Multi-Drug Resistant Pathogenic Escherichia coli Isolated from Wild Birds, Chicken, and the Environment in Malaysia.

Transmission of pathogenic microorganisms in the last decades has been considered a significant health hazard and pathogenic E. coli, particularly antibiotic-resistant strains, have long been identified as a zoonotic problem. This study aimed to investigate multidrug resistant pathogenic E. coli isolates from wild birds, chickens, and environment in selected Orang Asli and Malay villages in Peninsular Malaysia. The bacteriological culture-based technique, disc diffusion method, and multiplex Polymerase Chain Reaction (mPCR) assay was used to determine the occurrence of pathogenic E. coli strains in the several samples in the study. E. coli isolates showed a variety of multi-drug resistant (MDR) antibiotypes and Enteropathogenic E. coli (EPEC) and Enteroinvasive E. coli (EIEC) were the most predominantly identified pathogenic E. coli strains. The findings of this study demonstrated the significance of animal reservoirs and the environment as sources of pathogenic E. coli, resistant bacteria, and resistance genes. Hence, there is a need for adoption of a practical surveillance approach on MDR pathogens to control foodborne contamination.

Occurrence and Characteristics of Extended-Spectrum ß-Lactamase-Producing Escherichia coli from Dairy Cattle, Milk, and Farm Environments in Peninsular Malaysia.

The emergence and spread of antimicrobial resistance genes and resistant bacteria do not recognize animal, human, or geographic boundaries. Addressing this threat requires a multidisciplinary approach involving human, animal, and environmental health (One Health) sectors. This is because antimicrobial agents used in veterinary medicine have been reported to be the same or similar to those in human medicine use. Extended-spectrum ß-lactamase (ESBL) E. coli is a growing public health problem worldwide, and the agri-food industry is increasingly becoming a source of clinically important ESBL bacteria. Accordingly, the aim of this study was to investigate the occurrence and characteristics of ESBL-producing E. coli from dairy cattle, milk, and the farm environment. E. coli isolates were identified by their 16sRNA, and their ESBL production was confirmed using ESBL-CHROMagar media and the double disk diffusion method. Genotypes of ESBL producers were characterized using multiplex polymerase chain reaction (mPCR) assay. It was found that 18 (4.8%) of the total samples were positive for ESBL-producing E. coli. Of these, 66.7% were from milk, and 27.8% and 5.5% were from the farm environment and faecal samples, respectively. Predominant ESBL genotypes identified were a combination of both TEM and CTX-M in eight out of 18 (44.4%) isolates. Four (22.2%) isolates produced the CTX-M gene, two (11.1%) isolates produced the TEM gene, and four (22.2%) remaining isolates produced the ESBL genes other than TEM, SHV, CTX-M, and OXA. The SHV and OXA gene were not detected in all 18 isolates. In all, there were four profiles of genetic similarity. The occurrence of these genotypes in indicator organisms from dairy cattle, milk, and the farm environment further re-enforced the potential of food-animals as sources of ESBL-producing E. coli infection in humans via the food chain. Thus, there is the need for the adoption of a tripartite One Health approach in surveillance and monitoring to control antimicrobial resistance.

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.

Prevalence, antimicrobial resistance, virulence gene, and class 1 integrons of Enterococcus faecium and Enterococcus faecalis from pigs, pork and humans in Thai-Laos border provinces.

OBJECTIVES: This study aimed to determine prevalence, antimicrobial resistance, virulence genes, and Class 1 integrons of Enterococcus faecium (E. faecium) and Enterococcus faecalis (E. faecalis) from pigs, pork and humans in Thailand-Laos border provinces. METHODS: Six hundred and forty-eight rectal and carcass swab samples from border provinces of Thailand (n=359) and Lao PDR (n=289) were collected and examined from September 2013 to October 2014. RESULTS: The overall prevalence of Enterococcus species was 483 of 648 (75%), comprising E. faecium (359 of 483, 74.3%) and E. faecalis (124 of 483, 25.7%). The occurrence of E. faecium in pigs, pig carcasses, retail pork, and humans in Thailand was 80.6%, 73.8%, 77.6%, and 67%, respectively. The prevalence of E. faecium was higher in Laos (65.7%) than Thailand (47.1%) (P<0.001). Conversely, E. faecalis was more common in Thailand (24.2%) than Laos (12.8%) (P<0.001). The E. faecium and E. faecalis isolates were resistant to all antimicrobials except vancomycin. High resistance was first observed to tetracycline, erythromycin and streptomycin, followed by gentamicin, ampicillin and chloramphenicol. Both E. faecium (7%) and E. faecalis (0.8%) carried empty Class 1 integrons: E. faecium carried gel (6.4%) and esp (0.8%), while E. faecalis carried agg (41.9%), cylA (36.3%), gel (60.5%), and esp (42.7%). CONCLUSIONS: This study revealed a variable distribution of antimicrobial resistance and virulence genes among E. faecium and E. faecalis from pigs, pig products and humans in Thai-Laos border provinces. These pathogens may serve as potential reservoirs for the maintenance and widespread dissemination of antimicrobial resistance and virulence determinants from animals to humans via the food chain.

