Simple and Versatile Turbidimetric Monitoring of Bacterial Growth in Liquid Cultures Using a Customized 3D Printed Culture Tube Holder and a Miniaturized Spectrophotometer: Application to Facultative and Strictly Anaerobic Bacteria

ABSTRACT Clostridioides difficile is a major cause of diarrhea associated with antibiotherapy. After germination of C. difficile spores in the small intestine, vegetative cells are exposed to low oxygen (O2) tensions. While considered strictly anaerobic, C. difficile is able to grow in nonstrict anaerobic conditions (1 to 3% O2) and tolerates brief air exposure indicating that this bacterium harbors an arsenal of proteins involved in O2 detoxification and/or protection. Tolerance of C. difficile to low O2 tensions requires the presence of the alternative sigma factor, σB, involved in the general stress response. Among the genes positively controlled by σB, four encode proteins likely involved in O2 detoxification: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). As previously observed for FdpF, we showed that both purified revRbr1 and revRbr2 harbor NADH-linked O2- and H2O2-reductase activities in vitro, while purified FdpA mainly acts as an O2-reductase. The growth of a fdpA mutant is affected at 0.4% O2, while inactivation of both revRbrs leads to a growth defect above 0.1% O2. O2-reductase activities of these different proteins are additive since the quadruple mutant displays a stronger phenotype when exposed to low O2 tensions compared to the triple mutants. Our results demonstrate a key role for revRbrs, FdpF, and FdpA proteins in the ability of C. difficile to grow in the presence of physiological O2 tensions such as those encountered in the colon. IMPORTANCE Although the gastrointestinal tract is regarded as mainly anoxic, low O2 tension is present in the gut and tends to increase following antibiotic-induced disruption of the host microbiota. Two decreasing O2 gradients are observed, a longitudinal one from the small to the large intestine and a second one from the intestinal epithelium toward the colon lumen. Thus, O2 concentration fluctuations within the gastrointestinal tract are a challenge for anaerobic bacteria such as C. difficile. This enteropathogen has developed efficient strategies to detoxify O2. In this work, we identified reverse rubrerythrins and flavodiiron proteins as key actors for O2 tolerance in C. difficile. These enzymes are responsible for the reduction of O2 protecting C. difficile vegetative cells from associated damages. Original and complex detoxification pathways involving O2-reductases are crucial in the ability of C. difficile to tolerate O2 and survive to O2 concentrations encountered in the gastrointestinal tract.

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Mercury Methylation and Demethylation in Anoxic Lake Sediments and by Strictly Anaerobic Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.64.3.1013-1017.1998 ◽

1998 ◽

Vol 64 (3) ◽

pp. 1013-1017 ◽

Cited By ~ 119

Author(s):

K.-R. Pak ◽

R. Bartha

Keyword(s):

Organic Matter ◽

Anaerobic Bacteria ◽

Mercury Methylation ◽

Strictly Anaerobic ◽

Anoxic Sediment ◽

Acetogenic Bacteria ◽

High Potentials ◽

High Mercury ◽

Pure Cultures ◽

Sediment Ph

ABSTRACT After spiking anoxic sediment slurries of three acidic oligotrophic lakes with either HgCl2 at 1.0 μg/ml or CH3HgI at 0.1 μg/ml, both mercury methylation and demethylation rates were measured. High mercury methylation potentials were accompanied by high demethylation potentials in the same sediment. These high potentials correlated positively with the concentrations of organic matter and dissolved sulfate in the sediment and with mercury levels in fish. Adjustment of the acidic sediment pH to neutrality failed to influence either the methylation or the demethylation rate of mercury. The opposing methylation and demethylation processes converged to establish similar Hg2+-CH3Hg+equilibria in all three sediments. Because of their metabolic dominance in anoxic sediments, mercury methylation and demethylation in pure cultures of sulfidogenic, methanogenic, and acetogenic bacteria were also measured. Sulfidogens both methylated and demethylated mercury, but the methanogen tested only catalyzed demethylation and the acetogen neither methylated nor demethylated mercury.

