Adult immunizations effectively reduce morbidity, mortality, and transmission rates of multiple diseases; however, outpatient providers often a struggle to convince patients to accept vaccinations. This project’s aim is to address vaccination rates in our adult population, focusing first on the influenza vaccine in year one (2016), and then on pneumococcal vaccine in year two (2017), by 1) using a strong quality improvement strategy (known as a Champion Team) and 2) implementing a clinic program consisting of provider training, improved documentation, and informative posters targeted at patients. A quality improvement strategy known as a “Champion Team” provided a strong mechanism through which we developed and implemented the interventions across both years. Specifically, the Champion Team consisted of key stakeholders (nurses, residents, physician faculty, and informatics expert) who identified, developed, and evaluated the program. Programming included an annual health care professional training session for each vaccine (early fall of 2016 and 2017 for flu, spring 2017 for pneumococcal), improved documentation strategies and nursing uptake, and informative posters in the clinic. We assayed data from our patient electronic health record to evaluate: the percentage of our patient population for whom an immunization was documented relative to the number of unique patients seen in our clinic during that time frame. This approach in year one showed a marked increase in influenza vaccination rates in our clinic. During the 2014/2015 and 2015/2016 flu seasons our clinic vaccination rates were 39.98% and 42.05% respectively. After implementation of our champion team and clinic wide program to increase rates in 2016 our vaccination rates for the 2016/2017 flu seasons was 50.88%. Pneumonia data for a full year are under analyses and will be included in this presentation. We anticipate a similar increase in rates for our pneumococcal vaccinations. Our Champion Team and clinic wide program were perceived as relatively low-effort interventions yet appeared to increase vaccinations over the course of this study. The replication of these findings across pneumonia data (pending) and, in future work, with the herpes zoster vaccine (planned for Year 3), will increase our confidence that increases in rates were attributable to these very accessible interventions.

