aadA1 Gene Cassette Research Articles

Emergence of Salmonella Infantis carrying the pESI-like plasmid from eggs in egg grading and packing plants in Korea

The plasmid of emerging S. Infantis (pESI) or pESI-like plasmid in Salmonella enterica Infantis are consistently reported in poultry and humans worldwide. However, there has been limited research on these plasmids of S. Infantis isolated from eggs. Therefore, this study aimed to analyze the prevalence and characteristics of S. Infantis carrying the pESI-like plasmid from eggs in egg grading and packing plants. In this study, the pESI-like plasmid was only detected in 18 (78.3%) of 23 S. Infantis isolates, and it was absent in the other 9 Salmonella serovars. In particular, S. Infantis isolates carrying the pESI-like plasmid showed the significantly higher resistance to β-lactams, phenicols, cephams, aminoglycosides, quinolones, sulfonamides, and tetracyclines than Salmonella isolates without the pESI-like plasmid (p < 0.05). Moreover, all S. Infantis isolates carrying the pESI-like plasmid were identified as extended-spectrum β-lactamase (ESBL) producer, harboring the blaCTX-M-65 and blaTEM-1 genes, and carried non-β-lactamase resistance genes (ant(3′′)-Ia, aph(4)-Ia, aac(3)-IVa, aph(3′)-Ic, sul1, tetA, dfrA14, and floR) against five antimicrobial classes. However, all isolates without the pESI-like plasmid only carried the blaTEM-1 gene among the β-lactamase genes, and either had no non-β-lactamase resistance genes or harbored non-β-lactamase resistance genes against one or two antimicrobial classes. Furthermore, all S. Infantis isolates carrying the pESI-like plasmid carried class 1 and 2 integrons and the aadA1 gene cassette, but none of the other isolates without the pESI-like plasmid harbored integrons. In particular, D87Y substitution in the gyrA gene and IncP replicon type were observed in all the S. Infantis isolates carrying the pESI-like plasmid but not in the S. Infantis isolates without the pESI-like plasmid. The distribution of pulsotypes between pESI-positive and pESI-negative S. Infantis isolates was clearly distinguished, but all S. Infantis isolates were classified as sequence type 32, regardless of whether they carried the pESI-like plasmid. This study is the first to report the characteristics of S. Infantis carrying the pESI-like plasmid isolated from eggs and can provide valuable information for formulating strategies to control the spread of Salmonella in the egg industry worldwide.

Spring of Warta River as a Source of Antibiotic-Resistant Coliform Bacteria

The aim of the study was to evaluate the occurrence of integron-carrying coliform bacteria in the spring water of Warta river, a major river in Poland. We isolated 18 strains (12 Escherichia coli, three Pantoea sp., 2 Citrobacter freundii, and 1 Klebsiella oxytoca) harbouring class 1 and/or class 2 integrons. Class 1 integrons contained aadA1, dfrA7, dfrA17, dfrA1-aadA1, and dfrA17-aadA5 gene cassette arrays, whereas the genetic content of class 2 integrons was stable and consisted of sat2-aad1 gene cassette array. The strains carrying integrons were resistant to 4–11 antimicrobials, most frequently to sulfamethoxazole, ampicillin, piperacillin, trimethoprim, and trimethoprim/sulfamethoxazole.

High Frequency of Class I and II Integrons and the Presence of aadA2 and dfrA12 Gene Cassettes in the Clinical Isolates of Acinetobacter baumannii from Shiraz, Southwest of Iran

