Facultative Organisms Research Articles

Saccharomyces cerevisiae, a model to study sterol uptake and transport in eukaryotes

The molecular mechanisms that govern intracellular transport of sterols in eukaryotic cells are only poorly understood. Saccharomyces cerevisiae is a facultative anaerobic organism that requires supplementation with unsaturated fatty acids and sterols to grow in the absence of oxygen, as the synthesis of these lipids requires molecular oxygen. The fact that yeast grows well under anaerobic conditions indicates that lipid uptake is rapid and efficient. To identify components in this lipid uptake and transport pathway, we screened the yeast mutant collection for genes that are essential under anaerobic conditions. Out of the approx. 4800 non-essential genes represented in the mutant collection, 37 were required for growth under anaerobic conditions. Uptake assays using radiolabelled cholesterol revealed that 16 of these genes are required for cholesterol uptake/transport and esterification. Further characterization of the precise role of these genes is likely to advance our understanding of this elusive pathway in yeast and may prove to be relevant to understand sterol homoeostasis in higher eukaryotic cells.

The presence of four iron-containing superoxide dismutase isozymes in Trypanosomatidae: Characterization, subcellular localization, and phylogenetic origin in Trypanosoma brucei

Metalloenzymes such as the superoxide dismutases (SODs) form part of a defense mechanism that helps protect obligate and facultative aerobic organisms from oxygen toxicity and damage. Here, we report the presence in the trypanosomatid genomes of four SOD genes: soda, sodb1, sodb2, and a newly identified sodc. All four genes of Trypanosoma brucei have been cloned ( Tbsods), sequenced, and overexpressed in Escherichia coli and shown to encode active dimeric FeSOD isozymes. Homology modeling of the structures of all four enzymes using available X-ray crystal structures of homologs showed that the four TbSOD structures were nearly identical. Subcellular localization using GFP-fusion proteins in procyclic insect trypomastigotes shows that TbSODB1 is mainly cytosolic, with a minor glycosomal component, TbSODB2 is mainly glycosomal with some activity in the cytosol, and TbSODA and TbSODC are both mitochondrial isozymes. Phylogenetic studies of all available trypanosomatid SODs and 106 dimeric FeSODs and closely related cambialistic dimeric SOD sequences suggest that the trypanosomatid SODs have all been acquired by more than one event of horizontal gene transfer, followed by events of gene duplication.

Bacteriology of chronic maxillary sinusitis associated with nasal polyposis

Aspirates from 48 chronically inflamed maxillary sinuses from patients who had nasal polyposis were processed for aerobic and anaerobic bacteria. Bacterial growth was present in 46 (96%) specimens. Aerobic or facultative bacteria were present in 6 (13%) specimens, anaerobic bacteria alone in 18 (39%), and mixed aerobic and anaerobic bacteria in 22 (48%). There were 110 bacterial isolates (2.4 per specimen). Thirty-nine of the isolates were aerobic or facultative organisms (0.85 per specimen). The predominant aerobic or facultative organisms were: Staphylococcus aureus, microaerophilic streptococci, Haemophilus influenzae and Moraxella catarrhalis. Seventy-one anaerobes were isolated (1.5 per specimen), Peptostreptococcus spp., Prevotella spp., Porphyromonas asaccharolytica and Fusobacterium spp. being predominant. These findings illustrate for the first time the presence of polymicrobial aerobic-anaerobic flora in patients with chronic maxillary sinusitis who had nasal polyposis.

