Abstract
Fecal bacteria are considered to be a potential reservoir of antimicrobial resistance genes in the aquatic environment and could horizontally transfer these genes to autochthonous bacteria when carried on transferable and/or mobile genetic elements. Such circulation of resistance genes constitutes a latent public health hazard. The aim of this study was to characterize the variable region of the class 1 integron and relate its genetic content to resistance patterns observed in antimicrobial-resistant Escherichia coli isolated from the surface waters of Patos Lagoon, Southern Brazil. Genetic diversity of the isolates and presence of the qacEΔ1 gene, which confers resistance to quaternary ammonium compounds, were also investigated. A total of 27 isolates were analyzed. The variable region harbored dfrA17, dfrA1 and dfrA12 genes, which confer resistance to trimethoprim, and aadA1, aadA5 and aadA22 genes that encode resistance to streptomycin/spectinomycin. Most of the isolates were considered resistant to quaternary ammonium compounds and all of them carried the qacE Δ1 gene at the 3′ conserved segment of the integron. ERIC-PCR analyses of E. coli isolates that presented the integrons showed great genetic diversity, indicating diverse sources of contamination in this environment. These results suggest that fecal bacteria with class 1 integrons in aquatic environments are potentially important reservoirs of antibiotic-resistance genes and may transfer these elements to other bacteria that are capable of infecting humans.
Keywords
Antibiotic resistance; Class 1 integron; Superficial water; Escherichia coli
Introduction
The rapid spread of antimicrobial resistance in bacteria is an ever-increasing global concern. Further, antimicrobial resistance is no longer restricted to medical or veterinary settings and has also been reported in many aquatic environments such as rivers,11 Koczura R, Mokracka J, Jabłońska L, Gozdecka E, Kubek M, Kaznowski A. Antimicrobial resistance of integron-harboring Escherichia coli isolates from clinical samples, wastewater treatment plant and river water. Sci Total Environ. 2012;414:680-685. surface waters,22 Pereira A, Santos A, Tacão M, Alves A, Henriques I, Correia A. Genetic diversity and antimicrobial resistance of Escherichia coli from Tagus estuary (Portugal). Sci Total Environ.2013:461–462, 65–71. sewage treatment plants33 Ferreira da Silva M, Vaz-Moreira I, Gonzalez-Pajuelo M, Nunes OC, Manaia CM. Antimicrobial resistance patterns in Enterobacteriaceae isolated from an urban wastewater treatment plant. FEMS Microbiol Ecol. 2007;60:166-176. and hospital effluents.44 Ferreira AE, Marchetti DP, De Oliveira LM, Gusatti CS, Fuentefria DB, Corção G. Presence of OXA-23-producing isolates of Acinetobacter baumannii in wastewater from hospitals in Southern Brazil. Microb Drug Resist. 2011;17:221-227.–66 Spindler A, Otton LM, Fuentefria DB, Corção G. Beta-lactams resistance and presence of class 1 integron in Pseudomonas spp. isolated from untreated hospital effluents in Brazil. Antonie van Leeuwenhoek. 2012;102:73-81.
Antimicrobial resistance genes are mainly carried on mobile genetic elements such as plasmids and transposons. Integrons are natural recombination systems that are able to capture, mobilize and express several gene cassettes encoding antimicrobial resistance, and are often found inserted into these mobile elements.77 Cambray G, Guerout AM, Mazel D. Integrons. Annu Rev Genet. 2010;44:141-166. Three classes of integrons are responsible for multi-drug resistance and are classified based on the integrase gene sequence they carry. Class 1 integrons are widely found in Gram-negative bacteria and are associated with both an increase in and spread of antimicrobial multi-drug resistance throughout the world.88 Koczura R, Mokracka J, Barczak A, Krysiak N, Kaznowski A. Association between the presence of class 1 integrons, virulence genes, and phylogenetic groups of Escherichia coli isolates from river water. Microb Ecol. 2013;65:84-90.
Most integrons are of the class 1 type and harbor two conserved segment (CS) regions, one each in the 5′ and 3′ regions. The 5′-conserved segment (5′-CS) contains an intI gene that encodes an integrase (intI1), a recombination site (attI) and a common promoter that is responsible for the expression of the captured gene cassettes, whereas the 3′-conserved segment (3′-CS) includes a gene conferring resistance to quaternary ammonium compounds (qacEΔ1), a sulfonamide resistance gene (sul1) and an ORF (orf5) of unknown function.99 Lévesque C, Piché L, Larose C, Roy PH. PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob Agents Chemother. 1995;39:185-191.,1010 Sáenz Y, Vinué L, Ruiz E, et al. Class 1 integrons lacking qacEΔ1 and sul1 genes in Escherichia coli isolates of food, animal and human origins. Vet Microbiol. 2010;144:493-497. These segments are separated by a variable region, wherein one or more gene cassettes are inserted.
Investigations into antimicrobial resistance in aquatic environments have mainly focused on bacteria of fecal origin as they are used as indicators of pollution, may be associated with infectious diseases and are also capable of transferring resistance at the host–environment interface.1111 Costa PM, Loureiro L, Matos AJF. Transfer of multidrug-resistant bacteria between intermingled ecological niches: the interface between humans, animals and the environment. Int J Environ Res Public Health. 2013;10:278-294.,1212 Ozgumus OB, Sandalli C, Sevim A, Celik-Sevim E, Sivri N. Class 1 and class 2 integrons and plasmid-mediated antibiotic resistance in coliforms isolated from ten rivers in Northern Turkey. J Microbiol. 2009;47:19-27.Escherichia coli is a member of the natural gastrointestinal microbiota and is also a key indicator of fecal contamination in water bodies. Resistant E. coli may enter aquatic environments through the discharge of treated effluent from wastewater treatment plants,33 Ferreira da Silva M, Vaz-Moreira I, Gonzalez-Pajuelo M, Nunes OC, Manaia CM. Antimicrobial resistance patterns in Enterobacteriaceae isolated from an urban wastewater treatment plant. FEMS Microbiol Ecol. 2007;60:166-176. field runoffs, and from untreated effluents.1313 Peak N, Knapp CW, Yang RK, et al. Abundance of six tetracycline resistance genes in wastewater lagoons at cattle feedlots with different antibiotic use strategies. Environ Microbiol. 2007;9:143-151. It was recently found that some persistent strains of E. coli can survive for between 4 and 14 days in aquatic environments with low levels of fecal bacteria,1414 Berthe T, Ratajczak M, Clermont O, Denamur E, Petit F. Evidence for coexistence of distinct Escherichia coli populations in various aquatic environments and their survival in estuary water. Appl Environ Microbiol. 2013;79:4684-4693. and can thus, play an important role in the dissemination of antimicrobial resistance in this environment.
