Crohn’s disease is an inflammatory bowel disease that may involve any part of the gastrointestinal tract with a variety of extraintestinal features. A flare of Crohn’s disease may present as partial small bowel obstruction or peritonitis. Dehydration, infectious agents, and cigarette smoking are some of the factors linked to a relapse of Crohn’s disease. Plesimonas Shigelloides, a bacterium that belongs to the enterobacteriaceae group may rarely lead to a flare of Crohn’s disease. We describe the case of a 31-year-old male with Crohn’s disease who developed a flare triggered by Plesimonas Shigelloides infection presenting as partial small bowel obstruction with ileal narrowing, and regional lymphadenopathy that responded to immunosuppressants.

The prevalence of antibiotic resistant bacteria has made the choices for topical treatments for patients who experience burns wounds extremely limited. The Staphylococcus genus is naturally occurring in and on the human body but can become harmful once it enters the bloodstream. A novel antimicrobial gel has been shown by our laboratory to inhibit both the planktonic growth and biofilm formation of Staphylococcus aureus in previous studies. The antimicrobial gel is made of seven natural compounds including antioxidants (vitamin C and E). We wanted to examine the effects of the antimicrobial gel on numerous other Staphylococcal species because it is prevalent on the body and becomes harmful when the immune system is compromised. The species tested were Staphylococcus capitis, Staphylococcus epidermidis, and Staphylococcus saprophyticus. A planktonic broth challenge test, biofilm attachment test, and biofilm maturation test were all performed in order to test this hypothesis. These tests showed a significant inhibition of the Staphylococcus species as a result of the effects of the antimicrobial gel. The antimicrobial gel inhibited the attachment, maturation, and growth of Staphylococcus colonies in a 10% antimicrobial gel solution. The antimicrobial gel shows promise as an option in treating burn patients and should be considered in further testing for its uses in other areas of medicine.

Antibiotic Feed Additives (AFA) have been used to prevent many bacterial infections during weaning of livestock. The use of these AFA’s resulted in the development of multiresistant bacterial strains and was therefore banned by the European Union. The United States also restricted the use of these feed additives considerably. Many scientists have started to search for alternatives in the prophylactic and therapeutic treatment of bacterial infections. Plants have been used traditionally by resource poor people all over the world to treat many infections. Diarrhoea not only causes many problems in the high intensity production of livestock but also leads to many human deaths. A large number of plants have been used to treat diarrhoea in humans and animals. Several authors have selected one or two species based on traditional use to evaluate in depth. In this project several different relevant parameters of 53 plant species used traditionally to treat diarrhoea were investigated in order to develop a model that would identify the species with the highest chance of delivering a useful antibacterial product. Antibacterial activities against two pathogens important in diarrhoea (Staphylococcus aureus and Escherichia coli) were positive parameters in selecting species. Because tannins frequently have antibacterial activity, but are not useful as prophylactic agents due to their effect on production, high tannin content was considered to be a negative indication. Cellular toxicity was also used as a negative parameter at a later stage. In addition to the in vitro assays there are also other parameters to be investigated to evaluate the potential use of plants. The influence of season of collection was determined on the antibacterial activity and tannin content of acetone leaf extracts of five plant species traditionally used to treat diarrhoea. They were Acacia karroo, Acacia sieberiana var. woodii, Peltophorum africanum, Trichilia emetica and Ziziphus mucronata. The antibacterial activity varied depending on the season of collection with the best activity generally in the months of late summer to autumn (January to April). The activity of Acacia karoo against E. coli was best in the month of April (MIC = 0.11 mg/ml (TA = 332 ml/g)) and against S. aureus in the month of March (MIC = 0.06 mg/ml (TA = 334 ml/g)). Acacia sieberiana subsp. woodii extracts had the best activity against E. coli (MIC = 0.10 mg/ml (TA = 303 ml/g)) in March against S. aureus in April (MIC = 0.08 mg/ml (TA = 303 ml/g)). <i.Peltophorum africanum extracts were most active against E. coli in February (MIC = 0.05 mg/ml (TA = 1188 ml/g)) and against S. aureus in February and March (MIC = 0.04 mg/ml (TA = 1188 ml/g and 1075 ml/g)). Trichilia emetica extracts were generally not very active against the bacterial strains (best activity: MIC = 0.22 mg/ml (TA = 74 ml/g) against E. coli in May and MIC = 0.28 mg/ml (TA = 26 ml/g) against S. aureus in December). Ziziphus mucronata was most active against both bacterial strains and in May (E. coli: MIC = 0.10 mg/ml (TA = 589 ml/g); S. aureus: MIC = 0.04 mg/ml (TA = 1099 ml/g)). The tannin content varied in the extracts as well. The antibacterial activity however did not seem to be directly