Catfish is one of the most cultivated species worldwide. Antibiotics are usually used in catfish farming as therapeutic and prophylactic agents. In the USA, only oxytetracycline, a combination of sulfadimethoxine and ormetoprim, and florfenicol are approved by the Food Drug Administration for specific fish species (e.g., catfish and salmonids) and their specific diseases. Misuse of antibiotics as prophylactic agents in disease prevention, however, is common and contributes in the development of antibiotic resistance. Various studies had reported on antibiotic residues and/or resistance in farmed species, feral fish, water column, sediments, and, in a lesser content, among farm workers. Ninety percent of the world aquaculture production is carried out in developing countries, which lack regulations and enforcement on the use of antibiotics. Hence, efforts are needed to promote the development and enforcement of such a regulatory structure. Alternatives to antibiotics such as antibacterial vaccines, bacteriophages and their lysins, and probiotics have been applied to curtail the increasing emergence of antibiotic-resistant bacteria due to the imprudent application of antibiotics in aquaculture.
Strains of Aeromonas hydrophila isolates from skin lesions of the common freshwater fish, Telapia mossambica, were screened for the presence of plasmid DNA by agarose gel electrophoresis and tested for susceptibility to 10 antimicrobial agents. Of the 21 fish isolates examined, all were resistant to ampicillin and sensitive to gentamycin. Most isolates were resistant to streptomycin (57%), tetracycline (48%) and erythromycin (43%). While seven of 21 isolates harboured plasmids, with sizes ranging from 3 to 63.4 kilobase pair (kb), it was only possible to associate the presence of a plasmid with antibiotic resistance (ampicillin and tetracycline) in strain AH11. Both the plasmid and the associated antimicrobial resistance could be transferred to an Escherichia coli recipient by single-step conjugation at a frequency of 4.3 x 10(-3) transconjugants per donor cell.
Salmonella enterica serovar Paratyphi B (S. Paratyphi B) is a major foodborne pathogen distributed all over the world. However, little is known about the antibiotic resistance, genetic relatedness and virulence profile of S. Paratyphi B isolated from leafy vegetables and the processing environment in Malaysia. In this study, 6 S. Paratyphi B isolates were recovered from different vegetables and drain water of processing areas obtained from fresh food markets in Malaysia. The isolates were characterized by antibiogram, Pulsed-field gel electrophoresis (PFGE) and virulence genes. Antibiotic susceptibility test showed that 3 of the isolates were resistant to the antibiotics. These include S. Paratyphi B SP251 isolate, which was resistant to chloramphenicol, ampicillin, sulfonamides and streptomycin; Isolate SP246 which was resistant to chloramphenicol, sulfonamides and streptomycin and Isolate SP235 showing resistance to nalidixic acid only. PFGE subtyped the 6 S. Paratyphi B isolates into 6 distinct XbaI-pulsotypes, with a wide range of genetic similarity (0.55 to 0.9). The isolates from different sources and fresh food markets location were genetically diverse. Thirteen (tolC, orgA, spaN, prgH, sipB, invA, pefA, sofB, msgA, cdtB, pagC, spiA and spvB) out of the 17 virulence genes tested were found in all of the S. Paratyphi B isolates. Another gene (lpfC), was found only in one isolate (SP051). None of the isolates possessed sifA, sitC and ironN genes. In summary, this study provides unique information on antibiotic resistance, genetic relatedness, and virulotyping of S. Paratyphi B isolated from leafy vegetables and processing environment.
A total of 35 Burkholderia pseudomallei isolates from Thailand (16 clinical and eight soil isolates) and Malaysia (seven animal, two isolate each from clinical and soil) were investigated by their antimicrobial resistance, plasmid profiles and were typed by randomly amplified polymorphic DNA analysis. All isolates were found to be resistant to six or more of the 12 antimicrobial agents tested. Only two small plasmids of 1.8 and 2.4 megadalton were detected in two clinical isolates from Thailand. RAPD analysis with primer GEN2-60-09 resulted in the identification of 35 RAPD-types among the 35 isolates. The constructed dendrogram differentiated the 35 isolates into two main clusters and a single isolate. The wide genetic biodiversity among the 35 isolates indicate that RAPD-PCR can be a useful method to differentiate unrelated B. pseudomallei in epidemiological investigation.
