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Conventional versus molecular tests (Multiplex PCR and PCR mecA gene) for detection of methicillin resistant Staphylococcus aureus
Ribas, Rosineide Marques;Gontijo-Filho, Paulo Pinto;Darini, Ana Lúcia da Costa;
Brazilian Journal of Microbiology , 2003, DOI: 10.1590/S1517-83822003000500012
Abstract: in this study, for detection of methicillin-resistant s. aureus (mrsa), a meca multiplex pcr-based amplification was compared with the 1 μg oxacillin disk diffusion test, detection of minimal inhibitory concentration (mic), and screening in agar with 4% nacl and 6 μg/ml oxacillin. among 24 isolates obtained from blood, meca gene was detected in only 16 (66.7%) isolates by multiplex pcr. the mic test showed a range of resistance to oxacillin from 0.19 to 512 μg/ml, among these isolates. data obtained by screening and dilution tests showed that sensitivity to methicillin was 80.0% and 72.8%, respectively, when compared with the presence of meca gene (multiplex). all isolates, including the negatives, when revaluated for meca gene by pcr were positive. b-lactamase production was positive for 20/25 isolates (80.0%). about ? of patients died dispite most of them (83.3%) were adequately treated. the simultaneous identification of the bacteria and determination of this susceptibility to antibiotics are necessary for the choice of empiric antibiotic therapy in suspected staphylococcal sepse, but is important to considering the sensibility, specificity and validation of the available kits.
Simultaneous PCR detection of ica cluster and methicillin and mupirocin resistance genes in catheter-isolated Staphylococcus
Martín-López,Juana V.; Díez-Gil,Oscar; Morales,Manuel; Batista,Ninivé; Villar,Jesús; Claverie-Martín,Félix; Méndez-álvarez,Sebastián;
International Microbiology , 2004,
Abstract: recent data show that more than 50% of catheter-associated bloodstream infections are caused by staphylococci. staphylococcal infections produced by intercellular-adhesion cluster (ica) carriers can be even more problematic due to the presence of methicillin and mupirocin resistance genes. in the present study, a multiplex pcr protocol that allows the simultaneous identification of staphylococci and detection of both the ica and methicillin and/or mupirocin resistance genes was developed. furthermore, the method allows differential detection of the ica locus from staphylococcus aureus and staphylococcus epidermidis.
Detection of novel chromosome-SCCmec variants in Methicillin Resistant Staphylococcus aureus and their inclusion in PCR based screening
Anneke van der Zee, Lieuwe Roorda, Willem DH Hendriks, Jacobus M Ossewaarde, Johannes Buitenwerf
BMC Research Notes , 2011, DOI: 10.1186/1756-0500-4-150
Abstract: Efficient management of Methicillin Resistant Staphylococcus aureus (MRSA) in the hospital is needed to prevent dissemination. It is important that MRSA can be rapidly identified, and effective infection control measures can be initiated. Equally important is a rapid MRSA negative report, especially for patients in isolation. For negative screening we implemented fully automated high through-put molecular screening for MRSA.Fourteen variant chromosome-SCCmec junctions in MRSA, that are not detected in commercially available MRSA detection kits were added to our PCR to detect all currently known variant SCC-mec types of MRSA.The dissemination of Methicillin Resistant Staphylococcus aureus (MRSA) in hospitals is a growing problem worldwide. In The Netherlands, a search and destroy policy is implemented [1]. Patients colonized with MRSA are kept in isolation until they are culture negative. A MRSA negative report can faster be obtained by PCR. Therefore, a molecular approach for negative screening of MRSA was exploited. Molecular detection of the mecA gene, which confers resistance to all β-lactams, has often been used in combination with other S. aureus specific genes in a multiplex PCR. Genes that are specific for S. aureus comprise for example of the sequence published by Martineau et al. [2], the nuclease gene (nuc) [3,4], or the coagulase gene (coa) [5]. When clinical samples contain a mixture of coagulase negative staphylococci (CNS), methicillin sensitive S. aureus (MSSA), and MRSA, a positive mecA PCR can be generated by CNS while both MSSA and MRSA generate a positive PCR for the coa or the nuc gene. Only culturing could confirm MRSA. Another approach for detection of MRSA was presented by a multiplex PCR described by Huletsky et al. [6]. This PCR specifically targets the junction between a conserved open reading frame orfX in S. aureus, and the staphylococcal cassette chromosome containing the mecA gene (SCCmec). For MRSA, 8 different types of SCCmec elements
Staphylococcus aureus methicillin-resistance mechanisms  [PDF]
Petrovi?-Jeremi? Ljiljana,Kulji?-Kapulica Nada,Mirovi? Veljko,Koci? Branislava
Vojnosanitetski Pregled , 2008, DOI: 10.2298/vsp0805377p
