Effective treatment of tuberculosis requires at least six months of combination therapy involving four antibiotics. Alterations in the physiological state of Mycobacterium tuberculosis during infection may reduce drug efficacy and prolong treatment, but these adaptations are incompletely defined. To investigate the mechanisms limiting antibiotic efficacy, I performed a comprehensive genetic study to identify M. tuberculosis genes and pathways important for bacterial survival during antibiotic treatment in vivo. First, I identified mutants in the glycerol kinase enzyme, GlpK, that promote survival under combination therapy. Similar glycerol catabolic mutants are enriched in extensively drug-resistant clinical isolates, indicating that these mutations may promote survival and the development of resistance in humans. A majority of these mutations are frameshifts within a homopolymeric region of the glpK gene, leading to the hypothesis that M. tuberculosis may reversibly produce drug-tolerant phenotypes through genetic variation introduced at homopolymer sites as a strategy for survival during antibiotic treatment. Second, I identified bacterial mutants with altered susceptibility to individual first-line anti-mycobacterial drugs. Many of these mutations did not have obvious effects in vitro, demonstrating that a wide variety of natural genetic variants can influence drug efficacy in vivo without altering standard drug-susceptibility tests. A number of these genes are enriched in drug-resistant clinical isolates, indicating that these genetic variants influence treatment outcome. Together, these data suggest new targets for improving therapy, as well as mechanisms of genetic adaptations that can reduce antibiotic efficacy and contribute to the evolution of resistance.

Thesis (M.Sc.(Microbiology)) -- University of Limpopo, 2019 / Salmonella enterica serovar Gallinarum biovars Gallinarum, and Pullorum, Pasteurella multocida and Escherichia coli are among the most important pathogens in poultry and are the causal agents of fowl typhoid, pullorum disease, fowl cholera and collibacillosis in poultry. The present study was designed to identify and determine the distribution of these pathogens in household-raised chickens and their antibiotic and virulence profiles. For this purpose, 40 chickens were bought from household families at Ga-Dikgale, GaMolepo and Ga-Mphahlele in the Capricorn district of Limpopo Province and sacrificed for sampling. Tissues including breast meat, lungs, small and large intestines were harvested from each chicken. Bacteria associated with these samples were cultured in selective bacteriological media followed by biotyping using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) for identification. Out of a total of 160 tissue samples evaluated, E. coli and Salmonella were detected in these tissues. Furthermore, determination of the pathogenic E. coli and Salmonella strains at species level using primer sets that target selected genes of interest in the polymerase chain reaction (PCR) assay was employed. The invA gene, a confirmatory gene for Salmonella species was detected in all the Salmonella isolates using PCR. For the pathogenic E. coli, astA, eae, hlyA, fIiCH7, stxI and the fimbrial genes (F6 and F41) were detected in some of the E. coli isolates recovered from the samples. Disk diffusion test was also performed to determine the antibiotic susceptibility of the bacteria. The results from the current samples reveals that there is a high distribution of Salmonella and pathogenic E. coli in these areas and therefore further epidemiological and identification studies are needed to determine these organisms at species level and investigate their pathogenicity. The antimicrobial susceptibly data generated from this study can be a valuable reference to veterinarians for treating bacterial diseases in poultry.

Yeast probiotics and paraprobiotics, live and inactivated yeast cells, respectively, improve health and performance of livestock by stabilizing the intestinal microbial community. They have also been used for infection prevention and treatment. Despite much research already conducted, the mechanism of direct antagonism, or adhesion of bacteria to the probiotic/paraprobiotic, is under characterized. Additionally, it is unknown which probiotic/paraprobiotic is optimal to use for specific infections. The interactions between the yeast and certain pathogens were analyzed qualitatively with scanning electron microscopy (SEM) and quantitatively with membrane filtration assays. Gram-positive bacteria were found to exhibit specificity under SEM. Through membrane filtration, Listeria monocytogenes exhibited binding to all samples (P<0.05), while Salmonella Typhimurium exhibited binding (P<0.001) with all samples except with 2338. Escherichia coli O157:H7 only bound to the probiotics (P<0.001). With a better understanding of how specific yeast probiotics and paraprobiotics interact with bacteria, specific therapies can be administered to combat infections.

