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    Dynamics of drug resistance in environmental bacteria within an aquatic ecosystem = Umumo wokumelana nemithi kwamagciwane empilweni yasemanzini.
    (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. Iqoqa. Muva nje kunokukhula ngamandla kwezehlakalo nokwanda kwamagciwane angezwa mithi kanye nokungasebenzi kahle kwezinto ezilwa namagciwane endaweni okuhlalwa kuyo, ngenxa yengcindezi yobukhona bezilwimagciwane ezinjengama-antibiotics, amabiocides, kanye nokunye okusansimbi okusindayo namanye amakhemikhali acindezelayo anjengePoly aromatic hydrocarbons, pH, ukushisa nokubanda kanye nereactive oxygen. Yize kunjalo, amazinga okugxila kwalezi zilwimagciwane ezikwazi ukumelana nezimo ngayo ayikaziwa kahle. Lokhu kungabi khona kolwazi kunganqinda amandla okwakhiwa kwendlela yokuqapha izilwimagciwane okungaba nomthelela omubi ezimpilweni zabantu, izilwane nempilo yemvelo. Kulolu cwaningo, amasampula amanzi aqoqwa kuzona zonke izindawo ezinomthelela ngqo kuyona imvelo endaweni yokuhlanza amanzi eDarvill, okuyindawo lapho amanzi angcolile ephuma khona, enhla nasenzansi ekuphumeni kwamanzi. Ama-antibiotics, amaheavy metals, kanye namabiocides atholakala futhi akalwa ubungako bawo emasampuleni amanzi, kwase kuqinisekiswa umthelela wokushuba kwawo ekulweni nemithi kulawo aziwa ngobungozi njenge-Escherichia coli. Ubuningi bamaheavy metals batholakala kusetshenziswa i-United States Environmental Protection Agency (US EPA) method 200.7. Kwatholakala izinsalela zebiocide neze-antibiotic kusetshenziswa imithi ewuketshezi enamandla kakhulu eqinisekisiwe ehambisana nesisindo sezindlela ezigxile kwispectrometry. Amagciwane i-E. coli atholakala, kwakalwa nobuningi bawo kusetshenziswa uhlelo lweColilert-18TM yakwa-IDEXX, kanti ubungozi bezilwimagciwane bahlolwa kusetshenziswa indlela yedisc diffusion ngokwemigomo yeClinical and Laboratory Standards Institute. Ubuningi bama-antibiotics obabonakala kusukela kwisulfamethoxazole (286.180 µg/L) kuya kwipenicillin (2.2 µg/L); ngokwamametals, isodium (27.734 mg/L) kuya kwi-iron (0.001 mg/L); kanye namabiocides, ibenzalkonium chloride (BAC) 12 (7.805 µg/L) kuya kwiBenthEZ (0.035 µg/L). Kwabonakala ukukhula kwezinto ezingcolisayo emanzini angcolile kanye nakumasampula asemanzini ehlayo uma eqhathaniswa nalawo enyukayo, okuchaza ukuthi i-WWTP ingaba yisisusa sokumele kubhekwa, okwacacisa ukuthi izingcolisi zazingasukile kwi-WWTP. Ukuba sengozini ye-E. coli ATCC 25922 izinsuku ezingamashumi amathathu, endaweni ene-oxytetracycline, amoxicillin, zinc, copper, BAC 12 kanye ne-DADMAC 10 kwenziwa kodwa akukwazanga ukwenza ukumelana nesimo kuqale. Ukuhlaziya igenotypic analysis ye-WGS yalokho okwakusobala, kwathola imacrolide resistance mdf (A) gene kuphela (eyayikhona nakulokho okwakungeyona ingxenye yokuhlolwayo) kanye nedisinfectant resistance gene sitABCD. Uma kuqhutshekwa nokuhlaziya amanucleotide variants (SNV) awodwa, ukonakala kwabonakala kumajini ayi-19 uma kuqhathaniswa nalokho okungahlolwanga. Kwatholakala iresistance gene eyodwa, robA, eyilungu lomndeni we-ArcC/XylS, okuyiyona elawula i-ArcAB-TolC multi-drug efflux, enomthelelea ekungazwelini kwenhlanganisela yemithi. Amanye amajini ayi-18 atholakala ayebekezela okwaba yi-acnB, cusA, degQ, epmA, hsmP, mlc, purH, queG, srlE, tsaB, yddh kanye namayqhH, wonke ekulokhu okwakusobala. Amajini efilA ayekwi-oxytetracycline kuphela kanye ne-BAC 12-exposed isolates, nejini imutM ekwizinc, ijini inudK kuwona wonke ayevele obala ngaphandle kwe-DADMAC 10 exposed isolates, nejini iptsG eyayikwi-oxytetracycline-exposed isolates kuphela, kanye ne-ompD eyayikwi-DADMAC 12-exposed isolates kuphela. Wonke amajini atholakala kuma-exposed isolates atholakala futhi nakuma-environmental isolates, ngaphandle kwejini yerobA. Wonke la majini atholakala ene-oxidative stress, DNA repair, namamembrane proteins efflux systems, ukukhula kanye nesimo sokuphikelela. Ngaphezu kwalokhu, kwatholakala ukuthi ukuhlala izinsuku ezingama30 kuma-isolates kwadala ukukhula kokubekezela okuphezulu (25 x MIC) kwe-ampicillin ngama-30 kuya kuma-50% uma kuqhathaniswa nalokho okwakungahlolwanga. Ama-BAC 12-exposed isolates atholakala enokukhula kwezinga lokubekezela. Ukukhula kokubekezela kuqhamuka kumajini amaningi anesakhiwo sokuphikelela, kanye namatolerance gene expressions. Ama-MSW alawo ayeyamene ne-ampicillin kanye ne-amoxicillin, nawo akhula uma eqhathaniswa nalawo ayengahlolwanga okukhombisa amandla okubekezelela ukuba sobala ezinsukwini ezingama-30 kodwa i-MSW yahlala ifana kwi-oxytetracycline. Lokhu kukhombisa ukuthi ukuvezwa obala kumakhemikhali avimbayo ama-antibiotics, amaheavy metals kanye nezinsalela zebiocide, njengoba zibonakala endaweni enamanzi, azikwazi ukuthatha iphenotypic resistance kodwa ziyakwazi ukubona amajini adala ukukhula kokumelana kanye nokubekezelela amagciwane, okubukeka kuyindlela ebheke ekubekezeleni kwezilwimagciwane ezindaweni ezinamagciwane.