Screening of Class 1 and Class 2 Integrons in Clinical Isolates of Pseudomonas aeruginosa Collected from Seven Hospitals in Turkey: A Multicenter Study

Aysegul Copur Cicek, Aysegul Saral, Azer Ozad Duzgun, Zeynep Cizmeci, Tuba Kayman, Pervin Ozlem Balci, Tuba Dal, Mehmet Firat, Yelda Yazici, Metin Sancaktar, Osman Birol Ozgumus, Cemal Sandalli

Clinic of Infectious Diseases and Clinical Microbiology, Private OSM Ortadogu Hospital, Sanliurfa, Turkey.
Clinical Microbiology Laboratory, Ahi Evren Chest and Cardiovascular Surgery Research and Training Hospital, Trabzon, Turkey.
Clinical Microbiology Laboratory, Akcaabat Hackali Baba General State Hospital, Trabzon, Turkey.
Clinical Microbiology Laboratory, General State Hospital, Tokat, Turkey.
Clinical Microbiology Laboratory, Kecioren Research and Training Hospital, Ankara, Turkey.
Clinical Microbiology Laboratory, Research and Training Hospital, Kayseri, Turkey.
Department of Biology, Faculty of Arts and Sciences, Artvin Coruh University, Artvin, Turkey.
Department of Biology, Faculty of Arts and Sciences, Recep Tayyip Erdogan University, Rize, Turkey.
Department of Genetics and Bioengineering, Faculty of Engineering and Natural Sciences, Gümü?hane University, Gumushane, Turkey.
Department of Medical Microbiology, Faculty of Medicine, Dicle University, Diyarbakir, Turkey.
Department of Medical Microbiology, Faculty of Medicine, Recep Tayyip Erdogan University, Rize, Turkey.

DOI: 10.4236/ojmm.2013.34034                

Abstract

Pseudomonas aeruginosa is one of the leading nosocomial pathogens worldwide, and their infections are difficult to treat due to acquired resistance to many antibiotics. This study aimed to detect class 1 and 2 integrons and antibiotic susceptibility of clinical isolates of P. aeruginosa. Two hundred and five P. aeruginosa strains were collected from the seven general state hospitals in Turkey. They were characterized by antimicrobial susceptibility testing, screened for class 1 and class 2 integrons, and evaluated for the association between antibiotic resistance phenotypes and the presence of integrons. intI gene was amplified in 10 isolates (4.87%) by PCR and in seven isolates of them (70%) were found different gene cassettes. The aadA gene integrated into the class 1 integrons was most frequently found and it was followed by aac genes and blaOXA family genes. Sequence analysis of variable regions of the class 1 integrons showed five gene cassette arrays; aadA1(99%), aac(3)-Id(82%)-orf-aac(3”)-Ia(99%), aac(3)-Ie(83%)-blaoxa10(100%)- aadA1 (100%), aadA6(99%, 100%), aac(6’)-I(97%)-orf-aadA2(99%). No class 2 integron was detected. This study is the first multicenter study for class 1 integrons and it indicates the low rate of presence of class 1 gene cassette in P. aeruginosa.

Keywords

Pseudomonas aeruginosa; Antibiotic Resistance; Class 1 Integron; AadA; Multicenter Study

