Aims: This study was designed to determine the virulence genes and antibiotic resistance profiles of Staphylococcus aureus isolated from dogs, cats, chickens and horses. Methodology and results: A total of 15 S. aureus isolates were used in this study. Antibiogram and screening of virulence genes was carried out using disc diffusion method and polymerase chain reaction. The results obtained showed that a total of 9 S. aureus isolates were resistant towards oxacillin (60%), 9 isolates were resistant towards neomycin (60%) and 8 isolates were resistant towards tilmicosin (53%). Resistance to amoxicillin, tetracycline and vancomycin was also observed in 6 (40%) of the isolates. Additionally, 5 (33%) of the isolates showed resistance towards streptomycin and linzolide while 4 (27%) of the isolates were resistant towards rifampin, erythromycin and mupirocin. Lastly, 3 (20%) of the isolates were resistant towards doxycycline. Intermediate resistance to amoxicillin and doxycycline was also observed. Virulence gene profiling showed that 4 (26.7%) of the isolates were positive for hlβ and SspA, 9 of the isolates (60%) showed positive for geh and 12 of the isolates (80%) showed positive for Set-1. Similarly, 2 (13.3%) of the isolates showed positive for etA and Seu while only 1 isolate (6.7%) showed positive for PVL and hlα. None of the isolates were positive for tst-1 and etB. Conclusion, significance and impact of study: This study revealed reduced susceptibility and multiple drug resistance (MDR) in four isolates, and susceptibility to all antibiotics in two isolates in addition to low carriage rate of virulence gene in all isolates. Thus, indicating resistance development in majority of the isolates and the need to regulate indiscriminate use of antibiotics in animals.
Staphylococcus aureus

Aims:The aim of this study was to determine the occurrence of Escherichia coliand E. coliO157:H7 in the cattle, farm environment beef andmilk samples.Methodology and results:A total of 192 samples were collected from cattle (rectal faeces) (96) and their farm environment (96) including feed, floorof stall swabs, pooled flies and water samples and 30 milk samples from dairy cattle. A total of seven markets were selected to collect 60 beef samples. The presence of E. coli and E. coli O157:H7 was determined using culture method and confirmed using PCR assay. The overall occurrence rate of E. coli in the farm was 49%. In milk, the occurrence of E. coliwas 33.3% and in beef was 10%. The isolated E. coli were then screened for E. coli O157 using latex agglutination test (Oxoid) and 34.2% were found positive to the test. The identified E. coliO157 isolates were then subjected to multiplex PCR with five primer pairs. They were all negative except for 3.6% of the E. coliO157 (10.5%)isolates which were positive for the presence of fliCH7 genes. Conclusion, significance and impact of study: This study showed the absence of E. coli O157:H7 in the cattle, farm environment, milk and beef. This study may provide a base for conducting a broader spectrum study by having more risk factors included in the study of E. coliin cattle in Malaysia. Further detailed studies may provide a platform to control emergence and distribution of pathogenic bacteria and ensure good control and surveillance strategies and policies.

