The growing threat of drug- resistant Staphylococcus aureus (S. aureus) infections mandates the need to develop novel, effective and alternative antibacterial therapeutics. Despite infection prevention and control measures, methicillin resistant S. aureus (MRSA)-associated deaths reached 11,285 in 2011 in the USA (CDC, 2013). To counteract the threat of drug resistant S. aureus, we sought to construct and characterize a novel therapeutic based on the display of lytic antibacterial enzymes, termed endolysins. These endolysins were displayed on the surface of a specific bacterial virus, bacteriophage (phage), to generate lytic antibacterial nanoparticles. Endolysins are encoded individually by a variety of double-stranded DNA phage and act to direct host lysis and escape. These lytic enzymes confer a high degree of host specificity that could potentially substitute for, or be combined with, antibiotics in the treatment of gram-positive drug resistant bacterial infections such as MRSA. In this study, modular domains of the phage-encoded endolysin K enzyme, specific to S. aureus, were displayed on the capsid surface of phage lambda () via fusion with the λ major head (capsid) protein, gpD. The constructs of displayed endolysins were prepared in various combinations to maximize the functional display of gpD::X fusions on the phage. Phage lysates were generated, collected and purified and lysis was investigated by adding to fresh lawns of MRSA, vancomycin resistant S. aureus (VRSA) and bovine S. aureus. Phage preparations did not readily confer cell lysis, likely due to poor incorporation of the fusions onto the functional phage capsid. We purified the fusion proteins (gpD::X) and tested them for their lytic activity. We noted that the activity of the gpD::LysK protein was not impaired by the fusion and demonstrated lysis on live and dead (autoclaved) bovine S. aureus. In contrast to gpD::LysK, the gpD::CHAP protein fusion, expressing only the CHAP catalytic domain of endolysin K showed variable results in the lysis assays that we performed. In the zymogram assay, gpD::CHAP did not elicit any observable lysis on live bovine S. aureus cells, but did effectively lyse dead cells of the same S. aureus species; however, it was highly lytic in the inhibition assay on bovine S. aureus. The CHAP::gpD protein fusion, which is the CHAP domain fused to the N terminus of gpD only showed its ability to inhibit bovine S. aureus growth on the inhibition assay. The fusion of endolysin K or its CHAP domain to gpD protein does not seem to interfere with lytic activity, but may result in recalcitrant gpD fusions that compromise the ability to efficiently decorate the phage capsid. Suggestions for improved fusion capsid integration are discussed.

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

The food safety and human diagnostics markets are in need of faster working, reliable, sensitive, specific, low cost bioassays and biosensors for bacterial detection. This thesis reports the use of P22 bacteriophage tailspike proteins (TSP) immobilized on silanized silicon surfaces, roughened at a nano-scale, for specific capture and detection of Salmonella. Towards developing TSP biosensors, TSP immobilization characteristics were studied, and methods to improve bacterial capture were explored. Atomic force microscopy was used to count TSP immobilized on gold thin-films. Surface density counts are dependent on the immobilization scheme used. TSP immobilized on flat silicon (Si), silanized with 3-aminopropyltriethoxysilane and activated with glutaraldehyde, showed half the bacterial capture of gold thin-films. To improve bacterial capture, roughened mountain-shaped ridge-covered silicon (MSRCS) surfaces were coated with TSP and tested. Measurements of their bacterial surface density show that such MSRCS surfaces can produce bacterial capture close to or better than TSP-coated gold thin-films. / Biomedical Engineering

No description available.

