Microbiology

PHYSIOLOGICAL CHARACTERISTICS, AMYLASE PROPERTIES AND METAGENOMIC ANALYSES OF BACTERIAL AND FUNGAL COMMUNITIES FROM IKOGOSI WARM SPRINGS

Tue, 01 Aug 2023 00:00:00 GMT

PHYSIOLOGICAL CHARACTERISTICS, AMYLASE PROPERTIES AND METAGENOMIC ANALYSES OF BACTERIAL AND FUNGAL COMMUNITIES FROM IKOGOSI WARM SPRINGS FASESAN, Deborah Ebunoluwa Several warm and hot springs exist all over the world whose microbial diversity and physicochemical properties have been explored using both conventional and metagenomic methods to reveal biotechnologically relevant bacterial and fungal microbiomes. The Ikogosi Warm Springs (IWS) in Ekiti State, Nigeria is known for its unique warm and cold water outflow and tourism activities. Unlike other climes, there is paucity of information on the bacterial and fungal microbiome of IWS. This work was therefore designed to determine the taxonomic composition of the bacterial and fungal microbiome of IWS as well as prospect for alphaamylase of potential industrial importance. Sediment (20) and water (20) samples were collected from IWS during the wet and dry seasons for physicochemical analyses using standard procedures. The bacterial and fungal microbiome of IWS were evaluated using the V1–V3 regions of 16SrRNA and ITS1, respectively. Genomic libraries were constructed for paired-end sequencing and the microbial taxa present were analysed using DADA2 metagenomics pipeline, where alpha and beta diversity statistics and metrics were also computed. Bacteria were also isolated from samples employing culturedependent method and screened at 50°C for alpha-amylolytic properties using starchsupplemented media. Molecular identification of bacterial isolates, with the highest zone of hydrolysis, were done and alpha-amylase production and enzyme characteristics were determined using UV/visible spectrophotometry. Data were analysed using descriptive statistics, and ANOVA at α0.05. The pH and temperature of IWS ranged 6.36 to 7.93, and 28.3 to 38.0°C, in wet season, and 5.8 to 7.6, and 25.7 to 38.3°C, in dry season. In IWS water, Proteobacteria (84.4, 83.4%), Bacteroidetes (10.0, 16.0%), Firmicutes (2.4, 7.5%), Ascomycota (62.0, 89.0%) and Basidiomycota (37.7, 10.7%), were dominant in the wet and dry seasons; while the sediments (%) had 62.6 and 45.4; 21.4 and 53.5; 15.0 and 0.4; 65.1 and 65.1; 34.8 and 33.5, respectively. Alpha diversity measures indicated no significant phylogenetic diversity between the bacteriome and fungal microbiome of water and sediment, in both seasons. A significant difference occurred in the diversity between the bacteriome of water and sediment samples. Beta diversity showed significant differences in taxonomic similarity and relative abundance in water and sediment of both bacterial and fungal communities in both seasons. Vogesella, Aeromonas, Comamonas, Brevundimonas and Malassezia were present in both water and sediment samples. Culturedependent isolated amylolytic bacteria constituted 27.1% of 174 isolates. The best amylolytic isolates were Bacillus cereus-MPW3E and Bacillus siamensis-SW3F. Optimum alpha-amylase production of these bacilli was observed at 55°C; pH 7.0; Ca2+ supplementation; 1% seed inoculum and 48-hour incubation period. The alpha-amylase activities for Bacillus cereusMPW3E and Bacillus siamensis-SW3F were optimum at 1% substrate concentration, with pH 6.0 and 7.0, and at 50°C and 55°C, respectively. The Michaelis Menten constant was 0.197 and 0.052 mM, with maximum velocity of 6.305 and 25.974 µmol/min for Bacillus cereus-MPW3E and Bacillus siamensis-SW3F, respectively. Microbiome taxonomic diversity of Ikogosi warm springs predominantly contained phyla Proteobacteria, Bacteroidetes, Firmicutes, Ascomycota and Basidiomycota. Thermotolerant alpha-amylase-producing Bacillus cereus and Bacillus siamensis were also obtained.

