Oxacillin Disk Diffusion Method Research Articles

Evaluation of accuracy of Phenotypic methods in the detection of Methicillin Resistant Staphylococcus aureus

Abstract: Methicillin resistant Staphylococcus aureus (MRSA) is a variant of Staphylococcus aureus; responsible for a significant share of the infectious load. Objective: The current study was conducted to evaluate the accuracy of already in vogue phenotypic methods of identification of MRSA keeping in view the gold standard method of mec-A gene detection using Polymerase chain reaction (PCR) and to compare the sensitivity and specificity of different phenotypic methods (Cefoxitin Disc Diffusion, Oxacillin Disc Diffusion, Oxacillin Screen Agar, and MIC of Oxacillin by Agar Dilution Method) with the genotypic method (PCR). Method: A descriptive cross-sectional study was carried out at Microbiology Lab, Pakistan Railway Hospital, Rawalpindi from October 2021 to September 2022, after attaining the formal approval from Institutional Ethical Review Committee (Riphah/IIMC/IRC/21/73), using non-probability sampling technique. A total of 222 samples of Staphylococcus aureus were isolated from all the clinical specimens received at Microbiology lab at Pakistan Railway Hospital. Among those isolates, 150(67.5%) were Methicillin sensitive Staphylococcus aureus (MSSA) and 72(32.4%) were Methicillin Resistant Staphylococcus aureus (MRSA). The results of Phenotypic methods were compared with the results of PCR by using Chi-square formula. The sensitivity and specificity of Cefoxitin Disc Diffusion method was 62.5% and 96.6%, Oxacillin Disc Diffusion method was 65.3% and 93.3%, Oxacillin Screen Agar technique was 81.9% and 96% while that of Oxacillin MIC by Agar Dilution was 91.6% and 89.3% respectively. Oxacillin screen agar demonstrates the better phenotypic technique for detection of MRSA in routine practice.

Using Protein Fingerprinting for Identifying and Discriminating Methicillin Resistant Staphylococcus aureus Isolates from Inpatient and Outpatient Clinics.

In hospitals and other clinical settings, Methicillin-resistant Staphylococcus aureus (MRSA) is a particularly dangerous pathogen that can cause serious or even fatal infections. Thus, the detection and differentiation of MRSA has become an urgent matter in order to provide appropriate treatment and timely intervention in infection control. To ensure this, laboratories must have access to the most up-to-date testing methods and technology available. This study was conducted to determine whether protein fingerprinting technology could be used to identify and distinguish MRSA recovered from both inpatients and outpatients. A total of 326 S. aureus isolates were obtained from 2800 in- and outpatient samples collected from King Faisal Specialist Hospital and Research Centre in Riyadh, Saudi Arabia, from October 2018 to March 2021. For the phenotypic identification of 326 probable S. aureus cultures, microscopic analysis, Gram staining, a tube coagulase test, a Staph ID 32 API system, and a Vitek 2 Compact system were used. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), referred to as protein fingerprinting, was performed on each bacterial isolate to determine its proteomic composition. As part of the analysis, Principal Component Analysis (PCA) and a single-peak analysis of MALDI-TOF MS software were also used to distinguish between Methicillin-sensitive Staphylococcus aureus (MSSA) and MRSA. According to the results, S. aureus isolates constituted 326 out of 2800 (11.64%) based on the culture technique. The Staph ID 32 API system and Vitek 2 Compact System were able to correctly identify 262 (80.7%) and 281 (86.2%) S. aureus strains, respectively. Based on the Oxacillin Disc Diffusion Method, 197 (62.23%) of 326 isolates of S. aureus exhibited a cefoxitin inhibition zone of less than 21 mm and an oxacillin inhibition zone of less than 10 mm, and were classified as MRSA under Clinical Laboratory Standards Institute guidelines. MALDI-TOF MS was able to correctly identify 100% of all S. aureus isolates with a score value equal to or greater than 2.00. In addition, a close relationship was found between S. aureus isolates and higher peak intensities in the mass ranges of 3990 Da, 4120 Da, and 5850 Da, which were found in MRSA isolates but absent in MSSA isolates. Therefore, protein fingerprinting has the potential to be used in clinical settings to rapidly detect and differentiate MRSA isolates, allowing for more targeted treatments and improved patient outcomes.

