Stimulation of dissimilatory sulfate reduction in response to sulfate in microcosm incubations from two contrasting temperate peatlands near Ithaca, NY, USA.

Peatlands are responsible for over half of wetland methane emissions, yet major uncertainties remain regarding carbon flow, especially when increased availability of electron acceptors stimulates competing physiologies. We used microcosm incubations to study the effects of sulfate on microorganisms in two temperate peatlands, one bog and one fen. Three different electron donor treatments were used (13C-acetate, 13C-formate and a mixture of 12C short-chain fatty acids) to elucidate the responses of sulfate-reducing bacteria (SRB) and methanogens to sulfate stimulation. Methane production was measured and metagenomic sequencing was performed, with only the heavy DNA fraction sequenced from treatments receiving 13C electron donors. Our data demonstrate stimulation of dissimilatory sulfate reduction in both sites, with contrasting community responses. In McLean Bog (MB), hydrogenotrophic Deltaproteobacteria and acetotrophic Peptococcaceae lineages of SRB were stimulated, as were lineages with unclassified dissimilatory sulfite reductases. In Michigan Hollow Fen (MHF), there was little stimulation of Peptococcaceae populations, and a small stimulation of Deltaproteobacteria SRB populations only in the presence of formate as electron donor. Sulfate stimulated an increase in relative abundance of reads for both oxidative and reductive sulfite reductases, suggesting stimulation of an internal sulfur cycle. Together, these data indicate a stimulation of SRB activity in response to sulfate in both sites, with a stronger growth response in MB than MHF. This study provides valuable insights into microbial community responses to sulfate in temperate peatlands and is an important first step to understanding how SRB and methanogens compete to regulate carbon flow in these systems.

Response of Isovalerate-Degrading Methanogenic Microbial Community to Inhibitors.

Isovalerate is one of the key intermediates during anaerobic digestion treating protein-containing waste/wastewater. Investigating the effect of different kinds of inhibitors on isovalerate-degrading microbial community is necessary to develop measures for improving the effectiveness of the treatment plants. In the present study, dynamic changes in the isovalerate-degrading microbial community in presence of inhibitors (ammonium, sulfide, mixed ammonium and sulfide, and chlortetracycline (CTC)) were investigated using high-throughput sequencing of 16S rRNA gene. Our observations showed that the isovalerate-degrading microbial community responded differently to different inhibitors and that the isovalerate degradation and gas production were strongly repressed by each inhibitor. We found that sulfide inhibited both isovalerate oxidation followed by methanogenesis, while ammonium, mixed ammonium and sulfide, and CTC mainly inhibited isovalerate oxidation. Genera classified into Proteobacteria and Chloroflexi were less sensitive to inhibitors. The two dominant genera, which are potential syntrophic isovalerate oxidizers, exhibited different responses to inhibitors that the unclassified_Peptococcaceae_3 was more sensitive to inhibitors than the unclassified_Syntrophaceae. Upon comparison to acetoclastic methanogen Methanosaeta, hydrogenotrophic methanogens Methanoculleus and Methanobacterium were less sensitive to inhibitors.

The effect of pH and buffer concentration on anode biofilms of Thermincola ferriacetica.

We assessed the effects of pH and buffer concentration on current production and growth of biofilms of Thermincola ferriacetica - a thermophilic, Gram-positive, anode-respiring bacterium (ARB) - grown on anodes poised at a potential of -0.06V vs. SHE in microbial electrolysis cells (MECs) at 60°C. T. ferriacetica generated current in the pH range of 5.2 to 8.3 with acetate as the electron donor and 50mM bicarbonate buffer. Maximum current density was reduced by ~80% at pH5.2 and ~14% at 7.0 compared to pH8.3. Increasing bicarbonate buffer concentrations from 10mM to 100mM resulted in an increase in the current density by 40±6%, from 6.8±1.1 to 11.2±2.7Am(-2), supporting that more buffer alleviated pH depression within T. ferriacetica biofilms. Confocal laser scanning microscopy (CLSM) images indicated that higher bicarbonate buffer concentrations resulted in larger live biofilm thicknesses: from 68±20µm at 10mM bicarbonate to >150µm at 100mM, supporting that buffer availability was a strong influence on biofilm thickness. In comparison to mesophilic Geobacter sulfurreducens biofilms, the faster transport rates at higher temperature and the ability to grow at relatively lower pH allowed T. ferriacetica to produce higher current densities with lower buffer concentrations.

Microbial community response to addition of polylactate compounds to stimulate hexavalent chromium reduction in groundwater.

