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.

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.