Carlos C. Bezuidenhout

Isolation of Environmental Bacteria from Surface and Drinking Water in Mafikeng, South Africa, and Characterization Using Their Antibiotic Resistance Profiles

The aim of this study was to isolate and identify environmental bacteria from various raw water sources as well as the drinking water distributions system in Mafikeng, South Africa, and to determine their antibiotic resistance profiles. Water samples from five different sites (raw and drinking water) were analysed for the presence of faecal indicator bacteria as well as Aeromonas and Pseudomonas species. Faecal and total coliforms were detected in summer in the treated water samples from the Modimola dam and in the mixed water samples, with Pseudomonas spp. being the most prevalent organism. The most prevalent multiple antibiotic resistance phenotype observed was KF-AP-C-E-OT-K-TM-A. All organisms tested were resistant to erythromycin, trimethoprim, and amoxicillin. All isolates were susceptible to ciprofloxacin and faecal coliforms and Pseudomonas spp. to neomycin and streptomycin. Cluster analysis based on inhibition zone diameter data suggests that the isolates had similar chemical exposure histories. Isolates were identified using gyrB, toxA, ecfX, aerA, and hylH gene fragments and gyrB, ecfX, and hylH fragments were amplified. These results demonstrate that (i) the drinking water from Mafikeng contains various bacterial species and at times faecal and total coliforms. (ii) The various bacteria are resistant to various classes of antibiotics.

Interactions of the antimicrobial peptide nisin Z with conventional antibiotics and the use of nanostructured lipid carriers to enhance antimicrobial activity

Antimicrobial resistance is an imminent threat to the effective prevention and treatment of bacterial infections and alternative antimicrobial strategies are desperately needed. Antimicrobial peptides (AMPs) may be promising alternatives to current antibiotics or act as adjuvants to enhance antibiotic potency. Additionally, the use of biodegradable lipid nanoparticles can enhance the antibacterial activity of antibiotics and antimicrobial peptides. In this study, the interaction of the AMPs, nisin Z and melittin, with conventional antibiotics was investigated on Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli. The effectiveness of nanostructured lipid carriers (NLCs) for the entrapment of nisin Z was also evaluated. Findings revealed that nisin Z exhibited additive interactions with numerous conventional antibiotics. Notable synergism was observed for novobiocin-nisin Z combinations. The addition of the non-antibiotic adjuvant EDTA significantly improved the antimicrobial activity of free nisin Z towards E.coli. NLCs containing nisin Z were effective against Gram-positive species at physiological pH, with an increase in effectiveness in the presence of EDTA. Results indicate that nisin Z may be advantageous as an adjuvant in antimicrobial chemotherapy, while contributing in the battle against antibiotic resistance. NLCs have the potential to enhance the antibacterial activity of nisin Z towards Gram-positive bacterial species associated with skin infections.

Effects of Cry1Ab transgenic maize on lifecycle and biomarker responses of the earthworm, Eisenia andrei.

A 28-day study was conducted to determine the effects of the Bacillus thuringiensis Cry1Ab toxin on the earthworm Eisenia andrei. Previously, investigations have been limited to life-cycle level effects of this protein on earthworms, and mostly on E. fetida. In this study several endpoints were compared which included biomass changes, cocoon production, hatching success, a cellular metal-stress biomarker (Neutral Red Retention Time; NRRT) and potential genotoxic effects in terms of Randomly Amplified Polymorphic DNA sequences (RAPDs). NRRT results indicated no differences between treatments (p > 0.36), and NRRT remained the same for both treatments at different times during the experiment (p = 0.18). Likewise, no significant differences were found for cocoon production (p = 0.32) or hatching success (p = 0.29). Conversely, biomass data indicated a significant difference between the control treatment and the Bt treatment from the second week onwards (p < 0.001), with the Bt treatment losing significantly more weight than the isoline treatment. Possible confounding factors were identified that might have affected the differences in weight loss between groups. From the RAPD profiles no conclusive data were obtained that could link observed genetic variation to exposure of E. andrei to Cry1Ab proteins produced by Bt maize.

Molecular assessment of commercial and laboratory stocks of Eisenia spp. (Oligochaeta: Lumbricidae) from South Africa

ABSTRACT DNA barcoding was used to investigate laboratory and commercial stocks of Eisenia species from four provinces of South Africa. The COI gene was partially amplified and sequenced in selected earthworms from eight local populations (focal groups) and two European laboratory stocks (non-focal groups). Only nine COI haplotypes were identified from the 224 sequences generated. One of these haplotypes was found to belong to the megascolecid Perionyx excavatus. The remaining eight haplotypes belonged to the genus Eisenia, although only a single E. fetida haplotype, represented by six specimens, was found in one of the European populations. The other seven haplotypes, all occurring in South Africa, were E. andrei. One of the commercial stocks from South Africa and a laboratory culture from Europe were mixtures of E. andrei-P. excavatus and E. andrei-E. fetida, respectively. Previous allozyme studies have helped to suggest that some of the populations included in this study may be suffering from inbreeding depression, which could result in adverse consequences for both the vermiculture industry and ecotoxicological research in South Africa.

Antibiotic-resistant heterotrophic plate count bacteria and amoeba-resistant bacteria in aquifers of the Mooi River, North West province, South Africa.

