Performance of microcalorimetry for early detection of methicillin resistance in clinical isolates of Staphylococcus aureus.
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ABSTRACT:
SUBMITTER: Baldoni D
PROVIDER: S-EPMC2650961 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
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Project description:Staphylococcus intermedius is a zoonotic organism that can be associated with human disease. We report two separate cases of S. intermedius infection in which a false-positive rapid penicillin binding protein 2a latex test in conjunction with the phenotypic properties of beta-hemolysis and coagulase positivity allowed the clinical isolates to masquerade as methicillin-resistant Staphylococcus aureus. 16S rRNA gene sequencing and the absence of mecA revealed the strains to be methicillin-susceptible S. intermedius.
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Project description:β-Lactam antibiotics target penicillin-binding proteins and inhibit the synthesis of peptidoglycan, a crucial step in cell wall biosynthesis. Staphylococcus aureus acquires resistance against β-lactam antibiotics by producing a penicillin-binding protein 2a (PBP2a), encoded by the mecA gene. PBP2a participates in peptidoglycan biosynthesis and exhibits a poor affinity towards β-lactam antibiotics. The current study was performed to determine the diversity and the role of missense mutations of PBP2a in the antibiotic resistance mechanism. The methicillin-resistant Staphylococcus aureus (MRSA) isolates from clinical samples were identified using phenotypic and genotypic techniques. The highest frequency (60%, 18 out of 30) of MRSA was observed in wound specimens. Sequence variation analysis of the mecA gene showed four amino acid substitutions (i.e., E239K, E239R, G246E, and E447K). The E239R mutation was found to be novel. The protein-ligand docking results showed that the E239R mutation in the allosteric site of PBP2a induces conformational changes in the active site and, thus, hinders its interaction with cefoxitin. Therefore, the present report indicates that mutation in the allosteric site of PBP2a provides a more closed active site conformation than wide-type PBP2a and then causes the high-level resistance to cefoxitin.
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Project description:Ceftaroline is the first member of a novel class of cephalosporins approved for use in the United States. Although prior studies have identified eight ceftaroline-resistant methicillin-resistant Staphylococcus aureus (MRSA) isolates in Europe and Asia with MICs ranging from 4 to 8 mg/liter, high-level resistance to ceftaroline (>32 mg/liter) has not been described in MRSA strains isolated in the United States. We isolated a ceftaroline-resistant (MIC > 32 mg/liter) MRSA strain from the blood of a cystic fibrosis patient and five MRSA strains from the respiratory tract of this patient. Whole-genome sequencing identified two amino acid-altering mutations uniquely present in the ceftaroline-binding pocket of the transpeptidase region of penicillin-binding protein 2a (PBP2a) in ceftaroline-resistant isolates. Biochemical analyses and the study of isogenic mutant strains confirmed that these changes caused ceftaroline resistance. Thus, we identified the molecular mechanism of ceftaroline resistance in the first MRSA strain with high-level ceftaroline resistance isolated in the United States.
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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.
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Project description:To investigate the prevalence, location and genetic environments of fosfomycin-resistance (fos) genes in methicillin-resistant Staphylococcus aureus (MRSA) clinical strains, 67 fosfomycin-resistant MRSA strains were isolated from the blood and cerebrospinal fluid samples at a teaching hospital in Shanghai. The presence of fos genes in these clinical strains was detected by PCR and sequencing. The locations of fos genes were determined by Southern blotting and genetic environments were analyzed by primer walking sequencing. Multiple locus sequence typing (MLST) was used to characterize genetic diversity. Conjugation was performed to evaluate the transferability of fos genes. Among 67 fosfomycin-resistant MRSA strains, nine high level fosfomycin resistant strains (?128 ?g/ml) were fosB-positive. Three new subtypes of fosB, designated as fosB4, fosB5, and fosB6, were identified. fosB1, fosB4 or fosB6 genes were located on small plasmids (ca. 2.5 kb) and flanked by an analogous replication gene (rep). Differently, the fosB5 gene was surrounded by a shorter rep gene and two copies of a transposon gene (tnp) that shared high identity with the IS257-like transposon. Four MLST types were found among the nine fosB-positive strains. Transconjugants with the fosB genes were resistant to fosfomycin with MIC 64 or 128 ?g/ml. In conclusion, different subtypes and genetic environment of fosB genes indicate that gene heterogeneity for fosfomycin resistance in MRSA isolates.
| S-EPMC4856351 | biostudies-literature
Project description:We report the complete draft genome sequences of five individually isolated strains of methicillin-resistant Staphylococcus aureus (MRSA) and a Staphylococcus epidermidis strain. These clinically important isolates have staphylococcal cassette chromosome mec type A, while Panton-Valentine leukocidin (PVL) toxin coding genes were present in MRSA isolates only.
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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.
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Project description:A total of 71 fusidic acid-resistant Staphylococcus aureus (45 methicillin-resistant and 26 methicillin-susceptible) isolates were examined for the presence of resistance determinants. Among 45 fusidic acid-resistant methicillin-resistant S. aureus (MRSA), isolates, 38 (84%) had fusA mutations conferring high-level resistance to fusidic acid (the MIC was ≥128 μg/ml for 22/38), none had fusB, and 7 (16%) had fusC. For 26 fusidic acid-resistant methicillin-susceptible S. aureus (MSSA), only 3 possessed fusA mutations, but 15 (58%) had fusB and 8 (31%) had fusC. Low-level resistance to fusidic acid (MICs ≤ 32 μg/ml) was found in most fusB- or fusC-positive isolates. For 41 isolates (38 MRSA and 3 MSSA), with fusA mutations, a total of 21 amino acid substitutions in EF-G (fusA gene) were detected, of which R76C, E444K, E444V, C473S, P478S, and M651I were identified for the first time. The nucleotide sequencing of fusB and flanking regions in an MSSA isolate revealed the structure of partial IS257-aj1-LP-fusB-aj2-aj3-IS257-partial blaZ, which is identical to the corresponding region in pUB101, and the rest of fusB-carrying MSSA isolates also show similar structures. On the basis of spa and staphylococcal cassette chromosome mec element (SCCmec) typing, two major genotypes, spa type t037-SCCmec type III (t037-III; 28/45; 62%) and t002-II (13/45; 29%), were predominant among 45 MRSA isolates. By pulsed-field gel electrophoresis analysis, 45 MRSA isolates were divided into 12 clusters, while 26 MSSA isolates were divided into 15 clusters. Taken together, the distribution of fusidic acid resistance determinants (fusA mutations, fusB, and fusC) was quite different between MRSA and MSSA groups.
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Project description:Ceftobiprole is a fifth-generation cephalosporin with activity against methicillin-resistant Staphylococcus aureus (MRSA). One-year surveillance at the Regional Hospital of Ancona (Italy) disclosed a 12% ceftobiprole resistance rate (12/102 isolates; MIC, ?4?mg/liter). Epidemiological characterization demonstrated that the resistant isolates all belonged to different clones. Penicillin-binding protein (PBP) analysis showed substitutions in all PBPs and a novel insertion in PBP2a. The mecB and mecC genes were not detected. Ceftobiprole susceptibility screening is essential to avoid therapeutic failure and the spread of ceftobiprole-resistant strains.
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