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  Subjects -> BIOLOGY (Total: 2835 journals)
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BIOLOGY (1379 journals)            First | 1 2 3 4 5 6 7 8 | Last

DNA and Cell Biology     Hybrid Journal   (Followers: 4)
DNA Repair     Hybrid Journal   (Followers: 2)
DNA Research     Open Access   (Followers: 3)
Doklady Physics     Hybrid Journal   (Followers: 1)
Drug Discovery Today     Full-text available via subscription   (Followers: 98)
Drug Discovery Today: Disease Mechanisms     Full-text available via subscription   (Followers: 7)
Drug Discovery Today: Disease Models     Full-text available via subscription   (Followers: 6)
Drug Discovery Today: Technologies     Full-text available via subscription   (Followers: 11)
Drug Discovery Today: Therapeutic Strategies     Full-text available via subscription   (Followers: 9)
Drug Resistance Updates     Hybrid Journal   (Followers: 4)
e-Neuroforum     Hybrid Journal  
Ecohydrology & Hydrobiology     Full-text available via subscription   (Followers: 3)
Ecología en Bolivia     Open Access  
Ecological Engineering     Hybrid Journal   (Followers: 3)
Ecology and Society     Open Access   (Followers: 28)
Ecology Letters     Hybrid Journal   (Followers: 161)
Economics & Human Biology     Hybrid Journal  
Ecoprint : An International Journal of Ecology     Open Access   (Followers: 1)
Ecoscience     Full-text available via subscription   (Followers: 2)
Educational Technology Research and Development     Partially Free   (Followers: 82)
Egyptian Journal of Basic and Applied Sciences     Open Access  
Egyptian Journal of Biology     Open Access  
Egyptian Journal of Histology     Partially Free   (Followers: 1)
Egyptian Journal of Natural History     Open Access   (Followers: 1)
EJNMMI Research     Open Access   (Followers: 1)
Electromagnetic Biology and Medicine     Hybrid Journal   (Followers: 1)
eLife     Open Access   (Followers: 25)
el–Hayah     Open Access   (Followers: 1)
Embo Molecular Medicine     Open Access   (Followers: 4)
EMBO reports     Full-text available via subscription   (Followers: 18)
Emotion Review     Hybrid Journal   (Followers: 13)
Endocrine Connections     Open Access   (Followers: 4)
Endothelium: Journal of Endothelial Cell Research     Full-text available via subscription   (Followers: 4)
Engineering & Technology     Hybrid Journal   (Followers: 19)
Engineering Economist, The     Hybrid Journal   (Followers: 2)
Engineering in Life Sciences     Hybrid Journal   (Followers: 4)
Engineering Optimization     Hybrid Journal   (Followers: 8)
Ensaios e Ciência: Ciências Biológicas, Agrárias e da Saúde     Open Access  
Environmental Biology of Fishes     Hybrid Journal   (Followers: 5)
Environmental Biosafety Research     Hybrid Journal   (Followers: 1)
Environmental Microbiology     Hybrid Journal   (Followers: 14)
Environmental Science & Technology     Full-text available via subscription   (Followers: 135)
Enzyme and Microbial Technology     Hybrid Journal   (Followers: 5)
Enzyme Research     Open Access   (Followers: 4)
Epidemiology & Infection     Hybrid Journal   (Followers: 14)
Epigenetics in Cancer     Open Access   (Followers: 1)
EPMA Journal     Open Access  
ESHRE Monographs     Hybrid Journal  
Ethiopian Journal of Biological Sciences     Open Access   (Followers: 3)
Ethnobiology and Conservation     Open Access   (Followers: 2)
Ethnobotany Research & Applications : a journal of plants, people and applied research     Open Access   (Followers: 7)
Ethology     Hybrid Journal   (Followers: 6)
Ethology Ecology & Evolution     Hybrid Journal   (Followers: 13)
Eukaryotic Cell     Full-text available via subscription   (Followers: 6)
EuPA Open Proteomics     Open Access   (Followers: 2)
EURASIP Journal on Bioinformatics and Systems Biology     Open Access   (Followers: 7)
European Journal of Cell Biology     Hybrid Journal   (Followers: 3)
European Journal of Neuroscience     Hybrid Journal   (Followers: 28)
European Journal of Phycology     Hybrid Journal   (Followers: 5)
European Journal of Protistology     Hybrid Journal   (Followers: 3)
European Journal of Soil Biology     Hybrid Journal   (Followers: 2)
European Online Journal of Natural and Social Sciences     Open Access   (Followers: 14)
European Scientific Journal     Open Access   (Followers: 2)
Evidência - Ciência e Biotecnologia - Interdisciplinar     Open Access  
EvoDevo     Open Access   (Followers: 1)
Evolução e Conservação da Biodiversidade     Open Access  
Evolution     Partially Free   (Followers: 91)
Evolution and Human Behavior     Hybrid Journal   (Followers: 13)
Evolutionary Applications     Open Access   (Followers: 3)
Evolutionary Bioinformatics     Open Access   (Followers: 15)
Evolutionary Biology     Hybrid Journal   (Followers: 18)
Evolutionary Computation     Hybrid Journal   (Followers: 10)
Experimental & Molecular Medicine     Open Access   (Followers: 2)
Experimental and Applied Acarology     Hybrid Journal   (Followers: 1)
Experimental Cell Research     Hybrid Journal   (Followers: 4)
Expert Opinion on Biological Therapy     Hybrid Journal   (Followers: 10)
Expert Opinion on Environmental Biology     Hybrid Journal  
Expert Review of Proteomics     Hybrid Journal   (Followers: 2)
Extreme Life, Biospeology & Astrobiology - International Journal of the Bioflux Society     Full-text available via subscription   (Followers: 5)
Extremophiles     Hybrid Journal   (Followers: 1)
F1000Research     Open Access   (Followers: 4)
Facta Universitatis, Series : Medicine and Biology     Open Access  
Familial Cancer     Hybrid Journal   (Followers: 1)
Fatigue & Fracture of Engineering Materials and Structures     Hybrid Journal   (Followers: 14)
Fauna Norvegica     Open Access  
Febs Journal     Hybrid Journal   (Followers: 8)
Feddes Repertorium     Hybrid Journal  
Fems Yeast Research     Hybrid Journal   (Followers: 11)
Field Mycology     Full-text available via subscription   (Followers: 2)
Fish & Shellfish Immunology     Hybrid Journal   (Followers: 10)
Fitoterapia     Hybrid Journal   (Followers: 4)
Fly     Full-text available via subscription  
Folia Biologica     Free  
Folia Biologica et Oecologica     Open Access   (Followers: 1)
Folia Histochemica et Cytobiologica     Open Access  
Folia Microbiologica     Hybrid Journal   (Followers: 1)
Folia Primatologica     Full-text available via subscription   (Followers: 2)
Food and Bioproducts Processing     Hybrid Journal   (Followers: 2)
Food Webs     Hybrid Journal  
Forest Pathology     Hybrid Journal   (Followers: 1)

  First | 1 2 3 4 5 6 7 8 | Last

Journal Cover   Anaerobe
  [SJR: 1.09]   [H-I: 44]   [3 followers]  Follow
    
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 1075-9964 - ISSN (Online) 1095-8274
   Published by Elsevier Homepage  [2800 journals]
  • Aciduric microbial taxa including Scardovia wiggsiae and Bifidobacterium
           spp. in caries and caries free subjects
    • Abstract: Publication date: October 2015
      Source:Anaerobe, Volume 35, Part A
      Author(s): Karsten Henne, Anke Rheinberg, Beate Melzer-Krick, Georg Conrads
      Actinobacteria came into focus of being potential caries-associated pathogens and could, together with the established Streptococcus mutans and lactobacilli thus function as caries indicator species. Here we analyzed the role and diagnostic predictive value of the acidogenic–aciduric species Scardovia wiggsiae and Bifidobacterium dentium together with S. mutans, lactobacilli and bifidobacteria in biofilm of non-cavitated (n = 20) and cavitated (n = 6) caries lesions versus controls (n = 30). For the genus Bifidobacterium and for B. dentium new sets of primers were designed. Based on real-time quantitative PCR and confirmed by DNA sequencing we found a higher prevalence (61.5%) of S. wiggsiae in caries lesions than in controls (40%). However, among the controls we found three individuals with both the highest absolute and relative S. wiggsiae numbers. Testing for S. mutans revealed the same prevalence as S. wiggsiae in caries lesions (61.5%) but in controls its prevalence was only 10%. B. dentium was never found in healthy plaque but in 30.8% of clinical cases, with the highest numbers in cavitated lesions. The Bifidobacterium-genus specific PCR had less discriminative power as more control samples were positive. We calculated the relative abundances and applied receiver operating characteristic analyses. The top results of specificity (93% and 87%) and sensitivity (100% and 88%) were found when the constraint set was “Lactobacillus relative abundance ≥0.02%” and “two aciduric species with a relative abundance of each ≥0.007%”. Combinatory measurement of several aciduric taxa may be useful to reveal caries activity or even to predict caries progression.


