Systemic Analysis of the mecA Gene Using a Bioinformatics Tool
Shin‐Yi TsaiFu-Chieh ChangKevin Sheng‐KaiCheng-Wei HsuPo-Ya TungYan-Jiun HungYi-Ting ChouChien-Feng Kuo
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The mecA gene, carried by methicillin-resistant Staphylococcus aureus (MRSA), allows the bacterium to promotes bacterial resistance to antibiotics such as methicillin, penicillin, and other penicillin-like antibiotics. Our objectives are to use a bioinformatics tool to analyze the sequence of the mecA gene, which is spread on the SCCmec genetic element, and to investigate the relationship between each mecA gene. From 2008 to 2016, we collected 229 MRSA from bacteremia; we extracted DNA from the MRSA and designed specific primers to target mecA using PCR. The primer used are listed in mec A-1(5’-GGGATCATAGCGTCATTATTC-3’) and mec A-2(5’-AACGATTGTGACACGATAGCC-3’). We determined whether the mecA gene was present by using electrophoresis and then sequenced the MRSA samples in which it was present. The POWER tool was employed to analyze the mecA gene and compile a pedigree chart. Using the sequencing data, we created an MRSA database, and the BLAST findings demonstrated that most of the mecA genes were similar, with over 95% identified. The pedigree chart illustrates that there are four groups of mecA genes, and these groups were found to be not differentiated between the sources of the MRSA, whether from communities or hospital association infections. Our findings indicate that even though there were four groups with ancestors in the pedigree chart, no significant difference was found between MRSA from community- and hospital-associated infections. We plan to collect more MRSA samples for analysis and investigate the differences between MRSA groups and MRSA from various geographical regions. All authors: No reported disclosures.Keywords:
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Penicillin binding proteins
Context: Infections with methicillin-resistant Staphylococcus aureus (MRSA) greatly influence clinical outcome. Molecular characterisation of MRSA can help to predict their spread and to institute treatment and hospital protocols. Aim: The aim of this study is to understand the diversity of MRSA in a tertiary care hospital in Hyderabad, India. Settings and Design: Samples collected at Gandhi Medical College, Hyderabad, and designed to assess hospital-or community-associated MRSA (HA-MRSA or CA-MRSA). Subjects and Methods: MRSA were subjected to antibiotic susceptibility testing, pulsed-field gel electrophoresis (PFGE), spa typing, multi-locus sequence typing and staphylococcal cassette chromosome–mec (SCCmec) typing. Statistical Analysis Used: Discriminatory index and 95% confidence interval. Results: Of the 30 MRSA, (a) 18 and 12 were HA-MRSA and CA-MRSA, respectively, and (b) 23.3% and 6.6% displayed induced clindamycin and intermediate vancomycin resistance, respectively. Genetic diversity was evident from the presence of (a) 20 pulsotypes, (b) eight spa types, with the predominance of t064 (n = 9) and (c) seven sequence types (ST), with the preponderance of ST22 and ST8 (9 each). ST22 and ST8 were the most prevalent among HA-MRSA and CA-MRSA, respectively. SCCmec type IV was the most frequent (n = 8). 44.4% of HA-MRSA belonged to SCCmec IV and V, whereas 33.3% of CA-MRSA belonged to SCCmec I and III; 33.3% (5/15) of the isolates harbouring the pvl gene belonged to SCCmec IVC/H. Conclusions: ST8 was a dominant type along with other previously reported types ST22, ST239, and ST772 from India. The observations highlight the prevalence of genetically diverse clonal populations of MRSA, suggesting potential multiple origins.
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Earlier studies have shown that the highly penicillin-resistant South African Strains of pneumococci contain altered penicillin-binding proteins (PBPs) (S. Zighelboim and A. Tomasz, Antimicrob. Agents Chemother. 17:434-442, 1980). We now describe a detailed quantitative characterization of the reaction of radioactively labeled penicillin with the PBPs of the penicillin-susceptible and penicillin-resistant pneumococci and several intermediate-resistance-level genetic transformants as well. The altered binding of the antibiotic by the PBPs of resistant cells appears to be due to a combination of two factors: lower drug affinity and change in the cellular amounts of PBPs. No alteration in the rates of deacylation of the penicilloyl-PBPs of the resistant cells was detected.
