Chromosome Mediated Fluoroquinolone and Extended Spectrum Beta-lactamase Resistant Genes in E. coli of Poultry Origin in Ekiti State

Background: One health approach aimed at solving global health crisis links human, animal, and environmental health. This inclusive strategy has contributed to antibiotic classification in both human and animal medicine. Aims: The aims of this research work are to determine the phylogenetic relationship of E. coli isolated from poultry and waste sources. The presence of chromosome mediated fluoroquinolone and extended spectrum beta-lactamase resistant genes will also be detected in the isolates. Study Design: Experimental design. Methodology: Data on farming attitudes of poultry farmers were collected using a questionnaire. E. coli was isolated from fresh poultry droppings and waste disposal sites using eosine methylene blue agar. The antibiotic sensitivity profile of the isolates was determined using the modified Kirby Bauer disc diffusion method. Phenotypic expression of fluoroquinolone (qnrS) and beta-lactamase (blaCMY) resistant traits were further detected using Polymerase Chain Reaction. The 16S rRNA gene sequencing was carried out followed by sequence alignment of E. coli genes with those from GenBank sources to determine the molecular identity of the isolates. Spearman’s correlation coefficient (rs) was run to determine the relationship between antibiotic treatment and resistant profile of the isolates. The phylogenetic relationship of the isolates was determined using Bio edit and Mega 6 software. Results: Organic poultry farming was practiced by small-scaled, peasant farmers who raised free range birds while antibiotics were widely used on farms that adopted intensive mode of farming. The percentage occurrence of E. coli from waste disposal sources was lesser than that from fresh poultry droppings. Highest percentage of antibiotic resistance to the fluoroquinolones was found while the carbapenemase recorded the lowest. Statistical analysis shows that antibiotic treatment in poultry and resistant profile of isolates to antibiotics are directly related. The percentage similarity of gene sequence with those from Gene Data Bank (≥99.29%) validates the identity of the isolates as E. coli. About, 60% of the sampled population had the qnrS gene with a band size of approximately 322 base pair. Besides, 40% of the sampled isolates possessed the blaCMY gene with a band size of approximately 460 base pair. Both genes co-existed in the chromosome of 15% of the sampled isolates sourced from poultry droppings and waste sources. Phylogenetic classification links the origin of isolates from waste disposal sources to poultry production sites. Besides, variant strains of multiple antibiotic resistant E. coli from poultry with antibiotic treatment were more diverse compared to those obtained from birds raised without antibiotics. Conclusion: The qnrS and blaCMY genes found in multiple antibiotic resistant E. coli mediated resistance to critically important antibiotics. The co-existence of these genes in variants strains of E. coli occupying different phylogenetic clusters suggests that antibiotics were widely used on the birds. Antibiotic treatment regimen in poultry may be responsible for the expression of antibiotic resistant genes found in the chromosome of the variant strains of E. coli.

Sustainable Production of Bioethanol by Zymomonas mobilis and Saccharomyces cerevisiae using Rice Husk and Groundnut Shell as Substrates

