Variable Region Of Class Research Articles

Distribution diversity and expression regulation of class 1 integron promoters in clinical isolates of Morganella morganii.

Morganella morganii is an emerging nosocomial opportunistic pathogen with increasing multidrug resistance. Antibiotic resistance, driven primarily by the horizontal transfer of resistance genes, has become a global health crisis. Integrons, mobile genetic elements, are now understood to facilitate the transfer of these genes, contributing to the rapid proliferation of resistant strains. Understanding the regulatory role of integrons in drug resistance gene expression is crucial for developing novel strategies to combat this pressing public health issue. To investigate the distribution of promoter types in the variable regions of class 1 integrons isolated from clinical isolates of M. morganii and their regulatory role in the expression of downstream drug resistance gene cassettes. Ninety seven clinical isolates of M. morganii were screened for the presence of class 1 integrons (intI1) using polymerase chain reaction (PCR). Gene cassettes within the variable regions of positive isolates were characterized, and the gene cassette promoter Pc variants and downstream auxiliary promoter P2 were identified. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was employed for homology analysis. Recombinant plasmids containing different variable region promoters and gene cassettes were constructed to evaluate drug resistance genes and integrase (intI1) expression levels using reverse transcription-quantitative PCR (RT-qPCR) and antimicrobial susceptibility testing. Of the clinical isolates, 28.9% (n = 28/97) were positive for class 1 integrons. 24.7% (n = 24/97) of these isolates carried gene cassettes encoding resistance to aminoglycosides and trimethoprim. Three Pc promoter types (PcH1, PcS, and PcW) were identified, while all P2 promoters were inactive with a 14-base pair spacing between the -35 and -10 regions. ERIC-PCR analysis classified the integron-positive strains into 6 genotypes, with high consistency in promoter types and gene cassettes within each genotype. RT-qPCR and antimicrobial susceptibility testing demonstrated that strong promoters significantly enhanced the expression of downstream drug resistance gene cassettes compared to weak promoters. Additionally, RT-qPCR revealed a negative correlation between intI1 expression and Pc promoter strength. Class 1 integrons are prevalent in M. morganii. The promoter types within these integrons are diverse, and promoter strength is closely linked to downstream gene cassette expression. Integron-positive strains exhibit high homology, suggesting horizontal gene transfer and dissemination in clinical settings.

High rate of multidrug resistance and integrons in Escherichia coli isolates from diseased ducks in select regions of China

With the increasing number of ducks being raised and consumed, it is crucial to monitor the presence of multidrug resistant (MDR) bacteria in duck farming. Waterfowl, such as ducks, can contribute to the rapid dissemination of antibiotic resistance genes (ARGs). The objective of this study was to investigate the antimicrobial resistance (AMR), ARGs, and mobile genetic elements (MGEs), such as IS26, tbrC, ISEcp1 in Escherichia coli(E. coli) isolated from the intestinal contents of diseased ducks between 2021 and 2022 in Sichuan, Chongqing and Anhui, China. The AMR phenotypes of 201 isolated E. coli strains were determined using the minimum inhibitory concentrations (MICs) method. Subsequently, polymerase chain reaction and sequencing techniques were employed to screen for integron-integrase genes (intI1, intI2, intI3 genes), gene cassettes (GCs), MGEs, and ARGs. The results demonstrated that 96.5% of the E. coli isolates were resistant to at least 1 antibiotic, with 88.1% of the strains displaying MDR phenotype. The highest AMR phenotype observed was for trimethoprim-sulfamethoxazole (88.1%). Furthermore, class 1 and class 2 integrons were detected in 68.2% and 3.0% of all the isolates, respectively, whereas no class 3 integrons were found. Ten types of GCs were identified in the variable regions of class 1 and class 2 integrons. Moreover, 10 MGEs were observed in 46 combinations, with IS26 exhibiting the highest detection rate (89.6%). Among the 22 types of ARGs, tetA (77.1%) was the most frequently detected. In the conjugational transfer experiment, transconjugants were found to carry specific ARGs and MGEs, with their MIC values were significantly higher than those of recipient E. coli J53, indicating their status as MDR bacteria. This study emphasizes the necessity of monitoring MGEs, ARGs, and integrons in duck farms. It provides valuable insights into the complex formation mechanisms of AMR and may aid in preventing and controlling the spread of MDR bacteria in waterfowl breeding farm.

