Cefoxitin Disk Diffusion Test Research Articles

Subunit 76-kDa surface protein of Methicillin-Resistant Staphylococcus aureus (MRSA) is potentially useful for MRSA diagnostic tool

ObjectiveTo evaluate the diagnostic performance of the polyclonal antibody generated from the subunit surface protein of MRSA for MRSA detection. MethodsThe MRSA clinical isolates were identified by the cefoxitin disc diffusion test and confirmed by mecA PCR. The surface protein from the clinical isolates of MRSA was extracted and characterized with hemagglutination and adherence inhibition assays. Polyclonal antibody against the selected protein was produced in mice and then used for Western blot experiments. ResultsFour conserved surface protein bands (63, 76, 88, and 114-kDa) were found in each MRSA clinical isolate. Hemagglutination reaction was demonstrated by the subunit 76 and 114-kDa surface protein of MRSA at 1:32 dilution. Such proteins were identified as adhesive molecules in the enterocytes. The sensitivity and specificity of the polyclonal 76-kDa antibody in detecting MRSA were 94.59% and 85.14%, respectively, with the Kappa values fall under the interpretation of substantial agreement (0.752) with the gold standard, suggesting it is useful for MRSA detection. ConclusionSubunit 76 and 114-kDa surface proteins of MRSA exhibit adhesive properties in mediating MRSA infection. The polyclonal antibody of 76-kDa generated from the surface protein of MRSA could be used as an alternative for the identification of clinical isolates suspected with MRSA infection.

Occurrence of Methicillin-Resistant Staphylococcus aureus (MRSA) in Bovine Bulk Milk and Farm Workers in Smallholder Dairy Farming Systems in Northwestern Ethiopia.

Staphylococcus aureus is a common pathogen responsible for causing various human and animal infections and is well known for its ability to develop resistance to multiple antibiotics. This study aimed to evaluate the occurrence of methicillin-resistant Staphylococcus aureus (MRSA) in bulk milk and dairy farms in northwestern Ethiopia and to determine their phenotypic and genotypic antimicrobial susceptibility patterns. We collected 50 bulk milk samples from 50 dairy farms and 50 hand swabs from dairy milkers. The cefoxitin disk diffusion test and PCR-based assays were used to identify MRSA isolates. In addition, cefoxitin-resistant isolates were tested for susceptibility to other antibiotics using the Kirby-Bauer disk diffusion method. The results showed that MRSA was detected in 8 samples: 6 from bulk milk samples (12%) and 2 from hand swabs (4%). All MRSA isolates exhibited a high resistance rate to penicillin (100%), followed by tetracycline (75%), ciprofloxacin (25%), chloramphenicol (25%), erythromycin (25%), gentamycin (12.5%), and trimethoprim-sulfamethoxazole (12.5%). Moreover, 72% of the isolates showed resistance to three or more antibiotic classes and were classified as multidrug-resistant. This study identified methicillin-resistant Staphylococcus aureus and multidrug-resistant MRSA in bulk milk and dairy farms in northwestern Ethiopia. These findings highlight the potential risk of transmission of these antibiotic-resistant bacteria to humans and the need for improved antibiotic stewardship in the dairy sector using the One Health approach.

Methicillin, β-lactams, and Clindamycin Resistance Profiles of Staphylococcus aureus Strains Isolated from Patients with UTI in Bechar Province (Algeria)

