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Mutshiene Deogratias Ekwanzala,1 John Barr Dewar,2 Ilunga Kamika,3 Maggy Ndombo Benteke Momba1 1Department of Environmental, Water and Earth Sciences, Tshwane University of Technology, Pretoria, South Africa; 2Department of Life and Consumer Sciences, University of South Africa, Johannesburg, South Africa; 3Department of Environmental Sciences, University of South Africa, Johannesburg, South Africa Abstract: A systematic review was conducted to determine the distribution and prevalence of antibiotic-resistant bacteria (ARB), antimicrobial-resistant genes (ARGs), and antimicrobial-resistant gene determinants (ARGDs) in clinical, environmental, and farm settings and to identify key knowledge gaps in a bid to contain their spread. Fifty-three articles were included. The prevalence of a wide range of antimicrobial-resistant bacteria and their genes was reviewed. Based on the studies reviewed in this systematic review, mutation was found to be the main genetic element investigated. All settings shared 39 ARGs and ARGDs. Despite the fact that ARGs found in clinical settings are present in the environment, in reviewed articles only 12 were found to be shared between environmental and clinical settings; the inclusion of farm settings with these two settings increased this figure to 32. Data extracted from this review revealed farm settings to be one of the main contributors of antibiotic resistance in healthcare settings. ARB, ARGs, and ARGDs were found to be ubiquitous in all settings examined. Keywords: systematic review, ARB, clinical ARGs, environmental ARGs, ARGDs, South Africa

Synthetic estrogen analogues are among the most potent estrogenic contaminants in effluents from wastewater treatment plants. Although its effects have been well elucidated in the feminization of male fish and interference with the endocrine systems in humans, it has not been fully explored in the activated sludge (AS) microbiome, particularly EE2 (17α-ethynylestradiol). Therefore, in this study, the bacterial community shift in a 6-day laboratory-scale reactor in environmental (0, 5, 10, and 100 ng/L) and predictive elevated concentrations (5, 10, and 100 mg/L) of EE2 was investigated using culture-based and metagenomics approaches. Results showed significant changes ( t -test, all p  < 0.05) between initial and final physicochemical parameters (pH, DO, and EC). Although environmental concentrations showed a slight decrease in microbial counts (5.6 × 10 6 to 4.6 × 10 6  CFU/ml) after a 24-h incubation for the culturable approach, the predictive elevated concentrations (5 to 100 mg/L) revealed a drastic microbial counts reduction (5.6 × 10 6 to 8 × 10 2  CFU/ml). The metagenomic data analysis uncovered that bacterial communities in the control sample were dominated by Proteobacteria , followed by Bacteroidetes and Firmicutes . The taxonomic classification after exposure of microbial communities in various concentrations revealed significant differences in community composition between environmental concentration (Shannon indices between 2.58 to 3.68) and predictive elevated concentrations (Shannon indices between 2.24 and 2.84; t -test, all p  < 0.05). The EE2 enriched seven OTUs were Novosphingobium, Cloacibacterium, Stenotrophomonas, Enterobacteriaceae &#95;unclassified, Stenotrophomonas, Enterobacteriaceae &#95;unclassified and Rhodobacteraceae &#95;unclassified. These results were supported by a dehydrogenase activity (DHA) test, which demonstrated less (about 40%) DHA in predictive elevated concentrations than in environmental concentrations. Notwithstanding, these findings suggest that EE2 may possess potent hormetic effect as evidenced by promotion of microbiome richness and dehydrogenase activity of AS in lower EE2 doses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Budeli, Ekwanzala and Momba.)

Extended-spectrum beta-lactamase (ESBL)-producing bacteria are a major problem for public health worldwide because of limited treatment options. Currently, only limited information is available on ESBL-producing Shiga toxin-producing Escherichia coli (STEC) in cattle farms and the surrounding aquatic environment. This study sought to track and characterise ESBL-producing STEC disseminating from a cattle farm into the water environment. Animal husbandry soil (HS), animal manure (AM), animal drinking water (ADW), and nearby river water (NRW) samples were collected from the cattle farm. Presumptive ESBL-producing STEC were isolated and identified using chromogenic media and mass spectrophotometry methods (MALDI-TOF-MS), respectively. The isolates were subjected to molecular analysis, and all confirmed ESBL-producing STEC isolates were serotyped for their O serogroups and assessed for antibiotic resistance genes (ARGs) and for the presence of selected virulence factors (VFs). A phylogenetic tree based on the multilocus sequences was constructed to determine the relatedness among isolates of ESBL-producing STEC. The highest prevalence of ESBL-producing STEC of 83.33% was observed in HS, followed by ADW with 75%, NRW with 68.75%, and the lowest was observed in AM with 64.58%. Out of 40 randomly selected isolates, 88% ( n = 35) belonged to the serogroup O45 and 13% ( n = 5) to the serogroup O145. The multilocus sequence typing (MLST) analysis revealed four different sequence types (STs), namely ST10, ST23, ST165, and ST117, and the predominant ST was found to be ST10. All 40 isolates carried sul1 (100%), while bla OXA , bla CTX-M , sul 2, bla TEM , and qnrS genes were found in 98%, 93%, 90%, 83%, and 23% of the 40 isolates, respectively. For VFs, only stx2 was detected in ESBL-producing STEC isolates. The results of the present study indicated that a cattle environment is a potential reservoir of ESBL-producing STEC, which may disseminate into the aquatic environment through agricultural runoff, thus polluting water sources. Therefore, continual surveillance of ESBL-producing STEC non-O157 would be beneficial for controlling and preventing STEC-related illnesses originating from livestock environments.