Oxalate‐degrading Providencia rettgeri isolated from human stools

Oxalate-degrading bacteria are thought to metabolize intestinal oxalate and thus decrease the urinary excretion of oxalate by reducing its intestinal absorption. We have isolated several novel oxalate-degrading bacteria from human stools. Oxalate degrading bacteria were investigated to characterize their protein profiles with antibodies against oxalyl-coenzyme A decarboxylase (65 kDa) and formyl-coenzyme A transferase (48 kDa) purified from Oxalobacter formigenes. One of these isolates was identified as Providencia rettgeri, which showed two proteins (65 kDa and 48 kDa) on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) that were not found in non-oxalate-degrading P. rettgeri. Antibodies reacted with the 65 and 48 kDa proteins from the P. rettgeri strain on Western blotting. An Oxalobacter formigenes formyl-coenzyme A transferase gene probe reacted with chromosomal DNA from P. rettgeri on Southern blotting under high stringency conditions, while an Oxalobacter formigenes oxalyl-coenzyme A decarboxylase gene probe did not react under the same conditions. The mechamism of oxalate degradation by P. rettgeri appears to be similar to that of Oxalobacter formigenes. This is the first report of a facultative oxalate-degrading organism that is one of the Enterobacteriaceae.

Avian mycoplasmosis update

Avian mycoplasmas occur in a variety of bird species. The most important mycoplasmas for chickens and turkeys are Mycoplasma gallisepticum (MG), M. synoviae (MS), and M. meleagridis. Besides, M. iowe (MI) is an emerging pathogen in turkeys, but of little concern for chickens. Mycoplasmas are bacteria that lack cell wall and belong to the class Mollicutes. Although they have been considered extracellular agents, scientists admit nowadays that some of them are obligatory intracellular microorganisms, whereas all other mycoplasmas are considered facultative intracellular organisms. Their pathogenic mechanism for disease include adherence to host target cells, mediation of apoptosis, innocent bystander damage to host cell due to intimate membrane contact, molecular (antigen) mimicry that may lead to tolerance, and mitotic effect for B and/or T lymphocytes, which could lead to suppressed T-cell function and/or production of cytotoxic T cell, besides mycoplasma by-products, such as hydrogen peroxide and superoxide radicals. Moreover, mycoplasma ability to stimulate macrophages, monocytes, T-helper cells and NK cells, results in the production of substances, such as tumor necrosing factor (TNF-alpha), interleukin (IL-1, 2, 6) and interferon (<FONT FACE=Symbol>a, b, g</FONT>). The major clinical signs seen in avian mycoplasmosis are coughing, sneezing, snicks, respiratory rales, ocular and nasal discharge, decreased feed intake and egg production, increased mortality, poor hatchability, and, primarily in turkeys, swelling of the infraorbital sinus(es). Nevertheless, chronic and unapparent infections are most common and more threatening. Mycoplasmas are transmitted horizontally, from bird to bird, and vertically, from dam to offspring through the eggs. Losses attributed to mycoplasmosis, mainly MG and MS infections, result from decreased egg production and egg quality, poor hatchability (high rate of embryonic mortality and culling of day-old birds), poor feed efficiency, increase in mortality and carcass condemnations, besides medication costs. Mycoplasmas are diagnosed by serologic tests, culture and PCR and are sensitive to antimicrobials whose action sites are other than the bacterial cell wall, such as tetracyclines, macrolides, quinolones and tiamulin. However, mycoplasma control is more efficiently achieved by acquisition of birds free of MG, MS, MM and/or MI, vaccination of layers, and monitoring of breeder flocks, followed by elimination of the infected flocks that are detected.

Isolation and characterization of enteric bacteria from the hindgut of Formosan termite

The Formosan subterranean termite, Coptotermes formosanus Shiraki, is an aggressive, invasive termite species that has caused billions of dollars of damage across the United States for the past 50 years. Termites depend on intestinal microorganisms for cellulose digestion. Symbiotic microorganisms in the termite gut play key physiological functions such as cellulose and hemicellulose digestion, acetogenesis, hydrogenesis, methanogenesis, sulfate reduction, and nitrogen fixation. Additionally, intestinal microbes create suitable conditions for symbiotic protozoans through the production of nutrients and the maintenance of the pH and the anaerobic conditions in the gut. Although extensive research has been done on the symbiotic relationship of these termites and the microbes found in its gut, there is little information available on the role of facultative anaerobes in the gut. We isolated four enteric bacteria from the hindgut of Formosan subterranean termite, C. formosanus. All isolates were facultative anaerobes and G−. The isolates were identified as Serratia marcescens, Enterobacter aerogens, Enterobacter cloacae, and Citrobacter farmeri by using BIOLOG assay and fatty acid methyl ester analysis (FAME). Each isolate was characterized using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and biochemical study. This is the first report on the presence of facultative microbes in termite gut. Results of this first study on facultative microbes in the termite gut indicate that the role of facultative organisms in the Formosan termite gut may be to scavenge oxygen and create anaerobic conditions for the anaerobic microorganisms, which are essential for digestion of cellulose consumed by the termite.

