|Year : 2013 | Volume
| Issue : 2 | Page : 142-147
Detection of the common resistance genes in Gram-negative bacteria using gene chip technology
C Ting, A Jun, Z Shun
Clinical Research Center of Ningbo No. 2 Hospital, Ningbo, Zhejiang, 315010, China
|Date of Submission||25-May-2012|
|Date of Acceptance||13-Nov-2012|
|Date of Web Publication||19-Jul-2013|
Clinical Research Center of Ningbo No. 2 Hospital, Ningbo, Zhejiang, 315010
Source of Support: This work was fi nancially supported by Clinical research center of Ningbo No.2 Hospital, Conflict of Interest: None
Objective: To design a resistance gene detection chip that could, in parallel, detect common clinical drug resistance genes of Gram-negative bacteria. Materials and Methods: Seventy clinically significant Gram-negative bacilli (Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Pseudomonas aeruginosa, Acinetobacter baumannii) were collected. According to the known resistance gene sequences, we designed and synthesized primers and probes, which were used to prepare resistance gene detection chips, and finally we hybridized and scanned the gene detection chips. Results: The results between the gene chip and polymerase chain reaction (PCR) were compared. The rate was consistently 100% in the eight kinds of resistance genes tested (TEM, SHV, CTX-M, DHA, CIT, VIM, KPC, OXA-23). One strain of Pseudomonas aeruginosa had the IMP, but it was not found by gene chip. Conclusion: The design of Gram-negative bacteria-resistant gene detection chip had better application value.
Keywords: Gram-negative bacteria, gene chip, resistance gene
|How to cite this article:|
Ting C, Jun A, Shun Z. Detection of the common resistance genes in Gram-negative bacteria using gene chip technology. Indian J Med Microbiol 2013;31:142-7
|How to cite this URL:|
Ting C, Jun A, Shun Z. Detection of the common resistance genes in Gram-negative bacteria using gene chip technology. Indian J Med Microbiol [serial online] 2013 [cited 2020 Aug 5];31:142-7. Available from: http://www.ijmm.org/text.asp?2013/31/2/142/115230
| ~ Introduction|| |
With the third-and fourth-generation cephalosporins, carbapenems, and other extended-spectrum fluoroquinolone antimicrobial drugs widely used, bacterial resistance is becoming more serious all over the world.  Traditional culture-based pathogen detection method was used, through physiological and biochemical identification and antimicrobial susceptibility disk diffusion method, to determine the bacterial species and drug resistance. This method is complex and more time consuming. Sensitivity and specificity of detection depend on culture conditions, and, moreover, its results are lagging behind. Therefore, on the basis of epidemiological data, clinical symptoms, and signs and imaging characteristics, clinicians often estimate the potential and sensitivity of pathogens to drugs. Pathogens may be covered with the application of empirical antibiotics until culture results return, and then adjusted to the antimicrobial medication, which severely limits the clinicians' time and effective choice for patients sensitive to antibiotics, in particular, timely treatment of severe infectious diseases.
In recent years, the gene chip, also known as a DNA chip or a DNA microarray or a bio-chip, has been developed.  With the application of a known nucleic acid sequence as a probe, the gene chip hybridizes the complementary target nucleotide sequence and carries out qualitative and quantitative analysis of the genes by detecting hybridization signals. Chip technology has the advantages of high sensitivity, specificity, and simultaneous analysis of multiple genes to save time and improve detection efficiency. In this study, in order to establish a rapid and efficient detection method to support clinical guidance and help timely and accurate choice of antibiotics, a Gram-negative bacteria resistance gene chip was synthesized that could concurrently detect the current common clinical resistance genes (TEM, SHV, CTX-M, DHA, CIT, IMP, VIM, KPC, OXA-23).
| ~ Materials and Methods|| |
Five kinds of common clinical Gram-negative bacteria (Klebsiella pneumoniae, Escherichia More Details coli, Enterobacter cloacae, Pseudomonas aeruginosa, Acinetobacter baumannii) were selected for detection by the resistance gene chip. All strains were identified by the French Merieux VITEK-II microbial analysis systems purchased from French Merieux company. A total of 70 strains of multi-drug-resistant bacteria were selected and were not repeated, including 17 strains of imipenem-resistant K. pneumoniae, 17 strains of imipenem-resistant P. aeruginosa, 7 strains of imipenem-resistant A. baumannii, 16 strains of ceftazidime-resistant E. coli, and 13 strains of ceftazidime-resistant E. cloacae. Standard strains were E. coli ATCC25922 (negative control) and ATCC35218 (positive control). All bacteria were purchased from the Chinese Medical Culture Collection Management Center.