Phylogenetic characterization of Salmonella enterica from pig production and humans in Thailand and Laos border provinces.

BACKGROUND AND AIM: The genetic relationship among serotypes of Salmonella enterica from food animals, food of animal origin, and human is of interest as the data could provide an important clue for the source of human infection. This study aimed to determine the genetic relatedness of S. enterica from pig production and human in Thailand-Laos border provinces. MATERIALS AND METHODS: A total of 195 S. enterica serotypes isolated from pig and pork (n=178) and human (n=17) including four serotypes (Typhimurium, Rissen, Derby, and Stanley) were randomly selected to examine their genetic relatedness using highly conserved sequence of three genes (fim A, man B, and mdh). RESULTS: The results showed that 195 Salmonella isolates of four different serotypes were grouped into five different clusters, and members of the same Salmonella serotypes were found in the same cluster. Salmonella isolated from pig production and human in Thailand-Laos border provinces represented overlapping population and revealed a high degree of similarity, indicating close genetic relationship among the isolates. CONCLUSION: The results support that the determination of Salmonella serotyping combined with analysis of phylogenetic tree can be used track the clonal evolution and genetic diversity of Salmonella serotypes in different host species.

Establishment of lung auscultation scoring method and responses of acute phase proteins and heat shock proteins in vaccinated and non-vaccinated goats.

Pneumonic pasteurellosis is an economically important infectious disease in the small ruminant industry which causes sudden death and loss for farmers. Nonetheless, this disease is still a common sight in sheep and goats in Malaysia, probably due to the unpopular usage of pasteurellosis vaccine or inappropriate vaccination practices. The aim of this study was designed to classify the severity of pneumonia via the establishment of auscultation scoring method and to quantify the acute phase proteins and heat shock proteins responses from vaccinated and non-vaccinated goats. Goat farms, consist of vaccinated and non-vaccinated farms, were selected in this study: where 15 clinically normal healthy goats and 9 pneumonic goats were selected from vaccinated farms whereas 15 clinically normal healthy goats and 31 pneumonic goats from non-vaccinated farms were selected for this study. Crackle lung sounds were not detected in both vaccinated and non-vaccinated normal goats. However, vaccinated pneumonic goats showed mild crackle lung sound while non-vaccinated pneumonic goats exhibited moderate crackle lung sound. There were significant increases (p < 0.05) in acute phase proteins and heat shock proteins concentrations for the non-vaccinated pneumonic goats group. In this study, conclusion can be made that the vaccinated goats exhibited very mild clinical responses of pneumonia and non-significant biomarker responses compared to the non-vaccinated goats. Thus, vaccination is an effective preventive measure to control pneumonic pasteurellosis and acute phase proteins and heat shock proteins can be considered as future biomarkers in screening and rapid diagnostic method for this particular disease.

Dermatopathology of Orf Virus (Malaysian Isolates) in Mice Experimentally Inoculated at Different Sites with and without Dexamethasone Administration.

Orf is a clinical manifestation of parapoxvirus infection often fatal in goats and sheep especially when they are under stress or influenced by unfavorable environment. This study investigated the pathogenicity of two Orf virus isolates (ORFV UPM1/14 and UPM2/14) and host response in mouse model by using different inoculation sites with/without prior exposure to dexamethasone. Treatments with dexamethasone served as an immunosuppressant that may mimic stress situation in affected animals. Groups of five mice were given intradermal injection of 0.2 mL of tissue culture infective dose 50 (TCID50) of UPM1/14 (Group 1) and UPM2/14 (Group 2) at the dorsum (Group 1A; Group 2A), ear pinna (Group 1B; Group 2B), and labial commissure (Group 1C; Group 2C). An inoculum 0.2 mL of UPM1/14 was administered to animals treated with dexamethasone (n=5; 5 mg/kg/day intraperitoneally) and nondexamethasone (n=5) groups at the dorsum, ear pinna, and labial commissure. No significant difference (p>0.05) was observed in the mean lesion scores among the groups of different inoculation sites or between dexamethasone-treated and nontreated groups. However, there was a significant difference (p<0.05) in the mean stratum thickness of affected skin following inoculation with UPM2/14 isolate at the ear pinna and labial commissure. Histopathology examination revealed keratosis, acanthosis, and ballooning degeneration in the skin of affected mice. Orf virus DNA was detected in the skin samples by targeting F1L and B2L virus-specific genes in polymerase chain reaction (PCR) assay. Intradermal inoculation with UPM1/14 or UPM2/14 isolate produced a mild skin lesion in mice, and there was no significant difference in orf disease manifestation despite variation of inoculation sites. Similarly, short-term dexamethasone administration gave no adverse effects on pathogenicity of orf virus isolates.

