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. 2010 Jul;78(7):3136-43.
doi: 10.1128/IAI.01313-09. Epub 2010 Apr 19.

A Burkholderia pseudomallei deltapurM mutant is avirulent in immunocompetent and immunodeficient animals: candidate strain for exclusion from select-agent lists

Affiliations

A Burkholderia pseudomallei deltapurM mutant is avirulent in immunocompetent and immunodeficient animals: candidate strain for exclusion from select-agent lists

Katie L Propst et al. Infect Immun. 2010 Jul.

Abstract

Burkholderia pseudomallei causes the disease melioidosis in humans and is classified as a category B select agent. Research utilizing this pathogen is highly regulated in the United States, and even basic studies must be conducted in biosafety level 3 (BSL-3) facilities. There is currently no attenuated B. pseudomallei strain available that is excluded from select-agent regulations and can be safely handled at BSL-2 facilities. To address this need, we created Bp82 and Bp190, which are DeltapurM derivatives of B. pseudomallei strains 1026b and K96243 that are deficient in adenine and thiamine biosynthesis but replication competent in vitro in rich medium. A series of animal challenge studies was conducted to ensure that these strains were fully attenuated. Whereas the parental strains 1026b and K96243 and the complemented mutants Bp410 and Bp454 were virulent in BALB/c mice following intranasal inoculation, the DeltapurM mutants Bp82 and Bp190 were avirulent even when they were administered at doses 4 logs higher than the doses used for the parental strains. Animals challenged with high doses of the DeltapurM mutants rapidly cleared the bacterium from tissues (lung, liver, and spleen) and remained free of culturable bacteria for the duration of the experiments (up to 60 days postinfection). Moreover, highly susceptible 129/SvEv mice and immune incompetent mice (IFN-gamma-/-, SCID) were resistant to challenges with DeltapurM mutant Bp82. This strain was also avirulent in the Syrian hamster challenge model. We concluded that DeltapurM mutant Bp82 is fully attenuated and safe for use under BSL-2 laboratory conditions and thus is a candidate for exclusion from the select-agent list.

