Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 29, Num. 2, 2011, pp. 118-123

Indian Journal of Medical Microbiology, Vol. 29, No. 2, April-June, 2011, pp. 118-123

Original Article

High frequency of integrons related to drug-resistance in clinical isolates of Acinetobacter baumannii

Z Chang-Tai¹, L Yang², H Zhong-Yi³, Z Chang-Song², K Yin-Ze², L Yong-Ping², D Chun-Lei¹

¹ Department of Laboratory Medicine, Changzhou Tumor Hospital Soochow University, Changzhou 213001, China
² Department of Oncological Medicine, Changzhou Tumor Hospital Soochow University, Changzhou 213001, China
³ Department of Laboratory Medicine, Changzhou Tumor Hospital Soochow University, Changzhou 213001; Shanghai Key Laboratory of Tuberculosis, The Pulmonary Hospital, Tongji University School of Medicine, Shanghai 200433, China

Correspondence Address: D Chun-Lei Department of Laboratory Medicine, Changzhou Tumor Hospital Soochow University, Changzhou 213001 China dongchunlei116@163.com

Code Number: mb11029

PMID: 21654104

DOI: 10.4103/0255-0857.81784

Abstract

Keywords: Acinetobacter baumannii, drug resistance, multi-drug resistance, integrons

Introduction

It is reported that Acinetobacter baumannii could cause various nosocomial infectious diseases such as ventilator-associated pneumonia, septicemia, secondary meningitis, and urinary tract infections. ^[1] In addition, A. baumannii could cause the outbreaks of nosocomial infection. Up to now, more and more multi-drug-resistant A. baumannii strains were isolated from clinical species; especially, the appearance of carbapenem-resistant A. baumannii has caused the troubles of clinic treatment. ^[2] More seriously, pan-resistant A. baumannii strains have been found in a few regions recently. ^[3],[4] Therefore, it is very important to explore A. baumannii drug resistance and its relevant mechanisms. In this study, we analyzed the characteristics of the drug resistance in A. baumannii clinical isolates. Meanwhile, we explored the relationship between integrons and A. baumannii drug resistance.

Materials and Methods

Materials

Mueller Hinton agar and non-fermenting bacteria identification cards were provided by France bio-Merieux. Paper discs for determining the drug sensitivity was from Oxoid Company. DNA extraction kits, marker, and PCR-related reagents were kindly offered by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd.

Collection, identification of strains and antimicrobial susceptibility testing

A. baumannii isolates were collected from different clinical specimens of inpatients in East China (Changzhou and Tongling) between January 2006 and December 2008 (the repeated strains from the same patients were excluded). The samples include 69 sputa, 20 wounds, 4 throat swabs, and 3 bloods from 14 departments [Figure - 1]. All strains were identified using non-fermenting bacteria identification cards by Vitek-32 automicro-biological system. According to criteria of Clinical and Laboratory Standards Institute (CLSI) 2005 version, antimicrobial susceptibility testing was performed using disk-diffusion method (Kirby-Bauer). The Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC27853 strains were used for quality control.

DNA extraction

Fresh overnight cultures grown on Luria-Bertani (LB) agar plates were prepared for use as a source of template DNA. Extracted DNA was resolved with 100 μl of TE buffer (10 mM Tris, 1 mM EDTA [pH 8.0]) supplemented with 10 μg of RNase. After purifying DNA, the samples were stored at −20 °C.

Detection of integrons

Reaction volume of PCR amplifications was 20 μl containing 5 μl of template DNA, 0.2 mM (each) deoxynucleoside triphosphate (dNTP), 2 μl of 10× PCR buffer, 1 U of Taq polymerase (Perkin-Elmer [PE] Applied Biosystems, Foster City, Calif.), 1.5 mM MgCl ₂, and 1.25 μM each primer. PCR amplification was performed with the GeneAmp PCR System 9700 thermal cycler (PE Applied Biosystems). Amplified products were resolved by electrophoresis at 120 V for 2 h on 2% agarose gels with 0.5× Tris-borate-EDTA buffer containing ethidium bromide and were visualized under UV light.

