International Journal of Infectious Diseases
Volume 13, Issue 6 , Pages 717-721, November 2009

Identification and characterization of class 1 integrons among Pseudomonas aeruginosa isolates from patients in Zhenjiang, China

  • Jianguo Chen

      Affiliations

    • Clinical Laboratory, the Affiliated People's Hospital of Jiangsu University, Zhenjiang, Jiangsu Province, China
    • Department of Microbiology and Immunology, Institute of Clinical Laboratory Medicine, Jiangsu University, Xuefu Road 301, Zhenjiang, Jiangsu Province, 212013 China
    • ,
  • Zhaoliang Su

      Affiliations

    • Department of Microbiology and Immunology, Institute of Clinical Laboratory Medicine, Jiangsu University, Xuefu Road 301, Zhenjiang, Jiangsu Province, 212013 China
    • ,
  • Yingzhao Liu

      Affiliations

    • Clinical Laboratory, the Affiliated People's Hospital of Jiangsu University, Zhenjiang, Jiangsu Province, China
    • ,
  • Shengjun Wang

      Affiliations

    • Department of Microbiology and Immunology, Institute of Clinical Laboratory Medicine, Jiangsu University, Xuefu Road 301, Zhenjiang, Jiangsu Province, 212013 China
    • ,
  • Xiaoli Dai

      Affiliations

    • Department of Microbiology and Immunology, Institute of Clinical Laboratory Medicine, Jiangsu University, Xuefu Road 301, Zhenjiang, Jiangsu Province, 212013 China
    • ,
  • Yazhen Li

      Affiliations

    • Department of Microbiology and Immunology, Institute of Clinical Laboratory Medicine, Jiangsu University, Xuefu Road 301, Zhenjiang, Jiangsu Province, 212013 China
    • ,
  • Sufang Peng

      Affiliations

    • Department of Microbiology and Immunology, Institute of Clinical Laboratory Medicine, Jiangsu University, Xuefu Road 301, Zhenjiang, Jiangsu Province, 212013 China
    • ,
  • Qixiang Shao

      Affiliations

    • Department of Microbiology and Immunology, Institute of Clinical Laboratory Medicine, Jiangsu University, Xuefu Road 301, Zhenjiang, Jiangsu Province, 212013 China
    • ,
  • Haifang Zhang

      Affiliations

    • Department of Microbiology and Immunology, Institute of Clinical Laboratory Medicine, Jiangsu University, Xuefu Road 301, Zhenjiang, Jiangsu Province, 212013 China
    • ,
  • Ping Wen

      Affiliations

    • Clinical Laboratory, the Affiliated People's Hospital of Jiangsu University, Zhenjiang, Jiangsu Province, China
    • ,
  • Jianren Yu

      Affiliations

    • Clinical Laboratory, the Affiliated People's Hospital of Jiangsu University, Zhenjiang, Jiangsu Province, China
    • ,
  • Xinxiang Huang

      Affiliations

    • Department of Microbiology and Immunology, Institute of Clinical Laboratory Medicine, Jiangsu University, Xuefu Road 301, Zhenjiang, Jiangsu Province, 212013 China
    • ,
  • Huaxi Xu

      Affiliations

    • Department of Microbiology and Immunology, Institute of Clinical Laboratory Medicine, Jiangsu University, Xuefu Road 301, Zhenjiang, Jiangsu Province, 212013 China
    • Corresponding Author InformationCorresponding author. Tel.: +86 511 85038140; fax: +86 511 85038449.

Received 6 July 2008; received in revised form 22 September 2008; accepted 26 November 2008. published online 09 February 2009.

Corresponding Editor: J. Peter Donnelly, Nijmegen, The Netherlands

Article Outline

Summary 

Objectives

The role of integrons in the spread of antibiotic resistance has been well established. The aim of this study was to investigate the resistance profiles of Pseudomonas aeruginosa isolated from patients in Zhenjiang to 13 antibiotics, and to identify the structure and dissemination of class 1 integrons.

