scholarly journals Production of extracellular mucopolysaccharide and biofilm under different oxygen conditions by clinical isolates of Staphylococcus aureus non-susceptible to glycopeptides

2019 ◽  
pp. 487-498
Author(s):  
Ksenia Szymanek-Majchrzak ◽  
Sylwia Wodzyńska ◽  
Andrzej Młynarczyk ◽  
Grażyna Młynarczyk
Keyword(s):  
Staphylococcus Aureus ◽  
Regulatory System ◽  
Aerobic Condition ◽  
Etiologic Agent ◽  
Clinical Isolates ◽  
Control Group ◽  
Test Parameters ◽  
Oxygen Conditions ◽  
Mrsa Strains ◽  
Global Regulatory System

INTRODUCTION. Staphylococcus aureus, which is able to produce an extracellular mucopolysaccharide (MP) and biofilm (SP), is an important etiologic agent in persistent and implant-related infections. This phenotype may be expressed in different levels and character depending on various environmental and/or global intracellular regulatory mechanisms. It may also be induced by sub-inhibitory concentrations of some antibiotics, for example vancomycin. The main aim of the study was to assess the ability to produce MP and SP in different oxygen conditions by clinical isolates of S.aureus nonsusceptible to glycopeptides. MATERIALS AND METHODS. Clinical isolates of health-care associated methicillin resistant S. aureus (HA-MRSA) strains, non-susceptible to glycopeptides (GRSA, 47) and heterogeneous vancomycin intermediate S. aureus isolates (h-VISA, 8). Control group consisted of the following strains: 55 belonging to MRSA, vancomycin susceptible, VSSA and 19 as methicillin susceptible, MSSA/VSSA. The ability to produce MP was investigated according to Freeman method. SP production was tested by means of Christensen procedure. RESULTS. In aerobic conditions MRSA/GRSA and MRSA/h-VISA isolates were the strongest mucopolysaccharide (SMP) producers (12.2% and 28.6% SMP/MP), but MSSA/VSSA were the most frequent MP (100%). In anaerobic atmosphere, all isolates from all groups were MP-positive. MRSA/h-VISA were the strongest MP producers (75% SMP/MP), but MSSA/VSSA were the most susceptible to oxidative stress (the percentage of SMP among MP for MSSA/VSSA increased by 15.8 times). Each evaluated group of clinical S. aureus isolates in aerobic condition had representation in SP positive phenotype: MRSA/GRSA and MRSA/h-VISA, 63.9% and 62.5%; MRSA/VSSA and MSSA/VSSA, respectively 80% and 94.7%. For all mentioned groups of bacteria, SSP variants were present and the amount of values was higher than in similar results obtained in CRA method. The strongest slime producers (60%) were h-VISA strains. The results obtained in Christensen method for anaerobic conditions, were not conclusive due to insufficient optimization of the test parameters. SUMMARY AND CONCLUSIONS. Both methods reveal that MRSA isolates non-susceptible to glycopeptides are the strongest producers of both MP and SP. That is probably due to cell wall alterations and global regulatory system Agr disorders. The Christensen procedure allow to assess both ica- dependent and ica- independent (adhesive) mechanisms of slime production and allow to notice that, as a phenotyping “biofilm booster effect”. ica- dependent mechanism, which dominated in MSSA/VSSA strains, demonstrate phenotype with more susceptibility to oxygen stress conditions than adhesive one.

scholarly journals Variable Expressions of Staphylococcus aureus Bicomponent Leucotoxins Semiquantified by Competitive Reverse Transcription-PCR

2000 ◽  
Vol 66 (9) ◽  
pp. 3931-3938 ◽  
Author(s):  
St�phane Bronner ◽  
Patricia Stoessel ◽  
Alain Gravet ◽  
Henri Monteil ◽  
Gilles Pr�vost
Keyword(s):  
Staphylococcus Aureus ◽  
Reverse Transcription ◽  
Regulatory System ◽  
Optimization Procedure ◽  
Reverse Transcription Pcr ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Specific Mrnas ◽  
Casamino Acids ◽  
Global Regulatory System

