Vancomycin-resistant Staphylococcus Aureus Research Articles

Molecular characterization of Staphylococcus aureus isolated from hospital-acquired infections in Ilam, Iran.

This research study was undertaken to investigate antimicrobial resistance patterns and the prevalence of hospital-acquired infections (HAIs). The study focuses on common microorganisms responsible for HAIs and explores emerging challenges posed by antimicrobial drug-resistant isolates. A comprehensive analysis of 123 patients with HAIs, hospitalized in surgical department and intensive care unit (ICU) at Imam Khomeini Hospital, Ilam, Iran, was conducted over a six-month period. Pathogenic bacterial isolates, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA), were isolated and subjected to antibiotic susceptibility testing. The study findings revealed a significant prevalence of multidrug-resistant (MDR) isolates, of which 73.3% were MRSA. Notably, 6.7% of S. aureus isolates exhibited resistance to vancomycin, indicating the emergence of VRSA. Respiratory infections were identified as the most prevalent HAI, constituting 34.67% of cases, often arising from extended ICU stays and invasive surgical procedures. Furthermore, patients aged 60 and above, particularly those associated with MDR, exhibited higher vulnerability to HAI. This research sheds light on the intricate interplay between drug resistance and HAI, highlighting the imperative role of rational antibiotic use and infection control in addressing this critical healthcare challenge.

Lipophilic bioactive compounds from thermophilic cyanobacterium Leptolyngbya sp. HNBGU-004: Implications for countering VRSA resistance

Extremophiles thrive in extreme conditions, showcasing rich and unexplored diversity. This resilience hints at the existence of novel biochemical pathways and unique bioactive compounds. In contrast, the issue of drug resistance and excessive misuse of antibiotics in various settings, such as healthcare, agriculture, and veterinary medicine, has contributed to the emergence and spread of drug-resistant microorganisms.In the present research, Leptolyngbya sp. HNBGU-004, was obtained from an extreme location, a hot water spring in the Garhwal Himalayan region of India. The lipophilic fraction derived from Leptolyngbya sp. HNBGU-004 exhibited significant inhibitory effects against vancomycin-resistant Staphylococcus aureus (VRSA), displaying a bactericidal concentration of 0.5 mg mL−1. Furthermore, gas chromatography-mass spectrometry (GC-MS) analysis of the lipophilic extract unveiled the major constituents.Leptolyngbya sp. HNBGU-004 holds significant promise as a primary source of potent anti-vancomycin-resistant S. aureus components. These findings emphasize the importance of Leptolyngbya sp. HNBGU-004 as a foundational source for use as both a synergistic and alternative agent against VRSA.

Comparison of broth microdilution and E-test for susceptibility of MRSA to vancomycin

Introduction: Healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) developed soon after the introduction of methicillin. These bacteria have shown resistance to multiple drugs and therefore vancomycin became the antibiotic of choice for treatment of MRSA infections. Vancomycin is a bactericidal antibiotic that acts by inhibiting bacterial cell wall synthesis. This study aimed to compare broth microdilution (BMD) and E-test in determining vancomycin minimum inhibitory concentration (MIC) against MRSA.Methods: A total of 30 clinical isolates of MRSA were acquired from Colombo South Teaching Hospital, Sri Lanka. These MRSA strains were identified by cefoxitin disk diffusion and the vancomycin MIC was determined through BMD and E-test. The Kruskal-Wallis test was applied to test if the samples originated from the same distribution with post hoc determination of the correlation between methods using the Mann-Whitney U test.Results: All 30 MRSA isolates were 100% vancomycin susceptible (≤2 μg/mL) irrespective of methodology, according to the Clinical and Laboratory Standards Institute (CLSI) established breakpoints. However, the E-test MIC values were 1 to 2 dilutions higher than those of BMD. A statistically significant difference between vancomycin MICs of BMD and E-test (p = <0.00001) was calculated which indicated a difference in accuracy.Conclusion: Due to cost, convenience, and the ability to detect vancomycin intermediate Staphylococcus aureus (VISA), exploring the possibility of using E-test as an alternative to BMD is worthwhile.

