Detection Of Methicillin-resistant Staphylococcus Aureus Research Articles

Prevalence of Methicillin Resistant Staphylococcus Aureus In Khwaja Yunus Ali Medical College Hospital

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of healthcare-associated infections. To combat the challenge of life threatening MRSA remains a primary focus of most hospital infection control programs6. This study is undertaken in Khwaja Yunus Ali Medical College Hospital to identify the MRSA for determination of its prevalence and is considered a component of an infection control program in many countries5.Methods: Three hundred seventy hospitalized patients of surgery and medicine departments were studied during 2015. Clinical information of the patients and their pus, wound swab, sputum, throat swab and CSF were cultured.Results: Out of 370 patients, pus and wound swab of 217 (59%) had wound infection, sputum and throat swab of 141 had respiratory tract infection (38%) and CSF of 12 (3.2%) had meningitis were aseptically collected and cultured. Staphylococcus aureus were isolated in 51% cases and out of them MRSA was identified in 72 (73%) cases. MRSA isolated in 50% cases of meningitis, 49% cases of respiratory tract infection and 29% cases of wound infection.Conclusion: Methicillin-resistant Staphylococcus aureus appeared as a common cause of major illness and death and impose serious economic costs on patients and hospitals of our area like other developing countries. Detection of MRSA was associated with more severe clinical presentation.KYAMC Journal Vol. 7, No.-1, Jul 2016, Page 673-677

Evaluation of different phenotypic methods for the detection of methicillin resistant Staphylococcus aureus and antimicrobial susceptibility pattern of MRSA

Background: Rapid and accurate detection of methicillin resistant Staphylococcus aureus (MRSA) is an important role of clinical microbiology laboratories to avoid treatment failure. The aim of this study was to compare conventional methods against the cefoxitin disc diffusion method to determine the best phenotypic method. Methods: Study was carried out in the Department of Microbiology, National Institute of Medical Sciences & Research, Jaipur (India), between July 2016 – December 2016. The methods included were Oxacillin E-test MIC, Oxacillin screen agar, Oxacillin disk diffusion, Cefoxitin disk diffusion and CHROMagar- MRSA methods. Antimicrobial susceptibility performed as per CLSI guidelines.Results: Out of 142 isolates of S. aureus, fifty three (37.32%) strains of MRSA were isolated from clinical specimen. E-MIC test was selected as gold standard method. The sensitivity and specificity of Oxacillin screen agar and CHROMagar-MRSA were same 98.07% and 97.80%, respectively. The sensitivity and specificity of oxacillin disk diffusion were 94.23% and 98.89%. Fifty three strains of S. aureus were MRSA by cefoxitin disk diffusion method and Oxacillin Ezy MIC test. The sensitivity and specificity of cefoxitin disk diffusion method and Oxacillin Ezy MIC method was 100% and 100% respectively. All isolates including MRSA were susceptible to Vancomycin and Linezolid. Conclusions: All phenotypic methods had high sensitivity and specificity for detection of MRSA. However, cefoxitin disk diffusion method in comparison to other methods had higher sensitivity and specificity.

Accurate Detection of Methicillin-Resistant Staphylococcus aureus in Mixtures by Use of Single-Bacterium Duplex Droplet Digital PCR.

Accurate and rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) is needed to screen MRSA carriers and improve treatment. The current widely used duplex PCR methods are not able to differentiate MRSA from coexisting methicillin-susceptible S. aureus (MSSA) or other methicillin-resistant staphylococci. In this study, we aimed to develop a direct method for accurate and rapid detection of MRSA in clinical samples from open environments, such as nasal swabs. The new molecular assay is based on detecting the cooccurrence of nuc and mecA markers in a single bacterial cell by utilizing droplet digital PCR (ddPCR) with the chimeric lysin ClyH for cell lysis. The method consists of (i) dispersion of an intact single bacterium into nanoliter droplets, (ii) temperature-controlled release of genomic DNA (gDNA) by ClyH at 37°C, and (iii) amplification and detection of the markers (nuc and mecA) using standard TaqMan chemistries with ddPCR. Results were analyzed based on MRSA index ratios used for indicating the presence of the duplex-positive markers in droplets. The method was able to achieve an absolute limit of detection (LOD) of 2,900 CFU/ml for MRSA in nasal swabs spiked with excess amounts of Escherichia coli, MSSA, and other mecA-positive bacteria within 4 h. Initial testing of 104 nasal swabs showed that the method had 100% agreement with the standard culture method, while the normal duplex qPCR method had only about 87.5% agreement. The single-bacterium duplex ddPCR assay is rapid and powerful for more accurate detection of MRSA directly from clinical specimens.

