Analysis of Integrons and Antimicrobial Resistances of Multidrug Resistant Escherichia coli Isolated in Korea

Antibacterial drugs are one of the most important therapeutic agents of bacterial infections but multidrug resistant Escherichia coli (MDREC) is an increasing problem worldwide. Major resistance mechanism of MDREC is horizontal gene transfer of R plasmids harboring integrons, which the integron integrase (IntI) catalyzes gene cassette insertion and excision through site specific recombination. In this study, resistance profiles of integron harboring E. coli isolated in Korea and the genetic environments of integron gene cassettes were analyzed by PCR and direct sequencing to clarify the mechanisms of spread of integron harboring E. coli. Resistance rates of integron harboring E. coli, including β-lactams, aminoglycosides, and fluoroquinolones and MDR frequencies were significantly higher than that of E. coli without integron (p < 0.01). Majority (80%) of integron harboring E. coli showed resistance transfer by conjugation. Most (80%) of E. coli had dfrA17-aadA5 cassette array and PcH1 hybrid promoter; 16.7% of E. coli had dfrA12-orfF-aadA2 cassette array and PcW promoter. The higher prevalence of weak Pc variants among most (96.7%) of integron harboring MDREC suggests that a flexible cassette array is more important than enhanced expression. All the integrons had LexA binding motif suggests that SOS responses control the expression of these integrons. In conclusion, the genetic bases of integrons were diverse, and the spread and the expression of prevalent gene cassette arrays may be deeply related with strengths of Pc promoters in integrons. These informations will provide important knowledge to control the increase of integron harboring MDREC.

Association of glycerol kinase gene with class 3 integrons: A novel cassette array within Escherichia coli

Background: Integrons are genetic elements which are known for their role in capturing and spreading of antibiotic resistance determinants among Gram-negative bacilli. So far, there is no study regarding Class 3 integron and their genetic organisation in India. Objective: This study investigates the occurrence of Class 3 integron and their gene cassette array among Escherichia coli. Materials and Methods: In this study, a total of 200 E. coli isolates were collected from indoor and outdoor patients from Silchar Medical College and Hospital during September 2015 to February 2016. Detection of the integrase genes and gene cassettes within the Class 3 integron was performed by polymerase chain reaction which was further analysed by sequencing. Results: Twenty-seven isolates were found to harbour Class 3 integron. Sequencing of the gene cassettes and whole Class 3 integron revealed the presence of nine different types of cassettes array, out of which the arrangement with glycerol kinase gene cassette was found to be the most prevalent. Arrangement with blaCTX-Mgene cassette was also detected in few isolates. Conclusion: This study provides epidemiological profiling of Class 3 integrons in this geographical area. The data generated in this study are helpful in infection control programme, anti-infective research and search for epidemiological markers.

Structural analysis of atypical class 1 integrons in clinical Proteus isolates / 中华微生物学和免疫学杂志

Objective To analyze the structures of atypical class 1 integrons in clinical Proteus isolates. Methods This study included 26 class 1 integron integrase gene-positive clinical Proteus isolates, from which the variable regions of class 1 integrons could not be amplified. Six isolates were chosen from them to amplify the flanking DNA segments of class 1 integron integrase gene using inverse PCR. The se-quences of PCR products were analyzed with BLAST to identify the target homologous sequences as well as their accession numbers in GenBank. Primers for overlap PCR were designed according to the flanking se-quences. Then the 26 clinical Proteus isolates were analyzed with overlap PCR and sequencing analysis. Re-sults The variable regions of class 1 integrons in 25 out of the 26 clinical Proteus isolates were completely or partly identified by using inverse PCR,overlap PCR and sequencing analysis. A gene cassette array of estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3 was detected in 22 isolates. The variable regions of class 1 integrons in the other three isolates were identified to be estX-psp-aadA2-cmlA1,dfrA14 and IS26,respectively. All of the 25 isolates lacked the 3'conserved segements in class 1 integron. Conclusion Inverse PCR can be used to analyze the structures of atypical class 1 integrons. Gene cassette psp and gene cassette arrays of estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3 and estX-psp-aadA2-cmlA1 in clinical Proteus isolates are reported for the first time.