Methods

The Kirby–Bauer disk diffusion assay was used to determine the rate of P. aeruginosa resistance. Class 1 integrons from multidrug-resistant isolates were amplified by PCR, and their PCR products were sequenced. We also analyzed the integron structures containing the same gene cassettes by restriction fragment length polymorphism (RFLP). Isolates were genotyped by pulsed-field gel electrophoresis (PFGE).

Results

The resistance rates were between 29.6% and 90.1%. The prevalence of class 1 integrons was 38.0%. These integrons included five gene cassettes (aadB, aac6-II, blaPSE-1, dfrA17, and aadA5). The dfrA17 and aadA5 gene cassettes were found most often.

Conclusions

Class 1 integrons were found to be widespread in P. aeruginosa isolated from clinical samples in the Zhenjiang area of China. The antibiotic resistance rates in class 1 integron-positive strains of P. aeruginosa were noticeably higher than those in class 1 integron-negative strains. PFGE showed that particular clones were circulating among patients.

Keywords: Pseudomonas aeruginosa, Integrons, Gene cassettes, Multi-drug resistance

 

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Introduction 

The heavy selective pressure of antibiotics has promoted the rapid development of bacterial resistance. This bacterial resistance has become a severe and universal problem, leading to ineffective clinical treatment and increased economic burden, and also directly threatens the life of the patient.

Pseudomonas aeruginosa is an important opportunistic nosocomial pathogen, particularly evident in hospital-acquired pneumonia, especially in immunocompromised patients and those with a tracheal cannula, tracheotomy, or under mechanical ventilation.1 Its resistance mechanism is extremely complicated. Generally, bacterial resistance is caused by chromosomal gene mutations or plasmid acquisition, however, recently, more and more bacterial resistance has been found to be associated with integrons.2 Integrons, which consist of a 5′ conserved segment, a 3′ conserved segment, and a variable region between the two conserved segments, have been found to be mobilizable elements that capture external drug resistance gene cassettes and mediate bacterial resistance or multi-drug resistance. These have caused the rapid spread of resistance in bacteria, especially among the Gram-negative.3

To date, four classes of integrons have been described in Gram-negative bacterial isolates, however class 1 integrons have been found to be the most prevalent in clinical isolates, carrying single or multiple gene cassettes, which confer resistance to aminoglycosides, β-lactams, chloramphenicol, carbapenems, and macrolides.4, 5, 6, 7

In this study, we investigated the prevalence of resistance in 71 P. aeruginosa isolates to 13 antibiotics, and sought to identify the characteristics of class 1 integrons. At the same time, we also analyzed whether a particular clone was circulating among patients.

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Materials and methods 

Bacterial isolates 

From April 2006 to March 2007, 71 P. aeruginosa isolates were collected from different patients hospitalized at the Affiliated People's Hospital of Jiangsu University, a hospital with in excess of 900 beds. Identification of isolates was carried out using the VITEK32 GNI card (bioMérieux, Hazelwood, MO, USA). Bacterial isolates were stored as suspensions in a 10% (wt/vol) sterilized milk solution containing 10% (vol/vol) glycerol at −20°C until tests were performed.

Antimicrobial susceptibility testing 

Antimicrobial susceptibility was determined according to the Clinical and Laboratory Standards Institute (CLSI) using the Kirby–Bauer disk diffusion assay on Mueller–Hinton agar (Oxoid, UK).8 The susceptibility profiles were determined for 13 antibiotics: piperacillin (PIP, 100μg), piperacillin–tazobactam (TZP, 100/10μg), ceftriaxone (CRO, 30μg), cefepime (FEP, 30μg), cefotaxime (CTX, 30μg), ceftazidime (CAZ, 30μg), gentamicin (GEN, 10μg), amikacin (AMK, 30μg), tobramycin (TOB, 10μg), levofloxacin (LVX, 5μg), ciprofloxacin (CIP, 5μg), imipenem (IPM, 10μg), and trimethoprim–sulfamethoxazole (SXT, 12.5/23.7μg). Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as controls.

