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Detection of resistance genes and evaluation of water quality at zoo lakes in Brazil

Detecção de genes de resistência e qualidade da água dos lagos de zoológico, Brasil

ABSTRACT:

The investigation of the presence of antibiotic-resistance genes in aquatic environments is important to identify possible reservoirs of resistant microorganisms that could be a threat to human and animal health. The aims of this study were to analyze the presence of genes conferring resistance to antimicrobials in the aquatic environment and to assess the quality of water in zoo lakes. Results showed a pattern of genes conferring resistance to multiple antibiotics and turbidity, which was expected to be due to the presence of contaminants. The most frequent genes were sul I and sul II (sulfonamides), which were present in all the lakes, followed by genes encoding β-lactamases such as blaPSE I (77.8%) and ampC (66.7%). However, tet(K), tet(M), and ermC genes were not detected. There was a positive correlation between the number of Enterobacteriaceae and resistance genes. In conclusion, the source of contamination of all lakes was probably the neighboring urban sewage or wastewater that increased the frequency of the total coliforms and resistance genes, which in turn posed a threat to the conservation of the animal life inhabiting the zoo.

Key words:
multidrug resistance; zoo pollution; aquatic environment; Enterobacteriaceae

RESUMO:

A investigação da presença de genes de resistência a antibióticos (ARGs) em ambientes aquáticos é importante para identificação de possíveis reservatórios de microrganismos resistentes que podem ser uma ameaça para a saúde humana e animal. O objetivo deste estudo foi analisar a presença de genes de resistência a antimicrobianos e a qualidade da água de zoológico. Os resultados mostraram um padrão de genes de resistência a múltiplos antibióticos e turbidez da água, devido à presença de contaminantes. Os genes mais frequentes foram sul I e sul II (sulfonamidas) que estão presentes em todos os lagos, e β-lactamases como blaPSE I (77,8%) e ampC (66,7%). Os genes tet(K), tet(M) e ermC não foram detectados. Houve correlação entre o número deEnterobacteriaceae e aumento na detecção de genes de resistência. As fontes de contaminação dos lagos são, provavelmente, esgoto urbano vizinho ou de águas residuais que aumentam a presença de coliformes totais e a frequência dos genes de resistência, que podem ser um risco para vida de animais silvestres em cativeiro.

Palavras-chave:
resistência múltipla; poluição em zoo; ambiente aquático; Enterobacteriaceae

INTRODUCTION:

Water is one of the most important natural resource as it is indispensable for the survival of animal species (DUARTE, 2011DUARTE P.B. Microrganismos indicadores de poluição fecal em recursos hídricos - MG. 2011. 51f. Monografia (Especialização em Microbiologia) - Curso de Pós-graduação em Microbiologia, Universidade Federal de Minas Gerais, Belo Horizonte. ). It is considered as a critical factor for the successful performance and production of livestock, affecting various processes including the health status of animals (BARROS et al., 2010BARROS M.S. et al. Análise da Qualidade da Água em Estabelecimentos Leiteiros Associados da Cooperativa Agropecuária Batavo. 2010. Carambeí, PR. Available from: <Available from: http://www.emater.pr.gov.br/arquivos/File/Biblioteca_Virtual/Premio_Extensao_Rural/2_Premio_ER/05_Anal_Agua_Est_Leiteiros.pdf >. Accessed: 12 Sep. 2014.
http://www.emater.pr.gov.br/arquivos/Fil...
). Urban runoff water often has a poor quality and consequently may serve as a vehicle for several antibiotic-resistant microorganisms (GÕNI-URRIZA et al., 2000GÕNI-URRIZA, M. Impact of urban effluent on antibiotic resistance of riverine Enterobacteriaceae and Aeromonas sp. Applied Environmental Microbiology, v.66, n.1, p.125-132, Jan. 2000. Available from: <Available from: http://aem.asm.org/content/66/1/125.full.pdf+html >. Accessed: Dec. 20, 2014. doi: 10.1128/AEM.66.1.125-132.2000.
http://aem.asm.org/content/66/1/125.full...
; DUARTE, 2011DUARTE P.B. Microrganismos indicadores de poluição fecal em recursos hídricos - MG. 2011. 51f. Monografia (Especialização em Microbiologia) - Curso de Pós-graduação em Microbiologia, Universidade Federal de Minas Gerais, Belo Horizonte. ).

