Foodborne diseases caused by non-typhoidal Salmonella represent an important public health problem worldwide (Zhao et al., 2003). The transmission of Salmonella between animals and humans has been well established in epidemiological studies. In the case of complicated illness caused by Salmonella where antibiotics need to be administered, treatment can be compromised if the infecting organism is resistant to the prescribed antimicrobial agent. This study and earlier studies have shown that many Salmonella carry temperate bacteriophages as lysogens. Many of these bacteriophages are capable of mediating generalised transduction (Schicklmaier and Schmieger, 1995; Schicklmaier et al., 1998; Mmolawa et al., 2002). Schmieger and Schicklmaier (1999) demonstrated that bacteriophages ES18 and PDT17 are capable of transduction of antibiotic resistance genes from DT104. Phage-mediated transduction of antibiotic resistance genes has been largely neglected in the study of genetic transfer of antibiotic resistance in bacteria. This study investigates whether bacteriophages exist in antibiotic resistant Salmonella isolates. Such temperate phages in antibiotic resistant isolates could play a significant role in the transfer of resistance to other species of enteric bacteria, such as E. coli. Molecular epidemiology studies of antibiotic resistance genes were undertaken with Salmonella isolates from chicken, pig and human sources that were subjected to PCR for ampicillin (blaTEM-1), tetracycline (tetA, tetB) and streptomycin (aadA1, aadA2, strA, strB) resistance genes as well as Class 1 integrons. The blaTEM-1 gene was widely detected in isolates from pigs and chickens but rarely detected in human isolates. The tetB gene was more commonly found in pig isolates, while the tetA gene was associated with tetracycline resistance in chicken isolates. The strA and strB genes were responsible for streptomycin resistance in the S. Typhimurium isolates while the aadA1 gene was commonly detected in S. Kiambu and S. Virchow isolates. The aadA2 gene was associated with streptomycin resistance in the S. Ohio isolates from pigs. Class 1 integrons were widely distributed across serovars tested from chicken, pig and human sources. Temperate bacteriophages were induced using mitomycin C from antibiotic resistant Salmonella. These phages were able to infect antibiotic-sensitive Salmonella isolates from humans. Bacteriophages induced from one S. Sofia isolate also plaqued on Shigella flexneri. Bacteriophages induced from one S.Kiambu isolate and S. Typhimurium DB21 with an inserted Tn10 transposon (S. Typhimurium DB21 Tn10) were capable of transducing ampicillin and tetracycline resistance, respectively into S. Enteritidis PT1 isolates by in vitro methods. The molecular basis for resistance was established in subsequent PCR for antibiotic resistance genes in donor and recipient strains. This finding, in particular in the wild-type S. Kiambu strain, indicates that Salmonella from a natural source are able to infect and transfer antibiotic resistance by generalised transduction in controlled laboratory experiments. This current study has investigated the transfer of tetracycline and ampicillin resistance from a wild-type Salmonella strain and a laboratory strain of Salmonella to wild-type Salmonella bacteria as it occurs within the normal flora of the chicken gastrointestinal tract. It was demonstrated that the genetic transfer of tetracycline and ampicillin resistance genes as well as Class 1 integrons can occur within the chicken gastrointestinal tract. Transfer of tetracycline and ampicillin resistance could be demonstrated both in vitro and by using bacteriophage lysates obtained from in vivo studies in transduction experiments. It was clearly shown that bacteriophage isolated from chicken faeces and caeca could infect antibiotic sensitive recipient Salmonella. Interaction between phages of the administered Salmonella strains may be occuring with phages of bacteria in the normal flora allowing previously inactive phage in the indigenous flora to plaque on indicator strains. Additionally, strong evidence was presented to suggest that the environment of the chicken gastrointestinal tract could mediate phage type conversion in recipient and transductant strains. Phage typing of these recipient and transductant strains demonstrated a trend for recipient strains to become more resistant to phages in the S. Enteritidis typing panel. This led to weakened phage reactions such RDNC (reaction does not conform) and untypable. The acquisition of phages may be a way for Salmonella to enhance competitive fitness and generate new strains in order to evolve and diversify. Or the acquisition of plasmids either by transduction or conjugation may also mediate phage type conversion. MLVA typing was performed on selected recipient, donor and transductant strains. The changes to tandem repeat loci in Salmonella isolates that have passed through a chicken gastrointestinal tract have not been described before. The changes to fragment length suggest that the bacterial chromosome is undergoing rearrangement; this may be attributed to a number of factors including acquisition of phages, prophage integration into tRNA sites, slipped-strand mispairing or the adaption to changing environment, in this case the chicken gastrointestinal tract. This study has provided molecular epidemiological data on the antibiotic resistance genes and integrons present in Australian Salmonella isolates from human and animal sources. Information on the role of bacteriophages in the transfer of antibiotic resistance genes in vitro and in a chicken gastrointestinal tract has also been established. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2010
| Identifer | oai:union.ndltd.org:ADTP/288072 |
| Date | January 2010 |
| Creators | Tan, Sophia |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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