Background: Human-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) has mainly been reported in South African pig and chicken farms. The prevalence of antibiotic-resistant genes (ARGs), virulence factors (VFs), and multilocus sequence types (MLSTs) associated with HA-MRSA in cattle farms has not been reported. Consequently, this study characterised LA-MRSA and its spread from cattle farms into the environment., Method: Husbandry soil (HS), nearby river water (NRW), animal manure (AM) and animal drinking water (ADW) were collected on and around a cattle farm. Presumptive MRSA isolates were identified from these samples using CHROMagar media and genotyped as MRSA sequence types (STs), selected ARGs, and VFs, using polymerase chain reaction. An MLST-based dendrogram was generated to link the farm MRSA strains with those in a nearby river., Results: The prevalence of MRSA was 30.61% for HS, 28.57% for ADW, 22.44% for NRW, and 10.20% for AM. Isolates from HS harboured the highest number of resistant genes, with 100% for mecA , 91.66% for ermA , and 58.33% for blaZ . However, no ermC or tetM genes were detected. MRSA isolates from AM harboured the lowest number of resistant genes. Only sec and seq enterotoxins were found in all the assessed MRSA isolates. MRSA from the farm revealed six STs (ST80, ST728, ST1931, ST2030, ST3247, and ST5440); all of STs belonged to clonal complex 80 (CC80). An MLST-based dendrogram based on the concatenated sequences of MLST genes under the maximum likelihood criterion revealed four clades of amalgamated MRSA isolates from various livestock environmental matrices, including the NRW., Conclusion: The results suggest that livestock environmental matrices might be reservoirs of MRSA that could subsequently disseminate through runoff to pollute water resources. Therefore, continued surveillance of HA-MRSA in livestock environments is warranted.

The COVID-19 pandemic presents a serious public health challenge in all countries. However, repercussions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections on future global health are still being investigated, including the pandemic's potential effect on the emergence and spread of global antimicrobial resistance (AMR). Critically ill COVID-19 patients may develop severe complications, which may predispose patients to infection with nosocomial bacterial and/or fungal pathogens, requiring the extensive use of antibiotics. However, antibiotics may also be inappropriately used in milder cases of COVID-19 infection. Further, concerns such as increased biocide use, antimicrobial stewardship/infection control, AMR awareness, the need for diagnostics (including rapid and point-of-care diagnostics) and the usefulness of vaccination could all be components shaping the influence of the COVID-19 pandemic. In this publication, the authors present a brief overview of the COVID-19 pandemic and associated issues that could influence the pandemic's effect on global AMR., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

This study uncovered the prevalence, harboured species, and subtype diversity of Cryptosporidium species in river water and its sediment from the Apies River in South Africa. Cryptosporidium spp. concentrations in freshwater and its sediment were determined using Ziehl-Neelsen staining and quantitative Polymerase Chain Reaction (qPCR) techniques. Next-generation sequencing (NGS) targeting the 60 kDa glycoprotein (gp60) gene of Cryptosporidium spp. was performed to reveal the species, subtype families and subtypes harboured in freshwater and its sediment. Although the results revealed that water samples had a higher prevalence (30%) compared with sediment (28%), the number of observable Cryptosporidium spp. oocysts in sediment samples (ranging from 4.90 to 5.81 log 10 oocysts per 1 Liter) was higher than that of river water samples (ranging from 4.60 to 5.58 log 10 oocysts per 1 L) using Ziehl-Neelsen staining. The 18S ribosomal ribonucleic acid (rRNA) gene copy of Cryptosporidium in riverbed sediments ranged from 6.03 to 7.65 log 10 , whereas in river water, it was found to be between 4.20 and 6.79 log 10 . Subtyping results showed that in riverbed sediments, Cryptosporidium parvum accounted for 40.72% of sequences, followed by Cryptosporidium hominis with 23.64%, Cryptosporidium cuniculus with 7.10%, Cryptosporidium meleagridis with 4.44% and the least was Cryptosporidium wrairi with 2.59%. A considerable percentage of reads in riverbed sediment (21.25%) was not assigned to any subtype. River water samples had 45.63% of sequences assigned to C. parvum, followed by 30.32% to C. hominis, 17.99% to C. meleagridis and 5.88% to C. cuniculus. The data obtained are concerning, as Cryptosporidium spp. have intrinsic resistance to water treatment processes and low infectious doses, which can pose a risk to human health due to the various uses of water (for human consumption, leisure, and reuse)., (Copyright © 2021 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

