Genotypic insights to Panton-Valentine leukocidin positive methicillin-resistant Staphylococcus aureus isolated from cattle mastitis.

In this research communication we investigate the prevalence and antimicrobial susceptibility of S. aureus harboring virulent genes responsible for mastitis in cattle of Punjab, Pakistan. A total of 690 milk samples were collected from commercial dairy farms for analysis of the prevalence of subclinical and clinical mastitis and isolation of S. aureus. Virulence ability and methicillin resistance in S. aureus (MRSA) was determined by targeting the pvl (the gene for Panton-Valentine leukocidin) and mecA genes, respectively. A total of 175 S. aureus isolates exhibiting prevalence of pvl gene (6.28%) and mecA gene (22.28%) were determined. Antimicrobial susceptibility testing of pvl positive and negative MRSA against different classes of antibiotics revealed 100% resistance against ß-lactams while 100% sensitivity towards tylosin and linezolid.

Molecular insights into expression and silencing of resistance determinants in Staphylococcus aureus.

OBJECTIVE: This study aimed to investigate the status of antimicrobial-resistant strains of Staphylococcus aureus in Pakistan, their association in terms of co-occurrence with the biofilm-forming genes, resistance profiling and associated discrepancies in diagnostic methods. METHODOLOGY: A total of 384 milk samples from bovine was collected by using convenient sampling technique and were initially screened for subclinical mastitis, further preceded by isolation and confirmation of S. aureus. The S. aureus isolates were subjected to evaluation of antimicrobial resistance by phenotypic identification using Kirby-Bauer disc diffusion method, while the genotypic estimation was done by polymerase chain reaction to declare isolates as methicillin, beta-lactam, vancomycin, tetracycline, and aminoglycoside resistant S. aureus (MRSA, BRSA, VRSA, TRSA, and ARSA), respectively. RESULTS: The current study revealed an overall prevalence of subclinical mastitis and S. aureus to be 59.11% and 46.69%, respectively. On a phenotypic basis, the prevalence of MRSA, BRSA, VRSA, TRSA, and ARSA was found to be 44.33%, 58.49%, 20.75%, 35.84%, and 30.18%, respectively. The results of PCR analysis showed that 46.80% of the tested isolates were declared as MRSA, 37.09% as BRSA, and 36.36% as VRSA, while the occurrence of TRSA and ARSA was observed in 26.31% and 18.75%, respectively. The current study also reported the existence of biofilm-producing genes (icaA and icaD) in 49.06% and 40.57% isolates, respectively. Lastly, this study also reported a high incidence of discrepancies for both genotypic and phenotypic identification methods of resistance evaluation, with the highest discrepancy ratio for the accA-aphD gene, followed by tetK, vanB, blaZ, and mecA genes. CONCLUSION: The study concluded that different antibiotic resistance strains of S. aureus are prevalent in study districts with high potential to transmit between human populations. The study also determined that there are multiple resistance determinants and mechanisms that are responsible for the silencing and expression of antibiotic resistance genes.

THEILERIA EQUI INFECTION IN WORKING HORSES OF PAKISTAN: EPIDEMIOLOGY, MOLECULAR CHARACTERIZATION, AND HEMATOBIOCHEMICAL ANALYSIS.

