| Background and objectiveThe growing increase in the rates of antibiotic resistance is a major cause for concern in both nonfermenting bacilli and isolates of the Enterobacteriaceae family. The research of antibiotic resistance mechanisms has become one of the focus of microbial field. The antibiotic resistance mechanisms are complex, including alteration of target site, change in permeability, inactivation of drug, efflux pumps and biofilm formation. Horizontal transfer of resistance genes is an important cause of the rapid emergence of clinical multidrug-resistant bacterial isolates. Resistance genes can disseminate in different strains, through conjugation, transformation, transduction and transpostion.It was known that conjugative plsmids, transposons, temperate phages, integrons and ISCR elements are closely related to multidrug resistance.Integrons can capture and spread of gene cassettes by site-specific recombination in bacteria, especially in gram-negative bacilli. They can move gene cassettes by capture and excision, which are located in transposons, plasmids and other mobile genetic elements. Integrons were originally identified on mobile elements from pathogenic bacteria and were found to be a major reservoir of antibiotic-resistance genes. An integron is generally defined by the presence of an integrase gene. There are four main types of integrons. Class I intgrons are frequently identified in bacterial isolates from clinical specimens.ClassⅠintegron contains 5'-CS(conserved segment), 3'-CS and gene cassettes. The 5'-CS contains integraseⅠgene (intI), a recombination site (attI) and a promoter gene. The 3'-CS contains the qac△1, sull genes and ORF5, encoding resistance to quaternary ammonium salts, sulphonamides and unkown function, respectively. Gene cassettes mainly confer resistance to antibiotics.ISCR comes from "insert sequences" and "common regions", featured by IS element and common region. IS elements have high mobility, which can insert one or more genomic locus. ISCRs have two key features of IS91-like elements and lack terminal inverted repeats, thought to transpose adjacent DNA sequences by a mechanism termed rolling-circle transposition. ISCR1 is widely studied in the ISCR family and usually exists in the complex classⅠintegron. ISCR1 encodes a putative product of 513 amino acids and is responsible for the mobilization of many classes of antibiotic resistance genes(variable regions), including catA2, dfrA, qnr, blaCTX-M and blaCMY, which plays a role in the expression of nearby genes by providing a promoter. Together with noncassette resistance genes, ISCR1 is commonly found between truncated and full-length 3'-conserved sequences (3'-CS) of classⅠintegrons, mediating the formation of complex classⅠintegrons,such as In6 and In7. Alternatively, recombination into a class 1 integron already attached to a copy of ISCR1 would generate a direct duplication of the ISCR1 element following the duplication of the 3'-CS sequence.Non-fermenting bacilli are aerobic gram-negative spore-free bacteria, including pseudomonas, acinetobacter, flavobacterium and alkaligenes. They can not metabolize glucose as energy source or catalyze glucose other than through fermentation. With the development of clinical invasive diagnosis and treatment and the widespread use of new antibiotics and immunosuppressive agents, the rates of infection and antibiotic resistance about non-fermenting bacilli have increased. The purpose of this study is to examine the distribution of resistance genes related to class I integron and ISCR1 and preliminarily explore the putative architectures of comoplex class I integrons in non-fermenting bacilli from clinical specimens.Methods1. Bacterial strains and extraction of genomic DNA Eight hundred and nine isolates identified belonging to non-fermenting bacilli were studied. The isolates were randomly selected at clinical microbiology laboratory in NANFANG hospital during two years (January 2008 to December 2009). These organisms were identified as Acinetobacter baumannii (n=376), Pseudomonas aeruginosa (n=240), Stenotrophomo-nas maltophilia (n=106), Burkholderia cepacia (n=75) and Pseudomonas putida (n=12) by BD Phoenix 100. Genomic DNA was extracted by the method of SDS-protease K-alcohol phenyl-trichlormethane.2.Detection of conserved region and varible region of class I integronConserved region (class I integrase) and varible region (gene cassettes) were