MALAYSIAN JOURNAL OF MICROBIOLOGY
2020 - Volume 16
Aims: The coagulase-negative staphylococci (CoNS) are a group of Staphylococcus that is gaining clinical significance as major agents of nosocomial infections, especially amongst neonates and immuno-compromised patients. The identification of CoNS remains problematic, and there has been little information on their molecular genotyping. The overall aim of this study was to evaluate Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR) as a rapid and cost-effective tool for the genotyping of CoNS isolates from within a hospital setting.
Methodology and results: A total of 200 isolates of CoNS were collected from Hospital Tuanku Ampuan Rahimah, Klang, Malaysia and identified via sodA gene sequence analysis. Genetic diversity among the isolates was evaluated using the ERIC-PCR. The most frequently isolated species was S. epidermidis (37%) followed by S. haemolyticus (30%), S. hominis (18%) and S. capitis (8.5%). ERIC-PCR was found to be efficient for the differentiation of S. hominis isolates with a discriminatory index (DI) of 0.949 and satisfactory for S. epidermidis isolates at DI of 0.808. Poor discriminatory power was observed in S. haemolyticus (0.377) and S. capitis (0.111). The majority of the S. haemolyticus and S. capitis isolates were found to be genetically homogenous which imply that the source of these infections are due to hospital-derived contaminants. In contrast, the S. epidermidis and S. hominis strains displayed high genetic diversity suggesting the presence of different endemic strains and inflow of exogenous strains brought in by non-local residents.
Conclusion, significance and impact of study: ERIC-PCR is a useful tool to differentiate and track different selected species of CoNS.
Aims: Leptospirosis is an infectious disease that is endemic to many tropical regions. Large epidemics usually happen after heavy rainfall and flooding. This potentially fatal zoonosis is caused by pathogenic bacteria belonging to the genus Leptospira. Leptospirosis can be diagnosed using specific biomarkers such as target genes and virulence indicators that are well preserved across various Leptospira spp., including those that are prevalent in clinical samples and in the environment. To date, several pathogenicity-determinant genes, including lipL32 and lipL41, have been described and used for diagnosing leptospirosis. However, prevalence of these genes in leptospiral strains is unclear.
Methodology and results: In the present study, we assessed the distribution of eight pathogenicity-determinant genes in reference Leptospira strains and environmental isolates in Malaysia, by polymerase chain reaction (PCR). We found that only lipL32 and ligB were consistently expressed in all pathogenic Leptospira strains compared with the other tested genes. Moreover, our results suggested that the use of lipL41, lipL21, ompL1, lfb1, ligA, and ligC as biomarkers could incorrectly misdetect pathogenic Leptospira strains present in the environment.
Conclusion: Thus, our results suggest that the pathogenicity-determinant genes lipL32 and ligB can be used as biomarkers for detection pathogenic Leptospira.
Aims: The most common transmission route of hepatitis C virus (HCV) is via blood transfusion. Therefore, the screening of HCV is necessary to be performed regularly for all the volunteer blood donors. The prevalence of HCV subtypes varies in different geographical areas. The aim of this study is to identify the HCV genotypes of the HCV-RNA positive samples and performed serological and molecular characterization of HCV among blood donors from blood transfusion center of Tuban, East Java, Indonesia collected during the year of 2015.
Methodology and results: All blood donor samples were screened by enzyme-linked immunosorbent assay (ELISA) for anti-HCV. Reverse Transcription- Polymerase chain reaction (RT-PCR) was performed to detect the HCV-RNA. Subsequently, the HCV-RNA positive samples were genotyped using direct sequencing followed by subtype/genotype and phylogenetic analysis. Of the 500 blood samples, 7 were positive for anti-HCV antibody (1.4%) and 6 out of 7 (85.71%) were determined to be HCV-RNA positive. Among HCV-RNA carriers, genotyping showed genotypes 1 was the most prevalent. HCV subtypes 1a and 1b were detected in total of 4 out of 6 individuals (66.67%), two individuals for each. HCV subtypes 2a and genotype 1 were the least frequent among blood donors (each counted for 16.67%, respectively).
Conclusion, significance and impact for study: The prevalence of HCV found in this study is considerably low. The identification of genotypes 1a and 1b as major HCV genotypes circulating in blood donors in the region of Tuban may contribute in a better medical management towards HCV carriers.
Aims: Staphylococcus aureus is the most common pathogen found in humans, animals and foods worldwide. Vegetables contain essential vitamins, minerals, and fibres that may aid in protecting humans from chronic diseases and promote good health. This investigation aimed to find the prevalence of S. aureus contamination and antimicrobial resistance pattern of isolates from leafy vegetables in Peninsular Malaysia.
Methodology and results: A total of 397 samples, comprised of 16 different vegetables, were obtained from vendors in selected wet markets. Of the 397 samples, S. aureus was detected in 42 samples (10.6%) in which 9 (21.4%) were positive for methicillin-resistant Staphylococcus aureus (MRSA). The S. aureus isolates showed 52.3% resistance to tetracycline, followed by kanamycin (40.5%), penicillin G (35.7%), streptomycin (33.3%), oxacillin (21.4%) and cefoxitin (19.0%). The multiple antibiotic resistance indexes of S. aureus isolates varied from 0.21 to 0.50. The predominant antimicrobial resistance profiles of S. aureus were PDaOxSTeL (n=5), PDaSTeCe (n=4), TeKCnSxtC (n=3) and TeKCipQd, respectively.