Background: Acinetobacter baumannii is a global concern that causes healthcare-associated infections due to multidrug resistance against commercially available antimicrobial agents. Objectives: The present study was conducted to determine the antimicrobial susceptibility of A. baumannii isolates from clinical specimens in Shiraz, Iran. In addition, the possible relationship of susceptibility patterns with the presence of integrons and related gene cassettes is investigated. Methods: Acinetobacter baumannii isolates were collected, and their susceptibility to various antibiotics was tested using the Kirby-Bauer disk diffusion method. Moreover, molecular analysis were performed to detect the presence of the OXA-51-like gene, as well as class I, II, and III integrons, and associated gene cassettes. Results: The majority of isolates were resistant to imipenem (99.4%), piperacillin (98.2%), gentamycin (98.2%), meropenem (97.7%), ceftazidime (95.4%), amikacin (95.4%), and trimethoprim-sulfamethoxazole (90.8%). All strains showed multidrug resistance to the tested antibiotics. The distribution analysis of integrons genes revealed that 90.2, 72.4, and 12.1% of the isolates carried intI1, intI2, and intI3 genes, respectively. Moreover, two types of prevalent gene cassettes, including aad and dfr, were detected in class 1 integron-carrying strains. Conclusions: The current study showed the high prevalence of A. baumannii isolates harboring integrons in our investigated medical center, which may indicate the distribution of multidrug resistance events. The different gene cassette arrays highlight the remarkable role of geographical issues in disseminating multidrug-resistant (MDR) isolates. This could be attributed to distinct therapeutic interventions in different areas. The results demonstrate the necessity of continuous surveillance to prevent the distribution of multidrug resistance among A. baumannii strains in Iran.

Pathogenomics and Evolutionary Epidemiology of Multi-Drug Resistant Clinical Klebsiella pneumoniae Isolated from Pretoria, South Africa

Antibiotic-resistant Klebsiella pneumoniae is increasingly being implicated in invasive infections worldwide with high mortalities. Forty-two multidrug resistant (MDR) K. pneumoniae isolates were collected over a 4-month period. Antimicrobial susceptibility was determined using Microscan. The evolutionary epidemiology, resistome, virulome and mobilome of the isolates were characterised using whole-genome sequencing and bioinformatics analysis. All isolates contained the blaCTX-M gene, whilst 41/42(97%) contained blaTEM, 36/42(86%) contained blaOXA and 35/42(83%) harboured blaSHV genes. Other resistance genes found included blaLEN, aac(6′)-lb-cr, qnrA, qnrB, qnrS, oqxAB, aad, aph, dfr, sul1, sul2, fosA, and cat genes. Fluoroquinolone and colistin resistance-conferring mutations in parC, gyrAB, pmrAB, phoPQ and kpnEF were identified. The blaLEN gene, rarely described worldwide, was identified in four isolates. The isolates comprised diverse sequence types, the most common being ST152 in 7/42(17%) isolates; clone-specific O and K capsule types were identified. Diverse virulence genes that were not clone-specific were identified in all but one isolate. IncF, IncH and IncI plasmid replicons and two novel integrons were present. The blaCTX-M-15 and blaTEM-1 genes were bracketed by Tn3 transposons, ISEc9, a resolvase and IS91 insertion sequence. There were 20 gene cassettes in 14 different cassette arrays, with the dfrA and aadA gene cassettes being the most frequent. Phylogenetic analysis demonstrated that the isolates were evolutionarily associated with strains from both South Africa and abroad. These findings depict the rich resistome, mobilome and virulome repertoire in clinical K. pneumoniae strains, which are mainly transmitted by clonal, multiclonal and horizontal means in South Africa.

Manila clam (Ruditapes philippinarum) marketed in Korea as a source of vibrios harbouring virulence and β-lactam resistance genes.