“Evaluation Of Several Phenotypic Methods Of Antibiotic Susceptibility Pattern For The Detection Of MRSA With Molecular Profiling RT-PCR With The Detection Of MecA Gene”

INTRODUCTION:- Methicillin-resistant Staphylococcus aureus (MRSA) is a well-recognized public health problem throughout the world and community MRSA strains are now epidemic in India and others countries. The resistance rates of S. aureus infection and multidrug resistant strains are increasing, making the clinical anti-infective treatment and diagnosis more difficult.
 AIM:- To evaluate several phenotypic methods of the antibiotic susceptibility pattern for the detection of MRSA with Molecular Profiling Rt-PCR with the detection of MecA gene.
 MATERIALS AND METHODS:- The present study was a prospective observational study carried out at the Department of Microbiology, Santosh Medical College, Ghaziabad in a tertiary care hospital over a 12-month period from August 1, 2020 to July 31, 2021. Both IPD and OPD clinical sample comprised 385 clinical isolates as Staphylococcus aureus from various clinical specimens were included. Antibiotic susceptibility by the Kirby-Baure disc diffusion methods was performed according to the Clinical and Laboratory Standard Institute (CLSI) guidelines. Detection of MRSA by various methods like, Cefoxitin Disk diffusion Method, Oxacillin Disk diffusion method, E-Test method and detection of MecA gene by RT-PCR was carried out.
 RESULT:- In the present study out of 384 clinical samples, the majority of isolates from MRSA were found to be resistant to (E-Test) strip Oxacillin 114 (29.7%), followed by cefoxitin 113 (29.4%) and disc diffusion oxacillin 99 (25.8%). However, we observed a high incidence of resistance to other antibiotics such as Erythromycin 265 (69.0%), followed by Clotrimozole 228 (59.4%), Tetracyclin 144 (37.5), Vancomycin 102 (26.6%) and Refampicin 102 (26.6%). We also observed that Linezolid 354 (92.2%) followed by Teicoplanin 325 (84.6%), Gentamycin 272 (70.8%), Clindamycin 249 (64.8%) and Amoxyclave 281 (73.2%). In this study molecular RT-PCR test for the detection of mecA gene observed more in pus with 36 samples out of 113 followed by blood 17, urine 33, Sputum 05, pleural fluid 04, wound swab 16, Vaginal swab 06, CSF 01, Throat swab 2.
 CONCLUSION:- The gold standard assay for determining methicillin resistance is the PCR technique for detecting the mecA gene. But Because PCR is still time intensive and expensive, and it is not yet available in the 95% of routine clinical laboratories the phenotypic methods is still the choice where E-test is more reliable method than the disc diffusion method in detecting the drug resistance and can be utilised on a regular basis for better results.

PREVALENCE OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS IN WESTERN TAMILNADU

Humans are natural reservoir for Staphylococcus aureus, and asymptomatic colonization is far more common than infection. Colonization byStaph.aureusmay be persistent and can last for years. Recent reports of strains of Methicillin Resistant Staphylococcus aureus (MRSA) isolated from community have led speculation that the epidemiology of S. aureus is changing .Usually, MRSA infections havebeen a concern among hospitals for decades now and the reports revealthat the community acquired MRSA is increasing . The community acquired strains could possibly have arisen as a consequence of resistance gene transfer from a hospital acquired (nosocomial) donor into a susceptible recipient. With appropriate analysis of donor andrecipient chromosomes, it could be possible to determine whether these newly identified communityacquired strains are wild or self-supporting.The present study was conducted with a total sample of 1296 wound and other skin infection samples that were collected from different hospitals in western Tamilnadu. The specimens were inoculated in blood agar for isolation and identified as S.aureus by using standard method based on colony morphology, Gram's stain, catalase and coagulase test. A total 258 isolates were confirmed as S.aureus. These strains were processed by the following three techniques,(i) oxacillin, Methicillin and cefoxition disk diffusion method, (ii) cultured in MeRSA plate, (iii) Vitek 2 fully automated ID and AST system for detection mecA gene producer. 34 MRSA strains were identified out of 258 strains.

Comparison of Phenotypic and Genotypic Characterization Methods for the Detection of Methicillin-Resistant Staphylococcus Aureus.