MOLECULAR CHARACTERISATION OF MULTIPLE ANTIBIOTICS RESISTANT ENTEROBACTER SPECIES ISOLATED FROM POULTRY DROPPINGS OF SELECTED FARMS IN SOUTHWEST NIGERIA

Sat, 01 Jul 2023 00:00:00 GMT

MOLECULAR CHARACTERISATION OF MULTIPLE ANTIBIOTICS RESISTANT ENTEROBACTER SPECIES ISOLATED FROM POULTRY DROPPINGS OF SELECTED FARMS IN SOUTHWEST NIGERIA AMUSAN, Motunrayo Janet Indiscriminate use of antibiotics in poultry production is among the factors responsible for antibiotic resistance by microorganisms. Large amounts of poultry droppings are generated annually which are used in fish feeding and as manure in agricultural farms. However, there is a dearth of information on the antibiotic resistance profile of Enterobacter species, a member of pathogens on the priority list of the World Health Organisation for developing new antibiotics: Enterococcus faecium- Staphylococcus aureus- Klebsiella pneumoniae- Acinetobacter baumannii- Pseudomonas aeruginosa- Enterobacter species, from poultry origin. Therefore, the aim of this study was to determine the antibiotic resistance pattern of Enterobacter species isolated from poultry droppings of selected farms in southwest Nigeria. Poultry dropping samples from layer chickens (24), broiler chickens (16), cockerels (8) and Noilers (4) were aseptically collected from 27 farms across the six states of southwest, Nigeria. Total Heterotrophic Bacterial Count (THBC) was done using pour plate method, while the isolation of Enterobacter species was carried out using standard method. The isolates were identified using the conventional method and Matrix-Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry (MALDI-TOF-MS). Antibiotic susceptibility of the isolates on 20 antibiotics was determined using Kirby-Bauer’s disc diffusion method. Extended Spectrum Beta-Lactamase (ESBL) production of the isolates was determined using phenotypic methods. The ESBL and Antibiotic Resistance (AR) genes were detected with specific primers using polymerase chain reaction. Selected multiple antibiotic resistant isolates from chicken droppings were genome sequenced using Illumnia technology (Mi-Seq). Pathosystems Resource Integration and Centre for Genomic Epidemiology Database were used for genomic analysis. Data were analysed using descriptive statistics. The THBC ranged 8.8×106 ±0.3 (Noilers) to 9.6×106±2.1 CFU/g (layer chickens), while the 72 Enterobacter spp. isolated comprised E. cloacae (52), E. asburiae (12), E. kobei (7) and E. ludwigii (1). The resistance patterns of the Enterobacter spp. showed that all the isolates were resistant to cefpodoxime, cefixime and amoxicillin across the states, while the least resistance was to ciprofloxacin (8.3%). Forty-two of the Enterobacter spp. were ESBL producers out of which 71.4% haboured at least one of the ESBL genes (blaCTX-M, blaTEM and blaSHV). The ampC, qnrB, dfrA1 and ermB, were detected in 52.8% of the Enterobacter species, which are of public health importance. Enterobacter cloacae (ILB8) genome revealed a close relationship with the pathogenic E. hormaechei and E. mori from humans and plants, respectively and contained virulence genes of clinical importance. Forty AR genes were detected in the E. cloacae (ILB8). A class C betalactamase gene (blaACT-16_AB737978) had been identified in another E cloacae strain from a septicaemic neonate, while fosfomycin gene (fosA) had also been identified in E. mori from a diseased Morus alba plant. There is the possibility of the spread of AR genes from bacteria present in poultry droppings to humans and plants through contact with the environment. Enterobacter species from poultry droppings in the southwest Nigeria were multiple antibiotic resistant. Extended Spectrum Beta-Lactamase-producing Enterobacter species had antibiotic resistance genes.