Direct bacterial loop-mediated isothermal amplification detection on the pathogenic features of the nosocomial pathogen - Methicillin resistant Staphylococcus aureus strains with respiratory origins.

Loop-mediated isothermal amplification based detection assays using bacterial culture or colony for direct detection of methicillin resistant Staphylococcus aureus(MRSA) had been developed and evaluated, followed by its extensive application on a large scale of clinical MRSA isolated from respiratory origins, including nasal swabs and sputums. Six primers, including outer primers, inner primers and loop primers, were specifically designed for recognizing eight distinct sequences on four targets: 16SrRNA, femA, mecA and orfX. Twenty-seven reference strains were used to develop, evaluate and optimize this assay. Then, a total of 532 clinical MRSA isolates were employed for each detected targets. And the results were determined through both visual observation of the color change by naked eye and electrophoresis. The specific of each primer had been confirmed, and the optimal amplification was obtained under 65°C for 40min. The limit of detections (LOD) of bacteria culture LAMP assays were determined to be 104CFU/ml for 16S rRNA, femA, as well as orfX and 105CFU/ml for mecA, respectively. The established novel assays on MRSA detection may provide new strategies for rapid detection of foodborne pathogens.

Rapid Detection of Staphylococcus aureus and Methicillin-Resistant S. aureus in Atopic Dermatitis by Using the BD Max StaphSR Assay.

Eczematous lesions of atopic dermatitis (AD) patients are known to be a source of Staphylococcus aureus (SA) transmission and might be a reservoir for community-associated methicillin-resistant SA (MRSA). The BD Max StaphSR (BD-SR) is a fully automated, multiplex real-time PCR assay for the direct detection and differentiation of SA and MRSA from nasal swab samples. We evaluated the detection rates of SA and MRSA from skin lesions of outpatients with AD using the BD-SR assay, and determined the usefulness of the BD-SR assay. A total of 244 skin swab samples (skin lesions of 213 outpatients with AD and normal skin of 31 healthy controls) were tested directly by using the BD-SR assay. Of the 213 samples from patients with AD, 69 (32.4%) were positive for SA, 6 (8.7%) of which were positive for MRSA. Only 1 (3.2%) of 31 samples from healthy controls was positive for SA. The BD-SR assay is effective for the rapid detection of SA and MRSA from skin swab samples, which can provide important information for managing patients with AD and preventing the spread of MRSA.

A facile, rapid and sensitive detection of MRSA using a CRISPR-mediated DNA FISH method, antibody-like dCas9/sgRNA complex

Rapid and reliable diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) is crucial for guiding effective patient treatment and preventing the spread of MRSA infections. Nonetheless, further simplification of MRSA detection procedures to shorten detection time and reduce labor relative to that of conventional methods remains a challenge. Here, we have demonstrated a Clustered regularly interspaced palindromic repeats (CRISPR)-mediated DNA-FISH method for the simple, rapid and highly sensitive detection of MRSA; this method uses CRISPR associated protein 9/single-guide RNA (dCas9/sgRNA) complex as a targeting material and SYBR Green I (SG I) as a fluorescent probe. A dCas9/sgRNA-SG I based detection approach has advantages over monoclonal antibody in conventional immunoassay systems due to its ability to interact with the target gene in a sequence-specific manner. The detection limit of MRSA was as low as 10 cfu/ml and was found to be sufficient to effectively detect MRSA. Unlike conventional gene diagnosis methods in which PCR must be accompanied or genes are isolated and analyzed, the target gene can be detected within 30min with high sensitivity without performing a gene separation step by using cell lysates. We showed that the fluorescence signal of the MRSA cell lysate was more than 10-fold higher than that of methicillin-susceptible S. aureus (MSSA). Importantly, the present approach can be applied to any target other than MRSA by simply changing the single-guide RNA (sgRNA) sequence. Because dCas9/sgRNA-SG I based detection approach has proved to be easy, fast, sensitive, and cost-efficient, it can be applied directly at the point of care to detect various pathogens as well as MRSA in this study.

Is There a Need to Revise the Antibiotic Concentration in Clinical and Laboratory Standards Institute-Recommended Oxacillin Screen Agar?