This study aimed to uncover related strains of extended-spectrum beta-lactamase Escherichia coli O157:H7 between agricultural matrices (soil, manure and irrigation water) and nearby water sources using multilocus-based phylogenetic analysis. Resistant and nonresistant E. coli O157:H7 were isolated, identified and characterised using standard microbiological methods. The results showed that soil samples had a high prevalence of E. coli O157:H7 (31.73%) and ESBL-producing E. coli O157:H7 (22.11%). Multilocus sequencing typing (MLST) analysis revealed that all ESBL-producing E. coli O157:H7 were identified as ST11. Phylogenetic analysis of E. coli O157:H7 indicated that irrigation water might be a reservoir for E. coli O157:H7. For antibiotic-resistant genes (ARG), the most common was bla TEM in 85% (n = 34), followed by bla OXA in 70% (n = 28), bla NDM and sul1 30% (n = 12) and lastly mcr-1, which was only found in one soil isolate. The results showed that ESBL-producing E. coli O157:H7 isolates were intermixed in three clades, indicating close relatedness between isolated strains from different matrices., (Copyright © 2020. Published by Elsevier Ltd.)

The presence of antibiotic-resistant Salmonella spp. in the environment is of great public health interest, worldwide. Furthermore, its extended-spectrum β-lactamase (ESBL)-producing strains constitute an emerging global health concern due to their limited treatment options in hospital. Therefore, this study aimed at characterising and tracking nonresistant and ESBL-producing Salmonella spp. from agricultural settings to nearby water sources highlighting their antibiotic resistance genes (ARG) and virulence factor (VF) distribution using a combination of both culture-dependent and independent methods. Furthermore, this study investigated the diversity and shared serovars among sampled matrices using amplicon sequencing of the invasion gene A ( invA ) of Salmonella spp. The results showed that soil had the highest prevalence of Salmonella spp. (62.5%, 65/104) and ESBL-producing Salmonella (34.6%, 36/104). For typed ARG, the most commonly detected gene was bla OXA with 75% (30/40), followed by bla CTX-M 67.5% (27/40) , bla TEM 40% (16/40) and sul 1 30% (12/40) gene; bla SHV gene was not detected in isolated ESBL-producing Salmonella spp. For VF, the most detected gene was invA (96.9%, 38/40), followed by spa M (17.5%, 7/40), spi C (40%, 16/40), orf L (32.5%, 13/40), mis L 32.5% (13/40) and pip D 32.5 (13/40). For diversity analysis, soil, manure, irrigation water and nearby freshwater revealed 81, 68, 12 and 9 serovars, respectively. Soil, manure, irrigation water and freshwater stream samples shared five serovars, which indicated circulation of ESBL-producing Salmonella spp. within the agricultural environment and nearby water sources. Soil is therefore identified as one of the major reservoirs of ESBL-producing Salmonella spp. It is concluded that agricultural environment contamination may have a direct relationship with the presence of antibiotic-producing Salmonella in freshwater streams.