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	G. L. Winsor, D. K. W. Lam, L. Fleming, R. Lo, M. D. Whiteside, N. Y. Yu, R. E. W. Hancock and F. S. L. Brinkman, “Pseudomonas Genome Database: Improved Comparative Analysis and Population Genomics Capability for Pseudomonas Genomes,” Nucleic Acids Research, Vol. 39, 2011, pp. 596-600.
[2]	R. J. Gillis, K. G. White, K. H. Choi, V. E. Wagner, H. P. Schweizer and B. H. Iglewski, “Molecular Basis of Azithromycin-Resistant Pseudomonas aeruginosa Biofilms,” Antimicrobial Agents Chemotherapy, Vol. 49, No. 9, 2005, pp. 3858-3867. http://dx.doi.org/10.1128/AAC.49.9.3858-3867.2005
[3]	J. Patzer, M. A. Toleman, L. M. Deshpande, W. Kaminska, D. Dzierzanowska, P. M. Bennett, R. N. Jones and T. R. Walsh, “Pseudomonas aeruginosa Strains Harbouring an Unusual blaVIM-4 Gene cassette Isolated from Hospitalized Children in Poland (1998-2001),” Journal Antimicrobial Chemotherapy, Vol. 53, No. 3, 2004, pp. 451- 456. http://dx.doi.org/10.1093/jac/dkh095
[4]	J. Sarlangue, O. Brissaud and C. Labreze, “Clinical Features of Pseudomonas aeruginosa Infections,” Archives de Pediatrie, Vol. 13, No. 1, 2006, pp. 13-16.
[5]	P. D. Lister, D. J. Wolter and N. D. Hanson, “Antibacterial-Resistant Pseudomonas aeruginosa: Clinical Impact and Complex Regulation of Chromosomally Encoded Resistance Mechanisms,” Clinical Microbiology Reviews, Vol. 22, No. 4, 2009, pp. 582-610. http://dx.doi.org/10.1128/CMR.00040-09
[6]	http://textbookofbacteriology.net/themicrobialworld/Pseudomonas.html
[7]	J. K. Lutz and J. Lee, “Prevalence and Antimicrobial- Resistance of Pseudomonas aeruginosa in Swimming Pools and Hot Tubs,” International Journal of Environmental Research and Public Health, Vol. 8, No. 2, 2011, pp. 554-564. http://dx.doi.org/10.3390/ijerph8020554
[8]	E. L. Fonseca, V. V. Vieira, R. Cipriano and A. C. Vicente, “Class 1 Integrons in Pseudomonas aeruginosa Isolates from Clinical Settings in Amazon Region, Brazil,” FEMS Immunology and Medical Microbiology, Vol. 44, No. 3, 2005, pp. 303-309. http://dx.doi.org/10.1016/j.femsim.2005.01.004
[9]	F. Budak, M. Kasap, F. Kolayl?, A. Karadenizli and M. H. Vahabo?lu, “Integron-Associated Resistance Genes among Multidrug-Resistant Pseudomonas aeruginosa Isolated from Clinical Specimens,” Turkish Journal of Medical Science, Vol. 42, No. 1, 2012, pp. 149-156.
[10]	Y. Khosravi, S. T. Tay and J. Vadivelu, “Analysis of Integrons and Associated Gene Cassettes of Metallo- β-Lactamase-Positive Pseudomonas aeruginosa in Malaysia,” Journal of Medical Microbiology, Vol. 60, No. 7, 2011, pp. 988-994. http://dx.doi.org/10.1099/jmm.0.029868-0
[11]	D. Mazel, “Integrons: Agents of Bacterial Evolution,” Nature Reviews. Microbiology, Vol.4, No. 8, 2006, pp. 608-620.
[12]	G. D. Recchia and R. M. Hall, “Gene Cassettes: A New Class of Mobile Element,” Microbiology, Vol. 141, No. 12, 1995, pp. 3015-3027. http://dx.doi.org/10.1099/13500872-141-12-3015
[13]	C. M. Collis and R. M. Hall, “Gene Cassettes from the Insert Region of Integrons Are Excised as Covalently Closed Circles,” Molecular Microbiology, Vol. 6, No. 19, 1992, pp. 2875-2885. http://dx.doi.org/10.1111/j.1365-2958.1992.tb01467.x
[14]	R. M. Hall, D. E. Brookes and H. W. Stokes, “Site-Specific Insertion of Genes into Integrons: Role of the 59- Base Element and Determination of the Recombination Cross-Over Point,” Molecular Microbiology, Vol. 5, No. 8, 1991, pp. 1941-1959. http://dx.doi.org/10.1111/j.1365-2958.1991.tb00817.x
[15]	A. C. Fluit and F. J. Schmitz, “Resistance Integrons and Super-Integrons,” Clinical Microbiology and Infection, Vol. 10, No. 4, 2004, pp. 272-288. http://dx.doi.org/10.1111/j.1198-743X.2004.00858.x
[16]	C. Goldstein, M. D. Lee, S. Sanchez, C. Hudson, B. Phillips, B. Register, M. Grady, C. Liebert, A. O. Summers, D. G. White and J. J. Maurer, “Incidence of Class 1 and 2 Integrases in Clinical and Commensal Bacteria from Livestock, Companion Animals, and Exotics,” Antimicro- bial Agents and Chemotherapy, Vol. 45, No. 3, 2001, pp. 723-726. http://dx.doi.org/10.1128/AAC.45.3.723-726.2001
