scholarly journals Introduction of the mec Element (Methicillin Resistance) into Staphylococcus aureus Alters In Vitro Functional Activities of Fibrinogen and Fibronectin Adhesins

1998 ◽  
Vol 42 (3) ◽  
pp. 564-570 ◽  
Author(s):  
Pierre E. Vaudaux ◽  
Vincenza Monzillo ◽  
Patrice Francois ◽  
Daniel P. Lew ◽  
Tim J. Foster ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistance ◽  
Bacterial Colonization ◽  
Protein A ◽  
Methicillin Resistant ◽  
Functional Activities ◽  
Extracellular Matrix Components ◽  
Resistant Strains ◽  
The Impact

ABSTRACT Some methicillin-resistant strains of Staphylococcus aureus are defective in the production of major surface components such as protein A, clumping factor, or other important adhesins to extracellular matrix components which may play a role in bacterial colonization and infection. To evaluate the impact of methicillin resistance (mec) determinants on bacterial adhesion mediated by fibrinogen or fibronectin adhesins, we compared the in vitro attachment of two genetically distinct susceptible strains (NCTC8325 and Newman) to protein-coated surfaces with that of isogenic methicillin-resistant derivatives. All strains containing an intactmec element in their chromosomes were found to be defective in adhesion to fibrinogen and fibronectin immobilized on polymethylmethacrylate coverslips, regardless of the presence or absence of additional mutations in the femA,femB, or femC gene, known to decrease expression of methicillin resistance in S. aureus. Western ligand affinity blotting or immunoblotting of cell wall-associated adhesins revealed similar contents of fibrinogen- or fibronectin-binding proteins in methicillin-resistant strains compared to those of their methicillin-susceptible counterparts. In contrast to methicillin-resistant strains carrying a mec element in their genomes, methicillin-resistant strains constructed in vitro, by introducing the mecA gene on a plasmid, retained their adhesion phenotypes. In conclusion, the chromosomal insertion of themec element into genetically defined strains of S. aureus impairs the in vitro functional activities of fibrinogen or fibronectin adhesins without altering their production. This effect is unrelated to the activity of the mecA gene.

Staphylococcus aureus strains differ in their in vitro responsiveness to human urokinase: evidence that methicillin-resistant strains are predominately nonresponsive to the growth-enhancing effects of urokinase

1996 ◽  
Vol 42 (10) ◽  
pp. 1024-1031 ◽  
Author(s):  
David A. Hart ◽  
Carol Reno ◽  
Thomas Louie ◽  
Wallace Krulicki
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistance ◽  
Clinical Isolates ◽  
Growth Enhancement ◽  
Western Canada ◽  
Methicillin Resistant ◽  
Resistant Strains ◽  
The Uk ◽  
Regulation Of Growth

Clinical isolates of Staphylococcus aureus were found to exhibit strain-specific heterogeneity to the growth-enhancing effects of human urokinase (UK), a proteinase with plasminogen activator activity. Nine out of fourteen (64%) methicillin-sensitive strains of S. aureus were responsive to UK in "in vitro" cultures. In contrast, 3/29 (10%) methicillin-resistant strains were responsive to the proteinase. When only strains isolated from western Canada were considered, 6/11 methicillin-sensitive strains and 1/26 methicillin-resistant strains were responsive to UK. The single western Canadian methicillin-resistant strain (strain 456) responsive to UK was one of two isolated from the same patient, indicating that the two strains were phenotypically different. Strain 456, resistant to 32 μg mefhicillin/mL, was responsive to as little as 50 U UK/mL and enhancement of growth was evident by 9 h of incubation at 37 °C. This growth enhancement was specific to UK and not duplicated by equivalent concentrations of other proteins (bovine serum albumin, trypsin, plasminogen). The results presented indicate differences in the frequency of the UK-responsive phenotype between methicillin-sensitive and -resistant S. aureus. These findings indicate that the UK phenotype of S. aureus may have utility in both phenotyping clinical isolates, as well as providing insights into the regulation of growth in this clinically important organism.Key words: Staphylococcus aureus, growth, urokinase, methicillin resistance.

scholarly journals Detection of mecA gene by latex agglutination and its molecular analysis by PCR in methicillin resistant staphylococcus aureus.