Conclusion, significance and impact of study: The findings of this study revealed the consumption of raw or minimally prepared fresh leafy vegetables could be a possible source of infection with antimicrobial-resistant S. aureus.
Aim: Dandruff is characterized by the white flakes in the hairs and can be caused by dry skin and mainly by fungal growth of Malassezia yeasts. To treat this condition mainly synthetic type of active ingredient shampoos are used which gave severe adverse effect toward users like hair fall and weakening of the hair roots. In this study, we formulate a shampoo containing an active ingredient “Propolis” an antimicrobial agent which is also known as bee glue. Formed by the combination of bee wax and flower exudate collected from the flower bud.
Methodology and results: In this study, propolis extracts have been used as the antimicrobial agent in the shampoo formulation for treating dandruff-causing bacteria, Staphylococcus aureus. Interestingly, the developed propolis shampoo showed is 10-fold more effective against S. aureus compared to the propolis extracts alone. This is due to the presence of Tween 80 as the surfactant used in the formulation which adds to this antibacterial effect. The formulated shampoo was also compared with the commercially available shampoo (Safi Shayla brand) for physicochemical properties. Overall evaluation of the shampoo with propolis found to have pH (6-7), good foaming ability, less wetting time, a good percentage of solid content and viscosity. Also, the formulated shampoo has greater stability under accelerated room temperature and accelerated the ageing condition.
Conclusion, significance and impact of study:This study demonstrated the propolis extracted can be used as a potential antimicrobial agent. As it came from the natural resource the acceptance will high among the consumers.
Aims: Rice blast disease caused by Pyricularia oryzae is one of the major biotic diseases of rice in Sarawak, Malaysian Borneo. This study aims to isolate and characterize rice blast fungus obtained from infected leaf collected from four different divisions in Sarawak, viz, Miri, Serian, Sri Aman, and Kuching.
Methodology and results: Twelve succeeded isolates were pre-identified as P. oryzae by morphological characteristics of spores, followed by verification through (internal transcribed spacer) ITS sequencing. The isolates were evaluated for morphological characteristics, growth rate and sporulation rate, which were grown on two types of media, (filtered oatmeal agar) FOMA and (potato dextrose agar) PDA. Morphological characterization showed that the colony surface of the different isolates varied from smooth and fluffy to rough and flattened mycelia; some were with the present of concentric rings, and some with aerial mycelia. The growth rate and sporulation rate of each isolate varied based on types of media used. Most of the isolates grew faster on PDA than on FOMA but produced higher number of spores on FOMA as compared to PDA.
Conclusion, significance and impact of study: This preliminary study showed that there were variations observed based on morphological and physiological characterization for the different isolates collected in Sarawak, Malaysian Borneo. This study is the first step towards understanding variation in the population of P. oryzae from Sarawak.
Aims: Mycorrhiza has an important role as a biocontrol agent. Its association with Phalaenopsis amabilis was molecularly identified through rDNA-ITS sequence analysis. The aims of the study were to identify molecular of orchids mycorrhiza isolate from native tropical orchids in Indonesia, conducted as one of native orchid conservation efforts in Indonesia.
Methodology and results: One group of Ceratobasidium were isolated from the root of orchid plant in Yogyakarta based on morphological and microscopical analysis. The results of molecular analysis showed 600-750 bp of DNA products located on the ITS1-5.8S-ITS4 region. The sequenced products showed insertion and substitution occurances, which may result in strain diversity and possible variation. Reconstruction of phylogenetic trees using Maximum Parsimony and Bootstrap-1000 approach showed showed the Indonesian isolate is at the basal clade and already far apart from the other isolates.
Conclusion, significance and impact of study: Isolate Ceratobasidium from Yogyakarta, Indonesia successfully isolated based on identification of rDNA-ITS sequences. Results of this study were expected to become the basic information in an effort of native orchid cultivation and protection against infectious diseases in Indonesia. The study was the first to report regarding Ceratobasidium isolated from native tropical orchids in Indonesia.
Aims: Cordyceps militaris is a medicinal mushroom from Ascomycota. The aims of this study were to explore and identify the chemical compounds extracted in the non-polar fraction of the mushroom and to examine the biological potential of this extract.
Methodology and results: The n-hexane extract metabolites were identified using gas chromatography-mass spectrometry (GC-MS) and results revealed the presence of 37 compounds delivered from different chemical classes and were mainly comprised of fatty acids and their esters (72%), carboxylic acids and their esters (10.39%), and a sulphur compound (7.1%). The n-hexane extract recorded a promising antioxidant effect (80.9±1.5%) at 80 mg/mL total extract; potent cholesterol reduction activity (100%) was obtained after 96 h incubation by the total metabolites (4%). The cytotoxicity of the compounds revealed 50% cytotoxicity concentration (CC50) ˃ 1 mg/mL and anti-rotavirus SA-11 effect where inhibition of virus attachment and penetration into infected cells was recorded at 50% effective concentration (IC50) of 300±0.2 µg/mL.
Conclusion, significance and impact of study: This study confirmed the impact of the fatty acids produced by C. militaris as bioactive metabolites.
Aim: The aim of this study was to assay for the biogenic amine-producing capacity of bacteria isolated from proteinous food.