Introduction: Staphylococcus aureus is a bacterial pathogen responsible for many dis-eases. Multidrug resistance in S aureus has become common worldwide and is a serious clinical issue in the treatment and care of S. aureus-infected patients. S. aureus uropathogenic strains isolated from patients with UTI in Bechar province (Algeria) were phenotypically identified and evaluated for multiple drug resistance (MDR) patterns against recommended antimicrobial drugs. Methods: Through selective media, the uropathogenic S. aureus strains were isolated by analyzing a total of 211 urine samples, then subjected to various biochemical identification tests. Multidrug resistance pattern (MRSA, MLSB, and β-lactamase production) among Staphylococcus aureus uropathogenic strains was performed using phenotypic tests. : A total of 50 S. aureus strains were isolated, i.e., 40,48% of all the isolated uropathogenic agents estimated at 126 isolates. More than 69% of the women had a urinary tract infection, i.e., a sex ratio (SR) of 0,43. The susceptibility test results showed that the isolated S. aureus strains were resistant to various antibiotics of the β-lactams class (penicillin, cefoxitin, and oxacillin) and were susceptible to gentamicin, chloramphenicol, co-trimoxazole, vancomycin, ofloxacin, and ciprofloxacin. Cefoxitin disk diffusion and agar screening tests showed that all the isolated S. aureus strains were qualified as MRSA at the rate of 100% (50/50), whereas a rate of 74% (37/50) was granted to MRSA using oxacillin disk diffusion test. The highest percentage of MDR-MRSA was observed in class three of antibiotics 12 (24%). The lowest percentage was observed in classes 8 and 9 of antibiotics 1 (2%). For MDR-MRSA, the MAR index ranged from 0,27 to 1,0. Results:: Penicillin disk diffusion (DD) and DDST (PI/PIT) tests showed a production rate of β-lactamase in the range of 94% and 80%, respectively. 85,1% (40/50) of penicillin-resistant strains were positive for DDST. Thus, the D-test among MRSA strains shows a rate of 38% (19/50) of cMLSB and no strain attributed to the iMLSB phenotype. Conclusion: This is an alarming rate, confirming the concern that is still being discussed within the medical teams of the national health sector as well as the Algerian antimicrobial resistance network, which requires prompt intervention to update a new strategy to fight antimicrobial re-sistance.

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus among Patients Diagnosed with Surgical Site Infection at Four Hospitals in Ethiopia.

Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of severe surgical site infections (SSI). The molecular epidemiology of MRSA is poorly documented in Ethiopia. This study is designed to determine the prevalence of MRSA and associated factors among patients diagnosed with SSI. A multicenter study was conducted at four hospitals in Ethiopia. A wound culture was performed among 752 SSI patients. This study isolated S. aureus and identified MRSA using standard bacteriology, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), and cefoxitin disk diffusion test. The genes mecA, femA, vanA, and vanB were detected through PCR tests. S. aureus was identified in 21.6% of participants, with 24.5% of these being methicillin-resistant Staphylococci and 0.6% showing vancomycin resistance. Using MALDI-TOF MS for the 40 methicillin-resistant Staphylococci, we confirmed that 31 (77.5%) were S. aureus, 6 (15%) were Mammaliicoccus sciuri, and the other 3 (2.5%) were Staphylococcus warneri, Staphylococcus epidermidis, and Staphylococcus haemolyticus. The gene mecA was detected from 27.5% (11/40) of Staphylococci through PCR. Only 36.4% (4/11) were detected in S. aureus, and no vanA or vanB genes were identified. Out of 11 mecA-gene-positive Staphylococci, 8 (72.7%) were detected in Debre Tabor Comprehensive Specialized Hospital. Methicillin-resistant staphylococcal infections were associated with the following risk factors: age ≥ 61 years, prolonged duration of hospital stay, and history of previous antibiotic use, p-values < 0.05. Hospitals should strengthen infection prevention and control strategies and start antimicrobial stewardship programs.

On-farm epidemiology, virulence profiling, and molecular characterization of methicillin-resistant Staphylococcus aureus at goat farms