2020 ◽  
Vol 27 (07) ◽  
pp. 1363-1370
Author(s):  
Aneela Khawaja ◽  
Iffat Javed ◽  
Sohaila Mushtaq ◽  
Saeed Anwar ◽  
Faiqa Arshad ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Methicillin Resistance ◽  
Meca Gene ◽  
Latex Agglutination ◽  
Medical Institute ◽  
Cross Sectional ◽  
Methicillin Resistant ◽  
Agglutination Method ◽  
Resistant Strains

Antimicrobial resistance (AMR) is a devastating question that is threatening the health globally. The extensive and indiscriminative use of antibiotics has evolved a notorious resistance in Staphylococcus aureus.  This resistance developed through possession of mecA gene, which codes for modified penicillin binding protein (PBP2a) and the emergent strain being labeled “methicillin resistant Staphylococcus aureus”. Conventional phenotypic techniques for detection of MRSA rely on standardization of cultural characteristics. The drawbacks of diagnostic error to report MRSA include: poor prognosis, expensive treatment, dissemination of multi-drug resistant strains and even treatment failure. Latex agglutination method can be adopted as a more accurate and quick strategy for rapid detection of methicillin resistance. Objectives: To compare detection of mecA gene in methicillin resistant isolates of Staphylococcus aureus by latex agglutination and PCR; by assessing the sensitivity and specificity of both methods. Study Design: Descriptive Cross-Sectional study. Setting: Pathology Department, Post Graduate Medical Institute, Lahore. Period: From January 2015 to December 2015; according to standard operating procedures at Microbiology laboratory. Material & Methods: A total 713 consecutive, non-duplicate isolates of Staphylococcus aureus were processed. Methicillin resistance was determined using cefoxitin (30mg) by Kirby-Bauer method using CLSI guideline (2016), latex agglutination method; and PCR for mecA gene. Results: The results showed that out of 713 Staphylococcus aureus isolates, 92 (12.90%) isolates were resistant to cefoxitin and were labelled as MRSA. majority MRSA isolates recovered from pus (44.57%) and wound swab (20.65%), followed by blood (13.04%), fluid (8.70%), CSF (4.35%), CVP (3.26%), HVS (3.26%) and tracheal secretion (2.17%). By latex agglutination method, 87 (94.50%) were positive for PBP2a; while on PCR mecA gene was detected only in 82 (89.10%) MRSA isolates. When assessed with PCR (gold standard) the sensitivity and diagnostic accuracy of latex agglutination was 100% and 94.57%, respectively. Conclusion: Latex agglutination test can be employed as rapid and reliable diagnostic technique in MRSA isolates for mecA gene detection, where resources for molecular methods are inadequate. This can effectually lessen the misdiagnosis of resistant strains, and over/ ill-use of antibiotics.

scholarly journals Prevalence of Methicillin Resistant and Virulence Determinants in Clinical Isolates of Staphylococcus aureus

2018 ◽  
Vol 10 (1) ◽  
pp. 108-115
Author(s):  
Manjunath Chavadi ◽  
Rahul Narasanna ◽  
Ashajyothi Chavan ◽  
Ajay Kumar Oli ◽  
Chandrakanth Kelmani. R
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Morphology ◽  
Methicillin Resistance ◽  
Protein A ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Virulence Determinants ◽  
Methicillin Resistant ◽  
Alternative Medicines ◽  
Mrsa Strains

Introduction:Methicillin-resistantStaphylococcus aureus(MRSA) is the major threat that is a result of the uncontrolled use of antibiotics causing a huge loss in health, so understanding their prevalence is necessary as a public health measure.Objective:The aim of this study was to determine the prevalence of methicillin-resistant MRSA and virulence determinant among associatedS. aureusfrom the clinical samples obtained from various hospital and health care centers of the Gulbarga region in India.Materials and Methods:All the collected samples were subjected for the screening ofS. aureusand were further characterized by conventional and molecular methods including their antibiotic profiling. Further, the response of methicillin antibiotic on cell morphology was studied using scanning electron microscopy.Results:A total 126S. aureuswas isolated from the clinical samples which showed, 100% resistant to penicillin, 55.5% to oxacillin, 75.3% to ampicillin, 70.6% to streptomycin, 66.6% to gentamicin, 8.7% to vancomycin and 6.3% to teicoplanin. The selected MRSA strains were found to possessmecA(gene coding for penicillin-binding protein 2A) andfemA(factor essential for methicillin resistance)genetic determinants in their genome with virulence determinants such as Coagulase (coa) and the X region of the protein A (spa)gene. Further, the methicillin response in resistantS. aureusshowed to be enlarged and malformed on cell morphology.Conclusion:The molecular typing of clinical isolates ofS. aureusin this study was highly virulent and also resistant to methicillin; this will assist health professionals to control, exploration of alternative medicines and new approaches to combat Staphylococcal infections more efficiently by using targeted therapy.

scholarly journals Proteomic and Transcriptomic Profiling of Staphylococcus aureus Surface LPXTG-proteins: Correlation with agr Genotypes and Adherence Phenotypes

