Molecular Characterisation of Escherichia Coli Isolated from Raw Milk and Village Chicken and Broiler Litter

Thirty five litter samples of village chickens, 35 broilers and 32 samples of raw milk were examined for the presence of E. coli. All samples were positive for E. coli. Three hundred and five isolates of E. coli were isolated from litter samples of village chickens (105 isolates), broilers (105 isolates) and raw milk samples (95 isolates). All the isolates were examined for antibiotic resistance, plasmid profiles and polymorphism using random amplification of polymorphic DNA (RAPD) analysis. Isolates isolated from litter of village chickens and broilers had a multiple antibiotic resistance (MAR) index of 0.31 to 0.75 and 0.44 to 0.69, respectively. Isolates isolated from raw milk had a MAR index of 0.31 to 0.88. High MAR index suggests that all the isolates originated from high risk sources. The E. coli isolates isolated from village chickens, broilers and raw milk samples were grouped into 34,30 and 28 distinct antibiotypes, respectively. Eighty (76.2%) and 99 (94.3%) isolates were found to harbour plasmids ranging in size from 1.2 to 64 MDa and 1.2 to 80 MDa among isolates isolated from village chickens and broilers, respectively. Isolates isolated from raw milk harboured plasmids ranging in size from 1.4 to 68 MDa. Based on their plasmid profiles, the E. coli isolates isolated from village chickens, broilers and raw milk were grouped into 28, 57 and 5 plasmid patterns, respectively. Three l0-mer oligonucleotides primers (Gen1-50-02, Gen1-50-09 and Gen1-50-10) were used to amplify genomic DNA. The profiles observed after electrophoretic separation for the three primers when combined together were able to distinguish the E. coli isolates from village chickens, broilers and raw milk into 92, 96 and 50 RAPD patterns, respectively. The large number of subgroups within these isolates indicates that there is a high degree of diversity within E. coli isolates, isolated from village chickens, broilers and raw milk samples. Isolates, isolated from village chickens, broilers and raw milk are genotypically diverse as shown by RAPD pattern, suggesting that different strains have been brought into the geographic region and strains already present have continued to evolve. These results suggest that RAPD-PCR assay is more discriminating than plasmid profiling and antibiotyping, and could be a valuable tool for epidemiological studies

Prevalence and Characterisation of Listeria Monocytogenes Isolated From Chicken and Beef

