Citral modulates virulence factors in methicillin-resistant Staphylococcus aureus

AbstractMethicillin-resistant Staphylococcus aureus (MRSA) is responsible for high morbidity and mortality rates. Citral has been studied in the pharmaceutical industry and has shown antimicrobial activity. This study aimed to analyze the antimicrobial activity of citral in inhibiting biofilm formation and modulating virulence genes, with the ultimate goal of finding a strategy for treating infections caused by MRSA strains. Citral showed antimicrobial activity against MRSA isolates with minimum inhibitory concentration (MIC) values between 5 mg/mL (0.5%) and 40 mg/mL (4%), and minimum bactericidal concentration (MBC) values between 10 mg/mL (1%) and 40 mg/mL (4%). The sub-inhibitory dose was 2.5 mg/mL (0.25%). Citral, in an antibiogram, modulated synergistically, antagonistically, or indifferent to the different antibiotics tested. Prior to evaluating the antibiofilm effects of citral, we classified the bacteria according to their biofilm production capacity. Citral showed greater efficacy in the initial stage, and there was a significant reduction in biofilm formation compared to the mature biofilm. qPCR was used to assess the modulation of virulence factor genes, and icaA underexpression was observed in isolates 20 and 48. For icaD, seg, and sei, an increase was observed in the expression of ATCC 33,591. No significant differences were found for eta and etb. Citral could be used as a supplement to conventional antibiotics for MRSA infections.

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Virulence of methicillin-resistant Staphylococcus aureus modulated by the YycFG two-component pathway in a rat model of osteomyelitis

Journal of Orthopaedic Surgery and Research ◽

10.1186/s13018-019-1508-z ◽

2019 ◽

Vol 14 (1) ◽

Author(s):

Shizhou Wu ◽

Yunjie Liu ◽

Lei Lei ◽

Hui Zhang

Keyword(s):

Staphylococcus Aureus ◽

Western Blot ◽

Rat Model ◽

Biofilm Formation ◽

Bacterial Biofilm ◽

Invasive Ability ◽

Methicillin Resistant ◽

Two Component ◽

Mrsa Strains

Abstract Objectives Methicillin-resistant Staphylococcus aureus (MRSA) strains present an urgent medical problem in osteomyelitis cases. Our previous study indicated that the YycFG two-component regulatory pathway is associated with the bacterial biofilm organization of MRSA strains. The aim of this study was to investigate the regulatory roles of ASyycG in the bacterial biofilm formation and the pathogenicity of MRSA strains using an antisense RNA strategy. Methods An ASyycG-overexpressing MRSA clinical isolate was constructed. The bacterial growth was monitored, and the biofilm biomass on bone specimens was examined using scanning electron microscopy and confocal laser scanning microscopy. Furthermore, quantitative RT-PCR (QRT-PCR) analysis was used to measure the expression of yycF/G/H and icaA/D in the MRSA and ASyycG strains. The expression of the YycG protein was quantified by Western blot assays. We validated the role of ASyycG in the invasive ability and pathogenicity of the strains in vivo using histology and peptide nucleic acid fluorescent in situ hybridization. Results The results showed that overexpression of ASyycG lead to a reduction in biofilm formation and exopolysaccharide (EPS) synthesis compared to the control MRSA strains. The ASyycG strains exhibited decreased expression of the yycF/G/H and icaA/D genes. Furthermore, Western blot data showed that the production of the YycG protein was inhibited in the ASyycG strains. In addition, we demonstrated that ASyycG suppressed the invasive ability and pathogenicity of the strain in vivo using an SPF (specific pathogen free) rat model. Conclusion In summary, the overexpression of ASyycG leads to a reduction in biofilm formation and bacterial pathogenicity in vivo, which provides a potential target for the management of MRSA-induced osteomyelitis.

