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Item Differences in Compositions of Gut Bacterial Populations and Bacteriophages in 5-11 Year-Olds Born Preterm Compared to Full Term(Frontiers Media S.A., 2020-06-16) Jayasinghe TN; Vatanen T; Chiavaroli V; Jayan S; McKenzie EJ; Adriaenssens E; Derraik JGB; Ekblad C; Schierding W; Battin MR; Thorstensen EB; Cameron-Smith D; Forbes-Blom E; Hofman PL; Roy NC; Tannock GW; Vickers MH; Cutfield WS; O'Sullivan JM; Shkoporov APreterm infants are exposed to major perinatal, post-natal, and early infancy events that could impact on the gut microbiome. These events include infection, steroid and antibiotic exposure, parenteral nutrition, necrotizing enterocolitis, and stress. Studies have shown that there are differences in the gut microbiome during the early months of life in preterm infants. We hypothesized that differences in the gut microbial composition and metabolites in children born very preterm persist into mid-childhood. Participants were healthy prepubertal children aged 5-11 years who were born very preterm (≤32 weeks of gestation; n = 51) or at term (37-41 weeks; n = 50). We recorded the gestational age, birth weight, mode of feeding, mode of birth, age, sex, and the current height and weight of our cohort. We performed a multi'omics [i.e., 16S rRNA amplicon and shotgun metagenomic sequencing, SPME-GCMS (solid-phase microextraction followed by gas chromatography-mass spectrometry)] analysis to investigate the structure and function of the fecal microbiome (as a proxy of the gut microbiota) in our cross-sectional cohort. Children born very preterm were younger (7.8 vs. 8.3 years; p = 0.034), shorter [height-standard deviation score (SDS) 0.31 vs. 0.92; p = 0.0006) and leaner [BMI (body mass index) SDS -0.20 vs. 0.29; p < 0.0001] than the term group. Children born very preterm had higher fecal calprotectin levels, decreased fecal phage richness, lower plasma arginine, lower fecal branched-chain amino acids and higher fecal volatile (i.e., 3-methyl-butanoic acid, butyrolactone, butanoic acid and pentanoic acid) profiles. The bacterial microbiomes did not differ between preterm and term groups. We speculate that the observed very preterm-specific changes were established in early infancy and may impact on the capacity of the very preterm children to respond to environmental changes.Item Phenotypic characterization and genome analysis of a novel Salmonella Typhimurium phage having unique tail fiber genes.(Springer Nature Limited, 2022-04-06) Sattar S; Ullah I; Khanum S; Bailie M; Shamsi B; Ahmed I; Shah STA; Javed S; Ghafoor A; Pervaiz A; Sohail F; Shah NA; Imdad K; Bostan N; Altermann ESalmonella enterica serovar Typhimurium is a foodborne pathogen causing occasional outbreaks of enteric infections in humans. Salmonella has one of the largest pools of temperate phages in its genome that possess evolutionary significance for pathogen. In this study, we characterized a novel temperate phage Salmonella phage BIS20 (BIS20) with unique tail fiber genes. It belongs to the subfamily Peduovirinae genus Eganvirus and infects Salmonella Typhimurium strain (SE-BS17; Acc. NO MZ503545) of poultry origin. Phage BIS20 was viable only at biological pH and temperature ranges (pH7 and 37 °C). Despite being temperate BIS20 significantly slowed down the growth of host strain for 24 h as compared to control (P < 0.009). Phage BIS20 features 29,477-base pair (bp) linear DNA genome with 53% GC content and encodes for 37 putative ORFs. These ORFs have mosaic arrangement as indicated by its ORF similarity to various phages and prophages in NCBI. Genome analysis indicates its similarity to Salmonella enterica serovar Senftenberg prophage (SEStP) sequence (Nucleotide similarity 87.7%) and Escherichia virus 186 (~ 82.4% nucleotide similarity). Capsid genes were conserved however those associated with tail fiber formation and assembly were unique to all members of genus Eganvirus. We found strong evidence of recombination hotspot in tail fiber gene. Our study identifies BIS20 as a new species of genus Eganvirus temperate phages as its maximum nucleotide similarity is 82.4% with any phage in NCBI. Our findings may contribute to understanding of origin of new temperate phages.Item Characterization of two novel lytic bacteriophages having lysis potential against MDR avian pathogenic Escherichia coli strains of zoonotic potential.