Prevalence of classes 1 and 2 integrons in multidrug-resistant Acinetobacter baumanni isolates recovered from some aquatic environment in South Africa

The emergence of antibiotic-resistance genes (ARGs) by means of integrons in multidrug-resistant Acinetobacter baumannii (MDR A. baumannii) has become a significant challenge in the management of infections from this pathogen. In this paper, we report on the variable region of class 1 and 2 integrons observed in MDR A. baumanni isolates recovered from rivers in the Eastern Cape Province, South Africa. Class 1 and 2 integrons with their variable regions were evaluated with polymerase chain reaction techniques followed by sequencing. Antibiotic sensitivity testing, checkerboard assay, time-kill independent assay, and Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) were carried out using standard microbiological techniques. A total of fifty-six (56) isolates were examined, among which 45 (79%) tested positive for class 1 integron, and 7 (12.3%) had class 2 integron. None was found to be class 3 integron positive among the isolates. The variable region contained aadA1, aadA5, and aadA2 genes, which confer resistance against streptomycin and spectinomycin, aac(6′)-Ib against amikacin/ tobramycin and dfrA17 genes against trimethoprim. The minimum inhibitory concentrations of the antimicrobials for one of the tested organisms were resistant against meropenem, colistin sodium methanesulfonate, tetracycline, ceftazidime, and ciprofloxacin (16, > 16, > 8, > 256, and 128 ug/mL respectively). The impact of colistin combined with quinolones (ciprofloxacin), with the FICIs (0.31) indicated synergistic effects against MDR A baumanni. However, when colistin was combined with meropenem and ceftazidime, additive effects with fractional inhibitory concentration (FIC) index ranging from 0.52 to 1 were observed. No antagonistic effect was evaluated among the examined isolates. ERIC-PCR analyses of A. baumanni isolates revealed significant genetic diversity, suggesting various sources of environmental contamination. We conclude that A. baumanni harbouring class 1 integrons in aquatic milieus are a significant source of ARGs and can transmit these elements to other organisms and consequently to man with significant public health implications.

Antimicrobial Resistance in Escherichia coli from the Broiler Farm Environment, with Detection of SHV-12-Producing Isolates.

Antimicrobial resistance is an important One Health challenge that encompasses the human, animal, and environmental fields. A total of 111 Escherichia coli isolates previously recovered from manure (n = 57) and indoor air (n = 54) samples from a broiler farm were analyzed to determine their phenotypes and genotypes of antimicrobial resistance and integron characterization; in addition, plasmid replicon analysis and molecular typing were performed in extended-spectrum-beta-lactamase (ESBL) producer isolates. A multidrug-resistance phenotype was detected in 46.8% of the isolates, and the highest rates of resistance were found for ampicillin, trimethoprim–sulfamethoxazole, and tetracycline (>40%); moreover, 15 isolates (13.5%) showed susceptibility to all tested antibiotics. None of the isolates showed imipenem and/or cefoxitin resistance. Twenty-three of the one hundred and eleven E. coli isolates (20.7%) were ESBL producers and carried the blaSHV-12 gene; one of these isolates was recovered from the air, and the remaining 22 were from manure samples. Most of ESBL-positive isolates carried the cmlA (n = 23), tet(A) (n = 19), and aac(6′)-Ib-cr (n = 11) genes. The following genetic lineages were identified among the ESBL-producing isolates (sequence type-phylogroup-clonotype): ST770-E-CH116–552 (n = 12), ST117-B2-CH45–97 (n = 4), ST68-E-CH26–382/49 (n = 3), ST68-E-CH26–49 (n = 1), and ST10992-A/B1-CH11–23/41/580 (n = 4); the latter two were detected for the first time in the poultry sector. At least two plasmid replicon types were detected in the ESBL-producing E. coli isolates, with IncF, IncF1B, IncK, and IncHI1 being the most frequently found. The following antimicrobial resistance genes were identified among the non-ESBL-producing isolates (number of isolates): blaTEM (58), aac(6′)-Ib-cr (6), qnrS (2), aac(3)-II (2), cmlA (6), tet(A)/tet(B) (22), and sul1/2/3 (51). Four different gene-cassette arrays were detected in the variable region of class 1 (dfrA1-aadA1, dfrA12-aadA2, and dfrA12-orf-aadA2-cmlA) and class 2 integrons (sat2-aadA1-orfX). This work reveals the worrying presence of antimicrobial-resistant E. coli in the broiler farm environment, with ESBL-producing isolates of SHV-12 type being extensively disseminated.

