Pharmaceutical industrial wastewater exhibiting the co-occurrence of biofilm-forming genes in the multidrug-resistant bacterial community poses a novel environmental threat.

The interaction of the environment with the effluent of wastewater treatment plants, having antibiotics, multidrug-resistant (MDR) bacteria, and biofilm-forming genes (BFGs), has vast environmental risks. Antibiotic pollution bottlenecks environmental bacteria and has the potential to significantly lower the biodiversity of environmental bacteria, causing an alteration in ecological equilibrium. It can induce selective pressure for antibiotic resistance (AR) and can transform the non-resistant environmental bacteria into a resistant form through HGT. This study investigated the occurrence of MDR bacteria, showing phenotypic and genotypic characteristics of biofilm. The bacteria were isolated from the pharmaceutical wastewater treatment plants (WWTPs) of Dehradun and Haridwar (India), located in the pharmaceutical areas. The findings of this study demonstrate the coexistence of BFGs and MDR clinical bacteria in the vicinity of pharmaceutical industrial wastewater treatment plants. A total of 47 bacteria were isolated from both WWTPs and tested for antibiotic resistance to 13 different antibiotics; 16 isolates (34.04 %) tested positive for MDR. 5 (31.25 %) of these 16 MDR isolates were producing biofilm and identified as Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Burkholderia cepacia. The targeted BFGs in this study were ompA, bap and pslA. The most common co-occurring gene was ompA (80 %), with pslA (40 %) being the least common. A. baumannii contains all three targeted genes, whereas B. cepacia only has bap. Except for B. cepacia, all the biofilm-forming MDR isolates show AR to all the tested antibiotics and prove that the biofilm enhances the AR potential. The samples of both wastewater treatment plants also showed the occurrence of tetracycline, ampicillin, erythromycin and chloramphenicol, along with high levels of BOD, COD, PO4-3, NO3-, heavy metals and organic pollutants. The co-occurrence of MDR and biofilm-forming tendency in the clinical strain of bacteria and its environmental dissemination may have an array of hazardous impacts on human and environmental health.

Coexistence of multidrug resistance and ESBL encoding genes - blaTEM, blaSHV, and blaCTX-M; its amplification and dispersion in the environment via municipal wastewater treatment plant.

Municipal wastewater treatment plants (MWWTPs) are a global source of antibiotic resistance genes (ARGs), collecting wastewater from a variety of sources, including hospital wastewater, domestic wastewater, runoff from agricultural and livestock farms, etc. These sources are contaminated with organic and inorganic pollutants, ARGs and antibiotic-resistant bacteria (ARB). Such pollutants aided eutrophication and encouraged bacterial growth. During bacterial growth horizontal gene transfer (HGT) and vertical gene transfer (VGT) of ARGs and extended-spectrum ß-lactamase (ESBL) encoding genes may facilitate, resulting in the spread of antibiotic resistance exponentially. The current study investigated the prevalence of multidrug resistance (MDR) and ESBL encoding genes in various treatment units of MWWTP and their spread in the environment. A total of three sampling sites (BUT, BRO, and BFB) were chosen, and 33 morphologically distinct bacterial colonies were isolated. 14 of the 33 isolates tested positive for antibiotic resistance and were further tested for the coexistence of MDR and ESBL production. The selected 14 isolates showed the highest resistance to trimethoprim (85.71%), followed by ciprofloxacin, azithromycin, and ampicillin (71.42%), tetracycline (57.14%), and vancomycin, gentamicin, and colistin sulphate (50%). A total of 9 isolates (64.28%) were phenotypically positive for ESBL production (BUT2, BUT3, BUT5, BRO1, BRO2, BRO3, BRO4, BRO5 and BFB1). The molecular detection of ESBL encoding genes, i.e. blaTEM, blaSHV, and blaCTX-M was carried out. The most prevalent gene was blaTEM (69.23%), followed by blaSHV (46.15%), and blaCTX-M (23.07%). In this study, 9 isolates (64.28%) out of 14 showed the coexistence of MDR and ESBL encoding genes, namely BUT3, BUT4, BUT5, BUT6, BUT7, BRO1, BRO2, BRO4, and BFB1. The coexistence of ESBL encoding genes and resistance to other antibiotic classes exacerbates human health and the environment.

Municipal wastewater treatment plant showing a potential reservoir for clinically relevant MDR bacterial strains co-occurrence of ESBL genes and integron-integrase genes.

Municipal wastewater treatment plants (MWWTPs) are a milieu for co-occurrence of multiple antibiotic resistance genes (ARGs). This facilitates mixing and genetic exchange; and promotes dissemination of multidrug resistance (MDR) to wastewater bacterial communities which is hazardous for the effluent receiving environment. This study investigated the co-occurrence of extended-spectrum beta-lactamase (ESBL) genes (blaTEM, blaCTX-M, blaSHV, blaOXA), and integron-integrase genes (intI1, intI2, intI3) in MDR bacteria isolated from the Bharwara MWWTP in Lucknow, India. Thirty-one MDR bacterial colonies resistant to three or more antibiotics were isolated from three treatment stages of this MWWTP. Six of these: Staphylococcus aureus, Serratia marcescens, Salmonella enterica, Shigella sonnei, Escherichia coli, and Bacillus sp. Had co-occurrence of ESBL and integron-integrase genes. These six isolates were examined for the occurrence of MDR efflux genes (qacA, acrB) and ARGs (aac(3)-1, qnrA1, tetA, vanA) and tested for resistance against 12 different antibiotics. The highest resistance was against penicillin-G (100%) and lowest for chloramphenicol (16.66%). Bacillus sp. Isolate BWKRC6 had the highest co-occurrence of antibiotic resistance-determining genes and was resistant to all the 12 antibiotics tested. The co-occurrence of ESBL, integron-integrase, antibiotic resistance-determining and MDR efflux genes in bacteria isolated from the Bharwara MWWTP indicates that the wastewaters of this treatment plant may have become a hotspot for MDR bacteria and may present human and environmental health hazards. Therefore, there is need for a rapid action to limit the spread of this threat. Public regulatory authorities must urgently implement measures to prevent MWWTPs becoming reservoirs for evolution of antibiotic resistance genes and development of antibiotic resistance.