Theileria equi is 1 of the emerging and prevailing tick-borne hemoprotozoans adversely affecting the equids worldwide, including Pakistan. The current study aimed to investigate the prevalence and molecular characterization of T. equi in working horses (n = 194), the comparative efficacy of different diagnostic tests, associated risk factors, and hematobiochemical analysis. The blood samples of horses were subjected to microscopic examination, cELISA, and polymerase chain reaction (PCR) and the results revealed a prevalence of 9.79, 21.13, and 13.40%, respectively, for T. equi in working horses. The comparison of microscopy and cELISA results with PCR showed that cELISA had higher sensitivity (84.62%), but lower specificity (88.69%) and accuracy (88.14%) in comparison to microscopy (57.69, 97.62, and 92.27%). Molecular characterization of T. equi by phylogenetic analysis revealed a 61% resemblance of study isolates with each other OL662926, OL662925, and 82% similarity with isolate OL662924 while also showing homology with T. equi isolates of South Africa, South Korea, India, Pakistan, and Brazil. The risk factor analysis revealed a significant association (P < 0.05) of tick control status, previous tick history, tick infestation, house hygiene, deworming/vaccination, and the presence of other livestock species with T. equi infection in horses. The hematobiochemical profile revealed a significant (P < 0.05) decrease in red blood cells (RBCs), hemoglobin (Hb), packed cell volume (PCV), white blood cells (WBCs), platelet (PLT), phosphorus, and an increase in lymphocytes, granulocytes, aspartate aminotransferase (AST), glucose, bilirubin, blood urea nitrogen (BUN), and creatinine in T. equi-infected horses. The current study is the first comprehensive report for comparative evaluation of microscopy, cELISA, and PCR, assessment of epidemiological risk factors as well as hematobiochemical variations due to T. equi infection in Pakistan.

First report on genotypic estimation of MRSA load in udder of nomadic sheep flocks affected with subclinical mastitis in Pakistan.

Mastitis is one of the highly devastating issues responsible for production and economic losses in all dairy animals including sheep. This study was designed to investigate subclinical mastitis (SCM) associated with S. aureus in lactating nomadic ewes, along with the associated risk factors analysis. Furthermore, molecular characterization and antibiogram profiling of local methicillin-resistant S. aureus (MRSA) isolates of ovine origin were also performed. A total of 384 milk samples (n = 384) were collected from 13 nomadic sheep flocks using a convenient sampling technique. SCM was evaluated using a Surf Field Mastitis test and the S. aureus was isolated using standard microbiological techniques. Kirby-Bauer disc diffusion assay was used for phenotypic identification of MRSA while the mecA gene was tested through PCR. Study results revealed that SCM was prevalent at 34.37% while S. aureus association was recorded at 39.39%. MRSA prevalence was 36.53% and 21.15% using phenotypic and genotypic tests, respectively. The mecA gene sequences of study isolates showed maximum resemblance with already reported sequences from Pakistan, China, and Myanmar. MRSA isolates showed maximum resistance towards penicillin, ceftriaxone sodium, and trimethoprim + sulphamethoxazole while gentamicin, ciprofloxacin, and tylosin showed maximum efficacy. Risk factors analysis revealed that various flock management, housing, and host-related factors positively influenced the incidence of S. aureus-associated SCM. This study is the first report on the prevalence of S. aureus and MRSA associated with SCM in lactating ewes in Pakistan. This study will help to devise effective treatment and control strategies for S. aureus-associated SCM.

Discrepancies between phenotypic and genotypic identification methods of antibiotic resistant genes harboring Staphylococcusaureus.

Antimicrobial resistance is a global issue that limits therapeutic options for infections. S. aureus being a member of the ESKAPE group is capable of "escaping" the biocidal action of antimicrobial agents. There are phenotypic and genotypic methods used for the identification of antibiotic resistant genes harboring S. aureus but these methods do not always show concordant results. To address these discrepancies, a total of 335 equine nasal swab samples from four districts of Punjab were collected using a convenient sampling technique. These samples were first subjected to common microbial techniques to identify S. aureus. The disc diffusion assay was performed for the phenotypic identification of antibiotic resistant S. aureus by using discs of oxacillin, penicillin, vancomycin, gentamycin, and tetracycline. After this, PCR was performed by targeting mecA, blaZ, vanB, aaca-aphd, and tetK genes for genotypic identification of respective antibiotic-resistant S. aureus. Phenotypic discrepancies (number of antibiotic resistant isolates found from disc diffusion who appeared to be negative for the resistant gene), and genotypic discrepancies (number of antibiotic sensitive isolates found from disc diffusion who appeared to be positive for the resistant gene) were calculated. The discrepancy ratio for mecA, blaZ, vanB, aaca-aphd, and tetK genes were 3.09, 1.96, 2.67, 1.93, and 1.67 respectively. These discrepant results indicate that the absence or presence of only one gene is not a true marker of resistant or sensitive isolates. There are multiple resistance determinants and resistance mechanisms. This study also highlighted the phenomenon of silencing of antibiotic resistance determinants.