High incidence of multi-drug resistance and heterogeneity of mobile genetic elements in Escherichia coli isolates from diseased ducks in Sichuan province of China

Harmonious ecological environment is a major concern with rising feeding and consumption of ducks, as these waterfowl birds can promote the spread of antibiotic resistant genes (ARGs). Therefore, this study was conducted to know diversity of antimicrobial resistance (AMR), integrons, and mobile genetic elements (MGEs) in Escherichia coli (E. coli) isolated from intestinal contents or pericardial effusion of diseased ducks from 2018 to 2020 in Sichuan, China. The AMR phenotype was determined via disk diffusion test in 165 E. coli isolates. Further, the integrase genes of integron (intI1, intI2 and intI3 genes), gene cassettes (GCs) and MGEs were screened by PCR and sequencing. The results indicated 100% isolates were resistant to at least one antibiotic and 98.8% were multidrug-resistant strains. Highest AMR phenotype was recorded to rifampin (97.0%) followed by ampicillin (95.8%), chloramphenicol (89.7%), trimethoprim-sulfamethoxazole (84.2%), ciprofloxacin (83.0%), cefotaxime (80.0%), streptomycin (75.8%), doxycycline (49.7%), amikacin (10.3%), amoxicillin/clavulanic acid (3.6%), polymyxin B (1.2%) and ertapenem (0.6%). Further, class 1 and 2 integrons were found in 87.3% and 17.6% isolates, respectively. All isolates were negative for intI3 gene. The variable region of class 1 and 2 integrons contained total 13 different GCs, including arr-3+dfrA27, dfrA1+aadA1, dfrA17+aadA5, dfrA12, dfrA1+sat2+aadA1, dfrA12+aadA2, dfrA5, aadA2+ere(A)+dfrA32, aac(6’)-Ib-cr, aadA22, aadA5, dfrA17, and dfrA27. Moreover, 13 MGEs in 69 different combinations were observed with predominance of IS26 followed by tnpA/Tn21, trbC, ISEcp1, merA, ISAba1, tnsA, tnsB, tnsC, IS1133, tnsD, ISCR3/14, and tnsE. Thus, the monitoring of integrons, MGEs and ARGs is important to understand the complex mechanism of AMR, which might help to introduce interventions for prevention and control of AMR in duck farms in China.

Integron diversity in blavim-2-carrying carbapenem-resistant clinical Pseudomonas aeruginosa isolates.

The growing prevalence of metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa in nosocomial pathogen populations has been attributed to their clonal spread, and/or horizontal transfer of MBL determinants in mobile genetic elements, including integrons. To characterize the genetic background of the beta-lactamase VIM-2 encoding gene in the population of carbapenemresistant (Carba-R) P. aeruginosa clinical isolates.The detection of class 1 integrons was performed by PCR. Typing of the class 1 integrons containing the blaVIM gene cassette was performed by the PCR-restriction fragment length polymorphism (RFLP) approach followed by sequencing of variable regions of class 1 integrons. Five types of the blaVIM-2-carrying integrons were identified: ST654-isolates accounting for more than 50% of the Carba-R population harbored In56; ST235-isolates contained In559 (26% Carba-R isolates); ST111-isolates (19% Carba-R isolates) were characterized by carrying In59-like integron; two ST235-isolates harbored In59 and In249 each. Except In56, carrying the only blaVIM-2-gene cassette, all other identified integron types harbored the genes of resistance to trimethoprim and/or aminoglycosides. No new types of integrons were identified in the P. aeruginosa clinical isolates. The observed correlation of the integron type with specific STs indicates a clonal dissemination of significant resistance determinant producers - ST111, ST654 and ST235 epidemic lines. The features of the integron variable regions can be used for the epidemiological characterization of clinical P. aeruginosa isolates.

