Study On Carbapenemases Gene, Class â…  Integron And ISCR1among Carbapenem-resistant Gram Negative Bacteria

Background and objectiveInfections with carbapenem-resistant Gram negative bacteria are associated with therapeutic failure and high mortality rates. The production of carbapenemases, including Ambler classes A, D and B [metallo-(3-lactamases (MBLs)] enzymes, are the most common mechanism responsible for carbapenem resistance. The recently reported metallo-β-lactamase NDM-1(New Delhi metallo-β-lactamase), initially identified in Klebsiella pneumoniae and Escherichia coli isolates from a Swedish patient following hospitalization in India, is now emerging as a new mechanism by which bacteria manifest resistance to almost all antibiotic groups, including fluoroquinolones, aminoglycosides and β-lactams (especially carbapenems). NDM-1is now present among Enterobacteriaceae isolates worldwide and has been detected among non-fermentative bacilli, such as Pseudomonas aeruginosa and Acinetobacter species. Among class A enzymes, the most common are KPC β-lactamases, which hydrolyze all β-lactams except cephamycins. The enzyme has been reported in numerous genera of the Enterobacteriaceae and even in non-fermenting bacteria. The blaKPC (Klebsiella pneumoniae carbapenemase) gene is harbored on plasmids and carried within the transposon Tn4401, suggesting potential dissemination among different species. The geographic range of KPC-type enzymes has spread worldwide, since the report of the first isolate in2001in a clinical specimen of Klebsiella pneumoniae from a hospital in North Carolina.Increased usage of antimicrobial agents to treat Gram-negative bacterial infections has led to the emergence of carbapenem-resistant Gram negative bacteria. In recent years, infections caused by carbapenem-resistant Gram negative bacteria have increasingly gained worldwide attention due to its high morbidity and mortality among hospitalized patients. 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. A substantial proportion of these elements is integrons. The integrons in clinical isolates have been commonly found in plasmids and/or transposons that enhanced the spread of resistance genes. Typically, integrons are composed of a5’-conserved segment (5’CS) including three key elements necessary for the capture and expression of gene cassettes:an integrase gene encoding a site-specific integrase, IntI; a recombination site (attI) at which point gene cassettes are inserted; and a promoter (Pc) that controls the expression of gene cassettes; a3’conserved segment (3’CS) including the qacEΔ and sull genes; and an internal variable region (IVR) with one or more resistance gene cassettes. Different classes of integrons are distinguished by their highly conserved class-specific integrase genes. Class1integrons are most frequently isolated from MDR pathogens, and the ongoing use of antibiotics has swelled their numbers in recent years.ISCR elements are closely related to the IS element IS91family. These elements can transpose adjacent DNA sequences by a process called rolling-circle replication. ISCR elements are now recognized as powerful antibiotic resistance gene capture and movement systems and appear to have played a major role in the emergence and spread of antibiotic resistance genes. ISCR elements currently comprise23members that differ in the amino acid sequences of their cognate transposases and fit broadly into three groups based primarily on their G+C content. ISCR1are most frequently isolated from MDR pathogens, which are resistant to three or more antibiotics, and the ongoing use of antibiotics has swelled their numbers in recent years. ISCR1was discovered and reported in the early1990s as a2154bp DNA sequence, incorporating orf513(a putative gene of unknown function) inserted downstream the sull genes of class1integrons, In6and In7. ISCR1-linked genes are commonly located between ISCR1and the3’conserved segment (qacEΔ1/sull). ISCR1was found to be associated with genes encoding resistance to chloramphenicol, trimethoprim, quinolone, and P-lactamases.Methods1. Bacterial strains and extraction of genomic DNA Nine Enterobacteriaceae isolates and360non-fermenting bacteria with decreased susceptibility to carbapenems were isolated at Nanfang hospital from June2009to June2011. 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). Ertapenem (10μg, Merck&Co., Inc., Whitehouse Station, NJ, USA) and cefoperazone/sulbactam (105μg, Oxoid, Ltd., Basingstoke. Hampshire. UK) susceptibility testing was performed by the K-B method on Mueller-Hinton agar plates according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Zones of inhibition were measured in mm and categorized as sensitive or resistant according to the CLSI-defined breakpoints. Isolates were stored at-80℃in nutrient broth containing30%(v/v) glycerol. Control strains (E. coli ATCC25922and K. pneumonia ATCC700603, S. aureus ATCC25923, and P. aeruginosa ATCC27853) were included. Whole genomic DNA was extracted using a phenol-chloroform extraction method.2. carbapenemase gene detection and horizontal transmission mechanismThe blaNDM、blaKPC、blaVIM、blaIMP、blaSPM、blaGIM. blaOXA gene was identified by PCR. NDM or KPC-positive isolates were confirmed by16S rRNA sequence analysis, among them, Acinetobacter spp. were further confirmed by sequence analysis of the16S-23S rRNA gene spacer region. PCR amplifications were sequenced to compare them with sequences available in the NCBI database (www.ncbi.nlm.nih.gov). NDM or KPC-positive isolates were Detected whether located on the plasmid. 