Four Vibrio cholerae O139 Bengal strains isolated from surface water were characterized by antibiotic resistance, plasmid profile, presence of cholera toxin gene and random amplification of polymorphic DNA (RAPD) analysis. All four strains exhibit multiple resistance towards the antibiotics tested with a multiple antibiotic resistance index of 0.5-0.66, and harboured a 2.0 MDa non-conjugative plasmid. The Vibrio cholerae O139 Bengal were positive for the cholera toxin gene. Antibiotyping and random amplification of polymorphic DNA analysis with four primers proved to be useful in discriminating the isolates. RAPD proved to be more sensitive. These results reveal that there is significant genetic diversity among the Vibrio cholerae O139 Bengal strains studied.
A baseline study was carried out to assess the metal concentrations and microbial contamination at selected Lake waters in and around Miri City, East Malaysia. Sixteen surface water samples were collected at specific Lakes in the environs of major settlement areas and recreational centers in Miri City. The Physico-chemical parameters [pH, Electrical Conductivity (EC) and Dissolved Oxygen (DO)], metals (Fe, Mn, Cu, Cd, Ni and Zn) and Escherichia coli (E. coli) were analysed. The concentrations of Fe, Mn and Ni have been found to be above the permissible limits of drinking water quality standards. The metals data have also been used for the calculation of heavy metal pollution index. Higher values of E. coli indicate microbial contamination in the Lake waters.
The bioremediation of polluted groundwater, wastewater aeration pond and biopond sites was investigated using bacteria isolated from these sites located at the oil refinery Terengganu Malaysia. Out of 62 isolates, only 16 isolates from groundwater (8) and wastewater aeration pond (3) and biopond (5) were chosen based on growth medium containing 1% (v/v) Tapis crude oil. Only four isolates; Acinetobacter faecalis, Staphylococcus sp., Pseudomonas putida and Neisseria elongata showed percentage biodegradation of crude oil more than 50% after 5 days using Mineral Salts Medium (MSM). The effect of physical parameters (temperature, pH and agitation) on growth by all four strains showed a maximum growth in MSM medium with 1% Tapis crude oil at 37 degrees C with pH 7 and agitation of 130 rpm.
Hydrocarbon pollution in marine ecosystems occurs mainly by accidental oil spills, deliberate discharge of ballast waters from oil tankers and bilge waste discharges; causing site pollution and serious adverse effects on aquatic environments as well as human health. A large number of petroleum hydrocarbons are biodegradable, thus bioremediation has become an important method for the restoration of oil polluted areas. In this research, a series of natural attenuation, crude oil (CO) and dispersed crude oil (DCO) bioremediation experiments of artificially crude oil contaminated seawater was carried out. Bacterial consortiums were identified as Acinetobacter, Alcaligenes, Bacillus, Pseudomonas and Vibrio. First order kinetics described the biodegradation of crude oil. Under abiotic conditions, oil removal was 19.9% while a maximum of 31.8% total petroleum hydrocarbons (TPH) removal was obtained in natural attenuation experiment. All DCO bioreactors demonstrated higher and faster removal than CO bioreactors. Half life times were 28, 32, 38 and 58 days for DCO and 31, 40, 50 and 75 days for CO with oil concentrations of 100, 500, 1000 and 2000 mg/L, respectively. The effectiveness of Corexit 9500 dispersant was monitored in the 45 day study; the results indicated that it improved the crude oil biodegradation rate.
The degradation of animal carcasses can lead to rapid waste release (e.g., pathogenic bacteria, viruses, prions, or parasites) and also result in nutrient accumulation in the surrounding environment. However, how viral profile responds and influences nutrient pool (carbon (C), nitrogen (N), phosphorus (P) and sulfur (S)) in polluted water caused by animal carcass decomposition had not been explored. Here, we combined metagenomic analysis, 16S rRNA gene sequencing and water physicochemical assessment to explore the response of viral communities under different temperatures (23 °C, 26 °C, 29 °C, 32 °C, and 35 °C) in water polluted by cadaver, as well as compare the contribution of viral/bacterial communities on water nutrient pool. We found that a total of 15,240 viral species were classified and mainly consisted of Siphoviridae. Both temperature and carrion reduced the viral diversity and abundance. Only a small portion of the viruses (∼8.8 %) had significant negative correlations with temperature, while most were not sensitive. Our results revealed that the viruses had lager contribution on nutrient pool than bacteria. Besides, viral-related functional genes involved in C, N, P and S cycling. These functional genes declined during carcass decomposition and covered part of the central nutrient cycle metabolism (including carbon sugar transformation, denitrification, P mineralization and extracelluar sulfate transfer, etc.). Our result implies that human regulation of virus communities may be more important than bacterial communities in regulating and managing polluted water quality and nutrition.