Interaction of Klebsiella oxytoca and Burkholderia cepacia in Dual-Species Batch Cultures and Biofilms as a Function of Growth Rate and Substrate Concentration

Dual-species microbial interactions have been extensively reported for batch and continuous culture environments. However, little research has been performed on dual-species interaction in a biofilm. This research examined the effects of growth rate and substrate concentration on dual-species population densities in batch and biofilm reactors. In addition, the feasibility of using batch reactor kinetics to describe dual-species biofilm interactions was explored. The scope of the research was directed toward creating a dual-species biofilm for the biodegradation of trichloroethylene, but the findings are a significant contribution to the study of dual-species interactions in general. The two bacterial species used were Burkholderia cepacia PR1-pTOM(31c), an aerobic organism capable of constitutively mineralizing trichloroethylene (TCE), and Klebsiella oxytoca, a highly mucoid, facultative anaerobic organism. The substrate concentrations used were different dilutions of a nutrient-rich medium resulting in dissolved organic carbon (DOC) concentrations on the order of 30, 70, and 700 mg/L. Presented herein are single- and dual-species population densities and growth rates for these two organisms grown in batch and continuous-flow biofilm reactors. In batch reactors, planktonic growth rates predicted dual-species planktonic species dominance, with the faster-growing organism (K. oxytoca) outcompeting the slower-growing organism (B. cepacia). In a dual-species biofilm, however, dual-species planktonic growth rates did not predict which organism would have the higher dual-species biofilm population density. The relative fraction of each organism in a dual-species biofilm did correlate with substrate concentration, with B. cepacia having a greater proportional density in the dual-species culture with K. oxytoca at low (30 and 70 mg/L DOC) substrate concentrations and K. oxytoca having a greater dual-species population density at a high (700 mg/L DOC) substrate concentration. Results from this research demonstrate the effectiveness of using substrate concentration to control population density in this dual-species biofilm.

Anaerobic Pulmonary Infections in Children

Pulmonary infections due to anaerobic bacteria usually occur in children prone to aspiration. The source of the anaerobic bacteria is the oropharyngeal bacterial flora, where these organisms outnumber aerobic and facultative organisms in a 10:1 ratio. The most common lower respiratory tract infections where anaerobic bacteria are recovered mixed with aerobic organisms are aspiration pneumonia, lung abscess, and empyema. The predominant isolated anaerobic bacteria are Peptostreptococcus, Fusobacterium, pigmented Prevotella, and Porphyromonas spp. and Bacteroides fragilis group. Management of these infections includes the administration of antimicrobials effective against the anaerobic as well as the aerobic pathogens.

Applied and ecological aspects of oxidative-stress damage to bacterial spores and to oral microbes.