Background of Study: Plant waste such as rice husk and groundnut shell are generated in large amounts, these waste presents a tremendous pollution to the environment. Worldwide, these wastes are often simply dumped into landfills and oceans or used as animal feeds. The recovery of food processing wastes as renewable energy sources represents a sustainable option for the substitution of fossil energy in order to minimize environmental damages and to meet energy demands of the growing population. Aim: To produce bioethanol from rice husk and groundnut shell using local strains of Zymomonas mobilis and Saccharomyces cerevisiae. Place and Duration of Study: Conducted at the Microbiology Laboratory of Abubakar Tafawa Balewa University Bauchi, Bauchi state, Nigeria, between April to June, 2021. Methods: Groundnut shell and Rice husk were collected from local milling center. The wastes were powdered, sieved and used as carbon source. Proximate composition of the subsrate was done and the total carbohydrate was determined by difference. The sum of the percentage moisture, ash, crude lipid, crude protein and crude fibre was subtracted from 100. Zymomonas mobilis and Saccharomyces cerevisiae were isolated from rotten sweet oranges and locally fermented beverage (‘kunun-zaki’) respectively by growing them on Malt Yeast Peptone Glucose Agar (MYPGA) after which they were further screened for their ability to tolerate ethanol and they serve as organisms for fermentation. The enzyme α- amylase was used for hydrolysis. The fermented substrates were distilled at 78oC and the distillate was collected as bioethanol in a conical flask. UV-VIS spectrophotometer was used to determine the absorbance of each concentration (0, 0.2, 0.4, 0.6 and 0.8cm3) of reducing sugar content of the hydrolysates and the bioethanol produced by developing a standard curve at a wavelength of 491nm and 588nm respectively. The concentration of reducing sugar and bioethanol was determined using a reference line from the Standard curve. Results: Proximate analysis done shows that rice husk have 70.09% carbohydrates while groundnut shell has 65.09% carbohydrates. Groundnut shell yielded the highest reducing sugar of 5.096%. Rice husk yielded the lowest quantity of reducing sugar with a total yield of 2.962%. Maximum concentration of bioethanol of 0.971% was produced from the combination of Saccharomyces cerevisiae and Zymomonas mobilis from groundnut shell. The lowest concentration of 0.121% of bioethanol was produced when Saccharomyces cerevisiae was used on rice husk hydrolysates. The synergistic relationship of Saccharomyces cerevisiae and Zymomonas mobilis yielded the maximum bioethanol when compared with the yield obtained when the organisms were used singly. Zymomonas mobilis produced highest bioethanol content when the organisms are used single. Conclusion: This study demonstrates the potentiality of local strains of Saccharomyces cerevisiae and Zymomonas mobilis isolated from rotten sweet orange and locally fermented beverage (‘kunun-zaki’) to produce bioethanol by fermenting the rice husk and groundnut shell hydrolysates.

Evaluation of the Bacteriological Quality of Milk Sold in Nnewi, Nigeria

Introduction: Contamination of milk products can result to severe intestinal and extra-intestinal diseases in man. This study was aimed at evaluating the bacteriological quality of various milk products sold in Nnewi. Materials and Methods: Using the Cluster sampling technique, 30 milk samples (5 pasteurized skimmed milk, 5 powdered infant milk formulas, 5 powdered milk, 5 unsweetened evaporated milk, 5 branded soya milk and 5 unbranded soya milk) were purchased randomly from different shops and hawkers around Nnewi. Sample processing was done by serially diluting samples in sterile 1% peptone water before plating onto Mannitol Salt Agar, Violet Red Bile Glucose Agar, Blood Agar, MacConkey and Cysteine Lactose Electrolyte Deficient (CLED) agar using the Pour-plate technique. Bacterial count and identification were done using standard bacteriological as well as molecular techniques. The molecular techniques used were Polymerase Chain Reaction, Sanger Sequencing and BLAST analysis on the NCBI BLAST online. Results: This showed that 15 (50%) out of 30 milk samples were contaminated to varying degrees with bacteria. Nine (9) samples showed the presence of Escherichia coli with 32.14% of all the milk samples tested. E. coli was present in skimmed milk (20%), evaporated milk (20%), branded soyamilk (40%) and unbranded soyamilk (100%) but was not isolated from Infant formula and Powder milk. Klebsiella spp. showed the second highest prevalence (28.57%) and was present in evaporated milk (20%), branded soyamilk (40%), unbranded soyamilk (100%). Salmonella spp. (3.57%) was isolated from unbranded soyamilk,Enterobacter spp. (14.29%) was isolated from powder milk and in unbranded soyamilk samples, Staphylococcus aureus(3.57%) and Staphylococcus epidermidis (3.57%) were isolated only in unbranded soyamilk, Macrococcus caseolyticus (3.57%) was isolated from unbranded soyamilk. Novel species such as Aquitalea magnusonii (3.57%), Alishewanella fetalis (3.57%) and Lysinibacillus macroides (3.57%) were identified by molecular analysis to be present in infant formula, evaporated milk and unbranded soyamilk respectively. Conclusion: This research revealed that the bacteriological quality of some milk and milk products sold in Nnewi is not acceptable especially the unbranded soyamilk samples that showed gross contamination.

