# The Identification and Differentiation betweenBurkholderia mallei and Burkholderia pseudomallei Using One Gene Pyrosequencing

Authors: Damian H. Gilling; Vicki Ann Luna; Cori Pflugradt
Journal: International Scholarly Research Notices
 (2014)

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## Abstract

The etiologic agents for melioidosis and glanders,Burkholderia mallei andBurkholderia pseudomallei respectively, are genetically similar making identification and differentiation from otherBurkholderia species and each other challenging. We used pyrosequencing to determine the presence or absence of an insertion sequenceIS407A within the flagellin P (fliP) gene and to exploit the difference in orientation of this gene in the two species. Oligonucleotide primers were designed to selectively target theIS407A-fliP interface inB. mallei and thefliP gene specifically at the insertion point inB. pseudomallei. We then examined DNA from tenB. mallei, tenB. pseudomallei, 14B. cepacia, eight otherBurkholderia spp., and 17 other bacteria. Resultant pyrograms encompassed the target sequence that contained either thefliP gene with theIS407A interruption or the fully intactfliP gene with 100% sensitivity and 100% specificity. These pyrosequencing assays based upon a single gene enable investigators to reliably identify the two species. The information obtained by these assays provides more knowledge of the genomic reduction that created the new speciesB. malleifromB. pseudomallei and may point to new targets that can be exploited in the future.


