BACKGROUND
b. pseudomallei , the etiological agent of the disease melioidosis, is a gram-negative rod typically found in soil and water environments throughout the tropics  <cit> .
in thailand, numerous bp are found in the pooled surface waters of rice paddies  <cit>  coinciding with high disease and seropositivity rates in rural rice farmers.
compounding the problem is the quick progression to fatal sepsis; by the time patients seek medical intervention, the disease has progressed acutely, leading to a high mortality rate of  <dig> %  <cit> .
melioidosis is not just a public health challenge in northeastern thailand.
the disease is believed to be vastly underreported with ~ <dig>  cases worldwide and ~ <dig>  deaths  <cit> .
moreover, the us centers for disease control and prevention have listed bp as a tier- <dig>  select agent.
tier  <dig> organisms have the potential to pose a severe threat to us public health and safety and the us government agencies have invested resources to develop vaccines and therapeutics for them, including bp  <cit> .
tier  <dig> listing dictates numerous regulations and requires select agent compliant biosafety level  <dig>  facilities for manipulation of live organisms, driving up costs and increasing data collection time.
one tool that can benefit the researchers in this restricted environment is the use of biosafe surrogates, which allow both exclusion from the select-agent regulations and safe manipulation at biosafety level  <dig> .
burkholderia thailandensis  is an attenuated bacterium that is closely related to bp but most strains lack a capsular polysaccharide, among other genomic and virulence factor differences, that do not make it an ideal biosafe surrogate  <cit> .
bt strain e <dig> has the capsular polysaccharide and has been successfully evaluated as a live-attenuated vaccine against bp k <dig> challenge  <cit> .
there are currently three bp strains that are excluded form the select agent list .
the first is an aminoglycoside pump mutant that lacks a capsule, jw <dig>  another is a diaminopimelic acid  requiring Δasd mutant, b <dig>  and the third is an adenine requiring Δpurm mutant, bp <dig>  each individually has their drawbacks but cumulatively a major drawback is that all are made from the same strain background, bp 1026b, a serotype a strain.
as a species, bp contains a high level of genomic diversity that translates to phenotypic diversity.
the core genome of bp, the genes found in all members of the species, is composed of  <dig> genes  <cit> .
strain 1026b has  <dig> genes.
the available select-agent strains and bt strains partially fulfill the need for surrogates in some aspects of study but there is room to expand the toolset.
within-host, bp can infect most tissues and invades, then replicates inside the cytoplasm of many cell types .
to accomplish this feat, bp attaches to the host cell causing actin rearrangement and inducing bacterial phagocytosis  <cit> .
a myriad of virulence factors take part in the extra and intracellular lifestyle.
lipopolysaccharide  is the major component of the outer leaflet of the outer membrane and coats the surface of the gram-negative bacterium, including bp.
besides the ability of lps to strongly activate innate immunity, it has been shown to play a role in the intracellular survival of bp during invasion and mutants in the synthesis of the o-antigen of lps are attenuated in animal infection models  <cit> .
a major variable among strains can be the lipopolysaccharide .
previously published research identified the genomic differences that exist at the o-antigen biosynthetic operon and screened ~ <dig> bp strains for lps diversity and 90% of them were type a   <cit> .
strain 1026b has type a lps.
it has been found that the type a lps o-antigen is composed of repeating subunits of glucose and talose  <cit> .
of the remaining 10% with atypical lps; 9% were type b and 1% were type b <dig>  work presented in this study and in references cited above show a higher and larger banding pattern associated with the type b lps.
observations by western blot show antibodies from patients infected with strains of one type are not cross-reactive.
lack of cross-reactivity between a and b serotype strains was assumed due to be differences in glycosyl residues of the o-antigen  <cit> .
recently, work from the authors has shown that the type b o-antigen is composed of rhamnose, xylose, and galactose  synchronizing the genomic and structural aspects of bp lps data  <cit> .
beyond australia and southeast asia, the predominance of lps types in south asia, the middle east, and africa has been largely uncharacterized, but genomic data indicate a majority of strains identified in madagascar have the type b lps  <cit> .
even though the number of strains possessing atypical lps is a fraction of the typical, their impact on the study of bp and melioidosis is significant.
strain 576a was isolated from a fatal case of human melioidosis and has type b lps  <cit> .
the most effective melioidosis vaccine thus far is 2d <dig>  a branched chain amino acid auxotroph of bp strain 576a containing a transposon insertion in the ilvi gene.
it was an extremely effective vaccine in animal models, protecting balb/c mice from a 106 cfu challenge  <cit>  whereas 1026b based vaccines fail to illicit long-term protection against 5 ×  <dig> cfu challenge doses  <cit> .
the melioidosis vaccine steering committee has recommended that strain 2d <dig> be used as a positive control for vaccines in development  <cit> .
strain 2d <dig> is inaccessible to many researchers in the united states and is currently not a select-agent excluded strain.
as mentioned above, this mutant is an insertional mutant, thus posing inherent risks from recombination proficient bp.
we found significant differences between immunoreactivity of 1026b and 576a as well as organ loads and inflammation caused by these two wild-type strains in the balb/c murine melioidosis model.
the 1026b Δpurm strain, bp <dig>  has proven to be a valuable resource for studying antibiotic resistance mechanisms and vaccines .
this work aims to expand on the availability, familiarity, and utility of Δpurm biosafe strains for research by producing the bp 576a Δpurm strain that contains a 114 bp deletion in the purm gene.
the newly created bp 576a ∆purm strain, designated 576mn, was auxotrophic for adenine in minimal media, capable of wild-type growth in rich media with addition of adenine, and auxotrophy was partially abrogated with single-copy complementation.
bp 576mn was unable to replicate in human cells and was completely avirulent in balb/c mice following high-dose intranasal inoculation, similar to bp <dig>  organ loads indicated 576mn was unable to replicate in the organs tested.
this strain should prove a valuable addition to the biosafe study of bp and is an ideal candidate for select-agent exclusion and could serve as a safe background for creation of a live-attenuated double mutant vaccine strain.
RESULTS
strain background differences between bp 1026b and 576a
lps purified from bp <dig> and the 576a wcbb mutant show a fine laddering of o-antigen in the 1026b lps on silver stained gels  but the type b lps from 576a show a more pronounced laddering with a slightly higher molecular weight.
long and very-long lps structures at ~80 kda and beyond are also visible and of higher molecular weight in the 576a type b lps.
the lysates from bp <dig> and 576mn show a conservation of these high molecular weight lps structures.
the coomassie stained sds-page gel shows that protein production by these two strains shows some similarities but also differences .
western blots of the fplc-purified lps from 1026b and 576a with mabs to the respective lps types showed no cross-reactivity between the two strains .
a western blot using serum from a rhesus macaque 28 days after aerosol challenge with bp 1026b showed that the serum reacted strongly to the fplc - purified type a lps from 1026b and not at all to type b lps from 576a .
the highly immunogenic potential of bp lps during the humoral response is evident in the strength of macaque serum reactivity to bp <dig> lysate compared to reactivity with 576mn cell lysate .
there is faint reactivity to some proteins from 576mn but not to the major antigen lps.
the identity of these proteins will be determined in future work.
numerous serum samples from rhesus macaques were used in an elisa using purified lps from 1026b and 576a capsule mutants in two separate pure antigen assays.
all monkeys were challenged with bp strains that have type a lps.
the reactivity of the serum with the type a lps increases starting at 7 days post-infection and the mean od <dig> remained high in the serum samples of surviving monkeys .
in contrast, there was very little reactivity between the type b lps and all serum samples compared to serum from mice vaccinated with type b lps, agreeing with the blot in fig.
