JAC Advance Access originally published online on October 2, 2007
Journal of Antimicrobial Chemotherapy 2007 60(6):1243-1250; doi:10.1093/jac/dkm340
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Prevalence and characterization of integrons from bacteria isolated from a slaughterhouse wastewater treatment plant
1 CESAM & Department of Biology, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal 2 IperÁgua, Lda—Tratamento de Águas e Águas Residuais, Apartado 52, S. Bernardo, 3811-601 Aveiro, Portugal
* Corresponding author. Tel: +351-234370970; Fax: +351-234426408; E-mail: amoura{at}ua.pt
Received 9 March 2007; returned 6 April 2007; revised 10 August 2007; accepted 13 August 2007
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Abstract |
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Objectives: To investigate the presence and distribution of integron-carrying bacteria from a slaughterhouse wastewater treatment plant (WWTP).
Methods: Enterobacteriaceae and aeromonads were isolated at different stages of the wastewater treatment process and screened for the presence of integrase genes by dot-blot hybridization. Integrase-positive strains were characterized in terms of phylogenetic affiliation, genetic content of integrons and antimicrobial resistance profiles. Plasmid location of some integrons was established by Southern-blot hybridization. Strains containing integron-carrying plasmids were selected for mating experiments.
Results: Integrase genes were present in all samples, including the final effluent. The global prevalence was determined to be 35%, higher than in other aquatic environments. Forty-two integrase-positive isolates were further characterized. Nine distinct cassette arrays were found, containing genes encoding resistance to ß-lactams (blaOXA-30), aminoglycosides (aadA1, aadA2, aadA13, aadB), streptothricin (sat1, sat2), trimethoprim (dfrA1, dfrA12), a putative esterase (estX) and a protein with unknown function (orfF). Gene cassette arrays aadA1, dfrAI-aadA1 and estX-sat2-aadA1 were common to aeromonads and Enterobacteriaceae. The class 2 integron containing an estX-sat2-aadA1 cassette array was detected for the first time in Aeromonas sp. Nearly 12% (5 out of 43) of intI genes were located in plasmids. intI genes from isolates MM.1.3 and MM.1.5 were successfully conjugated into Escherichia coli at frequencies of 3.79 x 10–5 and 5.46 x 10–5 per recipient cell, respectively.
Conclusions: Our data support the hypothesis that WWTPs constitute a potential hot spot for horizontal gene transfer and for selection of antimicrobial resistance genes among aquatic bacteria. Moreover, water discharges represent a possible risk for dissemination of undesirable genetic traits.
Keywords: horizontal gene transfer , integrons , Aeromonas , Enterobacteriaceae
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Introduction |
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Integrons are natural recombination systems that mediate the capture and expression of gene cassettes. Their basic structure consists of an integrase encoding gene (intI), a recombination site (attI) and a promoter that controls the expression of gene cassettes (if present). Integrons are classified into classes according to the integrase sequence.1,2 Currently, nucleotide sequences representing at least nine classes are reported in the GenBank database.3
Gene cassettes comprise an open reading frame and a recombination site (attC), necessary for integration.1,2 More than 80 gene cassettes have been so far described, most of them encoding antibiotic resistance genes.4,5
The frequent association of integrons with mobile genetic elements, such as transposons and conjugative plasmids, contributes to their dissemination due to horizontal gene transfer events.1,2 Previous studies have been performed in order to assess the prevalence of integrons in several natural environments. In estuaries, class 1 integrons were found in 3.6% of Gram-negative bacteria and incidences of 25% and 3.6% of integrons from classes 1 and 2, respectively, were reported in ampicillin-resistant Enterobacteriaceae and Aeromonas/Pseudomonas isolates.6,7 In irrigation water and sediments, incidences of 4.18% and 0.31%, respectively, for class 1 and 2 integrons were found among Escherichia coli isolates.8 Class 1 integrons were present in 3.8% of Gram-positive and Gram-negative isolates from environments polluted by quaternary ammonia compounds.9 As hypothesized, these environmental reservoirs of integron-carrying bacteria may be linked to run-offs from agriculture fields and/or sewage inputs.6–9
Wastewater treatment of industrial, agricultural and domestic discharges plays an essential role in addressing the worldwide problem of increasing water pollution. Wastewater treatment allows waters to be reused for irrigation in agriculture or released directly in aquatic environments. Sludge produced throughout the detoxification processes may also be used afterwards as a fertilizer.