      PubDate: 2015-08-19T12:30:23Z
       
  • Penetration of Streptococcus sobrinus and Streptococcus sanguinis into
           dental enamel
    • Abstract: Publication date: October 2015
      Source:Anaerobe, Volume 35, Part A
      Author(s): Susanne Kneist, Sandor Nietzsche, Harald Küpper, Gerhard Raser, Brita Willershausen, Angelika Callaway
      The aim of this pilot study was to assess the difference in virulence of acidogenic and aciduric oral streptococci in an in vitro caries model using their penetration depths into dental enamel. 30 caries-free extracted molars from 11- to 16-year-olds were cleaned ultrasonically for 1 min with de-ionized water and, after air-drying, embedded in epoxy resin. After 8-h of setting at room temperature, the specimens were ground on the buccal side with SiC-paper 1200 (particle size 13–16 μm). Enamel was removed in circular areas sized 3 mm in diameter; the mean depth of removed enamel was 230 ± 60 μm. 15 specimens each were incubated anaerobically under standardized conditions with 24 h-cultures of Streptococcus sanguinis 9S or Streptococcus sobrinus OMZ 176 in Balmelli broth at 37 ± 2 °C; the pH-values of the broths were measured at the beginning and end of each incubation cycle. After 2, 4, 6, 8, and 10 weeks 3 teeth each were fixed in 2.5% glutaraldehyde in cacodylate buffer for 24 h, washed 3× and dehydrated 30–60min by sequential washes through a series of 30–100% graded ethanol. The teeth were cut in half longitudinally; afterward, two slits were made to obtain fracture surfaces in the infected area. After critical-point-drying the fragments were gold-sputtered and viewed in a scanning electron microscope at magnifications of ×20-20,000. After 10 weeks of incubation, penetration of S. sanguinis of 11.13 ± 24.04 μm below the break edges into the enamel was observed. The invasion of S. sobrinus reached depths of 87.53 ± 76.34 μm. The difference was statistically significant (paired t test: p = 0.033). The experimental penetration depths emphasize the importance of S. sanguinis versus S. sobrinus in the context of the extended ecological plaque hypothesis.


      PubDate: 2015-08-19T12:30:23Z
       
  • Effect of Bdellovibrio bacteriovorus HD100 on multispecies oral
           communities
    • Abstract: Publication date: October 2015
      Source:Anaerobe, Volume 35, Part A
      Author(s): Gitte Loozen, Nico Boon, Martine Pauwels, Vera Slomka, Esteban Rodrigues Herrero, Marc Quirynen, Wim Teughels
      The predation of Bdellovibrio bacteriovorus on different periodontal pathogens has already been described. However, it is necessary to consider the polymicrobial nature of periodontal disease. The current study explores the predation of Bdellovibrio on oral pathogens organized in multispecies communities. The effect of the predator was evaluated on in vitro six species communities with microbial culturing. Additionally, the effect on ex vivo subgingival plaque and saliva samples from periodontitis patients was assessed. In the latter experiment results were examined with microbial culturing, quantitative polymerase chain reaction (qPCR) and denaturing gradient gel electrophoresis (DGGE). The latter technique was used to get an overview of the whole mixed microbial population. Results showed that even in more complex models, B. bacteriovorus was still able to predate on Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans. However predation on Prevotella intermedia and Porphyromonas gingivalis could not be validated in multispecies models. The effect of Bdellovibrio was not restricted to the target bacteria. Changes in the overall ecology of the different models were evident. It could be concluded that the efficiency of predation decreased when complexity of the models increased. However, B. bacteriovorus was able to attack two important oral pathogens, F. nucleatum, and A. actinomycetemcomitans, even when present in ex vivo clinical samples. These effects still have to be validated in in vivo models to see the impact of Bdellovibrio on the whole bacterial ecology.


      PubDate: 2015-08-19T12:30:23Z
       
  • 16S rRNA based microarray analysis of ten periodontal bacteria in patients
           with different forms of periodontitis
    • Abstract: Publication date: October 2015
      Source:Anaerobe, Volume 35, Part A
      Author(s): Nursen Topcuoglu, Guven Kulekci
      DNA microarray analysis is a computer based technology, that a reverse capture, which targets 10 periodontal bacteria (ParoCheck) is available for rapid semi-quantitative determination. The aim of this three-year retrospective study was to display the microarray analysis results for the subgingival biofilm samples taken from patient cases diagnosed with different forms of periodontitis. A total of 84 patients with generalized aggressive periodontitis (GAP,n:29), generalized chronic periodontitis (GCP, n:25), peri-implantitis (PI,n:14), localized aggressive periodontitis (LAP,n:8) and refractory chronic periodontitis (RP,n:8) were consecutively selected from the archives of the Oral Microbiological Diagnostic Laboratory. The subgingival biofilm samples were analyzed by the microarray-based identification of 10 selected species. All the tested species were detected in the samples. The red complex bacteria were the most prevalent with very high levels in all groups. Fusobacterium nucleatum was detected in all samples at high levels. The green and blue complex bacteria were less prevalent compared with red and orange complex, except Aggregatibacter actinomycetemcomitas was detected in all LAP group. Positive correlations were found within all the red complex bacteria and between red and orange complex bacteria especially in GCP and GAP groups. Parocheck enables to monitoring of periodontal pathogens in all forms of periodontal disease and can be alternative to other guiding and reliable microbiologic tests.


      PubDate: 2015-08-19T12:30:23Z
       
  • Antibiotic susceptibility of 33 Prevotella strains isolated from Romanian
           patients with abscesses in head and neck spaces
    • Abstract: Publication date: October 2015
      Source:Anaerobe, Volume 35, Part A
      Author(s): Gabriela Bancescu, Andreea Didilescu, Adrian Bancescu, Maria Bari
      The purpose of this study was to investigate the susceptibility of a series of 33 Prevotella strains isolated from patients with abscesses in the head and neck spaces, presented to one Romanian hospital. The Etest was applied to determine the value of the minimum inhibitory concentrations for: penicillin G, ampicillin, amoxicillin-clavulanate, metronidazole and clindamycin. In addition, the beta-lactamase activity was detected by the chromogenic cephalosporin disc method. The results indicated that 11 isolates were resistant to both penicillin G and ampicillin due to the beta-lactamase production. All the 33 Prevotella strains were susceptible to the other 3 antimicrobial agents tested, except for only one penicillin G – ampicillin resistant isolate of Prevotella buccae (MIC > 32 and MIC = 12 mg/L, respectively), which showed high resistance to clindamycin (MIC > 256 mg/L) too. Our data underline the necessity for antimicrobial testing including monitoring of beta-lactamase production in cases of oro-maxillo-facial mixed anaerobic infections where antimicrobial treatment is required in addition to the surgical drainage. The results of the study indicated that amoxicillin-clavulanate, like metronidazole, was fully active against the tested Prevotella strains. However, local and multicentre surveys on drug resistance among the clinically significant anaerobic isolates should be carried out periodically.


      PubDate: 2015-08-19T12:30:23Z
       
  • Bacterial profiles and proteolytic activity in peri-implantitis versus
           healthy sites
    • Abstract: Publication date: October 2015
      Source:Anaerobe, Volume 35, Part A
      Author(s): J. Neilands, C. Wickström, B. Kinnby, J.R. Davies, J. Hall, B. Friberg, G. Svensäter
      Peri-implantitis is a biofilm-induced destructive inflammatory process that, over time, results in loss of supporting bone around an osseointegrated dental implant. Biofilms at peri-implantitis sites have been reported to be dominated by Gram-negative anaerobic rods with a proteolytic metabolism such as, Fusobacterium, Porphyromonas, Prevotella and Tannerella, as well as anaerobic Gram-positive cocci. In this study, we hypothesized that protease activity is instrumental in driving bone destruction and we therefore compared the microbial composition and level of protease activity in samples of peri-implant biofluid (PIBF) from 25 healthy subjects (H group) and 25 subjects with peri-implantitis (PI group). Microbial composition was investigated using culture techniques and protease activity was determined using a FITC-labelled casein substrate. The microbial composition was highly variable in subjects both in the H and PI groups but one prominent difference was the prevalence of Porphyromonas/Prevotella and anaerobic Gram positive cocci which was significantly higher in the PI than in the H group. A subgroup of subjects with peri-implantitis displayed a high level of protease activity in the PIBF compared to healthy subjects. However, this activity could not be related to the presence of specific bacterial species. We propose that a high level of protease activity may be a predictive factor for disease progression in peri-implantitis. Further longitudinal studies are however required to determine whether assessment of protease activity could serve as a useful method to identify patients at risk for progressive tissue destruction.


      PubDate: 2015-08-19T12:30:23Z
       
  • Hydrogen sulfide production from subgingival plaque samples
    • Abstract: Publication date: October 2015
      Source:Anaerobe, Volume 35, Part A
      Author(s): A. Basic, G. Dahlén
      Periodontitis is a polymicrobial anaerobe infection. Little is known about the dysbiotic microbiota and the role of bacterial metabolites in the disease process. It is suggested that the production of certain waste products in the proteolytic metabolism may work as markers for disease severity. Hydrogen sulfide (H2S) is a gas produced by degradation of proteins in the subgingival pocket. It is highly toxic and believed to have pro-inflammatory properties. We aimed to study H2S production from subgingival plaque samples in relation to disease severity in subjects with natural development of the disease, using a colorimetric method based on bismuth precipitation. In remote areas of northern Thailand, adults with poor oral hygiene habits and a natural development of periodontal disease were examined for their oral health status. H2S production was measured with the bismuth method and subgingival plaque samples were analyzed for the presence of 20 bacterial species with the checkerboard DNA–DNA hybridization technique. In total, 43 subjects were examined (age 40–60 years, mean PI 95 ± 6.6%). Fifty-six percent had moderate periodontal breakdown (CAL > 3 < 7 mm) and 35% had severe periodontal breakdown (CAL > 7 mm) on at least one site. Parvimonas micra, Filifactor alocis, Porphyromonas endodontalis and Fusobacterium nucleatum were frequently detected. H2S production could not be correlated to periodontal disease severity (PPD or CAL at sampled sites) or to a specific bacterial composition. Site 21 had statistically lower production of H2S (p = 0.02) compared to 16 and 46. Betel nut chewers had statistically significant lower H2S production (p = 0.01) than non-chewers. Rapid detection and estimation of subgingival H2S production capacity was easily and reliably tested by the colorimetric bismuth sulfide precipitation method. H2S may be a valuable clinical marker for degradation of proteins in the subgingival pocket.