Chitosan-Loaded Lagenaria siceraria and Thymus vulgaris Potentiate Antibacterial, Antioxidant, and Immunomodulatory Activities against Extensive Drug-Resistant Pseudomonas aeruginosa and Vancomycin-Resistant Staphylococcus aureus: In Vitro and In Vivo Approaches.

Antimicrobial resistance poses considerable issues for current clinical care, so the modified use of antimicrobial agents and public health initiatives, coupled with new antimicrobial approaches, may help to minimize the impact of multidrug-resistant (MDR) bacteria in the future. This study aimed to evaluate the antimicrobial, antioxidant, and immunomodulatory activities of Lagenaria siceraria, Thymus vulgaris, and their chitosan nanocomposites against extensive drug-resistant (XDR) Pseudomonas aeruginosa and vancomycin-resistant Staphylococcus aureus (VRSA) using both in vitro and in vivo assays. The in vitro antimicrobial susceptibilities of P. aeruginosa and VRSA strains revealed 100% sensitivity to imipenem (100%). All P. aeruginosa strains were resistant to cefoxitin, cefepime, trimethoprim + sulfamethoxazole, and fosfomycin. However, S. aureus strains showed a full resistance to cefoxitin, amoxicillin, ampicillin, erythromycin, chloramphenicol, and fosfomycin (100% each). Interestingly, all S. aureus strains were vancomycin-resistant (MIC = 32-512 μg/mL), and 90% of P. aeruginosa and S. aureus strains were XDR. The antimicrobial potential of Lagenaria siceraria and Thymus vulgaris nanocomposites with chitosan nanoparticles demonstrated marked inhibitory activities against XDR P. aeruginosa and VRSA strains with inhibition zones' diameters up to 50 mm and MIC values ranging from 0.125 to 1 μg/mL and 1 to 8 μg/mL, respectively. The results of the in vivo approach in male Sprague Dawley rats revealed that infection with P. aeruginosa and S. aureus displayed significant changes in biochemical, hematological, and histopathological findings compared to the negative control group. These values returned to the normal range after treatment by chitosan nanoparticles, either loaded with Lagenaria siceraria or Thymus vulgaris. Real-time quantitative polymerase chain reaction (RT-qPCR) findings presented significant upregulation of the relative expression of the IL10 gene and downregulation of the IFNG gene throughout the experimental period, especially after treatment with chitosan nanoparticles loaded either with Lagenaria siceraria or Thymus vulgaris in comparison to the positive control groups. In conclusion, this is the first report suggesting the use of Lagenaria siceraria and Thymus vulgaris nanocomposites with chitosan nanoparticles as a promising contender for combating XDR P. aeruginosa and VRSA infections as well as a manager for inflammatory situations and oxidative stress-related disorders.

Metabolic Profiling Reveals the Importance of Arginine Metabolism via the Arginine Deiminase Pathway in Vancomycin-intermediate S. aureus

Gene mutations located in VraSR and GraSR, two-component regulatory systems have been reported to cause vancomycin resistance in vancomycin-intermediate Staphylococcus aureus (VISA). Subsequent comparative proteomic profiling between vancomycin-susceptible S. aureus (VSSA) and VISAs of the Mu50 genetic lineage revealed up-regulated level of catabolic ornithine carbamoyltransferase (ArcB) in the latter, suggesting a role of arginine catabolism in VISA development. This study aimed to further investigate metabolic pathways associated with VISA development using liquid chromatography mass spectrometry (LCMS)-based untargeted metabolomic profiling. Metabolite profiles were compared among 3 isogenic strains of the Mu50 lineage: Mu50Ω (VSSA), Mu50Ω-vraSm (VISA) and Mu50Ω-vraSm-graRm (VISA). Intracellular levels of α-hydroxyglutaric acid, choline, lysine, malic acid, N-acetylornithine, nicotinamide, and cystathionine were found to be significantly different between VISAs compared to VSSA. VISA cells were found to have lower levels of intracellular citrulline and extracellular arginine, with higher extracellular level of ornithine compared to VSSA. These differences in metabolite levels between VISA and VSSA suggested the importance of arginine metabolism. Activation of the arginine deiminase pathway (ADI) in VISAs of the Mu50 lineage could be further explored as a target for antibiotic development.

