The antibacterial activity of vancomycin towards Staphylococcus aureus under aerobic and anaerobic conditions

AIMS: To investigate the antibacterial efficacy of vancomycin towards Staphylococcus aureus under aerobic and anaerobic conditions, and to assess the influence of oxygen on the duration of the post-antibiotic effect (PAE) after exposure to vancomycin. METHODS AND RESULTS: Culture-based techniques and flow cytometric measurements of 5-cyano-2,3-ditolyl tetrazolium chloride (an indicator of redox activity) and the membrane potential-sensitive fluorophore Sytox Green, were used to test four staphylococcal strains. The MICs for all strains, and the duration of PAE, were similar whether tested with or without oxygen. However, a fivefold logarithmic reduction in cell counts was observed in 10-15 h aerobically, depending on strain, compared with longer than 60 h in an anaerobic environment. Flow cytometric data correlated well with counts of colony-forming units under both aerobic and anaerobic conditions. CONCLUSIONS: The death rate of Staph. aureus exposed to vancomycin was greater in the presence of oxygen, although MIC values and PAE durations were similar whether tested aerobically or anaerobically. Also, flow cytometry provided a rapid and sensitive alternative to plate counts for the assessment of antibiotics in oxygen-free conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study underlines the need for further anaerobic testing using different strain/antibiotic combinations, the results of which will have clinical significance due to the anaerobic nature of some sites of infection.     

Proton motive force generation from stored polymers for the uptake of acetate under anaerobic conditions

The bacteria facilitating enhanced biological phosphorus removal gain a selective advantage from intracellularly stored polymer-driven substrate uptake under anaerobic conditions during sequential anaerobic : aerobic cycling. Mechanisms for these unusual membrane transport processes were proposed and experimentally validated using selective inhibitors and highly-enriched cultures of a polyphosphate-accumulating organism, Accumulibacter, and a glycogen-accumulating organism, Competibacter. Acetate uptake by both Accumulibacter and Competibacter was driven by a proton motive force (PMF). Stored polymers were used to generate the PMF -Accumulibacter used phosphate efflux through the Pit transporter, while Competibacter generated a PMF by proton efflux through the ATPase and fumarate reductase in the reductive TCA cycle.     

Metabolic profiling of Staphylococcus aureus cultivated under aerobic and anaerobic conditions with (1)H NMR-based nontargeted analysis

Staphylococcus aureus is a major pathogen in the medical area and food-producing sector. Detailed analyses of its basic cell physiology will help comprehensively understand this pathogen, which will be useful for developing novel diagnostic and treatment tools. Oxygen is one of the most crucial growth-limiting factors for S. aureus. In this study, to characterize and distinguish metabolic profiles of S. aureus cultivated under aerobic and anaerobic conditions, nontargeted analyses of both types of cultures were carried out using (1)H nuclear magnetic resonance spectroscopy. Fifty compounds were identified by Chenomx software. Characteristics of metabolic profiles were achieved by using principal components analysis. During aerobic growth, S. aureus mainly consumed glucose, alanine, arginine, glycine, isoleucine, leucine, phenylalanine, and acetate. Meanwhile, it accumulated 17 metabolites, mainly 2-oxoglutarate, isobutyrate, isovalerate, succinate, and ethanol. Under anaerobic condition, S. aureus mainly consumed glucose, arginine, and threonine. Meanwhile, it accumulated 13 metabolites, mainly ethanol, lactate, and ornithine. The representative metabolites that could most significantly differentiate metabolic profiles of S. aureus were isobutyrate, isovalerate, and succinate in aerobic cultivation; and lactate, ethanol, and ornithine in anaerobic cultivation. Among these metabolites, isobutyrate and ornithine were present only in aerobic and anaerobic culture, respectively.     

Interaction of H+ and K+ transport systems in E. coli growing under anaerobic and aerobic conditions

The interaction of H+-ATPase complex F1 X F0 with the Trk system of K+ accumulation in E. coli grown quasi-anaerobically in pepton media with glucose (anaerobia) and aerobically in the salt medium with succinate (aerobia) treated with cyanide was studied. The ratio of H+ fluxes via F1 X F0 and K+ fluxes via the Trk system is stable and equals 2 in anaerobia and is changed from 0.5 to 5.0 in aerobia treated with cyanide in response to pH variation, K+ activity and temperature variations. Q10 is about 2.8 both for F1 X F0 and the Trk system in anaerobia, but 2.4 and 1.0 respectively in aerobia. K+ distribution in anaerobia reaches high values, K+ equilibrium potential is much higher than the measured membrane potential. K+ distribution in aerobia is smaller, which is in conformity with the measured membrane potential. Structural association of F1 X F0 and the Trk system with the formation of H+--K+-pump is assumed to take place in anaerobia, and separate operation of these systems occurs in aerobia, transfer of K+ via Trk system being energized by the electric field on the membrane.     