ABSTRACT A competitive reverse transcription-PCR method was developed for the semiquantitation of the expression of genes encoding bicomponent leucotoxins of Staphylococcus aureus, e.g., Panton-Valentine leucocidin (lukPV), gamma-hemolysin (hlgA and hlgCB), and LukE-LukD (lukED). The optimization procedure included RNA preparation; reverse transcription; the use of various amounts of enzymes, antisense primer, and RNA; and the final amplification chain reaction. Reproducible results were obtained, with sensitivity for detection of cDNA within the range of 1 mRNA/104 CFU to 102 mRNA/CFU, depending on the gene. Both specific mRNAs were more significantly expressed at the late-exponential phase of growth. Expression was about 100-fold higher in yeast extract-Casamino Acids-pyruvate medium than in heart infusion medium. Expression of the widely distributed gamma-hemolysin locus in the NTCC 8178 strain was around 10-fold diminished compared with that in the ATCC 49775 strain. Because of the lower level of hlgA expression, the corresponding protein, which is generally not abundant in culture supernatant, should be investigated for its contribution to the leucotoxin-associated virulence. The agr, sar, and agr sar mutant strains revealed a great dependence with regard to leucotoxin expression on the global regulatory system inS. aureus, except that expression of hlgA was not affected in the agr mutant.

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 Efficacy of Human-Simulated Exposures of Tomopenem (Formerly CS-023) in a Murine Model of Pseudomonas aeruginosa and Methicillin-Resistant Staphylococcus aureus Infection

2011 ◽  
Vol 55 (11) ◽  
pp. 5004-5009 ◽  
Author(s):  
Kiyoshi Sugihara ◽  
Kazuhiro Tateda ◽  
Naotoshi Yamamura ◽  
Tetsufumi Koga ◽  
Chika Sugihara ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Clinical Isolates ◽  
Infection Model ◽  
Methicillin Resistant ◽  
Content Type ◽  
Target Values ◽  
Bactericidal Effects ◽  
Dosing Regimens ◽  
Mrsa Strains

ABSTRACTTomopenem (formerly CS-023) is a novel carbapenem with improved activity against diverse hospital pathogens, includingPseudomonas aeruginosaand methicillin-resistantStaphylococcus aureus(MRSA), and has a half-life about twice longer than the half-lives of other carbapenems such as imipenem and meropenem. Our objective in this study was to estimate the efficacy of tomopenem in humans by human-simulated exposures in a neutropenic murine thigh infection model against 9 clinical isolates ofP. aeruginosawith MICs of 4 to 32 μg/ml and 9 clinical isolates of MRSA with MICs of 4 to 16 μg/ml. Human-simulated dosing regimens in neutropenic mice were designed to approximate the cumulative percentage of a 24-h period that the free drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (f%TMIC) observed with tomopenem at 750 and 1,500 mg given as a 0.5-h infusion three times a day (TID) in humans. As reported previously, there was no difference between the target values ofP. aeruginosaand MRSA required for efficacy (K. Sugihara et al., Antimicrob. Agents Chemother.54:5298-5302, 2010). Tomopenem at 750 mg showed bactericidal or bacteriostatic effects against 10 of 11 strains ofP. aeruginosaand MRSA with MICs of ≤8 μg/ml (f%TMIC≥ 41), and tomopenem at 1,500 mg showed bactericidal effects against 16 of 17 strains ofP. aeruginosaand MRSA with MICs of ≤16 μg/ml (f%TMIC≥ 43). Meropenem at 1,000 mg TID was tested for comparison purposes and showed bactericidal or bacteriostatic effects against 3 of 4 strains ofP. aeruginosawith MICs of ≤4 μg/ml (f%TMIC≥ 33). From these results, tomopenem is expected to be effective with anf%TMICof over 40 againstP. aeruginosaand MRSA strains with MICs of ≤8 μg/ml at doses of 750 mg TID and strains with MICs of ≤16 μg/ml at doses of 1,500 mg TID.

scholarly journals Ability of biofilm production and molecular analysis of spa and ica genes among clinical isolates of methicillin-resistant Staphylococcus aureus