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Identification of ica-dependent biofilm production by Staphylococcus aureus clinical isolates and antibiofilm effects of ascorbic acid against biofilm production

Journal of Clinical Pathology ◽

10.1136/jclinpath-2019-206280 ◽

2020 ◽

Vol 73 (5) ◽

pp. 261-266

Author(s):

Sahra Kırmusaoğlu ◽

Havva Kaşıkçı

Keyword(s):

Staphylococcus Aureus ◽

Ascorbic Acid ◽

Biofilm Formation ◽

Surface Proteins ◽

Clinical Isolates ◽

High Morbidity ◽

Dependent Manner ◽

Independent Manner ◽

Biofilm Production ◽

Life Threatening

AimsStaphylococcus aureus (S. aureus) is a life-threatening pathogen with high morbidity and mortality rates which causes nosocomial and community-acquired infections. Biofilm, considered to be a common virulence factor for pathogens, plays a significant role in recurrent and untreatable infections. Biofilm formation of S. aureus is mediated by synthesis of either poly-N-acetylglucosamine in an ica-dependent manner or surface proteins in an ica-independent manner. In some cases treatment is impossible and recurrent. In this study, ica-dependent biofilm-producing S. aureus isolates were detected and the anti-biofilm effect of ascorbic acid against biofilm formation of isolates was investigated.MethodsA total of 21 methicillin-sensitive S. aureus (MSSA) clinical isolates stored in our bacterial stock were used to detect ica-dependent biofilm-producing MSSA isolates. The anti-biofilm study was undertaken with three ica-dependent biofilm-producing isolates (MSSA2–4) and ATCC 29213 (MSSA1). Biofilms and the anti-biofilm effect of ascorbic acid were detected using the microtitre plate (MtP) method. 16S-rRNA, nuc, icaA and icaD genes and expression levels of icaA and icaD of isolates were detected by RT-PCR.ResultsThe minimum inhibitory concentrations (MICs) of ascorbic acid prevented biofilm formation of MSSA1 and MSSA3. Also, 1/2 MIC of ascorbic acid prevented biofilm formation of MSSA3. It was observed that biofilm formation decreased with increased concentration. There was no significant increase in ica gene expression of MSSA1 and MSSA2. Expression of icaA and icaD of MSSA3 decreased 13% and 38%, respectively. Expression of icaA in MSSA4 decreased 12%.ConclusionThe results of our study show that ascorbic acid can be used as an anti-biofilm agent to prevent biofilm formation of S. aureus and thus biofilm-related infections.

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

BMC Research Notes ◽

10.1186/s13104-020-4885-9 ◽

2020 ◽

Vol 13 (1) ◽

Cited By ~ 3

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.

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Biofilm Formation and its Association with Antibiotic Susceptibility Pattern in Methicillin-resistant Staphylococcus aureus isolates

Journal of Pure and Applied Microbiology ◽

10.22207/jpam.15.4.26 ◽

2021 ◽

Author(s):

Bajarangi Lal Chaudhary ◽

Dakshina Bisht ◽

Sameer Singh Faujdar

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Methicillin Resistant Staphylococcus Aureus ◽

Antibiotic Sensitivity ◽

Diffusion Method ◽

Beta Lactamase ◽

Isolation And Identification ◽

Methicillin Resistant ◽

Clinical Specimens ◽

Biofilm Production

Methicillin-resistant Staphylococcus aureus is a clinically significant pathogen that causes infections ranging from skin and soft tissue infections to life-threatening sepsis. Biofilm formation by MRSA is one of the crucial virulence factor. Determination of beta-lactamase and biofilm production among Staphylococcus aureus was obtained from various clinical specimens. Standard bacteriological procedures were used for isolation and identification and antibiotic sensitivity was determined using the Kirby Bauer disc diffusion method according to CLSI guidelines. The cloverleaf method, acidometric, iodometric and chromogenic methods were used to detect beta-lactamase while the microtiter plate method and Congo red agar method were used to detect biofilm production. Of the 288 MRSA strains isolated from various clinical specimens,198 (67.07%) were biofilm producers. Cloverleaf and chromogenic (nitrocefin) disc shows 100% results for beta-lactamase detection. Vancomycin was 100% sensitive followed by teicoplanin (92.36%) and linezolid (89.93%). Cloverleaf and nitrocefin disc methods were the most sensitive for detection of beta-lactamase in S. aureus and there was no significant relation between biofilm production and antibiotic sensitivity pattern of S. aureus.

