Synthesis, spectral, and anti-microbial studies of thioiminium iodides and amine hydrochlorides

To avoid the undesired deprotonation during the addition of organolithium and organomagnesium reagents to ketones, the thioiminium salts, easily prepared from lactams and amides are converted into 2,2-disubstituted and 2-monosubstituted amines by reaction with simple nucleophiles such as organocerium and organocopper reagents. The reaction of thioiminium iodides with organocerium reagents derived by transmetalation of corresponding lithium reagents with anhydrous cerium(III) chloride has been investigated. These thioiminium iodides act as good electrophiles and accept alkylceriums towards bisaddition. The newly synthesized amines have been characterized by 1H and 13C NMR, IR and mass spectra. The amines have been converted into their hydrochlorides and characterized by COSY. These hydrochlorides have been subjected to antimicrobial screening with clinically isolated microorganisms, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi and Candida albicans. The hydrochlorides show quite good activity against these bacteria and fungus.

Shifts in microbial communities do not explain the response of grassland ecosystem function to plant functional composition and rainfall change

Ecosystem functioning in grasslands is regulated by a range of biotic and abiotic factors, and the role of microbial communities in regulating ecosystem function has been the subject of much recent scrutiny. However, there are still knowledge gaps regarding the impacts of rainfall and vegetation change upon microbial communities and the implications of these changes for ecosystem functioning. We investigated this issue using data from an experimental mesotrophic grassland study in south-east England, which had been subjected to four years of rainfall and plant functional composition manipulations. Soil respiration, nitrogen and phosphorus stocks were measured, and the abundance and community structure of soil microbes were characterised using quantitative PCR and multiplex-TRFLP analysis, respectively. Bacterial community structure was strongly related to the plant functional composition treatments, but not the rainfall treatment. However, there was a strong effect of both rainfall change and plant functional group upon bacterial abundance. There was also a weak interactive effect of the two treatments upon fungal community structure, although fungal abundance was not affected by either treatment. Next, we used a statistical approach to assess whether treatment effects on ecosystem function were regulated by the microbial community. Our results revealed that ecosystem function was influenced by the experimental treatments, but was not related to associated changes to the microbial community. Overall, these results indicate that changes in fungal and bacterial community structure and abundance play a relatively minor role in determining grassland ecosystem function responses to precipitation and plant functional composition change, and that direct effects on soil physical and chemical properties and upon plant and microbial physiology may play a more important role.

The outer mucus layer hosts a distinct intestinal microbial niche

The overall composition of the mammalian intestinal microbiota varies between individuals: within each individual there are differences along the length of the intestinal tract related to host nutrition, intestinal motility and secretions. Mucus is a highly regenerative protective lubricant glycoprotein sheet secreted by host intestinal goblet cells; the inner mucus layer is nearly sterile. Here we show that the outer mucus of the large intestine forms a unique microbial niche with distinct communities, including bacteria without specialized mucolytic capability. Bacterial species present in the mucus show differential proliferation and resource utilization compared with the same species in the intestinal lumen, with high recovery of bioavailable iron and consumption of epithelial-derived carbon sources according to their genome-encoded metabolic repertoire. Functional competition for existence in this intimate layer is likely to be a major determinant of microbiota composition and microbial molecular exchange with the host.

Effect of tympanic cavity evacuation and flushing on microbial isolates during total ear canal ablation with lateral bulla osteotomy in dogs.

