Integron-positive Isolates Research Articles

Multi-Drug Resistance Mediated by Class 1 Integrons in Aeromonas Isolated from Farmed Freshwater Animals.

Aeromonas is regarded as an important pathogen of freshwater animals but little is known about the genetics of its antimicrobial resistance in Chinese aquaculture. The aim of this study was to investigate the presence of integrons and characterize multidrug resistant Aeromonas spp. isolated from diseased farmed freshwater animals. These animal samples included fish, ornamental fish, shrimp, turtles, and amphibians which were collected from 64 farms in Guangdong province of South China. One hundred and twelve Aeromonas spp. isolates were examined for antimicrobial resistance phenotypes and the presence of class 1 integron sequences. Twenty-two (19.6%) of these isolates carried a class 1 integron comprising six different gene insertion cassettes including drfA12-orfF-aadA2, drfA12-orfF, aac(6′)-II-blaOXA-21-cat3, catB3, arr-3, and dfrA17. Among these, drfA12-orfF-aadA2 was the dominant gene cassette array (63.6%, 14/22) and this is the first report of aac(6′)-II-blaOXA-21-cat3 in an Aeromonas hydrophila isolate from a Chinese giant salamander (Andrias davidianus). All the integron-positive strains were resistant to more than five agents and 22 contained other resistance genes including blaCTX-M-3, blaTEM-1, aac(6′)-Ib-cr, and tetA. All integron-positive isolates also contained mutations in the quinolone resistance determining regions (QRDR). Our investigation demonstrates that freshwater animals can serve as a reservoir for pathogenic Aeromonas strains containing multiple drug-resistance integrons. This data suggests that surveillance for antimicrobial resistance of animal origin and a prudent and responsible use of antimicrobials in aquaculture is necessary in these farms.

Characterization of antimicrobial resistance in Klebsiella species isolated from chicken broilers

The prevalence of antimicrobial resistant Klebsiella pneumoniae in poultry products has been a public concern, as it severely endangers food safety and human health. In this study, we investigated 90 antimicrobial resistant Klebsiella strains that were isolated from a commercial broiler slaughter plant in Shandong province of China. Nearly all (89/90) of the isolates were identified as infectious phylogenetic group KpI-type K. pneumoniae. Out of these 90 strains, 87 (96.7%) were multidrug-resistant isolates, and 87 (96.7%) were extended-spectrum beta-lactamase (ESBL)-producing isolates. An analysis of the prevalence of quinolone resistance genes showed that 7.8%, 77.8%, 26.7%, and 2.2% of the strains carried the qnrA, qnrB, qnrS, and qepA genes, respectively. An analysis of beta-lactam resistance genes showed that a high percentage of the strains contain the blaTEM (76.7%), blaSHV (88.9%), and blaCTX-M (75.6%) genes, among which three blaSHV subtypes (blaSHV-1, n=30; blaSHV-11, n=38; blaSHV-12, n=12) and three blaCTX-M subtypes (blaCTX-M-14, n=14; blaCTX-M-15, n=35; blaCTX-M-55, n=19) were found. A further investigation of mobile genetic elements involved in horizontal multidrug resistance gene transfer showed the presence of class 1 and 2 integrons in 77 (85.6%) and five (5.6%) isolates, respectively, while no class 3 integrons were detected. Four types of class 1 integrons containing specific gene cassette arrays (dfrA12–orfF–aadA2, dfrA17–aadA5, dfrA1–aadA1, and empty) were identified. Only one gene cassette array (dfrA1–sat2–aadA1) was detected in the class 2 integrons. Furthermore, four different types of insertion sequence common region 1 (ISCR1)-mediated downstream structures were successfully identified in 46 class 1 integron-positive isolates, among which ISCR1–sapA-like–qnrB2–qacEΔ1 was the most commonly observed structure. Chi-square tests revealed a significant association between ESBL genes, plasmid-mediated quinolone resistance (PMQR) genes, and class 1 integrons (p<0.01). Additional conjugation experiments confirmed this relationship (p<0.01) in transconjugants by finding that a high percentage of PMQR genes (74.0%) and class 1 integrons (73.7%) were co-transferred with ESBL genes. Finally, multilocus sequence typing (MLST) was performed, and it revealed that the isolates from chickens are widely distributed in humans, and that antimicrobial resistance is not only disseminated by clonal spreading, but largely by horizontal gene transfer. These results suggest that horizontal transfer of antimicrobial resistance genes by mobile genetic elements, such as integrons, plays a major role in the spread of antimicrobial resistance. Therefore, elucidating the structures of drug resistance integrons is of great importance to the commercial broiler slaughter plant in Shandong, China.