The present study assessed the occurrence, virulence determinants and antimicrobial susceptibility patterns of Vibrio spp. isolated from live Manila clam (Ruditapes philippinarum). A total of 31 Vibrio spp. including 27 V. diabolicus, two V. fluvialis, one V. alginolyticus and one V. antiquarius were isolated and identified. Phenotypic detection of DNase, lipase, phospholipase, amylase and caseinase activities was 100%; and 87% gelatinase, 45% slime production and 6% haemolysin activities were also observed. The prevalence of toxin-related virulence genes for collagenase (94%), toxR (100%), tlh (68%), and VPI (71%) was detected by PCR. Additionally, two V. fluvialis isolates carried F-toxR and hupo genes. Moreover, 61% of the isolates showed multiple antimicrobial resistance indices >0·2. The resistance rates of ampicillin, piperacillin, colistin sulfate, rifampicin, and cephalothin were 100, 81, 71, 77 and 68% respectively. The prevalence of blaCTX-M (87%), blaTEM (55%) and Int1 (90%) genes was observed, whereas blaSHV, strA-strB, tetA, tetB, and aadA2 gene cassette were reported in varying combinations. However, armA, aac(3)-IIa and quinolone resistance genes (qnrA, qnrB, qnrS) were not amplified. Thus, the virulence along with extended-spectrum β-lactamases (ESBLs) and other antimicrobial resistance genes in multidrug-resistant Manila clam-borne vibrios may pose a public health threat for consumers. SIGNIFICANCE AND IMPACT OF THE STUDY: In Korea, eating raw seafood is considered a great delicacy. This has negatively affected the public by increasing health issues over the years due to the ingestion of vibrios. For the first time, we could identify Vibrio diabolicus and Vibrio antiquarius in marketed Manila clams in Korea. The prevalent Vibrio diabolicus isolates demonstrated the Vibrio parahaemolyticus and Vibrio cholerae homologous virulence genes (toxR, tlh, and VPI). Additionally, the abundance of extended-spectrum β-lactamases (ESBLs) and integron (IntI1) harboured by Manila clam-borne vibrios elucidate the potential health risk for consumers and may complicate health treatments in the case of infection.

Prevalence of virulence and extended-spectrum β-lactamase (ESBL) genes harbouring Vibrio spp. isolated from cockles (Tegillarca granosa) marketed in Korea.

The present study aimed to investigate the incidence, virulence and antibiotic properties in Vibrio spp. isolated from cockles (Tegillarca granosa) marketed in Korea. A total of 32 Vibrio spp. isolates including V. parahaemolyticus (n=4), V. alginolyticus (n=11), V. diabolicus (n=14) and V. harveyi (n=3) were detected using gyrB sequencing. The phenotypic pathogenicity revealed that the DNase, amylase and phospholipase activities were 100%, while lipase, slime production, gelatinase and caseinase were detected in 72, 88, 88 and 81% of the isolates respectively. The PCR amplification for the detection of V. parahaemolyticus species-specific tdh, tlh, trh and toxR genes were positive in 4 (13%), 16 (50%), 0 (0%) and 4 (13%) isolates respectively. The V. alginolytuicus species-specific tdh, tlh, trh, toxR and vac genes were carried by 15 (47%), 29 (91%), 0 (0%), 15 (47%) and 25 (78%) of the isolates respectively. In addition, multidrug resistance was observed by 27 (84%) isolates, whereas higher resistant rates were observed against ampicillin, piperacillin, streptomycin and cephalothin. The occurrence of blaCTX (78%), blaTEM (40%), blaSHV (22%) and aac(6')-Ib (94%) were prevalent, while strAB, tetB, aphAI-IAB, intl1 and aadA1 gene cassettes were also detected. The results signify the potential health risks resulting from the consumption of raw cockles in Korea. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrios are well known to cause human infections following consumption of raw or undercooked seafood. This phenomenon has undoubtedly increased the number of health issues over the past few years in Korea. Among the identified Vibrio spp., we could detect V. diabolicus and V. harveyi for the first time in marketed cockles in Korea. The presence of species-specific genes (tdh-VA, tlh-VP, tlh-VA and toxR-VA) in V. diabolicus exhibits the close genetic affinity among V. parahaemolyticus and V. alginolyticus. Furthermore, the prevalence of extended-spectrum β-lactamases (ESBLs) and other antibiotic resistance genes along with multidrug resistance signifies the potential threat for consumers.

Prediction of antibiotic resistance from antibiotic resistance genes detected in antibiotic-resistant commensal Escherichia coli using PCR or WGS.

