Integron Analysis Research Articles

Complete genome sequence of Pseudomonas aeruginosa PA99 clinical isolate from Thailand carrying two novel class 1 integrons, In2083 and In2084

The aim of this study was to identify and characterize multidrug resistance genes and the genetic contexts of integrons found in extensively drug resistant (XDR) Pseudomonas aeruginosa PA99 clinical isolate from Thailand. The sequencing of P. aeruginosa PA99 genomic DNA was done by using Pacific Biosciences RS II sequencing platform. The generated reads were de novo assembled by Canu version 1.4 and the annotation was performed using Prokka v1.12b. The complete genome sequence was subjected for identification of sequence type, serotype, integrons, and antimicrobial resistance genes by MLST 2.0, PAst 1.0, INTEGRALL, Resfinder 4.1, and CARD 3.2.5, respectively. Pseudomonas aeruginosa PA99 genome consisted of a 6,946,480-bp chromosomal DNA with 65.9% GC and belonged to ST964 and serotype O4. Twenty-one antimicrobial resistance genes conferring XDR phenotype were identified. Of special note were carbapenem resistance genes (blaIMP-1, blaPAO, blaOXA-21, and blaOXA-396) and colistin resistance gene basR with L71R mutation. Integron analysis revealed that P. aeruginosa PA99 harbored five class 1 integrons: two copies of In994 (blaIMP-1), an In1575 (aadB), and two novel integrons, In2083 (blaOXA-21 - aac(6')-Ib3 - aac(6')-Ib-cr - ere(A)1∆2 - dfrA1r) and In2084 (blaIMP-1 - aac(6')-Ib3 - aac(6')-Ib-cr). To the best of our knowledge, this is the first report of two novel class I integrons designated by INTEGRALL as In2083 and In2084 found in XDR-P. aeruginosa PA99 clinical isolate from Thailand. The characterization of genetic contexts of In2083 and In2084 provide the evidence of the assorting of resistance genes to evolve as novel integrons.

Comparison of antimicrobial resistant Escherichia coli isolated from Irish commercial pig farms with and without zinc oxide and antimicrobial usage

BackgroundThe prophylactic use of antimicrobials and zinc oxide (ZnO) in pig production was prohibited by the European Union in 2022 due to potential associations between antimicrobial and heavy metal usage with antimicrobial resistance (AMR) and concerns regarding environmental pollution. However, the effects of their usage on the bacterial AMR profiles on commercial pig farms are still not fully understood and previous studies examining the effect of ZnO have reported contrasting findings. The objective of this study was to examine the effects of antimicrobial and ZnO usage on AMR on commercial pig farms. Faecal and environmental samples were taken on 10 Irish commercial farms, of which 5 farms regularly used ZnO and antimicrobials (amoxicillin or sulphadiazine-trimethoprim) for the prevention of disease. The other 5 farms did not use ZnO or any other form of prophylaxis. Escherichia coli numbers were quantified from all samples using non-supplemented and supplemented Tryptone Bile X-glucuronide agar.ResultsIn total 351 isolates were phenotypically analysed, and the genomes of 44 AmpC/ESBL-producing E. coli isolates from 4 farms were characterised using whole-genome sequencing. Phenotypic analysis suggested higher numbers of multi-drug resistant (MDR) E. coli isolates on farms using prophylaxis. Furthermore, farms using prophylaxis were associated with higher numbers of isolates resistant to apramycin, trimethoprim, tetracycline, streptomycin, and chloramphenicol, while resistance to ciprofloxacin was more associated with farms not using any prophylaxis. Thirty-four of the 44 AmpC/ESBL-producing E. coli strains harboured the blaCTX-M-1 resistance gene and were multi drug resistant (MDR). Moreover, network analysis of plasmids and analysis of integrons showed that antimicrobial and biocide resistance genes were frequently co-located on mobile genetic elements, indicating the possibility for co-selection during antimicrobial or biocide usage as a contributor to AMR occurrence and persistence on farms.ConclusionsThe results of this study showed evidence that antimicrobial and ZnO treatment of pigs post-weaning can favour the selection and development of AMR and MDR E. coli. Co-location of resistance genes on mobile genetic elements was observed. This study demonstrated the usefulness of phenotypic and genotypic detection of antimicrobial resistance by combining sequencing and microbiological methods.