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## Body

## 1. Introduction

The proteobacteriaBurkholderia mallei andBurkholderia pseudomallei, distinct from theBurkholderia cepacia complex, cause melioidosis and glanders, respectively [1]. Inhalation of either organism can lead to pneumonia with reported mortality rates of 19–50% [2, 3]. Although human-to-human spread is extremely rare,B. mallei is highly infectious when aerosolized and resulting infections can be debilitating, painful, and difficult to diagnose in humans [4]. Historically,B. mallei was used as a bioweapon during the Civil War and the two World Wars with suspected use by the former USSR in Afghanistan [4–6]. Infections withB. pseudomalleiare more common especially in endemic areas such as Southeast Asia and Northern Australia [7]. In the United States, rare infections withB. pseudomallei have been reported; usually patients are military personnel who were exposed when they were on active duty in endemic areas either recently or even as long as 62 years before [8, 9]. Two active disease cases were reported in US civilians who had traveled to Honduras where they presumably were exposed [10] (CDC, 2006). Other cases have been reported in Arizona, Puerto Rico, and the British Virgin Islands as recently as 2011 where the source of infection appeared to be local soils and flood waters [11–15]. Laboratory exposure has also been reported in California and Florida [10, 16]. Today, bothB. mallei andB. pseudomallei are classified as Tier 1 select agents by the Centers for Disease Control and Prevention (CDC) due to their high virulence via the respiratory route, as well as potential to cause illness on a large scale if ever widely disseminated [6].Symptoms ofB. pseudomallei infection vary, ranging from severe pneumonia with concomitant abscesses in the liver and spleen to persistent infections of the skin, soft tissues, bones, and joints [2]. Often the symptomology ofB. pseudomalleiinfections parallel to those of infections such as tuberculosis which makes empiric diagnosis difficult unless the organism can be isolated, identified, and confirmed in the laboratory [2, 17, 18]. Another factor which makes the clinical identification and differentiation betweenB. pseudomallei andB. mallei even more challenging is the fact that human cases of glanders and melioidosis infections are not common in nonendemic parts of the world. As a result many medical professionals in clinics and laboratories lack experience with these bacteria [18, 19]. Additionally,B. pseudomallei has earned the nickname “the great mimicker” due to its wide repertoire of clinical manifestations which often prompt misdiagnosis. Several rapid detection/diagnostic biochemical methods have been introduced; yet problems have been reported such as the misidentification ofB. pseudomallei asB. cepacia [19–23]. Thus research has shifted focus toward molecular methods, but these too have had their problems, primarily assay specificity and sensitivities which are less than 100% [24, 25]. Culture methods have traditionally been the gold standard for identifyingB. mallei andB. pseudomallei [26, 27]. However these methods can be time consuming. Many molecular methods focus onB. mallei and the loss of a gene or phenotype and appear to be reliable in identifyingB. mallei but none of these methods can consistently distinguishB. pseudomallei from otherBurkholderia species [19, 25]. DNA fingerprinting assays such as multilocus sequence typing (MLST) have been proven efficacious at detection but are also time consuming, expensive, and technically challenging for many laboratories [28]. Identification ofB. mallei andB. pseudomallei through the sequencing of 16S DNA targets has been successfully accomplished; however, discrimination of the two closely related species requires additional sequencing [29, 30].Genetically,B. pseudomallei andB. mallei are so similar that differentiating between the two is a challenge. In factB. mallei evolved directly fromB. pseudomallei due to a process called reductive evolution wherein a massive invasion of many insertion sequence (IS) elements altered theB. pseudomallei genome through genomic rearrangements and deletions. This reduction evolution resulted in a smaller genome with enough key genotypic and phenotypic differences to create a separate species now known asB. mallei [31, 32]. The numerousIS elements in theB. malleigenome account for about 3.1% of the total genome; yet this smaller genome is only 80% in size of that ofB. pseudomallei [32, 33]. One specific insertion sequence, known asIS407A can be located in multiple sites in the genome and has been found to interrupt the flagellar gene (fliP) that encodes the flagellar P protein and rendersB. mallei nonmotile [33]. Additionally, the resultant truncatedfliP gene is completely flipped, having undergone a complete inversion on the DNA strand. This genomic structure and nonmotility is found in allB. mallei [32]. In comparison,B. pseudomallei does not contain theIS407A insertion sequence within itsfliP gene. This complete gene confers full motility and contains sequence regions that are distinct from thefliP gene in otherBurkholderia species such asB. cepacia.TheB. mallei fliP/IS407A region with its species wide conserved gene inversion and truncation provided the target forB. mallei used in this work. We hypothesized that when the target region was sequenced, that sequence would exhibit both the end of the IS407A insertion and a section of thefliP gene that would be in the reverse orientation to that same gene found inB. pseudomallei. We then chose aB. pseudomallei target that covered the same section of thefliP gene where theIS407A insertion sequence was located inB. mallei, using the rationale that, when sequenced, this target will exhibit either (A) a completefliP match in the correct orientation and thus can be identified asB. pseudomalleior (B) nothing at all if the gene is interrupted which precluded the primers from binding.  Thus this scheme is able to detect and differentiate betweenB. pseudomallei andB. mallei. We hypothesized that the targeting of thefliP gene with its clear differences betweenB. mallei andB. pseudomallei and distinctions from otherBurkholderia spp. should provide a reliable form of confirmation between inconclusive results.The purpose of this study was to explore the use of pyrosequencing to exploit the genetic difference of thefliP gene, its orientation, and surrounding sequences inB. mallei andB. pseudomallei as a single gene target method of detection and differentiation between the two species and otherBurkholderia species. We wanted to determine if the pyrosequencing platform, that is, putatively simple, quick, and trustworthy [34], would enable the consistent and reliable microbial typing by targeting conservedfliP regions ofB. pseudomallei in addition to the variablefliP/IS407A regions ofB. mallei.