det-ermined by PCR. PCR products of gene cassettes were digested with Hinf I and RasI, respectively. Amplicons with the same RFLP pattern were deemed to contain the same gene cassettes. One integron representative of each restriction profile were selected for DNA sequencing. The resulting DNA sequences were analysed with the BLAST algorithm in GenBank and submitted to Genbank.3.Detection of conserved region and varible region of ISCR1 elementConserved region (ISCR1 transposase gene) of ISCR1 element was detected by PCR. Varible region (antibiotic resistance genes carried by ISCR1) was examined in the ISCRl-postive strians by PCR. PCR products of varible region were digested with Hinf I and Ras I, respectively. Amplicons with the same RFLP pattern were deemed to contain the same antibiotic resistance genes. One representative of each restriction profile were selected for DNA sequencing. The resulting DNA sequences were analysed with the BLAST algorithm in GenBank and submitted to Genbank.4.Analysis of the putative structures of comoplex class I integronISCR1 usually mediates formation of complex class I integron. The region between internal truncated copy of 3'-CS and ISCR1 transposase gene was studied by PCR and sequenced. The putative structures of comoplex class I integron were analysed.Results1.358 strains carried conserved region of class I integron and 293 strains carried 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.Of 376 Acinetobacter baumannii isolates, six different gene cassette arrays were found, including aacA4+catB8+aadAl (n=159), catB3+qnrVC-like+aacA4 (n=18), aacC1+orfP+orfQ+aadA1(n=14), arr3+aacA4(n=9), dfrA15(n=4), aadB+aadA2 (n=1). Of 240 Pseudomonas aeruginosa isolates, ten different gene cassette arrays were found, including blaIMP-9+aacA4+blaoXA-10+aadA2 (n=32), aac(6')-II+aadA13+cmlA8+blaoxA-10a(n=10), aadB+blaPSE-1(n=8), aacA4+ blaIMP+blaoxA-30+catB3(n=6), dfrA12+orfF+aadA2(n=3), aacA4+aadA2(n=2), dfrA15 (n=2)5 aadB+aac6-II+pse-1(n=1), aacA4+catB3+dfrAl(n=1) and aadB+aadA2(n=1). Of 106 Stenotrophomonas maltophilia isolates, five different gene cassette arrays were found, including dfrA17+aadA5(n=3), aacA4+blaIMP-25+blaOXA-30+catB3(n=2), qacF(n=1), dfrA15(n=1) and aadB+aadA2 (n=1). Of 75 Burkholderia cepacia isoltes, two different gene cassette arrays were found, including aadB+aac6-Ⅱ+pse-1(n=7) and aacA4+catB8+aadAl(n=2). Of 12 Pseudomonas putida isolates, three different gene cassette arrays were found, including aadB+aac6-Ⅱ+pse-1(n=3), aadA1(n=1) and blaIMP-9+aacA4+blaoXA-10+aadA2(n=l).2.125 strains carried conserved region of ISCRl element and 18 strains carried variable region of ISCRl element.Of 376 Acinetobacter baumannii isolates,8 carreid ISCR1+qnrA1+ampR+ qacEAl and 6 carried ISCR1+blaPER-1+ABC transporter+qacEAl. Of 240 Pseudomonas aeruginosa isolates,3 carried ISCR1+qnrAl+ampR+qacE△1. Of 106 Stenotrophomonas maltophilia isolates,1 carried ISCR1+qnrA1+ampR+qacE△1.3.12 strains carried complex class I integron.Of 809 isolates, three different resistance genes arrays were found, including catB3+qnrVC-like+aacCA4+ISCR1+blaPER-1+ABC transporter+qacE△1(n=4), dfrA15+ISCR1+qnrAl+ampR+qacEA1(n=6) and aadB+aadA2+ISCR1+qnrA1+ ampR+qacE△1(n=2).Conclusion1. The detective rates of class I integrase and gene casettes of class I integron are from 14.2% to 61.7% and from 17.5% to 54.5% in 809 non-fermenting gram-negative bacilli from clinical specimens in NANFANG hospital, respectively.2. The detective rates of ISCR1 transposase genes are from 0 to 26.2% in 809 non-fermenting gram-negative bacilli from clinical specimens in NANFANG hospital. The detective rates of resistance genes carried by ISCR1 in ISCR1-postive isolates are from 0 to 16.7%.3. Class I integrons mainly contain aminoglycoside resistance genes, trimethoprim resistance genes, P-lactamases genes and chloramphenicol resistance genes in 809 non-fermenting gram-negative bacilli from clinical specimens in NANFANG hospital. 4. Resistance genes carried by ISCR1 mainly contain qnrA1 in 809 non-fermenting gram-negative bacilli from clinical specimens in NANFANG hospital.5. Resistance genes of complex class I integrons mainly contain catB, aacA4, aadA2, aadB, dfrA15 and qnr in 809 non-fermenting gram-negative bacilli from clinical specimens in NANFANG hospital.6. One noyel resistance gene array of complex I integron is reported for the first time in the word. |
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