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Rapid determination of methicillin resistance among Staphylococcus aureus clinical isolates by colorimetric methods.

ABSTRACT: In the present study, the effectiveness of a rapid and colorimetric nitrate reductase analysis (NRA) method and resazurin microplate assay (REMA) for rapid determination of methicillin resistance among Staphylococcus aureus was investigated. A total of 275 clinical isolates of S. aureus were included in the present study. Among these isolates, 151 had the mecA gene and were resistant to methicillin. The remaining 124 isolates were methicillin susceptible and did not have the mecA gene. Cefoxitin MICs of all isolates were detected by NRA, REMA, and reference broth microdilution methods. Category and essential agreement were determined as 100% and 99.6%, respectively, comparing both NRA and REMA with the reference method. No minor, major, or very major discrepancy was observed for either of the methods. The time necessary to have the MIC results was 5 h for NRA, whereas it was 6 h for REMA. Early detection of MRSA is an important public health concern, and the results of this study showed that both of the colorimetric methods are easy to perform and save time in the determination of MRSA. These methods have a potential use for early detection of MRSA for laboratories unable to use molecular techniques.

SUBMITTER: Coban AY

PROVIDER: S-EPMC3405631 | biostudies-other | 2012 Jul

REPOSITORIES: biostudies-other

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Rapid determination of methicillin resistance among Staphylococcus aureus clinical isolates by colorimetric methods.

Coban Ahmet Yilmaz AY

Journal of clinical microbiology 20120418 7

In the present study, the effectiveness of a rapid and colorimetric nitrate reductase analysis (NRA) method and resazurin microplate assay (REMA) for rapid determination of methicillin resistance among Staphylococcus aureus was investigated. A total of 275 clinical isolates of S. aureus were included in the present study. Among these isolates, 151 had the mecA gene and were resistant to methicillin. The remaining 124 isolates were methicillin susceptible and did not have the mecA gene. Cefoxitin ...[more]

PMID: 22518854

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Project description:BACKGROUND:Fosfomycin exhibits excellent in vitro activity against multidrug-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Increasing fosfomycin resistance among clinical MRSA isolates was reported previously, but little is known about the relative abundance of Fosfomycin resistance genes in MRSA isolates circulating in Taiwan. METHODS:All MRSA isolates, collected in 2002 and 2012 by the Taiwan Surveillance of Antimicrobial Resistance (TSAR) program, were used in this study. Susceptibility to various antimicrobial agents, including fosfomycin, was determined by broth microdilution. Genetic determinants of fosfomycin resistance, including fosB carriage and murA, glpT and uhpT mutations, were investigated using PCR and sequencing of amplicons. Staphylococcal protein A (spa) typing was also performed to determine the genetic relatedness of MRSA isolates. RESULTS:A total of 969 MRSA strains, 495 in the year 2002 and 474 in the year 2012, were analyzed. The overall in vitro susceptibility was 8.2% to erythromycin, 18.0% to clindamycin, 29.0% to tetracycline, 44.6% to ciprofloxacin, 57.5% to trimethoprim/sulfamethoxazole, 86.9% to rifampicin, 92.9% to fosfomycin and 100% to linezolid and vancomycin. A significant increase in the fosfomycin resistance rate was observed from 3.4% in 2002 to 11.0% in 2012. Of 68 fosfomycin-resistant MRSA isolates, several genetic backgrounds probably contributing to fosfomycin resistance were identified. Twelve isolates harbored the fosB gene, and various mutations in murA, uhpT, and glpT genes were noted in 11, 59, and 66 isolates, respectively. The most prevalent gene mutations were found in the combination of uhpT and glpT genes (58 isolates). The vast majority of the fosfomycin-resistant MRSA isolates belonged to spa type t002. CONCLUSIONS:An increased fosfomycin resistance rate of MRSA isolates was observed in our present study, mostly due to mutations in the glpT and uhpT genes. Clonal spread probably contributed to the increased fosfomycin resistance.

Project description:Rapid determination of antimicrobial susceptibility/resistance is an important factor in selecting an appropriate antimicrobial treatment and eradicating infections promptly. Conventional antimicrobial susceptibility tests (ASTs) are very time consuming. Thus, we developed a liquid chromatography-mass spectrometry (LC-MS/MS) method for rapidly determining the resistance of Staphylococcus aureus to penicillin-G in an animal-infection model. This technique will be able to detect those resistant strains whose resistance mechanism specifically controlled by penicillinase. The resistance status of S. aureus against penicillin-G was determined by conventional AST. Cultured S. aureus cells were inoculated to chicken for developing bacteraemia. The solution of penicillin-G was intravenously administered (10 mg/kg b.w.) to chickens just after infection detection. Blood samples were collected at different intervals after drug administration. The concentration of active penicillin-G and its metabolites were determined from the bacteria-free blood supernatant by utilizing the LC-MS/MS method. Evidence of infection in chicken was observed within 5 h of bacterial inoculation. The penicillinase enzyme generated by S. aureus transforms the active penicillin-G to an inactive metabolite by hydrolysis, which is evident by the mass shift from 335.10600 to 353.11579 Da as quantified using liquid chromatography quadrupole time-of-flight mass spectrometry (LC/Q-TOF/MS). The signal intensity of inactive/hydrolysed penicillin-G is several-fold greater than that of the active penicillin-G in the blood sample of chicken infected with resistant strain and treated with penicillin-G. The antimicrobial resistance index (ARI) value of resistant S. aureus strain was more than 1, demonstrating the penicillin-G-resistance pattern of that strain. This method is able to determine the extent of ?-lactam antimicrobial resistance within 1.5 h from the patient's blood and is complementary with those existing AST methods which are usually practicing in the evaluation of ?-lactam antibiotic resistance.

