Lesego Gertrude Molale-Tom

Agricultural antibiotic contamination poses increasing threats to crop productivity and ecosystem stability through disruption of the plant-associated microbiome. While antibiotic impacts on bulk soil and rhizosphere communities are documented, the extent to which spatial compartmentalization across the plant-soil continuum buffers these effects remains poorly understood. Here, we investigated how compartment-specific selective pressures influence bacterial community assembly, functional resilience, and interaction networks under antibiotic stress. Lettuce (Lactuca sativa) was grown under five treatments in a completely randomized greenhouse design: T1 (sulfamethoxazole [SMX], 3 mg kg⁻¹ + manure + plant), T2 (trimethoprim [TMP], 3 mg kg⁻¹ + manure + plant), T3 (manure + plant, antibiotic-free control), T4 (manure only, plant-free control), and T5 (soil only, negative control). Bacterial communities were profiled across bulk soil, rhizosphere, and endosphere compartments using full-length 16 S rRNA gene sequencing. Spatial compartmentalization emerged as the primary driver of bacteriome structure and functional potential, surpassing antibiotic treatment effects across all analytical approaches. PERMANOVA revealed significant compartment-driven community structuring (R² = 0.189, P = 0.001), while treatment effects were non-significant (R² = 0.145, P = 0.116). Endosphere communities exhibited substantially lower alpha diversity than bulk soil and rhizosphere (P = 0.0001), with significant treatment × compartment interactions (P = 0.007). Antibiotic treatments selectively enriched xenobiotic degradation (P = 0.042) and secondary metabolism functions, particularly in bulk soil, without systematically increasing pathogen-associated or resistance-related functions. Network analysis revealed reduced bacterial connectivity under antibiotic pressure, yet cooperative interactions dominated across all treatments. Compositional differential abundance testing (ALDEx2) detected no significantly altered taxa for primary antibiotic contrasts (T1 vs. T3, T2 vs. T3), indicating context-driven rather than antibiotic-driven compositional changes. Functional diversity was significantly structured by compartment (Shannon P = 0.0017; richness P = 0.0039), while core plant-beneficial functions remained stable across treatments, with large effect sizes (Cohen's d ≥ 0.8) restricted to antibiotic degradation and secondary metabolism pathways. Our findings demonstrate that plant-microbe spatial structuring provides an ecological buffer that maintains core bacteriome functions against pharmaceutical disturbance, preserving plant-beneficial capabilities despite compositional shifts. The selective enrichment of antibiotic degradation pathways suggests potential for microbiome-assisted mitigation of pharmaceutical residues in agricultural systems. These results provide insights for developing compartment-specific microbiome management strategies that integrate with One Health approaches to enhance agricultural resilience under increasing pharmaceutical pressure in agroecosystems.

Antibiotic resistance was, until recently, primarily documented as a clinical phenomenon, with limited consideration given to environmental settings in policymaking. Although literature has demonstrated the presence of antibiotic-resistant bacteria in water environments, there is limited information on the concentrations of antibiotic resistance genes (ARGs) and antimicrobial residues, particularly in sub-Saharan Africa. Hence, this study aimed to determine the concentrations of ARGs and antimicrobial residues in urban wastewater effluents and receiving waters in North West Province, South Africa. The physicochemical parameters of the water samples were determined, and the ARGs were screened and quantified using end-point and real-time PCR. Chemical analysis was performed to quantify the concentrations of antibiotics and fluconazole. High-throughput 16S rRNA sequencing was conducted to identify and profile bacterial communities. Correlations between bacterial communities and environmental parameters were determined. Physicochemical parameters indicate that the water quality from wastewater effluent and receiving waters poses no concern for livestock use. However, most were correlated with the presence of distinct microbial communities, of which Proteobacteria and Bacteroidota were the dominant groups. Elevated ARG levels, inducing multidrug resistance, were reported in river water, indicating the longevity and transfer of ARGs in the environment. Notably, river water and wastewater effluent were more contaminated with ampicillin compared to other antimicrobials. The presence of antimicrobials may select for the development of antimicrobial resistance. Despite the widespread presence of ARGs and antimicrobial residues in water environments, these contaminants are not routinely monitored or regulated. The presence of these contaminants in water poses human and ecological risks.

This study presents the empirical findings of an in-depth genomic analysis of Enterococcus faecalis and Enterococcus lactis isolates from South Africa. It offers valuable insights into their genetic characteristics and their significant implications for public health. The study uncovers nuanced variations in the gene content of these isolates, despite their similar GC contents, providing a comprehensive view of the evolutionary diversity within the species. Genomic islands are identified, particularly in E. faecalis, emphasizing its propensity for horizontal gene transfer and genetic diversity, especially in terms of antibiotic resistance genes. Pangenome analysis reveals the existence of a core genome, accounting for a modest proportion of the total genes, with 2157 core genes, 1164 shell genes, and 4638 cloud genes out of 7959 genes in 52 South African E. faecalis genomes (2 from this study, 49 south Africa genomes downloaded from NCBI, and E. faecalis reference genome). Detecting large-scale genomic rearrangements, including chromosomal inversions, underscores the dynamic nature of bacterial genomes and their role in generating genetic diversity. The study uncovers an array of antibiotic resistance genes, with trimethoprim, tetracycline, glycopeptide, and multidrug resistance genes prevalent, raising concerns about the effectiveness of antibiotic treatment. Virulence gene profiling unveils a diverse repertoire of factors contributing to pathogenicity, encompassing adhesion, biofilm formation, stress resistance, and tissue damage. These empirical findings provide indispensable insights into these bacteria’s genomic dynamics, antibiotic resistance mechanisms, and virulence potential, underlining the pressing need to address antibiotic resistance and implement robust control measures.

