In Vitro Evaluation of the Activity of Aztreonam-Avibactam against 341 Recent Clinical Isolates of Anaerobes.

Aztreonam-avibactam is under clinical development for multidrug-resistant Gram-negative infections. We evaluated in vitro activity against 341 recent clinical isolates. The addition of avibactam to aztreonam had no effect on the anaerobic activity of aztreonam. IMPORTANCE This work shows that aztreonam-avibactam lacks activity against anaerobic organisms.

Antimicrobial susceptibility testing of Eggerthella lenta blood culture isolates at a university hospital in Belgium from 2004 to 2018.

OBJECTIVES: Eggerthella lenta is a Gram-positive anaerobic bacillus that is an important cause of bloodstream infections. This study aims to test the susceptibility of Eggerthella lenta blood culture isolates to commonly used antibiotics for the empirical treatment of anaerobic infections. METHODS: In total, 49 positive blood cultures for Eggerthella lenta were retrospectively included from patients hospitalised at the Universitair Ziekenhuis Brussel, Belgium, between 2004 and 2018. Identification was done by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system. Antimicrobial susceptibility testing was performed using the reference agar dilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines with Brucella agar supplemented with 5 µg/mL hemin, 1 µg/mL vitamin K1 and 5% laked sheep blood. The minimal inhibitory concentrations were interpreted using the EUCAST breakpoints. Clinical characteristics were collected by reviewing the patient's medical records. RESULTS: All isolates were susceptible to amoxicillin-clavulanate, metronidazole and meropenem. Eighty-eight % of them were susceptible to clindamycin and 94% (20% S, 74% I) were susceptible to piperacillin-tazobactam. The mean age of the patients was 64 (±20) and they showed a 30-day mortality of 27%. The source of infection was in 65.3% of the cases abdominal, 20.4% were sacral pressure ulcers and 14.3% were unknown causes. While all isolates were fully susceptible at standard dosing regimen to amoxicillin-clavulanate, most were only susceptible at increased exposure or resistant to piperacillin-tazobactam. CONCLUSIONS: Our results suggest to be careful with the use of piperacillin-tazobactam and clindamycin in the empirical treatment of Eggerthella lenta infections.

[Progress in the treatment of intra-abdominal anaerobic infection].

Most abdominal infections are mixed infections caused by aerobic and anaerobic bacteria. Anaerobic infections are characterized by rancid secretions or abscess formation. Early implementation of source control is the key in the treatment of abdominal anaerobic infections. Damage control should be followed as one of the principles of surgical treatment. As the in vitro isolation and culture of anaerobic bacteria as well as its drug sensitivity test are time-consuming and sometimes inaccurate, the treatment of anaerobic bacteria infection is mostly empirical. Anti-infective therapy should be employed once anaerobic bacteria infection is confirmed. Ertapenem, Mosifloxacin, and Cefoperazone-sulbactam can be used for first-line monotherapy, while combination therapy can use second- or third-generation Cephalosporin, Quinolones plus Nitroimidazoles. Nutritional support and anti-shock treatment should not be neglected when implementing surgical control of infection source and antimicrobial therapy. Considering the increasing drug resistance of anaerobic bacteria, and the higher drug resistance rate in China as compared to western countries, the choice of antibiotics should be made rationally and based on epidemiological characteristics of anaerobic bacteria in different regions.

Petroleum hydrocarbon rich oil refinery sludge of North-East India harbours anaerobic, fermentative, sulfate-reducing, syntrophic and methanogenic microbial populations.