(Springer Nature Limited, 2023-06-20) Sattar S; Bailie M; Yaqoob A; Khanum S; Fatima K; Altaf AURB; Ahmed I; Shah STA; Munawar J; Zehra QA; Daud S; Arshad A; Imdad K; Javed S; Tariq A; Bostan N; Altermann EAvian pathogenic E. coli (APEC) is associated with local and systemic infections in poultry, ducks, turkeys, and many other avian species, leading to heavy economical losses. These APEC strains are presumed to possess zoonotic potential due to common virulence markers that can cause urinary tract infections in humans. The prophylactic use of antibiotics in the poultry sector has led to the rapid emergence of Multiple Drug Resistant (MDR) APEC strains that act as reservoirs and put human populations at risk. This calls for consideration of alternative strategies to decrease the bacterial load. Here, we report isolation, preliminary characterization, and genome analysis of two novel lytic phage species (Escherichia phage SKA49 and Escherichia phage SKA64) against MDR strain of APEC, QZJM25. Both phages were able to keep QZJM25 growth significantly less than the untreated bacterial control for approximately 18 h. The host range was tested against Escherichia coli strains of poultry and human UTI infections. SKA49 had a broader host range in contrast to SKA64. Both phages were stable at 37 °C only. Their genome analysis indicated their safety as no recombination, integration and host virulence genes were identified. Both these phages can be good candidates for control of APEC strains based on their lysis potential.Item ABAtE : active bacteriophages for AFB eradication : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Auckland, New Zealand(Massey University, 2023) Kok, DanielleThe European honey bee (Apis mellifera) is one of the most important livestock animals in New Zealand. Their value comes from a combination of pollination services and the production of honey for export, notably mānuka honey. American Foulbrood (AFB) is a disease of honey bee larvae and pupae and is caused by the bacterial pathogen, Paenibacillus larvae. AFB is the most serious disease that infects honey bees and is present in almost all countries where honey bees are found. AFB has been present in New Zealand since 1877 and spread to all parts of the country within 10 years. Unlike other countries, the use of antibiotics in hives infected with P. larvae is prohibited under New Zealand law and infected hives must be destroyed immediately. Bacteriophages (phages) are a well-studied alternative to antibiotics. Phages are simple viruses that kill specific bacteria and are highly abundant in the environment with an estimated 1031 globally. Phages have been shown to work effectively as a prophylactic to infection from certain diseases. With the growing antimicrobial resistance crisis, phages are becoming a well-studied and promising alternative to antibiotics. The aim of this research was to investigate the use of phages as a preventative measure against AFB. Previous work undertaken in other laboratories around the world has shown that phages can be isolated from healthy hives and nearby soil and that AFB pathogens are susceptible to destruction by these phages. In this work, we collected soil samples using citizen led science from hives throughout New Zealand. From soil samples provided we isolated 26 novel phages that are destructive to P. larvae. Selected phages were combined into a cocktail and tested against vegetative forms of P. larvae in in-lab testing. All phages were also sequenced and annotated and compared to other P. larvae phages that have been isolated around the world. This project: ABAtE (Active Bacteriophages for AFB Elimination), provides the groundwork study for an innovative approach to naturally protecting NZ beehives against AFB.Item Characterisation of the filamentous bacteriophages end-caps : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatu, New Zealand(Massey University, 2021) León Quezada, RayénFf (f1, fd, and M13) filamentous phage of Escherichia coli have collectively been the workhorse of phage display technology over the past few decades. Their use has expanded in the recent years into nanotechnology, where they serve as filament-like templates (≥ 880 nm x 6 nm) for assembly of nanostructures such as nanowires, nanorings, and more complex assemblies including nano-scale batteries, among others. The filament end-caps are the key to improving phage display applications and design of novel Ff-built nanostructures. Furthermore, proteins pIII and pVI, that form the distal end-cap, are of key importance for understanding the Ff biology. They mediate the Ff assembly-release and entry, two opposing processes that involve, respectively, excision and insertion of the virion out of and into the inner membrane of E. coli. The mechanisms of these two processes are a mystery, and the only path towards understanding is to determine the structures of the pIII-pVI complex. While the atomic resolution structure of the Ff filament shaft made of the major coat protein pVIII has been determined, the