MALAYSIAN JOURNAL OF MICROBIOLOGY
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.