Methicillin-resistant Staphylococcus Aureus Reference Strains Research Articles

Synergistic antimicrobial effect of ascorbic acid and nicotinamide with rifampicin and vancomycin against SCCmec type IV methicillin-resistant Staphylococcus aureus (MRSA).

Background. Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacteria involved in a wide spectrum of human diseases. Many virulence factors promote this widespread propagation. One important factor is acquiring antibiotic resistance genes, which leads to a reduction in the availability and efficacy of therapy options. Recently, research has suggested that the remarkable antimicrobial effect of antioxidants against superbugs such as MRSA shows synergistic effects when accompanied by antimicrobial therapy. This paper aims to examine the synergistic effects of ascorbic acid and nicotinamide with a panel of antibiotics used in antimicrobial therapy against MRSA. Material and Methods. Two SCCmec type IV MRSA reference strains (EMRSA-15 and USA300) and 10 MRSA clinical isolates feature in this paper. SCCmec typing was conducted on the 10 clinical isolates via multiplex PCR after identification. Synergy experiments on antioxidants and antibiotics were evaluated via checkerboard assay. The minimum inhibitory concentration (MIC) of each agent was determined in accordance with the Clinical and Laboratory Standards Institute (CLSI) M100 guidelines through twofold microdilution assay. Results and Discussion. Synergy (FIC <0.5) was demonstrated for ascorbic acid (1/2 to 1/4 MIC) with rifampicin (1/2 to 1/8 MIC), and also ascorbic acid (1/2 to 1/16 MIC) when associated with vancomycin (1/2 MIC). Similarly, nicotinamide (1/2 to 1/16 MIC) showed a synergistic effect when paired with low concentrations of rifampicin (1/2 to 1/16 MIC), and also (at 1/4 to 1/16 MIC) with vancomycin (1/2 MIC). All reduced MICs due to synergistic combinations demonstrated statistical significance (P<0.05). Conclusion. The synergistic activity demonstrated in associating antioxidants with antibiotics shows promise in managing superbugs. However, more research is required to better understand the mechanism of the synergy and for utilization in clinical care.

Identification of the antibacterial mechanism of cryptotanshinone on methicillin-resistant Staphylococcus aureus using bioinformatics analysis

Cryptotanshinone (CT) is an extract from the traditional Chinese medicine Salvia miltiorrhiza, which inhibits the growth of methicillin-resistant Staphylococcus aureus (MRSA) in vitro. This study aims to determine the antibacterial mechanisms of CT by integrating bioinformatics analysis and microbiology assay. The microarray data of GSE13203 was retrieved from the Gene Expression Omnibus (GEO) database to screen the differentially expressed genes (DEGs) of S. aureus strains that were treated with CT treatment. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to identify the potential target of CT. Data mining on the microarray dataset indicated that pyruvate kinase (PK) might be involved in the antimicrobial activities of CT. The minimum inhibition concentrations (MICs) of CT or vancomycin against the MRSA strain ATCC43300 and seven other clinical strains were determined using the broth dilution method. The effects of CT on the activity of PK were further measured. In vitro tests verified that CT inhibited the growth of an MRSA reference strain and seven other clinical strains. CT hampered the activity of the PK of ATCC43300 and five clinical MRSA strains. CT might hinder bacterial energy metabolism by inhibiting the activity of PK.

Antibacterial Activity of Selected Essential Oil Compounds Alone and in Combination with β-Lactam Antibiotics Against MRSA Strains.