Antimicrobial resistance (AMR) becomes a challenging issue that limits the therapeutic options for both veterinary and public health professionals. The current study aimed to investigate the on-farm epidemiology, antibiotics resisting profiling, virulence analysis, and molecular detection of methicillin-resistant Staphylococcus aureus (MRSA) at the caprine-human interface. A total of 768 goat milk samples and 94 skin swabs from farm personnel were collected from 30 goat flocks and processed for isolation of S. aureus. The study isolates were confirmed as MRSA based on the oxacillin and cefoxitin disc diffusion test and the presence of mecA gene. MRSA isolates of goats and human origin were characterized and further evaluated for the presence of virulence genes responsible for intramammary infections and public health hazards. The results revealed 26.82 % and 27.79 % goat milk samples and human samples positive for S. aureus, respectively. A higher MRSA prevalence of 35.92 % and 10.71 % was found in goat and human isolates respectively. The phylogenetic analysis revealed a lesser extent of homology in mecA gene of S. aureus isolates at the caprine-human interface. Moreover, this study revealed strong evolutionary connection between the study isolates and MRSA isolates of Pakistani cattle and buffalo while the in-silico protein analysis showed that all sequences have the same protein motifs resembling penicillin binding protein 2a. The risk factors analysis revealed that teat length, drainage system, hygienic measures during milking, use of teat dip, teat injury, and veterinary services were significantly associated with subclinical mastitis in goats. A total of 43.24 % of local MRSA isolates showed multi-drug resistance (MDR). The isolates showed higher resistance to oxytetracycline followed by gentamicin and vancomycin while moxifloxacin, and linezolid were among the susceptible antibiotics. Local MRSA isolates carried virulence markers (nuc and coag genes) and biofilm-associated icaA (43.24 %) and icaD (29.73 %) genes which are responsible for the intramammary infection. The local isolates also carried the virulence genes of public health concern including the enterotoxin C (sec) gene (24.3 %), enterotoxins B (seb) gene (5.41 %), and enterotoxin D (sed) gene (2.7 %). Enterotoxins A (sea) and E (see) genes were not detected in any isolate. The study concluded that MRSA is an emerging and prevailing pathogen in dairy goats with a high potential to transmit to associated human beings. The presence of a variety of virulence factors as well as the associated antibiotic resistance makes MRSA a potential threat at animal-human interface and thus demands further research.

Detection of Biofilm Production and Antibiotic Resistance Pattern in Clinical Isolates from Orthopaedic Implants Associated Infections

Introduction: Orthopaedic Implant associated infection is the major problem which leads to implant failure and economic burden to the patient. Implant-associated infections are due to the formation of biofilm at the implantation site leading to multidrug resistant organism. Aim: This study was done to evaluate the causative organisms, their antibiotic sensitivity pattern and their ability to form biofilm over the Implants used in Orthopaedic surgeries. Material and methods: This study was carried out in the department of Microbiology, Adichunchanagiri institute of Medical sciences from September 2019 to August 2022. Swabs from 100 patients who had undergone orthopaedic implant or prosthetic surgery and presented with signs and symptoms of infection were collected and processed as per standard procedures. All the isolates were subjected to detect biofilm production by Congo red agar method, MRSA detection by Cefoxitin disc diffusion test, vancomycin resistant Enterococcus by using Vancomycin (Minimum inhibitory concentration) MIC strips and Extended spectrum β lactamases (ESBL) production by phenotypic confirmatory combined-disc test. Results: Out of the 100 samples processed, culture positivity was observed in 72 specimens. Among them, Staphylococcus aureus 19(26%) was the predominant isolate and 44 (61%) are biofilm producers. 17 (89%) were MRSA (Methicillin resistance Staphylococcus aureus) strains and 15(37%) were ESBL producing strains. Conclusion: The appropriate pre- and post- operative care should be taken to prevent orthopaedic implant associated infections. Staphylococcus spp was the commonest isolate and its ability to produce biofilm indicates the need for an appropriate antibiotic policy and screening for MRSA carriers to reduce the infection. Keywords: Orthopaedic implant, Infection, Biofilm, MRSA, ESBL

Comparison of cefoxitin disc diffusion test, HiCrome rapid MRSA agar and PCR for&lt;em&gt; mec&lt;/em&gt;A, PVL and &lt;em&gt;fem&lt;/em&gt;B genes for detection of methicillinresistant &lt;em&gt;Staphylococcus aureus&lt;/em&gt; (MRSA)

This study evaluates the effectiveness of cefoxitin disk diffusion as a method to detect methicillin-resistant Staphylococcus aureus (MRSA), comparing with HiCrome MRSA screening agar and PCR-based mecA gene detection. Both PCR and the cefoxitin disk diffusion test exhibited 100% sensitivity and specificity, while HiCrome agar showed slightly lower sensitivity (90%) and specificity (95.24%) out of 72 Staphylococcus aureus isolates. The MRSA detection rate was found to be 41.66%, with femB and PVL genes present in 86.6% and 13.3% of MRSA isolates, respectively. Cefoxitin could serve as a reliable substitute marker for MRSA detection, particularly in settings with limited resources.