2012 ◽  
Vol 11 (11) ◽  
pp. 1123-1139 ◽  
Author(s):  
Mathilde Ythier ◽  
Grégory Resch ◽  
Patrice Waridel ◽  
Alexandre Panchaud ◽  
Aurélie Gfeller ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Regulatory Networks ◽  
Time Course ◽  
Methicillin Resistance ◽  
Binding Protein ◽  
Protein A ◽  
Surface Proteins ◽  
Protein Domain ◽  
Accessory Gene

Staphylococcus aureus infections involve numerous adhesins and toxins, which expression depends on complex regulatory networks. Adhesins include a family of surface proteins covalently attached to the peptidoglycan via a conserved LPXTG motif. Here we determined the protein and mRNA expression of LPXTG-proteins of S. aureus Newman in time-course experiments, and their relation to fibrinogen adherence in vitro. Experiments were performed with mutants in the global accessory-gene regulator (agr), surface protein A (Spa), and fibrinogen-binding protein A (ClfA), as well as during growth in iron-rich or iron-poor media. Surface proteins were recovered by trypsin-shaving of live bacteria. Released peptides were analyzed by liquid chromatography coupled to tandem mass-spectrometry. To unambiguously identify peptides unique to LPXTG-proteins, the analytical conditions were refined using a reference library of S. aureus LPXTG-proteins heterogeneously expressed in surrogate Lactococcus lactis. Transcriptomes were determined by microarrays. Sixteen of the 18 LPXTG-proteins present in S. aureus Newman were detected by proteomics. Nine LPXTG-proteins showed a bell-shape agr-like expression that was abrogated in agr-negative mutants including Spa, fibronectin-binding protein A (FnBPA), ClfA, iron-binding IsdA, and IsdB, immunomodulator SasH, functionally uncharacterized SasD, biofilm-related SasG and methicillin resistance-related FmtB. However, only Spa and SasH modified their proteomic and mRNA profiles in parallel in the parent and its agr- mutant, whereas all other LPXTG-proteins modified their proteomic profiles independently of their mRNA. Moreover, ClfA became highly transcribed and active in fibrinogen-adherence tests during late growth (24 h), whereas it remained poorly detected by proteomics. On the other hand, iron-regulated IsdA-B-C increased their protein expression by >10-times in iron-poor conditions. Thus, proteomic, transcriptomic, and adherence-phenotype demonstrated differential profiles in S. aureus. Moreover, trypsin peptide signatures suggested differential protein domain exposures in various environments, which might be relevant for anti-adhesin vaccines. A comprehensive understanding of the S. aureus physiology should integrate all three approaches.

scholarly journals MgrA Governs Adherence, Host Cell Interaction, and Virulence in a Murine Model of Bacteremia Due to Staphylococcus aureus

2019 ◽  
Vol 220 (6) ◽  
pp. 1019-1028 ◽  
Author(s):  
Liang Li ◽  
Genzhu Wang ◽  
Ambrose Cheung ◽  
Wessam Abdelhady ◽  
Kati Seidl ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Protein A ◽  
Virulence Gene ◽  
Toxin Production ◽  
Cell Interaction ◽  
Host Pathogen Interactions ◽  
Host Pathogen ◽  
Direct Binding ◽  
The Impact

AbstractBackgroundMgrA is an important global virulence gene regulator in Staphylococcus aureus. In the present study, the role of mgrA in host-pathogen interactions related to virulence was explored in both methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains.MethodsIn vitro susceptibilities to human defense peptides (HDPs), adherence to fibronectin (Fn) and endothelial cells (ECs), EC damage, α-toxin production, expression of global regulator (eg, agr RNAIII) and its downstream effectors (eg, α-toxin [hla] and Fn binding protein A [fnbA]), MgrA binding to fnbA promoter, and the effect on HDP-induced mprF and dltA expression were analyzed. The impact of mgrA on virulence was evaluated using a mouse bacteremia model.ResultsmgrA mutants displayed significantly higher susceptibility to HDPs, which might be related to the decreased HDP-induced mprF and dltA expression but decreased Fn and EC adherence, EC damage, α-toxin production, agr RNAIII, hla and fnbA expression, and attenuated virulence in the bacteremia model as compared to their respective parental and mgrA-complemented strains. Importantly, direct binding of MgrA to the fnbA promoter was observed.ConclusionsThese results suggest that mgrA mediates host-pathogen interactions and virulence and may provide a novel therapeutic target for invasive S. aureus infections.

scholarly journals Missense Mutations in PBP2A Affecting Ceftaroline Susceptibility Detected in Epidemic Hospital-Acquired Methicillin-Resistant Staphylococcus aureus Clonotypes ST228 and ST247 in Western Switzerland Archived since 1998