Implication of different replicons in the spread of the VIM-1-encoding integron, In110, in Enterobacterales from Czech hospitals.

VIM metallo-β-lactamases are enzymes characterized by the ability to hydrolyze all β-lactams. Usually, bla VIM-like genes are carried by class 1 integrons. In the Czech Republic, only sporadic cases of VIM-producing Enterobacterales have been reported in which those isolates carried the VIM-1 carbapenemase-encoding integron In110. However, during 2019-2020, an increased number was reported. Therefore, the aim of the current study was to characterize the genetic elements involved in the increased spread of bla VIM genes. 32 VIM-producing Enterobacterales collected between 2019 and 2020 were subjected to: antimicrobial susceptibility testing, integron analysis, and short reads sequencing. Based on the results, 19 isolates were selected as representative and sequenced using Sequel I platform. The 32 VIM-producing isolates exhibited variations in the MICs of carbapenems. Based on short-read data, 26 of the 32 sequenced isolates harbored the bla VIM-1 allele while six isolates carried the bla VIM-4 gene. The most prevalent was the In110 integron (n = 24) and two isolates carried the In4873 class 1 integron. The bla VIM-4 allele was identified in class 1 integrons In1174 (n = 3), In416 (n = 1), In2143 (n = 1) and In2150. Long reads sequencing revealed that the bla VIM was carried by: pKPC-CAV1193-like (n = 6), HI1 (pNDM-CIT; n = 4), HI2 (n = 3), FIB (pECLA; n = 2) and N (n = 1) incompatibility groups. Two bla VIM-carrying plasmids could not be typed by the database, while another one was integrated into the chromosome. We observed the spread of VIM-encoding integrons, mainly of In110, among Enterobacterales isolated from Czech hospitals, but also an increased number of novel elements underlining the ongoing evolution.

IntegronFinder 2.0: Identification and Analysis of Integrons across Bacteria, with a Focus on Antibiotic Resistance in Klebsiella.

Integrons are flexible gene-exchanging platforms that contain multiple cassettes encoding accessory genes whose order is shuffled by a specific integrase. Integrons embedded within mobile genetic elements often contain multiple antibiotic resistance genes that they spread among nosocomial pathogens and contribute to the current antibiotic resistance crisis. However, most integrons are presumably sedentary and encode a much broader diversity of functions. IntegronFinder is a widely used software to identify novel integrons in bacterial genomes, but has aged and lacks some useful functionalities to handle very large datasets of draft genomes or metagenomes. Here, we present IntegronFinder version 2. We have updated the code, improved its efficiency and usability, adapted the output to incomplete genome data, and added a few novel functions. We describe these changes and illustrate the relevance of the program by analyzing the distribution of integrons across more than 20,000 fully sequenced genomes. We also take full advantage of its novel capabilities to analyze close to 4000 Klebsiella pneumoniae genomes for the presence of integrons and antibiotic resistance genes within them. Our data show that K. pneumoniae has a large diversity of integrons and the largest mobile integron in our database of plasmids. The pangenome of these integrons contains a total of 165 different gene families with most of the largest families being related with resistance to numerous types of antibiotics. IntegronFinder is a free and open-source software available on multiple public platforms.

Methods for the targeted sequencing and analysis of integrons and their gene cassettes from complex microbial communities.