Resistência antimicrobiana em cepas de Escherichia coli isoladas de queijo Minas Frescal no município do Rio de Janeiro – Perfil fenotípico e genotípico

Introduction: Antimicrobial resistance is a public health problem. Foods may be carriers of antimicrobial resistant bacteria and resistance genes for humans. Objective: To isolate, identify, and evaluate the antimicrobial susceptibility and identification of resistance genes and integrons in Escherichia coli strains isolated from Minas Frescal cheese. Method: The presence of E. coli in thirty samples of Minas Frescal cheese was evaluated through the 3M™ Petrifilm plates. Thirty . coli isolates were evaluated through the Disk Diffusion Susceptibility Test for 17 antimicrobial agents. Through PCR and Multiplex PCR the isolates were examined for the presence of 24 antimicrobial resistance genes. Genes and variable regions of class 1, 2 and 3 integrons were also evaluated. Results: Phenotypic resistance to one or more classes of antimicrobial agents were found in 50% of E. coli isolates. One hundred percent of the isolates showed PCR amplification for the blaTEM gene and there was also amplification for blaSHV and tetB genes. In addition, 46.6% of the phenotypically resistant isolates amplified to one or more classes of integrons. This is one of the first studies to identify these genes in Minas Frescal cheese in Brazil. Conclusions: Cheese can be a source of multiresistant bacteria and those can disseminate their resistance genes to other bacteria being in the food and human gastrointestinal tract, emphasizing the importance of Good Manufacturing Practices and greater supervision on products on sale.

Class 1 integrons as predominant carriers in Escherichia coli isolates from waterfowls in Hainan, China

This study was conducted to determine the prevalence of integrons and associated gene cassettes in Escherichia coli (E. coli) isolates from waterfowls in Hainan, China. The antimicrobial resistance profile of the isolates was examined by using disc diffusion test. In addition, PCR, RFLP, plasmid replicon typing and DNA sequencing analyses were used for the characterization of integrase genes (class 1, 2 and 3) and associated gene cassettes. Approximatively, 90% of the isolates were positive for the integrase genes by PCR. Specifically, class 1 and class 2 integrons were found in 252 (81%) and 7 (2.3%) strains, respectively. While 21 (6.7%) isolates were positive for both class 1 and class 2 integrons. However, none of the isolate was positive for the class 3 integrons. In addition, 5 various cassette arrays, dfrA1-orfC, aadA2, aadA1, dfrA1-aadA1, and dfrA1-orfC- aadA1, were found within the variable regions (VRs) of class 1 integron isolates. While only single cassette array, dfrA1-sat2- aadA1, was identified within VRs of class 2 integron isolates. We identified incF plasmid as the most common plasmid type, which was detected in 81 of 243 VRs containing isolates. This study is the first report showing the baseline characteristics of integrons in E. coli isolates from waterfowls in Hainan, China. Our results provide evidence of the waterfowl birds as a reservoir of class 1 and class 2 integrons carrying antibiotic resistance gene cassettes. Therefore, strict preventive measures should be taken to avoid the spread of mobile genetic resistance elements in waterfowls in China.

High Prevalence of Antibiotic Resistance and Integrons inEscherichia coliIsolated from Urban River Water, India

The contamination of surface waters with multidrug resistant (MDR) coliforms is a major public health concern in developing countries. This study was aimed to evaluate the occurrence of antibiotic resistance and role of integrons in the spread of resistance genes in Escherichia coli isolated from urban waters of river Yamuna. One hundred and forty-one strains of E. coli were isolated and assessed for antibiotic resistance wherein high resistance was observed for Cefazolin. Integrons (class 1 and class 2) were detected in 32% of the isolates. Variable region of class 1 integron carried different gene cassettes, namely. dfrA17-aadA5, dfrA12-orfF-aadA2, blaOXA-1-aadA1, and unusual phage tail tape measure protein. These integron-positive isolates were further characterized by phylogrouping, serotyping, and BOX-PCR typing. Phylogroup B2 was found to be the most prevalent. Pathogenic E. coli O157 was also reported. Majority of the isolates (54%; 7/13) carrying "dfrA17-aadA5" gene cassette were clustered predominantly into a single BOX-PCR type (B8), suggesting a genetic relatedness among the isolates. This study thus depicts very high incidence of multidrug resistance and class 1 integrons in surface water of India. The prevalence of integrons in aquatic E. coli correlated well with resistance to increasing number of antibiotic classes and multiple antibiotic resistance (MAR) index at various sites. Integron-positive MDR E. coli isolates were found to be serologically and genetically diverse suggesting major role of integrons in the emergence and dissemination of resistance traits in waterborne E. coli.