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Mitra Omidi ◽  
Farzaneh Firoozeh ◽  
Mahmood Saffari ◽  
Hossein Sedaghat ◽  
Mohammad Zibaei ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cross Sectional Study ◽  
Clinical Isolates ◽  
Cross Sectional ◽  
Disk Diffusion Test ◽  
Methicillin Resistant ◽  
Isfahan Province ◽  
Biofilm Production ◽  
Mrsa Strains

Abstract Objective This study aimed to evaluate the phenotypic and genotypic characterization of biofilm formation and spa and ica genes among clinical isolates of methicillin-resistant Staphylococcus aureus. Result This cross-sectional study was performed on 146 Staphylococcus aureus isolates from hospitalized patients in Isfahan Province Hospitals. MRSA isolates were confirmed using disk diffusion test with oxacillin disk and amplification of mecA gene by PCR assays. Ability of biofilm production was evaluated targeting the icaA and icaD genes. Of 146 Staphylococcus aureus isolates, 24 (16.4%) carried mecA genes and identified as MRSA strains. Strong ability of biofilm production was seen among 76.02% (111/146) S. aureus isolates and 87.5% (21/24) MRSA strains, respectively. Also, 75.0% (18/24) MRSA isolates carried icaA and icaD was not detected in these strains. Analysis of spa gene showed 70.83% (17/24) MRSA strains were spa positive. From which 14 and 3 strains identified with one band (150, 270, 300, 360, 400 bp) and two bands (150–300 bp), respectively. According to data obtained, the prevalence of MRSA isolates from Isfahan Province Hospitals is relatively high and a remarkable percentage of them show strong power in biofilm production. Also analysis of spa gene showed a fairly large diversity among MRSA strains.

scholarly journals ThemecAHomologmecCConfers Resistance against β-Lactams in Staphylococcus aureus Irrespective of the Genetic Strain Background

2014 ◽  
Vol 58 (7) ◽  
pp. 3791-3798 ◽  
Author(s):  
Britta Ballhausen ◽  
André Kriegeskorte ◽  
Nina Schleimer ◽  
Georg Peters ◽  
Karsten Becker
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistance ◽  
Regulatory System ◽  
Wild Type ◽  
Beta Lactam ◽  
Beta Lactams ◽  
Content Type ◽  
Genetic Strain ◽  
Mrsa Strains ◽  
Strain Background

ABSTRACTIn staphylococci, methicillin resistance is mediated bymecA-encoded penicillin-binding protein 2a (PBP2a), which has a low affinity for beta-lactams. Recently, a novel PBP2a homolog was described as being encoded bymecC, which shares only 70% similarity tomecA. To prove thatmecCis the genetic determinant that confers methicillin resistance inStaphylococcus aureus, amecCknockout strain was generated. TheS. aureusΔmecCstrain showed considerably reduced oxacillin and cefoxitin MICs (0.25 and 4 μg/ml, respectively) compared to those of the corresponding wild-type methicillin-resistantS. aureus(MRSA) strain (8 and 16 μg/ml, respectively). Complementing the mutant intranswith wild-typemecCrestored the resistance to oxacillin and cefoxitin. By expressingmecCandmecAin differentS. aureusclonal lineages, we found thatmecCmediates resistance irrespective of the genetic strain background, yielding oxacillin and cefoxitin MIC values comparable to those withmecA. In addition, we showed thatmecCexpression is inducible by oxacillin, which supports the assumption that a functional beta-lactam-dependent regulatory system is active in MRSA strains possessing staphylococcal cassette chromosomemec(SCCmec) type XI. In summary, we showed thatmecCis inducible by oxacillin and mediates beta-lactam resistance in SCCmectype XI-carrying strains as well as in differentS. aureusgenetic backgrounds. Furthermore, our results could explain the comparatively low MICs for clinicalmecC-harboringS. aureusisolates.

scholarly journals Role of the Accessory Gene Regulatoragrin Community-Associated Methicillin-Resistant Staphylococcus aureus Pathogenesis