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We examined clinical isolates of Neisseria meningitidis relatively resistant to penicillin G (mean MIC, 0.3 micrograms/ml; range, 0.1 to 0.7 micrograms/ml), which were isolated from blood and cerebrospinal fluid for resistance mechanisms, by using susceptible isolates (mean MIC, less than or equal to 0.06 micrograms/ml) for comparison. The resistant strains did not produce detectable beta-lactamase activity, otherwise modify penicillin G, or bind less total penicillin. Penicillin-binding protein (PBP) 3 of the six resistant isolates tested uniformly bound less penicillin G in comparison to the same PBP of four susceptible isolates. Reflecting the reduced binding affinity of PBP 3 of the two resistant strains tested, the amount of 3H-labeled penicillin G required for half-maximal binding was increased in comparison with that of PBP 3 of the two susceptible isolates. We conclude that the mechanism of resistance in these meningococci relatively resistant to penicillin G was decreased affinity of PBP 3.
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A large number of pneumococcal isolates (over 80 strains) from a variety of geographic locales and representing a spectrum of resistance levels from a penicillin MIC of 0.003 microgram/ml up to an MIC of 16 micrograms/ml were analyzed for their penicillin-binding protein (PBP) patterns. With a few exceptions, the great majority of strains with penicillin MICs up to about 0.05 microgram/ml contained the same set of five PBPs with molecular sizes typical of those of susceptible pneumococci. In strains with penicillin MICs of about 0.1 microgram/ml and up, virtually all isolates showed two common features: (i) all isolates showed loss of PBP 1A (98 kilodaltons) with or without a parallel appearance of a "new" PBP that ranged in molecular size between 96 and 97 kilodaltons; and (ii) in strains with penicillin MICs of 0.5 microgram/ml or more, PBP 2B could not be detected on the fluorograms even with very high concentrations of radioactive penicillin. Beyond these two common features, resistant strains with similar penicillin MICs showed a surprising variety of PBP profiles (i.e., in the number and molecular sizes of PBPs), each characteristic of a given isolate. We suggest that in pneumococci remodeling of critical PBPs in more than one way may result in comparable levels of penicillin resistance.
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Abstract Methicillin Resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen worldwide. It is still one of the major problems of drug resistance and it should be a frequent and an important human pathogen both in community and in hospital. Methicillin - resistant Staphylococcus aureus (MRSA) has been known among the most important and intimidating bacteria involved in hospital infections in humans. MRSA resistance to methicillin has been attributed to a number of mechanisms, but the chief factor is reckon as its ability to produce specific binding protein 2a (PBP-2a) which renders β - lactamase resistant penicillins ineffective including all other β- lactam drugs. The Penicillin Binding Protein 2 has shown a usual low binding affinity for almost all beta - lactam antibiotics as compared to native PBPs. The Penicillin Binding Protein 2a (PBP2a) is coded and induced by the mec A gene a part of Staphylococcal cassette chromosome (SCCmec). The SCCmec is known to be present in MRSA but not in the MSSA strains. SCCmec is shown to be located in exactly the same region between spa and purA in the S. aureus chromosome. Another chromosomal gene called femA, working with mecA gene is required for the expression of MRSA and this gene is found to be absent in other Staphylococcus species giving S. aureus special feature to differentiate from other Staphylococci. Apart from mec A gene, a number of environmental and genetic factors have also shown to influence the methicillin resistance. The paper review the genetic and molecular mechanisms associated with beta - lactam antibiotics in MRSA. Keywords: Antibiotics, Staphylococcus aureus, MRSA, MecA, Resistance