Structural analysis of atypical class 1 integrons in clinical Proteus isolates

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

A Highly Promiscuous Integron, Plasmids, Extended Spectrum Beta Lactamases and Efflux Pumps as Factors Governing Multidrug Resistance in a Highly Drug Resistant Vibrio fluvialis Isolate BD146 from Kolkata, India.

In an earlier study from this laboratory, Vibrio fluvialis BD146, a clinical isolate from Kolkata, India, 2002, was found to be resistant to all the fourteen antibiotics tested. It harboured a high copy number plasmid pBD146 and a low copy number plasmid. In the present study, a more detailed analysis was carried out to unravel different resistance mechanisms in this isolate. Sequencing showed that variable region of class 1 integron located on low copy number plasmid harbored arr3-cmlA-blaOXA10-aadA1 gene cassettes. Analysis for extended spectrum beta lactamases (ESBLs) revealed that BD146 was ESBL positive. Efflux pumps were involved in the drug resistance phenotype for chloramphenicol, kanamycin, streptomycin and tetracycline. Sequence analysis of pBD146 revealed the presence of genes encoding BDint an integrase with a unique sequence having little similarity to other known integrases, toxin-antitoxin (parE/parD), a replicase, trimethoprim resistance (dfrVI) and quinolone resistance (qnrVC5). Presence of cmlA, putative novel integrase and toxin-antitoxin system in V. fluvialis has been documented for the first time in this report. pBD146 showed 99% sequence similarity with pVN84 from V. cholerae O1 of Vietnam, 2004 and a plasmid from V. parahaemolyticus v110 of Hong Kong, 2010. Conjugation experiments proved the ability of pBD146 and the low copy number plasmid, to get transferred to another host imparting their antibiotic resistance traits to the transconjugants. Therefore, present study has indicated that plasmids played an important role for dissemination of drug resistance.

Multidrug- and Extensively Drug-Resistant Uropathogenic Escherichia coli Clinical Strains: Phylogenetic Groups Widely Associated with Integrons Maintain High Genetic Diversity.

In recent years, an increase of uropathogenic Escherichia coli (UPEC) strains with Multidrug-resistant (MDR) and Extensively Drug-resistant (XDR) profiles that complicate therapy for urinary tract infections (UTIs) has been observed and has directly impacted costs and extended hospital stays. The aim of this study was to determine MDR- and XDR-UPEC clinical strains, their virulence genes, their phylogenetic groups and to ascertain their relationship with integrons and genetic diversity. From a collection of 500 UPEC strains, 103 were selected with MDR and XDR characteristics. MDR-UPEC strains were mainly associated with phylogenetic groups D (54.87%) and B2 (39.02%) with a high percentage (≥70%) of several fimbrial genes (ecpA, fimH, csgA, and papGII), an iron uptake gene (chuA), and a toxin gene (hlyA). In addition, a moderate frequency (40–70%) of other genes (iutD, tosA, and bcsA) was observed. XDR-UPEC strains were predominantly associated with phylogenetic groups B2 (47.61%) and D (42.85%), which grouped with ≥80 virulence genes, including ecpA, fimH, csgA, papGII, iutD, and chuA. A moderate frequency (40–70%) of the tosA and hlyA genes was observed. The class 1 and 2 integrons that were identified in the MDR- and XDR-UPEC strains were associated with phylogenetic groups D, B2, and A, while the XDR-UPEC strains that were associated with phylogenetic groups B2, D, and A showed an extended-spectrum beta-lactamase (ESBL) phenotype. The modifying enzymes (aadA1, aadB, aacC, ant1, dfrA1, dfrA17, and aadA4) that were identified in the variable region of class 1 and 2 integrons from the MDR strains showed resistance to gentamycin (56.25 and 66.66%, respectively) and trimethoprim-sulfamethoxazole (84.61 and 66.66%, respectively). The MDR- and XDR-UPEC strains were distributed into seven clusters and were closely related to phylogenic groups B2 and D. The diversity analysis by PFGE showed 42.68% of clones of MDR-UPEC and no clonal association in the XDR-UPEC strains. In conclusion, phylogenetic groups including virulence genes are widely associated with two integron classes (1 and 2) in MDR- and XDR-UPEC strains.