1e.
it was also found that in human lung epithelial a <dig> cells and mouse macrophage raw <dig>  cells that bp 576a attached better and formed larger and more numerous plaques , indicating further differences between these strains at the cellular infection level.fig.
<dig> strain background differences between bp 1026b and 576a.
a silver stain of highly pure lps from 1026b and 576a strain backgrounds and heat-killed lysate.
b coomassie stain of heat-killed cell lysate.
c and d western blots using lps type a specific mab 4c <dig> and lps type b specific mab 5b <dig>  e western blot using day  <dig> serum from a rhesus macaque after aerosol challenge by bp 1026b.
lanes:  <dig>  fplc-purified bp <dig> Δwcb lps;  <dig>  fplc-purified 576a Δwcb lps;  <dig> lys, heat-killed lysate of bp82;  <dig> lys, heat-killed lysate of 576mn.
chevrons indicate 80 kda and 25 kda as indicated
fig.
<dig>
a-b attachment and plaque sizes formed in a <dig> human lung epithelial cells and murine macrophage cell line raw <dig>  by bp 1026b and 576a.
c images of plaque formation by 1026b and 576a in a <dig> and raw <dig>  cells after infection with the cfu.
error bars represent the sem and differences found significant by one-way anova
bp 1026b and 576a performance in a murine melioidosis model
using the balb/c murine model of infection we also compared the organ dissemination characteristics of the parental wild-type bp strains 1026b and 576a.
mice challenged with 5000 cfu of either strain were sacrificed at 24 h post-infection and had the bacterial organ loads in blood, lung, liver, and spleen enumerated .
the data show that mean cfus of 576a bacteria in the blood, lung, and spleen trended higher but that the levels were not significantly different.
mean 576a bacterial levels in the liver were significantly higher.
ten times more cfus were recovered from 576a–infected mouse livers compared to livers from 1026b–infected mice and this difference was highly significant .
this may be due to the increased immune activation of 576a lps  <cit>  and activation of tlr4-dependent bacterial clearance and uptake by the liver during sepsis  <cit> .
although bacterial burden in the lung was not significantly different between 1026b and 576a infected mice, tnf-α levels were measured to characterize the acute-phase inflammatory responses to bacterial replication in the lung .
tnf-α levels in lung homogenate from 1026b–infected mice trended higher but were not significantly different than in uninfected mouse lungs.
tnf-α levels in lung homogenate from 576a infected mouse lung homogenates were significantly higher than those from the uninfected mice but not from 1026b infected mice.
this could be due to slightly higher levels of bacteria in the lung or to the higher immunogenicity associated with this strain.
regardless, in mouse survival curves following intranasal challenge with 5000 cfu of either wild-type strain indicate all mice are moribund prior to 3 days post-infection and survival of mouse groups infected by the two strains are not significantly different .fig.
<dig> bacterial burdens and tnf-α levels are different in the organs of balb/c mice infected with bp 1026b or bp 576a.
a bacterial cfu/ml of blood and bacterial cfu/organ, i.e.
, lung; , liver; , spleen.
statistical significance determined by mann-whitney statistical testing of the medians of the two groups.
** = p <  <dig>  e), tnf-α levels in lung homogenate from 1026b are not significantly  different than uninfected mice while tnf-α levels from 576a treated lungs were significantly different than untreated mice lungs by the one-way anova test for comparing the three groups.
** = p <  <dig> .
black circles indicate 1026b–infected mice.
white squares indicate 576a–infected mice.
white triangles indicate uninfected mice.
f balb/c survival proportions following intranasal challenge with 5000 cfu of each strain indicated.
the black arrow indicates the 24 h time point for removal of organs during the pre-determined endpoint study in , the survival data is also presented in fig.
<dig> for clarity.
ns = not significant by the log-rank test of survival curves
construction and growth characterization of a 576a Δpurm mutant
allelic recombination was utilized to delete a 125 bp fragment internal to the purm gene by insertion of the frt2-ble-frt <dig> marker and selection on zeocin.
insertion of the marker was verified and three isolates were screened by pcr and patching as described in methods.
the presence of the mutant allele was first characterized by pcr.
replacement of the internally deleted purm dna sequences with the frt2-ble-frt <dig> fragment resulted in a shift in pcr product size from  <dig> to  <dig>  kb .
this mutant genotype was accompanied with the correct adenine auxotrophy and zeocin resistance phenotypes.
after flp-mediated selection marker excision and curing of the flp expression plasmid, the size of the pcr product obtained with the same primer set from the resulting zeocin susceptible adenine auxotrophs was reduced to 930 bp .
as expected, cells of bp 576a Δpurm::frt2-ble-frt <dig> did not grow on minimal glucose media unless supplemented with adenine and thiamine .
the observed growth phenotypes and pcr product patterns were consistent with those previously observed for bp <dig>  <cit> .
growth analysis by growth curve was carried out using strains 1026b and bp <dig> as positive controls .
without adenine bp <dig> was unable to replicate well in rich lb media.
addition of adenine to the media restored wild-type growth to bp <dig> .
in minimal media, thiamine partially restored growth.
supplementation with adenine alone afforded bp <dig> growth similar to wild-type 1026b and both adenine and thiamine increased the growth rate a little further.
in rich lb media the markerless bp 576a Δpurm::frt <dig> strain, henceforth called 576mn, required adenine supplementation to grow the same as wild-type 576a.
without adenine the growth rate was halved .
in minimal media bp 576mn required both adenine and thiamine for restoration of wild-type growth rates.
thiamine alone was not sufficient while adenine alone only partly complemented .
bp 576mn was complemented using a mini-tn <dig> vector with a wild-type bp purm gene.
in rich media the complementation was complete and 576mn comp was able to grow the same as wild-type 576a .
the single-copy complementation was incomplete in minimal glucose media but did allow the growth at half the rate of wild-type 576a without supplementation .
by increasing the primary dilution factor to 1: <dig> and time of study we sought to ensure the strain was unable to grow in minimal media without adenine and thiamine supplementation.
in fig.
4g and e, show that strain 576mn grows poorly in lb without adenine and is unable to grow after 48 h in minimal glucose media without adenine and thiamine supplementation.
these data show that 576mn is an adenine auxotroph.fig.
<dig> pcr verification of bp 576a ∆purm::frt2-ble-frt <dig>  ble selection marker removal, and adenine auxotrophy screening.
a agarose gel showing the pcr product from the wild-type  576a purm region and the shift-up after deletion by insertion of the frt2-ble-frt <dig> cassette in three mutants .
b agarose gel showing the pcr product from the wild-type  576a purm region, the shift to a larger size after deletion of an internal purm fragment and insertion of the frt2-ble-frt <dig> cassette and the shift to a smaller size after flp-excision of the ble cassette in two isolates.
genotypes and phenotypes are indicated below panels  and .
patch plates showing the phenotype of 576a wild-type  and 576mn patched on minimal glucose  + thiamine media  and on mg + adenine + thiamine media
fig.