Groundwater in the Mooi River catchment is prone to mining, agricultural, municipal and septic tank pollution. In this study physico-chemical and microbiological parameters were determined using appropriate methods. Bacterial isolates were identified by 16S rRNA sequencing (heterotrophic plate count (HPC) bacteria and amoeba-resistant bacteria (ARB)) and multiplex polymerase chain reaction (Escherichia coli). Antibiotic resistance tests were also performed. Physico-chemical parameters were generally within target water quality ranges for drinking water. HPC bacteria ranged between 10(5) and 10(7) colony-forming units (cfu)/ml. E. coli were enumerated from Trimpark, School and Cemetery. The Blaauwbank borehole was negative for faecal streptococci. Pseudomonas spp. were most abundant in the bulk water. Opportunistic pathogens isolated included Pseudomonas aeruginosa, Acinetobacter, Aeromonas, Alcaligenes, Flavobacterium, Bacillus cereus and Mycobacterium spp. Varying patterns of antibiotic resistance were observed. Most HPC bacterial isolates were resistant to cephalothin and/or amoxicillin and a few were resistant to erythromycin and streptomycin. Pseudomonas spp. was also the most abundant ARB. Other ARBs included Alcaligenes faecalis, Ochrobactrum sp. and Achromobacter sp. ARBs were resistant to streptomycin, chloramphenicol, cephalothin, and/or amoxicillin compared to HPCs. The presence of E. coli and ARB in these groundwater sources indicates potential human health risks. These risks should be further investigated and quantified, and groundwater should be treated before use.

Inadequate Taxonomy and Highly Divergent COI Haplotypes in Laboratory and Field Populations of Earthworms used in Ecotoxicology. A Case Study

DNA barcoding was used to investigate the phylogenetic delimitations of Eisenia sp. populations used in ecotoxicological research in South Africa. A total of three focal groups (used in published works) and two non-focal groups were assessed. These focal groups, including two laboratory cultures and one field population, have been referred to as Eisenia fetida in the literature. A previous molecular study had already helped to establish that one of the two laboratory groups was a population of E. fetidas sister species E. andrei. In the present contribution, analyses of the COI gene revealed that the taxonomy of the remaining laboratory and field populations had also been incorrectly assigned since all the generated sequences grouped unequivocally with published sequences of E. andrei. Very high sequence divergence (>25% K2P) found within E. andrei could signal the occurrence of hitherto undescribed cryptic species. These findings are discussed with an emphasis on the possible consequences of using poorly identified earthworms or specimens with high molecular divergence in ecotoxicological bioassays. It is not clear whether unbeknownst to the researchers, the use of cryptic species in bioassays could jeopardise the quality of ecotoxicological investigations. Early evidence suggests that cryptic oligochaete species may respond differently to metal toxicity. The need for comparative ecotoxicological studies between E. andrei and E. fetida is also evidenced, especially in the light of recent numerous reports of cryptic oligochaete species. Ecotoxicologists are consequently encouraged, whenever possible, to make use of available genomic technologies to screen their laboratory stocks and available field populations for any molecular distinctiveness.

Ecological guild and enzyme activities of rhizosphere soil microbial communities associated with Bt‐maize cultivation under field conditions in North West Province of South Africa

Insecticidal proteins expressed by genetically modified Bt maize may alter the enzymatic and microbial communities associated with rhizosphere soil. This study investigated the structure and enzymatic activity of rhizosphere soil microbial communities associated with field grown Bt and non‐Bt maize. Rhizosphere soil samples were collected from Bt and non‐Bt fields under dryland and irrigated conditions. Samples were subjected to chemical tests, enzyme analyses, and next generation sequencing. Results showed that nitrate and phosphorus concentrations were significantly higher in non‐Bt maize dryland soils, while organic carbon was significantly higher in non‐Bt maize irrigated field soil. Acid phosphatase and β‐glucosidase activities were significantly reduced in soils under Bt maize cultivation. The species diversity differed between fields and Bt and non‐Bt maize soils. Results revealed that Actinobacteria, Proteobacteria, and Acidobacteria were the dominant phyla present in these soils. Redundancy analyses indicated that some chemical properties and enzyme activities could explain differences in bacterial community structures. Variances existed in microbial community structures between Bt and non‐Bt maize fields. There were also differences between the chemical and biochemical properties of rhizosphere soils under Bt and non‐Bt maize cultivation. These differences could be related to agricultural practices and cultivar type.

Analysis of physico-chemical and bacteriological quality of drinking water in Mafikeng, South Africa

Mafikeng, the capital of the North West Province, receives water from two sources, namely the Molopo eye and the Modimola dam. Once treated, the potable water is mixed and supplied to the city via distribution systems. This study was designed to assess the quality of drinking water in Mafikeng and also to determine whether the water from the two sources has an impact on the mixed water quality. Physico-chemical parameters and bacteriological quality (faecal coliforms (FCs), total coliforms (TCs), heterotrophic bacteria and Peudomonas spp.) was monitored at three drinking water sites weekly for 4 months. The results revealed that the physico-chemical quality of the water was generally acceptable. The pH ranged from 5.7 ± 0.18 to 8.6 ± 0.14, the temperature ranged from 18.3 ± 0.69 to 25.1 ± 0.69 °C and the total dissolved solids (TDS) ranged from 159.9 ± 22.44 to 364.4 ± 12.44 mg/l. These values are within the target water quality range for drinking water as prescribed by WHO, Department of Water Affairs and SANS 241. What is of concern was the microbial quality of the water. FCs, TCs, heterotrophic bacteria and Pseudomonas spp. were present in some of the treated water samples. The most significant finding of this study is that all drinking water samples were positive for Pseudomonas spp. (>100/100 ml).