Abstract
The aim of the present study was to investigate the resistance profile, to detect the presence of beta-lactam resistance genes, phenotypic expression of efflux pump systems and class 1 integrons in Pseudomonas spp. strains obtained from untreated hospital effluents. Effluent samples were collected from four hospitals in Porto Alegre, RS, Brazil. Pseudomonas were isolated on MacConkey agar plates and the identification was confirmed by 16S rRNA PCR and biochemical tests. Susceptibility testing was determined by disk-diffusion method using 11 different beta-lactams and MIC assays were performed on isolates resistant to imipenem and ceftazidime. The beta-lactamase genes bla IMP, bla VIM, bla SPM-1, bla OXA-23-like, bla OXA-24-like, bla OXA-51-like and the intl1 gene from class 1 integron were analysed by PCR. One hundred and twenty-four isolates were recovered and the most common species was Pseudomonas pseudoalcaligenes. The resistance found among the isolates was considered high, 62 (50%) isolates were multiresistant. No isolate carrying the beta-lactamase genes tested was found among the strains. Seven isolates showed reduction of MIC for imipenem and ceftazidime in the presence of cyanide m-chlorophenylhydrazone, indicating the hyper expression of efflux pumps. From the 124 isolates, 52 (41.9%) were identified as carrying the class 1 integron gene, intI1. Untreated hospital effluents could be a source of environmental contamination due to discharge of antimicrobial resistant bacteria which can carry integron class 1 and act as a reservoir of resistance genes and have efflux pump systems.
Similar content being viewed by others
References
Almuzara M, Radice M, de Gárate N, Kossman A, Cuirolo A, Santella G, Famiglietti A, Gutkind G, Vay V (2007) VIM-2-producing Pseudomonas putida, Buenos Aires. Emerg Infect Dis 13:668–669
Bogaerts P, Huang TD, Rodriguez-Villalobos H, Bauraing C, Deplano A, Struelens MJ, Glupczynski Y (2008) Nosocomial infections caused by multidrug-resistant Pseudomonas putida isolates producing VIM-2 and VIM-4 metallo-beta-lactamases. J Antimicrob Chemother 61:749–751
Clinical and Laboratory Standards Institute/NCCLS (2005) Performance standards for antimicrobial susceptibility testing; 15th informational supplement. Clinical and Laboratory Standards Institute, Wayne
Empel J, Filczak K, Mrowka A, Hryniewicz W, Livermore DM, Gniadkowski M (2007) Outbreak of Pseudomonas aeruginosa with PER-1 extended-spectrum beta-lactamase in Warsaw, Poland; further evidence for an international clonal complex. J Clin Microbiol 45:997–1007
Ferreira AE, Marchetti DP, Oliveira LM, Gusatti CS, Fuentefria DB, Corção G (2011) Presence of oxa-23-producing isolates of Acinetobacter baumannii in wastewater from hospitals in Southern Brazil. Microb Drug Resist 17(2):221–227
Fonseca EL, Vieira VV, Cipriano R, Vicente ACP (2005) Class 1 integrons in Pseudomonas aeruginosa isolates from clinical settings in the Amazon region, Brazil. FEMS Immunol Med Microbiol 44:303–309
Fuentefria DB, Ferreira, Gräf T, Corção G (2008) Pseudomonas aeruginosa: disseminação de resistência antimicrobiana em efluente hospitalar e água superficial. Rev Soc Bras Med Trop 41:470–473
Fuentefria DB, Ferreira AE, Gräf T, Corção G (2009) Spread of metallo-beta-lactamases: screening reveals the presence of a bla SPM-1 gene in hospital sewage in southern Brazil. Braz J Microbiol 40:82–85
Fuentefria DB, Ferreira AE, Corção G (2011) Antibiotic-resistant Pseudomonas aeruginosa from hospital wastewater and superficial water: Are they genetically related? J Environ Manag 92:250–255
Garrity GM (1984) Bergey’s manual of systematic bacteriology. Springer, New York
Gräf T, Fuentefria DB, Corção G (2008) Occurrence of multiresistant strains of Pseudomonas aeruginosa producing metallo-b-lactamase bla SPM-1 in clinical samples. Rev Soc Brasil Med Trop 41:306–308
