ABSTRACT
Aim: Salmonella is an important food-borne pathogen in humans and a broad range of animals. Antimicrobial resistance in Salmonella spp. is a serious health problem in human and veterinary medicine worldwide. The aim of this study was to detect integrons, the natural recombination systems that can be transferred in companion with mobile genetic elements and play a major role in spreading antibiotic resistance genes in clinical isolates. Place and Duration of Study: Salmonella clinical isolates were provided by a number of institutes and hospitals over the country through the years 2008-2009. Methodology: Antimicrobial susceptibility testing and serotyping were carried out for eighty four epidemiologically unrelated clinical isolates of Salmonella serovars collected from different provinces of Iran through the years 2008-2009. PCR assays were carried out to detect intI2 gene (integrase I attributed to class 2 integron) and internal variable regions (IVRs) of class 2 integron. These sequences were deposited in EMBL/GenBank database (www.ncbi.nlm.nih.gov). Results: Eleven isolates (13.1%) which were resistant to at least 4 groups of antimicrobial agents were considered as MDR (multidrug resistant) Salmonella serovars. PCR assays detected intI2 gene (integrase I attributed to class 2 integron) and internal variable regions (IVRs) of class 2 integron in Fourteen (16.7%) and eleven (78.6%) of Salmonella clinical isolates respectively. Analysis of the sequence data revealed 3 gene cassette arrays deposited in Genbank databases including the dhfrA1 (0.75 kb), dfrA14- lsp (1 kb), dhfrA1- sat2-aadA1 (3 kb) with three IVR distribution patterns. An artifact PCR product of 2 kb was reported in this study to be amplified together with IVRs of class 2 integrons which was associated with the fhuE- ptsG genes. Conclusions: Presence of MDR Salmonella serovars demonstrates that antimicrobial selection pressure is widespread in our clinical settings. Detection of class 2 integron carrying gene cassettes which confer resistance to different classes of antibiotics such as aminoglycosides, and trimethoprim confirms that integron-mediated antimicrobial gene cassettes are prevalent in Salmonella serovars isolated in Iran.
ABSTRACT
BACKGROUND: The occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied recently. As screening and detection of integrons are concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for integrons detection. RESULTS: In this study, three loop-mediated isothermal amplification (LAMP) assays targeting on class 1, 2 and 3 integrons were implemented and evaluated. Optimization of these detection assays were performed, including studing on the reaction temperature, volume, time, sensitivity and specificity (both primers and targets). Application of the established LAMP assays were further verified on a total of 1082 isolates (previously identified to be 397 integron-positive and 685 integron-negative strains). According to the results, the indispensability of each primer had been confirmed and the optimal reaction temperature, volume and time were found to be 65°C, 45 min and 25 µL, respectively. As application was concerned, 361, 28 and 8 isolates carrying intI1, intI2 and intI3 yielded positive amplicons, respectively. Other 685 integron-negative bacteria were negative for the integron-screening LAMP assays, totaling the detection rate and specificity to be 100%. CONCLUSIONS: The intI1-, intI2- and intI3-LAMP assays established in this study were demonstrated to be the valid and rapid detection methodologies for the screening of bacterial integrons.
Subject(s)
DNA, Bacterial/isolation & purification , Nucleic Acid Amplification Techniques/methods , Integrons , Organic Chemicals , Salmonella/genetics , Serratia marcescens/genetics , Staphylococcus/genetics , Vibrio cholerae/genetics , Colony Count, Microbial , Microbial Sensitivity Tests , Polymerase Chain Reaction/methods , Sensitivity and Specificity , DNA, Complementary , DNA Primers , Integrases/genetics , Drug Resistance, Bacterial/genetics , Electrophoresis, Agar Gel , Escherichia coli/genetics , Fluorescent Dyes , Hot TemperatureABSTRACT
This study identified and characterised class 1 and 2 integrons in clinical and environmental Vibrio cholerae O1 and non-O1/non-O139 strains isolated from the Brazilian Amazon. The aadA2 and aadA7 gene cassettes were found in class 1 integrons in two genotypes of environmental V. cholerae non-O1/non-O139. Empty integrons were found in strains from the Brazilian cholera epidemic. A class 2 integron was detected in one strain from the V. cholerae Amazonia lineage harbouring sat1 and aadA1 genes. All isolates were resistant to aminoglycosides, indicating aadA functionality. These findings suggest that environmental bacteria act as cassette reservoirs that favour the emergence of resistant pathogens.