Antibiotic susceptibility and class I integron analysis of imipenem-resistant Pseudomonas aeruginosa / 实用医学杂志

Objective To investigate the class I integrons and their gene cassettes of imipenem-resistant Pseudomonas aeruginosa (IRPA) , and to analyze the correlation between integrons and drug resistance. Methods PCR was used to determine the presence of integrase genes and class I integrons. The variable regions were detected by sequencing. Resistance genes of integron gene cassettes including metal-β-lactamases, aminoglycoside modifying enzymes (AMEs), 16SrRNA methylating enzyme and the OprD2 genes were detected by PCR. The VITEK-2 automated system was used to determine the antibiotic susceptibility of integron-positive IRPA strains. Results The positive rates of integrase genes and class I integrons were 23.3%(20/86)and 8.14%(7/86) , and five kinds of gene cassettes were detected in 86 IRPA strains. The class I integrons-positive bacterial strains exhibited different resistant patterns to 12 antibiotics with large number of resistance genes. Conclusion The class I integrons and their gene cassettes are associated with multiple drug resistance of IRPA.

Distribution of Class II integrons and their contribution to antibiotic resistance within Enterobacteriaceae family in India.

Background: Integrons are the main contributors to the development of multidrug resistance (MDR) among Gram‑negative bacilli. There is a lack of knowledge about the molecular relation between gene cassettes and antibiotic resistance in India. Objective: In this study, we have investigated the occurrence of Class II integron and their cassette array among Enterobacteriaceae. Materials and Methods: A total of 268 MDR non‑duplicate strains of Enterobacteriaceae were collected from Silchar Medical College and Hospital, Silchar, Assam, India, during June 2012 to May 2013. Polymerase chain reaction was performed for detection of the integrase genes and gene cassettes within the Class II integron which were further analysed by sequencing. Results: Class II integron was observed in 47 isolates. Four different gene cassette arrangements were detected: dfrA1‑sat2‑aadA1; dfrA1‑sat2‑aadA1‑orfX‑ybeA‑ybfA‑ybfB‑ybgA; dfrA12‑sat2‑aadA1; and dfrA1‑linF‑aadA1. The most prevalent cassette combination was dfrA1‑sat2‑aadA1. This study has also identified a set of gene cassette associated with linF gene instead of sat2 gene. Conclusion: Further investigation is required to determine the current situation and important reservoir of Class II integron for the transmission of drug resistance among Enterobacteriaceae and their contribution to antimicrobial resistance in hospital environment.

Emergence of integron borne PER-1 mediated extended spectrum cephalosporin resistance among nosocomial isolates of Gram-negative bacilli.

Background & objectives: Pseudomonas extended resistant (PER) enzymes are rare type of extended-spectrum beta lactamases (ESBLs) that confer third generation cephalosporin resistance. These are often integron borne and laterally transmitted. The aim of the present study was to investigate the emergence of integron borne cephalosporin resistant PER-1 gene in diverse incompatibility (Inc) group plasmids among gram-negative bacteria. Methods: a total of 613 consecutive, non-duplicate, Gram-negative bacteria of Enterobacteriaceae family and non-fermenting Gram-negative bacteria were isolated from different clinical specimens during a period of 18 months. For amplification and detection of blaPER, multiplex PCR was done. For understanding the genetic environment of blaPER-1, integrase gene PCR and cassette PCR (59 be) was performed. Gene transferability experiment was carried out and PCR based replicon typing was performed for incompatibility group typing of plasmids using 18 pairs of primers. An inhibitor based method was used for phenotypic detection of intrinsic resistance. Results: Multiplex PCR and sequencing confirmed that 45 isolates were harbouring blaPER-1. Both class 1 and class 2 integrons were observed among them. Integrase and cassette PCR (59 be) PCR results confirmed that the resistant determinant was located within class 1 integron. Transformation and conjugation experiments revealed that PER-1 was laterally transferable and disseminated through diverse Inc plasmid type. Efflux pump mediated carbapenem resistance was observed in all isolates. All isolates belonged to heterogenous groups. Interpretation & conclusions: This study demonstrates the dissemination of cephalosporins resistant, integron borne blaPER-1 in hospital setting in this part of the country and emphasizes on the rational use of third generation cephalosporins to slow down the expansion of this rare type of ESBL gene.