[17]	D. A. Rowe-Magnus, A. M. Guerout and D. Mazel, “Bacterial Resistance Evolution by Recruitment of Super- Integron Gene Cassettes,” Molecular Microbiology, Vol. 43, No. 6, 2002, pp. 1657-1669. http://dx.doi.org/10.1046/j.1365-2958.2002.02861.x
[18]	A. Carattoli, “Importance of Integrons in the Diffusion of Resistance,” Veterinary Research, Vol. 32, No. 3-4, 2001, pp. 243-259. http://dx.doi.org/10.1051/vetres:2001122
[19]	J. Chen, Z. Su, Y. Liu, S. Wang, X. Dai, Y. Li, S. Peng, Q. Shao , H. Zhang , P. Wen , J. Yu , X. Huang and H. Xu, “Identification and Characterization of Class 1 Integrons among Pseudomonas aeruginosa Isolates from Patients in Zhenjiang, China,” International Journal of Infectious Diseases, Vol. 13, No. 6, 2009, pp. 717-721. http://dx.doi.org/10.1016/j.ijid.2008.11.014
[20]	D. Girlich, T. Naas, A. Leelaporn, L. Poirel, M. Fennewald and P. Nordmann, “Nosocomial Spread of the Integron-Located Veb-1-Like Cassette Encoding an Extended-Spectrum Beta-Lactamase in Pseudomonas aeruginosa in Thailand,” Clinical Infectious Diseases, Vol. 34, No. 5, 2002, pp. 603-611. http://dx.doi.org/10.1086/338786
[21]	K. Lee, J. B. Lim, J. H. Yum, D. Yong, Y. Chong, J. M. Kim and D. M. Livermore, “Bla(VIM-2) Cassette Containing Novel Integrons in Metallo-Beta-Lactamase-Producing Pseudomonas aeruginosa and Pseudomonas putida Isolates Disseminated in a Korean Hospital, “ Antim- icrobial Agents and Chemotherapy, Vol. 46, No. 4, 2002, pp. 1053-1058. http://dx.doi.org/10.1128/AAC.46.4.1053-1058.2002
[22]	L. Pallecchi, M. L. Riccio, J. D. Docquier, R. Fontana and G. M. Rossolini, “Molecular Heterogeneity of blaVIM-2-Containing Integrons from Pseudomonas aeruginosa Plasmids Encoding the VIM-2 Metallo-Beta-Lactamase,” FEMS Microbiology Letters, Vol. 195, No. 2, 200, pp. 145-150.
[23]	L. Poirel, T. Lambert, S. Türko?lu, E. Ronco, J. Gaillard and P. Nordmann, “Characterization of Class 1 Integrons from Pseudomonas aeruginosa that Contain the Blavim-2 Carbapenem-Hydrolyzing β-Lactamase Gene and of Two Novel Aminoglycoside Resistance Gene Cassettes,” Antimicrobial Agents and Chemotherapy, Vol. 45, No. 2, 2001, pp. 546-552. http://dx.doi.org/10.1128/AAC.45.2.546-552.2001
[24]	E. L. Schnabel and A. L. Jones, “Distribution of Tetracycline Resistance Genes and Transposons among Phylloplane Bacteria in Michigan Apple Orchards,” Applied and Environmental Microbiology, Vol. 65, No. 11, 1999, pp. 4898-4907.
[25]	P. Severino and V. D. Magalhaes, “The Role of Integrons in the Dissemination of Antibiotic Resistance among Clinical Isolates of Pseudomonas aeruginosa from an Intensive Care unit in Brazil,” Research in Microbiology, Vol. 153, No. 4, 2002, pp. 221-226. http://dx.doi.org/10.1016/S0923-2508(02)01310-4
[26]	H. Yan, L. Shi, S. Yamasaki, X. Li, Y. Cao, L. Li, L. Yang and S. Miyoshi, “A Plasmidic Class 1 Integron from Five Pseudomonas aeruginosa Clinical Strains Harbored aacA4 and Nonsense-Mutated cmlA1 Gene Cas-settes,” Journal of Health Science, Vol. 53, No. 6, 2007, pp. 750-755. http://dx.doi.org/10.1248/jhs.53.750
[27]	S. Yousefi, M. R. Nahaei, S. Farajnia, M. Ghojazadeh, M. T. Akhi, Y. Sharifi, M. Milani and R. Ghostaslou, “Class 1 Integron and Imipenem Resistance in Clinical Isolates of Pseudomonas aeruginosa: Prevalence and Antibiotic Susceptibility,” Iranian Journal of Microbiology, Vol. 2, No. 3, 2010, pp. 113-119.
[28]	K. Hansson, L. Sundstr?m, A. Pelletier and P. H. Roy, “IntI2 Integron Integrase in Tn7,” Journal of Bacteriology, Vol. 184, No. 6, 2002, pp. 1712-1721.
[29]	Z. Xu, L. Li, M. E. Shirtliff, M. J. Alam, S. Yamasaki and L. Shi, “Occurrence and Characteristics of Class 1 and 2 Integrons in Pseudomonas aeruginosa Isolates from Patients in Southern China,” Journal of Clinical Microbiology, Vol. 47, No.1, 2009, pp. 230-234. http://dx.doi.org/10.1128/JCM.02027-08