Introduction: In routine diagnostic microbiology laboratories, Clinical and Laboratory Standards Institute (CLSI) recommends the use of cefoxitin disc, in addition to oxacillin screen agar (OSA) of 6 μg/ml for the detection of methicillin-resistant Staphylococcus aureus (MRSA), whereas minimum inhibitory concentration values of oxacillin for S. aureus are ≤2 μg/ml (susceptible) and ≥4 μg/ml (resistant). Hence, the study was carried out to evaluate the ability of screen agar with lower concentrations of oxacillin to identify the isolates of MRSA and to compare this with cefoxitin disc diffusion (CDD). Materials andMethods: Six hundred and seventy-six isolates of S. aureus were screened for methicillin resistance by OSA with 2 μg/ml and 4 μg/ml and 6 μg/ml of oxacillin concentration as well as CDD. Polymerase chain reaction for mecA gene was carried out for all isolates which grew on OSA 2, 4 and 6 μg/ml regardless of their cefoxitin susceptibility. Latex agglutination test for penicillin-binding protein 2a was performed for the isolates which grew on OSA 2 and or 4 μg/ml but not on OSA 6 μg/ml. Results: Eight per cent of MRSA isolates was missed by using OSA 6 μg/ml, when compared with other methods. Sensitivities of OSA 2 μg/ml, OSA 6 μg/ml and CDD were found to be 100%, 92.5% and 97.5%, respectively, and specificities for the same were found to be 100%, 100% and 98%, respectively. As per FDA criteria, categorical agreement for OSA 2 μg/ml was found to be 100% in comparison with the reference broth microdilution method. No major and very major discrepancies were documented. Conclusion: Similar findings on a larger and more heterogeneous collection of isolates may indicate the need to revise the concentration of OSA to 2 μg/ml for the detection of MRSA.

Comparison of conventional and rapid methods for the detection of methicillin resistance in Staphylococcus aureus from blood cultures

Background: Considering the increasing rate of infections caused by methicillin-resistant Staphylococcus aureus (MRSA), reliable, accurate, and rapid testing for the detection of MRSA is essential for both initiation of antimicrobial therapy and infection control measures. Materials and Methods: Fifty clinical isolates of S. aureus from patients with bacteremia were confirmed bacteriologically and subjected to antimicrobial susceptibility testing, followed by the detection of methicillin resistance by oxacillin and cefoxitin disc diffusion methods, oxacillin agar screen test, and direct cefoxitin disc diffusion method from blood culture bottles. Results: On comparing the methods under study, 36% MRSA were detected by disc diffusion methods, whereas 44% and 46% were detected by oxacillin agar screen and direct cefoxitin disc diffusion blood culture, respectively. Conclusions: The emergence of MRSA underscores the need for programs to prevent the spread of antimicrobial-resistant organisms and control the use of antimicrobial drugs in health-care settings.

Rapid MRSA PCR on respiratory specimens from ventilated patients with suspected pneumonia: a tool to facilitate antimicrobial stewardship.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of pneumonia in ventilated patients. Our objective was to evaluate the GeneXpert MRSA/SA SSTI Assay (Xpert MRSA/SA) (Cepheid, Sunnyvale, CA) for use in lower respiratory tract (LRT) specimens for rapid MRSA detection and to determine the potentially saved antibiotic-days if a culture-based identification method was replaced by this assay. Remnant LRT samples from ventilated patients submitted to the microbiology laboratory for routine culture were tested using conventional culture and Xpert MRSA/SA. One hundred of 310 LRT specimens met the inclusion criteria. Ten samples were positive for MRSA by Xpert MRSA/SA, while six were positive by routine culture methods. Xpert MRSA/SA correctly identified 5/6 positive and 89/94 negative MRSA specimens, for a sensitivity of 83.3%, specificity of 94.7%, positive predictive value of 45.6%, and negative predictive value of 98.9%. The assay also correctly detected 3/3 positive and 90/97 negative methicillin-susceptible S. aureus (MSSA) specimens, for a sensitivity of 100%, specificity of 92.8%, positive predictive value of 30%, and negative predictive value of 100%. A total of 748 vancomycin and 305 linezolid antibiotic-days were associated with the enrolled specimens. Vancomycin and linezolid utilization could decrease by 68.4% and 83%, respectively, if discontinued 1day after negative polymerase chain reaction (PCR) results. The Xpert MRSA/SA SSTI rapid MRSA PCR assay performed well in respiratory samples from ventilated patients with suspected pneumonia and has the potential to facilitate stewardship efforts such as reducing empiric vancomycin and linezolid therapy.