IntroductionMethicillin-resistant Staphylococcus aureus (MRSA) is associated with high morbidity and mortality due to the development of antimicrobial resistance secondary to irrational use of antibiotics, nonadherence to infection control practices, and increased use of intravascular devices in healthcare systems. Detection of MRSA is critical in clinical microbiology laboratories as it helps identify MRSA carriers and avoid treatment failure in patients. Hence, this study compared various phenotypic methods with the standard genotyping method to determine a method that permits rapid and accurate detection of MRSA.Materials & MethodsStaphylococcus aureus (S. aureus) was initially identified based on colony morphology, Gram staining, standard biochemical tests, and antibiotic susceptibility using disk diffusion. MRSA was identified based on the detection of the mecA gene by polymerase chain reaction (PCR) and subsequent gel electrophoresis. Disk diffusion using cefoxitin or oxacillin and mannitol salt agar with 6-µg/ml oxacillin were used for phenotypic detection of MRSA. The D test was used to detect inducible clindamycin resistance in S. aureus isolates.ResultsOf the 100 S. aureus isolates analyzed, 37% were identified as MRSA by PCR and the cefoxitin disk diffusion method; however, only 31% were detected by the oxacillin disk diffusion method and 29% by the mannitol salt agar method. The sensitivity of the cefoxitin disk diffusion test, oxacillin disk diffusion, and mannitol salt agar methods was 86.05%, 83.78%, and 70.73%, respectively. Specificity was 100% for all the three phenotypic methods (p < 0.001). Notably, inducible clindamycin resistance was found in 37.2% of the MRSA isolates, indicating potential challenges in treatment.ConclusionAmong the three phenotypic methods tested, the cefoxitin disk diffusion method had 100% sensitivity and specificity, which is similar to that of PCR-based MRSA detection. Hence, the cefoxitin disk diffusion method is recommended for use in clinical laboratories, where molecular methods are not available as it is both cost-effective and easy to perform.

Comparison of Different Phenotypic Methods Including E-Test, Cefoxitin and Oxacillin Disk Diffusion for Detection of Methicillin Resistant Staphylococcus aureus

Introduction: Methicillin-Resistant Staphylococcus aureus (MRSA) has spread throughout the world as a hospital and communityacquired illness. Although a variety of strategies have been employed, laboratory identification of MRSA remains a difficulty. Aim: To examine several phenotypic approaches for accuracy results, with an (Epsilometer) E-test based method serving as the gold standard for MRSA identification. Materials and Methods: A prospective observational study was conducted in the Microbiology department of Santosh Medical College, Ghaziabad, Uttar Pradesh, India, from August 2020 to July 2021. Total of 384 isolates S.aureus were identified by using the required samples including pus, swab, blood, wound and urine, etc., which were collected from the Microbiology department and the comparison was done between E-test serving as the gold standard for MRSA identification with Cefoxitin Disk Diffusion (CDD)/ Oxacillin Disk Diffusion method (ODD). The diagnostic kit for using E-Test in collected samples was purchased from Himedia Laboratries Pvt., Ltd., Mumbai, India (EM0065). The data was calculated by using MS-Excel. Results: A total of 113 strains were revealed to be MRSA in clinical specimens out of 384 isolated S.aureus. The gold standard method was chosen to be the E-Test, which had found a high sensitivity of 79.8% and a specificity of 94.2%. Compared to the cefoxitin/oxacillin disc diffusion method, isolates including MRSA were highly susceptible to Tiecoplanin and Linezolid. Conclusion: The present study concludes that E-Test (strip) method is a high sensitivity and highly specific for detecting MRSA in comparison to other disk methods used in this study. Due to less number of sample size and lesser time period more studies are needed to establish this fact.