Elizabethkingia Meningoseptica (E. Meningoseptica) an oxidase-positive gram-negative aerobic rod.1-2 Although ubiquitous in nature and widely distributed in soil and water, it is not a part of normal human flora. Cases of outbreaks of meningitis in premature neonates or infants have been reported, however, very few cases have been reported in adults.3 Infection is primarily nosocomial, or hospital acquired and has been implicated in bacteremia, meningitis, pneumonia, endocarditis especially in immunocompromised individuals.2-4 We report a 29-year-old male with past medical history significant for intravenous drug abuse, hepatitis C, oxymorphone induced hemolytic uremic syndrome, who presented to hospital with altered mental status. On admission, patient was unresponsive to vocal commands, febrile (102.3 F), tachycardic and tachypneic. He had pinpoint pupils and diffuse petechiae. In addition, he had erythematous flat macular lesions on his palms and dorsum of hands as well as injection marks in left cubital fossa. Cardiac examination was significant for a grade III systolic murmur at apical region and diastolic murmur at left second intercostal space. Laboratory studies revealed thrombocytopenia (43,000m/microL), lactic acidosis (4.9mmol/L), serum creatinine (Cr) of 6.6 mg/dL, glomerular filtration rate (GFR) of 10 ml/min. Transthoracic echocardiogram (TTE) revealed large mobile vegetation on aortic valve measuring 3.6 x 0.72 cm. Patient’s presentation was consistent with infective endocarditis with the vegetation seen on TTE and patient’s physical findings. Magnetic Resonance Imaging of the brain showed numerous small hemorrhagic infarcts, likely secondary to emboli from aortic valve vegetation. Patient required intubation for airway protection and started on hemodialysis. He was initially started on Meropenem and Vancomycin for infective endocarditis and later switched to Ciprofloxacin based on blood cultures and sensitivities which revealed methicillin sensitive staphylococcus aureus and multi-drug resistant E. Meningoseptica. Patient was transferred to long term care facility after acute care at the hospital. The increasing incidence of polymicrobial infective endocarditis and increasing resistance to antibiotic therapy pose challenges to the rapid assessment and treatment to mitigate the multi-organ involvement with septic emboli. Reports of pathogenicity associated with native valve endocarditis with this organism is scarce and exist primarily in a very few case reports and is resistant to many traditional antibiotics.5,6 E. Meningoseptica has shown antimicrobial susceptibility to the newer quinolones, rifampin, trimethoprim/sulfamethoxazole and ciprofloxacin with reasonable activity.7 Due to the unusual pattern of antibiotic resistance, early switching to appropriate antibiotics based on sensitivities is crucial for survival in patients with E. Meningoseptica. References 1..Kim KK, Kim MK, Lim JH, Park HY, Lee ST. Transfer of Chryseobacterium meningosepticum and Chryseobacterium miricola to Elizabethkingia gen. nov. as Elizabethkingia meningoseptica comb. nov. and Elizabethkingia miricola comb. nov. Int J Syst Evol Microbiol.2005 May;55(Pt 3):1287-93. 2:Shinha T, Ahuja R. Bacteremia due to Elizabethkingia meningoseptica. IDCases. 2015 Jan 17;2(1):13-5. doi: 10.1016/j.idcr.2015.01.002. eCollection 2015. 3..Jung SH, Lee B, Mirrakhimov AE, Hussain N. Septic shock caused by Elizabethkingia meningoseptica: a case report and review of literature. BMJ Case Rep. 2013 Apr 3;2013. pii: bcr2013009066. doi: 10.1136/bcr-2013-009066. 4.Ratnamani MS, Rao R. Elizabethkingia meningoseptica: Emerging nosocomial pathogen in bedside hemodialysis patients. Indian J Crit Care Med. 2013 Sep;17(5):304-7. 5.Bomb K, Arora A, Trehan N. Endocarditis due to Chryseobacterium meningosepticum. Indian J Med Microbiol. 2007 Apr;25(2):161-2. 6.Yang J, Xue W, Yu X. Elizabethkingia meningosepticum endocarditis: A rare case and special therapy. Anatol J Cardiol. 2015 May;15(5):427-8. 7. Hsu MS, Liao CH, Huang YT, Liu CY, Yang CJ, Kao KL, Hsueh PR. Clinical features, antimicrobial susceptibilities, and outcomes of Elizabethkingia meningoseptica (Chryseobacterium meningosepticum) bacteremia at a medical center in Taiwan,1999-2006. Eur J Clin Microbiol Infect Dis. 2011 Oct;30(10):1271-8.

Tuberculosis (TB) is primarily a pulmonary disease caused by Mycobacterium tuberculosis (Mtb). Mtb is highly infectious, but studies have shown that only 5–15% of Mtb-infected individuals develop TB disease. The Bacille Calmette-Gu.rin (BCG) vaccine is the only commercially available Mtb vaccine, but its efficacy varies based on the strain used. The Mtb PhoPR-mutant variant, MTBVAC, has been tested as a possible attenuated live vaccine against Mtb. Although it has successfully conferred durable CD4+ T-cell responses in infants, it has also resulted in adverse effects. Our goal is to identify PhoPR-regulated gene(s) that mediate Mtb-induced burst size necrosis in infected cells. PhoPR is a two-component system in mycobacteria. PhoR responds to environmental cues, such as changes in pH, and phosphorylates the PhoP transcription factor, which then activates or suppresses the expression of approximately 40 Mtb genes. The Mtb PhoPR-mutant strain is able to replicate in infected macrophages, but it does not induce the horizontal spread of Mtb to other immune cells. Our lab has previously shown that virulent, cytopathic strains of Mtb, such as H37Rv, suppress early apoptosis, have faster replication rates in macrophages, and trigger cell death at a lethal load threshold of approximately 25 bacteria. Cell death of infected macrophages primarily occurs via necrosis, which involves nuclear pyknosis without DNA fragmentation and general disruption of lipid bilayer membranes. Viable bacilli are released to infect other macrophages and neutrophils recruited to the developing TB lesion. Here, we show that PhoP contributes to burst size necrosis in macrophages and that the PhoP-regulated genes, fadD21 and pks3, are potential drivers of this necrosis. FadD21 and pks3 are involved in the generation of diacyl trehalose/penta-acyl trehalose (DAT/PAT) for cell wall synthesis, suggesting that Mtb cell wall composition may determine virulence. Therefore, we have uncovered potential targets for early intervention or vaccinations to avoid granuloma formation or tissue damage in response to Mtb-induced macrophage necrosis.