Methodology and results: Previously characterized bacterial isolates (Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae) obtained from proteinous food samples (smoked fish and yoghurt) were subjected to proteolytic analysis using nutrient agar supplemented with 0.2 g/mL casein and decarboxylase activity using nutrient broth supplemented with 0.004 g/mL amino acids (histidine, tyrosine, asparagines, leucine and lysine). Isolates that expressed proteolytic and decarboxylase activities were screened for biogenic amine producing capacity using decarboxylase broth which was supplemented with an amino acid (tyrosine). Biogenic amines obtained in this research were classified into primary amine and secondary amine based on their qualitative characteristics. Confirmatory and quantitative analysis of biogenic amines produced was done using high-performance liquid chromatography. The confirmatory screening revealed the presence of methylamine, ethylamine, putrescine, cadaverine, histamine, spermidine, phernylethylamine, spermine, agmatine, tyramine, dopamine, tryptamine, norepinephrine and serotonin respectively. Total biogenic amines produced by S. aureus was 70.12 mg/kg, K. pneumoniae (62.58 mg/kg) and E. coli (56.57 mg/kg) respectively.
Conclusion, significance and impact of study: Enzymatic decarboxylation of free amino acids and other metabolic processes by the test organisms (S. aureus, E. coli and K. pneumoniae) leads to production of biogenic amines which can be used as a quality indicator in food in terms of degree of spoilage, use of non-hygienic raw material and poor manufacturing environment. Thus, effect of biogenic amines obtained in this research would be determined by individual toxicological threshold which can be extremely variable from few mg/kg in sensitive person to several hundred mg/kg in healthy person. The concentrations of each biogenic amine quantified are within the limit but their toxic effects depend on the type of amine, the presence of modulating compounds and the efficiency of an individual’s detoxification mechanism.
Aims: The present study deals with the isolation and identification of lactase producing probiotic strains from camel and sheep milk, determination of the enzyme activity by β-galactosidase assay (Miller Assay) in the presence of garlic, peas, onion and leeks extracts containing inulin as a prebiotic component.
Methodology and results: The two isolates were screened for lactase producing ability to degrade lactose on MRS agar at 37 °C. These were identified as Lactococcus lactis from camel (Marecha) milk and Lactobacillus casei from sheep (Kajli) milk through morphological and biochemical tests using MRS medium. The optimized pH and temperature of both strains were 6 and 35 °C, respectively. Among the three concentrations used (0.2 %, 0.4 %, 0.8 %), the optimal concentration of inulin rich onion and leeks extracts was 0.8 % for maximum growth of L. casei and of the peas extract for L. lactis growth. 0.2% garlic extract was more effective prebiotic source for L. lactis growth. 0.8% commercial inulin used as a positive control was less effective as compared to plant extracts used in the study. With o-nitrophenyl-β-D-galactoside) used as a substrate in the enzyme assay, maximum lactase activity obtained with 0.8 % concentration of garlic extract is 7.10 Miller Units as compared to the peas extract with 6.17 Miller Units from L. lactis. Lactobacillus casei has produced more lactase, 6.85 Miller units with onion extract than with leeks extract, 6.43 Miller Units. Pure commercial inulin used as a control has given maximum enzyme activity as 9.14 Miller Units at 0.2 % concentration.
Conclusion, significance and impact of the study: It is concluded that the extracted prebiotic may enhance lactase activity of the probiotics to supplement the development of food products for lactose intolerant patients.
Aim: This study aimed to isolate, express and characterize the lipase derived Psychrobacter sp. S1B in Escherichia coli expression system.
Methodology and results: Exploration towards S1B lipase characteristic was conducted where shotgun cloning method was applied to obtain lipase encoded gene and E. coli expression system through pET28a was used to overexpress S1B lipase. Lipase activity was measured by using p-nitrophenol method. The S1B lipase gene is 1005 bp in length with molecular weight of 46 kDa, optimum pH was 10.0, showed hydrolytic activity preference toward p-nitrophenyl caprylate (C8) and p-nitrophenyl hexanoate (C6) substrates (C6 < C8). The best temperature for S1B lipase activity was at 30 ⁰C while exhibited high activity at lower temperature (10-25 ⁰C) with above 90% of maximum activity, therefore it is classified as cold adaptive lipase. In addition, S1B lipase showed stability against various metal ions, including Cu2+ and Zn2+ which commonly act as inhibitors of lipases derived from Psychrobacter species. Moreover, S1B lipase exhibited great tolerance against up to 50% (v/v) hexane and some non-ionic detergents such as 1% (v/v) DMSO and 1% (v/v) Triton X-100.
Conclusion, significance and impact of study: The study proposes a novel cold-adapted lipase which has potential as a biocatalyst for synthesis caprylic acid ester.
Aim: Multiple drug resistant bacteria are serious health problems worldwide, with carbapenem resistant and extended spectrum-β-lactamase (ESBL) producing Enterobacteriaceae classified by Centers for Disease Control and Prevention under the category of “Urgent Threats” and “Serious Threats”, respectively. The study characterized Escherichia coli from Oreochromis niloticus procured from two wet markets in Metro Manila in January and September 2016 for their drug resistance.