Characterization of Integrons and qnr Genes in Proteeae from a Teaching Hospital in China

Background: Proteeae isolates displaying multidrug-resistance (MDR) are the second most common causes of hospital-associated infections. The aim of this study was to screen class 1-3 integrons and plasmid-mediated quinolone resistance (PMQR) genes in Proteeae isolates from the First Affiliated Hospital of the Wenzhou Medical University. Materials and Methods: 176 Proteeae isolates were collected from clinical specimens of inpatients between January 2011 and December 2013. Susceptibility testing was determined by the agar dilution method. Class 1-3 integrons and PMQR genes were amplified by polymerase chain reaction, and the variable regions of integrons were determined by restriction fragment length polymorphisms. Results: 68.2% Proteeae isolates exhibited MDR phenotypes: 46.6 and 10.8% Proteeae isolates were positive for intI1 and intI2, respectively. The resistance rate of integron-positive isolates to aminoglycosides, fluoroquinolones, and trimethoprim/sulfamethoxazole was significantly higher than integron-negative isolates. Sequence analysis revealed that dfrA1-sat2-aadA1, dfrA1-catB2-sat2-aadA1, and sat2-aadA1 were first detected in Morganella morganii strains isolated from China. PMQR was determined by qnrD in 40 strains (22.7%). Conclusion: Our results indicate that class 1 and 2 integrons are common among Proteeae isolates. Meanwhile, qnrD are highly prevalent in Proteeae isolated from our hospital.

Carriage of Class 1 and Class 2 Integron in Multidrug Resistant Pseudomonas aeruginosa Isolated from Burn Patients in Tehran Hospitals, Iran.

To investigate the antimicrobial susceptibility patterns of Pseudomonas aeruginosa clinical isolates and their associations with the existence of integrons. During a 12-month study, 140 clinically significant Pseudomonas aeruginosa isolates were collected from patients hospitalized in the burn ward of different hospitals in Tehran. Pseudomonas aeruginosa isolates were identified using standard laboratory procedures. Antimicrobial susceptibility testing was performed for 13 antimicrobial agents according to the standard Kirby-Bauer disk diffusion method and Clinical and Laboratory Standards Institute (CLSI) guidelines. The frequency of Class 1, 2 and 3 integrons was detected using a polymerase chain reaction (PCR) method. The resistance rates of Pseudomonas aeruginosa isolates to 13 antimicrobial agents were between 34.7% and 90.8%. Ceftriaxone and imipenem had good activity against the isolates. Of 140 tested isolates, 91 (65%) were multidrug resistant. The most predominant resistance profile among the isolates included resistance to 10 (12.14%), 9 (12.14%) and 8 (12.14%) antibiotics. Class 1 and 2 integrons were detected in 57.2% (56/98) and 30.6% (30/98) of tested Pseudomonas aeruginosa isolates, respectively. Of 98 (70%) integron positive isolates, only 12 (12.2%) isolates were positive for both classes of integrons. Resistance of the isolates to cefotaxime, aztreonam, imipenem, tobramycin, ticarcillin, ciprofloxacin and cloxacillin was observed to be significantly associated with the existence of integrons. These data confirmed high prevalence of Class 1 integrons among Pseudomonas aeruginosa isolates from burn patients in this study. Based on these results, integrons may play an important role in the possible transmission of resistance genes to the clinical Pseudomonas aeruginosa isolates.

Occurrence of classes I and II integrons in Enterobacteriaceae collected from Zagazig University Hospitals, Egypt.

