Pharmaceutical Sciences

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Dynamics of drug resistance in environmental bacteria within an aquatic ecosystem.
(2023) Chukwu, Kelechi Benedict.; Akebe, Luther King Abia.; Essack, Sabiha Yusuf.
Recently there has been a rapid increase in the incidence and prevalence of drug-resistant bacteria and antimicrobial resistance genes in the environment, largely attributed to selection pressure from the environmental presence of antimicrobials such as antibiotics, biocides, and heavy metals, as well as other physicochemical stressors. such as Poly aromatic hydrocarbons, pH, temperature, and reactive oxygen. However, the concentrations at which these antimicrobials could elicit resistance are poorly understood. Such lack of information could hamper the development of standards for the environmental surveillance of antmicrobials with potential adverse effects on human, animal and environmental health. In this study, Water samples were collected from all the points that impact the environment directly around the Darvill wastewater treatment plant, namely the treatment plant effluent discharge point, the upstream and downstream from the effluent discharge point. Antibiotics, heavy metals, and biocides were identified and quantified from the water samples, and we ascertained the effect of environmental concentrations of some of these selected stressors on the antibiotic resistance in previously susceptible Escherichia coli. Heavy metals concentrations were determined using the United States Environmental Protection Agency (US EPA) method 200.7. Biocide and antibiotic residue concentrations were determined using validated ultra-high-performance liquid chromatography with tandem mass spectrometry-based methods. E. coli was identified and quantified using the Colilert-18TM system from IDEXX, while antimicrobial susceptibility was performed using the disc diffusion method according to the Clinical and Laboratory Standards Institute guidelines. The concentration of antibiotics observed ranged from sulfamethoxazole (286.180 μg/L) to penicillin (2.2 μg/L); for metals, sodium (27.734 mg/L) to iron (0.001 mg/L); and for biocides, benzalkonium chloride (BAC) 12 (7.805 μg/L) to BenthEZ (0.035 μg/L). There was observed increase in the pollutant concentrations in the effluent and downstream samples compared to the upstream samples, suggesting that the WWTP might be a potential source of interest, indicating that these pollutants, were not completely removed at the WWTP. Thirty days' exposure of wholly susceptible E. coli ATCC 25922 strains, to environmental and sub-inhibitory concentrations of oxytetracycline, amoxicillin, zinc, copper, BAC 12 and DADMAC 10 was conducted but could not trigger phenotypic resistance. Genotypic analysis of the WGS on the exposed isolates, found only the macrolide resistance mdf (A) gene (which was also present in the control) and the disinfectant resistance gene sitABCD. With further analysis for single nucleotide variants (SNV), mutations were detected for 19 genes compared to the control. Only one resistance gene was detected, robA, a member of the ArcC/XylS family, that regulates the ArcAB-TolC multi-drug efflux, that contributes to multi-drug resistance. The other 18 genes we detected were tolerance conferring genes, acnB, cusA, degQ, epmA, hsmP, mlc, purH, queG, srlE, tsaB, yddh and yqhH genes, in all the exposed isolates. filA genes in only the oxytetracycline and BAC 12-exposed isolates, mutM gene in zinc exposed isolates, nudK gene in all the exposed isolates except the DADMAC 10 exposed isolates, ptsG gene in only the oxytetracycline-exposed isolates, and ompD in only DADMAC 12-exposed isolates. All the genes detected in the exposed isolates were also detected in the environmental isolates, except the robA gene. These genes detected encode for oxidative stress, DNA repair, membrane proteins efflux systems, growth and persister formations. In addition, we observed that the 30-days exposed isolates developed increased tolerance to high (25 x MIC) concentrations of ampicillin by 30 to 50% when compared to unexposed control. BAC 12-exposed isolates had the highest tolerance increase. The increased tolerance seems to emanate from multi gene induced persister cells formations, as well as tolerance gene expressions. The MSW of the exposed isolates to ampicillin and amoxicillin, also slightly increased compared to the control indicating the amplification of persister cells during the 30-day exposure but the MSW remained same to oxytetracycline. This indicates that exposure to sub-inhibitory concentrations of antibiotics, heavy metals and biocide residues, as observed in the aquatic environment, cannot induce phenotypic resistance but can encode for genes responsible for the development of persistence and tolerance in bacteria, which seems to be the pathway towards eventual antimicrobial resistance in environmental bacteria.
Molecular epidemiology of antibiotic-resistant Enterococcus spp. from farm-to-fork in intensive pig production in KwaZulu-Natal, South Africa.
(2021) Badul, Sasha.; Essack, Sabiha Yusuf.; Bester, Linda Antionette.; Amoako, Daniel Gyamfi.; Akebe, Luther King Abia.