Methodology and results: Antimicrobial susceptibility profiles were determined using standard disc diffusion method. Extended-spectrum β-lactamase production was confirmed using clavulanate double disc synergy assay, carbapenemase production was tested using modified Hodge test, and MBL (metallo-β-lactamase) production was tested using EDTA double disc synergy assay. Results show that of the 25 isolated E. coli, 24 or 96% were resistant to at least one antimicrobial, with 60% being multiple drug resistant. These strains exhibited 20 different resistance phenotypes, suggesting these were different strains. Fifteen of the isolates (60%) screened positive for ESBL. Among these, 11 lost their resistance, indicating the instability of the resistance genes in the host, a characteristic of plasmid-mediated ESBL production. The ESBL suspects tested were confirmed to be ESBL producers. A high 48% of isolates were found to be resistant to carbapenems, with eight of the 11 tested (73%) being positive for carbapenemase production. MBL positive isolates carried the blaIMP gene as determined by multiplex PCR and nucleotide sequencing.
Conclusion, significance and impact of study: Study showed a high prevalence of multiple drug resistant E. coli isolates from the commonly-consumed Tilapia procured from the wet markets. This result is compounded by the alarmingly high prevalence of carbapenem resistant and ESBL-producing strains among these isolates. Considering that the genes coding for these resistances are found in mobile genetic elements such as plasmids and integrons that can be transferred to other bacteria resulting to a rapid increase in drug resistant strains, it is highly imperative for all the concerned government units to establish a well-coordinated national surveillance program to monitor and address the occurrence and increase in drug resistant microorganisms in man, animals and the environment. In addition, prudent use of antimicrobials among these should be seriously instituted.
Aims: The CRISPR locus in Salmonella genome is comprised of three main components which are the (CRISPR-associated) cas genes, an AT-rich leader sequence and the CRISPR array. The length of CRISPR array is determined by the number of spacers within it and varies not only among different organisms but also varies among the bacterial serotypes and strains. This present study aimed at determining if the CRISPR array in Salmonella spp. could be applied to establish a correlation between serogroup type and the fingerprint generated by CRISPR typing.
Methodology and results: A total of 30 Salmonella samples were obtained from the Veterinary Diagnostic Laboratory, Kota Kinabalu, Sabah. Salmonella serogroup was determined using the slide agglutination test. Four different serogroups were identified which were serogroup B, C, D, and E. Deoxyribonucleic acid (DNA) was extracted and polymerase chain reaction (PCR) was performed using primers which were designed to amplify the CRISPR array in Salmonella genome. Our results indicate that there is a positive correlation between serogroup results obtained using slide agglutination test and the profile generated by CRISPR typing.
Conclusion, significance and impact of study: CRISPR typing has the potential to be applied for the genotyping of Salmonella bacteria.
Aims: The study was carried out to investigate Staphylococcus aureus in clinical and subclinical mastitis in small ruminant and to identify the antibiotic sensitivity profiles of the isolates.
Methodology and result: A total of 171 milk samples from lactating sheep and goats were collected from Besut and Setiu districts in Terengganu, Peninsular Malaysia. All animals were screened for mastitis using the California Mastitis Test (CMT). Phenotypic identification of S. aureus was determined using Gram-staining, Catalase test, Coagulase test, and Oxidase test. The genotypic identification was conducted using Polymerase Chain Reaction (PCR) to detect the nuc gene. The susceptibility of S. aureus to the antibiotic was tested by using the Kirby-Bauer method. In this study, subclinical and clinical mastitis were detected in 66/171 (39%) and 41/171 (24%) respectively. The cultures and PCR results showed that 18/39 (46%) samples (9 subclinical and 9 clinical mastitis) were positive for S. aureus. The antimicrobial susceptibility tests profiles shows 4/18 (22%) and 2/18 (11%) isolates were resistant to penicillin and tetracycline, respectively. However, all isolates were tetK and tetM negative. On the other hand, these isolates susceptible to amoxicillin, gentamicin, nitrofurantoin, oxacillin, cefoxitin, norfloxacin, chloramphenicol, amikacin, kanamycin, doxycycline and cefotaxime.
Conclusion, significance and impact of study: The presence of S. aureus from milk samples of both clinical and subclinical mastitis goats indicates, potential hazard on the livestock as well as public health settings. The occurrence of penicillin and tetracycline resistance should not be undermined. Milk from mastitis samples may play an important role as potential reservoir and transmission of this pathogen in posing disease regardless of antibiotics resistance background.
Prevalence and antimicrobial sensitivity pattern of Staphylococcus aureus isolated from clinical and subclinical mastitis in small ruminant in Besut and Setiu, Terengganu, Malaysia
- Ariffin, M. F. T., Hasmadi, N., Hian, C. M., Ghazali, M. F., Suhaili, Z., Ariffin, S. M. Z.
Aims: Tuberculosis and other mycobacterial infections occur worldwide especially in patients with immunodeficiency. Typically, an empirical treatment for disseminated disease is required for initial therapy due to slow growing nature of most mycobacterial species. Therefore, species distribution and average time to positivity of blood culture is crucial. However, such information is limited for blood culture and, therefore, were determined.
Methodology and results: The blood culture data using the BACTEC FX system and drugs susceptibility testing (DST) pattern was recovered during 2012-2017 from a large teaching hospital in Bangkok, Thailand. Overall, 7.8% of 4,838 blood and 6.4% of 1,056 bone marrow (BM) samples were positive for mycobacterial growth. Mycobacterium tuberculosis complex (MTBC), Mycobacterium avium, and Mycobacterium abscessus, were the most three common species to be isolated from blood (3.8%, 2.1%, and 0.9%, respectively) and BM (2.4%, 2.4%, and 0.9%, respectively). The average time to positivity for MTBC, M. avium, and M. abscessus was 25.7, 16.1, and 3.8 days, respectively. From 209 antimycobacterial susceptibility testing (AST)-available MTBC strains, 6 (2.87%) strains were multi-drugs resistant (MDR-TB). From 35 AST-available M. avium complex (MAC) isolates, 6 (17.14%), 33 (94.29%), and 28 (80%) isolates were resistant to clarithromycin, moxifloxacin, and linezolid, respectively. BM MAC isolates were significantly more resistant to clarithromycin than the blood isolates (44.5% vs 7.69%; p= 0.027).
