Dataset Information

In Vitro Cultivation of 'Unculturable' Oral Bacteria, Facilitated by Community Culture and Media Supplementation with Siderophores.

ABSTRACT: Over a third of oral bacteria are as-yet-uncultivated in-vitro. Siderophores have been previously shown to enable in-vitro growth of previously uncultivated bacteria. The objective of this study was to cultivate novel oral bacteria in siderophore-supplemented culture media. Various compounds with siderophore activity, including pyoverdines-Fe-complex, desferricoprogen and salicylic acid, were found to stimulate the growth of difficult-to-culture strains Prevotella sp. HOT-376 and Fretibacterium fastidiosum. Furthermore, pyrosequencing analysis demonstrated increased proportions of the as-yet-uncultivated phylotypes Dialister sp. HOT-119 and Megasphaera sp. HOT-123 on mixed culture plates supplemented with siderophores. Therefore a culture model was developed, which incorporated 15 ?g siderophore (pyoverdines-Fe-complex or desferricoprogen) or 150 ?l neat subgingival-plaque suspension into a central well on agar plates that were inoculated with heavily-diluted subgingival-plaque samples from subjects with periodontitis. Colonies showing satellitism were passaged onto fresh plates in co-culture with selected helper strains. Five novel strains, representatives of three previously-uncultivated taxa (Anaerolineae bacterium HOT-439, the first oral taxon from the Chloroflexi phylum to have been cultivated; Bacteroidetes bacterium HOT-365; and Peptostreptococcaceae bacterium HOT-091) were successfully isolated. All novel isolates required helper strains for growth, implying dependence on a biofilm lifestyle. Their characterisation will further our understanding of the human oral microbiome.

SUBMITTER: Vartoukian SR

PROVIDER: S-EPMC4713201 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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In Vitro Cultivation of 'Unculturable' Oral Bacteria, Facilitated by Community Culture and Media Supplementation with Siderophores.

Vartoukian Sonia R SR Adamowska Aleksandra A Lawlor Megan M Moazzez Rebecca R Dewhirst Floyd E FE Wade William G WG

PloS one 20160114 1

Over a third of oral bacteria are as-yet-uncultivated in-vitro. Siderophores have been previously shown to enable in-vitro growth of previously uncultivated bacteria. The objective of this study was to cultivate novel oral bacteria in siderophore-supplemented culture media. Various compounds with siderophore activity, including pyoverdines-Fe-complex, desferricoprogen and salicylic acid, were found to stimulate the growth of difficult-to-culture strains Prevotella sp. HOT-376 and Fretibacterium ...[more]

PMID: 26764907

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Project description:BackgroundBacteria produce small molecule iron chelators, known as siderophores, to facilitate the acquisition of iron from the environment. The synthesis of more than one siderophore and the production of multiple siderophore uptake systems by a single bacterial species are common place. The selective advantages conferred by the multiplicity of siderophore synthesis remains poorly understood. However, there is growing evidence suggesting that siderophores may have other physiological roles besides their involvement in iron acquisition.Methods and principal findingsHere we provide the first report that pyochelin displays antibiotic activity against some bacterial strains. Observation of differential sensitivity to pyochelin against a panel of bacteria provided the first indications that catecholate siderophores, produced by some bacteria, may have roles other than iron acquisition. A pattern emerged where only those strains able to make catecholate-type siderophores were resistant to pyochelin. We were able to associate pyochelin resistance to catecholate production by showing that pyochelin-resistant Escherichia coli became sensitive when biosynthesis of its catecholate siderophore enterobactin was impaired. As expected, supplementation with enterobactin conferred pyochelin resistance to the entE mutant. We observed that pyochelin-induced growth inhibition was independent of iron availability and was prevented by addition of the reducing agent ascorbic acid or by anaerobic incubation. Addition of pyochelin to E. coli increased the levels of reactive oxygen species (ROS) while addition of ascorbic acid or enterobactin reduced them. In contrast, addition of the carboxylate-type siderophore, citrate, did not prevent pyochelin-induced ROS increases and their associated toxicity.ConclusionsWe have shown that the catecholate siderophore enterobactin protects E. coli against the toxic effects of pyochelin by reducing ROS. Thus, it appears that catecholate siderophores can behave as protectors of oxidative stress. These results support the idea that siderophores can have physiological roles aside from those in iron acquisition.