2015 ◽  
Vol 59 (4) ◽  
pp. 1922-1930 ◽  
Author(s):  
William L. Kelley ◽  
Ambre Jousselin ◽  
Christine Barras ◽  
Emmanuelle Lelong ◽  
Adriana Renzoni
Keyword(s):  
Staphylococcus Aureus ◽  
University Hospital ◽  
Surveillance Program ◽  
Missense Mutations ◽  
Unknown Parameters ◽  
Methicillin Resistant ◽  
Content Type ◽  
Resistant Strains ◽  
Mrsa Strains

ABSTRACTThe development and maintenance of an arsenal of antibiotics is a major health care challenge. Ceftaroline is a new cephalosporin with activity against methicillin-resistantStaphylococcus aureus(MRSA); however, no reports concerning MRSA ceftaroline susceptibility have been reported in Switzerland. We tested thein vitroactivity of ceftaroline against an archived set of 60 MRSA strains from the University Hospital of Geneva collected from 1994 to 2003. Our results surprisingly revealed ceftaroline-resistant strains (MIC, >1 μg/ml in 40/60 strains; EUCAST breakpoints, susceptible [S], ≤1 μg/ml; resistant [R], >1 μg/ml) were present from 1998 to 2003. The detected resistant strains predominantly belonged to sequence type 228 (ST228) (South German clonotype) but also to ST247 (Iberian clonotype). A sequence analysis of these strains revealed missense mutations in the penicillin-binding protein 2A (PBP2A) allosteric domain (N146K or E239K and N146K-E150K-G246E). The majority of our ST228 PBP2A mutations (N146K or E150K) were distinct from ST228 PBP2A allosteric domain mutations (primarily E239K) recently described for MRSA strains collected in Thailand and Spain during the 2010 Assessing Worldwide Antimicrobial Resistance Evaluation (AWARE) global surveillance program. We also found that similar allosteric domain PBP2A mutations (N146K) correlated with ceftaroline resistance in an independent external ST228 MRSA set obtained from the nearby University Hospital of Lausanne, Lausanne, Switzerland, collected from 2003 to 2008. Thus, ceftaroline resistance was observed in our archived strains (including two examples of an MIC of 4 µg/ml for the Iberian ST247 clonotype with the triple mutation N146K/E150K/G246E), at least as far back as 1998, considerably predating the commercial introduction of ceftaroline. Our results reinforce the notion that unknown parameters can potentially exert selective pressure on PBP2A that can subsequently modulate ceftaroline resistance.

scholarly journals Changes in Antibiotic Susceptibility Pattern of Atypical Staphylococcus Aureus Strains Isolated from Cows of the same Herd in 2008-2010

2012 ◽  
Vol 56 (2) ◽  
pp. 139-143
Author(s):  
Magdalena Małkińska-Horodyska ◽  
Joanna Kubiak ◽  
Henryka Lassa ◽  
Edward Malinowski
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistance ◽  
Penicillin G ◽  
Diffusion Method ◽  
Conventional Pcr ◽  
Antibiotic Susceptibility Pattern ◽  
Microdilution Method ◽  
Resistant Strains ◽  
One Year

Abstract The isolates of Staphylococcus aureus strains were examined phenotypically by cultural features, tube coagulase test and clumping factor (CF), and genotypically by conventional PCR. The strains had positive reaction in CF test, but were negative in tube coagulase test. The analysed strains from the same cows in each year expressed also nuc and coa genes. About 25% of the strains were examined by the disc diffusion method for their sensitivity to antibiotics. During three years, the strains were highly susceptible in vitro to amoxicillin with clavulanic acid, oxacillin, bacitracin, and cefoperazone (more than 90%), and highly resistant to tetracycline, neomycin, and streptomycin. Forty randomly chosen strains, and eight strains from the same cows in each year were analysed for minimal inhibitory concentration of penicillin G using microdilution method. An increasing resistance to the penicillin was noted. Moreover, eight strains, the same in each year, were also examined for β-lactamase production and methicillin resistance. No β-lactamase producers and no methicillin resistant strains were found using phenotypic and genotypic methods. In conclusion, it can be stated that antimicrobial susceptibility can change from one year to another.

scholarly journals Prevalence of fosfomycin resistance and gene mutations in clinical isolates of methicillin-resistant Staphylococcus aureus

2020 ◽  
Author(s):  
Yi-Chien Lee ◽  
Pao-Yu Chen ◽  
Jann-Tay Wang ◽  
Shan-Chwen Chang
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Agents ◽  
Genetic Relatedness ◽  
Protein A ◽  
Gene Mutations ◽  
Resistance Rate ◽  
Staphylococcal Protein A ◽  
Methicillin Resistant ◽  
Fosfomycin Resistance

Abstract 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 genetic mechanisms of fosfomycin resistance.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, 12 harbored the fosB gene, and expression of murA, uhpT, and glpT mutations was noted in 11, 59, and 66 isolates, respectively. Combination of mutations of uhpT and glpT genes (58 isolates) was the most prevalent resistant mechanism. 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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