Thesis

Prevalence and characterization of integrons in multidrug-resistant non-clinical enteric bacterial isolates

Antibiotic resistance in bacteria has been a concern in the medical field for almost as long as antibiotics have been available. The last several decades have seen marked increases in antibiotic resistance, leading to the discovery of multidrug-resistant (MDR) bacteria, which can be resistant to several antibiotics. MDR bacteria are a major problem in the healthcare industry, creating to numerous challenges such as reduced treatment options, increased mortality rates, longer hospital stays, and increased costs. The increasing dissemination of resistance genes is believed to be the result of horizontal gene transfer via mobile genetic elements, including plasmids and transposons. Several studies have also shown that integrons play a significant role in the spread of resistance, acting as gene capture and expression mechanisms that are often associated with mobile genetic elements. However, most of the studies investigating the role of integrons in the dissemination of antibiotic resistance utilized bacterial samples from environmental sources or hospitalized patients. Far fewer studies have examined the role of integrons in the propagation of multidrug-resistance in bacteria from the lower intestinal tract of healthy individuals. The purpose of this study was to determine whether or not integrons play a significant role in the proliferation of multidrug-resistance in enteric bacteria isolated from healthy, non-hospitalized adults. Attempts were also made to identify the gene cassettes and organization of cassettes within the identified integrons. Over the course of five years (2005-2009), a total of 92 enteric bacterial samples were collected from students at CSUS via a rectal swab, and isolated on MacConkey agar. These samples were isolated and subjected to a variety of antibiotics and biochemical tests to determine antibiotic resistance profiles and species. PCR amplification of class 1 and class 2 integrase genes (intI1 and intI2) yielded 19 (out of 84 unique samples) class 1 positive isolates, one of which was also found to be class 2 positive. Resistance to trimethoprim/sulfamethoxazole, ampicillin, and piperacillin was found to be significantly greater in class 1 positive isolates compared to class 1 negative isolates (P<0.05). Resistance to two or more classes of antibiotics was also significantly higher in class 1 positive isolates compared to class 1 negative isolates. Resistance to two or more antibiotics, regardless of class was also significantly higher in class 1 positive isolates. PCR amplification of the variable regions of intI1 and intI2 samples yielded seven unique amplicons ranging in size from approximately 250bp to >3kbp. Subsequent sequencing and nucleotide BLAST searches led to the identification of eight different gene cassettes organized in six unique arrays.

Thesis (M.S., Biological Sciences (Molecular and Cellular Biology))--California State University, Sacramento, 2013.

Antibiotic resistance in bacteria has been a concern in the medical field for almost as long as antibiotics have been available. The last several decades have seen marked increases in antibiotic resistance, leading to the discovery of multidrug-resistant (MDR) bacteria, which can be resistant to several antibiotics. MDR bacteria are a major problem in the healthcare industry, creating to numerous challenges such as reduced treatment options, increased mortality rates, longer hospital stays, and increased costs. The increasing dissemination of resistance genes is believed to be the result of horizontal gene transfer via mobile genetic elements, including plasmids and transposons. Several studies have also shown that integrons play a significant role in the spread of resistance, acting as gene capture and expression mechanisms that are often associated with mobile genetic elements. However, most of the studies investigating the role of integrons in the dissemination of antibiotic resistance utilized bacterial samples from environmental sources or hospitalized patients. Far fewer studies have examined the role of integrons in the propagation of multidrug-resistance in bacteria from the lower intestinal tract of healthy individuals. The purpose of this study was to determine whether or not integrons play a significant role in the proliferation of multidrug-resistance in enteric bacteria isolated from healthy, non-hospitalized adults. Attempts were also made to identify the gene cassettes and organization of cassettes within the identified integrons. Over the course of five years (2005-2009), a total of 92 enteric bacterial samples were collected from students at CSUS via a rectal swab, and isolated on MacConkey agar. These samples were isolated and subjected to a variety of antibiotics and biochemical tests to determine antibiotic resistance profiles and species. PCR amplification of class 1 and class 2 integrase genes (intI1 and intI2) yielded 19 (out of 84 unique samples) class 1 positive isolates, one of which was also found to be class 2 positive. Resistance to trimethoprim/sulfamethoxazole, ampicillin, and piperacillin was found to be significantly greater in class 1 positive isolates compared to class 1 negative isolates (P<0.05). Resistance to two or more classes of antibiotics was also significantly higher in class 1 positive isolates compared to class 1 negative isolates. Resistance to two or more antibiotics, regardless of class was also significantly higher in class 1 positive isolates. PCR amplification of the variable regions of intI1 and intI2 samples yielded seven unique amplicons ranging in size from approximately 250bp to >3kbp. Subsequent sequencing and nucleotide BLAST searches led to the identification of eight different gene cassettes organized in six unique arrays.

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