To evaluate the efficacy of bioimmobilization of Cr(VI) in groundwater at the Department of Energy Hanford site, we conducted a series of microcosm experiments using a range of commercial electron donors with varying degrees of lactate polymerization (polylactate). These experiments were conducted using Hanford Formation sediments (coarse sand and gravel) immersed in Hanford groundwater, which were amended with Cr(VI) and several types of lactate-based electron donors (Hydrogen Release Compound, HRC; primer-HRC, pHRC; extended release HRC) and the polylactate-cysteine form (Metal Remediation Compound, MRC). The results showed that polylactate compounds stimulated an increase in bacterial biomass and activity to a greater extent than sodium lactate when applied at equivalent carbon concentrations. At the same time, concentrations of headspace hydrogen and methane increased and correlated with changes in the microbial community structure. Enrichment of Pseudomonas spp. occurred with all lactate additions, and enrichment of sulfate-reducing Desulfosporosinus spp. occurred with almost complete sulfate reduction. The results of these experiments demonstrate that amendment with the pHRC and MRC forms result in effective removal of Cr(VI) from solution most likely by both direct (enzymatic) and indirect (microbially generated reductant) mechanisms.

Inhibiting mild steel corrosion from sulfate-reducing and iron-oxidizing bacteria using gramicidin-S-producing biofilms.

A gramicidin-S-producing Bacillus brevis 18-3 biofilm was shown to reduce corrosion rates of mild steel by inhibiting both the sulfate-reducing bacterium Desulfosporosinus orientis and the iron-oxidizing bacterium Leptothrix discophora SP-6. When L. discophora SP-6 was introduced along with D. orientis to a non-antimicrobial-producing biofilm control, Paenibacillus polymyxa ATCC 10401, a corrosive synergy was created and mild steel coupons underwent more severe corrosion than when only D. orientis was present, showing a 2.3-fold increase via electrochemical impedance spectroscopy (EIS) and a 1.8-fold difference via mass-loss measurements. However, when a gramicidin-S-producing, protective B. brevis 18-3 biofilm was established on mild steel, the metal coupons were protected against the simultaneous attack of D. orientis and L. discophora SP-6. EIS data showed that the protective B. brevis 18-3 biofilm decreased the corrosion rate about 20-fold compared with the non-gramicidin-producing P. polymyxa ATCC 10401 biofilm control. The mass loss for the protected mild steel coupons was also significantly lower than that for the unprotected ones (4-fold decrease). Scanning electron microscope images corroborated the corrosion inhibition by the gramicidin-S-producing B. brevis biofilm on mild steel by showing that the metal surface remained untarnished, i.e., the polishing grooves were still visible after exposure to the simultaneous attack of the sulfate-reducing bacterium and the iron-oxidizing bacterium.

Inhibiting mild steel corrosion from sulfate-reducing bacteria using antimicrobial-producing biofilms in Three-Mile-Island process water.

Biofilms were used to produce gramicidin S (a cyclic decapeptide) to inhibit corrosion-causing, sulfate-reducing bacteria (SRB). In laboratory studies these biofilms protected mild steel 1010 continuously from corrosion in the aggressive, cooling service water of the AmerGen Three-Mile-Island (TMI) nuclear plant, which was augmented with reference SRB. The growth of both reference SRB (Gram-positive Desulfosporosinus orientis and Gram-negative Desulfovibrio vulgaris) was shown to be inhibited by supernatants of the gramicidin-S-producing bacteria as well as by purified gramicidin S. Electrochemical impedance spectroscopy and mass loss measurements showed that the protective biofilms decreased the corrosion rate of mild steel by 2- to 10-fold when challenged with the natural SRB of the TMI process water supplemented with D. orientis or D. vulgaris. The relative corrosion inhibition efficiency was 50-90% in continuous reactors, compared to a biofilm control which did not produce the antimicrobial gramicidin S. Scanning electron microscope and reactor images also revealed that SRB attack was thwarted by protective biofilms that secrete gramicidin S. A consortium of beneficial bacteria (GGPST consortium, producing gramicidin S and other antimicrobials) also protected the mild steel.

[Betadine for antisepsis of the conjunctival sac]. / Betadyna w antyseptyce worka spojówkowego.

12.5% solution of betadine is used for conjunctival sac antiseptics. To estimate effectiveness of betadine in 50 eyes, bacteriological examination was performed before and after local betadine application. Betadine is effective against Staphylococcus. Peptococcaceae were cultured after betadine application, in 3 cases.

Enumeration of transconjugated Ruminococcus albus and its survival in the goat rumen microcosm.

