Antibiotic resistance is a growing problem worldwide. For this reason, clinical laboratories often determine the susceptibility of the bacterial isolate to a number of different antibiotics in order to establish the most effective antibiotic for treatment. Unfortunately, current susceptibility assays are time consuming. Antibiotic resistance often involves the chemical modification of an antibiotic to an inactive form by an enzyme expressed by the bacterium. Selected reaction monitoring (SRM) has the ability to quickly monitor and identify these chemical changes in an unprecedented time scale. In this work, we used SRM as a technique to determine the susceptibility of several different antibiotics to the chemically modifying enzymes β-lactamase and chloramphenicol acetyltransferase, enzymes used by bacteria to confer resistance to major classes of commonly used antibiotics. We also used this technique to directly monitor the effects of resistant bacteria grown in a broth containing a specific antibiotic. Because SRM is highly selective and can also identify chemical changes in a multitude of antibiotics in a single assay, SRM has the ability to detect organisms that are resistant to multiple antibiotics in a single assay. For these reasons, the use of SRM greatly reduces the time it takes to determine the susceptibility or resistance of an organism to a multitude of antibiotics by eliminating the time-consuming process found in other currently used methods.

We designed, built, tested, space-qualified, launched, and collected telemetered data from low Earth orbit from PharmaSat, a 5.1-kg free flying "nanosatellite" that supported microbial growth in 48 microfluidic wells, dosed microbes with multiple concentrations of a pharmaceutical agent, and monitored microbial growth and metabolic activity using a dedicated 3-color optical absorbance system at each microwell. The PharmaSat nanosatellite comprised a structure approximately 10 x 10 x 35 cm, including triple-junction solar cells, bidirectional communications, power-generation and energy-storage system, and a sealed payload 1.2-L containment vessel that housed the biological organisms along with the fluidic, optical, thermal, sensor, and electronic subsystems. Growth curves for S. cerevisiae (Brewer's yeast) were obtained for multiple concentrations of the antifungal drug voriconazole in the microgravity conditions of low Earth orbit. Corresponding terrestrial control experiments were conducted for comparison.

We designed, built, tested, space-qualified, launched, and downlinked bioanalytical data from PharmaSat, the first fully autonomous outer-space pharmaceutical dose-response bioanalytical system on a free-flying satellite.PharmaSat tracks microorganism culture population density and metabolic activity in 48 microwells via 3-color optical absorbance.Its 5.1-kg total mass includes solar cells, integrated spacecraft "bus" module (power/batteries/ control/communications), and a sealed containment vessel housing the biofluidic, optical, thermal, and sensor subsystems.Data were obtained from all subsystems over several days following nutrient introduction to initiate growth of Saccharomyces cerevisiae, followed by challenges with three dose levels of an antifungal agent.

We develop integrated instruments and platforms suitable for economical, frequent space access for autonomous life science experiments and processes in outer space. The technologies represented by three of our recent free-flyer small-satellite missions are the basis of a rapidly growing toolbox of miniaturized biologically/biochemically-oriented instrumentation now enabling a new generation of in-situ space experiments. Autonomous small satellites (~ 1 50 kg) are less expensive to develop and build than fullsize spacecraft and not subject to the comparatively high costs and scheduling challenges of human-tended experimentation on the International Space Station, Space Shuttle, and comparable platforms. A growing number of commercial, government, military, and civilian space launches now carry small secondary science payloads at far lower cost than dedicated missions; the number of opportunities is particularly large for so-called cube-sat and multicube satellites in the 1 10 kg range. The recent explosion in nano-, micro-, and miniature technologies, spanning fields from telecommunications to materials to bio/chemical analysis, enables development of remarkably capable autonomous miniaturized instruments to accomplish remote biological experimentation. High-throughput drug discovery, point-of-care medical diagnostics, and genetic analysis are applications driving rapid progress in autonomous bioanalytical technology. Three of our recent missions exemplify the development of miniaturized analytical payload instrumentation: GeneSat-1 (launched: December 2006), PharmaSat (launched: May 2009), and O/OREOS (organism/organics exposure to orbital stresses; scheduled launch: May 2010). We will highlight the overall architecture and integration of fluidic, optical, sensor, thermal, and electronic technologies and subsystems to support and monitor the growth of microorganisms in culture in these small autonomous space satellites, including real-time tracking of their culture density, gene expression, and metabolic activity while in the space environment. Flight data and results will be presented from GeneSat-1, which tracked gene expression levels of GFP-labeled E. coli and from PharmaSat, which monitored the dose dependency of an antifungal agent against S. cerevisiae. The O/OREOS SESLO instrument, which will study the effects of radiation and microgravity upon the viability and growth characteristics of B. subtilis and the halophile Halorubrum chaoviatoris for periods of 0 - 6 months in space, will be described as well. The ongoing expansion of the small satellite toolbox of biological technologies will be summarized.