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Antibacterial Activity of Saponin Extracted from Phyllanthus niruri on Methicillin-Resistant Staphylococcus aureus (MRSA)

Journal of Complementary and Alternative Medical Research ◽

10.9734/jocamr/2019/v7i130092 ◽

2019 ◽

pp. 1-9

Author(s):

V. A. Ajibade ◽

V. O. Oluwasusi ◽

M. F. Ibiyemi ◽

O. A. Ajenifuja ◽

O. Famurewa

Keyword(s):

Staphylococcus Aureus ◽

Nuclear Magnetic Resonance ◽

Antimicrobial Activity ◽

Methicillin Resistant Staphylococcus Aureus ◽

Inhibitory Effect ◽

Diffusion Method ◽

Phyllanthus Niruri ◽

Methicillin Resistant ◽

Agar Diffusion Method ◽

Conventional Antibiotics

The antimicrobial activity of saponin extracted from Phyllanthus niruri was investigated on methicillin-resistant Staphylococcus aureus (MRSA). The nuclear magnetic resonance (NMR) was used to determine the structure spectra of the extracted purified saponin. The 13carbon NMR predicted on the basis of chemical shift that appeared in the resonances of 20 – 60 ppm gave a structure named Phylagenin-13-O-α-D-glucopyranoside and Phylagenin-25-O-β-D-glucopyra-noside. The susceptibility profile of MRSA determined by the agar-diffusion method showed that 97.0% and 90.0% of the test bacterium were resistant to Tetracycline and Cotrimoxazole respectively and 60% of the bacterium was susceptible to saponin extract. The ability of saponin extracted from P. niruri to treat clinical manifestation like chest congestion and skin desquamation from which S. aureus resistant to conventional antibiotics have been isolated has been confirmed in this study. The fact that this extract exerted an inhibitory effect on MRSA indicates that they can potentially be further developed into antimicrobial clinically used agents.

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5-Dodecanolide interferes with biofilm formation and reduces the virulence of Methicillin-resistant Staphylococcus aureus (MRSA) through up regulation of agr system

Scientific Reports ◽

10.1038/s41598-019-50207-y ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 16

Author(s):

Alaguvel Valliammai ◽

Sivasamy Sethupathy ◽

Arumugam Priya ◽

Anthonymuthu Selvaraj ◽

James Prabhanand Bhaskar ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Bacterial Infections ◽

Pcr Analysis ◽

High Morbidity ◽

Healthy Human ◽

Methicillin Resistant ◽

Gene Expression Studies ◽

Human Infections

Abstract Methicillin resistant Staphylococcus aureus (MRSA) is a predominant human pathogen with high morbidity that is listed in the WHO high priority pathogen list. Being a primary cause of persistent human infections, biofilm forming ability of S. aureus plays a pivotal role in the development of antibiotic resistance. Hence, targeting biofilm is an alternative strategy to fight bacterial infections. The present study for the first time demonstrates the non-antibacterial biofilm inhibitory efficacy of 5-Dodecanolide (DD) against ATCC strain and clinical isolates of S. aureus. In addition, DD is able to inhibit adherence of MRSA on human plasma coated Titanium surface. Further, treatment with DD significantly reduced the eDNA synthesis, autoaggregation, staphyloxanthin biosynthesis and ring biofilm formation. Reduction in staphyloxanthin in turn increased the susceptibility of MRSA to healthy human blood and H2O2 exposure. Quantitative PCR analysis revealed the induced expression of agrA and agrC upon DD treatment. This resulted down regulation of genes involved in biofilm formation such as fnbA and fnbB and up regulation of RNAIII, hld, psmα and genes involved in biofilm matrix degradation such as aur and nuc. Inefficacy of DD on the biofilm formation of agr mutant further validated the agr mediated antibiofilm potential of DD. Notably, DD was efficient in reducing the in vivo colonization of MRSA in Caenorhabditis elegans. Results of gene expression studies and physiological assays unveiled the agr mediated antibiofilm efficacy of DD.