<dig> growth analysis of bp 576mn in rich and minimal media.
growth phenotypes of bp strains 1026b and bp <dig> were carried out as controls after 1: <dig> dilutions in lb  and minimal glucose media  +/− adenine and thiamine supplements as indicated.
growth phenotypes of bp strains 576a and 576mn in lb  and minimal glucose media  +/− adenine and thiamine supplements as indicated.
growth curves of the 576mn comp compared to 576a and 576mn in lb  and minimal glucose media  showing full complementation in lb but partial complementation in minimal glucose media.
growth phenotypes of 576a and 576mn in rich media  or minimal media  after 1: <dig> dilutions out to 48 h +/− adenine and thiamine as indicated
intracellular replication of Δpurm mutants in the human cell-line hek <dig>
in a crucial step in demonstrating the biosafe utility of this strain, we wanted to ensure that the new bp 576mn strain was unable to grow in human cells.
hek <dig>  a human embryonic kidney cell line used for bp intracellular infection experiments , was used to determine if 576mn was capable of invasion and intracellular replication in human cells .
all strains showed no significant difference in invasion efficiency .
demonstration of bp <dig> replicative ability in human cells was never investigated so we included bp <dig> in the hek <dig> infection model and compared it to wild-type 1026b.
bp <dig> invaded the same as 1026b but showed over a log reduction in intracellular replication at 24 h post-infection that remained on the verge of undetectable for the 48 h time point.
strain 576a replicated similarly to 1026b but with a slightly higher cfu at 24 h. previous experiments have shown that total lysis of monolayers by intracellular bp can occur after 24 h of infection.
the drop in intracellular wild-type cfu between  <dig> and 48 h is indicative of lysis of host-cells.
strain 576mn invaded the hek <dig> cells at a similar efficiency as the wild-type bacteria and intracellular cfu levels among all strains were not significantly different at 2 h .
intracellular cfu of 576mn at 24 h were decreased in comparison to the 2 h time point and mirrored the log reduction in levels from the  <dig> to 24 h seen with bp <dig>  differences in cfu of wild-type 576a and 576mn at 24 h were significant.
in comparing the complemented 576mn strain, 576mn comp, the complementation allowed some intracellular replication but there was still  <dig> times less bacteria than wild-type at 24 h. the single chromosomal copy of purm was only able to partially complement the defect in intracellular replication of the 576mn mutant.
the intracellular cfu of the 576mn complement at 24 h slightly increased in relation to the 2 h time point and by 48 h showed a 2-log increase in intracellular cfu.
data from the growth curve indicated a reduction in growth rate of 576mn comp in rich media that possibly translated to inefficient growth within hek <dig> cells.fig.
<dig>
bp 576mn is unable to replicate in the human cell line hek <dig> intracellular replication model and is not significantly cytotoxic.
a aminoglycoside protection assay revealed that bp 576mn  was unable to replicate inside hek <dig> cells compared to 576a wt .
strain 1026b  and bp <dig>  were included as controls.
strain 576mn comp was only partially returned to wild-type levels of replication .
the symbols are the average of experimental data acquired in biological triplicate.
the sem is not visible at the scale presented.
the differences between all strains at 2 h were not significant.
significance determined by two-way anova.
ns = not significant, ** = p <  <dig>  **** = p <  <dig> .
b cytotoxicity of hek <dig> cells after 48 h infection with the indicated strains.
significance determined by one-way anova.
ns = not significant, ** = p <  <dig>  **** = p <  <dig>
cytotoxicity in hek <dig> cells was also measured at the 48 h time point .
hek <dig> cells infected with either wild-type 1026b or 576a exhibited almost 100% cytotoxicity.
in comparison, both purm mutants had levels hovering around 10%, significantly lower than their respective wild-type strains.
the complemented 576mn strain caused ~50% cytotoxicity that was in between the wild-type and purm mutants.
strain attenuation testing in the murine melioidosis model
most importantly, the new adenine auxotrophic strain, 576mn, was tested for attenuation in mice.
a control experiment was first carried out where 1026b and bp <dig> were compared in the balb/c inhalation mouse model.
5000 cfu  of wild-type 1026b and 1 ×  <dig> cfu of bp <dig> were intranasally inoculated into the nares of anesthetized mice and observed for moribundity .
mice challenged with ~ <dig> times the ld <dig> of wild-type 1026b  became moribund 2–3 days post-infection.
mice challenged with 1 ×  <dig> cfu bp <dig>  appeared healthy until the study endpoint, 14 days post-infection.
substantiation of the attenuation of bp <dig> in the balb/c intranasal model led us to testing 576mn in the same manner .
mice infected with 5000 cfu wild-type bp 576a succumbed quickly to infection.
similar to bp <dig>  mice challenged with 1 ×  <dig> cfu of 576mn  showed no sign of illness and survived until the study endpoint, 14 days post-infection.
as a comparison the capsule mutant 576a Δwcbb was used to infect balb/c mice intranasally at the same dose as 576mn  .
at the dose used, the 576a Δwcbb capsule mutant was just as virulent as wild-type.
at the end of the study, organs from the bp <dig> and 576mn mice were removed and the bacterial organ loads were determined .
bacteria were not found in the spleens or livers of any of the mice.
two mice challenged with bp <dig> had low amounts of bacteria still present in the lungs following high dose lethal challenge .
the  <dig> and 100 cfu counts in two of the mice represent an average ~  <dig> times decrease in bacterial load compared to the 1 ×  <dig> cfu inoculum, indicating  <dig> of the mice completely cleared the initial challenge dose.
lungs of one mouse from the 576mn challenged group had 100 cfu bacteria present at the end of the study, representing a  <dig>  times decrease in cfu compared to the inoculum cfu.
the presence of 576mn bacteria in one mouse at low levels and the absence of bacteria in  <dig> out of  <dig> mice suggests it was more efficiently cleared than the bp <dig> strain.fig.
<dig>
bp strain 576mn is attenuated in the balb/c mouse model of melioidosis and is efficiently cleared from the organs.
a balb/c mice challenged intranasally with 5 ×  <dig> cfu of wild-type bp 1026b succumbed to infection within 3 days while mice intranasally challenged with 1 ×  <dig> cfu of bp <dig> survived until the end of the study.
b balb/c mice intranasally challenged with 5 ×  <dig> cfu of wild-type bp 576a succumbed to infection within 3 days while mice challenged intranasally with 1 ×  <dig> cfu of 576mn survived until the end of the study.
the increased survival by mice challenged with the mutant strains was highly significant in  and .
c a 1 ×  <dig> cfu intranasal challenge of the bp 576a Δwcbb mutant exhibited the same lethality as wild-type bp 576a.
d organ loads from bp <dig> challenged mice at the end of the 14 day study show 2/ <dig> mice have detectable numbers of bacteria in their lungs.
e organ loads from 576mn challenged mice at the end of the 14 day study show 1/ <dig> mice have detectable numbers of bacteria in their lungs, a 4-log reduction in bacterial numbers.
significance was determined by the log-rank mantel-cox test.
ns = not significant, ** = p <  <dig>
discussion
the bp type strain 1026b has dominated vaccine studies, antibiotic resistance mechanism studies, virulence related studies and many others.
the accessibility of the biosafe 1026b derivative strain bp <dig> has been a boon to such studies by reducing the need for bsl <dig> manipulation and exclusion from select agent regulations.
it has also proven to be an ideal source of material for immunological and vaccinological studies.
major differences in antigenic and genetic properties exist amongst bp strains.
another type strain, 576a, has been used for numerous studies by groups in the uk.
these two strains have many genetic differences, the most notable of which is the o-antigen structure.
antibodies raised against the lps  appeared to be a major component of the humoral response and of the three measured  elevated levels of anti-type ii ops  antibodies were found to be the only antibody correlated with patient survival  <cit> .
lps subunit vaccines were also found to produce high levels of igm and igg and protected 60% of mice from ip challenge to day  <dig> but not from aerosol challenge  <cit> .