Streptococcus pneumoniae, a facultative human pathogen associated with wide variety of diseases, such as pneumonia, meningitis, sepsis and otitis media. Factors involved in the initial colonization, survival and etiology of this commensal bacteria in the human host from the ex vivo environment is still not clearly understood. Here, we report alterations in global transcriptional profiles of S. pneumoniae 6304 serotype 4 after 50 passages (50P) and 100 passages (100P) on laboratory media to better understand gene expression strategies employed by the bacterium during progression from the nasopharynx to the blood. The results show that six-fold more genes were differentially expressed after 100P as compared to 50P. After 100P on blood agar plates, 726 genes (33%) of 2192 genes in the S. pneumoniae genome were differentially expressed. Moreover, the majority of these genes (68%) were expressed at higher levels with increasing passage number and from different functional groups. Significantly, all the genes present in Region of Diversity 10 (RD10) are required for virulence during blood stream infection showed enhanced expression after passage. However, there was no significant decrease in the LD(50) of serial passage strains compare to single passage strain in a mouse challenge model. Overall, our data suggest that bacteria adapt to extended laboratory passage by substantially altering gene expression. Furthermore, extended passage on blood agar plates reduces the expression of genes associated with initial colonization and adherence but enhances the expression of genes needed for systemic infection.

The impact of low-shear stress (LSS) was evaluated on an Adherent-invasive Escherichia coli clinical isolate (AIEC strain O83:H1) from a Crohn's disease patient. High-aspect ratio vessels (HARVs) were used to model LSS conditions to characterize changes in environmental stress resistance and adhesion/invasive properties. Low-shear stress-grown cultures exhibited enhanced thermal and oxidative stress resistance as well as increased adherence to Caco-2 cells, but no changes in invasion were observed. An AIEC rpoS mutant was constructed to examine the impact of this global stress regulator. The absence of RpoS under LSS conditions resulted in increased sensitivity to oxidative stress while adherence levels were elevated in comparison with the wild-type strain. TnphoA mutagenesis and rpoS complementation were carried out on the rpoS mutant to identify those factors involved in the LSS-induced adherence phenotype. Mutagenesis results revealed that one insertion disrupted the tnaB gene (encoding tryptophan permease) and the rpoS tnaB double mutant exhibited decreased adherence under LSS. Complementation of the tnaB gene, or medium supplemented with exogenous indole, restored adhesion of the rpoS tnaB mutant under LSS conditions. Overall, our study demonstrated how mechanical stresses such as LSS altered AIEC phenotypic characteristics and identified novel functions for some RpoS-regulated proteins.