structure of the phage end-caps remains unresolved, as they constitute only 2% of the virion mass. To enable the end-cap structural analyses we developed methodology for high-efficiency production and purification of short Ff-derived nanorods, where the end-caps are highly enriched, accounting for up to 38% of the total virion mass. Furthermore, methods for Ff-derived nanorod production have been majorly improved in this thesis by engineering a novel system, resulting in at least 200-fold increase relative to the systems described previously. The nanorod purification was also improved by including an anion exchange chromatography step. Highly pure and concentrated 50 nm and 80 nm nanorods were analysed structurally and biochemically to characterise the pIII-pVI complex. Intact nanorods were structurally characterised by cryo-EM single-particle analysis (cryo-EM SPA) that resulted in 2D classes of the filament end-caps. Furthermore, a preliminary 3D model of the pIII-pVI cap was generated at a resolution of 5 Å. Further refinement of the 3D model is under way. Besides the intact particles, analysis was expanded to purified pIII-pVI complex obtained from the DOC-chloroform-disassembled nanorods by size exclusion chromatography. Under native conditions, protein pIII could be detected in a complex larger than 720 kDa, indicating that multiple copies of pVI and pIII form a multimer-dimer that includes a substantial amount of the shaft protein, pVIII. Applications of nanorods benefit from precise control of the nanorod lengths, which is very difficult to achieve when it comes to non-biological materials. In this thesis, Ff-derived nanorods of novel sizes were designed by eliminating specific DNA regions from the nanorod replication cassettes that controls the length of the nanorod ssDNA backbone. This work showed that the DNA segment between the packaging signal and (-) ori is not essential for replication and resulted in production of 40 nm nanorods, shortest ever constructed to date. Two novel lengths, 40 and 70 nm, were added to the of sub-100-nm nanorod collection produced using this system.Item Development and applications of filamentous phage-derived particles in immunotherapy and diagnostics : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University (Manawatū), New Zealand(Massey University, 2020) Rajič, MarinaMost vaccines that are currently in clinical use induce antibody-mediated responses. However, for many infectious diseases, T cells are an essential part of naturally acquired protective immune responses. T cell-inducing vaccines, such as the one developed in this research, could additionally be used for treatments in cancer or chronic viral infections. One way to target the immune cells and stimulate their responses is to use filamentous phage particles. Filamentous phages (Ff) are ssDNA viruses that infect Escherichia coli, which have been adapted and used extensively in phage display technology and nanotechnology. In this research, a filamentous phage (Ff)-based vaccine carrier was constructed to allow the tuneable display of non-protein immune adjuvant molecules (BODIPY-α-GalCer) and antigenic peptides (OVA; MHC I + MHC II) on the same particle. For the first time, azide groups were incorporated into the recombinant pVIII phage coat proteins that expressed recombinant peptides. Azide groups were subsequently used to attach fluorescently labelled adjuvant molecules, which were successfully presented to NKT cells in vivo. Additionally, high induction of in vitro proliferation of OVA-specific CD8+ T-cells was achieved. However, the Ff-derived particle use outside the laboratory is hindered because they are genetically modified viruses, which in addition, carry antibiotic resistance genes that can be horizontally transferred to gut bacteria. These limitations were overcome by developing a system for efficient production of non-replicating, controllable-length protein-DNA nanorods, derived from Ff, named BSFnano (~100 × 6 nm). In this research, functionalised BSFnano particles were constructed, and their application in diagnostics was tested in a proof of concept dipstick assay for detection of a soluble analyte (fibronectin). For the first time, an ultrasensitive dipstick assay was achieved with Ff-derived nanorods, detecting the test-analyte at a concentration of as low as 0.04 pg/μL, equivalent to only 100,000 molecules/μL. Overall, while the phage-based vaccine produced in this research elicited CD8+ T-cell responses in vitro, but not in vivo, the Ff-derived nanorods were successfully functionalised and tested in lateral flow immunoassay, with promising implications for use in point of care diagnostics.Item Testing the potential of mycobacteriophage