A Highly Promiscuous Integron,Plasmids, Extended Spectrum Beta Lactamases and Efflux Pumps as Factors Governing Multidrug Resistance in a Highly Drug Resistant <Emphasis Type="Italic">Vibrio fluvialis</Emphasis> Isolate BD146 from Kolkata,India

In an earlier study from this laboratory, Vibrio fluvialis BD146, a clinical isolate from Kolkata, India, 2002, was found to be resistant to all the fourteen antibiotics tested. It harboured a high copy number plasmid pBD146 and a low copy number plasmid. In the present study, a more detailed analysis was carried out to unravel different resistance mechanisms in this isolate. Sequencing showed that variable region of class 1 integron located on low copy number plasmid harbored arr3-cmlA-bla _OXA10-aadA1 gene cassettes. Analysis for extended spectrum beta lactamases (ESBLs) revealed that BD146 was ESBL positive. Efflux pumps were involved in the drug resistance phenotype for chloramphenicol, kanamycin, streptomycin and tetracycline. Sequence analysis of pBD146 revealed the presence of genes encoding BDint an integrase with a unique sequence having little similarity to other known integrases, toxin–antitoxin (parE/parD), a replicase, trimethoprim resistance (dfrVI) and quinolone resistance (qnrVC5). Presence of cmlA, putative novel integrase and toxin–antitoxin system in V. fluvialis has been documented for the first time in this report. pBD146 showed 99% sequence similarity with pVN84 from V. cholerae O1 of Vietnam, 2004 and a plasmid from V. parahaemolyticus v110 of Hong Kong, 2010. Conjugation experiments proved the ability of pBD146 and the low copy number plasmid, to get transferred to another host imparting their antibiotic resistance traits to the transconjugants. Therefore, present study has indicated that plasmids played an important role for dissemination of drug resistance.     

Analysis of a novel class 1 integron containing metallo-β-lactamase gene VIM-2 in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Carbapenems such as imipenem are stable to most β-lactamases. Recently, increased numbers of carbapenemase producing Gram-negative bacterial strains have been isolated because of the increased use of cabapenems. In this respect, control of these infectious carbapenemase producing Gram-negative bacteria and understanding their resistance mechanism are becoming more important. These carbapenem-hydrolyzing β-lactamase genes have been reported to exist mostly as gene cassettes in an integron. This implies that antibiotic resistance genes may be transferred to other bacteria via the integron. In the present study, we identified and analyzed an integron containing VIM-2 type metallo-β-lactamase gene in a carbapenemase producing Pseudomonas aeruginosa. In addition, the possibility of resistance spread by integron located in a plasmid was tested. Among glucose non-fermenting Gram-negative bacilli with reduced imipenem susceptibility (MIC≥8 μg/ml) isolated from Korean patients, P. aeruginosa 1082 showed resistance to most β-lactams, cephalosporin, and aminoglycoside. We found that P. aeruginosa 1082 was inhibited by EDTA in EDTA double disk synergy test which means that this strain produces metallo-β-lactamase. Class 1 integron containing bla _VIM-2 (carbapenem resistance gene), qacF (quaternary ammonium compound resistance gene), aacA4 (aminoglycoside resistance gene), catB3 (chloramphenicol resistance gene), bla _oxa-30 (extended-spectrum β-lactam resistance gene), and aadAl (aminoglycoside resistance gene) gene cassettes was detected in P. aeruginosa 1082. The size of the integron was 5,246 bp and the structure and arrangement of the integron was a novel one in comparison with other integrons found in other P. aeruginosa. The integron could be transferred to Escherichia coli JM109 from P. aeruginosa 1082 possibly via self-transferable plasmid DNA. The integron and a bla _VIM-2 gene were detected in the plasmid DNA of the transconjugants whose imipenem resistance was slightly increased as a result of accepting the integron from the donor strain.     

