DNA extraction of bacterial cells using a semi-automated filtration system.

The COVID-19 pandemic lockdown created problems with importing of commercial kits resulting in extended turnaround times for consumable deliveries. One way to circumvent this was to use an inexpensive optimized in-house method for DNA extraction from water. • The DNA extraction methods were optimized on a 96-well plate using a semi-automated filtration system to increase the number of samples from 24 to 96 at a time in 2 h. The DNA extraction method optimizations included: (a) Guanidium thiocyanate method plus dilution series of celite to determine DNA binding capacity; (b) QIamp 96 Qiacube HT kit (Qiagen®); (c) Guanidium thiocyanate with the celite replaced with a binding buffer. • The in-house DNA extraction methods and adapted in-house DNA extraction method were compared to QIamp 96 Qiacube HT kit (Qiagen®), which is used on a 96-well semi-automated filtration system. The results showed maximum capacity of the 96-well filter plates was 400 µâ„“ broth (OD600 = 0.45 = 3.6 × 108 cells/mℓ) before the 96-well filters blocked. • When the methods were compared, there was no significant difference between the in-house DNA extraction method with 1:420 celite dilution (P-value = 0.126) and the adapted in-house method with binding buffer (P-value = 0.298) DNA yield or amplification of PCR products.

Culture independent DNA extraction method for bacterial cells concentrated from water.

•To bypass the problem of viable but non-culturable bacteria that cannot be isolated by culturable methods would be to isolate DNA from bacterial cells concentrated from water samples used as a template for the polymerase chain reactions (PCR). DNA extraction protocol (Omar et al. 2010) was used as a foundation for extracting Escherichia coli DNA from water. The method combinations i.e., guanidium thiocyanate, celite and home-made spin column were chosen because it has been shown to be reliable, rapid, simple, and inexpensive for routine analysis in developing country settings.•The following optimizations were included: (a) Polycarbonate (Poly) was statistically compared with Polyether sulphone (PES), Nitrocellulose acetate (NA) and Nitrocellulose (NC) membranes; (b) Various housing containers for the membranes were tested: plastic/glass petri-dish, Falcon tubes, Ogreiner cryovials; (c) various solutions was tested to add to the membrane to remove cells from membranes; (d) celite was chosen to bind the DNA because it had a higher DNA binding capacity compared to silicon dioxide; (e) incubation times and rotation speed were tested when adding reagents.•The optimized in-house DNA extraction method was validated with environmental water samples, high (dam water) and low (borehole) bacterial load to determine upper and lower detection limits of the method.

Absolute quantification of E. coli virulence and housekeeping genes to determine pathogen loads in enumerated environmental samples.

Quantifying pathogenic genes with q-PCR in complex samples to determine the pathogen loads is influenced by a wide range of factors, including choice of extraction method, standard curve, and the decision to use relative versus absolute quantification of the genes. The aim was to investigate the standardisation of q-PCR methods to determine enumerated E. coli gene ratios grown with the IDEXX Colilert® Quanti-Trays® using enteropathogenic E. coli as the model pathogen. q-PCR targeting the eaeA and gadAB genes was used to calculate the eaeA: gadAB ratios for clinical strains collected between [2005-2006 (n = 55)] and [2008-2009 (n = 19)] using the LinRegPCR software and Corbett Research Thermal cycler software. Both programs grouped the isolates into two distinct groups based on the gene ratios although the Corbett Research Thermal cycler software gave results one log higher than the LinRegPCR program. Although the eaeA: gadAB ratio range was determined using extracted E. coli DNA, the impact of free DNA and other bacteria present in the sample needed to be understood. Standard curve variations using serially diluted extracted E. coli DNA, serially diluted pure E. coli culture followed by DNA extraction from each dilution with or without other bacteria was tested using the eaeA q-PCR to quantify the genes. Comparison of the standard curves showed no significant difference between standard curves prepared with diluted DNA or with cells diluted before the DNA is extracted (P = 0.435). Significant differences were observed when background DNA was included in the diluent or Coliform cells added to the diluent to dilute cells before the DNA is extracted (P < 0.001). The "carrier" DNA and Coliform cells enhanced the DNA extraction results resulting in better PCR efficiency. This will have an influence on the quantification of gene ratios and pathogen load in samples containing lower numbers of E. coli.

Mycotoxigenic potentials of the genera: Aspergillus, Fusarium and Penicillium isolated from houseflies (Musca domestica L.).