Studies that investigate antimicrobial resistance in natural ecosystems are of great importance in identifying environmental reservoirs of resistance and also contribute toward understanding the routes of dissemination of these resistant bacteria. Therefore, in order to develop a better understanding of the spread of antimicrobial resistance genes in surface waters, this study aimed to characterize the variable region of the class 1 integron and relate the gene cassettes found therein to the antimicrobial susceptibility pattern of resistant E. coli isolated from the surface water of the Patos Lagoon in Southern Brazil. The presence of the qacEΔ1 gene and susceptibility to quaternary ammonium compounds were also assessed.
Materials and methods
Isolation and characterization of E. coli strains
In a previous study, 441 E. coli were isolated from surface water at five sampling sites on the Patos Lagoon in Southern Brazil (Fig. 1).1515 Canal N [M.Sc. Dissertation. Instituto de Ciências Básicas da Saúde] Caracterização de Resistência a Antimicrobianos e Diversidade Genética em Escherichia coli Isolada de Amostras de Água da Lagoa dos Patos, RS. Porto Alegre, Brazil: UFRGS; 2010. The Patos Lagoon is located between parallels 30°30′ S and 32°12′ S, is a semi-closed microtidal coastal lagoon, and its water circulation is mainly controlled by the northeast–southwest wind regime and freshwater input tributaries. The lagoon surface area is around 10,230 km2 and extends in a northeast–southwest direction from the delta of the Guaíba Lake to the Atlantic Ocean near Rio Grande.1717 Möller OO, Castaing P, Salomon JC, Lazure P. The influence of local and non-local forcing effects on the subtidal circulation of Patos Lagoon. Estuaries. 2001;24:297-311. This water system receives a variety of urban, industrial, and agricultural inputs, including those from the petrochemical industry in the cities of Porto Alegre and Rio Grande. Uncontrolled settlements, the use of agricultural fertilizers and pesticides near its shores, discharge of domestic and industrial effluents into the estuarine zone of the lagoon, among other factors, have contributed to the degradation of this lagoon; and the resultant adverse effects have intensified in the recent decades. Organic and fecal-bacterial contamination and excessive concentration of chemical compounds such as ammonium and phosphate due to the discharge of untreated domestic sewage along the western shore of the lagoon have led to eutrophication; nevertheless, this area is also very popular for fishing and recreation.1818 Seeliger U, Costa CSB. The Patos-Mirim basins, lagoons and estuary: natural and human forcing factors. LOICZ Rep Stud. 2002;21:105-112.
Sampling sites in Patos Lagoon, Brazil. Sampling site Barra (B), Saco da Mangueira (SM) and Museum (M) located at the estuary of Patos Lagoon, at Rio Grande city; city of São Lourenço do Sul (SL); city of Tapes (T) (adapted).1616 Yunes JS. Florações de Microcystis na Lagoa dos Patos e o seu estuário: 20 anos de estudos. Oecol Bras. 2009;13:313-318.
Surface water samples were collected four times from each sampling site between June 2007 and March 2008. The samples (4 L; at 0.3 m depth) were collected in sterile plastic bottles and stored at 4 °C in the dark until analysis. All samples were analyzed within 8 h after collection. Each water sample was diluted in 0.1% peptone water (10-1, 10-2, 10-3, 10-4, 10-5, 10-6) and filtered through a 0.45 µm mixed-ester filter; this filter was then placed onto m-Endo Les Agar (Himedia) and incubated at 44.5 °C for 24 h.1919 American Public Health Association (APHA). Standard Methods for the Examination of Water and Wastewater. Washington, DC: American Public Health Association; 1980. Pink colonies with a metallic sheen were inoculated onto Eosin Methylene Blue Agar (EMB, Himedia) and incubated at 37 °C for 24 h. After isolation of characteristic colonies, presumptive E. coli colonies were cryopreserved in Brain Heart Infusion Broth (BHI, Himedia) containing 15% glycerol and stored at -20 °C.
Isolates were identified using Gram staining and biochemical tests (indole test, methyl red test, Voges–Proskauer test, citrate test, triple sugar iron agar, motility test, urease test and growth on presence of D-sorbitol). Antimicrobial resistance patterns were determined by the agar diffusion method on Mueller-Hinton Agar (Oxoid, Basingstoke, UK) using the following 17 antimicrobial agents at concentrations indicated in parentheses: ampicillin (10 µg), amoxicillin/clavulanic acid (20 µg/10 µg), piperacillin/tazobactam (100 µg/10 µg), aztreonam (30 µg), imipenem (30 µg), cefoxitin (30 µg), ceftazidime (30 µg), cefotaxime (30 µg), cefepime (30 µg), amikacin (30 µg), gentamicin (30 µg), chloramphenicol (30 µg), tetracycline (30 µg), trimethoprim/sulfamethoxazole (1.25 µg/23.75 µg), norfloxacin (30 µg), sulfonamide (250 µg), streptomycin (10 µg) and spectinomycin (100 µg).2020 Clinical and Laboratory Standards Institute (CLSI). Performance Standard for Antimicrobial Susceptibility Testing Document Approved Standard M100-S24. Wayne, PA, USA; 2014. Isolates that showed resistance to at least three different classes of antimicrobials were considered multiresistant.11 Koczura R, Mokracka J, Jabłońska L, Gozdecka E, Kubek M, Kaznowski A. Antimicrobial resistance of integron-harboring Escherichia coli isolates from clinical samples, wastewater treatment plant and river water. Sci Total Environ. 2012;414:680-685. The presence of the class 1 integron was tested by PCR using primers for the integrase (intI1) gene2121 Sandvang D, Diggle M, Platt DJ. Translocation of integron-associated resistance in a natural system: acquisition of resistance determinants by Inc P and Inc W plasmids from Salmonella enterica Typhimurium DT104. Microb Drug Res. 2002;8:151-160. with PCR conditions were previously established.66 Spindler A, Otton LM, Fuentefria DB, Corção G. Beta-lactams resistance and presence of class 1 integron in Pseudomonas spp. isolated from untreated hospital effluents in Brazil. Antonie van Leeuwenhoek. 2012;102:73-81.