BACKGROUND: Sustainable management of voluminous and hazardous oily sludge produced by petroleum refineries remains a challenging problem worldwide. Characterization of microbial communities of petroleum contaminated sites has been considered as the essential prerequisite for implementation of suitable bioremediation strategies. Three petroleum refinery sludge samples from North Eastern India were analyzed using next-generation sequencing technology to explore the diversity and functional potential of inhabitant microorganisms and scope for their on-site bioremediation. RESULTS: All sludge samples were hydrocarbon rich, anaerobic and reduced with sulfate as major anion and several heavy metals. High throughput sequencing of V3-16S rRNA genes from sludge metagenomes revealed dominance of strictly anaerobic, fermentative, thermophilic, sulfate-reducing bacteria affiliated to Coprothermobacter, Fervidobacterium, Treponema, Syntrophus, Thermodesulfovibrio, Anaerolinea, Syntrophobacter, Anaerostipes, Anaerobaculum, etc., which have been well known for hydrocarbon degradation. Relatively higher proportions of archaea were detected by qPCR. Archaeal 16S rRNA gene sequences showed presence of methanogenic Methanobacterium, Methanosaeta, Thermoplasmatales, etc. Detection of known hydrocarbon utilizing aerobic/facultative anaerobic (Mycobacterium, Pseudomonas, Longilinea, Geobacter, etc.), nitrate reducing (Gordonia, Novosphigobium, etc.) and nitrogen fixing (Azovibrio, Rhodobacter, etc.) bacteria suggested niche specific guilds with aerobic, facultative anaerobic and strict anaerobic populations. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) predicted putative genetic repertoire of sludge microbiomes and their potential for hydrocarbon degradation; lipid-, nitrogen-, sulfur- and methane- metabolism. Methyl coenzyme M reductase A (mcrA) and dissimilatory sulfite reductase beta-subunit (dsrB) genes phylogeny confirmed methanogenic and sulfate-reducing activities within sludge environment endowed by hydrogenotrophic methanogens and sulfate-reducing Deltaproteobacteria and Firmicutes members. CONCLUSION: Refinery sludge microbiomes were comprised of hydrocarbon degrading, fermentative, sulfate-reducing, syntrophic, nitrogen fixing and methanogenic microorganisms, which were in accordance with the prevailing physicochemical nature of the samples. Analysis of functional biomarker genes ascertained the activities of methanogenic and sulfate-reducing organisms within sludge environment. Overall data provided better insights on microbial diversity and activity in oil contaminated environment, which could be exploited suitably for in situ bioremediation of refinery sludge.

Eggerthella lenta Bloodstream Infections Are Associated With Increased Mortality Following Empiric Piperacillin-Tazobactam (TZP) Monotherapy: A Population-based Cohort Study.

Background: Eggerthella lenta is a anaerobic gram-positive bacilli associated with polymicrobial intraabdominal infections. Recently, E. lenta was recognized as an important cause of anaerobic bloodstream infections (BSIs) associated with high mortality. Eggerthella lenta has been reported to have high minimal inhibitory concentrations (MICs) to piperacillin-tazobactam (TZP), a broad-spectrum antibiotic with anaerobic coverage commonly used in multiple centers for empiric treatment of abdominal sepsis. Methods: We describe a retrospective population-based analysis of invasive E. lenta infections from 2009 through 2015. A logistic regression analysis for 30-day mortality risk factors was conducted. Results: We identified 107 E. lenta infections, 95 (89%) were BSIs, 11 (10%) skin and soft tissue infections, and 1 intraabdominal abscess. Polymicrobial infections were found in 40%; 72% of isolates were from a gastrointestinal source, most commonly appendicitis (33%) of which two-thirds were perforated. TZP MIC50 and MIC90 for E. lenta isolates were 32 µg/mL and 64 µg/mL, respectively. The overall 30-day mortality for BSI was 23% and was independently associated with empiric TZP monotherapy (odds ratio [OR], 4.4; 95% confidence interval [CI], 1.2-16; P = .02) and intensive care unit stay (OR, 6.2; 95% CI, 1.4-27.3; P = .01). Thirty-day mortality rates were significantly influenced by the use of different TZP MIC breakpoints. Conclusions: Our results demonstrate the increased recognition of E. lenta as an anaerobic opportunistic pathogen and highlight the need for improved empiric antimicrobial guidelines and TZP MIC breakpoints with better correlation to clinical outcomes to guide appropriate management of invasive E. lenta infections.

Effectiveness of meibomian gland massage combined with topical levofloxacin against ocular surface flora in patients before penetrating ocular surgery.