The environmental dissemination of selected antibiotics from hospital wastewater into municipal wastewater and lastly to a receiving water body was investigated. Selected antibiotics (azithromycin (AZM), ciprofloxacin (CIP), clindamycin (CDM), doxycycline (DXC) and sulfamethoxazole (SMZ)) present in effluents of academic hospital wastewater, influents, sewage sludge, and effluents of municipal wastewater, receiving water, and its benthic sediment samples were quantified using the Acquity ® Waters Ultra-Performance Liquid Chromatography System hyphenated with a Waters Synapt G2 coupled to a quadrupole time-of-flight mass spectrometer. The overall results showed that all assessed antibiotics were found in all matrices. For solid matrices, river sediment samples had elevated concentrations with mean concentrations of 34,834, 35,623, 50,913, 55,263, and 41,781 ng/g for AZM, CIP, CDM, DXC, and SMZ, respectively, whereas for liquid samples, hospital wastewater and influent of wastewater had the highest concentrations. The lowest concentrations were observed in river water, with mean concentrations of 11, 97, 15, and 123 ng/L, except for CDM, which was 18 ng/L in the effluent of wastewater. The results showed that the highest percentages of antibiotics removed was SMZ with 90%, followed by DXC, AZM and CIP with a removal efficiency of 85%, 83%, and 83%, respectively. The antibiotic that showed the lowest removal percentage was CDM with 66%. However, the calculated environmental dissemination analysis through the use of mass load calculations revealed daily release of 15,486, 14,934, 1526, 922, and 680 mg/d for SMZ, CIP, AZM, DXC, and CDM, respectively, indicating a substantial release of selected antibiotics from wastewater to the river system, where they are possibly adsorbed in the river sediment. Further research into the efficient removal of antibiotics from wastewater and the identification of antibiotic sources in river sediment is needed.

In this paper, we deciphered the core resistome disseminating from hospital wastewater to the aquatic environment by characterising the resistome, plasmidome, mobilome and virulome using metagenomic analysis. This study also elucidated different environmental resistome risks using shotgun-metagenomic assembly. The results showed that clinically relevant taxa were found in assessed matrices (Salmonella spp., Acinetobacter spp, Escherichia-Shigella spp., Pseudomonas spp., Staphylococcus spp. and Vibrio spp.). For the plasmidome, we found 249 core plasmidome sequences that were shared among all assessed matrices. The core mobilome of 2424 mobile genetic elements shared among all assessed matrices was found. Regarding the virulome, we found 148 core virulence factors shared among all assessed samples, and the core virulome content was consistently shared across the most abundant bacterial genera. Although influent of wastewater showed considerable higher relative bacterial abundance (P = 0.008), hospital wastewater showed significant higher environmental resistome risk scores against all other assessed matrices, with an average of 46.34% (P = 0.001). These results suggest hospital wastewater, effluent and sewage sludge should be subjected to stringent mitigating measures to minimise such dissemination., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The rise of vancomycin-resistant Enterococcus spp. (VRE) has led to treatment challenges in hospital settings worldwide. Hospital wastewater (HW) might disseminate this threat to the aquatic environment. Thus, this study elucidates the VRE resistance quotient (RQ) of different environmental matrixes in wastewater and compares genomic determinants of VRE strains recovered from HW to water resources. Presumptive Enterococcus spp. and VRE were quantified and isolated using standard microbiological procedures. Fourteen VRE genomes were then sequenced using an Illumina HiSeq X™ Ten platform. Subsequently, VRE genomes were compared based on antibiotic resistance genes, plasmids, bacteriophages, insertion sequences, transposons, virulence and pathogenicity. Wastewater effluent showed the highest RQ among all sampled matrixes. The phylogeny of vancomycin-resistant E. faecalis (VREfs) and E. faecium (VREfm) revealed a tree structure based on their respective sequence type. A comparative genomic analysis of 14 genomes highlighted regions encoding phage protein, phage holin, phage integrase, integrase and transposase on both query genomes and the reference genome. Acquired resistance to vancomycin was conferred by vanA, vanN, vanL, vanG and the intrinsic resistance vanC operons. Plasmids were dominated by the presence of conserved areas of the replication initiating genes (rep). The Tn3-like and Tn917 transposons were present in all erythromycin-carrying erm(B) isolated VRE genomes. All VRE genomes expect one were putatively predicted as human pathogens with varying degrees of virulence. The presence of such resistant bacteria in African water resource is of great public health concern. It is, therefore, recommended that these bacteria be tracked and characterised from different environments to contribute to improved epidemiological containment action., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objectives: This study reported the resistome content of sewage sludge-isolated carbapenem-resistant Citrobacter koseri (C. koseri) carrying bla OXA-181 . It also provided a general phylogenomic analysis highlighting antibiotic resistance genes (ARGs), plasmids and pathogenicity of C. koseri genomes., Methods: The carbapenem-resistantC. koseri AS1 strain was isolated from sewage sludge on CHROMagar™ mSuperCARBA™ media. Whole genome sequencing of C. koseri AS1 was performed using an HiSeq X™ Ten instrument. Additional C. koseri genomes were downloaded from National Center for Biotechnology Information (NCBI). Phylogenomic analysis was established through CSI Phylogeny. ARGs, plasmids and pathogenicity were identified using ResFinder 3.1, PlasmidFinder 2.0 and PathogenFinder 1.1, respectively., Results: The phylogenomic tree indicated a polyclonal pattern ofC. koseri genomes. Resistome analysis of C. koseri AS1 revealed β-lactam resistance genes (bla MAL-1 and bla OXA-181 ) as well as a fosfomycin resistance gene (fosA7). Three plasmids (ColKP3, ColRNAI and IncX30) were identified in the C. koseri AS1 genome. In addition, 25 ARGs were found in downloaded genomes. Of these, clinically significant ARGs such as bla KPC-2 and bla OXA-48 were found in two and four genomes, respectively. Assessment of the genomes using PathogenFinder revealed all genomes as putative human pathogens., Conclusions: It is believed that noC. koseri genome has been reported to carry bla OXA-181 ; therefore, C. koseri AS1 is the first of its kind. This study also highlighted the resistome contents of C. koseri genomes., (Copyright © 2019 International Society for Antimicrobial Chemotherapy. Published by Elsevier Ltd. All rights reserved.)