Proton motive force in washed cells of Rhizobium japonicum and bacteroids from Glycine max.

The components of the proton motive force (delta p), namely the membrane potential and the transmembrane pH gradient, were measured in washed cells of Rhizobium japonicum CC705 grown in cultures (5% O2-95% N2) in the presence of 10 mM KNO3 and in bacteroids from Glycine max. The delta p and its components remained reasonably constant in cells as well as in bacteroids at various stages of growth. The effects of uncouplers and ATPase inhibitors on the delta p and its components were determined in both cultured cells and bacteroids. The data indicated that a respiration-driven H+ translocation is the source of the delta p in both cultured cells and bacteroids.     

Measurements of bovine sperm velocities under true anaerobic and aerobic conditions.

Velocities of bovine spermatozoa in a medium containing glucose were similar under true anaerobic and aerobic conditions. Spermatozoa were not able to sustain motility under anaerobic conditions when glycolysis was inhibited, but regained motility when re-aerated. This demonstrates that immobilisation was due to lack of oxygen and that conditions under which motility was analysed were truly anaerobic. Sperm motility parameters were not significantly different in the presence and absence of 4 microM antimycin A and 4 microM rotenone when glucose was present in the medium. After each incubation, functionality of sperm mitochondria was assayed by washing sperm into the medium which supported respiration but not glycolysis, and motility was visually assessed. All sperm samples were highly motile in this medium indicating that their mitochondria were functional. When glycolysis was inhibited, antimycin and rotenone abolished sperm motility immediately after addition. Bovine sperm can maintain similar levels of motility aerobically and anaerobically if a glycolysable substrate is available. Available data on bovine sperm energetics support this view.     

Hydrogen evolution by strictly aerobic hydrogen bacteria under anaerobic conditions.

When strains and mutants of the strictly aerobic hydrogen-oxidizing bacterium Alcaligenes eutrophus are grown heterotrophically on gluconate or fructose and are subsequently exposed to anaerobic conditions in the presence of the organic substrates, molecular hydrogen is evolved. Hydrogen evolution started immediately after the suspension was flushed with nitrogen, reached maximum rates of 70 to 100 mumol of H2 per h per g of protein, and continued with slowly decreasing rates for at least 18 h. The addition of oxygen to an H2-evolving culture, as well as the addition of nitrate to cells (which had formed the dissimilatory nitrate reductase system during the preceding growth), caused immediate cessation of hydrogen evolution. Formate is not the source of H2 evolution. The rates of H2 evolution with formate as the substrate were lower than those with gluconate. The formate hydrogenlyase system was not detectable in intact cells or crude cell extracts. Rather the cytoplasmic, NAD-reducing hydrogenase is involved by catalyzing the release of excessive reducing equivalents under anaerobic conditions in the absence of suitable electron acceptors. This conclusion is based on the following experimental results. H2 is formed only by cells which had synthesized the hydrogenases during growth. Mutants lacking the membrane-bound hydrogenase were still able to evolve H2. Mutants lacking the NAD-reducing or both hydrogenases were unable to evolve H2.     

Amine production by Streptococcus lactis under different growth conditions

The highest amount of histamine, tyramine and tryptamine were produced by S. lactis at 30 degrees C in 24 h at pH = 5.0. Maximum amount of different amines was noted in a growth medium lacking NaCl. After addition of NaCl even at 0.5% concentration, slight inhibitory effect on the synthesis of these amines was obtained.     

Swarming characteristics of Proteus mirabilis under anaerobic and aerobic conditions

Nutrients have a pronounced effect on the growth and swarming behaviour of Proteus mirabilis 7002. Iron, zinc, amino acids, and dioxygen are important for rapid growth and normal swarming. Anaerobically grown cultures of P. mirabilis 7002 were unable to swarm on anaerobically maintained rich nutrient agar. Upon exposure to aerobic conditions, P. mirabilis 7002 resumed swarming behaviour. Scanning electron microscopy was used to demonstrate the presence of community organization and mature rafts during normal swarming. These results support the importance of dioxygen and redox status in cell differentiation.     