Relationship between resistance and integron of multidrug-resistant Acine-tobacter baumannii / 中国感染控制杂志

Objective To investigate the expression and resistant gene of integron in multidrug-resistant Acinetobacter baumannii (MDR-Ab).Methods 51 strains of MDR-Ab isolated from a hospital in August-October 2012 were collected, antimicrobial susceptibility testing was performed.Class I(Int I),II (Int II)and III (Int III)of integrase genes and inte-gron variable region gene cassettes were detected by polymerase chain reaction (PCR),and the homology of integron varia-ble region was analyzed by detection results of restriction fragment length polymorphism (RFLP)and DNA sequencing. Results Positive rate of integrase gene in MDR-Ab was 78.43%(40/51).All genes belonged to Int I,while IntⅡand IntⅢ were not found.Variable region cassettes were detected in 97.50% (n=39)of Int I,there were 5 types of integron gene cassettes:aacA4 in 14 strains,aacA4+catB8 in 22 strains,arr-3 +aacA4 in 1 strain,dfrA15 in 1 strain and arr-3 in 1 strain.Conclusion MDR-Ab isolated from this hospital may be related with Int I expression.Int I carried gene cassettes as follows:aacA4,aacA4+catB8,arr-3+aacA4,dfrA15 and arr-3.

Sewage treatment plant serves as a hot-spot reservoir of integrons and gene cassettes.

This study investigated the occurrence and abundance of class 1 integrons and related antibiotic resistance genes (ARGs) in a sewage treatment plant (STP) of China. Totally, 189 bacterial strains were isolated from influent, activated sludge and effluent, and 40 isolates contained the integons with a complete structure. The intI1-carrying isolates were found to harbor two types of gene cassettes: dfr17-aadA5 and aadA2, conferring resistances to trimethoprim and streptomycin, which were further confirmed by antimicrobial susceptibility analysis. Many other gene cassettes were carried on integron, including qnrVC1, catB-8-blaoxa-10-aadA1-aac(6'), aadB-aacA29b, aadA2, aac(6')-1b, aadA6 and aadA12, which were detected using DNA cloning. Quantitative real time PCR showed that over 99% of the integrons was eliminated in activated sludge process, but average copy number of integrons in given bacterial cells was increased by 56% in treated sewage. Besides integrons, other mobile gene elements (MGEs) were present in the STP with high abundance. MGEs and the associated ARGs may be wide-spread in STPs, which constitute a potential hot spot for selection of antibiotic resistant bacteria and horizontal transfer of ARGs.

Determining the start codons of aadA2 gene in class 1 integron / 中华微生物学和免疫学杂志

ObjectiveTo determine whether aadA2 gene can be translated from the ATG triplet,which there was no plausible ribosome binding site preceding it,and synthetized a functional protein in class 1 integron.MethodsSite-specific mutagenesis was used to construct aadA2 gene cassette with different start codons,together with their upstreamed promoters of variable regions were cloned into plasmid pACYC184 respective.The constructed plasmids were then transfored into Escherichia coli JM109,Western blot was used to detect the translation products of aadA2 gene with different start codons.Broth microdilution method was used to detect the minimal inhibitory concentrations to streptomycin in Escherichia coli JM109 containing aadA2 gene with different start codons.ResultsaadA2 gene can initiate translation from both ATG and GTG triplets in aminoacyl -3-adenylyltransferase protein synthesis,though there was no plausible ribosome binding site preceding the ATG triplet.Besides GTG and ATG triplets,there was other start codon downstream of the GTG triplet in aadA2 gene.The translated products that initiated from the start codons that described above were all functional AAD(3) proteins that can be detected by anti- aminoacyl -3-adenylyltransferase polyclonal antisera in Western blot and conferred different resistance levels to streptomycin in E.coli.ConclusionWhen inserted as the first gene cassette in class 1 integron,aadA2 gene can initiate translation from ATG triplet and synthetized a functional protein,though there was no plausible ribosome binding site preceding it.This structural characterization of class 1 integron can initiate translation of the open reading frame harbored in gene cassette that integrated into class 1 integron,though there was no plausible RBS preceding the start codon.This make class 1 integron be more convenience to express the genes that capture from environment.