Henrichfreise B, Wiegand I, Pfister W, Wiedemann B (2007) Resistance mechanisms of multiresistant Pseudomonas aeruginosa strains from Germany and correlation with hypermutation. Antimicrob Agents Chemother 51:4062–4070
Henriques IS, Fonseca F, Alvesa A, Saavedra MJ, Correia A (2006) Occurrence and diversity of integrons and beta-lactamase genes among ampicillin-resistant isolates from estuarine waters. Res Microbiol 157:938–947
Hsueh PR, Teng LJ, Pan HJ, Chen YC, Sun CC, Ho SW, Luh KT (1998) Outbreak of Pseudomonas fluorescens bacteremia among oncology patients. J Clin Microbiol 36:2914–2917
Jeannot K, Elsen S, Kohler T, Attree I, van Delden C, Plesiat P (2008) Resistance and virulence of Pseudomonas aeruginosa clinical strains overproducing the mexCD-oprJ efflux pump. Antimicrob Agents Chemother 52:2455–2462
Kim IS, Lee NY, Ki CS, Oh WS, Peck KR, Song JH (2005) Increasing prevalence of imipenem-resistant Pseudomonas aeruginosa and molecular typing of metallo-beta-lactamase producers in a Korean hospital. Microb Drug Resist 11:355–359
Koh TH, Wang GCY, Sng LH (2004) IMP-1 and a novel metallo-beta-lactamase, VIM-6, in fluorescent Pseudomonads isolated in Singapore. Antimicrob Agents Chemother 48:2334–2336
Li D, Yang M, Hu J, Zhang J, Liu R, Gu X, Zhang Y, Wang Z (2009) Antibiotic-resistance profile in environmental bacteria isolated from penicillin production wastewater treatment plant and the receiving river. Environ Microbiol 11:1506–1517
Livermore DM (2002) Multiple mechanisms of antimicrobial resistance in Pseudomonas aeruginosa: Our worst nightmare? Clin Infect Dis 34:634–640
Livermore DM, Winstanley TG, Shannon KP (2004) Interpretative reading: recognizing the unusual and inferring resistance mechanisms from resistance phenotypes. J Antimicrob Chemother 48:87–102
Mah TF, Pitts B, Pellock B, Walker GC, Stewart PS, O’Toole GA (2003) A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance. Nature 426:306–310
Martinez JL, Sanchez MB, Solano LM, Hernandez A, Garmendia L, Fajardo A, Ortega CA (2009) Functional role of bacterial multidrug efflux pumps in microbial natural ecosystems. FEMS Microbiol Rev 33:430–449
Meirelles-Pereira F, Pereira MAS, Silva MCG (2002) Ecological aspects of the antimicrobial resistance in bacteria of importance to human infections. Braz J Microbiol 33:287–293
Moura A, Soares M, Pereira C, Leitão N, Henriques I, Correia A (2009) INTEGRALL: a database and search engine for integrons, integrases and gene cassettes. Bioinformatics 25:1096–1098
Paterson DL (2006) The epidemiological profile of infections with multidrug-resistant Pseudomonas aeruginosa and Acinetobacter species. Clin Infect Dis 43:S43–S48
Pellegrini C, Mercuri PS, Celenza G, Galleni M, Segatore B, Sacchetti E, Volpe R, Amicosante G, Perilli M (2009) Identification of bla(IMP-22) in Pseudomonas spp. in urban wastewater and nosocomial environments: biochemical characterization of a new IMP metallo-enzyme variant and its genetic location. J Antimicrob Chemother 63:901–908
Poirel I, Carrër A, Pitout JD, Nordmann P (2009) Integron mobilization unit as a source of mobility of antibiotic resistance genes. Antimicrob Agents Chemother 53:2492–2498
Poole K (2005) Efflux-mediated antimicrobial resistance J Antimicrob Chemother 56:20–51
Prado T, Pereira WC, Silva DM (2008) Detection of extended-spectrum-β-lactamase-producing Klebsiella pneumoniae in effluents and sludge from a hospital sewage treatment plant. Lett Appl Microbiol 46:136–141
Quinteira S, Ferreira H, Peixe L (2005) First isolation of bla VIM-2 in an environmental isolate of Pseudomonas pseudoalcaligenes. Antimicrob Agents Chemother 49:2140–2141
