Abstract
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.
Introduction
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Among the multiple environmental sources of antibiotic resistance, hospital effluents deserve special attention given the high prevalence of resistance and, in particular, the risk of propagation of resistance genes common in clinical environments [e.g., vanA, mecA, and aac(6′)-Ib-cr genes].7,19,29,32,39,45 Hospital effluents, classified as domestic effluents, are not subjected to any special categorization that would require a previous treatment before its discharge in the municipal collector and urban wastewater treatment facilities. However, hospital effluents can be important reservoirs for the dissemination of antibiotic-resistant bacteria.
Although municipal wastewater treatment can reduce the bacterial density in water in up to four logarithmic cycles, the prevalence of antibiotic-resistant bacteria, in general, does not decrease.13,22,38 On the contrary, some resistance phenotypes can even become more prevalent after wastewater treatment, as was reported for ciprofloxacin-resistant enterobacteria, probably due to selection occurring during treatment.9,10,27
The increased dissemination of quinolone resistance observed in clinical settings and in the environment is probably due to the extensive use of quinolones to treat human and veterinary bacterial infections, to their persistence in the environment and to the multiplicity of acquired resistance mechanisms.31,38 Since 1998, the year in which plasmid-mediated quinolone resistance was described for the first time, it has been demonstrated that not only vertical, but also horizontal gene transfer may play an important role on the propagation of this resistance phenotype. 20
The potential effect of quinolones as selectors may be explained based on its behavior as chemical contaminants, described as persistent and immobile, detected in hospital effluents, sewage sludge, and rivers.1,38,44 These evidences and the frequent association of ciprofloxacin resistance with multidrug resistance (MDR) phenotypes, suggest that quinolone-resistant bacteria may represent one of the major targets of selective pressures in the environment.
Therefore, this study is focused on quinolone-resistant bacteria isolated from urban wastewater and hospital effluent, exhibiting resistance to antibiotics of six or more distinct classes or resistance phenotypes still not common in domestic wastewater, 30 in particular meropenem and ceftazidime. The identification of important bacterial carriers of resistance is fundamental to investigate major pathways of transmission between distinct environmental compartments, in particular, from hospital and municipal wastewater that have the potential to contaminate surface water or soils.
Although the indicators of fecal contamination, Escherichia coli and enterococci are the most studied,2,4,9,13,22,39 other environmental bacteria may play an important role as resistance carriers. This aspect was the underlying question of this study, which aimed at identifying the taxonomic groups of quinolone-resistant bacteria with the highest MDR indices, which are released by hospital effluent and/or detected in the municipal wastewater treatment plant receiving those effluents.
Materials and Methods
Sampling, bacterial enumeration, and isolation
The strains analyzed in this study were isolated from samples collected between October 2010 and July 2011 from a hospital effluent and from the raw inflow and the treated effluent of the receiving urban wastewater treatment plant (UWTP). 38 The hospital, with a capacity of 1,120 beds, and the UWTP, serving a population of 200,000 inhabitants and receiving domestic and hospital sewage and storm water, are located in the northern region of Portugal.
Bacterial enumeration was performed on Plate Count Agar (PCA), membrane Fecal Coliforms agar (mFC), or Glutamate Starch Phenol Red Agar (GSP) and on these culture media supplemented with 4 mg/L of ciprofloxacin (CIP), as described before. 38 Because quinolone resistance is a promising track to trace antibiotic resistance in water environments,9,27,43 quinolone-resistant isolates recovered from culture media supplemented with ciprofloxacin, and exhibiting distinct morphotypes, were selected for this study. In addition, the study was focused on MDR bacteria, whose occurrence in water will be neglected by most of the water quality control protocols. Therefore, Gram-negative cytochrome c oxidase-negative isolates that formed typical E. coli colonies on the culture medium RAPID'E. coli 2 agar (BioRad), were presumptively identified as E. coli and excluded from this study.