Cultivation of strains and DNA extraction
According to protocols from ATCC and Chinese Medical Culture Collection Management Center, cryopreserved bacteria were inoculated and cultured in blood agar, were subjected to passage once, and a single colony was picked to modulate bacterial suspension turbidity of 1.0. In accordance with DNeasy Tissue Kit (Qiagen, Crawley, UK), DNA was extracted and the concentration and quality were measured using a nucleic acid concentration detector ND-1000 Spectrophotometer purchased from NanoDrop company.
Design and synthesis of resistance gene primers
The current common clinical resistance genes (TEM, SHV, CTX-M, DHA, CIT, IMP, VIM, KPC, OXA-23) were selected for the microarray detection range of resistance genes. Polymerase chain reaction (PCR) primers and probes were designed by Shanghai Biochip National Engineering Research Center and synthesized by Shanghai Invitrogen Corporation.
PCR amplification system: 1.5 μl 10 × PCR buffer, 0.6 μl MgCl 2 (25 mM), 0.2 μl dNTP mixture (10 mM), 0.2 μl primer 1, primer 2 (20 pm), 0.2 μl Taq DNA polymerase (5 U/μl), 1.0 μl (200 ng/μl) template DNA, and then sterile distilled water was added for 15 μl. PCR conditions were maintained at 95 °C 5 min, (95 °C 30s, 56 °C 40 s, 72 °C 50 s) × 30 cycles, 72°C 10 min. PCR product was run on 2% agarose gel electrophoresis, EB staining, with GIS2010 gel imaging system observations, and a corresponding size of the band was judged as positive. Primer names and stripe sizes are shown in [Table 1] (primers probe sequences could be obtained from the authors).
Two groups for multiplex PCR are shown in [Table 1]. PCR amplification system set at 1.5 μl 10 × Titanium Taq PCR buffer, 0.2 μl dNTP mixture (10 mM), 1.0 μl primer mixture (geometric mixing), 0.1 μl Titanium Taq DNA polymerase, 1.0 μl (200 ng/μl) template DNA, and finally sterile distilled water was added for 15 μl. PCR conditions were maintained at 95°C 5 min, (95°C 30 s, 68°C 30 s) × 30 cycles, 68°C 3 min. PCR product was run on 2% agarose gel electrophoresis, EB staining, with GIS2010 gel imaging system observations.
Preparation and hybridization of microarray
Design and synthesis of hybridization probes
These probes were designed by Shanghai Biochip National Engineering Research Center and synthesized by Shanghai Invitrogen Corporation.
The test sample on the chip
Chips were purchased from Boao Biotechnology Compay, Shanghai city, in China. After probe synthesis, a concentration of 100 pmol/μl was developed. According to the array arrangement of gene chip [Table 2], 5 μl from each of the probe solution were mixed with the hybridization buffer and loaded into 384-well plates with the chip spotting instrument (OmniGridTM 100 microarrayer, USA) gene chip point system. There were ten reactions per chip area, and each reaction was repeated three times. The resistance genes chip arrangement is shown in [Table 2].
Preliquid configuration: 312 μl water, 40 μl 20 × SSC, 40 μl 100 × BSA, 8 μl 10% SDS, and finally water up to 400 μl was added. Preliquid was evenly added to the microarray hybridization region, at room temperature for 30 min.
PCR product purification and SBE markers
SAP and ExoI were used for purifying the multiplex PCR products as follows: Take 4 μl DNA, add 1 μl SAP and 1 μl ExoI, incubate at 37°C for 30 min, and finally at 85°C for 10 min. The total volume of SBE labeling reaction was 15 μl, and the reaction system was 3 μl purified product, 1.5 μl 10 × ThermoPol Reaction Buffer, 0.2 μl Thermo Sequenase DNA polymerase (sequencing enzymes), 0.2 μl labeled primers, 0.2 μl ddATP-Cy3 (1/10). SBE labeling reaction conditions were maintained at 95°C for 5 min, (95°C 30 s, 60°C 30 s, 72°C 20 s) × 40 cycles, 72°C for 5 min.