Pollution in urban zoos usually comes from small point sources (ZHAO et al., 2007ZHAO, J.W. et al. Pollutant loads of surface runoff inWuhan City Zoo, an urban tourist area. Journal of Environmental Sciences, v.19, n.4, p.464-468, May. 2007. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S1001074207600771 >. Accessed: Dec. 01, 2014. doi:10.1016/S1001-0742(07)60077-1.
http://www.sciencedirect.com/science/art...
). These sources, such as wastewater or untreated sewage, can have a profound effect on the ecological health of streams and reservoirs (DELETIC, 1998 DELETICA. The first flush load of urban surface runoff. Water Research, v.32, n.8, p.2462-2470, Aug. 1998. Available from: <Available from: https://www.researchgate.net/publication/223024586_The_First_Flush_Load_of_Urban_Surface_Runoff >. Accessed: Nov. 20, 2015. doi: 10.1016/S0043-1354(97)00470-3.
https://www.researchgate.net/publication...
; SCHREIBER et al., 2001SCHREIBER J.D. et al. Dynamics of diffuse pollution from us southern catchments., Water Researchv.35, n.10, p.2534-2542. Aug. 2001. Available from: <Available from: https://www.researchgate.net/publication/11944325_Dynamics_of_diffuse_pollution_from_US_southern_watersheds >. Accessed: Nov. 20, 2015. doi: 10.1016/S0043-1354(00)00510-8.
https://www.researchgate.net/publication...
). Several studies have reported pollution due to runoff from urban-residential and industry-dominated areas (GROMAIRE-MERTZ et al., 1999GROMAIRE-MERTZ M.C. et al. Characterization of urban runoff pollution in Paris., Water Science Technologyv.39, p.1-8. Apr. 1999. Available from: <Available from: http://wst.iwaponline.com/content/70/3/397 >. Accessed: May. 01, 2015. doi:10.1016/S0273-1223(99)00002-5.
http://wst.iwaponline.com/content/70/3/3...
; LEE & BANG., 2000LEE J.H., BANG K.W. Characterization of urban stormwater runoff. Water research, v.34, n.6, p.1773-1780. Apr. 2000. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0043135499003255 >. Accessed: May 01, 2015. doi:10.1016/S0043-1354(99)00325-5.
http://www.sciencedirect.com/science/art...
; CHOE et al., 2002CHOE J.S. et al. Characterization of urban runoff in urban areas. Water Science Technology, v.45, n.9, p. 249-254, May. 2002. Available from: <Available from: http://wst.iwaponline.com/content/45/9/249.full.pdf >. Accessed: 23 May. 2015.
http://wst.iwaponline.com/content/45/9/2...
). Considering the paucity of data regarding antimicrobial resistance in the aquatic environment and zoos in Brazil as well as throughout the world, this study was aimed to investigate the presence of resistance genes in order to identify the possible reservoirs of resistant microorganisms.

MATERIALS AND METHODS:

The study was conducted at the Zoo of Universidade Federal do Mato Grosso (UFMT), located in the city of Cuiabá-MT, Brazil (near residential neighborhoods), which has an area of 11 hectares and is inhabited by approximately 500 animals of 80 different species including birds, reptiles, and mammals.

From November 2013 to April 2014, water samples from nine different points of the zoo were collected as follows: stream before Jaguar's enclosure (P1); stream after the Jaguar's enclosure (P2); Big Lake (P3); Nesting Alligator (P4); monkey's lake (P5); tortoise's enclosure (P6); Albino Alligator's Lake (P7); seasonal spring stream (P8) and Tapir's lake (P9) (Figure 1).

Figure 1
Stream before Jaguar's enclosure (P1); stream after the Jaguar's enclosure (P2); Big Lake (P3); Nesting Alligator (P4); monkey's lake (P5); tortoise's enclosure (P6); Albino Alligator's Lake (P7); seasonal spring stream (P8) and Tapir's lake (P9).

For the detection of resistance genes, approximately 1-L water samples were collected in sterile plastic bags. The samples were filtered in a vacuum pump system (Sigma-AldrichTM) through a polyethersulfone membrane of 0.2μm pore size (PallTM), with a maximum interval of 3 hours between sample collection and processing.