A new tetronomycin analog, broad-spectrum and potent antibiotic against drug-resistant Gram-positive bacteria.

We discovered a new tetronomycin analog, C-32-OH tetronomycin (2) from the Streptomyces sp. K20-0247 strain, which produces tetronomycin (1). After NMR analysis of 2, we determined the planar structure. Futhermore, the absolute stereochemistry of 2 was deduced based on the biosynthetic pathway of 1 in the K20-0247 strain and a comparison of experimental electronic circular dichroism (ECD) results of 1 with 2. While 2 exihibits potent antibacterial activity aganist Gram-positive baceria including vancomycin-intermediate Staphylococcus aureus (VISA) strains and vancomycin-resistant Enterococci (VRE), the antibacterial activity of 2 shows 16-32-folds weaker than that of 1 suggesting that the C-34 methyl group in 1 is one of the very important functinal group. Moreover, we evaluated the ionophore activity of 1 and 2 and neither compound shows ionophore activity at reasonable concetrations. Our research suggests that 1 and 2 would have different target(s) from an ionophore mechanism in the antibacterial activity and tetronomycins are promising natural products for broad-spectrum antibiotics.

Antimicrobial activity of nature-inspired molecules against multidrug-resistant bacteria.

Multidrug-resistant bacterial infections present a serious challenge to global health. In addition to the spread of antibiotic resistance, some bacteria can form persister cells which are tolerant to most antibiotics and can lead to treatment failure or relapse. In the present work, we report the discovery of a new class of small molecules with potent antimicrobial activity against Gram-positive bacteria and moderate activity against Gram-negative drug-resistant bacterial pathogens. The lead compound SIMR 2404 had a minimal inhibitory concentration (MIC) of 2 μg/mL against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-intermediate Staphylococcus aureus (VISA). The MIC values against Gram-negative bacteria such as Escherichia coli and Actinobacteria baumannii were between 8-32 μg/mL. Time-kill experiments show that compound SIMR 2404 can rapidly kill tested bacteria. Compound SIMR 2404 was also found to rapidly kill MRSA persisters which display high levels of tolerance to conventional antibiotics. In antibiotic evolution experiments, MRSA quickly developed resistance to ciprofloxacin but failed to develop resistance to compound SIMR 2404 even after 24 serial passages. Compound SIMR 2404 was not toxic to normal human fibroblast at a concentration of 4 μg/mL which is twice the MIC concentration against MRSA. However, at a concentration of 8 μg/mL or higher, it showed cytotoxic activity indicating that it is not ideal as a candidate against Gram-negative bacteria. The acceptable toxicity profile and rapid antibacterial activity against MRSA highlight the potential of these molecules for further studies as anti-MRSA agents.

Tackling strong biofilm and multi-virulent vancomycin-resistant Staphylococcus aureus via natural alkaloid-based porous nanoparticles: perspective towards near future eradication.

As a growing direction, nano-based therapy has become a successful paradigm used to address the phytogenic delivery-related problems in overcoming multivirulent vancomycin-resistant Staphylococcus aureus (VRSA) infection. Hence, our aim was to develop and assess a novel nanocarrier system (mesoporous silica nanoparticles, MPS-NPs) for free berberine (Free-BR) as an antimicrobial alkaloid against strong biofilm-producing and multi-virulent VRSA strains using in vitro and in vivo mouse model. Our outcomes demonstrated vancomycin resistance in 13.7% of Staphylococcus aureus (S. aureus) strains categorized as VRSA. Notably, strong biofilm formation was observed in 69.2% of VRSA strains that were all positive for icaA gene. All strong biofilm-producing VRSA strains harbored a minimum of two virulence genes comprising clfA and icaA with 44.4% of them possessing all five virulence genes (icaA, tst, clfA, hla, and pvl), and 88.9% being multi-virulent. The study findings affirmed excellent in vitro antimicrobial and antibiofilm properties of BR-loaded MPS-NPs. Real-time quantitative reverse transcription PCR (qRT-PCR) assay displayed the downregulating role of BR-loaded MPS-NPs on strong biofilm-producing and multi-virulent VRSA strains virulence and agr genes in both in vitro and in vivo mice models. Additionally, BR-loaded MPS-NPs supplementation has a promising role in attenuating the upregulated expression of pro-inflammatory cytokines' genes in VRSA-infected mice with attenuation in pro-apoptotic genes expression resulting in reduced VRSA-induced apoptosis. In essence, the current study recommends the future scope of using BR-loaded MPS-NPs as auspicious alternatives for antimicrobials with tremendous antimicrobial, antibiofilm, anti-quorum sensing (QS), and anti-virulence effectiveness against problematic strong biofilm-producing and multi-virulent VRSA-associated infections.