The ability to protect mice against respiratory infections with virulent Francisella tularensis has been problematic and the role of antibody-versus-cell-mediated immunity controversial. In this study, we tested the hypothesis that protective immunity can develop in mice that were given antibiotic therapy following infection via the respiratory tract with F. tularensis SCHU S4. We show that mice infected with a lethal dose of SCHU S4, via an intra-nasal challenge, could be protected with levofloxacin treatment. This protection was evident even when levofloxacin treatment was delayed 72h post-infection. At early time points after levofloxacin treatment, significant numbers of bacteria could be recovered from the lungs and spleens of mice, which was followed by a dramatic disappearance of bacteria from these tissues. Mice successfully treated with levofloxacin were later shown to be almost completely resistant to re-challenge with SCHU S4 by the intra-nasal route. Serum antibody appeared to play an important role in this immunity. Normal mice, when given sera from animals protected by levofloxacin treatment, were solidly protected from a lethal intra-nasal challenge with SCHU S4. The protective antiserum contained high titers of SCHU S4-specific IgG2a, indicating that a strong Th1 response was induced following levofloxacin treatment. Thus, this study describes a potentially valuable animal model for furthering our understanding of respiratory tularemia and provides suggestive evidence that antibody can protect against respiratory infections with virulent F. tularensis.

Garland D. Anderson, MD Dean of Medicine Tetsuo Ashizawa, MD Chair, Neurology Howard A. Brody, MD, PhD Director, Institute for the Medical Humanities Harvey Bunce, III, PhD Chair, Preventive Medicine & Community Health Martin Colman, MD, FACR Chair, Radiation Oncology Henry F. Epstein, MD Chair, Neuroscience & Cell Biology Bernard F. Godley, MD, PhD, FACS Ophthalmology & Visual Sciences James R. Halpert, PhD Chair, Pharmacology & Toxicology Gary D.V. Hankins, MD Chair, Obstetrics & Gynecology Robert M.A. Hirschfeld, MD Chair, Psychiatry & Behavioral Sciences Stanley M. Lemon, MD Director, Institute for Human Infections & Immunity Ronald W. Lindsey, MD Chair, Orthopaedic Surgery & Rehabilitation Shawn D. Newlands, MD, PhD, MBA Chair, Otolaryngology David W. Niesel, PhD Chair, Microbiology & Immunology J. Regino Perez-Polo, PhD Chair, Biochemistry & Molecular Biology Donald S. Prough, MD Chair, Anesthesiology Sharon S. Raimer, MD Chair, Dermatology Lawrence R. Stanberry, MD, PhD Chair, Pediatrics Gregory L. Katzman, MD Chair, Radiology Barbara L. Thompson, MD Chair, Family Medicine Courtney M. Townsend, Jr., MD Chair, Surgery Randall J. Urban, MD Chair , Internal Medicine David H. Walker, MD Chair, Pathology MEMBERS – AT – LARGE (elected to one year term) Karl Anderson, MD Preventive Medicine & Community Health 2007-2008

To facilitate ground-based microgravity analogue studies, NASA developed high aspect ratio vessels (HARVs) which model low-shear microgravity conditions (LSMMG). To investigate how LSMMG affects global transcriptional activity in opportunistic bacterial pathogens, DNA microarray analysis was carried out on Streptococcus pneumoniae grown in HARVs under LSMMG and compared to 1 x g and static conditions. Analysis of the array data was carried out using multiple analysis methods (Genepix Pro 6.0, Spotfire 7.3, SAM, and ANOVA). SAM analysis revealed 138 significant LSMMG-responsive genes. Among the responsive genes (threshold = 1.5-fold), 81 genes representing different functional groups were down-regulated under LSMMG. Distinct expression patterns were revealed using hierarchical clustering methods (Cluster/Treeview, CLUSFAVOR 6.0, and Spotfire 7.3). Quantitative RT-PCR was used to confirm gene expression changes. Computational analyses revealed 101 genes which were differentially expressed under static versus 1 x g conditions. Among these genes, 46 were altered between 1 x g and LSMMG conditions while 9 were altered between static and LSMMG conditions. SAM analysis revealed 147 genes which exhibited significant changes between static and 1 x g groups. These results indicate LSMMG represents a unique environment which can alter the transcriptional profile and subsequent physiology of bacteria. Further, differences in gene expression between the static and 1 x g controls clearly impact the analysis and must be taken into consideration in the assignment of LSMMG responsive genes.

