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More Easily Cultivated Than Identified: Classical Isolation With Molecular Identification of Vaginal Bacteria.

ABSTRACT: Women with bacterial vaginosis (BV) have complex communities of anaerobic bacteria. There are no cultivated isolates of several bacteria identified using molecular methods and associated with BV. It is unclear whether this is due to the inability to adequately propagate these bacteria or to correctly identify them in culture.Vaginal fluid from 15 women was plated on 6 different media using classical cultivation approaches. Individual isolates were identified by 16S ribosomal RNA (rRNA) gene sequencing and compared with validly described species. Bacterial community profiles in vaginal samples were determined using broad-range 16S rRNA gene polymerase chain reaction and pyrosequencing.We isolated and identified 101 distinct bacterial strains spanning 6 phyla including (1) novel strains with <98% 16S rRNA sequence identity to validly described species, (2) closely related species within a genus, (3) bacteria previously isolated from body sites other than the vagina, and (4) known bacteria formerly isolated from the vagina. Pyrosequencing showed that novel strains Peptoniphilaceae DNF01163 and Prevotellaceae DNF00733 were prevalent in women with BV.We isolated a diverse set of novel and clinically significant anaerobes from the human vagina using conventional approaches with systematic molecular identification. Several previously "uncultivated" bacteria are amenable to conventional cultivation.

SUBMITTER: Srinivasan S

PROVIDER: S-EPMC4957512 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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More Easily Cultivated Than Identified: Classical Isolation With Molecular Identification of Vaginal Bacteria.

Srinivasan Sujatha S Munch Matthew M MM Sizova Maria V MV Fiedler Tina L TL Kohler Christina M CM Hoffman Noah G NG Liu Congzhou C Agnew Kathy J KJ Marrazzo Jeanne M JM Epstein Slava S SS Fredricks David N DN

The Journal of infectious diseases 20160801

<h4>Background</h4>Women with bacterial vaginosis (BV) have complex communities of anaerobic bacteria. There are no cultivated isolates of several bacteria identified using molecular methods and associated with BV. It is unclear whether this is due to the inability to adequately propagate these bacteria or to correctly identify them in culture.<h4>Methods</h4>Vaginal fluid from 15 women was plated on 6 different media using classical cultivation approaches. Individual isolates were identified by ...[more]

PMID: 27449870

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Project description:BackgroundThe present study dealt with the screening of soil bacteria with antibacterial activity from different locations in Bangalore, India. Antibiotics play the role of self-defense mechanism for the bacteria and are produced as secondary metabolites to protect themselves from other competitive microorganisms. The need for new antibiotics arose as the pathogenic bacteria acquire resistance to various antibiotics meant for treating human diseases. Given the importance of antibiotics of bacterial origin, standard techniques have been used to isolate and characterize the soil bacteria which showed antibacterial activity.ResultsThe isolated bacteria were tested against human pathogenic bacteria like Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae by primary and secondary screening methods. The isolates PR1, PR2, and PR3 were confirmed to have antibacterial activity against S. aureus, E. coli, P. aeruginosa, and K. pneumoniae by both methods. Studies on the effect of filter sterilization, autoclaving, and proteinase K treatment on culture filtrates showed filter sterilization as the best method. The effect of different carbon and nitrogen sources on the antibacterial activity showed that preference by each isolate differed for carbon and nitrogen requirements. The isolates PR1, PR2, and PR3 were identified as Bacillus aryabhattai strain PR-D07, Arthrobacter humicola strain PR-F07, and Neomicrococcus lactis strain PR-F11 through 16S rRNA sequencing.ConclusionFindings from this research work are encouraging and could proceed further to applied aspects. Only 3 bacterial isolates out of 263 isolates from soil samples displayed antibacterial activity against human pathogens S. aureus, E. coli, P. aeruginosa, and K. pneumoniae. They were identified as B. aryabhattai, A. humicola, and N. lactis by 16S rRNA studies and all of them are Gram-positive. Each isolate preferred different carbon and nitrogen sources for their enhanced antibacterial activity. Efficacy of the culture filtrates of these isolates was tested by filter sterilization, autoclaving, and proteinase K treatment. Filter-sterilized culture filtrates showed higher antibacterial activity than other treatments. A comparison of the antibacterial activity of culture filtrates and antibiotic streptomycin produced an inhibition zone of 18.5 mm and 15.5 mm respectively. This is the first report on the antibacterial activity of all the 3 bacterial strains (B. aryabhattai strain PR-D07, A. humicola strain PR-F07, and N. lactis strain PR-F11), against all the human pathogens, mentioned earlier. It is also found that the antibiotic factor is proteinaceous as proteinase K considerably reduced the antibacterial activity of the culture filtrates. With the above significant results, these 3 bacteria are considered to be promising candidates for the isolation of new antibacterial agents.

Project description:The poultry industry is facing continuous challenges with regard to increased feed costs and loss due to infectious disease. To overcome this challenge, several antibiotics have been used along with chicken feeds to promote growth. Nevertheless, the use of antibiotics as growth promoter has been banned in many countries, due to the concerns associated with potential risks of emerging and horizontal transfer of multidrug resistant genes to bacteria in animal tissues. The objective of this study was to identify and characterize potential probiotic bacteria strains from the gastrointestinal tract of free-range locally selected chickens. The bacterial isolates were screened, purified and characterized based on morphological, biochemical and molecular characteristics from 12 well-adopted free-range healthy young chickens. Low pH and bile salt tolerance, antagonistic activity, antibiotic activity, hemolysis activity, adhesion to the chicken intestine and carbohydrate fermentation tests was conducted to identify potential probiotic bacteria. Twelve bacterial isolates were screened based on their ability for their tolerance to low pH and bile salt. The isolates were identified by using 16S rRNA gene partial sequencing method. All screened isolates showed great survival percentage at low pH, that is (89.2 ± 0.75 to 97.1 ± 0.64) survived at 3 h and (83.6 ± 0.75 to 95.2 ± 0.63) at 6 h challenge at pH2. Isolate GCM112 was the least tolerant strain in 6.0% salt concentration at 12 and 24 h exposure time (82.1 ± 1.28 and 79.9 ± 1.96%) respectively. The result revealed no strain tests in this study exhibited α- and β-hemolytic activity when cultured in sheep blood agar. Most isolated strains showed best growth at 37°C temperature and up to 4% NaCl concentration. Based on the reported result from in vitro data, GCH212 and GCM412 isolates were recognized as best potential probiotic bacteria for chicken against pathogens but further studies are needed on in vivo assessment on the health benefits in the real life situation.

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More Easily Cultivated Than Identified: Classical Isolation With Molecular Identification of Vaginal Bacteria.

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More Easily Cultivated Than Identified: Classical Isolation With Molecular Identification of Vaginal Bacteria.

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