Conclusion, significance and impact of study: In summary, an emergence of M. abscessus and unusually high moxifloxacin and linezolid resistance of MAC isolates were reported in this study. Additional information of this study benefits physicians for anti-mycobacterial drug selection for initial treatment of mycobacteremia while blood and BM culture is pending.
Aim: Di-(2-ethylhexyl) phthalate (DEHP) has been identified as an endocrine-disrupting chemical, commonly found in the environment. The aim of this study was to isolate bacteria from municipal solid waste (MSW) leachates in Nigeria and its ability to degrade DEHP.
Methodology and results: The DEHP degrading bacterium was isolated and identified. The degradation process was monitored aerobically at varying temperature and pH and the metabolites were determined using High Performance-Liquid Chromatography and Gas Chromatography-Mass Spectrometry, respectively. Based on the morphology and the 16S rDNA sequence, the bacterial isolate was identified as Bacillus aquimaris. B aquimaris was able to degrade 99% of 200 mg/L DEHP within 12 days. The optimum pH and temperature for its biodegradation were 8 and 25 °C, respectively and the intermediate metabolites were identified as butyl octyl phthalate and phthalic acid.
Conclusion, significance and impact of study: This study showed that B. aquimaris could be a useful tool for the biodegradation of DEHP in the environment.
Aim: Biofilm is the major causative factor of infectious diseases. Difficulty in combating biofilm-related diseases is typically due to persisters, heterogeneous microbial population and viscoelastic extracellular polymeric substances (EPS) matrix. Antibiofilm activities of Chromolaena odorata extracts have previously been demonstrated, however, the effects of its treatment on the biofilm proteome expression remains not well understood. Thus, this study was carried out to profile changes in biofilm proteome of Pseudomonas aeruginosa following treatment with chloroform and ethanol extracts of C. odorata.
Methodology and results: Biofilm was developed in 6-well microplate in the presence or absence of C. odorata extracts overnight at 37 °C. Whole-cell proteome analysis was carried out by combining two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Treatment with C. odorata extracts triggered changes in two-dimensional proteome profiles of P. aeruginosa biofilm under aerobic and anaerobic conditions. The differentially expressed proteins were successfully identified and were assigned to various functional categories including protein metabolism, carbohydrate metabolism, peptidoglycan metabolism, electron transport and iron transport.
Conclusion, significance and impact of study: The present study demonstrates differential proteome expression in P. aeruginosa biofilm following treatment with C. odorata extracts. This suggests that C. odorata extracts may target multiple biological processes to control P. aeruginosa biofilm. C. odorata extracts may be useful for development of novel antibiofilm agents.
Aim: The study was designed to evaluate the physio-chemical properties and microbial load of the soil polluted with coffee processing wastes such as coffee husk and coffee pulp.
Methodology and results: A total of ten soil samples were taken from three taluks of Coorg district of Karnataka, India. Out of which five soil samples were taken from places where the coffee processing wastes were dumped as landfills. The other five soil samples were taken from places free from coffee processing wastes which represent the control soil samples. The physical and chemical properties of the soil were measured using standard protocols. The highlight of the study was quantification of chemicals of ecotoxicological concern such as caffeine, polyphenols and tannin in soil samples. The identification and enumeration of soil bacteria, fungi, actinomycetes, yeast and plant growth promoting microorganisms were also done. The pollution with the coffee processing wastes make the soil acidic. The concentration of chemicals of ecotoxicological concern such as caffeine, polyphenols and tannins were significantly high in polluted soil. The colony forming units of plant growth promoting microorganism were declined significantly in the polluted soil. Instead of all these detrimental factors, the organic carbon, nitrogen, potassium, phosphorus and micronutrient content of the polluted soil was significantly high.
Conclusion, significance and impact of study: This study revealed the fact that the unscientific disposal of coffee processing wastes as landfill make the soil less fertile, damage the normal microbial diversity of the soil and would cause severe pollution problems.
Aims: This study aims to characterize the intestinal carp (Cyprinus carpio L.) bacteria, especially lactic acid bacteria (LAB) and its potential as immune-stimulant to be applied in the prevention of diseases in fish.
Methodology and result: The bacteria were isolated from carp intestine and cultured in de Mann Rogosa Sharpe (MRS) and glucose yeast peptone agar + calcium carbonate (GYPA+CaCO3) media. The obtained LABs were identified and characterized by 16S rRNA gene primers. The phylogenetic analysis on DNA sequence was performed by using BioEdit and MEGA 7.0 software. The potential immunostimulant were derived from its ability to resist the growth of Aeromonas sp. and Vibrio sp. as pathogenic bacteria by the paper disc agar diffusion method. The clear zone diameter around the paper disc were measured by using calipers. Forty bacteria that isolated from the carp were selected for lactic acid production and clear zone around the colonies were formed from the GYPA+CaCO3. For further analysis, a total of ten LABs were selected based on different colony forms and the largest clear zone around the colonies. Based on phylogenetic analysis, Enterococcus, Lactobacillus, Streptococcus and Lactococcus were found as the genera of lactic acid bacteria.