Epidemiology of clinically isolated methicillin-resistant Staphylococcus aureus (MRSA) and its susceptibility to linezolid and vancomycin in Egypt: a systematic review with meta-analysis

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen that causes severe morbidity and mortality worldwide. For the establishment of national strategies to combat MRSA infection in each country, accurate and current statistics characterizing the epidemiology of MRSA are essential. The purpose of this study was to determine the prevalence of MRSA among Staphylococcus aureus clinical isolates in Egypt. In addition, we aimed to compare different diagnostic methods for MRSA and determine the pooled resistance rate of linezolid and vancomycin to MRSA. To address this knowledge gap, we conducted a systematic review with meta-analysis.MethodsA comprehensive literature search from inception to October 2022 of the following databases was performed: MEDLINE [PubMed], Scopus, Google Scholar, and Web of Science. The review was conducted following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) Statement. Based on the random effects model, results were reported as proportions with a 95% confidence interval (CI). Analyses of the subgroups were conducted. A sensitivity analysis was conducted to test the robustness of the results.ResultsA total of sixty-four (64) studies were included in the present meta-analysis, with a total sample size of 7171 subjects. The overall prevalence of MRSA was 63% [95% CI: 55–70]. Fifteen (15) studies used both PCR and cefoxitin disc diffusion for MRSA detection, with a pooled prevalence rate of 67% [95% CI: 54–79] and 67% [95% CI: 55–80], respectively. While nine (9) studies used both PCR and Oxacillin disc diffusion for MRSA detection, the pooled prevalences were 60% [95% CI: 45–75] and 64% [95% CI: 43–84], respectively. Furthermore, MRSA appeared to be less resistant to linezolid than vancomycin, with a pooled resistance rate of 5% [95% CI: 2–8] to linezolid and 9% [95% CI: 6–12] to vancomycin, respectively.ConclusionOur review highlights Egypt's high MRSA prevalence. The cefoxitin disc diffusion test results were found to be consistent with PCR identification of the mecA gene. A prohibition on antibiotic self-medication and efforts to educate healthcare workers and patients about the proper use of antimicrobials may be required to prevent further increases.

Isolation and molecular identification of methicillin resistant (MRSA) from different type of wound (Cuts, burn and chemical

Three hundred of clinical wound samples (Cuts, burn and chemical) were collected from Murtala Muhammad Specialist Hospital, Kano State. The isolation and identification of was done through culture on Nutrient agar and Mannitol salt agar, Gram staining, microscopic and standard biochemical tests such as (catalase, coagulase, oxidase and hemolysis test) were carried out. Cefoxitin disc diffusion test and molecular analysis was done for the detection of methicillin resistant strain of . A total of one hundred and one isolates were identified in the present study in which 13(12.90%) were MRSA positive and 88(87.10%) were negative MRSA. Polymerase chain reaction (PCR) analysis reveals the presence of mec A gene under 310base pairs nucleotide sequence in the positive MRSA.

Phenotypic and Genotypic Characterization of Methicillin Resistance in Staphylococci Isolated from an Egyptian University Hospital.

Methicillin-resistant in Staphylococci is a serious public health issue. It is mostly encoded by the mecA gene. The mecC gene is a new mecA analog responsible for resistance to methicillin in some Staphylococcal clinical isolates. This mecC gene is still underestimated in Egypt. The aim of the current study was to detect mecA and mecC genes in clinical Staphylococci isolates from a tertiary care university hospital in Egypt compared to the different phenotypic methods. A total of 118 Staphylococcus aureus (S. aureus) and 43 coagulase-negative Staphylococci (CoNS) were identified from various hospital-acquired infections. Methicillin resistance was identified genotypically using the PCR technique and phenotypically using the cefoxitin disc diffusion test, oxacillin broth microdilution and the VITEK2 system in all Staphylococcal isolates. The mecA gene was detected in 82.2% of S. aureus and 95.3% of CoNS isolates, while all of the isolates tested negative for the mecC gene. Interestingly, 30.2% of CoNS isolates showed the unique character of inducible oxacillin resistance, being mecA-positive but oxacillin-susceptible (OS-CoNS). The dual use of genotypic and phenotypic methods is highly recommended to avoid missing any genetically divergent strains.