## 2. Materials and Methods

### 2.1. Bacterial Strains

We examined tenBurkholderia mallei, tenBurkholderia pseudomallei, and 40 other bacterial strains, including 15B. cepacia, 3B. thailandensis, and five variousBurkholderia species (Table 1). TheB. mallei,B. pseudomallei, and 2B. thailandensis were received from the NIH Biodefense and Emerging Infections Research Resources Repository (BEI Resources, Bethesda, MD, USA) as either live strains or purified DNA. Access to both isolates and DNA of bothB. mallei andB. pseudomalleiwere limited due to restrictions on how many Tier 1 isolates and DNA can be purchased yearly from BEI Resources. Of the 15B. cepacia isolates, one was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), three were collected from the Florida Department of Health, Bureau of Laboratories, Tampa, FL (FDOH) and 11 were procured from the University of Washington Medical Center in Seattle, WA. The other 23 bacterial strains including six variousBurkholderia species were part of our large bacterial collection and were either collected previously as clinical isolates from FDOH or received from ATCC (Table 1).Table 1
List of bacteria used in this study.
Organism
CBD numbera
Historical strain identifier
Burkholderia
Burkholderia mallei
BB 0044
China 5 (MM-A, NBL 4) ATCC 10399
Burkholderia mallei
BB 0045
Ivan (NCTC 10230) ATCC 15310
Burkholderia mallei
BB 0046
China 7 (NBL 7) ATCC 23344
Burkholderia mallei
BB 0050
GB8 horse 4
Burkholderia mallei
DD-675
BURK014
Burkholderia mallei
DD-671
BURK005 (SR092700A)
Burkholderia mallei
DD-372
BURK007 (SR092700C)
Burkholderia mallei
DD-673
BURK009 (SR092700E)
Burkholderia mallei
DD-677
BURK062 (Turkey 1)
Burkholderia mallei
DD-178
BURK010 (2344)
Burkholderia pseudomallei
BB 0047
China 3 (MP-H, NBL 104)
Burkholderia pseudomallei
BB 0048
S 397 (NRRL B-1112, CCEB 472)
Burkholderia pseudomallei
BB 0049
286 (MP-S, NBL 121)
Burkholderia pseudomallei
BB 0051
K96243
Burkholderia pseudomallei
BB 0052
1026b
Burkholderia pseudomallei
BB 0053
1106b
Burkholderia pseudomallei
BB 0054
1710a
Burkholderia pseudomallei
BURK088
BURK088
Burkholderia pseudomallei
BURK099
BURK099
Burkholderia pseudomallei
NR-9320
K96423
Burkholderia cepacia
CBD 1341
FL-M-05-0506072
Burkholderia cepacia
CBD 1342
FL-M-05-B24210
Burkholderia cepacia
CBD 1343
FL-M-05-B25995R
Burkholderia cepacia
CBD 1442
ATCC BAA-245
Burkholderia cepacia
CBD 1450
F68492
Burkholderia cepacia
CBD 1456
H27659
Burkholderia cepacia
CBD 1455
H35975
Burkholderia cepacia
CBD 1449
M25311
Burkholderia cepacia
CBD 1458
M27066
Burkholderia cepacia
CBD 1451
M42544
Burkholderia cepacia
CBD 1453
M52455
Burkholderia cepacia
CBD 1454
M74393
Burkholderia cepacia
CBD 1457
T10400
Burkholderia cepacia
CBD 1452
T33589
Burkholderia cepacia
CBD 1448
T47491
Burkholderia graminis
CBD 1440
ATCC 700544
Burkholderia multivorans
CBD 1443
ATCC BAA-247
Burkholderia stabilis
CBD 1441
ATCC BAA-67
Burkholderia vietnamiensis
CBD 1438
ATCC 55792
Burkholderia vietnamiensis
CBD 1444
ATCC BAA-248
Burkholderia thailandensis
CBD 1439
ATCC 700388
Burkholderia thailandensis
BURK254
E254
Burkholderia thailandensis
BURK43
MSMB043
Other bacteria
Achromobacter xylosoxidans
CBD 0307
ATCC 27061
Acinetobacter baumannii
CBD 1323b
ATCC 19606
Acinetobacter calcoaceticus
CBD 1336b
ATCC 23055
Bacillus badius
CBD 0091
ATCC 6462
Cedecea neteri
CBD 0309
ATCC 33855
Citrobacter freundii
CBD 0553b
ATCC 8090
Enterobacter cloacae
CBD 0556b
ATCC 13047
Enterococcus faecalis
CBD 1406
ATCC 49532
Enterococcus faecalis
CBD 1407
ATCC 49533
Klebsiella pneumoniae
CBD 0555b
ATCC 35657
Lactobacillus rhanmosus
CBD 1409
ATCC 53103
Pseudomonas aeruginosa
CBD 0551b
ATCC 15442
Serratia marcescens
CBD 1239
ATCC 13880
Staphylococcus aureus
CBD 0534
WA-HMC-03-4905
Staphylococcus aureus
CBD 1276
ATCC BAA-976
Stenotrophomonas maltophilia
CBD 0552b
ATCC 51331
Streptococcus pneumoniae
CBD 1405
ATCC 700669
aCBD: CBD stands for “Center for Biological Defense” and is the starting designation we use for bacterial strains that are in our BSL2 bacterial collection. Besides the CBD letter designation, each strain in our collection is also given a number to identify it. Each isolate in our BSL3 bacterial collection has a BB letter designation and then assigned a number as the BSL2 strains.
bdenotes an isolate carries a homolog of the fliP gene.All manipulations of cultures ofB. malleiandB. pseudomalleistrains were performed in a biological safety cabinet in a biological safety level 3 (BSL3) laboratory. All safety protocols followed “Biosafety in Microbiological and Biomedical Laboratories, 5th Edition” (BMBL) practices including the use of protective laboratory clothing and respiratory equipment [35]. The safety and security requirements by US federal regulation DHHS 42 CFR 73 were strictly adhered to. Manipulations of the other bacterial strains were performed in a biological safety level 2 (BSL2) laboratory in a biological safety cabinet following safety practices outlined in BMBL as above.


