Browsing by Author "Pillay, Balakrishna."

Now showing 1 - 16 of 16

Application of bacterial bioflocculants for wastewater and river water treatment.
(2008) Buthelezi, Simphiwe P.; Pillay, Balakrishna.
Dyes are often recalcitrant organic molecules that produce a colour change and contribute to the organic load and toxicity of textile industrial wastewater. Untreated effluent from such sources is harmful to aquatic life in the rivers and lakes due to reduced light penetration and the presence of highly toxic metal complex dyes. The use of alum as flocculant/coagulant in wastewater treatment is not encouraged as it induces Alzheimer’s disease in humans and results in the production of large amounts of sludge. Therefore, the development of safe and biodegradable flocculating agents that will minimize environmental and health risks may be considered as an important issue in wastewater treatment. Bioflocculants are extracellular polymers synthesized by living cells. In this study, bacterial bioflocculants were assessed for their ability to remove dyes from textile wastewater as well as reducing the microbial load in untreated river water. The bacteria were isolated from a wastewater treatment plant and identified using standard biochemical tests as well as the analysis of their 16S rDNA gene sequences. Six bacterial isolates were identified viz. Staphylococcus aureus, Pseudomonas plecoglossicida, Pseudomonas pseudoalcaligenes, Exiguobacterium acetylicum, Bacillus subtilis, and Klebsiella terrigena. The flocculating activities of the bioflocculants produced by these isolates were characterized. The effect of temperature, pH, cations and bioflocculant concentration on the removal of dyes, kaolin clay and microbial load was also determined. The amount of bioflocculants produced by the bacterial isolates ranged between 5 and 27.66 g/l. According to the findings of the present study, bacterial bioflocculants were composed of carbohydrates, proteins, uronic acid, and hexosamine in varying quantities. The bioflocculants were effective to varying degrees in removing the dyes in aqueous solution, in particular whale dye, medi-blue, fawn dye and mixed dyes, with a decolourization efficiency ranging between 20-99.9%. Decolourization efficiency was influenced by the bioflocculant concentration, pH, temperature, and cations. The bacterial bioflocculants were also capable of reducing both the kaolin clay and the microbial load from river water. The flocculating activity ranged between 2.395–3.709 OD-1 while up to 70.84% of kaolin clay and 99% of the microbial load from the river water was removed. The efficiency of kaolin clay flocculation increased with higher concentration of bacterial bioflocculants. The optimum pH for the flocculating activity was observed between 6 and 9. The best flocculating activity was observed at 28oC. Divalent cations such as Mg2+ and Mn2+ improved the flocculation while salts such as K2HPO4, CH2COONa, and Na2CO3 did not. The findings of this study strongly suggest that microbial bioflocculants could provide a promising alternative to replace or supplement the physical and chemical treatment processes of river water and textile industry effluent.
Clinical strains of mycobacterium tuberculosis induce strain-specific patterns of cytokine production, gene expression and pathway changes in pulmonary alveolar epithelial cells.
(2016) Mvubu, Nontobeko Eunice.; Pillay, Manormoney.; Pillay, Balakrishna.
The epidemiological success of M. tuberculosis strains, dominant in different geographic regions globally, may be ascribed to a subversion of the host‟s protective immune response. The increasing prevalence of F15/LAM4/KZN, Beijing, F11 and F28 Mycobacterium tuberculosis strain families, coupled with rapidly evolving drug resistance within the KwaZulu-Natal province of South Africa population has resulted in a need to characterize host response associated with infection by these strains. Therefore, in this study, cytokine/chemokine production and host transcriptomics were investigated in A549 pulmonary epithelial cells infected with the F15/LAM4/KZN, Beijing, F28, F11, Unique and H37Rv strains. Cytokines/chemokines were quantified using the Bio-Plex Pro Human Cytokine 27-Plex assay at 0, 24, 48 and 72 hr post-infection. Changes in host gene expression were determined by whole genome RNA Sequencing (RNA-Seq) using the Illumina HiSeq 2000 platform. The 50 bp reads were mapped to the human genome (hg19) using Tophat (2.0.10). Differential expression was quantified using Cufflinks (2.1.0) with false discovery rate (FDR) of 0.05 and a log fold change cutoff of ≥2. R commands (Bioconductor), MeV and Ingenuity Pathway Analysis (IPA) were used to generate heat maps, network and pathways analysis. Twenty-three out of 27 analytes were detected. All strains, except the F28 strain induced an increased production of 18, and a decrease in 5 cytokines/chemokines at 24, 48 and 72 hr post-infection, compared to the uninfected control. Increased production of all 23 analytes by the F28 strain occurred at 48 and 72 hr. Among the 23 cytokines/chemokines that were detected, anti-inflammatory and pro-inflammatory cytokines, as well as chemokines were produced at the different time intervals. Compared to the other strains, high cytokine levels were induced by the F28 strain at 48 hr and F15/LAM4/KZN strain at 72 hr for most analytes. A lower cytokine production was induced by the Beijing and Unique strains at all time intervals. In the case of the laboratory strain H37Rv either a higher or lower cytokine/chemokine production was observed, compared to the clinical strains. RNA-Seq revealed differential gene expression that varied among the strains with respect to both up- and down-regulated genes: F15/LAM4/KZN (1187), Beijing (1252), F11 (1639), F28 (870), Unique (886) and H37Rv (1179). A total of 292 genes were commonly induced by all strains, of which 52 were down-regulated and 240 were up-regulated. Different strain combinations induced different genes that were involved in a variety of pathways, including immune response and apoptosis pathways. Furthermore, strain specific genes were activated by each strain as follows: F15/LAM4/KZN (138), Beijing (52), F11 (255), F28 (55), Unique (185) and H37Rv (125). The F15/LAM4/KZN, Unique and H37Rv were the only strains that had molecular signatures with overlapping functional Kegg and Reactome pathways for their specific genes. IPA analysis revealed canonical pathways that differed among the strains, with the interferon signalling and hepatic fibrosis/hepatic stellate cell activation pathways being among the top 5 pathways in all the strains. Cholesterol biosynthesis and immune related pathway enrichment was similar in the Beijing and Unique strains whilst the F15/LAM4/KZN strain showed closer relatedness to the F11 strain, and the F28 strain closely clustered to the H37Rv strain. The Beijing and Unique strains highly enriched cholesterol biosynthesis pathways compared to other clinical and laboratory H37Rv strain. The top scoring networks induced by these clinical strains varied among the strains with the associated functions. These gene networks were involved in antimicrobial response, developmental disorder, organismal injury, infectious disease and cellular development. Among the transcriptional factors, only EHL, IRF7, PML, STAT1, STAT2 and VDR were induced by all clinical strains, while other factors were strain specific. In conclusion, low cytokine/chemokine production and activation of immune associated pathways by the Beijing and Unique strains suggest a higher virulence for these strains compared to the F15/LAM4/KZN, F11 and F28 strains. These characteristics may explain the high transmissibility and prevalence of the Beijing strains. A similar pattern exhibited by the less prevalent, non-clustering Unique strain, may suggest some virulence attributes in common with the Beijing strain. Findings in this study have the potential to reveal useful biomarkers that can be used as targets for alternative TB therapeutics including immunomodulators that take into consideration network regulations and strain-specific pathways and molecular signatures.