      PubDate: 2015-08-19T12:30:23Z
       
  • Quantitation of biofilm and planktonic life forms of coexisting
           periodontal species
    • Abstract: Publication date: October 2015
      Source:Anaerobe, Volume 35, Part A
      Author(s): Maribasappa Karched, Radhika G. Bhardwaj, Anandavalli Inbamani, Sirkka Asikainen
      Background Complexity of oral polymicrobial communities has prompted a need for developing in vitro models to study behavior of coexisting bacteria. Little knowledge is available of in vitro co-growth of several periodontitis-associated species without early colonizers of dental plaque. The aim was to determine temporal changes in the quantities of six periodontal species in an in vitro biofilm model in comparison with parallel planktonic cultures. Material and methods Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Parvimonas micra, Campylobacter rectus and Fusobacterium nucleatum were anaerobically grown as multispecies and monospecies biofilms and parallel planktonic cultures using cell culture plates and microfuge tubes, respectively. After incubating 2, 4, 6, 8 days, biofilms and planktonic cultures were harvested, DNA extracted and the target species quantified using qPCR with species-specific 16S rDNA primers. Biofilm growth as monocultures was visualized at day 2 and 8 with confocal microscopy and crystal violet staining. Results The six species were found throughout the test period in all culture conditions, except that P. gingivalis and F. nucleatum were not detected in multispecies planktonic cultures at day 8. In multispecies biofilm, P. gingivalis qPCR counts (cells/ml) increased (P<0.05) from day 2–8 and were then higher (P<0.05) than those of A. actinomycetemcomitans and C. rectus, whereas in monospecies biofilm, P. gingivalis counts were lower (P<0.05) than those of the other species, except A. actinomycetemcomitans. When multi- and monospecies biofilm cultures were compared, P. gingivalis counts were higher (P<0.05) but those of the other species, except P. intermedia, lower (P<0.05) in multispecies biofilm. Comparison between planktonic and biofilm cultures showed that A. actinomycetemcomitans, P. micra and C. rectus had higher (P<0.05) counts in planktonic cultures no matter whether grown in mono- or multispecies environment. Conclusions Six periodontal species were able to form multispecies biofilm up to 8 days in vitro without pioneer plaque bacteria. P. gingivalis seemed to prefer multispecies biofilm environment whereas P. micra and A. actinomycetemcomitans planktonic culture.


      PubDate: 2015-08-19T12:30:23Z
       
  • Relationship between methanogenic archaea and subgingival microbial
           complexes in human periodontitis
    • Abstract: Publication date: October 2015
      Source:Anaerobe, Volume 35, Part A
      Author(s): H.P. Horz, N. Robertz, M.E. Vianna, K. Henne, G. Conrads
      We compared the amounts of methanogenic archaea with ten of the most important periodontal pathogens in 125 clinical samples. Correlation analysis suggests that the support of the periodontitis-associated bacterial consortium by methanogenic archaea may be driven through direct or indirect interactions with Prevotella intermedia.


      PubDate: 2015-08-19T12:30:23Z
       
  • Editorial board
    • Abstract: Publication date: October 2015
      Source:Anaerobe, Volume 35, Part A




      PubDate: 2015-08-19T12:30:23Z
       
  • LuxS signaling in Porphyromonas gingivalis-host interactions
    • Abstract: Publication date: October 2015
      Source:Anaerobe, Volume 35, Part A
      Author(s): Nina Scheres, Richard J. Lamont, Wim Crielaard, Bastiaan P. Krom
      Dental plaque is a multispecies biofilm in the oral cavity that significantly influences oral health. The presence of the oral anaerobic pathogen Porphyromonas gingivalis is an important determinant in the development of periodontitis. Direct and indirect interactions between P. gingivalis and the host play a major role in disease development. Transcriptome analysis recently revealed that P. gingivalis gene-expression is regulated by LuxS in both an AI-2-dependent and an AI-2 independent manner. However, little is known about the role of LuxS-signaling in P. gingivalis-host interactions. Here, we investigated the effect of a luxS mutation on the ability of P. gingivalis to induce an inflammatory response in human oral cells in vitro. Primary periodontal ligament (PDL) fibroblasts were challenged with P. gingivalis ΔluxS or the wild-type parental strain and gene-expression of pro-inflammatory mediators IL-1β, IL-6 and MCP-1 was determined by real-time PCR. The ability of P. gingivalis ΔluxS to induce an inflammatory response was severely impaired in PDL-fibroblasts. This phenotype could be restored by providing of LuxS in trans, but not by addition of the AI-2 precursor DPD. A similar phenomenon was observed in a previous transcriptome study showing that expression of PGN_0482 was reduced in the luxS mutant independently of AI-2. We therefore also analyzed the effect of a mutation in PGN_0482, which encodes an immuno-reactive, putative outer-membrane protein. Similar to P. gingivalis ΔluxS, the P. gingivalis Δ0482 mutant had an impaired ability to induce an inflammatory response in PDL fibroblasts. LuxS thus appears to influence the pro-inflammatory responses of host cells to P. gingivalis, likely through regulation of PGN_0482.


      PubDate: 2015-08-19T12:30:23Z
       
  • Growth characteristics of Lactobacillus brevis KB290 in the presence of
           bile
    • Abstract: Publication date: Available online 10 August 2015
      Source:Anaerobe
      Author(s): Hiromi Kimoto-Nira, Shigenori Suzuki, Hiroyuki Suganuma, Naoko Moriya, Chise Suzuki
      Live Lactobacillus brevis KB290 have several probiotic activities, including immune stimulation and modulation of intestinal microbial balance. We investigated the adaptation of L. brevis KB290 to bile as a mechanism of intestinal survival. Strain KB290 was grown for 5 days at 37°C in tryptone–yeast extract–glucose (TYG) broth supplemented with 0.5% sodium acetate (TYGA) containing 0.15%, 0.3%, or 0.5% bile. Growth was determined by absorbance at 620 nm or by dry weight. Growth was enhanced as the broth’s bile concentration increased. Bile-enhanced growth was not observed in TYG broth or with xylose or fructose as the carbon source, although strain KB290 could assimilate these sugars. Compared with cells grown without bile, cells grown with bile had twice the cell yield (dry weight) and higher hydrophobicity, which may improve epithelial adhesion. Metabolite analysis revealed that bile induced more lactate production by glycolysis, thus enhancing growth efficiency. Scanning electron microscopy revealed that cells cultured without bile for 5 days in TYGA broth had a shortened rod shape and showed lysis and aggregation, unlike cells cultured for 1 day; cells grown with bile for 5 days had an intact rod shape and rarely appeared damaged. Cellular material leakage through autolysis was lower in the presence of bile than in its absence. Thus lysis of strain KB290 cells cultured for extended periods was suppressed in the presence of bile. This study provides new role of bile and sodium acetate for retaining an intact cell shape and enhancing cell yield, which are beneficial for intestinal survival.


      PubDate: 2015-08-14T12:00:29Z
       
  • Performance of BacT/Alert resin-based FN Plus bottles compared with
           BacT/Alert charcoal-based FN bottles for the detection of anaerobes in
           experimentally seeded blood cultures
    • Abstract: Publication date: Available online 5 August 2015
      Source:Anaerobe
      Author(s): Steven De Keukeleire, Ingrid Wybo, Kristof Emmerechts, Denis Piérard
      Recently new resin-based BacT/Alert FAN Plus bottles containing antibiotic-binding polymeric beads, were introduced as an improvement of the charcoal-based FAN bottles for the recovery of bacteria and fungi. To assess the performance of the novel anaerobic FN plus bottles in the detection of anaerobic organisms, we compared the detection rate and the time to detection (TTD) in spiked resin-based FN Plus bottles and charcoal-based FN bottles. The bottles were experimentally seeded with reference strains or clinical strains collected from positive blood cultures. Five reference strains and fifty-five clinically significant anaerobic isolates were investigated, of which 91% (61/67) showed showed growth in both types of bottles within an incubation time of 5 days. A significant prolonged median TTD of 45 h for anaerobic microorganisms was observed in the resin-based bottles versus 29 h in the charcoal-based bottles (P < 0.0001). Bacteroides spp., associated with higher virulence and higher mortality rates in bloodstream infections, were detected faster in the charcoal-based bottles as compared to the resin-based bottles. In conclusion recently improved resin FN bottles showed a significantly increased median TTD for the recovery of anaerobic isolates.


      PubDate: 2015-08-06T11:20:13Z
       
  • Antibiotic profiling of Clostridium difficile ribotype 176 - a multidrug
           resistant relative to Clostridium difficile ribotype 027
    • Abstract: Publication date: Available online 6 August 2015
      Source:Anaerobe
      Author(s): Marcela Krutova, Jana Matejkova, Jan Tkadlec, Otakar Nyc
      Antibiotic profiling of twenty Czech C. difficile PCR-ribotype 176 isolates revealed a high level of resistance to erythromycin, ciprofloxacin and moxifloxacin (n=20) and to rifampicin (n=13). Accumulation of resistance mechanisms to multiple antibiotics highlight that PCR-ribotype 176 belong to problematic epidemic strains.