The filters were then washed in sterile buffered water, cut into several pieces, placed in polystyrene tubes and resuspended in 1.8mL of SET solution (20% sucrose, 50mM EDTA and 50mM Tris-HCl [pH 8.0]) and subsequently frozen (SOMERVILLE et al., 1989SOMERVILLE, C.C. et al. Simple, rapid method for direct isolation of nucleic acids from aquatic environments., Applied Environmental Microbiology v.55, n.3, p.548-554, Mar. 1989. Available from: <Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC184158 >. Accessed: Aug. 10, 2104.
http://www.ncbi.nlm.nih.gov/pmc/articles...
). The DNA was extracted using 360µL of lysozyme buffer (5mg mL-1, 1mM EDTA, 10mM NaCl) and incubated at 37°C for 1h. Sodium dodecyl sulfate (SDS 1.0%) and 25µL of proteinase K (20mg mL-1) were subsequently added and the samples were further incubated at 37°C for 2h with horizontal shaking at 150rpm. Finally, 280µL of CTAB (cetyltrimethylammonium bromide 10%) was added, followed by incubation at 65°C for 20min.

DNA was purified using phenol-chloroform-isoamyl alcohol and precipitated using isopropanol, washed with 70% ethanol and resuspended in ultrapure 200µL water (SOMERVILLE et al., 1989SOMERVILLE, C.C. et al. Simple, rapid method for direct isolation of nucleic acids from aquatic environments., Applied Environmental Microbiology v.55, n.3, p.548-554, Mar. 1989. Available from: <Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC184158 >. Accessed: Aug. 10, 2104.
http://www.ncbi.nlm.nih.gov/pmc/articles...
; RIVIERA et al., 2003RIVERA, I.N.G. et al. Method of DNA extraction and application of multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae O1 and O139 from aquatic ecosystems. Environmental Microbiology, v.5, n.7, p.599-606, Jun. 2003. Available from: <Available from: http://onlinelibrary.wiley.com/doi/10.1046/j.1462-2920.2003.00443.x/pdf. >. Accessed: Oct. 11, 2014. doi: 10.1046/j.1462-2920.2003.00443.x.
http://onlinelibrary.wiley.com/doi/10.10...
).

The detection of 16 resistance genes was performed using the Polymerase Chain Reaction (PCR) in a final volume of 25μL, along with a positive control, and these genes were confirmed by DNA sequencing. The primer sequences of the antimicrobial resistance genes are listed in table 1. The amplification products were analyzed by electrophoresis on 2% agarose gel (InvitrogenTM) at 5 volts per cm, stained with Gel Red(tm) (Biotium(r)) and observed under ChemiDoc XRS(tm) system using ImageLab(tm) software. We used a standard molecular weight marker consisting of 100 bases pairs (Fermentas(r)).

Table 1
Sequence of oligonucleotides used for amplification of resistance genes.

Approximately 2L of water from P3, P4, P5, P6, P7, P8, and P9 was collected in individual bottles and sent for microbiological and physicochemical analyses, following the methodology of the American Public Health Association (RICE et al., 2012RICE, E.W. et al. Standard methods for the enumeration of water and wastewater. 2.ed. Washington, D.C.: American Public Health Association, 2012. 1496p.). Local data to assess the type of environment, communication between the enclosures, presence, and type of contaminants, and occupation density were collected according to the instructions of IBAMA 169/2008 (BRASIL, 2008BRASIL. Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis - IBAMA - Instrução Normativa n. 169, 20 de fevereiro de 2008. Brasília: Ministério do Meio Ambiente 11p. Available from: <Available from: http://www.mma.gov.br/port/conama/legiabre.cfm?codlegi=585 >. Online. Accessed: Aug. 20, 2014.
http://www.mma.gov.br/port/conama/legiab...
). Statistical analysis was performed using software R version 3.0.1. (FOX, 2005FOX, J. The R commander: a basic statistics graphical user interface to R. Journal of Statistical Software, v.14, n.9, p.1-42. 2005. Available from: <Available from: http://www.jstatsoft.org/v14/i09/paper >. Accessed: Nov. 12, 2014.
http://www.jstatsoft.org/v14/i09/paper...
; R DEVELOPMENT CORE TEAM, 2011R DEVELOPMENT CORE TEAM. R: a language and environment for statistical computing. R Foundation for Statistical Computing. Vienna, Austria, 2011. ISBN 3-900051-07-0. Statistical software found on the website: < Statistical software found on the website: http://www.R-project.org/ >. Accessed: Nov. 12, 2014.
http://www.R-project.org/...
). Associations between the resistance and other variables were calculated using a binomial regression between genes, microbiological examination, and physical and chemical examination. Spearman correlation coefficient was used to assess the association between microbiological analysis (total and thermotolerant coliforms) and the presence of resistance genes. In all cases, p-value ≤0.05 was defined as significant.