Antibiotic susceptibility, plasmid profiles and random amplification of polymorphic DNA (RAPD) were used to study strains of Vibrio vulnificus isolated from cockles (Anadara granosa). Thirty-six isolates were analyzed. The prevalent biotypes were 1 (72.2% of the isolates) and 2 (27.8%). Among these, 21 strains of biotype 1 and two strains of biotype 2 contained plasmid DNA bands ranging in size from 1.4 to 9.7 MDa. Thirty-one (83.3%) were found to be resistant to one or more of the antimicrobial agents tested, however no specific correlation between antimicrobial resistance patterns and a single biotype was found. In addition, no particular plasmid profile was predictive of a particular pattern of antibiotic susceptibility. Two primers produced polymorphisms in all strains tested, producing bands ranging from 0.25 to 2.7 kb, indicating a high variability among both biotype 1 and biotype 2 of the V. vulnificus strains investigated. RAPD identity across biotypes was also observed among Vibrio vulnificus strains.
Composition, physicochemical properties and enzyme inactivation kinetics of coconut water were compared between immature (IMC), mature (MC) and overly-mature coconuts (OMC). Among the samples studied, pH, turbidity and mineral contents for OMC water was the highest, whereas water volume, titratable acidity, total soluble solids and total phenolics content for OMC water were the lowest. Maturity was found to affect sugar contents. Sucrose content was found to increase with maturity, and the reverse trend was observed for fructose and glucose. Enzyme activity assessment showed that polyphenol oxidase (PPO) in all samples was more heat resistant than peroxidase (POD). Compared to IMC and MC, PPO and POD from OMC water showed the lowest thermal resistance, with D83.3°C=243.9s (z=27.9°C), and D83.3°C=129.9s (z=19.5°C), respectively.
This article describes the Pulsed-field gel electrophoresis clustering of the predominantSalmonellastrains (Salmonellaser. Albany,Salmonellaser. Brancaster, andSalmonellaser. Corvallis) isolated from poultry and processing environment in wet market and small-scale processing plant in Penang and Perlis, the northern states of Malaysia. Agar disk diffusion assay was performed to determine the phenotypic antibiotic resistance of theseSalmonellastrains. The most common antibiograms among the three predominantSalmonellaserovars were reported. The presence of integrase genes and antibiotic resistance genes conferring to resistance against β-lactams, aminoglycosides, tetracyclines, quinolones, sulphonamides and chloramphenicol, was detected via PCR amplification.
Paralactobacillus selangorensis gen. nov., sp. nov. is described. This organism, isolated from a Malaysian food ingredient called chili bo, is an obligatory homofermentative, rod-shaped lactic acid bacterium. The G+C content is 46.1-46.2+/-0.3 mol%. Earlier 16S rRNA studies showed that this organism constitutes a new taxon distantly related to the Lactobacillus casei-Pediococcus group. A phenotypic description that distinguishes Paralactobacillus selangorensis from other genera of lactic acid bacteria is presented. The type strain of Paralactobacillus selangorensis is LMG 17710T.
A total of 57 Vibrio vulnificus isolates from coastal water were characterized for their antimicrobial resistance, plasmid profiles and were typed by the PCR-based techniques: a random amplification of polymorphic DNA (RAPD) method and the enterobacterial repetitive intergenic consensus sequence (ERIC) method. All isolates were susceptible to chloramphenicol, nalidixic acid, tetracycline and trimethoprim-sulfamethoxazole. Fifty-one isolates were resistant to one or more of the other antibiotics tested. Plasmid analysis indicated that only 18 isolates carried small plasmids of 1.6 to 16 megadaltons. Analysis of the RAPD and ERIC DNA fingerprints of the V. vulnificus isolates with Gel Compare and cluster analysis software revealed significant genetic heterogeneity among these isolates. The combination of RAPD and ERIC analysis allowed us to distinguish all isolates. Thus, the combination of the two techniques is recommended for epidemiological investigation.
