Occurrence of Campylobacter species from broiler chickens and chicken meat in Malaysia.

Campylobacter is reported as a major cause of foodborne illness worldwide. Consumption of contaminated chicken meat is considered a significant risk factor of Campylobacter infection in humans. This study investigated the occurrence of non-Campylobacter jejuni-Campylobacter coli, in broiler chickens (n = 210) and chicken meat (n = 109). The samples were collected from seven broiler chicken farms (n = 210 cloacal swabs), 11 markets (n = 84 chicken meat), and 5 supermarkets (n = 25 chicken meat) located in different districts of Selangor State. Campylobacter were isolated from cloacal swabs using the Cape Town Protocol and from meat samples using the method of Duffy et al. (2007) with some modifications for Campylobacter isolations which were reported effective in the isolation of non-C. jejuni-C. coli Campylobacter species. The isolates were identified by Gram staining for cellular morphology, wet mount for motility and biochemical tests. Confirmation of presumed Campylobacter isolates was carried out using multiplex PCR (mPCR). One hundred seven (107/210) or 50.9% and twenty-nine (29/109) or 26.6% of chickens and chicken meat samples respectively were positive for Campylobacter species. Among the Campylobacter isolates from chickens, C. jejuni was the most predominantly isolated species (69.5%), followed by C. coli (16.2%). Campylobacter fetus and C. upsaliensis were the non-C. jejuni-C. coli Campylobacter species isolated in this study, at 9.3% and 2.5% respectively. Overall, the findings indicated broiler chickens were colonized not only by the common Campylobacter species but also by other Campylobacter species. We found the Cape Town Protocol useful to detect the occurrence of non-C. jejuni-C. coli isolates in chickens.

Determining the Efficacy of CRISPR-Cas9 Mechanisms on the uppP Gene of Klebsiella pneumonia using Lysozyme Assay

@#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.

Staphylococcal cassette chromosome mec (SCCmec) and characterization of the attachment site (attB) of methicillin resistant Staphylococcus aureus (MRSA) and methicillin susceptible Staphylococcus aureus (MSSA) isolates.

This study was designed to screen for SCCmec types and to characterize the attachment site (attB) and universal insertion site (orfX) of SCCmec in a collection of 27 isolates (n = 11) methicillin resistant S. aureus and (n = 16) methicillin susceptible S. aureus isolates in Malaysia. Screening of SCCmec types and characterization of the attachment site was carried out using PCR amplification and Sanger's sequencing method. The result showed that a large proportion of the MRSA isolates carried SCCmec type III 7/11 (63%). Three isolates 3/11 (27%) and 1/11 (9.0%) carried SCCmec type II and IVd respectively. Amplification of the universal insertion site of the SCCmec (orfX) and attachment site (attB) showed that all 16 S. aureus isolates were positive for the orfX gene, while only 7 were positive for the attB gene. Phylogenetic diversity showed that the isolates clustered around strains with features similar to a community acquired MRSA. In conclusion, a high carriage rate of SCCmec type III was observed. The result also showed that all the S. aureus isolates have the orfX structure; however, not all isolates possesses the attB site on the 3' end of the orfX region.

In vitro treatment of lipopolysaccharide increases invasion of Pasteurella multocida serotype B:2 into bovine aortic endothelial cells

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.

Occurrence of Escherichia coliand Escherichia ColiO157:H7 in cattle, farm environment, milk and beef

@#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.

In vitro transfer of methicillin resistance determinants mecA from methicillin resistant Staphylococcus aureus (MRSA) to methicillin susceptible Staphylococcus aureus (MSSA).

BACKGROUND: Staphylococcus aureus more than any other human pathogen is a better model for the study of the adaptive evolution of bacterial resistance to antibiotics, as it has demonstrated a remarkable ability in its response to new antibiotics. This study was designed to investigate the in vitro transfer of mecA gene from methicillin resistant S. aureus to methicillin susceptible S. aureus. RESULT: The recipient transconjugants were resistant to erythromycin, cefpodoxime and were mecA positive. PCR amplification of mecA after mix culture plating on Luria Bertani agar containing 100 µg/mL showed that 75% of the donor and 58.3% of the recipient transconjugants were mecA positive. Additionally, 61.5% of both the donor cells and recipient transconjugants were mecA positive, while 46.2% and 41.75% of both donor and recipient transconjugants were mecA positive on LB agar containing 50 µg/mL and 30 µg/mL respectively. CONCLUSION: In this study, the direction of transfer of phenotypic resistance as well as mecA was observed to have occurred from the donor to the recipient strains. This study affirmed the importance of horizontal transfer events in the dissemination of antibiotics resistance among different strains of MRSA.

New sites of localisation of Pasteurella multocida B:2 in buffalo surviving experimental haemorrhagic septicaemia.

BACKGROUND: Haemorrhagic septicaemia (HS) is an acute septicaemic disease of buffalo and cattle caused by Pasteurella multocida B:2 and E:2. Field outbreaks of HS are known to result in localisation of bacteria in the tonsils of surviving buffalo, confirming that animals can become carriers and the role of respiratory tract in the transmission of the disease. This report describes additional sites of localisation of P. multocida B:2 in surviving buffalo following experimental induction of HS. RESULTS: Following P. multocida B:2 infection, all calves in group 1 and one calf in group 2 that was allowed to commingle with infected calves from group 1 were euthanised within 48 h. Pasteurella multocida B:2 was detected from the nasal and rectal swab samples on days 5 and 6 from the remaining calves in group 2. The first injection of dexamethasone into the carrier animals resulted in reemergence in samples from the nose, rectum and vagina. However, subsequent dexamethasone injections failed to re-activate P. multocida B:2. When surviving carrier calves in group 2 were euthanised at the end of the experiment, P. multocida B:2 was detected in the lungs and various organs of the respiratory, gastrointestinal and urinary tracts. CONCLUSIONS: Commingling naive buffalo calves with calves acutely infected with P. multocida B:2 resulted in carriers among surviving buffalo. Pasteurella was found in various organs of the respiratory, gastrointestinal and urinary tracts, suggesting their role in the pathogenesis of HS.