DNA extraction 

Genomic DNA used as template was obtained from bacterial suspensions grown overnight in Luria broth with shaking, suspended in 100μl of sterile water, and boiled for 10min.9

PCR amplifications and sequencing analysis 

PCR was used to amplify the class 1 integrons, and the primers were designed as follows: forward primer: 5′-GGCATCCAAGCAGCAAG-3′, and reverse primer: 5′-AAGCAGACTTGACCTGA-3′.10 Primers were synthesized by Shanghai GeneCore BioTechnologies Co., Ltd. A Mastercycler instrument (Eppendorf, Hamburg, Germany) was used with the following reaction conditions: 94°C for 5min, 30 cycles of 94°C for 30s, 55°C for 45s, and 72°C for 2min, and finally, 72°C for 10min. The amplicons were separated on 1% agarose gel prepared in TAE buffer and visualized using ultraviolet light after staining with ethidium bromide. PCR positive products were directly sequenced on both strands by the primer walking sequence strategy using the BigDye Terminator Cycle Sequencing Kit on an ABI PRISM 3730 automated DNA sequencer. The sequences were aligned in GenBank (http://www.ncbi.nlm.nih.gov/BLAST).

PCR-restriction fragment length polymorphism (RFLP) 

To determine whether different isolates carried identical integrons, the amplicons of similar sizes were compared by RFLP typing using the restriction endonucleases HinfI and EcoRV. If the amplicons from two strains yielded the same RFLP pattern, the two integrons were considered to be identical. If the PCR products contained a different RFLP pattern, the new product was sequenced as well.

Pulsed-field gel electrophoresis (PFGE) 

P. aeruginosa isolates carrying class 1 integrons were genotyped by PFGE with the restriction enzyme SpeI. DNA-PFGE marker (Amersham) was used as the size marker. The DNA fragments were separated on 1.0% agarose gels in 0.5× Tris–borate–EDTA buffer with a CHEF Mapper system (Bio-Rad Laboratories, Hercules, CA, USA) at 6V/cm for 20h. Clonal relatedness based on the PFGE patterns was interpreted according to the criteria proposed by Tenover et al.11

Nucleotide sequence accession numbers 

GenBank accession numbers are as follows: aadB–aac6-II–pse-1, DQ266447; dfrA17–aadA5, DQ838665; aac6-II, EU723083.

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Results 

Antimicrobial susceptibility of P. aeruginosa isolates 

Susceptibility testing of the 71 P. aeruginosa isolates showed that all the isolates were resistant to at least one of the antibiotics, and 64 (90.1%) isolates were resistant to three or more antimicrobials. Resistance was most often observed to cefotaxime (90.1%), followed by ceftriaxone (88.7%), trimethoprim–sulfamethoxazole (84.5%), and piperacillin (69.0%), and to a lesser extent tobramycin (35.2%), amikacin (31.0%), ceftazidime (29.6%), and imipenem (29.6%).

Integrons and characterization of gene cassettes 

PCR-based examination for the presence of the class 1 integrons was carried out on the entire collection. Of 71 isolates, 27 (38.0%) carried detectable class 1-related integrons, showing the existence of amplicons of 2500bp in three, 1600bp in 22, and 750bp in two of the class 1 integron-positive isolates. Amplicons of the same size gave identical restriction patterns with HinfI and EcoRV (Figure 1), which is indicative of their structural homogeneity. Sequence analysis of the class 1 integron variable region revealed that the 2500bp amplicon harbored gene cassettes aadB, aac6-II, and blaPSE-1, among which aadB and aac6-II gene cassettes conferred resistance to aminoglycosides;12, 13 the blaPSE-1 gene cassette contained the blaPSE-1 beta-lactamase gene, conferring resistance to ampicillin, carbenicillin, cefoxitin, cefamandole, and piperacillin.14 The 1600bp amplicon harbored genes dfrA17 and aadA5, conferring resistance to trimethoprim, as well as to streptomycin and spectinomycin,15 and the 750bp amplicon contained the aac6-II gene, conferring resistance as described previously (Figure 2). In this study, the most frequent variable region length among the class 1 integron-positive P. aeruginosa isolates was 1600bp.

  • View full-size image.
  • Figure 1. 

    Schematic representation of the 1.6-kb integron digested with HinfI and EcoRV. M, DNA marker; 1, 1.6-kb integron PCR amplicon; 2–4, ZJ070, ZJ199, and ZJ374 digested with HinfI; 5–7, ZJ070, ZJ199, and ZJ374 digested with EcoRV.