There is a rapid spread of antibiotic resistance in the environment. However, the impact of antibiotic resistance in drinking water is relatively underexplored. Thus, this study aimed to quantify antibiotic resistance genes (ARGs) and antibiotic residues in two drinking water production facilities (NW-E and NW-C) in North West Province, South Africa and link these parameters to bacterial communities. Physicochemical and ARG levels were determined using standard procedures. Residues (antibiotics and fluconazole) and ARGs were quantified using ultra-high performance liquid chromatography (UHPLC) chemical analysis and real-time PCR, respectively. Bacterial community compositions were determined by high-throughput 16S rRNA sequencing. Data were analysed using redundancy analysis and pairwise correlation. Although some physicochemical levels were higher in treated than in raw water, drinking water in NW-E and NW-C was safe for human consumption using the South African Water Quality Guideline (SAWQG). ARGs were detected in raw and treated water. In NW-E, the concentrations of ARGs (sul1, intl1, EBC, FOX, ACC and DHA) were higher in treated water than in raw water. Regarding antimicrobial agents, antibiotic and fluconazole concentrations were higher in raw than in treated water. However, in NW-C, trimethoprim concentrations were higher in raw than in treated water. Redundancy analysis showed that bacterial communities were not significantly correlated (Monte Carlo simulations, p-value >0.05) with environmental factors. However, pairwise correlation showed significant differences (p-value Armatimonas, CL500-29 marine group, Clade III, Dickeya and Zymomonas genera with environmental factors. The presence of ARGs and antibiotic residues in the current study indicated that antibiotic resistance is not only a clinical phenomenon but also in environmental settings, particularly in drinking water niches. Consumption of NW-E and NW-C treated water may facilitate the spread of antibiotic resistance among consumers. Thus, regulating and monitoring ARGs and antibiotic residues in drinking water production facilities should be regarded as paramount.

Human activity affects the quality of potable water sources and their associated bacterial communities. Here, we discuss the heterotrophic Bacillus altitudinis 2R-9 draft isolated from the raw source of a drinking water distribution system in South Africa.

Human activities contribute to the contamination of drinking water sources, thereby impacting both the quality of the water and the composition of the bacterial communities present. We report the draft genome sequences of two pathogenic heterotrophic Bacillus bombysepticus strains harboring various antibiotic resistance genes; the strains were isolated from distribution water in South Africa.

Water quality affected by polluted surface runoff from agricultural settings, storm-water as well as sewage from urban locations, and mining in the North West Province are reasons for concern. Similar situations may exist in areas elsewhere that have similar water issues and impacts. This study aimed to evaluate the water quality of the Loopspruit River by analysing the physico-chemical and microbiological parameters of the water. Bacterial diversity was the highest in less polluted areas of the Loopspruit River. Dominating at all the sites tested were Proteobacteria, followed by Bacteroidetes, Cyanobacteria, Actinobacteria and Verrucomicrobia. Predicted metagenome analysis revealed a correlation between the physico-chemical parameters and observed taxonomic units (OTU). Temperature had a negative correlation with Patescibacteria, Nanoarchaeaeota and Firmicutes (p < 0.05). The negative correlation was strongest with Patescibacteria. Sulphate had a strong correlation with Fusobacteria (p < 0.05). There were was also evidence of faecal and agricultural pollution. Analysing and visually representing the water quality of the Loopspruit River demonstrated the value of combining physico-chemical, microbiology and geospatial data for an overview of understanding the potential environmental health risks. Such data could potentially be used for management strategies.

The Wonderfonteinspruit, South Africa, is highly impacted by a century of gold mining activities. The aim of this study was to investigate the physico-chemical properties of the Wonderfonteinspruit and the receiving Mooi River system, the levels of antimicrobial (metals and antibiotics) resistance characteristics and heterotrophic bacteria levels in these water systems. Various physico-chemical parameters were determined. R2A agar and R2A agar supplemented with antimicrobials were used to enumerate heterotrophic bacteria. Morphologically distinct antimicrobial-resistant isolates were purified and screened for antibiotic susceptibility by a disc diffusion method. Selected isolates were identified, and minimum inhibitory concentration ranges determined. Among the antimicrobial resistant isolates, 87% were resistant to at least one antibiotic. Of these, almost 50% were resistant to more than 3 antibiotic classes. A large proportion was resistant to all 7 antibiotics tested. Phyla detected were Proteobacteria, Firmicutes and Bacteriodetes. High MIC levels for metals and antibiotics were detected among all the genera. Results demonstrate potential impacts of physico-chemical properties on levels of antimicrobial-resistant bacteria. Metal-resistant bacteria were also resistant to multiple antibiotics, suggesting that metal pollution from mining may be responsible for co-selection and maintenance of antibiotic-resistant bacteria in this aquatic system.