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Reduced Vancomycin Susceptibility in anIn VitroCatheter-Related Biofilm Model Correlates with Poor Therapeutic Outcomes in Experimental Endocarditis Due to Methicillin-Resistant Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02073-12 ◽

2013 ◽

Vol 57 (3) ◽

pp. 1447-1454 ◽

Cited By ~ 42

Author(s):

Wessam Abdelhady ◽

Arnold S. Bayer ◽

Kati Seidl ◽

Cynthia C. Nast ◽

Megan R. Kiedrowski ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Wall Thickness ◽

Biofilm Formation ◽

Methicillin Resistant ◽

Content Type ◽

Biofilm Model ◽

Mrsa Strains

ABSTRACTStaphylococcus aureusis the most common cause of endovascular infections, including catheter sepsis and infective endocarditis (IE). Vancomycin (VAN) is the primary choice for treatment of methicillin-resistantS. aureus(MRSA) infections. However, high rates of VAN treatment failure in MRSA infections caused by VAN-susceptible strains have been increasingly reported. Biofilm-associated MRSA infections are especially prone to clinical antibiotic failure. The present studies examined potential relationships between MRSA susceptibility to VAN in biofilmsin vitroand nonsusceptibility to VAN in endovascular infectionin vivo. Using 10 “VAN-susceptible” MRSA bloodstream isolates previously investigated for VAN responsiveness in experimental IE, we studied the mechanism(s) of suchin vivoVAN resistance, including: (i) VAN binding to MRSA organisms; (ii) the impact of VAN on biofilm formation and biofilm composition; (iii) VAN efficacy in anin vitrocatheter-related biofilm model; (iv) effects on cell wall thickness. As a group, the five strains previously categorized as VAN nonresponders (non-Rsp) in the experimental IE model differed from the five responders (Rsp) in terms of lower VAN binding, increased biofilm formation, higher survival in the presence of VAN within biofilms in the presence or absence of catheters, and greater biofilm reduction upon proteinase K treatment. Interestingly, sub-MICs of VAN significantly promoted biofilm formation only in the non-Rsp isolates. Cell wall thickness was similar among all MRSA strains. These results suggest that sublethal VAN levels that induce biofilm formation and reduce efficacy of VAN in thein vitrocatheter-associated biofilms may contribute to suboptimal treatment outcomes for endovascular infections caused by “VAN-susceptible” MRSA strains.

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Increased Incidence and Plasma-Biofilm Formation Ability of SCCmec Type IV Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated From Patients With Bacteremia

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.602833 ◽

2021 ◽

Vol 11 ◽

Author(s):

Masakaze Hamada ◽

Tetsuo Yamaguchi ◽

Ayami Sato ◽

Daisuke Ono ◽

Kotaro Aoki ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Detection Rate ◽