Of the isolates, 157 were resistant to at least one antimicrobial agent and were tested for the presence of intI1 gene. The intI1 gene was detected in 62 isolates, and 43 of these isolates that were resistant to two or more antimicrobials agents were selected for analyzing the variable region of the class 1 integron.
Evaluating genetic diversity of E. coli isolates using ERIC-PCR (Enterobacterial Repetitive Intergenic Consensus Sequence PCR)
DNA was extracted as described previously (Misbah et al., 2005)2222 Misbah S, Hassan H, Yusof MY, Hanifah YA, Abubakar S. Genomic species identification of Acinetobacter of clinical isolates by 16S rDNA sequencing. Singapore Med J. 2005;46:461-464. and the variable region was amplified using primers ERIC1 and ERIC2.2323 Versalovic J, Koeuth T, Lupski JR. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 1991;19:6823-6831. PCRs were performed in a volume of 25 µL containing 1× reaction buffer, 6.5 mM MgCl2, 125 µM dNTPs (Biolabs), 400 ng of each primer, 1 U Taq DNA polymerase (Invitrogen Life Technologies) and 7 µL of genomic DNA (approximately 100 ng). The amplification conditions were as follows: initial denaturation at 94 °C for 7 min, followed by 35 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 51 °C, and extension for 3 min at 72 °C, with a final extension of 15 min at 72 °C. In all ERIC-PCRs E. coli (ATCC 25922) was used as a positive control and also for standardization and evaluation of reproducibility.
The amplified products were separated on a 2% agarose gel in Tris–borate buffer for 4 h at 80 V. The gels were stained with ethidium bromide (0.5 µg/mL), visualized on a UV transilluminator and documented using the Kodak 1D image analysis software. The ERIC-PCR fingerprints were transformed onto a similarity matrix based on the presence or absence of each band. Only visible bands in the ERIC-PCR fingerprints were used to construct the similarity matrix and the dendrogram. Similarity among profiles was determined using the Jaccard coefficient and dendrograms were generated using the unweighted pair-group with mathematic average (UPGMA) method. All calculations were performed using the NTSYSPC version 2.0 software (Applied Biostatistics).
Amplification and sequencing of the variable region of the class 1 integron and amplification of the qacΔE1 gene
The variable region of the class 1 integron was amplified using previously established primers 5CS′/3CS′.99 Lévesque C, Piché L, Larose C, Roy PH. PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob Agents Chemother. 1995;39:185-191. The extraction of DNA was performed as previously described and PCRs were performed in a volume of 50 µL containing 2 µL genomic DNA (approximately 100 ng), 1× reaction buffer, 1 U of Taq DNA polymerase, 200 µM of each dNTP, 2.5 mM MgCl2 and 5 µM of each primer. PCR conditions were as follows: initial denaturation of 5 min at 94 °C, followed by 35 cycles of 1 min at 94 °C, 1 min at 50 °C, and 2 min at 72 °C, with a final extension of 10 min at 72 °C. E. coli ATCC 25922 and Pseudomonas aeruginosa blaVIM-1 were used as negative and positive controls, respectively. The PCR products were analyzed on a 1% agarose gel containing 0.5 µg/mL ethidium bromide, visualized with UV light and analyzed using image analysis Kodak 1D software (version 3.5.2). PCR product size was estimated using a 100 bp Ladder (New England Biolabs).
The PCR products were purified using the Invisorb Fragment CleanUp kit (Stratec Molecular, Berlin). DNA sequencing was performed on both strands using primers used for amplification using an ABI Prism Big Dye Terminator Cycle Sequencing Kit and analyzed on an ABI Prism 310 Genetic Analyzer (Applied Biosystems). The sequences were analyzed using BioEdit and aligned with GenBank data using the nucleotide Basic Local Alignment Search Tool (BLAST) software. A sequence was tagged as that of a gene if the similarity with GenBank sequence was at least 98%. Nucleotide sequences were deposited in the GenBank database under accession numbers KJ569553, KJ569554, KJ569555, KJ569556, KJ569557, KJ569558, KJ569559, KJ569560, KJ569561, KJ547712, KJ547713, KJ547714, KJ547715, KJ547716, KJ547717, KJ547718, KJ547719, KJ547720, KJ547721, KJ547722, KJ547723, KJ547724, KJ547725, KJ547726, KJ594081, KJ594082, KJ594083. The integron sequences were also registered within the INTEGRALL database (http://integrall.bio.ua.pt/).2424 Moura A, Soares M, Pereira C, Leitão N, Henriques I, Correia A. INTEGRALL: a database and search engine for integrons, integrases and gene cassettes. Bioinformatics. 2009;25:1096-1098.
The qacΔE1 gene was amplified using the primers 3′CSF2 TCAGGTCAAGTCTGCTT) and qacR (GGCACTGCATCCATGACG). The PCRs contained 1 U Taq DNA polymerase (Invitrogen), 1× reaction buffer, 2.5 mM MgCl2 (Invitrogen), 200 µM dNTP (Biolabs) and 1 µM of each primer in a total volume of 25 µL. Amplification conditions were denaturing at 94 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, 57 °C for 1 min, and 72 °C for 2 min, with a final extension step at 72 °C for 10 min. The PCR products were separated by gel electrophoresis on a 1% agarose gel in TAE buffer stained with ethidium bromide (0.5 µg/mL) and visualized using UV transillumination. The strains of E. coli ATCC 25922 and P. aeruginosa blaVIM-1 were used as negative and positive controls, respectively.