3. Detection of class I integron Class I integrase and variable region (gene cassettes) were determined by PCR. Distinction gene cassette arrays were identified by restriction fragment length polymorphism (RFLP) with Hinf I and Rasl and DNA sequencing analysis. The resulting DNA sequences were analysed with the BLAST algorithm in GenBank.4. Detection of ISCRl element PCR was used to detect the presence of ISCR1and ISCR1-linked genes. The ISCR1-linked genes amplicons were then characterized by restriction fragment length polymorphism (RFLP) with Hinf I and Ras1and DNA sequencing analysis. The resulting DNA sequences were analysed with the BLAST algorithm in GenBank.5. Analysis of the putative structures of comoplex class I integron ISCR1usually mediates formation of complex class I integron. The region between internal truncated copy of3’-CS and ISCR1transposase gene was studied by PCR and sequenced. The putative structures of comoplex class I integron were analysed.6. study on genotyping We have established ERIC-PCR genotyping methods for molecular epidemiological studies of pan-resistant Pseudomonas aeruginosa and Acinetobacter. spp. isolated from clinical samples. Multilocus sequence typing (MLST) has been established to genotype the NDM-1or KPC-positive isolates.Results1. detection of carbapenemase geneAmong carbapenem-resistant Gram negative bacteria, two isolates, including Acinetobacter genomic species3and13TU. were found to be blaNDM-1positive. Nine K. pneumonia isolates were positive for blaKPC-2-Nine K. pneumonia isolates were positive for blaKPC-2.31Pseudomonas aeruginosa isolates were positive for blaIMP-1.15P. aeruginosa isolates were positive for blaIMP-9,1P. aeruginosa isolate was positive for blaIMP-25.16P. aeruginosa isolates were positive for blaVIM-1.180A. baumannii isolates were positive for blaoxA-23,148A. baumannii isolates were positive for blaOXA-51.8A. baumannii isolates were positive for blaOXA-58. NDM and KPC genes are located on the plasmid. 2.239strains carried conserved region of class I integron and153strainscarried variable region of class I integron. In this study, aminoglycoside resistance genes, trimethoprim resistance genes, β-lactamases genes and chloramphenicol resistance genes were detected in gene cassettes of class I integrons. Of121Acinetobacter baumannii isolates, three different gene cassette arrays were found, including aacA4+catB8+aadAl (n=115), aacCl+orfP+orfQ+aadAl(n=4), arr3+aacA4(n=2). Of35P. aerugmosa isolates, seven different gene cassette arrays were found, including blaIMP-9+aacA4+blaOXA-10+aadA2(n=15), aac(6’)-II+aadA13+cmlA8+blaOXA-10a(n=7), aadB+blaPSE-1(n=6), aacA4+blaIMP+blaOXA-30+catB3(n=3), dfrAl2+orfF+aadA2(n=2), aacA4+aadA2(n=2), dfrA15(n=l),and aadB+aadA2(n=l).3.121strains carried conserved region of ISCRl element and23strains carried variable region of ISCRl element.Of201Acinetobacter baumannii isolates,79strains carried ISCRl+qnrAl+ampR+qacEΔl and11carried ISCRl+blapER-1+GST+ABC transporter+qacEAlsul. Of159P. aeruginosa isolates,2strains carried ISCRl+qnr A1+ampR+qacE A1.4.1P. aeruginosa strain carried complex class I integron.Of169isolates, two different complex class I integron arrays were found, including dfrA15+ISCR1+qnrAl+ampR+qacEΔl and aadB+aadA2+ISCRl+qnr A1+ampR+qacE A1.5.ERIC-PCR resultsERIC-PCR results showed that: P. aeruginosa and Acinetobacter baumannii disseminate, Acinetobacter baumannii strains has dominant clonal. Nine strains of Klebsiella pneumoniae MLST typing were ST11.Conclusion1.This work was the first to describe NDM-1presence in Acinetobacter genomic species3and13TU. This is the first identification of KPC-2-producing K. pneumoniae in Guangdong.2.The prevalence of class1integron associated integrase gene (Intll) was64.8%. Of239integrase-positive strains,153(41.5%) appeared to carry gene cassettes. The gene cassettes included those encoding resistance to trimethoprim, aminoglycosides, the β-lactamase, chloramphenicol and rifampicin.3. The prevalence of ISCR1associated ORF513was32.8%. Of121orf513-positive strains,23(19.0%) appeared to carry resistance gene.4. two different complex class I integron arrays were first found from one P. aeruginosa strain, including intI+dfrA15+ISCR1+qnrAl+ampR+qacEA1and intI+aadB+aadA2+ISCR1+qnrAl+ampR+qacEA1.5. P. aeruginosa and Acinetobacter.spp were disseminate, Acinetobacter.spp strains has dominant clonal. The monitor should be strengthened to prevent the occurrence and spread of strains of the advantages of cloning, to prevent of hospital infection.