OBJECTIVE: To investigate the bacterial profile in the conjunctiva and meibomian glands in patients before penetrating ocular surgeries, and to compare the anti-bacterial efficacy of 0.5% levofloxacin and its combination with meibomian gland massage. DESIGN: Hospital-based, case-control study. PARTICIPANTS: Two hundred and twenty-six eyes from 226 patients with non-infective ocular diseases and scheduled for penetrating ocular surgeries. METHODS: Tested eyes were administered topical 0.5% levofloxacin (4 times daily) for 2 days. Among them, 91 eyes received meibomian gland massage before levofloxacin application. Samples from the conjunctival sac and meibomian glands were collected for aerobic and anaerobic cultures. MAIN OUTCOME MEASURES: Culture-positivity and bacterial strains. RESULTS: Before treatment, aerobes and anaerobes were cultured from 38.5% and 11.0% of the conjunctival samples respectively, compared with 38.5% and 8.8% in the meibomian secretions respectively. Staphylococcus epidermidis and Propionibacterium acnes were the commonest isolated aerobe and anaerobe. Two-day application of levofloxacin reduced aerobic growth to 29.6% in the conjunctiva and 19.3% in the meibomian glands. It had no effect on the anaerobes in these regions (13.3% in the conjunctiva and 10.4% in the meibomian glands). Combined levofloxacin with meibomian gland massage further reduced aerobic growth to 19.8% in the conjunctiva and 11.0% in the meibomian glands. It also drastically decreased anaerobic growth in the meibomian glands (1.1%). CONCLUSIONS: Meibomian glands carrying considerable bacteria should be considered as a potential source of contamination in ocular surgery. Meibomian gland massage shows additional anti-bacterial effects to topical levofloxacin and could be recommended as a complementary preoperative prophylaxis.

Isolation of bacteria from diabetic foot ulcers with special reference to anaerobe isolation by simple two-step combustion technique in candle jar.

BACKGROUND & OBJECTIVES: Although polymicrobial infections involving both aerobic and anaerobic bacteria are very common in diabetic foot ulcers, in many centres of developing countries, anaerobes are rarely isolated due to technical difficulties. This can be overcome by using a new simple, innovative technique of a combination of candle combustion and use of acidified copper-coated steel wool, as reported here. METHODS: In-house developed method was used in a prospective clinico-microbiological study for anaerobes from randomly selected 43 patients with diabetic foot ulcers along with conventional method of anaerobic culture in GasPak system and aerobic culture by standard laboratory procedures. For primary isolation of anaerobes, Brucella blood agar supplemented with hemin (5 µg/ml) and menadione (1 µg/ml) was used. Antibiotic sensitivity tests were performed by the standard disc diffusion method for aerobes and E-test method for anaerobes. RESULTS: All the 43 samples were culture positive, of which aerobic Gram-negative bacteria (GNB) predominated, followed by Staphylococcus aureus, Enterococcus and diphtheroids. Anaerobes isolated from 21 samples were Peptostreptococcus, Bacteroides, Porphyromonas, Veillonella spp. and Clostridium perfringens by both GasPak and in-house developed and modified candle jar techniques. Imipenem and metronidazole were most sensitive while clindamycin, penicillin and cefoxitin were least sensitive drugs for anaerobes. Aerobic GNB were found to be multidrug resistant, especially to penicillin and cephalosporins. The most sensitive drug was piperacillin-tazobactam. INTERPRETATION & CONCLUSIONS: For isolation of anaerobes from clinical specimens such as diabetic foot ulcers, modified candle jar technique was found to be as reliable as GasPak system. This modified technique needs to be tested for many other clinical materials which are not yet evaluated.

A quasi-universal medium to break the aerobic/anaerobic bacterial culture dichotomy in clinical microbiology.