Prevalence of integrons and analysis of resistance genes in integrons among Acinetobacter baumannii isolates from Nanjing region / 中国感染与化疗杂志

Objective To investigate the prevalence of integrons in A.baumannii isolates,analyze the correlation between inte- grons and resistance of A.baumannii,and study the resistance genes in integrons.Methods A total of 106 strains of A.bau- mannii were collected to test the antibiotic susceptibility by disk diffusion method.The classification of integrons was per- formed by analyzing the positive PCR products using restriction fragment length polymorphism (RFLP).The variable region of integrons was amplified by integron PCR.RFLP and DNA sequencing were used to analyze the resistance genes in integrons. Results About 52.8% (56/106) of the isolates showed integron positive.PCR-RFLP analysis revealed that they were all class I integrons.About 94.6%(53/56) of the positive strains with integrons owned the variable region,which was confirmed by integron PCR.The sizes of the amplicons ranged from 0.15 kb to 2.8 kb.All together 7 different cassette arrays were detec- ted,including genes coding resistance to aminoglycosides (aadA1,aadA2,aadA5,aadB,aacA4),sulphonamides (dfrⅫ, dfr17),?-lactam compounds (bla_(ara-10)),chloramphenicol (catB-like,catB8),and two open reading frames (orfF,orfI) with unknown function.A novel cassette array orfI-aadA1 was reported,and its GenBank accession number was DQ092497.Conclu- sions Class I integrons are widespread in A.baumannii isolates in Nanjing.The integrons are closely associated with the resist- ance and multidrug resistance in A.baumannii isolates.

Three strains of class I integrons with novel cassette arrays in Pseudomonas aeruginosa isolates / 临床检验杂志

Objectives To investigate the mechanism of integron mediated resistance and multidrug-resistance in P.aeruginosa.Methods The variable region of integron was amplified by integron PCR.Restriction fragment length polymorphism(RFLP)and DNA sequencing were used to investigate the resistance genes in the variable region of integron.Results Of the 98 strains 35(35.7%)were the variable region positive,and size of the amplicons ranged from 1.0 kb to 4.0 kb.A total of 6 different cassette arrays were detected,including genes coding resistance to aminoglycosides,?-lactam compounds and sulfanilamides.Of the 5 cassette arrays 3 were novel,including aadA6-orfD,aadB-blaP1 and aadB-aac6-Ⅱ-blaCARB-8,and the Genbank numbers were DQ 091179,DQ 141316 and DQ 288251 respectively.Conclusions Integron mediates the resistance and multidrug resistance in P.aeruginosa.The majority of the genes located in integrons are those coding resistance to aminoglycosides.Three strains of class I integron with novel cassette arrays are reported.

Association of class 1 integrons of Acinetobacter baumannii with resistance / 临床检验杂志

Objective To monitor the distribution of class 1 integrons of Acinetobacter baumannii and interpret the association of class 1 integrons with the resistance in clinical isolates.Methods Susceptibility to 14 different antimicrobial agents was determined by agar dilution method.DNA of all clinical isolates was extracted to detect class 1 integrons by PCR.Results The differences between integron-positive isolates and integron-negative isolates was significant in the resistance to all tested antibiotics except for Imipenem,Cefepime and Cefoperazone plus Sulbactam.Different types of class 1 integrons showed different resistance types.Conclusions There were some close relations of class 1 integrons with multi-drug resistance as well as resistance types in Acinetobacter baumannii.

Detection of a new gene cassette aadA23 in Escherichia coli / 中华传染病杂志

Objective Since integrons play an important role in the spread of antibiotic resistance genes in bacteria,the characterization of a new resistance gene cassette in class 1 integron positive strains of E.coli was analyzed. Methods The presence and genetic content of class 1 integron were examined by PCR and sequencing.The sequence was analyzed by using some bioinformatics softwares.Results 5 class 1 integron positive strains were amplified by primers of in-F and in-B which were set for amplifying the region of antibiotics resistance genes.Among the 5 strains,an amplicon of 1009 bp was yielded.Sequencing analysis revealed that amplicon of 1009 bp harbored a 780 bp ORF.Further analysis with bioinformatics software showed that it was 99.6% and 99.5% identical to the known aadA23 and aadA21 cassette,and was just 66.4% identical to the known aadA5 cassette.It was conferring resistant to spectinomycin and streptomycin,and was given a new name aadA23b.Conclusions Multi-drug resistance genes has been proved changeable in E.coli clinical strains.The result not only stressed the need for continuing surveillance of antibiotic resistance in the molecular level,but also the need caring for genetic variation of drug resistance gene cassettes.