2011 ◽  
Vol 79 (5) ◽  
pp. 1927-1935 ◽  
Author(s):  
Gordon Y. C. Cheung ◽  
Rong Wang ◽  
Burhan A. Khan ◽  
Daniel E. Sturdevant ◽  
Michael Otto
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistance ◽  
Regulatory System ◽  
Accessory Gene ◽  
Methicillin Resistant ◽  
Content Type ◽  
Fibrinogen Binding ◽  
Gene Regulatory System ◽  
Differential Gene ◽  
Mrsa Strains

ABSTRACTThe molecular basis underlying the pathogenic success of community-associated methicillin-resistantStaphylococcus aureus(CA-MRSA) is not completely understood, but differential gene expression has been suggested to account at least in part for the high virulence of CA-MRSA strains. Here, we show that theagrgene regulatory system has a crucial role in the development of skin infections in the most prevalent CA-MRSA strain USA300. Importantly, our data indicate that this is due to discrepancies between theagrregulon of CA-MRSA and those of hospital-associated MRSA and laboratory strains. In particular,agrregulation in strain USA300 led to exceptionally strong expression of toxins and exoenzymes, upregulation of fibrinogen-binding proteins, increased capacity to bind fibrinogen, and increased expression of methicillin resistance genes. Our findings demonstrate thatagrfunctionality is critical for CA-MRSA disease and indicate that an adaptation of theagrregulon contributed to the evolution of highly pathogenic CA-MRSA.

scholarly journals Fate of Mutation Rate Depends onagrLocus Expression during Oxacillin-Mediated Heterogeneous-Homogeneous Selection in Methicillin-Resistant Staphylococcus aureus Clinical Strains

2011 ◽  
Vol 55 (7) ◽  
pp. 3176-3186 ◽  
Author(s):  
Konrad B. Plata ◽  
Roberto R. Rosato ◽  
Adriana E. Rosato
Keyword(s):  
Staphylococcus Aureus ◽  
Mutation Rate ◽  
Selection Process ◽  
Critical Role ◽  
Ectopic Expression ◽  
Regulatory System ◽  
Methicillin Resistant ◽  
Content Type ◽  
Heterogeneous Expression ◽  
Mrsa Strains

ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) strains are characterized by a heterogeneous expression of resistance. We have previously shown in clinical oxacillin-susceptible,mecA-positive MRSA strains that selection from a very heterogeneous (HeR) to highly homogeneous (HoR) resistant phenotype was mediated by acquisition of mutations through an oxacillin-induced SOS response. In the present study, we used a spotted DNA microarray to evaluate differential gene expression during HeR-HoR selection and found increased expression of theagrtwo-component regulatory system. We hypothesized that increased expression ofagrrepresents a mechanistically relevant component of this process. We demonstrated that inactivation ofagrduring the HeR-HoR selection process results in a significant increase in mutation rate; these effects were reversed by complementing theagrmutant. Furthermore, we found that extemporal ectopic expression ofagrand, more specifically, RNAII inagr-null mutant HeR cells suppressed mutation frequency and the capacity of these cells to undergo the HeR-HoR selection. These findings sustain the concept that increased expression ofagrduring HeR-HoR selection plays a critical role in regulating the β-lactam-induced increased mutation rate in very heterogeneous MRSA strains. Moreover, they indicate that a temporally controlled increase inagrexpression is required to tightly modulate SOS-mediated mutation rates, which then allows for full expression of oxacillin homogeneous resistance in very heterogeneous clinical MRSA strains.

scholarly journals Mutated Response Regulator graR Is Responsible for Phenotypic Conversion of Staphylococcus aureus from Heterogeneous Vancomycin-Intermediate Resistance to Vancomycin-Intermediate Resistance

2007 ◽  
Vol 52 (1) ◽  
pp. 45-53 ◽  
Author(s):  
Hui-min Neoh ◽  
Longzhu Cui ◽  
Harumi Yuzawa ◽  
Fumihiko Takeuchi ◽  
Miki Matsuo ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Response Regulator ◽  
Regulatory System ◽  
Vancomycin Resistance ◽  
Whole Genome ◽  
Two Component ◽  
Intermediate Resistance ◽  
Mrsa Strains

