Breaking membrane barriers to neutralize E. coli and K. pneumoniae virulence with PEGylated branched polyethylenimine.

Bacterial infections caused by Gram-negative pathogens, such as those in the family Enterobacteriaceae, are among the most difficult to treat because effective therapeutic options are either very limited or non-existent. This raises serious concern regarding the emergence and spread of multi-drug resistant (MDR) pathogens in the community setting; and thus, creates the need for discovery efforts and/or early-stage development of novel therapies for infections. Our work is directed towards branched polyethylenimine (BPEI) modified with polyethylene glycol (PEG) as a strategy for targeting virulence from Gram-negative bacterial pathogens. Here, we neutralize lipopolysaccharide (LPS) as a barrier to the influx of antibiotics. Data demonstrate that the ß-lactam antibiotic oxacillin, generally regarded as ineffective against Gram-negative bacteria, can be potentiated by 600 Da BPEI to kill some Escherichia coli and some Klebsiella pneumoniae. Modification of 600 Da BPEI with polyethylene glycol (PEG) could increase drug safety and improves potentiation activity. The ability to use the Gram-positive agent, oxacillin, against Gram-negative pathogens could expand the capability to deliver effective treatments that simplify, reduce, or eliminate some complicated treatment regimens.

Detection of Borrelia miyamotoi and Powassan Virus Lineage II (Deer Tick Virus) from Odocoileus virginianus Harvested Ixodes scapularis in Oklahoma.

Odocoileus virginianus (white-tailed deer) is the primary host of adult Ixodes scapularis (deer tick). Most of the research into I. scapularis has been geographically restricted to the northeastern United States, with limited interest in Oklahoma until recently as the I. scapularis populations spread due to climate change. Ticks serve as a vector for pathogenic bacteria, protozoans, and viruses that pose a significant human health risk. To date, there has been limited research to determine what potential tick-borne pathogens are present in I. scapularis in central Oklahoma. Using a one-step multiplex real-time reverse transcription-PCR, I. scapularis collected from white-tailed deer was screened for Anaplasma phagocytophilum, Borrelia burgdorferi, Borrelia miyamotoi, Babesia microti, and deer tick virus (DTV). Ticks (n = 394) were pooled by gender and life stage into 117 samples. Three pooled samples were positive for B. miyamotoi and five pooled samples were positive for DTV. This represents a minimum infection rate of 0.8% and 1.2%, respectively. A. phagocytophilum, B. burgdorferi, and B. microti were not detected in any samples. This is the first report of B. miyamotoi and DTV detection in Oklahoma I. scapularis ticks. This demonstrates that I. scapularis pathogens are present in Oklahoma and that further surveillance of I. scapularis is warranted.

Detection of Rickettsia amblyommatis and Ehrlichia chaffeensis in Amblyomma americanum Inhabiting Two Urban Parks in Oklahoma.

For the past 30 years, the number of people infected with causative agents of ehrlichiosis, Rocky Mountain spotted fever, and spotted fever group rickettiosis (SFGR) has increased in Oklahoma. However, there is a lack of data on pathogen prevalence within urban environments. To assess the prevalence of tick-borne pathogens in different environments, 434 Amblyomma americanum (lone star) ticks were collected from the environment in two parks in Edmond, Oklahoma. The presence of Ehrlichia spp. and spotted fever group (SFG) Rickettsia spp. was determined using quantitative real-time polymerase chain reaction (qPCR). 33.6% (146/434) of the A. americanum ticks were positive for Rickettsia amblyommatis and 15.2% (66/434) were positive for Ehrlichia chaffeensis. No ticks were positive for other SFG Rickettsiae (R. rickettsii, R. parkeri) or other Ehrlichiae (E. ewingii, and Panola Mountain Ehrlichia). These studies provide increased understanding of the potential risk for encountering tick-borne pathogens in urban environments.

Expanding the Spectrum of Antibiotics Capable of Killing Multidrug-Resistant Staphylococcus aureus and Pseudomonas aeruginosa.

Infections from antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa are a serious threat because reduced antibiotic efficacy complicates treatment decisions and prolongs the disease state in many patients. To expand the arsenal of treatments against antimicrobial-resistant (AMR) pathogens, 600-Da branched polyethylenimine (BPEI) can overcome antibiotic resistance mechanisms and potentiate ß-lactam antibiotics against Gram-positive bacteria. BPEI binds cell-wall teichoic acids and disables resistance factors from penicillin binding proteins PBP2a and PBP4. This study describes a new mechanism of action for BPEI potentiation of antibiotics generally regarded as agents effective against Gram-positive pathogens but not Gram-negative bacteria. 600-Da BPEI is able to reduce the barriers to drug influx and facilitate the uptake of a non-ß-lactam co-drug, erythromycin, which targets the intracellular machinery. Also, BPEI can suppress production of the cytokine interleukin IL-8 by human epithelial keratinocytes. This enables BPEI to function as a broad-spectrum antibiotic potentiator, and expands the opportunities to improve drug design, antibiotic development, and therapeutic approaches against pathogenic bacteria, especially for wound care.