Integrons are genetic units characterized by the ability to capture and incorporate gene cassettes, thus can contribute to the emergence and transfer of antibiotic resistance. The objectives of this study were: (1) to investigate the presence and distribution of class I and class II integrons and the characteristics of the gene cassettes they carry in Enterobacteriaceae isolated from nosocomial infections at Zagzig University Hospital in Egypt, (2) to determine their impact on resistance, and (3) to identify risk factors for the existence of integrons. Relevant samples and full clinical history were collected from 118 inpatients. Samples were processed; isolated microbes were identified and tested for antibiotic susceptibilities. Integrons were detected by polymerase chain reaction (PCR) and were characterized into class I or II by restriction fragment length polymorphism (RFLP). Integron-positive isolates were subjected to another PCR to detect gene cassette, followed by gene cassette sequencing. Risk factors were analyzed by logistic regression analysis. Seventy-six Enterobacteriaceae isolates were recognized, 41 of them (53.9%) were integron-positive; 39 strains carried class I and 2 strains carried class II integrons. Integrons had gene cassettes encoding different combinations and types of resistance determinants. Interestingly, blaOXA129 gene was found and ereA gene was carried on class I integrons. The same determinants were carried within isolates of the same species as well as isolates of different species. The presence of integrons was significantly associated with multidrug resistance (MDR). No risk factors were associated for integron carriage. We conclude that integrons carrying gene cassettes encoding antibiotic resistance are significantly present among Enterobacteriaceae causing nosocomial infection in our hospital. Risk factors for acquisition remain to be identified.

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major factor in the spread of diverse resistance mechanisms. Amongst the DNA elements facilitating lateral transfer, the class 1 integrons have largely been responsible for spreading antibiotic resistance determinants amongst Gram negative pathogens. In total, these integrons have acquired and disseminated over 130 different antibiotic resistance genes. With continued antibiotic use, class 1 integrons have become ubiquitous in commensals and pathogens of humans and their domesticated animals. As a consequence, they can now be found in all human waste streams, where they continue to acquire new genes, and have the potential to cycle back into humans via the food chain. This protocol details a streamlined approach for detecting class 1 integrons and their associated resistance gene cassettes in foodstuffs, using culturing and PCR. Using this protocol, researchers should be able to: collect and prepare samples to make enriched cultures and screen for class 1 integrons; isolate single bacterial colonies to identify integron-positive isolates; identify bacterial species that contain class 1 integrons; and characterize these integrons and their associated gene cassettes.

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes.

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major factor in the spread of diverse resistance mechanisms. Amongst the DNA elements facilitating lateral transfer, the class 1 integrons have largely been responsible for spreading antibiotic resistance determinants amongst Gram negative pathogens. In total, these integrons have acquired and disseminated over 130 different antibiotic resistance genes. With continued antibiotic use, class 1 integrons have become ubiquitous in commensals and pathogens of humans and their domesticated animals. As a consequence, they can now be found in all human waste streams, where they continue to acquire new genes, and have the potential to cycle back into humans via the food chain. This protocol details a streamlined approach for detecting class 1 integrons and their associated resistance gene cassettes in foodstuffs, using culturing and PCR. Using this protocol, researchers should be able to: collect and prepare samples to make enriched cultures and screen for class 1 integrons; isolate single bacterial colonies to identify integron-positive isolates; identify bacterial species that contain class 1 integrons; and characterize these integrons and their associated gene cassettes.

Class 1 and class 2 integrons in avian pathogenic Escherichia coli from poultry in Italy

The aim of this study was to investigate the occurrence of class 1 and 2 integrons in avian pathogenic Escherichia coli (APEC) from poultry in northern Italy. Strains were tested for phenotypic resistance to aminoglycosides and sulphonamides, and the association between the presence of integrons and the resistance to these antimicrobials was evaluated. A total of 299 isolates (158 from turkeys, 110 from broilers, and 31 from layer hens) were collected from 200 industrial farms. Antimicrobial susceptibility test by the disk diffusion method was performed in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. All strains were screened for the presence of class 1 and 2 integrons by PCR and sequencing. About 55% of APEC contained integrons (class 1, 49.8%; class 2, 10.4%). Different variants of the aadA (5 variants) and the dfrA (4 variants) genes, encoding for streptomycin and trimethoprim resistance respectively, were detected in integron-positive isolates. Less common gene cassettes, such as sat, estX, and orfF, were also identified. Fifteen and 4 gene cassette arrays were found among class 1 and 2 integrons, respectively. High levels of resistance were observed for triple sulphonamides (79.3%), streptomycin (67.2%), and sulfamethoxazole combined with trimethoprim (62.2%), whereas resistance against gentamycin (16.7%), kanamycin (14.7%), and apramycin 3.0%) was low. Integron positivity was significantly higher in isolates phenotypically resistant to aminoglycosides (63.6% vs. 37.8%, P < 0.001) and sulfonamides (64.1% vs. 21.1%, P < 0.001) than in susceptible ones. Integron-borne aminoglycoside and sulfonamide resistance in APEC represents a concern for the poultry industry in Italy, since they are among the most commonly used antimicrobials in poultry therapy.

