Hydrochemical,bacteriological assessment,and classification of groundwater quality in Thulamela Municipality,South Africa: potential health risk

Abstract

Potential effects of anthropogenic activities on different boreholes around Thulamela Municipality, South Africa, were evaluated by quantifying the bacteria indicators and physicochemical parameters during summer, autumn, and winter. The purpose was to determine whether the borehole water in this region is safe for domestic use across the seasons. The concentrations of physicochemical (Temperature, pH, electrical conductivity (EC), turbidity, and nitrate) and bacteriological (both Escherichia coli and Enterococcus faecalis) contaminants in the borehole water samples were determined using standard microbiology methods. The mean concentration of NO₃ for most of the boreholes failed to comply with the recommended guidelines throughout the season. High microbial load of E. coli (2.0?×?10¹ – 4.6?×?10³ CFU/100?ml) and E. faecalis (2.0?×?10° – 6.0?×?10² CFU/100?ml) was recorded in the wet season than in the dry season (0.0 – 7.0?×?10² and 0.0 – 1.0?×?10¹ CFU/100?ml, respectively). Sanitary inspection and water source classification showed that most boreholes are prone to chemical contaminants during summer and autumn due to rainfall and this correlates with the measured microbial contamination. The result is significantly important because water from these boreholes is used for domestic purposes without treatment and could pose major public health risks to the consumers.     

Development of a combined immunochromatographic lateral flow assay for accurate and rapid Escherichia coli O157:H7 detection

Escherichia coli O157:H7 is an important pathogenic Bacterium that threatens human health. A convenient, sensitive and specific method for the E. coli O157:H7 detection is necessary. We developed two pairs of monoclonal antibodies through traditional hybridoma technology, one specifically against E. coli O157 antigen and the other specifically against E. coli H7 antigen. Using these two pairs of antibodies, we developed two rapid test kits to specifically detect E. coli O157 antigen and E. coli H7 antigen, respectively. The detection sensitivity for O157 positive E. coli is 1 × 10³ CFU per ml and for H7 positive E. coli is 1 × 10⁴ CFU per ml. Combining these two pairs of antibodies together, we developed a combo test strip that can specifically detect O157: H7, with a detection sensitivity of 1 × 10⁴ CFU per ml, when two detection lines are visible to the naked eye. This is currently the only rapid detection reagent that specifically detects O157: H7 by simultaneously detecting O157 antigen and H7 antigens of E. coli. Our product has advantages of simplicity and precision, and can be a very useful on-site inspection tool for accurate and rapid detection of E. coli O157:H7 infection.     

Fate of Escherichia coli O157:H7 in ground beef following high‐pressure processing and freezing

Aims: The purposes of this study were to evaluate the efficacy of high pressure to inactivate Escherichia coli O157:H7 in ground beef at ambient and subzero treatment temperatures and to study the fate of surviving bacteria postprocess and during frozen storage. Methods and Results: Fresh ground beef was inoculated with a five‐strain cocktail of E. coli O157:H7 vacuum‐packaged, pressure‐treated at 400 MPa for 10 min at ?5 or 20°C and stored at ?20 or 4°C for 5–30 days. A 3‐log CFU g^?1 reduction of E. coli O157:H7 in the initial inoculum of 1 × 10⁶ CFU g^?1 was observed immediately after pressure treatment at 20°C. During frozen storage, levels of E. coli O157:H7 declined to <1 × 10² CFU g^?1 after 5 days. The physiological status of the surviving E. coli was affected by high pressure, sensitizing the cells to pH levels 3 and 4, bile salts at 5% and 10% and mild cooking temperatures of 55–65°C. Conclusions: High‐pressure processing (HPP) reduced E. coli O157:H7 in ground beef by 3 log CFU g^?1 and caused substantial sublethal injury resulting in further log reductions of bacteria during frozen storage. Significance and Impact of the Study: HPP treatment of packaged ground beef has potential in the meat industry for postprocess control of pathogens such as E. coli O157:H7 with enhanced safety of the product.     