Listeria monocytogenes is an opportunistic haemolytic pathogen of humans and animals involved in several outbreaks and sporadic cases of listeriosis associated with the consumption of contaminated food. Growing antibiotic resistance demands the constant reassessment of antimicrobial efficacy, particularly in countries with wide antibiotic used in veterinary science, where higher resistance prevalence is often found and it has been reported that antibiotic resistant is associated with plasmid in bacteria. The purpose of this study is to characterize L. monocytogenes isolated from chicken and beef meat by antibiotic resistance test, plasmid DNA profile and arbitrarily primed (AP)- and repetitive sequence (RS)-PCR analysis and to determine whether there is any correlation between the antibiotic resistant and the incidence of plasmid. A total of 112 samples of chicken meat and 101 samples of beef were collected from different wet markets and night markets in Malaysia. Listeria spp. were detected in 39 (34.8%) of the chicken meat and 21 (20.8%) of the beef samples, respectively. L. monocytogenes were detected in 27 (24.1%) and 17 (16.8%) of the chicken meat and beef, respectively. Out of all the Listeria spp., 42 and 20 isolates from the chicken meat and beef, respectively, were confirmed as L. monocytogenes. The antibiotic susceptibility test with sixteen different types of antibiotics revealed that the highest prevalence of resistance among the L. monocytogenes isolates was observed against nalidixic acid (100%). None (0%) of the isolates were resistant to vancomycin. The L. monocytogenes strains showed resistance to at least two or more of the antibiotics tested with their multiple antibiotic resistance index (MARI) ranging between 0.13 to 0.63. Thirty-three different patterns of resistance were observed among all the L. monocytogenes isolates. Plasmid analysis of the L. monocytogenes strains revealed that 24 (38.7%) of the strains carried plasmid DNA ranging in sizes from 1.75 to 104.0 megadalton (MDa). Spearman’s rho correlation analysis was utilised to determine the correlation coefficient between MARI and the incidence of plasmid in the L. monocytogenes isolates. The result shows that there were no significant correlation between the MARI and the incidence of the plasmid (r= 0.143, p= 0.269). The AP- and RS-PCR analyses generated diverse PCR patterns with multiple DNA fragments in sizes ranging between 250 and 3000 bp. Dendrograms generated from the PCR patterns clustered the isolates into several groups and subgroups. PCR analyses with primers GEN1-50-02, GEN1-50-10 and repetitive primer clustered the chicken isolates into 3, 13 and 6 main groups; and the beef isolates into 2, 4 and 5 main groups; respectively. These main groups were further clustered into several subgroups. Isolation of L. monocytogenes from the beef and chicken meat suggests that there is a risk of acquiring listeriosis through these popular meat sources in Malaysia. L. monocytogenes will be killed by cooking and raw or semi raw meat are not consumed in Malaysia. However, L. monocytogenes in raw beef and chicken meat may pose a health hazard in kitchen if contaminating cooked food or other ready to eat food. Considering outbreaks of listeriosis associated with different foods, avoidance of consumption of insufficiently cooked meat by at-risk populations is recommended. Hygiene quality control of meat and its products must be recommended during the slaughtering, transportation carriage, other used devices and stuff carriers. Diligent enforcement of sanitary conditions of food contact surface and handling areas, and personal hygiene practices should reduce the potential contamination of meat products by L. monocytogenes at the retail level. Resistance to two or more antibiotics among these isolates was common. It is suggested that incorrect use of these antimicrobial agents for therapeutical purposes in veterinary science may lead to the development of antibiotic resistance. The high MARI value indicated that the L. monocytogenes strains were originated from high risk sources of contamination in the geographical area. L. monocytogenes can no longer be thought to be uniformly susceptible to antibiotics active against Gram-positive bacteria. Continued surveillance of emerging antimicrobial resistance among L. monocytogenes is important to ensure effective treatment of human listeriosis. The non-significant correlation between the antibiotic resistance and the incidence of plasmid suggest that the plasmids could be cryptic plasmids which have no apparent effect on the phenotype, especially the antibiotic resistance of the host. The presence of hemolysin gene in the beef and chicken meat isolates is of public health concern, as this virulence gene is associated with pathogenicity of the bacteria in human listeriosis. Both AP-PCR and RS-PCR, having high discriminatory power, have revealed the high diversity of food related L. monocytogenes isolates and their suitability to track down the contamination sources. The results suggest how complex the epidemiology of the L. monocytogenes in the study area, as a result of several strains as opposed to the widespread transmission of a single type. The results do not support that certain genetically related strains are better adapted to a particular food source. The genomic heterogeneity of the L. monocytogenes found in this study confirms the usefulness of the AP- and RS-PCR analyses for the rapid differentiation and grouping of this organism