Evaluation of minimal inhibitory concentration (MIC) for quaternary ammonium compounds (QAC)
The MIC of QAC (10%, w/v, benzalkonium chloride, monoquaternary mixture of alkyldimethylbenzylammonium chloride) was determined by using the broth microdilution method2525 Penna TCV, Mazzola PG, Martins AMS. The efficacy of chemical agents in cleaning and disinfection programs. BMC Infect Dis. 2001;1:1-16. with a concentration range of 14.75–118 mg/L. The cultures at 105 cfu/mL were added to each well of a microtiter plate containing 300 µL Muller Hinton broth and different concentrations of QAC. Negative control used only Muller Hinton broth and positive control used broth with known bacterial inoculum. The assays were carried out in duplicate, for both samples and controls. The MIC was defined as the lowest concentration of the QAC at which complete inhibition of growth was observed at 24 h and under optimal incubation conditions. The isolates were considered QAC resistant when the MIC was ≥59 mg/L.2525 Penna TCV, Mazzola PG, Martins AMS. The efficacy of chemical agents in cleaning and disinfection programs. BMC Infect Dis. 2001;1:1-16. To facilitate visualization of MIC, 20 µL of a 1% solution of 2,3,5-triphenyl tetrazolium chloride (TTC) was added to each well of the microplate after the incubation period; E. coli ATCC 25922 was used as positive control.
Results
In a previous study, 518 colonies were isolated from m-Endo Les Agar and inoculated on Eosine Methylene Blue Agar resulting in 441 presumptive E. coli colonies.1515 Canal N [M.Sc. Dissertation. Instituto de Ciências Básicas da Saúde] Caracterização de Resistência a Antimicrobianos e Diversidade Genética em Escherichia coli Isolada de Amostras de Água da Lagoa dos Patos, RS. Porto Alegre, Brazil: UFRGS; 2010. The intI1 gene was detected in 62 isolates and 43 that were resistant to two or more antimicrobials agents were selected for variable region of the class 1 integron analyses. ERIC-PCR was performed to assess the genetic diversity of the E. coli isolates that carried the class 1 integron and to exclude clones.
The isolates showed great genetic diversity and a dendrogram was constructed based on 43 isolates which generated 19 groups with a similarity of 70%. Most clusters were represented by a single isolate, except two clusters which comprised 3 (cluster 10) and 23 isolates (cluster 15). The largest cluster, cluster 15, comprising 23 isolates with 100% similarity and identical resistance pattern (AmSxTeChSmSu) were all from the same sampling site (Museum). As these isolates were considered clones, only 7 isolates were randomly chosen for analysis of the integron variable region. Cluster 10 was the second largest group and consisted of three isolates with 71% similarity; all from the same sampling site (Tapes). However, these isolates had a variable antimicrobial susceptibility pattern even though they shared resistance to tetracycline, spectinomycin, sulfonamide and trimethoprim–sulfamethoxazole, apart from resistance to other antimicrobials (ampicillin, norfloxacin, chloramphenicol and amikacin). These isolates also presented distinct integron gene cassettes (Fig. 2; Table 1). Contrarily, isolates M83 and M85, although from the same sampling site and with identical resistance pattern (AmSxTeChSmSu) and integron gene cassette, were genetically distinct with less than 50% similarity to cluster 15 (Fig. 2; Table 1).
Dendrogram for the ERIC-PCR pattern of E. coli isolates resistant to antibiotics with class 1 integron from superficial water calculated using the Jaccard coefficient and UPGMA clustering. Isolates T – city of Tapes; SL – city of São Lourenço do Sul; B, SM, M – estuary of Patos Lagoon. The axis indicates the similarity index and the vertical line indicates the 70% similarity cut-off. *Asterisk indicates cluster 10.
Resistance pattern, gene cassettes, integron, MIC quaternary ammonium and ERIC-PCR group in E. coli isolates with a variable region class 1 integron from the surface water of Patos Lagoon, Brazil.
Among the 43 isolates of resistant E. coli studied here, the genetic content of the class 1 integron was analyzed in 27 isolates, of which 18 isolates were considered multiresistant. As shown in Table 1, the size of the variable region PCR product ranged between 0.570 kb and 1.850 kb and consisted of previously reported integron sequences such as In2, In54, In27, In377, In681 and In751. The isolate T357 and the seven isolates from cluster 15 did not display the complete sequence of the aadA1b gene cassette, and thus, these integrons could not be numbered, even if they were very likely to be identical to In377. A similar scenario occurred in isolates T337 and T334 which presented an incomplete sequence of the dfrA17 gene, and we hypothesize that this probably occurred due to an insufficient extension time (2 min) used during PCR.
Gene cassettes encoding the dihydrofolate reductase genes (dfrA1, dfrA12 and dfrA17), which confer resistance to trimethoprim, and those encoding aminoglycoside adenyltransferase genes (aadA1, aadA5 and aadA22) responsible for resistance to streptomycin/spectinomycin, were also found among the isolates (Table 1).
The MICs for QACs ranged between 118 mg/L and 29 mg/L. While most isolates were resistant to QAC (MIC ≥ 59 mg/L), 36 isolates showed an MIC of 59 mg/L, one isolate had an MIC of 118 mg/L and 6 isolates had an MIC of 29 mg/L; these 6 isolates were thus considered susceptible to QACs (Table 1). All the 43 isolates displayed the qacEΔ1 gene.