Molecular Detection and Characterization of the Staphylococcus epidermidis and Staphylococcus haemolyticus Isolated from Hospitalized Patients and Healthcare Workers in Iran.

The present study is aimed at surveying the antibiotics resistance profile, biofilm formation ability, staphylococcal cassette chromosome mec (SCCmec) types, and molecular epidemiology of Staphylococcus epidermidis and Staphylococcus haemolyticus isolated from hospitalized patients and healthcare workers in four teaching hospitals in Iran. In total, 43 Staphylococcus epidermidis and 12 Staphylococcus haemolyticus were isolated from hospitalized patients, and 19 Staphylococcus epidermidis and 7 Staphylococcus haemolyticus isolated from healthcare workers were included in the present study. The antimicrobial resistance profile of isolates was determined using the disk diffusion method. Moreover, the resistance of isolates to methicillin was identified using the cefoxitin disk diffusion test. The microtiter-plate test was used for quantifying biofilm formation. Moreover, the frequency of icaA and icaD genes was determined using PCR assay. The molecular epidemiology of methicillin-resistant isolates was determined using SCCmec typing and pulsed-field gel electrophoresis methods. Among all coagulase-negative staphylococci isolates, the highest resistance rate (81.5%) was seen for cefoxitin and cotrimoxazole. All of the isolates were susceptible to linezolid. Out of the 66 mecA-positive isolates, the most common SCCmec type was the type I (n = 23; 34.8%) followed by type IV (n = 13; 19.7%). Using pulsed-field gel electrophoresis (PFGE) assay, 27 PFGE types including 14 common types and 13 singletons were obtained among 51 methicillin-resistant S. epidermidis (MRSE) isolates. Moreover, among 12 methicillin-resistant S. haemolyticus (MRSH) isolates, 8 PFGE types were detected, of which 5 PFGE types were singletons. The high rate of resistance to antibiotics as well as the possibility of cross-infection shows the importance of a pattern shift in the management and controlling programs of coagulase-negative staphylococci, especially in healthcare centers. Clinical trial registration. The present study is not a clinical trial study. Thus, a registration number is not required.

Distribution of chlorhexidine resistance genes among Staphylococcus aureus clinical isolates: the challenge of antiseptic resistance.

Chlorhexidine is an antiseptic agent which is extensively used to prevent nosocomial infections; however, this could result in reduction of its susceptibility. The aim of this work was to determine chlorhexidine susceptibility among Staphylococcus aureus isolates and to detect qacA/B and smr antiseptic resistance genes among these isolates. Furthermore, we aimed to identify possible risk factors for the reduction of chlorhexidine susceptibility among S. aureus isolates. Various clinical samples were collected from patients with evidence of S. aureus infection. Antimicrobial susceptibilities of identified S. aureus isolates were determined by disk diffusion method. Resistance to methicillin was identified by cefoxitin disk diffusion test besides mecA gene detection by PCR. Chlorhexidine minimum inhibitory concentration (MIC) values were measured by broth microdilution method while qacA/B and smr resistance genes were detected by multiplex PCR. A total percentage of 25.9% of S. aureus isolates showed reduced susceptibility to chlorhexidine. Methicillin resistant S. aureus (MRSA) had a reported percentage of 39.5%, which was significantly higher than the 11.3% reported for methicillin susceptible S. aureus (MSSA), p<0.001. S. aureus isolates were found to harbor qacA/B and smr genes at 23.2% and 7.7% respectively. Risk factors for reduced susceptibility to chlorhexidine included; ICU setting (OR=2.02, 95%CI: 0.3-1.6), prolonged ICU stay (OR=1.7, 95%CI: 0.4-1.1), presence of central vascular catheter (OR=2.3, 95%CI: 0.2-1.9), mechanical ventilation (OR=1.88, 95%CI: 0.4-1.7) and acquisition of qacA/B (OR=15.7, 95%CI: 3.4-12.1) or smr gene (OR=15.7, 95%CI: 3.4-12.1). Our work highlighted the current challenge of antiseptic resistance in our locality. The frequencies of qacA/B and smr genes were significantly higher among MRSA than MSSA isolates. About two thirds of chlorhexidine tolerant isolates displayed an MDR profile. To maintain chlorhexidine efficiency, biocidal stewardship program and ongoing surveillance are essential.