Evaluation of Oxacillin and Cefoxitin Disk Diffusion and Microbroth Dilution Methods for Detecting mecA -Mediated β-Lactam Resistance in Contemporary Staphylococcus epidermidis Isolates

Methicillin (β-lactam) resistance in Staphylococcus epidermidis is mediated by the mecA gene, with resistance reported to be as high as 90%. The goal of this study was to evaluate oxacillin and cefoxitin disk diffusion (DD) and broth microdilution (BMD) methods for the detection of mecA-mediated β-lactam resistance in 100 human isolates of S. epidermidis (48 mecA-positive isolates and 52 mecA negative isolates). Oxacillin DD tests using the Clinical and Laboratory Standards Institute (CLSI) M100-S28 breakpoints for S. pseudintermedius/S. schleiferi accurately differentiated mecA-positive and -negative S. epidermidis isolates, with categorical agreement (CA) of 100% and no very major errors (VMEs) or major errors (MEs) identified. Likewise, oxacillin BMD and cefoxitin DD tests using the coagulase-negative Staphylococcus species (CoNS) breakpoints were highly reliable for detecting mecA-mediated β-lactam resistance in S. epidermidis isolates. For cefoxitin DD and BMD results interpreted using S. aureus/S. lugdunensis breakpoints, the CA was 97.6% and 96.2%, respectively. There were 4.9% VMEs for cefoxitin DD with 0% MEs, and 3.6% VMEs and 3.9% MEs for cefoxitin BMD. Oxacillin BMD using S. aureus/S. lugdunensis breakpoints yielded the highest VMEs at 17.4% and 90% CA. Our findings demonstrate that oxacillin DD tests using the CLSI M100-S28 breakpoints for S. pseudintermedius/S. schleiferi and oxacillin BMD and cefoxitin DD tests using the CoNS breakpoints reliably identified mecA-mediated β-lactam resistance in S. epidermidis Using mecA PCR as the gold standard, the PBP2a SA culture colony test (Abbott Diagnostics) exhibited 100% sensitivity and specificity whereas 2 false negatives were identified using the PBP2' latex agglutination test kit (Thermo Fisher Scientific) with sensitivity and specificity of 95.8% and 100%, respectively.

Prevalence of methicillin-resistant Staphylococcus aureus in healthy Chinese population: A system review and meta-analysis.

ObjectiveTo comprehensively determine the prevalence of MRSA in healthy Chinese population, the influencing factors of MRSA colonization and its antibiotic resistance.MethodsArticles that studied prevalence or influencing factors of MRSA carriage in healthy Chinese population were retrieved from PubMed, Ovid database, three Chinese electronic databases. The pooled prevalence of MRSA, its antibiotic resistance and influencing factors were analyzed by STATA12.0.Results37 studies were included. The pooled prevalence of MRSA was 21.2% (95% CI: 18.5%-23.9%), and the prevalence of S.aureus was 15% (95% CI: 10%-19%), with a significant heterogeneity (MRSA: I2 = 97.6%, P<0.001; S.aureus: I2 = 98.4%, P < 0.001). In subgroup analysis, the pooled prevalence of MRSA was 28% (95%CI: 10%-51%) for Livestock-related workers, 18% (95%CI: 11%-26%) for children, 20% (95%CI: 12%-29%) for healthcare workers, 7% (95%CI: 3%-13%) for community residents. The prevalence of MRSA in studies with oxacillin disk diffusion method (28%, 95%CI: 21%-35%) seemed higher than that with the mecA gene method(12%, 95%CI: 7%-19%). MRSA in studies conducted in Taiwan was more common than in Mainland China and Hong Kong. Similar results were found in meta-regression. Influencing factors for MRSA colonization were noted in seven eligible studies, they included younger age (OR: 3.54, 95% CI: 2.38–5.26; OR: 2.24, 95% CI: 1.73–2.9), attending day care centers (DCCs) (OR: 1.95, 95% CI: 1.4–2.72; OR: 1.53, 95% CI: 1.2–1.95), flu vaccination (OR:1.73, 95% CI: 1.28–2.35), using antibiotics within the past year (OR: 2.05, 95% CI:1.35–3.11), residing in northern Taiwan (OR: 1.45, 95% CI: 1.19–1.77), regular visits to health care facility (OR: 23.83, 95% CI: 2.72–209.01), household member working in health care facility (OR: 8.98, 95% CI:1.4–55.63), and contact with livestock (OR: 6.31, 95% CI: 3.44–11.57). Moreover, MRSA was found to be highly resistant to penicillin, ampicillin, erythromycin, and clindamycin, with a pooled resistance ratio of 100, 93, 88, and 75%, respectively. However, no resistance were noted to vancomycin.ConclusionThe pooled prevalence of MRSA was considerably high in health Chinese population. Additionally, these strains showed extreme resistance to penicillin, ampicillin, erythromycin and clindamycin. Public MRSA protection measures and the surveillance of MRSA should be strengthened to reduce the spread of MRSA among hospitals, communities, and livestock.