correlated to the tannin content. Another important parameter in the use of plant species is to determine the interspecies variation of plants based on genetic or environmental influences. Leaves from 42 plants of Combretum molle were collected at different locations during the same season. The average MIC against E. coli was 0.227 mg/ml. The low standard deviation of 0.07 indicates that there was very little variation in activity. The average value against S. aureus was 0.399 mg/ml with a slightly higher standard deviation of 0.16. However due to the fact that the samples from different areas extracted different amounts, the total activity varied. The tannin assays revealed that there was with one exception no correlation between the antibacterial activity and the tannin content. So it can be safe to assume that genetic variation does not influence the activity too much at least in C. molle leaves Fifty three plant species traditionally used to treat diarrhoea in published literature were then ranked using a novel system in order to determine which species had the most potential value. Ranking was based on the lowest MIC value against E. coli, lower activity against S. aureus (to limit selecting for general metabolic toxins), low tannin concentration and high extract yield. From this ranking, five plants were chosen to investigate their potential value further: Acacia sieberiana var. woodii (E. coli: MIC = 0.13 mg/ml, TA = 108 ml/g; S. aureus: MIC = 0.13 mg/ml, TA = 108 ml/g; Yield = 14 mg), Albizia adianthifolia (E. coli: MIC = 0.14 mg/ml, TA = 239 ml/g; S. aureus: MIC = 0.04 mg/ml, TA = 765 ml/g; Yield = 34 mg), Deinbollia oblongifolia (E. coli: MIC = 0.17 mg/ml, TA = 158 ml/g; S. aureus: MIC = 0.08 mg/ml, TA = 338 ml/g; Yield = 27 mg), Spirostachys africana (E. coli : MIC = 0.13 mg/ml, TA = 300 ml/g; S. aureus: MIC = 0.09 mg/ml, TA = 438 ml/g; Yield = 38 mg) and Tetradenia riparia (E. coli : MIC = 0.09 mg/ml, TA = 214 ml/g; S. aureus: MIC = 0.13 mg/ml, TA = 149 ml/g; Yield = 20 mg). None of the plants contained any tannin. The next step towards the recommendation of a plant for the development of a commercial product was to evaluate the cytotoxicity of the selected five species. The following values were obtained: Acacia sieberiana var. woodii LC50 = 0.026 mg/ml, Albizia adianthifolia LC50 = 0.068 mg/ml, Deinbollia oblongifolia LC50 = 0.078 mg/ml, Spirostachys africana LC50 = 0.025 mg/ml and Tetradenia riparia LC50 = 0.028 mg/ml. Deinbollia oblongifolia (for its low LC50 value) and Spirostachys africana (for its good antibacterial activity and total activity) were potentized by removing inactive compounds through solvent-solvent fractionation. The antibacterial activity against E. coli was increased this way (MIC = 0.08 mg/ml for Deinbollia oblongifolia (chloroform fraction) and MIC = 0.08 mg/ml for Spirostachys africana (chloroform fraction)) The LC50 values for both chloroform fractions were determined (LC50 = 0.188 mg/ml for Deinbollia oblongifolia and LC50 = 0.062 mg/ml for Spirostachys africana ). The selectivity index (SI) was also determined and proved that the potentization was indeed successful (Deinbollia oblongifolia SI = 2.35 compared to a value of 0.45 for the crude extract; Spirostachys africana SI = 0.78 compared to a value of 0.19 for the crude extract). Based on these values, the chloroform fraction of Deinbollia oblongifolia was chosen as the less toxic one with similar activity and a higher selectivity index to be worked on further. One of the active compounds was isolated and evaluated for its activity against E. coli (MIC = 0.74 mg/ml) The LC50 value of 0.042 mg/ml indicated that the activity of the extract was a result of synergism rather than being due to a single active compound (the selectivity index (SI) was 0.06 compared to the values of 0.45 for the crude and 2.35 for the potentized extract). The plant extracts should of course be as effective against pathogenic strains as they were against the ATCC strains and so the extracts and pure compound of Deinbollia oblongifolia were tested for their activity against four different pathological E. coli strains. The results showed that the crude extract and the fraction were as active as in the preliminary screening results against only one of the four pathological strains. The pure compound on the other hand was more active against all four pathological strains than against the ATCC strain. The next step was to test the safety of the extracts of Deinbollia oblongifolia in mammals. Unfortunately neither the crude extract nor the chloroform fraction of Deinbollia oblongifolia could be used safely in a living organism or in an isolated organ study. A part of the problem may have been caused by the vehicle used in the study despite reports in the literature that an acetone water mixture is safe to use. In general all the species investigated had good antibacterial activity against E. coli this supports the traditional use of these species although we used acetone as extractant rather than the water used traditionally. There were major differences in antibacterial activity over a season indicating that mature leaves were more active than young leaves before senescence started. At least in the case of Combretum molle there was little difference in the antibacterial activity of many plants collected at different locations during the same season. The results obtained in this study could be useful in further studies to develop extracts that can be used to control diarrhoea in animals. Possibly more emphasis should be put on the difference in activity towards E. coli and S. aureus to eliminate the presence of general metabolic toxins. Such an approach would lead to a different priority order for species to examine. A major first step would probably be to test the in vitro and in vivo toxicity of selected species. / Thesis (PhD)--University of Pretoria, 2010. / Paraclinical Sciences / unrestricted