This study aimed to determine the effect of selected essential oil compounds (EOCs) on the antibacterial activity of β-lactam antibiotics (βLAs) against methicillin-resistant Staphylococcus aureus (MRSA) strains. The following parameters were studied: antibiotic susceptibility testing, detection of mecA gene and evaluation of genotypic relativity of isolates using molecular techniques, analysis of chemical composition applying Fourier-transform infrared (FTIR) spectroscopy, and determination of antibacterial activity of EOCs alone and in combination with βLAs against MRSA strains using microdilution and checkerboard methods. It was found that all isolates expressed MRSA and resistance phenotypes for macrolides, lincosamides, and streptogramins B. All isolates harbored the mecA gene and belonged to three distinct genotypes. Eight of the 10 EOCs showed efficient antimicrobial activity against the MRSA reference strain. The analysis of interaction between EOCs and βLAs against the MRSA reference strain revealed a synergistic and additive effect of the following combinations: methicillin (Met)-linalyl acetate (LinAc), penicillin G (Pen)-1,8-cineole (Cin), and Pen-LinAc. Analysis of EOC-βLA interactions showed a synergistic and additive effect in the following combinations: Met-LinAc (against low- and high-level βLAs resistance strains), Pen-Cin, and Pen-LinAc (against low-level βLAs resistance strains). It was also confirmed that changes in phosphodiester, -OH, -CH2 and -CH3 groups may change the interactions with βLAs. Moreover, the presence of two CH3O- moieties in the Met molecule could also play a key role in the synergistic and additive mechanism of LinAc action with Met against MRSA strains. Direct therapy using a Met-LinAc combination may become an alternative treatment method for staphylococcal infections caused by MRSA. However, this unconventional therapy must be preceded by numerous cytotoxicity tests.

Improved DNA extraction and purification with magnetic nanoparticles for the detection of methicillin-resistant Staphylococcus aureus

Molecular methods offer fast, safe and cost-efficient detection of pathogenic bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). These tests depend on a rapid extraction of bacterial DNA. The aim of this study was to compare an optimized DNA extraction and purification protocol for MRSA using magnetic nanoparticles with the original method. The purity of the extracted DNA was assessed by photometric measurements and the amount of DNA was determined by real-time PCR. Three MRSA reference strains (S. aureus ATCC® 70699, S. aureus ATCC® 43300; S. aureus ATCC® 33592) and eleven MRSA field strains, which include SCCmec elements of types I to XI, were used in this study. The optimized protocol can save approximately 20 min time compared to the original method and the DNA yield was higher with the new protocol. Therefore, this new protocol allows a faster DNA extraction from MRSA cultures.

Disruption of methicillin-resistant Staphylococcus aureus protein synthesis by tannins.

Methicillin-resistant Staphylococcus aureus (MRSA) is a worldwide public health threat, displaying multiple antibiotic resistance that causes morbidity and mortality. Management of multidrug-resistant (MDR) MRSA infections is extremely difficult due to their inherent resistance to currently used antibiotics. New antibiotics are needed to combat the emergence of antimicrobial resistance. The in vitro effect of tannins was studied against MRSA reference strain (ATCC 43300) and MRSA clinical strains utilizing antimicrobial assays in conjunction with both scanning and transmission electron microscopy. To reveal the influence of tannins in MRSA protein synthesis disruption, we utilized next-generation sequencing (NGS) to provide further insight into the novel protein synthesis transcriptional response of MRSA exposed to these compounds. Tannins possessed both bacteriostatic and bactericidal activity with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 0.78 and 1.56 mg/mL, respectively, against all tested MRSA. Scanning and transmission electron microscopy of MRSA treated with tannins showed decrease in cellular volume, indicating disruption of protein synthesis. Analysis of a genome-wide transcriptional profile of the reference strain ATCC 43300 MRSA in response to tannins has led to the finding that tannins induced significant modulation in essential ribosome pathways, which caused a reduction in the translation processes that lead to inhibition of protein synthesis and obviation of bacterial growth. These findings highlight the potential of tannins as new promising anti-MRSA agents in clinical application such as body wash and topical cream or ointments.

Synergistic effect of non-steroidal anti-inflammatory drugs (NSAIDs) on antibacterial activity of cefuroxime and chloramphenicol against methicillin-resistant Staphylococcus aureus.