RESULTS AND DISCUSSION:

The results of the microbiological analysis of the total and thermotolerant coliforms revealed high counts of Enterobacteriaceae in all sampling sites (Table 2), with P5 showing the lowest and P6 showing the highest counts. There was a positive correlation between total microbial (r=0.763, P=0.0457) as well as thermotolerant (r=0.807, P=0.0281) counts and increased numbers of detected antibiotic-resistance genes. Previously it was shown that the discharge of urban waste resulted in an increase in resistant strains and antibiotic-resistance genes, and that these discharges were usually derived from domestic sewage (GÕNI-URRIZA et al., 2000GÕNI-URRIZA, M. Impact of urban effluent on antibiotic resistance of riverine Enterobacteriaceae and Aeromonas sp. Applied Environmental Microbiology, v.66, n.1, p.125-132, Jan. 2000. Available from: <Available from: http://aem.asm.org/content/66/1/125.full.pdf+html >. Accessed: Dec. 20, 2014. doi: 10.1128/AEM.66.1.125-132.2000.
http://aem.asm.org/content/66/1/125.full...
; STOLL et al., 2012STOLL, C. et al. Prevalence of clinically relevant antibiotic resistance genes in surface water Samples collected from Germany and Australia. Environmental Science Technology, v.46, n.17, p.9716-9726, 2012. Available from: <Available from: http://pubs.acs.org/doi/full/10.1021/es302020s >. Accessed: Jul. 20, 2015. doi: 10.1021/es302020s.
http://pubs.acs.org/doi/full/10.1021/es3...
).

Tabela 2
Microbiological, physicochemical and epidemiological characteristics in water samples from collection points of UFMT Zoo, November /2013 - April /2014.

During the physicochemical analyses (Table 2), changes were observed in turbidity in P3 (90.1NTU), and in OD (Oxygen Demand) (0.2mg L-1) and BOD (Biological Oxygen Demand) (6.4mg L-1) in P6, based on CONAMA Resolution 357/05 (BRASIL, 2005BRASIL. Conselho Nacional do Meio Ambiente - CONAMA - Resolução n. 357, de 17 março 2005. Brasília, 17 de março de 2005, p.23. Available from: <Available from: http://www.mma.gov.br/port/conama/legiabre.cfm?codlegi=459 >. Online. Accessed: Aug. 20. 2014.
http://www.mma.gov.br/port/conama/legiab...
). High turbidity may indicate an excessive amount of microorganisms or organic matter. Moreover, at point P3, the water surface was covered by aquatic vegetation and was associated with a slight change in OD. Although this site has not yet fully undergone eutrophication, care is needed so that the local situation does not get worse. In a similar previous study, changes in OD and BOD were reported at some sites, especially where there was a discharge of wastewater (GÕNI-URRIZA et al., 2000GÕNI-URRIZA, M. Impact of urban effluent on antibiotic resistance of riverine Enterobacteriaceae and Aeromonas sp. Applied Environmental Microbiology, v.66, n.1, p.125-132, Jan. 2000. Available from: <Available from: http://aem.asm.org/content/66/1/125.full.pdf+html >. Accessed: Dec. 20, 2014. doi: 10.1128/AEM.66.1.125-132.2000.
http://aem.asm.org/content/66/1/125.full...
).

All the nine samples tested were positive for resistance to at least three of the antimicrobial classes. Moreover, 13 of the 16 antimicrobial-resistance genes tested were detected; only ermC, tet(K), and tet(M) were not detected.