Identification of Two Multidrug-Resistant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Clonal Lineages with a Countrywide Distribution in Hungary

The aim of this study was to identify class 1 integrons from extended-spectrum and metallo-β-lactamase-negative, multidrug-resistant Pseudomonas aeruginosa clinical isolates from Hungary and to characterize the isolates by phenotypic and molecular methods. Fourteen selected P. aeruginosa isolates resistant to ceftazidime, gentamicin, and ciprofloxacin were subjected to serotyping, random amplification of polymorphic DNA (RAPD), integron content analysis, and a phenotypic test to detect high-level production of AmpC. Four representative isolates were further analyzed by multilocus sequence typing. Two P. aeruginosa multidrug-resistant clonal lineages were identified with a countrywide distribution. The first lineage is characterized by serotype O4, RAPD genotype A, sequence type ST175, and the presence of a class 1 integron harbouring aadB and aadA13 gene cassettes in its variable region. The second lineage is characterized by serotype O6, RAPD genotype B, sequence type ST395, and a class 1 integron carrying a single aadB cassette. The corresponding isolates were recovered from altogether 11 towns in Hungary. ST175 and ST395 are the presently calculated founders of two distinct P. aeruginosa clonal complexes that appear to have a wide geographical distribution also outside Hungary. The multidrug-resistant phenotype associated with these two clonal lineages might have contributed to an increase in their frequency and to their subsequent diversification. Both P. aeruginosa lineages displayed ≥8-fold synergy with boronic acid/ceftazidime combinations, suggesting an AmpC-mediated resistance to ceftazidime. Our observations underscore the role of class 1 integrons in the spread of aminoglycoside resistance by clonal dissemination among P. aeruginosa clinical isolates in Hungary.     

Genetic diversity analyses of class 1 integrons and their associated antimicrobial resistance genes in Enterobacteriaceae strains recovered from aquatic habitats in China

Aims: To characterize the molecular diversity of class 1 integrons and antibiotic resistance (AR) genes of Enterobacteriaceae strains recovered from aquatic habitats in Jinan, Shandong Province, China. Methods and Results: Six hundred and thirty‐eight antimicrobial‐resistant Enterobacteriaceae isolated from wastewater were examined for class 1 integron. Of these, 293 were positive for the class 1 integrase gene intI1; among these, 34 gene cassettes and 29 AR genes were detected. Twenty‐nine distinct gene cassette arrays were identified by restriction fragment length polymorphism (RFLP). Seven strains harboring novel gene cassette arrays were subjected to further study, in which antimicrobial susceptibility profiles were determined, and the presence of other AR genes outside of the integrons was assayed. Several of the resistance determinants were found to be transferable by conjugation or transformation. Conclusions: This study established the assessment of class 1 integron and antimicrobial resistance gene patterns among environmental Enterobacteriaceae. Also, a restriction enzyme EcoRII was employed to develop a rapid and simple method for characterizing gene cassette arrays by RFLP analysis, which facilitated further study of novel gene cassette arrays. Significance and Impact of Study: These data not only illustrated the diversity of class 1 integron gene cassettes but also provided direct evidence that integrons mobilized gene cassettes, generating new linkages of resistance genes, and they could be integrated in gene transfer units such as conjugative plasmids to contribute to the dissemination of AR genes by horizontal gene transfer (HGT) in aquatic environments.     

Class 1 integrons in Pseudomonas aeruginosa isolates from clinical settings in Amazon region, Brazil

A hundred and six Pseudomonas aeruginosa isolates from clinical cases were screened using PCR for the presence of integrons and associated resistance gene cassettes. Forty-four isolates harboured class 1 integrons (41.5%), of which 29 isolates (66%) also carried gene cassettes. The aacA gene was most frequently found within class 1 integrons (69%), followed by blaOXA family genes (52%). From class 1 integron-positive strains, we detected a total of 15 isolates (34%) carrying no gene cassettes. Restriction fragment-length polymorphism analysis of the integrons variable region revealed some identical structures, as well as distinct profiles indicating heterogeneity among these cassette regions. Multiresistance was observed in 71% of isolates, nevertheless no strong correlation was observed between integron presence and multiresistance. This is the first report showing class 1 integron prevalence and gene cassette content in P. aeruginosa isolates from clinical settings in the Brazilian Amazon.     

Evidence for stable transformation of Pseudomonas aeruginosa with a pUC-based plasmid

Pseudomonas aeruginosa was transformed with pUC8:16, a pUC-based plasmid bearing the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). Transformation was initially indicated by an increase in ampicillin resistance from 1500 to 2500 mg l^–1. Presence of the plasmid in P. aeruginosa was confirmed by amplification of a portion of vgb from and detection of VHb in the transformant but not the untransformed host. Southern blot analysis further indicated that pUC8:16 existed as an autonomous plasmid rather than integrated into the chromosome of the P. aeruginosa transformant.     