Discussion
ERIC-PCR analysis revealed a high degree of diversity among the E. coli isolates, probably due to the different sources of contamination draining into the lagoon. There was low overall similarity (<40%) and clustering, indicating that the isolates from the Tapes sampling site were genetically distinct from those of the Museum sampling site. Nevertheless, the presence of isolates with 100% similarity, identical resistance patterns and integron gene cassette sequence indicate that either a clonal population is being released into the aquatic environment from a common source or that these strains might be persistent at the sampling sites. The latter scenario seems possible because it has been reported that some E. coli strains can persist in secondary habitats such as riverbank soils and temperate soils.2626 Solo-Gabriele HM, Wolfert MA, Desmarais TR, Palmer CJ. Sources of Escherichia coli in a coastal subtropical environment. Appl Environ Microbiol. 2000;66:230-237.,2727 Gordon DM, Bauer S, Johnson JR. The genetic structure of Escherichia coli populations in primary and secondary habitats. Microbiology. 2002;148:1513-1522. Antimicrobial resistance attributable to the presence of integrons has been frequently reported in clinical E. coli isolates2828 Vinué L, Sáenz Y, Rojo-Bezares B, et al. Genetic environment of sul genes and characterization of integrons in Escherichia coli isolates of blood origin in a Spanish hospital. Int J Antimicrob Agents. 2010;35:492-496. and in environmental isolates.1212 Ozgumus OB, Sandalli C, Sevim A, Celik-Sevim E, Sivri N. Class 1 and class 2 integrons and plasmid-mediated antibiotic resistance in coliforms isolated from ten rivers in Northern Turkey. J Microbiol. 2009;47:19-27.,2929 Laroche E, Pawlak B, Berthe T, Skurnik D, Petit F. Occurrence of antibiotic resistance and class 1, 2 and 3 integrons in Escherichia coli isolated from a densely populated estuary (Seine, France). FEMS Microbiol Ecol. 2009;68:118-130. Integrons promote the spread of antimicrobial resistance, not only within a species but also between species and genera.3030 Mokracka J, Koczura R, Kaznowski A. Multi-resistant Enterobacteriaceae with class 1 and class 2 integrons in a municipal wastewater treatment plant. Water Res. 2012;46:3353-3363. However, the prevalence of antimicrobial resistance in aquatic environments has received relatively little attention.
The occurrence of antimicrobial-resistant bacteria in aquatic environments in Brazil has considerably increased in recent years. A study on a hospital wastewater treatment plant in Rio de Janeiro, Brazil, detected ESBL (Extended Spectrum Beta Lactamases) and KPC (Klebsiella pneumoniae carbapenemase) producing Enterobacteriaceae in both untreated and treated sewage.3131 Chagas TP, Seki LM, da Silva DM, Asensi MD. Occurrence of KPC-2-producing Klebsiella pneumoniae strains in hospital wastewater. J Hosp Infect. 2011;77:281. Recently, Enterobacteriaceae and Aeromonas spp. KPC-producing were recovered from the sewage of a tertiary teaching hospital and also from a municipal wastewater treatment plant located in São Paulo, the largest city in Brazil.3232 Picão RC, Cardoso JP, Campana EH, et al. The route of antimicrobial resistance from the hospital effluent to the environment: focus on the occurrence of KPC-producing Aeromonas spp. and Enterobacteriaceae in sewage. Diagn Microbiol Infect Dis. 2013;76:80-85. This report also showed that KPC-producing bacteria were present in the effluent of the wastewater treatment plant which was being discharged into an urban river.3232 Picão RC, Cardoso JP, Campana EH, et al. The route of antimicrobial resistance from the hospital effluent to the environment: focus on the occurrence of KPC-producing Aeromonas spp. and Enterobacteriaceae in sewage. Diagn Microbiol Infect Dis. 2013;76:80-85. Additionally, KPC-producing K. pneumoniae has also been recovered from other urban rivers in São Paulo.3333 Bonelli RR, Moreira BM, Picão RC. Antimicrobial resistance among Enterobacteriaceae in South America; history, current dissemination status and associated socioeconomic factors. Drug Resist Updat. 2014;17:24-36.,3434 Oliveira S, Moura RA, Silva KC, et al. Isolation of KPC-2-producing Klebsiella pneumoniae strains belonging to the high-risk multiresistant clonal complex 11 (ST437 and ST340) in urban rivers. J Antimicrob Chemother. 2013;69:849-852.
Coutinho et al. (2014)3535 Coutinho FH, Silveira CB, Pinto LH, et al. Antibiotic resistance is widespread in urban aquatic environments of Rio de Janeiro, Brazil. Microb Ecol. 2014;68:441-452. analyzed the diversity of antibiotic-resistant bacteria occurring in aquatic environments subjected to distinct degrees of anthropogenic effects in the city of Rio de Janeiro and detected microbial communities capable of tolerating antibiotic concentrations up to 600 times higher than the clinical dose. Potentially pathogenic species were among the resistant organisms detected and some of them were multiresistant. Basso et al. (2014)3636 Basso AP, Martins PD, Nachtigall G, Sand SV, Moura TM, Frazzon AP. Antibiotic resistance and enterotoxin genes in Staphylococcus sp. isolates from polluted water in Southern Brazil. An Acad Bras Cienc. 2014;86:1813-1820. report that polluted water from the Dilúvio stream (Porto Alegre, Southern Brazil) may act as a reservoir for the dissemination of antibiotic-resistance and enterotoxin producing Staphylococcus species into the community at large. Together, these studies demonstrate a high degree of antibiotic resistance among environmental bacteria in Brazil and provide evidence that pollution influences the observed diversity among drug-resistant bacteria in these aquatic habitats.3535 Coutinho FH, Silveira CB, Pinto LH, et al. Antibiotic resistance is widespread in urban aquatic environments of Rio de Janeiro, Brazil. Microb Ecol. 2014;68:441-452.
In the environment, bacteria may exchange genes through horizontal gene transfer, which is one of the main mechanisms used to spread resistance traits. Aquatic habitats that receive wastewater discharge are hotspots for horizontal gene transfer; and in such sites, distinct bacterial communities from several sources come together, thus creating an ideal environment for gene exchange.3535 Coutinho FH, Silveira CB, Pinto LH, et al. Antibiotic resistance is widespread in urban aquatic environments of Rio de Janeiro, Brazil. Microb Ecol. 2014;68:441-452.
In the present study, the variable region of the integrons showed little diversity and only genes encoding resistance to streptomycin, spectinomycin (aadA) and trimethoprim (dfr) were observed. Sequence analyses revealed the presence of six different integrons and In2 and In54 as most common. These two are also the more frequently found integrons among the E. coli strains in the INTEGRALL database, and are followed by In27, In377, and In751. Even though strains T258 and SM100 had an identical resistance pattern and showed integron PCR products that were very similar in size, sequence analysis revealed that they were very different integrons. The threshold used to distinguish between gene cassettes was a single amino-acid variation, thus two similar arrays of gene cassettes differing by only one amino acid would actually consist of different combinations of genes and are, therefore, assigned different In numbers. For example, while In54 designates reference dfrA17/reference aadA5, In751 indicates the dfrA17g allele/reference aadA5 (dfrA17g is reference dfrA17 with a Gly124Ala mutation).