ABSTRACT Multistep genetic alteration is required for methicillin-resistant Staphylococcus aureus (MRSA) to achieve the level of vancomycin resistance of vancomycin-intermediate S. aureus (VISA). In the progression of vancomycin resistance, strains with heterogeneous vancomycin resistance, designated hetero-VISA, are observed. In studying the whole-genome sequencing of the representative hetero-VISA strain Mu3 and comparing it with that of closely related MRSA strains Mu50 (VISA) and N315 (vancomycin-susceptible S. aureus [VSSA]), we identified a mutation in the response regulator of the graSR two-component regulatory system. Introduction of mutated graR, designated graR*, but not intact graR, designated graRn, could convert the hetero-VISA phenotype of Mu3 into a VISA phenotype which was comparable to that of Mu50. The same procedure did not appreciably increase the vancomycin resistance of VSSA strain N315, indicating that graR* expression was effective only in the physiological milieu of hetero-VISA cell to achieve a VISA phenotype. Interestingly, the overexpression of graR* increased the daptomycin MICs in both Mu3 and N315 and decreased the oxacillin MIC in N315.

scholarly journals VanA-Type Vancomycin-Resistant Staphylococcus aureus

2009 ◽  
Vol 53 (11) ◽  
pp. 4580-4587 ◽  
Author(s):  
Bruno Périchon ◽  
Patrice Courvalin
Keyword(s):  
Staphylococcus Aureus ◽  
Suicide Gene ◽  
The United States ◽  
Clinical Isolates ◽  
Delivery Vector ◽  
Drug Classes ◽  
Resistance Pathway ◽  
Mrsa Strains ◽  
Competition Growth

ABSTRACT Since 2002, nine methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) strains that are also resistant to vancomycin (VRSA) have been reported in the United States, including seven clinical isolates from Michigan. The strains harbor a plasmid-borne Tn1546 element following conjugation from a glycopeptide-resistant Enterococcus strain. In the second step, Tn1546 transposed to a resident plasmid in five strains; the acquired plasmid behaved as a suicide gene delivery vector, and the incoming DNA had been rescued by illegitimate recombination. Surprisingly, combination of a glycopeptide with a β-lactam has a strong synergistic effect against VRSA, both in vitro and in an animal model, despite resistance of the strains to both drug classes when administered separately. This results from the fact that the late peptidoglycan precursors ending in d-alanine-d-lactate (d-Ala-d-Lac) that are mainly synthesized in the presence of glycopeptide inducers are not substrates for PBP2′, which is the only transpeptidase that remains active in the presence of oxacillin. One VRSA strain is partially dependent on vancomycin for growth due to a mutation in the host d-Ala:d-Ala ligase, thus having to rely on the inducible resistance pathway for cell wall synthesis. Competition growth experiments in the absence of inducer between the MRSA recipient and isogenic VRSA transconjugant revealed a disadvantage for the transconjugant, accounting, in part, for the low level of dissemination of the VRSA clinical isolates. The association of multiple molecular and environmental factors has been implicated in the regional emergence of VRSA in Michigan.

scholarly journals Analysis of Diversity of Mutations in the mecI Gene and mecA Promoter/Operator Region of Methicillin-Resistant Staphylococcus aureus andStaphylococcus epidermidis

1998 ◽  
Vol 42 (3) ◽  
pp. 717-720 ◽  
Author(s):  
Nobumichi Kobayashi ◽  
Koki Taniguchi ◽  
Shozo Urasawa
Keyword(s):  
Staphylococcus Aureus ◽  
Staphylococcus Epidermidis ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Genomic Diversity ◽  
Clinical Isolates ◽  
Base Substitution ◽  
Gene Sequences ◽  
Methicillin Resistant ◽  
Single Base ◽  
Mrsa Strains

ABSTRACT Genomic diversity of mutation in the mecI gene ormecA promoter/operator region was analyzed for clinical isolates of methicillin-resistant Staphylococcus aureus(MRSA) and Staphylococcus epidermidis (MRSE). In most MRSA strains, a single base substitution was detected in either themecI (three different positions) or the mecApromoter (two different positions), while a 28-base deletion inmecI was found in one strain. In contrast, no mutation was detected in these gene sequences of MRSE strains.

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