Influence of Some Antibiotics and Essential Oils Used Alone or in Combination on the Vitality of Presumptive Probiotic Lactic acid Bacteria

Aims: The aim of this study was to assess the in vitro antibacterial activity of selected antibiotics and essential oils alone or in combination, on selected presumptive probiotic lactic acid bacteria. Study Design: Experimental studies. Place and Duration of Study: Department of Microbiology of the University of Yaounde I between August 2017 and December 2017 (5 months). Methodology: The chemical composition of five essential oils was determined by gas chromatography coupled with Solid-phase micro extraction. Then the sensitivity of four lactic acid bacteria to the essential oils and four antibiotics was assessed by the well diffusion and macrodilution method. Subsequently, two essential oils active on these bacteria and broad spectrum antibiotics were combined according to the central composite design plan. Results: In general, the chemical composition of essential oils is very diverse, with the example of carvacrol found only in Origanum compactum at 53.24% and thymol in Thymus vulgaris at 56.19% and in Origanum compactum at 15.28%. The antibacterial activity shows that the majority of antibiotics used are active on the bacteria in the study compared to the essential oils where two were active (Origanum compactum and Cymbopogon winterianus). The evaluation of the combinations of essential oils and antibiotics in terms of kinetics has given us three cases: the first case is the one with no acidity or no growth at all; the second is the one where growth is normal; the third where growth is delayed with a more pronounced latency phase. Conclusion: This study suggest that the effect of essential oils and medicinal plant used alone or in combination to antibiotics on the gut microbiota have to be evaluated for validation as well as their toxicity activities before using them for human therapy.

Bacteriological Quality Assessment of Well and Stream Water Sources from Ikwerre, Emohua and Etche Local Government Areas of Rivers State

Background: Majority of the human population in semi-urban and urban areas in Nigeria are heavily reliant on well and stream water as the main source of water supply for drinking and domestic use due to the inadequate provision of potable pipe borne water. These groundwater sources can easily be fecally contaminated and thus, increase the incidence and outbreaks of preventable waterborne diseases. This study was carried out to determine the bacteriological quality of some well and stream waters sources in Ikwerre, Emohua and Etche Local Government Areas of Rivers State. Methodology: Twenty-four sampling sites with 8 from each Local Government Area were randomly selected for this study. A total of 48 samples, 2 from each of the sites were collected aseptically for 12 months. Samples were analysed using membrane filtration technique for total heterotrophic bacterial count and total coliform count. The well and stream water samples were also cultured for E. coli, Salmonella, and Vibrio sp. Results: The results show that Emohua Local Government Area had the highest total heterotrophic bacterial count of 5.2 x 103 cfu/ml. This was followed by Ikwerre LGA with total bacterial count of 4.3 x 102 cfu/ml, while Etche LGA had 3.1 x 102 cfu/ml. The total coliform count ranged from 25 cfu/100ml to 50 cfu/100ml for the three LGA’s, whereas the faecal coliform count was between 12 cfu/100ml and 20 cfu/100ml. The total coliform, E. coli, Salmonella and Vibrio counts from the three local government areas occurred above the WHO stipulated zero count in 100ml of water. Conclusion: The results from this study showed contamination of all the wells studied with faecal coliforms thus, indicating the presence of other enteric pathogens and a potential source for waterborne disease outbreaks. Well water in Ikwerre, Emohua and Etche Local Government Areas of Rivers State are not safe for drinking without additional treatment like disinfection or boiling. Periodic testing and constant monitoring of these water sources should also be done to meet up with the World Health Organization Standards in the provision of safe, clean drinking water.