Occurrence and transmission of class 1 and 2 integrons among phenotypic highly ampicillin-resistant avian <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates from Pakistan

Thirty four avian Escherichia coli isolates were collected from different cities of Punjab province, Pakistan during 2008–2009. Twenty five phenotypic highly ampicillin-resistant (MICs ≥ 256 μg/ml) avian E. coli strains were selected for the investigation of occurrence and transmission of class 1, 2 and 3 integrons and β-lactamase genes. Amoxicillin, sulfonamide, trimethoprim, enrofloxacin, pefloxacin and tetracycline were the most common phenotypic resistant among ampicillin-resistant avian E. coli strains. Integrons and β-lactamase were found 60 and 72% respectively. Class 1 and 2 integrons were found 52 and 8%, while class 3 integrons were not found in all strains. All class 1 positive strains had variable fragments associated with gene cassettes dfrA7, dfrA1-aadA1, aadA1, aadA22 and dfrA12-orfF-aadA2 respectively, which confer resistance to trimethoprim and streptomycin. Class 2-positive strains had similar gene cassettes array dfrA1-sat1-aadA1 conferring resistance to trimethoprim, streptothricin and spectinomicin/streptomycin. Integrons are frequently found in β-lactamase positive isolates and widely disseminate multidrug resistance genes but they do not play role in the spreading of β-lactamase genes. Class 1 integrons gene cassette aadA22 is reported for the first time in avian E. coli. Findings of this study may provide important and useful information reflecting specific antibiotic selective pressure in Punjab province, Pakistan.     

Molecular characterization of In50, a class 1 integron encoding the gene for the extended-spectrum β-lactamase VEB-1 in Pseudomonas aeruginosa

A clinical isolate of Pseudomonas aeruginosa, JES, was resistant to extended-spectrum cephalosporins with a marked synergistic effect with clavulanic acid on a routine antibiogram. Preliminary PCR analysis revealed the presence of blaVEB-1, an integron-located gene encoding an extended-spectrum beta-lactamase previously identified in Escherichia coli MG-1. Using class 1 integron primers and blaVEB-1 intragenic primers, the insert region of the blaVEB-1 containing integron along with some flanking sequence from P. aeruginosa JES was amplified and subsequently sequenced. In50 contains within its variable region, in addition to qacE delta 1 and sull genes commonly found in class 1 integrons, two gene cassettes, veb1 and aadB. In50 is peculiar since its attI1 site is interrupted by two novel insertion sequences, IS1999 and IS2000. P. aeruginosa JES and Escherichia coli MG-1 strains were isolated from patients previously hospitalized in south east Asian countries. The finding of blaVEB-1 in these strains and on different integrons underlines the interspecies spread of this integron-located extended-spectrum beta-lactamase gene.     

Changing Trends in the Prevalence of Shigella Species: Emergence of Multi-Drug Resistant Shigella sonnei Biotype g in Bangladesh

Shigellosis, caused by Shigella species, is a major public health problem in Bangladesh. To determine the prevalence and distribution of different Shigella species, we analyzed 10,827 Shigella isolates from patients between 2001 and 2011. S. flexneri was the predominant species isolated throughout the period. However, the prevalence of S. flexneri decreased from 65.7% in 2001 to 47% in 2011, whereas the prevalence of S. sonnei increased from 7.2% in 2001 to 25% in 2011. S. boydii and S. dysenteriae accounted for 17.3% and 7.7% of the isolates respectively throughout the period. Of 200 randomly selected S. sonnei isolates for extensive characterization, biotype g strains were predominant (95%) followed by biotype a (5%). Resistance to commonly used antibiotics including trimethoprim-sulfamethoxazole, nalidixic acid, ciprofloxacin, mecillinam and ampicillin was 89.5%, 86.5%, 17%, 10.5%, and 9.5%, respectively. All isolates were susceptible to ceftriaxone, cefotaxime, ceftazidime and imipenem. Ninety-eight percent of the strains had integrons belonging to class 1, 2 or both. The class 1 integron contained only dfrA5 gene, whereas among class 2 integron, 16% contained dhfrAI-sat1-aadA1-orfX gene cassettes and 84% harbored dhfrA1-sat2 gene cassettes. Plasmids of ∼5, ∼1.8 and ∼1.4 MDa in size were found in 92% of the strains, whereas only 33% of the strains carried the 120 MDa plasmid. PFGE analysis showed that strains having different integron patterns belonged to different clusters. These results show a changing trend in the prevalence of Shigella species with the emergence of multidrug resistant S. sonnei. Although S. flexneri continues to be the predominant species albeit with reduced prevalence, S. sonnei has emerged as the second most prevalent species replacing the earlier dominance by S. boydii and S. dysenteriae in Bangladesh.     