so, it stands to reason that lps structure impacts vaccine design and efficacy.
existence of a biosafe strain from the 576a background would allow for an increased ability to produce an array of immunogenic material from bp and increase the biosafe toolkit available to investigators working under time, regulatory and cost limitations.
the data show that both lps and protein content are different between the two strains.
we demonstrated significant differences in immunogenicity using non-human primate serum and performance between 1026b and 576a in the animal model.
we have also shown that the lps of 576a is more immunogenic than 1026b  <cit>  that may lead to increased virulence of this strain.
we consistently found the proportion of acylation  and degree of hydroxylation  were different in the lipid a of 576a compared to 1026b.
in other work in preparation we found that in response to increased growth temperature, strain 1026b begins to modify the lipid a structures while 576a does not.
many intracellular pathogens reduce the immunogenicity of the lipid a portion of lps to avoid activating the immune response in this manner .
while there may be some similarities in the pathogenesis of tularemia, plague, and melioidosis, yersinia pestis and francisella tularensis require a host for replication.
wild-type bp can grow freely in diverse environments.
so replication in the host is not an essential part of the lifecycle but more of an accidental occurrence and modification of the lipid a may be an adaptation to the host environment.
our hypothesis is that some bp strains receive selective pressure while in the host and attempt to decrease immune recognition.
others do not and the increased stress response enhances production of virulence factors.
this requires more work in the future, but the modifications are strain specific and most likely depend on the natural history of each strain.
1026b was isolated form a non-fatal case of septicemic melioidosis and has an ld <dig> by intraperitoneal injection in balb/c mice of  <dig>  ×  <dig> cfu  <cit> .
strain 576a was isolated from a fatal disseminated melioidosis case and has an ld <dig> of 80 cfu in the same murine melioidosis model  <cit> .
in our balb/c intranasal challenge model, 5000 cfu of either strain was used in a pre-determined endpoint study and showed that 576a induced higher levels of inflammation, by measure of tnf-α, in the lungs of infected mice than 1026b when compared to the pbs control.
for consistency, 5000 cfu was also used to produce an acute infection and survival in the two groups of mice was not significantly different.
the decision to recapitulate the ∆purm mutation specifically in the bp 576a strain background was based on the following criteria:  <dig>  bp <dig>  is a popular widely utilized strain and a whole cell vaccine based on the strain is effective .
this work sought to build on the familiarity and accessibility of bp <dig>   <dig>  strain 576a has lps o-antigen that is different than the other exempt strains.
<dig>  besides all being of the same strain background, the other select agent exempt strains have the following drawbacks.
strain b <dig> is a Δasd mutant but the growth rate is affected in rich media compared to wild-type, while the capsule mutant, jw <dig>  may still be virulent at the same challenge doses as seen in our capsule mutant strain made in this study.
the capsule is a major virulence factor and immunogenic polysaccharide that would be important in the utility of a biosafe strain.
<dig>  the previously published success of a vaccine based on strain 576a made this a desirable strain background for isolation of immunologic material and for comparison to the large amount of published data using strain 576a.
<dig>  the aforementioned 2d <dig> strain is not excluded from select agent regulations, difficult to obtain, and is an insertion mutant that poses a risk of genetic reversion, thus necessitating the present work.
we propose that any live attenuated vaccine strain based on 576a with actual potential should include a combination of purm and ilvi mutations.
successful creation of the Δpurm mutation in the 576a strain background was verified by a strict requirement for adenine in minimal media by the patching of colonies.
the effect of the Δpurm mutation on the growth of 576a was modest in rich media and could be overcome by the addition of adenine to lb media.
the ability of thiamine to overcome the purm mutation is variable and may be due to small amounts of adenine crossover during growth preparation or growth stage of the bacteria used for inoculum dilution.
growth lag by the complement in minimal glucose media is more than likely due to the absence of important upstream regulatory elements not cloned into the single-copy complementation vector.
even though growth is delayed, the final cell density is equal to the wild-type.
the phenotype of 576mn is consistent with bp <dig>  as previously published and in our hands.
further extension of growth curves to 48 h showed there was no measureable growth in minimal media without adenine and thiamine supplementation.
each of the two Δpurm strains was unable to grow within the cytosol of hek <dig> cells.
after  <dig> cells invading, intracellular cfu of both strains decreased by a log and a half and hovered near our limit of detection out to 48 h. they were able to invade the cytosol but failed to replicate.
the wild-type strains both had an ~ <dig> increase in intracellular cfu by 24 h post-infection while it took the 576mn comp 48 h to reach ~105 cfu.
cytotoxicity measurements showed the wild-type strains  and comp  afforded significantly higher levels of cellular damage upon the hek <dig> cells compared to bp <dig> or 576mn .
the relatively mild cytotoxic effect of the purm mutants agrees with the intracellular cfu data.
we demonstrated that bp <dig> and 576mn are fully attenuated in the intranasal balb/c mouse model even when inoculated with  <dig> times more cfu than wild-type.
the 576a Δwcbb mutant was also tested in the same approach.
the difference in mouse survival between the mice challenged with 5000 cfu wild-type 576a and 1 ×  <dig> cfu of the 576a Δwcbb mutant was not significantly different with all mice becoming moribund by 3 days post-infection.
even without a capsule, bp 576a can be virulent at higher doses, similar to the select agent excluded strain jw <dig> and b. thailandensis  <cit> .
these survival experiments indicate that 576mn behaves the same as bp <dig> and that it is more attenuated than a Δwcbb capsule mutant in the same background.
one of five mice contained detectable bacteria in the lung after 14 days infection with a massive dose of 576mn, indicating the mutant is steadily cleared and would possibly be completely absent in a longer-term study.
the absence of any detectable cfu in livers and spleens of any animals challenged with either Δpurm mutant indicate inability to disseminate and establish infections at secondary sites.
CONCLUSIONS
it has been demonstrated that 1026b and 576a are very different strains of bp especially in terms of the lps o-antigen and total cellular protein patterns.
besides being a major virulence factor and a protective antigen, lps is a highly immunogenic potentiator of immune responses.
at the doses tested, strains 1026b and 576a are no different in their lethality in the balb/c mouse model of melioidosis.
select-agent excluded strains have been crucial to the development of vaccines, particularly as sources of immunogenic material, such as outer membrane vesicles.
available select agent excluded bp are all based on the same 1026b strain background, limiting the tools available for studying a highly diverse species.
we are aiming to fill that gap by engineering and testing the 576a Δpurm::frt <dig> mutant, 576mn.
this work demonstrated the auxotrophy of the mutant and its ability to grow in rich media, while being unable to replicate in minimal media after sufficient dilution or within human cells compared to the parental wild-type bp strains.
strain 576mn is unable to significantly damage human cells and is avirulent in mice.
it exhibits higher attenuation than the 576a Δwcbb capsule mutant in the balb/c mouse model.
strain jw <dig>  which is 1026b with the acapsular phenotype, has already been excluded from select agent regulations even though it was as virulent as the 576a acapsular mutant tested in this work.