Use of zero-valent iron biosand filters to reduce Escherichia coli O157:H12 in irrigation water applied to spinach plants in a field setting

Aims:  Zero‐valent iron (ZVI) filters may provide an efficient method to mitigate the contamination of produce crops through irrigation water. Methods:  A field‐scale system was utilized to evaluate the effectiveness of a biosand filter (S), a biosand filter with ZVI incorporated (ZVI) and a control (C, no treatment) in decontaminating irrigation water. An inoculum of c. 8·5 log CFU 100 ml^?1 of Escherichia coli O157:H12 was introduced to all three column treatments in 20‐l doses. Filtered waters were subsequently overhead irrigated to ‘Tyee’ spinach plants. Water, spinach plant and soil samples were obtained on days 0, 1, 4, 6, 8, 10, 13 and 15 and analysed for E. coli O157:H12 populations. Results:  ZVI filters inactivated c. 6 log CFU 100 ml^?1E. coli O157:H12 during filtration on day 0, significantly (P < 0·05) more than S filter (0·49 CFU 100 ml^?1) when compared to control on day 0 (8·3 log CFU 100 ml^?1). On day 0, spinach plants irrigated with ZVI‐filtered water had significantly lower E. coli O157 counts (0·13 log CFU g^?1) than spinach irrigated with either S‐filtered (4·37 log CFU g^?1) or control (5·23 log CFU g^?1) water. Soils irrigated with ZVI‐filtered water contained E. coli O157:H12 populations below the detection limit (2 log CFU g^?1), while those irrigated with S‐filtered water (3·56 log CFU g^?1) were significantly lower than those irrigated with control (4·64 log CFU g^?1). Conclusions:  ZVI biosand filters were more effective in reducing E. coli O157:H12 populations in irrigation water than sand filters. Significance and Impact of the Study:  Zero‐valent ion treatment may be a cost‐effective mitigation step to help small farmers reduce risk of foodborne E. coli infections associated with contamination of leafy greens.     

A Novel GFP-Fused Eukaryotic Membrane Protein Expression System in Lactococcus lactis and Its Application to Overexpression of an Elongase

Ultraviolet (UV) irradiation has high potential to inactivate a wide range of biologic agents and is one of several nonadditive technologies being studied. The photoinactivation property of pulsed UV laser radiation (at wavelengths of 355 and 266 nm), used as an effective physical means to inactivate two typical microorganisms, prokaryotic (Escherichia coli K12) and eukaryotic (Saccharomyces cerevisiae), with respect to dose and exposure times, was examined. An E. coli population of 1.6 × 10⁴ colony-forming units (CFU)/ml was inactivated with a dose of 16.7 J/cm² energy at 355-nm wavelength. However, E. coli cells at higher concentrations were inactivated by only 98% using the same dose. Interestingly, an E. coli population of 2 × 10⁷ CFU/ml was completely inactivated using only 0.42 J/cm² at 266-nm wavelength (P ≤ 0.05). With respect to S. cerevisiae, the results were similar to those of E. coli irradiation considering that S. cerevisiae is 100 times larger than E. coli. A dose of 16.7 J/cm² completely inactivated an S. cerevisiae population of 6 × 10³ CFU/ml at 355-nm wavelength. Exposure to 266-nm wavelength, with energy doses of 1.67, 0.835, and 0.167 J/cm², successfully inactivated S. cerevisiae populations of 3 × 10⁶, 1.4 × 10⁵, and 1.5 × 10⁴ CFU/ml, respectively (P ≤ 0.05). In conclusion, compared with 355-nm wavelength, a pulsed UV laser at 266-nm wavelength inactivated a high titer of bacterial and yeast indicator standards suspended in phosphate-buffered saline-A.     

Behavior of Some Episomal Elements in a Recombination-Deficient Mutant of Escherichia coli

Conjugal transfer and autonomous replication of some episomes occurred normally in a recombination-deficient (Rec^–) mutant of Escherichia coli K-12. Transduction with phage Plbt of an R factor also occurred normally in this Rec^– mutant, but complete or abortive transduction with Plbt of chromosomal genes did not occur. In contrast, transduction of galactose genes by phage λ_dg occurred in the Rec^– bacteria as frequently as in the Rec⁺ strain. It was shown that phage Plbt does not grow at all on the Rec–bacteria. Recombination between two different R factors, two mutants of phage λ and two mutants of phage T4 occurred normally in the Rec^– bacteria, but did not give a Rec⁺ phenotype to the host bacteria. Colicinogenic factor I made the Rec^– host bacteria more resistant to ultraviolet light but the colicinogenic strain was still infertile in the crosses with the Hfr srains of E. coli K-12.     