Prevalence of integrons and a new dfrA17 variant in Gram‐negative bacilli which cause community‐acquired infections

A hundred and eleven Gram‐negative bacilli from community‐acquired infections were characterized by antimicrobial susceptibility testing, screened for class 1 and 2 integrons, and statistically evaluated for the association between antibiotic profile and the presence of integrons. The frequency with which integrons were harbored was 28.8%. Three E. coli strains contained a dfrA17 variant inserted in a class 1 integron. Results of PFGE indicated that some E. coli strains carrying integrons were clonally related. Carriage of gene cassettes was significantly associated with resistance to certain antibiotics (P < 0.05).     

Unnoticed spread of class 1 integrons in gram-positive clinical strains isolated in Guangzhou, China

A total of 46 gram-positive bacteria isolated from clinical specimens collected in China were subjected to PCR analysis with the intI1-specific primers, and the intI1-positive strains were further analyzed for their resistance gene cassette. All isolates possessed the class 1 integron in their genomes and the array of gene cassettes was dhfrXII-orfF-aadA2, which is very similar to other organisms except in one isolate carrying an additional copy of the class 1 integron containing the aadA2 gene cassette. Altogether, the results indicate that the class 1 integron is widespread in gram-positive clinical strains isolated in Guangzhou, China.     

Gene disruption in Lactobacillus plantarum strain 80 by site-specific recombination: isolation of a mutant strain deficient in conjugated bile salt hydrolase activity

A chloramphenicol-resistance gene (cml) was introduced into the Lactobacillus plantarum gene encoding conjugated bile acid hydrolasc (cbh) on a ColEl replicon. This plasmid which is nonreplicative in Lactobacillus was used to transform L. plantarum strain 80. A homologous double cross-over recombination event resulted in replacement of the chromosomal cbh gene by the cml-containing cbh gene. The transformants obtained were unable to synthesize active conjugated bile acid hydrolase (Cbh). The Cbh-Cml^R phenotype was stably maintained for more than 100 generations under nonselective conditions.This paper is dedicated with great appreciation to Dr. Frits Berends on the occasion of his retirement as Head of the Biochemistry Department of the TNO Medical Biological Laboratory     

Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination

An integron is a genetic unit that includes the determinants of the components of a site-specific recombination system capable of capturing and mobilizing genes that are contained in mobile elements called gene cassettes. An integron also provides a promoter for expression of the cassette genes, and integrons thus act both as natural cloning systems and as expression vectors. The essential components of an integron are an int gene encoding a site-specific recombinase belonging to the integrase family, an adjacent site, attl, that is recognized by the integrase and is the receptor site for the cassettes, and a promoter suitably oriented for expression of the cassette-encoded genes. The cassettes are mobile elements that include a gene (most commonly an antibiotic-resistance gene) and an integrase-specific recombination site that is a member of a family of sites known as 59-base elements. Cassettes can exist either free in a circularized form or integrated at the attl site, and only when integrated is a cassette formally part of an integron. A single site-specific recombination event involving the integron-associated attl site and a cassette-associated 59-base element leads to insertion of a free circular cassette into a recipient integron. Multiple cassette insertions can occur, and integrons containing several cassettes have been found in the wild. The integrase also catalyses excisive recombination events that can lead to loss of cassettes from an integron and generate free circular cassettes. Due to their ability to acquire new genes, integrons have a clear role in the evolution of the genomes of the plasmids and transposons that contain them. However, a more general role in evolution is also likely. Events involving recombination between a specific 59-base-element site and a nonspecific secondary site have recently been shown to occur. Such events should lead either to the insertion of cassettes at non-specific sites or to the formation of stable cointegrates between different plasmid molecules, and a cassette situated outside the integron context has recently been identified.     