of the two strains evaluated, strain 576mn is the superior candidate for select-agent exclusion and will build on the utility and success of strain bp <dig>

 b. pseudomallei, the etiological agent of the disease melioidosis, is a gram-negative rod typically found in soil and water environments throughout the tropics. in thailand, numerous bp are found in the pooled surface waters of rice paddies coinciding with high disease and seropositivity rates in rural rice farmers. compounding the problem is the quick progression to fatal sepsis; by the time patients seek medical intervention, the disease has progressed acutely, leading to a high mortality rate of %. melioidosis is not just a public health challenge in northeastern thailand. the disease is believed to be vastly underreported with ~ cases worldwide and ~ deaths. moreover, the us centers for disease control and prevention have listed bp as a tier- select agent. tier organisms have the potential to pose a severe threat to us public health and safety and the us government agencies have invested resources to develop vaccines and therapeutics for them, including bp. tier listing dictates numerous regulations and requires select agent compliant biosafety level facilities for manipulation of live organisms, driving up costs and increasing data collection time. one tool that can benefit the researchers in this restricted environment is the use of biosafe surrogates, which allow both exclusion from the select-agent regulations and safe manipulation at biosafety level. burkholderia thailandensis is an attenuated bacterium that is closely related to bp but most strains lack a capsular polysaccharide, among other genomic and virulence factor differences, that do not make it an ideal biosafe surrogate. bt strain e has the capsular polysaccharide and has been successfully evaluated as a live-attenuated vaccine against bp k challenge. there are currently three bp strains that are excluded form the select agent list. the first is an aminoglycoside pump mutant that lacks a capsule, jw another is a diaminopimelic acid requiring Δasd mutant, b and the third is an adenine requiring Δpurm mutant, bp each individually has their drawbacks but cumulatively a major drawback is that all are made from the same strain background, bp 1026b, a serotype a strain. as a species, bp contains a high level of genomic diversity that translates to phenotypic diversity. the core genome of bp, the genes found in all members of the species, is composed of genes. strain 1026b has genes. the available select-agent strains and bt strains partially fulfill the need for surrogates in some aspects of study but there is room to expand the toolset. within-host, bp can infect most tissues and invades, then replicates inside the cytoplasm of many cell types. to accomplish this feat, bp attaches to the host cell causing actin rearrangement and inducing bacterial phagocytosis. a myriad of virulence factors take part in the extra and intracellular lifestyle. lipopolysaccharide is the major component of the outer leaflet of the outer membrane and coats the surface of the gram-negative bacterium, including bp. besides the ability of lps to strongly activate innate immunity, it has been shown to play a role in the intracellular survival of bp during invasion and mutants in the synthesis of the o-antigen of lps are attenuated in animal infection models. a major variable among strains can be the lipopolysaccharide. previously published research identified the genomic differences that exist at the o-antigen biosynthetic operon and screened ~ bp strains for lps diversity and 90% of them were type a. strain 1026b has type a lps. it has been found that the type a lps o-antigen is composed of repeating subunits of glucose and talose. of the remaining 10% with atypical lps; 9% were type b and 1% were type b work presented in this study and in references cited above show a higher and larger banding pattern associated with the type b lps. observations by western blot show antibodies from patients infected with strains of one type are not cross-reactive. lack of cross-reactivity between a and b serotype strains was assumed due to be differences in glycosyl residues of the o-antigen. recently, work from the authors has shown that the type b o-antigen is composed of rhamnose, xylose, and galactose synchronizing the genomic and structural aspects of bp lps data. beyond australia and southeast asia, the predominance of lps types in south asia, the middle east, and africa has been largely uncharacterized, but genomic data indicate a majority of strains identified in madagascar have the type b lps. even though the number of strains possessing atypical lps is a fraction of the typical, their impact on the study of bp and melioidosis is significant. strain 576a was isolated from a fatal case of human melioidosis and has type b lps. the most effective melioidosis vaccine thus far is 2d a branched chain amino acid auxotroph of bp strain 576a containing a transposon insertion in the ilvi gene. it was an extremely effective vaccine in animal models, protecting balb/c mice from a 106 cfu challenge whereas 1026b based vaccines fail to illicit long-term protection against 5 ×  cfu challenge doses. the melioidosis vaccine steering committee has recommended that strain 2d be used as a positive control for vaccines in development. strain 2d is inaccessible to many researchers in the united states and is currently not a select-agent excluded strain. as mentioned above, this mutant is an insertional mutant, thus posing inherent risks from recombination proficient bp. we found significant differences between immunoreactivity of 1026b and 576a as well as organ loads and inflammation caused by these two wild-type strains in the balb/c murine melioidosis model. the 1026b Δpurm strain, bp has proven to be a valuable resource for studying antibiotic resistance mechanisms and vaccines. this work aims to expand on the availability, familiarity, and utility of Δpurm biosafe strains for research by producing the bp 576a Δpurm strain that contains a 114 bp deletion in the purm gene. the newly created bp 576a ∆purm strain, designated 576mn, was auxotrophic for adenine in minimal media, capable of wild-type growth in rich media with addition of adenine, and auxotrophy was partially abrogated with single-copy complementation. bp 576mn was unable to replicate in human cells and was completely avirulent in balb/c mice following high-dose intranasal inoculation, similar to bp organ loads indicated 576mn was unable to replicate in the organs tested. this strain should prove a valuable addition to the biosafe study of bp and is an ideal candidate for select-agent exclusion and could serve as a safe background for creation of a live-attenuated double mutant vaccine strain.  strain background differences between bp 1026b and 576a lps purified from bp and the 576a wcbb mutant show a fine laddering of o-antigen in the 1026b lps on silver stained gels but the type b lps from 576a show a more pronounced laddering with a slightly higher molecular weight. long and very-long lps structures at ~80 kda and beyond are also visible and of higher molecular weight in the 576a type b lps. the lysates from bp and 576mn show a conservation of these high molecular weight lps structures. the coomassie stained sds-page gel shows that protein production by these two strains shows some similarities but also differences. western blots of the fplc-purified lps from 1026b and 576a with mabs to the respective lps types showed no cross-reactivity between the two strains. a western blot using serum from a rhesus macaque 28 days after aerosol challenge with bp 1026b showed that the serum reacted strongly to the fplc - purified type a lps from 1026b and not at all to type b lps from 576a. the highly immunogenic potential of bp lps during the humoral response is evident in the strength of macaque serum reactivity to bp lysate compared to reactivity with 576mn cell lysate. there is faint reactivity to some proteins from 576mn but not to the major antigen lps. the identity of these proteins will be determined in future work. numerous serum samples from rhesus macaques were used in an elisa using purified lps from 1026b and 576a capsule mutants in two separate pure antigen assays. all monkeys were challenged with bp strains that have type a lps. the reactivity of the serum with the type a lps increases starting at 7 days post-infection and the mean od remained high in the serum samples of surviving monkeys. in contrast, there was very little reactivity between the type b lps and all serum samples compared to serum from mice vaccinated with type b lps, agreeing with the blot in fig. 1e. it was also found that in human lung epithelial a cells and mouse macrophage raw cells that bp 576a attached better and formed larger and more numerous plaques, indicating further differences between these strains at the cellular infection level.fig. strain background differences between bp 1026b and 576a. a silver stain of highly pure lps from 1026b and 576a strain backgrounds and heat-killed lysate. b coomassie stain of heat-killed cell lysate. c and d western blots using lps