In the mid-19th century, the dichotomy between aerobic and anaerobic bacteria was introduced. Nevertheless, the aerobic growth of strictly anaerobic bacterial species such as Ruminococcus gnavus and Fusobacterium necrophorum, in a culture medium containing antioxidants, was recently demonstrated. We tested aerobically the culture of 623 bacterial strains from 276 bacterial species including 82 strictly anaerobic, 154 facultative anaerobic, 31 aerobic and nine microaerophilic bacterial species as well as ten fungi. The basic culture medium was based on Schaedler agar supplemented with 1 g/L ascorbic acid and 0.1 g/L glutathione (R-medium). We successively optimized this media, adding 0.4 g/L uric acid, using separate autoclaving of the component, or adding haemin 0.1 g/L or α-ketoglutarate 2 g/L. In the basic medium, 237 bacterial species and ten fungal species grew but with no growth of 36 bacterial species, including 22 strict anaerobes. Adding uric acid allowed the growth of 14 further species including eight strict anaerobes, while separate autoclaving allowed the growth of all tested bacterial strains. To extend its potential use for fastidious bacteria, we added haemin for Haemophilus influenzae, Haemophilus parainfluenzae and Eikenella corrodens and α-ketoglutarate for Legionella pneumophila. This medium allowed the growth of all tested strains with the exception of Mycobacterium tuberculosis and Mycobacterium bovis. Testing primoculture and more fastidious species will constitute the main work to be done, but R-medium coupled with a rapid identification method (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) will facilitate the anaerobic culture in clinical microbiology laboratories.

Development of antimicrobial resistance in the normal anaerobic microbiota during one year after administration of clindamycin or ciprofloxacin.

Thirty healthy subjects (15 males and 15 females) were randomly assigned in three groups and clindamycin (150 mg qid) or ciprofloxacin (500 mg bid) or placebo was given for a 10-day period. Skin, nasal, saliva, faeces samples were collected at day - 1, day 11, 1 month, 2 months, 4 months and 12 months post administration for microbiological analysis. Ciprofloxacin or clindamycin had no impact on the anaerobic skin microbiota and the proportions of antibiotic resistant anaerobic bacteria were similar as in the placebo group. Ciprofloxacin had impact on the Propionibacterium acnes in the nasal microbiota that normalized after 1 month, however, ciprofloxacin-resistant P. acnes strains increased at month 2 and month 12. Clindamycin had no impact on the nasal microbiota. In the oropharyngeal microbiota, a higher proportion of ciprofloxacin resistant Veillonella was found, it lasting up to 12 months post dosing. In the clindamycin group, clindamycin-resistant Prevotella spp. were found in increased proportions compared to placebo at various time points except month 4 in the saliva samples. The relative proportion of ciprofloxacin-resistant Bifidobacteria increased in the faecal samples on day 11, 1 month, 4 months and 12 months post dosing compared to placebo. The proportion of clindamycin-resistant Bacteroides spp. increased at 1, 2, 4 and 12 months post dosing compared to placebo in the faecal samples. No Clostridium difficile was recovered from any of the samples from any of the volunteers at any visit. The concentrations of ciprofloxacin or clindamycin in the faeces were higher than the MICs for most of the organisms present in the normal microbiota. No obvious correlation between the groups in resistant patterns for anaerobic bacteria was observed. In conclusion, based on the microbiological data of the microbiota as well as the results of the bioassays for ciprofloxacin and clindamycin concentrations in the faecal samples, oral administration of ciprofloxacin and clindamycin has an impact on the anaerobic microbiota and may have a long-term effect on the development and persistence of antibiotic-resistant anaerobes in the normal microbiota.

Antibacterial activity of Pinus elliottii against anaerobic bacteria present in primary endodontic infections.