      PubDate: 2015-08-06T11:20:13Z
       
  • Comparative analysis of extractable proteins from Clostridium perfringens
           type A and type C strains showing varying degree of virulence
    • Abstract: Publication date: Available online 31 July 2015
      Source:Anaerobe
      Author(s): Pratistha Dwivedi, Syed Imteyaz Alam, Om Kumar, Ravi Bhushan Kumar
      The prevailing scenario of bioterrorism warrants development of medical countermeasures with expanded coverage of select agents. Clostridium perfringens is a pathogen of medical, veterinary and military importance, and has been listed as Validated Biological Agent. We employed 2DE-MS approach to identify a total of 134 unique proteins (529 protein spot features) from the extractable proteome of four type A and type C strains. Proteins showing altered expression under host-simulated conditions from virulent type A strain (ATCC13124) were also elucidated. Significant among the differentially expressed proteins were elongation factor, molecular chaperones, ribosomal proteins, carbamoyl phosphate synthase, clpB protein, choloylglycine hydrolase, phosphopyruvate hydratase, and trigger factor. Predictive elucidation, of putative virulence associated proteins and sequence conservation pattern of selected candidates, was carried out using homologous proteins from other bacterial select agents to screen for the commonality of putative antigenic determinants. Pathogens (17 select agents) were observed to form three discrete clusters; composition of I and II being consistent in most of the phylogenetic reconstructions. This work provides a basis for further validation of putative candidate proteins as prophylactic agents and for their ability to provide protection against clusters of pathogenic select bacterial agents; aimed at mitigating the shadows of biothreat.
      Graphical abstract image

      PubDate: 2015-08-02T03:26:01Z
       
  • The Prevalence of Enterotoxin and Antibiotic Resistance Genes In Clinical
           and Intestinal Bacteroides fragilis group isolates in Turkey
    • Abstract: Publication date: Available online 26 July 2015
      Source:Anaerobe
      Author(s): Achille Aime Kangaba, Filiz Yarimcam Saglam, Hrisi Bahar Tokman, Mert Torun, Muzeyyen Mamal Torun
      This study was conducted to measure the antibiotic susceptibilities, corresponding gene contents, and the enterotoxin gene bft, in 50 Bacteroides fragilis group isolates, 25 of which were clinical and 25 intestinal. The resistance rates to amoxicillin/clavulanic acid, imipenem and metronidazole were low; ampicillin and tetracyclin resistance was high; clindamycin resistance and ermF gene presence was also high. Regarding phenotypical bacterial resistance and the presence of resistance genes, there was not statistically significant difference between clinical and intestinal isolates and bft positive and negative isolates.


      PubDate: 2015-07-28T20:48:37Z
       
  • Functional analysis of the type II toxin-antitoxin systems of the MazEF
           and RelBE families in Bifidobacterium longum subsp. infantis ATCC 15697
    • Abstract: Publication date: Available online 23 July 2015
      Source:Anaerobe
      Author(s): Olga Averina, Maria Alekseeva, Andrei Shkoporov, Valery Danilenko
      Analysis of the Bifidobacterium longum subsp. infantis ATCC 15697 genome sequence for the presence of toxin-antitoxin genes revealed two relBE-like operons, three relB-mazF-like operons, one relB-vapC-like operon, one solitary gene coding for the MazF toxin and one gene coding for the RelB antitoxin. An attempt to clone the selected relE and mazF toxin genes from B. longum subsp. infantis ATCC 15697 revealed their toxic effects on Escherichia coli, which could be neutralized by coexpression of these toxins with their cognate antitoxins. The only two toxin proteins, RelE and VapC, that were found to be non-toxic to E. coli, were overproduced and purified. Electrophoretic assays showed that both RelE and VapC possessed direct endoribonuclease activity. The expression levels of toxin genes in B. longum subsp. infantis ATCC 15697 increased during the nutrient starvation and entry into the late stationary phase. The two relBE bicistronic operons relE2-relB1 and relE1-relB4 from B. longum subsp. infantis ATCC 15697 were cloned and overexpressed in B. longum subsp. longum NCC2705 strain. The strain B. longum NCC2705 [pCESH80::relE1-relB4] showed a significantly decreased growth rate with later onset of the log phase and decreased cells density in the stationary phase.


      PubDate: 2015-07-25T08:03:03Z
       
  • cfxA expression in oral clinical Capnocytophaga isolates
    • Abstract: Publication date: Available online 21 July 2015
      Source:Anaerobe
      Author(s): Zohreh Tamanai-Shacoori, Clarisse Monfort, Nolwenn Oliviero, Philippe Gautier, Martine Bonnaure-Mallet, Anne Jolivet-Gougeon
      Capnocytophaga spp. are commensal bacteria involved in oral and systemic diseases, with a variable susceptibility to beta-lactams. The cfxA gene expression level was assessed using quantitative RT-PCR, and reasons of the observed misexpression were discussed, as insertion of foreign genetic material, contributing to dissemination and evolution of antibiotic resistance genes.


      PubDate: 2015-07-21T07:51:22Z
       
  • Gut Bifidobacterium microbiota in one-month-old Brazilian newborns
    • Abstract: Publication date: Available online 20 July 2015
      Source:Anaerobe
      Author(s): Łukasz Grześkowiak, Tatiana Fiche Sales Teixeira, Solange Mara Bigonha, Guilherme Lobo, Seppo Salminen, Celia Lucia de Luces Fortes Ferreira
      Gut colonisation with bifidobacteria in early infancy is essential for the well-being of the infant. Gestational age and mode of delivery are among the factors influencing the colonisation process. The aim was to characterise the bifidobacterial composition in the gut of one-month-old full-term and pre-term Brazilian infants, both being delivered vaginally or by caesarean section. Fourthy nine Brazilian (Viçosa, Minas Gerais state) one-month-old infants were divided in two groups: full-term (n=24) and pre-term (n=25), and compared to each other. Each group was then characterised according to its mode of delivery. Infant stool samples were available for bifidobacterial characterisation by quantitative polymerase chain reaction (qPCR) method. All study infants were colonised by bifidobacteria. Bifidobacterium longum colonised all full-term and pre-term newborns. Differences were observed in counts of Bifidobacterium genus and Bifidobacterium longum between full-term and pre-term infants (8.8 log cells/g, IQR 7.9-9.1 vs. 7.1 log cells/g, IQR 6.6-8.6, p=0.02 and 8.3 log cells/g, IQR 6.7-9.1 vs. 6.4 log cells/g, IQR 6.1-6.7, p=0.001, respectively). Furthermore, the prevalence of Bifidobacterium lactis differed between pre-term caesarean and pre-term vaginally born infants (50.0 % vs. 93.8 %, p=0.023). Gut bifidobacterial composition of one-month-old full-term infants differs from that of pre-term newborns in Viçosa, Minas Gerais state, Brazil. Gestational age is a factor influencing bacterial numbers and species, while mode of delivery have an impact on the prevalence and quantity of bifidobacteria in studied infants. Bifidobacteria may have an impact on later health of the infants and the species B. longum and B. lactis might provide clues on the potential probiotic applications in pre-term newborns at the risk of developing postnatal complications.


      PubDate: 2015-07-21T07:51:22Z
       
  • A Clostridium hathewayi isolate in blood culture of a patient with an
           acute appendicitis
    • Abstract: Publication date: Available online 15 July 2015
      Source:Anaerobe
      Author(s): Adrien Randazzo, Anne Kornreich, Bénédicte Lissoir
      Introduction Clostridium species is a group of anaerobic bacteria constituting the colonic microflora of the intestinal tract. Since molecular methodologies based on 16 rRNA have been established for the classification and the recognition of bacterial species, more than 150 species of Clostridium have been described. Most are considered harmless saprophytes; however, these bacteria may be involved in a wide variety of infections and may be a common cause of enteritis and enterotoxemias in humans. Case presentation We present the case of a 60-year-old Asian patient admitted in the emergency room with an acute appendicitis where a blood culture showed the presence of a Clostridium hathewayi. This microorganism is an anaerobic bacteria described in 2001 as a Gram negative end-pointed bacillus, usually endospore-forming. It was reclassified in 2014 as Hungatella hathewayi. A literature review has been performed to find articles relating to this bacteria in a clinical case. Conclusion Clostridium hathewayi is microorganism recently reclassified as Hungatella hathewayi. Its growth in blood cultures has been reported in a few cases in the literature. Although only a few articles have reported its involvement in clinical infections, we assess that its part in the cause of the illness should be evaluated.


      PubDate: 2015-07-17T07:34:18Z
       
  • First Clinical Description of Eggerthia catenaformis Bacteremia in a
           Patient with Dental Abscess
    • Abstract: Publication date: Available online 11 July 2015
      Source:Anaerobe
      Author(s): Hayarpi H. Kordjian , Joyce D.J.H. Schultz , Flemming Schønning Rosenvinge , Jakob Møller , Rune M. Pedersen
      We present a case of Eggerthia catenaformis bacteremia originating from a dental abscess and imitating necrotizing fasciitis in a previously healthy adult. The isolates were easily identified by MALDI-TOF MS. The clinical course, surgical and antibiotic treatment as well as the successful outcome are reported.