ENVIRONMENTAL HYGIENE AND MICROBIOLOGICAL ASSESSMENT OF FOOD SERVICE ESTABLISHMENTS IN SELECTED BOARDING HIGH SCHOOLS IN IBADAN, NIGERIA

Sat, 01 Jul 2023 00:00:00 GMT

ENVIRONMENTAL HYGIENE AND MICROBIOLOGICAL ASSESSMENT OF FOOD SERVICE ESTABLISHMENTS IN SELECTED BOARDING HIGH SCHOOLS IN IBADAN, NIGERIA ADEBAYO, KAFAYAT ADENRELE Schools’ Food Service Establishments (FSEs) have been incriminated in numerous foodborne diseases outbreaks globally and have been linked to the environment and food handling procedures in the establishments. Despite this, FSEs in Nigerian boarding schools have been poorly investigated. In order to provide baseline data for infection control, this study was designed to assess environmental hygiene and food handlers’ Knowledge, Attitude and Practices (KAP) and investigate food-related microbial contamination from selected boarding schools’ FSEs in Ibadan, Nigeria. Observational checklist and interviewer-administered questionnaire were used to evaluate environmental hygiene parameters, food handlers’ KAP in four schools’ FSEs out of forty-three schools by inclusion criteria and balloting. Swabs from Food Contact Surfaces (FCS): utensils and surfaces; 20 food handlers’ hands and samples of Readyto-Eat (RTE) foods were examined for Aerobic Plate Count (APC), Total Coliform (TC), Faecal Coliform (FC) and selected important foodborne pathogens counts using standard methods. Isolated bacteria were characterised phenotypically and subjected to 16S rRNA sequencing. Antibiotic susceptibility testing was determined using disc diffusion and E-strip techniques based on CLSI and EUCAST standards, respectively. Data were analysed using descriptive statistics and ANOVA at α0.05. Schools FSEs’ compliance mean scores for environmental hygiene parameters were 82.2, 56.8, 52.7 and 65.6% for toilets, dining areas, kitchens and observed food handlers at work, respectively. The food handlers had good knowledge (61.9%), positive attitude (81.4%) to ensure food safety, but poor hygiene practices (52.6%) which differed significantly among schools (p=0.012, χ2=10.15). Major unsanitary practices observed were: use of basins and buckets for dish washing, uncovered solid waste receptacles, non-availability of sanitising agents and inadequate handwashing. Mean logCFU/cm2 of APC for counter tops, chopping boards, grinders, trays and knives were 5.59±1.56, 4.38±2.62, 4.01±0.77, 2.47±2.23 and 2.38±1.75, respectively. Food handlers’ hands’ mean logCFU/cm2 of APC, TC, FC, Staphylococcus and Bacillus species were 3.10±1.78, 2.62±1.23, 2.80±1.74, 1.94±1.04 and 1.97±1.39, respectively. Seventy-eight percent of RTE foods conformed to acceptable limit of < 4logCFU/g for APC. The distribution of bacteria from schools FSEs were 62.0% (FCS), 19.0% (food handlers’ hands) and 19.0% (RTE foods). The identified food-related bacteria were Alcaligenes faecalis, Achromobacter xylosoxidans, Bacillus cereus, Ochrobactrum anthropi,viii Proteus mirabilis, Serratia marcescens, Staphylococcus saprophyticus and Bordetella species. Alcaligenes faecalis resistance (%) to cefixime, cefuroxime, ceftazidime, gentamicin, augmentin, nitrofurantoin, ofloxacin and ciprofloxacin were 76.2, 71.4, 66.7, 61.9, 57.1, 42.9, 4.8 and 4.8, while for Bacillus cereus, they were 85.7,100.0, 57.1, 85.7, 28.6, 57.1, 0.0 and 0.0, respectively. The minimum inhibitory concentration of colistin for Alcaligenes faecalis ranged from 1.5 µg/mL to >256 µg/mL which was highly significant (F=9.194, p<0.05) compared to other antibiotics. Two Bacillus cereus were resistant to imipenem, 81.0% were multi-antibiotic resistant, while none of the identified bacteria showed resistance to piperacillin/tazobactam. Food contact surfaces and food handlers’ hands were grossly contaminated. The presence of colistin-resistant Alcaligenes faecalis and resistance of Bacillus cereus to imipenem in boarding schools’ food service establishments is a serious public health concern. These findings will be useful in policy formulation and the development of food safety guidelines in boarding schools.