Endodontic infections have a polymicrobial nature, but anaerobic bacteria prevail among the infectious microbes. Considering that it is easy to eliminate planktonic bacteria, biofilm-forming bacteria still challenge clinicians during the fight against endodontic diseases. The chemical constituents of the oleoresin of Pinus elliottii, a plant belonging to the family Pinaceae, stand out in the search for biologically active compounds based on natural products with potential application in the treatment of endodontic infections. Indeed, plant oleoresins are an abundant natural source of diterpenes that display significant and well-defined biological activities as well as potential antimicrobial action. In this context, this study aimed to (1) evaluate the in vitro antibacterial activity of the oleoresin, fractions, and subfractions of P. elliottii as well as the action of dehydroabietic acid against 11 anaerobic bacteria that cause endodontic infection in both their planktonic and biofilm forms and (2) assess the in vitro antibiofilm activity of dehydroabietic acid against the same group of bacteria. The broth microdilution technique helped to determine the minimum inhibitory concentration (MIC) of the oleoresin and fractions. This same technique aided determination of the MIC values of nine subfractions of Fraction 1, the most active fraction. The MIC, minimum bactericidal concentration, and antibiofilm activity of dehydroabietic acid against the tested anaerobic bacteria were also examined. The oleoresin and fractions, especially fraction PE1, afforded promising MIC values, which ranged from 0.4 to 50 µg/mL. Concerning the nine evaluated subfractions, PE1.3 and PE1.4 furnished the most noteworthy MIC values, between 6.2 and 100 µg/mL. Dehydroabietic acid displayed antibacterial activity, with MIC values lying from 6.2 to 50 µg/mL, as well as bactericidal effect for all the investigated bacteria, except for Prevotella nigrescens. Assessment of the antibiofilm activity revealed significant results--MICB50 lay between 7.8 and 62.5 µg/mL, and dehydroabietic acid prevented all the evaluated bacteria from forming a biofilm. Hence, the chemical constituents of P. elliottii are promising biomolecules to develop novel therapeutic strategies to fight against endodontic infections.

Prevalence and antimicrobial susceptibilities of anaerobic bacteria isolated from perforated corneal ulcers by culture and multiplex PCR: an evaluation in cases with keratitis and endophthalmitis.

BACKGROUND: Anaerobic bacteria play an important role in eye infections; however, there is limited epidemiologic data based on the the role of these bacteria in the etiology of keratitis and endophthalmitis. The aim of this re- search is to determine the prevalence of anaerobic bacteria in perforated corneal ulcers of patients with keratitis and endophthalmitis and to evaluate their antimicrobial susceptibilities. METHODS: Corneal scrapings were taken by the ophthalmologist using sterile needles. For the isolation of anaerobic bacteria, samples were inoculated on specific media and were incubated under anaerobic conditions obtained with Anaero-Gen (Oxoid & Mitsubishi Gas Company) in anaerobic jars (Oxoid USA, Inc. Columbia, MD, USA). The molecular identification of anaerobic bacteria was performed by multiplex PCR and the susceptibilities of an- aerobic bacteria to penicillin, chloramphenicol, and clindamycin were determined with the E test (bioMerieux). RESULTS: 51 strains of anaerobic bacteria belonging to four different genuses were detected by multiplex PCR and only 46 strains were isolated by culture. All of them were found susceptible to chloramphenicol whereas penicillin resistance was found in 13.3% of P.anaerobius strains, clindamycin resistance was found in 34.8% of P.acnes and 13.3% of P. anaerobius strains. Additionnaly, one strain of P. granulosum was found resistant to clindamycin, one strain of B. fragilis and one strain of P.melaninogenica were found resistant to penicillin and clindamycin. CONCLUSIONS: Routine analyses of anaerobes in perforated corneal ulcers is inevitable and usage of appropriate molecular methods, for the detection of bacteria responsible from severe infections which might not be deter- mined by cultivation, may serve for the early decision of the appropriate treatment. Taking into account the in- creasing antimicrobial resistance of anaerobic bacteria, alternative eye specific antibiotics effective against anaer- obes are needed to achieve a successful treatment.

[Unsterile urine in health human--new paradigm in medicine].

The 3-fold urine culture evaluation in healthy women (24) and men (28) was performed. In 100% of cases, various types of multicomponent aerobic-anaerobic microorganism associations were found. Dominant clusters in the urine of women incleded coagulase-negative staphylococci, Corynebacterium sp., Lactobacillus sp., Peptococcus sp., Propionibacterium sp., in men--coagulase negative staphylococci, Corynebacterium sp., Eubacterium sp. For aerobic microorganisms, level of bacteriuria in both groups was 10(2) CFU/ml, for most anaerobes--≥10(3) CFU/ml. Spectrum of microorganisms isolated from the urine was quite wide and variable. In women, number of assiociates in urine ranged from 3 to 10, in men--from 6 to 9, as well as individually--in each subject, the original range of microorganisms was not repeated in any other case.