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Decarboxylating and Nondecarboxylating Glutaryl-Coenzyme A Dehydrogenases in the Aromatic Metabolism of Obligately Anaerobic Bacteria

Journal of Bacteriology ◽

10.1128/jb.00205-09 ◽

2009 ◽

Vol 191 (13) ◽

pp. 4401-4409 ◽

Cited By ~ 29

Author(s):

Simon Wischgoll ◽

Martin Taubert ◽

Franziska Peters ◽

Nico Jehmlich ◽

Martin von Bergen ◽

...

Keyword(s):

Coenzyme A ◽

Anaerobic Bacteria ◽

Oxidative Decarboxylation ◽

Kinetic Properties ◽

Strictly Anaerobic ◽

Amino Acid Residues ◽

Geobacter Metallireducens ◽

Sulfate Reducing ◽

Reverse Transcription Pcr ◽

Gene Coding

ABSTRACT In anaerobic bacteria using aromatic growth substrates, glutaryl-coenzyme A (CoA) dehydrogenases (GDHs) are involved in the catabolism of the central intermediate benzoyl-CoA to three acetyl-CoAs and CO2. In this work, we studied GDHs from the strictly anaerobic, aromatic compound-degrading organisms Geobacter metallireducens (GDHGeo) (Fe[III] reducing) and Desulfococcus multivorans (GDHDes) (sulfate reducing). GDHGeo was purified from cells grown on benzoate and after the heterologous expression of the benzoate-induced bamM gene. The gene coding for GDHDes was identified after screening of a cosmid gene library. Reverse transcription-PCR revealed that its expression was induced by benzoate; the product was heterologously expressed and isolated. Both wild-type and recombinant GDHGeo catalyzed the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA at similar rates. In contrast, recombinant GDHDes catalyzed only the dehydrogenation to glutaconyl-CoA. The latter compound was decarboxylated subsequently to crotonyl-CoA by the addition of membrane extracts from cells grown on benzoate in the presence of 20 mM NaCl. All GDH enzymes were purified as homotetramers of a 43- to 44-kDa subunit and contained 0.6 to 0.7 flavin adenine dinucleotides (FADs)/monomer. The kinetic properties for glutaryl-CoA conversion were as follows: for GDHGeo, the Km was 30 ± 2 μM and the V max was 3.2 ± 0.2 μmol min−1 mg−1, and for GDHDes, the Km was 52 ± 5 μM and the V max was 11 ± 1 μmol min−1 mg−1. GDHDes but not GDHGeo was inhibited by glutaconyl-CoA. Highly conserved amino acid residues that were proposed to be specifically involved in the decarboxylation of the intermediate glutaconyl-CoA were identified in GDHGeo but are missing in GDHDes. The differential use of energy-yielding/energy-demanding enzymatic processes in anaerobic bacteria that degrade aromatic compounds is discussed in view of phylogenetic relationships and constraints of overall energy metabolism.

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Cyclohexa-1,5-Diene-1-Carbonyl-Coenzyme A (CoA) Hydratases of Geobacter metallireducens and Syntrophus aciditrophicus: Evidence for a Common Benzoyl-CoA Degradation Pathway in Facultative and Strict Anaerobes

Journal of Bacteriology ◽

10.1128/jb.01467-06 ◽

2006 ◽

Vol 189 (3) ◽

pp. 1055-1060 ◽

Cited By ~ 49

Author(s):

Franziska Peters ◽

Yoshifumi Shinoda ◽

Michael J. McInerney ◽

Matthias Boll

Keyword(s):