PRODUCTION, PURIFICATION AND CHARACTERISATION OF LASPARAGINASE FROM ACTINOMYCETES AND EVALUATION OF ITS ANTI-CANCER AND ACRYLAMIDE REDUCTION ACTIVITIES

Wed, 01 Sep 2021 00:00:00 GMT

PRODUCTION, PURIFICATION AND CHARACTERISATION OF LASPARAGINASE FROM ACTINOMYCETES AND EVALUATION OF ITS ANTI-CANCER AND ACRYLAMIDE REDUCTION ACTIVITIES SALAMI, MOJISOLA OLAJUMOKE The potential of L-asparaginase to inhibit acrylamide formation (a carcinogenic compound from fried starchy foods) and the growth of cancer cells have attracted scientific attention in biomedical fields. Utilisation of L-asparaginase from several sources has been limited due to high rate of allergic reactions, after a prolonged use because of the presence of glutaminase activity which deamidates glutamine to glutamic acid and ammonia, thereby increasing the enzyme toxicity. Hence, it is necessary to search for other L-asparaginase sources with no Lglutaminase activity as alternatives. Production trials with Actinomycetes and their characterisation have been limited. The work aim was to produce, purify and characterize Lasparaginase with anticancer and acrylamide reduction potential from Actinomycetes. Actinomycetes were isolated from rhizospheric soil of Moringa plants in the botanical garden, University of Ibadan, and were screened for L-asparaginase activity. The selected Lasparaginase producers were screened for L-glutaminase activity using plate assay method. The L-asparaginase producers with lower glutaminase activity were identified using molecular methods. Effects of temperature, pH, metal ions, incubation time, carbon and nitrogen sources, agitation and medium composition on the growth and L-asparaginase production by identified isolates were determined. The L-asparaginase produced was purified by ammonium sulphate precipitation, dialysis and column chromatography using Sephadex G-50. The molecular weight of the enzyme produced was determined using SDS-PAGE. Effect of pH, temperature, metal ions, inducers and inhibitors and substrate concentration on activity and stability of the partially purified enzyme was determined. The in-vitro anticancer activity of the partially purified L-asparaginase on colon cancer cell line at different concentrations using 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was done. The acrylamide reduction activity of L-asparaginaseinpotato after heat treatment (above 120oC) was determined using standard methods. Data were analysed using descriptive statistics. One hundred and forty-five bacteria were isolated, 46% of them were L-asparaginase producers. The best six L-asparaginase producers with lower glutaminase activity were identified as Amycolatopsis japonica, Sphingobium yanoikuyae, Sphingobacterium caenis, Stenotrophomonas pavani, Paenibacillus cineris and Actinomycetal bacterium. Amycolatopsis japonica showed highest L-asparaginase activity (0.2772±0.001 U/mL) and no glutaminase activity after 25 days of screening. The highest L-asparaginase was produced at pH 7.0, 35oC, 7 days incubation time, Mg2+, 150rpm, M9 medium, maltose (0.2% w/v) and yeast extract (0.2% w/v). Partially purified L-asparaginase from Amycolatopsis japonica had total activity of 1968.98 U/mL, total protein (26.69 mg), specific activity (73.75 U/mg), purification fold (6.42), recovery yield (42.86%) and molecular weight of 37.5 kDa. Ethylenediamine Triacetic Acid, Mg2+, pH 8, 45oC supported optimum activity and stability of the enzyme. The Michaelis constant (Km) and maximum velocity (Vmax) were 7.874 mM and 2.57 U/mL, respectively. The enzyme showed high anticancer activity against colon cancer cell line with half Maximum Inhibitory Concentration (IC50) of 36 μg/mL. It also had 11% reduction of acrylamide formation in potato which is a carcinogenic compound in starchy food. Amycolatopsis japonica L-asparaginase exhibited anticancer potential and reduced formation of acrylamide in fried starchy food. This enzyme has the potential to be used as drug to complement the ones currently in use.