Phenotypic detection of methicillin resistance, biofilm production, and inducible clindamycin resistance in Staphylococcus aureus clinical isolates in Kathmandu, Nepal

IntroductionMethicillin resistance, inducible clindamycin resistance (ICR), biofilm production, and increased minimum inhibitory concentration (MIC) of vancomycin in Staphylococcus aureus are major causes of antibiotic treatment failure and increased morbidity and mortality. The surveillance of such isolates and the study of their antimicrobial pattern are essential in managing the infections caused by these isolates. This study aimed to determine methicillin resistance, biofilm production, and ICR in S. aureus isolates from a tertiary care hospital in Kathmandu, Nepal.Materials and methodsA total of 217 S. aureus isolated from different samples were processed following standard laboratory procedures. Antibiotic susceptibility testing was performed by the Kirby–Bauer disk diffusion technique. Methicillin-resistant S. aureus (MRSA) were identified by the cefoxitin disk diffusion test, and biofilm producers were examined using the microtiter plate technique. D-test and E-test were performed to determine inducible clindamycin resistance and minimum inhibitory concentration of vancomycin, respectively.ResultsAmong the 217 S. aureus isolates, 78.3% were multidrug-resistant (MDR), 47.0% were MRSA, 62.2% were biofilm producers, and 50.7% showed ICR. All MRSA isolates exhibited MIC levels of vancomycin within the susceptible range. Biofilm producers and MRSA isolates showed elevated antimicrobial resistance. MRSA was significantly associated with MDR. Biofilm-producing and multidrug-resistant MRSA isolates showed significantly higher MIC levels of vancomycin (p = 0.0013 and < 0.0001, respectively), while ICR was significantly higher in MDR (p = 0.0001) isolates.ConclusionHigh multidrug resistance, MRSA, and ICR in this study call for routine evaluation of antibiotic susceptibility patterns of S. aureus. Vancomycin can be used to treat serious staphylococcal infections. Clindamycin should be prescribed only after performing the D-test. Drugs like teicoplanin, chloramphenicol, doxycycline, amikacin, and levofloxacin can treat MRSA infections.

Comparison of Different Phenotypic Methods of Detection of Methicillin-Resistant Staphylococcus aureus with Polymerase Chain Reaction

Objectives: The objective of the current study was to compare the diagnostic methods of Oxacillin Disk Diffusion, Cefoxitin Disk Diffusion, Oxacillin Resistance Screening Agar Base, and CHROM Agar MRSA with the gold-standard method of Polymerase Chain Reaction for detection of Methicillin-resistant Staphylococcus aureus.&#x0D; Methods: Two hundred pus samples were included in the study from which Staphylococcus strains were evaluated. The isolates of Staphylococcus aureus were subjected to the Oxacillin Disk Diffusion test, Cefoxitin Disk Diffusion test, Oxacillin Resistance Screening Agar Base, and CHROM Agar MRSA to detect MRSA with PCR, the reference standard. The diagnostic techniques were compared to their sensitivity, specificity, positive predictive, and negative predictive values.&#x0D; Results: The sensitivity of the Cefoxitin Disk Diffusion test was 100%, followed by CHROM Agar MRSA at 96.7%, Oxacillin Disk Diffusion at 90%, and Oxacillin Resistance Screening Agar Base at 86.7%. Most specific was the Cefoxitin Disk Diffusion test (99.4%), followed by Oxacillin Resistance Screening Agar Base (98.8%), CHROM Agar MRSA (97.7%), and the least specific was the Oxacillin Disk Diffusion test (96.5%).&#x0D; Conclusion: The Cefoxitin Disk Diffusion test was the most sensitive and specific of all four methods, next to the Polymerase Chain Reaction. However, future multicentric studies are recommended to test this method across all prevalent centers of methicillin resistance. J Microbiol Infect Dis 2022; 12(3):116-126.