Methicillin resistance Staphylococcus aureus (MRSA) and Vancomycin Resistant Staphylococcus aureus (VRSA) problem in human and livestock and solutions.

Methicillin resistance Staphylococcus aureus (MRSA) and Vancomycin Resistant Staphylococcus aureus (VRSA) problem in human and livestock and solutions.

Molecular distribution of biocide resistance genes and susceptibility to biocides among vancomycin resistant Staphylococcus aureus (VRSA) isolates from intensive care unit (ICU) of cardiac hospital- A first report from Pakistan

BackgroundThe study was conducted with the aim to investigate the VRSA isolates in terms of their susceptibility to routinely used biocides influenced by the co-occurrence of biocide resistant gene (BRGs) and efflux pumps genes. MethodologyFrequently touched surfaces within intensive care unit (ICU) of cardiac hospital were classified into three primary sites i.e., structure, machines and miscellaneous. Over a period of six months (January 2021 to July 2021) twenty three swabs samples were collected from these sites. Subsequently, these samples underwent both phenotypic and molecular methods for VRSA isolation and identification. Susceptibility and efficacy testing of biocides (benzalkonium chloride (BAC), cetrimide (CET) and chlorhexidine gluconate (CHG)) were evaluated using microdilution broth and suspension method. Furthermore, specific primers were used for singleplex PCR targeting BRGs (cepA, qacA, and qacE) and efflux pump (norA, norB, norC, sepA, mepA and mdeA) associated genes. ResultsWe found that 72.2 % S. aureus demonstrate the presence of vanA or vanB genes with no significant difference among three sites (p > 0.05). cepA is the most dominant BRGs followed by qacA and qacE from structure site as compared to other sites (p < 0.05). BAC showed reduced biocide susceptibility and MIC50. There was no significant difference between presence or absence of BRGs and high MIC values of VRSA isolates from all three sites. However, efflux pump genes (EFPGs) particularly norA and norA + sepA had a significant association with BRGs and reduced biocide. ConclusionBAC is the most effective disinfectant against VRSA. Proper and controlled use of BAC is required to overcome the VRSA contamination. We recommend continuous monitoring of the BRGs prevalence for better prevention of microorganism dissemination and infection control in hospitals.

Characterization of Brazilian green propolis as a photosensitizer for LED light-induced antimicrobial photodynamic therapy (aPDT) against methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin-intermediate Staphylococcus aureus (VISA)

Characterization of Brazilian green propolis as a photosensitizer for LED light-induced antimicrobial photodynamic therapy (aPDT) against methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin-intermediate Staphylococcus aureus (VISA)

Multiplex System: Identification of Vancomycin (Vana) And Methicillin (Meca) Resistance Genes In Staphylococcus Aureus

Introduction: Emergence of Methicillin resistant Staphylococcus aureus (MRSA) and Vancomycin resistant Staphylococcus aureus (VRSA) strain from different regions of the world poses a grave concern to human health. Antibiotic resistance in S. aureus is mainly because of the genetic factors which modify or disrupt their target site on bacteria. Methods: This study is focused to identify the vancomycin and methicillin resistance gene in antibiotic sensitive and resistant S. aureus. The pure cultures of S. aureus were isolated, subjected to morphological and biochemical characterization. Antibiotic susceptibility testing was done to check the resistance pattern. DNA isolation was followed by genotyping of the antibiotic resistance genes (VanA and MecA) and housekeeping gene (AroE) was done through multiplex PCR method. All the strains showed the colonial, microscopic and biochemical characteristics (catalase and coagulase positive) specific for S. aureus. Results: Majority of the strains were resistant to cefixime (80 %) and least resistance was observed with fusidic acid (0%), while resistance frequency of the remaining antibiotics falls between them. All the strains showed the presence of housekeeping AroE gene with frequency of VanA is 2% and MecA is 24% which coincides with the findings of antibiotic resistance testing. For VanA, there might be other resistance genes of vancomycin cassette which confer the resistance against it. Conclusion: The study will help to discriminate the vancomycin and methicilin sensitive and resistance strains of S. aureus based on their respective genetic factors and help to validate the underlying mechanism in the acquisition of antibiotic resistance.