Effect of fermentation and nutritional conditions on the profile of flavour active ester compounds in beer.
(2011) Hiralal, Lettish.; Olaniran, Ademola Olufolahan.; Pillay, Balakrishna.
During fermentation, the yeast Saccharomyces cerevisiae produces a broad range of aroma-active esters that are important for the desirable complex flavour of beer. The sensory threshold levels of these esters in beer are low, ranging from 0.2 ppm for isoamyl acetate to 15-20 ppm for ethyl acetate. Although esters are only present in trace amounts in beer, they are extremely important as minor changes in their concentration may have dramatic effects on beer flavour. Therefore, optimization of the concentrations of these aroma-active esters in beer is of interest in beer brewing. The number and concentration of esters in beer may be influenced by the fermentation parameters, nutritional composition of fermentation medium and yeast strain type. Therefore, this study investigated the influence of fermentation temperature, pH, and wort nutritional supplements (amino acids and zinc) on the production of yeast-derived ester compounds. In addition, the overall fermentation performance was evaluated based on the reducing sugar and Free Amino Nitrogen (FAN) utilization, ethanol production and yeast cell density. These parameters were analysed using the Dinitrosalicyclic acid method, Ninhydrin assay, Gas Chromatography and standard spread plate technique. The concentration and stability of ethyl acetate, isoamyl acetate, phenyl ethyl acetate, ethyl hexanoate, ethyl decanoate and ethyl octanoate was monitored during storage at 4 °C and room temperature (RT), in the final beer by Chromatography. The expression levels of the ester synthetase genes under conditions that resulted in the highest increase in ester production were quantified by Real-Time PCR. For the lager beer, the best fermentation performance was achieved at RT (±22.5°C), resulting in the utilization of the highest amount of nutrients and production of 4.86% (v/v) ethanol. This was accompanied by the highest production of acetate and ethyl esters, which were 40.86% and 87.21%, respectively, higher than that of the control. Spent yeast density ranged from 2.492 to 3.358 mg/ml for all parameters tested, with the highest yield produced when wort was supplemented with 0.120 g/l zinc sulphate. Fermentations at 14 °C yielded the highest foam head stability and spent yeast viability with a foam head rating of 2.67 and a spent yeast viability of 3.85 × 107 cfu/ml. Ester compounds were relatively stable at 4 °C than at room temperature decreasing by only 7.93% after three months. Of all the volatile esters produced, ethyl decanoate was the least stable, with a 36.77% decrease in concentration at room temperature. For the ale beer, the best fermentation performance which resulted in the highest nutrient utilization was achieved when wort was supplemented with 0.75 g/l L-leucine resulting in the utilization of the highest amount of nutrients (51.25% FAN and 69.11% reducing sugar utilization) and production of 5.12% (v/v) ethanol. At the optimum fermentation pH of 5, 38.27% reducing sugars and 35.28% FAN were utilized, resulting in 4.32% ethanol (v/v) production. Wort supplemented with 0.12 g/l zinc sulphate resulted in 5.01% ethanol (v/v) production and 54.32% reducing sugar utilization. Spent yeast density ranged from 1.985 to 2.848 mg/ml for all parameters tested with the highest yield produced when wort was supplemented with 0.120 g/l zinc sulphate. This was also accompanied by the highest yeast viability of 2.12 × 107 cfu/ml achieved on day 3 of fermentation. Supplementation with 0.75 g/l L-leucine yielded the highest foam head stability with a rating of 2.67. Overall, ester compounds were relatively more stable at 4 °C than at RT decreasing by only 6.93% after three months, compared to a decrease of up to 16.90% observed at RT at the same time. Of all the volatile esters produced, ethyl octanoate was the least stable, with a 32.47% decrease in concentration at RT, phenyl ethyl acetate was the most stable ester at RT, decreasing by 9.82% after three months. Wort supplemented with 0.75 g/l L-leucine resulted in an increase in isoamyl acetate and phenyl ethyl acetate production by 38.69% and 30.40%, respectively, with a corresponding high expression of alcohol acetyltransferases, ATF2 (133.49-fold higher expression than the control). Elevation of fermentation temperature to RT resulted in the upregulation of ATF2 (27.11-fold), and producing a higher concentration of isoamyl acetate. These findings indicate that ester synthesis during fermentation is linked to both substrate availability and the regulation of gene expression. Therefore, it would be possible to manipulate the expression of certain ester synthestase genes to create new yeast strains with desirable ester production characteristics. Results from this study also suggest that supplementing wort with essential nutrients required for yeast growth and optimizing the fermentation conditions could be effective in controlling aroma-active ester concentrations to a desired level in beer.
Effects of biochar addition on soil nitrogen retention and vegetable uptake in intensive production systems, China.
(2017) Yu, Ying Liang.; Odindo, Alfred Oduor.; Pillay, Balakrishna.; Yang, Linzhang.