Conclusion, significance, and impact of study: We discovered that there is a wide diversity among the 40 isolated bacteria. This result indicates that Enterococcus, Lactobacillus, Streptococcus and Lactococcus were the common genera. The most potential LAB as an immuno-stimulant was Lactobacillus sp.
Aims: Betanin is a red plant pigment belonging to the group called betalain. This present study aimed at investigating the effect betanin from beetroot (Beta vulgaris subsp. vulgaris) as a potential anti-infective agent against methicillin-resistant Staphylococcus aureus (MRSA) using a Caenorhabditis elegans infection model.
Methodology and results: The minimum inhibitory concentration of betanin against MRSA strain ATCC33591 was determined to establish the non-inhibitory concentration. The minimum inhibitory concentration of betanin against MRSA was > 20 mg/mL. C. elegans were then infected with MRSA and treated with betanin at different concentrations (100, 200, 300 and 400 µg/mL). Betanin at 200 µg/mL significantly improved worm survival following infection whereby the mean time to death was extended about 76 h upon treatment. Intestinal colonization by MRSA of worms exposed to betanin extract was similar to non-betanin-treated infected worms.
Conclusion, significance and impact of study: The enhanced survival of MRSA-infected worms upon betanin treatment was not a result of the activation of the host antimicrobial mechanism. Betanin from beetroot can be potentially used as a natural anti-infective agent as a mean to reduce antimicrobial resistance of S. aureus or used in combination with established antimicrobials to increase their effectiveness.
Aims: Nitrile compounds are cyanogenic intermediates, products, byproducts and waste products of agriculture, chemical and pharmaceutical industries and fossil fuels degradation. The enzymatic hydrolysis of nitriles to non-toxic carboxylic acids or amides plays an increasingly important role in environment remediation. This study aimed at exploring the potential of Aspergilli in the detoxification nitrile compounds at two selected dump sites in Lagos, Nigeria.
Methodology and results: Decomposing solid waste leachate samples (SWL) were randomly collected at two selected dump sites namely Olusosun (Ojota) and Isolo (Oke-Afa). Samples per site were pooled, processed by selective enrichment and screened for the presence of Aspergilli by culture technique and intergenic spacer sequencing (ITS). Biomass generation and pH changes in the culture fluids were monitored at 4-days interval by dry weight measurements. Nitrilase production was determined spectrophotometrically. Two nitrilase producing Aspergillus strains: Aspergillus fumigatus strain WO2 with accession number MF78882 and Aspergillus niger strain WO7 with accession number MH542673 were identified. Growth investigation revealed biomass generations of 17.8 g and 23.8 g dry weight per one liter media for A. fumigatus strain WO2 and A. niger strain WO7 respectively. Progressive pH monitoring showed decline from 7.2 to 4.5 and 7.2 to 6.2 was obtained for strains WO2 and WO7 respectively, during nitrilase production at different yields of 0.0150 and 0.0161 mg/mL/min respectively.
Conclusion, significance and impact of study: This study supports the studied dump sites as important sources of nitrilase-producing A. fumigatus and A niger strains with potentials as cost-effective environmental bioremediation agents in Nigeria.
Aims: Pathogenic Escherichia coli is one of global concern because of their effect on public health. The aims of this study were to determine the prevalence of E. coli, i.e. enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorragic E. coli (EHEC), and enteroinvasive E. coli (EIEC) in ice-based beverages sold in street food vendors in Bogor and to determine virulence factor and quantity of labile toxin (LT), stable toxin (ST), intimin (EAE), shiga-like toxin1 (STX1), shiga-like toxin 2 (STX2), and plasmid invasion (INV).
Methodology and results: Identification and quantification of pathogenic E. coli in ice-based beverages (n=85) were performed using real-time PCR (RT-PCR) assay adding propidium monoazide (PMA) prior to samples extraction. Extracted samples were analyzed using electrophoresis gel to define positive genome. The analyzed result showed 55.3% samples were positive genome and those samples were analyzed using RT-PCR to define virulence factor and determined concentration of bacteria. The result showed virulence factor of pathogenic on ice-based beverages (pudding with coconut milk ice, flavored ice, mix fruits ice, coconut ice, and orange ice) were only labile toxin (LT). The prevalence value was 8.2% with varied cycle threshold (Ct) numbers between 28.2 ± 0.3 to 32.8 ± 3.0 and the melting temperature (Tm) was 78.5 °C. The enterotoxigenic E. coli (ETEC) concentration in samples were 1.8 x 10^1-2.0 x 10^2 cells/mL.
Conclusion, significance and impact study: Iced-based beverage samples in Bogor contained 8.2% of ETEC. Based on the consumption data and the concentration of ETEC in iced-based beverages, the number of ETEC exposure in ice-based beverages consumed by the consumer in Bogor (2.9 x 10^3-2.4 x 10^4 cells) was lower than infection dose set by FDA. However, this result could be considered as an early warning of health problems.
Aims: Most of the industrial processes require thermostable alkaline proteases. Thus, a search was initiated to isolate and characterize a bacterium which can produce thermostable alkaline protease.
