Monitoring bacterial resistance to chloramphenicol and other antibiotics by liquid chromatography electrospray ionization tandem mass spectrometry using selected reaction monitoring.

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 ß-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.

Global transcription profiling and virulence potential of Streptococcus pneumoniae after serial passage.

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.

Levofloxacin rescues mice from lethal intra-nasal infections with virulent Francisella tularensis and induces immunity and production of protective antibody.

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.

The effects of low-shear stress on Adherent-invasive Escherichia coli.

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.

Global profiling of Streptococcus pneumoniae gene expression at different growth temperatures.

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 degrees C. Here, we found that 658 genes (29%) out of 1717 genes were differently expressed (>or=1.5-fold change) in at least one growth temperature relative to 37 degrees C. The percentages of genes whose expression was altered in each growth temperature, respectively, were: 21 degrees C: 53% upward arrow, 47% downward arrow; 29 degrees C: 44% upward arrow, 56% downward arrow; 33 degrees C: 27% upward arrow, 73% downward arrow and 40 degrees C: 44% upward arrow, 56% downward arrow. 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 degrees C. Cluster B included 260 genes whose expression increased with growth temperature. Cluster C had 28 genes with 68% showing enhanced expression at 29 degrees C while cluster D had 289 genes with 74% genes showing enhanced expression at 21 degrees C relative to 37 degrees 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.