Roe MT, Vega E, Pillai SD (2003) Antimicrobial resistance markers of class 1 and class 2 integron-bearing Escherichia coli from irrigation water and sediments. Emerg Infect Dis 9:822–829
Sandvang D, Diggle M, Platt DJ (2002) 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 8:151–160
Sekiguchi JI, Asagi T, Miyoshi-Akiyama T, Kasai A, Mizuguchi Y, Araake M, Fujino T, Kikuchi H, Sasaki S, Watari H, Kojima T, Miki H, Kanemitsu K, Kunishima K, Kikuchi Y, Kaku M, Yoshikura H, Kuratsuji T, Kirikae T (2007) Outbreaks of multidrug-resistant Pseudomonas aeruginosa in community hospitals in Japan. J Clin Microbiol 45:979–989
Smalla K, Sobecky PA (2002) The prevalence and diversity of mobile genetic elements in bacterial communities of different environmental habitats: insights gained from different methodological approaches. FEMS Microbiol Ecol 42:165–175
Spilker T, Coenye T, Vandamme P, LiPuma JJ (2004) PCR-based assay for differentiation of Pseudomonas aeruginosa from other Pseudomonas species recovered from cystic fibrosis patients. J Clin Microbiol 42:2074–2079
Strateva T, Yordanov D (2009) Pseudomonas aeruginosa—a phenomenon of bacterial resistance. J Med Microbiol 58:1133–1148
Tuméo E, Gbaguidi-Haore H, Patry I, Bertrand X, Thouverez M, Talon D (2008) Are antibiotic-resistant Pseudomonas aeruginosa isolated from hospitalized patients recovered in the hospital effluents? Int J Hyg Environ Health 21:200–204
Walsh TR, Toleman MA, Poirel L, Nordmann P (2005) Metallo-β-lactamases: The quiet before the storm? Clin Microbiol Rev 18:306–325
White PA, McIver CJ, Rawlinson WD (2001) Integrons and gene cassettes in the Enterobacteriaceae. Antimicrob Agents Chemother 45:2658–2661
Wolter DJ, Schmidtke AJ, Hanson ND, Lister PD (2007) Increased expression of ampC in Pseudomonas aeruginosa mutants selected with ciprofloxacin. Antimicrob Agents Chemother 51:2997–3000
Woodford N, Ellington MJ, Coelho JM, Turton JF, Ward ME, Brown S, Amyes SG, Livermore DM (2006) Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp. Int J Antimicrob Agents 27:351–353
Wright MS, Austin CB, Lindell AH, Stepanauskas R, Stokes HW, McArthur JV (2008) Influence of industrial contamination on mobile genetic elements: class 1 integron abundance and gene cassette structure in aquatic bacterial communities. Int Soc Microb Ecol J J 2:417–428
Yang CM, Lin MF, Liao PC, Yeh HW, Chang BV, Tang TK, Cheng C, Sung CH, Liou ML (2009) Comparison of antimicrobial resistance patterns between clinical and sewage isolates in a regional hospital in Taiwan. Lett Appl Microbiol 48:560–565
Zavascki AP, Gaspareto PB, Martins AF, Gonçalves AL, Barth AL (2005) Outbreak of carbapenem-resistant Pseudomonas aeruginosa producing SPM-1 metallo-beta-lactamase in a teaching hospital in southern Brazil. J Antimicrob Chemother 56:1148–1151
Ziha-Zarifi I, Llanes C, Köhler T, Pechere JC, Plesiat P (2007) In vivo emergence of multidrug-resistant mutants of Pseudomonas aeruginosa overexpressing the active efflux system mexA-mexB-oprM. Antimicrob Agents Chemother 43:287–291
Acknowledgments
This research was supported by CAPES (Brazilian Government Supporting Agency) and Rio Grande do Sul State Supporting Agency FAPERGS. The authors would like to thanks Dr. Ana Cristina Gales who kindly provided the strains used as positive controls in this study. The authors are also grateful to Clarissa Branco Haas, Lyvia Moreira de Oliveira and Gabriela Rosa da Cunha for the technical assistance.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Spindler, A., Otton, L.M., Fuentefria, D.B. et al. Beta-lactams resistance and presence of class 1 integron in Pseudomonas spp. isolated from untreated hospital effluents in Brazil. Antonie van Leeuwenhoek 102, 73–81 (2012). https://doi.org/10.1007/s10482-012-9714-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10482-012-9714-2