  • View full-size image.
  • Figure 2. 

    Schematic representation of the class 1 integrons from P. aeruginosa isolates. These integrons had approximate sizes of (A) 2.5kb, (B) 1.6kb, and (C) 0.75kb. Arrows indicate the direction of transcription. The core is represented by black circles.

Susceptibility data from integron-positive and integron-negative P. aeruginosa isolates 

Compared with the 44 integron-negative isolates, the 27 integron-positive clinical isolates were statistically more often resistant to certain antibiotics, including cefepime, ceftazidime, gentamicin, amikacin, tobramycin, levofloxacin, and ciprofloxacin (p<0.05). Although there was no statistically significant difference (p>0.05), the percentage of isolates susceptible to piperacillin, piperacillin–tazobactam, and trimethoprim–sulfamethoxazole was also lower among the integron-positive isolates (Table 1).

Table 1. Antibiotic susceptibility of integron-positive and integron-negative strains of P. aeruginosa
AntibioticIntegron-positive (N=27)Integron-negative (N=44)p-Value
%S%R%S%R
PIP18.581.538.661.4NS
TZP29.670.452.347.7NS
CRO11.188.911.488.6NS
FEP37.063.075.025.0<0.01
CTX7.492.611.488.6NS
CAZ51.948.181.818.2<0.05
GEN7.492.656.843.2<0.01
AMK33.366.790.99.1<0.01
TOB33.366.784.115.9<0.01
LVX29.670.479.520.5<0.01
CIP40.759.370.529.5<0.05
IPM74.125.968.231.8NS
SXT7.492.620.579.5NS

%S, percentage susceptible; %R, percentage resistant; PIP, piperacillin; TZP, piperacillin–tazobactam; CRO, ceftriaxone; FEP, cefepime; CTX, cefotaxime; CAZ, ceftazidime; GEN, gentamicin; AMK, amikacin; TOB, tobramycin; LVX, levofloxacin; CIP, ciprofloxacin; IPM, imipenem; SXT, trimethoprim–sulfamethoxazole; NS, not statistically significant.

Statistical significance (p) was calculated using the Pearson Chi-square test in terms of the number of resistant strains and susceptible strains in the integron-positive and integron-negative groups. Intermediate isolates were considered as non-susceptible isolates.

PFGE 

To assess whether the high frequency of 1.6 kb-integrons in our isolates from different sources was caused by the spread of a specific clone, 22 isolates of 1.6kb integron-positive P. aeruginosa were analyzed by PFGE profile. The PFGE profiles obtained after SpeI digestion showed 15 to 20 DNA fragments. Ten strains (3–5, 6–8, 15 and 16, 21 and 22) were divided into four groups (namely groups A, B, C, and D) and each group had an identical DNA fingerprinting pattern; the others had different restriction patterns (Figure 3). The six isolates in clones A and B were obtained from the intensive care unit (ICU), and the four isolates in clones C and D came from the critical care unit (CCU), which indicates the spread of particular clones among the patients.

  • View full-size image.
  • Figure 3. 

    PFGE of SpeI-digested chromosomal DNA. M, molecular size marker; lanes 1–22, P. aeruginosa isolates from different patients. PFGE patterns A, B, C, and D of the isolates are shown.

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Discussion 

Susceptibility testing of 71 P. aeruginosa isolates showed that all isolates were resistant to at least one of the antibiotics, and most of the isolates were multi-resistant. Sixty-four isolates (90.1%) were resistant to three or more antimicrobials. Resistance to cefotaxime, ceftriaxone, trimethoprim–sulfamethoxazole, and piperacillin was often detected.