The highest gene frequency was observed in P6 (62.5%), followed by that in P1 (50%), P3 (50%), and P4 (50%), as depicted in table 3. A pattern of genes conferring resistance to multiple antibiotics was expected due to the presence of external contaminants (wastewater and sewage) discharged into the zoo lakes. The presence of resistance genes are linked to the presence of sewage and other sources of contamination such as residual water, which increases the amount of resistant bacteria (GÕNI-URRIZA et al., 2000GÕNI-URRIZA, M. Impact of urban effluent on antibiotic resistance of riverine Enterobacteriaceae and Aeromonas sp. Applied Environmental Microbiology, v.66, n.1, p.125-132, Jan. 2000. Available from: <Available from: http://aem.asm.org/content/66/1/125.full.pdf+html >. Accessed: Dec. 20, 2014. doi: 10.1128/AEM.66.1.125-132.2000.
http://aem.asm.org/content/66/1/125.full...
).

Tabela 3
Results of antimicrobial class's analyses and resistance genes detection in water samples from UFMT Zoo, November /2013 - April /2014.

Sulfonamide-resistance genes (sul I and sul II) were present in all samples. COLUMER-LLUCH et al. (2014COLUMER-LLUCH, M. et al. Antibiotic resistance genes and bacteriofhage fractions of Tunisian and Spanish wastewater as markes to compare the antibiotic resistance patterns in each population. Environment International, v.73, p.1767-175. Dec. 2014. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0160412014002086 >. Accessed: Jan. 16, 2015. doi: 10.1016/j.envint.2014.07.003.
http://www.sciencedirect.com/science/art...
) demonstrated that the sul I gene was more prevalent in water samples in Tunisia and DOBIASOVA et al. (2013DOBIASOVA, H. et al. Extended spectrum beta-lactamase and fluorquinolone resistance genes and plasmids among Escherichia coli isolates from zoo animals, Czech Republic. FEMS Microbiology Ecology, v.85, n.3, p.604-611, Sept. 2013. Available from: <Available from: http://dx.doi.org/10.1111/1574-6941.12149 >. Accessed: Nov. 25, 2014. doi: 10.1111/1574-6941.12149.
http://dx.doi.org/10.1111/1574-6941.1214...
) reported that the sul I and sul II genes were mostly detected in Escherichia coli isolates from stool samples from zoo animals. Although sulfonamides are widely used in veterinary medicine, their use is even more widespread in human medicine (SKÖLD, 2000SKÖLD, O. Sulfonamide resistance: mechanisms and trends. Drugs Resistance Updates, v.3, n.3, p.155-160, Jun. 2000. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S1368764600901468 >. Accessed: Dec. 10, 2014. doi: 10.1054/drup.2000.0146.
http://www.sciencedirect.com/science/art...
). Since the genes encoding resistance to sulfonamides are present in all lakes, it is probable that contamination of these lakes occurred from human contaminants.

β-Lactam-resistance genes were detected in all samples, except P7. The frequency of resistance genes varied between 77.8% (blaPSE I) and 11.1% (blaZ). Studies have also demonstrated the presence of the β-lactamases (ampC) genes from the DNA samples of biofilms in wastewater treatment stations, drinking water, and hospital water (SCHWARTZ et al., 2003SCHWARTZ, T. et al. Detection of antibiotic-resistant bacteria and their resistance genes in wastewater, surface water, and drinking water biofilms. FEMS Microbiology Ecology, v.43, n.3, p.325-335, Apr. 2003. Available from: <Available from: http://femsec.oxfordjournals.org/content/43/3/325.long >. Accessed: Sept. 12, 2014. doi: 10.1111/j.1574-6941.2003.tb01073.x.
http://femsec.oxfordjournals.org/content...
), probably because of the presence of Enterobacteriaceae from wastewater or sewage contamination (HENRIQUES et al., 2006HENRIQUES, I.S. et al. Occurrence and diversity of integrons and beta-lactamase genes among ampicillin-resistant isolates from estuarine waters. Research in Microbiology, v.157, p.938-947, Oct. 2006. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0958166908000591 >. Accessed: Jul. 10, 2015. doi: 10.1016/j.copbio.2008.05.006.
http://www.sciencedirect.com/science/art...
).