### 2.2. DNA Extraction

Bacteria were grown on tryptic soy agar supplemented with 5% sheep red blood cells (blood agar (BA)) (Remel, Lenexa, KS, USA). All culture plates were incubated at 35°C overnight (18–24 hours) before performing DNA extractions. Following manufacturers’ instructions, all genomic DNA extractions were either performed using the Epicentre (Qiagen Inc, Madison, WI) extraction kits, a MagNaPure Compact automated instrument (Roche, Inc, Indianapolis, IN, USA), or a boil preparation method used by FDOH. The boil preparation, (adapted from a reference lab in the Laboratory Response Network) is described as follows: bacterial growth from an overnight cultured media plate was removed and placed into 100μL of sterile water and boiled for 5 minutes and then placed onto ice for 2 minutes, followed by centrifugation at 12,000 ×g for 10 minutes at 4°C. The supernatant was transferred to a 0.1 μM filter tube (Millipore Corporation Billerica, MA) and centrifuged for 2 minutes at 8,000 ×g. To prove sterility of all filtrates and DNA extractions (following the University of South Florida Institutional Biosafety Committee guidelines), 10 μL was sacrificed and used to inoculate a BA media culture plate and incubated at 35°C for 48 hours. Extracts and lysates having no growth after two days were allowed out of the BSL3 environment and made available for molecular work. The DNA was stored at 4°C or −30°C until used. Before use, the DNA was diluted 1 : 20 in molecular-grade water (Fisher Scientific, Pittsburg, PA) to achieve a working concentration of 0.5 to 2 ng/μL.


