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.

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.

Susceptibility of anaerobic bacteria to cefoperazone and other beta-lactam antibiotics.

The in vitro activity of cefoperazone was tested against 155 clinical isolates of anaerobic bacteria and compared to that of cefoxitin, LY127935 (moxalactam), cefotaxime, cefamandole, and cephalothin. Against the Bacteroides fragilis group, the compound was as active as LY127935, but less active than cefoxitin. Cefotaxime was less active than cefoperazone, cefoxitin, and LY127935. Cefoperazone, as well as cefoxitin, LY127935, and cefotaxime, exhibited excellent activity against all other anaerobic organisms tested. Cefamandole and cephalothin showed inferior activity when compared to the other four drugs.

In vitro activity of tiamulin (81.723 HFU), a new pleuromulin derivative, against clinically significant anaerobes.

The susceptibility of more than 40 strains of Gram-negative and Gram-positive anaerobes to tiamulin (Sandoz 81.723 hfu), a new pleuromulin (pleuromutilin) derivative, was determined by broth dilution and agar dilution tests. The influences of density of the inoculum upon MICs was studied by a specially designed pour plate-technique. Bacteroides fragilis, B. vulgatus, B. splanchnicus, B. oralis, B. asaccharolyticus, B. melaninogenicus, Fusobacterium fusiforme (F. nucleatum), Sphaerophorus necrophorus, Clostridium perfringens, C. fallax, Propionibacterium acnes and several species of Peptococcaceae showed broth dilution MICs of 0.03 similar to 1 microgram/ml. Members of B. thetaiotaomicron, B. distasonis and S. freundii (F. mortiferum) were inhibited by 8 similar to 32 microgram/ml and 2 strains of S. varius had a broth dilution MIC of 256 microgram/ml. With most strains, the agar dilution MICs were 2 similar to 4 similar to 8 times the broth dilution MICs. In pour plate-tests, the MICs were not considerably influenced influenced by varying initial concentrations of viable cells. With most anaerobes, the MBCs of tiamulin were more than 100-fold higher than the MICs. The results obtained indicated that, apart from S. varius, B. thetaiotaomicron, B. distasonis and S. freundii (F. mortiferum), members of 16 other anaerobic species including B. fragilis were without exception sensitive to tiamulin.

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.

[Changes of antimicrobial susceptibility of anaerobic bacteria from clinical specimens (author's transl)].

The susceptibility to several commonly used antimicrobial agents was examined on approximately 900 isolates of various anaerobes from clinical specimens between October 1965 to May 1975, and the changes of susceptibility were investigated. Antimicrobial agents tested were penicillin G (PC-G), ampicillin (AB-PC), cephaloridine (CER), erythromycin (EM), josamycin (JM), lincomycin (LCM), clindamycin (CLDM), chloramphenicol (CP) and tetracycline (TC). And thiamphenicol (TP) and doxycycline (DOTC) were also used against recently isolated organisms. Minimal inhibitory concentrations (MIC) were determined with agar plate dilution method. The activity of PC-G, AB-PC and CER against almost all strains of anaerobes was good at 12.5 mug/ml or less, but a marked increase of resistant strains of Bacteroides was recognized. EM, JM and LCM had similar activity against anaerobes, and were effective to most strains of anaerobes except for Veillonella, Bacteroides and Fusobacterium. CLDM was highly active against almost all strains of anaerobes, and more active than the two macrolides and LCM. But recently, significant increase in resistant strains was observed among Bacteroides. CP had good activity against all strains of anaerobes, and the increase of resistant strains was not recognized. TP had a similar good activity as CP. While, activity of TC against all anaerobes was poor at the level of 25 mug/ml or more. DOTC was more active than TC against all strains tested.

[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).

[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.

[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.

[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.

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.

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.

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.

[Susceptibility testing of anaerobes (author's transl)]. / Resistenzbestimmung bei Anaerobiern.

The methodology of susceptibility testing of anaerobes has not yet been standardized and many individual test methods are in use. Among the factors which may influence the outcome of susceptibility tests are test organisms, antimicrobials and methods of susceptibility testing. Comparative studies have shown that the density of the inoculum largely influences the MICs of sulphonamides and certain other antimicrobials. In testing susceptibility of anaerobes to doxycycline, the MICs determined by agar dilution tests were 2--4--8 times the MICs obtained by broth dilution technique. However, with certain cephalosporins and beta-lactamase-producing strains of Bacteroides fragilis and other Bacteroides species the MICs obtained by the two methods differed by four to five dilution steps. Judging by the broth dilution test, the majority of the beta-lactamase-producing Bacteroides strains would be classified as resistant, whereas with beta-lactamase-negative strains of Sphaerophorus varius the broth dilution test gave much lower MICs than the agar dilution technique. These findings pose the rather embarrassing problem as to whether strains are to be regarded as resistant or sensitive. As is demonstrated by lack of stochastic linearity and/or low correlation coefficients, the results of standardized anaerobic agar diffusion tests are for the most part not correlated to MICs determined by either method of quantitative susceptibility testing.