Currently, only a few antibiotics are available to treat methicillin-resistant Staphylococcus aureus (MRSA). One alternative approach includes adjuvants to antibiotic therapy. Non-steroidal anti-inflammatory drugs (NSAIDs) are non-antibiotic drugs reported to exhibit antibacterial activity. The objective of this study was to investigate the interaction between NSAIDs with selected antibiotics (cefuroxime and chloramphenicol) against strains of S. aureus. The antibacterial activity of four NSAIDs (aspirin, ibuprofen, diclofenac and mefenamic acid) were tested against ten pathogenic bacterial strains using the microdilution broth method. The interaction between NSAIDs and antibiotics (cefuroxime/chloramphenicol) was estimated by calculating the fractional inhibitory concentration (FICI) of the combination. Aspirin, ibuprofen and diclofenac exhibited antibacterial activity against the selected pathogenic bacteria. The interaction between ibuprofen/aspirin with cefuroxime was demonstrated to be synergistic against methicillin-sensitive S. aureus (MSSA) and the MRSA reference strain, whereas for MRSA clinical strains additive effects were observed for both NSAIDs and cefuroxime combinations. The combination of chloramphenicol with ibuprofen/aspirin was synergistic against all of the tested MRSA strains and displayed an additive effect against MSSA. A 4-8192-fold reduction in the cefuroxime minimum inhibitory concentration (MIC) and a 4-64-fold reduction of the chloramphenicol MIC were documented. Overall, the NSAIDs ibuprofen and aspirin showed antibacterial activity against strains of S. aureus. Although individually less potent than common antibiotics, these NSAIDs are synergistic in action with cefuroxime and chloramphenicol and could potentially be used as adjuvants in combating multidrug-resistant MRSA.

Transcriptome analysis of methicillin-resistant Staphylococcus aureus in response to stigmasterol and lupeol.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen with multiple antibiotic resistance that causes morbidity and mortality worldwide. Multidrug-resistant (MDR) MRSA with increased resistance to currently available antibiotics has challenged the world to develop new therapeutic agents. Stigmasterol and lupeol, from the plant Phyllanthus columnaris, exhibit antibacterial activities against MRSA. The aim of this study was to utilise next-generation sequencing (NGS) to provide further insight into the novel transcriptional response of MRSA exposed to stigmasterol and lupeol. Time-kill analysis of one MRSA reference strain (ATCC 43300) and three clinical isolates (WM3, BM1 and KJ7) for both compounds was first performed to provide the bacteriostatic/bactericidal profile. Then, MRSA ATCC 43300 strain treated with both compounds was interrogated by NGS. Both stigmasterol and lupeol possessed bacteriostatic properties against all MRSA tested; however, lupeol exhibited both bacteriostatic and bactericidal properties within the same minimum inhibitory concentration and minimum bactericidal concentration values against BM1 (12.5mg/mL). Transcriptome profiling of MRSA ATCC 43300 revealed significant modulation of gene expression with multiple desirable targets by both compounds, which caused a reduction in the translation processes leading to inhibition of protein synthesis and prevention of bacterial growth. This study highlights the potential of both stigmasterol and lupeol as new promising anti-MRSA agents.

Direct detection of methicillin-resistant Staphylococcus aureus in sputum specimens from patients with hospital-associated pneumonia using a novel multilocus PCR assay.

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant cause of hospital-associated pneumonia (HAP). The rapid identification of MRSA would be beneficial for early diagnosis. The study aimed to evaluate a multilocus, fluorescence-based PCR assay based on the detection of mecA and nuc genes for identification of S. aureusin lower respiratory tract (LRT) specimens. Sensitivity and specificity of the PCR assay were analyzed. Clinical evaluation for the assay was performed using LRT specimens from patients with HAP, and the sensitivity, specificity, positive and negative predictive values (PPV and NPV) were evaluated in comparison with semi-quantitative culture methods. The result showed the assay provided positive identification of all MRSA reference strains with a limit of detection for MRSA of 4 × 103 CFU/mL. Compared with semi-quantitative culture, the sensitivity, specificity, PPV and NPV were 100%, 89.6%, 75.0%, and 100%, respectively. A positive correlation between MRSA bacterial colonies and PCR copy number was found. The specificity and PPV reached 96.6% and 89.7% respectively, if the PCR copy number reached a definite positive threshold of 5.96 × 105. It suggested that this novel multilocus, fluorescence-based PCR assay proved to be a fast, sensitive and specific tool for direct detection of MRSA from LRT specimens.