The presence of antibiotic-resistant bacteria in effluents10–12 as well as high levels of antibiotic compounds in wastewater treatment plants (WWTPs)13 have been addressed in several studies, creating a growing concern about their impact on animal and human health. These conditions may exert selective pressure on sewage bacteria, providing the opportunity for further dissemination of undesirable genetic elements by horizontal gene transfer in these particular environments. However, little is known regarding the presence and dissemination of integrons in WWTPs.
The aim of this study was to investigate the presence and dissemination of integrons in a wastewater environment at different stages of the treatment process, in order to gather data pertaining to the contribution of these environments to horizontal gene transfer and spread of antibiotic resistance determinants.
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Materials and methods |
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Sampling and bacterial isolation
Sampling was performed in October 2005 in a WWTP located in Mirandela, Portugal, that receives wastewater exclusively from a slaughterhouse. Treatment consists of an activated sludge process and includes the following steps: mechanical screening, fat separation, equalization (homogenization), biological aeration, pH correction and precipitation. Sludge produced is dried and further used as a fertilizer in agriculture.
Water samples were collected from raw water (RW), homogeny tank (HT), aeration tank (AT), sludge recirculation (SR) and final effluent (FE). Biochemical oxygen demand, chemical oxygen demand, total suspended solids, volatile suspended solids, oils and pH were determined by standard methods.14
Serial decimal dilutions of water samples were prepared in 0.9% NaCl. Undiluted or diluted samples (10 mL) were filtered through 0.45 µm pore size cellulose sterile filters (Pall Life Sciences, MI, USA). Filters were placed onto agar plates selective for Aeromonas [glutamate starch phenol red (GSP) agar, Merck, Darmstadt, Germany] and Enterobacteriaceae (MacConkey agar, Merck, Darmstadt, Germany). Duplicate sets of plates were incubated for 24 h at 30°C and 37°C. All individual colonies used in further experiments were picked from the following dilutions: 10–3 and 10–2 in RW; 10–3 and 10–1 in HT; 10–3 and 10–1 in AT; 10–3 and 10–2 in SR and 10–2 and 10–1 in FE, for MacConkey and GSP agar, respectively.
Integron screening by dot-blot hybridization
Integrase genes intI1, intI2 and intI3 were amplified from genomic DNA of positive control strains [Salmonella enterica serovar Typhimurium (intI1+), Escherichia coli (intI2+) and Klebsiella pneumoniae (intI3+)] as described previously7,15 using the primer pairs listed in Table 1. The amplification products were labelled by incorporation of digoxygenin-11-dUTP (Roche Molecular Biochemicals, Indianapolis, IN, USA) during PCR. Isolates were screened by dot-blot hybridization. Bacterial colonies were picked from fresh Luria-Bertani agar plates and boiled in 20 µL of sterile distilled water for 15 min at 100°C. Cell suspensions were denatured with 0.5 M NaOH, incubated at 50°C for 5 min and equilibrated in 20 x SSC. Denatured DNA was transferred onto positively charged nylon membranes (Hybond N+; Amersham, Freiburg, Germany) using a Minifold I system (Schleicher and Schuell, Dassel, Germany) and subsequently cross-linked under UV irradiation for 5 min. Hybridizations were performed overnight in 50% formamide hybridization buffer at 42°C and detections were carried out using the DIG Nucleic Acid Detection Kit following instructions provided by the manufacturer. Positive and negative controls were included in all experiments to confirm the specificity of detection. Positive detections were further confirmed by PCR to exclude the presence of false-positive results.