Methodology and results: Best higher titer thermostable alkaline protease producing wild type organism was screened from beef, dog and fish decaying soil samples. Among the 92 bacterial strains, three strains which produced alkaline proteases having activities at pH 10.5 and above 70 °C were selected. Among the three strains, the one from the dog decaying soil (Strain DDS2 ; DDS-dog decaying soil) which produced the protease showing highest activity at pH 10.5 and 73 °C and stability (half-life: 10.5 h) without additives was selected and identified. Based on the biochemical and morphological studies, strain DDS2 could be either Paenibacillus dendritiformis or Paenibacillus thiaminolyticus. From 16S rDNA sequencing, the strain DDS2 was confirmed as P. dendritiformis.
Conclusion, significance and impact: This is the first report published to show that P. dendritiformis is a protease producer and the organism was named as P. dendritiformis DDS2.
Aims: A number of reports have implicated the role of the symbiotic bacterial communities associated with toxic dinoflagellates in the biosynthesis of saxitoxin during harmful algal blooms (HABs). However, the exact mechanisms by which the bacteria facilitate toxin production remain inconclusive. The toxic dinoflagellate, Pyrodinium bahamense var. compressum, is the causative organism responsible for paralytic shellfish poisoning in the coastal waters of Sabah, and it is caused by the consumption of filter-feeding shellfish contaminated with the neurotoxin, saxitoxin. The present study aimed at characterizing the species diversity of symbiotic bacteria occurring within a monoalgal culture of P. bahamense var. compressum.
Methodology and results: The total bacterial DNA was amplified using paired-end 16S community sequencing on the Illumina platform, targeting the V3-V4 region of the 16S ribosomal RNA gene. Bacteria were classified into 20 classes, 43 orders, 60 families, and 105 genera. A total of 10 phyla were present, where the major phylum was Proteobacteria (69.5%). The major genera were Pseudoruegeria (32%), Roseibium (16%), Hyphomonas (16%), Phaeobacter (7%), Lutimaribacter (5%) and Methylophaga (5%). This study showed that the previous method of assessing microbial diversity occurring in P. bahamense var. compressum has underestimated the actual species diversity.
Conclusion, significance and impact of study: The high-throughput sequencing of the 16S metagenomes revealed hitherto unreported bacterial taxa associated with P. bahamense var. compressum. The findings of the present work will pave the way for further studies aimed at isolating and characterizing symbiotic bacteria that are likely to be associated with the biosynthesis of toxins.
Aims: Hot springs are major sources of useful thermophilic microbes. Our study therefore aimed at documenting and analysing the microbial communities at Ulu Slim hot spring in Perak, Malaysia, for bacteria with beneficial enzymes for industrial applications.
Methodology and results: The study sampled water and sediment materials from the Ulu Slim hot spring at two sublocations of different temperatures, 45 °C and 72 °C. The samples were then extracted for genomic DNA, followed by paired-end metagenome sequencing using prokaryotic-conserved, locus specific primers for 16S rRNA V3 and V4 domains, and Illumina MiSeq (2 300) sequencing platform. Raw data were optimized using Trimmomatic and FLASH, followed by OTU determination and clustering. 16S rRNA gene sequences were then analysed against Silva (SSU123) 16S rRNA gene databases for phylum and genus classifications. Data analysis showed relevant alignment of present bacteria phyla against data from a previous study on Ulu Slim hot springhead, though the different sample temperatures significantly affected phylum and genus composition. Our study also confirmed the presence of a site-specific genus, Fervidobacteria, which are anaerobic bacteria with a unique ability in degrading keratin.
Conclusion, significance and impact of study: The study further completes and complement the findings on microbial communities at the Ulu Slim hot spring. The findings help to reveal the associations between microbes and environmental factors at thermophilic locations, as well as potential thermostable enzymes like lipase and amylase that can be isolated from thermophilic sample as they are indispensable for industry.
Aims: Urinary tract infection (UTI) is a common infection caused by many virulent bacteria. Multidrug resistance (MDR) by bacteria represents a major therapeutic challenge worldwide. MRD bacteria have different mechanisms to avoid antibiotics; one of them is horizontal gene transfer. Such genes, encoding antimicrobial resistance, are easily transferred from one bacterium to another. Magnesium and calcium chloride (MgCl2 and CaCl2) have an effect on the permeability of bacterial cell membrane. We aimed these chemical materials could increase the antibiotics efficiency on multidrug resistance bacteria. 250 UTI specimens were collected to isolate multidrug resistant bacteria. Depending on antibiotics resistance, we selected three species of virulent bacteria: Escherichia coli, Staphylococcus aureus and Proteus mirabilis. Then, we tested the effect of MgCl2 and CaCl2 on their antibiotics resistance.
Methodology and results: The results showed that percentage of E. coli in UTI infection is the highest (45%), while Enterococcus faecalis is the lowest (3%). The effect of MgCl2 and CaCl2 on bacterial antibiotics resistance has been tested using different types of antibiotics. The findings showed that MgCl2 has significant effect to aid antibiotics against bacteria. In particular, nalidixic acid has shown more efficiency against E. coli and S. aureus but not P. mirabilis. Using different concentrations of CaCl2 increased the efficiency of gentamycin, amoxicillin and trimethoprim against S. aureus, while has increased the efficiency of ampicillin and nalidixic acid against E. coli. However, CaCl2 has no effect on the efficiency of antibiotics against P. mirabilis. In addition, MgCl2, and CaCl2 had no toxic effects in both T24 and 5637 urinary bladder cell lines. Finally, plasmids were isolated from these species to detect any antimicrobial resistance genes such as qnr-A.
