Severe staphylococcal infections treated with vancomycin.
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Adult↗
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Results for “microbial function”
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A cutaneous gene therapy approach to treat infection through keratinocyte-targeted overexpression of antimicrobial peptides.
Infection represents a major associated problem in severely burned patients, as it causes skin graft failure and increases the risk of mortality. Topical and systemic antibiotic treatment is limited by the appearance of resistant bacterial strains. Antimicrobial peptides (AMPs) are gene-encoded "natural antibiotics" that form part of the innate mechanism of defense and may be active against such antibiotic-resistant microorganisms. Several microbicidal peptides are expressed in human skin under inflammatory conditions, and their function is not only limited to microbial killing but also influences tissue repair and adaptive immunity. Protein delivery through cutaneous gene therapy is a promising therapeutic tool for both skin and nonskin diseases. Here we present a gene transfer approach aimed at delivering antimicrobial peptides from keratinocytes. Adenoviral vectors encoding antimicrobial peptide genes were used to infect human keratinocytes growing either on plastic or as part of cultured skin equivalents. Inhibition of bacterial growth occurred both in conditioned media and in direct contact with AMPs gene-transduced keratinocytes. In addition, we showed cooperative effects after transfer of combinations of genes encoding for AMPs with structural differences. Combined cutaneous tissue engineering in conjunction with (microbicidal) gene therapy emerges as a tailored therapeutic approach that is useful for wound coverage and, in this case, concomitantly combating infection.
Adenoviridae↗
Autoantibodies directed against bactericidal/permeability-increasing protein in patients with cystic fibrosis: association with microbial respiratory tract colonization.
BACKGROUND: Cystic fibrosis (CF) is associated with the appearance of serum autoantibodies directed against bactericidal/permeability-increasing protein (BPI). OBJECTIVES: To determine the age-specific seroprevalence rates of anti-BPI-IgG and IgA in a population of patients with CF and to correlate anti-BPI antibody concentrations with microbial respiratory tract colonization and pulmonary function variables at the time of serum sampling and 6 years thereafter. METHODS: Determination of BPI antibodies of the IgG and IgA isotypes using a commercial enzyme-linked immunosorbent assay in sera of a CF serum bank of 1992; correlation of anti-BPI antibody concentrations with age, clinical score, pulmonary function variables in 1992 and 1998, total serum immunoglobulin isotype concentrations and respiratory tract colonization with Pseudomonas aeruginosa and Aspergillus spp. RESULTS: Seventy-one patients (age in 1992, 14.1 +/- 7.5 years) were studied. Reactivities for anti-BPI-IgG and IgA were found in 28 (39%) and 26 (37%) patients, respectively. The seroprevalence of anti-BPI-IgA, but not IgG, increased significantly with age. P. aeruginosa colonization was associated with elevated concentrations of anti-BPI-IgG (P = 0.003) and IgA (P = 0.037). There were significant negative correlations between pulmonary function variables (vital capacity, forced expiratory volume in 1 s) in 1992 and 1998, respectively, and concentrations of anti-BPI-IgG or IgA in a multiple regression analysis. Anti-BPI-IgG, but not IgA, remained significantly associated with P. aeruginosa colonization (P = 0.006) and with reduced vital capacity (P = 0.01) in 1998 after correction for total serum isotype concentration. CONCLUSIONS: Anti-BPI-IgG are strongly associated with concurrent P. aeruginosa colonization and with long term restrictive pulmonary function abnormalities.
Adolescent↗
Chromobacterium violaceum infection.
Chromobacterium violaceum infection is confined to the tropical and subtropical areas, with almost all reported cases occurring in the Southeast. The most common feature of this infection is sepsis, followed by cutaneous involvement and liver abscesses. Treatment consists of surgical drainage of purulent collections and appropriate antimicrobial therapy, such as chloramphenicol, gentamicin, imipenem, trimethoprim-sulfamethoxazole, or ciprofloxacin. Although C violaceum infection is rare, it is potentially fatal and remains an important entity for clinicians to suspect and treat appropriately.
Anti-Bacterial Agents↗
Source control in the management of severe sepsis and septic shock: an evidence-based review.