The multiple resistance mechanism of P. aeruginosa is extremely complicated, and mainly involves plasmids and transposons, changes of outer membrane permeability, biofilm formation, and up-regulation of multi-drug efflux pumps, as well as integrons and gene cassette-mediated resistance.16 In this study, we examined 71 P. aeruginosa isolates from patients in Zhenjiang and found that 38.0% contained class 1 integrons. This proportion is comparable to that found in other studies.17, 18 Integrons are known to be associated with multi-drug resistance, especially class 1 integrons, which are widely distributed among Gram-negative bacteria; our study also shows that multiple resistance often appears in isolates containing integrons. The dfrA17–aadA5 array was detected in 22 P. aeruginosa strains and confers resistance to trimethoprim, as well as to streptomycin and spectinomycin. This array has been reported in other bacteria and in other regions, for example in China,19, 20 Korea,21 Australia,15 Norway,22 and Hungary.23 However, it was the first to be reported in P. aeruginosa isolates, which may indicate that the dfrA17–aadA5 array can disseminate by self-transferable plasmids in humans or animals and in different areas of the world.24 Therefore, the increase in clinically resistant isolates may be explained by antibiotic selective pressure and the widespread presence of integrons.16 In addition, we also identified two aminoglycoside genes within the integrons, aadB and aac6-II, and one beta-lactamase gene, blaPSE-1, coding for aminoglycoside resistance and beta-lactamase resistance, respectively. Many isolates also had other antibiotic resistances not encoded by integron-associated resistance gene cassettes. These resistances may have resulted from chromosomal mutation, plasmid acquisition, or the presence of other integrons besides class 1 types.

From 22 isolates carrying the 1.6-kb integron, 10 were genetically related (from the ICU and CCU). These results show that there were four drug-resistant strains disseminated in our hospital through different patients and clinical wards, indicating that an infection control omission was occurring at the hospital. PFGE demonstrated that inter-hospital transmission might have occurred prior to our study. A number of factors might have contributed to the dissemination of clonal isolates in the ICU and CCU, including the use of suboptimal aseptic techniques and infection control practices by healthcare workers, inadequate cleaning and disinfection of the environment and medical equipment, and understaffing. Moreover, the infected patients in the ICU and CCU were often exchanged. We have no information to support any of these possibilities at this time.

In conclusion, integrons were prevalent and played an important role in multidrug-resistant P. aeruginosa, which may provide some important surveillance information reflecting the antibiotic selective pressure in this specific region. It is necessary to focus on tracing the source of the infections to lessen the occurrence of multi-resistant strains.

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Acknowledgments 

We thank Shihe Shao (Jiangsu University) for helpful suggestions on this work.

Funding: This work was supported by the Foundation of Social Development Item of Jiangsu Province (grant No. BS2007041) and the National Nature Science Foundation of China (grant Nos 30471627 and 30300169).

Conflict of interest: No conflict of interest to declare. We received no financial support or benefits with the exception of those noted under the heading ‘funding’.

Ethical approval: Ethical approval for this study and agreement to participate from all patients were obtained from the Affiliated People's Hospital of Jiangsu University, which abides by the Helsinki Declaration on ethical principles for medical research involving human subjects.