To assess the effectiveness of bioinformatic detection of resistance genes in whole-genome sequences in correctly predicting resistance phenotypes. Genomes of a collection of well-characterized commensal Escherichia coli were sequenced using Illumina HiSeq technology and assembled with SPAdes. Antibiotic resistance genes identified by PCR, SRST2 analysis of reads and ResFinder analysis of SPAdes assemblies were compared with known resistance phenotypes. Generally, the antibiotic resistance genes detected using bioinformatic methods were concordant, but only ARG-ANNOT included sat2 . However, the presence or absence of genes was not always predictive of the phenotype. In one strain, trimethoprim resistance was due to a known mutation in the chromosomal folA gene. In cases where the copy number was low, the aadA5 gene downstream of dfrA17 did not confer streptomycin or spectinomycin resistance. Resistance genes were found in the genomes that were not detected previously by PCRs targeting a limited gene set and gene cassettes in class 1 or class 2 integrons. In one isolate, the aadA1 gene cassette in the estX - aadA1 cassettes pair was outside an integron context and was not expressed. The qnrS1 gene, conferring reduced susceptibility to fluoroquinolones, and the bla CMY-2 gene, encoding an ESBL, were each detected in a single isolate and mphA (macrolide resistance) was present in six isolates surrounded by IS 26 and IS 6100 . WGS analysis detected more genes than PCR. Some were not expressed, causing inconsistencies with the experimentally determined phenotype. An unpredicted chromosomal folA mutation causing trimethoprim resistance was found.

Antimicrobial resistance and molecular subtypes of Salmonella enterica serovar Typhi isolates from Kolkata, India over a 15 years period 1998–2012

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), remains an unresolved public health problem in India. Emergence of antimicrobial resistant strains poses a great concern for typhoid treatment and influences reshaping of current S. Typhi population. We included representative S. Typhi strains (n=164) from retrospective studies, both community and hospital based, conducted at National Institute of Cholera and Enteric Diseases, Kolkata during 15 years period (1998–2012) to analyze their antimicrobial resistance (AMR) profiles, mechanism of AMR and molecular subtypes of the strains. More than 60% of the S. Typhi isolates were obtained from community based studies. During the study period, steady decline (46.4%–15.6%) in isolation of multidrug-resistant (MDR, resistant to ampicillin, chloramphenicol and co-trimoxazole) S. Typhi was noticed with parallel increase of nalidixic acid-resistant (NALR) strains (60.7%–93.8%) and ciprofloxacin resistant (CIPR) strains (0%–25%). Of 53 MDR strains, 46 (86.8%) were NALR showing decreased ciprofloxacin susceptible (DCS) (MIC for ciprofloxacin 0.12-0.5μg/ml) phenotype. Conjugative IncHI1 (230kb) and non-conjugative non-IncHI1 (180kb) plasmids were found in 23 (43.4%) and 14 (26.4%) MDR strains respectively, plasmid was absent in 16 (30.2%) MDR strains. MDR strains with or without plasmid shared the same set of resistance genes (blaTEM-1, catA1, sul1, sul2, strA and strB) and class 1 integron possessing dfrA7 gene cassette. Two S. Typhi strains harbored 50kb transferrable plasmids carrying dfrA15 and aadA1 gene cassettes in class 1 integron. The majority of the strains (135/164, 82.3%) belonged to H58 haplotype. Among the MDR isolates, fluoroquinolone resistant or combined resistant isolates (n=147), 127 (86.4%) were H58 and 20 (13.6%) belonged to non-H58. NALRS. Typhi strains with decreased susceptibility or resistance to ciprofloxacin had point mutation(s) in quinolone resistance-determining region of gyrA and parC genes. Pulsed-field gel electrophoresis showed more diversity among NALRS. Typhi than MDR strains. Results of this study generated information useful for better understanding of the disease epidemiology and its control in endemic settings.