China has a limited area of cultivated land per capita and an increasing population. Maintaining a high crop yield is essential to meet the large food demand and to assure grain self-sufficiency. With the pace of economic development, the demand for vegetables keeps growing. In Southern China, many of the fields used to grow vegetables were previously under paddy production. Compared to the paddy production system, the vegetable production system is intensive with excessive use of nitrogen fertilizer. Excessive nitrogen fertilizer application has changed soil chemical properties and nutrient dynamics, and thus created a negative impact on sustainable agricultural development. A preliminary study was conducted in the absence of nitrogen fertilizer to determine the effect of field utilization conversion on soil nitrogen uptake by pakchoi. It was found that soil pH values and organic matter content decreased with intensive vegetable planting and nitrogen leaching loss was higher from vegetable soils compared to that from paddy soils. Although the soil mineral nitrogen content in vegetable soils was higher than that in paddy soils, nitrogen uptake by plants from vegetable soils was lower than that from paddy soils, and decreased quickly in the later growing seasons. The lower plant nitrogen uptake was attributed to the high nitrogen leaching loss and soil acidity caused by the excessive application of nitrogen fertilizer in vegetable production systems. Therefore, it is imperative to find suitable approaches to mitigate nitrogen leaching loss and soil acidity in vegetable production systems and promote nitrogen retention and vegetable nitrogen uptake for sustainable productivity. Biochar is a fine-grained and porous substance produced through pyrolysis processes, under oxygen-free conditions, from a wide range of biomass. In recent years, biochar has received more attention with regard to its capacity to increase crop yields by ameliorating the soil environment and regulating nutrient processes. According to previous studies, biochar is an option for mitigating soil acidity and nitrogen leaching problems in vegetable soils due to its alkalinity and adsorption properties. However, studies of biochar addition to vegetable production systems have not been well documented. The effect of biochar addition on leachate volume is still lacking. Whether the mineral nitrogen retained by biochar can be re-used by plants is still unknown. Few studies have investigated the effect of biochar addition on nitrogen processes and soil acidity under continuous growing conditions. Therefore, with the aim of determining the effect of biochar on soil nitrogen retention and vegetable nitrogen uptake, pakchoi was planted in a pot experiment during four continuous growing seasons with three biochar addition rates (0, 1% w/w and 5% w/w). In the 1st, 2nd and 3rd seasons, pakchoi was applied with 15N-labelled urea and in the 4th season no nitrogen fertilizer was provided. The results of this study were presented in four parts (soil nitrogen retention, soil acidity, vegetable nitrogen uptake and a distinction between two nitrogen sources in vegetable nitrogen uptake i.e. nitrogen left in the soil and nitrogen loss). The main conclusions are as follows: Biochar addition significantly increased the soil mineral nitrogen content by enhancing nitrogen retention in soils and soil nitrogen mineralization. Part of the mineral nitrogen retained by biochar was still bioavailable for plant uptake in the soil. Biochar significantly reduced nitrogen leaching loss by decreasing leachate volumes and nitrate concentrations in the leachate. Biochar addition significantly ameliorated or retarded soil acidity by promoting soil pH buffering capacity, reducing soil acidification rates and maintaining soil bases contents induced by biochar. The mitigation of soil acidity was not only as a result of biochar’s natural alkalinity but can also be attributed to the altered nitrogen processes (promotion of plant nitrate uptake, reduction of nitrification and nitrate leaching and maintaining soil bases contents) with the addition of biochar. Biochar’s mitigation of soil acidity was partly dependent on its effect on soil properties (such as bases contents) and processes (such as nitrification, nitrate leaching and plant nitrate uptake) rather than its natural alkalinity. Biochar maintained pakchoi yields and nitrogen uptake during four growing seasons. The fertilizer nitrogen recovery efficency was improved with an increase in the recovery of fertilizer nitrogen in the soil and the decrease in the recovery of fertilizer nitrogen in leachate. Fertilizer nitrogen was the major source for pakchoi nitrogen uptake, soil residual nitrogen and nitrogen leaching loss, while nitrogen from soil mineralization was the major nitrogen source for biochar retention. When nitrogen fertilizer was absent in the 4th season, the nitrogen fertilizer left in the soil from the 1st to 3rd seasons decreased sharply and fertilizer nitrogen retained by biochar was simultaneously released. The conclusion was that biochar addition could promote soil nitrogen retention and maintain high nitrogen uptake by vegetables in continuous growing seasons. However, the comprehensive effect of biochar on nitrogen loss still needs to be assessed before recommending extended utilization of biochar in vegetable production systems in China.
Evaluation of a synthetic peptide for the detection of anti-Mycobacterium tuberculosis curli pili IgG antibodies in patients with pulmonary tuberculosis.
(Elsevier., 2018) Naidoo, Natasha.; Pillay, Balakrishna.; Bubb, Martin Owen.; Pym, Alexander S.; Chiliza, Thamsanqa Emmanuel.; Naidoo, Kogieleum.; Ndung'u, Peter Thumbi.; Kasprowicz, Victoria.; Pillay, Manormoney.
Abstract available in pdf.
Genetic characterisation of an indigenous plasmid in xanthomonas albilineans.
(1999) Permaul, Kugenthiren.; Pillay, Balakrishna.; Pillay, D.
Abstract available in PDF file.
Impact of irrigation water on the quality of fresh produce.
(2011) Naidoo, Aileen.; Pillay, Balakrishna.; Olaniran, Ademola Olufolahan.