Evaluation of the Diagnostic Utility of Geohelminths from Environmental and Stool Samples in Nnewi Metropolis

Background: Geohelminths are groups of four intestinal soil transmitted parasites.They are of public health concern due to their serious morbidity they cause in children which are the major groups affected. Aim/Objective: The aim of this study is to evaluate the diagnostic performance of wet mount and concentration techniques of stool and soil samples for identification of geohelminths. Methodology: Eighty stool samples were collected from four primary schools, while forty soil samples were collected from the important sites (playground, and classroom areas) in each of the school accessed. The stool samples were examined with the wet preparation and formol-ether concentration technique, while the soil samples were examined with the formol-ether concentration technique. Ethical approval was sought from the Faculty of Health Sciences and Technology. Results: The results of geohelminths showed an overall prevalence of 6.25% (5/80) from the stool samples, and 27.5% (11/40) obtained from the soil. Recovery rates from the stool showed the presence of only two geohelminth;Hookworm (5%) and Strongyloides stercoralis (1.25%). Out of the four schools examined only three showed positive results with geohelminth infections, while the remaining school showed positive results with only Giardia.lamblia. The recovery rates from the soil also showed the same pattern of distribution as with the stool but with a higher prevalence of Hookworm (20.7%) and Strongyloides stercoralis (7.5%) with the toilet areas showing the highest prevalence (50%). The dominant specie of geohelminth observed in both stool and soil was Hookworm. The results also showed that both male (6%) and female (6.7%) are susceptible to the infection with no significant difference between their prevalances (p>0.05). Conclusion: The prevalence of Hook worm was poorly reported by wet mount technique.The diagnostic performance of concentration methods for the diagnosis of Hook worms in the samples was notably high as compared to wet mount techniques.

Physicochemical Indices of Phyto Remediated Hydrocarbon Polluted Soil and the Effect of Bio-nutrient Amendment