The trend of enteropathogenic Escherichia coli towards atypical multidrug resistant genotypes

The aim of this study was to investigate the prevalence of multidrug resistance (MDR) in atypical enteropathogenic Escherichia coli (EPEC) genotypes among 547 diarrheal children. All E. coli isolates with eae+stx1−stx2− genotypes included in this study and atypical property of EPEC was characterized by the absence of Bundle-forming pili (bfpA gene). Bacterial pathogens were detected in 70 patients (12.8%) among which atypical EPEC (5.3%) were the most common. The higher resistance rate was seen to tetracycline (70%), cotrimoxazole (60%) and nalidixic acid (53.3%) related to MDR phenotype in 63.3% of isolates. The presence of class 1 and 2 integrons was 30% and 6.6% with the dominance of dfrA, aadA gene cassettes among the isolates. Eleven out of 21 phenotypically tetracycline-resistant isolates (52.38%) harboured one or two tetracycline resistance genes (A–D) which shows the incapability of known data to reasonable tetracycline resistance phenotypes among EPEC isolates. A high level of genotypic diversity was seen among the isolates by Pulsed-field gel electrophoresis method which ranged from 89.7 to 29% and no clear correlation was obtained between tetracycline resistance or integron carriage and specific pulsotypes. In conclusion, the data presented here add to knowledge about the heterogeneous nature of MDR EPEC population in Iran which has a growing tendency towards atypical genotypes. The distribution of integrons among EPEC isolates in Iran is decreasing; although, the resistance gene content is almost stable through years.

Salmonella enterica resistant to antimicrobials in wastewater effluents and black-headed gulls in the Czech Republic, 2012

We investigated the presence and epidemiological relatedness of Salmonella isolates from a wastewater treatment plant (WWTP) in Brno, Czech Republic and from nestlings of black-headed gulls (Chroicocephalus ridibundus) at the Nove Mlyny waterworks, situated 35km downstream from the WWTP. During 2012, we collected 37 wastewater samples and 284 gull cloacal swabs. From wastewater samples, we obtained 89 Salmonella isolates belonging to 19 serotypes. At least one resistant strain was contained in 89% of those samples. Ten different serotypes of Salmonella were detected in 38 young gulls, among which 14 (37%) were resistant to antimicrobials. Wastewater isolates were mostly resistant to sulphonamides and tetracycline, gull isolates to tetracycline and ampicillin. We detected the occurrence of blaTEM-1,tet(A), tet(B), tet(G), sul1, sul2, sul3, floR and strA resistance genes. For the first time, we identified a class 1 integron with the dfrA12–orfF–aadA2 gene cassette in S. Infantis. Using pulsed-field gel electrophoresis, we confirmed the presence of identical clusters of S. Agona, S. Enteritidis PT8, S. Infantis and S. Senftenberg in wastewater and black-headed gulls, thus indicating the possibility of resistant Salmonella isolates spreading over long distances in the environment.

Detection of blaCTX-M-type genes in complex class 1 integrons carried by Enterobacteriaceae isolated from retail chicken meat in Brazil

CTX-M-type extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae have been increasingly identified in humans and animals, and their potential transmission by contaminated food has been highlighted. In this study, we report for the first time the isolation of multidrug-resistant (MDR) Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis strains harboring blaCTXM-2 or blaCTXM-8 gene variants in chicken meat sold in markets in southeast Brazil. In this regard, the genetic environment of the blaCTX-M-2 gene is composed of a complex class 1 integron and an ISCR1-associated sequence with dfr and/or aadA gene cassettes located within the variable region. In summary, chicken meat may be a reservoir of MDR Enterobacteriaceae harboring blaCTX-M-type genes, which is a public health concern.