Molecular evidence of ß-lactam resistant Staphylococcus aureus in equids with respiratory tract infections: Frequency and resistance modulation strategy.

The emergence of antimicrobial-resistant strains in Staphylococcus aureus (ß-lactam and methicillin-resistant) is an overwhelming issue worldwide. Using the purposive sampling technique, 217 equids samples were collected from district Layyah which were subjected to culturing followed by genotypic identification of mecA and blaZ genes by PCR. This study revealed that by phenotypic methods, a prevalence of 44.24%, 56.25%, and 47.92% was found for S. aureus, MRSA, and ß-lactam resistant S. aureus in equids. While genotypically, MRSA was found in 29.63% and ß-lactam resistant S. aureus in 28.26% of equids. In-vitro antibiotic susceptibility testing against S. aureus isolates harboring both mecA and blaZ genes showed a high resistance against Gentamicin (75%), followed by Amoxicillin (66.67%) and Trimethoprim+sulfamethoxazole (58.34%). In an attempt to re-sensitize the resistant bacteria to antibiotics, a combination of antibiotics and non-steroidal anti-inflammatory drugs (NSAIDs) was used which revealed synergistic effect of Gentamicin and Trimethoprim+sulfamethoxazole with Phenylbutazone; and Amoxicillin with Flunixin meglumine. Analysis of risk factors revealed significant association with the S. aureus-associated respiratory infection in equids. Phylogenetic analysis of mecA and blaZ genes showed a high resemblance of study isolate's sequences with each other and variable resemblance with already reported isolates obtained from different samples of neighboring countries. This study reports the first molecular characterization and phylogenetic analysis of ß-lactam and methicillin resistant S. aureus in equids in Pakistan. Moreover, this study will help in the resistance modulation of resistant antibiotics (Gentamicin, Amoxicillin, Trimethoprim+sulfamethoxazole) and provide a good insight into planning an effective therapeutic regime.

Evidence and Molecular Characterization of Multidrug Resistant Staphylococcus aureus Isolated From Equines in Pakistan.

The purpose of the current study was to investigate the nasal colonization and drug resistance profile of Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and vancomycin-resistant S. aureus (VRSA) in donkeys (n =63), mules (n = 42), and horses (n = 98). MRSA and VRSA were confirmed based on phenotypic and molecular methods, followed by phylogenetic analysis. Furthermore, the association of various animal and management-based risk factors with S. aureus colonization was also evaluated. The presence of nuc gene on polymerase chain reaction showed an overall prevalence of 42.86% of S. aureus in equines. Based on Kirby-Bauer disc diffusion test, 26.44% of isolates showed resistance to vancomycin. Further, mecA and vanB genes were targeted which showed an overall 14.94% and 13.79% prevalence of methicillin and vancomycin-resistant isolates, respectively. The phylogenetic analysis revealed a significant variation of the study isolates with each other and with already reported sequences of mecA and vanB genes. Risk factor analysis revealed that raising purpose (P = .011), work intensity (P < .001), stocking density (P = .006), presence of other livestock animals in surroundings (P = .043), and common drinking water source (P = .023) as significant. Antimicrobial susceptibility testing of MRSA and VRSA isolates showed high resistance to various commonly used antibiotics. Furthermore, all the tested isolates showing resistance to three or more than three antibiotics were considered multiple drug-resistant. The current study manifests the molecular evidence of MRSA and VRSA isolated from equines in Pakistan which will help to address the emerging issue of multidrug resistance in S. aureus in equines and emphasizes the need for possible measures to tackle this issue.