Integrons are microbial genetic elements that can integrate mobile gene cassettes. They are mostly known for spreading antibiotic resistance cassettes among human pathogens. However, beyond clinical settings, gene cassettes encode an extraordinarily diverse range of functions important for bacterial adaptation. The recovery and sequencing of cassettes has promising applications, including: surveillance of clinically important genes, particularly antibiotic resistance determinants; investigating the functional diversity of integron-carrying bacteria; and novel enzyme discovery. Although gene cassettes can be directly recovered using PCR, there are no standardised methods for their amplification and, importantly, for validating sequences as genuine integron gene cassettes. Here, we present reproducible methods for the amplification, sequence processing, and validation of gene cassette amplicons from complex communities. We describe two different PCR assays that either amplify cassettes together with integron integrases, or gene cassettes together within cassette arrays. We compare the performance of Nanopore and Illumina sequencing, and present bioinformatic pipelines that filter sequences to ensure that they represent amplicons from genuine integrons. Using a diverse set of environmental DNAs, we show that our approach can consistently recover thousands of unique cassettes per sample and up to hundreds of different integron integrases. Recovered cassettes confer a wide range of functions, including antibiotic resistance, with as many as 300 resistance cassettes found in a single sample. In particular, we show that class one integrons are collecting and concentrating resistance genes out of the broader diversity of cassette functions. The methods described here can be applied to any environmental or clinical microbiome sample.

Drug Resistance and Integron Genes in Escherichia coli Isolated from Urinary Tract Infection.

Escherichia coli (E. coli) is a major cause of urinary tract infections. Treatment of these infections with antibiotics is often not effective due to the acquisition of drug-resistance genes by the bacteria. This process is mediated by integrons which belong to bacterial mobile genetic elements. Therefore, the present study addressed the issue of the relation between antibiotic resistance and integron genes in E. coli isolated from patients affected by urinary tract infection. Multiplex PCR assay employed to detect the E. coli integrase gene demonstrated that out of 49 bacterial strains, 26 were carrying class 1 integron and there was no case of bacteria harboring class 2 or class 3 integrons. Correlation analysis documented that E. coli strains harboring class 1 integron exhibited higher resistance towards tobramycin. The variable region gene cassette contained combinations of four genes responsible for antibiotic resistance: dfr17, aadA2, aadA5, and aac(6')-Ib-cr, of which the latter conferred tobramycin resistance. Together, the collected data underscore the need for identification and analysis of integrons in E. coli-induced urinary infections.

Molecular analysis of integrons and antimicrobial resistance profile in Shigella spp. isolated from acute pediatric diarrhea patients.

Introduction: Shigella spp. is a growing global health concern due to increasing multiple drug resistance, commonly resulting in therapeutic failure. Integrons are gene expression systems run by integrase genes. The aims of this study were detection of class I, II and III integrons and assessment of antimicrobial resistance in Shigella spp. isolated from acute pediatric diarrhea patients.Materials and methods: From January to December 2015, 16 Shigella spp. were isolated from 310 non-duplicative diarrheal stool samples in Children’s Medical Center, Tehran, Iran. The isolates were analyzed for their antibiotic susceptibility using CLSI guidelines M100-S14. Multiplex PCR was used for amplification of I, II and III integron-associated integrase (intl) genes.Results: Of 310 stool samples, 16 (5.2%) were positive for Shigella spp., in 7 of them S. sonnei and in 9 of them S. flexneri were identified. Results of the antimicrobial susceptibility test showed that 6.2%, 50%, 31.2%, 6.2%, 81.2%, 56.2% and 31.2% of the isolates were resistant to gentamicin, chloramphenicol, nalidixic acid, ciprofloxacin, tetracycline, ampicillin and trimethoprim-sulfamethoxazole, respectively. Multiplex PCR results revealed that 6.2% (1/16), 31.2% (5/16), 50% (8/16) of Shigella isolates carried intlI, intlII and both intlI/intllI genes. No class 3 integrons were detected.Discussion: In this study, multidrug resistance was seen in Shigella isolates similar to that in isolates from other geographical areas. This is possible due to inappropriate use of antimicrobials. Furthermore, prevalence of multidrug resistance was significantly linked to the presence of integrin genes. Conclusion: A class 2 integron plays a role in presence of multidrug resistance in Shigella spp. It is vital to prevent the spread of antibiotic resistance through continuous monitoring.

Molecular analysis of multidrug resistance in clinical isolates of Shigella spp. from 2001-2010 in Kolkata, India: role of integrons, plasmids, and topoisomerase mutations.