Low antibiotic resistance rates and high genetic heterogeneity of Escherichia coli isolates from urinary tract infections of diabetic patients in Tunisia

Antimicrobial resistance phenotypes, tetracycline, sulphonamide resistance genes, and integrons were analysed in 48 Escherichia coli isolates recovered from urine cultures of diabetic patients in Tunisia. Twenty-one were susceptible to all antibiotics tested. High rates of resistance were observed for amoxicillin (39.5%), trimethoprim-sulphamethoxazole (33.3%), sulphonamide (33.3%), and tetracycline (31.2%). Resistance to imipenem was not detected, and ESBL producing isolates were not recovered. Our analysis assigned 26, 13, 3, and 5 isolates to phylogroups A, B1, B2, and D, respectively. It is worthy to note that all the resistant isolates belonged to phylogroups A (15 isolates) and B1 (12 isolates), while for the 21 susceptible isolates, phylogroups A, B1, B2, and D were found in 11, 2, 3, and 5 isolates, respectively. Among 15 tetracycline-resistant isolates, the tetA and tetB genes were detected in three and four isolates, respectively. Among 17 sulphonamide resistant isolates, 12, 3, and 1 isolates harboured sul1, sul2, and sul3, respectively. sul1 and sul2 genes occurred simultaneously in three isolates. Integrons were detected in 11 isolates. Ten isolates harboured the class 1 integron and three the class 2 integron. The variable regions (VRs) of the class 1 integrons were analysed in the 10 in1-positive isolates, and the following gene cassette arrangements were detected: dfrA12-orfF-aadA2-cmIA1-aadA1-qacH-IS440-sul3 (one isolate), dfrA15-aadA1 (three isolates), dfrA5 (one isolate), dfrA17- aadA5 (one isolate), dfrA1-aadA1 (one isolate), and dfrA14 (one isolate). The VR of class 2 integron was analysed in the in2-positive isolates, and only one gene cassette arrangement was detected, dfrA1-sat-aadA1. Pulsed-field gel electrophoresis (PFGE) analysis of resistant isolates showed that all were unrelated. Our results highlight the moderate antibiotic resistance in the clinical isolates as well as their heterogeneous genetic background.

Identification of integrons and phylogenetic groups of drug-resistant Escherichia coli from broiler carcasses in China

The dissemination of drug-resistant Escherichia coli in poultry products is becoming a public concern, as it endangers food security and human health. It is very common for E. coli to exhibit drug resistance in the poultry industry in China due to the excessive use of antibiotics. However, few studies have examined the drug resistance endowed by integrons and integron-associated gene cassettes in different phylogenetic groups of E. coli isolated from broiler carcasses. In this study, 373 antibiotic-resistant E. coli strains were isolated from the surfaces or insides of broiler carcasses from a slaughterhouse in Shandong Province, China. According to phylogenetic assays of chuA, yjaA, and an anonymous DNA fragment, TSPE4-C2, these isolates belong to four phylogenetic groups (A, B1, B2, and D) and seven subgroups (A0, A1, B1, B21, B22, D1, and D2). Of the tested isolates, 95.71% (n=357) are multi-drug resistant, among which group B1 was predominant, accounting for 33.51% (n=125) of the tested isolates. A high percentage of the E. coli isolates were resistant to amoxicillin–clavulanic acid (99.20%, n=370), doxycycline (92.23%, n=344), sulfamethoxazole–trimethoprim (90.88%, n=339), ciprofloxacin, (64.61%, n=241), sulbactam–cefoperazone (51.21%, n=191), and amikacin (33.78%, n=126). Furthermore, among the 373 isolates, class 1 and 2 integrons were identified in 292 (78.28%) and 49 (13.14%) of the isolates, respectively, while no class 3 integrons were detected. The most prevalent gene cassette arrays were dfrA17–aadA5 and dfrA12–orfF–aadA2 in the variable region of class 1 integrons, while only one gene cassette array (dfrA1–sat2–aadA1) was detected in the variable region of class 2 integrons. Class 1 integrons were distributed in various physiological subtypes, whereas no predominant phylogenetic groups could be identified. The presence of class 2 integrons in the B21 subtype was significantly higher than in the other subtypes, and it coexisted with the class 1 integron. This study suggests that broiler products are potential sources of multi-drug resistant E. coli, and that resistance genes could be spread by lateral gene transfer.