Prevalence of integrons and Antimicrobial Resistance Genes Among Clinical Isolates of Enterobacter spp. From Hospitals of Tehran

Background: Enterobacter infections are increasingly recognized as an important nosocomial infection. Here we describe the prevalence of three classes of integrons in clinical isolates of Enterobacter spp. and the prevalence of antibiotic resistance genes among isolates with integron. Objectives: Here we describe the prevalence of integrons genes among clinical isolates of Enterobacter spp. and antibiotic susceptibility pattern, ESBL production and the prevalence of resistance genes among clinical isolates of Enterobacter spp. Materials and Methods: A total of 110 Enterobacter isolates collected from four hospitals in Tehran during 2012-2013. Enterobacter species were identified by using API 20E system. The existence of integron classes was investigated by PCR assay through the amplification of integrase genes. Then, antibacterial susceptibility and confirmation of ESBL phenotype was determined. Then, the bla groups, blaTEM, blaSHV, blaCTX-M-1 and aminoglycoside modifying enzymes genes were identified by PCR with specific primers. Results: The prevalence of Enterobacter species were E. cloacae (78.2 %), E. aerogenes (13.6 %) and E. sakazakii (8.2%). They were from different clinical sources. Forty five of Enterobacter isolates have integron but there was not detected class 3 of integrons. All isolates with integron were susceptible to imipenem. Ten isolates of Enterobacter with integron showed ESBL phenotype. The frequency of blaTEM, blaSHV and blaCTX-M-1 genes are 20%, 0% and 15.6%, respectively. The frequency of genes encoding ANT (2˝)-Ia, APH (3΄)-Ia, AAC (6΄)-Ib and AAC (3)-IIa were 11.1%, 13.3%, 13.3 % and 20 %, respectively. Conclusions: The high prevalence of integron-positive isolates in our MDR Enterobacter isolates indicates that these mobile genetic elements are common among different Enterobacter spp. and associate with reduced susceptibility to the first-line antimicrobial drugs. This so highlight the continued monitoring of drug resistance in clinical settings.

Molecular clonality and detection of class 1 integron in multidrug-resistant Salmonella enterica isolates from animal and human in Iran.

A total of 70 multidrug-resistant (MDR) Salmonella isolates (44 human and 26 poultry) were examined. The conserved segment-PCR, restriction fragment length polymorphism-PCR analysis, and DNA sequencing were used to determine the presence and cassette content of integrons. The genetic relatedness among the isolates was examined by pulsed-field gel electrophoresis (PFGE). The rate of integron carriage for MDR Salmonella isolates was 91.4% and integron-positive isolates belonged to six distinct serovars. Out of 64 integron-positive isolates, only four Salmonella Paratyphi C isolates could transfer integrons to Escherichia coli K12 by conjugation. Thirty-three PFGE types were detected in 52 integron-positive isolates, including 22, 4, 3, 2, 1, and 1 patterns among Salmonella serovars Enteritidis, Typhimurium, Paratyphi C, Paratyphi B, Paratyphi A, and Havana, respectively. The human and poultry Salmonella Enteritidis isolates from different regions with identical integrons had closely related PFGE patterns. Of the four integron-positive Salmonella Typhimurium isolates, the two poultry isolates with identical integron had very closely related PFGE patterns whereas the two human isolates with different integrons showed unrelated PFGE patterns. PFGE showed undistinguishable patterns in Salmonella Paratyphi C isolates with identical cassettes but revealed relatively unrelated patterns in those with different cassettes. Relatively unrelated and identical PFGE patterns were found in two Salmonella Paratyphi B and three Salmonella Paratyphi A isolates with the same integrons, respectively. In conclusion, PFGE patterns demonstrated more genetic relatedness among each Salmonella serovar with identical class 1 integrons than the same serovar with different class 1 integrons.