OBJECTIVE: In 2003, critical care and infectious disease experts representing 11 international organizations developed management guidelines for source control in the management of severe sepsis and septic shock that would be of practical use for the bedside clinician, under the auspices of the Surviving Sepsis Campaign, an international effort to increase awareness and to improve outcome in severe sepsis. DESIGN: The process included a modified Delphi method, a consensus conference, several subsequent smaller meetings of subgroups and key individuals, teleconferences, and electronic-based discussion among subgroups and among the entire committee. METHODS: The modified Delphi methodology used for grading recommendations built on a 2001 publication sponsored by the International Sepsis Forum. We undertook a systematic review of the literature graded along five levels to create recommendation grades from A to E, with A being the highest grade. Pediatric considerations to contrast adult and pediatric management are in the article by Parker et al. on p. S591. CONCLUSION: Source control represents a key component of success in therapy of sepsis. It includes drainage of infected fluids, debridement of infected soft tissues, removal of infected devices or foreign bodies, and finally, definite measures to correct anatomic derangement resulting in ongoing microbial contamination and to restore optimal function. Although highly logical, since source control is the best way to reduce quickly the bacterial inoculum, most recommendations are, however, graded as D or E due to the difficulty to perform appropriate randomized clinical trials in this respect. Appropriate source control should be part of the systematic checklist we have to keep in mind in setting up the therapeutic strategy in sepsis.
Abdomen↗
Systematic evaluation of metatranscriptomic differential gene expression in silico, in vitro, and in vivo enables elucidation of inter-species cross-feeding.
Metatranscriptomic (MTX) sequencing quantifies gene expression from the collective genomes of microbial communities (microbiomes), enabling assessment of functional activity rather than functional potential. While differential expression testing is instrumental to RNA-sequencing analysis, current metatranscriptomic approaches have been benchmarked only on simulated data and not under real operating conditions, resulting in a lack of standard practices. Here, we evaluate the performance of statistical differential expression methods on both simulated datasets and data collected from real bacterial 'mock communities' designed for this purpose. We assess the robustness of individual methods to organisms' low relative abundance, differential abundance, low prevalence, and transcription rate changes, showing that no existing methods perform adequately across all confounding conditions. We then apply the same approaches to metatranscriptomic datasets generated from gnotobiotic mice colonized with defined consortia of human bacterial strains and show that the method nominated by our mock community comparisons successfully inferred cross-feeding dynamics which were validated in vitro. We conclude that MTX method benchmarking on real, not simulated, datasets can and should optimize model implementation, enabling inference and validation of cross-feeding and other inter-species and host-microbe dynamics from in vivo studies.
Journal Article↗
The molecular basis of connective tissue pathology.
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Animals↗
Enteroaggregative Escherichia coli flagellin-induced interleukin-8 secretion requires Toll-like receptor 5-dependent p38 MAP kinase activation.
Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen that causes acute and chronic diarrhoea in a number of clinical settings. EAEC diarrhoea involves bacterial aggregation, adherence to intestinal epithelial cells and elaboration of several toxigenic bacterial mediators. Flagellin (FliC-EAEC), a major bacterial surface protein of EAEC, causes interleukin (IL)-8 release from several epithelial cell lines. The host response to flagellins from E. coli and several other bacteria is mediated by Toll-like receptor 5 (TLR5), which signals through nuclear factor kappa B (NF-kappaB) to induce transcription of pro-inflammatory cytokines. p38 mitogen-activating protein (MAP) kinase (MAPK) is a member of a family of stress-related kinases that influences a diverse range of cellular functions including host inflammatory responses to microbial products. We studied the role of p38 MAPK in FliC-EAEC-induced IL-8 secretion from Caco-2 human intestinal epithelial cells and THP-1 human monocytic cells. We found that IL-8 secretion from both cell types is dependent on p38 MAPK, which is phospho-activated in response to FliC-EAEC. The role of TLR5 in p38 MAPK-dependent IL-8 secretion was verified in HEp-2 cells transiently transfected with a TLR5 expression construct. Activation of interleukin-1 receptor-associated kinase (IRAK) was also observed in Caco-2 and TLR5-transfected HEp-2 cells after exposure to FliC-EAEC. Finally, we demonstrated that pharmacological inhibition of p38 MAPK reduced IL-8 transcription and mRNA levels, but did not affect NF-kappaB activation. Collectively, our results suggest that TLR5 mediates p38 MAPK-dependent IL-8 secretion from epithelial and monocytic cells incubated with FliC-EAEC, and that this effect requires IL-8 promoter activation independent of NF-kappaB nuclear migration.
Caco-2 Cells↗
Global physiological analysis of carbon- and energy-limited growing Escherichia coli confirms a high degree of catabolic flexibility and preparedness for mixed substrate utilization.