Molecular and phylogenetic characterization of multidrug resistant extended spectrum beta-lactamase producing Escherichia coli isolated from poultry and cattle in Odisha, India

The present study was undertaken to determine the occurrence and characterization of extended spectrum beta-lactamase (ESBL) producing Escherichia coli isolated from cattle and poultry in Odisha, India. Of 316 E. coli isolated from 305 samples (170 fecal samples from poultry and 135 milk samples from cattle), a total of 18 E. coli isolates were confirmed as ESBL producers by combination disc method and ESBL E-test. The isolates were resistant to oxyimino cephalosporins and monobactam as revealed by disc diffusion assay and determination of minimum inhibitory concentration. Resistance against other antibiotics was frequently noted as well. Further, beta-lactamase genes viz., blaSHV, blaCTXM, blaTEM and blaampC were detected in 17, 13, 9 and 2 isolates, respectively in PCR. Of the 18 ESBL strains, 16 were positive for class I integron (int1), nine of them carried sulphonamide resistance gene (sul1) and one harbored quinolone resistance gene (qnrB). Virulence markers for extraintestinal pathogenic E. coli like astA, tsh and iucD were also present in 4, 3 and 3 isolates, respectively. All the PCR amplified products were cloned and subjected to sequencing for homology analysis and data were submitted to gene bank. Sequence analysis of the amplified variable regions of class 1 integron of four representative isolates revealed the presence of aadA2 and dfrA12 gene cassettes conferring resistance to aminoglycosides and trimethoprim, respectively. Most of the ESBL producing strains emerged as single lineage through phylogenetic analysis by RAPD and ERIC PCR. This is the first ever systemic study on multidrug resistant ESBL producing E. coli in food producing animals from India.

Integron-bearing Gram-negative bacteria in lake waters

The aim of the study was to determine the occurrence of integron-bearing Gram-negative bacteria in the water of four lakes located in Wielkopolski National Park, Poland. Altogether, 17 isolates harbouring class 1 or class 2 integrons were found. The integron-bearing bacteria were identified as Escherichia coli, Klebsiella pneumoniae, Pasteurella multocida and Aeromonas hydrophila. The variable regions of the class 1 integrons contained aadA1 and dfrA1-aadA1 gene cassettes, whereas class 2 integrons carried dfrA1-sat2-aadA1 gene cassette array. The isolates were resistant to 3-20 antimicrobials. One of them produced SHV-type extended-spectrum β-lactamase. Integrons play a major role in the spread of antibiotic resistance among bacteria. They are frequently found in clinical bacterial strains, but are also detected in environmental isolates in sites affected by anthropogenic pressure. Little is known, however, about the presence and characteristics of integrons in bacteria living in water environments in areas of nature preservation. To the best of our knowledge, this is the first study focused on detection and characterization of integrons in bacteria living in water ecosystems in a national park.

Class 1 integrons and antibiotic resistance of clinical Acinetobacter calcoaceticus-baumannii complex in Poznań, Poland.

Sixty-three clinical isolates of Acinetobacter calcoaceticus–baumannii complex were analyzed for the presence of integrons and antimicrobial resistance. Class 1 integrons were detected in 40 (63.5 %) isolates. None of them had class 2 or class 3 integrons. The majority of the integrons contained aacC1–orfA–orfB–aadA1 gene cassette array. The presence of integrons was associated with the increased frequency of resistance to 12 of 15 antimicrobials tested, multi-drug resistance phenotype, and the overall resistance ranges of the strains.Electronic supplementary materialThe online version of this article (doi:10.1007/s00284-014-0581-0) contains supplementary material, which is available to authorized users.

Determination of a novel integron‐located variant (blaOXA‐320) of Class D β‐lactamase in Proteus mirabilis

Proteus mirabilis (P. mirabilis) is one of Gram-negative pathogens encountered in clinical specimens. A clinical isolate (TRP41) of P. mirabilis was isolated from a Turkish patient in Turkey. The isolate was identified using the API 32GN system and 16S rRNA gene sequencing and it was found resistant to ampicillin/sulbactam, piperacillin, tetracycline, and trimethoprim/sulfamethoxazole. This isolate was harboring a Class 1 integron gene cassette and its DNA sequence analysis revealed a novel blaOXA variant exhibiting one amino acid substitution (Asn266Ile) from blaOXA-1 . This new variant of OXA was located on Class 1 integron together with aadA1 gene encoding aminoglycoside-modifying enzymes. According to sequence records, the new variant was named as blaOXA-320 . Cassette array and size of integron were found as blaOXA-320 -aadA1 and 2086 bp, respectively. The blaOXA-320 gene is not transferable according to conjugation experiment. In this study, we report the first identification of blaOXA-320 -aadA1 gene cassette, a novel variant of Class D β-lactamase, in P. mirabilis from Turkey.