Participation of RNase I in MS2 Phage Growth

Participation of RNase I in the growth of phage on infection with bacteriophage MS2 was studied.

Some strains of uracil-requiring E. coli were isolated, grown in MS broth, and transferred to a minimal medium to exhaust the pool of nucleotides. The phage was then added to the cells grown in uracil-deficient medium. The growth of phage was observed to occur at the burst size of two hundreds in strains of E. coli K12S (F⁺) U^? and C600 (F⁺) U^?, which possessed RNase I, but not in strains, A19 (Hfr) U^? and Q13 (Hfr) U^?, which lacked RNase I.

A marked increase in acid-soluble fraction was observed with E. coli K12S (F⁺) U^? and C600 (F⁺) U^?, whereas the increase was little with E. coli A10 (Hfr) U^? and Q13 (Hfr) U^? Conditions for the growth of phage in uracil-deficient medium were investigated and the effect of antibiotics were also investigated.     

Distribution of Coliphages Against Four <Emphasis Type="Italic">E. Coli</Emphasis> Virotypes in Hospital Originated Sewage Sample and a Sewage Treatment Plant in Bangladesh

The distribution of coliphages infecting different Escherichia coli virotypes (EHEC, EIEC, EPEC, ETEC) and an avirulent strain (K-12) in sewage system of a hospital and a sewage treatment plant (STP) was investigated by culture-based agar overlay methods. Coliphages were found in all the samples except stool dumping site in the sewage system of the hospital and lagoon of the STP. Bacteriophage count (pfu/ml) infecting E. coli strains showed the following ascending pattern (EHEC < EIEC < EPEC < ETEC < E coli K-12) in all the collected samples except one. Phages capable of infecting avirulent E. coli K-12 strains were present in the highest number among all the examined locations. Phages specific for E. coli K-12 presented high diversity in plaque size on the bacterial lawn. Virulent E. coli specific coliphages rarely produced plaques with diameter of 1–2 mm or over. Conventional agar overlay method was found to be not satisfactory for phage community analysis from primary stool dumping site of the hospital, probably due to the presence of high concentration of antimicrobial substances. The gradual decrease seen in the five groups of coliphage quantity with the ongoing treatment process and then the absolute absence of coliphages in the outlet of the examined treatment plant is indicative of the usefulness of the treatment processes practiced there.     

The slow dissociation rate of K-1602 contributes to the enhanced inhibitory activity of this novel alkyl–aryl-bearing fluoroketolide

Ketolides belong to the latest generation of macrolides and are not only effective against macrolide susceptible bacterial strains but also against some macrolide resistant strains. Here we present data providing insights into the mechanism of action of K-1602, a novel alkyl–aryl-bearing fluoroketolide. According to our data, the K-1602 interacts with the ribosome as a one-step slow binding inhibitor, displaying an association rate constant equal to 0.28?×?10^4?M^?1 s^?1 and a dissociation rate constant equal to 0.0025?min^?1. Both constants contribute to produce an overall inhibition constant K_i equal to 1.49?×?10^?8?M, which correlates very well with the superior activity of this compound when compared with many other ketolides or fluoroketolides.     

Nano-scale probe fabrication using self-assembly technique and application to detection of<Emphasis Type="Italic">Escherichia coli</Emphasis> O 157∶H7

A self-assembled monolayer of protein G was fabricated to develop an immunosensor based on surface plasmon resonance (SPR), thereby improving the performance of the antibody-based biosensor through immobilizing the antibody molecules (IgG). As such, 11-mercaptoundecanoic acid (11-MUA) was adsorbed on a gold (Au) support, while the non-reactive hydrophilic surface was changed through substituting the carboxylic acid group (-COOH) in the 11-MUA molecule using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrocholide (EDAC). The formation of the self-assembled protein G layer on the Au substrate and binding of the antibody and antigen were investigated using SPR spectroscopy, while the surface topographies of the fabricated thin films were analyzed using atomic force microscopy (AFM). A fabricated monoclonal antibody (Mab) layer was applied for detectingE. coli O157∶H7. As a result, a linear relationship was achieved between the pathogen concentration and the SPR angle shift, plus the detection limit was enhanced up to 10² CFU/mL.     