Coenzyme A ◽

Anaerobic Bacteria ◽

Degradation Pathway ◽

Ring Cleavage ◽

Strictly Anaerobic ◽

Significant Activity ◽

Geobacter Metallireducens ◽

Sulfate Reducing ◽

Coa Reductase ◽

Syntrophus Aciditrophicus

ABSTRACT In the denitrifying bacterium Thauera aromatica, the central intermediate of anaerobic aromatic metabolism, benzoyl-coenzyme A (CoA), is dearomatized by the ATP-dependent benzoyl-CoA reductase to cyclohexa-1,5-diene-1-carbonyl-CoA (dienoyl-CoA). The dienoyl-CoA is further metabolized by a series of β-oxidation-like reactions of the so-called benzoyl-CoA degradation pathway resulting in ring cleavage. Recently, evidence was obtained that obligately anaerobic bacteria that use aromatic growth substrates do not contain an ATP-dependent benzoyl-CoA reductase. In these bacteria, the reactions involved in dearomatization and cleavage of the aromatic ring have not been shown, so far. In this work, a characteristic enzymatic step of the benzoyl-CoA pathway in obligate anaerobes was demonstrated and characterized. Dienoyl-CoA hydratase activities were determined in extracts of Geobacter metallireducens (iron reducing), Syntrophus aciditrophicus (fermenting), and Desulfococcus multivorans (sulfate reducing) cells grown with benzoate. The benzoate-induced genes putatively coding for the dienoyl-CoA hydratases in the benzoate degraders G. metallireducens and S. aciditrophicus were heterologously expressed and characterized. Both gene products specifically catalyzed the reversible hydration of dienoyl-CoA to 6-hydroxycyclohexenoyl-CoA (Km , 80 and 35 μM; V max, 350 and 550 μmol min−1 mg−1, respectively). Neither enzyme had significant activity with cyclohex-1-ene-1-carbonyl-CoA or crotonyl-CoA. The results suggest that benzoyl-CoA degradation proceeds via dienoyl-CoA and 6-hydroxycyclohexanoyl-CoA in strictly anaerobic bacteria. The steps involved in dienoyl-CoA metabolism appear identical in all nonphotosynthetic anaerobic bacteria, although totally different benzene ring-dearomatizing enzymes are present in facultative and obligate anaerobes.

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A Culture Tube for the Separation of Anaerobic Bacteria

American Journal of Clinical Pathology ◽

10.1093/ajcp/37.6_ts.667 ◽

1962 ◽

Vol 37 (6_ts) ◽

pp. 667-668

Author(s):

E. M. Stapert ◽

W. N. DeWolff ◽

W. T. Sokolski

Keyword(s):

Anaerobic Bacteria ◽

Culture Tube

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Dynamics of nasopharyngitis in children

Otolaryngology ◽

10.1067/mhn.2000.105417 ◽

2000 ◽

Vol 122 (5) ◽

pp. 696-700

Author(s):

Itzhak Brook ◽

Alan E. Gober

Keyword(s):

Bacterial Growth ◽

Anaerobic Bacteria ◽

Purulent Discharge ◽

Nasal Discharge ◽

Bacterial Interaction ◽

Prevotella Species ◽

Group 2 ◽

Aerobic And Anaerobic ◽

Fusobacterium Species ◽

Group 1

PURPOSE: Our goal was to characterize the dynamics and bacterial interaction of the aerobic and anaerobic flora of nasal discharge of children at different stages of uncomplicated nasopharyngitis. METHODS AND PATIENTS: Serial semiquantitative nasopharyngeal (NP) and quantitative nasal discharge (ND) cultures were taken every 3 to 5 days from 20 children in whom purulent discharge eventually developed (group 1), and a single culture was obtained from a group of 20 who had only clear discharge (group 2). RESULTS: Aerobic and anaerobic bacteria were isolated from all NP cultures. Bacterial growth was present in 8 (40%) NDs of group 2. Only 7 (35%) of the clear NDs of group 1 showed bacterial growth; the number increased to 14 (70%) at the mucoid stage and 20 (100%) at the purulent stage. It declined to 6 (30%) at the final clear stage. The number of species and total number of organisms increased in the NDs of group 1. Group 1 patients had higher recovery rates of Streptococcus pneumoniae and Haemophilus influenzae in their NP cultures than group 2 patients ( P < 0.05). During the purulent stage, Peptostreptococcus species were isolated in 15 (75%), Fusobacterium species in 10 (50%), Prevotella species in 9 (45%), H influenzae in 8 (40%), S pneumoniae in 6 (30%), and β-hemolytic streptococci in 5 (25%) of group 1 NDs. This was higher than their recovery in the clear stages of both groups and the mucoid stage of group 1. A total of 8 organisms capable of interfering with the growth of potential pathogens were isolated from the NPs of group 1, as compared with 35 from group 2 ( P < 0.001). CONCLUSIONS: The development of purulent nasopharyngitis is associated with the pre-existing presence of potential pathogens and the absence of interfering organisms.