The increasing incidence of infections due to antibiotic resistant bacteria has led to renewed interest in bacteriophages (= phages) and phage therapy. Although phage therapy has been applied to control bacterial diseases in plants, poultry, livestock and humans, its application in aquaculture is still relatively limited. The emergence of phage-resistant bacterial mutants has been considered to be one of the major limitations of phage therapy. This study aimed to (i) isolate and characterise phages; (ii) select phages and their bacterial hosts to set up in vivo phage therapy models with aquaculture animals, and estimate the efficiency of phage therapy; (iii) investigate the generation and characteristics of phage-resistant mutants, and thus estimate the consequence of applying phage therapy when phage-resistant mutants emerge; and (iv) discuss the prospects for application of phages in aquaculture. Two Vibrio isolates and their phages were isolated from a Scottish marine fish farm. Based on the results of conventional phenotype testing and 16S rRNA gene sequencing analysis, the two vibrios, V9 and V13, were identified as Vibrio splendidus and Vibrio cyclitrophicus, respectively. The bacterial characteristics including morphology, temperature and salinity range of growth, production of extracellular enzymes, and the possession of virulence genes were examined. According to the morphological characteristics observed using transmission electron microscopy by negative staining, phage PVS9 of V. splendidus V9 was identified as a myophage, while phage PVC13 of V. cyclitrophicus V13 was identified as a siphophage. The phages could only lyse one bacterial host strain and their genomic DNA was double stranded with a size of ~46 kb. The two Vibrio isolates were found to be non- or of low virulence to rainbow trout, goldsinny wrasse and Artemia in pathogenicity experiments. Thus an in vivo phage therapy model could not be set up using these Vibrio isolates and their phages. Two phages pAS-3 and pAS-6 were isolated using the Aeromonas salmonicida subsp. salmonicida Hooke strain as the host. Phages pAS-3 and pAS-6 had a similar genome size of ~50 kb, and the same relatively narrow host range within A. salmonicida subsp. salmonicida strains. The siphophage pAS-3 formed clear plaques and inhibited A. salmonicida Hooke growth in vitro completely for at least 18 hours when using MOI = 1,000, whereas the podophage pAS-6 formed turbid plaques and weakly inhibited Hooke growth. Rainbow trout exposed by intraperitoneal injection with 0.1 mL of the raw phage preparations at a concentration of 108 PUF mL-1 showed no adverse effects over 14 days. In the phage therapy trial, fish were firstly injected with 1 x 102 CFU fish-1 of A. salmonicida Hooke, then immediately injected with phage preparations of pAS-3 and pAS-6, respectively, using MOI = 10,000. Compared with the control group (which did not receive phage treatment), phage treated groups showed a delay in the time to death, and lower mortalities. However, the mortalities and time to death between phage treated and non-treated groups were not significantly different. Phage-resistant mutants of pathogenic A. salmonicida strain Hooke were induced by repeatedly challenging with phage pAS-3. One of the mutants, termed HM, was chosen to compare the characteristics with the parental wild-type strain Hooke. Test results including the formation of ‘smooth’ colonies on TSA, autoagglutination negative, the formation of creamy colonies on Coomassie Brilliant Blue agar, and the degradation of a thick/furry layered structure on the cell surface indicated a deficiency of the A-layer in the phage-resistant mutant HM. Therefore, it was deduced that the A-layer either directly acted as the receptor of A. salmonicida phage pAS-3, or was affected indirectly by the change of an unknown phage receptor. The greater wax moth larvae model was used to compare the virulence of the phage-resistant mutant HM and the parental wild-type strain Hooke, as it is an ethically acceptable animal model, which has the advantages of being low cost and convenient for injection, and is also a recognised alternative model for bacterial pathogens of fish. The results showed that virulence of the phage-resistant mutant HM did not decline in the greater wax moth larvae model compared with that of the parental wild-type strain Hooke. In conclusion, different approaches were used to isolate and characterise phages from different aquaculture environments for potential use in phage therapy. A rainbow trout model was set up using pathogenic A. salmonicida strain Hooke and two A. salmonicida phages pAS-3 and pAS-6. The use of phage treatment led to lower cumulative mortalities and delay to the time of death, although the differences between the groups were not significant, futher work is required to determine if these phages have potential in phage therapy. The consequence of applying phage therapy when phage-resistant mutants emerge was estimated based on their characteristics and virulence, and no decline in virulence of the phage-resistant mutant from this study indicates the importance of fully testing the virulence of phage-resistant mutants before carrying out large scale field trials of phage therapy. It appears feasible to use phage therapy as an alternative approach to control bacterial infections in aquaculture, but further studies are required to focus on improving effectiveness, and also to overcome the concrete limitations and hurdles in application and commercialisation. Moreover, a broader range of applications of phages in aquaculture should be explored.

Isolating novel phages using Streptomyces azureus, which produces antibiotic thiostrepton, as a host, and characterizing the genomes may help us to find new tools that could be used to develop antibiotics in addition to contribute to the databases of phages and specifically, Streptomyces phages. Streptomyces phages Alsaber, Omar, Attoomi, Rowa, and ZamZam were isolated using during this study. They were isolated from enriched soil and sequenced by Illumina sequencing method. They were isolated from three different geographical regions. They are siphoviridae phages that create small clear plaques with a diameter of approximately 0.5-1 mm, except for Rowa which has cloudy plaques, and they have varied sizes of their heads and tails. ZamZam was not characterized at this time. The sequencing shows that they are circular genome with 3' sticky overhang and various genomes' sizes with high percentage of GC content with the average of 66%. Alsaber was classified under sub-cluster BD3, while Omar was categorized under sub-cluster BD2. They share the same cluster of Cluster BD. Rowa was placed in Cluster BL and Attoomi is currently a singleton that does not fit into an established cluster. Alsaber yields 76 putative genes with no tRNA, Omar 81 putative genes with 1 tRNA. Attoomi 53 putative genes with no tRNA, and Rowa with 61 orfs and 7 tRNA. Rowa also was a putative temperate phage due to its lysogenic activity, and Row was not able to reinfect the lysogenic strain, S. azureus (Rowa). All of the isolated phages infected S. indigocolor, while only Attoomi and Rowa were able to infect S. tricolor. Upon completion of this project, we acquired more data and understanding of S. azureus phages and Actinobacteriophage in general, which will expand the scale of future research of Streptomyces bacteriophages.