Integrons PCR amplification was performed with: P1 5′-TGCGGGTYAARGATBTKGATTT-3′; P2 5′-CARCACATGCGTRTARAT-3′. ^[5] The primers could amplify three classes of integrons and the expected amplification fragment band size was 491 bp. PCR amplification was performed for 35 cycles: 30 s of denaturation at 94 °C, 30 s of annealing at 55 °C, and 30 s of extension at 72 °C. Amplified DNA products were analyzed by conventional agarose gel [1.5%, w/v)] electrophoresis. The PCR products were digested with 5 U Hinf I at 37 °C for 4 h to distinguish the types of integrons (Classes 2,3 integrons could be digested by restriction enzyme Hinf I).

Identification of class 1 integrons

In order to improve specificity, two pairs of class 1 integrons specific primers were use to identified the positive integrons by PCR amplification. Primer 1: P1 5′-CCCGAGGCATAGACTGTA-3′; P2 5′-CAGTGGACATAA GCCTGTTC-3′. ^[6] The expected band size of PCR products is 160 bp. Primer 2: P1 5′-TGATGGCGACGCACGAC-3; P2 5′-TTGGGCAGCAGCGAAGT-3′. ^[7] The expected band size is 587 bp. PCR Amplification used 50 μl mixed reaction volume containing: 10× buffer 5 μl, dNTPs 200 nmol/l, MgC1₂ 2 mmol/l, primer 10 pmol, Taq DNA polymerase 1.5 μl, template 2 μl. Cycle parameters were as following: 95 °C degeneration 5 min; 94 °C 30 s, 50 °C 40 s, 72 °C 50 s, cycle 40; at 72 °C for 5 min. Amplified PCR products were assayed with the 20 g/l agarose gel electrophoresis and purified by PCR commercial purification kits. Finally, five PCR products of 587 bp selected randomly were sequenced commercially by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd.

Amplification and sequencing of gene cassettes

Gene cassettes of the integrons were amplified with primer: P1 5′-GGCATCCAAGCAGCAAG-3; P2 5′-AAGCAGACTTGACCTGA-3′. ^[8] 50 μl PCR reaction volume contained: 10× buffer 5 μl, dNTPs 200 nmol/L, MgC12 2 mmol/L, primer 20 pmol, Taq DNA polymerase 1.5 μl, template 2 μl. Cycle conditions were as follows: 95 °C5 min; 94 °C 30 s, 50 °C 40 s, 72 °C 50 s, cycle 38; 72 °C for 5 min. PCR products were detected by agarose gel electrophoresis and purified by purification kits. PCR amplified products of 12 integrons gene cassettes were sequenced commercially by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd., China.

Statistical analysis

The analyses of drug sensitivity data used WHONET 5.3 software. Statistical analyses were conducted by using SPSS 11.5.

Results

Antimicrobial susceptibility testing

Overall, the drug-resistance rates in integrons-positive A. baumannii to 14 kinds of antibacterials were higher than those in integrons-negative A. baumannii. Except imipenem and cefoperazone/sulbactam, the drug-resistance rates of the A. baumannii clinical isolates to other 15 kinds of antibacterials all surpassed 30% [Table - 1]. The multi-drug-resistance rate in integrons-negative A. baumannii was only 23.3% (7/30), while the rate in integrons-positive group was 77.3% (51/66). There was a significant difference between two groups (P < 0.05).

Integrons PCR detection

Sixty six of 96 strains were confirmed to carry integrons. PCR amplification band size was 491 bp [Figure - 2]. In addition, all the PCR amplified products could not be digested by Hinf I, which showed that classes 2 or 3 integrons did not be found in our experiments.

Classification of integrons

Integrons classification amplification of two pairs of primers for detecting class 1 integrons both showed that all the above 66 strains emerged positive bands by agarose gel electrophoresis. The products sizes were 160 bp and 587 bp, respectively [Figure - 3] and [Figure - 4]. Via blast search in GenBank, the sequencing results of the PCR amplification products confirmed that the sequenced genes had higher consensuses with class 1 integrons [Figure - 5], [Figure - 6]. A new gene sequence was submitted to the GenBank sequence database and assigned the accession number FJ573247 ( http://www.ncbi.nlm.nih.gov/ nuccore/221145747).

Amplification and sequencing of class 1 integrons gene cassettes

PCR amplification bands size ranged from 0.3 to 2.5 kb [Figure - 5]. Via blast search in GenBank, DNA sequencing results confirmed that 9 of 12 integrons harboured 7 different gene cassettes (aacA4, catB3, dfrA1, blam-1, orfX, aadA1 and sat2). The cassette arrays aacA4-catB3-dfrA1 were found in five integrons-positive strains. A new gene sequence was submitted to the GenBank sequence database and assigned the accession number FJ573248 ( http://www.ncbi.nlm.nih.gov/nuccore/221145748).