Detection of Methicillin Resistance and β-Lactamase Production in Staphylococcus aureus Isolates: A Cross Sectional Study from a Tertiary Care Hospital

Nosocomial infections are proving to be a menace for the whole health care system, with methicillin resistant Staphylococcus aureus (MRSA) being a very notorious causative agent. Along with the role of mecA gene producing penicillin-binding protein (PBP2a), production of beta-lactamase enzyme by Staphylococcus aureus makes the organism resistant to all β-lactam agents. This study aims at phenotypic detection of methicillin resistance and β-Lactamase production in all S. aureus isolates by Cefoxitin disk diffusion test and Penicillin zone-edge test, respectively. In this prospective cross-sectional study, samples were obtained from patients admitted to different departments and were processed according to the standard laboratory protocols. As per the CLSI guidelines, phenotypic detection of resistance to methicillin was done by Cefoxitin Disk Diffusion test, whereas production of beta-lactamase enzyme was phenotypically detected by penicillin zone edge test. Among 179 isolates of S. aureus, 116 (64.8%) were MRSA, whereas the remaining 63 (35.2%) isolates were methicillin-sensitive Staphylococcus aureus (MSSA). Staphylococcus aureus infection among ICU and non-ICU patients were found to be 24(13%) and 155(86.6%) respectively. Among 24 ICU patients, 19 had MRSA infection, whereas among 155 non-ICU patients, 97 had MRSA infection. Out of 63 MSSA isolates, only 2 (3.17%) were found to be sensitive to Penicillin by Zone-edge test, 04 isolates showed presence of β-lactamase production, while remaining 57 isolates were resistant to Penicillin. At the same time, several class of antibiotics were found to be ineffective against these MRSA isolates. Cases of methicillin-resistant and b–lactamase producing Staphylococcus aureus infections are on the rise in our hospital settings.

Diagnostic Accuracy of CHROMagar MRSA for Detection of Methicillin-Resistant Staphylococcus Aureus (MRSA) from Screening Swab Specimens

Objective: To determine the diagnostic accuracy of CHROMagar MRSA for detecting MRSA from screening swab specimens keeping the Cefoxitin disk diffusion test as the reference method.&#x0D; Study Design: A cross-sectional validation study.&#x0D; Place and duration of study: Department of Microbiology, Armed Forces Institute of Pathology (AFIP), Rawalpindi Pakistan, from Mar to Aug 2019.&#x0D; Methodology: A total of 243 screening swab specimens, e.g., axillary, nasal and web swabs, each of hospitalized patients and healthcare workers (HCW) submitted for MRSA screening were included in the study and were processed simultaneously on blood agar, MacConkey agar and CHROMagar MRSA. The agar plates were incubated at 35°C ± 2°C for 18-24 hours in ambient air. The cefoxitin disk diffusion test followed the isolation and identification of MRSA according to the latest Clinical and Laboratory Standards Institute (CLSI) guidelines. In CHROMagar MRSA screening, after incubation, plates were examined for the presence of mauve colonies (MRSA detected), and the results were obtained and validated against the reference method of Cefoxitin disk diffusion.&#x0D; Results: Overall, the diagnostic accuracy of CHROMagar MRSA for detecting MRSA was 97.53%. Diagnostic accuracy of CHROMagar MRSA was 95.1%, 97.5% and 100% in axillary, nasal and web specimens, respectively. The rate of MRSA detection was maximum in axillary swabs, i.e., 40.7%, followed by 29.6% and 9.8% in nasal and web swabs, respectively.&#x0D; Conclusion: CHROMagar MRSA is found to be accurate for the detection of MRSA. It is reliable, easy to perform, less timeconsuming, and cost-effective. It is an affordable alternative to the conventional..............