Abstract: Background/Aim. In many hospitals in the world and in our country, the spread of methicillin-resistant Staphylococcus aureus (MRSA) is so wide that nowdays vancomycin is recommended for empiric treatment of staphylococcal life threatening infections (sepsis, pneumonia) instead of beta-lactam antibiotics. The aim of this study was to determine the production of beta-lactamases in hospital and community isolates of staphyloococus aureus, i. e. hospital associated MRSA (HA-MRSA) and community associated MRSA (CA-MRSA), the presence of homogeneous and heterogeneous type of methicillin resistance, and border-line resistance in Staphylococcus aureus (BORSA). The aim of this study was also to determine if there was a statistically significant difference between mechanisms of resistance in HA-MRSA and CA-MRSA. Methods. A total 216 clinical Staphylococcus aureus isolates from the General Hospital in the town of Cuprija and 186 ambulance Staphylococcus aureus isolates from the community were examined for the presence of methicillin-resistance using disk-diffusion test with penicillin disk (10 ij), oxacillin disk (1 μg) and cefoxitin disk (30 μg). Betalactamases production was detected by nitrocefin disk and betalactamase tablets. Determination of oxacillin minimum inhibitory concentracion (MIC) was done by agar-dilution method. Results. The prevalence of HA-MRSA was 57.4%, and CA-MRSA was 17.7% (p < 0.05). There was a higher rate of heterogeneous type of resistance among clinical MRSA isolates (11.1%) compared with ambulance ones (3.8%) (p < 0.05). The rates of beta-lactamases production were similar among hospital associated isolates (97.5%), as well as in the community associated isolates (95.5%) (p > 0.05). There were 4.6 % of BORSA hospital isolates and 3.3 % of BORSA ambulance isolates (p > 0.05). Conclusion. The frequency of MRSA isolates in hospital was significantly higher than in community, as well as the heterogeneous type of resistance. The frequency of BORSA isolates and production of betalactamases were higher among hospital Staphylococcus aureus isolates, but the difference is not significant.
Frequency of methicillin resistant Staphylococcus aureus in health care  [cached]
Somayeh Rahimi-Alang,Mehdi Asmar,Fatemeh Cheraghali,Sarah Yazarlou
Zahedan Journal of Research in Medical Sciences , 2011,
Abstract: Background: Methicillin resistant Staphylococcus aureus (MRSA) is one of the most important pathogen in hospitals. Healthcare personnel are the main source of nosocomial infections and identification and control of MRSA carriers can reduce incidence of infections. The aim of this study was to determine the prevalence of MRSA and their antibiotic susceptibility profile among healthcare workers in Gorgan.Materials and Method: 333 healthcare workers were participated in this cross-sectional study in 2009. Samples were taken with sterile cotton swabs from both anterior nares and hands. Swabs were plated immediately on to the mannitol salt agar. Suspected colonies were confirmed as S. aureus by Gram staining, catalase, coagulase and DNase tests. Minimum inhibition concentration by micro dilution broth method was used to determine methicillin resistant strains. Antimicrobial susceptibility to other antibiotics was performed according to NCCLS guidelines by disc diffusion method.Result: Frequency of S.aureus and MRSA carriers among healthcare workers was 24% and 3% respectively. The highest rate of S. aureus and MRSA carriers were observed in operating room staff. Resistance to penicillin was seen in 97.5% of isolates and all strains were sensitive to vancomycin.Conclusions: Frequency of S. aureus and MRSA in healthcare workers was median and rather low respectively. Continual monitoring and control of carriers can reduce distribution of this organism and their infections
M. Conter,P. Di Ciccio,E. Zanardi,S. Ghidini
Italian Journal of Food Safety , 2012, DOI: 10.4081/ijfs.2012.3.25
Abstract: The aims of this study were (i) to estimate the prevalence of Staphylococcus aureus (S.a) in pig farm environments; (ii) to evaluate the presence of S.a in pork processing environments (iii) to detect the presence of methicillin-resistant (MRSA) among isolated strains. Samples of pig stool, farm environment and pork processing environment were collected. These samples were submitted to detection of S.a following the international method: UNI EN ISO 6888-2 and the Minimum Inhibitory Concentrations (MIC) tests were performed by using the automated VITEK 2 system. In addition, a PCR for the detection of the mecA gene was applied. Overall, S. aureus were more frequently detected from pig farms than from pork processing environments. Among the n.51 isolated strains, n. 49 (96%) were methicillin resistant (MRSA) and only n.2 strains were methicillin sensitive (MSSA). The results of the present study highlighted that further studies are needed to elucidate transmission routes of MRSA in pig production chain.