Characterization of Methicillin-Resistant Staphylococcus aureus Isolated from Healthy Turkeys and Broilers Using DNA Microarrays.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major human health problem and recently, domestic animals are described as carriers and possible reservoirs. Twenty seven S. aureus isolates from five turkey farms (n = 18) and two broiler farms (n = 9) were obtained by culturing of choana and skin swabs from apparently healthy birds, identified by Taqman-based real-time duplex nuc-mecA-PCR and characterized by spa typing as well as by a DNA microarray based assay which covered, amongst others, a considerable number of antibiotic resistance genes, species controls, and virulence markers. The antimicrobial susceptibility profiles were tested by agar diffusion assays and genotypically confirmed by the microarray. Five different spa types (3 in turkeys and 2 in broilers) were detected. The majority of MRSA isolates (24/27) belonged to clonal complex 398-MRSA-V. The most frequently occurring spa types were accordingly t011, t034, and t899. A single CC5-MRSA-III isolated from turkey and CC398-MRSA with an unidentified/truncated SCCmec element in turkey and broiler were additionally detected. The phenotypic antimicrobial resistance profiles of S. aureus isolated from both turkeys and broilers against 14 different antimicrobials showed that all isolates were resistant to ampicillin, cefoxitin, oxacillin, doxycycline, and tetracycline. Moreover, all S. aureus isolated from broilers were resistant to erythromycin and azithromycin. All isolates were susceptible to gentamicin, chloramphenicol, sulphonamides, and fusidic acid. The resistance rate against ciprofloxacin was 55.6% in broiler isolates and 42.1% in turkey isolates. All tetracycline resistant isolates possessed genes tetK/M. All erythromycin-resistant broiler isolates carried ermA. Only one broiler isolate (11.1%) carried genes ermA, ermB, and ermC, while 55.6% of turkey isolates possessed ermA and ermB genes. Neither PVL genes (lukF/S-PV), animal-associated leukocidin (lukM and luk-P83) nor the gene encoding the toxic shock syndrome toxin (tst1) were found in turkey and broiler isolates. In conclusion, the detection of MRSA in healthy turkeys and broilers with even additional antibiotic resistance markers is of major public health concern. The difference in antibiotic resistance and virulence markers between MRSA isolates from turkeys and broilers was addressed.

Rapid detection of methicillin-resistant Staphylococcus aureus in pork using a nucleic acid-based lateral flow immunoassay

Methicillin-resistant Staphylococcus aureus (MRSA) is considered as one of the leading causes of food poisonings worldwide. Due to the high prevalence and extensive challenges in clinical treatment, a rapid and accurate detection method is required to differentiate MRSA from other S. aureus isolated from foods. Since the methicillin resistance of S. aureus is due to the acquisition of the mecA gene from staphylococcal chromosome cassette, the presence of the mecA gene is interpreted as a marker for the identification of MRSA. In this study, a low-cost lateral flow immunoassay (LFI) strip was used to detect the mecA amplicons subsequent to polymerase chain reaction (PCR). The specificity of this PCR-LFI assay was tested between MRSA and methicillin-susceptive S. aureus. Both the test line and control line were shown up on the LFI strip for MRSA, whereas only the control line developed for methicillin-susceptive S. aureus. The detection limit of PCR-LFI assay was 20fg for genomic DNA (100 times more sensitive than gel electrophoresis) and 2×100CFU per 100g of pork products after enrichment at 37°C for 48h. The total detection time of using LFI was 3min, which was faster than the conventional electrophoresis (~45min). With the performance of PCR-LFI, 7 out of 42 S. aureus isolates were identified to be MRSA from imported pork products, which was consistent to the standardized minimum inhibitory concentration assay. This mecA-based PCR-LFI strip can be used for rapid and accurate detection of MRSA isolated from commercial pork products.

PERFORMANCE OF CHROM AGAR MEDIUM AND CONVENTIONAL METHODS FOR DETECTION OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS.