<p>Antimicrobial resistant bacteria pose a serious threat to global public health. With the development of antibioticresistance far outpacing the discovery of new antibiotics, there is a need to  develop  alternative  strategies  to  control  bacterial  infections without increasing  antibiotic resistance. This study focused on developing bacteriophage therapy as a non-antibiotic means to control colibacillosis in poultry. Avian colibacillosis causes significant mortality and economic loss to poultry industries around the world. The etiological agent is Avian Pathogenic<em> E.  coli</em> (APEC), with serotypes are O78, O1, O2 and O5 most often associated with infections. Here, seven bacteriophages (AHP, MP1, MP2, AKA, MKA, AHC and MIA) were isolated from human and poultry wastewater samples to target these four APEC serotypes.</p> <p>The host-spectrum analysis of these phages revealed that all seven phages lysed at least two different APEC strains, with four phages lysing four or more distinct APEC serotypes. Taken together, the isolated phages covered 90% (9/10) of O-serotyped APEC strains targeted in our study. When co-cultured with the targeted APEC strain, bacterial concentrations in phage treated APEC cultures (average OD₆₀₀= 0.09) were significantly (P < 0.05) lower than those of untreated cultures  (average  OD₆₀₀=  1.22)  after  4  hr  incubation.  However,  exposure  of  the  phages  to simulated gastric fluid (pH 2.2–2.5) reduced viability of three of the seven phages by 2.35 –4.01 log PFU/mL after 90 min and to undetectable levels after 5 min for other four phages. In contrast, phage viability was not impacted by simulated intestinal fluid (SIF) with no reductions in phage concentrations after exposure to SIF for 3 hr. All seven bacteriophages were encapsulated in sodium  alginate  microcapsules  with  encapsulation  efficiencies  between  94.4%  to  98.9%.  In contrast to unprotected phages, viability of encapsulated phages was reduced by only0.74 –1.21  log PFU/mL when exposed to SGF for 90 min. Incubating the encapsulated phages sequentially in SGF (1 hr) and SIF (3 hr) indicated that 85% -90% of phages were released from the microcapsules after1 hr incubation in SIF with the maximum release of phages from the microcapsules occurring after 3 hr of incubation in SIF. To assess the in vivoefficacy of the phage treatment, broiler chicks were challenged with APEC and treated with a mixture of unprotected and encapsulated phages. Concentrations of the APEC in the ceca of phage treated birds (2.79 log CFU/g) were significantly lower (P < 0.05) than those of untreated birds (6.18 log CFU/g) by 4 d post-challenge. Additionally, in  most  cases,  APEC  was  not  recovered  from  the  lungs  of  phage  treated  birds  whereas concentrations of APEC in lungs of untreated birds was 4.81 log CFU/g. Hence, these results indicate that phage treatmenteffectively controlled APEC colonization and replication in the ceca and lungs of APEC-challenged chickens and provide further evidence of the viability of phage-based treatments as a non-antibiotic means of controlling bacterial infections in chickens.</p>

The decrease in subtherapeutic antibiotic administration in poultry has increased the need to address production challenges caused by pathogens, such as E. coli. One potential way to improve bird health and reduce bacterial infection is through the addition of litter amendments that absorb moisture. Biochar (BC) has previously been shown to increase water holding capacity in poultry litter, but its effects on E. coli mitigation are unknown. The objectives of this research were to 1) evaluate water activity of poultry litter amended with pine and miscanthus BC, and 2) determine the effects of different BC inclusion rates on litter E. coli populations. The studies found that BC increased water activity when mixed with broiler litter, and pine BC resulted in lower E. coli counts over time than miscanthus BC. An inclusion rate of 30% by weight of pine BC was most effective at reducing E. coli populations in broiler litter.