The consumption of minimally processed fresh fruit and vegetables has increased over the past years, mostly because of consumers awareness that fresh produce serves as a good source of vitamins, minerals and fibre. Although fresh produce is important for the human diet it may provide an optimal environment for the growth and proliferation of pathogenic microorganisms, from cultivation to processing. Several outbreaks of disease, associated with the consumption of fresh produce, have been reported worldwide. In addition, fresh produce can become contaminated by heavy metals imposing a public health concern. One of the major sources of contamination is irrigation water, as it may contain pathogens and heavy metals from upstream operations. Irrigation water has been previously shown to be associated with the contamination of fresh produce. Therefore the objective of this study was to evaluate the microbial- and heavy metal- content of irrigation water used by local farmers in KwaZulu-Natal (KZN) over a 12- month period, in order to establish a link between the water quality and the safety of fresh produce, and to develop a suitable method to reduce the microbial contamination of fresh produce during both pre- and post-harvest phases. The microbial quality of the water and fresh produce samples was determined using the membrane filtration and standard spread-plate techniques, respectively. The heavy metal content of the water and fresh produce samples were analysed using inductively coupled plasma optical emission spectrophotometry (ICP-OES). Presumptive Escherichia coli, Salmonella spp., Shigella spp. and coliform counts in the water samples were high during the sampling period. Presumptive E. coli exceeded the DWAF limit of 2×10³ cfu/100 ml for E. coli in irrigation water, in some instances. High counts of presumptive coliforms, Shigella spp. and Campylobacter spp. were recorded in the fresh produce, throughout the sampling period. The roots of the plant demonstrated the highest microbial and heavy metal contamination. Leafy vegetables such as spinach and lettuce were more contaminated than the other fresh produce sampled; for example, Campylobacter spp. exceeded 4.5×10⁵ cfu/g in crisphead lettuce. With regard to the heavy metal content of the irrigation water and the fresh produce, mercury (Hg) exceeded the FAO and WHO limit of 0.001 mg/L, throughout the sampling period, with the highest concentration of 0.057 mg/L obtained from irrigation water. Since the concentrations of Hg in both the irrigation water and fresh produce were the highest during the same period, such as in winter, a clear link can be seen between the irrigation water and fresh produce. The method used during the pre-harvest phase, in order to reduce pathogens from produce, was the effect of Pseudomonas aeruginosa on the uptake of pathogens to the fresh produce. Inhibition assays were employed to determine whether P. aeruginosa could inhibit the pathogens (E. coli, Listeria monocytogenes, Salmonella spp. and Shigella spp.) tested. Only L. monocytogenes was found to be inhibited by P. aeruginosa. A greenhouse experiment was employed to prove that P. aeruginosa could prevent the uptake of this pathogen, via the roots, into the fresh produce by monitoring the concentration of L. monocytogenes in the soil and fresh produce by standard spread-plating. Denaturing gradient gel electrophoresis (DGGE) was also used to monitor the populations of L. monocytogenes and P. aeruginosa in the soil. Colony counts of L. monocytogenes decreased from 6 to 3.5 log cfu/g in the soil during the first 3 weeks of sampling. This decrease was confirmed by DGGE and suggested that this pathogen was inhibited by P. aeruginosa in the soil; hence, this pathogen was also not detected in the plant. During the post-harvest phase the effect of different treatment methods on the quality of the final produce was evaluated using tap water, NaCl, chlorine, hydrogen peroxide, blanching and ultraviolet (UV) light. UV light showed the most promise as the quality of this treated produce was better as compared to the other treated produce. A link between irrigation water qualities with that of produce was evident in this study as the highest microbial counts were recorded in summer for both the water and fresh produce samples. The pre-harvest method for the reduction of pathogens from the produce, which was the effect of P. aeruginosa on the uptake of pathogens to the produce, was limited as this organism had only inhibited L. monocytogenes, of the pathogens tested. Of the post-harvest treatment methods, UV treatment had caused the highest reduction in the microbial load of the fresh produce, with tap water treatment aiding in the survival of these presumptive pathogens. The presence of P. aeruginosa and the use of UV light in reducing microbial counts on fresh produce had both shown promise in this study. However, further studies need to be employed in order to optimise these methods before application. In addition, irrigation water should be routinely monitored and properly decontaminated, if necessary, to prevent the transmission of food-borne pathogens to crops. This may curb the problem of food-borne associated disease outbreaks world-wide as irrigation water has been shown, by the current study, as a link to the contaminated state of fresh produce.
Implementation of ISA S88 batch control standards on a traditional microbrewery system.
(2016) Dlodlo, Snqobizizwe Buphilo.; Pillay, Balakrishna.
Beer production dates back to the Babylonian and Monks age in 1000 BC and had no standard manufacturing protocols for a very long time. It was the passing of the purity law by the Germans in 1514 that advanced beer production and handling into a more industrial approach. In the years that followed, beer production has evolved into a very technical and delicate procedure that has high quality control measures. This investigation set out to prove the hypothesis that the implementation of the ISA S88 batch control standards in a traditional microbrewery system would increase process efficiency, as well as product consistency and stability. The study commenced with modifications to an existing traditional microbrewery system that included additional stirring flaps in the mash tun, construction of a wort/water recirculation pipeline coupled with a sprinkler in the lauter tun, and additional heating belts and temperature probes as well as a removable cooling coil in the kettle. Thereafter, an experimental plan was developed to brew Premium English pale ale under consistent conditions defined by the proposed ISA S88 model where quality defining parameters included specific gravity, pH and total dissolved solids, colour, batch volumes, reducing sugars content, free amino nitrogen content, simple sugars and flavour compound concentrations. Six identical batches were brewed and apportioned for fermentation at 14, 16, and 18 °C, respectively. Racking of all fermented batches was performed at 0 °C for two weeks before bottling, conditioning and final storage of all batches of beer at 0, 4, and 18 °C, respectively. A HACH HQ 40d multimeter probe was used for all physico chemical measurements with its various probes whilst a Shimadzu UV – 1800 spectrophotometer coupled with a Shimadzu CPS temperature controller was used for all colourimetric and optical density measurements. Simple sugars and beer flavour compound concentrations were measured by means of an Agilent 7890 A gas chromatography system coupled with an Agilent GC 80 sampler and an inert mass spectrophotometry detector. In the mashing process, the final gravity of the wort was observed to be 14.06 ± 0.18 °P, reducing sugars were found to be 89.47 ± 2.39 g/l. In the lautering stage, the three runnings resulted in 7.92 ± 0.51 °P, 3.95 ± 0.60 °P and 1.67 ± 0.15 °P gravities, corresponding to 7.67 ± 0.55 l, 7.58 ± 0.48 l and 5.45 ± 0.42 l volumes, respectively. The collective volume was 35.71 ± 0.51 l and 167.61 ± 1.71 g reducing sugars were recovered from the spent grain. In the kettle, gravity increased to 12.10 ± 0.46 °P. Upon addition of 462 ± 68.87 g maltose syrup and boiling, the final reducing sugars amount was found to be 1632.97 ± 12.64 g in 26.45 ± 1.34 l of wort. Optimum fermentation and beer storage conditions were noted to be 16 °C and 0 °C, respectively. Flavour compounds formed during this fermentation period were found to be at concentration levels of 4.52 ± 0.24 % v/v, 119.05 ± 9.66 mg/l, and 64.02 ± 7.72 mg/l for ethanol, total fusel alcohols and total esters, respectively. Beer fermented at 16 °C depleted the total simple sugars from 12.99 ± 1.25 g/l to 5.23 ± 0.24 g/l, 10.61 ± 1.61 g/l to 5.24 ± 0.29 g/l, and 8.56 ± 3.12 g/l to 4.84 ± 0.47 g/l for storage temperatures of 0 °C, 4 °C, and 18 °C, respectively. The ethanol concentrations increased during the storage period from 4.57 ± 0.39 % v/v to 5.12 ± 0.43 % v/v, 4.70 ± 0.37 % v/v to 5.24 ± 0.29 % v/v, and 4.82 ± 0.43 % v/v to 5.39 ± 0.22 % v/v for beer stored at 0 °C, 4 °C, and 18 °C, respectively. The primary fermentation temperature of 16 ºC was found to be the most ideal (r² = 0.9551), as it produced a very steady and predictable fermentation trend. There were no pH changes in the beer fermented at 16 ºC, implying that no mouth feel changes in the product‟s taste were significantly possible. The physical and chemical property trends, statistical analyses, and literature comparison of the produced wort and beer proved that ISA S88 batch controlling standards, even in a basic traditional microbrewery, can improve process-product quality and guarantee product quality consistency.