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Efficiency of various bacterial suspensions derived from cecal floras of conventional chickens in reducing the population level of Salmonella typhimurium in gnotobiotic mice and chicken intestines

Canadian Journal of Microbiology ◽

10.1139/m85-155 ◽

1985 ◽

Vol 31 (9) ◽

pp. 832-838 ◽

Cited By ~ 27

Author(s):

Sylvie Hudault ◽

Hilaire Bewa ◽

Chantal Bridonneau ◽

Pierre Raibaud

Keyword(s):

Salmonella Typhimurium ◽

Volatile Fatty Acids ◽

Anaerobic Bacteria ◽

Population Level ◽

Antagonistic Effect ◽

Strictly Anaerobic ◽

Antagonistic Bacteria ◽

Fecal Flora ◽

Intestinal Colonization ◽

Gnotobiotic Mice

The antagonistic effect exerted towards Salmonella typhimurium by the flora issued from conventional chickens was studied in gnotobiotic animals. In germfree chickens and mice inoculated with S. typhimurium, the highest bacterial counts were observed in ceca, and were not significantly different in either host. The protection afforded by the inoculation of cecal flora issued from a conventional chicken was more effective when this flora was inoculated first into germfree chickens than when it was given only after inoculation with S. typhimurium. Administration of a cecal flora from a 15-day-old chick to gnotobiotic mice and chicken resulted in the inhibition of a further intestinal colonization by S. typhimurium in both hosts. Sixteen strains were isolated among the predominant populations of the fecal flora from chicken flora recipient mice. Association of 14 strains of strictly anaerobic bacteria with 2 strains of Escherichia coli and Streptococcus faecium only decreased the number of S. typhimurium in the ileum of gnotobiotic mice, but not in their cecum. Anaerobe cultures were obtained from 10−6 and 10−8 dilutions prepared from the fecal flora of gnotobiotic recipient mice. Antagonistic bacteria were present only in cultures from the 10−6 dilution. Cecal concentrations of volatile fatty acids were shown not to be the sole factor implicated in the antagonistic effect against S. typhimurium.

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Anaerobic bacteria as indicators of faecal pollution

Proceedings of the Royal Society of Edinburgh Section B Biological Sciences ◽

10.1017/s0269727000003110 ◽

1980 ◽

Vol 78 (3-4) ◽

pp. s161-s169 ◽

Cited By ~ 1

Author(s):

D. W. F. Wheater ◽

D. Mara ◽

A. Opara ◽

P. Singleton

Keyword(s):

Intestinal Tract ◽

Anaerobic Bacteria ◽

Anaerobic Growth ◽

Strictly Anaerobic ◽

Gram Positive ◽

E Coli ◽

Faecal Pollution ◽

Aqueous Environments ◽

Intestinal Pathogens ◽

Special Circumstances

SynopsisIn several well-authenticated instances intestinal pathogens, includingSalmonellaspecies, have been isolated from water in the absence of bacteria, such asEscherichia coli,commonly used to detect faecal pollution. The present study examines certain anaerobic, non-sporing commensals of the intestinal tract as alternative ‘indicator’ bacteria. The numbers of bifidobacteria andBacteroides fragilisrecovered from faecal specimens were between 109 and 1010 per gram while the Gram-positive, anaerobic cocci numbered only about 106per gram. In sewage, this numerical difference was eliminated by a rapid loss of viability of bifidobacteria andB. fragilisso that the counts of all three types of bacteria approximated to those ofE. coli.Storage tests with aerated and non-aerated sewage established that further loss of viability of bifidobacteria andB. fragilisoccurred only fairly slowly. In samples of water from the Dighty Water and River Tay Estuary, shown to be faecally polluted, the three anaerobic types of bacteria were recovered in numbers roughly equal to those ofE. coliand their persistence in the surface of the bed of the Dighty Water was also at least equal to that ofE. coli.These results demonstrated that, in spite of their strictly anaerobic growth requirements, Bifidobacteria,B. fragilisand the Gram-positive, anaerobic cocci persist in aerobic, aqueous environments. If their habitat can be shown to be reasonably restricted to the intestinal tract they are likely, under special circumstances, to be useful indicators of faecal pollution.

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