BIO-GUIDED STUDY ON &lt;i&gt;MELASTOMA MALABATHRICUM&lt;/i&gt; LINN LEAVES AND ELUCIDATION OF ITS BIOLOGICAL ACTIVITIES

The present study aimed to isolate and identify con stituents with antibacterial activity from the meth anol extract of Melastoma malabathricum leaves (MMML) through bioassay guided fractionation. Furthermore, the study scoped to evaluate the cytotoxic effects of M. malabathricum leaves fractions for possible protective effect of antoxidative constituents pres ent in the fractions. The MMML extract was fractionated by Vacuum Liquid Chromatography (VLC) to afford M. malabathricum leaves fractions (ML1-ML6) of increasing polarities. Antibacterial activity of pl ant extract, six fractions and two bioactive consti tuents against Staphylococcus aureus reference strain, methicillin-resistant Staphylococcus aureus reference strain (MRSA), eleven clinical MRSA isolates, three clinic al Pseudomonas aeruginosa isolates and P. aeruginosa reference strain was evaluated using Minimum Inhibitory Concentration (MIC). The bioactive constituent s responsible for antibacterial activity of MMML, fra ctions were detected by direct TLC-bioautography. T he data obtained from MIC assay showed that ML5 effectively inhibited growth of all test bacterial pathog ens. Direct TLC-bioautography revealed that ML5 had the highest number of antibacterial compounds. Following bioassay-guided fractionation, Kaempferol-3-O-(2”, 6”-di-O-p-trans -coumaroyl)-βglucopyranoside and Kaempferol were isolated from ML5. The structures of bioactive compounds were elucidated using Nuclear Magnetic Resonance (NMR) spectroscopy from 1 HNMR and 13 CNMR. In addition, the cytotoxic effect of M. malabathricum leaves fractions (ML1-ML6) revealed that ML5 had the highest Cytotoxicity Concentration (CC 50 ) at 0.75 mg mL -1 , an observation traceable to the presence of flavonoids constituents with antioxidant properties . Therefore, the results clearly indicate that MMML , fractions and bioactive constituents have profound antibacterial activity. Furthermore, antioxidative compounds from ML5 fraction exert their effects by enhancing the level of CC 50 .

Identification of a Highly Transmissible Animal-Independent Staphylococcus aureus ST398 Clone with Distinct Genomic and Cell Adhesion Properties

ABSTRACTA methicillin-resistant Staphylococcus aureus (MRSA) clone known as ST398 has emerged as a major cause of acute infections in individuals who have close contact with livestock. More recently, the emergence of an animal-independent ST398 methicillin-sensitive S. aureus (MSSA) clone has been documented in several countries. However, the limited surveillance of MSSA has precluded an accurate assessment of the global spread of ST398 and its clinical relevance. Here we provide evidence that ST398 is a frequent source of MSSA infections in northern Manhattan and is readily transmitted between individuals in households. This contrasts with the limited transmissibility of livestock-associated ST398 (LA-ST398) MRSA strains between humans. Our whole-genome sequence analysis revealed that the chromosome of the human-associated ST398 MSSA clone is smaller than that of the LA-ST398 MRSA reference strain S0385, due mainly to fewer mobile genetic elements (MGEs). In contrast, human ST398 MSSA isolates harbored the prophage φ3 and the human-specific immune evasion cluster (IEC) genes chp and scn. While most of the core genome was conserved between the human ST398 MSSA clone and S0385, these strains differed substantially in their repertoire and composition of intact adhesion genes. These genetic changes were associated with significantly enhanced adhesion of human ST398 MSSA isolates to human skin keratinocytes and keratin. We propose that the human ST398 MSSA clone can spread independent of animal contact using an optimized repertoire of MGEs and adhesion molecules adapted to transmission among humans.

Detection of methicillin-resistant Staphylococcus aureus (MRSA) from nasal samples by multiplex real-time PCR based on dual priming AT-rich primers