Twenty-five and three strains of Escherichia coli O157:H7 were identified from 25 tenderloin beef and three chicken meat burger samples, respectively. The bacteria were recovered using the immunomagnetic separation procedure followed by selective plating on sorbitol MacConkey agar and were identified as E. coli serotype O157:H7 with three primer pairs that amplified fragments of the SLT-I, SLT-II and H7 genes in PCR assays. Susceptibility testing to 14 antibiotics showed that all were resistant to two or more antibiotics tested. Although all 28 strains contained plasmid, there was very little variation in the plasmid sizes observed. The most common plasmid of 60 MDa was detected in all strains. We used DNA fingerprinting by randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) to compare the 28 E. coli O157:H7 strains. At a similarity level of 90%, the results of PFGE after restriction with XbaI separated the E. coli O157:H7 strains into 28 single isolates, whereas RAPD using a single 10-mer oligonucleotides separated the E. coli O157:H7 strains into two clusters and 22 single isolates. These typing methods should aid in the epidemiological clarification of the E. coli O157:H7 in the study area.
Twelve strains of Escherichia coli O157:H7 were isolated from 9 of 25 beef samples purchased from retail stores in Malaysia. These strains produced Shiga toxin 2 with or without Shiga toxin 1 and had the eae gene and a 60-MDa plasmid. The antibiograms and the profiles of the arbitrarily primed PCR of the strains were diverse, suggesting that the strains may have originated from diverse sources.
Twenty-eight isolates of E. faecalis and 5 isolates of E. hirae were isolated from chicken samples obtained from markets in Sri Serdang, Selangor. They were tested for susceptibility to vancomycin and other antimicrobial agents. All of the isolates showed multiple resistance to the antibiotic tested. All Enterococcus spp. were resistant (100%) to ceftaxidime, cephalothin, erythromycin, gentamicin, kanamycin, nalidixic acid and streptomycin. Resistance was also observed to norfloxacin (97%), tetracycline (91%), penicillin (85%), bacitracin (82%), chloramphenicol (61%) and the least resistance was to ampicillin (27%). High prevalence to vancomycin resistance was detected among the E. faecalis (27of 28) and E. hirae (4 of 5) isolates. The multiple antibiotic resistance index ranging between 0.64 to 1.0 showed that all strains tested originated from high-risk contamination. Plasmid profile analysis of Enterococcus spp. revealed plasmid DNA bands ranging in size from 1.3 to 35.8 megadalton but some isolates were plasmidless. No correlation could be made between plasmid patterns and antibiotic resistance.
Sugarcane juice deteriorates rapidly as enzymatic browning and microbial spoilage take place soon after juice extraction. Therefore, the effects of pasteurization treatment (70ºC for 10 minutes) and addition of calamansi juice at different concentrations (0, 1.0, 1.5, and 3.0%, v/v) on the physicochemical (color, pH, total soluble solid, titratable acidity, and peroxidase enzymatic activity), microbiological (total plate count, yeast and mold), and sensory characteristics of black stem sugarcane juice were investigated. Quantitative descriptive analysis (QDA®) was conducted by eight trained panelists to determine the intensity of the sensory attributes of sugarcane juice, such as brownish color, grassy aroma, citrus aroma, sweetness, sourness, sweet aftertaste, and overall acceptability using a 15 cm unstructured line scale. The results showed that the pasteurization treatment significantly (p ˂ 0.05) increased the L* value (lightness) of sugarcane juice at 0% (v/v) of calamansi juice. Increasing the concentration of calamansi juice decreased the pH value and increased the titratable acidity of the sugarcane juice at 5% significance level. The relative effectiveness in reducing the peroxidase (POD) enzymatic activity and microbial load of sugarcane juice was shown after the addition of calamansi juice and pasteurization treatment, respectively. Significant (p ˂ 0.05) changes in the intensity of brownish color, grassy aroma, and sweet aftertaste of sugarcane juice were observed after the pasteurization treatment at 0% (v/v) calamansi juice; whereas, the addition of calamansi juice at different concentrations increased the intensity of citrus aroma and sourness, but decreased the intensity of grassy aroma and sweet aftertaste of sugarcane juice at 5% significance level. Hence, pasteurization treatment coupled with the addition of 1.5% (v/v) calamansi juice was effective in achieving microbial stability and consumer’s acceptability on sugarcane juice.