The contribution of class 1 integron to antimicrobial resistance in Stenotrophomonas maltophilia.

Two hundred clinical isolates of Stenotrophomonas maltophilia were examined for the presence of class 1 integron and for the susceptibility to 12 different antimicrobials and detergents. The prevalence of class 1 integron in S. maltophilia isolates was 11%. The class 1 integron-positive isolates exhibited a higher resistance to kanamycin, tobramycin, and trimethoprim-sulfamethoxazole (SXT) than the class 1 integron-negative ones. Polymerase chain reaction (PCR), amplifying the variable region of the class 1 integron, showed the existence of six different amplicon sizes, indicating that there are at least six different class 1 integrons distributed in the 23 class 1 integron-positive isolates. Sequence analysis of six representative PCR amplicons revealed that qacK, aac(6')-Ib', qacK-aac(6')-Ib, qacK-aac(6')-Ib-aac(6')-Ib, and qacL-aadB-cmlA-aadA2 were identified in the 550-, 800-, 1,200-, 1,800, and 3,600-bp amplicons, respectively. The sequence analysis of the 150-bp PCR amplicon demonstrated no additional resistance-associated genes except the basic genetic elements of class 1 integron. The impact of class 1 integron acquisition on the antimicrobials susceptibility was assayed by isogenic integron deletion mutant construction and the susceptibility test. The most significant contribution of the class 1 integron acquisition to S. maltophilia is the increased resistance to SXT.

Antibiotic resistance and prevalence of class 1 and 2 integrons in Escherichia coli isolated from two wastewater treatment plants, and their receiving waters (Gulf of Gdansk, Baltic Sea, Poland).

In this study, antimicrobial-resistance patterns were analyzed in Escherichia coli isolates from raw (RW) and treated wastewater (TW) of two wastewater treatment plants (WWTPs), their marine outfalls (MOut), and mouth of the Vistula River (VR). Susceptibility of E. coli was tested against different classes of antibiotics. Isolates resistant to at least one antimicrobial agent were PCR tested for the presence of integrons. Ampicillin-resistant E. coli were the most frequent, followed by amoxicillin/clavulanate (up to 32 %), trimethoprim/sulfamethoxazole (up to 20 %), and fluoroquinolone (up to 15 %)-resistant isolates. Presence of class 1 and 2 integrons was detected among tested E. coli isolates with rate of 32.06 % (n = 84) and 3.05 % (n = 8), respectively. The presence of integrons was associated with increased frequency of resistance to fluoroquinolones, trimethoprim/sulfamethoxazole, amoxicillin/clavulanate, piperacillin/tazobactam, and presence of multidrug-resistance phenotype. Variable regions were detected in 48 class 1 and 5 class 2 integron-positive isolates. Nine different gene cassette arrays were confirmed among sequenced variable regions, with predominance of dfrA1-aadA1, dfrA17-aadA5, and aadA1 arrays. These findings illustrate the importance of WWTPs in spreading of resistance genes in the environment and the need for inclusion of at least monitoring efforts in the regular WWTP processes.

Polymorphism of gene cassette promoter variants of class 1 integron harbored in S. Choleraesuis and Typhimurium isolated from Taiwan.

Integrons, mobile genetic units, capture and incorporate antibiotic resistance gene cassette by site-specific recombination. Class 1 integrons are widespread and associated with dispersion of antibiotic resistance among Gram-negative bacteria. The expression of gene cassette in Class 1 can vary, based on the Pc promoter but seldom from another promoter hiding downstream of Pc, called P2. To probe distribution and prevalence of gene cassette promoter variants, we analyzed 169 S. Choleraesuis and 191 S. Typhimurium isolates from humans and animals, finding 95.27% occurrence of integrin among S. Choleraesuis, 83.25% among S. Typhimurium. PCR-RFLP analysis identified four promoters (PcS+P2, PcWTGN-10+P2, PcH1+P2, and PcWTGN-10+P2-GGG) in said integron-positive isolates; major types in S. Choleraesuis and S. Typhimurium were PcS+P2 and PcWTGN-10+P2, respectively. Likewise, β-galactosidase assay rated promoter strength of variants by transcriptional fusion constructs to show extended -10 promoter (TGn/-10 promoter) in Pc and three-nucleotide insertion (GGG) between -35 and -10 region of P2 improving promoter strength of gene cassette.