Nasal Carriage of Methicillin Resistant Staphylococcus aureus Among Health Care Workers At Tertiary Care Hospital In Dhaka.

Nasal carriage of MRSA among hospital stuff act as a source of endogenous infection and becomes a source for hospital and community acquired infection. The study was conducted to determine the rate antibiotic resistance pattern of nasal carriage of MRSA among the hospital stuff of Sir Salimullah Medical College and Mitford Hospital, Dhaka. Pre moistened nasal swabs from hospital stuff (doctor, nurses, lab technicians and other helping stuff were obtained. These swabs were inoculated into Blood agar and Mannitol salt agar media. Antibiogram was done by modified Kirby Bauer disc diffusion method. MRSA were detected by oxacillin and cefoxitin disc diffusion method. The resistance was confirmed by MIC of oxacillin agar dilution method. Out of 142 samples 34 strains of Staphylococcus were isolated among them 07 (4.93%) were MRSA and 27 (19.01%) were MSSA. The carriage rate of MRSA was higher among nurse than other healthcare provider. Nasal carriage of MRSA is responsible for spreading infection from healthcare personnel to normal individual. So, regular screening of carrier is required from prevention of hospital acquired infection.&#x0D; Bangladesh J Med Microbiol 2017; 11 (2): 17-19

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.

개와 고양이에서 분리한 methicillin 내성 및 감수성 Staphylococcus pseudintermedius

Staphylococcus pseudintermedius is an important opportunistic pathogen of dog and cats. Since 2006 there has been a significant emergence of methicillin-resistant S. pseudintermedius (MRSP) mainly due to clonal spread. The aim of this study was to investigated the prevalence of antibiotic resistance and presence of mecA and femA gene in 91 S. pseudintermedius isolates isolated from dogs and cats associated with various clinic infections. Methicillin resistance was confirmed by oxacillin disc diffusion method. MRSP isolate was detected 19 isolates (20.9%). MRSP and methicillin-resistant S. pseudintermedius (MSSP) isolates were highly resistant to penicillin, kanamycin, tetracycline, erythromycin, trimethoprim-sulfamethoxazole, clindamycin, ciprofloxacin, enrofloxacin and choloramphenicol (100~47.3% and 90.3~33.3%, respectively). About 90% of MRSP isolates were multi-drug resistance (resistance to at least five or more antimicrobials), and MSSP isolates was ca 74%. Among the 91 isolates, mecA gene was detected in 25 isolates (27.5%, 19 in MRSP isolates and 6 in MSSP isolates), but none carried the femA gene. Our results indicated MRSA isolates show a strong resistance to antimicrobials commonly used in veterinary medicine. A continuous surveillance and monitoring should be called for to prevent the contamination and spread of MRSP in dogs and cats.

Determination of vancomycin and methicillin resistance in clinical isolates of Staphylococcus aureus in hospitals of Ilam city

Introduction: In this study, using the phenotypic and genotypic methods, oxacillin susceptibility in Staphylococcus aureus (S. aureus) strains isolated from patients at two government hospitals in Ilam, Iran was tested. Materials and methods: Out of 200 S. aureus isolates from different human clinical specimens consisting of blood (31%), wound (20%), urine (21%), catheters (7%), sputum (12%), others (9%) were collected. The methicillin resistant S. aureus isolates were investigated using disk diffusion methods and oxacillin (1μg) and cefoxitin (30μg), on MuellerHinton agar were used, and MecA and vanA genes were detected by PCR. In addition, the isolates were tested for their antibiogram profiles. Results: Among 200 S. aureus strains included in this study, 35.96% were MRSA. The percentage of resistance by disk diffusion method was as below: penicillin 85.96%, vancomycin 0%, ampicillin 87.71%, gentamicin 48.25% erythromycin 54.25%, clindamycin 32.45%, amikacin 21.05%, ciprofloxacin 42.10%, tetracycline 51.75% and co-trimoxazole 42.10%. Phenotyping method by disk diffusion method using oxacillin and cefoxitin for detecting of MRSA showed sensitivity and specificity of about 33.33% and 35.96%, respectively. Presence of MecA and vanA genes in MRSA isolates by PCR were 35.96% and 0%, respectively. The oxacillin and cefoxitin disk diffusion methods showed 92.68% and 100% sensitivity, respectively, and 98.8% specificity. Conclusion: Our finding showed that, the cefoxitin disk diffusion method is better in compared to the oxacillin disk diffusion similar to results from detecting of MecA gene in PCR as a golden test.