Molecular identification of Methicillin-resistant staphylococcus aureus (MRSA) and Vancomycin- resistant staphylococcus aureus (VRSA) strains isolated from milk and milk products.

Molecular identification of Methicillin-resistant staphylococcus aureus (MRSA) and Vancomycin- resistant staphylococcus aureus (VRSA) strains isolated from milk and milk products.

Distribution and Prevalence of Multidrug-Resistant Organisms (MDROs) Among MDRO-Positive Individuals at Dr. Kariadi Hospital

Background: Multidrug-resistant organisms (MDRO) pose a significant challenge to healthcare facilities globally, impacting patient outcomes and healthcare costs. Understanding the distribution and prevalence of MDRO is crucial for effective infection control and prevention strategies. This study aimed to investigate the distribution and prevalence of MDROs, among MDRO-positive individuals at Dr. Kariadi Hospital.Methods: A retrospective analysis of secondary data was conducted, encompassing a diverse range of MDROs, including ESBL, carbapenem-resistant Enterobacteriaceae (CRE), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Staphylococcus aureus (VRSA). The study population consisted of individuals who tested positive for MDRO within the hospital.Results: A total of 100 MDRO-positive cases were identified during the study period. The most prevalent MDRO identified was ESBL-producing organisms, accounting for 59% of all cases. Other significant findings included the presence of carbapenem-resistant Enterobacteriaceae (CRE) and MRSA, which constituted 4% and 3% of the MDRO-positive cases, respectively. Notably, a diverse range of MDRO species, such as MRCoNS (methicillin-resistant coagulase-negative Staphylococci), was also detected.Conclusion: This study provides valuable insights into the distribution and prevalence of MDRO at Dr. Kariadi Hospital. The findings underscore the urgent need for robust infection control measures and targeted interventions to mitigate the spread of MDRO. Implementing effective surveillance, promoting antibiotic stewardship, and enhancing preventive strategies are crucial for controlling MDRO infections. Future research should focus on exploring the molecular characteristics and resistance mechanisms of the identified MDRO to inform tailored prevention and treatment approaches.

Identification of Vancomycin Resistance in Methicillin-resistant Staphylococcus aureus in two macaque species and decolonization and long-term prevention of recolonization in Cynomolgus Macaques (Macaca fascicularis).

Methicillin-resistant Staphylococcus aureus (MRSA) is a S. aureus strain with resistance to beta-lactam antibiotics, making it a global human and veterinary health concern. Specifically, immunosuppressed patients have a remarkably higher risk of clinical MRSA infections with significantly increased rates of prolonged clinical recovery, morbidity, and mortality. The current treatment of choice for MRSA is vancomycin. Importantly, we report the first known vancomycin-resistant S. aureus (VRSA) carriers in a cohort of Mauritian cynomolgus macaques (CM) imported to the Oregon National Primate Research Center (ONPRC), with a MRSA carrier rate of 76.9% (10/13 animals). All MRSA isolates also demonstrated resistance to vancomycin with prevalence of vancomycin-intermediate Staphylococcus aureus (VISA) at 30% (3/10 MRSA-positive CMs) and VRSA at 70% (7/10 MRSA-positive CMs). Additionally, we identified VRSA in a rhesus macaque (RM) housed within the same room as the VRSA-positive CMs and identified a MRSA/VISA carrier rate of 18.8% in RMs (3/16 positive for both MRSA and VISA) in unexposed recently assigned animals directly from the ONPRC RM breeding colony. Considering that the MRSA and VRSA/VISA-positive CMs future study aims included significant immunosuppression, MRSA/VRSA/VISA decolonization treatment and expanded "MRSA-free" practices were employed to maintain this status. We report the first controlled study using in-depth analyses with appropriate diagnostic serial testing to definitively show an MRSA decolonization therapy (90% success rate) and expanded barrier practice techniques to successfully prevent recolonization (100%) of a cohort of CMs MRSA-free (up to 529 days with a total of 4,806 MRSA-free NHP days).