Background:- Methicillin resistant Staphylococcus aureus (MRSA) are responsible for hospital and community acquired infections. There are many laboratory methods for detection of MRSA. Chromogenic media have been used for the last few years for the quick detection of MRSA. Objective:- Aim of this study was to compare the performance of conventional methods and chromogenic media for the detection of MRSA in a tertiary care hospital. Material and method: - 200 consecutive isolates of S. aureus confirmed by conventional methods, collected in a tertiary care hospital were used for this study. Cefoxitin and oxacillin disc diffusion test used as conventional methods and Chromogenic media i.e. oxacillin resistant screen agar base (ORSAB) was used for detection of methicillin resistant Staphylococcus aureus. All confirmed MRSA were checked by gold standard mecA base PCR method. Result: - Out of 200 isolates of Staphylococcus aureus, 50,52 and 47 strains were MRSA by Cefoxitin disc diffusion method, oxacillin disc diffusion method and oxacillin resistant screen agar base (ORSAB) method respectively. Specificity was 100%, 98.66%, 98.66% by Cefoxitin disc diffusion, oxacillin disc diffusion and ORSAB method respectively. Conclusion: - In conclusion, cefoxitin disc diffusion was the best for the phenotypic detection of MRSA because their sensitivity and specificity were better than oxacillin and ORSAB.

Genotyping of methicillin-resistant Staphylococcus aureus (MRSA) isolated from milk and dairy products in South Italy

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen emerging in hospitals as well as community and livestock. MRSA is a significant and costly public health concern because it may enter the human food chain and contaminate milk and dairy products causing foodborne illness.This study aimed to determine the occurrence and the characteristics of MRSA isolated from 3760 samples of milk and dairy products in a previous survey conducted in southern Italy during 2008–2014. Overall out of 484 S. aureus strains isolated, 40 (8.3%) were MRSA and were characterized by spa-typing, Multi-Locus Sequence Typing, SCCmec typing, Staphylococcal enterotoxins (SEs) genes, Panton-Valentine Leukocidin (PVL) genes and ability to form biofilm.The most frequently recovered STs were ST152 (t355–67.5%), followed by ST398 (t899, t108–25%), ST1 (t127–5%) and ST5 (t688–2.5%). All isolates harboured the SCCmec type V (92.5%) or IVa (25%). In one isolate (2.5%), ST398/t899, the SCCmec resulted not detected. Three isolates (7.5%) carried one or more enterotoxin encoding genes (one strain had seg, sei, sem, sen and seo genes; two strains had seh gene). The 50% of isolated strains harboured PVL-encoding genes. Molecular analysis for icaA and icaD genes showed: 72.5% icaA and icaD positive, 25% only icaD gene and one icaA and icaD negative. The detection of MRSA in food of animal origin is a potential health hazard, thus it is necessary monitoring of food-producing animals and improving hygiene standards in food practices in order to reduce the microbiological risk to minimum.

Isolation, Characterization, and Antibacterial Activity of Bacteriophages Against Methicillin-Resistant Staphylococcus aureus in Pakistan.

BackgroundIn recent years, antibiotic resistance has been indicated as a paramount threat to public health. The use of bacteriophages appears to be a safer alternative for the control of bacterial infections.ObjectivesThe present study aims to explore sewage water for the presence of indigenous bacteriophages, and to investigate their antibacterial potential against Methicillin-resistant Staphylococcus aureus (MRSA).MethodsBacterial isolates were first collected and identified from pus samples taken from the surgical and burn units using standard microbiological procedures. A cefoxitin disk screen test was then used and interpreted according to the clinical laboratory standards institute (CLSI) guidelines for the detection of MRSA. The sewage samples were processed and the phages enriched using S. aureus as a host organism. Turbid and clear plaques of different sizes were isolated using an overlay method, purified, and then enumerated by means of a dilution method.ResultsThe phages exhibited good lytic activity against MRSA when tested in-vitro, and the highest activity was attained within three to six hours of phage infection. The isolated phage pq/48 was also found efficient in decreasing the bacterial count during an in-vivo trial in rabbits. A protein analysis using SDS-PAGE revealed 10 proteins of between 20 kDa and 155 kDa in size.ConclusionsThe overall results indicated that bacteriophages isolated from sewage exhibited excellent lytic activity against MRSA strains. In conclusion, bacteriophages can be further characterized and appear to be a promising candidate for phage therapy against MRSA in the future.

Minimum inhibitory concentration of vancomycin to methicillin resistant Staphylococcus aureus isolated from different clinical samples at a tertiary care hospital in Nepal.