Siderophore production by Pseudomonas stutzeri under aerobic and anaerobic conditions

The siderophore production of the facultative anaerobe Pseudomonas stutzeri, strain CCUG 36651, grown under both aerobic and anaerobic conditions, was investigated by liquid chromatography and mass spectrometry. The bacterial strain has been isolated at a 626-m depth at the Äspö Hard Rock Laboratory, where experiments concerning the geological disposal of nuclear waste are performed. In bacterial culture extracts, the iron in the siderophore complexes was replaced by gallium to facilitate siderophore identification by mass spectrometry. P. stutzeri was shown to produce ferrioxamine E (nocardamine) as the main siderophore together with ferrioxamine G and two cyclic ferrioxamines having molecular masses 14 and 28 atomic mass units lower than that of ferrioxamine E, suggested to be ferrioxamine D₂ and ferrioxamine X₁, respectively. In contrast, no siderophores were observed from anaerobically grown P. stutzeri. None of the siderophores produced by aerobically grown P. stutzeri were found in anaerobic natural water samples from the Äspö Hard Rock Laboratory.In order to facilitate iron(III) acquisition, plants and microorganisms, such as fungi and bacteria, produce and excrete strong iron(III) chelators, i.e., siderophores (18, 22, 23, 33, 34). While fungal siderophores bind to iron(III) by hydroxamate ligands, bacterial siderophores are more structurally diverse, and common ligands are catecholates, hydroxamates, and carboxylates (21). The iron(III) stability constants for bacterial siderophores vary in the range of 10²⁰ to 10⁵² (6). In addition to iron(III), other metals can be complexed by siderophores. For the trihydroxamate siderophore desferrioxamine B, sometimes called proferrioxamine B (10), some actinides have been shown to have stability constants in the same range as the ferric stability constant (10^30.6), e.g., 10^26.6 with thorium(IV) and 10^30.8 with plutonium(VI) (32), while the stability constant for uranium(VI) was lower, i.e., 10¹⁸ (2).Concerning bacteria, there are several reports on siderophore production by Pseudomonas spp. (1, 3, 4, 19). More than 50 structurally related siderophores, i.e., pyoverdins, produced by the fluorescent Pseudomonas spp., especially Pseudomonas fluorescens and Pseudomonas aeruginosa, have been characterized (3). All pyoverdins emit yellow fluorescent light due to the presence of a 5-amino-2,3-dihydro-8,9-dihydroxy-1-H-pyrimido-quinoline-carboxylic chromophore, to which a peptide chain and a carboxyl chain are attached (1, 3). Nonfluorescent Pseudomonas has also been shown to produce siderophores, such as ferrioxamine E, also called nocardamine (Fig. ​(Fig.1),1), which was produced by one strain of Pseudomonas stutzeri (19). In addition to ferrioxamines, the P. stutzeri strain KC produced a smaller siderophore, i.e., pyridine-2,6-bis(thiocarboxylic acid) (35). Conversely, a catecholate-type siderophore was shown to be produced by another strain of P. stutzeri, which did not produce any hydroxamate siderophores (4).Open in a separate windowFIG. 1.Structures, molecular masses (mw), and stability constants (K_s) of ferric complexes of the three ferrioxamines: ferrioxamine B (B), ferrioxamine E (E), and ferrioxamine G (G) (5, 18).Most of the studies on bacterial siderophore production have been conducted on microorganisms growing under aerobic conditions. One field-based report, however, indicates the occurrence of putative siderophores in anaerobic environments also (29). In the present study, siderophore production has been studied with both aerobic and anaerobic cultures of P. stutzeri. This species is a facultative aerobe, able to grow with oxygen or nitrate as the electron acceptor, meaning that it can be active under both anaerobic and aerobic conditions. The P. stutzeri strain CCUG 36651, studied here, has been isolated from a depth of 626 m below ground at the Äspö Hard Rock Laboratory (16), where research concerning the geological disposal of nuclear waste is performed. The possibility of mobilizing radionuclides by complexing compounds from bacteria is an important research area in the context of nuclear waste disposal research. It is unknown if such compounds are produced in aquifers under conditions relevant to a disposal site, which would be approximately 500 m underground in granitic rock (27).A study from 2004 shows that P. stutzeri growing aerobically in the presence of uranium-containing shale leached Fe, Mo, V, and Cr from the shale material (17). More recently it was shown that the supernatant of aerobically and anaerobically cultured P. stutzeri was able to increase the partitioning of added Fe, Pm, Am, and Th into the aqueous phase in samples where quartz sand was used as a solid surface (16). Aerobic supernatants maintained 60% or more of the added metals in solution, while anaerobic supernatants were best at maintaining Am in solution, reaching a value of 40% in solution. The increased partitioning to the aqueous phase in the presence of the supernatants was ascribed to the production of organic ligands. Supernatants of both aerobically and anaerobically grown P. stutzeri strain CCUG 36651 yielded a positive response on the universal siderophore assay, the CAS assay (16). This assay is based on ligand competition for iron bound to the colored chrome azurol complex (25, 30).In this study, siderophore production by P. stutzeri strain CCUG 36651 was investigated using mass spectrometry (MS) and liquid chromatography (LC) followed by mass spectrometric detection. Electrospray ionization mass spectrometry (ESI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) are useful tools in characterizing siderophores such as ferrioxamines (10, 13, 14, 28, 31). In order to detect iron(III)-chelating compounds, the ferric iron can be replaced by gallium(III) through ascorbate-mediated reduction of iron(III) (8, 20). In mass spectra, gallium-bound substances are easily recognized due to the characteristic isotope pattern of gallium, where the intensity of the ⁷¹Ga signal is about 66% of that of the ⁶⁹Ga signal. The use of ESI provides so-called soft ionization; thus, information about the molecular weight is obtained. However, by employing MS/MS, fragmentation is achieved, providing more information about the compound structure.In order to verify the chemical difference between the siderophores found by ESI-MS, chromatographic separation was performed. In this case, one reversed-phase C₁₈ column and one column containing a porous graphitic carbon (PGC) stationary phase were used. The separation mechanism of PGC is a combination of hydrophobic interactions, as in C₁₈, and electrostatic interactions between π-electrons. In order to detect substances at low concentrations, column-switched capillary chromatography with MS detection was used. The detection limits of the combined LC-MS/MS system used in this study are in the range of 1 to 5 nM for hydroxamate siderophores of the ferrichrome and ferrioxamine families (9). In order to facilitate analysis of lower concentrations of ferrioxamines, natural water samples were preconcentrated by solid-phase extraction (SPE), resulting in minimum detectable concentrations in the range of 0.02 to 0.1 nM, depending on the initial sample volume.     