A total of 101 nonrepetitive isolates, 15 from PCA, 74 from mFC, and 12 from GSP, corresponding to 36 from hospital effluent (HE), 36 from raw wastewater (RWW), and 29 from treated wastewater (TWW), were characterized for their antibiotic resistance profiles. Selected isolates (∼40%) were identified based on the 16S rRNA gene sequence analysis through the EzTaxon database.9,18
Antibiotic resistance phenotypes and selection of MDR isolates
Isolates were characterized for their antibiotic resistance profiles according to the standard disc diffusion method, as recommended by CLSI. 5 The antibiotics tested were amoxicillin (AML, 25 μg), ticarcillin (TIC, 75 μg), cephalothin (CP, 30 μg), ceftazidime (CEF, 30 μg), meropenem (MER,10 μg), colistin sulfate (CT, 50 μg), sulfamethoxazole (SUL, 25 μg), sulfamethoxazole/trimethoprim (SXT, 23.75/1.25 μg), ciprofloxacin (CIP, 5 μg), tetracycline (TET, 30 μg), gentamicin (GEN, 10 μg), and streptomycin (STR, 10 μg). In each assay, the reference strains E. coli ATCC 25922 and Pseudomonas aeruginosa DSM 1117 (=ATCC 27853) were used as quality controls. The ciprofloxacin minimum inhibitory concentration (MIC) was determined, using the M.I.C. Evaluator (0.002–32 mg/L; Oxoid) according to the manufacturer's instructions. Antibiotic resistance prevalence values were compared between sites using the chi-squared test.
The isolates nonsusceptible to at least one agent in six or more antibiotic classes (MDR ≥6; with MDRn phenotype corresponding to resistance to antibiotics belonging to n distinct classes), or to meropenem or ceftazidime were considered relevant tracers of acquired antibiotic resistance in wastewater and were further characterized (n = 25).
Plasmid-mediated quinolone resistance genes and other genetic elements
The selected MDR isolates (n = 25) were screened for the presence of plasmid-mediated quinolone resistance (PMQR) genes qnrA, qnrB, qnrS, qnrC, qnrD, qepA, oqxAB, and aac(6′)-Ib-cr. Genes qnr's, qepA, and aac(6′)-Ib-cr were tested as described by Figueira et al., 12 and oqxAB as described by Liu et al., 21 using an annealing temperature of 55°C. The nucleotide sequence of positive polymerase chain reaction (PCR) was determined to confirm the authenticity of the amplicon, and for the gene aac(6′)-Ib, it was also used to detect the mutations characteristic of the cr variant, responsible for low-level ciprofloxacin resistance. 36
Because some plasmids can be mobilized between bacteria of different taxonomic groups, plasmid replicons commonly found in enterobacteria and other Gram-negative bacteria were screened for in the MDR isolates. 3 The replicon types FIA, FIB, FIC, HI1, HI2, I1-Iγ, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIS, and the integrase genes and the variable region of class 1 and class 2 integrons were screened for as previously described.9,17,34 The nucleotide sequences of the gene cassettes inserted in the CS5-CS3 variable regions with more than 1000 bp were identified, based on alignment search in the GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
The capacity of each of the 25 MDR isolates to conjugate was assayed through broth mating, using the sodium azide-resistant E. coli J53 strain as recipient. Transconjugants were selected on Luria-Bertani agar supplemented with sodium azide (100 mg/L) and amoxicillin (32 mg/L) or ciprofloxacin (0.06 mg/L), confirmed through genotyping and characterized for antibiotics susceptibility, including ciprofloxacin MIC, and detection of genetic determinants harbored by the donors and gyrA gene mutations. 10
Accession numbers
The 16S rRNA and aac(6′)-Ib(-cr) gene sequences were deposited in the EMBL database with the accession numbers LN624790-LN624814 and LN624772-LN624789, respectively.
Results
Prevalence of resistance
Part of the isolates recovered on ciprofloxacin-supplemented (4 mg/L) culture media 17.8% (18/101) presented MIC values that ranged between 0.008 and 3 mg/L, which, according to the CLSI criteria, corresponded to susceptibility or intermediary phenotypes. Resistance to first-generation drugs, such as amoxicillin, ticarcillin, cephalothin, sulfamethoxazole, and streptomycin, was common in these isolates, with prevalence values above 50% (Table 1). Less common was resistance to ceftazidime and meropenem with prevalence values of 19.8% and 12.9%, respectively, and prevalence values significantly lower in municipal wastewater than in hospital effluent (p < 0.01) (Table 1).
The prevalence of intermediary resistance phenotypes is indicated within parenthesis.
Location: HE, hospital effluent; RWW, municipal raw wastewater; TWW, municipal treated wastewater.
Culture medium supplemented with 4 mg/L ciprofloxacin: P, Plate Count Agar; F, membrane Fecal Coliforms agar (mFC); G, Glutamate Starch Phenol Red Agar (GSP).