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative facultative anaerobe that induces severe inflammation resulting in gastroenteritis. In the case of S. Typhimurium infection, induction of an inflammatory response has been linked to its primary virulence mechanism, the type III secretion system (T3SS). The T3SS secretes protein effectors that exploit the host’s cell biology to facilitate bacterial entry and intracellular survival, and to modulate the host immune response. One such effector, SifA, is a bi-functional T3SS effector protein that plays an important role in Salmonella virulence. The N-terminal domain of SifA binds SifA-Kinesin-Interacting-Protein (SKIP), and via an interaction with kinesin, forms tubular membrane extensions called Sif filaments (Sifs) that emanate from the Salmonella Containing Vacuole (SCV). The C-terminal domain of SifA harbors a WxxxE motif that functions to mimic active host cell GTPases. Taken together, SifA functions in inducing endosomal tubulation in order to maintain the integrity of the SCV and promote bacterial dissemination. Since SifA performs multiple, unrelated functions, the objective of this study was to determine how each functional domain of SifA becomes processed. In the present study, we demonstrate that a linker region containing a caspase-3 cleavage motif separates the two functional domains of SifA. To test the hypothesis that processing of SifA by caspase-3 at this particular site is required for function and proper localization of the effector protein domains, we developed two tracking methods to analyze the intracellular localization of SifA. We first adapted a fluorescent tag called phiLOV that allowed for T3SS mediated delivery of SifA and observation of its intracellular colocalization with caspase-3. Additionally, we created a dual-tagging strategy that permitted tracking of each of the SifA functional domains following caspase-3 cleavage to different subcellular locations. The results of this study reveal that caspase-3 cleavage of SifA is required for the proper localization of functional domains and bacterial dissemination. Considering the importance of these events in Salmonella pathogenesis, we conclude that caspase-3 cleavage of effector proteins is a more broadly applicable effector processing mechanism utilized by Salmonella to invade and persist during infection.

Synthetic biology, a field that sits between Biology and Engineering disciplines, has come into its own in the last decade. The decreasing cost of DNA synthesis has lead to the creation of larger and more complex synthetic gene networks, engineered with functional goals rather than simple demonstration. While many methods have been developed to reduce the time required to produce complex networks, none focus upon the considerable tuning needed to turn structurally correct networks into functional gene networks. To this end, we created a Plug-and-Play synthetic gene network assembly that emphasizes character-driven iteration for producing functional synthetic gene networks. This platform enables post-construction modification and easy tuning of networks through its ability to swap individual parts. To demonstrate this system, we constructed a functional bistable genetic toggle and transformed it into two functionally distinct synthetic networks. Once these networks have been created and tuned at the bench, they next must be delivered to bacteria in their target environment. While this is easy for industrial applications, delivering synthetic networks as medical therapeutics has a host of problems, such as competing microbes, the host immune system, and harsh microenvironments. Therefore, we employed bacteriophage technologies to deliver functional synthetic gene networks to specific bacterial strains in various microenvironments. We first sought to deliver functional genetic networks to bacteria present in the gut microbiome. This allows for functionalization of these bacteria to eventually sense disease states and secrete therapeutics. As a proof of concept a simple circuit was created using the Plug-and-Play platform and tested before being moved into the replicative form plasmid of the M13 bacteriophage. Bacteriophage particles carrying this network were used to infect gut bacteria of mice. Infection and functionality of the synthetic network was monitored from screening fecal samples. Next, we employed phagemid technologies to deliver high copy plasmids expressing antibacterial networks to target bacteria. This allows for sustained expression of antibacterial genes that cause non-lytic bacterial death without reliance upon traditional small molecule antibiotics. Phagemid particles carrying our antibacterial networks were then tested against wild type and antibiotic-resistant bacteria in an in vitro and in vivo environment.

