Investigation And Diversity Analysis Of The Antibiotic Resistant Integron-bearing Bacteria In Aquatic Environment

To date, antibiotic agents are wildely used to treat infection in our daily life and boost growth in livestock breeding. The mechanism of resistance to antibiotics is becoming exceedingly complex under selective presure. What is more, multi-drug resistant microbes with complex resistance patterns have posed an increasing threat to the health. So intensive research leading to greater understanding is imperative.Integrons are mobile DNA elements that can capture and excise genes, particularly those responsible for antibiotic resistance (AR). Integrons do this by site-specific recombination. While integrons mobilize AR gene cassettes, they themselves are not mobile; however, they are frequently associated with transposons or transferable plasmids that allow them to spread among bacterial populations. On the basis of integrase amino acid sequence similarity, integrons are divided into five classes. Class 1 integrons are composed of a 5'-conserved segment (5'-CS), a 3'-conserved segment (3'-CS), and a variable region containing the gene cassettes. Research conducted over the past decade suggests that class 1 integrons play a vital role in the dissemination of antibiotic resistance and that gene cassettes encoding dihydrofolate reductases (dfr), chloramphenicol acetyltransferases (cat, cml), P-lactamases (bla), aminoglycoside-modifying enzymes (aac, aad, aphA), and ADP-ribosyl transferases (arr) are frequently identified within such mobile genetic elements.Previous studies have focused primarily on investigating the molecular diversity of class 1 integrons and antibiotic resistant phenotypes in clinical strains, particularly the Enterobacteriaceae. Clinical pathogenic bacteria with various resistance genes released into the environment by different ways, and the resistance mechanism can be transferred by daughter colony and the other bacteria can obtain the resistance genes through the horizontal gene transfer. Recombinations or mutations was occurred frequently in the process of gene transfer to form the new resistant genes, and novel arrays of multiple drug resistance genes were formed by recombination involving integrases. Consequently, these new resistance factors may be transferred to the clinical pathogenic bacteria from the environment and through air, food or water to infect the human body to form new resistance factors in clinical and threat to human health.In this study, we investigated antibiotic-resistant Enterobacteriaceae isolated from the wastewater of a hospital in Jinan, Shandong Province, as this source presented a constant selective pressure on the resident bacteria due to the presence of various antimicrobials. The prevalence of class 1 integrons was assessed and their gene cassette arrays were characterized to assess the dissemination of antibiotic resistance genes.In this study, we detected 29 kinds of gene cassettes arrays in class 1 integron,of these contained 7 kinds of novel gene cassettes arrays:orfⅠ(693 bp); aadB—cat—blaoxA-10—aadA1—orfⅡ-unknown (5193 bp); catB8—linF(1877 bp); aadB—cat—blaOXA-10—aadA1—dfrA1—aacA4 (4336 bp); arr-3—dfrA27 (1393 bp); dfrA1—aadB—aphA15—catB3—blaOXA-1—aadA15 (4797 bp); dhfrV—aac(6')-Ⅱ—nit1—nit2—catB3—blaOXA-1(3800 bp). We detected 5 kinds of gene cassettes arrays in class 2 integron, of these contained 3 kinds of novel gene cassettes arrays:linF—dfrA1—aadA1 (2460 bp); dfrA1—catB2—sat2—aadA1 (2892 bp); linF—dfrA1—aadA1—orf441 (4297 bp). Both class 1 integrons and several antibiotic-resistance genes outside of the integrons were characterized. The transferability of integrons and antimicrobial resistance genes was determined.In addition, we report a rapid and simple method for characterizing gene cassette arrays by restriction fragment length polymorphism (RFLP) analysis and sequencing.