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Figures

FIG. 1.
FIG. 1.
purM mutant alleles and in vitro growth of B. pseudomallei ΔpurM mutants Bp82 and Bp190 and their purM+ derivatives Bp410 and Bp454. (A) Sequence of the FRT scar region in Bp82. Lowercase letters indicate purM sequences. Uppercase letters indicate the 86-bp FRT scar sequence with the XbaI site and the Flp recombination sequences, which are enclosed in boxes. The residual PurM amino acid sequence is shown below the nucleotide sequence. NruI sites indicate the original junction sequences of purM and a zeocin resistance-encoding cassette from pFZE1 and resulted from fusion of an NruI half-site with a T4 DNA polymerase-blunted SacI site. (B) Sequence of the deletion junction in Bp190. Deletion of an internal NruI fragment from purM resulted in deletion of 38 amino acids from PurM. The purM open reading frame terminates with the stop codon that naturally occurs after valine 351. (C to F) Growth of strains in minimal media. The following strains were tested: 1026b and its ΔpurM derivative Bp82 and K96243 and its ΔpurM derivative Bp190. The strains were inoculated into 200 μl of M9-glucose medium (M9G) and into this medium supplemented with 0.6 mM adenine (M9G+A), 0.0005% thiamine (M9G+T), or both adenine and thiamine (M9G+A+T), and growth at 37°C was monitored by determining the optical densities at 600 nm (OD600nm) of the cultures at 30-min intervals. The symbols indicate the means of three independently monitored wells, and the error bars indicate standard deviations. (G and H) Growth of prototype, mutant, and complemented strains in minimal medium. The following strains were tested: 1026b and its ΔpurM derivative Bp82 and K96243 and its ΔpurM derivative Bp190. Bp410 and Bp454 are Bp82 and Bp190, respectively, with the ΔpurM allele replaced by purM+ from 1026b. The strains were grown in 200 μl of M9-glucose minimal medium without supplements, and growth at 37°C was monitored as described above.
FIG. 2.
FIG. 2.
B. pseudomallei ΔpurM mutants Bp82 (A) and Bp190 (B) are attenuated in BALB/c mice. (A) Mice (5 animals per group) were challenged i.n. with either 5 ×ばつ 103 CFU B. pseudomallei 1026b (wild-type strain), 1 ×ばつ 106 CFU or 1 ×ばつ 108 CFU ΔpurM strain Bp82, or 5 ×ばつ 103 CFU Bp410 (Bp82 ΔpurM lesion repaired with purM gene sequence from 1026b). Animal survival was assessed as described in Materials and Methods. The statistical significance of differences in survival times was determined by using Kaplan-Meier curves, followed by a log rank test **, P < 0.01 for comparison of Bp82 and 1026b or for comparison of Bp82 and Bp410. (B) Mice (5 animals per group) were challenged i.n. with either 5 ×ばつ 103 CFU B. pseudomallei K96243 (wild-type strain), 1 ×ばつ 106 CFU ΔpurM strain Bp190, or 5 ×ばつ 103 CFU Bp454 (Bp190 ΔpurM lesion repaired with purM gene sequence from 1026b). **, P < 0.01 for comparison of Bp190 and K92643 or for comparison of Bp190 and Bp454. WT, wild type.
FIG. 3.
FIG. 3.
Bacterial burdens in the lungs, liver, and spleen 48 h after i.n. challenge with wild-type B. pseudomallei 1026b or ΔpurM mutant Bp82. (A) BALB/c mice (3 mice per group) were challenged with 6 ×ばつ 103 CFU of B. pseudomallei 1026b or Bp82. The bacterial burden in each organ was determined 48 h after challenge. The statistical significance of differences was evaluated using a one-sample t test (**, P < 0.01; *, P < 0.05). WT, wild type. (B and C) BALB/c mice (3 mice per group) were challenged with 1 ×ばつ 106 or 1 ×ばつ 108 CFU Bp82 (challenge doses are indicated), and bacterial burdens were determined 48 h later. The dagger indicates that there was a single colony on the liver plate for one mouse.
FIG. 4.
FIG. 4.
B. pseudomallei ΔpurM strains Bp82 and Bp190 are avirulent in 129/SvEv mice. Mice (5 animals per group) were challenged i.n. with either 100 CFU B. pseudomallei 1026b or 1 ×ばつ 106 CFU Bp82. Mice (5 animals per group) were also challenged with either 200 CFU B. pseudomallei K92643 or 1 ×ばつ 106 CFU Bp190. The statistical significance of differences in survival times was determined by using Kaplan-Meier curves, followed by a log rank test (**, P < 0.01 for comparison of Bp82 and 1026b or for comparison of Bp190 and K92643).
FIG. 5.
FIG. 5.
B. pseudomallei ΔpurM mutant Bp82 is avirulent in immunodeficient mice. (A) IFN-γ−/− mice (3 or 4 mice per group) were challenged i.n. with either 500 CFU of B. pseudomallei 1026b or 1 ×ばつ 106 CFU of ΔpurM strain Bp82. (B) SCID mice (5 mice per group) were challenged i.n. with either 1 ×ばつ 104 CFU of B. pseudomallei 1026b or 1 ×ばつ 106 CFU of Bp82. The statistical significance of differences in the survival times of Bp82 and 1026b was determined by using Kaplan-Meier curves, followed by a log rank test (**, P < 0.01; *, P < 0.05).
FIG. 6.
FIG. 6.
B. pseudomallei ΔpurM strain Bp82 is avirulent in Syrian hamsters. Hamsters (5 animals per group) were challenged i.n. with 400 CFU of B. pseudomallei 1026b or 1 ×ばつ 106 CFU of ΔpurM mutant Bp82. The statistical significance of differences in the survival times of Bp82 and 1026b was determined by using Kaplan-Meier curves, followed by a log rank test (**, P < 0.01).

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