Sccmec Type ◽

Bloodstream Infections ◽

Molecular Characteristics ◽

Type Ii ◽

Methicillin Resistant ◽

Type Iv ◽

Mrsa Strains

In Japan, Staphylococcal cassette chromosome mec (SCCmec) type IV methicillin-resistant Staphylococcus aureus (MRSA) is an increasingly prominent cause of bacteremia, but the virulence of most of these strains is unclear. We aimed to investigate the relationship between the molecular characteristics and the ability to form biofilms in the presence of blood plasma (plasma-biofilms) of MRSA strains isolated from bloodstream infections. In this study, the molecular characteristics and biofilms of MRSA strains isolated from blood cultures between 2015 and 2017 were analyzed by PCR-based assays, crystal violet staining, and confocal reflection microscopy methods. Among the 90 MRSA isolates, the detection rate of SCCmec type II clones decreased from 60.7 to 20.6%. The SCCmec type IV clone replaced the SCCmec type II clone as the dominant clone, with a detection rate increasing from 32.1 to 73.5%. The plasma-biofilm formation ability of the SCCmec type IV clone was higher than the SCCmec type II clone and even higher in strains harboring the cna or arcA genes. Plasma-biofilms, mainly composed of proteins, were formed quickly and strongly. Our study demonstrated the increased plasma-biofilm formation ability of SCCmec type IV strains.

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icaA/D Genes and Biofilm Formation of Methicillin-Resistant Staphylococcus aureus in Dr. Soetomo Hospital, Surabaya

Folia Medica Indonesiana ◽

10.20473/fmi.v54i4.10709 ◽

2018 ◽

Vol 54 (4) ◽

pp. 263

Author(s):

Putu Arya Suryanditha ◽

Yoeke Dewi Rasita ◽

Kartuti Debora ◽

K Kuntaman

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Methicillin Resistant Staphylococcus Aureus ◽

Meca Gene ◽

Clinical Isolates ◽

Health Concern ◽

Microtiter Plate Assay ◽

Methicillin Resistant ◽

Biofilm Production ◽

Icad Gene

Methicillin-resistant Staphylococcus aureus (MRSA) is a global health concern. One of the factors causing hospital infection is related to the ability of MRSA bacteria to form biofilms. Polysaccharide intercellular adhesin (PIA), encoded by ica gene, have an important role in S. aureus intracellular accumulation and aggregation. The aims of this study was to analyze the relationship between icaA, icaD genes and biofilm production in MRSA carrier and clinical isolate in Dr. Soetomo Hospital Surabaya. This study was an observational study using cross sectional approach. The sample was 47 MRSA isolates is as follow 28 isolates from carrier and 19 were clinical isolates. All of MRSA isolates carried mecA gene. PCR was performed to detect icaA and icaD genes. Biofilm formation was detected using microtiter plate assay (MTP). icaA gene was detected in all isolates whereas icaD gene in 96,4% carrier isolates and all (100%) of clinical isolates. Positive MTP results showed in all (100%) of carrier isolates and 57,9% of clinical isolates. Statistic result was significantly different in biofilm formation between carrier and clinical MRSA isolates. The proportion of positive biofilm formation in isolate with positive icaA/D genes was 82.6%. There was not any association between icaA and icaD gene with biofilm production.

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Biofilm Formation by and Accessory Gene Regulator Typing of Methicillin-ResistantStaphylococcus aureusStrains Recovered From Patients With Nosocomial Infections

Infection Control and Hospital Epidemiology ◽

10.1086/500620 ◽

2006 ◽

Vol 27 (2) ◽

pp. 188-190 ◽

Cited By ~ 19

Author(s):

Kunihiro Manago ◽

Junichiro Nishi ◽

Naoko Wakimoto ◽

Hiroaki Miyanohara ◽

Jav Sarantuya ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Nosocomial Infections ◽

Biofilm Formation ◽

Accessory Gene ◽

Infection Group ◽

Methicillin Resistant ◽

Accessory Gene Regulator ◽

Mrsa Strains

The association between biofilm formation and the accessory gene regulator (agr) types of methicillin-resistantStaphylococcus aureus(MRSA) strains in our hospital were investigated. The biofilm index and the incidence of MRSA strains carryingagr-2in the infection group (n= 91) were significantly higher than were those in the carrier group (n= 225), suggesting that biofilm formation andagrtype are associated with nosocomial MRSA infections.

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