Bacterial cells have adapted in a variety of ways to resist oxidative stresses and damage in their everyday lives in a predominantly aerobic world. The nearly universal occurrence of resistance mechanisms against oxidative stresses, particularly those due to reactive oxygen species (ROS), suggests that most, if not all, bacteria have to deal with oxidative assaults. A primary source of oxidative stress is aerobic metabolism, which leads to production of ROS such as hydrogen peroxide, superoxide radical, perhydroxyl radical, hydroxyl radical and a variety of other toxic metabolites, including organic peroxides and other organics or inorganics able to transfer electrons to sites of oxidative damage. Anaerobes as well as aerobic and facultative organisms are subject to oxidative stresses, often as a result of their own metabolism of O2 or that of associated facultative organisms. If anaerobes would just ignore oxygen instead of metabolizing it, they would not have to deal with toxic metabolites of their own making. Another major source of oxidative stress comes from the use of oxidative agents in the disinfection-sterilization industry. Notable examples are hypochlorite for water purification and hydrogen peroxide used for industrial sterilization. Antimicrobials such as isoniazide and mitomycin C also act oxidatively to cause damage. In this article, aseptic packaging and processing involving use of hydrogen peroxide for sterilization of packaging materials is reviewed as an example of oxidative stress imposed on bacterial spores and vegetative cells from outside the organisms or the microbial community. The other example considered is related to oral microbiology and infectious disease in which oxidative stress may arise from the metabolism of the oral microbiota or may come from outside through use of oral care products.

Inhibition of Apoptosis in Sheep Monocytes by Brucella melitensis 16M

Bishor, V.I., Singh, D.K., Sinha, D.K. and Goswami, T.K. 2004. Inhibition of apoptosis in sheep monocytes by Brucella melitensis 163VI. J. Appl. Anim. Res., 25: 65–68. Brucella is a facultative intracellular organism of domestic animals and man. The mechanism for its survival is not well understood. The effect of Brucella in modulation of monocytes and neutrophil apoptosis in sheep was analysed. The apoptosis of neutrophils was not affected by the Brucella melitensis 16M (live or killed). However, the live B. melitensis 16M prolonged apoptosis of monocytes/macrophages separated from sheep in vitro.

Antimicrobial Use in Septic Shock

Journal of Gynecologic SurgeryVol. 20, No. 1 Infectious DiseasesAntimicrobial Use in Septic ShockNewton G. OsborneNewton G. OsborneSearch for more papers by this authorPublished Online:6 Jul 2004https://doi.org/10.1089/104240604323022025AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetails Volume 20Issue 1Mar 2004 To cite this article:Newton G. Osborne.Antimicrobial Use in Septic Shock.Journal of Gynecologic Surgery.Mar 2004.29-31.http://doi.org/10.1089/104240604323022025Published in Volume: 20 Issue 1: July 6, 2004PDF download

The intestinal microbiota and probiotics in irritable bowel syndrome

Irritable bowel syndrome (IBS) is a poorly understood gastrointestinal condition affecting approximately onefifth of the UK population, with a higher prevalence in women and accounting for up to half of referrals to gastroenterology clinics in the UK. It is characterized by abdominal pain, excessive flatus, variable bowel habit and abdominal bloating with no evidence of organic disease. IBS commonly occurs after gastroenteritis or following a course of antibiotics. Suggested aetiologies include motility and psychological disorders and psychophysiological phenomena, although there is also evidence that disruption to the intestinal microbiota can play a role in IBS. Episodes such as hysterectomy, first incidence of bacterial gastroenteritis or a course of antibiotics have been shown to contribute to symptoms compatible with those of IBS. The intestinal microflora in IBS has been shown to differ from that of healthy individuals. Faecal microfloras of IBS patients har bour higher numbers of facultative organisms, such as Klebsiella spp. and enterococci, and low numbers of lactobacilli and bifidobacteria. The role of probiotics in IBS has not been clearly defined. Some studies have shown improvements in pain and flatulence in response to probiotic supplementation, while others have shown no symptomatic or objective improvements. Administration of probiotics reduced caecal and faecal yeast proliferation in IBS patients treated with antibiotics, suggesting that they prevent the disruption of the intestinal microbiota known to be associated with IBS. It is possible that the role of probiotics in the management of IBS will lie in prevention, rather than cure. Keywords: IBS; microflora; probiotics

Comparison of linezolid activities under aerobic and anaerobic conditions against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium.

Methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium were exposed to linezolid (MIC of 2 mg/liter) under aerobic or anaerobic conditions in an in vitro pharmacodynamic model. Drug concentration and half-life were adjusted to simulate clinical dosing (600 mg twice daily) of linezolid. Linezolid produced a 2-log(10) killing at 24 h, and rates of killing against each of these facultative organisms as measured by mean survival time appeared similar under aerobic and anaerobic conditions.