Revisiting methicillin-resistant Staphylococcus aureus infections  [cached]
Waness Abdelkarim
Journal of Global Infectious Diseases , 2010,
Abstract: Within less than 50 years, methicillin-resistant Staphylococcus aureus (MRSA) made a tremendous impact worldwide. It is not limited to medical facilities and healthcare institutions anymore. Indeed since two decades, cases of MRSA infections arising from the community among apparently healthy individuals are increasing. In this paper, I will present a case of community-associated MRSA sepsis followed by a comprehensive review about the history, pathogenesis, epidemiology, clinical presentations, diagnostic modalities, therapeutic options, contributing factors, growing cost and other pertinent elements of this newly evolving epidemic of MRSA infections.
Multiplex PCR Assays for the Detection of Clinically Relevant Antibiotic Resistance Genes in Staphylococcus aureus Isolated from Malaysian Hospitals
E. Amghalia,Nagi A. AL-Haj,Mariana N. Shamsudin,Son Radu,Rozita Rosli,V. Neela,Raha A. Rahim
Research Journal of Biological Sciences , 2012,
Abstract: Multiple drug resistant Staphylococcus aureus is one the most common nosocomial pathogen worldwide. The timely identification of this hospital acquired pathogen and detection of the various antibiotic resistant genes harbored is one of the most important function of the microbiology laboratory. In this study, we report the development of a multiplex PCR system for the diagnosis of S. aureus and the detection of clinically relevant antibiotic resistance genes harbored by some isolates. This system was designed to identify S. aureus at species level and to detect methicillin, gentamycin, erythromycin, vancomycin and mupirocin resistant genes, respectively from a single colony in a single tube reaction. All isolates amplified a 108 bp fragment (conserved in S. aureus) confirming the identity of S. aureus, 23 isolates produced a band at the position of 533 bp, 28 isolates at 139 bp and 30 isolates at 174 bp evidencing the presence of mecA (methicillin or oxacillin resistance), ermA (erythromycin resistance), aac (6`)-aph (2``) (gentamycin resistance) genes. None of the isolates amplified van A (vancomycin resistance) and ileS-2 (mupirocin resistance) genes showing the absence of their resistance in the isolates studied. These genotypic results when compared with classical antibiotic susceptibility tests showed less correlation. Overall, we found a correlation between phenotypic and genotypic methods of 60% for methicillin, 36.7% for gentamycin, 43.3% for erythromycin, 100% for vancomycin and mupirocin. This suggests that classical antibiotic sensitivity test is not accurate, but need to be supplemented with other methods to be applied in a clinical laboratory. The system developed in this study offers a rapid, simple specific and accurate detection of multiple antibiotic resistant genes in clinical S. aureus isolates and thus could be systematically applied as a diagnostic test in clinical microbiology laboratories, facilitating the design and use of antibiotic therapy.
Nasal carriage of methicillin-resistant Staphylococcus aureus in university students
Prates, Karina Aparecida;Torres, Ana Maria;Garcia, Lourdes Botelho;Ogatta, Sueli Fumie Yamada;Cardoso, Celso Luiz;Tognim, Maria Cristina Bronharo;
Brazilian Journal of Infectious Diseases , 2010, DOI: 10.1590/S1413-86702010000300021
Abstract: in a study of university students, the percentage nasal carriage of staphylococcus aureus was 40.8% (102/250). of the isolates, mic50 of methicillin was 0.5 μg/ml and mic90 was 1 μg/ml. six (5.8%) isolates were methicillin-resistant and carried the meca gene. these results suggest that community-associated methicillin-resistant s. aureus may be spreading in brazil.
Methicillin-resistant Staphylococcus aureus in human milk
Novak, FR;Almeida, JAG;Warnken, MB;Ferreira-Carvalho, BT;Hagler, AN;
Memórias do Instituto Oswaldo Cruz , 2000, DOI: 10.1590/S0074-02762000000100003
Abstract: we collected and analyzed 500 samples of human milk, from five brazilian cities (100 from each) to detect methicillin-resistant strains of staphylococcus aureus (mrsa) producing enterotoxins. we found 57 strains of mrsa, and the meca gene, responsible for resistance, was detected in all of them using a specific molecular probe. we examined 40 strains for the presence of four enterotoxins, after selecting a subset that included all strains from each region, except for the largest sample, from which 10 were randomly selected. among these two presented enterotoxin b, and growth in human colostrum and trypicase soy broth. after 5 h of incubation at 37°c, population sizes were already higher than 9.4 x 105 ufc/ml and enterotoxin was released into culture medium and colostrum. our results stress the importance of hygiene, sanitary measures, and appropriate preservation conditions to avoid the proliferation of s. aureus in human milk.
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