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.

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.

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.

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.

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.

Previous research discovered the existence of sedimentation-resistant mutants of E. coli. Genomic studies revealed that these mutants resisted sedimentation due to independent modifications to genes that influenced the Rcs signal transduction pathway, causing increased secretion of an exopolysaccharide capsule comprised primarily of colanic acid. The Rcs system is responsible for detecting envelope stressors; consequently, ampicillin and osmotic stress were used to perturb the cellular envelope and study the response of the mutants compared to wild-type cells. It was found that the overproduction of colanic acid in the mutants confers some resistance to envelope stress; however, the mutants still behaved similarly to wild-type cells. The doubling times of the strains grown in sodium chloride solutions were calculated. A wavelength scan from 400 nm to 800 nm was performed on strains grown in different salt concentrations to determine if there were significant differences in light scattering between the wild-type and mutant cells. Further analysis was performed that, along with the doubling time data, suggested that wild-type cells may have turned on genes for capsule production in response to being grown in high salt concentrations. Additional research could be conducted to test this hypothesis, perhaps through the quantification of colanic acid through a methyl pentose assay for wild-type cultures grown with high salt concentrations. The idea that wild-type cells could digest colanic acid as a carbon source when lacking resources was also investigated with different preparations of colanic acid. One preparation of colanic acid showed promising results, which could indicate that bacteria are able to digest their capsule in a novel method to produce energy when starved. Again, additional investigation should be conducted to confirm these results. Other future experiments could study the metabolome of these mutants to determine if they have increased quantities of alarmones related to biofilm formation.

Anaplasma phagocytophilum and A. marginale are the etiologic agents of bovine anaplasmosis and human granulocytic anaplasmosis, respectively. As obligate intracellular pathogens, binding and entry of host cells is a prerequisite for survival. The molecular events associated with these processes are poorly understood. Identifying the adhesins mediating binding, delineating their key functional domains, and determining the molecular determinants to which they bind not only benefits better understanding of Anaplasma spp. pathobiology, but could also benefit the development of novel approaches for protecting against infection. We previously demonstrated that A. phagocytophilum outer membrane protein A (ApOmpA) is critical for bacterial binding and entry host through recognition of α2,3-sialic acid and α1,3-fucose of its receptors, including 6-sulfo-sLex. In this study, we determined that two amino acids, G61 and K64, within its binding domain (ApOmpA59-74), are essential for ApOmpA function. We also confirmed the ability of ApOmpA to act as an adhesin and invasin as it conferred adhesiveness and invasiveness to inert beads. We next extended our studies to A. marginale as it also expresses OmpA (AmOmpA) and its role in infection has not been studied. Molecular models of ApOmpA and AmOmpA were nearly identical, especially in the ApOmpA binding domain and its counterpart in AmOmpA. Antisera raised against AmOmpA or its putative binding domain inhibit A. marginale infection. AmOmpA G55 and K58 are contributory and K59 is essential for AmOmpA to bind to host cells. AmOmpA binding is dependent on α2,3-sialic acid and α1,3-fucose. Coating inert beads with AmOmpA conferred the ability to bind to and be taken up by host cells, confirming that it acts as an adhesin and invasin. 6-sulfo-sLex is dispensable for AmOmpA binding and A. marginale infection. ApOmpA works cooperatively with Asp14 (14-kDa A. phagocytophilum surface protein) to promote optimal infection of host cells. We found that Asp14 is conserved across A. phagocytophilum strains and in A. marginale and confirmed the ability of Asp14 to act as an adhesin and invasin as it conferred adhesiveness and invasiveness to inert beads. Collectively, this work advances our understanding of A. phagocytophilum and A. marginale adhesion and invasion of host cells.