endolysins fused to biodegradable nanobeads for controlling mycobacteria : a thesis presented in partial fulfilment of the requirements for the degree of Master of Natural Sciences at Massey University, Albany, New Zealand(Massey University, 2019) Davies, Courtney Grace10.4 million people are diagnosed with tuberculosis, worldwide, every year, according to the World Health Organisation. Mycobacterium tuberculosis is a Gram-positive bacterial pathogen that can easily be transmitted to health care professionals and people supplying aid in these nations. Fortunately, over the last 100 years, the bacteriophage has gained traction as a suitable therapeutic, antibiotic-alternative against bacterial pathogens, such as M. tuberculosis. Herein I describe my work utilising endolysins the lytic protein that bacteriophages usually employ to burst out of the cell, but instead using these proteins to lyse “from without”. In order to develop a proof-of-principle product, we used the expansive M. smegmatis bacteriophage collection and chose eight endolysin candidates for testing against M. smegmatis. These endolysins were bioinformatically analysed for active domains before being synthesised and inserted into an expression vector to produce fused biodegradable nanobeads made of polyhydroxyalkanoate. These nanobeads were tested for activity against M. smegmatis, a safe mycobacterium closely related to pathogenic tuberculosis. Four distinct tests were carried out to test the efficiency of these beads in causing cell death in different situations (45 minutes and 5 hours, across concentrations of 10mg/ml, 20mg/ml and 80mg/ml. Ultimately the nanobead fusions of endolysin Inca (lysin B) caused the most cell death at 80mg/ml after exposure to M. smegmatis for 5 hours in a standing culture, at 78.87% cell death ± 5.21. When the nanobeads were applied to filter paper to mimic application to a hospital mask as a proof-of-concept approach before spraying with a bacterial aerosol, we saw that endolysin nanobead Jaws (lysin B) caused the most cell death with 75.54% ± 3.15 at 80mg/ml. These results are promising and present a unique opportunity to take advantage of an existing natural mechanism to use as a prophylactic defense against pathogenic bacteria in hospital settings.Item Filamentous phage derived biological nanorods : development of a novel display system : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Manawatū, New Zealand(Massey University, 2018) Davey, Georgia RoseThe Ff filamentous bacteriophage are filament-like bacterial viruses approximately 900 nm in length. The F-pilus-specific filamentous phage are resistant to heat, pH-extremes, and detergents in combination with their structural properties and amenability to DNA recombinant engineering has enabled their extensive use in modern biotechnology. However, the use of Ff-phage in vaccines and other such biological uses is controversial due to their ability to replicate in gut Escherichia coli, and the possibility of mobilisation and horizontal gene transfer of antibiotic resistance-encoding genes among the gut bacteria. As such, the novel system was established to create short, stable particles that cannot replicate, called NanoZap particles. However, this system has the disadvantage of often producing multiple-length particles, rather than the desired single-length particles; another disadvantage is that during packaging, one particle in a million packages the entire plasmid due to recombination that removes the terminator copy of the (+) ori, and given that these plasmids contain antibiotic resistance genes, this likely would spread antibiotic resistance throughout the surrounding environment. In this thesis several variations upon the original pNanoZap vector were created and tested to obtain monodisperse unit-length particles. The deletion of the complete multiple cloning site (MCS) that lied between the initiator and terminator of replication from the original pNanoZap vector achieved this aim. To eliminate rare antibiotic resistant particles that package the complete pNanoZap vector, the antibiotic resistance gene was removed from the pNanoZap 537 vector and replaced with an auxotrophic marker nadC; this vector was named pNanoZap 537N. It is yet to be seen if this new pNanoZap vector is capable of producing NanoZap particles. For high-sensitivity diagnostics it is desirable to construct high-avidity particles, containing large number of detector molecules. To achieve this a double-display (detector displayed on phage which in turn is displayed on the surface of florescent E. coli) was designed and tested. When the E. coli expressing red fluorescent protein TinselPurple were infected with the bacteriophage, the chromogenic