Bacterial Diseases Transmitted Through Water Environment and Zoonotic Exposures

The health of the people is no longer separated from the health of the animals and the surrounding environments such as water, soil and air. The ongoing interactions among the people, animals and environments have changed because of certain factors, among others are the growing human population, climate change and the increasing movement of people and animals as well as animal product across borders. The changes have linked to the outbreak of known and emerging infectious zoonotic diseases, which are the diseases that spread between animals and human. A diverse range of microbial diseases can spread from wild and pet animals to human population. These includes bacteria, viruses, parasites, prions and other microbial agents. Water is one of the most vital resources for all life on earth. Contaminated water and poor sanitation have been associated with transmission of bacterial pathogens such as cholera, diarrhoea, dysentry, typhoid and many other diseases. The presence of diverse bacterial pathogens in the water environment with their characteristic of exhibiting multiple antibiotic resistance has become a global health issue. Water has become the reservoir for the antibiotic resistant bacteria and the transmission of the bacteria could be possible through certain routes including the drinking of contaminated water and through direct contact during recreational activities. The bacterial pathogens from the animal and water environment have caused significant morbidity and mortality among human population. Over a billion of the people have been afflicted and millions of annual deaths. The outbreak of zoonoses have not only affecting human health but also have adverse impact on regional economics. In this presentation, the presence of bacteria in the water environment and animals as well as the characteristics of the bacterial pathogens from the water and animals will be discussed. The possible routes of the transmission of the bacterial diseases form both sources to human will also be highlighted

Virulence genes detection among the antibiotic resistant Enterococcus faecalis isolated from bird industry in Borneo

The abuse of antibiotics usage in bird industry has resulted in the emerging antibiotic resistant Enterococci worldwide which has posed a threat clinically to human health. The present study was to screen and identify the potential virulence agents in antibiotic resistance E. faecalis in bird industry in Borneo. Enterococcus bacteria collected from the birds’ faeces and indoor air inside ten birdhouses were identified to species level and their antibiotic resistance was checked using antibiotic susceptibility discs. Specific primers using PCR assay were intended for the detection of four potential virulence genes (ace, AS, efaA, gelE). Out of the thirty-seven Enterococci faecal bacteria, the prevailing bacteria found were Enterococcus qallinacum (51%), Enterococcus faecalis (35%) and Enterococcus harae (8%). The airborne bacteria were reported as Enterococcus faecalis (5%) and Enterococcus qallinacum (1%). Twenty-seven percent of isolates were reported to have Multiple Antibiotic Resistance (MAR) index ≥ 0.2 with 9 distinct resistance patterns formed. E. faecalis showed higher resistance to vancomycin. Virulence genes were successfully reported in the 15 E. faecalis isolates. Sixty-seven percent of isolates were detected positive for four virulence genes, 27% possessed three (AS, efaA, gelE) genes and 6% possessed two (ace, AS) genes. Antibiotic resistance and virulence genes detection were significantly correlated. These virulence genes or antibiotic resistance genes were important in the pathogenesis of E. faecalis infections

The Rampancy of Multiple-Drug Resistant Bacteria Isolated from Surface Water of Satow Waterfall, Bau Sarawak

Antimicrobial resistance (AMR) is universally acknowledged as a serious threat to human health, wellbeing, and prosperity. Many therapeutically important antibiotic resistance genes are thought to have arisen in the environment. Antibiotics are no longer effective in treating infections caused by multi-and pan-resistant bacteria (dubbed "superbugs") that have spread rapidly around the world [1]. Furthermore, AMR bacteria and antimicrobial resistant genes can thrive and proliferate in natural waterbodies because they can act as both recipients and natural reservoirs for the bacteria and genes. Toxic exposure to AMR bacteria and ARGs puts recreationists in danger, reducing their ability to combat illnesses. Considering Satow Waterfall, Bau is a renowned water-based recreational destination, an assessment of their physiochemical water quality characteristics and the prevalence of AMR bacteria was conducted to corroborate the safety of the water. A total of nine water samples were collected at the surface water (0–30 cm depth) of the upstream, midstream, and downstream. The water quality index (WQI) of the sampling stations ranged from 92.60 to 95, classifying the water under Class I. However, the water is microbiologically polluted. During Trip 2, the highest level of coliform was found in the middle stream at 2.59x105 cfu/mL, which is above the regulatory standard set by the Malaysian Department of Environment (DOE). A total of 100 isolated bacteria were subjected to (GTG)5- genomic fingerprinting analysis to evaluate the degree of genetic similarity among the samples and thus enable the selection of appropriate clonal representatives. Fifty-four bacterial isolates were chosen as representatives and identified by 16SrDNA sequencing, which confirmed the existence of 21 bacterial genera as shown in Figure 1. All identified isolates were tested against 15 antibiotics of various classes, namely, streptomycin (S), Kanamycin (K), cefotaxime (CTX), cephalothin (KF), tetracycline (TE), doxycycline (DO), carbenicillin (CAR), sulfamethoxazole (SXT), ofloxacin (OFX), levofloxacin (LEV), erythromycin (E), meropenem (MEM), ertapenem (ETP), vancomycin (VA) and chloramphenicol (C) employing the Clinical and Laboratory Standards Institute (CLSI) protocols. The antibiotic susceptibility test (AST) demonstrated substantial resistance to SXT (41%), E (41%) and CAR (39%), as well as high susceptibility to C (85%), followed by LEV (78%) and OFX (72%). The Multiple Antibiotic Resistance Index (MARI) evaluation demonstrated the MARI varied from 0–0.60, with 33% of the isolates having a MARI greater than 0.20