A study on the potential of houseflies (Musca domestica L.) to spread fungal spores in Gauteng Province, South Africa proved that houseflies are vectors for fungal spores. Therefore, there is a need to determine the toxigenic potentials and to identify the mycotoxins produced by fungal isolates derived from this study. In total 377 potentially toxigenic isolates of Aspergillus (186), Fusarium (85) and Penicillium (106) species (spp.) were isolated. These isolates were further tested for their ability to produce aflatoxins (AFs) [aflatoxin B1, B2, G1 and G2], deoxynivalenol (DON), fumonisin B1 (FB1) ochratoxin A (OTA), and zearalenone (ZEA) by high-performance liquid chromatography (HPLC) respectively. Strains of A. flavus and A. parasiticus belonging to the genera of Aspergillus were found to be the main producers of AFB1, AFB2, AFG1, and AFG2, while A. carbonarius, A. niger and A. ochraceus produced OTA. Fumonisin B1 was produced by F. verticillioides and F. proliferatum with concentrations ranging from 20 to 1834µg/kg and 79 to 262µg/kg respectively. Deoxynivalenol produced mainly by F. culmorum (2-6µg/kg), F. graminearum (1-4µg/kg), F. poae (1-3µg/kg), and F. sporotrichioides (2-3µg/kg) species was the least detected toxin in this study. The high mycotoxins levels produced in isolates from houseflies in this study are regarded as unsafe, especially when international legislated tolerance levels for mycotoxins are considered. Thus, possible human exposure to mycotoxins may pose concerns with respect to human health and demands constant and consistent investigation.

Coexistence of free-living amoebae and bacteria in selected South African hospital water distribution systems.

Pathogenic free-living amoebae (FLA), such as Naegleria fowleri, Balamuthia mandrillaris and Acanthamoeba species isolated from aquatic environments have been implicated in central nervous system, eye and skin human infections. They also allow the survival, growth and transmission of bacteria such as Legionella, Mycobacteria and Vibrio species in water systems. The purpose of this study was to investigate the co-occurrence of potentially pathogenic FLA and their associated bacteria in hospital water networks in Johannesburg, South Africa. A total of 178 water (n = 95) and swab (n = 83) samples were collected from two hospital water distribution systems. FLA were isolated using the amoebal enrichment technique and identified using PCR and 18S rDNA sequencing. Amoebae potentially containing intra-amoebal bacteria were lysed and cultured on blood agar plates. Bacterial isolates were characterized using the VITEK®2 compact System. Free-living amoebae were isolated from 77 (43.3 %) of the samples. Using microscopy, PCR and 18S rRNA sequencing, Acanthamoeba spp. (T3 and T20 genotypes), Vermamoeba vermiformis and Naegleria gruberi specie were identified. The Acanthamoeba T3 and T20 genotypes have been implicated in eye and central nervous system infections. The most commonly detected bacterial species were Serratia marcescens, Stenotrophomonas maltophilia, Delftia acidovorans, Sphingomonas paucimobilis and Comamonas testosteroni. These nosocomial pathogenic bacteria are associated with systematic blood, respiratory tract, the urinary tract, surgical wounds and soft tissues infections. The detection of FLA and their associated opportunistic bacteria in the hospital water systems point out to a potential health risk to immune-compromised individuals.

Fungal dissemination by housefly (Musca domestica L.) and contamination of food commodities in rural areas of South Africa.

Several insects that act as vectors, including houseflies (Musca domestica L.), are often considered to be an important source of fungal contamination in human foods. Houseflies are also involved in the transmission of bacterial pathogens that may pose a serious hazard to human health. Thus, the rural population of South Africa, as typified by that in the Gauteng Province investigated in this study, is at high risk from fungal exposure disseminated by houseflies and it is therefore important to assess the role of flies in contaminating various food commodities. Eighty four samples of houseflies (captured from households and pit toilets) were studied for their potential to carry fungal spores into food commodities. The fungi occurring in samples of raw maize (15) and porridge (19) were also assessed. Fungal isolates were identified based on morphological characteristics by conventional identification methods. Fifteen genera of fungi were isolated and identified, of which Aspergillus, Fusarium, Penicillium, Cladosporium, Moniliella and Mucor were the most prevalent in all three sample types analysed. The incidence rates of fungal contamination per total fungal count isolated in houseflies, maize and porridge were recorded with mean fungal load of 2×10(8) CFU/ml, 1×10(7)CFU/g and 2×10(7)CFU/g respectively. Additionally, A. flavus, A. parasiticus, F. verticillioides, F. proliferatum, P. verrucosum, P. aurantiogriseum and M. suaveolens were the most frequent fungal isolates in houseflies with incidence rate of 34%, 11%, 27%, 21%, 22%, 17% and 32% respectively. F. verticillioides, A. flavus, A. niger and P. oslonii were the most prevalent species contaminating porridge and maize with incidence rate of 23%, 32%, 16% and 28% in maize samples, while incidence rates of 59%, 15% and 29% were recorded in porridge samples with the exception of F. verticillioides. The prevalence of these genera of fungi may pose serious health risks.