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Molecular typing and phylogenetic affiliation of integrase-positive isolates
Molecular typing of integrase-positive isolates was performed by REP-PCR, using primers REP1R and REP2I (Table 1) to amplify repetitive extragenic palindromic sequences from 1 µL of cell suspension prepared in 100 µL of distilled water (turbidity of suspension equivalent to a 1.0 McFarland standard). The composition of reaction mixtures, the PCR program and gel analysis were as described previously.7
Isolates displaying different REP patterns were subsequently identified by 16S rRNA gene sequencing analysis. Amplification was performed with universal bacterial primers 27F and 1492R (Table 1), as described previously.21 PCR products were purified with Jetquick PCR Product Purification Spin Kit (Genomed, Löhne, Germany). Sequencing reactions were carried out using an ABI PRISM® BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Foster City, CA, USA). Samples were then analysed in an automatic DNA sequencer (ABI PRISM® 310 Genetic Analyser, PE Applied Biosystems). The sequence similarity search and phylogenetic affiliation were performed using the BLAST program.22
Integron characterization was carried out through PCR amplification with primer pairs targeting integron variable regions (Table 1), as described previously.18 PCR products with the same size were digested overnight with 1 U of HaeIII (MBI Fermentas, Vilnius, Lithuania) and 1 U of Sau3A1 (Roche Molecular Biochemicals) at 37°C. Products showing different sizes or different digestion profiles were used for subsequent sequence analysis of integron variable regions.
DNA extraction, plasmid isolation and integron genomic location
In order to determine the integron location, total genomic DNA and plasmid DNA were extracted and purified using the Genomic DNA Purification Kit (MBI Fermentas) and the EZNA Plasmid Mini Kit II (Omega Bio-tek, GA, USA), respectively, according to manufacturer’s instructions. Aliquots were loaded onto 0.8% agarose gels and separated by electrophoresis at 80 V for 75 min. Gels were then stained and documented with Molecular Imager FXTM System (Bio-Rad, Hercules, CA, USA). DNA was transferred to a nylon membrane (Hybond N+; Amersham) and hybridized with integrase probes, as described above.
Antibiotic susceptibility testing
Integrase-positive isolates were tested for antibiotic susceptibility by using the disc diffusion method according to CLSI (formerly NCCLS) recommendations.23
The following antimicrobials were tested: ampicillin (10 µg), aztreonam (30 µg), ceftazidime (30 µg), cefalotin (30 µg), ciprofloxacin (5 µg), chloramphenicol (30 µg), erythromycin (15 µg), gentamicin (10 µg), imipenem (10 µg), nalidixic acid (30 µg), streptomycin (10 µg), tetracycline (30 µg) and trimethoprim/sulfamethoxazole (25 µg) (Oxoid, Basingstoke, UK).
Integrase-positive strains containing integron-carrying plasmids (MM.1.3, MM.1.5, MM.2.2, MM.2.11) were included as donors in mating assays. Rifampicin-resistant E. coli CV601-GFP was used as recipient strain.24 Overnight cultures of donor and recipient strains were adjusted to an optical density of 0.6 at 600 nm. Recipient and donor strains were mixed (donor to recipient ratio of 1:1) in 0.9% NaCl solution and filtered through 0.45 µm pore size nitrocellulose filters. Filters were placed on TSA plates and incubated at 37°C overnight. Cells were washed off the filter by vortexing in 10 mL of 0.9% NaCl. Serial dilutions were prepared and aliquots of 100 µL were spread on TSA plates containing rifampicin (100 mg/L), kanamycin (30 mg/L) and streptomycin (50 mg/L). Assays were run in duplicate. Donor and recipient were also placed on the selective plates for mutant detection. Putative transconjugants were confirmed by REP-PCR and amplification of integrase genes as described above.