Leptospirosis, caused by the Leptospira spp., is endemic in humid subtropical and tropical climates. Several environmental settings can harbour the survival of Leptospira; hence, continuous surveillance of the high-risk areas is critical. This study was conducted to detect the presence of Leptospira in wet markets and paddy fields in Penang, Malaysia. A total of 62 soil and water samples were collected aseptically from three wet markets and two paddy fields. The samples were cultured twice on the sampling day and after two weeks of storage in the dark at ambient temperature. All environmental samples and positive cultures were subjected to deoxyribonucleic acid (DNA) extraction and polymerase chain reaction (PCR) targeting 16S rRNA, lipL32 and rrs genes. 16S rRNA-positive samples were further sent for sequencing. Of 62 samples, 21 (21/62, 33.9%) tested positive via culture and PCR. Wet markets significantly had a higher detection rate of leptospires (29%) than paddy fields (4.8%). Four Leptospira species (L. selangorensis, L. dzoumogneensis, L. mtsangambouensis, and L. meyeri) were identified in wet markets and three (L. meyeri, L. wolffii, and L. kmetyi) in paddy fields. Storing the environmental samples for two weeks before isolation yielded a higher positivity (11/14) than culturing samples on the sampling day (6/14). The presence of Leptospira in the environment of wet markets and paddy fields in Penang although primarily intermediate and saprophyte indicates the crucial need to implement preventive measures to prevent the possibility of leptospirosis infection among the workers.
Molecular characterization of a total of 54 isolates of Salmonella typhi from Santiago, Chile, was performed by pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with three restriction endonucleases: XbaI (5'-TCTAGA-3'), AvrII (5'-CCTAGG-3'), and SpeI (5'-ACTAGT-3'). Thirteen of the 54 isolates were obtained from environmental sources (sewage and river water), and the rest were isolates from clinical cases of typhoid fever. Considerable genetic diversity was detected among the human isolates obtained in 1994, as evidenced by the presence of 14 to 19 different PFGE patterns among 20 human isolates, with F (coefficient of similarity) values ranging from 0.69 to 1.0 (XbaI), 0.61 to 1.0 (AvrII), and 0.70 to 1.0 (SpeI). A total of eight phage types were detected among these 20 isolates, with 50% possessing the E1 or 46 phage type. There was no correlation between PFGE pattern and phage types. Similar diversity was seen among 21 isolates obtained in 1983, with 17 to 19 PFGE patterns detected and F values of 0.56 to 1.0 (XbaI), 0.55 to 1.0 (AvrII), and 0.67 to 1.0 (SpeI). Comparison of these two groups of human isolates obtained 11 years apart indicated that certain molecular types of S. typhi are shared and are able to persist for considerable periods. A similar degree of genetic diversity was also detected among the environmental isolates of S. typhi, for which 10 to 12 different PFGE patterns were detected among the 13 isolates analyzed, with F values ranging from 0.56 to 1.0 (XbaI), 0.52 to 1.0 (AvrII), and 0.69 to 1.0 (SpeI). Certain molecular types present among the environmental isolates of S. typhi were also found among the human isolates from the same time period, providing evidence for the epidemiological link between environmental reservoirs and human infection.
Adventure travel is becoming more popular, increasing the likelihood of contact with unusual pathogens. We investigated an outbreak of leptospirosis in "Eco-Challenge" multisport race athletes to determine illness etiology and implement public health measures. Of 304 athletes, we contacted 189 (62%) from the United States and 26 other countries. Eighty (42%) athletes met our case definition. Twenty-nine (36%) case-patients were hospitalized; none died. Logistic regression showed swimming in the Segama River (relative risk [RR]=2.0; 95% confidence interval [CI]=1.3 to 3.1) to be an independent risk factor. Twenty-six (68%) of 38 case-patients tested positive for leptospiral antibodies. Taking doxycycline before or during the race was protective (RR=0.4, 95% CI=0.2 to 1.2) for the 20 athletes who reported using it. Increased adventure travel may lead to more frequent exposure to leptospires, and preexposure chemoprophylaxis for leptospirosis (200 mg oral doxycycline/week) may decrease illness risk. Efforts are needed to inform adventure travel participants of unique infections such as leptospirosis.
Matched MeSH terms: Fresh Water/microbiology; Water Microbiology
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.