Growth conditions for heterotrophic bacteria in the environment are characterized by low concentrations of carbon and energy sources and complex substrate mixtures. While mechanisms of starvation-survival in the absence of carbon substrates have been studied in considerable detail, information on the physiology of slow growth under oligotrophic conditions is limited. We intended to elucidate general strategies by which Escherichia coli adapts to low concentrations of a mixed carbon and energy source pool. A new screening method based on BIOLOG AN MicroPlates, which allowed us to distinguish repressed and induced catabolic functions in E. coli, was combined with the analysis of periplasmic high-affinity binding proteins. Extending previous findings for E. coli and other microbial species, we found that numerous alternative catabolic functions and high-affinity binding proteins are derepressed under either glucose- or arabinose-limited growth conditions, in spite of the absence of the respective inducers. Escherichia coli cells growing in carbon-limited complex medium chemostat cultures exhibited an even higher degree of catabolic flexibility and were able to oxidize 43 substrates. The BIOLOG respiration pattern indicated simultaneous dissimilation of diverse sugars, amino acids and dipeptides (mixed substrate growth). The observed physiological adaptations of E. coli to low concentrations of carbon and energy substrates presumably are advantageous in many natural growth situations and also offer an explanation why many heterotrophic bacteria have and maintain such a broad carbon substrate range.
Bacteriological Techniques↗
Flow cytometric detection of specific gene expression in prokaryotic cells using in situ RT-PCR.
Prokaryotic in situ RT-PCR was coupled with flow cytometry to detect mRNA transcripts of the toluene dioxygenase (todC1) gene in intact cells of the bacterium Pseudomonas putida F1. Recovery efficiency of fixed cells over the course of the entire in situ detection procedure was approximately 81% for both P. putida F1 and AC10R cells. It appeared that lysozyme treatment and PCR thermal cycling were the steps responsible for most of observed cell loss. Bacterial cells expressing the todC1 gene could be discriminated from negative control cells of the same size based on flow cytometrically-measured fluorescence and forward angle light scatter. According to flow cytometric analysis, the fluorescence intensity of positive cells was 4-5 times brighter than that of negative cells. The combination of flow cytometry and a prokaryotic in situ reverse transcription-PCR (RT-PCR) approach make possible the rapid detection and enumeration of functional (based on mRNA) populations of microbial cells.
Flow Cytometry↗
Doxycyclinum NFN: a pharmacological and bacteriological investigation.
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Doxycycline↗
Impact of the order of initiation of fluconazole and amphotericin B in sequential or combination therapy on killing of Candida albicans in vitro and in a rabbit model of endocarditis and pyelonephritis.
In vitro time-kill studies and a rabbit model of endocarditis and pyelonephritis were used to define the impact that the order of exposure of Candida albicans to fluconazole (FLC) and amphotericin B (AMB), as sequential and combination therapies, had on the susceptibility of C. albicans to AMB and on the outcome. The contribution of FLC-induced resistance to AMB for C. albicans also was assessed. In vitro, AMB monotherapy rapidly killed each of four C. albicans strains; FLC alone was fungistatic. Preincubation of these fungi with FLC for 18 h prior to exposure to AMB decreased their susceptibilities to AMB for 8 to >40 h. Induced resistance to AMB was transient, but the duration of resistance increased with the length of FLC preincubation. Yeast sequentially incubated with FLC followed by AMB plus FLC (FLC-->AMB+FLC) showed fungistatic growth kinetics similar to that of fungi that were exposed to FLC alone. This antagonistic effect persisted for at least 24 h. Simultaneous exposure of C. albicans to AMB and FLC [AMB+FLC(simult)] demonstrated activity similar to that with AMB alone for AMB concentrations of > or =1 microg/ml; antagonism was seen using an AMB concentration of 0.5 microg/ml. The in vitro findings accurately predicted outcomes in our rabbit infection model. In vivo, AMB monotherapy and treatment with AMB for 24 h followed by AMB plus FLC (AMB-->AMB+FLC) rapidly sterilized kidneys and cardiac vegetations. AMB+FLC(simult) and FLC-->AMB treatments were slower in clearing fungi from infected tissues. FLC monotherapy and FLC-->AMB+FLC were both fungistatic and were the least active regimens. No adverse interaction was observed between AMB and FLC for the AMB-->FLC regimen. However, FLC-->AMB treatment was slower than AMB alone in clearing fungi from tissues. Thus, our in vitro and in vivo studies both demonstrate that preexposure of C. albicans to FLC reduces fungal susceptibility to AMB. The length of FLC preexposure and whether AMB is subsequently used alone or in combination with FLC determine the duration of induced resistance to AMB.