Molecular characterisation and detection of xanthomonas albilineans, the sugarcane leaf scald pathogen.
(1994) Permaul, Kugenthiren.; Pillay, Balakrishna.; Pillay, D.
No abstract available.
Mycobacterium tuberculosis pili (MTP) modulates pathogen and host metabolomic changes in an A549 epithelial cell model of infection.
(2020) Reedoy, Kajal Soulakshana.; Pillay, Manormoney.; Pillay, Balakrishna.
Background/Aim: Mycobacterium tuberculosis, the causative organism of tuberculosis, continues to drive research efforts in the quest to develop novel diagnostics and therapeutics. The complexities associated with drug-resistant strains (multidrug-resistant (MDR), extensively drug-resistant (XDR) and totally drug-resistant tuberculosis (TDR-TB)) and co-infection with human immunodeficiency virus/acquired immune deficiency virus (HIV/AIDS) further cripple the fight against tuberculosis (TB). Hence, a comprehensive understanding of the M. tuberculosis genome, transcriptome, proteome and metabolome is required to gain different perspectives on potential target points, such as novel biomarkers, for intervention. The M. tuberculosis curli pili (MTP), a surface-located adhesin is involved in the first point of contact with the host cell, and has shown diagnostic and therapeutic potential based on previous genomic, transcriptomic and proteomic findings. Understanding the metabolome of Mycobacterium tuberculosis and its target host cell during infection will provide further insights into the role of MTP and its metabolic influence. This study aimed to determine the role of MTP in modulating bacterial and host metabolic pathways of M. tuberculosis and A549 epithelial cells, respectively, using a two-dimensional gas chromatography time-of-flight mass spectrometry (GCxGC-TOFMS) approach coupled with bioinformatic analyses. Methods: The wild-type (WT), mtp deletion mutant (Δmtp) and mtp-complemented strains were confirmed by genomic DNA extraction and PCR. For the pathogen model investigation, ten biological replicates of each of the three strains were individually cultured in supplemented Middlebrook 7H9 broth till an OD600 of 1 was reached. Cultures were centrifuged, subjected to a washing procedure and the resulting pellets were stored at - 80 °C. For the infection model investigation, A549 epithelial cells were grown till confluent, and seeded at a concentration of 5 x 105 cells/mL. A549 cells were infected with each M. tuberculosis strain at a multiplicity of infection of approximately 5. After the 2 hr infection period, cells underwent a washing procedure and the resulting pellets were stored at - 80°C prior to extraction for GCxGC-TOFMS metabolomic analysis. A whole metabolome extraction method was applied to extract metabolites from various metabolite classes using chloroform:methanol:water (1:3:1). The samples were analysed by GCxGC-TOFMS which underwent first and second dimensional separation. ChromaTOF software, MATLAB software along with the Eigenvector PLS_Toolbox 8.7 were used to identify differentiating metabolites. Parametric univariate analysis included independent samples t-test with its associated Cohen’s d-value. Correcting for multiple testing was done by the Benjamini & Hochberg (BH) adjustment to control the rate of false discovery. Multivariate analyses included quality assurance based on Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA). Variable Importance in the Projection (VIP) values, BH-adjusted p-values, Cohen’s d-values and fold changes were considered as criteria for shortlisting metabolites. In order to better understand the metabolomic changes at a transcriptomics level, RT-qPCR was performed on the bacterial strains. The resulting gene expression data was normalised using 16S rRNA and analysed using the relative standard curve method. GraphPad Prism version 8 software was used to determine significance values. Results/Discussion: The results from the bacterial model investigation were significant as 27 metabolites were found to be altered in concentration between the mtp-deficient cells and the WT, while 7 metabolites were deemed significantly different between the WT and mtp-complemented strains. Three of the 4 categories were produced in higher relative concentrations by Δmtp; carbohydrates in cell wall biogenesis, fatty acid metabolism and peptidoglycan synthesis, indicating an overall reduced ability in the utilisation of these metabolites for natural cellular processes in the Δmtp compared to the WT. Metabolites involved in amino acid and protein synthesis were produced in relatively lower concentrations in Δmtp, again suggesting defective pathways in Δmtp. The infection model analysis investigated five different M. tuberculosis infection models, of which only one validated. The first three models, which revealed minimal differences, were the infected and uninfected models of comparison to determine whether any significant differences existed in the M. tuberculosis-infected A549 cell model compared to the respective uninfected model. There were no major differences between the WT-infected and mtp-complement-infected strain showing functional restoration of mtp. Significant differences were observed between the WT-infected and Δmtp-infected A549 cells. These included a total of 46 metabolites produced in significantly lower relative concentrations in the Δmtp-infected cells. The deletion of the MTP adhesin led to a perturbation in nucleic acid metabolism, which was found to be less efficient in the Δmtp-infected cells. A similar observation was seen for lysine metabolism and degradation. Nitrogen assimilation was also found to be less prominent in Δmtp-infected cells arising from aspartate, alanine and glutamate metabolism. Metabolites involved in glutathione metabolism, oxidative stress and lipid metabolism were produced in lower relative concentration in Δmtp-infected cells, potentially resulting in a compromised mycobacterial cell envelope in the deletion mutant. Lanthionine was an unusual metabolite detected in the present study. These metabolic alterations were indicative of lowered pathogenicity of the M. tuberculosis mutant strain, as a result of the absence of MTP. Conclusion: The significant findings of this study confirm previous reports that MTP has potential as a biomarker that can be targeted for intervention. The first investigation revealed a total of 27 metabolites to be biologically significant between the Δmtp and WT strains. These were associated with reduced cell wall biogenesis, fatty acid metabolism, amino acid and protein synthesis, and peptidoglycan synthesis. Between the WT and mtp-complemented strains, seven metabolites were biologically significant and corresponded with various cell envelope functions. In the second investigation, all 46 metabolites were produced in a relatively lower concentration by the Δmtp-infected cells compared to the WT-infected cells and were associated with a decrease in nucleic acid synthesis, amino acid metabolism, glutathione metabolism, oxidative stress, lipid metabolism and a peptidoglycan anomaly. The MTP adhesin is associated with various changes to the pathogen and host metabolome, highlighting its importance as a virulence factor that further substantiates its potential as a suitable biomarker for corrective intervention in the fight against TB.