Rapid translocation of polarized MDCK cell monolayers by Leptospira interrogans, an invasive but nonintracellular pathogen.

Pathogenic spirochetes of the genus Leptospira are a major cause of human zoonotic infectious disease worldwide. After gaining entry through the skin, the organism causes disease by hematogenously disseminating to multiple organs. The mechanism by which it penetrates the mammalian cell barriers to disseminate is not well understood. In this study, we used a low-passage-number isolate of Leptospira interrogans to elucidate the invasive potential of this spirochete. Quantification of bacteria by dark-field microscopy revealed that pathogenic spirochetes were able to translocate through polarized MDCK cell monolayers at a rate significantly greater than that of nonpathogenic Leptospira or a recognized invasive bacterial pathogen, Salmonella: In contrast to Salmonella, L. interrogans did not alter transepithelial electrical resistance during cell translocation. Both transmission and scanning electron microscopy revealed tight association of the extracellular spirochetes with the host cell plasma membrane, without membrane perturbations suggestive of cytoskeletal rearrangement. Spirochetes were not observed within intercellular junctions or membrane-bound compartments inside cells. They were found within the cytoplasm of only 8% of the counted cells. These results indicate that Leptospira is an invasive but not a facultative intracellular organism. We propose that the rapid translocation of mammalian cells by pathogenic Leptospira is a mechanism designed to evade killing by host cells that permits the organism to quickly reach the bloodstream and disseminate to multiple organs.

A review of the role of the gut microflora in irritable bowel syndrome and the effects of probiotics.

Irritable bowel syndrome (IBS) is a multi-factorial gastrointestinal condition affecting 8-22 % of the population with a higher prevalence in women and accounting for 20-50 % of referrals to gastroenterology clinics. It is characterised by abdominal pain, excessive flatus, variable bowel habit and abdominal bloating for which there is no evidence of detectable organic disease. Suggested aetiologies include gut motility and psychological disorders, psychophysiological phenomena and colonic malfermentation. The faecal microflora in IBS has been shown to be abnormal with higher numbers of facultative organisms and low numbers of lactobacilli and bifidobacteria. Although there is no evidence of food allergy in IBS, food intolerance has been identified and exclusion diets are beneficial to many IBS patients. Food intolerance may be due to abnormal fermentation of food residues in the colon, as a result of disruption of the normal flora. The role of probiotics in IBS has not been clearly defined. Some studies have shown improvements in pain and flatulence in response to probiotic administration, whilst others have shown no symptomatic improvement. It is possible that the future role of probiotics in IBS will lie in prevention, rather than cure.

Bacteriology of acute and chronic frontal sinusitis.

Aspirates of 15 acutely and 13 chronically infected frontal sinuses were processed for aerobic and anaerobic bacteria. A total of 20 isolates (1.3 per specimen) were recovered from the 15 cases of acute frontal sinusitis, 16 aerobic and facultative isolates (1.1 per specimen) and 4 anaerobic isolates (0.3 per specimen). Aerobic and facultative organisms alone were recovered in 13 specimens (87%), and mixed aerobic and anaerobic bacteria were recovered in 2 (13%). The predominant aerobic and facultative organisms were Haemophilus influenzae (6), Streptococcus pneumoniae (5), and Moraxella catarrhalis (3). A total of 32 isolates were recovered from the 13 cases (2.5 per patient) of chronic frontal sinusitis, 12 aerobic and facultative isolates (0.9 per specimen) and 20 anaerobic isolates (1.5 per specimen). Aerobic and facultative organisms only were recovered in 3 instances (23%), anaerobes only in 7 instances (54%), and mixed aerobic and anaerobic bacteria in 3 instances (23%). The predominant aerobic bacteria were gram-negative bacilli (H influenzae, Klebsiella pneumoniae, and Pseudomonas aeruginosa). The predominant anaerobes included Prevotella species (8), Peptostreptococcus species (6), and Fusobacterium species (4). These findings illustrate the microbiologic features of acute and chronic frontal sinusitis.