Background: The current conventional serotyping based on antigen-antisera agglutination could not provide a better understanding of the potential pathogenicity of Salmonella enterica subsp. enterica serovar Brancaster. Surveillance data from Malaysian poultry farms indicated an increase in its presence over the years. Objective: This study aims to investigate the virulence determinants and antimicrobial resistance in S. Brancaster isolated from chickens in Malaysia. Methods: One hundred strains of archived S. Brancaster isolated from chicken cloacal swabs and raw chicken meat from 2017 to 2022 were studied. Two sets of multiplex polymerase chain reaction (PCR) were conducted to identify eight virulence genes associated with pathogenicity in Salmonella (invasion protein gene [invA], Salmonella invasion protein gene [sipB], Salmonella-induced filament gene [sifA], cytolethal-distending toxin B gene [cdtB], Salmonella iron transporter gene [sitC], Salmonella pathogenicity islands gene [spiA], Salmonella plasmid virulence gene [spvB], and inositol phosphate phosphatase gene [sopB]).Antimicrobial susceptibility assessment was conducted by disc diffusion method on nine selected antibiotics for the S. Brancaster isolates. S. Brancaster, with the phenotypic ACSSuT-resistance pattern (ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracycline), was subjected to PCR to detect the corresponding resistance gene(s). Results: Virulence genes detected in S. Brancaster in this study were invA, sitC, spiA, sipB, sopB, sifA, cdtB, and spvB. A total of 36 antibiogram patterns of S. Brancaster with a high level of multidrug resistance were observed, with ampicillin exhibiting the highest resistance. Over a third of the isolates displayed ACSSuT-resistance, and seven resistance genes (β-lactamase temoneira [blaTEM ], florfenicol/chloramphenicol resistance gene [floR], streptomycin resistance gene [strA], aminoglycoside nucleotidyltransferase gene [ant(3″)-Ia], sulfonamides resistance gene [sul-1, sul-2], and tetracycline resistance gene [tetA]) were detected. Conclusion Multidrug-resistant S. Brancaster from chickens harbored an array of virulenceassociated genes similar to other clinically significant and invasive non-typhoidal Salmonella serovars, placing it as another significant foodborne zoonosis.

Pasteurella multocida serotype B:2 causes hemorrhagic septicemia in cattle and buffalo. The invasion mechanism of the bacterium when invading the bloodstream is unclear. This study aimed to characterize the effects of immunomodulatory molecules, namely dexamethasone and lipopolysaccharide, on the invasion efficiency of P. multocida serotype B:2 toward bovine aortic endothelial cells (BAECs) and the involvement of actin microfilaments in the invasion mechanism. The results imply that treatment of BAECs with lipopolysaccharide at 100 ng/mL for 24 h significantly increases the intracellular bacteria number per cell (p < 0.01) compared with those in untreated and dexamethasone-treated cells. The lipopolysaccharide-treated cells showed a significant decrease in F-actin expression and an increase in G-actin expression (p < 0.001), indicating actin depolymerization of BAECs. However, no significant differences were detected in the invasion efficiency and actin filament reorganization between the dexamethasone-treated and untreated cells. Transmission electron microscopy showed that P. multocida B:2 resided in a vacuolar compartment of dexamethasone-treated and untreated cells, whereas the bacteria resided in cellular membrane of lipopolysaccharide-treated cells. The results suggest that lipopolysaccharide destabilizes the actin filaments of BAECs, which could facilitate the invasion of P. multocida B:2 into BAECs.
Actin Cytoskeleton ; Actins ; Animals ; Bacteria ; Buffaloes ; Cattle ; Dexamethasone ; Endothelial Cells ; Hemorrhagic Septicemia ; In Vitro Techniques ; Membranes ; Microscopy, Electron, Transmission ; Pasteurella multocida ; Pasteurella ; Serogroup

Introduction: Multidrug resistance bacteria is alarming worldwide. A lot of research were done and are ongoing to search for the best, convenient and economically affordable ways to fight them. With the latest genome editing tool; Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, this research was performed to develop a novel strategy to genetically modify the genome and inhibit the growth of Klebsiella pneumoniae (UPM ESBLKP1), an Extended Spectrum Beta Lactamases (ESBL) organism. Methods: A CRISPR-Cas9 vector was constructed together with guide RNAs designed specifically for the targeted uppP gene, a gene responsible for bacterial cell growth and protection. Results: The growth and cell wall integrity of the modified Klebsiella pneumoniae (ΔUPM ESBLKP1) were significantly inhibited and reduced, respectively. Interestingly, wild type Klebsiella pneumoniae showed a normal growth curve while modified strains showed a faster doubling rate when supplemented with Luria-Bertani media. In contrast, slower growth rate of modified strain was observed in the M9 minimal media. This explained the higher doubling rate of mutants on nutrient rich medium earlier is being related to gene recovery. They grew slowly in the minimal media as they were adapting to a new environment while recovering the uppP gene and surviving, proving the success of its gene modification. Conclusion: The developed CRISPR-gRNA system was able to modify the targeted Klebsiella pneumoniae gene hence providing an opportunity to develop a new drug for Klebsiella pneumoniae infection as an alternative to antibiotics.