Aim: To assess the Physicochemical indices of Phytoremediated Crude Oil polluted amended soil using grass plant Cyperus esculentus (Cyp) and Phyllanthus amarus (Phy). Study Design: The study employs experimental design, statistical analysis of the data and interpretation. Place and Duration of Study: Rivers State University demonstration farmland in Nkpolu- Oroworukwo, Mile 3 Diobu area of Port Harcourt, was used for this study. The piece of land is situated at Longitude 4°48’18.50” N and Latitude 6ᵒ58’39.12” E measuring 5.4864 m x 5.1816 m with a total area of 28.4283 square meter. Phytoremediation process monitoring lasted for 240 days; analyses were carried out monthly at 30 days’ interval. Methodology: The study was carried out on Crude Oil Polluted soil (PS) amended with bio-nutrient supplements (Spent Mushroom Substrate (SMS) and selected fungi (Aspergillus niger(AN) andMucor racemosus (MR)) used to stimulate and augment the indigenous microbial population present in a crude oil polluted soil thereby enhancing hydrocarbon reduction in pari per sue with phytoremediation (uptake of Crude oil by test plants) over a period of 240 days. Ten (10) experimental plots (two Control (Unpolluted and polluted soil without amendment) and eight polluted amended/treated plots) employing Randomized Block Design (each having dimensions: 100 x 50 x 30 cm LxBxH); formed and mapped out on agricultural soil and left fallow for 6 days before contamination on the seventh day; after which it was allowed for 21 days for proper contamination and exposure to natural environmental factors (to mimic soil crude oil spill site); thereafter nutrients/organics (biostimulating agents) and bioaugmenting organisms were applied. Baseline studies were carried out on soil profile before and after contamination, major parameters monitored and assayed were Total Petroleum Hydrocarbon (TPH) uptake by plant roots and stem, Polycyclic Aromatic Hydrocarbons (PAHs) and TPH reduction in soil. Other physicochemical properties analyzed in the soil from different plots were pH, Electrical Conductivity, Moisture Content, Total Nitrogen, Available Phosphorus, Potassium, Total Organic Carbon, Plant Height, Iron, Lead and Zinc at regular intervals; days 1, 60, 90, 120, 150, 180, 210 & 240. The rate of phytoremediation was estimated from percentage (%) uptake of Total petroleum hydrocarbon (TPH) in plant roots and stem from day 1 -240; while percentage (%) reduction of TPH and PAHs in soil was estimated from day 1 to the residual at day 240. Results: The test plants decreased significant amount of crude oil as revealed in TPH uptake in their roots and Stem. Mean amount and percentage Total Petroleum Hydrocarbon (TPH) uptake by Cyperus esculentus roots and stem were; 152.33±50.34mg/kg, 12.57±4.16% and 201.13±8.80mg/kg, 13.27±0.58% respectively; while that of Phyllanthus amarusroots and stem were 141.50±35.62mg/kg, 11.68±2.94% and 174.44±19.98mg/kg, 11.51±1.32% respectively; revealing higher Uptake of TPH in plant stem than roots. From the initial TPH contamination value of 5503.00mg/kg, it was observed that plots planted with Cyperus esculentus (TPH 5492.75±76.36mg/kg) showed higher reduction of TPH from soil than those planted with Phyllanthus amarus(TPH 5449.72±18.27mg/kg); while PAHs degradation/reduction showed a reverse trend with plots planted with Phyllanthus amarus (PAHs 28.72±2.74mg/kg; 60.46±5.77%) higher than plots planted with Cyperus esculentus s (PAHs 25.77±2.12mg/kg, 54.24±4.47%). Conclusion: Plots planted with Cyperus esculentus showed higher reduction of TPH from soil than those planted with Phyllanthus amarus while PAHs degradation/reduction in plots planted with Phyllanthus amarus was higher than plots planted with Cyperus esculentus. TPH uptake was higher in plant stems than roots; more so, plots amended with nutrient supplements showed significant higher percentage reduction in hydrocarbon in the polluted soil than unamended polluted soil. It is therefore recommended that Cyperus esculentus is a suitable plant species for phytoremediation of crude oil contaminated soil with high TPH value while Phyllanthus amarusis the best option in phytoremediation of polluted soil with high PAHs value, both in combination with bio-nutrient supplement.

Isolation, Identification and Antibiotic Susceptibility of Escherichia coli Isolated from Raw Meat from Modake and Ile-Ife, Osun State, Nigeria

The study reported isolation, identification and antibiotic susceptibility of Escherichia coli isolated from raw meat from Modakeke and Ile-ife, Osun State, Nigeria, with the view to determining the antibiogram profiling of the bacterial isolates. In this study, five samples of fresh meat were collected from different abattoirs in Ile-Ife and Modakeke, Osun State. Isolates of Escherichia coli were isolated, identified morphologically based on their growth on nutrient agar and subjected to antibiotic susceptibility test on Mueller Hinton agar. The mean microbial load from the meat samples ranged from 8.85 x 102cfu/ml to 5.77 x 104cfu/ml. A total of 69 E. coli isolates were recovered from the meat sampled. All the isolates appeared cream, translucent, entire, convex, circular, smooth and glistering. The isolates were identified as Gram negative rods, non-motile, lactose fermenters, positive for indole test and negative for citrate utilization test. All the E. coli isolates were resistant to augmentin, ceftriazone, nitrofurantoin and gentamycin. 98.55% of E. coli isolated was resistant to amoxillin and the least resistant was recorded in ofloxacin (8.70%). However, 91.30% of the E. coli isolates was sensitive to ofloxacin, 81.16% to ciprofloxacin and 36.23% to pefloxacin while none was sensitive to augmentin, ceftriazone, nitrofurantoin and gentamycin. A total of 19 different multiple antibiotic resistance patterns were observed among the isolates. Thirty isolates (43.48%) showed multiple antibiotic resistance to 5 and 10 different antibiotic types each. The study concluded that occurrence of E. coli infection is high in the study area with high level of multiple antibiotic resistance.