Background: Substantial antibiotic use and high population densities in intensive farming systems results in the emergence and spread of antibiotic-resistant commensals and pathogens. This study investigated the molecular epidemiology of antibiotic resistance (ABR) and virulence in Enterococcus spp. from pigs in an intensive food production continuum from farm-to-fork in the uMgungundlovu district, Kwa-Zulu Natal. Methods: A total of 174 samples obtained along the pig farm-to-fork continuum (farm, transport, abattoir, and retail meat) were subjected to the quantification and putative identification of Enterococcus spp. using the IDEXX Enterolert® method and selective media, respectively. Up to three presumptive enterococcal colonies were picked per sampling point for molecular confirmation by real-time PCR, targeting the genus- and species-specific (tuf and sodA) genes, respectively. Antibiotic resistance profiles were determined by the Kirby-Bauer disk diffusion method against a panel of antibiotics for Enterococcus spp. recommended by the WHO-AGISAR using EUCAST guidelines. Selected antibiotic resistance and virulence genes were detected by real-time PCR. Clonal relatedness between isolates across the continuum was evaluated by REP-PCR. Results: A total of 284 isolates constituted the final sample. Real-time PCR confirmed 79.2% of the isolates as E. faecalis, 6.7% as E. faecium, 2.5% as E. casseliflavus, 0.4% as E. gallinarum, and 11.2% as other Enterococcus spp. Antibiotic susceptibility testing revealed resistance to sulfamethoxazole-trimethoprim (78.8%), tetracycline (76.9%), erythromycin (68.1%), streptomycin (62.6%), chloramphenicol (27.0%), ciprofloxacin (8.5%), gentamicin (8.1%), and levofloxacin (5.6%) but no vancomycin, teicoplanin, tigecycline or linezolid resistance was detected. E. faecium displayed 44.4% resistance to quinupristin-dalfopristin. A total of 78% of enterococcal isolates were MDR. Phenotypic resistance to tetracycline, aminoglycosides, and macrolides was corroborated by the presence of the tetM, aph(3’)-IIIa, and ermB genes in 99.1%, 96.1%, and 88.3% of the isolates, respectively. The most commonly detected virulence genes were: gelE, efaAfs, and cpd in 89.1%, 78.5%, and 77.1% of isolates conferring autolysin and biofilm formation capabilities, cell adhesion, and conjugative plasmid accumulation, respectively. Clonality evaluated by REP-PCR revealed that E. faecalis isolates belonged to diverse clones along the continuum with major REP-types, largely consisting of isolates from the same sampling source but different sampling rounds (on the farm). E. faecium isolates revealed a less diverse profile. There was minimal evidence of clonal transmission across the continuum. Conclusion: Multi-drug resistant Enterococcus spp. were isolated along the farm-to-fork continuum. Isolates harboured a diversity of antibiotic resistance and virulence genes in different combinations forming reservoirs for the potential transfer of these genes from pigs to occupationally exposed workers and consumers via direct contact with animals and animal products/food, respectively. The results highlight the need for more robust guidelines for antibiotic use in intensive farming practices and the necessity of including Enterococcus spp. as an indicator in antibiotic resistance surveillance systems in food animals.
Molecular epidemiology of carbapenem-resistant Enterobacterales colonization in an intensive care unit.
(2021) Madni, Osama.; Essack, Sabiha Yusuf.; Rout, Joan Allison.; Amoako, Daniel Gyamfi.; Akebe, Luther King Abia.
Background: Due to the high association with mortality and morbidity, carbapenem-resistant Enterobacterales (CRE) in general, and carbapenem-resistant Klebsiella pneumoniae, in particular, have been listed as high-priority pathogens by the World Health Organization (WHO) for the research and development of new antibiotics. Concomitant resistance to multiple antibiotics of different classes, impedes efficient clinical management of CRE infections. We characterized carbapenemase-producing K. pneumoniae (CPKP) isolates from sequential rectal screening of patients in a single intensive care unit (ICU) in a public hospital in the uMgungundlovu District of Kwazulu-Natal, South Africa, collected over one month. Method: Ninety-seven rectal swabs collected from consenting adult patients (n=31) on day 1, 3, 7 and weekly thereafter were screened for carbapenemase-production using Chrome-ID selective media. Fourteen CPKP were subjected to speciation and antibiotic susceptibility testing using the VITEK 2® automated system and their clonality was ascertained by ERIC/PCR. A sub-sample of eight isolates from five patients underwent whole genome sequencing (WGS) on the Illumina MiSeq platform followed by bioinformatics analysis to delineate the resistome, virulome, mobilome, clonality and phylogeography. Results: All isolates (100%) were resistant to ertapenem and meropenem and 71.4% (n=10) were resistant to imipenem. All isolates harbored the blaOXA-181 carbapenemase (100%, n=8) and also carried other β-lactamase genes such as OXA-1, CTX-M-15, TEM-1B and SHV-1. IncF, IncX3, and Col plasmid replicons groups and class I integrons (ln191 and ln27) were detected. All isolates belonged to the same sequence type ST307 and capsular serotypes (K102, O2v2) and several were associated with a single bed located in the ICU. All but one isolate carried the same plasmid multilocus sequence type [K7:A-:B-] and the same virulence repertoire was identified reflecting the epidemiological relationships between isolates. BlaOXA-181 were presumably located on a multi-replicon plasmid similar to that of E. coli p010_B-OXA181, and isolates were aligned with several South African and international clades, demonstrating horizontal and vertical transboundary distribution. Conclusion: OXA-181-producing K. pneumoniae belonging to ST307 was found to be potentially endemic in the hospital ICU environment of a public hospital in KwaZulu-Natal South Africa. The presence of a myriad of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in different permutations and combinations presents challenges to clinical management and infection prevention and control measures. This necessitates a CRE screening programme and strict infection prevention and control measures to detect and eliminate this endemic clone.

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Browsing Pharmaceutical Sciences by Author "Akebe, Luther King Abia."