Tick species establishment in Oklahoma County, Oklahoma, U.S.A., identified by seasonal sampling in residential and non-residential sites.

In recent years, human tick-borne disease occurrence has risen in Oklahoma, U.S.A., but year-round data on tick presence in frequently used recreational areas is not widely available. In this study, ticks were collected monthly for one year at residential and non-residential sites in a suburban area of Oklahoma County, OK, U.S.A. At each trapping site, dry ice traps were used in both woodland and grassland areas and fabric tick drags were used in grassland areas. Four species were collected from each park: Amblyomma americanum, Amblyomma maculatum, Dermacentor variabilis, and Ixodes scapularis. Prior to this study, A. americanum was the only species with an established population in Oklahoma County. Consistent with this, A. americanum was collected in all months of the year and accounted for over 90% of ticks collected at each site. Based on our tick survey, we report that A. maculatum, D. variabilis, and I. scapularis, which were each collected in numbers greater than six within a single sampling occasion, are now each confirmed as established populations in Oklahoma County.

Trypanosoma cruzi in a Mexican Free-Tailed Bat ( Tadarida brasiliensis) in Oklahoma, USA.

Trypanosoma cruzi is a vector-borne protozoan parasite that infects seven million individuals in Central and South America and is the etiologic agent of Chagas disease. There are increasing reports of endemic transmission within the southern US. Trypanosoma cruzi occurs in wild raccoons and dogs in Oklahoma, but its endemicity in the state is poorly studied. We suspected Mexican free-tailed bats ( Tadarida brasiliensis) contributed to the endemicity of T. cruzi in Oklahoma due to their annual migration from Central America to their North American maternity roosts. During the summer of 2017, we sampled 361 Mexican free-tailed bats for T. cruzi at three maternity roosts in Oklahoma. We collected wing tissues, extracted T. cruzi DNA, amplified target DNA by PCR using the primers TCZ1/TCZ2, and observed amplification by gel electrophoresis. One juvenile Mexican free-tailed bat was positive for T. cruzi resulting in a 0.27% prevalence in the 361 sampled bats. Our finding of a wild bat naturally infected with T. cruzi in Oklahoma provided insight on the endemicity of T. cruzi in underrepresented endemic areas. The positive sample was sequenced, confirmed as T. cruzi, and uploaded to GenBank (no. MG869732). Future research will focus on monitoring T. cruzi prevalence in wild bats and insect vectors to better understand the enzootic emergence of this neglected tropical parasite.

Development, Validation, and Application of the Microbiology Concept Inventory.

If we are to teach effectively, tools are needed to measure student learning. A widely used method for quickly measuring student understanding of core concepts in a discipline is the concept inventory (CI). Using the American Society for Microbiology Curriculum Guidelines (ASMCG) for microbiology, faculty from 11 academic institutions created and validated a new microbiology concept inventory (MCI). The MCI was developed in three phases. In phase one, learning outcomes and fundamental statements from the ASMCG were used to create T/F questions coupled with open responses. In phase two, the 743 responses to MCI 1.0 were examined to find the most common misconceptions, which were used to create distractors for multiple-choice questions. MCI 2.0 was then administered to 1,043 students. The responses of these students were used to create MCI 3.0, a 23-question CI that measures students' understanding of all 27 fundamental statements. MCI 3.0 was found to be reliable, with a Cronbach's alpha score of 0.705 and Ferguson's delta of 0.97. Test item analysis demonstrated good validity and discriminatory power as judged by item difficulty, item discrimination, and point-biserial correlation coefficient. Comparison of pre- and posttest scores showed that microbiology students at 10 institutions showed an increase in understanding of concepts after instruction, except for questions probing metabolism (average normalized learning gain was 0.15). The MCI will enable quantitative analysis of student learning gains in understanding microbiology, help to identify misconceptions, and point toward areas where efforts should be made to develop teaching approaches to overcome them.

Concept Inventory Development Reveals Common Student Misconceptions about Microbiology.