Thermal processing of green coconut water (GCW) caused non-enzymic browning and development of rancidity. Effect of the addition of several combinations of ascorbic acid (AA) (0 to 100 ppm) and sodium metabisulfite (SMB) (0 to 30 ppm) on brown discolouration and rancidity of GCW during elevated thermal processing (121°C for 5 min at 15 psi) was investigated. Addition of AA and/or SMB significantly (P
The objective of the present study was to evaluate the effect of electroporation on the membrane properties of lactobacilli and their ability to remove cholesterol in vitro. The growth of lactobacilli cells treated at 7.5 kV/cm for 4 ms was increased by 0.89 to 1.96 log(10) cfu/mL upon fermentation at 37 °C for 20 h, the increase being attributed to the reversible and transient formation of pores and defragmentation of clumped cells. In addition, an increase of cholesterol assimilation as high as 127.2% was observed for most cells electroporated at a field strength of 7.5 kV/cm for 3.5 ms compared with a lower field strength of 2.5 kV/cm. Electroporation also increased the incorporation of cholesterol into the cellular membrane, as shown by an increased cholesterol:phospholipids ratio (50.0-59.6%) upon treatment at 7.5 kV/cm compared with treatment at 2.5 kV/cm. Saturation of cholesterol was observed in different regions of the membrane bilayer such as upper phospholipids, apolar tail, and polar heads, as indicated by fluorescence anisotropy using 3 fluorescent probes. Electroporation could be a useful technique to increase the ability of lactobacilli to remove cholesterol for possible use as cholesterol-lowering adjuncts in the future.
The effectiveness of two different rapid methods involving the 3M™ molecular detection assay Listeria and the 3M™ Petrifilm environmental Listeria Plate were evaluated for the rapid detection of Listeria from naturally contaminated vegetables and chicken-processing environments against the standard culture-based method. A total of 178 samples were examined for the presence of Listeria. A total of 47/178 (26.4%) by standard ISO culture-based method (EN ISO 11290-1), 42/178 (23.6%) by 3M™ MDA Listeria and 40/178 (22.5%) by 3M™ Petrifilm EL Plate showed positive results, respectively. The accuracy, sensitivity, specificity, positive predictive value, and negative predictive value for 3M™ MDA Listeria and 3M™ Petrifilm EL Plate were 97.2, 89.4, 99.3, 97.7, 96.4% and 96.1, 85.1, 100.0, 100.0, 94.9%, respectively. Based on the Cohen's Kappa value, there was a complete and robust concordance between 3M™ MDA Listeria (0.911) and 3M™ Petrifilm EL Plates (0.894) as compared to the standard culture-based method.