Seven different gene cassettes were detected and eight strains showed a dfrA1b–aadA1b arrangement, while other eight displayed the aadA1 gene, and most of the isolates presented at least one kind of aadA cassette in the variable region (aadA1, aadA1b, aadA5 and aadA22), either alone or in combination with other cassettes. It is known that these two gene cassettes are commonly found in the integrons of E. coli isolated from various environments.33 Ferreira da Silva M, Vaz-Moreira I, Gonzalez-Pajuelo M, Nunes OC, Manaia CM. Antimicrobial resistance patterns in Enterobacteriaceae isolated from an urban wastewater treatment plant. FEMS Microbiol Ecol. 2007;60:166-176.,3737 Ribeiro AF, Laroche E, Hanin G, et al. Antibiotic-resistant Escherichia coli in karstic systems: a biological indicator of the origin of fecal contamination?. FEMS Microbiol Ecol. 2012;81:267-280.
38 Soufi L, Abbassi MS, Sáenz Y, et al. Prevalence and diversity of integrons and associated resistance genes in Escherichia coli isolates from poultry meat in Tunisia. Foodborne Pathog Dis. 2009;6:1067-1073.-3939 Vinué L, Sáenz Y, Somalo S, et al. Prevalence and diversity of integrons and associated resistance genes in fecal Escherichia coli isolates of healthy humans in Spain. J Antimicrob Chemother. 2008;62:934-937.
The variable region of the class 1 integron was analyzed in 27 E. coli isolates and all the isolates were resistant to sulfonamide while 22 isolates were resistant to trimethoprim–sulfamethoxazole. Among these, 18 isolates had the dfrA12, dfrA17, dfrA17g, dfrA1b or dfrA1f gene in the variable region, corroborating previous observations that class 1 integrons are important for the spread of resistance to trimethoprim–sulfamethoxazole.4040 Sukumaran DP, Durairaj S, Abdulla MH. Antibiotic resistance of Escherichia coli serotypes from Cochin estuary. Interdiscip Perspect Infect Dis. 2012;2012:124879. On the other hand, four isolates with trimethoprim–sulfamethoxazole resistance did not display the dfrA genes in the variable region of the integron. A possible reason for this observation is that the strains probably carry larger integrons which were not amplified by the PCR conditions used in this study or that the sulI gene was present in the 3′conserved segment of the integron; the latter would be a key factor in determining resistance to trimethoprim–sulfamethoxazole and sulfonamide in these E. coli isolates.
We also observed that genes not associated with resistance to β-lactam antibiotics (ampicillin, piperacillin–tazobactam, amoxicillin–clavulanate), or other antibiotics (amikacin, tetracycline, chloramphenicol and norfloxacin) were present in the variable region of the class 1 integrons analyzed. This finding implies that resistance pattern need not always be linked only to integrons carrying antibiotic-resistance gene cassettes, and that other mobile genetic elements such as plasmids and transposons may also carry resistance genes.2929 Laroche E, Pawlak B, Berthe T, Skurnik D, Petit F. Occurrence of antibiotic resistance and class 1, 2 and 3 integrons in Escherichia coli isolated from a densely populated estuary (Seine, France). FEMS Microbiol Ecol. 2009;68:118-130.,4141 Li D, Yu T, Zhang Y, et al. Antibiotic resistance characteristics of environmental bacteria from an oxytetracycline production wastewater treatment plant and the receiving river. Appl Environ Microbiol. 2010;76:3444-3451.,4242 Stalder T, Barraud O, Casellas M, Dagot C, Ploy MC. Integroninvolvement in environmental spread of antibiotic resistance. Front Microbiol. 2012;3:119. Plasmids carrying the ESBL genes are frequently conjugative and also carry determinants of resistance to non-beta-lactams such as tetracyclines, quinolones or aminoglycosides. Furthermore, we have previously observed that some of these isolates show a reduction in MIC for tetracycline and ampicillin in the presence of efflux pump inhibitors such as carbonyl-m-chlorofenilidrazone (unpublished data), reinforcing an idea that other mechanisms of antimicrobial resistance potentially collaborate to yield the phenotype of multidrug resistance.
Finally, a majority of isolates were resistant to QACs although they showed relatively low MIC, and all isolates displayed the qacEΔ1 gene. The qacEΔ1 gene is a typical component in the 3′-CS region of the class integron 1 and confers resistance to quaternary ammonium compounds; nevertheless class 1 integrons lacking this normal 3′-CS have also been reported.1010 Sáenz Y, Vinué L, Ruiz E, et al. Class 1 integrons lacking qacEΔ1 and sul1 genes in Escherichia coli isolates of food, animal and human origins. Vet Microbiol. 2010;144:493-497.,4343 Sunde M, Solheim H, Slettemeås JS. Genetic linkage between class 1 integrons with the dfrA12–orfF–aadA2 cassette array, and sul3 in Escherichia coli. Vet Microbiol. 2008;130:422-425. Quaternary ammonium compounds are commonly found in cationic biocide formulations; therefore, the use of various cationic biocides may also play an important but indirect role in the selection of these antibiotic-resistant bacteria.4444 Hegstad K, Langsrud S, Lunestad BT, Scheie AA, Sunde M, Yazdankhah SP. Does the wide use of quaternary ammonium compounds enhance the selection and spread of antimicrobial resistance and thus threaten our health?. Microb Drug Resist. 2010;16:91-104. Contrarily, six isolates carrying this gene were susceptible to QAC; it has been reported that resistance to quaternary ammonium compounds is strongly affected by the conditions under which bacteria are exposed to QACs and that the mere presence of the qacEΔ1 gene does not necessarily imply resistance.4545 Cervinkova D, Babak V, Marosevic D, Kubikova I, Jaglic Z. The role of the qacA gene in mediating resistance to quaternary ammonium compounds. Microb Drug Resist. 2013;19:160-167. For example, have demonstrated that the qacEΔ1 gene does not play an important role in resistance to benzalkonium chloride, a QAC, in P. aeruginosa.4646 Romao C, Miranda CA, Silva J, Clementino MM, de Filippis I, Asensi M. Presence of qacEΔ1 gene and susceptibility to a hospital biocide in clinical isolates of Pseudomonas aeruginosa resistant to antibiotics. Curr Microbiol. 2011;63:16-21. One explanation for this observation could lie in the fact that qac -mediated resistance targets a broad spectrum of distinct cationic compounds which in turn results in the exclusion of a high-level of specificity to certain QACs.4747 Jaglic Z, Cervinkova D. Genetic basis of resistance to quaternary ammonium compounds – the qac genes and their role: a review. Vet Med. 2012;57:275-281. Further, as mentioned above, it is also known that qac -mediated resistance could be affected by specific conditions under which the bacteria were exposed to the QACs.