To understand the genetic basis of high drug resistance in Shigella, 95 clinical isolates of Shigella spp. (2001–2010) were obtained from the Infectious Diseases Hospital, Kolkata, India. Ninety-three isolates were resistant to three or more antibiotics. Resistance to nalidixic acid, trimethoprim, streptomycin, and co-trimoxazole was most common in this population. Dendrogram analysis showed that S. sonnei strains were more clonally related when compared to the other Shigella species. The role of mobile genetic elements and chromosome-borne resistance factors was analyzed in detail. Integron analysis indicated the preponderance of class 2 and atypical class 1 integrons in that population. Typical class 1 integron was present in only one S. sonnei isolate and harbored trimethoprim resistance-encoding gene dfrV, while atypical class 1 integrons harbored dfrA1–aadA or blaOXA-aadA gene cassettes responsible for resistance to trimethoprim, aminoglycosides, and β-lactams. Class 2 integrons harbored either dfrA1-sat-aadA or dfrA1-sat gene cassettes. Most importantly, a novel gene cassette array InsE-InsO-dfrA1-sat was found in class 2 integron of S. sonnei NK4846. Many of the resistance traits for antibiotics such as trimethoprim, co-trimoxazole, kanamycin, ampicillin, and tetracycline were transferred from parent Shigella isolates to recipient Escherichia coli during conjugation, establishing the role of plasmids in horizontal transfer of resistance genes. Multiple mutations such as S80→I, S83→L, and D87→G/N/Y in quinolone resistance determining regions of topoisomerases from the representative quinolone-resistant isolates could explain the spectrum of minimal inhibitory concentration values for various quinolones. To the best of our knowledge, this is the first comprehensive report that describes the contribution of mobile (plasmids, integrons, and quinolone resistance genes named qnr) and innate genetic elements (mutations in topoisomerases) in determining the resistance phenotype of all the four species of Shigella over a span of ten years.

Identification and analysis of integrons and cassette arrays in bacterial genomes

Integrons recombine gene arrays and favor the spread of antibiotic resistance. Their broader roles in bacterial adaptation remain mysterious, partly due to lack of computational tools. We made a program – IntegronFinder – to identify integrons with high accuracy and sensitivity. IntegronFinder is available as a standalone program and as a web application. It searches for attC sites using covariance models, for integron-integrases using HMM profiles, and for other features (promoters, attI site) using pattern matching. We searched for integrons, integron-integrases lacking attC sites, and clusters of attC sites lacking a neighboring integron-integrase in bacterial genomes. All these elements are especially frequent in genomes of intermediate size. They are missing in some key phyla, such as α-Proteobacteria, which might reflect selection against cell lineages that acquire integrons. The similarity between attC sites is proportional to the number of cassettes in the integron, and is particularly low in clusters of attC sites lacking integron-integrases. The latter are unexpectedly abundant in genomes lacking integron-integrases or their remains, and have a large novel pool of cassettes lacking homologs in the databases. They might represent an evolutionary step between the acquisition of genes within integrons and their stabilization in the new genome.

Molecular Characterization of Salmonella enterica Serovars Isolated from a Turkey Production Facility in the Absence of Selective Antimicrobial Pressure.

This study evaluated antimicrobial resistance and virulence factors in Salmonella enterica isolated from a turkey flock in which the birds were raised in an environment where antimicrobials were not administered to the birds, either through feed or water. Salmonella was isolated from turkeys and various environmental samples in the facility using conventional microbiological procedures. Isolates were serotyped and analyzed phenotypically by antimicrobial resistance profiling and genotypically by pulsed-field gel electrophoresis (PFGE) fingerprinting, integron analysis, plasmid profiling, replicon-based incompatibility (Inc) group typing, and virulence gene profiling. Ninety-five S. enterica isolates were isolated from cecal contents (n = 29), feed (n = 22), leftover feed (n = 13), litter (n = 12), drinkers (n = 10), environment (n = 8), and an insect. The following serotypes were identified: Montevideo (24%), Anatum (22%), Agona (17%), Kentucky and Worthington (12%), Senftenberg (11%), and rough phenotypes (3%). The majority of isolates (61/95; 64%) were susceptible to 12 antimicrobials tested; however, despite the absence of antimicrobials in the facility, approximately 36% of the isolates were resistant to two to five antimicrobials. Class 1 integrons were detected in 8% of the isolates. The integron sequence analysis revealed dihydrofolate reductase (dhfr) and aminoglycoside adenylyl transferase (aadA2) genes, which encode trimethoprim and streptomycin resistance, respectively. Furthermore, 71% of the isolates had at least one plasmid. There were five plasmid replicon types identified among the isolates, including IncI1, IncHI2, IncFIIA, IncB/O, and IncP, with variable prevalence among the serotypes. All 95 isolates tested polymerase chain reaction-positive for 19 virulence genes and negative for virD4 and virB4. The virulence gene profiles were similar within the isolates from the same serotype. Within particular serotypes, PFGE patterns revealed 100% similarity, even when the bacterial strains were isolated from different sources, indicating cross-colonization of sources within the turkey facility. On this antibiotic-free turkey farm, turkeys and feed appeared to be the major reservoirs of multidrug-resistant Salmonella, which harbored multiple virulence genes.