### 2.3. Validation PCR Assays and Gel Electrophoresis

Based upon thefliP gene GenBank sequence BMA2686, PCR assays were designed to target sequences of thefliP gene inB. mallei(NCBI GenBank Accession #:NC_006348) andB.pseudomallei (NCBI GenBank Accession #:NC_009076). Figure 1 shows a schematic of the target regions in both species. All oligonucleotides used in the PCR assays forB. malleiandB pseudomallei are listed in Table 2. Primer-BLAST analysis (via NCBI) predicted 217 bp and 397 bp amplicons for thefliP—IS407A interface inB. malleiand thefliP gene inB psudomallei, respectively. The PCR reaction mixture with a final volume of 10 μL volume consisted of 1 μL of DNA template (at a concentration of 0.5–2 ng/μL), 1 μL of the Takara (Takara, Madison, WI, USA) 10X buffer containing 1.5 mM MgCl2, 0.82 μL of dNTPs at 25 μM, 0.05 μL of Takara HS Taq enzyme, and 2 μL of each specific primer at a concentration of 1 μM. Universal primers for the 16S rDNA sequence ofBurkholderia spp. previously described [20, 30] were later incorporated in the assays and produced a 1500 bp product to act as a control. The universal primers were IAC-16S-F (5′-AGAGTTTGATCCTGGCTCAG-3′) and IAC-rDNA-R (5′-ACGGCTACCTTGTTACGACTT-3′). All PCR reactions were carried out on a T1 Biometra Thermocycler (Biometra Inc., Göttingen, Germany) with the following conditions: initial denaturation at 95°C for 15 minutes, followed by 35 cycles of 94°C for 30 seconds, 58°C for 1 minute, and 72°C for 3 minutes, and lastly a final extension of 72°C for 10 minutes.Table 2
List of oligonucleotide primers used in pyrosequencing assays.
Organism
Target
Primer
Sequence (5′-3′)
Location (bp)a
B. mallei
fliP
BmfliP-F1b
ACGAACAGCGTGAGGAAGAG
2786291–2786310
IS407A
BmIS407A-R1
CTAGAAGCCCATTGGCCCTAT
2786443–2786423
Interface
Bm-S1
GGGGCAGGTCAACGA
2786417–2786403
Interface
Bm-S3
GGCAGGTCAACGAGC
2786415–2786401
B. mallei
fliP
BmfliP-F2b
CGAACAGCGTGAGGAAGAG
2786292–2786310
IS407A
BmIS407A-R1
CTAGAAGCCCATTGGCCCTAT
2786443–2786423
Interface
Bm-S3
GGCAGGTCAACGAGC
2786415–2786401
B. pseudomallei
fliP
BpfliP-F1
AGACGATGCTGCTGCTCAC
31112–31130
fliP
BpfliP-R1b
CCCGACGAGCACCTGATTC
31257–31239
fliP
BpfliP-R2b
GAACAGCGTGAGGAAGAGGG
31281–31262
fliP
BpfliP-S4
GCTGTCGTTCCTGCC
31134–31148
B. pseudomallei
fliP
BpfliP-F2b
GACGATGCTGCTGCTCAC
31113–31130
fliP
BpfliP-R3
AACAGCGTGAGGAAGAGGG
31280–31262
fliP
BpfliP-S5
AGCAGCGACAGCACG
31205–31191
Pyrosequencing primer sets forB. mallei and B. pseudomallei with respective target regions. Primers were designed using the design software supplied by the manufacturer.
adenotes location on the published sequences of either B. mallei GenBank accession number NC_006348 or B. pseudomallei GenBank accession number NC_009076.
bdenotes a biotinylated primer.
“Interface” denotes the sequence whereIS407A interrupts the fliP gene.
“F” denotes forward primer used for PCR.
“R” denotes reverse primer used for PCR.
“S” denotes the pyrosequencing primers.
The primers were first rehydrated in molecular grade water to bring them to a 100μM concentration for each primer. Forward and reverse primers were used at 1 μM concentration while the sequence primers were used at 100 μM.
The target sequence for the primer sets forB. mallei was: GCCTGCCGCAGCAGCGACAGCACGACGATGATCCGCGTGA, located at 2786360–2786399 bp in the sequence NCBI Genbank Accession #: NC_006348. The target sequence for the primer sets for B. pseudomallei was: GCGATGCTGCTGATGATGACGAGCTTCACGCGGATCATCA, located in the NCBI Genbank Accession #: NC_009076 at 31150–31189 bp.  PCR primer targets for both B. mallei and B. pseudomallei are all located on their respective chromosome 1. All primer coordinates were last verified on April 1, 2014.Figure 1
(a) Schematic ofBurkholderia mallei fliP gene withIS407A interruption that caused both gene inversion and loss of function. The large arrow within the block designates the orientation of the two parts of thefliP gene on the DNA strand. The location and direction of the various primers are noted by the small arrows. The pyrosequencing target inB. mallei is the interface between the insertion sequence element andfliP gene. (b) Schematic of theBurkholderia pseudomallei intact and functionalfliP gene. The large arrow within the block designates the orientation of the intact gene while the location and directions of the primers used are denoted by the small arrows. The pyrosequencing target inB. pseudomallei covers both sides of the point of thefliP gene that corresponds to where the insertion sequence is found inB. mallei.PCR amplicons were electrophoresed on a 1% agarose gel containing 15μg of ethidium bromide (final concentration of 0.2 μg/mL) in 1X TBE (44.5 mmol Tris-borate and 1 mmol EDTA, pH 8.3) for 60 min at 100 mV constant voltage. DNA bands were visualized with UV light and photographed using the GelDoc (Bio-Rad, Hercules, CA, USA).