In summary, this study characterized the variable region of the class 1 integron in antimicrobial-resistant E. coli strains isolated from an aquatic environment and compared their genetic profile to find antibiotic resistance patterns. It was found that the resistance pattern could not be entirely attributed to the majority of the genes present in the variable region. Nevertheless, integrons probably contribute to the spread of resistance and also possibly synergize with other mechanisms of resistance, thereby explaining the multiresistance patterns observed among the isolates. Environmental water habitats receive fecal contamination from different sources and are ideal environments for the dissemination of antimicrobial resistance among various species. The results of this study reflect the fact that the presence of the class 1 integron in fecal bacteria isolated from an aquatic environment can be considered a risk to public health as these bacteria are important reservoirs of antibiotic-resistance genes and can transfer these genetic determinants of resistance to other bacteria capable of infecting humans. The resistance to quaternary ammonium compounds is also of concern since these are often used in many biocidal formulations and may contribute to the selection of antibiotic-resistant bacteria in these environments.
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Associate Editor: Jorge Luiz Mello Sampaio
Acknowledgments
This study was supported by CAPES (Brazilian Government Supporting Agency). The authors would like to thanks Dr. Thomas Jové (INTEGRALL curator) who kindly provided the information about the integrons.
References
-
1Koczura R, Mokracka J, Jabłońska L, Gozdecka E, Kubek M, Kaznowski A. Antimicrobial resistance of integron-harboring Escherichia coli isolates from clinical samples, wastewater treatment plant and river water. Sci Total Environ 2012;414:680-685.
-
2Pereira A, Santos A, Tacão M, Alves A, Henriques I, Correia A. Genetic diversity and antimicrobial resistance of Escherichia coli from Tagus estuary (Portugal). Sci Total Environ.2013:461–462, 65–71.
-
3Ferreira da Silva M, Vaz-Moreira I, Gonzalez-Pajuelo M, Nunes OC, Manaia CM. Antimicrobial resistance patterns in Enterobacteriaceae isolated from an urban wastewater treatment plant. FEMS Microbiol Ecol 2007;60:166-176.
-
4Ferreira AE, Marchetti DP, De Oliveira LM, Gusatti CS, Fuentefria DB, Corção G. Presence of OXA-23-producing isolates of Acinetobacter baumannii in wastewater from hospitals in Southern Brazil. Microb Drug Resist 2011;17:221-227.
-
5Fuentefria DB, Ferreira AE, Corção G. Antibiotic-resistant Pseudomonas aeruginosa from hospital wastewater and superficial water: are they genetically related?. J Environ Manag 2011;92:250-255.
-
6Spindler A, Otton LM, Fuentefria DB, Corção G. Beta-lactams resistance and presence of class 1 integron in Pseudomonas spp. isolated from untreated hospital effluents in Brazil. Antonie van Leeuwenhoek 2012;102:73-81.
-
7Cambray G, Guerout AM, Mazel D. Integrons. Annu Rev Genet 2010;44:141-166.
-
8Koczura R, Mokracka J, Barczak A, Krysiak N, Kaznowski A. Association between the presence of class 1 integrons, virulence genes, and phylogenetic groups of Escherichia coli isolates from river water. Microb Ecol 2013;65:84-90.
-
9Lévesque C, Piché L, Larose C, Roy PH. PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob Agents Chemother 1995;39:185-191.
-
10Sáenz Y, Vinué L, Ruiz E, et al. Class 1 integrons lacking qacEΔ1 and sul1 genes in Escherichia coli isolates of food, animal and human origins. Vet Microbiol 2010;144:493-497.
-
11Costa PM, Loureiro L, Matos AJF. Transfer of multidrug-resistant bacteria between intermingled ecological niches: the interface between humans, animals and the environment. Int J Environ Res Public Health 2013;10:278-294.
-
12Ozgumus OB, Sandalli C, Sevim A, Celik-Sevim E, Sivri N. Class 1 and class 2 integrons and plasmid-mediated antibiotic resistance in coliforms isolated from ten rivers in Northern Turkey. J Microbiol 2009;47:19-27.
-
13Peak N, Knapp CW, Yang RK, et al. Abundance of six tetracycline resistance genes in wastewater lagoons at cattle feedlots with different antibiotic use strategies. Environ Microbiol 2007;9:143-151.
-
14Berthe T, Ratajczak M, Clermont O, Denamur E, Petit F. Evidence for coexistence of distinct Escherichia coli populations in various aquatic environments and their survival in estuary water. Appl Environ Microbiol 2013;79:4684-4693.
-
15Canal N [M.Sc. Dissertation. Instituto de Ciências Básicas da Saúde] Caracterização de Resistência a Antimicrobianos e Diversidade Genética em Escherichia coli Isolada de Amostras de Água da Lagoa dos Patos, RS Porto Alegre, Brazil: UFRGS; 2010.
-
16Yunes JS. Florações de Microcystis na Lagoa dos Patos e o seu estuário: 20 anos de estudos. Oecol Bras 2009;13:313-318.
-
17Möller OO, Castaing P, Salomon JC, Lazure P. The influence of local and non-local forcing effects on the subtidal circulation of Patos Lagoon. Estuaries 2001;24:297-311.
-
18Seeliger U, Costa CSB. The Patos-Mirim basins, lagoons and estuary: natural and human forcing factors. LOICZ Rep Stud 2002;21:105-112.
-
19American Public Health Association (APHA). Standard Methods for the Examination of Water and Wastewater Washington, DC: American Public Health Association; 1980.
-
20Clinical and Laboratory Standards Institute (CLSI). Performance Standard for Antimicrobial Susceptibility Testing Document Approved Standard M100-S24. Wayne, PA, USA; 2014.