The emergence and dissemination of infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) are of great concern worldwide, as there are limited options for their treatment. Thus, in this study, whole-genome sequencing (WGS) was applied to assess CRKP distribution and dissemination from hospital settings to the aquatic environment in order to identify the extent of the problem. Samples were collected from hospital wastewaters and receiving water bodies. Susceptible K. pneumoniae and CRKP were enumerated and isolated using standard methods. Seventeen CRKP were DNA-sequenced using an Illumina HiSeq X™ platform. De novo assembly and annotation were performed using SPAdes and RAST, respectively. The study analysed antibiotic resistance traits (antibiotic resistant genes, mobile genetic elements, and virulence genes) in CRKP isolates. Although influent of wastewater harboured the highest CRKP, wastewater treatment plants were efficient in reducing the threat. In terms of resistance per matrix, benthic sediment proved to harbour more CRKP (22.88%) versus susceptible K. pneumoniae, as revealed by their resistant quotient analysis, while effluent of wastewaters (4.21%) and water bodies (4.64%) had the lowest CRKP loads. The disseminating CRKP consisted of six sequence types (ST) - ST307 (n = 7), a novel ST3559 (n = 5), ST15 (n = 2), and one isolate of each of ST39, 152 and 298. All CRKP isolates harboured β-lactams (bla CTX-M-15 and bla OXA-1 ), quinolone (oqxA and oqxB) and fosfomycin (fosA) resistance genes as well as virulence genes. This study highlights the dissemination of 'high' importance and novel ST CRKP from hospital wastewater to waterbodies. This is concerning, particularly in the African context where a sizable number of people still rely on direct water resources for household use, including drinking. Further research is needed to systematically track the occurrence and distribution of these bacteria so as to mitigate their threat., (Copyright © 2019. Published by Elsevier B.V.)

Here, we report a novel sequence type, 3559, from four genomes of Klebsiella pneumoniae isolates from South African hospital wastewater, influent wastewater, river water, and riverbed sediment. The genome annotation indicated a wide variety of resistance genes, including bla KPC-2 , and virulence factors revealing their possible pathogenicity., (Copyright © 2019 Ekwanzala et al.)

To date, the microbiological quality of river sediments and its impact on water resources are not included in the water quality monitoring assessment. Therefore, the aim of this study was to establish genetic relatedness between faecal coliforms and enterococci isolated from the river water and riverbed sediments of Apies River to better understand the genetic similarity of microorganisms between the sediment and water phases. Indicator bacteria were subjected to a molecular study, which consisted of PCR amplification and sequence analysis of the 16S rRNA and 23S rRNA gene using specific primers for faecal coliforms and enterococci, respectively. Results revealed that the Apies River had high faecal pollution levels with enterococci showing low to moderate correlation coefficient (r 2 values ranged from 0.2605 to 0.7499) compared to the faecal coliforms which showed zero to low correlation (r 2 values ranged from 0.0027 to 0.1407) indicating that enterococci may be better indicator than faecal coliforms for detecting faecal contamination in riverbed sediments. The phylogenetic tree of faecal coliforms revealed a 98% homology among their nucleotide sequences confirming the close genetic relatedness between river water and riverbed sediment isolates. The phylogenetic tree of the enterococci showed that Enterococcus faecalis and Enterococcus faecium are the predominant species found in both river water and riverbed sediments with bootstrap values of ≥99%. A high degree of genetic relatedness between sediment and water isolates indicated a possible common ancestry and transmission pathway. We recommend the microbial monitoring of riverbed sediments as it harbours more diverse microbial community and once resuspended may cause health and environmental problems.