The production of monoclonal antibodies against esat6 of mycobacterium tuberculosis.
(2016) Pillay, Nethi.; Pillay, Balakrishna.
Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis (TB), was responsible for 9.6 million cases and 1.5 million deaths globally. Therefore, early TB diagnosis remains a high priority to mitigate the consequences of poor health outcomes and continuous disease transmission. The strategy in detecting the TB antigen instead of antibodies elicited against TB is preferred as immunosuppression in patients co-infected with HIV, makes antibody detection unfavourable. Monoclonal antibodies (MAbs) to TB-specific markers are therefore an attractive option for use in ELISAs and lateral flow tests that effectively meet the criteria for a rapid antigen detection test. Consequently, the objective for the present study was to produce TB-specific MAbs that would aid in the development and optimization of an ELISA for the rapid detection of TB. In order to meet these objectives this study focused on the production and characterization of MAbs that specifically target the early secretory antigenic target 6 (ESAT6) protein from M. tuberculosis. PCR was performed on M. tuberculosis H37Ra DNA, using primers specific to the esat6 gene. PCR products were inserted into pGEM-T vector followed by ligation into the expression vector pGEX6P-1 for transformation into Escherichia coli (E. coli) strain XL-1 Blue. ESAT6 GST protein was expressed and purified by glutathione sepharose affinity chromatography. Thereafter, recombinant ESAT6 GST protein was used to immunise 10 Balb/C mice and stable hybridoma cell lines were generated. The specificity of three monoclonal antibodies were confirmed and identified as anti-ESAT6 DE2-1, anti- ESAT6 KE10-1 and KE10-2. Hybridomas showing cross-reactivity to non-specific antigens, were excluded from the study. Ouchterloney Double-Diffusion was employed and the three MAbs were subtyped as an IgG and two IgM’s respectively. The multi-epitopic nature of ESAT6 is a desirable characteristic in diagnostic assay development. This characteristic was demonstrated by ELISA using anti-ESAT6 DE2-1 as a coating MAb and anti-ESAT6 KE10-1, conjugated to horse radish peroxidase, as the detection antibody. The generation of anti-ESAT6 MAbs in this study, have demonstrated their potential for use in the development of a rapid TB diagnostic test. This is critical in TB management, treatment of the disease, reducing TB transmission and incidence. Future work must therefore be aimed at the development of a diagnostic test, for use at the point-of-care, which would complement the TB diagnostic algorithm.
Role of marine nitrifying bacteria in a closed system with Penaeus monodon.
(1984) Pillay, Balakrishna.; Roth, G.
In recent years there has been widespread interest in rearing aquatic organisms of nutritional and commercial value (Calaprice, 1976). The most hopeful prospect for marine prawn culture in the United Kingdom (Wickins, 1976), the Americas (Hanson & Goodwin, 1977) and South Africa probably lies in intensive culture under controlled conditions. A closed system approach, in which a captive body of water is circulated, provides the scope for water quality management which results ~n maximum water utilization and minimal discharge. On the other hand, direct utilization of sea-water in open systems presents problems for aquaculture since this water is subjected to diurnal and seasonal fluctuations in temperature, salinity and turbidity, as well as contamination from industrial, agricultural and maritime sources. Furthermore, large mariculture farms release enormous amounts of organic wastes which result in eutrophication and could lead to environmental deterioration of coastal waters (Gerhardt, 1978). It is well established that circulated sea-water develops an unusual ~on~c composition as a result of the metabolic activity of the prawns and of the nitrifying bacteria in the biological filter. The changes include elevated levels of ammonia, nitrite and nitrate and reduced pH. The presence of even sublethal levels of these nitrogenous compounds ~n closed systems have been found to affect growth of penaeid spec~es (Wickins, 1976). Ammonia and nitrite, which rapidly accumulate in the water, are usually maintained at nontoxic levels by nitrification in the biological filters (Spotte, 1974; Johnson & Sieburth, 1974). The chemolithotrophic bacteria responsible for nitrification are presently classified by their · cellular morphology and by the oxidation of either ammonia and nitrite (Watson, 1974). The predominant ammonia- and nitrite-oxidizing bacteria isolated from natural environments are Nitrosomonas europaea and Nitrobacter winogradskyi, respectively (Watson et aZ., 1981). Direct observation of nitrifying bacteria in natural environments, however, has been limited to studies involving light microscopy with immunofluorescent techniques (Fliermans et aZ., 1974; Fliermans & Schmidt, 1975). The electron microscopic observation of nitrifying bacteria ~sdifficult in natural microcosms with low levels of nitrification and with the presence of sunlight and anaerobic conditions conducive to the enrichment of other bacteria with a similar ultrastructure. However, in closed systems with extremely active nitrification but poor light conditions, the occurrence of morphologically similar forms in numbers that could be easily detected by electron microscopy is unlikely (Johnsort & Sieburth, 1976). Furthermore, the cyst-like colonies of the nitrifiers are unique and are not found with the methane-oxidizing bacteria with a similar ultrastructure (Davies & Whittenbury, 1970; Smith & Ribbons, 1970), whereas the thick cell wall of the cyanobacteria (Carr & Whitton, 1973) and the distinctive cell morphologies of the purple sulphur and purple nonsulphur bacteria (Pfennig, 1967) separate them from the nitrifiers. Therefore, closed systems with active nitrification provide the ideal environment to study the activities of nitrifiers in conjunction with their relative abundance, nature and diversity. In spite of the opportunity offered by closed systems, previous studies (Kawai et aZ., 1965; Wickins, 1976; Gerhardt, 1978; Mevel & Chamroux, 1981) on nitrification have been primarily indirect observations on rates of ammonia and nitrite oxidation to nitrate (Johnson & Sieburth, 1976). Studies on the enumeration and identification of nitrifiers ~n closed systems have been seriously neglected. Kawai et aZ. (1964) included the enumeration of nitrifiers in their study on nitrification while,in a qualitative study, an attempt to identify the in situ nitrifiers 1n closed systems (Johnson & Sieburth, 1976) was not very successful. This study was undertaken to investigate the three basic aspects of nitrification necessary for the understanding of such a process in closed systems, viz., the oxidation of ammonia and nitrite to nitrate, and the enumeration and identification of the nitrifying bacteria. Prior to determining the concentrations of the nitrogenous compounds in the culture water, various methods were evaluated for their accuracy and reproducibility with both sea-water and culture water samples. This approach is necessary in order to gauge the accuracy of results obtained by such methods. Enumeration of nitrifying bacteria was preceded by an investigation on the effect of incubation time on the maximum most probable number , estimate. Such an investigation was necessary because of the inconsistent approach to the enumeration of nitrifiers in previous studies (Wilson, 1927; Walker et al., 1937; Lewis & Pramer, 1958; Molina & Rovira, 1964; Meiklejohn, 1965; Smith et al., 1968). Incubation periods appear to have been chosen arbitrarily in previous investigations. Identifi~ation of nitrifying bacteria necessitates the isolation and purification of these organisms. Isolation of nitrifiers 1S a difficult and time-consuming task (Watson et al., 1981) and could be the main reason for not being included in previous studies on nitrification. Since the success of this study depended upon the isolation and purification of these chemolithotrophs, this aspect is de~lt with in detail. The changes most likely to be associated with nitrification in a closed system were also monitored 1n the culture water. These included pH, dissolved oxygen and biochemical oxygen demand. Apart from a biological sand filter, no other form of culture water treatment was effected during the investigation. The effect of growing the "sugpo" or jumbo tiger prawn, Penaeus monodon (Kinne, 1977) for 22 weeks in a captive body of sea-water was evaluated by comparing the survival and wet mass with those reported by other workers. This study differs greatly from previous reports on nitrification in closed systems because both the "causes" and "symptoms" of this important detoxifying process are investigated. It is intended that the findings of such a study would aid culturists in exploiting the nitrifying potential of closed systems to its utmost.