The contribution of antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates: an emphasis on efflux pump activity.

Due to the limited information of the contribution of various antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates, Antibiotic resistance mechanisms, including integron analysis, identification of quinolone resistance-determining region mutations, measurement of efflux pump activity, and sequence analysis of efflux pump regulators, were investigated in 66 clinical B. cepacia complex isolates. Species were identified via recA-RFLP and MALDI-TOF. Four genomovars were identified by recA-RFLP. B. cenocepacia (genomovar III) was the most prevalent genomovar (90.1%). Most isolates (60/66, 90.9%) were correctly identified by MALDI-TOF analysis. Clonal relatedness determined by PFGE analysis revealed 30 pulsotypes, including two major pulsotypes that comprised 22.7% and 18.2% of the isolates, respectively. Seventeen (25.8%) isolates harboured class 1 integron with various combinations of resistance genes. Among six levofloxacin-resistant isolates, five had single-base substitutions in the gyrA gene and three demonstrated efflux pump activities. Among the 42 isolates exhibiting resistance to at least one antimicrobial agent, 94.4% ceftazidime-resistant isolates (17/18) and 72.7% chloramphenicol-resistant isolates (16/22) demonstrated efflux pump activity. Quantitation of efflux pump RNA level and sequence analysis revealed that over-expression of the RND-3 efflux pump was attributable to specific mutations in the RND-3 efflux pump regulator gene. In conclusion, high-level expression of efflux pumps is prevalent in B. cepacia complex isolates. Mutations in the RND-3 efflux pump regulator gene are the major cause of efflux pump activity, resulting in the resistance to antibiotics in clinical B. cepacia complex isolates.

Analysis of integrons and associated gene cassettes in clinical isolates of multidrug resistant Pseudomonas aeruginosa from Southwest Nigeria

BackgroundMultidrug resistant Pseudomonas aeruginosa harbours integrons and other mobile genetic elements such as plasmids and transposons, which easily disseminate antibiotic resistance genes among clinical strains of P. aeruginosa.MethodologyPlasmid extraction of 54 clinical isolates of P. aeruginosa was carried out by alkaline lysis method; and plasmid size estimation was done by using E. coli V517 standard plasmid marker. Fifty-four clinical strains of P. aeruginosa were isolated from 5 hospitals in 3 Southwestern states of Nigeria between March and September 2010. Plasmid extraction of isolates was carried out by alkaline lysis method; and plasmid size estimation was done by using E. coli V517 standard plasmid marker. PCR amplification for the 3 classes of resistance integrons, and gene cassette characterization were carried out using specific primers and by sequencing of PCR products. Conjugal mating of the integron positive P. aeruginosa strains with E. coli DH5α was performed to demonstrate transferability of integrons and gene cassettes.ResultAgarose gel electrophoresis of plasmid DNA revealed that all the 54 P. aeruginosa harboured 1–4 plasmids with sizes ranging from 2.2 – >58 kb. Class 1 integron was identified in 31 (57%) strains; but none of them carried class 2 and class 3 integrons. High prevalence of aadA gene conferring resistance to streptomycin/spectinomycin was detected in the strains positive for class 1 integron. Sequencing of the 1.6 kb and 1.2 kb amplified band of gene cassettes revealed the presence of aadA6-orfD and aadA13 respectively.ConclusionThis study demonstrates the presence of plasmids and integrons harbouring resistance gene cassettes, which may collectively constitute an efficient system for dissemination of resistance genes in P. aeruginosa. Disturbingly, the rapid and unabated spread of class 1 integron-associated multidrug resistant P. aeruginosa in Southwest Nigeria may greatly hamper successful treatment of infections caused by such strains. This necessitates the establishment of functional antimicrobial resistance surveillance programmes in Nigeria.