      PubDate: 2015-07-13T07:07:23Z
       
  • Occurrence and new mutations involved in rifampicin-resistant
           Propionibacterium acnes strains isolated from biofilm or device-related
           infections
    • Abstract: Publication date: August 2015
      Source:Anaerobe, Volume 34
      Author(s): Ulrika Furustrand Tafin , Guillaume Ghislain Aubin , Gerhard Eich , Andrej Trampuz , Stéphane Corvec
      We described for the first time the amino acid substitutions conferring rifampicin resistance in eight Propionibacterium acnes strains isolated from patients with biofilm or device-related infections. We identified different mutations in cluster I and one mutation, never reported, in cluster II of the rpoB gene (I480V) associated with the most frequent one in cluster I (S442L). Half of the patients previously received treatment with rifampicin.


      PubDate: 2015-07-08T06:30:23Z
       
  • Fusobacterium necrophorum and other Fusobacterium spp. isolated from head
           
    • Abstract: Publication date: August 2015
      Source:Anaerobe, Volume 34
      Author(s): Erlangga Yusuf , Stijn Halewyck , Ingrid Wybo , Denis Piérard , Frans Gordts
      Background Fusobacterium spp. from clinical specimens are increasingly reported. We sought to describe the epidemiology, the microbiological, and the clinical characteristics of head and neck infections caused by Fusobacterium necrophorum and other Fusobacterium spp. Materials and methods Retrospective cohort study between October 1st, 2004 and September 30th, 2014 performed in an academic hospital. Electronic patient charts and the laboratory information system were reviewed for demographic and microbiological data. The number and percentages of specific diagnosis and treatment among patients with positive Fusobacterium spp. culture were calculated. The incidence was calculated based on the number of specimens investigated each year. Results Included were 230 cultures of 230 patients (median age of 28 years, 61.7% men). F. necrophorum was often found in young patients with high C-reactive protein (CRP) and high number of leukocytes in blood. Other Fusobacterium spp. were often found in middle aged patients with relatively high CRP and slightly increased leukocytes. Three major causes of the isolation of F. necrophorum and other Fusobacterium spp. were acute tonsillitis (n = 18, incidence of 0.2%), peritonsillar abscess (n = 39, 0.5%) and acute otitis (n = 45, 0.1%). While F. necrophorum was found in majority (37/57) of patients with acute tonsillitis or peritonsillar abscess, Fusobacterium spp. other than F. necrophorum were found in the majority (35/45) of patients with acute otitis. Isolated fusobacteria were susceptible to beta-lactam antibiotics, clindamycin and metronidazole. The outcomes of patients with Fusobacterium spp. were good. Conclusion F. necrophorum and other Fusobacterium spp. are rare cause of head and neck infections. The infections are well treated by combination of antibiotics and surgery.


      PubDate: 2015-07-08T06:30:23Z
       
  • Recurrent Clostridium difficile infection: From colonization to cure
    • Abstract: Publication date: August 2015
      Source:Anaerobe, Volume 34
      Author(s): Kelsey Shields , Roger V. Araujo-Castillo , Thimmaiah G. Theethira , Carolyn D. Alonso , Ciaran P. Kelly
      Clostridium difficile infection (CDI) is increasingly prevalent, dangerous and challenging to prevent and manage. Despite intense national and international attention the incidence of primary and of recurrent CDI (PCDI and RCDI, respectively) have risen rapidly throughout the past decade. Of major concern is the increase in cases of RCDI resulting in substantial morbidity, morality and economic burden. RCDI management remains challenging as there is no uniformly effective therapy, no firm consensus on optimal treatment, and reliable data regarding RCDI-specific treatment options is scant. Novel therapeutic strategies are critically needed to rapidly, accurately, and effectively identify and treat patients with, or at-risk for, RCDI. In this review we consider the factors implicated in the epidemiology, pathogenesis and clinical presentation of RCDI, evaluate current management options for RCDI and explore novel and emerging therapies.


      PubDate: 2015-07-08T06:30:23Z
       
  • Molecular characterization and antimicrobial susceptibilities of
           Clostridium difficile clinical isolates from Victoria, Australia
    • Abstract: Publication date: August 2015
      Source:Anaerobe, Volume 34
      Author(s): Kate E. Mackin , Briony Elliott , Despina Kotsanas , Benjamin P. Howden , Glen P. Carter , Tony M. Korman , Thomas V. Riley , Julian I. Rood , Grant A. Jenkin , Dena Lyras
      Some Australian strain types of Clostridium difficile appear unique, highlighting the global diversity of this bacterium. We examined recent and historic local isolates, finding predominantly toxinotype 0 strains, but also toxinotypes V and VIII. All isolates tested were susceptible to vancomycin and metronidazole, while moxifloxacin resistance was only detected in recent strains.


      PubDate: 2015-07-08T06:30:23Z
       
  • Actinobaculum schaalii bacteremia: A report of two cases
    • Abstract: Publication date: August 2015
      Source:Anaerobe, Volume 34
      Author(s): Lemuel R. Non , Allison Nazinitsky , Mark D. Gonzalez , Carey-Ann D. Burnham , Rupa Patel
      We report two cases of bacteremia with Actinobaculum schaalii, a rarely reported, anaerobic, Gram-positive bacterium. The first case was a patient with renal cancer who developed pyelonephritis after cryoablation, and the second was a patient who developed sepsis after a urogenital procedure. Bacteremia resolved after administration of empiric antibiotic therapy.


      PubDate: 2015-07-08T06:30:23Z
       
  • Antibacterial activity of Lactobacillus spp. isolated from the feces of
           healthy infants against enteropathogenic bacteria
    • Abstract: Publication date: August 2015
      Source:Anaerobe, Volume 34
      Author(s): Abolfazl Davoodabadi , Mohammad Mehdi Soltan Dallal , Abbas Rahimi Foroushani , Masoumeh Douraghi , Mohammad kazem Sharifi Yazdi , Farzaneh Amin Harati
      Lactobacilli are normal microflora of the gastrointestinal (GI) tract and are a heterogeneous group of lactic acid bacteria (LAB). Lactobacillus strains with Probiotic activity may have health Benefits for human. This study investigates the probiotic potential of Lactobacillus strains obtained from the feces of healthy infants and also explores antibacterial activity of Lactobacillus strains with probiotic potential against enteropathogenic bacteria. Fecal samples were collected from 95 healthy infants younger than 18 months. Two hundred and ninety Lactobacillus strains were isolated and assessed for probiotic potential properties including ability to survive in gastrointestinal conditions (pH 2.0, 0.3% oxgall), adherence to HT-29 cells and antibiotic resistance. Six strains including Lactobacillus fermentum (4 strains), Lactobacillus paracasei and Lactobacillus plantarum showed good probiotic potential and inhibited the growth of enteropathogenic bacteria including ETEC H10407, Shigella flexneri ATCC 12022, Shigella sonnei ATCC 9290, Salmonella enteritidis H7 and Yersinia enterocolitica ATCC 23715. These Lactobacillus strains with probiotic potential may be useful for prevention or treatment of diarrhea, but further in vitro and in vivo studies on these strains are still required.


      PubDate: 2015-07-08T06:30:23Z
       
  • Editorial board
    • Abstract: Publication date: August 2015
      Source:Anaerobe, Volume 34




      PubDate: 2015-07-08T06:30:23Z
       
  • The role of anaerobes in diabetic foot infections
    • Abstract: Publication date: August 2015
      Source:Anaerobe, Volume 34
      Author(s): Patrick G.P. Charles , Ilker Uçkay , Benjamin Kressmann , Stéphane Emonet , Benjamin A. Lipsky
      Diabetic foot infections (DFI) are a common cause of morbidity and, on occasion, even mortality. Infection can be either mono- or polymicrobial, with a wide variety of potential pathogens. Anaerobes may be involved, particularly in wounds that are deeper or more chronic, and are more frequently identified when using modern molecular techniques, such as 16s PCR and pyrosequencing. It remains unclear whether the presence of anaerobes in DFI leads to more severe manifestations, or if these organisms are largely colonizers associated with the presence of greater degrees of tissue ischemia and necrosis. Commonly used empiric antibiotic therapy for diabetic foot infections is generally broad-spectrum and usually has activity against the most frequently identified anaerobes, such as Peptostreptococcus and Bacteroides species. Adequate surgical debridement and, when needed, foot revascularization may be at least as important as the choice of antibiotic to achieve a successful treatment outcome.


      PubDate: 2015-07-08T06:30:23Z
       
  • A new medium containing mupirocin, acetic acid, and norfloxacin for the
           selective cultivation of bifidobacteria
    • Abstract: Publication date: August 2015
      Source:Anaerobe, Volume 34
      Author(s): Eva Vlková , Hana Salmonová , Věra Bunešová , Martina Geigerová , Vojtěch Rada , Šárka Musilová
      Various culture media have been proposed for the isolation and selective enumeration of bifidobacteria. Mupirocin is widely used as a selective factor along with glacial acetic acid. TOS (transgalactosylated oligosaccharides) medium supplemented with mupirocin is recommended by the International Dairy Federation for the detection of bifidobacteria in fermented milk products. Mupirocin media with acetic acid are also reliable for intestinal samples in which bifidobacteria predominate. However, for complex samples containing more diverse microbiota, the selectivity of mupirocin media is limited. Resistance to mupirocin has been demonstrated by many anaerobic bacteria, especially clostridia. The objective was to identify an antibiotic that inhibits the growth of clostridia and allows the growth of bifidobacteria, and to use the identified substance to develop a selective cultivation medium for bifidobacteria. The susceptibility of bifidobacteria and clostridia to 12 antibiotics was tested on agar using the disk diffusion method. Only norfloxacin inhibited the growth of clostridia and did not affect the growth of bifidobacteria. Using both pure cultures and faecal samples from infants, adults, calves, lambs, and piglets, the optimal concentration of norfloxacin in solid cultivation media was determined to be 200 mg/L. Our results showed that solid medium containing norfloxacin (200 mg/L) in combination with mupirocin (100 mg/L) and glacial acetic acid (1 mL/L) is suitable for the enumeration and isolation of bifidobacteria from faecal samples of different origins.