BackgroundMethicillin resistant Staphylococcus aureus (MRSA) has evolved as a serious threat to public health. It has capability to cause infections not only in health care settings but also in community. Due to the multidrug resistance shown by MRSA, there are limited treatment options for the infections caused by this superbug. Vancomycin is used as the drug of choice for the treatment of infections caused by MRSA. Different studies from all around the world have documented the emergence of strains of S. aureus those are intermediate sensitive or resistant to vancomycin. And recently, there have been reports of reduced susceptibility of MRSA to vancomycin, from Nepal also. So the main purpose of this study was to determine the minimum inhibitory concentration (MIC) of vancomycin to methicillin resistant S. aureus isolated from different clinical specimens.MethodsTotal 125 strains of S. aureus isolated from different clinical samples at KIST Medical College and Teaching Hospital, Lalitpur, Nepal from Nov 2012 to June 2013, were subjected to MRSA detection by cefoxitin disc diffusion method. The minimum inhibitory concentrations of vancomycin to confirmed MRSA strains were determined by agar dilution method. Yellow colored colonies in mannitol salt agar, which were gram positive cocci, catalase positive and coagulase positive were confirmed to be S. aureus.ResultsAmong, total 125 S. aureus strains isolated; 47(37.6%) were MRSA. Minimum inhibitory concentrations of vancomycin to the strains of MRSA ranged from 0.125 μg/ml to 1 μg/ml.ConclusionFrom our findings we concluded that the rate of isolation of MRSA among all the strains of S. aureus isolated from clinical samples was very high. However, none of the MRSA strains were found to be vancomycin intermediate-sensitive or vancomycin-resistant.

Eradication of methicillin resistant Staphylococcus aureus detected for the first time in cystic fibrosis: A single center observational study.

To retrospectively identify CF patients with methicillin resistant Staphylococcus aureus (MRSA) and to assess the long-term success of an eradication scheme introduced in 2002 for all newly colonized patients. All microbiological results from all 505 CF patients followed between 2002 and 2012 were analyzed focusing on the detection of MRSA. Retrospective patient record analysis of MRSA positive CF patients regarding eradication and clinical outcome. We identified 57 patients with MRSA, mean age 15.3 years (range: 0.6-36.9, incidence 0.9%/year). Of these, nine patients were lost to follow-up; seven chronically colonized patients were excluded from the intervention. Eradication was suggested to all patients, 37/41 gave their consent to the following two-step approach: (i) dual iv antibiotic treatment over 3 weeks, accompanied by hygienic directives and topical therapy for 5 days followed by a 6-week period with dual oral antibiotic therapy and inhalation with vancomycin. (ii) Each new MRSA detection was treated with 6 weeks inhalation of vancomycin and topical therapy for 5 days. Long-term eradication was rated by the microbiological status in the third year after first detection. MRSA was eradicated in 31 of 37 patients (84%) whose clinical course was stable (mean FEV1 one year before MRSA 80.4%, 3 years after MRSA 81.0%). MRSA colonization mandates complex and expensive hygienic measures which are not well accepted by patients. Therefore, MRSA eradication is desirable. Intensive therapy regimens may be successful in patients with CF and might help to maintain a stable clinical course. Pediatr Pulmonol. 2016;51:1010-1019. © 2016 Wiley Periodicals, Inc.

Molecular Detection of Methicillin-Resistant Staphylococcus aureus by Non-Protein Coding RNA-Mediated Monoplex Polymerase Chain Reaction.

Non-protein coding RNA (npcRNA) is a functional RNA molecule that is not translated into a protein. Bacterial npcRNAs are structurally diversified molecules, typically 50–200 nucleotides in length. They play a crucial physiological role in cellular networking, including stress responses, replication and bacterial virulence. In this study, by using an identified npcRNA gene (Sau-02) in Methicillin-resistant Staphylococcus aureus (MRSA), we identified the Gram-positive bacteria S. aureus. A Sau-02-mediated monoplex Polymerase Chain Reaction (PCR) assay was designed that displayed high sensitivity and specificity. Fourteen different bacteria and 18 S. aureus strains were tested, and the results showed that the Sau-02 gene is specific to S. aureus. The detection limit was tested against genomic DNA from MRSA and was found to be ~10 genome copies. Further, the detection was extended to whole-cell MRSA detection, and we reached the detection limit with two bacteria. The monoplex PCR assay demonstrated in this study is a novel detection method that can replicate other npcRNA-mediated detection assays.