### 2.4. Sanger Sequencing

The amplicons from the validation PCR and the initial pyrosequencing PCR reactions were purified using the Wizard DNA Purification Kit (Promega, Madison, WI, USA). Cycle sequencing was performed using the Beckman-Coulter Dye Terminator Cycle Sequencing (DTCS) Quick Start kit protocol (Beckman-Coulter, Fullerton, CA, USA). Cycle sequencing reactions with a final volume of 20μL consisted of 8 μL DTCS Quick Start master mix (Beckman-Coulter), 1.5 μL sequence primer at 25 pmol, and 8 μL of DNA. Cycle sequencing PCR was performed with the Biometra T3 Thermocycler (Biometra Inc.,) for 30 cycles under the following conditions: 96°C for 20 seconds, 50°C for 20 seconds, and 60°C for 4 minutes. The DNA produced from the cycle sequencing reaction was precipitated as per the Beckman-Coulter ethanol precipitation protocol. Finally, Sanger sequencing was performed on a Beckman-Coulter CEQ-8000 (Fullerton, CA) following the manufacturer’s directions. The resulting nucleotide sequences were compared to the public databases via NCBI nucleotide nBLAST alignment which confirmed or negated the sequences’ identity for eitherB. mallei orB. pseudomallei.


### 2.5. Pyrosequencing

Initial pyrosequencing PCR assays with the validation primers described above forB. mallei andB. pseudomalleiwere carried out in 50 μL reaction volumes. The amplicons were then used as template for the pyrosequencing assays using primers that nested within the amplicon sequence (Table 2 and Figure 1). One primer from each primer set carried a biotin tag at the 5′ end (Table 2). Different primer sets were used to examine if the target sequence could be produced consistently or if some primer locations were better than others. Finally, sepharose beads were used to capture the remaining biotinylated PCR product, after which the product-bead mixture was washed using a series of solutions that removed impurities and stripped the double-stranded DNA down to single-stranded DNA. This was done using the Pyromark vacuum prep worktable (Qiagen, Valencia, CA, USA). Specific pyrosequencing primers (Table 2) designed to target the regions of interest onB. mallei andB. pseudomallei were then annealed to the single-stranded DNA template before analysis on the Q96 ID Pyrosequencer (Qiagen). This procedure was performed as per the manufacturer’s instructions with each sample sequenced at least twice. The assay was conducted in the sequence analysis (SQA) mode and the pyrosequencing results were graded as pass or fail based on the test samples’ adherence to the predetermined qualifications as per the manufacturer’s directions. All strains were tested a minimum of two times to confirm results.