Re-identification of Aeromonas isolates from rainbow trout and incidence of class 1 integron and β-lactamase genes

Forty-eight Aeromonas isolates from rainbow trout previously identified by the 16S rDNA-RFLP technique were re-identified using 2 housekeeping genes (gyrB and rpoD). After sequencing the prevalences of the species were A. veronii (29.2%), A. bestiarum (20.8%), A. hydrophila (16.7%), A. sobria (10.4%), A. media (8.3%), A. popoffii (6.2%), A. allosaccharophila (2.1%), A. caviae (2.1%), A. salmonicida (2.1%) and one isolate (2.1%) belongs to a candidate new species “Aeromonas lusitana”. Coincident identification results to the 16S rDNA-RFLP technique were only obtained for 68.8% of the isolates. PCR amplification of the enterobacterial repetitive intergenic consensus (ERIC-PCR) indicated that the 48 isolates belonged to 33 different ERIC genotypes. Several genotypes were isolated from different farms and organs in the same fish, indicating a systemic dissemination of the bacteria. The presence of genes (blaIMP, blaCphA/IMIS, blaTEM, blaSHV and intI1) that encode extended-spectrum beta-lactamases (ESBLs), metallo-beta-lactamases (MBLs) and class 1 integrons were studied by PCR. Only 39.6% (19/48) of the strains showed the presence of one or more resistance genes. The gene blaCphA/IMIS was detected in 29.2% of the isolates, followed by the intI1 (6.2%) and blaSHV (4.2%) genes. The variable region of class 1 integrons of the 3 positive isolates was sequenced revealing the presence of the gene cassette aadA1 (aminoglycoside transferase) that plays a role in streptomycin/spectinomycin resistance.

A novel functional class 2 integron in clinical Proteus mirabilis isolates

To describe a novel functional class 2 integron that was found in clinical Proteus mirabilis isolates. Class 1 and 2 integrons were screened by PCR in 153 clinical Proteus isolates. The variable regions of class 1 and 2 integrons were determined by restriction analysis and sequencing. The mutations of internal stop codons in class 2 integrons and their common promoters were also determined by sequencing. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was used to analyse the phylogenetic relations of class 2 integron-positive P. mirabilis isolates. Class 1 integrons were detected in 96 (63%) of 153 Proteus isolates: eight different gene cassette arrays were detected, including dfrA32-ereA1-aadA2, which was detected for the first time in P. mirabilis. Class 2 integrons were detected in 101 (66%) of 153 Proteus isolates: four different gene cassette arrays were detected, including dfrA1-catB2-sat2-aadA1, which was detected for the first time in a class 2 integron. A novel functional class 2 integron was detected in 38 P. mirabilis isolates with a common promoter (-35 TTTAAT|16 bp|-10 TAAAGT). The variable region of this functional class 2 integron contained dfrA14 and three novel open reading frames with unknown functions. Very similar ERIC-PCR fingerprinting patterns were detected in these 38 P. mirabilis isolates and were different from other class 2 integron-positive isolates. A novel functional class 2 integron was found for the first time in P. mirabilis. These functional class 2 integron-harbouring P. mirabilis isolates were likely to be clonally spread in our hospital.

Class 1 integron element in Thai Acinetobacter baumannii reveals a linkage to the European clone I

BACKGROUND: Class 1 integron element is innate to most of the multidrug resistant Acinetobacter baumannii and its spread is common among international clones worldwide. The aim of this study was to document the presence of blaVEB-1 harboring class 1 integron element and its gene cassettes in Thai A. baumannii in relation to A. baumannii European clone I, AYE strain. MATERIALS AND METHODS: Thirty seven carbapenem resistant A. baumannii isolates identified in routine microbiology laboratory of Siriraj Hospital, Bangkok were studied. The dot blot hybridization was performed to detect class 1 integron element integrase gene. PCR was used to amplify blaVEB-1, arr2, cmlA, blaOXA-10 resistance cassettes, and variable region of class 1 integron element. blaVEB-1 gene was localized by southern blot hybridization. RESULTS: The prevalence of class 1 integron element was 86.48% in the isolates studied. The blaVEB-1 was present in 7 isolates however the location of blaVEB-1 gene was different in different isolate. Four isolates (Ab03-168, Ab04-28, Ab08-20, and Ab08-22) harbored calss 1 integron element variable region sized 5.5 kb as described in strain AYE. However, blaVEB-1 was only amplified from Ab03-168. The cassette organization in this isolate was 5’CS-aadB-blaVEB-1-arr2-cmlA-blaOXA- 10-aadA1-3’CS. The class 1 integron element similar to the element identified in genomic resistance island, AbaRI of European clone I, AYE was identified in Thai A. baumannii. CONCLUSIONS: blaVEB-1 harboring class 1 integron element with minor cassette variation was identified in Thai A. baumanni isolate which might suggest the spread of this resistant cassette or the spread of the European clone I in Thailand. Monitoring of the global spread of multi-resistant A. baumannii is mandatory to control the spread of resistant genes and this multi-resistant pathogen. DOI: http://dx.doi.org/10.3126/ijim.v1i1.6715Int J Infect Microbiol 2012;1(1):24-28