OBJECTIVE To evaluate differences in bacterial numbers, identity, and susceptibility in samples obtained from the tympanic cavity on entry (preflush) and after evacuation and lavage (postflush) and assess perioperative and empiric antimicrobial selection in dogs that underwent total ear canal ablation (TECA) with lateral bulla osteotomy (LBO) or reoperation LBO. DESIGN Prospective clinical study. ANIMALS 34 dogs. PROCEDURE TECA with LBO or reoperation LBO was performed on 47 ears. Pre- and postflush aerobic and anaerobic samples were obtained from the tympanic cavity. Isolates and antimicrobial susceptibility patterns were compared. RESULTS Different isolates (31/44 [70%] ears) and susceptibility patterns of isolate pairs (6/44 [14%] ears) were detected in pre- and postflush samples from 84% of ears. Evacuation and lavage of the tympanic cavity decreased the number of bacterial isolates by 33%. In 26% of ears, bacteria were isolated from post-flush samples but not preflush samples. Only 26% of isolates tested were susceptible to cefazolin. At least 1 isolate from 53% of dogs that received empirically chosen antimicrobials postoperatively was resistant to the selected drugs. Anaerobic bacteria were recovered from 6 ears. CONCLUSIONS AND CLINICAL RELEVANCE Accurate microbiologic assessment of the tympanic cavity should be the basis for selection of antimicrobials in dogs undergoing TECA with LBO. Bacteria remain in the tympanic cavity after evacuation and lavage. Cefazolin was a poor choice for dogs that underwent TECA with LBO, as judged on the basis of culture and susceptibility testing results.

The identification and characterization of the Staphylococcus aureus microbial immunomodulatory molecules (MIMS) Map and Efb

Staphylococcus aureus is an opportunistic pathogen that is a major health threat in the clinical and community settings. An interesting hallmark of patients infected with S. aureus is that they do not usually develop a protective immune response and are susceptible to reinfection, in part because of the ability of S. aureus to modulate host immunity. The ability to evade host immune responses is a key contributor to the infection process and is critical in S. aureus survival and pathogenesis. This study investigates the immunomodulatory effects of two secreted proteins produced by S. aureus, the MHC class II analog protein (Map) and the extracellular fibrinogen-binding protein (Efb). Map has been demonstrated to modulate host immunity by interfering with T cell function. Map has been shown to significantly reduce T cell proliferative responses and significantly reduce delayed-type hypersensitivity responses to challenge antigen. In addition, the effects of Map on the infection process were tested in a mouse model of infection. Mice infected with Map− S. aureus (Map deficient strain) presented with significantly reduced levels of arthritis, osteomyelitis and abscess formation compared to mice infected with the wild-type Map+S. aureus strain suggesting that Map−S. aureus is much less virulent than Map+S. aureus. Furthermore, Map−S. aureus-infected nude mice developed arthritis and osteomyelitis to a severity similar to Map +S. aureus-infected controls, suggesting that T cells can affect disease outcome following S. aureus infection and Map may attenuate cellular immunity against S. aureus. The extracellular fibrinogen-binding protein (Efb) was identified when cultured S. aureus supernatants were probed with the complement component C3. The binding of C3 to Efb resulted in studies investigating the effects of Efb on complement activation. We have demonstrated that Efb can inhibit both the classical and alternative complement pathways. Moreover, we have shown that Efb can inhibit complement mediated opsonophagocytosis. Further studies have characterized the Efb-C3 binding interaction and localized the C3-binding domain to the C-terminal region of Efb. In addition, we demonstrate that Efb binds specifically to a region within the C3d fragment of C3. This study demonstrates that Map and Efb can interfere with both the acquired and innate host immune pathways and that these proteins contribute to the success of S. aureus in evading host immunity and in establishing disease. ^

Effect of the emergence of community acquired methicillin resistant Staphylococcus aureus on microbial resistance patterns seen during a seven year study of minocycline/rifampin catheter use

Background. Health care associated catheter related blood stream infections (CRBSI) represent a significant public health concern in the United States. Several studies have suggested that precautions such as maximum sterile barrier and use of antimicrobial catheters are efficacious at reducing CRBSI, but there is concern within the medical community that the prolonged use of antimicrobial catheters may be associated with increased bacterial resistance. Clinical studies have been done showing no association and a significant decrease in microbial resistance with prolonged minocycline/rifampin (M/R) catheter use. One explanation is the emergence of community acquired methicillin resistant Staphylococcus aureus (MRSA), which is more susceptible to antibiotics, as a cause of CRBSI.^ Methods. Data from 323 MRSA isolates cultured from cancer patients at The University of Texas MD Anderson Cancer center from 1997-2007 displaying MRSA infection were analyzed to determine whether there is a relationship between resistance to minocycline and rifampin and prolonged wide spread use of minocycline (M/R) catheters. Analysis was also conducted to determine whether there was a significant change in the prevalence community acquired MRSA (CA-MRSA) during this time period and if this emergence act as a confounder masquerading the true relationship between microbial resistance and prolonged M/R catheter use.^ Results. Our study showed that the significant (p=0.008) change in strain type over time is a confounding variable; the adjusted model showed a significant protective effect (OR 0.000281, 95% CI 1.4x10 -4-5.5x10-4) in the relationship between MRSA resistance to minocycline and prolonged M/R catheter use. The relationship between resistance to rifampin and prolonged M/R catheter use was not significant.^ Conclusion. The emergence of CA-MRSA is a confounder and in the relationship between resistance to minocycline and rifampin and prolonged M/R catheter use. However, despite the adjustment for the more susceptible CA-MRSA the widespread use of M/R catheters does not promote microbial resistance. ^