-
21Sandvang D, Diggle M, Platt DJ. Translocation of integron-associated resistance in a natural system: acquisition of resistance determinants by Inc P and Inc W plasmids from Salmonella enterica Typhimurium DT104. Microb Drug Res 2002;8:151-160.
-
22Misbah S, Hassan H, Yusof MY, Hanifah YA, Abubakar S. Genomic species identification of Acinetobacter of clinical isolates by 16S rDNA sequencing. Singapore Med J 2005;46:461-464.
-
23Versalovic J, Koeuth T, Lupski JR. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res 1991;19:6823-6831.
-
24Moura A, Soares M, Pereira C, Leitão N, Henriques I, Correia A. INTEGRALL: a database and search engine for integrons, integrases and gene cassettes. Bioinformatics 2009;25:1096-1098.
-
25Penna TCV, Mazzola PG, Martins AMS. The efficacy of chemical agents in cleaning and disinfection programs. BMC Infect Dis 2001;1:1-16.
-
26Solo-Gabriele HM, Wolfert MA, Desmarais TR, Palmer CJ. Sources of Escherichia coli in a coastal subtropical environment. Appl Environ Microbiol 2000;66:230-237.
-
27Gordon DM, Bauer S, Johnson JR. The genetic structure of Escherichia coli populations in primary and secondary habitats. Microbiology 2002;148:1513-1522.
-
28Vinué L, Sáenz Y, Rojo-Bezares B, et al. Genetic environment of sul genes and characterization of integrons in Escherichia coli isolates of blood origin in a Spanish hospital. Int J Antimicrob Agents 2010;35:492-496.
-
29Laroche E, Pawlak B, Berthe T, Skurnik D, Petit F. Occurrence of antibiotic resistance and class 1, 2 and 3 integrons in Escherichia coli isolated from a densely populated estuary (Seine, France). FEMS Microbiol Ecol 2009;68:118-130.
-
30Mokracka J, Koczura R, Kaznowski A. Multi-resistant Enterobacteriaceae with class 1 and class 2 integrons in a municipal wastewater treatment plant. Water Res 2012;46:3353-3363.
-
31Chagas TP, Seki LM, da Silva DM, Asensi MD. Occurrence of KPC-2-producing Klebsiella pneumoniae strains in hospital wastewater. J Hosp Infect 2011;77:281.
-
32Picão RC, Cardoso JP, Campana EH, et al. The route of antimicrobial resistance from the hospital effluent to the environment: focus on the occurrence of KPC-producing Aeromonas spp. and Enterobacteriaceae in sewage. Diagn Microbiol Infect Dis 2013;76:80-85.
-
33Bonelli RR, Moreira BM, Picão RC. Antimicrobial resistance among Enterobacteriaceae in South America; history, current dissemination status and associated socioeconomic factors. Drug Resist Updat 2014;17:24-36.
-
34Oliveira S, Moura RA, Silva KC, et al. Isolation of KPC-2-producing Klebsiella pneumoniae strains belonging to the high-risk multiresistant clonal complex 11 (ST437 and ST340) in urban rivers. J Antimicrob Chemother 2013;69:849-852.
-
35Coutinho FH, Silveira CB, Pinto LH, et al. Antibiotic resistance is widespread in urban aquatic environments of Rio de Janeiro, Brazil. Microb Ecol 2014;68:441-452.
-
36Basso AP, Martins PD, Nachtigall G, Sand SV, Moura TM, Frazzon AP. Antibiotic resistance and enterotoxin genes in Staphylococcus sp. isolates from polluted water in Southern Brazil. An Acad Bras Cienc 2014;86:1813-1820.
-
37Ribeiro AF, Laroche E, Hanin G, et al. Antibiotic-resistant Escherichia coli in karstic systems: a biological indicator of the origin of fecal contamination?. FEMS Microbiol Ecol 2012;81:267-280.
-
38Soufi L, Abbassi MS, Sáenz Y, et al. Prevalence and diversity of integrons and associated resistance genes in Escherichia coli isolates from poultry meat in Tunisia. Foodborne Pathog Dis 2009;6:1067-1073.
-
39Vinué L, Sáenz Y, Somalo S, et al. Prevalence and diversity of integrons and associated resistance genes in fecal Escherichia coli isolates of healthy humans in Spain. J Antimicrob Chemother 2008;62:934-937.
-
40Sukumaran DP, Durairaj S, Abdulla MH. Antibiotic resistance of Escherichia coli serotypes from Cochin estuary. Interdiscip Perspect Infect Dis 2012;2012:124879.
-
41Li D, Yu T, Zhang Y, et al. Antibiotic resistance characteristics of environmental bacteria from an oxytetracycline production wastewater treatment plant and the receiving river. Appl Environ Microbiol 2010;76:3444-3451.
-
42Stalder T, Barraud O, Casellas M, Dagot C, Ploy MC. Integroninvolvement in environmental spread of antibiotic resistance. Front Microbiol 2012;3:119.
-
43Sunde M, Solheim H, Slettemeås JS. Genetic linkage between class 1 integrons with the dfrA12–orfF–aadA2 cassette array, and sul3 in Escherichia coli Vet Microbiol 2008;130:422-425.
-
44Hegstad K, Langsrud S, Lunestad BT, Scheie AA, Sunde M, Yazdankhah SP. Does the wide use of quaternary ammonium compounds enhance the selection and spread of antimicrobial resistance and thus threaten our health?. Microb Drug Resist 2010;16:91-104.
-
45Cervinkova D, Babak V, Marosevic D, Kubikova I, Jaglic Z. The role of the qacA gene in mediating resistance to quaternary ammonium compounds. Microb Drug Resist 2013;19:160-167.
-
46Romao C, Miranda CA, Silva J, Clementino MM, de Filippis I, Asensi M. Presence of qacEΔ1 gene and susceptibility to a hospital biocide in clinical isolates of Pseudomonas aeruginosa resistant to antibiotics. Curr Microbiol. 2011;63:16-21.
-
47Jaglic Z, Cervinkova D. Genetic basis of resistance to quaternary ammonium compounds – the qac genes and their role: a review. Vet Med 2012;57:275-281.
Publication Dates
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Publication in this collection
Apr-Jun 2016
History
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Received
26 Sept 2014 -
Accepted
17 Aug 2015