TRACKING ANTIBIOTIC RESISTANCE FROM HOSPITAL EFFLUENTS TO THE SURROUNDING ENVIRONMENT

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Insights into the relationship between antimicrobial residues and bacterial populations in a hospital-urban wastewater treatment plant system

Publication . Varela, Ana Rita; André, Sandra; Nunes, Olga C.; Manaia, Célia M.

The relationship between antimicrobial residues, antibiotic resistance prevalence and bacterial community composition in hospital effluent and in the receiving wastewater treatment plant was studied. Samples from hospital effluent, raw inflow and final effluent of the receiving wastewater treatment plant were characterized for amoxicillin and ciprofloxacin resistance prevalence, content of heavy metals and antimicrobial residues and bacterial community structure, based on 16S rRNA gene PCR-DGGE analysis. The concentration of fluoroquinolones, arsenic and mercury was in general higher in hospital effluent than in raw inflow, while the opposite was observed for tetracyclines, sulfonamides and penicillin G. The prevalence of ciprofloxacin resistance was significantly higher in hospital effluent than in raw inflow. The concentration of antimicrobial residues was observed to be significantly correlated with the prevalence of antibiotic resistant bacteria and with variations in the bacterial community. Hospital effluent was confirmed as a relevant, although not unique, source of antimicrobial residues and antibiotic resistant bacteria to the wastewater treatment plant. Moreover, given the high loads of antibiotic residues and antibiotic resistant bacteria that may occur in hospital effluents, these wastewater habitats may represent useful models to study and predict the impact of antibiotic residues on bacterial communities.

2014Journal article

Open access

blaTEM and vanA as indicator genes of antibiotic resistance contamination in a hospital–urban wastewater treatment plant system

Publication . Narciso-da-Rocha, Carlos; Varela, Ana R.; Schwartz, Thomas; Nunes, Olga C.; Manaia, Celia M.

Four indicator genes were monitored by quantitative PCR in hospital effluent (HE) and in the raw and treated wastewater of the municipal wastewater treatment plant receiving the hospital discharge. The indicator genes were the class 1 integrase gene intI1, to assess the capacity of bacteria to be involved in horizontal gene transfer processes; blaTEM, one of the most widespread antibiotic resistance genes in the environment, associated with Enterobacteriaceae; vanA, an antibiotic resistance gene uncommon in the environment and frequent in clinical isolates; and marA, part of a locus related to the stress response in Enterobacteriaceae. Variation in the abundance of these genes was analysed as a function of the type of water, and possible correlations with cultivable bacteria, antimicrobial residue concentrations, and bacterial community composition and structure were analysed. HE was confirmed as an important source of blaTEM and vanA genes, and wastewater treatment showed a limited capacity to remove these resistance genes. The genes blaTEM and vanA presented the strongest correlations with culturable bacteria, antimicrobial residues and some bacterial populations, representing interesting candidates as indicator genes to monitor resistance in environmental samples. The intI1 gene was the most abundant in all samples, demonstrating that wastewater bacterial populations hold a high potential for gene acquisition

2014Journal article

Open access

Genetic characterization of fluoroquinolone resistant Escherichia coli from urban streams and municipal and hospital effluents

Publication . Varela, Ana Rita; Macedo, Gonçalo N.; Nunes, Olga C.; Manaia, Célia M.

Escherichia coli with reduced susceptibility to ciprofloxacin, isolated from urban streams, wastewater treatment plants and hospital effluent between 2004 and 2012, were compared based on multilocus sequence typing (MLST), quinolone and beta-lactam resistance determinants and plasmid replicon type. Isolates from the different types of water and isolation dates clustered together, suggesting the persistence and capacity to propagate across distinct aquatic environments. Escherichia coli with reduced susceptibility to ciprofloxacin, isolated from urban streams, wastewater treatment plants and hospital effluent between 2004 and 2012, were compared based on multilocus sequence typing (MLST), quinolone and beta-lactam resistance determinants and plasmid replicon type. Isolates from the different types of water and isolation dates clustered together, suggesting the persistence and capacity to propagate across distinct aquatic environments. The most prevalent MLST groups were ST10 complex and ST131. Almost all isolates (98%) carried mutations in the chromosomal genes gyrA and/or parC, and 10% possessed the genes qepA, aac(6')-Ib-cr and/or qnrS1. Over 80% of the isolates were resistant to three or more classes of antibiotics (MDR ≤3). The most prevalent beta-lactamase encoding gene was blaTEM, followed by blaCTX-M-15, co-existing with plasmid mediated quinolone resistance. The plasmid replicon types of the group IncF were the most prevalent and distributed by different MLST groups. The genes aac(6')-Ib-cr and/or qnrS1 could be transferred by conjugation in combination with the genes blaTEM,blaSHV-12 or blaOXA-1 and the plasmid replicon types I1-Iγ, K, HI2 and/or B/O. The potential of multidrug resistant E. coli with reduced susceptibility to ciprofloxacin, harboring mobile genetic elements and with ability to conjugate and transfer resistance genes, to spread and persist across different aquatic environments was demonstrated.