Misconceptions, or alternative conceptions, are incorrect understandings that students have incorporated into their prior knowledge. The goal of this study was the identification of misconceptions in microbiology held by undergraduate students upon entry into an introductory, general microbiology course. This work was the first step in developing a microbiology concept inventory based on the American Society for Microbiology's Recommended Curriculum Guidelines for Undergraduate Microbiology. Responses to true/false (T/F) questions accompanied by written explanations by undergraduate students at a diverse set of institutions were used to reveal misconceptions for fundamental microbiology concepts. These data were analyzed to identify the most difficult core concepts, misalignment between explanations and answer choices, and the most common misconceptions for each core concept. From across the core concepts, nineteen misconception themes found in at least 5% of the coded answers for a given question were identified. The top five misconceptions, with coded responses ranging from 19% to 43% of the explanations, are described, along with suggested classroom interventions. Identification of student misconceptions in microbiology provides a foundation upon which to understand students' prior knowledge and to design appropriate tools for improving instruction in microbiology.

Cloning, expression, and characterization of a Coxiella burnetii Cu/Zn Superoxide dismutase.

BACKGROUND: Periplasmically localized copper-zinc co-factored superoxide dismutase (SodC) enzymes have been identified in a wide range of Gram-negative bacteria and are proposed to protect bacteria from exogenously produced toxic oxygen radicals, which indicates the potential significance of a Coxiella burnetii SodC. RESULTS: Assays for SOD activity demonstrated that the cloned C. burnetii insert codes for a SOD that was active over a wide range of pH and inhibitable with 5 mM H2O2 and 1 mM sodium diethyldithiocarbamate, a characteristic of Cu/ZnSODs that distinguishes them from Fe or Mn SODs. The sodC was expressed by C. burnetii, has a molecular weight of approximately 18 kDa, which is consistent with the predicted molecular weight, and localized towards the periphery of C. burnetii. Over expression of the C. burnetii sodC in an E. coli sodC mutant restored resistance to H2O2 killing to wild type levels. CONCLUSIONS: We have demonstrated that C. burnetii does express a Cu/ZnSOD that is functional at low pH, appears to be excreted, and was able to restore H2O2 resistance in an E. coli sodC mutant. Taken together, these results indicate that the C. burnetii Cu/ZnSOD is a potentially important virulence factor.

Both inducible nitric oxide synthase and NADPH oxidase contribute to the control of virulent phase I Coxiella burnetii infections.

Host control of Coxiella burnetii infections is believed to be mediated primarily by activated monocytes/macrophages. The activation of macrophages by cytokines leads to the production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) that have potent antimicrobial activities. The contributions of ROI and RNI to the inhibition of C. burnetii replication were examined in vitro by the use of murine macrophage-like cell lines and primary mouse macrophages. A gamma interferon (IFN-gamma) treatment of infected cell lines and primary macrophages resulted in an increased production of nitric oxide (NO) and hydrogen peroxide (H2O2) and a significant inhibition of C. burnetii replication. The inhibition of replication was reversed in the murine cell line J774.16 upon the addition of either the inducible nitric oxide synthase (iNOS) inhibitor NG-monomethyl-L-arginine (NGMMLA) or the H2O2 scavenger catalase. IFN-gamma-treated primary macrophages from iNOS-/- and p47phox-/- mice significantly inhibited replication but were less efficient at controlling infection than IFN-gamma-treated wild-type macrophages. To investigate the contributions of ROI and RNI to resistance to infection, we performed in vivo studies, using C57BL/6 wild-type mice and knockout mice lacking iNOS or p47phox. Both iNOS-/- and p47phox-/- mice were attenuated in the ability to control C. burnetii infection compared to wild-type mice. Together, these results strongly support a role for both RNI and ROI in the host control of C. burnetii infection.

Evaluation of Coxiella burnetii antibiotic susceptibilities by real-time PCR assay.

Coxiella burnetii is an obligate intracellular bacterium. The inability to cultivate this organism on axenic medium has made calculation of infectious units challenging and prevents the use of conventional antibiotic susceptibility assays. A rapid and reliable real-time PCR assay was developed to quantify C. burnetii cells from J774.16 mouse macrophage cells and was applied to antibiotic susceptibility testing of C. burnetii Nine Mile, phase I. For calculation of bacterial replication, real-time PCR performed equally as well as immunofluorescent-antibody (IFA) assay when J774.16 cells were infected with 10-fold serial dilutions of C. burnetii and was significantly (P < 0.05) more repeatable than IFA when 2-fold dilutions were used. Newly infected murine macrophage-like J774.16 cells were treated with 8 microg of chloramphenicol per ml, 4 microg of tetracycline per ml, 4 microg of rifampin per ml, 4 microg of ampicillin per ml, or 1 microg of ciprofloxacin per ml. After 6 days of treatment, tetracycline, rifampin, and ampicillin significantly (P < 0.01) inhibited the replication of C. burnetii, while chloramphenicol and ciprofloxacin did not. In general, these results are consistent with those from prior reports on the efficacy of these antibiotics against C. burnetii Nine Mile, phase I, and indicate that a real-time PCR-based assay is an appropriate alternative to the present methodology for evaluation of the antibiotic susceptibilities of C. burnetii.