Genetic studies of the ribosomal proteins in Escherichia coli. 7. Mapping of several ribosomal protein components by transduction experiments between different strains of Escherichia coli

Summary Two 50s (50-10 and 50-12) and two 30s (30-4 and 30-7) ribosomal proteins could be distinguished between Shigella dysenteriae Sh/s and Escherichia coli K-12 JC411 with CMC column chromatography. On the other hand, E. coli K-12 AT2472 was shown to have a 30s ribosomal protein, 30-6(AT), which is specific to this strain and distinguishable from 30-6 of other E. coli K-12 strains. Transduction experiments by phage Plkc between Sh. dysenteriae Sh/s and E. coli ATSPCO1, a spectinomycin resistant mutant derived from AT2472 in which the 30-4 protein is altered, indicated that the genes specifying the above five ribosomal protein components are located in the streptomycin region on the E. coli chromosome.The gene order for three 50s (50-8, 50-10 and 50-12) and three 30s [str (30-?), 30-4 and 30-6] ribosomal proteins on the chromosome was determined by transduction technique between Sh. dysenteriae Sh/s and E. coli ATSPC01, between E. coli ATSPC01 and E. coli ER05 (an erythromycin resistant strain in which the 50-8 protein is altered), and between Sh. dysenteriae Sh/s and E. coli ERSPC14 (str ^s spc ^r ery ^r), respectively. It was found that these protein genes are arranged on the chromosome in the order of str (30-?)-30-4-30-6-50-8-50-10-50-12.     

Potential for gene transfer from recombinantEscherichia coli K-12 used in bovine somatotropin production to indigenous bacteria in river water

Summary This study examined the transfer of the plasmid pBGH1, an expression vector for bovine somatotropin (BST), fromEscherichia coli K-12 strain W3110G [pBGH1] to indigenous microorganisms present in flasks containing Missouri River water. Strain LBB269 is a nalidixic acid-resistant derivative of W3110G which was used as a plasmid-free control strain in these studies. Water samples were inoculated with strains W3110G [pBGH1] and LBB269; after 21 days of incubation the number of viable colony-forming units (CFU) of W3110G [pBGH1] and LBB269 were reduced from an initial level of about 1×10⁷ CFU per ml to less than 1 CFU per 100 ml. At this time indigenous microbes resistant to both ampicillin and tetracycline (the antibiotic resistance markers on pBGH1) were isolated from 100 ml of water from each of the flasks inoculated with either strain W3110G [pBGH1] or LBB269. Plasmid DNA was isolated from these organisms and examined for sequences containing the gene for BST from pBGH1, using a polymerase chain reaction (PCR) assay. As expected, the day 0 sample from the flask inoculated withE. coli K-12 strain W3110G [pBGH1] gave a positive PCR response and the day 0 sample from the flask inoculated withE. coli K-12 strain LBB269 gave a negative PCR response. All of the day 21 samples containing indigenous microbes isolated from flasks that were inoculated with either W3110G [pBGH1] or LBB269 were negative in the PCR assay, indicating that the target sequence from pBGH1 was not present in any of these indigenous microorganisms. The results of this particular assay indicate that pBGH1 or the portion of pBGH1 including the BST structural gene had not been transferred from W3110G [pBGH1] to indigenous microbial inhabitants of the Missouri River water flasks during this study.     