2015Journal article

Restricted access

Multidrug resistance in quinolone-resistant gram-negative bacteria isolated from hospital effluent and the municipal wastewater treatment plant

Publication . Vaz-Moreira, Ivone; Varela, Ana Rita; Pereira, Thamiris V.; Fochat, Romário C.; Manaia, C. M.

This study is aimed to assess if hospital effluents represent an important supplier of multidrug-resistant (MDR) Gram-negative bacteria that, being discharged in the municipal collector, may be disseminated in the environment and bypassed in water quality control systems. From a set of 101 non-Escherichia coli Gram-negative bacteria with reduced susceptibility to quinolones, was selected a group of isolates comprised by those with the highest indices of MDR (defined as nonsusceptibility to at least one agent in six or more antimicrobial categories, MDR ‡6) or resistance to meropenem or ceftazidime (n = 25). The isolates were identified and characterized for antibiotic resistance phenotype, plasmid-mediated quinolone resistance (PMQR) genes, and other genetic elements and conjugative capacity. The isolates with highest MDR indices were mainly from hospital effluent and comprised ubiquitous bacterial groups of the class Gammaproteobacteria, of the genera Aeromonas, Acinetobacter, Citrobacter, Enterobacter, Klebsiella, and Pseudomonas, and of the class Flavobacteriia, of the genera Chryseobacterium and Myroides. In this group of 25 strains, 19 identified as Gammaproteobacteria harbored at least one PMQR gene (aac(6¢)-Ib-cr, qnrB, qnrS, or oqxAB) or a class 1 integron gene cassette encoding aminoglycoside, sulfonamide, or carbapenem resistance. Most of the E. coli J53 transconjugants with acquired antibiotic resistance resulted from conjugation with Enterobacteriaceae. These transconjugants demonstrated acquired resistance to a maximum of five classes of antibiotics, one or more PMQR genes and/or a class 1 integron gene cassette. This study shows that ubiquitous bacteria, other than those monitored in water quality controls, are important vectors of antibiotic resistance and can be disseminated from hospital effluent to aquatic environments. This information is relevant to support management options aiming at the control of this public health problem.

2016Journal article

Restricted access

Quinolone resistant Aeromonas spp. as carriers and potential tracers of acquired antibiotic resistance in hospital and municipal wastewater

Publication . Varela, Ana Rita; Nunes, Olga C.; Manaia, Célia M.

Members of the genus Aeromonas are recognized carriers of antibiotic resistance in aquatic environments. However, their importance on the spread of resistance from hospital effluents to the environment is poorly understood. Quinolone resistant Aeromonas spp. (n = 112) isolated from hospital effluent (HE) and from raw (RWW) and treated wastewater (TWW) of the receiving urban wastewater treatment plant (UWTP) were characterized. Species identification and genetic intraspecies diversity were assessed based on the 16S rRNA, cpn60 and gyrB genes sequence analysis. The antibiotic resistance phenotypes and genotypes (qnrA, qnrB, qnrC, qnrD, qnrS, qnrVC; qepA; oqxAB; aac(6′)-Ib-cr; blaOXA; incU) were analyzed in function of the origin and taxonomic group. Most isolates belonged to the species Aeromonas caviae and Aeromonas hydrophila (50% and 41%, respectively). The quinolone and the beta-lactamase resistance genes aac(6′)-Ib-cr and blaOXA, including gene blaOXA-101, identified for the first time in Aeromonas spp., were detected in 58% and 56% of the isolates, respectively, with identical prevalence in HE and UWTP wastewater. In contrast, the gene qnrS2was observedmainly in isolates from the UWTP (51%) and rarely in HE isolates (3%), suggesting that its origin is not the clinical setting. Bacterial groups and genes that allowthe identification of major routes of antibiotic resistance dissemination are valuable tools to control this problem. In this study, itwas concluded that members of the genus Aeromonas harboring the genes aac(6′)-Ib-cr and blaOXA are relevant tracers of antibiotic resistance dissemination inwastewater habitats, while those yielding the gene qnrS2 allow the traceability from non-clinical sources.

2016Journal article

Restricted access