Study On Carbapenemase Genes And Structure Of Mobile Genetic Resistance Elements Among Gram Negative Superbugs

Background and objectiveIn recent years, large usage of carbapenems to treat Gram-negative bacterial infections has led to the increasing emergence of carbapenem-resistant Gram negative superbugs. It is a challenge for clinical infection treatment, and the carbapenems resistance mechanismshas of bacteria have gained worldwide attention. The carbapenem resistance mechanisms of bacterial are complex, but carbapenemases is the most important reason. The evolution of MDR is relatively fast, as the main driving force is lateral gene transfer (LGT), a process influenced by a wide range of mobile genetic elements, such as conjugative plasmid, transposons, integrons or insertion sequence common region (ISCR). At home and abroad, research mainly focus on the integration carrying a variety of drug-resistant gene cassette and insertion sequence common region (ISCR1) carrying more gene cassette and allowing a greater ability to spread.This study mainly detected the common carbapenemase gene, class I integron and ISCCR1among clinical gram-negative superbug isolates, aiming to understand the resistance mechanism of superbugs. NDM or KPC positive isolates were subjected to analyse the location of NDM and KPC genes, genetic environment of KPC-2gene and MLST analysis in order to understand transfer mechanism of resistance gene. In addition, we established a rapid, sensitive and specific duplex real time PCR for simultaneous detection of two genes NDM and KPC in a single system.Methods1. Bacterial strains, antimicrobial susceptibility test and extraction of genomic DNA Enterobacteriaceae, Acinetobacter and Pseudomonas aeruginosa isolates were collected at Nanfang hospital from July2011to July2012. The isolates were collected from various clinical specimens from diverse units of the hospital. All bacterial identification and susceptibility testing were performed using the BD Phoenix100Automated Microbiology System (Benton, Dickinson and Co., Franklin Lakes, NJ, USA). Whole genomic DNA was extracted using a phenol-chloroform extraction method.2. carbapenemase gene detection The NDM, KPC, VIM, IMP, SPM, GES and OXA-48gene was identified by PCR in all collected isolates. As for Acinetobacter isolates, OXA-23-like, OXA-24-like, OXA-51-like and OXA-58-like genes were also detected. NDM or KPC-positive isolates were confirmed by16S rRNA sequence analysis. PCR amplifications were sequenced to compare them with sequences available in the NCBI database (www.ncbi.nlm.nih.goy).3. Horizontal transmission patterns of special resistance genes plasmid conjugation experiment was performed on NDM or KPC positive strains to explore the location of these genes. Genetic environment of KPC gene and MLST analysis on KPC-positive isolates were carried out in order to compare with data reported at home and abroad.4. Detection of mobile genetic elements Class I integrase and ISCR1were determined by PCR. The positive isolates were further detect the presence of class I integron and ISCR1-linked genes. The variable region of class I integron and ISCR1-linked genes amplicons were then characterized by restriction fragment length polymorphism (RFLP) with Hinf I and RasI and DNA sequencing analysis. The resulting DNA sequences were analysed with the BLAST algorithm in GenBank. The region between internal truncated copy of 3’-CS of class I integron and ISCCR1transposase gene was studied by PCR and sequenced. The putative structures of comoplex class I integron were analysed.5. The established of duplex real-time PCR for simultaneous detection of NDM and KPC genes Based on the comprehensive analyses and alignments of both the two gene families, primers and probes for the duplex real-time PCR assay were specifically designed to amplify all alleles of each gene family described above using Beacon Designer software (Premier Biosoft, USA). Recombinant plasmid pNDM-1and pKPC-2were used for qPCR optimization. Sensitivity and reproducibility of the of the assay were estimated by serial10-fold dilution experiments using the mixture of recombinant plasmid pNDM-1and pKPC-2. The plasmids of NDM and KPC with two different concentrations were used as templates in the real-time PCR reactions to evaluate the reproducibility. Each concentration was repeated three times on three consecutive days, the repeatability and stability were evaluated by calculating CV of CT values. The analytical specificity was applied on several standard strain, including E. coli ATCC25922, K. pneumoniae ATCC700603,S. aureus ATCC25923,P. aeruginosa ATCC27853).220previously identified by conventional PCR clinical isolates were tested by duplex qPCR.Results1. detection of carbapenemase gene Among16bapenem-resistant Enterobacteriaceae isolates,10isolates, including Enterobacter cloacae(n=4), Klebsiella pneumoniae(n=3), Klebsiella oxytoca(n=1), Enterobacter aerogenes(n=1) and Enterobacter hormaechei(n=1) were found to be blaNDM-1positive. One K. pneumonia isolate were positive for both KPC-2and IMP-4. One Enterobacter aerogenes isolates carried IMP-4. A total of six distinct carbapenemase genes were detected among811non-fermentative bacterial. For Acinetobacter baumanmii,25isolates carrying OXA-23-like,139isolates carrying OXA-51-like and6isolates carrying OXA-58-like were found among strains with susceptible to carbapenem and129isolates carrying OXA-23-like,2isolates carrying OXA-24-like,126isolates carrying OXA-51-like and15isolates carrying OXA-58-like were found among strains with non-susceptible to