EFFECTIVENESS OF MRSA DETECTION METHODS IN THE LABORATORY PRACTICE – A BRIEF REVIEW

Methicillin-resistant Staphylococcus aureus (MRSA) are bacteria, responsible for severe and hard-to-manage infections in human. They are resistant to beta-lactam antibiotics - penicillins (methicillin, dicloxacillin, nafcillin, and oxacillin), cephalosporins and carbapenems, but can also be resistant to the new-generation MRSA-active cephalosporins (such as ceftaroline) or other groups of antibiotics, including aminoglycosides, macrolides, clindamycin, amphenicols, quinolones and tetracyclines. MRSA bacteria are pandemic and are often isolated in medical practice and nosocomial infections. The MRSA detection is a challenge to any clinical microbiology laboratory and demands implementation of strict protocols for active screening. While more expensive molecular techniques have the potential of offering highly sensitive and rapid results, the cultural methods require longer time but can achieve a comparable sensitivity for lower price.

A Systematic Review on Prevention of Methicillin-Resistant Staphylococcus Aureus Infection by Pre-Admission Screening: The Cost Effectiveness and Practicality

Background: Methicillin Resistant Staphylococcus aureus (MRSA) is a common source of nosocomial infection, which is spreading through the community and hospitals across the countries. The performance of screening program really needs major effort related to laboratory capacity and ethical consideration, among other costly components. Significant literature research was conducted to review the cost, effectiveness and practicality of different methods of pre-admission MRSA screening in the hospital setting. A systematic literature review was conducted with search strategy using the PubMed Medline, Scopus and the Science Direct databases. The relevant data was abstracted from all studies based on various countries which in line with the finalized eligibility criteria. Results: PCR method was reported to have high sensitivity with low turnaround time as compared to culture method. A review of selected studies found the increasing annual costs of screening from standard culture, chromogenic agar to rapid PCR. In the meantime, other studies reported the total costs for labor and materials was lower for rapid PCR screening compared to culture methods. The culturing method offers a high level of variability due to time consumption and additional costs. Whereas PCR was reported as advantageous in term of saving time to identify MRSA positive patients, which involved isolation, thus increase the effectiveness of screening programs. It can pick up false negative results by conventional methods in the early condition of disease. Conclusion: Most studies verified that PCR is the most accurate method for detection of MRSA with Xpert MRSA having the best performance. Otherwise, oxacillin agar screen was revealed as a good alternative method to PCR. Targeted screening on high risk patients using rapid PCR may be the best choice to be implemented, in order to balance the economic and practicality of screening. We recommend that further clinical studies should be done to provide a sharp evidence of MRSA screening.

Characterization of Extended Spectrum Β-Lactamase Producing Enterobacteria and Methicillin-Resistant Staphylococcus aureus Isolated from Raw Pork and Cooked Pork Products in South China.

In this study, we assessed the co-colonization with extended spectrum β-lactamase producing Enterobacteria (ESBL-E) and methicillin-resistant Staphylococcus aureus (MRSA) in raw pork and cooked pork products in south China. In total, 240 raw pork and 240 cooked pork samples collected from supermarkets (n = 20) and local butcher shops (n = 20) in the city of Guangzhou (China) were investigated. Raw pork and cooked pork was more frequent colonization with ESBL-E (7.5% in raw pork and 0.4% in cooked pork products) than with MRSA (4.2% in raw pork). Two of samples were contaminated with both tested types of multidrug-resistant bacteria. High antibiotic-resistance rate with wide spectrums of both ESBL-E and MRSA isolated were observed. In ESBL-E isolates, TEM (n = 15), CTX-M-1 (n = 3), CTX-M-9 (n = 1), and SHV (n = 1) genes were detected. TEM and SHV genes were associated with CTX-M-1 in 2 isolates, respectively. The CTX-M-9 gene of 1 isolate from cooked pork samples was found to be transferred to Escherichia coli J53 by conjugation. Detected MLST-types of MRSA were livestock-associated ST7 (n = 5) and ST9 (n = 4), as well as hospital-acquired ST239 (n = 1), suggesting contamination from human source(s) during meat processing. These findings confirmed a contamination of raw pork and cooked pork with ESBL-E and MRSA and emphasized the necessity of enforcing hygienic practices and specific detection of MRSA and ESBL-producing bacteria in meat processing and storage.