Three strains of Gram-staining-positive, coccus-shaped, lactic acid bacteria, designated as HibF3T, HibF2 and HibF5 were isolated from fresh flowers of hibiscus, and a fourth, DF1T, was isolated from fresh flowers of durian tree, in Penang, Malaysia. Taxonomic characterisation was performed by polyphasic analysis. Sequence similarities of the 16S rRNA gene and the housekeeping rpoA and pheS genes of these strains with their closely-related lactococcal and streptococcal relatives were 92-94, 78 and 81 %, respectively. The results of phylogenetic analysis indicated that strains DF1T, HibF2, HibF5 and HibF3T were clustered together but were clearly separated from species of the genera Streptococcus and Lactococcus, indicating that they represent members of a novel genus of the family Streptococcaceae. Calculation of average nucleotide identity (ANI) values between the genomes of DF1T and HibF3T yielded values of 92.50-92.93 %. ANI values below the cut-off value and distinctive chemotaxonomic characteristics supported the hypothesis that these strains represented two novel species. Major cellular fatty acids in DF1T, HibF2 and HibF5 were C18 : 1ω7c and C16 : 0, while C12 : 0 and C14 : 0 were also dominant, in addition to C18 : 1ω7c and C16 : 0, in HibF3T. A novel genus is proposed with the name Floricoccus gen. nov. which consists of two species, Floricoccus tropicus sp. nov as the type species, and Floricoccus penangensis sp. nov. The respective type strains are DF1T (=LMG 29833T=JCM 31733T) and HibF3T (=LMG 29831T=DSM 31735T).
A total of 43 Salmonella enterica isolates belonging to different serovars (Salmonella Albany, Salmonella Agona, Salmonella Corvallis, Salmonella Stanley, Salmonella Typhimurium, Salmonella Mikawasima, and Salmonella Bovismorbificans) were isolated from catfish (Clarias gariepinus) and tilapia (Tilapia mossambica) obtained from nine wet markets and eight ponds in Penang, Malaysia. Thirteen, 19, and 11 isolates were isolated from 9 of 32 catfish, 14 of 32 tilapia, and 11 of 44 water samples, respectively. Fish reared in ponds were fed chicken offal, spoiled eggs, and commercial fish feed. The genetic relatedness of these Salmonella isolates was determined by random amplified polymorphic DNA PCR (RAPD-PCR) using primer OPC2, repetitive extragenic palindromic PCR (REP-PCR), and pulsed-field gel electrophoresis (PFGE). Composite analysis of the RAPD-PCR, REP-PCR, and PFGE results showed that the Salmonella serovars could be differentiated into six clusters and 15 singletons. RAPD-PCR differentiated the Salmonella isolates into 11 clusters and 10 singletons, while REP-PCR differentiated them into 4 clusters and 1 singleton. PFGE differentiated the Salmonella isolates into seven clusters and seven singletons. The close genetic relationship of Salmonella isolates from catfish or tilapia obtained from different ponds, irrespective of the type of feed given, may be caused by several factors, such as the quality of the water, density of fish, and size of ponds.
The incidence of food-borne salmonellosis due to Salmonella enterica serotype Weltevreden is reported to be on the increase in Malaysia. The pulsed-field gel electrophoresis (PFGE) subtyping method was used to assess the extent of genetic diversity and clonality of Salmonella serotype Weltevreden strains from humans and the environment. PFGE of XbaI-digested chromosomal DNA from 95 strains of Salmonella serotype Weltevreden gave 39 distinct profiles with a wide range of Dice coefficients (0.27 to 1.00), indicating that PFGE is very discriminative and that multiple clones of Salmonella serotype Weltevreden exist among clinical and environmental isolates. Strains of one dominant pulsotype (pulsotype X1/X2) appeared to be endemic in this region, as they were consistently recovered from humans with salmonellosis between 1996 and 2001 and from raw vegetables. In addition, the sharing of similar PFGE profiles among isolates from humans, vegetables, and beef provides indirect evidence of the possible transmission of salmonellosis from contaminated raw vegetables and meat to humans. Furthermore, the recurrence of PFGE profile X21 among isolates found in samples of vegetables from one wet market indicated the persistence of this clone. The environment in the wet markets may represent a major source of cross-contamination of vegetables with Salmonella serotype Weltevreden. Antibiotic sensitivity tests showed that the clinical isolates of Salmonella serotype Weltevreden remained drug sensitive but that the vegetable isolates were resistant to at least two antibiotics. To the best of our knowledge, this is the first study to compare clinical and environmental isolates of Salmonella serotype Weltevreden in Malaysia.