Amphotericin B↗
Intracellular expression of Peptide fusions for demonstration of protein essentiality in bacteria.
We describe a "protein knockout" technique that can be used to identify essential proteins in bacteria. This technique uses phage display to select peptides that bind specifically to purified target proteins. The peptides are expressed intracellularly and cause inhibition of growth when the protein is essential. In this study, peptides that each specifically bind to one of seven essential proteins were identified by phage display and then expressed as fusions to glutathione S-transferase in Escherichia coli. Expression of peptide fusions directed against E. coli DnaN, LpxA, RpoD, ProRS, SecA, GyrA, and Era each dramatically inhibited cell growth. Under the same conditions, a fusion with a randomized peptide sequence did not inhibit cell growth. In growth-inhibited cells, inhibition could be relieved by concurrent overexpression of the relevant target protein but not by coexpression of an irrelevant protein, indicating that growth inhibition was due to a specific interaction of the expressed peptide with its target. The protein knockout technique can be used to assess the essentiality of genes of unknown function emerging from the sequencing of microbial genomes. This technique can also be used to validate proteins as drug targets, and their corresponding peptides as screening tools, for discovery of new antimicrobial agents.
Bacterial Proteins↗
Combination therapy with intravenous colistin for management of infections due to multidrug-resistant Gram-negative bacteria in patients without cystic fibrosis.
Colistin, an antibiotic almost abandoned for intravenous administration for many years due to its reported toxicity, has been recently reintroduced in clinical practice due to the emergence of multidrug-resistant gram-negative bacteria and the lack of development of new antibiotics to combat them. To assess the safety and effectiveness of intravenous colistin, in combination with other antimicrobial agents, in the treatment of serious infections in patients without cystic fibrosis, a retrospective cohort study in a 450-bed tertiary-care hospital in Athens, Greece, was performed. Patients who were hospitalized from 1 October 2000 to 31 January 2004 and received intravenous colistin for more than 72 h were further analyzed. The primary outcome measure was the in-hospital mortality; secondary end points were the clinical outcome of the infections and the occurrence of colistin toxicity. Fifty patients received intravenous colistin with a median (mean) daily dose of 3 (4.5) million IU for 16.5 (21.3) days for the management of 54 episodes of infections due to multidrug-resistant gram-negative bacteria. The predominant infections were pneumonia (33.3%), bacteremia (27.8%), urinary tract infection (11.1%), and intra-abdominal infection (11.1%). The responsible pathogens were Acinetobacter baumannii (51.9%), Pseudomonas aeruginosa (42.6%), and Klebsiella pneumoniae (3.7%) strains (no pathogen was isolated from one case). In-hospital mortality was 24% (12/50 patients). Clinical response (cure or improvement) of the infection was observed in 66.7% of episodes (36/54). In the studied group, serum creatinine levels were decreased, at the end of colistin treatment, by an average of 0.2 +/- 1.3 mg/dl compared to baseline levels. Deterioration of renal function during colistin therapy was observed in 4/50 patients (8%). Coadministration of other antimicrobial agents with spectrum against gram-negative microorganisms and the absence of a control group constitute the major limitations of this study. The use of intravenous colistin for the treatment of infections due to multidrug-resistant gram-negative bacteria appears to be safe and effective.
Adult↗
Influence of alternative electron acceptors on the anaerobic biodegradability of chlorinated phenols and benzoic acids.
Nitrate, sulfate, and carbonate were used as electron acceptors to examine the anaerobic biodegradability of chlorinated aromatic compounds in estuarine and freshwater sediments. The respective denitrifying, sulfidogenic, and methanogenic enrichment cultures were established on each of the monochlorinated phenol and monochlorinated benzoic acid isomers, using sediment from the upper (freshwater) and lower (estuarine) Hudson River and the East River (estuarine) as source materials. Utilization of each chlorophenol and chlorobenzoate isomer was observed under at least one reducing condition; however, no single reducing condition permitted the metabolism of all six compounds tested. The anaerobic biodegradation of the chlorophenols and chlorobenzoates depended on the electron acceptor available and on the position of the chlorine substituent. In general, similar activities were observed under the different reducing conditions in both the freshwater and estuarine sediments. Under denitrifying conditions, degradation of 3- and 4-chlorobenzoate was accompanied by nitrate loss corresponding reasonably to the stoichiometric values expected for complete oxidation of the chlorobenzoate to CO2. Under sulfidogenic conditions, 3- and 4-chlorobenzoate, but not 2-chlorobenzoate, and all three monochlorophenol isomers were utilized, while under methanogenic conditions all compounds except 4-chlorobenzoate were metabolized. Given that the pattern of activity appears different for these chlorinated compounds under each reducing condition, their biodegradability appears to be more a function of the presence of competent microbial populations than one of inherent molecular structure.