A transconjugant Ruminococcus albus A3 culture was released into a goat rumen, and the extent of its survival in the rumen microcosm was measured by distinguishing this bacterium from indigenous microbes by antibiotic resistance. A3 cells remained roughly constant for 14 days in this goat rumen.

[A cassette micromethod of determining the sensitivity of anaerobic microorganisms to antimicrobial drugs]. / Kassetnyi mikrometod opredeleniia chuvstvitel'nosti anaérobnykh mikroorganizmov k antibakterial'nym preparatam.

A micromethod based on the use of plates with wells and microquantities of microbial suspensions is described. It provides determination of MICs of antibacterial drugs and sensitivity of clinical strains of anaerobes of 2 or 3 species predominating in pathological materials as well as to preliminarily identify some anaerobic bacteria by their antibiotic sensitivity.

[Effects of antibiotics and anti-bacterial components of preparations for local treatment of suppurative wounds on pathogens of odonto- genic infections]. / Deistvie na vozbuditelei odontogennoi infektsii antibiotikov i antibakterial'nykh komponentov, vkhodiashchikh v sostav preparatov dlia mestnogo lecheniia gnoinykh ran.

The results of identification and sensitivity assay of 156 strains of pathogens causing odontogenic infections are presented. In the sensitivity assay antibacterial drugs were used. 42.3 percent of the strains were obligate anaerobes belonging to Bacteroides, Fusobacterium, Peptococcus, Peptostreptococcus, Veillonella and Actinomyces. Significant differences in the microbial sensitivity to the drugs used for general and local therapy were detected. There was observed high sensitivity of the obligate anaerobes to gramicidin (0.02 micrograms/ml), nitazol (10 micrograms/ml), levomycetin and tetracycline (60 micrograms/ml). Antiseptics such as dioxidine and chlorhexidine used locally showed satisfactory results. The above mentioned drugs and especially levomycetin were also rather active against facultative organisms in associations of pathogens causing odontogenic infections: Bacillus coagulans, B. licheniformis, Pseudomonas sp., Acinetobacter calcoaceticus, Staphylococcus sp. and Streptococcus sp.

In vitro activity of LY264826 compared to other glycopeptides and daptomycin.

LY264826 a new naturally occurring glycopeptide inhibited 90% of methicillin-susceptible and -resistant Staphylococcus aureus at 1 micrograms/ml. LY264826 had similar activity against methicillin-susceptible and -resistant coagulase-negative staphylococci. The LY264826 MIC90 for Streptococcus pyogenes was 0.25 microgram/ml, twofold more active than vancomycin and twofold less active than teicoplanin. LY264826 was eightfold more active than vancomycin and twofold more active than teicoplanin against enterococci. LY264826 inhibited Streptococcus pneumoniae at 0.25 microgram/ml and Listeria monocytogenes at 0.5 microgram/ml. Clostridium were inhibited by less than or equal to 0.25 microgram/ml of LY264826 and peptococci, peptostreptococci, and Fusobacterium were inhibited by less than 0.5 microgram/ml. Bacteroides species were LY284826 -resistant as were all Enterobacteriaceae, Flavobacterium, and Neisseria spp. Minimum bactericidal and inhibitory concentrations (MBCs and MICs) were within a dilution for S. aureus, S. pyogenes, and S. pneumoniae, but greater than or equal to 32-fold greater for enterococci.

Cellobiose uptake and metabolism by Ruminococcus flavefaciens.

The cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 utilizes cellobiose but not glucose as a substrate for growth. Cellobiose uptake by R. flavefaciens FD-1 was measured under anaerobic conditions (N2), using [G-3H]cellobiose. The rate of cellobiose uptake for early- or late-log-phase cellobiose-grown cells was 9 nmol/min per mg of whole-cell protein. Cellobiose uptake was inhibited by electron transport inhibitors, iron-reactive compounds, proton ionophores, sulfhydryl inhibitors, N,N-dicyclohexylcarbodiimide, and NaF, as well as lasalocid and monensin. The results support the existence of an active transport system for cellobiose. Transport of [U-14C]glucose was not detected with this system. Phosphorylation of cellobiose was not by a phosphoenolpyruvate-dependent system. Cellobiose phosphorylase activity was detected by both a coupled spectrophotometric assay and a discontinuous assay. The enzyme was produced constitutively in cellobiose-grown cells at a specific activity of 329 nmol/min per mg of cell-free extract protein.

Effects of methanol on the growth of gastrointestinal anaerobes.