The role of Mycobacterium tuberculosis curli pili (MTP) and heparin-binding hemagglutinin adhesin (HBHA) on global in vitro bacterial transcriptomics.
(2021) Naidoo, Tarien Jael.; Pillay, Manormoney.; Pillay, Balakrishna.
Background/Aim: Tuberculosis (TB), is an infectious, airborne disease caused by Mycobacterium tuberculosis (M. tuberculosis). TB remains one of the most devastating bacterial causes of human mortality, especially in low-income countries. Surface located adhesins are crucial for M. tuberculosis survival, as they initiate and perpetuate host-pathogen interactions. The adhesin, M. tuberculosis curli pili (MTP), plays a role in adhesion and invasion of host cells and biofilm formation, whilst heparin-binding hemagglutinin adhesin (HBHA) promotes M. tuberculosis dissemination from the site of infection. The use of transcriptomics promises to enhance current knowledge on MTP and HBHA as virulence factors, thereby substantiating their role as biomarkers for the development of accurate TB diagnostics and therapeutics. Therefore, this study aimed to elucidate the role of MTP and HBHA in the regulation of M. tuberculosis transcriptome, and to identify novel biomarkers. This was achieved by analysing the transcriptomic perturbations in the strains lacking the MTP adhesin, HBHA adhesin or both MTP-HBHA adhesins and the strains containing the aforementioned adhesins. Methods: Polymerase chain reaction (PCR) confirmed strains of M. tuberculosis wild-type (WT), mtp-deletion mutant (Δmtp), hbhA-deletion mutant (ΔhbhA), mtp-hbhA-deletion mutant (Δmtp-hbhA), and the respective complemented strains, were standardized and cultured until log phase. Thereafter, the bacterial cultures were prepared for RNA extraction. RNA was extracted via an optimized TRIzol method and sequenced using the Illumina 2×150 HiSeq ×10 platform. The sequenced reads were analysed by FastQC toolkit (version 0.11.8), pre-processed using Trimmomatic (version 0.36), mapped to the custom-built M. tuberculosis H37Rv genome index using hierarchical indexing for spliced alignment of transcripts (HISAT version 2.1.0), assembled by Stringtie (version 1.2.1), and further annotated and assembled the transcripts into known and novel categories by Gffcompare, located within Stringtie. The output files were annotated in R (version 1.2.1578) using the Ballgown package to generate the respective fold changes (FC) between the deletion mutants and the WT, and q-values and p-values for the differential expression. The generated results were filtered using a FC cut-off value ≥ 1.3 (to indicate a 1.3-fold up-regulation) and ≤ 0.5 (to indicate a 2-fold down-regulation) to identify significant genes and pathways. Thereafter, relevant databases and literature were reviewed to categorize the genes into pathways. Real time quantitative PCR (RT-qPCR) was performed on 10 selected genes, as a genotypic method to functionally confirm the RNA sequencing data. A bacterial bioluminescence cell viability assay was performed to elucidate the concentration of adenosine triphosphate (ATP) in the deletion mutants and complements, relative to the WT. Results: A total of 43 genes were significantly differentially expressed amongst the deletion mutants. These genes were functionally categorized into: intermediary metabolism and respiration metabolism, cell wall biosynthesis, cell wall transport and processes, lipid metabolism, and virulence; stable RNA’s; conserved hypotheticals; proline-glutamate (PE) or proline-proline-glutamate (PPE); insertion sequences and phages; and information pathways. The bioluminescence assay functionally confirmed the increased utilization of ATP in the absence of MTP and HBHA. Discussion/Conclusion: Adhesin gene deletions caused major perturbations to the central carbon metabolism, cell wall biosynthesis, cell transport process, lipid biosynthesis, and virulence pathways, leading to potentially increased energy requirements; compensatory transport of proteins to the cell wall, altered cell wall biosynthesis and decreased virulence and pathogenicity. Additionally, deletion of these adhesins resulted in the disruption of many processes potentially attenuating growth and replication. Thus, this study further corroborates the adhesins, MTP and HBHA, and associated pathway genes as potential suitable targets for TB diagnostic/therapeutic interventions.
Selection and identification of novel Mycobacterium tuberculosis phage-displayed biomarkers by immunoscreening against patients’ serum samples.
(2018) Chiliza, Thamsanqa Emmanuel.; Pillay, Balakrishna.; Pillay, Manormoney.
Abstract available in pdf.
Soil microbial responses to simulated climate change drivers.
(2014) Rajpal, Desireé Alvika.; Pillay, Balakrishna.