MULTIDRUG-RESISTANCE AND PRESENCE OF CLASS 1 INTEGRONS IN CLINICAL ISOLATES OF SALMONELLA ENTERICA SEROTYPE ENTERITIDIS, CIRCULATING IN ARMENIA

Abstract. The aim of this work was detection of class 1 integrons and their contribution to the antimicrobial resistance phenotypes in strains of subspecies enterica serotype Enteritidis. S. Enteritidis strains (n = 29) were isolated from patients with salmonellosis at “Nork” Clinical Hospital of Infectious Diseases, Yerevan, Republic of Armenia. High prevalence of multi-drug resistance (MDR) phenotypes was revealed and isolates with MDR phenotypes which are rare in the S. Enteritidis serotype were observed. Class 1 integrons were detected in 27,6% of isolates, with the prevalence of a variable region of 1000 bp. Occurrence of the MDR phenotype was more frequent in integron-positive isolates compared to integron-negative isolates of S. Enteritidis. Further studies are necessary to reveal the genetic background of MDR phenotypes and to estimate the genetic kinship among the isolates. Our results suggest a rapid and large-scale penetration of antibiotic resistance genes into populations of S. Enteritidis, which complicates infection control. More rigorous regulations should be imposed on antibiotic use, together with a vigilant epidemiological surveillance, to prevent the emergence and spread of MDR S. Enteritidis.

Antimicrobial resistance patterns and characterization of integrons in clinical isolates of Shigella from China

One hundred fifty-three Shigella isolates were examined for multiple antibiotic resistance phenotypes and prevalence of class 1 and class 2 integron sequences. The gene cassettes dfrA17-aadA5, dfrA12-orfF-aadA2, and arr-3-aacA4 were found in typical class 1 integrons. The gene cassettes blaOXA-1-aadA1 and dfrA1-sat1-aadA1 were detected in atypical class 1 integrons and in class 2 integrons, respectively. This is the first report of arr-3-aacA4 cassette detected in typical class 1 integrons among Shigella isolates. Rates of antibiotic resistance were different between integron-positive and integron-negative strains (P < 0.05), and all integron-positive isolates were resistant to at least 3 different antimicrobial agents. Typical class 1 integron-positive isolates showed higher resistance rates to cefotaxime and ciprofloxacin than did integron-negative ones (P < 0.05). Typical class 1 integrons and β-lactamase genes were found in conjugative plasmids, otherwise class 2 and atypical class 1 integrons were located on chromosome. This study demonstrated the wide distribution of class 1 integrons in Shigella spp., which may lead resistance to cefotaxime and ciprofloxacin in China.

The consequences of a sudden demographic change on the seroprevalence pattern, virulence genes, identification and characterisation of integron-mediated antibiotic resistance in the Salmonella enterica isolated from clinically diarrhoeic humans in Egypt