Evaluation of Petrifilm™ EC method for enumeration of E. coli from soil

Aims: To evaluate the suitability of commercially available Petrifilm? EC plates for enumeration of Escherichia coli from soil. Methods and Results: A confirmed E. coli strain isolated from liquid swine manure was inoculated into sterilized sandy clay loam and loam soils at the concentrations of 10², 10³, 10⁵ CFU g^?1 of soil. The efficiency of recovery on Petrifilm? EC plates for soils spiked with E. coli was compared with standard membrane filtration techniques on m‐FC basal medium supplemented with 3‐bromo‐4‐chloro‐5‐indoyl‐β‐d ‐glucopyranoside (BCIG) and most probable numbers (MPN) techniques in E. coli medium with 4‐methylumbelliferyl‐β‐d ‐glucuronide (EC‐MUG) broth. Petrifilm? EC and m‐FC (BCIG) methods were then assessed for the ability to recover E. coli from field soils applied with swine manure. No significant differences (P > 0·05) were observed between Petrifilm? EC, m‐FC (BCIG) and MPN methods for the recovery of E. coli from spiked samples, irrespective of soil type. However, recovery of E. coli from manure‐applied field soil samples showed a significant difference (P < 0·05) between the Petrifilm? EC method and the m‐FC method in enumerating E. coli possibly as a result of false positives on m‐FC. Conclusion: The Petrifilm? EC method is suitable for the enumeration of E. coli from soil with a detection limit of 10 CFU g^?1 soil. Significance and Impact of the Study: The commercially available Petrifilm? EC method is comparatively low cost, easy to use method for the enumeration of E. coli from soil without the need for further confirmation tests.     

Single Walled Carbon Nanotube-Based Junction Biosensor for Detection of Escherichia coli

Foodborne pathogen detection using biomolecules and nanomaterials may lead to platforms for rapid and simple electronic biosensing. Integration of single walled carbon nanotubes (SWCNTs) and immobilized antibodies into a disposable bio-nano combinatorial junction sensor was fabricated for detection of Escherichia coli K-12. Gold tungsten wires (50 µm diameter) coated with polyethylenimine (PEI) and SWCNTs were aligned to form a crossbar junction, which was functionalized with streptavidin and biotinylated antibodies to allow for enhanced specificity towards targeted microbes. In this study, changes in electrical current (ΔI) after bioaffinity reactions between bacterial cells (E. coli K-12) and antibodies on the SWCNT surface were monitored to evaluate the sensor''s performance. The averaged ΔI increased from 33.13 nA to 290.9 nA with the presence of SWCNTs in a 10⁸ CFU/mL concentration of E. coli, thus showing an improvement in sensing magnitude. Electrical current measurements demonstrated a linear relationship (R² = 0.973) between the changes in current and concentrations of bacterial suspension in range of 10²–10⁵ CFU/mL. Current decreased as cell concentrations increased, due to increased bacterial resistance on the bio-nano modified surface. The detection limit of the developed sensor was 10² CFU/mL with a detection time of less than 5 min with nanotubes. Therefore, the fabricated disposable junction biosensor with a functionalized SWCNT platform shows potential for high-performance biosensing and application as a detection device for foodborne pathogens.     

Variable agronomic practices, cultivar, strain source and initial contamination dose differentially affect survival of Escherichia coli on spinach

Aims: Greenhouse and field trials were conducted under different agronomic practices and inoculum doses of environmental Escherichia coli and attenuated E. coli O157:H7, to comparatively determine whether these factors influence their survival on leaves and within the rhizosphere. Methods and Results: Hydroponic conditions: E. coli spray‐inoculated at log 4 CFU ml^?1 was recovered from leaf surfaces at a mean population of 1·6 log CFU g^?1 at 15 days. E. coli O157:H7 sprayed at log 2 or 4 CFU ml^?1 levelled off on spinach leaf surfaces at a mean average population of 1·4 log CFU g^?1 after 14 days, regardless of initial dose. Quantitative recovery was inconsistent across leaf developmental age. Field conditions: Average populations of E. coli O157:H7 spray‐inoculated at log 1·45 or 3·4 CFU m^?2 levelled off at log 1·2 CFU g^?1 over a 14‐day period. Pathogen recovery from leaves was inconsistent when compared to regularly positive detection on basal shoot tissue. Pathogen recovery from soil was inconsistent among sampling locations. Moisture content varied up to 40% DW and was associated with 50% (P < 0·05) decrease in positive locations for E. coli O157:H7 but not for E. coli. Conclusions: Overall, similar populations of environmental E. coli and E. coli O157:H7 were recovered from plants despite differences in inoculum dose and agronomic conditions. Strain source had a significant impact on the quantitative level and duration of survival on leaves and in soil. Water availability appeared to be the determinant factor in survival of E. coli and E. coli O157:H7; however, E. coli showed greater environmental fitness. Significance and Impact of the Study: Persistence of surrogate, indicator E. coli and E. coli O157:H7, irrespective of variable growing conditions in spinach is predominantly limited by water availability, strain source and localization within the plant. These findings are anticipated to ultimately be adopted into routine and investigative pathogen testing protocols and mechanical harvest practices of spinach.     