Climate change is one of the biggest environmental challenges being experienced in the 21st century and is expected to continue to cause drastic alterations to the hydrological, biological and ecological ecosystems. Soil, the second largest carbon pool after the oceans, is a major factor in the global response towards climate change. The ability of soil to act as a sink or source of carbon as climate change increases can be influenced by soil microbial activity. Soil microbial activity is a key driver of terrestrial ecosystem functions and is extremely sensitive towards climate changes. Therefore, the main objective of this study was to investigate the effects of individual and/or interactive global change factors on soil microbial activity and diversity under elevated or ambient temperature incubations during the spring and summer seasons. This was accomplished by the addition of carbon dioxide (CO2), methane (CH4) or simulated rainfall to soil over a 60-day period using Screen Aided Carbon Dioxide Control experiments. Soil microbial dehydrogenase, urease, arylsulphatase and β-glucosidase activities were determined using standard enzyme assays over the 60-day period. In spring, the soil dehydrogenase and arylsulphatase activities increased by 28.07% and 28.48%, respectively, after the addition of elevated CO2 under elevated temperature. Lower dehydrogenase activities were observed at day 60 for most plots during summer while β-glucosidase activity was unaffected by the addition of single or multiple global change drivers during spring. In summer however, all treatments resulted in 28.05 - 36.39% higher β-glucosidase activity by day 15, compared to day 0. Urease enzyme activity was higher during spring at both temperature conditions indicating that moisture limitation and temperature change constrained the urease enzyme production during the summer period. Neither the combination of elevated CO2 and rainfall nor the combination of elevated CO2, rainfall and methane induced substantial changes to the enzyme activities during both seasons, suggesting an antagonistic effect of the combination of these global change factors. However, differences observed from a combination of elevated CO2 at higher temperature clearly reflect a potential for interaction that will affect soil enzymes and subsequent nutrient cycling. This study also investigated the changes in the soil bacterial RuBisCo gene (cbbL), important for CO2 fixation and the corresponding changes in soil organic carbon (SOC), upon exposure to various single or multiple global change drivers. Lowest cbbL gene copy numbers were observed during summer, while, during spring, the cbbL gene copy numbers increased (90.9 – 93.09%) by day 60 compared to day 0, under elevated temperatures. The combination of global change drivers did not result in a substantial variation in cbbL gene copy numbers across seasons suggesting a counteractive effect of the factors, similar to changes in soil microbial enzyme activity. No direct correlation between changes in copy number and SOC was observed, although lower SOC in summer at elevated temperature did result in overall lower cbbL gene copies. Denaturing Gradient Gel Electrophoresis, used to investigate changes in soil microbial community structure, revealed seasonal variability changes in microbial diversity during spring. Soil moisture was a key factor in determining microbial responses during both seasons, with the elevated rainfall treatments able to counteract the adverse effects of elevated temperature during the spring season, with communities in these plots appearing more robust. Increased temperatures and lower soil moisture during the summer period had a negative effect on microbial diversity; however, sequence analysis of excised bands revealed the dominance of thermotolerant bacterial species. A combination of all the global change factors did not induce substantial change in community structure during spring at both temperature regimens. During summer at elevated temperature, growth of certain microbial species were inhibited by a combination of all the global change factors, highlighting the interactive effect between temperature, greenhouse gases and soil moisture. Furthermore, the loss of methanotrophic bacteria, (Methylosinus and Methylocystis) during both seasons can negatively impact greenhouse gas flux and consequently the carbon cycle at large. In this study, seasonal changes linked to variations in soil moisture, substrate availability and temperature strongly influenced soil microbial responses towards climate change. Considering that climate change is a multifactorial process, this study also clearly highlights the necessity for multi-factor global change studies, especially across different seasons in order to accurately predict the fate of soil ecosystem as climate changes continue to increase. Climate change studies often disregard microbial contributions and if carbon sequestration strategies are to be successful, we must fully understand microbial responses under various environmental conditions. An in depth understanding of factors that can lead to changes in soil microbial community activity and structure which influence nutrient and greenhouse gas cycling is essential towards enhancing knowledge of climate change mitigation strategies. Despite the drastic increases in greenhouse gases, temperature and/or rainfall simulated in the present study, it was evident that certain species of soil microorganisms were still able to survive and mediate biochemical activities that are beneficial to the community as a whole.
Treated wastewater effluent as a potential source of virulent and antibiotic resistant Yersinia species in receiving surface water.
(2015) Khumalo, Gcinile Zamantungwa.; Pillay, Balakrishna.; Olaniran, Ademola Olufolahan.
Yersinia enterocolitica is a potentially pathogenic bacterium transmitted through the faecal-oral route. Typical symptoms include those associated with gastrointestinal disease, although infection can also lead to more serious and invasive illnesses, particularly in sensitive populations. Previous studies have detected Y. enterocolitica in surface water in various parts of the world, and studies have reported the intake of untreated water to be one of the potential risk factors for Y. enterocolitica infection. This study investigated the antibiotic resistant patterns and the virulence determinants of the previously identified Y. enterocolitica in treated wastewater effluents and the receiving rivers. In addition, the antibiogram and virulence factors of these isolates were determined in order to establish the possible effects posed by these isolates to the users of receiving surface waters. Finally, the genetic relatedness of the isolates was established by Random Amplified Polymorphic DNA (RAPD-PCR). The antibiotic susceptibility assays revealed that the isolates were resistant to ampicillin (100%), amoxicillin (98%), cefuroxime (96%), cefalothin (90%), streptomycin (93%), chloramphenicol (100%), tetracycline (100%) and trimethoprim (100%). The calculated multiple antibiotic resistance (MAR) indices of the Y. enterocolitica isolates ranged from 0.5-0.66, suggesting high multi-antibiotic resistance among the isolates. A high prevalence (59%) of class 2 integrin was found among the isolates, with 26 and 6% of the isolates in possession of class1 and class 3, respectively. The integrase genes detection showed that the isolates possessed 3 classes of integrons, detected in 59%, 26% and 6% of the isolates, respectively. The virulence determinant assays using crystal violet staining showed that only 21% (15/70) of the isolates could retain the purple colour suggesting that they may be the virulent strain of Y. enterocolitica. The negative MBL activity suggests that the tested isolates do not demonstrate any hydrolytic activity for the degradation of cephalosporins. Virulence gene detection via PCR showed that the most abundant gene is the ystA (56%) followed by ail (34%), both chromosomally located. The plasmid located genes were detected in 3% of the isolates for both Vir/Lcr and yadA. The genotypic characterization of the tested isolates revealed two main clusters (A and B), with cluster A comprising the majority of the isolates (68%) and include the Y. enterocolitica positive control, whilst cluster B grouped 31% of the isolates. had 31% similarity to the control.