The present study was undertaken to identify and characterise integrons and integrated resistance gene cassettes among eight multidrug-resistant (MDR) Salmonella serovars isolated from humans in Egypt. Virulotyping by polymerase chain reaction (PCR) was used for the detection of the presence of virulence genes. Integron PCR was used to detect the presence of class 1 in the MDR strains. The associated individual resistance gene cassettes were identified using specific PCRs. The isolated serovars were Salmonella Grampian (C1; 2/5), Larose (C1; 1/5), Hato (B; 1/5) and Texas (B; 1/5). Among the Salmonella serovars, five Salmonella isolates showed the highest resistance to amoxicillin, ampicillin, chloramphenicol, lincomycin, gentamicin, nalidixic acid, streptomycin and trimethoprim (100%), followed by neomycin, norfloxacin and tetracycline (80%), while the lowest resistance was recorded to colistin sulphate and ciprofloxacin in percentages of 20 and 40%, respectively. The invA, avrA, ssaQ, mgtC, siiD and sopB genes were detected in all isolates (100%), while the spvC and gipA genes were totally (100%) absent from all isolates. The remaining three virulence genes were diversely distributed as follows: the bcfC gene was detected in all isolates except Salmonella Hato (80%); the sodC1 gene was detected only in Salmonella Grampian and Salmonella Texas (60%); and the sopE1 gene was detected only in Salmonella Grampian, Hato and Texas (60%). Class 1 integrons were detected in 90% of the MDR isolates, comprising serovars Muenster, Florian, Noya, Grampian, Larose, Hato and Texas. Of the class 1 integron-positive isolates, 45% harboured Salmonella genomic island 1 (SGI1) either right junction or right and left junction having an A-C-S-T phenotype. Of the class 1 integron-positive isolates, 44% harboured integron gene cassette aadA2, while 11% harboured the floR gene present in multidrug resistance flanked by two integrons of SGI1. The results of the present study indicate that class 1 integrons carrying gene cassettes conferring resistance mainly to aminoglycosides are widespread among the MDR Salmonella serovars isolated from humans in Egypt, indicating the important role of these genetic elements in the dissemination of multidrug resistance.

Class 1 integrons contributes to antibiotic resistance among clinical isolates of Escherichia coli producing extended-spectrum beta-lactamases

Objectives: The objective of this study is to determine the prevalence of antibiotic resistance factors, including the production of extended-spectrum beta-lactamases (ESBLs) and the presence of class 1 integrons among Escherichia coli isolated from clinical specimens. Materials and Methods: Bacterial species identification was performed using a VITEK-2 system (VITEK2 GN-card; bioMérieux, France). Antimicrobial susceptibility testing was determined using the disk diffusion method according to the 2010 Clinical and Laboratory Standards Institute guidelines. Polymerase chain reaction (PCR) was used to detect integrons and amplify variable regions of the blaTEM, blaSHV and blaCTX-M genes. Gene cassettes were detected by deoxyribonucleic acid sequencing. Results: In this study, 58% (100/172) of clinical E. coli isolates were identified as ESBL producers. We found that 90% of the ESBL-producing E. coli isolates harbored the blaCTX-M gene, whereas only 59% and 32% possessed the blaTEM and blaSHV genes respectively. The presence of class 1 integrons was based on the detection of the integrase gene by PCR. A total of 69% of the ESBL-producing isolates were integron-positive. Resistance to 10 antibiotics, including quinolones, sulfonamides and β-lactam/enzyme inhibitors, was significantly higher in the class 1 integron-positive isolates (P < 0.05). The occurrence of class 1 integrons in blaTEM, blaSHV and blaCTX-M gene carriers was 72.9%, 84.4% and 68.9%, respectively. Class 1 integrons were detected in 61.5% of the isolates with only one ESBL genotype, but in 69.0% and 92.3% of the isolates with two or three different ESBL genotypes, respectively. Conclusions: Our findings indicate that clinical strains of bacteria with multiple ESBL genotypes may have greater opportunities to carry class 1 integrons.

개와 고양이에서 분리된 Enterobacteriaceae와 Pseudomonas aeruginosa의 항균제 내성 및 내성 유전자의 분포

This study was carried out to investigate the antimicrobial resistance pattern and distribution of resistance gene in 44 Enterobacteriaceae and 21 Pseudomonas (P) aeruginosa isolated from hospitalized dogs and cats in animal hospital from 2010 to 2011 in Daegu. Among Enterobacteriaceae, Escherichia (E) coli was highly resistant to ampicillin (56.7%), followed by tetracycline (53.3%), cephalothin, streptomycine, sulfamethoxazole/trimethoprim, gentamicin and norfloxacin (40.0~43.3%). The remaining isolates of Enterobacteriaceae had high resistance to ampicillin (64.3%) and streptomycin (42.9%). Whereas, P. aeruginosa was low resistant to all antimicrobials tested (less than 15%). int I 1 gene was detected in 20 (57.1%) of 35 antimicrobial resistant Enterobacteriaceae and 2 (9.5%) of 21 P. aeruginosa., but int I 2 gene was not detected in all isolates. The eight resistance genes were found either alone or combination with other gene (s): <TEX>$bla_{TEM}$</TEX>, aadA, strA-strB, clmA, tetA, tetB, sul I and sul II. About 78% of integron-positive isolates were resistance to more than four antimicrobial agents. The findings suggest that class I integrons are widely distributed in E. coli among Enterobacteriaceae from dogs and cats and multi-drug resistance related to the presence of class I integrons. The prudent use of antimicrobials and continuous monitoring for companion animals are required.