## 2.1. Bacterial Strains

We examined tenBurkholderia mallei, tenBurkholderia pseudomallei, and 40 other bacterial strains, including 15B. cepacia, 3B. thailandensis, and five variousBurkholderia species (Table 1). TheB. mallei,B. pseudomallei, and 2B. thailandensis were received from the NIH Biodefense and Emerging Infections Research Resources Repository (BEI Resources, Bethesda, MD, USA) as either live strains or purified DNA. Access to both isolates and DNA of bothB. mallei andB. pseudomalleiwere limited due to restrictions on how many Tier 1 isolates and DNA can be purchased yearly from BEI Resources. Of the 15B. cepacia isolates, one was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), three were collected from the Florida Department of Health, Bureau of Laboratories, Tampa, FL (FDOH) and 11 were procured from the University of Washington Medical Center in Seattle, WA. The other 23 bacterial strains including six variousBurkholderia species were part of our large bacterial collection and were either collected previously as clinical isolates from FDOH or received from ATCC (Table 1).Table 1
List of bacteria used in this study.
Organism
CBD numbera
Historical strain identifier
Burkholderia
Burkholderia mallei
BB 0044
China 5 (MM-A, NBL 4) ATCC 10399
Burkholderia mallei
BB 0045
Ivan (NCTC 10230) ATCC 15310
Burkholderia mallei
BB 0046
China 7 (NBL 7) ATCC 23344
Burkholderia mallei
BB 0050
GB8 horse 4
Burkholderia mallei
DD-675
BURK014
Burkholderia mallei
DD-671
BURK005 (SR092700A)
Burkholderia mallei
DD-372
BURK007 (SR092700C)
Burkholderia mallei
DD-673
BURK009 (SR092700E)
Burkholderia mallei
DD-677
BURK062 (Turkey 1)
Burkholderia mallei
DD-178
BURK010 (2344)
Burkholderia pseudomallei
BB 0047
China 3 (MP-H, NBL 104)
Burkholderia pseudomallei
BB 0048
S 397 (NRRL B-1112, CCEB 472)
Burkholderia pseudomallei
BB 0049
286 (MP-S, NBL 121)
Burkholderia pseudomallei
BB 0051
K96243
Burkholderia pseudomallei
BB 0052
1026b
Burkholderia pseudomallei
BB 0053
1106b
Burkholderia pseudomallei
BB 0054
1710a
Burkholderia pseudomallei
BURK088
BURK088
Burkholderia pseudomallei
BURK099
BURK099
Burkholderia pseudomallei
NR-9320
K96423
Burkholderia cepacia
CBD 1341
FL-M-05-0506072
Burkholderia cepacia
CBD 1342
FL-M-05-B24210
Burkholderia cepacia
CBD 1343
FL-M-05-B25995R
Burkholderia cepacia
CBD 1442
ATCC BAA-245
Burkholderia cepacia
CBD 1450
F68492
Burkholderia cepacia
CBD 1456
H27659
Burkholderia cepacia
CBD 1455
H35975
Burkholderia cepacia
CBD 1449
M25311
Burkholderia cepacia
CBD 1458
M27066
Burkholderia cepacia
CBD 1451
M42544
Burkholderia cepacia
CBD 1453
M52455
Burkholderia cepacia
CBD 1454
M74393
Burkholderia cepacia
CBD 1457
T10400
Burkholderia cepacia
CBD 1452
T33589
Burkholderia cepacia
CBD 1448
T47491
Burkholderia graminis
CBD 1440
ATCC 700544
Burkholderia multivorans
CBD 1443
ATCC BAA-247
Burkholderia stabilis
CBD 1441
ATCC BAA-67
Burkholderia vietnamiensis
CBD 1438
ATCC 55792
Burkholderia vietnamiensis
CBD 1444
ATCC BAA-248
Burkholderia thailandensis
CBD 1439
ATCC 700388
Burkholderia thailandensis
BURK254
E254
Burkholderia thailandensis
BURK43
MSMB043
Other bacteria
Achromobacter xylosoxidans
CBD 0307
ATCC 27061
Acinetobacter baumannii
CBD 1323b
ATCC 19606
Acinetobacter calcoaceticus
CBD 1336b
ATCC 23055
Bacillus badius
CBD 0091
ATCC 6462
Cedecea neteri
CBD 0309
ATCC 33855
Citrobacter freundii
CBD 0553b
ATCC 8090
Enterobacter cloacae
CBD 0556b
ATCC 13047
Enterococcus faecalis
CBD 1406
ATCC 49532
Enterococcus faecalis
CBD 1407
ATCC 49533
Klebsiella pneumoniae
CBD 0555b
ATCC 35657
Lactobacillus rhanmosus
CBD 1409
ATCC 53103
Pseudomonas aeruginosa
CBD 0551b
ATCC 15442
Serratia marcescens
CBD 1239
ATCC 13880
Staphylococcus aureus
CBD 0534
WA-HMC-03-4905
Staphylococcus aureus
CBD 1276
ATCC BAA-976
Stenotrophomonas maltophilia
CBD 0552b
ATCC 51331
Streptococcus pneumoniae
CBD 1405
ATCC 700669
aCBD: CBD stands for “Center for Biological Defense” and is the starting designation we use for bacterial strains that are in our BSL2 bacterial collection. Besides the CBD letter designation, each strain in our collection is also given a number to identify it. Each isolate in our BSL3 bacterial collection has a BB letter designation and then assigned a number as the BSL2 strains.
bdenotes an isolate carries a homolog of the fliP gene.All manipulations of cultures ofB. malleiandB. pseudomalleistrains were performed in a biological safety cabinet in a biological safety level 3 (BSL3) laboratory. All safety protocols followed “Biosafety in Microbiological and Biomedical Laboratories, 5th Edition” (BMBL) practices including the use of protective laboratory clothing and respiratory equipment [35]. The safety and security requirements by US federal regulation DHHS 42 CFR 73 were strictly adhered to. Manipulations of the other bacterial strains were performed in a biological safety level 2 (BSL2) laboratory in a biological safety cabinet following safety practices outlined in BMBL as above.