Molecular characterization of multidrug-resistant extended-spectrum β-lactamase-producing Enterobacteriaceae isolated in Antananarivo, Madagascar

BackgroundWe investigated the molecular characteristics of multidrug-resistant, extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolated in community settings and in hospitals in Antananarivo, Madagascar.ResultsForty-nine E. coli, K. pneumoniae, K. oxytoca and E. cloacae ESBL-producing isolates were studied. In antimicrobial susceptibility analyses, many of the isolates exhibited resistance to aminoglycosides, fluoroquinolones and trimethoprim-sulfamethoxazole. Gene amplification analysis and sequencing revealed that 75.5% (n=37) of the isolates harbored blaCTX-M-15 and 38.7% (n=19) harbored blaSHV-12. The non-ESBLs resistance genes detected were blaTEM-1, blaOXA-1, aac(6′)-Ib,aac(6′)-Ib-cr, tetA, sul-1, sul-2, qnrA, qnrB and catB-3. We found dfrA and aadA gene cassettes in the class 1 integron variable regions of the isolates, and the combination of dfrA17-aadA5 to be the most prevalent. All blaCTX-M-15 positive isolates also contained the ISEcp1 insertion element. Conjugation and transformation experiments indicated that 70.3% of the antibiotic resistance genes resided on plasmids. Through a PCR based replicon typing method, plasmids carrying the blaSHV-12 or blaCTX-M-15 genes were assigned to either the IncFII replicon type or, rarely, to the HI2 replicon type. All isolates were subtyped by the rep-PCR and ERIC-PCR methods.Phylogenetic grouping and virulence genotyping of the E. coli isolates revealed that most of them belonged to group A1. One isolate assigned to group B2 harbored blaCTX-M-15 and five virulence genes (traT, fyuA, iutA, iha and sfa) and was related to the O25b-ST131 clone.ConclusionsOur results highlight the dissemination of multidrug resistant Enterobacteriaceae isolates in Antananarivo. These findings underline the need for a rational use of antibiotic and for appropriate methods of screening ESBL in routine laboratories in Antananarivo.

Antimicrobial Resistance, Virulence, and Phylogenetic Characteristics ofEscherichia coliIsolates from Clinically Healthy Swine

A total of 344 commensal Escherichia coli isolates from clinically healthy pigs were examined for antimicrobial resistance phenotypes, class 1 integrons, resistance genes, virulence gene profile, and phylogenetic groups. The majority of E. coli isolates were resistant to tetracycline (96.2%) and ampicillin (91.6%). Up to 98% were multidrug resistant. Seventy-three percent of the isolates carried class 1 integrons. Inserted-gene cassette arrays in variable regions included incomplete sat, aadA22, aadA1, dfrA12-aadA2, and sat-psp-aadA2, of which the aadA2 gene cassette was most prevalent (42.9%). Horizontal transfer was detected in eight E. coli isolates carrying class 1 integrons with dfrA12-aadA2 gene cassette array. Sixteen resistance genes were identified among the E. coli isolates with corresponding resistance phenotype. Ten virulence genes (including elt, estA, estB, astA, faeG, fasA, fedA, eaeA, paa, and sepA) were detected, of which fasA was most commonly found (98.3%). Most of the E. coli isolates belonged to phylogenetic group B1. Significantly positive associations were observed between some virulence genes and some resistance phenotypes and genotypes (p < 0.05). The results support a finding that commensal E. coli have a role as reservoirs for antimicrobial resistance-encoding genes and virulence determinants.