Pseudomonas aeruginosa, a Gram-negative bacillus, commonly infects immunocompromised individuals and uses a variety of virulence factors to persist in these hosts. The posttranscriptional regulator, RsmA, plays a role in the expression of many virulence factors in P. aeruginosa. RsmA up regulates virulence factors used in colonizing hosts. However, regulation of rsmA is not well elucidated. Transposon mutagenesis was performed on P. aeruginosa containing a transcriptional rsmA-lacZ fusion to answer this question. Mutants were screened via β-galactosidase assay and transposon insertions identified via arbitrary PCR. A probable MFS transporter, we named mtpX, was one significant transposon mutant identified. A ΔmtpX mutant containing the rsmA-lacZ transcriptional fusion was constructed to confirm our results. Further analysis of rsmA, looking at RNA and protein levels, revealed varying results in nonmucoid versus mucoid backgrounds. Phenotypic assays were performed to characterize this unknown transporter and develop a putative mechanism as to how MtpX affects rsmA expression.

Drug resistance is unfortunately becoming a prevalent issue in the course of patient treatment, ranging from chemotherapy resistance to antimicrobial resistance. The Centers for Disease Control and Prevention (CDC) estimated in 2016 that at least 23,000 people die every year in the United States from an infection with an antibiotic-resistant organism (Munita, et al, 2016). Carl Friedlander was the first scientist to describe Klebsiella pneumoniae in 1882 as an encapsulated bacillus after isolating the bacterium from the lungs of patients who had died from pneumonia (Ashurst and Dawson, 2022). Klebsiella pneumoniae is the type species for the Klebsiella genus and is the bacterium of interest for this project. It is one of the very few Gram-negative bacilli that can cause primary pneumonia, commonly affecting patients with compromised immune systems, alcohol use disorder, or diabetes mellitus (Ristuccia and Burke, 1984). However, microbes are able to produce a wide range of microbial defense systems including classic antibiotics, metabolic byproducts, and lytic agents. Bacteriocins are some of the most common defense mechanisms produced, which are different from antibiotics in that they have a narrow killing spectrum and are toxic only to bacteria that is closely related to the strain that is producing it. It has been estimated that 99% of all bacteria possibly make a minimum of one bacteriocin (Riley and Wertz, 2002). Because of the rapidly growing number of infections that are caused by antibiotic-resistant bacteria along with the harm that broad-spectrum antibiotics can cause to the human microbiome, these bacteriocins are being studied as potential alternatives to tradition antibiotics. In this study, we will assess and characterize a Klebsiella bacteriocin that may work synergistically with antibiotics so that antibiotic dosage might be reduced. In this study, we have isolated the plasmids from a possible Klebsiella bacteriocin and transformed them into E. coli to characterize the plasmid. This potential bacteriocin demonstrates efficacy towards Citrobacter, Enterobacter, and Klebsiella species and could offer an alternative treatment option for the highly drug resistant Enterobacteriaceae family.