Molecular and Bacteriological Study of Vancomycin Resistant Staphylococcus Aureus (VRSA) Isolated from Buffalo Raw Milk in Baghdad

One hundred buffalo raw milk samples from different locations in Baghdad governorate, Iraq, were tested for Vancomycin-resistant Staphylococcus aureus (VRSA) by streaking on a plate of mannitol salt agar plate and incubated in 37 for 24hr then inoculated on blood agar. The isolates were confirmed according to colony morphology, Gram staining and conventional biochemical techniques and polymerase chain reaction (PCR) to detect vanB gene. The disc diffusion testing was done to determine antimicrobial resistance including VRSA. Staphylococcus aureus (S. aureus) was found in 46 (46%) of raw buffalo milk. The isolates exhibited high resistance (80%) to methicilin, 75% to Oxacillin and Cefoxitin, 55% to gentamycin, and 50% to vancomycin. The phenotypic analyses revealed that out of 46 S. aureus, 23 (50%) were VRSA. While the genotypic identification by PCR showed that out of the 23 VRSA, 18 (78.2%) isolates had vanB genes. Results highlight the potential zoonotic risk of buffalo milk as a potential source of VRSA, which may be transmitted to human beings working in close contact with the buffalos.

Evolutionary history of Staphylococcus aureus influences antibiotic resistance evolution

Antibiotic resistance often confers a fitness cost to the resistant cell and thus raises key questions of how resistance is maintained in the absence of antibiotics and, if lost, whether cells are genetically primed for re-evolving resistance. To address these questions, we have examined vancomycin-intermediate Staphylococcus aureus (VISA) strains that arise during vancomycin therapy. VISA strains harbor a broad spectrum of mutations, and they are known to be unstable both in patients and in the laboratory. Here, we show that loss of resistance in VISA strains is correlated with a fitness increase and is attributed to adaptive mutations, leaving the initial VISA-adaptive mutations intact. Importantly, upon a second exposure to vancomycin, such revertants evolve significantly faster to become VISA, and they reach higher resistance levels than vancomycin-naive cells. Further, we find that sub-lethal concentrations of vancomycin stabilize the VISA phenotype, as do the human β-defensin 3 (hBD-3) and the bacteriocin nisin that both, like vancomycin, bind to the peptidoglycan building block, lipid II. Thus, factors binding lipid II may stabilize VISA both invivo and invitro, and in case resistance is lost, mutations remain that predispose to resistance development. These findings may explain why VISA infections often are re-occurring and suggest that previous vancomycin adaptation should be considered a risk factor when deciding on antimicrobial chemotherapy.

Evaluation of Rosmarinus officinalis Leaves Essential Oils Activity Against Vancomycin Intermediate Staphylococcus aureus (VISA) Isolated from Baghdad Hospital Patients

Rosemary is a well-known aromatic and medicinal plant used to treat various ailments. This study evaluated Rosmarinus officinalis essential oil for its phytochemical and antibacterial properties. The essential oil was analysed by using a gas chromatography-mass Spectrometry (GC-MS) that revealed the common chemicals containing verbenone 36.20% and 1,8-cineol (Eucalyptol) 12.14%. Extracted essential oils were tested for antibacterial activity against vancomycin intermediate Staphylococcus aureus (VISA), a strain of bacteria obtained locally from bacteremia patients. Three isolates were found to be VISA positive using the E-test (strips) and the population analysis profile method (PAP). VISA showed lower resistance to the essential oils (MIC: Minimum inhibitory concentration, MIC = 1.5 mg/ml). Overall, the genetic influence of VISA was evaluated by looking at the hld gene expression after being isolated from blood samples and RNA extraction for qRT-PCR to determine hld gene expression. R. officinalis gene expression was analysed and calculated by using real-time PCR of RNA which revealed that it had decreased.