Nucleotide sequence accession numbers
Sequences of the 16S rRNA gene and integron gene cassette arrays were deposited in the GenBank public database under the accession numbers EF550539 [GenBank] –EF550580 [GenBank] and EU089665 [GenBank] –EU089673 [GenBank] , respectively.
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Sampling and bacterial isolation
FE concentrations were in conformity with the Portuguese emission standards. Waters were filtered through membranes and placed in selective media for Aeromonas and Enterobacteriaceae. A total of 286 bacterial isolates were obtained (158 on MacConkey agar and 128 on GSP agar) from each of the following treatment stages: RW (n = 61), HT (n = 41), AT (n = 35), SR (n = 55) and FE (n = 94). Bacterial isolates were further screened for the presence of integrons.
Screening of integrons and phylogenetic affiliation of integrase-positive isolates
Figure 1 represents the prevalence of integron-positive isolates along the wastewater treatment process. IntI1-carrying bacteria corresponded to 30.7% (88 out of 286) of the isolates and were detected in all stages of the wastewater treatment process, being more abundant in the FE. The intI2 gene was detected in 4.5% (13 out of 286) of the bacterial isolates, being present only in RW, AT and FE. No positive detection was obtained for intI3. One isolate (MM.2.1, from RW) possessed simultaneously a class 1 and a class 2 integrase.
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Forty-two different REP-PCR profiles out of 100 integrase-positive isolates were obtained. Subsequent sequencing of the 16S rRNA gene allowed us to affiliate integrase-positive strains to Aeromonas sp. (69%), isolated from all samples except RW, Escherichia sp. (28.6%), isolated from all samples except AT, and Morganella morganii (2.4%), only found in RW (Table 2).
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Detection and characterization of integron gene cassettes
The genetic content of 43 integrons was determined through PCR amplification of integron variable regions. Results obtained are summarized in Table 2. PCR products were obtained for 25 intI1-positive strains and 5 intI2-positive strains, ranging from 
1–2.5 kb. No amplification products were detected in 31% (13 out of 42) of the strains. Digestion of PCR products with restriction enzymes (HaeIII and Sau3A1) allowed the identification of seven distinct cassette arrays in class 1 integrons and two in integrons belonging to class 2. As determined by subsequent sequencing analysis, all cassette arrays contained genes encoding for aminoglycoside adenyltransferases conferring resistance to streptomycin and spectinomycin (aadA1, aadA2 and aadA13), alone or in combination with other resistance genes. Genes encoding dihydrofolate reductases (dfrA1 and dfrA12), conferring resistance to trimethoprim, were also frequently (46.5%) found in intI-positive isolates and always together with an aadA-type gene. Cassettes encoding streptothricin acetyltransferases (sat1, sat2), a ß-lactamase (blaOXA-30), a putative esterase (estX) and a protein with unknown function (orfF) were also detected (Table 2). Common gene cassette arrays were found in both Aeromonas sp. and Enterobacteriaceae (e.g. dfrA1-aadA1 in class 1 integrons and estX-sat2-aadA1 in class 2 integrons).
Most integrons (38) were exclusively located in chromosomal DNA, whereas 2 integrons (in isolates MM.1.5 and MM.2.11) were exclusively located in plasmid DNA. Three isolates (MM.1.3, MM.2.2 and MM.2.6) gave positive hybridization signals in both chromosomal and plasmid DNA. In isolates MM.1.3 and MM.2.2, amplification of integron variable regions and subsequent digestion with restriction enzymes detected only one type of gene cassette array present, indicating the presence of at least two copies of those integrons.
The intI-positive plasmids of isolates MM.1.3 (Aeromonas sp.) and MM.1.5 (E. coli), both carrying dfrA1-aadA1 gene cassette arrays, were successfully transferred into E. coli CV601 at transfer frequencies of 3.79 x 10–5 and 5.46 x 10–5 transconjugants/recipient, respectively. No transconjugants were obtained from experiments using MM.2.2 (E. coli) and MM.2.11 (E. coli) as donor strains, although the detection limit was 7.59 x 10–7 transconjugants/recipient.