Antibiotics: AML, amoxicillin; TIC, ticarcillin; CP, cephalothin; CEF, ceftazidime; MER, meropenem; CT, colistin sulfate; SUL, sulfamethoxazole; SXT, sulfamethoxazole/trimethoprim; CIP, ciprofloxacin; TET, tetracycline; GEN, gentamicin; STR, streptomycin.
MDR ≥ 3, bacteria resistant to at least one antibiotic belonging to three or more distinct classes.
About 40% of the 101 isolates were identified as Klebsiella spp., Citrobacter freundii, Enterobacter spp., Aeromonas spp., Acinetobacter spp., Pseudomonas aeruginosa, Chryseobacterium spp. and Myroides odoratus.
a and b indicate sampling sites with significantly different prevalence values of antibiotic resistance (p < 0.01).
MDR, multidrug resistance.
The prevalence of resistance to at least one antibiotic of three or more distinct classes (MDR ≥3) ranged from 91.7% to 72.4% and could be ranked as hospital effluent>raw wastewater>treated wastewater (Table 1). However, only the simultaneous resistance to antibiotics belonging to six or more different classes (MDR ≥6) was significantly higher in hospital effluent than in municipal wastewater (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/mdr), suggesting that these phenotypes may indicate recently acquired resistance. This situation contrasted with what was observed for the phenotypes MDR3 and MDR4, which occurred mainly in municipal raw wastewater (15–30%) or MDR5 with identical prevalence in hospital effluent and in municipal wastewater (∼30%; Supplementary Fig. S1). Based on these findings, isolates with MDR ≥6 were further characterized.
Characterization of the MDR isolates
Isolates with a MDR ≥6 phenotype were in total 25, 21 of which were from hospital effluent. These isolates comprised Gammaproteobacteria of the genera Aeromonas, Acinetobacter, Citrobacter, Enterobacter, Klebsiella, and Pseudomonas and Flavobacteriia of the genera Myroides and Chryseobacterium (Table 2). The genes aac(6′)-Ib-cr, qnrB, qnrS, or oqxAB were detected in 16 of these 25 isolates, all belonging to the class Gammaproteobacteria (Table 2).
In the cases that an isolate presented more than one class I integron, both integrons are presented.
Contains the plasmid replicon type IncFIC.
Contains the plasmid replicon type incHI2.
PMQR, plasmid-mediated quinolone resistance; +, detected; −, not detected; nt, not tested.
Antibiotics: AML, amoxicillin 25 μg; TIC, ticarcillin 75 μg; CP, cephalothin 30 μg; CEF, ceftazidime 30 μg; MER, meropenem 10 μg; CT, colistin sulfate 50 μg; SUL, sulfamethoxazole 25 μg; SXT, sulfamethoxazole/trimethoprim 23.75/1.25 μg; CIP, ciprofloxacin 5 μg; TET, tetracycline 30 μg; GEN, gentamicin 10 μg; STR, streptomycin 10 μg.
The most common gene was aac(6′)-Ib-cr, observed in 14 isolates, followed by qnrB, detected in 4 hospital effluent isolates. The strains HE3, HE9, HE10, and HE21 harbored the gene aac(6′)-Ib encoding resistance to aminoglycosides, but not the cr variant conferring resistance to ciprofloxacin. The gene qnrS was detected only in Enterobacter ludwigii HE15, and coexisted with the genes qnrB and aac(6′)-Ib-cr (Table 2). The efflux pump oqxAB was detected only in members of the genus Klebsiella, in which the gene oqxAB is described as being chromosomal 46 and coexisted with the genes qnrB or aac(6′)-Ib-cr. In two Enterobacter spp. isolated from the hospital effluent it was possible to detect the gene oqxA, although for the associated gene oqxB no PCR amplification was observed. The genes qnrA, qnrC, qnrD, and qepA were not detected, and in 9 out of the 25 MDR isolates none of the searched PMQR genes was detected.
Genetic elements associated with horizontal gene transfer
The gene encoding class 1 integron integrase was detected in 17 Gammaproteobacteria. In most of those isolates the variable region of the class 1 integron contained genes related to aminoglycoside (aad, aac) and trimethoprim (dfr) resistance. The class 1 integron gene cassettes of three hospital effluent isolates contained also carbapenemases encoding genes (blaIMP, blaOXA, blaVIM) (Table 2). Although class 1 integrons were mainly detected in isolates harboring PMQR, these genes were not inserted in the variable region of the class 1 integrons. Of the plasmid replicons surveyed, only FIC and HI2 were detected in Enterobacteriaceae of the genera Enterobacter and Citrobacter (Table 2).