Effects of grape seed extract on the oxidative and microbial stability of restructured mutton slices.

The antioxidant and antimicrobial efficacy of grape seed extract (GSE) was studied in restructured mutton slices (RMS) under aerobic and vacuum packaging conditions during refrigerated storage. The RMS treated with grape seed extract (GSE) had significantly (P<0.05) lower thiobarbituric acid reactive substance (TBARS) values and free fatty acids (FFA) % compared to control (C) and butylated hydroxy anisole (BHA) treated RMS during storage at 4±1°C. Addition of GSE significantly (P<0.05) reduced the total psychrophilic and coliform counts in RMS during refrigerated storage. The GSE treated mutton slices recorded significantly (P<0.05) superior scores of color, flavor, juiciness and overall palatability than C and BHA treated RMS. The TBARS values, FFA % and microbial counts increased significantly (P<0.05) during storage. It can be concluded that GSE has excellent antioxidant and antimicrobial properties compared to control and BHA treated RMS during refrigerated storage under aerobic and vacuum conditions.

Effects of bamboo charcoal and bamboo vinegar as antibiotic alternatives on growth performance, immune responses and fecal microflora population in fattening pigs.

This study was carried out to investigate the effects of bamboo charcoal and bamboo vinegar as alternatives of antibiotics in the diet of fattening pigs and their influence on growth performance, immune responses and fecal microflora populations. Crossed pigs (n = 144, 79 kg body weight) were divided into 12 heads per pen, four diets and three replications. The basal diet (negative control: NC) was supplemented with 0.3% antibiotics (positive control: PC), 0.3% bamboo charcoal (BC) and 0.3% bamboo vinegar (BV). Average daily weight gain and feed efficiency were higher (P < 0.05) in PC, BC and BV. The concentration of lactate dehydrogenase and cortisol were lower (P < 0.05), but the concentration of immunoglobulin G (IgG) and IgA were higher (P < 0.05) in PC, BC and BV. Counts of coliform bacteria and Salmonella spp. were lower (P < 0.05), while the counts of fecal anaerobic total bacteria and lactic acid bacteria were higher (P < 0.05) in PC, BC and BV. A reasonable inclusion of bamboo charcoal or bamboo vinegar as antibiotics in the diet of fattening pigs leads to a better growth performance, immune responses and fecal microflora populations. The results of the present study suggest that bamboo charcoal or bamboo vinegar could be a potential additives in animal production as an alternative to antibiotics.

[C-ring cleavage of liquiritigenin extracted from licorice roots by an oxygen-tolerant bovine rumen bacterium strain Aeroto-Niu-O16].

Aeroto-Niu-O16, an oxygen-tolerant bovine rumen bacterium, is capable of aerobically reducing isoflavones daidzein and genistein to dihydrodaidzein and dihydrogenistein through catalytic hydrogenation. In this study, it was found that bacterium strain Aeroto-Niu-O16 was able to cleavage the C-ring of liquiritigenin (LG), which is one of the main biologically active components of licorice roots, in the presence of atmospheric oxygen. LG was prepared by acid hydrolysis of the crude extract of licorice roots. The metabolite of LG obtained in strain Aeroto-Niu-O16 was identified as davidigenin (DG) based on the data of UV, MS, 1H and 13C NMR. The maximal concentration of LG that the strain Aeroto-Niu-O16 was able to transform effectively was 0.8 mmol x L(-1) and the average productivity of the metabolite DG was 71.7%. Furthermore, when 0.1% (m/v) of L-cysteine or sodium thiosulfate was added in the cultural medium, the average bioconversion rate of LG was increased from 71.7% to 78.3% and 77.2%, respectively. The in vitro antioxidant investigation showed that 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity of DG was significantly or extremely significantly higher than that of LG at the concentrations from 0.2 mmol x L(-1) to 1.6 mmol x L(-1). We discoverd for the first time that LG can be converted to DG, which has stronger and wider biological activities, through microbial biotransformation method.