protein was lost, thereby showing that a different method of colouring E. coli will need to be used in order to construct the double-display particles.Item The host specificities of Phage Mu : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University(Massey University, 1983) Moody, Judith MavisPhage Mu, a temperate phage, possesses an invertible region of DNA 3kb in length, the G region. The orientation of the G region determines the host specificities of phage Mu (Van de Putte et al . , 1980). The G region codes for tail polypeptides, the products of the S and U genes. In one orientation ( + ) the phage adsorbs to and infects E. coli K12 (Mu.K). In the opposite orientation (-) the phage adsorbs to and infects a strain of C. freundii (Mu.F). However Mu also plates on E. coli C, Serratia marcescens, E. cloacae and several other hosts. Rice (1980) was able to raise specific antisera to the Mu.K form and the W.C (Mu.C equivalent) form of Mu. However no antisera was raised to the Mu.F form. We attempted to isolate gin̄ mutants, in which the G region is not inverted; such a mutant would provide a specific antigen for the G(-) Mu.F phage. Presumptive gin̄ mutants were identified as phage that could plate only on one host, E. coli K12 or C. freundii. A specific antisera was raised to the MH₄₄₀₀gin̄ G(-) phage. Attempts were made to isolate the Mu.KC phage as seen by Jamieson (1971) and Rice (1980). For this purpose, heat induced lysogens of E. coli K12 were used, as opposed to lytic Mu.K lysates; the former give high titre lysates of G(-) and G(+) phage. The resulting Mu.KC lysogens were examined for their plating behaviour and neutralisation by the anti W.C serum. Two classes of phage were isolated, Mu.KC'' and Mu.KC'''. These phage differed from the Mu.KC phage seen by Jamieson and Rice as judged from their limited neutralisation by the anti W.C serum, and they also differed from each other with respect to their plating on C. freundii and the extent to which they are neutralised by the anti W.C serum. The Mu.KC" forms do not plate on C. freundii , e.o.p. <8 x 10̄⁹, and are not neutralised by the anti W.C serum. Whereas the Mu.KC''' forms do plate on C. freundii, e.o.p. 10̄¹-10̄², and exhibit a low level of neutralisation with the anti W.C serum. E. coli C lysogens of phage Mu.KFC were also isolated. These also fell into two distinct classes as judged by their plating ability on C. freundii ; Mu.KFC'' forms do not plate on this host, e.o.p. <5 x 10̄⁸, while the Mu.KFC''' forms do plate on C. freundii, with an e.o.p. 1 x 10̄¹ (comparable to the Mu.KC'' and Mu.KC''' mentioned above). During the study, differences in the plating ability of lytic and induced lysates propagated on the same host were observed. For example, compare the E. coli K12 system; lytic Mu.K phage plate on C. freundii with an e.o.p. of <10̄⁹, while induced Mu.K plate on this host with an e.o.p. of 1. Again, lytic Mu.KF lysates plate on E. coli K12, e.o.p. 10̄¹ , and an E. coli C, e.o.p. 2 x 10̄⁵. However, induced Mu.KF lysates plate on E. coli K12, e.o.p. 10¹, but these lysates were not observed to plate on E. coli c, e.o.p. <2.5 x 10̄⁹ . And yet again, lytic Mu.KC'' and Mu.KC''' plate on E. coli K12 with an e.o.p. of 10̄² -10̄³, however induced Mu.KF'' and Mu.KFC''' lysates plate on this host with a e.o.p. of 2.0 - 8.5. The MH₄₄₀₀ strain, gin̄ (albeit leaky), enabled the detection of the role of G inversion during plating Mu onto different hosts. This phage plates with a reduced frequency when G inversion is involved, e.g. induced MH₄₄⁰⁰.K plated on C. freundii with an e.o.p. of 10̄³. The e.o.p. of induced MH₄₄⁰⁰ .KFC on E. coli K12 (4.5 x 10̄⁴), was observed to be significantly lower than the wild type Mu (Mucts61) on this host (e.o.p. 2.0 - 8.5).Item Prophages and genetic restriction in Escherichia coli, strain W.: a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University(Massey University, 1987) Doherty, Timothy MarkE.coli strain W (ATCC 9637) is lysogenic for 2 phages which propagate on either E.coli C or E.coli K12. Each phage adsorbs specifically to only one of these hosts and the phages are therefore designated W0 and Wk respectively. These phages display a large number of similarities to phage Mu but restriction endonuclease digestion and probing with radioactively labelled DNA show that the W-phage are not identical to phage Mu. Lysogens of E.coli C carrying either of the W-phage exhibit strong restriction towards phages P, and Lambda; lysogens of E.coli K12 carrying the same phage have not been shown to exhibit such restriction. Conjugation experiments between E.coli W, E.coli C, and E.coli K12 allowed the mapping of the insertion sites of the 2 W-prophages on the E.coli W genome