Heterotrimeric G protein -mediated signal transduction in Neurospora crassa

Heterotrimeric G protein-mediated signal transduction is one of numerous means that cells utilize to respond to external stimuli. G proteins consist of α, β andγ subunits. Extracellular ligands bind to seven-transmembrane helix receptors, triggering conformational changes. This is followed by activation of coupled G proteins through the exchange of GDP for GTP on the Gα subunit. Once activated, Gα-GTP dissociates from the βγ dimer. Both of these two moieties can interact with downstream effectors, such as adenylyl cyclase, phospholipase C, phosphodiesterases, or ion channels, leading to a series of changes in cellular metabolism and physiology. ^ Neurospora crassa is a eukaryotic multicellular filamentous fungus, with asexual/vegetative and sexual phases to its life cycle. Three Gα (GNA-1, GNA-2, GNA-3) and one Gβ (GNB-1) proteins have been identified in this organism. This dissertation investigates GNA-1 and GNB-1 mediated signaling pathways in N. crassa. ^ GNA-1 was the first identified microbial Gα that belongs to a mammalian superfamily (Gαi). Deletion of GNA-1 leads to multiple defects in N. crassa. During the asexual cycle, Δgna-1 strains display a slower growth rate and delayed conidiation on solid medium. In the sexual cycle, the Δgna-1 mutant is male-fertile but female-sterile. Biochemical studies have shown that Δ gna-1 strains have lower adenosine 3′–5 ′ cyclic monophosphate (cAMP) levels than wild type under conditions where phenotypic defects are observed. In this thesis work, strains containing one of two GTPase-deficient gna-1 alleles (gna-1 R178C, gna-1Q204L) leading to constitutive activation of GNA-1 have been constructed and characterized. Activation of GNA-1 causes uncontrolled aerial hyphae proliferation, elevated sensitivity to heat and oxidative stresses, and lower carotenoid synthesis. To further study the function of GNA-1, constructs to enable expression of mammalian Gαi superfamily members were transformed into a Δ gna-1 strain, and complementation of Δgna-1 defects investigated. Gαs, which is not a member of Gα i superfamily was used as a control. These mammalian Gα genes were able to rescue the vegetative growth rate defect of the Δ gna-1 strain in the following order: Gαz > Gα o > Gαs > Gαt > Gαi. In contrast, only Gαo was able to complement the sexual defect of a Δgna-1 strain. With regard to the thermotolerance phenotype, none of the mammalian Gα genes restored the sensitivity to a wild type level. These results suggest that GNA-1 regulates two independent pathways during the vegetative and sexual cycles in N. crassa. ^ GNB-1, a G protein β subunit from N. crassa, was identified and its functions investigated in this thesis work. The sequence of the gnb-1 gene predicts a polypeptide of 358 residues with a molecular mass of 39.7 kDa. GNB-1 exhibits 91% identity to Cryphonectria parasitica CPGB-1, and also displays significant homology with human and Dictyostelium Gβ genes (∼66%). A Δ gnb-1 strain was constructed and shown to exhibit defects in asexual spore germination, vacuole number and size, mass accumulation and female fertility. A novel role for GNB-1 in regulation of GNA-1 and GNA-2 protein levels was also demonstrated. ^