Conclusion, significance and impact of study: MDR distribution in the worldwide was increased, we highly recommend the avoidance of the random antibiotic usages. The salts CaCl2 and/or MgCl2 can be used at specific concentration to enhance the antibiotics permeability and therefore to decrease the antibiotic resistance.
Aims: This study aimed to evaluate and compare the antibacterial activity (AA) of Origanum dubium and Cinnamomum cassia essential oils dissolved in different ratios of three different solvents against Streptococcus mutans.
Methodology and results: The essential oils were obtained from O. dubium leaves and C. cassia barks using hydro-distillation method and gas chromatography/mass spectrometry (GC/MS) analyses were performed to identify the compositions. The obtained essential oils were diluted in DMSO, Tween 20 and ethanol with concentrations of 10, 20 and 60% respectively. Each oil mixture was furtherdiluted with distilled water to provide different ratios. Then, the AA of the dilutions were evaluated. Comparison of AA of essential oils showed that C. cassia had higher AA than O. dubium for each dilution ratio for Tween and ethanol.
Conclusion, significance and impact of this study: The results obtained in our study lead us to affirm that the AA of both oils are dependent on dilution ratios of the solvents and the AA of C. cassia is higher than that of O. dubium except for the 1:8 and 1:16 dilution ratios of DMSO. Increasing ratios of solvents used to dilute the O. dubium and C. cassia essential oils resulted in a decrease in the antimicrobial activity against S. mutans.
Aims: Today, probiotic bacteria can be used as safe and suitable candidates for the prevention of pathogenic bacterial infections. They produce antimicrobial components and compete with pathogens for host cell receptors. The present study aimed at evaluating probiotic lactobacilli antagonistic and antiadhesive activities against multi-antibiotic resistant Pseudomonas aeruginosa isolated from burned patients.
Methodology and results: This study examined 100 strains of multidrug-resistant (MDR) P. aeruginosa isolated from patients, who were admitted to the burn units of hospitals. Antagonistic activity of Lactobacillus casei and Lactobacillus plantarum against P. aeruginosa was determined by a modified double layer and well diffusion methods. The anti-adhesive effects of two probiotic lactobacilli and co-aggregation assay were determined by the microtiter plate method. Finally, the expression of two main adhesion genes, algD, and pelA was investigated in 100 MDR P. aeruginosa isolates via real-time polymerase chain reaction (RT-PCR) assay. Based on the results, the algD gene was detected in 97 out of 100 P. aeruginosa isolates, and the pelA gene was found in all of the isolates. Based on our findings, lactobacilli bacteria exerted good inhibitory effects on P. aeruginosa, especially on algD and pelA expression. Also, co-aggregation and antiadhesive results showed the positive effects of lactobacilli on P. aeruginosa.
Conclusion, significance, and impact of study: Probiotic lactobacilli can be a new suitable candidate for controlling P. aeruginosa infections.
Aims: To investigate time-kill curve and morphological changes of Proteus mirabilis cells exposed to ethyl acetate crude extract of endophytic fungus, Lasiodiplodia pseudotheobromae IBRL OS-64, isolated from Ocimum sanctum.
Methodology and results: Inhibitory effect of the fungal extract against the test bacteria via disc diffusion assay showed a fair antibacterial activity with diameter of inhibition zone was 12.0 ± 0.4 mm. The Minimal Inhibition Concentration (MIC) and Minimal Bactericidal Concentration (MBC) values of the ethyl acetate extract against P. mirabilis was 250 and 500 µg/mL, respectively. The value of MBC which is two-fold higher than MIC value indicated that the fungal extract exerted bactericidal effect on bacterial cells of P. mirabilis. Time-kill curve study revealed that the bactericidal effect of the crude extract towards test bacteria was both dose and time dependent. Scanning electron microscope (SEM) observation revealed that the bacterial cells of P. mirabilis exposed to fungal crude extract resulted in formation of pits, irregular shape of the bacterial cell and ultimately cell death beyond repair.
Conclusion, significance and impact of the study: The time-kill curve study, and cell morphological changes suggested the potential of ethyl acetate extract of L. pseudotheobromae IBRL OS-64 against P. mirabilis infection by formation of cavities, irregular bacterial cell that leads to ultimate cell death and the extract may have pharmaceutical potential to be develop as antibacterial agent.
Aims: Toxoplasma gondii is one of the most significant food-borne protozoan pathogens and has a significant effect on public health globally. This study aimed to determine the prevalence of T. gondii among retail sheep meat in Erbil Governorate, Iraq.
Methodology and results: During January to June 2019, a total of 375 samples were aseptically collected from retail meat shops. The ELISA test was used for the detection of antibodies against T. gondii from meat juice samples. The overall prevalence of T. gondii antibodies was 16.5%. Late winter and early spring (January-March) showed the highest prevalence, while the prevalence decreased in the subsequent months of the study. There were slightly, but insignificant, increase in the rate of occurrence in suburban area compared with urban area. Samples from sheep carcasses of 1-2 years of age showed the highest prevalence (20.7%) of T. gondii antibodies.
Conclusion and impact of the study: The prevalence of T. gondii in sheep meat sold in Erbil Governorate is high. Thus, proper cooking and strict adherence to hygiene policies are necessary to prevent transmission of toxoplasmosis to human from sheep meat.