Antibiotic susceptibility testing
The antimicrobial agents used for susceptibility testing were chosen to cover different antibiotic groups and/or based on resistance genes associated with integrons.4,25 Nearly 50% of integrase-positive strains (20 out of 42) showed a pattern of multiresistance to five or more antibiotics (Table 2). Among Aeromonas sp., the most frequent resistances were to ampicillin (100%), cefalotin (76%), streptomycin (69%), tetracycline (48%) and trimethoprim/sulfamethoxazole (45%), whereas none of the isolates showed resistance to aztreonam, ciprofloxacin or nalidixic acid. Among Enterobacteriaceae, there was a higher resistance to erythromycin (100%), tetracycline (92%) and streptomycin (85%); no isolates were found to be resistant to aztreonam, ceftazidime, ciprofloxacin or imipenem (Figure 2).
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Discussion |
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In this study, the prevalence of integrons was investigated in a collection of bacterial isolates from a WWTP receiving waters from a slaughterhouse. Two bacterial groups were selected to represent aquatic naturally occurring bacteria (Aeromonas) and bacteria mainly introduced due to human activity (Enterobacteriaceae).
The prevalence of classes 1 and 2 integrons among Aeromonas sp. and Enterobacteriaceae isolates in the WWTP of Mirandela was found to be of 30.7% and 4.5%, respectively. IntI3 genes, originally identified in clinical Serratia marcescens and Klebsiella pneumoniae,15,26 were not detected among the wastewater isolates included in the present study. The global prevalence of integron-carrying bacteria was 35%, a value superior to those usually reported for other types of aquatic environments.6–9
Integrase genes were detected in all wastewater samples. The relative presence of integrons among Enterobacteriaceae and Aeromonas sp. was expected to be higher at earlier stages of the treatment process (i.e. in RW, HT and AT), due to higher selective pressures, such as lower pH and higher levels of oils and greases, and higher abundance of microorganisms at these stages. However, this was true only for class 2 integrons. Isolates harbouring class 1 integrons were more prevalent in the FE, drawing attention to the risk of wastewaters to act as a proper place for horizontal gene transfer, contributing to the spread of harmful genetic determinants in natural environments.
Integrase-positive bacteria were further characterized in terms of phylogenetic affiliation, gene cassette content and antibiotic susceptibility (Table 2). No amplification product was obtained in 31% of the integrase-positive isolates, probably due to the lack of a 3'-conserved region. Fifty-eight per cent (25 out of 43) of class 1 and 11.6% (5 out of 43) of class 2 integrons harboured at least one gene cassette. Nine arrays were detected containing one to three cassettes, out of which seven distinct arrays were present in integrons of class 1 and two arrays in class 2 integrons. Common gene cassette arrays were detected in both Aeromonas sp. and Enterobacteriaceae isolates. By comparison to other sequences deposited in databases, the following gene cassettes were identified: streptomycin and spectinomycin resistance genes (aadA1, aadA2 and aadA13), trimethoprim resistance genes (dfrA1, dfrA12), streptothricin resistance genes (sat1, sat2), a ß-lactam resistance gene (blaOXA-30), a putative esterase (estX) and an orfF encoding a protein with unknown function. Although aminoglycosides and trimethoprim were often used in the past years to prevent diarrhoea in calves,27 their use is now limited. Despite this, the array dfrA1-aadA1 in class 1 integrons was found along the entire process, being the most common array. Previous studies have reported aadA- and dfr-like genes as frequently found gene cassettes both in clinical28,29 and environmental7,8,30 isolates. Additionally, the dfrA1-aadA1 array is thought to be considerably stable and that its transfer occurs by the mobilization of the complete integron structure in larger mobile elements, such as transposons or plasmids, rather than individual resistance gene cassettes.31
Integron location within the bacterial genomes was determined by plasmid and chromosomal DNA hybridizations with intI probes. Most integrases were located on bacterial chromosomes. The total prevalence of integrases carried in plasmids (11.9%) was similar to that previously reported in wastewaters.32 Nevertheless, we are aware that the prevalence obtained may be biased due to the fact that plasmid and chromosomal DNA can display the same electrophoretic mobility.