Molecular typing among poultry isolates of Salmonella agona by PCR-based techniques and pulsed field gel electrophoresis (PFGE)

Nine genomic DNA of Salmonella agona isolated from poultry sources were characterised by three variations of PCR-based techniques [polymerase chain reaction (PCR)-ribotyping, enterobacterial repetitive intergenic consensuspolymerase chain reaction (ERIC-PCR), and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR)] and pulsed field gel electrophoresis (PFGE). These isolates originated from three different states of Peninsular Malaysia (Pulau Pinang, Negeri Sembilan and Selangor). The results of PCR-ribotyping, ERIC-PCR, RADP-PCR and PFGE to differentiate between nine isolates of S. agona were compared. The isolates were separated into three different genome types by PCR-ribotyping, nine and eight genome types by ERIC-PCR and PFGE, respectively

Presence of Bacillus cereus s.l. from ready-to-eat cereals (RTE) products in Sarawak

Bacillus cereus is a soil inhabitant gram positive bacterium, and is known to cause severe food poisoning. The objective of this study was to isolate and identify the presence of Bacillus cereus s.l. from selected ready to eat cereals purchased randomly from local supermarkets in Kuching and Kota Samarahan, Sarawak. The result showed that four of the 30 food samples were detected to be contaminated by B. cereus s.l.. Our findings suggested that it is important for the public to be aware of the safety of RTE cereals consumption, as it is possible that B. cereus s.l. may be present in high count number and pose hazardous health effects to the consumers

Isolation of indigenous arbuscular mycorrhizal fungi and selection of host plant for inoculum production

The objective of this study was to select a suitable host plant for mass production of indigenous arbuscular mycorrhizal (AM) fungi. Lemongrass and onion were compared for mass multiplication of Glomus species viz; Gl. mossea, Gl. geosporum and Gl. etunicatum. Spore count ranges from 17.67 (Gl. etunicatum) to 26.33 (Gl. mossea) g-1 soil under onion and lemongrass respectively. There was no significant difference (0.05) between Gl. Mossea and Gl. Geosporum in onion. Similarly, no significant difference was observed between Gl. Geosporum and Gl.etunicatum in lemongrass. Gl.mossea recorded the highest spore number followed by Gl.geosporum in both plant species. Root colonization % ranges from 67.33% (Gl.mossea) in onion to 80% (Gl.geosporum) in lemongrass. Colonization % of Gl.mossea and Gl.geosporum were statistically similar under individual plant species. Despite the lowest spore counts recorded by Gl.etunicatum, % root colonization was significantly (0.05) higher compared to Gl.mossea and Gl.geosporum in onion. Lemongrass recorded the highest average mean (77.33%) of root colonization % and spore counts (23.44) compared to onion (68.44%, 19.67). The study showed that AMF-plant interaction was host preference. Lemongrass favored the mass multiplication of Gl. mossea, Gl.geosporum and Gl.etunicatum thus, was the most suitable host plant compared to onion for inoculum productio