Characterization of Pc Promoter Variants of Class 1 Integrons inEscherichia coliIsolates from Poultry Meat

Integrons are important genetic elements implicated in acquisition and expression of antimicrobial resistance genes. Gene cassettes of class 1 integrons may be differently expressed depending on the Pc promoter variant. Thirty-four Escherichia coli isolates (carrying 38 class 1 integrons), recovered from poultry meat in previous studies in Tunisia and selected by their specific traits, were further characterized in this study. Integron promoter variants and the pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) of isolates were determined. Three types of promoter variants were identified among the 38 class 1 integrons (PcW, PcH1, and PcS); the weak promoters were the most predominant. A high clonal diversity of the E. coli strains was demonstrated by PFGE or by MLST. Fifteen PFGE profiles were detected among 19 integron-positive isolates of phylogroup B2, and 12 different sequence types were identified by MLST among the remaining 15 isolates (ST48, ST88, ST101, ST117, ST155, ST189, ST351, ST359, ST410, ST641, ST665, and one new ST). These data reflect that the presence of integrons in these isolates is not due to the clonal dispersion but to dissemination of genetic structures carrying integrons in different clones. To the best of our knowledge, this is the first report examining the gene cassette promoter variants in class 1 integrons of E. coli isolates from poultry meat origin. The predominance of promoters implicated in weak expression/high excision activity of gene cassette arrays is of interest because they could theoretically enhance the capacity of integrons to adapt to antibiotic pressure.

Diverse Gene Cassettes in Class 1 Integrons of Facultative Oligotrophic Bacteria of River Mahananda, West Bengal, India

BackgroundIn this study a large random collection (n = 2188) of facultative oligotrophic bacteria, from 90 water samples gathered in three consecutive years (2007–2009) from three different sampling sites of River Mahananda in Siliguri, West Bengal, India, were investigated for the presence of class 1 integrons and sequences of the amplification products.Methodology/Principal FindingsReplica plating method was employed for determining the antibiotic resistance profile of the randomly assorted facultative oligotrophic isolates. Genomic DNA from each isolate was analyzed by PCR for the presence of class 1 integron. Amplicons were cloned and sequenced. Numerical taxonomy and 16S rRNA gene sequence analyses were done to ascertain putative genera of the class 1 integron bearing isolates. Out of 2188 isolates, 1667 (76.19%) were antibiotic-resistant comprising of both single-antibiotic resistance (SAR) and multiple-antibiotic resistant (MAR), and 521 (23.81%) were sensitive to all twelve different antibiotics used in this study. Ninety out of 2188 isolates produced amplicon(s) of varying sizes from 0.15 to 3.45 KB. Chi-square (χ2) test revealed that the possession of class 1 integron in sensitive, SAR and MAR is not equally probable at the 1% level of significance. Diverse antibiotic-resistance gene cassettes, aadA1, aadA2, aadA4, aadA5, dfrA1, dfrA5, dfrA7, dfrA12, dfrA16, dfrA17, dfrA28, dfrA30, dfr-IIe, blaIMP-9, aacA4, Ac-6′-Ib, oxa1, oxa10 and arr2 were detected in 64 isolates. The novel cassettes encoding proteins unrelated to any known antibiotic resistance gene function were identified in 26 isolates. Antibiotic-sensitive isolates have a greater propensity to carry gene cassettes unrelated to known antibiotic-resistance genes. The integron-positive isolates under the class Betaproteobacteria comprised of only two genera, Comamonas and Acidovorax of family Comamonadaceae, while isolates under class Gammaproteobacteria fell under the families, Moraxellaceae, Pseudomonadaceae, Aeromonadaceae and Enterobacteriaceae.ConclusionsOligotrophic bacteria are good sources of novel genes as well as potential reservoirs of antibiotic resistance gene casettes.