Phenotypic and genotypic characterization of oxacillin-susceptible and mecA positive Staphylococcus aureus strains isolated in Uruguay

The aim of this study was to characterize phenotypically and genotypically 27 mecA positive Staphylococcus aureus strains with oxacillin MICs of ≤2μg/ml by Vitek 2, isolated in different regions of Uruguay. Susceptibility to oxacillin and cefoxitin was studied by gradient diffusion, disk diffusion to cefoxitin, and Phoenix and MicroScan systems. PBP2a was determined. SCCmec typing was performed and the isolates were compared by PFGE. Twenty-six isolates were susceptible to oxacillin; one strain was susceptible to cefoxitin by disk diffusion and 3 strains by gradient diffusion. Phoenix and MicroScan panels detected methicillin resistance in 25 and 27 strains, respectively. Twenty-six strains tested positive for PBP2a. Twenty-six strains carried SCCmec V and 24 belonged to pulsotype A. One strain carried SCCmec IV and did not belong to pulsotype A. Cefoxitin disk diffusion test and PBP2a detection correctly identified 26 of these 27 strains as MRSA. PFGE results suggest the dissemination of a cluster of MRSA carrying SCCmec V.

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 Methods for Detecting Methicillin-Resistant Coagulase-Negative Staphylococci in Device-Associated Infections

Background: Coagulase-negative Staphylococci (CoNS) is one of the major causes of infections in hospitals. Methicillin resistance has greatly increased in different CoNS species in previous years. Here, we evaluated the performance of four phenotypic tests for detecting mecA-mediated methicillin resistance rate in CoNS isolated from different device-associated infections in Tanta University Hospitals, Egypt. Methodology: One hundred and fifteen CoNS isolates were examined for mecA-mediated methicillin resistance using polymerase chain reaction (PCR) as the standard gold test. Susceptibility to methicillin was investigated using VITEK 2 assay, oxacillin broth microdilution, and cefoxitin disc diffusion tests. Results: Of all isolates, 98.3% were mecA-positive. The sensitivities of the different methods examined were as follows: 100% for the VITEK cefoxitin test, 97.4% for the VITEK oxacillin test, 93.8% for the oxacillin broth microdilution, and 82.3% for the cefoxitin disc diffusion test. The test specificity could not be accurately determined because of the small number of mecA-negative isolates (n = 2). Conclusion: The rate of methicillin resistance reached 98.3% among CoNS isolates. Our results demonstrate that the VITEK 2 assay is rapid and has high sensitivity compared to other phenotypic methods for detecting methicillin resistance among different species of CoNS. Therefore, we recommend the dual measurement of both cefoxitin and oxacillin susceptibilities using the VITEK 2 assay for best results.

Biofilm-forming ability and methicillin resistance among Staphylococci isolated from various surfaces in community settings

Biofilm-forming ability and sensitivity to antibiotic methicillin of staphylococci outside the healthcare facilities is not sufficiently explored. The aim of this study was to examine the biofilm-forming capacity of staphylococci on various metal, plastic and leather surfaces in our living and working environment and the association between their biofilm production and methicillin resistance. Detection of methicillin resistance in staphylococci isolates were examined by cefoxitin disc diffusion test. Production of bacterial biofilms was investigated by two phenotypic methods: crystal violet (CV) tube-adherence method and Congo red agar (CRA) plate method. A total of 88 staphylococci isolates, 47 (53.41%) of S. aureus and 41 (46.59 %) of S. epidermidis strains were recovered from 100 collected samples. Among them, 35 methicillin-resistant strains (20 MRSA and 15 MRSE) were detected. Applying the CV tube adherence method, 21 (39.62%) MRSA and MRSE isolates were positive for biofilm production, while using the CRA method 15 (44.12%) MRSA and MRSE strains were detected as biofilm producers. Statistical analysis did not find association between biofilm production ability and methicillin resistance in both applied methods for a significance level of 0.05. The study shows that different objects and surfaces contaminated with biofilm-forming staphylococci, including MRSA and MRSE, may serve as environmental source of infection for long periods of time.