      PubDate: 2015-07-08T06:30:23Z
       
  • Study of the combined effect of electro-activated solutions and heat
           treatment on the destruction of spores of Clostridium sporogenes and
           Geobacillus stearothermophilus in model solution and vegetable puree
    • Abstract: Publication date: October 2015
      Source:Anaerobe, Volume 35, Part B
      Author(s): Viacheslav Liato , Steve Labrie , Catherine Viel , Marzouk Benali , Mohammed Aïder
      The combined effect of heat treatment and electro-activated solution (EAS) on the heat resistance of spores of Clostridium sporogenes and Geobacillus stearothermophilus was assessed under various heating and exposure time combinations. The acid and neutral EAS showed the highest inhibitory activity, indicating that these solutions may be considered as strong sporicidal disinfectants. These EAS were able to cause a reduction of ≥6 log of spores of C. sporogenes at 60 °C in only 1 min of exposition. For G. stearothermophilus spores, a reduction of 4.5 log was observed at 60 °C in 1 min, while in 5 min, ≥7 log CFU/ml reduction was observed. Inoculated puree of pea and corn were used as a food matrix for the determination of the heat resistance of these spores during the treatments in glass capillaries. The inactivation kinetics of the spores was studied in an oil bath. Combined treatment by EAS and temperature demonstrated a significant decrease in the heat resistance of C. sporogenes. The D100°C in pea puree with NaCl solution was 66.86 min while with acid and neutral EAS it was reduced down to 3.97 and 2.19 min, respectively. The spore of G. stearothermophilus displayed higher heat resistance as confirmed by other similar studies. Its D130°C in pea puree showed a decrease from 1.45 min in NaCl solution down to 1.30 and 0.93 min for acid and neutral EAS, respectively. The differences between the spores of these species are attributable to their different sensitivities with respect to pH, Redox potential and oxygen.


      PubDate: 2015-07-08T06:30:23Z
       
  • Evaluation of immunomodulatory activity of two potential probiotic
           Lactobacillus strains by in vivo tests
    • Abstract: Publication date: October 2015
      Source:Anaerobe, Volume 35, Part B
      Author(s): Dayong Ren , Chang Li , Yanqing Qin , Ronglan Yin , Shouwen Du , Hongfeng Liu , Yanfang Zhang , Cuiyan Wang , Fengjun Rong , Ningyi Jin
      Here we evaluate the immunomodulatory function of two potential probiotic strains, Lactobacillus salivarius CICC 23174 and Lactobacillus plantarum CGMCC 1.557. Mice were fed with each Lactobacillus strain at different doses for several consecutive days. The effects of the two probiotic strains on immune organs, immune cells and immune molecules were investigated on days 10 and 20. Both Lactobacillus strains increased the spleen index, improved the spleen lymphocyte transformation rate, enhanced sIgA production and improved the number of CD11c+ CD80+ double-positive cells. L. plantarum CGMCC 1.557 was the more active strain in enhancing the phagocytic activity of macrophages, while, L. salivarius CICC 23174 was the more effective strain at maintaining the Th1/Th2 balance. This study suggests that these two Lactobacillus strains have beneficial effects on regulation of immune responses, which has promising implications for the development of ecological agents and functional foods.


      PubDate: 2015-07-08T06:30:23Z
       
  • 16S rDNA analysis of archaea indicates dominance of Methanobacterium and
           high abundance of Methanomassiliicoccaceae in rumen of Nili-Ravi buffalo
    • Abstract: Publication date: October 2015
      Source:Anaerobe, Volume 35, Part B
      Author(s): S.S. Paul , S.M. Deb , A. Dey , S.P.S. Somvanshi , D. Singh , R. Rathore , J. Stiverson
      The molecular diversity of rumen methanogens was investigated using 16S rDNA gene library prepared from the rumen contents of Nili-Ravi buffaloes. Microbial genomic DNA was isolated from four adult male fistulated buffaloes and PCR conditions were set up using specific primers. Amplified product was cloned into a suitable vector, and the inserts of positive clones were sequenced. A total of 142 clones were examined, and the analysis revealed 46 species level (0.01 distance) operational taxonomic units (OTUs). Twenty six OTUs comprising 89 clones (63% of the total clones) were taxonomically assigned to Methanobacterium genus and the majority of them had highest percent identity with Methanobacterium flexile among cultured methanogens. Five OTUs comprising 27 clones (19% of total clones) were taxonomically assigned to Methanomicrobium genus and these clones showed highest sequence identity with Methanomicrobium mobile. Only two OTUs comprising 6 clones (4% of total clones) were assigned to Methanobrevibacter genus. A total of 17 clones belonging to 10 species level OTUs showed highest percent identity (ranging from 85 to 95%) with Methanomassilicoccus luminyensis and were taxonomically classified as Methanomassiliicocaceae. Out of the 142 rDNA clones, 112 clones, which constitute 79% of the total clones representing 42 OTUs, had less than 98.5% sequence identity with any of the cultured strains of methanogens and represent novel species of methanogens. This study has revealed the largest assortment of hydrogenotrophic methanogen phylotypes ever identified from the rumen of Nili-Ravi buffaloes. The study indicates that Methanobacterium is the most dominant methanogen in the rumen of Nili-Ravi buffalo. This is also the first report on the presence of methanogens phylogenetically close to M. luminyensis, an H2 dependent methylotrophic methanogen, in the rumen of buffaloes at such a high level of abundance.


      PubDate: 2015-07-08T06:30:23Z
       
  • Actinomycosis of eye: Forgotten but not uncommon
    • Abstract: Publication date: October 2015
      Source:Anaerobe, Volume 35, Part B
      Author(s): Neha Mehrotra , Ankita Baidya , Megha Brijwal , Rita Aggarwal , Rama Chaudhry
      Actinomyces species are known to cause a variety of human infections. Ocular actinomycosis is a rare disease. We report an unusual case of bilateral actinomycotic blepharoconjunctivitis in the absence of canaliculitis that presented with forniceal masses in eye. The case report is discussed here along with Indian literature.


      PubDate: 2015-07-08T06:30:23Z
       
  • Spondylodiscitis due to anaerobic bacteria about a case of Parvimonas
           micra infection
    • Abstract: Publication date: August 2015
      Source:Anaerobe, Volume 34
      Author(s): B. Pilmis , J. Israel , A. Le Monnier , A. Mizrahi
      Parvimonas micra is a rare isolate in clinical specimens. We report a case of spondylodiscitis caused by P. micra, a rarely reported Gram positive cocci. The case was an elderly patient with joint surgery and ischaemic heart disease history. Infection resolved after adequate antibiotic therapy.


      PubDate: 2015-07-08T06:30:23Z
       
  • Bacteremic meningitis caused by Parvimonas micra in an immunocompetent
           host
    • Abstract: Publication date: August 2015
      Source:Anaerobe, Volume 34
      Author(s): Jae-Hoon Ko , Jin Yang Baek , Cheol-In Kang , Woo Joo Lee , Ji Yong Lee , Sun Young Cho , Young Eun Ha , So Hyun Kim , Doo Ryeon Chung , Kyong Ran Peck , Nam Yong Lee , Jae-Hoon Song
      A 61-year-old man with chronic hepatitis B and dyslipidemia visited the emergency department with a fever and severe headache. He was diagnosed with bacterial meningitis after a lumbar puncture, and blood culture revealed Parvimonas micra bacteremia. Although he had a history of extraction of a molar two weeks before symptom onset, there was no evidence of abscess formation on physical examination or imaging studies. He was successfully treated with oral metronidazole for 12 days after 9 days of treatment with IV ceftriaxone and vancomcycin. This is the first report of primary bacterial meningitis caused by this organism, which indicates that this organism is capable of being a bacterial meningitis pathogen.


      PubDate: 2015-07-08T06:30:23Z
       
  • Pomegranate ellagitannins stimulate growth of gut bacteria in vitro:
           Implications for prebiotic and metabolic effects
    • Abstract: Publication date: August 2015
      Source:Anaerobe, Volume 34
      Author(s): Zhaoping Li , Paula H. Summanen , Tomoe Komoriya , Susanne M. Henning , Ru-Po Lee , Eliisa Carlson , David Heber , Sydney M. Finegold
      The present study investigated the effect of pomegranate extract (POMx) and pomegranate juice (POM juice) on the growth of major groups of intestinal bacteria: Enterobacteriaceae, Bacteroides fragilis group, clostridia, bifidobacteria, and lactobacilli, and the utilization of pomegranate polyphenols by Bifidobacterium and Lactobacillus. The total phenolic content of the pomegranate extract and juice was determined using the Folin-Ciocalteau colorimetric method and reported as gallic acid equivalent (GAE). The polyphenol composition was determined by HPLC. Stool specimens were incubated with 400, 100, and 25 μg/ml GAE POMx and POM juice and subjected to selective culture. Bifidobacterium and Lactobacillus strains were incubated with 400 μg/ml GAE POMx and POM juice and metabolites were analyzed. POMx and POM juice increased the mean counts of Bifidobacterium and Lactobacillus and significantly inhibited the growth of B. fragilis group, clostridia, and Enterobacteriaceae in a dose–response manner. Bifidobacterium and Lactobacillus utilized ellagic acid and glycosyl ellagic acid but little or no punicalin was utilized. Neither POMx nor POM juice was converted to urolithins by the test bacteria or the in vitro stool cultures. The effect of pomegranate on the gut bacteria considered to be beneficial (Bifidobacterium and Lactobacillus) suggests that pomegranate may potentially work as a prebiotic. The concept that polyphenols such as those in pomegranate impact gut microbiota populations may establish a new role for polyphenols in human health.