In this study, we reported on the design of a multiplex real-time PCR assay based on SYBR Green I, incorporating dual priming adenine-thymine (AT)-rich primers for direct detection of MRSA from nasal samples. The multiplex real-time polymerase chain reaction (RT-PCR) assay reported in this study is based on SYBR Green I with incorporation of six dual priming AT-rich primers designed from the SCCmec/orf junction. A string (4-6 bp) of low-melting bases, such as adenine and thymine, was incorporated into the primers, which virtually divided a single primer in two functional regions, thus decreasing non-specific PCR products. The analytical sensitivity and specificity of the RT-PCR assay was determined with genomic DNA of reference strains (MRSA, MSSA, and MRCoNS). RT-PCR assay was performed for analysis of 72 nasal swab specimens, and the results were confirmed by use of a culture method. Furthermore, the results of RT-PCR were compared with LightCycler MRSA advance test. The multiplex RT-PCR assay reproducibly detected a minimum of 1 pg genomic DNA (31.5 copy of genome) of MRSA reference strains and clinical isolates, with a specific melting peak at 83.5 ± 1.5°C, and neither fluorescence nor a melting peak was detected in non-target isolates. The concordance rate between RT-PCR assay and culture method was 87.5% with Cohen's kappa value (κ) 0.75, which showed good agreement between the two assays. The sensitivity, specificity, positive predictive value, and negative predictive value of the assay were 93.5%, 82.9%, 80.5%, and 94.4%, respectively. In a comparative study for the detection of 72 nasal samples, the sensitivity, specificity, positive predictive value, and negative predictive value of the multiplex RT-PCR assay with respect to LightCycler MRSA advance test was 84.2%, 88.2%, 89%, and, 83.3%, respectively. The results of RT-PCR assay demonstrated high specificity (88.2%) and positive predictive value (89%) for the direct detection of MRSA from nasal samples.

Real-time multiplex PCR for direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in clinical samples enriched by broth culture

A real-time multiplex PCR using the orfX and staphylococcal cassette chromosome (SCC) mec of Staphylococcus aureus was developed. The aim was to achieve a rapid and sensitive high-throughput method for direct detection of heterogeneous methicillin-resistant S. aureus (MRSA) in clinical samples, present in a low-endemic population, such as in Sweden. Consecutive broth enriched pooled clinical screening samples (nares, throat and/or perineum/groin) (n = 541 pools), broth enriched clinical samples showing growth of methicillin-sensitive S. aureus (MSSA) (n = 95 pools), clinical MRSA isolates (n = 173), MRSA reference strains (n = 43) and various coagulase-negative staphylococcal isolates (n = 33) were analyzed. The multiplex PCR detected all heterogeneous MRSA strains (n = 173) obtained in our area as well as all pooled consecutive broth enriched clinical samples with MRSA, i. e. 36 of 541 pools. None of the CoNS were positive. However, 18 out of 541 pools (3.3%) were positive in the multiplex PCR but no growth of MRSA could be detected by subculture and were regarded as false positive. Furthermore, the assay is rapid and reliable negative results can be delivered to the clinician within 18 h that will facilitate the infection control management of patients and hospital staff.

Rapid Identification of Methicillin-Resistant Staphylococcus aureus Transmission in Hospitals by Use of Phage-Derived Open Reading Frame Typing Enhanced by Multiplex PCR and Reverse Line Blot Assay

The relatively high-level clonality of methicillin-resistant Staphylococcus aureus (MRSA) and its frequent high-level endemicity in nosocomial settings complicate the development of methods for rapid subtyping of MRSA strains that are capable of identifying person-to-person transmission in hospitals. Phage-derived open reading frame (PDORF) typing is an MRSA typing method targeting mobile genetic elements that was recently described and evaluated using a geographically restricted set of isolates. The objective of this study was to develop a multiplex PCR-reverse line blot (mPCR/RLB) assay for PDORF typing and to test its applicability on a broad range of isolates and in an environment where MRSA is highly endemic. The 16 targets were identified using a 23-primer-pair mPCR/RLB assay with two probes for each target. The method was evaluated using 42 MRSA reference strains, including those representing major international clones, and 35 isolates from episodes of suspected nosocomial transmission. In vivo stability was explored using 81 isolate pairs. Pulsed-field gel electrophoresis (PFGE) and spa typing were performed for comparison. Among the 42 reference strains, there were 33 PFGE subtypes, 30 PDORF types, and 22 spa types. Simpson's index of diversity was 0.987, 0.971, and 0.926 for PFGE subtyping, PDORF typing, and spa typing, respectively. Typing of clinical isolates by PDORF typing and PFGE demonstrated concordant results. mPCR/RLB-based PDORF typing has similar discriminatory power to that of PFGE, can assist in tracking MRSA transmission events in a setting of high-level endemicity, and has the advantage of being a high-throughput technique.