Analyses of n-alkanes degrading community dynamics of a high-temperature methanogenic consortium enriched from production water of a petroleum reservoir by a combination of molecular techniques.

Despite the knowledge on anaerobic degradation of hydrocarbons and signature metabolites in the oil reservoirs, little is known about the functioning microbes and the related biochemical pathways involved, especially about the methanogenic communities. In the present study, a methanogenic consortium enriched from high-temperature oil reservoir production water and incubated at 55 °C with a mixture of long chain n-alkanes (C(15)-C(20)) as the sole carbon and energy sources was characterized. Biodegradation of n-alkanes was observed as methane production in the alkanes-amended methanogenic enrichment reached 141.47 µmol above the controls after 749 days of incubation, corresponding to 17 % of the theoretical total. GC-MS analysis confirmed the presence of putative downstream metabolites probably from the anaerobic biodegradation of n-alkanes and indicating an incomplete conversion of the n-alkanes to methane. Enrichment cultures taken at different incubation times were subjected to microbial community analysis. Both 16S rRNA gene clone libraries and DGGE profiles showed that alkanes-degrading community was dynamic during incubation. The dominant bacterial species in the enrichment cultures were affiliated with Firmicutes members clustering with thermophilic syntrophic bacteria of the genera Moorella sp. and Gelria sp. Other represented within the bacterial community were members of the Leptospiraceae, Thermodesulfobiaceae, Thermotogaceae, Chloroflexi, Bacteroidetes and Candidate Division OP1. The archaeal community was predominantly represented by members of the phyla Crenarchaeota and Euryarchaeota. Corresponding sequences within the Euryarchaeota were associated with methanogens clustering with orders Methanomicrobiales, Methanosarcinales and Methanobacteriales. On the other hand, PCR amplification for detection of functional genes encoding the alkylsuccinate synthase α-subunit (assA) was positive in the enrichment cultures. Moreover, the appearance of a new assA gene sequence identified in day 749 supported the establishment of a functioning microbial species in the enrichment. Our results indicate that n-alkanes are converted to methane slowly by a microbial community enriched from oilfield production water and fumarate addition is most likely the initial activation step of n-alkanes degradation under thermophilic methanogenic conditions.

Anaerocella delicata gen. nov., sp. nov., a strictly anaerobic bacterium in the phylum Bacteroidetes isolated from a methanogenic reactor of cattle farms.

A strictly anaerobic bacterial strain (WN081(T)) was isolated from rice-straw residue in a methanogenic reactor treating waste from cattle farms in Japan. Cells were Gram-staining negative, non-motile, non-spore-forming straight rods. The strain grew rather well on PY agar slants supplemented with a B-vitamin mixture as well as sugars (PYV4S medium) and made translucent and glossy colonies. Growth in liquid medium with the same composition, however, was scanty, and growth was not improved in spite of various additives to the medium. Strain WN081(T) produced small amounts of acetate, propionate, isobutyrate, butyrate, isovalerate and H(2) from PYV liquid medium. The strain did not use carbohydrates or organic acids. The pH range for growth was narrow (pH 6.8-8.2), having a pH optimum at 6.8-7.5. The temperature range for growth was 10-37°C, the optimum being 25-30°C. The strain was sensitive to bile, and did not have catalase or oxidase activities. Hydrogen sulfide was produced from L-cysteine and L-methionine as well as peptone. Indole was produced from L-tryptophan and peptone. The strain had iso-C(15:0) as the exclusively predominant cellular fatty acid (70%) together with some branched chain components (such as iso-C(15:0) DMA, iso-C(17:0) 3-OH and iso-C(15:0) aldehyde) as minor components. The genomic DNA G+C content was 32.3 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence placed strain WN081(T) in the phylum Bacteroidetes with rather low sequence similarities with the related species such as Rikenella microfusus (85.7% sequence similarity), Alistipes putredinis (85.5%) and Alistipes finegoldii (85.5%) in the family Rikenellaceae. Based on the phylogenetic, physiological and chemotaxonomic analyses, the novel genus and species Anaerocella delicata gen. nov., sp. nov. is proposed to accommodate the strain. The type strain is WN081(T) (= JCM 17049(T) = DSM 23595(T)).