To assess the potential of the selected 25 MDR strains to promote the transfer of their resistance genes to other bacteria, conjugation assays were performed with the sodium azide-resistant E. coli J53. Under the conjugation conditions used, it was observed that for seven donor strains transconjugants showed acquired antibiotic resistance, demonstrated at the phenotypic or genotypic levels (Table 3). Other putative transconjugants (14/19) presented only an increase in the MIC to ciprofloxacin, from 0.012 mg/L, observed for E. coli J53, to 0.060–0.250 mg/L, which was associated with mutations in the gyrA gene of the recipient strain.
MIC 0.012 for Escherichia coli J53.
Putative transconjugants from strains HE1, HE3, HE5, HE6, HE7, HE8, HE9, HE11, HE12, HE13, HE14, and HE21 presented an increase in the MIC value to CIP, from 0.012 mg/L, observed for E. coli J53, to 0.06–0.25 mg/L, however were not included in this table because in those transconjugants were detected mutations in the gyrA gene (Ser83Leu or Asp87Tyr), which were probably due to a spontaneous mutation in the recipient strain rather than a gene transfer event.
MIC, minimum inhibitory concentration.
All donors of the transconjugants that featured most of the resistance traits were Enterobacteriaceae from hospital effluent, identified as members of the species E. ludwigii and Klebsiella pneumoniae, or from raw wastewater, Citrobacter freundii (Table 3). These transconjugants had acquired resistance to a maximum of five classes of antibiotics and one or more PMQR genes, associated with measured increases of MIC to ciprofloxacin. In strain E. ludwigii HE15, the plasmid replicon genotype HI2 was cotransferred with the three PMQR genes, suggesting that those genes were located on that plasmid. Klebsiella spp. strains transferred plasmids related to a pir-type plasmid (data not shown). 28
Discussion
Most of the ciprofloxacin-resistant bacteria (86.1%) recovered from the three types of wastewater (hospital effluent, raw and treated municipal wastewater), presented an MDR phenotype (Table 1). This complies with the hypothesis proposed in this study that the ciprofloxacin-resistant bacteria may be important targets of selective pressures, being the resistance to ciprofloxacin frequently associated with resistance to other classes of antibiotics.
Bacteria of distinct phylogenetic groups, MDR phenotypes (six or higher), and resistance to antibiotics used in hospitals, such as ceftazidime or meropenem, were observed to be discharged by hospital effluent (Supplementary Fig. S1).
The bacteria identified in this study, comprising members of the genera Aeromonas, Citrobacter, Enterobacter, Klebsiella, Acinetobacter, and Pseudomonas (class Gammaproteobacteria) and Chryseobacterium and Myroides (class Flavobacteriia) are ubiquitous in water environments, including in drinking water.11,16,26,33,41 Overwhelmingly, when conventional bacteriological control methods, based on conventional indicator bacteria such as E. coli and enterococci, are used, these bacterial groups may pass unnoticed. Nevertheless, bacteria belonging to these groups include recognized vectors of antibiotic resistance9,12,14,15,23,24 (e.g., Aeromonas, Klebsiella, Acinetobacter, Pseudomonas, Chryseobacterium, Myroides) and have been detected in the human microbiome. 42
Ubiquitous bacteria that can cross different environmental compartments and have the ability to acquire and transfer antibiotic resistance, being simultaneously well fitted to integrate the human microbiome, may have a crucial role on antibiotic resistance transmission to humans. Indeed, bacteria with such characteristics may have a significant advantage under antibiotic-induced selective pressure, explaining that members of the bacterial groups identified in this study (e.g., Citrobacter, Klebsiella, Pseudomonas, Enterobacter, Acinetobacter) are also frequently reported as causative agents of healthcare-associated infections. 8
In contrast to the Gammaproteobacteria, not much is known about antibiotic resistance genes and associated horizontal gene transfer processes in Chryseobacterium and Myroides. Although it can be argued that members of these genera are probably intrinsically MDR, their resistance profiles deserve attention. Since these bacteria have an advantage to colonize habitats with high selective pressure imposed by antibiotics, it is worthy of investigation if they may have any role on resistance propagation, mainly if their opportunistic character is taken into account. 6
Acquired antibiotic resistance genes located on mobile genetic elements, such as plasmids, are considered major drivers for antibiotic resistance dissemination. The transfer of PMQR associated to plasmids HI2 and pir-type was demonstrated in this study, suggesting that hospital effluent MDR bacteria have the potential to spread their resistance genes to other environmental bacteria (Table 3). Other PMQR detected in this study could not be transferred to a recipient and therefore it is possible to argue about their location in the chromosome. The occurrence of topoisomerase gene mutations conferring resistance to high ciprofloxacin concentration (4 mg/L) or other unknown or not tested resistance mechanisms may also be involved.