Novel Class 1 Integrons in Multi-drug Resistant Isolates from Eastern China

Integrons are mobile genetic elements able to capture, express and excise resistance genes, playing an important role in the spread of bacterial resistance. The present study was to investigate the occurrence and diversity of integrons in 120 clinical multi-drug resistant Gram-negative isolates from eastern China. Screening of integrons was performed by PCR and gene cassettes were further characterized by PCR–RFLP and sequencing. Class 1 integrons were detected in 70.8 % of isolates and no class 2 and class 3 integrons were detected in any isolates. A total of 19 resistant gene cassettes were identified, four representative of novel gene cassettes: an aacA3 variant (aacA3c), an aacA4 variant (aacA4′-17), a bla _OXA variant (bla _OXA-251 ), and a catB8 gene cassette interrupted by an insertion sequence IS10 (catB8::IS10). In addition, 14 cassette arrays were detected, including three novel integrons: gcuD1-aacA4′-17-gcu38B-catB8::IS10 (In712), aacA3c-aadA13-bla _OXA-251 (In713) and dfrA1-gcu37-aadA5 (In714). The presence of novel integron structures in clinical isolates suggests hospital environments may favor the formation of novel combination of gene cassettes. Moreover, the high prevalence of integrons in multi-drug resistant isolates highlights the urgent need to employ effective means to avoid dissemination of drug-resistant bacteria.     

The integron In1 in plasmid R46 includes two copies of the oxa2 gene cassette

The sequence of the insert region of the integron In1 found in the IncN plasmid R46 was completed. The insert region is 2929 bases long and includes four gene cassettes, two of which are identical copies of the oxa2 gene cassette flanking an aadA1 cassette. The fourth cassette encodes an open reading frame orfD. From comparison of these data with published maps and sequences it is argued that the integrons found in the IncN plasmids pCU1 and R1767 and in the transposon Tn2410 are closely related to In1 from R46. Both site-specific gene insertion and recA-dependent recombination are likely to have contributed to the evolution of these integrons.     

Characterization of antimicrobial resistance of Pseudomonas aeruginosa isolated from canine infections

Aims: To determine the prevalence of Pseudomonas aeruginosa among dogs with suspected soft tissue infections and to characterize these isolates. Methods and Results: Swabs were taken from infected soft tissues of 402 dogs. Pseudomonas aeruginosa strains were confirmed phenotypically and tested for susceptibility to 11 antimicrobial agents and genotyped by SpeI pulsed‐field gel electrophoresis (PFGE). The genetic basis of fluoroquinolone (FQ) resistance and the presence of integrons were also characterized. A total of 27 (6·7%) dogs tested positive for Ps. aeruginosa. Fourteen different SpeI patterns were observed in 25 typeable strains. Among the β‐lactams, three isolates presented resistance to ticarcillin and carbenicillin, while only one isolate exhibited resistance to ceftazidime. Among the aminoglycosides (AGs), three strains showed resistance to amikacin, and four strains exhibited resistance to gentamicin and tobramycin. Four strains with mutations that led to the substitution of Thr at position 83 with Ile in GyrA and the exchange of Ser at position 87 with Leu in ParC displayed resistance to all tested FQs. These strains also carried class 1 integrons and showed resistance to between 6 and 10 antimicrobials. These integrons included four different gene cassettes (aacA4‐aadA1, bla_OXA‐31‐aadA2, aadA1‐arr‐3‐catB3 and cmlA5‐cmlA‐aadA1). Conclusions: A small proportion of infected dogs treated in two animal hospitals in Beijing, China carried Ps. aeruginosa isolates. Low levels of resistance to anti‐pseudomonal agents were observed in these strains. Significance and Impact of the Study: This study is the first report on the antimicrobial resistance profiles of Ps. aeruginosa isolated from infected canine origin in China. Additionally, this is the first report of the oxacillin resistance gene bla_OXA‐31 in a canine Ps. aeruginosa isolate.     

Four Novel Resistance Integron Gene-Cassette Occurrences in Bacterial Isolates from Zhenjiang, China

Integrons, which are widely distributed among bacteria and are strongly associated with resistance, are specialized genetic elements that are capable of capturing, integrating, and mobilizing gene cassette. In this work, we investigated classes 1, 2, and 3 integrons associated integrases genes in 365 bacteria isolates, amplified and analyzed the structure of class 1 integron, detected 8 resistant gene cassettes [dfr17, aadA5, aadA1, aadA2, dhfrI, aadB, aac(6′)-II, and pse-I], and found four novel gene-cassette arrays. We also found that commensal bacteria in the common microenvironment had the same integron gene cassette, which provided direct evidence that integron was an important horizontal transmission element.