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References 

  1. Sarlangue J, Brissaud O, Labrèze C. Clinical features of Pseudomonas aeruginosa infections. Arch Pediatr. 2006;13(Suppl 1):S13–S16
  2. Aarestrup FM, Wegener HC. The effects of antibiotic usage in food animals on the development of antimicrobial resistance of importance for humans in Campylobacter and Escherichia coli. Microbes Infect. 1999;1:639–644
  3. Rowe-Magnus DA, Mazel D. Resistance gene capture. Curr Opin Microbiol. 1999;2:483–488
  4. Weldhagen GF. Integrons and β-lactamases—a novel perspective on resistance. Int J Antimicrob Agents. 2004;23:556–562
  5. Thungapathra M, Amita , Sinha KK, Chaudhuri SR, Garg P, Ramamurthy T, et al. Occurrence of antibiotic resistance gene cassettes aac(6′)-Ib, dfrA5, dfrA12, and ereA2 in class I integrons in non-O1, non-O139 Vibrio cholerae strains in India. Antimicrob Agents Chemother. 2002;46:2948–2955
  6. Héritier C, Dubouix A, Poirel L, Marty N, Nordmann P. A nosocomial outbreak of Acinetobacter baumannii isolates expressing the carbapenem-hydrolysing oxacillinase OXA-58. J Antimicrob Chemother. 2005;55:115–118
  7. Leverstein-van Hall MA, Blok HE, Donders AR, Paauw A, Fluit AC, Verhoef J. Multidrug resistance among Enterobacteriaceae is strongly associated with the presence of integrons and is independent of species or isolate origin. J Infect Dis. 2003;187:251–259
  8. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. 16th Information supplement; M100-S16. Wayne, PA: CLSI; 2006.
  9. Hall RM, Collis CM. Antibiotic resistance in Gram-negative bacteria: the role of gene cassettes and integrons. Drug Resist Updates. 1998;1:109–119
  10. Shahada F, Chuma T, Tobata T, Okamoto K, Sueyoshi M, Takase K. Molecular epidemiology of antimicrobial resistance among Salmonella enterica serovar Infantis from poultry in Kagoshima, Japan. Int J Antimicrob Agents. 2006;28:302–307
  11. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol. 1995;33:2233–2239
  12. Tenover FC, Filpula D, Phillips KL, Plorde JJ. Cloning and sequencing of a gene encoding an aminoglycoside 6′-N-acetyltransferase from an R factor of Citrobacter diversus. J Bacteriol. 1988;170:471–473
  13. Casin I, Bordon F, Bertin P, Coutrot A, Podglajen I, Brasseur R, et al. Aminoglycoside 6′-N-acetyltransferase variants of the Ib type with altered substrate profile in clinical isolates of Enterobacter cloacae and Citrobacter freundii. Antimicrob Agents Chemother. 1998;42:209–215
  14. Livermore DM. beta-Lactamases in laboratory and clinical resistance. Clin Microbiol Rev. 1995;8:557–584
  15. White PA, McIver CJ, Deng Y, Rawlinson WD. Characterization of two new gene cassettes, aadA5 and dfrA17. FEMS Microbiol Lett. 2000;182:265–269
  16. Tenover FC. Mechanisms of antimicrobial resistance in bacteria. Am J Infect Control. 2006;34:S3–S10
  17. Gu B, Tong M, Zhao W, Liu G, Ning M, Pan S, et al. Prevalence and characterization of class I integrons among Pseudomonas aeruginosa and Acinetobacter baumannii isolates from patients in Nanjing, China. J Clin Microbiol. 2007;45:241–243
  18. Fonseca EL, Vieira VV, Cipriano R, Vicente AC. Class 1 integrons in Pseudomonas aeruginosa isolates from clinical settings in Amazon region, Brazil. FEMS Immunol Med Microbiol. 2005;44:303–309
  19. Zhang H, Shi L, Li L, Guo S, Zhang X, Yamasaki S. Identification and characterization of class 1 integron resistance gene cassettes among Salmonella strains isolated from healthy humans in China. Microbiol Immunol. 2004;48:639–645
  20. Chang LL, Chang TM, Chang CY. Variable gene cassette patterns of class 1 integron-associated drug-resistant Escherichia coli in Taiwan. Kaohsiung J Med Sci. 2007;23:273–280
  21. Kang HY, Jeong YS, Oh JY, Tae SH, Choi CH, Moon DC, et al. Characterization of antimicrobial resistance and class 1 integrons found in Escherichia coli isolates from humans and animals in Korea. J Antimicrob Chemother. 2005;55:639–644
  22. Lindstedt BA, Heir E, Nygard I, Kapperud G. Characterization of class I integrons in clinical strains of Salmonella enterica subsp. enterica serovars Typhimurium and Enteritidis from Norwegian hospitals. J Med Microbiol. 2003;52:141–149
  23. Nógrády N, Gadó I, Tóth A, Pászti J. Antibiotic resistance and class 1 integron patterns of non-typhoidal human Salmonella serotypes isolated in Hungary in 2002 and 2003. Int J Antimicrob Agents. 2005;26:126–132
  24. Guerra B, Junker E, Schroeter A, Malorny B, Lehmann S, Helmuth R. Phenotypic and genotypic characterization of antimicrobial resistance in German Escherichia coli isolates from cattle, swine and poultry. J Antimicrob Chemother. 2003;52:489–492

PII: S1201-9712(09)00003-4

doi:10.1016/j.ijid.2008.11.014

International Journal of Infectious Diseases
Volume 13, Issue 6 , Pages 717-721, November 2009

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