Use of RAPD-PCR, plasmid profiling, class 1 integron analysis, and antimicrobial resistance for molecular characterisation of Salmonellastrains isolated from Turkey

Forty-one Salmonella strains, isolated from food samples collected from different parts of Turkey were characterized by various biochemical and genotypic tests. Among 41 isolates, 10 strains harboured at least one plasmid, sizes ranging from 3 kb to 400 kb. The antibiotic resistance profiles of Salmonella isolates were studied against 16 commonly used antibiotics. The highest level of resistance was observed against sulfonamide and neomycin, respectively. Moreover, resistance to multiple antibiotics (5) was also observed among 59% of isolates. Each isolate was also screened for the presence of class 1 integrons and nearly 88% of strains contained class 1 integron with different variable regions (VRs). Further examination of the Salmonella isolates was carried out by testing their clonal relation by RAPD-PCR analyses. With the amplification of primer P1254 the isolates classified into 15 groups and with primer P1283, they classified into 8 groups, on the basis of statistical analyses.

Multi-drug Resistance and Characteristic of Integrons in Shigella spp.Isolated from China

Objective To investigate the characteristic of integrons and the relationship between integrons and antimicrobial resistance in Shigella spp. Methods Ninety Shigella strains (83 S. flexneri and 7 S. sonnei) were isolated from the stools of patients in China. Susceptibility to 8 antimicrobials was tested for all isolated strains. PCR, RFLP and sequencing analysis of integrons were applied to all of them. Results High prevalence of multi-drug resistance (95.6%) was identified. Of the isolates 79 (87.8%) carried integrase genes of class 1 integron (3.3%), class 2 integron (10.0%) or both (74.4%). No intI3 was detected in the tested isolates. The prevalence of intI2 was significantly higher in isolates with multi-drug resistance to at least 3 antibiotics than that in isolates with resistance to 2 and less antibiotics ( P<0.05). Gene cassettes dfrA17-aadA5, dfrA12-orfF-aadA2 of class 1 integron and dfrA1-sat1-aadA1 of class 2 integron were identified. Conclusion The class 2 integron may play a role in the emergence of multi-drug resistance in Shigella spp.

Salmonella enterica isolates from pasture-raised poultry exhibit antimicrobial resistance and class I integrons

While considerable foodborne pathogen research has been conducted on conventionally produced broilers and turkeys, few studies have focused on free-range (organic) or pastured poultry. The current surveillance study was designed to isolate, identify and genetically characterize Salmonella from pastured poultry farm environment and from retail samples. In this study, 59 isolates were collected from two pastured poultry farms (n = 164; pens, feed, water and insect traps) and retail carcasses (n = 36) from a local natural foods store and a local processing plant. All isolates were serotyped and analysed phenotypically (antimicrobial resistance profiles) and genotypically (DNA fingerprints, plasmid profiles and integron analysis). Salmonella enterica was detected using standard microbiological methods. Salmonella Kentucky was the most prevalent serotype detected from the sampled sources (53%), followed by Salmonella Enteritidis (24%), Bareilly (10%), Mbandaka (7%), Montevideo (5%) or Newport (2%). All isolates were resistant to sulfisoxazole and novobiocin, and the majority (40/59) possessed class I integrons shown by PCR detection. Each Salmonella serotype elicited a distinct pulsed-field gel electrophoresis fingerprint profile, and unique differences were observed among the serotypes. The findings of this study show that Salmonella serotypes isolated from pasture-raised poultry exhibit antimicrobial resistance and class I integrons. This study demonstrates that despite the cessation of antibiotic usage in poultry production, antibiotic resistant Salmonella may still be recovered from the environment and poultry products.