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.

A retrospective study of bovine tuberculosis at the municipal abattoir of Bauchi State, Northeastern Nigeria.

BACKGROUND AND AIM: Bovine tuberculosis (bTB) still remains a major zoonotic bacterial disease affecting livestock and humans worldwide. The disease remains a poorly managed tropical disease in most developing countries of the world; where in addition to productivity losses and significance in international trade, it posed a major public health threat to both humans and animals. A retrospective study was designed to investigate the occurrence of bTB lesions at Bauchi municipal abattoir. MATERIALS AND METHODS: The study utilized abattoir records spanning a period of 10 years (2004-2013). The records indicated that a total of 1,08,638 heads of cattle comprising n = 56,070 males and n = 52,570 females were slaughtered at the municipal abattoir during the study period. RESULT: Of these heads, n = 1230 (1.13%) (95% confidence interval [CI]: 1.07, 1.19) had tuberculous lesions. The annual occurrence during the study period varied significantly (p<0.001) from 0.53% (95% CI: 0.40, 0.67) to 1.87% (95% CI: 1.66, 2.10) in 2010 and 2012, respectively. Females had a significantly higher (p<0.001) prevalence of 2.10% (95% CI: 1.98, 2.23) compared with the males 0.23% (95% CI: 0.19, 0.27). The distribution of suspected gross bTB lesions in different organs showed 11.87% in the lungs, 5.93% in the liver, 1.14% in the heart, and 0.49% accounted for generalized bTB. However, none was observed on the lymph nodes and intestines. CONCLUSION: It can be concluded that bTB persists in Bauchi State with annual variations during the study period. This study highlights the importance of meat inspection as an important tool for detecting the presence of bTB lesions.

Seroprevalence of small ruminant caprine arthritis encephalitis lentivirus among goats from selected small ruminant farms in Selangor, Malaysia.

BACKGROUND AND AIM: Caprine arthritis encephalitis (CAE) is an important viral disease of small ruminants particularly in dairy goats with severe social and economic implication. Hence, this study was designed to determine the seroprevalence of CAE virus (CAEV) among goat population in selected small ruminant farms in Selangor and the risk factors associated with the occurrence of the disease. MATERIALS AND METHODS: Blood samples were collected from a total of 91 goats selected at random. Blood serum was harvested and used for competitive enzyme-linked immunosorbent assay test to detect antibodies against CAE virus. RESULTS: The result obtained showed that 8/91 (8.8%) of the goats were seropositive for CAEV. In addition, biosecurity management, source of origin and sex of the animal were observed to be important risk factors associated with the occurrence of CAE in goats. CONCLUSION: The findings of this study affirmed that the seroprevalence of CAEV infection among goat population in small ruminant farms in Selangor, Malaysia, is low. However, there is need to institute strict control measures such as testing and culling positive animals or separation of infected animals from those that tested negative to the disease for effective eradication of the disease.

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.

Seroprevalence of brucellosis among cattle slaughtered in three municipal abattoirs of Gombe state, Northeastern Nigeria.

AIM: A cross-sectional study was conducted to determine the seroprevalence of bovine brucellosis among cattle slaughtered at three municipal abattoirs of Gombe State, Nigeria. MATERIALS AND METHODS: A total of 200 blood samples collected from slaughtered cattle of different breeds (Sokoto Gudali - 50, White Fulani - 102, Red bororo - 34, and Crossbreeds - 14), sex (males - 19 and females - 181), and from different locations (Billiri - 30, Yamaltu Deba - 50, and Gombe - 120) were screened for brucellosis using rose bengal plate test (RBPT), serum agglutination test (SAT), and microtiter agglutination test (MAT). RESULTS: Of the 200 serum samples analyzed, 7 (3.5%), 10 (5.0%) and 18 (9.0%) were positive by RBPT, SAT and MAT, respectively. The results showed no statistically significant association between sex and seropositivity to bovine brucellosis. However, seropositivity of bovine brucellosis was higher in females than in males. Similarly, no statistically significant association was observed between breed and occurrence of bovine brucellosis. Moreover, the prevalence of brucellosis was higher in Sokoto Gudali as compared with the other breeds. Based on the study locations, higher seroprevalence was observed in animals screened from Billiri as compared with those from other locations (p<0.05). CONCLUSION: The presence of Brucella abortus antigen in the sera of slaughtered cattle in Gombe state poses a significant public health risk. Therefore, it is important to carry out further epidemiological studies on fulani herdsmen and cattle herds in the study area, in order to explore the risk factors associated with the occurrence and perpetuation of brucellosis among cattle herds, ascertain the prevalence and status of the disease among both farms and nomadic herds.