Emergence of -lactamases OXA-10, VEB-1 and CMY in Providencia spp. from Nigeria

Emergence of -lactamases OXA-10, VEB-1 and CMY in Providencia spp. from Nigeria

A multiple antibiotic and serum resistant oligotrophic strain, Klebsiella pneumoniae MB45 having novel dfrA30, is sensitive to ZnO QDs

BackgroundThe aim of this study was to describe a novel trimethoprim resistance gene cassette, designated dfrA30, within a class 1 integron in a facultatively oligotrophic, multiple antibiotic and human serum resistant test strain, MB45, in a population of oligotrophic bacteria isolated from the river Mahananda; and to test the efficiency of surface bound acetate on zinc oxide quantum dots (ZnO QDs) as bactericidal agent on MB45.MethodsDiluted Luria broth/Agar (10-3) media was used to cultivate the oligotrophic bacteria from water sample. Multiple antibiotic resistant bacteria were selected by employing replica plate method. A rapid assay was performed to determine the sensitivity/resistance of the test strain to human serum. Variable region of class 1 integron was cloned, sequenced and the expression of gene coding for antibiotic resistance was done in Escherichia coli JM 109. Identity of culture was determined by biochemical phenotyping and 16S rRNA gene sequence analyses. A phylogenetic tree was constructed based on representative trimethoprim resistance-mediating DfrA proteins retrieved from GenBank. Growth kinetic studies for the strain MB45 were performed in presence of varied concentrations of ZnO QDs.Results and conclusionsThe facultatively oligotrophic strain, MB45, resistant to human serum and ten antibiotics trimethoprim, cotrimoxazole, ampicillin, gentamycin, netilmicin, tobramycin, chloramphenicol, cefotaxime, kanamycin and streptomycin, has been identified as a new strain of Klebsiella pneumoniae. A novel dfr gene, designated as dfrA30, found integrated in class 1 integron was responsible for resistance to trimethoprim in Klebsiella pneumoniae strain MB45. The growth of wild strain MB45 was 100% arrested at 500 mg/L concentration of ZnO QDs. To our knowledge this is the first report on application of ZnO quantum dots to kill multiple antibiotics and serum resistant K. pneumoniae strain.

Antimicrobial resistance and class I integrons in Salmonella enterica isolates from wild boars and Bísaro pigs.

The antibiotic resistance phenotype and genotype and the integron type were characterized in 58 Salmonella enterica isolates recovered from Bísaro pigs and wild boars (20 S. Typhimurium, 17 S. Rissen, 14 S. Enteritidis and 7 S. Havana). Most S. Typhimurium isolates (15/20 of Bísaro pigs and wild boars) showed ampicillin, chloramphenicol, streptomycin, tetracycline, sulfonamide, and amoxicillin-clavulanic acid resistances. Of the 17 S. Rissen isolates of both origins, 13 were resistant to ampicillin, tetracycline and trimethoprim-sulfamethoxazole. Among the S. Enteritidis isolates of Bísaro pigs, eight were nalidixic acid-resistant and three were sulfonamide-resistant. The tet(A) or tet(G) genes were detected in most tetracycline-resistant isolates. The intI1 gene was identified in 72.5% of S. enterica isolates in which the conserved region 3' of class 1 integrons (qacEΔ1+sul1) was also amplified, whereas none had the intI2 gene. The dfrA12+orfF+aadA2 gene cassette arrangement was found in the variable region of class 1 integrons in 14 S. Rissen isolates. Fifteen S. Typhimurium isolates had two integrons with variable regions of 1000 and 1200 bp that harbored the aadA2 and blaPSE-1 gene cassettes, respectively. In these isolates the floR and tet(G) genes were also amplified, indicative of the genomic island 1 (SGI1). Salmonella Typhimurium and S. Rissen of animal origin frequently show a multi-antimicrobial resistant phenotype, which may have implications in public health.