type a specific mab 4c and lps type b specific mab 5b e western blot using day serum from a rhesus macaque after aerosol challenge by bp 1026b. lanes: fplc-purified bp Δwcb lps; fplc-purified 576a Δwcb lps; lys, heat-killed lysate of bp82; lys, heat-killed lysate of 576mn. chevrons indicate 80 kda and 25 kda as indicated fig. a-b attachment and plaque sizes formed in a human lung epithelial cells and murine macrophage cell line raw by bp 1026b and 576a. c images of plaque formation by 1026b and 576a in a and raw cells after infection with the cfu. error bars represent the sem and differences found significant by one-way anova bp 1026b and 576a performance in a murine melioidosis model using the balb/c murine model of infection we also compared the organ dissemination characteristics of the parental wild-type bp strains 1026b and 576a. mice challenged with 5000 cfu of either strain were sacrificed at 24 h post-infection and had the bacterial organ loads in blood, lung, liver, and spleen enumerated. the data show that mean cfus of 576a bacteria in the blood, lung, and spleen trended higher but that the levels were not significantly different. mean 576a bacterial levels in the liver were significantly higher. ten times more cfus were recovered from 576a–infected mouse livers compared to livers from 1026b–infected mice and this difference was highly significant. this may be due to the increased immune activation of 576a lps and activation of tlr4-dependent bacterial clearance and uptake by the liver during sepsis. although bacterial burden in the lung was not significantly different between 1026b and 576a infected mice, tnf-α levels were measured to characterize the acute-phase inflammatory responses to bacterial replication in the lung. tnf-α levels in lung homogenate from 1026b–infected mice trended higher but were not significantly different than in uninfected mouse lungs. tnf-α levels in lung homogenate from 576a infected mouse lung homogenates were significantly higher than those from the uninfected mice but not from 1026b infected mice. this could be due to slightly higher levels of bacteria in the lung or to the higher immunogenicity associated with this strain. regardless, in mouse survival curves following intranasal challenge with 5000 cfu of either wild-type strain indicate all mice are moribund prior to 3 days post-infection and survival of mouse groups infected by the two strains are not significantly different.fig. bacterial burdens and tnf-α levels are different in the organs of balb/c mice infected with bp 1026b or bp 576a. a bacterial cfu/ml of blood and bacterial cfu/organ, i.e., lung;, liver;, spleen. statistical significance determined by mann-whitney statistical testing of the medians of the two groups. ** = p <  e), tnf-α levels in lung homogenate from 1026b are not significantly different than uninfected mice while tnf-α levels from 576a treated lungs were significantly different than untreated mice lungs by the one-way anova test for comparing the three groups. ** = p < . black circles indicate 1026b–infected mice. white squares indicate 576a–infected mice. white triangles indicate uninfected mice. f balb/c survival proportions following intranasal challenge with 5000 cfu of each strain indicated. the black arrow indicates the 24 h time point for removal of organs during the pre-determined endpoint study in, the survival data is also presented in fig. for clarity. ns = not significant by the log-rank test of survival curves construction and growth characterization of a 576a Δpurm mutant allelic recombination was utilized to delete a 125 bp fragment internal to the purm gene by insertion of the frt2-ble-frt marker and selection on zeocin. insertion of the marker was verified and three isolates were screened by pcr and patching as described in methods. the presence of the mutant allele was first characterized by pcr. replacement of the internally deleted purm dna sequences with the frt2-ble-frt fragment resulted in a shift in pcr product size from to  kb. this mutant genotype was accompanied with the correct adenine auxotrophy and zeocin resistance phenotypes. after flp-mediated selection marker excision and curing of the flp expression plasmid, the size of the pcr product obtained with the same primer set from the resulting zeocin susceptible adenine auxotrophs was reduced to 930 bp. as expected, cells of bp 576a Δpurm::frt2-ble-frt did not grow on minimal glucose media unless supplemented with adenine and thiamine. the observed growth phenotypes and pcr product patterns were consistent with those previously observed for bp. growth analysis by growth curve was carried out using strains 1026b and bp as positive controls. without adenine bp was unable to replicate well in rich lb media. addition of adenine to the media restored wild-type growth to bp. in minimal media, thiamine partially restored growth. supplementation with adenine alone afforded bp growth similar to wild-type 1026b and both adenine and thiamine increased the growth rate a little further. in rich lb media the markerless bp 576a Δpurm::frt strain, henceforth called 576mn, required adenine supplementation to grow the same as wild-type 576a. without adenine the growth rate was halved. in minimal media bp 576mn required both adenine and thiamine for restoration of wild-type growth rates. thiamine alone was not sufficient while adenine alone only partly complemented. bp 576mn was complemented using a mini-tn vector with a wild-type bp purm gene. in rich media the complementation was complete and 576mn comp was able to grow the same as wild-type 576a. the single-copy complementation was incomplete in minimal glucose media but did allow the growth at half the rate of wild-type 576a without supplementation. by increasing the primary dilution factor to 1: and time of study we sought to ensure the strain was unable to grow in minimal media without adenine and thiamine supplementation. in fig. 4g and e, show that strain 576mn grows poorly in lb without adenine and is unable to grow after 48 h in minimal glucose media without adenine and thiamine supplementation. these data show that 576mn is an adenine auxotroph.fig. pcr verification of bp 576a ∆purm::frt2-ble-frt ble selection marker removal, and adenine auxotrophy screening. a agarose gel showing the pcr product from the wild-type 576a purm region and the shift-up after deletion by insertion of the frt2-ble-frt cassette in three mutants. b agarose gel showing the pcr product from the wild-type 576a purm region, the shift to a larger size after deletion of an internal purm fragment and insertion of the frt2-ble-frt cassette and the shift to a smaller size after flp-excision of the ble cassette in two isolates. genotypes and phenotypes are indicated below panels and. patch plates showing the phenotype of 576a wild-type and 576mn patched on minimal glucose  + thiamine media and on mg + adenine + thiamine media fig. growth analysis of bp 576mn in rich and minimal media. growth phenotypes of bp strains 1026b and bp were carried out as controls after 1: dilutions in lb and minimal glucose media +/− adenine and thiamine supplements as indicated. growth phenotypes of bp strains 576a and 576mn in lb and minimal glucose media +/− adenine and thiamine supplements as indicated. growth curves of the 576mn comp compared to 576a and 576mn in lb and minimal glucose media showing full complementation in lb but partial complementation in minimal glucose media. growth phenotypes of 576a and 576mn in rich media or minimal media after 1: dilutions out to 48 h +/− adenine and thiamine as indicated intracellular replication of Δpurm mutants in the human cell-line hek in a crucial step in demonstrating the biosafe utility of this strain, we wanted to ensure that the new bp 576mn strain was unable to grow in human cells. hek a human embryonic kidney cell line used for bp intracellular infection experiments, was used to determine if 576mn was capable of invasion and intracellular replication in human cells. all strains showed no significant difference in invasion efficiency. demonstration of bp replicative ability in human cells was never investigated so we included bp in the hek infection model and compared it to wild-type 1026b. bp invaded the same as 1026b but showed over a log reduction in intracellular replication at 24 h post-infection that remained on the verge of undetectable for the 48 h time point. strain 576a replicated similarly to 1026b but with a slightly higher cfu at 24 h. previous experiments have shown that total lysis of monolayers by intracellular bp can occur after 24 h of infection. the drop in intracellular wild-type cfu between and 48 h is indicative of lysis of host-cells. strain 576mn invaded the hek cells at a similar efficiency as the wild-type bacteria and intracellular cfu levels among all strains were not significantly different at 2 h. intracellular cfu of 576mn at 24 h were decreased in comparison to the 2 h time point and mirrored the log reduction in levels from the to 24 h seen with bp differences in cfu of wild-type 576a and 576mn at 24 h were significant. in comparing the complemented 576mn strain, 576mn comp, the complementation allowed