Detection of free-living amoebae using amoebal enrichment in a wastewater treatment plant of Gauteng Province, South Africa.

Free-living amoebae pose a potential health risk in water systems as they may be pathogenic and harbor potential pathogenic bacteria known as amoebae resistant bacteria. Free-living amoebae were observed in 150 (87.2%) of the environmental water samples. In particular, Acanthamoeba sp. was identified in 22 (12.8%) using amoebal enrichment and confirmed by molecular analysis. FLA were isolated in all 8 stages of the wastewater treatment plant using the amoebal enrichment technique. A total of 16 (9.3%) samples were positive for FLA from influent, 20 (11.6%) from bioreactor feed, 16 (9.3%) from anaerobic zone, 16 (9.3%) from anoxic zone, 32 (18.6%) from aerators, 16 (9.3%) from bioreactor effluent, 11 (6.4%) from bioreactor final effluent, and 45 (26.2%) from maturation pond. This study provides baseline information on the occurrence of amoebae in wastewater treatment plant. This has health implications on receiving water bodies as some FLA are pathogenic and are also involved in the transmission and dissemination of pathogenic bacteria.

Fungi in housefly (Musca domestica L.) as a disease risk indicator-A case study in South Africa.

Houseflies are the commonest insects which have increasingly overcrowded human dwellings, particularly in rural areas and constitute a health hazard. In the environment they move back and forth by feeding and breeding on food commodities and filth. This may lead to the spread of diseases and also mycotoxin-producing fungi. Thus frequent exposure to the activity of such houseflies will have an impact on the welfare of humans. The study investigated the natural occurrence of fungal contamination in housefly samples captured from different households and pit toilets from a rural community in South Africa. Fungal contamination data were based on the prevalence, contamination level and morphological characteristics of the different identified species. A total of 497 fungal isolates of 15 genera including Aspergillus (37%), Fusarium (17%), Penicillium (21%), Alternaria (1.4%), Chrysosporium (2%), Cladosporium (0.2%), Curvularia (0.4%), Epicoccum (1%), Eupenicillium (1%), Moniliella (9%), Mucor (2%), Nigrospora (1%), Rhizopus (2%), Scopulariopsis (2%) and Yeasts (3%) were identified from the external surfaces of both female and male houseflies. The range of fungal contamination per total fungal count isolated from female and male houseflies were recorded with mean fungal load of 4.1×10(6), 8.4×10(6), 4.4×10(6), 3.3×10(5), 9.8×10(6), 2.2×10(4), 5.6×10(4), 2.9×10(6), 5.2×10(6), 4.7×10(6), 4.5×10(7), 4.6×10(6), 2.3×10(6), 4.9×10(7) and 6.4×10(6)CFU/ml, respectively. However, the range from The most dominant fungal isolates of the female housefly samples were Aspergillus flavus, Fusarium verticillioides, Penicillium verrucosum and Moniliella suaveolens, while A. flavus, Aspergillus parasiticus, F. verticillioides, Fusarium proliferatum and Penicillium aurantiogriseum were most prevalent in male samples. The study proves that housefly is a vector for fungal spores. Therefore, it is crucial to implement housefly-control measures to curb the spread of diseases.

Use of a bio-wipe kit to detect fumonisin B1 in faecal materials.

Fusarium toxins with reference to fumonisin B1 (FB1) have long been regarded as contaminants of maize and maize-based related products. However, when consumed they can cause intoxication, especially in humans. Therefore, effective quantitative methods for assessing the dietary exposure of this toxic fungal metabolite are required. The objective of this investigation was to evaluate the effect on the use of a bio-wipe kit, which is a faecal material collection kit, to detect the presence of FB1. Faecal materials were collected from a rural farming community in Gauteng Province, South Africa. In total, 200 samples of faecal material were analysed for Fusarium species using a serial dilution method, while FB1 was further analysed and quantified by reversed-phase TLC and HPLC. The study showed the presence of 11 different Fusarium species grown on potato dextrose agar culture medium of which F. verticillioides and F. proliferatum, producers of FB1, and F. oxysporum were the dominant species. Fumonisin B1 was recorded at an incidence rate of 65% of the total using TLC. Results from HPLC showed that 84% were positive at different ranges of concentration for FB1. This study supports the use of a bio-wipe as a rapid method to determine human exposure to FB1.

