2-bis(6-bromo-1H-indol-3-yl)ethanamine (1) and its fluorinated analogue (2) were tested for their potential use as antibiotic adjuvants and antibiofilm agents against S. aureus CH 10850 (MRSA) and S. aureus ATCC 29213 (MSSA). Both compounds showed antimicrobial activity and bisindole 2 enabled 256-fold reduction (ΣFICs = 0.5) in the minimum inhibitory concentration (MIC) of oxacillin for the clinical MRSA strain. In addition, these molecules inhibited biofilm formation of S. aureus strains, and compound 2 showed greater eradicating activity on preformed biofilm compared to 1. None of the tested molecules exerted a viable but non-culturable cells (VBNC) inducing effect at their MIC values. Moreover, both compounds exhibited no hemolytic activity and a good stability in plasma, indicating a non-toxic profile, hence, in particular compound 2, a potential for in vivo applications to restore antibiotic treatment against MRSA infections.

Synergistic Potential of Antimicrobial Combinations Against Methicillin-Resistant Staphylococcus aureus.

The chemotherapeutic options for methicillin-resistant Staphylococcus aureus (MRSA) infections are limited. Due to the multiple resistant MRSA, therapeutic failure has occurred frequently, even using antibiotics belonging to different categories in clinical scenarios, very recently. This study aimed to investigate the interactions between 11 antibiotics representing different mechanisms of action against MRSA strains and provide therapeutic strategies for clinical infections. Susceptibilities for MRSA strains were determined by broth microdilution or agar dilution according to CLSI guideline. By grouping with each other, a total of 55 combinations were evaluated. The potential synergism was detected through drug interaction assays and further investigated for time-killing curves and an in vivo neutropenic mouse infection model. A total of six combinations (vancomycin with rifampicin, vancomycin with oxacillin, levofloxacin with oxacillin, gentamycin with oxacillin, clindamycin with oxacillin, and clindamycin with levofloxacin) showed synergistic activity against the MRSA ATCC 43300 strain. However, antibacterial activity against clinical isolate #161402 was only observed when vancomycin combined with oxacillin or rifampicin in time-killing assays. Next, therapeutic effectiveness of vancomycin/oxacillin and vancomycin/rifampicin was verified by an in vivo mouse infection model inoculated with #161402. Further investigations on antimicrobial synergism of vancomycin plus oxacillin and vancomycin plus rifampicin against 113 wild-type MRSA strains were evidenced by combined antibiotic MICs and bacterial growth inhibition and in vitro dynamic killing profiles. In summary, vancomycin/rifampicin and vancomycin/oxacillin are the most potential combinations for clinical MRSA infection upon both in vitro and in vivo tests. Other synergetic combinations of levofloxacin/oxacillin, gentamycin/oxacillin, clindamycin/oxacillin, and clindamycin/fosfomycin are also selected but may need more assessment for further application.

Prevalence of fosfomycin resistance and gene mutations in clinical isolates of methicillin-resistant Staphylococcus aureus

BackgroundFosfomycin exhibits excellent in vitro activity against multidrug-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Increasing fosfomycin resistance among clinical MRSA isolates was reported previously, but little is known about the relative abundance of Fosfomycin resistance genes in MRSA isolates circulating in Taiwan.MethodsAll MRSA isolates, collected in 2002 and 2012 by the Taiwan Surveillance of Antimicrobial Resistance (TSAR) program, were used in this study. Susceptibility to various antimicrobial agents, including fosfomycin, was determined by broth microdilution. Genetic determinants of fosfomycin resistance, including fosB carriage and murA, glpT and uhpT mutations, were investigated using PCR and sequencing of amplicons. Staphylococcal protein A (spa) typing was also performed to determine the genetic relatedness of MRSA isolates.ResultsA total of 969 MRSA strains, 495 in the year 2002 and 474 in the year 2012, were analyzed. The overall in vitro susceptibility was 8.2% to erythromycin, 18.0% to clindamycin, 29.0% to tetracycline, 44.6% to ciprofloxacin, 57.5% to trimethoprim/sulfamethoxazole, 86.9% to rifampicin, 92.9% to fosfomycin and 100% to linezolid and vancomycin. A significant increase in the fosfomycin resistance rate was observed from 3.4% in 2002 to 11.0% in 2012. Of 68 fosfomycin-resistant MRSA isolates, several genetic backgrounds probably contributing to fosfomycin resistance were identified. Twelve isolates harbored the fosB gene, and various mutations in murA, uhpT, and glpT genes were noted in 11, 59, and 66 isolates, respectively. The most prevalent gene mutations were found in the combination of uhpT and glpT genes (58 isolates). The vast majority of the fosfomycin-resistant MRSA isolates belonged to spa type t002.ConclusionsAn increased fosfomycin resistance rate of MRSA isolates was observed in our present study, mostly due to mutations in the glpT and uhpT genes. Clonal spread probably contributed to the increased fosfomycin resistance.