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Analyses of the Distribution Patterns of Burkholderia pseudomallei and Associated Phages in Soil Samples in Thailand Suggest That Phage Presence Reduces the Frequency of Bacterial Isolation

View Article: PubMed Central - PubMed

ABSTRACT

Background: Burkholderia pseudomallei is a soil saprophytic bacterium that causes melioidosis. The infection occurs through cutaneous inoculation, inhalation or ingestion. Bacteriophages (phages) in the same ecosystem may significantly impact the biology of this bacterium in the environment, and in their culturability in the laboratory.

Methods/principal findings: The soil samples were analysed for the presence of bacteria using culture methods, and for phages using plaque assays on B. pseudomallei strain 1106a lawns. Of the 86 soil samples collected from northeastern Thailand, B. pseudomallei was cultured from 23 (26.7%) samples; no phage capable of infecting B. pseudomallei was detected in these samples. In contrast, phages capable of infecting B. pseudomallei, but no bacteria, were present in 10 (11.6%) samples. B. pseudomallei and their phages were co-isolated from only 3 (3.5%) of soil samples. Since phage capable of infecting B. pseudomallei could not have appeared in the samples without the prior presence of bacteria, or exposure to bacteria nearby, our data suggest that all phage-positive/bacteria-negative samples have had B. pseudomallei in or in a close proximity to them. Taken together, these findings indicate that the presence of phages may influence the success of B. pseudomallei isolation. Transmission electron microscopy revealed that the isolated phages are podoviruses. The temperate phages residing in soil-isolated strains of B. pseudomallei that were resistant to the dominant soil borne phages could be induced by mitomycin C. These induced-temperate phages were closely related, but not identical, to the more dominant soil-isolated phage type.

Conclusion/significance: The presence of podoviruses capable of infecting B. pseudomallei may affect the success of the pathogen isolation from the soil. The currently used culture-based methods of B. pseudomallei isolation appear to under-estimate the bacterial abundance. The detection of phage capable of infecting B. pseudomallei from environmental samples could be a useful preliminary test to indicate the likely presence of B. pseudomallei in environmental samples.

No MeSH data available.


Restriction enzyme analyses of B. pseudomallei phages.Genomic DNA extracted from soil isolated (ΦBp-RE4-5) and MMC-induced (ΦBp-RE1-3) B. pseudomallei phages were digested with restriction enzyme BstBI (A) or MluI (B) and analyzed using agarose gel electrophoresis. Different DNA patterns were observed when digested with the MluI restriction enzyme. A 1-kb DNA ladder was included as a DNA marker.
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pntd.0005005.g003: Restriction enzyme analyses of B. pseudomallei phages.Genomic DNA extracted from soil isolated (ΦBp-RE4-5) and MMC-induced (ΦBp-RE1-3) B. pseudomallei phages were digested with restriction enzyme BstBI (A) or MluI (B) and analyzed using agarose gel electrophoresis. Different DNA patterns were observed when digested with the MluI restriction enzyme. A 1-kb DNA ladder was included as a DNA marker.

Mentions: To further compare the genetic features between MMC-induced B. pseudomallei phages (ΦBp-RE1-3) and two newly isolated free phages (ΦBp-RE4-5), BstBI and MluI restriction enzyme digestion analyses of the phage genomic DNA were carried out. No significant difference between the BstBI restriction enzyme digestion patterns among the three MMC-induced phages and free phages were observed and these phages have the same calculated genome size of approximately 45 Kb (Fig 3A). However, different DNA patterns were observed when the MluI restriction digestion profiles were analyzed (Fig 3B). It appears that additional MluI recognition sites are present in the genomes of the induced phages, resulting in the alterations of the MluI digestion profile of the induced phages compared to that of the dominant soil-isolated ones.


Analyses of the Distribution Patterns of Burkholderia pseudomallei and Associated Phages in Soil Samples in Thailand Suggest That Phage Presence Reduces the Frequency of Bacterial Isolation
Restriction enzyme analyses of B. pseudomallei phages.Genomic DNA extracted from soil isolated (ΦBp-RE4-5) and MMC-induced (ΦBp-RE1-3) B. pseudomallei phages were digested with restriction enzyme BstBI (A) or MluI (B) and analyzed using agarose gel electrophoresis. Different DNA patterns were observed when digested with the MluI restriction enzyme. A 1-kb DNA ladder was included as a DNA marker.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036839&req=5

pntd.0005005.g003: Restriction enzyme analyses of B. pseudomallei phages.Genomic DNA extracted from soil isolated (ΦBp-RE4-5) and MMC-induced (ΦBp-RE1-3) B. pseudomallei phages were digested with restriction enzyme BstBI (A) or MluI (B) and analyzed using agarose gel electrophoresis. Different DNA patterns were observed when digested with the MluI restriction enzyme. A 1-kb DNA ladder was included as a DNA marker.
Mentions: To further compare the genetic features between MMC-induced B. pseudomallei phages (ΦBp-RE1-3) and two newly isolated free phages (ΦBp-RE4-5), BstBI and MluI restriction enzyme digestion analyses of the phage genomic DNA were carried out. No significant difference between the BstBI restriction enzyme digestion patterns among the three MMC-induced phages and free phages were observed and these phages have the same calculated genome size of approximately 45 Kb (Fig 3A). However, different DNA patterns were observed when the MluI restriction digestion profiles were analyzed (Fig 3B). It appears that additional MluI recognition sites are present in the genomes of the induced phages, resulting in the alterations of the MluI digestion profile of the induced phages compared to that of the dominant soil-isolated ones.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Burkholderia pseudomallei is a soil saprophytic bacterium that causes melioidosis. The infection occurs through cutaneous inoculation, inhalation or ingestion. Bacteriophages (phages) in the same ecosystem may significantly impact the biology of this bacterium in the environment, and in their culturability in the laboratory.

Methods/principal findings: The soil samples were analysed for the presence of bacteria using culture methods, and for phages using plaque assays on B. pseudomallei strain 1106a lawns. Of the 86 soil samples collected from northeastern Thailand, B. pseudomallei was cultured from 23 (26.7%) samples; no phage capable of infecting B. pseudomallei was detected in these samples. In contrast, phages capable of infecting B. pseudomallei, but no bacteria, were present in 10 (11.6%) samples. B. pseudomallei and their phages were co-isolated from only 3 (3.5%) of soil samples. Since phage capable of infecting B. pseudomallei could not have appeared in the samples without the prior presence of bacteria, or exposure to bacteria nearby, our data suggest that all phage-positive/bacteria-negative samples have had B. pseudomallei in or in a close proximity to them. Taken together, these findings indicate that the presence of phages may influence the success of B. pseudomallei isolation. Transmission electron microscopy revealed that the isolated phages are podoviruses. The temperate phages residing in soil-isolated strains of B. pseudomallei that were resistant to the dominant soil borne phages could be induced by mitomycin C. These induced-temperate phages were closely related, but not identical, to the more dominant soil-isolated phage type.

Conclusion/significance: The presence of podoviruses capable of infecting B. pseudomallei may affect the success of the pathogen isolation from the soil. The currently used culture-based methods of B. pseudomallei isolation appear to under-estimate the bacterial abundance. The detection of phage capable of infecting B. pseudomallei from environmental samples could be a useful preliminary test to indicate the likely presence of B. pseudomallei in environmental samples.

No MeSH data available.