DNA microarray hybridization
The SBE reaction products, the positive quality control, and the hybridization solution were mixed for resistance gene chip hybridization reaction, and hybridized at 48°C for 2 h, and finally the chip was cleaned after hybridization.
Scan the chip and analyze
With a microarray scanner (GenePix 4000B), scan the chip and analyze the fluorescence signal intensity by means of the GenePix Pro 6.0 software.
| ~ Results|| |
In vitro susceptibility test results
The study involved five kinds of strains of Gram-negative bacteria, which were pan-resistant to β-lactamases antibiotics such as ampicillin, cefazolin, ceftazidime, ceftriaxone, aztreonam, quinolones, amino glycosides, and carbapenem antibiotic. The resistance rates of these bacteria were above 50%, and only the rate of sulfa drug resistance was relatively low. Among them, imipenem-resistant K. pneumoniae and P. aeruginosa were not seriously resistant to cotrimoxazole, and the resistance rate of imipenem-resistant K. pneumoniae and P. aeruginosa to other drugs was more than 80%; 7 strains of imipenem-resistant A. baumannii were generally resistant to 17 kinds of antimicrobial drugs; ceftazidime-resistant E. coli was relatively sensitive to cefotetan, piperacillin/tazobactam, imipenem, kanamycin, and nitrofurantoin; the sensitivity of the ceftazidime-resistant E. cloacae to cefepime and imipenem was higher than that of the other four strains. Susceptibility test results are shown in [Table 3].
|Table 3: Drugs resistance of 5 kinds of Gram-negative bacilli to antimicrobial drugs|
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Single-plex polymerase chain reaction results
In five kinds of experimental strains, the detection rate of β-lactamases resistance genes was higher; the TEM-type resistance gene was generally detected in all the strains, and detection rates of imipenem-resistant A. baumannii and K. pneumoniae were 100% and 94.12%, respectively; the SHV-type resistance gene was mainly present in the imipenem-resistant K. pneumoniae and ceftazidime-resistant E. cloacae, and was not detected in the ceftazidime-resistant E. coli and imipenem-resistant A. baumannii; the DHA and CIT-type resistance genes were highly detected only in imipenem-resistant K. pneumoniae, and their detection rates were 88.24% and 94.12%, respectively; the CTX-M and KPC-type resistance genes were mainly present in the imipenem-resistant K. pneumoniae and ceftazidime-resistant E. coli; the OXA-23-type resistance gene was detected in all seven imipenem-resistant A. baumannii; the metal type β-lactamases resistance genes IMP and VIM were highly detected only in P. aeruginosa. PCR results are shown in [Table 4].
Multiplex polymerase chain reaction results
A variety of drug-resistant genes coexisted in imipenem-resistant K. pneumoniae, and six or more resistance genes were detected in 15 of 17 strains; two or three kinds of resistance genes simultaneously existed in ceftazidime-resistant E. coli. Five strains had a resistance gene for TEM or CTX-M type; and two or more β-lactamases resistance genes existed in ceftazidime-resistant E. cloacae; only 1 of 17 strains of imipenem-resistant P. aeruginosa had three kinds of resistance genes, TEM, SHV, and VIM, at the same time, and there were eight strains in which none of the resistance genes involved in the study were detected, and others had only one resistance gene VIM or TEM detected; there were simultaneous TEM and OXA-23-type resistance genes in all seven strains of imipenem-resistant A. baumannii.
Gene chip hybridization
The study designed and prepared the resistance gene chip, which, except the resistance gene IMP, detected all other resistance genes, and these results were consistent with the PCR results. These results are shown in [Table 5].
|Table 5: Gene chip detection of five kinds of bacterial resistance genes|
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Comparison and analysis
Evaluation of the reliability of experimental methods
The gene chip result had 98.57% the same to PCR test of the resistance gene IMP, and the remaining resistance genes had 100% the same to PCR tests; the Kappa value of IMP was 0 and the Kappa values of the other resistance genes were 1. In summary, except for the resistance gene IMP, consistency of both methods of the other resistance gene was high.