When presented with certain unfavorable environmental conditions, C. trachomatis reticulate bodies (RBs) enter into a viable, yet noncultivable state called persistence. Two hallmarks of persistent chlamydiae are swollen, aberrantly shaped RBs, as viewed by transmission electron microscopy and a decrease in infectious progeny. Several models of chlamydial persistence have been described, including interferon-γ (IFN-γ), IFN-α, IFN-β, and tumor necrosis factor-α-exposure and nutrient deprivation. Previously, we established an in vitro co-infection model of two of the most common sexually transmitted pathogens in the United States, C. trachomatis and Herpes Simplex Virus-2 (HSV). Data from this tissue culture model indicate that: i) viral co-infection stimulates the formation of persistent chlamydiae and ii) productive HSV replication is not required for persistence induction. Further studies indicate that, co-infection-induced persistence is not mediated by: i) any known anti-chlamydial cytokine; ii) activation of inducible nitric oxide synthase or indoleamine 2, 3-dioxygenase; iii) inhibition of vesicular trafficking or sphingomyelin transport to the inclusion or; iv) amino acid, iron or glucose deprivation. These data demonstrate that co-infection-induced persistence is mediated by a previously undescribed, novel mechanism. During long-term co-infection with UV-inactivated HSV-2, chlamydiae recover following an initial suppression of chlamydial infectivity. These data indicate that HSV-induced persistence, like other persistence models, is reversible. Co-incubation of fixed, HSV-2-infected inducer cells with viable, C. trachomatis infected responder cells suppresses production of infectious chlamydial progeny and stimulates the formation of swollen, aberrantly shaped RBs. Antibody neutralization of HSV glycoprotein D (gD), which prevents viral attachment to one of four known HSV co-receptors on the host cell surface, also prevents co-infection-induced persistence, suggesting that HSV gD interaction with host cell surface receptors can provide the necessary stimulus to alter C. trachomatis development. Finally, exposure of C. trachomatis infected cells to soluble, recombinant HSV-2 gD:Fc fusion proteins decreases production of infectious EBs to a similar degree observed in co-infected cultures. Thus, we hypothesize that interaction of HSV gD with the host cell surface triggers a novel host anti-chlamydial pathway that restricts chlamydial development.

Several antibiotics that prevent translation by binding to ribosomal subunits have been shown to also inhibit ribosomal subunit assembly (Champney and Tober 2003). The aminoglycoside hygromycin B was examined in Escherichia coli cells for inhibitory effects on translation and ribosomal subunit assembly. The streptogramin antibiotics quinupristin-dalfopristin and XRP 2868 (NXL 103) were examined for similar effects on these 2 cellular functions in antibiotic-resistant strains of Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae. Pulse chase experiments were performed which verified slower rates of ribosomal subunit formation in drug treated cells. Hygromycin B exhibited a concentration dependent inhibitory effect on viable cell number, growth rate, protein synthesis and 30S and 50S subunit formation. 16S rRNA specific probes hybridized to rRNA fragments in cells treated with hygromycin B. RNase II and RNase III deficient strains of E. coli exhibited the most accumulation of 16S rRNA fragments upon treatment with hygromycin B. Examination of total RNA from treated cells showed an increase in RNA corresponding to precursor to the 16S rRNA while 16S rRNA decreased. There was also an increase in small fragment RNA. Hygromycin B was a more effective inhibitor of translation than ribosomal subunit formation in E. coli. Two streptogramin antibiotics were compared for inhibitory effects in antibiotic-resistant Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae. IC50 values for XRP 2868 were several fold lower than those of quinupristin-dalfopristin for inhibition of cell viability, protein synthesis, and ribosomal subunit formation. Both antibiotics revealed a concentration dependent inhibitory effect on cellular functions including 50S ribosomal subunit formation in the three organisms examined. XRP 2868 inhibited both 50S ribosomal subunit assembly and translation. XRP 2868 was effective against MRSA and was a better inhibitor in each of the antibiotic resistant strains examined compared with quinupristin-dalfopristin.

Excerpt: The title and conclusions of the study recently published by Jones et al. (1) concluded that monkeys were protected from dying from a form of simian-human immunodeficiency virus (SHIV) by an psoralen- and ultraviolet-inactivated vaccinia virus in a multi-envelope DNA-VV-protein (DVP). However, the findings in the study are more equivocal than indicated by the title because the effectiveness of the modified vaccinia virus was not decisively demonstrated.

The morphogenesis of C. albicans is a major aspect of its virulence and is regulated by quorum sensing (QS) molecules they produce, as well as the presence of neighboring microbes.Two mutant transporters, SSU1 and CDR4, were characterized for their ability to form biofilms in the presence of cyclic-di-GMP and 3-oxo-12-homoserine lactone. While homoserine lactone showed a decrease in biofilm density of both mutants compared to the wild-type strain, wild-type and ssu1 biofilm densities increased considerably in the presence of cyclic-di-GMP while testing lower inocula. Additionally, it has been shown that C. albicans mutants lacking the hybrid histidine kinase, Chk1, are refractory to the effects of farnesol, a QS molecule that inhibits morphogenesis.We determined both CDR4 and SSU1 expression is reduced or highly repressed in the chk1, ypd1, and skn7 null strains. Our results suggest these two genes are downstream targets in a pathway regulated by Chk1p.

No description available.