Transfer of ampicillin resistance from Salmonella Typhimurium DT104 to Escherichia coli K12 in food

Aims: To investigate the transfer of antibiotic resistance from a donor Salmonella Typhimurium DT104 strain to a recipient Escherichia coli K12 strain. Methods and Results: Mating experiments were conducted in broth, milk and ground meat (beef) at incubation temperatures of 4, 15, 25 and 37°C for 18 and 36 h. Ampicillin‐resistance transfer was observed at similar frequencies in all transfer media at 25 and 37°C (10^?4 to 10^?5 log₁₀ CFU ml g^?1, transconjugants per recipient) for 18 h. At 15°C, transfer was observed in ground meat in the recipient strain (10^?6, log₁₀ CFU g^?1, transconjugants per recipient), but not in broth or milk. At 4°C, transfer did not occur in any of the examined mediums. Further analysis of the E. coli K12 nal^R transconjugant strain revealed the presence of a newly acquired plasmid (21 kbp) bearing the β‐lactamase gene bla_TEM. Transconjugants isolated on the basis of resistance to ampicillin did not acquire any other resistant markers. Conclusion: This study demonstrates the transfer of antibiotic resistance in food matrices at mid‐range temperatures. Significance and Impact of the Study: It highlights the involvement of food matrices in the dissemination of antibiotic‐resistant genes and the evolution of antibiotic‐resistant bacteria.     

Chromosome transfer and R-prime formation by an RP4::mini-Mu derivative in Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, and Proteus mirabilis

We have introduced into the wide host range conjugative plasmid RP4, a mini-Mu derivative which was known to be able to transpose spontaneously in E. coli K-12, and to induce in such a host several kinds of chromosomal rearrangements including replicon fusions. Unlike RP4, RP4::mini-Mu can mediate the transfer of the host chromosome to a recipient bacterium and generate R primes at high frequencies (10^?4 for the transfer of a given marker, 10^?5 for the formation of R primes carrying a given marker). Two such RP4::mini-Mu plasmids were introduced into one Salmonella typhimurium strain, one Klebsiella pneumoniae strain, and one Proteus mirabilis strain. Each of these three strains were mated with an E. coli K-12 recipient and transconjugants carrying R primes were recovered in all three cases at frequencies ranging from 5 × 10^?6 to 10^?7.     

Application of Bacteriophages To Control Intestinal Escherichia coli O157:H7 Levels in Ruminants

A previously characterized O157-specific lytic bacteriophage KH1 and a newly isolated phage designated SH1 were tested, alone or in combination, for reducing intestinal Escherichia coli O157:H7 in animals. Oral treatment with phage KH1 did not reduce the intestinal E. coli O157:H7 in sheep. Phage SH1 formed clear and relatively larger plaques on lawns of all 12 E. coli O157:H7 isolates tested and had a broader host range than phage KH1, lysing O55:H6 and 18 of 120 non-O157 E. coli isolates tested. In vitro, mucin or bovine mucus did not inhibit bacterial lysis by phage SH1 or KH1. A phage treatment protocol was optimized using a mouse model of E. coli O157:H7 intestinal carriage. Oral treatment with SH1 or a mixture of SH1 and KH1 at phage/bacterium ratios ≥10² terminated the presence of fecal E. coli O157:H7 within 2 to 6 days after phage treatment. Untreated control mice remained culture positive for >10 days. To optimize bacterial carriage and phage delivery in cattle, E. coli O157:H7 was applied rectally to Holstein steers 7 days before the administration of 10¹⁰ PFU SH1 and KH1. Phages were applied directly to the rectoanal junction mucosa at phage/bacterium ratios calculated to be ≥10². In addition, phages were maintained at 10⁶ PFU/ml in the drinking water of the phage treatment group. This phage therapy reduced the average number of E. coli O157:H7 CFU among phage-treated steers compared to control steers (P < 0.05); however, it did not eliminate the bacteria from the majority of steers.