Bacteria, Anaerobic↗
Identification and functional characterization of arylamine N-acetyltransferases in eubacteria: evidence for highly selective acetylation of 5-aminosalicylic acid.
Arylamine N-acetyltransferase activity has been described in various bacterial species. Bacterial N-acetyltransferases, including those from bacteria of the gut flora, may be involved in the metabolism of xenobiotics, thereby exerting physiopathological effects. We characterized these enzymes further by steady-state kinetics, time-dependent inhibition, and DNA hybridization in 40 species, mostly from the human intestinal microflora. We report for the first time N-acetyltransferase activity in 11 species of Proteobacteriaceae from seven genera: Citrobacter amalonaticus, Citrobacter farmeri, Citrobacter freundii, Klebsiella ozaenae, Klebsiella oxytoca, Klebsiella rhinoscleromatis, Morganella morganii, Serratia marcescens, Shigella flexneri, Plesiomonas shigelloides, and Vibrio cholerae. We estimated apparent kinetic parameters and found that 5-aminosalicylic acid, a compound efficient in the treatment of inflammatory bowel diseases, was acetylated with a catalytic efficiency 27 to 645 times higher than that for its isomer, 4-aminosalicylic acid. In contrast, para-aminobenzoic acid, a folate precursor in bacteria, was poorly acetylated. Of the wild-type strains studied, Pseudomonas aeruginosa was the best acetylator in terms of both substrate spectrum and catalytic efficiency. DNA hybridization with a Salmonella enterica serovar Typhimurium-derived probe suggested the presence of this enzyme in eight proteobacterial and four gram-positive species. Molecular aspects together with the kinetic data suggest distinct functional features for this class of microbial enzymes.
Acetylation↗
The molecular biology of leukocyte chemoattractant receptors.
Leukocytes migrate from the blood to sites of inflammation in response to locally produced chemoattractants that activate specific cell surface receptors. The primary structures of leukocyte receptors for N-formyl peptides, C5a, platelet-activating factor, and 8 of the 18 known human chemokines (interleukin-8 and related molecules) have been deduced from cloned cDNAs. All of these are seven-transmembrane-domain rhodopsin-like G protein-coupled receptors. Biochemical and molecular genetic analysis of the chemoattractant receptors indicates that the chemoattractants may have both broadly overlapping as well as specialized roles in the regulation of acute and chronic inflammation. Interestingly, the chemokine receptors have functional homologues in human cytomegalovirus and Herpesvirus saimiri. Moreover, the Duffy antigen, which mediates invasion of erythrocytes by Plasmodium vivax, a major cause of malaria, is also a chemokine binding protein. These surprising developments suggest that in addition to leukocyte-mediated inflammation, the chemokines may also be involved in erythrocyte function and, through molecular mimicry, in microbial pathogenesis.
Amino Acid Sequence↗
Memories of microbes and metabolism.
As Jackie Gleason was wont to say: "How sweet it really is!" And--reflecting on the 1940s-1980s, when studies of microbial nutrition revealed exciting structure-function relationships of the B-complex vitamins with relevance to metabolism in humans--it really is. A chemistry degree from Beloit College and a doctorate in biochemistry from the University of Wisconsin set the stage for my life's work at Lederle Laboratories, the University of Illinois, and Vanderbilt University. At Lederle my research contributed to folic acid chemistry: coenzyme forms and function; antimetabolites and cancer chemotherapy. My subsequent university studies centered on lysine biosynthesis and metabolism, e.g. its precursor role in carnitine and in indolizidine alkaloids of physiological interest. There were also many opportunities to reach out and give something back to the system via teaching and diverse service activities, all of which has led to a happy, fulfilling career, one for which I am ever thankful.
Animal Nutritional Physiological Phenomena↗