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ABSTRACT Molecular methods for the rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) are generally based on the detection of an S. aureus -specific gene target and the mecA gene. However, such methods cannot be applied for the direct detection of MRSA from nonsterile specimens such as nasal samples without the previous isolation, capture, or enrichment of MRSA because these samples often contain both coagulase-negative staphylococci (CoNS) and S. aureus , either of which can carry mecA. In this study, we describe a real-time multiplex PCR assay which allows the detection of MRSA directly from clinical specimens containing a mixture of staphylococci in <1 h. Five primers specific to the different staphylococcal cassette chromosome mec (SCC mec ) right extremity sequences, including three new sequences, were used in combination with a primer and three molecular beacon probes specific to the S. aureus chromosomal orfX gene sequences located to the right of the SCC mec integration site. Of the 1,657 MRSA isolates tested, 1,636 (98.7%) were detected with the PCR assay, whereas 26 of 569 (4.6%) methicillin-susceptible S. aureus (MSSA) strains were misidentified as MRSA. None of the 62 nonstaphylococcal bacterial species or the 212 methicillin-resistant or 74 methicillin-susceptible CoNS strains (MRCoNS and MSCoNS, respectively) were detected by the assay. The amplification of MRSA was not inhibited in the presence of high copy numbers of MSSA, MRCoNS, or MSCoNS. The analytical sensitivity of the PCR assay, as evaluated with MRSA-negative nasal specimens containing a mixture of MSSA, MRCoNS, and MSCoNS spiked with MRSA, was ∼25 CFU per nasal sample. This real-time PCR assay represents a rapid and powerful method which can be used for the detection of MRSA directly from specimens containing a mixture of staphylococci.
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Objectives: To identify the staphylococcal cassette chromosome mec (SCCmec) types of methicillin-resistant Staphylococcus aureus (MRSA) isolated from bovine milk, and examine the genetic relatedness between MRSA from bovine milk and MRSA from human isolates.
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Since the discovery in 1965 that penicillin inhibits the transpeptidation reaction in peptidoglycan synthesis, a considerable effort has been put into the purification of enzymes that catalyse this reaction. This has resulted in the recognition that bacteria possess multiple forms of these penicillin-sensitive enzymes and has made it difficult to identify the precise target that penicillin inactivates to kill the organism. Recently penicillin-sensitive enzymes have been detected and studied as penicillin-binding proteins on sodium dodecyl sulphate polyacrylamide gels. The availability of this convenient method for identifying penicillin-sensitive enzymes has allowed biochemical and genetical approaches to be used to dissect their roles in the lethal effects of penicillin and other β-lactam antibiotics. Three penicillin-binding proteins (1B, 2 and 3) have been identified as killing targets for penicillin in Escherichia coli , whereas four other binding proteins are not implicated in the mechanism of action of the antibiotic. The complex biological effects that β-lactam antibiotics produce on the growth of E. coli can be explained by their interaction with the three killing targets. Progress in the correlation of penicillin-binding proteins with penicillin-sensitive enzymes and in the development of strains of E. coli that overproduce penicillin-binding proteins is discussed.
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Journal Article Molecular aspects of methicillin resistance in Staphylococcus aureus Get access Herminia de Lencastre, Herminia de Lencastre aThe Rockefeller University1230 York Avenue, New York, NY10021, USAbInstituto de Tecnologia Quimica e Biólogica, Universidade Nova de LisboaOeiras, Portugal Search for other works by this author on: Oxford Academic PubMed Google Scholar Boudewijn L. M. de Jonge, Boudewijn L. M. de Jonge aThe Rockefeller University1230 York Avenue, New York, NY10021, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Peter R. Matthews, Peter R. Matthews aThe Rockefeller University1230 York Avenue, New York, NY10021, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Alexander Tomasz Alexander Tomasz * aThe Rockefeller University1230 York Avenue, New York, NY10021, USA *Corresponding author. Search for other works by this author on: Oxford Academic PubMed Google Scholar Journal of Antimicrobial Chemotherapy, Volume 33, Issue 1, January 1994, Pages 7–24, https://doi.org/10.1093/jac/33.1.7 Published: 01 January 1994 Article history Received: 12 August 1993 Accepted: 01 October 1993 Published: 01 January 1994
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