carbapenem. Especially,48isolates co-produce OXA-23-like and OXA-51-like, one co-produce OXA-24-like and OXA-51-like and14isolates co-produce OXA-58-like and OXA-51-like. For Pseudomonas aeruginosa, four isolates with susceptible to carbapenem harbour IMP,33and10isolates with non-susceptible to carbapenem harbour IMP and VIM respectively.2. Horizontal transmission patterns of the special genes Four of ten NDM-positive isolates successfully transferred their resistance gene to the recipient strain, furthermore, for the complex class1integron identified in Enterobacter hormaechei located on the plasmid carrying NDM-1. the gene KPC-2but IMP-4identified in Klebsiella pneumoniae was found in a conjugational plasmid. the Klebsiella pneumoniae producing KPC-2belong to sequence type530and this rare ST type is for the first report.3. Characteristic of class I integron Ten of sixteen carbapenem resistant Enterobacteriaceae strains harboured integrase and integron. Sequence analysis showed that five different gene cassette arrays were found. Klebsiella pneumoniae Car1, Klebsiella oxytoca Car4and Enterobacter cloacae Car12carried aar-3+dfrA27gene cassette array; four Enterobacter cloacae carried aadB+aadAl gene cassette array; Enterobacter hormaechei Car9carried dfrA16+aadA2gene cassette array; Enterobacter cloacae Car14carried arr3+aadAl gene cassette array; Klebsiella pneumoniae Car16carried dfrA17+aadA5gene cassette array. Two hundred and thirty of391Acinetobacter baumannii isolates harboured6distinct gene cassette arrays. The details are as follows:one hundred and sixty-four isolates carried aacA4+catB8+aadAl; forty-seven isolates carried aacC1+orfP+orfP; twelve isolates harboured arr-3+aacA4; one harboured blaPSE; three isolates harboured dfrA17+aadA5; two isolates harboured aacA4. Sixty of420Pseudomonas aeruginosa harboured14distinct gene cassette arrays. The details are as follows:ten isolates carried aacA4+aadA2; three isolates carried aadAl; the arrays aadA2, cmlAl+aadA2, tetR and oxa-10each for one was found; the arrays aadA4and aacA4each for five was found; the arrays aadB+cm1A6, aadA7and dfrA17+aadA5each for four was found; thirteen isolates carried IMP-9+aadA2; seven isolates carried aacA4+catB8+aadAl.4. Characteristic of variable region of ISCCR1element. Eight of sixteen carbapenem resistant Enterobacteriaceae strains were positive for ISCCR1and ISCCR1-linked genes carryig three distinct arrays, one hundred and eighty-five of811non-fermentative bacterial, including162Acinetobacter baumannii and23Pseudomonas aeruginosa, were positive for ISCR1-linked genes. All of them harboured the same array ISCR1+blaPER-1+GST-like+ABC transporter.5. complex class I integron. Three different complex class I integron arrays were found, including intIl+dfrA16+aadA2+qacdelsul+ISCR1+qnrAl+ampR+qacdelsul, intIl+aar-3+dfrA27+qacdelsul-ISCR1+qnrAl+ampR and intI1+aac A4+catB8+aadAl+qacdelsul+ISCR1+blaPER-1+GST-like+ABC transporter+qacdelsul.6. The duplex qPCR No amplification for neither NDM nor KPC was observed with DNA extracted from any of our specificity test panel of4organisms. Thus, the specificity of our duplex PCR is considered satisfactory.The linearity and limit of detection of the assay were determined by performing serial10-fold dilutions mixture of recombinant plasmid pNDM-1and pKPC-2from10to108copies/μL. Our assay was found to correlate well and the limit of detection both on NDM and KPC were10copies per20μl reaction. The intra-and inter-batch variation coefficients for two genes were all below5%, as demonstrated by repeated testings, indicating a good repeatability.when tested retrospectively against a collection of200isolates, the duplex PCR assay showed100%concordance with conventional PCR previously identified.Conclusion1. This work was the first to describe NDM-1presence in Enterobacter hormaechei. This is the first report that NDM-1gene and complex class1integron coexist in one plasmid in Enterobacter hormaechei. A novel sequence type530of Klebsiella pneumoniae co-producing KPC-2and IMP-4is for the first report.2. In our hospital, Enterobacteriaceae isolates with resistance to carbapenem mainly produce NDM-1, followed by the KPC and IMP-4; Acinetobacter baumannii mainly produce OXA carbapenemase and p.aeruginosa mainly produce IMP and VIM enzymes.3. The frequency of class1integron and ISCR1were high in Enterobacteriaceae and Acinetobacter baumannii isolates with resistant to carbapenem, but low in p. aeruginosa isolates with resistant to carbapenem. Two novel complex class1integrons were found among the isolates.4. the production of carbapenemase is the main reason for carbapenem resistance, in particular for NDM and KPC type enzyme, which have extensive resistant spectrum and strong ability of hydrolysis. Integron and ISCR play a role in transfer of resistance genes, especial for quinolone and aminoglycoside resistance. In total, the mechanism of drug resistance is a complex process that is usually a result of combined action of multiple mechanisms.5. A rapid, sensitive and specific duplex real-time PCR for simultaneous detection of two carbapenemase genes NDM and KPC in a single resction was established. successfully. The assay is able to detect all known subtypes of the two genes.6. The emergence of new resistant genes and drug-resistant forms are the result of antibiotic selection pressure, so the appropriate use of antibiotics is crucial. In addition, the rapid emergence and spread of strains producing carbapenemase is threat to global public health, timely detection and surveillance of carbapenemase genes is required to prevent their spread.