      PubDate: 2015-07-08T06:30:23Z
       
  • Screening of Propionibacterium spp. for potential probiotic properties
    • Abstract: Publication date: August 2015
      Source:Anaerobe, Volume 34
      Author(s): Daniela Campaniello , Antonio Bevilacqua , Milena Sinigaglia , Clelia Altieri
      The main topic of this paper is the evaluation of adhesion of propionibacteria to IPEC-J2 cells and the survival at pH 2.5 and with 0.3% bile salts added, bioactivity towards pathogens and antibiotic resistance of Propionibacterium freudenreichii subsp. shermanii, Propionibacterium jensenii, Propionibacterium acidipropionici and Propionibacterium thoenii. Adhesion to IPEC-J2 cell lines was ca. 25–35% and significantly increased with CaCl2. Moreover, propionibacteria showed a reduction of cell count of ca. 0.5% at pH 2.5 after 3 h, whereas cell count increased after 24 h with bile salts; finally, they significantly inhibited Escherichia coli O157:H7.


      PubDate: 2015-07-08T06:30:23Z
       
  • Insight into alteration of gut microbiota in Clostridium difficile
           infection and asymptomatic C. difficile colonization
    • Abstract: Publication date: August 2015
      Source:Anaerobe, Volume 34
      Author(s): Lihua Zhang , Danfeng Dong , Cen Jiang , Zhen Li , Xuefeng Wang , Yibing Peng
      Clostridium difficile is well recognized as the common pathogen of nosocomial diarrhea, meanwhile, asymptomatic colonization with C. difficile in part of the population has also drawn public attention. Although gut microbiota is known to play an important role in the pathogenesis of C. difficile infection (CDI), whether there is any alteration of gut microbial composition in asymptomatic C. difficile carriers hasn't been clearly described. The purpose of this study was to explore the differences in gut microbiome among CDI patients, asymptomatic C. difficile carriers and healthy individuals. We performed fecal microbiota analysis on the samples of eight CDI patients, eight asymptomatic C. difficile carriers and nine healthy subjects using 16S rRNA gene pyrosequencing. CDI patients and asymptomatic carriers showed reduced microbial richness and diversity compared with healthy subjects, accompanied with a paucity of phylum Bacteroidetes and Firmicutes as well as an overabundance of Proteobacteria. Some normally commensal bacteria, especially butyrate producers, were significantly depleted in CDI patients and asymptomatic carriers. Furthermore, the differences observed in microbial community structure between CDI patients and asymptomatic carriers suggested that the gut microbiota may be a potential factor of disease state for CDI. Our study demonstrates the characterization and diversity of gut microbiota in CDI and asymptomatic C. difficile colonization, which will provide new ideas for surveillance of the disease state and development of microbiota-targeted agents for CDI prevention and treatment.


      PubDate: 2015-07-08T06:30:23Z
       
  • Microbiota and probiotics in canine and feline welfare
    • Abstract: Publication date: August 2015
      Source:Anaerobe, Volume 34
      Author(s): Łukasz Grześkowiak , Akihito Endo , Shea Beasley , Seppo Salminen
      Dogs and cats have been cohabiting with us for thousands of years. They are the major human companions. Today, dogs and cats live in urban areas. Cats and most dogs are on high carbohydrate diets and face similar life-style challenges as the human beings. The health and well-being of companion animals, just as their owners, depends on the gut microbes. Providing a proper care and nutritionally balanced diet to companion animals is recognised as a part of our responsibility to maintain the health and well being of our pet. However, as microbiota differences may facilitate exposure to pathogens and harmful environmental influences, it is prudent to search for novel tools to protect dogs and cats and at the same time the human owners from pathogens. Specific probiotic strains and/or their defined combinations may be useful in the canine and feline nutrition, therapy, and care. Probiotic supplementations have been successful in the prevention and treatment of acute gastroenteritis, treatment of IBD, and prevention of allergy in companion animals. New challenges for probiotic applications include maintenance of obesity and overweight, urogenital tract infections, Helicobacter gastritis and parasitic infections. The probiotics of human origin appear to be among the new promising tools for the maintenance of pets' health. However, the host-derived microorganisms might be the most appropriate probiotic source. Therefore, more controlled trials are needed to characterise new and safe probiotic preparations with an impact on general health and well being as well as health maintenance in dogs and cats.


      PubDate: 2015-07-08T06:30:23Z
       
  • Non-classical azoreductase secretion in Clostridium perfringens in
           response to sulfonated azo dye exposure
    • Abstract: Publication date: August 2015
      Source:Anaerobe, Volume 34
      Author(s): Jessica M. Morrison , Gilbert H. John
      Clostridium perfringens, a strictly anaerobic microorganism and inhabitant of the human intestine, has been shown to produce an azoreductase enzyme (AzoC), an NADH-dependent flavin oxidoreductase. This enzyme reduces azo dyes into aromatic amines, which can be carcinogenic. A significant amount of work has been completed on the activity of AzoC. Despite this, much is still unknown, including whether azoreduction of these dyes occurs intracellularly or extracellulary. A physiological study of C. perfringens involving the effect of azo dye exposure was completed to answer this question. Through exposure studies, azo dyes were found to cause cytoplasmic protein release, including AzoC, from C. perfringens in dividing and non-dividing cells. Sulfonation (negative charge) of azo dyes proved to be the key to facilitating protein release of AzoC and was found to be azo-dye-concentration-dependent. Additionally, AzoC was found to localize to the Gram-positive periplasmic region. Using a ΔazoC knockout mutant, the presence of additional azoreductases in C. perfringens was suggested. These results support the notion that the azoreduction of these dyes may occur extracellularly for the commensal C. perfringens in the intestine.


      PubDate: 2015-07-08T06:30:23Z
       
  • Development of a triplex real-time PCR assay for the simultaneous
           detection of Clostridium beijerinckii, Clostridium sporogenes and
           Clostridium tyrobutyricum in milk
    • Abstract: Publication date: August 2015
      Source:Anaerobe, Volume 34
      Author(s): Stefano Morandi , Paola Cremonesi , Tiziana Silvetti , Bianca Castiglioni , Milena Brasca
      Clostridium beijerinckii, Clostridium sporogenes and Clostridium tyrobutyricum are considered the leading bacteria implicated in late blowing defects affecting semi-hard and hard cheese production. The aim of this study was to develop a multiplex Real-Time PCR (qPCR) analysis for a rapid and simultaneous detection of C. beijerinckii, C. sporogenes and C. tyrobutyricum, using specific primers respectively targeting the nifH, gerAA and enr genes. The limits of detection in raw milk were 300 CFU/50 mL in the case of C. beijerinckii, 2 CFU/50 mL for C. sporogenes and 5 CFU/50 mL for C. tyrobutyricum spores. The qPCR method was applied to artificially contaminated raw milk samples, and molecular quantification showed good correlation (R2 = 0.978) with microbiological counting. Our results demonstrate that this method, combined with a DNA extraction protocol optimized for spore lysis, could be a useful tool for the direct quantification of the considered clostridia species.


      PubDate: 2015-07-08T06:30:23Z
       
  • Evaluation of composition and individual variability of rumen microbiota
           in yaks by 16S rRNA high-throughput sequencing technology
    • Abstract: Publication date: August 2015
      Source:Anaerobe, Volume 34
      Author(s): Wei Guo , Ying Li , Lizhi Wang , Jiwen Wang , Qin Xu , Tianhai Yan , Bai Xue
      The Yak (Bos grunniens) is a unique species of ruminant animals that is important to agriculture of the Tibetan plateau, and has a complex intestinal microbial community. The objective of the present study was to characterize the composition and individual variability of microbiota in the rumen of yaks using 16S rRNA gene high-throughput sequencing technique. Rumen samples used in the present study were obtained from grazing adult male yaks (n = 6) in a commercial farm in Ganzi Autonomous Prefecture of Sichuan Province, China. Universal prokaryote primers were used to target the V4–V5 hypervariable region of 16S rRNA gene. A total of 7200 operational taxonomic units (OTUs) were obtained after sequence filtering and chimera removal. Within these OTUs, 0.56% belonged to Archaea (40 OTUs), 7.19% to unassigned species (518 OTUs), and the remaining OTUs (6642) in all samples were of bacterial origin. When examining the community structure of bacteria, we identified 23 phyla within 159 families after taxonomic summarization. Bacteroidetes and Firmicutes were the predominant phyla accounting for 39.68% (SD = 0.05) and 45.90% (SD = 0.06), respectively. Moreover, 3764 OTUs were identified as shared OTUs (i.e. represented in all yaks) and belonged to 35 genera, exhibiting highly variable abundance across individual samples. Phylogenetic placement of these genera across individual samples was examined. In addition, we evaluated the distance among the 6 rumen samples by adding taxon phylogeny using UniFrac, representing 24.1% of average distance. In summary, the current study reveals a shared rumen microbiome and phylogenetic lineage and presents novel information on composition and individual variability of the bacterial community in the rumen of yaks.