Occurrence and Distribution of Various Genetic Structures of Class 1 and Class 2 Integrons inSalmonella entericaIsolates from Foodborne Disease Patients in Korea for 16 Years

We have investigated the distribution of integrons among 752 multidrug-resistant Salmonella isolates from human febrile and/or diarrheal patients during 1992-2007 and analyzed their genetic characteristics. Here, we report extensive integron analysis results within human isolates during the last 16 years. The gene or gene cassette(s) in the class 1 integrons found in the isolates were dfrA7, dfrA12-orfF-aadA2, aadA2, bla(PSE1), dfrA1-aadA1, dfrA17-aadA5, bla(OXA1)-aadA1, aadB-aadA1, aadA22, aadA1, and aac6'Ib-bla(OXA1)-aadA2. Class 2 integrons harboring dfrA1-sat2-aadA1 gene cassette were also found in four isolates. Twenty-nine isolates including one Salmonella Schleissheim isolate had two integrons harboring aadA2 and bla(PSE1) in their variable regions of 1.0 and 1.2 kb amplicons, respectively, which have been also found in Salmonella genomic island 1 (SGI1) of multidrug-resistant Salmonella Typhimurium DT104. The presence of SGI1 in Salmonella Schleissheim isolate was proved by SGI1-specific polymerase chain reaction. We first report a Salmonella Schleissheim having SGI1, Salmonella Typhimurium and Salmonella Heidelberg having the class 2 integron with dfrA1-sat2-aadA1 cassettes, Salmonella London with the aac6'Ib-bla(OXA1)-aadA2 gene cassette, Salmonella Chailey with the gene cassette of aadA22, and coexistence of two class 1 integrons carrying aadA22 and dfrA12-orfF-aadA2 in Salmonella Typhimurium.

Antibiotic resistance integrons and extended-spectrum -lactamases among Enterobacteriaceae isolates recovered from chickens and swine in Portugal

To investigate the diversity of integrons and extended-spectrum beta-lactamases (ESBLs) among Enterobacteriaceae from chickens and swine in Portugal and analyse the clonal relationships between Portuguese ESBL-producing isolates of animal and human origin. We analysed samples from faeces of healthy swine (HSF, n = 35), from uncooked chicken carcasses (CM, n = 20) and from faeces of healthy chickens (HCF, n = 20). Samples were plated on MacConkey agar with and without ceftazidime (1 mg/L) or cefotaxime (1 mg/L). ESBLs were characterized by PCR and DNA sequencing. Bacterial identification, antibiotic susceptibility and conjugation assays were performed by standard procedures. Isolate clonal relatedness was established by PFGE and by RAPD for PFGE non-typeable isolates. Escherichia coli phylogenetic groups were identified by a multiplex PCR. Integron analysis was accomplished by PCR-RFLP and sequencing. ESBL-producing Enterobacteriaceae were identified in 60% of CM, 10% of HCF and 5.7% of HSF samples, respectively, mostly corresponding to E. coli (phylogroups A, D and B1). TEM-52, SHV-2 and CTX-M-1 were detected from chicken and SHV-12 from swine samples. High clonal diversity was observed and most bla(ESBL) genes were transferable (67%). Class 1 and/or class 2 integrons were identified in 80% of CM, 10% of HCF and 63% of HSF samples, with class 1 integrons more common than class 2 integrons (36% versus 12% of the isolates recovered, respectively). Ten class 1 integron types are described, aadA1 and dfrA1-aadA1 being the most frequently found. Two class 1 integron types (aadA13-estX and dfrA14-aadA1-catB2) and one class 2 integron (aadA1) are first reported here. This study is the first report of ESBLs and integrons from chickens and swine in Portugal and highlights the antibiotic-resistant bacteria and/or resistance genes that might be acquired by humans through the food chain.