Space flight has been shown to result in altered immune responses. The current study was designed to investigate this possibility by using the bed rest model of some space flight conditions. A large number of women are included as subjects in the study. The hypothesis being tested is: 60 days head-down tilt bed rest of humans will affect the immune system and resistance to infection. Blood, urine and saliva samples will be obtained from bed rest subjects prior to, at intervals during, and after completion of 60 days of head-down tilt bed rest. Leukocyte blastogenesis, cytokine production and virus reactivation will be assessed. The ability of the subjects to respond appropriately to immunization with the neoantigen bacteriophage φX-174 will also be determined. Bed rest is being carried out at MEDES, Toulouse France, and the University of Texas Medical Branch, Galveston, TX. The studies to be carried out in France will also allow assessment of the effects of muscle/bone exercise and nutritional countermeasures on the immune system in addition to the effects of bed rest.

High-aspect rotating vessels (HARVs) are used to study the effects low-shear modeled microgravity (LSMMG) on bacterial gene expression. LSMMG is generated by orienting HARVs with the axis of rotation perpendicular to the gravity vector while gravitational controls are oriented with the axis of rotation parallel to the gravity vector. Microarray analysis was performed on Streptococcus pneumoniae TIGR4 grown in HARVs under three conditions (LSMMG, 1×g, and static) to determine if global transcriptional activity is altered between different gravitational controls and LSMMG. Results revealed 101 differentially expressed genes under static conditions compared to 1×g, 46 genes between 1×g and LSMMG, and nine genes between static and LSMMG. Hierarchical cluster analysis revealed 15 genes exhibiting similar expression patterns under static conditions compared to 1×g. These results indicate that rotation, in addition to low-shear forces, might contribute to bacterial adaptation to the LSMMG.

Streptococcus pneumoniae is a common commensal of the upper respiratory tract of healthy humans and is an important pathogen in young children, immunocompromised adults, and the elderly. To better understand the strategies employed by this bacterial species in adapting to conditions present at different infection sites in the host, global transcription profiling was used to study gene expression at different growth temperatures: 21, 29, 33, 37, and 40 °C. Here, we found that 658 genes (29%) out of 1717 genes were differently expressed (≥1.5-fold change) in at least one growth temperature relative to 37 °C. The percentages of genes whose expression was altered in each growth temperature, respectively, were: 21 °C: 53% ↑, 47% ↓; 29 °C: 44% ↑, 56% ↓; 33 °C: 27% ↑, 73% ↓ and 40 °C: 44% ↑, 56% ↓. Hierarchical clustering (HC) of the temperature regulated genes resulted in four clusters, namely A–D of differently expressed genes grouped by bacterial growth temperature. Cluster A represented 81 genes reflecting enhanced expression at 33 °C. Cluster B included 260 genes whose expression increased with growth temperature. Cluster C had 28 genes with 68% showing enhanced expression at 29 °C while cluster D had 289 genes with 74% genes showing enhanced expression at 21 °C relative to 37 °C. Principal component (PC) analysis also divided differentially expressed genes into four groups and was highly correlated with HC, suggesting that temperature regulated expression is not random but coordinated. Overall, these results indicated substantial reprogramming of transcription in response to growth temperature. Functional characterization of differential gene expression at different temperatures provides further information on the molecular mechanism(s) that allows S. pneumoniae to adapt to various host environments.