Project description:Objective:? To determine the presence of unculturable bacteria using polymerase chain reaction (PCR) in infertile men with pyosperrmia. Design:? Perspective clinical study. Setting:? The study took place at the Department of Reproductive Medicine, Owaisi Hospital and Research Center; In vitro Fertilization Unit, Mahavir Hospital and Research Center; Center for Cellular and Molecular Biology; and Bharat Biotech Foundation, Hyderabad, India. Patients:? A total of 68 infertile men and 15 donors, all with no symptoms of genito-urinary tract infections and sterile semen cultures were included in the study. Interventions:? None. Main outcome measures:? Screening bacteria using routine bacterial cultures and PCR based screening with universal eubacterial primers. Results:? The statistical analysis of all the semen parameters in asthenazoospermic, azoospermic, ceyptazoospermic, severe oligospermic and mild oligospermic patients were found to be significant compared with the controls. All the groups were found to be significant compared with the controls (P < 0.05) except for volume and pus cells in the cryptozoospermia group. The Student's t-test also was significant for the seminal parameters before and after treatment of 68 selected individuals with pyospermia and sterile cultures. A total of 44.11% (30/68) samples were collected from the negative culture of pyospermic infertile men have shown the presence of bacteria on amplification using PCR with universal eubacterial primers. The DNA was purified and sequenced. The sequences were checked for homology using DNASTAR and Ribosomal DataBase Project II. A total of 90% of the samples have shown the nearest evolutionary relation to Pantoea P102 (AF394539) and 10% of samples have shown close relation with Burkholderia cepacia (AF042161). Conclusion:? The routine bacteriological cultures were unable to detect certain bacterial species particularly with members of enterobacteriaceae family (Pantoea species). Polymerase chain reaction, when used for screening bacteria, can detect the unculturable form of bacteria in infertile men. No amplification for bacterial DNA was obtained in control samples (fertile men with sterile semen cultures.) (Reprod Med Biol 2004; 3: 77-?84).

Project description:The rumen microbiota is critical in cattle digestion. Still, its low cultivability makes it difficult to study its ecological function and biotechnological potential. To improve the recovery of ruminal microorganisms, this study combined the evaluation of several cultivation parameters with metabarcoding analysis. The parameters tested comprised eight media cultures, three sample dilutions (10-2, 10-6, 10-12), and two incubation times (3 and 7 days). Bacterial populations were determined through Illumina sequencing of 16S rRNA from three biological replicates. The results indicate that none of the culture media recovered all rumen populations and that there was an altered relative abundance of the dominant phyla. In the rumen, Bacteroidetes and Firmicutes comprised 75% and 15% of the relative abundance, respectively, while in the culture media, these were 15% and 60%, respectively. Principal coordinate analysis (PCoA) of the bacterial community revealed significant shifts in population composition due to dilution, with 10-2 and 10-6 dilutions clustered closely while the 10-12 dilution differed markedly. In contrast, incubation duration did not influence population diversity. According to the results, two media, CAN and KNT, were selected based on their ability to recover more similar populations compared to the rumen sample. The metataxonomic study showed that CAN media had consistent reproducibility over time, while KNT showed enrichment of different taxa due to the use of rumen fluid as a substrate. From these, 64 pure cultures were obtained and 54 were identified through 16S rRNA gene sequencing. Being Streptococcus the most frequently isolated genus, this prevalence contrasts with the liquid media composition, underscoring the importance of refining single colony isolation strategies. Although no culture medium could replicate the native rumen bacterial population perfectly, our findings highlight the potential of CAN and KNT media in recovering populations that are more closely aligned to natural rumen conditions. In conclusion, our study emphasizes the importance of integrating molecular approaches in selecting suitable cultivation media and parameters to depict rumen bacteria accurately.

Dataset Information

In Vitro Cultivation of 'Unculturable' Oral Bacteria, Facilitated by Community Culture and Media Supplementation with Siderophores.

Publications

In Vitro Cultivation of 'Unculturable' Oral Bacteria, Facilitated by Community Culture and Media Supplementation with Siderophores.

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