Pulsed field, ribotype and integron analysis of multidrug resistant isolates of Salmonella enterica serovar Newport

Twenty-one isolates of Salmonella enterica serovar Newport were evaluated for their antimicrobial resistance, pulsed field gel electrophoresis (PFGE) profiles, ribotype profiles, and their integron profiles. Antimicrobial resistance profiles indicated that 20 of the 21 isolates were resistant to the following antibiotics: amoxicillin-clavulanic acid (AMOX/CA), ampicillin (AMPC), cefoxitin (CFOX), ceftiofur (TIO), cephalothin (CRIN), chloramphenicol (CHL), streptomycin (STR), tetracycline (TET), and sulfamethoxazole (SMX). Five isolates showed resistance to gentamycin (GEN) and kanamycin (KAN). Trimethoprim-sulfamethoxazole (SMX/TMP) resistance was observed in six isolates. Eight of the twenty one isolates showed intermediate resistance to ceftriaxone (CTRX), with one isolate exhibiting complete resistance. PFGE clearly resolved the Salmonella Newport isolates into nine distinct clusters, and a good congruence was observed between PFGE and antibiotic resistance patterns. Automated riboprinting clearly distinguished between antibiotic resistant and sensitive strains of Salmonella Newport, and resolved the isolates into two ribogroups. One group consisted of the multidrug resistant isolates, and the other grouping contained the sensitive isolate. Three different integrons (1.0, 1.2, and 1.8 kb) were observed in many of the isolates, and several isolates contained more than one integron. Restriction fragment length polymorphisms (RFLP) indicated that integrons of the same size were indistinguishable. When integron analysis and ribotype analysis were used in conjunction, four subtypes of multidrug resistant Salmonella Newport isolates were clearly defined. These results demonstrate the possibility of utilizing automated ribotyping and integron analysis to rapidly subtype multidrug resistant Salmonella Newport isolates.

Emergence of PER and VEB extended-spectrum -lactamases in Acinetobacter baumannii in Belgium

Strains of Acinetobacter baumannii producing the extended-spectrum beta-lactamase (ESBL) PER-1 are widespread in Turkey and have also been reported from Korea and France. In contrast, A. baumannii producing the ESBL VEB-1 have only been reported from France, where one strain was responsible for a nationwide outbreak in 2003-2004. Here we describe the emergence of strains of A. baumannii producing VEB-1 and PER- 1 in Belgium. Belgian hospitals were alerted in December 2003 to the emergence in France of VEB-1-producing A. baumannii susceptible only to meropenem and colistin. Isolates with a compatible susceptibility profile were sent to a single central laboratory for VEB-1 confirmation, molecular characterization and typing. From December 2003 to March 2005, three hospitals located close to the French border and one in the Brussels area reported isolation of eight A. baumannii isolates compatible with the French epidemic clone. Using PCR, six were identified as VEB-1-positive and two as PER-1-positive. All the VEB-1-positive isolates were clonally related by PFGE and by integron analysis to the French epidemic strain. The PER-1-positive strains were indistinguishable by PFGE but not related to the known French isolate or to several Turkish isolates. Both genes were chromosomally encoded. This work illustrates the inter-country spread of VEB-1-producing A. baumannii isolates as well as the emergence of PER-1-producing A. baumannii strains in Belgium.

Analysis of integrons in clinical isolates ofEscherichia coliin China during the last six years

A multiple PCR for the detection of the integrase genes of the three classes of integrons was carried out, and their gene cassettes were characterized in 111 clinical strains of Escherichia coli isolated in Guangzhou City, China during the last 6 years. IntI1 and intI2 genes were detected in 95 isolates (85.6%) and four isolates (3.6%), respectively. No intI3 gene was detected. Six different gene cassettes were found in these strains, and a high prevalence of dfr and aad genes was observed. The E. coli isolates that contained a 1664-bp amplicon of dfrA17-aadA5 in class 1 integron were found to be phylogenetically unrelated to each other by using the enterobacterial repetitive intergenic consensus PCR, as the cassette could be transferred to recipient strains, indicating that the gene cassettes might be disseminated in the clinical strains by a horizontal gene transfer. Therefore, it is important that guidelines for the prudent use of antimicrobial agents are adopted and surveillance programs are established.