The application of an HPLC bioactivity profiling/microtiter plate technique in conjunction with microprobe NMR instrumentation and access to the AntiMarin database has led to the isolation of a new 1. In this example, 1 was isolated from a cytotoxic fraction of an extract obtained from marine-derived Streptomyces sp. cultured on Starch Casein Agar (SCA) medium. The 1D and 2D (1)H NMR and ESIMS data obtained from 20 μg of compound 1 fully defined the structure. The known 2 was also isolated and readily dereplicated using this approach.
We report the first study on the occurrence of high-level aminoglycoside-resistant (HLAR) Enterococci in coastal bathing waters and beach sand in Malaysia. None of the encountered isolates were resistant to high levels of gentamicin (500 μg/mL). However, high-level resistance to kanamycin (2,000 μg/mL) was observed in 14.2 % of tested isolates, the highest proportions observed being among beach sand isolates. High-level resistance to kanamycin was higher among Enterococcus faecalis and Enterococcus faecium than Enterococcus spp. Chi-square analysis showed no significant association between responses to tested antibiotics and the species allocation or source of isolation of all tested Enterococci. The species classification of encountered Enterococci resistance to vancomycin was highest among Enterococcus spp. (5.89 %) followed by E. faecium (4.785) and least among E. faecalis. A total of 160 isolates were also tested for virulence characteristics. On the whole, caseinase production was profoundly highest (15.01 %) while the least prevalent virulence characteristic observed among tested beach Enterococci was haemolysis of rabbit blood (3.65 %). A strong association was observed between the source of isolation and responses for each of caseinase (C = 0.47, V = 0.53) and slime (C = 0.50, V = 0.58) assays. Analysis of obtained spearman's coefficient showed a strong correlation between caseinase and each of the slime production (p = 0.04), gelatinase (p = 0.0035) and haemolytic activity on horse blood (p = 0.004), respectively. Suggestively, these are the main virulent characteristics of the studied beach Enterococci. Our findings suggest that recreational beaches may contribute to the dissemination of Enterococci with HLAR and virulence characteristics.
Photodynamic therapy (PDT) is a promising cancer treatment which involves activation of a photosensitizing drug with light to produce reactive oxygen species that kill tumors without causing damage to unirradiated normal tissues. To date, only Photofrin, Foscan and Levulan have been approved for clinical treatment of cancer. Tropical habitats such as those found in Malaysia are attractive sources of new therapeutic compounds as tremendous chemical diversity is found in a large number of plants, animals, marine- and micro-organisms. In our screening program for novel photosensitizers from nature, colorful strains of fungi (from Aspergillus and Penicillium genus) and bacteria (including actinomycetes and photosynthetic bacteria) were collected from various habitats in Peninsular Malaysia, such as coastal soil, peat soil, marine sponges and wastewater ponds. Methanolic extracts from a total of 85 different species were evaluated with a short-term cell viability assay for photo-cytotoxicity, where a promyelocytic leukemia cell-line, HL60 incubated with 20 microg/ml of extracts was irradiated with 9.6 J/cm(2) of a broad spectrum light. Two of these extracts, one from Rhodobacter sphaeroides (PBUM003) and one from Rhodopseudomonas palustris (PBUM001) showed moderate to strong photo-cytotoxicity. Subsequent bioassay guided isolation of the PBUM001 extract yielded known photosensitisers that are based on bacteriochlorophyll-a by comparing their molecular weight data, HPLC profiles and UV-vis absorption spectra with literature values, thereby demonstrating the validity of our screening approach.
Twenty-one Vibrio parahaemolyticus isolates representing 21 samples of coastal seawater from three beaches in peninsular Malaysia were found to be sensitive to streptomycin, norfloxacin and chloramphenicol. Resistance was observed to penicillin (100%), ampicillin (95.2%), carbenicilin (95.2%), erythromycin (95.2%), bacitracin (71.4%), cephalothin (28.6%), moxalactam (28.6%), kanamycin (19.1%), tetracycline (14.3%), nalidixic acid (9.5%) and gentamicin (9.5%). Plasmids of 2.6 to 35.8 mDa were detected among plasmid-containing isolates. All isolates carried the Vp-toxR gene specific to V. parahaemolyticus and were negative for the tdh gene, but only one isolate was positive for the trh gene. DNA fingerprinting of the isolates using ERIC-PCR and PFGE showed that the isolates belong to two major clonal groups, with several isolates from different locations in the same group, indicating the presence of similar strains in the different locations.