Isolates with a putative location of integrons on plasmids were included as donors in mating assays using E. coli CV601 as recipient strain. The dfrA1-aadA1 gene cassette arrays were successfully transferred using isolates MM.1.3 (Aeromonas sp.) and MM.1.5 (E. coli) as donor cells, which could explain the high prevalence of this array in the WWTP of Mirandela. No transconjugants were obtained using isolate MM.2.11 (E. coli), harbouring the array estX-sat2-aadA1, as donor. However, the presence of the estX-sat2-aadA1 array in Aeromonas sp. due to horizontal gene transfer is not excluded. Class 2 integron variable regions were amplified using primers that bind to attI2 and to orfX, situated downstream of the cassette region within transposon Tn7.19 Therefore, the presence of such an integron in Aeromonas sp. may have had its origin in a transposition event. To the best of our knowledge, this is the first report of a gene cassette array detected in class 2 integrons from Aeromonas sp.
Antimicrobial resistance patterns revealed that 47.6% of the integrase-positive strains were multiresistant (i.e. resistant to five or more antibiotics). Previously, classes 1 and 2 integrons were reported to be present together in 10% of E. coli isolates, being most prevalent among strains with resistance profiles to five or more antibiotics.17 Although 46% and 48% of Enterobacteriaceae and Aeromonas sp., respectively, displayed a multiresistance pattern to five or more antibiotics, only one E. coli strain possessed simultaneously intI1 and intI2 genes. The gene cassettes found in this study did not explain the totality of resistance phenotype observed, suggesting the probable existence of other resistance mechanisms encoded in the chromosome and/or in other mobile DNA elements not detected in this study.
Isolates MM.1.10 and MM.1.11 exhibited only reduced susceptibility to aminoglycosides, although they possessed aadA gene cassettes in their integron structure. The lack of resistance to these antibiotics may be due to differences in the promoter sequence that can cause weakness in expression of the gene cassette arrays, as discussed elsewhere.31
The use of selective media clearly limits the diversity of wastewater bacteria obtained and leads consequently to an underestimation of true diversity of integrons present in wastewaters. Nevertheless, this approach provided us with valuable data regarding the taxonomic affiliation, antibiotic resistance phenotype and gene cassette arrays of integron-carrying bacteria accessible to cultivation among two important bacterial groups.
WWTPs constitute potential hot spots for horizontal gene transfer and may represent a major concern regarding the dissemination of undesirable genetic traits in the environment. The results obtained in this study strongly support this hypothesis. Additionally, this study highlights the need to control antibiotic-resistant bacteria in treated effluents, in order to avoid the risk of spreading of harmful genetic determinants through discharges in aquatic ecosystems.
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This work was supported by Fundação para a Ciência e a Tecnologia (POCTI/BME/45881/2002) and in the form of grants to A. M. (SFRH/BD/19443/2004) and I. H. (SFRH/BPD/21384/2005).
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None to declare.
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Acknowledgements |
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We wish to thank John Maurer and Marie Maier (University of Georgia, Greece), Séamus Fanning (University College Dublin, Ireland), Sónia Ferreira and Sónia Mendo (University of Aveiro, Portugal) and Kornelia Smalla (Institute for Plant Virology, Germany) for providing the integrase-positive control strains and recipient strains. We are also grateful to Artur Alves (University of Aveiro, Portugal) for assistance with phylogenetic analyses and Ana Pamplona (Instituto de Medicina Molecular and Instituto Gulbenkian de Ciência, Portugal) for reviewing the manuscript prior to submission.
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References |
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