some intracellular replication but there was still times less bacteria than wild-type at 24 h. the single chromosomal copy of purm was only able to partially complement the defect in intracellular replication of the 576mn mutant. the intracellular cfu of the 576mn complement at 24 h slightly increased in relation to the 2 h time point and by 48 h showed a 2-log increase in intracellular cfu. data from the growth curve indicated a reduction in growth rate of 576mn comp in rich media that possibly translated to inefficient growth within hek cells.fig. bp 576mn is unable to replicate in the human cell line hek intracellular replication model and is not significantly cytotoxic. a aminoglycoside protection assay revealed that bp 576mn was unable to replicate inside hek cells compared to 576a wt. strain 1026b and bp were included as controls. strain 576mn comp was only partially returned to wild-type levels of replication. the symbols are the average of experimental data acquired in biological triplicate. the sem is not visible at the scale presented. the differences between all strains at 2 h were not significant. significance determined by two-way anova. ns = not significant, ** = p <  **** = p < . b cytotoxicity of hek cells after 48 h infection with the indicated strains. significance determined by one-way anova. ns = not significant, ** = p <  **** = p <  cytotoxicity in hek cells was also measured at the 48 h time point. hek cells infected with either wild-type 1026b or 576a exhibited almost 100% cytotoxicity. in comparison, both purm mutants had levels hovering around 10%, significantly lower than their respective wild-type strains. the complemented 576mn strain caused ~50% cytotoxicity that was in between the wild-type and purm mutants. strain attenuation testing in the murine melioidosis model most importantly, the new adenine auxotrophic strain, 576mn, was tested for attenuation in mice. a control experiment was first carried out where 1026b and bp were compared in the balb/c inhalation mouse model. 5000 cfu of wild-type 1026b and 1 ×  cfu of bp were intranasally inoculated into the nares of anesthetized mice and observed for moribundity. mice challenged with ~ times the ld of wild-type 1026b became moribund 2–3 days post-infection. mice challenged with 1 ×  cfu bp appeared healthy until the study endpoint, 14 days post-infection. substantiation of the attenuation of bp in the balb/c intranasal model led us to testing 576mn in the same manner. mice infected with 5000 cfu wild-type bp 576a succumbed quickly to infection. similar to bp mice challenged with 1 ×  cfu of 576mn showed no sign of illness and survived until the study endpoint, 14 days post-infection. as a comparison the capsule mutant 576a Δwcbb was used to infect balb/c mice intranasally at the same dose as 576mn. at the dose used, the 576a Δwcbb capsule mutant was just as virulent as wild-type. at the end of the study, organs from the bp and 576mn mice were removed and the bacterial organ loads were determined. bacteria were not found in the spleens or livers of any of the mice. two mice challenged with bp had low amounts of bacteria still present in the lungs following high dose lethal challenge. the and 100 cfu counts in two of the mice represent an average ~  times decrease in bacterial load compared to the 1 ×  cfu inoculum, indicating of the mice completely cleared the initial challenge dose. lungs of one mouse from the 576mn challenged group had 100 cfu bacteria present at the end of the study, representing a times decrease in cfu compared to the inoculum cfu. the presence of 576mn bacteria in one mouse at low levels and the absence of bacteria in out of mice suggests it was more efficiently cleared than the bp strain.fig. bp strain 576mn is attenuated in the balb/c mouse model of melioidosis and is efficiently cleared from the organs. a balb/c mice challenged intranasally with 5 ×  cfu of wild-type bp 1026b succumbed to infection within 3 days while mice intranasally challenged with 1 ×  cfu of bp survived until the end of the study. b balb/c mice intranasally challenged with 5 ×  cfu of wild-type bp 576a succumbed to infection within 3 days while mice challenged intranasally with 1 ×  cfu of 576mn survived until the end of the study. the increased survival by mice challenged with the mutant strains was highly significant in and. c a 1 ×  cfu intranasal challenge of the bp 576a Δwcbb mutant exhibited the same lethality as wild-type bp 576a. d organ loads from bp challenged mice at the end of the 14 day study show 2/ mice have detectable numbers of bacteria in their lungs. e organ loads from 576mn challenged mice at the end of the 14 day study show 1/ mice have detectable numbers of bacteria in their lungs, a 4-log reduction in bacterial numbers. significance was determined by the log-rank mantel-cox test. ns = not significant, ** = p <  discussion the bp type strain 1026b has dominated vaccine studies, antibiotic resistance mechanism studies, virulence related studies and many others. the accessibility of the biosafe 1026b derivative strain bp has been a boon to such studies by reducing the need for bsl manipulation and exclusion from select agent regulations. it has also proven to be an ideal source of material for immunological and vaccinological studies. major differences in antigenic and genetic properties exist amongst bp strains. another type strain, 576a, has been used for numerous studies by groups in the uk. these two strains have many genetic differences, the most notable of which is the o-antigen structure. antibodies raised against the lps appeared to be a major component of the humoral response and of the three measured elevated levels of anti-type ii ops antibodies were found to be the only antibody correlated with patient survival. lps subunit vaccines were also found to produce high levels of igm and igg and protected 60% of mice from ip challenge to day but not from aerosol challenge. so, it stands to reason that lps structure impacts vaccine design and efficacy. existence of a biosafe strain from the 576a background would allow for an increased ability to produce an array of immunogenic material from bp and increase the biosafe toolkit available to investigators working under time, regulatory and cost limitations. the data show that both lps and protein content are different between the two strains. we demonstrated significant differences in immunogenicity using non-human primate serum and performance between 1026b and 576a in the animal model. we have also shown that the lps of 576a is more immunogenic than 1026b that may lead to increased virulence of this strain. we consistently found the proportion of acylation and degree of hydroxylation were different in the lipid a of 576a compared to 1026b. in other work in preparation we found that in response to increased growth temperature, strain 1026b begins to modify the lipid a structures while 576a does not. many intracellular pathogens reduce the immunogenicity of the lipid a portion of lps to avoid activating the immune response in this manner. while there may be some similarities in the pathogenesis of tularemia, plague, and melioidosis, yersinia pestis and francisella tularensis require a host for replication. wild-type bp can grow freely in diverse environments. so replication in the host is not an essential part of the lifecycle but more of an accidental occurrence and modification of the lipid a may be an adaptation to the host environment. our hypothesis is that some bp strains receive selective pressure while in the host and attempt to decrease immune recognition. others do not and the increased stress response enhances production of virulence factors. this requires more work in the future, but the modifications are strain specific and most likely depend on the natural history of each strain. 