Distribution of class 1 integrons among enteropathogenicEscherichia coli

The aim of this study was to investigate the incidence of and resistance gene content of class 1 integrons among enteropathogenic Escherichia coli (EPEC) and non-EPEC and to investigate intraspecies genetic diversity of EPEC strains isolated from children with diarrhea in Iran. Twenty-eight EPEC and 16 non-EPEC strains isolated from children with diarrhea were tested for the presence of a class 1 integron associated integrase gene (int1). Sequence analysis was performed to identify the resistance gene content of integrons. Genetic diversity and cluster analysis of EPEC isolates were also investigated using enterobacterial repetitive intergenic concensus-polymerase chain reaction (ERIC-PCR) fingerprinting. Twenty-three (82%) EPEC isolates and 11 (68.7%) non-EPEC isolates harbored the int1 gene specific to the conserved integrase region of class 1 integrons. Sequence analysis revealed the dominance of dfrA and aadA gene cassettes among the isolates of both groups. ERIC-PCR fingerprinting of EPEC isolates revealed a high diversity among these isolates. The widespread distribution of 2 resistance gene families (dfrA and aadA) among both groups of EPEC and non-EPEC isolates indicates the significance of integrons in antibiotic resistance transfer among these bacteria. Furthermore, clonal diversity of EPEC isolates harbouring a class 1 integron also suggests the circulation of these mobile elements among a diverse population of EPEC in this country.

Molecular Characterization of a 21.4 Kilobase Antibiotic Resistance Plasmid from an α-Hemolytic Escherichia coli O108:H- Human Clinical Isolate

This study characterizes the 21.4 kilobase plasmid pECTm80 isolated from Escherichia coli strain 80, an α hemolytic human clinical diarrhoeal isolate (serotype O108:H-). DNA sequence analysis of pECTm80 revealed it belonged to incompatibility group X1, and contained plasmid partition and toxin-antitoxin systems, an R6K-like triple origin (ori) replication system, genes required for replication regulation, insertion sequences IS1R, ISEc37 and a truncated transposase gene (Tn3-like ΔtnpA) of the Tn3 family, and carried a class 2 integron. The class 2 integron of pECTm80 contains an intact cassette array dfrA1-sat2, encoding resistance to trimethoprim and streptothricin, and an aadA1 gene cassette truncated by the insertion of IS1R. The complex plasmid replication system includes α, β and γ origins of replication. Pairwise BLASTn comparison of pECTm80 with plasmid pE001 reveals a conserved plasmid backbone suggestive of a common ancestral lineage. Plasmid pECTm80 is of potential clinical importance, as it carries multiple genes to ensure its stable maintenance through successive bacterial cell divisions and multiple antibiotic resistance genes.

Group IIC Intron with an Unusual Target of Integration in Enterobacter cloacae

A potential role of group IIC-attC introns in integron gene cassette formation, that is, the way in which they could provide the attC sequence essential for recombination, has been proposed. Group IIC introns usually target the attC site of gene cassettes and more specifically their inverse core. Here we characterized a novel group IIC intron targeting the core site of the aadA1 gene cassette attC site (aadA1-qacEΔ1 gene cassette junction) from enterobacterial isolates. Intron mobility (retrohoming) was analyzed using a two-plasmid assay performed in Escherichia coli. Intron mobility assays confirmed the mobilization-integration of the group II intron into the core site of the aadA2, bla(VIM-2), bla(CARB-2), aac(6')-Ib, dfrXVb, arr2, cmlA4, and aadB gene cassettes but not into the attI site. This mobility was dependent on maturase activity. Reverse transcriptase PCR showed that this intron was transcriptionally active, and an intermediate circular form was detected by inverse PCR. This element was linked to the bla(VEB-1) extended-spectrum β-lactamase gene in a high number of enterobacterial isolates. A phylogenetic tree showed that the identified element was located in a branch separate from group IIC-attC introns, being an IIC intron possessing the ability to integrate using the core site of the attC sites as target.