Virulence Factors in Staphylococci Isolated From Nasal Cavities of Footballers

AimThis study aimed to investigate the rate of Panton-Valentine Leukocidin producing Staphylococcus aureus and methicillin (mecA) and slime (icaA/icaD) genes in staphylococcal strains isolated from nasal cavities of footballers. Materials and MethodsNasal swab samples were taken from each footballers and a healthy control group for the isolation of staphylococcal strains. The polymerase chain reaction technique was used to determine Panton-Valentine Leukocidin, mecA and icaA/icaD genes in staphylococcal isolates. ResultsAmong 91 S. aureus strains, the presence of mecA gene was detected as 9.9%. This ratio was 17.9% (27 of 151) among the coagulase-negative staphylococci. A significant difference was found between coagulase-negative staphylococci and S. aureus isolates regarding the presence of mecA gene (P < 0.001). As for the genes of the slime, icaA/icaD genes were detected in 198 of 242 (81.8%) strains. The occurrence of slime genes was 91.2% and 89.4% among the S. aureus coagulase and negative staphylococci, respectively (P > 0.05). There was a statistically significant difference between the frequency of the mecA and slime genes when compared with the healthy control group and the football players (P < 0.01). Of 91 isolates, 22 were found to be methicillin resistant by the oxacillin disc diffusion method, whereas the remaining (220) were methicillin susceptible. Methicillin resistance was detected as 14.9% by the polymerase chain reaction method, whereas it was found as 9.1% by phenotypic methods. ConclusionsEarly and accurate diagnosis of virulent staphylococcal strains is crucial because the virulent coagulase-negative and coagulase-positive staphylococcal strains in the nasal floras of footballers may be major potential sources of superficial and deep tissue infections.

Prevalence and antimicrobial susceptibility pattern of methicillin-resistant, vancomycin-resistant, and Panton-Valentine leukocidin positive Staphylococcus aureus in a tertiary care hospital Dhaka, Bangladesh

ObjectivesTo observe the prevalence of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant S. aureus (VRSA), and Panton-Valentine leukocidin (PVL)-positive S. aureus, this study was carried out in a tertiary care hospital in Dhaka, Bangladesh. Materials and methodsS. aureus strains were recovered from 200 postoperative wound swab samples from patients hospitalized in Dhaka Medical College Hospital between July 2011 and June 2012. Methicillin resistance was determined by the oxacillin and cefoxitin disc diffusion method, the minimum inhibitory concentration (MIC) of oxacillin, and mecA gene detection. VRSA resistance was determined by the disc diffusion method, the MIC of vancomycin, and screening for the vanA and vanB genes. The PVL gene was also detected in MRSA strains. ResultsFifteen of the 44 isolated strains of S. aureus were MRSA (2 of them were VRSA) and 29 were methicillin-sensitive S. aureus. All MRSA isolates were highly resistant to oxacillin (MIC ≥ 256 μg/mL). When compared with polymerase chain reaction (PCR), the sensitivity and specificity of the oxacillin disc diffusion method were 93.33% and 100% respectively; for the cefoxitin disc diffusion method and MIC of oxacillin both the sensitivity and specificity were 100%. Four (26.67%) MRSA isolates were positive for PVL genes which were also mecA positive. The MRSA strains were highly resistant to ciprofloxacin (93.33%), ceftriaxone (86.63%), azithromycin (73.33%), gentamycin (73.33%), and amoxiclav (66.67%). All (100%) MRSA strains were sensitive to linezolid and 86.67% were sensitive to vancomycin. The VRSA strains had an MIC ≥256 μg/mL for vancomycin and were positive for the vanB gene but negative for the vanA gene. ConclusionThe results of this study provide insight into the high proportion of MRSA and presence of VRSA in Bangladesh.