Detoxification of furfural in Corynebacterium glutamicum under aerobic and anaerobic conditions

The toxic fermentation inhibitors in lignocellulosic hydrolysates raise serious problems for the microbial production of fuels and chemicals. Furfural is considered to be one of the most toxic compounds among these inhibitors. Here, we describe the detoxification of furfural in Corynebacterium glutamicum ATCC13032 under both aerobic and anaerobic conditions. Under aerobic culture conditions, furfuryl alcohol and 2-furoic acid were produced as detoxification products of furfural. The ratio of the products varied depending on the initial furfural concentration. Neither furfuryl alcohol nor 2-furoic acid showed any toxic effect on cell growth, and both compounds were determined to be the end products of furfural degradation. Interestingly, unlike under aerobic conditions, most of the furfural was converted to furfuryl alcohol under anaerobic conditions, without affecting the glucose consumption rate. Both the NADH/NAD⁺ and NADPH/NADP⁺ ratio decreased in the accordance with furfural concentration under both aerobic and anaerobic conditions. These results indicate the presence of a single or multiple endogenous enzymes with broad and high affinity for furfural and co-factors in C. glutamicum ATCC13032.     

A blood-free enrichment medium for growing Campylobacter spp. under aerobic conditions

Detection limits for Campylobacter jejuni strains JH93 and ATCC 29428 in a new blood-free enrichment broth (BFEB) were investigated under aerobic conditions. Cultures of Camp. jejuni were inoculated into 50 ml BFEB containing 10% food homogenate in 50 ml screw-cap tubes. After 24 h enrichment under aerobic conditions, Camp. jejuni were isolated on four selective agar media. The least squares means of the detection limit 50% endpoint (DL₅₀) values were 0·4 (plain BFEB), 0·9 (crabmeat), 1·7 (mushroom), 1·7 (raw milk) and 2·1 (oyster) colony forming units (cfu) 5 g⁻¹ food. The efficiency of the BFEB was significantly affected ( P < 0·05) by food type and bacterial strain. Overall, the BFEB enrichment compared favourably with the existing US Food and Drug Administration method under modified atmosphere. In addition, the BFEB method did not require the use of blood, special equipment or Oxyrase^® to reduce oxygen tensions.