Description of Anaerobaculum hydrogeniformans sp. nov., an anaerobe that produces hydrogen from glucose, and emended description of the genus Anaerobaculum.

A novel anaerobic, moderately thermophilic, NaCl-requiring fermentative bacterium, strain OS1T, was isolated from oil production water collected from Alaska, USA. Cells were Gram-negative, non-motile, non-spore-forming rods (1.7-2.7×0.4-0.5 µm). The G+C content of the genomic DNA of strain OS1T was 46.6 mol%. The optimum temperature, pH and NaCl concentration for growth of strain OS1T were 55 °C, pH 7 and 10 g l(-1), respectively. The bacterium fermented D-fructose, D-glucose, maltose, D-mannose, α-ketoglutarate, L-glutamate, malonate, pyruvate, L-tartrate, L-asparagine, Casamino acids, L-cysteine, L-histidine, L-leucine, L-phenylalanine, L-serine, L-threonine, L-valine, inositol, inulin, tryptone and yeast extract. When grown on D-glucose, 3.86 mol hydrogen and 1.4 mol acetate were produced per mol substrate. Thiosulfate, sulfur and L-cystine were reduced to sulfide, and crotonate was reduced to butyrate with glucose as the electron donor. 16S rRNA gene sequence analysis indicated that strain OS1T was related to Anaerobaculum thermoterrenum (99.7 % similarity to the type strain), a member of the phylum Synergistetes. DNA-DNA hybridization between strain OS1T and A. thermoterrenum DSM 13490T yielded 68 % relatedness. Unlike A. thermoterrenum, strain OS1T fermented malonate, maltose, tryptone, L-leucine and L-phenylalanine, but not citrate, fumarate, lactate, L-malate, glycerol, pectin or starch. The major cellular fatty acid of strain OS1T was iso-C15:0 (91 % of the total). Strain OS1T also contained iso-C13:0 3-OH (3 %), which was absent from A. thermoterrenum, and iso-C13:0 (2 %), which was absent from Anaerobaculum mobile. On the basis of these results, strain OS1T represents a novel species of the genus Anaerobaculum, for which the name Anaerobaculum hydrogeniformans sp. nov. is proposed. The type strain is OS1T (=DSM 22491T=ATCC BAA-1850T). An emended description of the genus Anaerobaculum is also given.

Anaerosalibacter bizertensis gen. nov., sp. nov., a halotolerant bacterium isolated from sludge.

A strictly anaerobic, halotolerant and thermotolerant strain, designated C5BEL(T), was isolated in north Tunisia from storage tanks holding waste generated by the recycling of discarded motor oils. Cells of strain C5BEL(T) were Gram-stain-positive, motile by laterally inserted flagella, straight, and spore-forming. Their two major fatty acids were iso-C(15 : 0) and iso-C(15 : 0) dimethyl acetal. Growth was observed at temperatures of 25-55 °C (optimum, 40 °C) and at pH 6-9 (optimum, pH 7.5). The salinity range for growth was 0-100 g l(-1) NaCl (optimum, 5 g l(-1)). Yeast extract was required for growth. Strain C5BEL(T) was heterotrophic, able to use glucose, pyruvate, succinate, yeast extract, bio-trypticase and peptone, but unable to grow on Casamino acids. Sulfate, thiosulfate, sulfite, elemental sulfur, fumarate, nitrate and nitrite were not reduced. The DNA G+C content of strain C5BEL(T) was 31.1 mol%. 16S rRNA gene sequence analysis indicated that strain C5BEL(T) was a member of the family Clostridiaceae, class Clostridia, phylum Firmicutes and was most closely related to Sporanaerobacter acetigenes Lup33(T) ( = DSM 13106(T)) (92.4 % similarity). On the basis of 16S rRNA gene sequence comparisons and physiological characteristics, strain C5BEL(T) can be classified as a novel species in a new genus, for which the name Anaerosalibacter bizertensis gen. nov., sp. nov. is proposed. The type strain of the type species is C5BEL(T) ( = DSM 23801(T) = JCM 17239(T)).