PCR-based identification of methicillin–resistant Staphylococcus aureus strains and their antibiotic resistance profiles

To evaluated the PCR for mecA gene compared with the conventional oxacillin disk diffusion method for methicillin-resistant Staphylococcus aureus (S. aureus) identification. A total of 292 S. aureus strains were isolated from various clinical specimens obtained from hospitalized patients. Susceptibility test to several antimicrobial agents was performed by disk diffusion agar according to Clinical and Laboratory Standards Institute guidelines. The PCR amplification of the mecA gene was carried out in all the clinical isolates. Among antibiotics used in our study, penicillin showed the least anti-staphylococcal activity and vancomycin was the most effective. The rate of methicillin-resistant S. aureus prevalence determined by oxacillin disk diffusion method was 47.6%; whereas, 45.1% of S. aureus isolates were mecA- positive in the PCR assay. This study is suggestive that the PCR for detection of mecA gene is a fast, accurate and valuable diagnostic tool, particularly in hospitals in areas where methicillin-resistant S. aureus is endemic.

Comparison of antibiotic susceptibility pattern of community- and hospital-acquired methicillin-resistant Staphylococcus aureus with special reference to inducible clindamycin resistance in a tertiary care hospital in southern India

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial and community infections. Its prevalence varies markedly across different countries and among hospitals of the same country. Aims and Objectives: To estimate the prevalence of MRSA strains and investigate their antibiogram with special reference to inducible clindamycin resistance. Materials and Methods: All S. aureus isolates obtained from the clinical samples of microbiology laboratory were included in the study. All the isolates were identified by standard methods, and antimicrobial susceptibility testing was performed by Kirby Bauer disk diffusion method. Methicillin resistance was detected by combined cefoxitin and oxacillin disk diffusion method. Results were interpreted as per the Clinical and Laboratory Standards Institute (CLSI) guidelines. Results: A total of 362 S. aureus strains were isolated, of which 36.1% (131/362) isolates were MRSA. Of these, 79.4% (104/131) were hospital-acquired MRSA (HA-MRSA) and 20.6% (27/131) were community-acquired MRSA (CA-MRSA). All the isolates were sensitive to vancomycin. Inducible clindamycin [macrolide-lincosamide-streptogramin B (iMLS B )] resistance (D test) among MRSA isolates was 12.3% (16/131). HA-MRSA isolates showed 12.5% (13/104) D test positivity, as compared to 11.2% (3/27) seen in CA-MRSA isolates. Conclusion: The reported rate of MRSA incidence is alarming. Regular surveillance of hospital-acquired infections, isolation nursing of patients who carry MRSA, monitoring of antimicrobial susceptibility pattern, and formulation of a definite antibiotic policy may be helpful.

English

The emergence of heterogeneous populations of methicillin-resistant&nbsp;Staphylococcus aureus&nbsp;(MRSA) causes major problems in routine screening for MRSA. Cefoxitin is a potent inducer of the&nbsp;mecA&nbsp;regulatory system and can be use for detection of heterogeneous populations of MRSA. Detection of the&nbsp;mecA&nbsp;gene by PCR was considered to be the &ldquo;gold standard&rdquo;. In this study we determined the sensitivity and specificity of the oxacillin disk diffusion test, cefoxitin disk diffusion test and oxacillin agar screening for detection of MRSA. A total of 124 non-duplicate isolates of&nbsp;S. aureus&nbsp;were included in the study. Methicillin resistance was measured using oxacillin (1&mu;g) and cefoxitin(30&mu;g) disc diffusion method and oxacillin agar screening test (6 mg/ml oxacillin) according to CLSI guideline and PCR for the&nbsp;mecA gene. Compared with the molecular detection of methicillin resistance the overall sensitivities and specificities of the phenotypic tests for cefoxitin disc diffusion were 100%, for oxacillin disc diffusion were 91.7 and 92.8% and for oxacillin agar screening were 95 and 95.5%, respectively. We concluded that in the absence of availability of molecular biology techniques, the cefoxitin disc was the best detector of methicillin resistance in&nbsp;S. aureus&nbsp;related to the other phenotypic tests. &nbsp; Key words:&nbsp;Methicillin-resistant&nbsp;Staphylococcus aureus, mecA, Cefoxitin, polymerized chain reaction