Detection of diarrhoeagenic Escherichia coli in clinical and environmental water sources in South Africa using single-step 11-gene m-PCR.

Escherichia coli (E. coli) consists of commensal (ComEC) and diarrhoeagenic (DEC) groups. ComEC are detected using traditional culture methods. Conformational steps are performed after culturing if it is required to test for the presence of DEC, increasing cost and time in obtaining the results. The aim of this study was to develop a single-step multiplex polymerase chain reaction (m-PCR) that can simultaneously amplify genes associated with DEC and ComEC, with the inclusion of controls to monitor inhibition. A total of 701 samples, taken from clinical and environmental water sources in South Africa, were analysed with the optimised m-PCR which targeted the eaeA, stx1, stx2, lt, st, ial, eagg, astA and bfp virulence genes. The mdh and gapdh genes were included as an internal and external control, respectively. The presence of the external control gapdh gene in all samples excluded any possible PCR inhibition. The internal control mdh gene was detected in 100 % of the environmental and 85 % of the clinical isolates, confirming the classification of isolates as E. coli PCR positive samples. All DEC types were detected in varying degrees from the mdh positive environmental and clinical isolates. Important gene code combinations were detected for clinical isolates of 0.4 % lt and eagg. However, 2.3 % of eaeA and ial, and 8.7 % of eaeA and eagg were reported for environmental water samples. The E. coli astA toxin was detected as positive at 35 and 17 % in environmental isolates and clinical isolates, respectively. Interestingly, 25 % of the E. coli astA toxin detected in environmental isolates and 17 % in clinical isolates did not contain any of the other virulence genes tested. In conclusion, the optimised single-step 11-gene m-PCR reactions could be successfully used for the identification of pathogenic and non-pathogenic E. coli types. The m-PCR was also successful in showing monitoring for PCR inhibition to ensure correct reporting of the results.

Applicability of Bio-wipes for the collection of human faecal specimens for detection and characterisation of enteric viruses.

OBJECTIVE: To determine whether gastroenteritis viruses and other enteric viruses could be detected in faecal specimens collected with Bio-wipes. METHODS: Faecal specimens, self-collected with Bio-wipes, from 190 individuals (94 diarrhoeal, 93 non-diarrhoeal, 3 unknown) were screened for eight human enteric viruses (enterovirus, hepatitis A virus, adenovirus, astrovirus, norovirus GI and GII, sapovirus and rotavirus) by real-time (reverse transcription)-polymerase chain reaction. Rotaviruses and noroviruses from positive specimens were genotyped. RESULTS: At least one enteric virus could be detected in 82.6% (157/190) of faecal specimens. Mixed infections of up to four different viruses could be detected in both diarrhoeal and non-diarrhoeal specimens. Enteroviruses were detected most frequently (63.7%), followed by adenoviruses (48.4%) and noroviruses (32.2%). Genotyping was successful for 78.6% of rotaviruses and 44.8% of noroviruses. CONCLUSIONS: Bio-wipes provide a user friendly, easier method for stool collection that facilitates enteric virus detection and genetic characterisation.

Pathogenic Escherichia coli in rural household container waters.

Plastic containers in the range of 5-20 L are widely used - especially in rural African settings - to collect, transport and store water for domestic use, including drinking, bathing and hygiene. The pathogen content of the waters in these containers has not been adequately characterized as yet. This paper presents the primary findings of a synoptic survey of drinking water quality samples from these containers and involved collection of bacterial indicator and pathogenicity gene data. In total, 571 samples of a variety of waters were taken in rural communities in South Africa and the Escherichia coli numbers measured. Of the E. coli positive samples, 46% (n = 148) were screened for the presence of E. coli pathogen gene markers. Though synoptic, the survey provided many insights into the issues that drove the study. Container use markedly degraded water quality as judged by indicator counts, even where improved water supply services were in place. Household container use also appeared to promote regrowth or contamination of containers with pathogenic E. coli strains. Polymerase chain reaction (PCR) analysis also showed that the diversity of potential pathogenic E. coli carrying virulence genes was great. All seven genes screened for (Ial, Stx1, Stx2, EaeA, Eagg, ST, LT) were found in the waters, alone or as mixtures (number of different combinations = 31) including those characteristic of the more dangerous invasive and haemorrhagic E. coli strains. Given the central role of containers in the management of water supply to rural communities, it is clear the microbiology of these waters requires much further characterization.