A novel bacterium, strain MUSC 273(T), was isolated from mangrove sediments of the Tanjung Lumpur river in the state of Pahang in peninsular Malaysia. The bacterium was yellow-pigmented, Gram-negative, rod-shaped and non-spore-forming. The taxonomy of strain MUSC 273(T) was studied by a polyphasic approach and the organism showed a range of phenotypic and chemotaxonomic properties consistent with those of the genus Novosphingobium. The 16S rRNA gene sequence of strain MUSC 273(T) showed the highest sequence similarity to those of Novosphingobium indicum H25(T) (96.8 %), N. naphthalenivorans TUT562(T) (96.4 %) and N. soli CC-TPE-1(T) (95.9 %) and lower sequence similarity to members of all other species of the genus Novosphingobium. Furthermore, in phylogenetic analyses based on the 16S rRNA gene sequence, strain MUSC 273(T) formed a distinct cluster with members of the genus Novosphingobium. DNA-DNA relatedness of strain MUSC 273(T) to the type strains of the most closely related species, N. indicum MCCC 1A01080(T) and N. naphthalenivorans DSM 18518(T), was 29.2 % (reciprocal 31.0 %) and 17 % (reciprocal 18 %), respectively. The major respiratory quinone was ubiquinone Q-10, the major polyamine was spermidine and the DNA G+C content was 63.3±0.1 mol%. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidyldimethylethanolamine, phosphatidylcholine and sphingoglycolipid. The major fatty acids were C18 : 1ω7c, C17 : 1ω6c, C16 : 0, C15 : 0 2-OH and C16 : 1ω7c. Comparison of BOX-PCR fingerprints indicated that strain MUSC 273(T) represented a unique DNA profile. The combined genotypic and phenotypic data showed that strain MUSC 273(T) represents a novel species of the genus Novosphingobium, for which the name Novosphingobium malaysiense sp. nov. is proposed. The type strain is MUSC 273(T) ( = DSM 27798(T) = MCCC 1A00645(T) = NBRC 109947(T)).
A sacred ritual well with continuously discharging of methane gas through its water body was studied for physicochemical and microbiological quality in three seasons and during ritual mass bathing. Most of the physicochemical parameters showed significant seasonal variations (P<0.05) and a sharp fluctuation during mass bathing. Dissolved oxygen (DO) was found negatively correlated with temperature (r=-0.384, P<0.05), biochemical oxygen demand (BOD) (r=-0.58, P<0.001) and ammonia (r=-0.738, P<0.001), while BOD showed positive correlation with chemical oxygen demand (COD) (r=0.762, P<0.001) and ammonia (r=0.83, P<0.001). Simple regression analysis also yielded significant linear relationship in DO vs. temperature (r(2)=0.147, P<0.05), DO vs. ammonia (r(2)=0.544, P<0.001) and BOD vs. DO (r(2)=0.336, P<0.001). A total of eight microbial indicators were studied and found that all of them increased unusually during mass bathing comparing with their respective seasonal values. Total coliforms (TC) were found positively correlated with fecal coliforms (FC) (r=0.971), FC with Escherichia coli (EC) (r=0.952), EC with intestinal enterococci (IE) (r=0.921), fecal streptococci (FS) with IE (r=0.953) and Staphylococcus aureus (SA) with Pseudomonas aeruginosa (PA) (r=0.946), which were significant at P<0.001. Some regression models showed significant linear relationship at P<0.001 with r(2) value of 0.943 for FC vs. TC, 0.907 for EC vs. FC, 0.869 for FS vs. FC, 0.848 for IE vs. EC and 0.909 for IE vs. FS. The overall results found in this study revealed that well water is suitable for bathing purpose but the religious activity considerably worsen its quality.
Klebsiella pneumoniae is an opportunistic pathogen that is responsible for causing nosocomial and community-acquired infections. Despite its common presence in soil and aquatic environments, the virulence potential of K. pneumoniae isolates of environmental origin is largely unknown. Hence, in this study, K. pneumoniae isolated from the estuarine waters and sediments of the Matang mangrove estuary were screened for potential virulence characteristics: antibiotic susceptibility, morphotype on Congo red agar, biofilm formation, presence of exopolysaccharide and capsule, possession of virulence genes (fimH, magA, ugE, wabG and rmpA) and their genomic fingerprints. A total of 55 strains of K. pneumoniae were isolated from both human-distributed sites (located along Sangga Besar River) and control sites (located along Selinsing River) where less human activity was observed, indicated that K. pneumoniae is ubiquitous in the environment. However, the detection of potentially virulent strains at the downstream of Kuala Sepetang village has suggested an anthropogenic contamination source. In conclusion, the findings from this study indicate that the Matang mangrove estuary could harbor potentially pathogenic K. pneumoniae with risk to public health. More studies are required to compare the environmental K. pneumoniae strains with the community-acquired K. pneumoniae strains.