Effect of Arbuscularmycorrhizal Fungi and Poultry Manure on Growth and Nutrients Uptake by Maize under Field Condition

Field experiment was conducted to study the effect of arbuscularmycorrhizal fungi (AMF) and poultry manure (PM) on the growth and nutrients uptake by maize. The experiment was laid out in a randomized complete block design with 6 levels of PM in tones ha-1 (0, 4, 6, 8, 10, & 12) and 2 levels of AMF; (inoculated , +AMF and un-inoculated, -AMF) + recommended dose of NPK (RD NPK) chemical fertilizer; making 13 treatment combinations replicated 3 times. Plant growth (height, leaf area, root volume, shoot, and root dry weight biomass), root colonization (RC %) and uptakes of N, P, & K were assessed after six weeks growing period of maize. Inoculated plants and RD NPK recorded higher values for plant growth biometrics and nutrients uptake compared to un-inoculated plants.Poultry manure application enhanced RC % and AMF spore density with maximum value (43.00±38.70 %, 17.33±1.202 g-1 soil) at 12 t PM ha-1. Applying 12 t PM+AMF produced plants with the highest shoot and root biomass weights (199.00±3.055, 9.57±0.713 g) that were comparable to RD NPK (194.33±2.404, 9.27±0.376 g). Increase in shoot dry biomass due to AMF revealed, 19.3%, 20%, 12.2%, 7.2%, 7.9%, and 15.2% over 0, 4, 6, 8, 10 & 12 t PM ha-1 of un-inoculated plants. Applying 12 t PM+AMF recorded higher shoot dry biomass by 2.3% over RD NPK. RC % correlated positively with shoot biomass (R2= 0.753). Maximum N, P, & K uptake were recorded at 12 t PM+AMF compared to all the treatments. It could be concluded that PM have potentially stimulated AMF symbiosis, enhanced maize growth and nutrients uptake under normal field condition compared to chemical fertilizer, thus could be considered for maize production

Effects of short-term application of arbuscular mycorrhizal fungi and poultry manure on improvement of soil quality

A field study was conducted to investigate the short-term effect of arbuscular mycorrhizal fungi (AMF) and poultry manure (PM) application on improvements of some selected soil properties compared to NPK chemical fertilizer cropped to maize. PM was applied in tones (t) ha-1 (0, 4, 6, 8, 10 & 12) inoculated with AMF (+AMF) and without AMF (-AMF).Soil samples (0-15 cm) were collected from field according to treatments after maize growth to determine; physical (bulk density, water-stable aggregate [WSA]), chemical (pH, NO3-N, K, organic carbon[OC]& P) and biological properties (dehydrogenase, urease, phosphatase, AMF spore counts and root colonization [RC]). Results revealed significant improvement in soil properties due to application of poultry manure with and without AMF compared to control and chemical fertilizer. Integration of PM and AMF at 12 t PM ha-1 significantly increased soil pH, NO3-N, OC, available P, K, % WSA, dehydrogenase, urease and phosphatase activities with reduced bulk density compared to all the treatments. Residual soil NO3-N, available P & K content at 12 t PM+AMF indicated increment by 11.4% N, 5.8% P, 15.2% K, 25.9% OC, and 21.4% WSA over RD NPK. Spore counts and RC % increased with addition of poultry, the highest recorded at 12 t PM+AMF (17.33±1.202 g -1 soil, 43.00±38.70 %). There was strong positive correlations between RC% and dehydrogenase (R2= 0.854), phosphatases (R2= 0.894), urease (R2= 0.935) activities and WSA (R2= 0.958). Results suggested that application of poultry manure and AMF inoculation could improve soil physical, chemical and biological properties thus could be regarded as a reliable option for maintenance of soil quality and sustainability of crop production