Project description:The significant increase in resistance of methicillin-resistant Staphylococcus aureus (MRSA) to fusidic acid (FA) is a worrying public concern. However, the data on the prevalence of FA-resistant MRSA isolates in China is still limited. This study aims to investigate the prevalence of FA resistance and resistance determinants among MRSA isolates from six tertiary hospitals in different regions of China between 2016 and 2020. The antimicrobial susceptibility of MRSA isolates was performed by disk diffusion test and broth microdilution method. Whole-genome sequencing was conducted to evaluate the determinants of FA resistance and molecular characterization of FA-resistant MRSA isolates. In this study, a total of 74 (74/457, 16.2%) isolates were identified to be FA-resistant among 457 non-duplicate MRSA isolates. The prevalence of 74 FA-resistant isolates was as follows: Hubei (28/70, 40%), Shanghai (18/84, 21.4%), Jiangxi (7/58, 12.1%), Inner Mongolia Autonomous Region (6/38, 15.8%), Guangdong (12/112, 10.7%), and Sichuan (3/95, 3.2%). The mutations in fusA were present in 79.7% (59/74) of FA-resistant MRSA isolates, with 54 (54/74, 73%) having L461K mutation and conferring high-level resistance [Minimum Inhibitory Concentration (MIC)>128 μg/ml]. Acquired gene, fusB, with low-level resistance (MIC <16 μg/ml) was found in 20.3% (15/74) FA-resistant MRSA isolates. ST5-MRSA-II-t2460 was the most prevalence clone with high-level resistance, accounting for 51.4% (38/74), which was distributed in Hubei (24/28, 85.7%), Inner Mongolia Autonomous Region (4/6, 66.7%), Shanghai (7/18, 38.9%), and Guangdong (3/12, 25%). ST630-t4549 MRSA isolates with low-level resistance were the most common in Jiangxi (3/7, 42.9%) and Sichuan (2/3, 66.7%). In brief, the prevalence of FA resistance among MRSA isolates in China was relatively high with geographic differences. High-level FA resistance was associated mostly with fusA mutations, especially the L461K mutation, whereas fusB usually conferred the low-level resistance to FA. The spread of ST5-MRSA-II-t2460 clone with high-level resistance to FA contributed greatly to the increase of FA-resistant MRSA isolates in most regions, especially in Hubei.

Project description:BackgroundStaphylococcus aureus is responsible for large numbers of hospital-related and community-acquired infections. In this study, we investigated the presence of S. aureus and methicillin-resistant S. aureus (MRSA) in 100 samples from animals (55 cattle, 36 dogs, and 9 cats) and 150 samples from hospitalized human patients. The samples were collected from healthy and diseased animals and from diseased humans and included milk, wound swab, pus, exudates, nasal swab and diabetic ulcer. Initially, S. aureus was isolated and identified by colony morphology, Gram staining, and biochemical tests (catalase and coagulase tests). The S. aureus-positive samples were examined by polymerase chain reaction (PCR) to determine their MRSA status.ResultsOf the 100 animal samples, 29 were positive for S. aureus. Four samples (13.8%) from dogs were MRSA-positive, but samples from cattle and cats were MRSA-negative. Of the 150 human samples we collected, 64 were S. aureus-positive and, of these, 34 (53.1%) were MRSA-positive. Most (28%) of the MRSA samples were isolated from surgical wound swabs, followed by the pus from skin infections (11%), exudates from diabetic ulcers (6%), exudates from burns (4%), and aural swabs (3%). By contrast, a low MRSA detection rate (n?=?4) was seen in the non-human isolates, where all MRSA bacteria were isolated from nasal swabs from dogs. The antimicrobials susceptibility testing results showed that S. aureus isolates with mecA genes showed resistance to penicillin (100%), oxacillin (100%), erythromycin (73.5%), ciprofloxacin (70.6%), and gentamicin (67.7%). The lowest resistance was found against ceftazidime, and no vancomycin-resistant isolates were obtained.ConclusionsWe detected S. aureus and MRSA in both human and canine specimens. Isolates were found to be resistant to some of the antimicrobials available locally. MRSA carriage in humans and animals appears to be a great threat to effective antimicrobials treatment. The prudent use of antimicrobials will reduce the antimicrobial resistance. Our findings will help to find the most appropriate treatment and to reduce antimicrobial resistance in the future by implementing prudent use of antimicrobials. Further studies are required to better understand the epidemiology of MRSA human-animal inter-species transmission in Bangladesh.

Dataset Information

Rapid determination of methicillin resistance among Staphylococcus aureus clinical isolates by colorimetric methods.

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Rapid determination of methicillin resistance among Staphylococcus aureus clinical isolates by colorimetric methods.

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