Relationships between drug resistances with integrons in A. baumannii

The drug-resistance rates in integrons-positive A. baumannii strains to 14 kinds of antimicrobial agents were higher than those in integrons-negative strains [Table - 2].

Discussion

A. baumannii is an aerobic, glucose-non-fermenting, gram-negative bacterium, which has recently emerged as a serious opportunistic pathogen. In this study, 96 A. baumannii isolates were mainly from ICU, neurosurgery, neurology and burn unit, which indicated that patients with severe diseases were more likely to be infected by A. baumannii Our investigation demonstrated that, except imipenem and cefoperazone/sulbactam, the resistance rates of 96 A. baumannii isolates to other 15 kinds of antimicrobial agents all surpassed 30%, which showed severe A. baumannii drug resistance appeared in East China. Previous studies confirmed that A. baumannii drug resistance involve a variety of mechanisms, including the acquisition of lactamases, AmpC stable depression, decreased permeability, altered penicillin-binding proteins (PBPs) and efflux pump over expression. ^[9],[10] In this study, we concluded that integrons could play an important role in A. baumannii resistance in East China. In fact, integrons is a kind of movable gene elements containing three highly conserved regions at 5′ and 3′ ends and the gene codes between the 5′ and 3′ ends form a gene horizontal transmission system. Previous results revealed that integrons could capture and integrate the exogenous gene cassettes of determining drug resistance and transmit drug resistance among different strains and even among different bacteria species. ^{[11],[12],[13]} Integrons could local in a plasmid or on the chromosome. Based on the homology differences of 5′-conserved DNA sequence of integrase, integrons were classified into four classes (class 1-4 integrons). Homology among four classes integrons vary from 45% to 58%. ^{[14],[15],[16]} Commonly class 4 integrons only appeared in vibrio cholerae, so classes 1, 2 and 3 integrons were more often taken as research objectives about drug resistance. ^{[16],[17],[18]} However, the distributions frequencies and the characteristics of integrons encoded gene cassettes in A. baumannii in detail still need to further research.

In this study, we demonstrated that A. baumannii clinical isolates in East China carried high frequencies integrons (66/96, 68.8%), which were higher than those in the previous reports. ^[19],[20] It indicated that the distributions of integrons in A. baumannii just like phenotypic resistance might emerge geographic differences. The analyses of the results showed that the drug resistance in integrons-positive A. baumannii to most common antibacterial agents increased compared to integrons-negative, which revealed that integrons was closely related with A. baumannii drug resistance. In addition, the multi-drug-resistance rate in integrons-negative A. baumannii was only 23.3% (7/30), while the multi-drug-resistance rate in integrons-positive group was 77.3% (51/66). It demonstrated integrons was also associated with A. baumannii multi-drug resistance.

By restriction enzyme digestion and gene sequencing, all the integrons belonged to class 1 integrons and no classes 2 or 3 integrons were found in our investigation. DNA sequencing of integrons cassettes showed that 9 of 12 integrons contained 7 different gene cassettes (aacA4, catB3, dfrA1, blam-1, orfX, aadA1 and sat2). The cassette arrays aacA4-catB3-dfrA1 were most frequently found. We also found that the drug resistance features prognosticated by the integrons cassettes were completely identical to phenotypic resistance. Thus, we believed that integrons could be taken as a marker of drug-resistance and multi-drug resistance for the surveillance of A. baumannii clinical strains. Meanwhile, we also noticed some phenotypic resistances couldn′t be prognosticated by integrons cassettes gene. So, we believed that integrons could not prognosticate all the drug resistance proprieties of A. baumannii, only reveal partial antimicrobial resistance profiles.

In addition, we also believed that integron plays an important role in A. baumannii genome evolution. However, further exploring the function of integrons in A. baumannii is important for clarifying the molecular mechanisms of A. baumannii resistance and preventing the spreads of drug resistance. In future, the following subjects about integrons are expected to be explored: A. under what conditions are integrons easier to capture or remove gene cassettes? B. why are the combination frequencies of integrons gene cassettes different? C. what is the relationship between integrons evolution and inductive drug resistance.

References

Copyright 2011 - Indian Journal of Medical Microbiology

The following images related to this document are available:

Photo images