The effects of methanol on the growth of representative, predominant, anaerobic gut bacteria were studied. Growth yields and rates were determined in a base medium to which methanol was added to produce media with methanol concentrations varying, in twofold steps, over a concentration range of 0.01 to 25%, by volume. The growth of many of the organisms was completely inhibited by a methanol concentration equal to, or less than, 6.2%. Isolates representing cellulolytic species were completely inhibited at a methanol concentration of 3.1%, and inhibitory effects on the yield of some cellulolytic isolates were found at a methanol concentration as small as 0.01%. Although most of the organisms studied were inhibited at relatively small methanol concentrations, isolates of Selenomonas ruminantium, Bacteroides ovatus, and Fusobacterium necrophorum were relatively methanol resistant. A methanol concentration of 12.5% was required to completely inhibit S. ruminantium. Substantial growth of B. ovatus was obtained in media containing 12.5% methanol, and for F. necrophorum, substantial growth occurred in media containing 25% methanol. The yields of F. necrophorum strain B85 and S. ruminantium strain PC18 were enhanced by relatively small methanol concentrations and reduced with further methanol concentration increase Anaerobic, nonsporing gut bacteria exhibit a diversity of responses to methanol.

In vitro activity of flomoxef compared to moxalactam, cefoxitin, cefotaxime, and clindamycin against anaerobes.

To assess the in vitro activity of flomoxef (6315-S), moxalactam, cefoxitin, cefotaxime, and clindamycin against anaerobes 197 clinical isolates (27 Bacteroides fragilis, 42 B. thetaiotaomicron, 10 B. vulgatus, 7 B. ovatus, 6 B. uniformis, 6 B. distasonis, 7 Bacteroides melaninogenicus group, 11 Bacteroides oralis group, 21 Clostridium difficile, 7 C. perfringens, 3 C. sporogenes, 3 Clostridium spp., 33 Propionibacterium acnes, 14 Peptococcaceae) were studied by means of agar dilution tests. The MIC90 of B. fragilis was less than 2 micrograms/ml for flomoxef, less than 4 micrograms/ml for moxalactam, less than 16 micrograms/ml for cefoxitin, less than 128 micrograms/ml for cefotaxime and less than 2 micrograms/ml for clindamycin. The respective MIC90's of B. thetaiotaomicron were less than 64, less than 128, less than 32, less than 256 and 8 micrograms/ml. Strains of the other Bacteroides species and groups were more susceptible to flomoxef and the other antibiotics than B. thetaiotaomicron. Against Clostridium difficile flomoxef (MIC90 less than 4 micrograms/ml) proved to be superior to the other agents tested. Most of the Clostridium strains other than C. difficile were also susceptible to flomoxef; anaerobic grampositive cocci and Propionibacterium acnes were very sensitive (MIC90's less than 1 and less than or equal to 0.125 micrograms/ml, respectively). Its anti-anaerobic activity, together with its efficacy against aerobes, should make flomoxef a useful adjunct to the arsenal of modern antibiotic therapy.

Analysis of antibiotic susceptibility and extrachromosomal DNA content of Ruminococcus albus and Ruminococcus flavefaciens.

Seventeen Ruminococcus albus and Ruminococcus flavefaciens strains have been screened for naturally occurring antibiotic resistance, as determined by zones of inhibition from antibiotic disks. These strains were also examined for extrachromosomal DNA content. All strains screened are resistant to low levels (10-200 micrograms/mL) of streptomycin. In contrast to the previously reported data, we have found that R. flavefaciens C-94 is now susceptible to both kanamycin and tetracycline. However, R. flavefaciens FD-1 is not susceptible to kanamycin (minimum inhibitory concentration (MIC) = 40 micrograms/mL). Furthermore, R. albus 8 is resistant to tetracycline (MIC = 40 micrograms/mL), and erythromycin (MIC = 100 micrograms/mL). Six freshly isolated strains showed resistance to tetracycline (35-70 micrograms/mL), and all tetracycline-resistant strains also showed resistance to minocycline. None of these Ruminococcus determinants share homology with the streptococcal tetL, tetM, or tetN determinants. All 17 strains were screened for extrachromosomal DNA content. Nine different techniques for the detection and isolation of extrachromosomal DNA were tested. However, owing to difficulties in demonstrating or isolating plasmid DNA, it has not been possible to determine if these antibiotic resistance genes are plasmid borne. Evidence is presented to suggest that the presence of oxygen may affect the quality of the DNA obtained from Ruminococcus.

[In vitro activity of an ofloxacin-metronidazole combination against anaerobic bacteria. Kinetics of the action of metronidazole against Bacteroides fragilis]. / Activité in vitro de l'association ofloxacine-métronidazole sur les anaérobies stricts. Cinétique d'action du métronidazole sur Bacteroides fragilis.