Prediction of metal-adsorption behaviour in the remediation of water contamination using indigenous microorganisms.

In recent years, the adsorption of heavy metal cations onto bacterial surfaces has been studied extensively. This paper reports the findings of a study conducted on the heavy metal ions found in mine effluents from a mining plant where Co(2+) and Ni(2+) bearing minerals are processed. Heavy metal ions are reported to be occasionally present in these mine effluents, and the proposed microbial sorption technique offers an acceptable solution for the removal of these heavy metals. The sorption affinity of microorganisms for metal ions can be used to select a suitable microbial sorbent for any particular bioremediation process. Interactions of heavy metal ions (Co(2+) and Ni(2+)) and light metal ions (Mg(2+) and Ca(2+)) with indigenous microbial cells (Brevundimonas spp., Bacillaceae bacteria and Pseudomonas aeruginosa) were investigated using the Langmuir adsorption isotherm, pseudo second-order reaction kinetics model and a binary-metal system. Equilibrium constants and adsorption capacities derived from these models allowed delineation of the effect of binding affinity and metal concentration ratios on the overall adsorption behaviour of microbial sorbents, as well as prediction of performance in bioremediation systems. Although microbial sorbents used in this study preferentially bind to heavy metal ions, it was observed that higher concentrations (>90 mg/ℓ) of light metal ions in multi-metal solutions inhibit the adsorption of heavy metal ions to the bacterial cell wall. However, the microbial sorbents reduced Ni(2+) levels in the mine-water used (93-100% Ni(2+) removal) to below the maximum acceptable limit of 350 µg/ℓ, established by the South African Bureau of Standards. Competition among metal ions for binding sites on the biomaterial surface can occur during the bioremediation process, but microbial sorption affinity for heavy metal ions can enhance their remediation in dilute (<5 mg/ℓ heavy metal) wastewaters.

Detection of Vibrio cholerae and Vibrio parahaemolyticus by molecular and culture based methods from source water to household container-stored water at the point-of-use in South African rural communities.

Detection methods for Vibrio cholerae and Vibrio parahaemolyticus which included the culture based approach with polymerase chain reaction (PCR) confirmation, PCR detection without enrichment and PCR with a pre-enrichment were developed and their performance evaluated. PCR assays targeted the SodB (V. cholerae species), Flae (V. parahaemolyticus species), 16S rRNA (Vibrio and Enterobacteriacea species) genes (Multiplex 1) and V. cholerae O1 and V. cholerae O139 rfb genes, ctxA (cholera toxin) gene and 16S rRNA gene (Multiplex 2). These methods were used to determine the occurrence of selected Vibrios in source water as well as in household container-stored water. The combination of filtration, enrichment and PCR method provided a sensitive and specific method for the detection of selected Vibrios in water samples. The PCR with a pre-enrichment method detected as few as 4-10 cfu/100 mL of selected Vibrios and PCR detection without the enrichment method detected as few as 40-100 cfu/100 mL of selected Vibrios. The inclusion of an enrichment period allows detection of culturable bacteria. As an application of the developed methods, V. cholerae and V. parahaemolyticus were detected in the source water used by the population and in the water-storage containers. The results indicate that Vibrio species in the containers could have originated from the source water and survive in biofilms inside the containers.

Haemophilus paragallinarum haemagglutinin: role in adhesion, serotyping and pathogenicity.

It is suggested that Haemophilus paragallinarum requires at least three haemagglutinins for adhesion during infection. This paper reports the partial purification and characterization of the HA-L haemagglutinin from H. paragallinarum strain 46-C3, a heat sensitive, trypsin sensitive haemagglutinin that has been shown to be the serovar specific haemagglutinin in this organism. Using the pl and molecular mass obtained, it was shown that this protein shares similarities with other types of adhesins found in Gram-negative bacteria. The haemagglutination assay conditions were optimized at pH 7.5 at 37 degrees C. It was also shown that activity is enhanced by the addition of Ca2+ and Mn2+ ions.