Evaluation of the authenticity of experimental methods
DNA microarray results were in good agreement with PCR test results, and all resistance genes, except that the sensitivity of detection of IMP was 0, had 100% sensitivity; its specificity was 100%. The resistance gene chip designed in the study could have a better application.
| ~ Discussion|| |
The clinical tests of microbial resistance were mainly based on the basic phenotypic testing. Owing to the diversity of resistance mechanisms, the expression of some resistance genes may be silent in the bacteria, but the bacteria had the potential to resistance and, in the appropriate environmental conditions, could be transformed into drug-resistant bacteria or could spread the resistance genes to other bacteria. PCR hybridization analysis detecting bacterial resistance gene is the most common method. However, to screen one or several resistance genes in numerous resistance genes, this method is usually time consuming and requires repeated detection. Microarray assay has high throughput and can detect multiple resistance gene simultaneously, which significantly increases the detection efficiency.
Domestic and foreign researchers have tried a different detection range of gene chips. SHEN D X  designed gene chips detecting ESBLs and AmpC enzymes produced by E. coli, K. pneumoniae, and acid-producing Klebsiella, and they detected a total of 225 of the above three strains, except for some Amp C-positive phenotype of E. coli and K. pneumoniae, and the chip was able to detect all phenotypes of Extended Spectrum Beta-Lactamases (ESBL)-positive strains, while classifying the CTX-M genes. Jan Weile et al.,  developed a kind of gene chip detecting P. aeruginosa antibiotic resistance and virulence factor and could complete the whole process in 5 h, including DNA extraction, gene amplification, fluorescent labeling, and hybridization, and its the sensitivity and specificity were 89% and 83%, respectively. Recent studies , reported micro-chips for drug resistance genes of E. coli and Salmonella More Details that could detect 47 resistance genes including genes resistant to aminoglycosides, trimethoprim, sulfonamides, tetracycline, and β-lactams as well as extended-spectrum-β-lactamase, and the test results of the micro-chips had no significant difference compared with the PCR method. Cassone et al.,  designed a kind of gene chip covering 65 macrolide resistance genes of eight species of bacteria. HongJu et al., designed a kind of gene chip that could detect several types of resistant genes SHV and CTX-M in the super broad-spectrum β-amine enzymes, and this method only takes 6-8 h to complete from the specimen processing to the bacteria identification and resistance spectrum testing to save the time for the etiology testing for patients with severe infection.  Yajie et al., designed a kind of gene chip that could synchronously detect the common Gram-positive bacteria identification and resistance testing, and its sensitivity and specificity were higher, and the gene chip was a better assistant to clinical physicians for diagnosis and treatment. 
In the present study, classic molecular biology method served as a control and was used to evaluate the application value of present microarray in the detection of resistance genes. The bacteria in the present study were confirmed by the French Merieux VITEK-II Microbiology Analysis System to analyze sensitivity. The bacteria were found to be resistant or multi-resistant. Microarray analysis showed that the detection rate of super-resistant extended-spectrum β-lactamases genes (TEM, SHV, CTX-M) were the highest and more than 90% in K. pneumoniae, and AmpC-resistant genes were mainly detected in K. pneumoniae. In this study, the detection rates of the carbapenem-resistant gene KPC in K. pneumoniae, E. coli, and E. cloacae were 100.00%, 37.50%, and 7.69%, respectively, but the carbapenem-resistant gene KPC was not detected in A. baumannii and P. aeruginosa. Less resistance genes were detected in P. aeruginosa, and only the metal-type β-lactamases resistance genes IMP and VIM were detected in the bacteria, but the detection rate was low, indicating that in P. aeruginosa of the region, the β-lactam antibiotics could still be of clinical use for P. aeruginosa infection as the preferred antimicrobial agents.
In summary, in the study, we designed a kind of resistance gene chip that could detect resistance genes of a variety of common clinical Gram-negative bacteria in parallel, except that IMP gene was not detected, and other resistance gene detection results were consistent with the PCR results. In future studies, we will increase the number of resistance genes in the microarray and attempt to simultaneously identify bacteria and detect the resistance gene. This could broaden the range of detection with microarray and the gene chip could be a useful tool for rapid diagnosis and treatment of clinical infectious diseases.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]