Among the macrolide-resistance genes, the ermC gene was earlier reported to be isolated from the dominant Staphylococcus epidermidis from biofilms (HONG-JING et al., 2014HONG-JING, H. et al. Erythromicin resistance features and biofilm formation affected by subinhibitory erythromycin in clinical isolates of Staphylococcus epidermidis. Journal Microbiology Immunology and Infection, v.xx, p.1-8. Apr. 2014. Available from: <Available from: http://dx.doi.org/10.1016/j.jmii.2014.03.001 >. Accessed: Nov. 20, 2014. doi: doi.org/10.1016/j.jmii.2014.03.001.
http://dx.doi.org/10.1016/j.jmii.2014.03...
) and ermA and ermC genes from samples of a wastewater treatment station (FARIA et al., 2009FARIA, C. et al. Antibiotic resistance in coagulase negative Staphylococcus isolated from wastewater and drinking water. Science Total Environmental, v.407, n.12 p.3876-3882, Jun. 2009. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0048969709002125 >. Accessed: Jan. 15, 2015. doi: 10.1016/j.scitotenv.2009.02.034.
http://www.sciencedirect.com/science/art...
). Contrary to this, no samples were positive for the ermC gene in the present study.

The aac(6')-aph(2''), aph3'-IIIa and ant(4')-Ia genes have been previously detected in Staphylococcus aureus isolates (DURAN et al., 2012DURAN, N. et al. Antibiotic resistance genes and susceptibility patterns in Staphylococci. Indian Journal of Medical Research, v.135, n.3, p.389-396. Mar. 2012. Available from: <Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3361877/ >. Accessed: Jan. 15, 2015.
http://www.ncbi.nlm.nih.gov/pmc/articles...
). However, in the present study, the low rate of detection of aph3'-IIIa and ant(4')-Ia genes and absence of ermC, tet(K) and tet(M) may have been observed since no bacterial isolation was performed. Another factor to be considered in this regard is the low concentration of bacteria hosting these resistance genes (SCHWARTZ et al., 2003SCHWARTZ, T. et al. Detection of antibiotic-resistant bacteria and their resistance genes in wastewater, surface water, and drinking water biofilms. FEMS Microbiology Ecology, v.43, n.3, p.325-335, Apr. 2003. Available from: <Available from: http://femsec.oxfordjournals.org/content/43/3/325.long >. Accessed: Sept. 12, 2014. doi: 10.1111/j.1574-6941.2003.tb01073.x.
http://femsec.oxfordjournals.org/content...
).

The tet(K) and tet(M) genes that encode tetracycline resistance were not detected in this study, probably because of the geographical location, as has also been previously described for rivers in German and Australia (STOLL et al., 2012STOLL, C. et al. Prevalence of clinically relevant antibiotic resistance genes in surface water Samples collected from Germany and Australia. Environmental Science Technology, v.46, n.17, p.9716-9726, 2012. Available from: <Available from: http://pubs.acs.org/doi/full/10.1021/es302020s >. Accessed: Jul. 20, 2015. doi: 10.1021/es302020s.
http://pubs.acs.org/doi/full/10.1021/es3...
). This result may be associated with the fact that these genes are not expected in aquatic environments because of the precipitation and/or hydrolysis of tetracyclines and penicillins in such environments (KEMPER, 2008KEMPER, N. Veterinary antibiotics in the aquatical and terrestrial environment. Ecological Indicators, v.8, n.1, p.1-13, Jan. 2008. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S1470160X07000647 >. Accessed: Nov. 16, 2014. doi:10.1016/j.ecolind.2007.06.002.
http://www.sciencedirect.com/science/art...
).

CONCLUSION:

Zoo lakes serve as reservoir for multiple antibiotic-resistance genes, which could be transferred to animal and human pathogens. The source of contamination of all these lakes is probably the neighboring urban sewage or wastewater. Thus, the findings of this study may assist in developing strategies to avoid the spread of antibiotic-resistant bacteria.

ACKNOWLEDGEMENT

We are grateful to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), for granting Doctoral scholarship

REFERENCES:

  • 1
    CR-2015-0827.R2

Publication Dates

  • Publication in this collection
    May 2016

History

  • Received
    06 Sept 2015
  • Accepted
    28 Oct 2015
  • Reviewed
    25 Feb 2016
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