ABSTRACT Lipopolysaccharide (LPS) and Braun (murein) lipoprotein (Lpp) are major components of the outer membrane of gram-negative enteric bacteria that function as potent stimulators of inflammatory and immune responses. In a previous paper, we provided evidence that two functional copies of the lipoprotein gene (lppA and lppB) located on the chromosome of Salmonella enterica serovar Typhimurium contributed to bacterial virulence. In this study, we characterized lppA and lppB single-knockout (SKO) mutants and compared them with an lpp double-knockout (DKO) mutant using in vitro and in vivo models. Compared to the lpp DKO mutant, which was nonmotile, the motility of the lpp SKO mutants was significantly increased (73 to 77%), although the level of motility did not reach the level of wild-type (WT) S. enterica serovar Typhimurium. Likewise, the cytotoxicity was also significantly increased when T84 human intestinal epithelial cells and RAW264.7 murine macrophages were infected with the lpp SKO mutants compared to the cytotoxicity when cells were infected with the lpp DKO mutant. The level of interleukin-8 (IL-8) in polarized T84 cells infected with the lppB SKO mutant was significantly higher (two- to threefold higher), reaching the level in cells infected with WT S. enterica serovar Typhimurium, than the level in host cells infected with the lppA SKO mutant. The lpp DKO mutant induced minimal levels of IL-8. Similarly, sera from mice infected with the lppB SKO mutant contained 4.5- to 10-fold-higher levels of tumor necrosis factor-α and IL-6; the levels of these cytokines were 1.7- to 3.0-fold greater in the lppA SKO mutant-infected mice than in animals challenged with the lpp DKO mutant. The increased cytokine levels observed with the lppB SKO mutant in mice correlated with greater tissue damage in the livers and spleens of these mice than in the organs of animals infected with the lppA SKO and lpp DKO mutants. Moreover, the lppB SKO mutant-infected mice had increased susceptibility to death. Since the lpp DKO mutant retained intact LPS, we constructed an S. enterica serovar Typhimurium triple-knockout (TKO) mutant in which the lppA and lppB genes were deleted from an existing msbB mutant (msbB encodes an enzyme required for the acylation of lipid A). Compared to the lpp DKO and msbB SKO mutants, the lpp-msbB TKO mutant was unable to induce cytotoxicity and to produce cytokines and chemokines in vitro and in vivo. These studies provided the first evidence of the relative contributions of Lpp and lipid A acylation to Salmonella pathogenesis.

Septic shock due to Salmonella and other gram-negative enteric pathogens is a leading cause of death worldwide. The role of lipopolysaccharide in sepsis is well studied; however, the contribution of other bacterial outer membrane components, such as Braun (murein) lipoprotein (Lpp), is not well defined. The genome of Salmonella enterica serovar Typhimurium harbors two copies of the lipoprotein (lpp) gene. We constructed a serovar Typhimurium strain with deletions in both copies of the lpp gene (lpp1 and lpp2) by marker exchange mutagenesis. The integrity of the cell membrane and the secretion of the effector proteins through the type III secretion system were not affected in the lpp double-knockout mutant. Subsequently, the virulence potential of this mutant was examined in a cell culture system using T84 intestinal epithelial and RAW264.7 macrophage cell lines and a mouse model of salmonellosis. The lpp double-knockout mutant was defective in invading and inducing cytotoxic effects in T84 and RAW264.7 cells, although binding of the mutant to the host cell was not affected when compared to the wild-type (WT) serovar Typhimurium. The motility of the mutant was impaired, despite the finding that the number of flagella was similar in the lpp double knockout mutant and the WT serovar Typhimurium. Deletion in the lpp genes did not affect the intracellular survival and replication of Salmonella in macrophages and T84 cells. Induction of the proinflammatory cytokines tumor necrosis factor alpha and interleukin-8 (IL-8) was significantly reduced in macrophages and T84 cells infected with the lpp double-knockout mutant. The levels of IL-8 remained unaffected in T84 cells when infected with either live or heat-killed WT and lpp mutant, indicating that invasion was not required for IL-8 production and that Toll-like receptor 2 signaling might be affected in the Lpp double-knockout mutant. These effects of the Lpp protein could be restored by complementation of the isogenic mutant. The lpp double-knockout mutant was avirulent in mice, and animals infected with this mutant were protected from a lethal challenge dose of WT serovar Typhimurium. The severe combined immunodeficient mice, on the other hand, were susceptible to infection by the lpp double-knockout mutant. The serovar Typhimurium mutants from which only one of the lpp (lpp1 or lpp2) genes was deleted were also avirulent in mice. Taken together, our data indicated that Lpp specifically contributed to the virulence of the organism.