1026b was isolated form a non-fatal case of septicemic melioidosis and has an ld by intraperitoneal injection in balb/c mice of  ×  cfu. strain 576a was isolated from a fatal disseminated melioidosis case and has an ld of 80 cfu in the same murine melioidosis model. in our balb/c intranasal challenge model, 5000 cfu of either strain was used in a pre-determined endpoint study and showed that 576a induced higher levels of inflammation, by measure of tnf-α, in the lungs of infected mice than 1026b when compared to the pbs control. for consistency, 5000 cfu was also used to produce an acute infection and survival in the two groups of mice was not significantly different. the decision to recapitulate the ∆purm mutation specifically in the bp 576a strain background was based on the following criteria: bp is a popular widely utilized strain and a whole cell vaccine based on the strain is effective. this work sought to build on the familiarity and accessibility of bp strain 576a has lps o-antigen that is different than the other exempt strains. besides all being of the same strain background, the other select agent exempt strains have the following drawbacks. strain b is a Δasd mutant but the growth rate is affected in rich media compared to wild-type, while the capsule mutant, jw may still be virulent at the same challenge doses as seen in our capsule mutant strain made in this study. the capsule is a major virulence factor and immunogenic polysaccharide that would be important in the utility of a biosafe strain. the previously published success of a vaccine based on strain 576a made this a desirable strain background for isolation of immunologic material and for comparison to the large amount of published data using strain 576a. the aforementioned 2d strain is not excluded from select agent regulations, difficult to obtain, and is an insertion mutant that poses a risk of genetic reversion, thus necessitating the present work. we propose that any live attenuated vaccine strain based on 576a with actual potential should include a combination of purm and ilvi mutations. successful creation of the Δpurm mutation in the 576a strain background was verified by a strict requirement for adenine in minimal media by the patching of colonies. the effect of the Δpurm mutation on the growth of 576a was modest in rich media and could be overcome by the addition of adenine to lb media. the ability of thiamine to overcome the purm mutation is variable and may be due to small amounts of adenine crossover during growth preparation or growth stage of the bacteria used for inoculum dilution. growth lag by the complement in minimal glucose media is more than likely due to the absence of important upstream regulatory elements not cloned into the single-copy complementation vector. even though growth is delayed, the final cell density is equal to the wild-type. the phenotype of 576mn is consistent with bp as previously published and in our hands. further extension of growth curves to 48 h showed there was no measureable growth in minimal media without adenine and thiamine supplementation. each of the two Δpurm strains was unable to grow within the cytosol of hek cells. after cells invading, intracellular cfu of both strains decreased by a log and a half and hovered near our limit of detection out to 48 h. they were able to invade the cytosol but failed to replicate. the wild-type strains both had an ~ increase in intracellular cfu by 24 h post-infection while it took the 576mn comp 48 h to reach ~105 cfu. cytotoxicity measurements showed the wild-type strains and comp afforded significantly higher levels of cellular damage upon the hek cells compared to bp or 576mn. the relatively mild cytotoxic effect of the purm mutants agrees with the intracellular cfu data. we demonstrated that bp and 576mn are fully attenuated in the intranasal balb/c mouse model even when inoculated with times more cfu than wild-type. the 576a Δwcbb mutant was also tested in the same approach. the difference in mouse survival between the mice challenged with 5000 cfu wild-type 576a and 1 ×  cfu of the 576a Δwcbb mutant was not significantly different with all mice becoming moribund by 3 days post-infection. even without a capsule, bp 576a can be virulent at higher doses, similar to the select agent excluded strain jw and b. thailandensis. these survival experiments indicate that 576mn behaves the same as bp and that it is more attenuated than a Δwcbb capsule mutant in the same background. one of five mice contained detectable bacteria in the lung after 14 days infection with a massive dose of 576mn, indicating the mutant is steadily cleared and would possibly be completely absent in a longer-term study. the absence of any detectable cfu in livers and spleens of any animals challenged with either Δpurm mutant indicate inability to disseminate and establish infections at secondary sites.  it has been demonstrated that 1026b and 576a are very different strains of bp especially in terms of the lps o-antigen and total cellular protein patterns. besides being a major virulence factor and a protective antigen, lps is a highly immunogenic potentiator of immune responses. at the doses tested, strains 1026b and 576a are no different in their lethality in the balb/c mouse model of melioidosis. select-agent excluded strains have been crucial to the development of vaccines, particularly as sources of immunogenic material, such as outer membrane vesicles. available select agent excluded bp are all based on the same 1026b strain background, limiting the tools available for studying a highly diverse species. we are aiming to fill that gap by engineering and testing the 576a Δpurm::frt mutant, 576mn. this work demonstrated the auxotrophy of the mutant and its ability to grow in rich media, while being unable to replicate in minimal media after sufficient dilution or within human cells compared to the parental wild-type bp strains. strain 576mn is unable to significantly damage human cells and is avirulent in mice. it exhibits higher attenuation than the 576a Δwcbb capsule mutant in the balb/c mouse model. strain jw which is 1026b with the acapsular phenotype, has already been excluded from select agent regulations even though it was as virulent as the 576a acapsular mutant tested in this work. of the two strains evaluated, strain 576mn is the superior candidate for select-agent exclusion and will build on the utility and success of strain bp 
the ∆purm mutation was recapitulated in the bp 576a strain, a serotype b background. in the united states, bp is listed as a tier- select agent by the centers for disease control and prevention, thus requiring high levels of regulation and biosafety level facilities for experimental manipulation of live organisms. in this work, the new biosafe strain 576mn with atypical type b lps was generated. strain 576mn is an ideal candidate for select-agent exclusion. an important difference between strains 1026b and 576a is the lipopolysaccharide, a major virulence factor and protective antigen. bp 576mn was unable to replicate in human cells and was avirulent in balb/c mice following high-dose intranasal inoculation, similar to bp organ loads indicated a significant reduction in bacterial replication. the newly created bp 576a ∆purm strain, designated 576mn, was auxotrophic for adenine in minimal media, capable of wild-type growth in rich media with addition of adenine, and auxotrophy was abrogated with single-copy complementation. the work was undertaken to expand the tools available for researching burkholderia pseudomallei, the etiological agent of the tropical disease melioidosis. melioidosis has the potential to pose a severe threat to public health and safety. strain 576a replicates to higher levels in mouse organs and induces more tnf-α in the lungs of balb/c mice compared to 1026b. an avirulent ∆purm derivative of strain 1026b has proven to be a valuable tool for biosafe research as a select-agent excluded strain, but the high level of genetic diversity between bp strains necessitates an expansion of the biosafe toolset. this strain should prove a valuable addition to the toolkit for biosafe studies of bp and development of therapeutic and preventative strategies aimed at combatting melioidosis.
 the work was undertaken to expand the tools available for researching burkholderia pseudomallei, the etiological agent of the tropical disease melioidosis. melioidosis has the potential to pose a severe threat to public health and safety. in the united states, bp is listed as a tier- select agent by the centers for disease control and prevention, thus requiring high levels of regulation and biosafety level facilities for experimental manipulation of live organisms. an avirulent ∆purm derivative of strain 1026b has proven to be a valuable tool for biosafe research as a select-agent excluded strain, but the high level of genetic diversity between bp strains necessitates an expansion of the biosafe toolset.  the ∆purm mutation was recapitulated in the bp 576a strain, a serotype b background. an important difference between strains 1026b and 576a is the lipopolysaccharide, a major virulence factor and protective antigen. polyclonal sera from 1026b–challenged non-human primates showed no cross reactivity with strain 576a lps and low reactivity with whole cell lysate. strain 576a replicates to higher levels in mouse organs and induces more tnf-α in the lungs of balb/c mice compared to 1026b. the newly created bp 576a ∆purm strain, designated 576mn, was auxotrophic for adenine in minimal media, capable of wild-type growth in rich media with addition of adenine, and auxotrophy was abrogated with single-copy complementation. bp 576mn was unable to replicate in human cells and was avirulent in balb/c mice following high-dose intranasal inoculation, similar to bp organ loads indicated a significant reduction in bacterial replication.  in this work, the new biosafe strain 576mn with atypical type b lps was generated. this strain should prove a valuable addition to the toolkit for biosafe studies of bp and development of therapeutic and preventative strategies aimed at combatting melioidosis. strain 576mn is an ideal candidate for select-agent exclusion. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. http://dx.doi.org/ /100008287science and technology directoratehshqdc-10-c-00135tuanyok apichai http://dx.doi.org/ /100007698university of floridaepi 16-3norris michael h. university of floridaepi startup fundstuanyok apichai issue-copyright-statement© the author 2017 