Microbiota of Periapical Lesions Refractory to Endodontic Therapy

The periapical microbiota of 36 teeth with refractory apical periodontitis was investigated. None of the teeth had responded to conventional endodontic or long-term (> 6 months), calcium-hydroxide treatment. Eight patients had received antibiotics systemically. After anaerobic culture, a total of 148 microbial strains were detected among 67 microbial species. One of the 36 lesions was culture-negative. Approximately half (51.0%) of the bacterial strains were anaerobic. Gram-positive species constituted 79.5% of the flora. Facultative organisms, such as Staphylococcus, Enterococcus, Enterobacter, Pseudomonas, Stenotrophomonas, Sphingomonas, Bacillus, or Candida species were recovered from 27 of the lesions (75%). Sulfur granules were found in 9 lesions (25%). In these granules Actinomyces israelii, A. viscosus, A. naeslundii, and A. meyeri were identified. Other bacterial species, both gram-positive and gram-negative, were detected in the granules as well. Two sulfur granules did not contain Actinomyces. Scanning electron microscopy demonstrated rod- and spirochete-like cells in the granules, and transmission electron microscopy revealed organisms with copious amounts of extracellular material. Outer membrane vesicles were also seen. Some of the granules were calcified. This study demonstrated a wide variety of microorganisms, particularly gram-positive ones, in the periapical lesions of teeth with refractory apical periodontitis.

A novel application of gene arrays: Escherichia coli array provides insight into the biology of the obligate endosymbiont of tsetse flies.

Symbiotic associations with microorganisms are pivotal in many insects. Yet, the functional roles of obligate symbionts have been difficult to study because it has not been possible to cultivate these organisms in vitro. The medically important tsetse fly (Diptera: Glossinidae) relies on its obligate endosymbiont, Wigglesworthia glossinidia, a member of the Enterobacteriaceae, closely related to Escherichia coli, for fertility and possibly nutrition. We show here that the intracellular Wigglesworthia has a reduced genome size smaller than 770 kb. In an attempt to understand the composition of its genome, we used the gene arrays developed for E. coli. We were able to identify 650 orthologous genes in Wigglesworthia corresponding to approximately 85% of its genome. The arrays were also applied for expression analysis using Wigglesworthia cDNA and 61 gene products were detected, presumably coding for some of its most abundant products. Overall, genes involved in cell processes, DNA replication, transcription, and translation were found largely retained in the small genome of Wigglesworthia. In addition, genes coding for transport proteins, chaperones, biosynthesis of cofactors, and some amino acids were found to comprise a significant portion, suggesting an important role for these proteins in its symbiotic life. Based on its expression profile, we predict that Wigglesworthia may be a facultative anaerobic organism that utilizes ammonia as its major source of nitrogen. We present an application of E. coli gene arrays to obtain broad genome information for a closely related organism in the absence of complete genome sequence data.

Hemochromatosis as a Risk Factor for Fulminant Staphylococcus aureus Pneumonia*

Hemochromatosis as a Risk Factor for Fulminant Staphylococcus aureus Pneumonia*

Coping with formaldehyde during C1 metabolism of Paracoccus denitrificans

Methylotrophic bacteria are capable of growth using reduced one-carbon (C1) compounds like methanol or methylamine as free energy sources. Paracoccus denitrificans, which is a facultative methylotrophic organism, switches to this type of autotrophic metabolism only when it experiences a shortage of available heterotrophic free energy sources. Since the oxidation of C1 substrates is energetically less favourable than that of the heterotrophic ones, a global regulatory circuit ensures that the enzymes involved in methylotrophic growth are repressed during heterotrophic growth. Once the decision is made to switch to methylotrophic growth, additional regulatory proteins ensure the fine-tuned expression of the participating enzymes such that the steady-state concentration of formaldehyde, the oxidation product of C1 substrates, is kept below cytotoxic levels.