As in previous studies, it was not clear the role of the class 1 integrons on the resistance phenotypes of these isolates,9,11 since the variable region of class 1 integrons included mainly genes related to aminoglycosides and trimethoprim resistance (Table 2). However, in Acinetobacter johnsonii HE10 and P. aeruginosa HE21 the integron variable region contained carbapenemase genes frequently detected in clinical environments such as blaVIM-2 or blaIMP. 47 Moreover, class 1 integrons prevailed in isolates with PMQR, suggesting that these genetic elements may have a role on the process of resistance acquisition.
The release of MDR bacteria from hospital effluent is of concern, since these bacteria may survive and proliferate in different environmental compartments, being also able to transfer their resistance genes to other bacterial hosts. Indeed, it was demonstrated that MDR strains discharged by hospital effluents were able to transfer PMQR genes and other traits that may confer their host an important advantage to survive and acquire new resistance determinants.
It was not possible to provide evidences in this study that resistance discharged by the hospital effluent could be found in the municipal wastewater.35,40 However, since the hospital effluent is diluted in the municipal effluent, 38 it could be probabilistically almost impossible to detect the same strains in the municipal wastewater. Nevertheless, it is noteworthy that bacteria with MDR ≥6 phenotype were still present in the final municipal effluent that is discharged in the receiving water bodies.
In a recent study of Varela et al., 40 based on multilocus sequence typing of E. coli recovered from the same locations used in this study, it was observed that the genetically closely related strains could be detected in the hospital effluent and also in the raw or treated wastewater. Also, Slekovec et al. 35 using multilocus sequencing typing observed that some sequence types of P. aeruginosa released by an hospital were found in the urban wastewater and in the river water downstream from the wastewater treatment plant discharge.
Conceptually, MDR bacteria discharged by hospital effluents can either transfer genetic determinants leading to MDR phenotypes or simply contribute to the evolution toward MDR of bacteria that live in the same habitat. A better understanding of which carriers and what conditions favor antibiotic resistance evolution and spread is needed to support management options aiming at the control of this public health problem.
These results allow to conclude that MDR bacteria, displaying resistance to at least one antibiotic of three, four or five classes of antibiotics are nonsignificantly more prevalent in hospital effluent than in municipal wastewater (Supplementary Fig. S1). However, hospital effluent was confirmed as a source of MDR bacteria combining resistance to six or more classes of antibiotics. These phenotypes were detected in ubiquitous bacteria, belonging to the genera Aeromonas, Acinetobacter, Citrobacter, Enterobacter, Klebsiella, Pseudomonas, Chryseobacterium and Myroides. Among these, the Gammaproteobacteria were able to transfer their resistance genes to a susceptible recipient.
These results demonstrate that ubiquitous bacteria that are not commonly monitored in routine water quality controls are important vectors of antibiotic resistance dissemination and should not be neglected. Once entering the urban water cycle, these bacteria may reach other water compartments, including drinking water.
Footnotes
Acknowledgments
The authors gratefully acknowledge Prof. P. Nordmann, Dr. L.M. Cavaco, Prof. M. Wang, and Dr. A. Carattoli for the positive controls for the detection of the genes qnrA, qnrB and qnrS; qnrD and aac(6′)-Ib-cr; qnrC; and plasmid replicons, respectively. The authors also acknowledge Prof. L. Martínez-Martínez and Dr. J. Calvo for the azide-resistant E. coli strain J53 used in conjugation experiments, and Dr. Conceição Egas from BIOCANT for the plasmid sequencing assistance. This work was supported by National Funds from Fundação para a Ciência e Tecnologia through project PEst-OE/EQB/LA0016/2013 and PTDC/AAC-AMB/113840/2009, the IVM grant (SFRH/BPD/87360/2012) and ARV grant (SFRH/BD/70986/2010). TVP and RCF grants were financed through the National Council for Scientific and Technological Development (CNPq), Brazil.
Disclosure Statement
No competing financial interests exist.
References
Supplementary Material
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