Molecular characterization of vancomycin-resistant Staphylococcus aureus isolated from bovine milk.

Vancomycin-resistant Staphylococcus aureus (VRSA) is a zoonotic life-threatening pathogen. Vancomycin exhibits anti-bacterial activity by inhibiting peptidoglycan synthesis by binding to the D-ala-D-ala terminus of the peptidoglycan. But in VRSA, D-ala-D-ala is replaced by D-ala-D-lactate due to the presence of vanA, vanB or vanD genes. This study was intended to identify the molecular prevalence of VRSA in 768 bovine milk samples, risk factor association, antibiogram profile and bioinformatics analysis of VRSA by targeting vanB gene. Out of a total of 248 S. aureus isolates from mastitic milk samples, the phenotypic and genotypic prevalence of VRSA was estimated to be 17.74% and 10.89%, respectively. Farm-level risk factors including use of improper milking technique, lack of milker's care during milking, unhygienic conditions during milking and no dry cow therapy were found to be significantly associated (p < 0.05). Anti-microbial susceptibility testing of VRSA isolates exhibited the highest resistance to oxytetracycline, followed by oxacillin and Trimethoprim+sulfamethoxazole. The current study sequences showed more resemblance with reported sequences from Iraq (MN747834) and Egypt (MK095504, MK087830), which belong to vanB gene from S. aureus as compared to sequences from other countries, which belong to vanB gene from the genus Enterococcus. The Genetic Algorithm for Recombination Detection (GARD) found 234 potential breakpoints, translating into a search room of 123,883,305 models with up to 4 breakpoints. The phylogenetic motif profiling method discovered evolutionarily conserved residues across target genes' homologous protein sequences. These residues were discovered to be conserved in drug-resistant target proteins over the evolutionary process and may play a key role in their function. The current study revealed a molecular prevalence of VRSA in dairy animals, along with molecular analysis of vancomycin resistance in S. aureus by targeting the vanB gene using standard bioinformatics tools. The occurrence of VRSA in animals requires serious attention because this pathogen has zoonotic potential, so it can become a greater risk to consumer health.

Antimicrobial Resistance and Major Virulence Gene Detection in Methicillin Resistant Staphylococcus aureus in Humans and Livestock Animals of Assam: A North Eastern State of India

Methicillin resistant Staphylococcus aureus (MRSA) is highly divergent antibiotic resistant bacteria earmarked as “High” in global pathogens’ priority list varying the severity and resistance geographically. Here, MRSA were screened using mecA gene with Cefoxitin and other 27 antibiotics of 19 classes using disc diffusion method from a highly humid climate of India. Multiple Antibiotic Resistance (MAR) index was calculated. Minimum Inhibitory Concentration (MIC) was determined against 11 classes of antibiotics. Detection of major virulence genes tst-1 and lukPV were done. A total of 95.24% Hospital Associated (HA)-MRSA, 56.14% Community Associated (CA)-MRSA and 82.53 % Livestock Associated (LA)-MRSA were detected. Cefoxitin, Oxacillin, Ciprofloxacin, Fusidic acid and Ticarcillin-Clavulinic acid resistance was observed in more than 60% of HA-MRSA, CA-MRSA and LA-MRSA. Across the hosts, Mupirocin, Gentamicin, Linezolid, Co-trimoxazole, and Rifampicin were found effective. Vancomycin Intermediate Staphylococcus aureus (VISA) detected in CA-MRSA &amp; LA-MRSA. Multidrug Resistant (MDR) was found very high but extensively drug-resistant (XDR) was detected moderately. No pan drug-resistant (PDR) was detected. Virulence gene tst-1 and lukPV were detected in 7.69% and 32.69% MRSA isolates. The gene tst-1 is reported for the first time in pre and post-caesarian samples from Gynaecology department in this region with high MDR. This study showed S. aureus and subsequent prevalence of MRSA is higher in this region then national data. 2nd generation Cephalosporins were found effective which is very encouraging due to their limited uses. Detection of tst-1 in caesarian samples is a serious threat as neonatal transmission of MRSA from mother is reported.