      PubDate: 2015-07-08T06:30:23Z
       
  • Optimal spore germination in Clostridium botulinum ATCC 3502 requires the
           presence of functional copies of SleB and YpeB, but not CwlJ
    • Abstract: Publication date: August 2015
      Source:Anaerobe, Volume 34
      Author(s): Carolyn A. Meaney , Stephen T. Cartman , Peter J. McClure , Nigel P. Minton
      Germination, the process by which dormant endospores return to vegetative growth, is a critical process in the life cycle of the notorious pathogen Clostridium botulinum. Crucial is the degradation by hydrolytic enzymes of an inner peptidoglycan spore layer termed the cortex. Two mechanistically different systems of cortex lysis exist in spores of Clostridium species. C. botulinum ATCC 3502 harbours the Bacillus-like system of SleB, CwlJ and YpeB cortex lytic enzymes (CLEs). Through the construction of insertional gene knockout mutants in the sleB, cwlJ and ypeB genes of C. botulinum ATCC 3502 and the production of spores of each mutant strain, the effect on germination was assessed. This study demonstrates a reduced germination efficiency in spores carrying mutations in either sleB or ypeB with an approximate 2-fold reduction in heat resistant colony forming units (CFU/OD600) when plated on rich media. This reduction could be restored to wild-type levels by removing the spore coat and plating on media supplemented with lysozyme. It was observed that cwlJ spores displayed a similar germination efficiency as wild-type spores (P > 0.05). An optimal germinant commixture was identified to include a combination of l-alanine with sodium bicarbonate as it resulted in a 32% drop in OD600, while the additional incorporation of l-lactate resulted in a 57% decrease. Studies of the germination efficiency of spores prepared from all three CLE mutants was performed by monitoring the associated decrease in optical density but a germination defect was not observed in any of the CLE mutant strains. This was likely due to the lack of specificity of this particular assay. Taken together, these data indicate that functional copies of SleB and YpeB, but not CwlJ are required for the optimal germination of the spores of C. botulinum ATCC 3502.


      PubDate: 2015-07-08T06:30:23Z
       
  • Mesophilic hydrogen production in acidogenic packed-bed reactors (APBR)
           using raw sugarcane vinasse as substrate: Influence of support materials
    • Abstract: Publication date: August 2015
      Source:Anaerobe, Volume 34
      Author(s): Antônio Djalma Nunes Ferraz Júnior , Claudia Etchebehere , Marcelo Zaiat
      Bio-hydrogen production from sugarcane vinasse in anaerobic up-flow packed-bed reactors (APBR) was evaluated. Four types of support materials, expanded clay (EC), charcoal (Ch), porous ceramic (PC), and low-density polyethylene (LDP) were tested as support for biomass attachment. APBR (working volume – 2.3 L) were operated in parallel at a hydraulic retention time of 24 h, an organic loading rate of 36.2 kg-COD m−3 d−1, at 25 °C. Maximum volumetric hydrogen production values of 509.5, 404, 81.4 and 10.3 mL-H2 d−1 L−1 reactor and maximum yields of 3.2, 2.6, 0.4 and 0.05 mol-H2 mol−1 carbohydrates total, were observed during the monitoring of the reactors filled with LDP, EC, Ch and PC, respectively. Thus, indicating the strong influence of the support material on H2 production. LDP was the most appropriate material for hydrogen production among the materials evaluated. 16S rRNA gene by Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis and scanning electron microscopy confirmed the selection of different microbial populations. 454-pyrosequencing performed on samples from APBR filled with LDP revealed the presence of hydrogen-producing organisms (Clostridium and Pectinatus), lactic acid bacteria and non-fermentative organisms.


      PubDate: 2015-07-08T06:30:23Z
       
  • The detection of Dichelobacter nodosus and Fusobacterium necrophorum from
           ovine footrot in Kashmir, India
    • Abstract: Publication date: Available online 6 July 2015
      Source:Anaerobe
      Author(s): Shaheen Farooq , Shakil A. Wani , Mir Nadeem Hassan , Nazima Nazir , Qazi Javed Nyrah
      In a study conducted, a total of 450 swab samples from footrot lesions of naturally infected sheep were collected in all the ten districts of the Kashmir valley and were examined for the presence of Dichelobacter nodosus (D. nodosus) and Fusobacterium necrophorum (F. necrophorum), in order to determine if F. necrophorum was associated with ovine footrot. The detection of F. necrophorum and D. nodosus was carried out by polymerase chain reaction targeting the leukotoxin (lktA) and 16S rRNA genes, respectively. In this study, only less than 50% of positive samples contained both the bacteria, so it is not possible to conclude with certainty that both bacteria are together required for the disease manifestation.


      PubDate: 2015-07-08T06:30:23Z
       
  • Single-tube nested PCR assay for the detection of avian botulism in cecal
           contents of chickens
    • Abstract: Publication date: Available online 6 July 2015
      Source:Anaerobe
      Author(s): Il Jang , Jae-Il Lee , Yong-Kuk Kwon , Min-Su Kang , Hye-Ryoung Kim , Ji-Young Park , Song-Hyun Lee , Hee-Soo Lee , You-Chan Bae
      This paper describes a novel diagnostic method for the detection of avian botulism caused by Clostridium botulinum type C and C/D, using single-tube nested PCR assay. This assay was developed to overcome the disadvantages of bioassays used in experiments with mice. Three primer pairs including an antisense primer were designed to target the N-terminal of the toxin gene from C. botulinum types C and C/D. The specificity of the PCR assay was confirmed by using 33 bacterial strains and chicken cecal contents from farms that experienced botulism outbreaks. The detection limit for purified DNA was 1.1 fg/μl, and for bacterial spores was 4.3 spores/200 mg of cecal contents. While checking for specificity of the PCR assay, the reactions with the templates form C. botulinum type C and C/D which were tested became positive, but the rest of the reactions turned negative. However, the results for all clinical samples (n=8) were positive. The PCR assay results for cecal samples obtained from 300 healthy chickens (150 Korean native chickens and 150 broilers) were all negative. This assay is rapid and straightforward and evades ethical issues associated with mouse bioassay. Moreover, it is more economical than real-time PCR.


      PubDate: 2015-07-08T06:30:23Z
       
  • A prospective study of community-associated Clostridium difficile
           infection in Kuwait: epidemiology and ribotypes
    • Abstract: Publication date: Available online 2 July 2015
      Source:Anaerobe
      Author(s): Wafaa Jamal , Eunice Pauline , Vincent Rotimi
      Clostridium difficile infection (CDI) is increasingly recognized as a significant community acquired pathogen that cause disease in the community. The aim of the study was to investigate prospectively the incidence of community-acquired-CDI (CA-CDI) in Kuwait. Of the 2584 patients with diarrhea, 16 (0.62%) were confirmed cases of CA-CDI. The other notable pathogens were Salmonella spp. (0.39%) and Campylobacter spp. (0.23%). The mean age was 39 years and the CDI was mild. Exposure to antibiotics in the previous 12 weeks, contact with infant aged < 2 years and history of foreign travel was significantly associated with CA-CDI (P < 0.001; P < 0.0001; P < 0.002, respectively). Detected PCR ribotypes were 139 (n = 4) and 014, 056, 070, 097 and 179 (each n =2). CA-CDI in Kuwait is more likely to occur in younger age and associated with ribotype 139. CA-CDI is not a common problem in Kuwait however extra vigilance must be maintained to detect it in the community even without traditional predisposing factors.


      PubDate: 2015-07-08T06:30:23Z
       
  • Clostridium amazonense sp. nov. an obliqately anaerobic bacterium isolated
           from a remote Amazonian community in Peru
    • Abstract: Publication date: Available online 26 June 2015
      Source:Anaerobe
      Author(s): Lindsey O’Neal , Alexandra J. Obregón-Tito , Raul Y. Tito , Andrew T. Ozga , Susan I. Polo , Cecil M. Lewis Jr. , Paul A. Lawson
      A strictly anaerobic Gram-stain positive, spore-forming, rod-shaped bacterium designated NE08VT, was isolated from a fecal sample of an individual residing in a remote Amazonian community in Peru. Phylogenetic analysis based on the 16S rRNA gene sequence showed the organism belonged to the genus Clostridium and is most closely related to Clostridium vulturis (97.4% sequence similarity) and was further characterized using biochemical and chemotaxonomic methods. The major cellular fatty acids were anteiso C13:0 and C16:0 with a genomic DNA G + C content of 31.6 mol%. Fermentation products during growth on glucose were acetate and butyrate. Based on phylogenetic, phenotypic and chemotaxonomic information, strain NE08V was identified as representing a novel species of the genus Clostridium, for which the name Clostridium amazonense sp. nov. is proposed. The type strain is NE08VT (DSM 23598 T = CCUG 59712T).


      PubDate: 2015-07-08T06:30:23Z
       
  • Oral anaerobes in health and disease
    • Abstract: Publication date: Available online 3 June 2015
      Source:Anaerobe
      Author(s): Georg Conrads



      PubDate: 2015-07-08T06:30:23Z
       
 
 
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