English

Methicillin-resistant&nbsp;Staphylococcus aureus&nbsp;(MRSA) has become a frequent cause of serious infections worldwide. We identified clinical isolates of&nbsp;S. aureus&nbsp;using conventional methods based on morphological and biochemical characteristics. Resistance of isolates to oxacillin was tested by growth on Oxacillin Resistance Screening Agar Base (ORSAB) and disc diffusion method. Oxacillin MICs were determined by agar dilution method. Sensitivity of isolates to a range of antibiotics was also tested by disc diffusion method. We further confirmed methicillin resistance using a PCR-based molecular approach. Data revealed that among 120 clinical bacterial samples tested 81 were confirmed as&nbsp;S. aureus. Out of these 81 isolates, 72 were MRSA (88.9%). The distribution of resistance among MRSA isolates was alarming.&nbsp;Twenty (20)&nbsp;MRSA isolates (27.8%) showed the highest level of resistance detected in this study with oxacillin MIC &gt;6400 &mu;g/ml. Most isolates were also resistant to multiple antibiotics. PCR results revealed the detection of&nbsp;mecA&nbsp;gene responsible for resistance in all tested isolates and therefore confirmed the conventional identification of MRSA isolates. The present study provides additional evidence that the rate of emergence of MRSA is in a continuous increase. &nbsp; Key words:&nbsp;Staphylococcus aureus, MRSA,&nbsp;mecA&nbsp;gene, methicillin resistance, multi-drug resistance.

Oxacillin Resistance and Antimicrobial Susceptibility Profile of Staphylococcussaprophyticus and Other Staphylococci Isolated from Patients with Urinary Tract Infection

Background:Staphylococcus saprophyticus is the second most frequent community-acquired causative agent of urinary tract infection (UTI). The objective of this study was to evaluate the susceptibility profile and resistance detection in Staphylococcus species. isolated from patients with UTI. Materials and Methods: The isolates were investigated using the disk diffusion method, Vitek I system, E-test®, and detection of the mecA gene. Results: Most isolates (76.2%) were resistant to oxacillin by the disk diffusion method, followed by those resistant to penicillin (72.2%). The oxacillin disk diffusion method, E-test, and Vitek I method showed higher sensitivity (94.4%) and lower specificity (28.9, 26.5, and 24.0%, respectively) than the cefoxitin disk diffusion test (sensitivity: 83.5%, specificity: 85.5%) for the detection of oxacillin resistance. Conclusions: The large number of oxacillin-resistant isolates indicates that the breakpoint value recommended by the Clinical and Laboratory Standards Institute may overestimate oxacillin resistance in S. saprophyticus. Thus, changes in these guidelines are necessary for the correct detection of this resistance.

Detecting methicillin resistance in Staphylococcus aureus: comparison of different phenotypic methods and the polymerase chain reaction

Cefoxitin is a more potent inducer of the mecA regulatory system compared to oxacillin in Staphylococcus aureus. It has been recommended for the detection of methicillin-resistant Staphylococcus aureus (MRSA) when using disk-diffusion testing. The aim of this study is to compare the results of cefoxitin and oxacillin disk-diffusion methods against the polymerase chain reaction (PCR) for the detection of MRSA. A total of 75 strains of S. aureus isolated from different clinical specimens (e.g., pus, blood, wound swabs, tracheal aspirates, eye swabs and cerebrospinal fluid [CSF]) were screened for methicillin resistance by PCR and cefoxitin and oxacillin disk-diffusion tests. The antibiotic susceptibility testing was performed by the Baur disk-diffusion method using oxacillin (1 |ig) and cefoxitin (30 |ig) disks on Mueller Hinton agar (MHA), and zone diameters as recommended by CLSIs were read at 18 h and 24 h. All MRSA isolates detected phenotypically were confirmed by PCR for the amplification of mecA and nucA genes. Of the 75 isolates screened, 27 were resistant to oxacillin, 30 were resistant to cefoxitin using the disk-diffusion method, while 30 isolates were confirmed as MRSA by PCR. Performing the cefoxitin disk-diffusion method using a 30 | g disk could be a reliable and more accurate method to detect methicillin resistance in S. aureus strains in situations where mecA PCR cannot be performed.