A novel electrochemical DNA biosensor for ultrasensitive and selective quantitation of Escherichia coli DNA based on aminated hollow silica spheres (HSiSs) has been successfully developed. The HSiSs were synthesized with facile sonication and heating techniques. The HSiSs have an inner and an outer surface for DNA immobilization sites after they have been functionalized with 3-aminopropyltriethoxysilane. From field emission scanning electron microscopy images, the presence of pores was confirmed in the functionalized HSiSs. Furthermore, Brunauer-Emmett-Teller (BET) analysis indicated that the HSiSs have four times more surface area than silica spheres that have no pores. These aminated HSiSs were deposited onto a screen-printed carbon paste electrode containing a layer of gold nanoparticles (AuNPs) to form a AuNP/HSiS hybrid sensor membrane matrix. Aminated DNA probes were grafted onto the AuNP/HSiS-modified screen-printed electrode via imine covalent bonds with use of glutaraldehyde cross-linker. The DNA hybridization reaction was studied by differential pulse voltammetry using an anthraquinone redox intercalator as the electroactive DNA hybridization label. The DNA biosensor demonstrated a linear response over a wide target sequence concentration range of 1.0×10-12-1.0×10-2 μM, with a low detection limit of 8.17×10-14 μM (R2 = 0.99). The improved performance of the DNA biosensor appeared to be due to the hollow structure and rough surface morphology of the hollow silica particles, which greatly increased the total binding surface area for high DNA loading capacity. The HSiSs also facilitated molecule diffusion through the silica hollow structure, and substantially improved the overall DNA hybridization assay. Graphical abstract Step-by-step DNA biosensor fabrication based on aminated hollow silica spheres.
Monitoring the groundwater chemical composition and identifying the presence of pollutants is an integral part of any comprehensive groundwater management strategy. The present study was conducted in a part of West Tripura, northeast India, to investigate the presence and sources of trace metals in groundwater and the risk to human health due to direct ingestion of groundwater. Samples were collected from 68 locations twice a year from 2016 to 2018. Mixed Ca-Mg-HCO3, Ca-Cl and Ca-Mg-Cl were the main groundwater types. Hydrogeochemical methods showed groundwater mineralization due to (1) carbonate dissolution, (2) silicate weathering, (3) cation exchange processes and (4) anthropogenic sources. Occurrence of faecal coliforms increased in groundwater after monsoons. Nitrate and microbial contamination from wastewater infiltration were apparent. Iron, manganese, lead, cadmium and arsenic were above the drinking water limits prescribed by the Bureau of Indian Standards. Water quality index indicated 1.5% had poor, 8.7% had marginal, 16.2% had fair, 66.2% had good and 7.4% had excellent water quality. Correlation and principal component analysis reiterated the sources of major ions and trace metals identified from hydrogeochemical methods. Human exposure assessment suggests health risk due to high iron in groundwater. The presence of unsafe levels of trace metals in groundwater requires proper treatment measures before domestic use.
Matched MeSH terms: Water Microbiology; Waste Water/microbiology
The membrane-filter assay, GM1-ELISA, and DNA-DNA hybridization assay, were used to detect enterotoxigenic Escherichia coli (ETEC) in samples of water, weaning food, food preparation surface swabs, fingerprints of mothers, and the fingerprints and stools of children under 5 years of age, in 20 households in a Malaysian village. Weaning food and environmental samples were frequently contaminated by faecal coliforms, including ETEC. The membrane-filter assay detected and enumerated faecal coliforms and LT-ETEC in all types of water and weaning food samples. Highest concentrations of faecal coliforms and LT-ETEC were found in weaning food, followed by well-water, stored water and stored drinking water. The GM1-ELISA detected LT-ETEC in weaning food, food preparation surfaces, fingerprints and stool samples. The DNA-DNA hybridization assay detected a larger proportion of STa2-ETEC than the other toxotypes, either singly or in combination. All the assays in combination detected the presence of ETEC in all types of samples on at least one occasion in each household. It was not possible to classify households as consistently more or less contaminated with ETEC. On individual occasions it was possible to show a significant association of the presence of LT-ETEC between the fingerprints of children and their stools, fingerprints of mothers and children, and weaning food and the stools of the child consuming the food.