[30]	National Committee for Clinical Laboratory Standards, “Performance Standards for Antimicrobial Susceptibility testing” NCCLS Antim-Icrobial Susceptibility Testing Standards M100-S22, 2012.
[31]	F. M. Ausubel, R. Brient, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith and J. Struhl, “Short Protocols in Molecular Biology: 2nd ed.,” John Willey and Sons, New York, 1995.
[32]	S. J. Rosser and H. K. Young, “Identification and Characterization of Class 1 Integrons in Bacteria from an Aquatic Environment,” The Journal of Antimicrobial Chemotherapy, Vol. 44, No. 1, 1999, pp. 11-18. http://dx.doi.org/10.1093/jac/44.1.11
[33]	C. Lévesque, L. Piche, C. Larose and P. H. Roy, “PCR Mapping of Integrons Reveals Several Novel Combinations of Resistance Genes,” Antimicrobial Agents and Chemotherapy, Vol. 39, No. 1, 1995, pp. 185-191. http://dx.doi.org/10.1128/AAC.39.1.185
[34]	J. Sambrook, E. F. Fritsch and T. Maniatis, “Molecular Cloning. A Laboratory Manual,” Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989.
[35]	S. F. Altschul, T. L. Madden, A. A. Sch?ffer, J. Zhang, Z. Zhang, W. Miller and D. J. Lipman, “Gapped BLAST and PSI-BLAST: A New Generation of Protein Database Search Programs,” Nucleic Acids Research, Vol. 25, No. 17, 1997, pp. 3389-3402. http://dx.doi.org/10.1093/nar/25.17.3389
[36]	B. Gu, M. Tong, W. Zhao, G. Liu, M. Ning, S. Pan and W. Zhao, “Prevalence and Characterization of Class I Integrons among Pseudomonas aeruginosa and Acinetobacter baumannii Isolates from Patients in Nanjing, China,” Journal of Clinical Microbiology, Vol. 45, No. 1, 2007, pp. 241-243. http://dx.doi.org/10.1128/JCM.01318-06
[37]	T. Strateva and D. Yordanov, “Pseudomonas aeruginosa —A Phenomenon of Bacterial Resistance,” Journal of Medical Microbiology, Vol. 58, No. 9, 2009, pp. 1133- 1148. http://dx.doi.org/10.1099/jmm.0.009142-0
[38]	O. B. Ozgumus, R. Caylan, I. Tosun, C. Sandalli, K. Aydin and I. Koksal, “Molecular Epidemiology of Clinical Pseudomonas aeruginosa Isolates Carrying the IMP-1 Metallo-β-Lactamase Gene in a University Hospital in Turkey,” Microbial Drug Resistance, Vol. 13, No. 3, 2007, pp. 191-198. http://dx.doi.org/10.1089/mdr.2007.748
[39]	K. Poonsuk, C. Tribuddharat and R. Chuanchuen, “Class 1 Integrons In Pseudomonas Aeruginosa And Acinetobacter Baumannii Isolated from Clinical Isolates,” The Southeast Asian Journal of Tropical Medicine Public Health, Vol. 43, No. 2, 2012, pp. 376-384.
[40]	A. Bito and M. Susani, “Revised Analysis of aadA2 Gene of Plasmid pSa,” Antimicrobial Agents Chemotherapy, Vol. 38, No. 5, 1994, pp. 1172-1175. http://dx.doi.org/10.1128/AAC.38.5.1172
[41]	T. Naas, L. Poirel and P. Nordmann, “Molecular Characterisation of In51, a Class 1 integron Containing a Novel Aminoglycoside Adenylyltransferase Gene Cassette, aadA6, in Pseudomonas aeruginosa,” Biochimica et Biophysica Acta, Vol. 1489, No. 2-3, 1999, pp. 445-451. http://dx.doi.org/10.1016/S0167-4781(99)00202-X
[42]	M. L. Riccio, J. D. Docquier, E. Dell’Amico, F. Luzzaro, G. Amicosante and G. M. Rossolini, “Novel 3-N-Ami- noglycoside Acetyltransferase Gene, aac(3)-Ic, from a Pseudomonas aeruginosa Integron,” Antimicrobial Agents and Chemotherapy, Vol. 47, No. 5, 2003, pp. 1746-1748. http://dx.doi.org/10.1128/AAC.47.5.1746-1748.2003
[43]	A. E. Sorour, I. E. Wali and S. K. El-Hodaky, “OXA-Type-Beta-Lactamases among Extended-Spectrum-Cephalosporin Non-Susceptible Pseudomonas Aeruginosa Isolates Collected from a Large Teaching Hospital in Cairo,” Egyptian Journal of Medical Microbiology, Vol. 17, No. 4, 2008, pp. 565-572.
[44]	G. H. Miller, F. J. Sabatelli, R. S. Hare, Y. Glupczynski, P. Mackey, D. Shlaes, K. Shimizu and K. J. Shaw, “The Most Frequent Aminoglycoside Resistance Mechanisms —Changes with Time and Geographic Area: A Reflection of Aminoglycoside Usage Patterns?” Clinical Infectious Diseases, Vol. 24, No. 1, 1997, pp. 46-62. http://dx.doi.org/10.1093/clinids/24.Supplement_1.S46