The activity of metronidazole combined with ofloxacin was investigated by the checkerboard method in liquid medium against 60 obligate anaerobes. The bacteriostatic effect of the combination was assessed by calculating the FIC index. Two metronidazole resistant strains of Propionibacterium acnes (MIC greater than 32 mg/l) were inhibited by 0.125 mg/l of this former antibiotic in presence of an ofloxacin concentration equal to half the value of the MIC. On the 58 other anaerobic strains, the combination of metronidazole plus ofloxacin had an additive bacteriostatic effect (30 strains) or a synergistic effect (26 strains). No antagonism was noted with any strain. For selected anaerobic or mixed infections the combination of metronidazole and ofloxacin may be useful. Killing curves demonstrated that, under good anaerobic conditions, metronidazole acted rapidly against Bacteroides fragilis. At a concentration of 8 mg/l, a decrease of log10 of the population was observed after 2 hours. After a contact ranging from 1 to 8 hours, depending of the investigated strains, a bactericidal effect was observed with metronidazole.

[Sensitivity of clinical strains of facultatively anaerobic bacteria to antimicrobial drugs]. / Chuvstvitel'nost' klinicheskikh shtammov obligatno anaérobnykh bakterii k antimikrobnym preparatom.

Six hundred and sixty five samples of clinical materials from patients with various pyoinflammatory diseases were tested for obligatory anaerobes. Anaerobes were detected in 148 samples which amounted to 22.3 per cent of the total number of the samples and to 33.2 per cent of the samples with microbial growth. A total of 171 strains of obligatory anaerobes were isolated. Among them 58.5, 24.5, 16.4 and 0.6 per cent were nonsporulating gramnegative bacilli, grampositive cocci, grampositive bacilli and gramnegative cocci respectively. Sensitivity of the isolated anaerobes was tested with the disk diffusion method. The most active drugs against the tested strains were: nitroxoline, rifampicin, metronidasole, erythromycin, carbenicillin and cefotaxim (4.2, 4.5, 9.3, 10.6, 11.5 and 11.7 per cent of the resistant strains respectively). Gentamicin, polymyxin M, novobiocin and cefazoline were the least active drugs (94.6, 78.9, 65.4 and 50.0 per cent of the resistant strains respectively). Metronidasole, levomycetin, nitroxolin, rifampicin and furazolidone showed the highest activity against bacteroids of the fragilis group (0, 0, 0, 8 and 12.5 per cent of the resistant strains respectively) while gentamicin, polymyxin M, cefazolin, oxacillin, novobiocin and penicillin showed the lowest activity (100, 100, 100, 100, 87.0 and 66.7 per cent of the resistant strains respectively).

Selective suppression of the anaerobic oropharyngeal microflora with local metronidazole.

Anaerobic microorganisms of the normal oropharyngeal flora have been shown to be the main pathogens in orofacial infections of odontogenic origin. Reduction of the density of anaerobes in the oral cavity as a prophylactic measure before oral surgery may be a rational way to reduce the frequency of post-operative infections. This report describes a local antibiotic regimen that can reduce anaerobic pathogens in the oral cavity. Over a period of 7 days, 10 healthy individuals applied locally 1.5-2 g of an antibiotic preparation consisting of 0.5% metronidazole in 99.5% Orabase paste three times daily. The paste had antibacterial activity against obligate anaerobic micro-organisms such as bacteroides, fusobacteria and leptotrichia, known pathogens in orofacial infections. Fusobacteria and leptotrichia were eliminated in all subjects. Bacteroides species were eliminated in five subjects and significantly decreased in three subjects. The aerobic microflora was not affected. No new colonisation of the oropharynx was observed during the treatment period. The pre-treatment numbers of different micro-organisms were re-established within 2-9 days after the withdrawal of metronidazole treatment.

In vitro activity of ampicillin plus sulbactam against anaerobes compared to ampicillin and cefoxitin.

The antimicrobial susceptibility of 195 recent clinical isolates of anaerobic bacteria was studied to ampicillin alone, ampicillin + 1 mg/l sulbactam, ampicillin + 5 mg/l sulbactam, and cefoxitin by means of agar dilution tests. The ampicillin-sulbactam combinations were the most effective drugs against species of the Bacteroides fragilis group, the MIC90 of ampicillin + 5 mg/l sulbactam for B. fragilis being less than 1 mg/l, compared to 256 mg/l of ampicillin, 4 mg/l of ampicillin + 1 mg/l sulbactam, and 8 mg/l of cefoxitin. No significant difference between ampicillin alone and in combination with sulbactam was observed against gram-positive anaerobic rods, Peptococcus spp. and Peptostreptococcus spp. with MIC's less than 2 mg/l.