Phenylacetic acid stimulation of cellulose digestion by Ruminococcus albus 8.

The rate of cellulose digestion by Ruminococcus albus 8 grown on a defined medium could be increased by adding a minimum of 6.6% (vol/vol) rumen fluid. Strain 8 was grown on half this concentration, and the culture medium before and after growth was analyzed by gas chromatography-mass spectrometry to determine which components of the rumen fluid were used. Phenylacetic acid was identified as the component needed to make the defined medium nutritionally equivalent to one supplemented with rumen fluid. [14C]phenylacetic acid fed to cultures of strain 8 was primarily incorporated into protein. Hydrolysis of protein samples and separation of the resulting amino acids showed that only phenylalanine was labeled. The results indicate that cellulose digestion by strain 8 was probably limited by phenylalanine biosynthesis in our previously reported medium. The data obtained on the utilization of other rumen fluid components, as well as on the production of metabolites, illustrate the potential usefulness of this method in formulating defined media to simulate those in nature.

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.

Collaborative evaluation of the micro-media systems anaerobe susceptibility panel: comparisons with reference methods and test reproducibility.

The Micro-Media Systems (MMS) anaerobe susceptibility testing panel results from four laboratories were compared for interlaboratory and intralaboratory variations and for the results with the reference agar dilution and a broth microdilution method. The interlaboratory agreement was 98.0% and intralaboratory agreement was 97.3% (+/- 1 log2 dilution). When interpretive criteria for each antimicrobial agent (susceptible, intermediate, and resistant) were assigned, the MMS anaerobic minimum inhibitory concentration data showed an interpretive accuracy of 91.0 and 95.5% for comparisons to the reference agar dilution and the broth methods, respectively. Most significant interpretive errors were considered minor, and nearly half of all errors involved tetracycline, a drug rarely used for serious anaerobic infections. The MMS anaerobe panels appear to be acceptable for selected use in clinical microbiology laboratories.

Comparison of three methods of susceptibility testing of bacteroidaceae, peptococcaceae and other anaerobes to doxycycline.

The in vitro activity of doxycycline against 147 strains of gram-positive and gram-negative anaerobes was determined by broth dilution, agar dilution and agar diffusion tests. The strains were isolated from clinical specimens in 1977. Saccharolytic Bacteroides strains (39 B. fragilis, 6 B. thetaiotaomicron, 4 B. vulgatus) showed broth dilution MICs of less than or equal to 0.0625-4.0 microgram/ml after 5 h incubation and MICs of less than or equal to 16 microgram/ml after 15 h incubation at 37 degrees C. With strains of B. oralis, B. melaninogenicus ss. intermedius, B. corrodens, Veillonella sp. and Megasphaera elsdenii the broth dilution MICs were less than or equal to 0.0625-32 microgram/ml after 15 h incubation. Peptostreptococcus spp., Propionibacterium acnes, Eubacterium sp., Bifidobacterium sp. and Clostridium sp. had broth dilution MICs of less than or equal to 2 microgram/ml, whereas Peptococcus spp. were inhibited by less than or equal to 0.0625 to greater than 32 microgram/ml. With the great majority of strains tested, MICs were 2 to 256 times higher in agar than in broth dilution tests. Clinical and experimental studies seem to be needed to determine which of the in vitro data are correlated to the outcome of doxycycline therapy. Several groups and species were separately considered for statistical analysis of the relationship between zone size and MIC. With gram-positive anaerobes, correlation was poor between broth dilution MIC and zone size (correlation coefficients r = - 0.168 for Peptococcaceae, and r = - 0.108 for P. acnes). When calculating the regression lines for agar dilution MICs and zone diameters, a higher correlation was found (r = - 0.9 for Peptococcaceae; r = - 0.397 for P. acnes). With B. fragilis and other Bacteroides species correlation coefficients were r = - 0.807 to r = - 0.891 for broth and agar dilution MICs and zone size but stochastic linearity was lacking.