Antioxidant and Antimicrobial Activities of Various Extracts from Safou (Dacryodes edulis) Cultivated in Côte d’Ivoire

Safou (Dacryodes edulis) is a fruit with very interesting nutritional and pharmacological properties. However, this fruit remains rather unknown to the Ivorian population. Aims: The objective of this study was to highlight the antioxidant and antimicrobial potential of the seed and pulp of Safou (Dacryodes edulis) cultivated in Côte d'Ivoire for its valorization. Study Design: Microbiological and biochemical studies Place and Duration of Study: Laboratory of Biotechnology and Food Microbiology,Abidjan, Côte d’Ivoire, between September 2020 and February 2021. Methodology: Aqueous, ethanolic and methanolic extracts of the pulp and seed of Safou were prepared. Moisture content, pH, yield, total polyphenols,diphényl-2-picrylhydrazyle (DPPH) radical scavenging testof the different extracts were determined.Aqueous, ethanolicand methanolicextracts of the seed and pulp at varying concentrations of 200 mg/mL, 100 mg/mL, 50 mg/mL, 25 mg/mL, 12.5mg/mL and 6.25 mg/mL was tested against human pathogens such asEscherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiellapneumoniae, Proteus mirabilis, Aspergillus fumigatus and Penicilliumchrysogenum. Results: pH, moisture content and yield ranged from 3.16 to 4.74; 9 to 18.30%; and 10.79 to 18.78%, respectively. Total polyphenol content ranged from 1.45 mgEAG/g to 4.56 mgEAG/g. The highest levels of total polyphenols were observed for the methanolic extracts of pulp. The different extracts strongly scavenged the DPPH radical with percentages of anti-free radical activity that varied from 50.76 to 64.43%.The antimicrobial susceptibility results of the methanolic extracts of D. edulis seed showed highest zones of inhibition to the microbial isolates tested. The microorganisms were resistant to the aqueous extracts of pulp and seed. Conclusion: The presence of bioactive compounds and the antimicrobial activity of Safou extracts could justify its use in the food and pharmaceutical fields.

Molecular Characterisation and Plasmid Profiling of Hydrocarbon Utilizing Bacteria Isolates from Wetlands in Rivers State, Southern Nigeria

Wetlands can intercept runoff from surfaces prior to reaching open water and remove pollutants through physical, chemical, and biological processes thereby protecting and preserving the environment. Because of unsustainable oil exploration activities, most wetlands in Rivers State, Southern Nigeria have suffered severe petroleum-damaged ecosystems. This research was carried out to characterize and identify the hydrocarbon utilizing bacteria associated with crude oil polluted wetlands and to screen for the presence of plasmids that could confer resistance to antibiotics using both cultural and molecular methods. Soil samples were collected from three different wetlands across the state with hand auger at two depths of 0-15cm and 15-30cm twice monthly for three months. The presence of microbial activity was determined by the enumeration and isolation of total heterotrophic and hydrocarbon utilizing bacteria. Eight (8) most occuring hydrocarbon utilizing bacterial isolates were isolated and identified culturally and phenotypically from the 54 wetland soil samples. These bacteria isolates were confirmed to be Bacillus flexus, Bacillus subtilis, Lysinibacillus macroides, Staphylococcus aureus, Chryseobacterium aquifrigidense, Pseudomonas aeruginosa and Salmonella enterica molecularly via sequencing of the 16S rRNA gene. The most common bacteria isolated were Bacillus species, followed by Pseudomonas at a dilution of 106. Seven (7) out of the eight (8) isolates (except Salmoella enterica) showed the presence of the 25kb plasmids at various intensities.