Pseudomonas aeruginosa is an important nosocomial infection that has the potential to infect almost every tissue of the human body though it is mainly opportunistic, due to the organism’s intrinsic antibiotic resistance is becoming increasingly difficult to treat. Two-component systems (TCS) rely on a signal sensed from the outside environment by the sensor histidine kinase to initiate phosphotransfer to the response regulator, which may then regulate virulence factors in the organism in response to a changing environment. One important two-component system in P. aeruginosa is the AlgZ/R system. AlgZ, the sensor histidine kinase, has been shown to be co-transcribed with its’ response regulator, AlgR, to affect a myriad of virulence factors, including those related to motility. Pseudomonas species is capable of four types of motility: twitching, swimming, swarming, and gliding. Twitching motility is achieved through the expression of the FimU operon and Type VI pilli, and is most useful when attaching to a solid surface in the initial step of pathogenesis: colonization. Conversely, the swimming phenotype relies on the production of a single polar flagellum upon the activation of the FleQ operon, and allows the organism to move through a fluid environment. A previously unidentified regulator of AlgZ, but not AlgR, has been identified via transposon mutagenesis screening, PA2771, which has a GGDEF domain and predicted diguanylate cyclase activity. The mechanism of PA2771’s action within P. aeruginosa has not been previously studied. The nonpolar deletion mutant was first characterized via various phenotypic assays (including biofilm, rhamnolipid, swimming, and swarming assays) and transcriptional fusions to propose a mechanism in which this predicted diguanylate cyclase (DGC) works with AlgZ to determine the switch in motility from twitching to swimming. When PA2771 is present and active in the cell, cyclic di-GMP levels should be high, leading to the production of Type VI pilli and the upregulation of the FimU operon. In the PA2771 mutant a significant decrease in the expression of the FimU operon, and an increase in the expression of the flagellar genes. Subsequent alterations in swimming and swarming phenotypes were observed, as well as the restoration of these effects via complementation studies. Overexpression of AlgZ in the 2771 mutant showed a restoration of AlgZ expression in the nonmucoid background, and the predicted DGC activity was indirectly verified via a cdrA-lacZ transcriptional fusion.

Myeloid-derived suppressor cells (MDSC) expand during sepsis, suppress both innate and adaptive immunity, and promote chronic immunosuppression, which characterizes the late/chronic phase of sepsis. We previously reported that the transcription factors Stat3 and C/EBPb synergize to induces the expression of microRNA (miR)-21 and miR-181b to promote MDSC expansion in a mouse model of polymicrobial sepsis that progresses from an early/acute proinflammatory phase to a late/chronic immunosuppressive stage. We also showed that Gr1+CD11b+ cells, the precursors of MDSCs, from mice genetically deficient in the inflammatory protein S100A9 lack miR-21 or miR-181b in late sepsis, and are not immunosuppressive. In the present study, we show that S100A9 induces miR-21 and miR-181b during the late sepsis phase. We find that S100A9 associates with and stabilizes the Stat3-C/EBPb protein complex that activates the miRNA promoters. Reconstituting Gr1+CD11b+ cells from the S100A9 knockout mice with late sepsis with S100A9 protein restores the Stat3-C/EBPb protein complex and miRNA expressions, and switches the Gr1+CD11b+ cells into the immunosuppressive, MDSC phenotype. Importantly, we find that this process requires IL-10 mediated signaling, which induces S100A9 translocation from the cytosol to the nucleus. These results demonstrate that S100A9 promotes MDSC expansion and immunosuppression in late/chronic sepsis by inducing the expression of miR-21 and miR-181b.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen responsible for human genital and ocular infections. Species of Chlamydia utilize a type-III secretion system to deliver bacterial effector proteins into the host cell in order to promote invasion and establish residence within a parasitophorous vacuole called an inclusion. The effector protein Tarp has been previously implicated as an important effector for promoting invasion during Chlamydia trachomatis infection by directing the formation of new actin filaments and bundles. Intriguingly, the significance of Tarp mediated cytoskeletal changes has not been fully explored in vivo. Host-pathogen interaction studies that replicate the human infection can be performed with mouse adapted Chlamydia, Chlamydia muridarum. However, the genetic tools to create gene deletions in C. muridarum have been lacking. Recently, our collaborators in the Fields and Wolf Laboratories developed a novel genetic tool for creating Tarp deletion mutants and complement clones in Chlamydia muridarum. Through the use of this tool, we were able to study the significance of Tarp in a murine infection model. In addition to Tarp, two other early effectors TmeA and TmeB are hypothesized to play a role in invasion, but a full account of their involvement remained unknown. In our studies, we were able to determine the roles of TmeA and TmeB in remodeling the host cytoskeleton. Using biochemical crosslinking assays, and actin polymerization studies, we discovered that TmeA has the ability to activate host protein N-Wasp in order to increase Arp2/3-dependent actin polymerization, while TmeB can in turn inhibit Arp2/3-directed actin polymerization via direct interactions with Arp2/3. Collectively, these are important findings as our studies have revealed how a collection of early chlamydial effectors work to modulate the host cytoskeleton to facilitate Chlamydia infections.