## 2.2. DNA Extraction

Bacteria were grown on tryptic soy agar supplemented with 5% sheep red blood cells (blood agar (BA)) (Remel, Lenexa, KS, USA). All culture plates were incubated at 35°C overnight (18–24 hours) before performing DNA extractions. Following manufacturers’ instructions, all genomic DNA extractions were either performed using the Epicentre (Qiagen Inc, Madison, WI) extraction kits, a MagNaPure Compact automated instrument (Roche, Inc, Indianapolis, IN, USA), or a boil preparation method used by FDOH. The boil preparation, (adapted from a reference lab in the Laboratory Response Network) is described as follows: bacterial growth from an overnight cultured media plate was removed and placed into 100μL of sterile water and boiled for 5 minutes and then placed onto ice for 2 minutes, followed by centrifugation at 12,000 ×g for 10 minutes at 4°C. The supernatant was transferred to a 0.1 μM filter tube (Millipore Corporation Billerica, MA) and centrifuged for 2 minutes at 8,000 ×g. To prove sterility of all filtrates and DNA extractions (following the University of South Florida Institutional Biosafety Committee guidelines), 10 μL was sacrificed and used to inoculate a BA media culture plate and incubated at 35°C for 48 hours. Extracts and lysates having no growth after two days were allowed out of the BSL3 environment and made available for molecular work. The DNA was stored at 4°C or −30°C until used. Before use, the DNA was diluted 1 : 20 in molecular-grade water (Fisher Scientific, Pittsburg, PA) to achieve a working concentration of 0.5 to 2 ng/μL.


## 2.3. Validation PCR Assays and Gel Electrophoresis

Based upon thefliP gene GenBank sequence BMA2686, PCR assays were designed to target sequences of thefliP gene inB. mallei(NCBI GenBank Accession #:NC_006348) andB.pseudomallei (NCBI GenBank Accession #:NC_009076). Figure 1 shows a schematic of the target regions in both species. All oligonucleotides used in the PCR assays forB. malleiandB pseudomallei are listed in Table 2. Primer-BLAST analysis (via NCBI) predicted 217 bp and 397 bp amplicons for thefliP—IS407A interface inB. malleiand thefliP gene inB psudomallei, respectively. The PCR reaction mixture with a final volume of 10 μL volume consisted of 1 μL of DNA template (at a concentration of 0.5–2 ng/μL), 1 μL of the Takara (Takara, Madison, WI, USA) 10X buffer containing 1.5 mM MgCl2, 0.82 μL of dNTPs at 25 μM, 0.05 μL of Takara HS Taq enzyme, and 2 μL of each specific primer at a concentration of 1 μM. Universal primers for the 16S rDNA sequence ofBurkholderia spp. previously described [20, 30] were later incorporated in the assays and produced a 1500 bp product to act as a control. The universal primers were IAC-16S-F (5′-AGAGTTTGATCCTGGCTCAG-3′) and IAC-rDNA-R (5′-ACGGCTACCTTGTTACGACTT-3′). All PCR reactions were carried out on a T1 Biometra Thermocycler (Biometra Inc., Göttingen, Germany) with the following conditions: initial denaturation at 95°C for 15 minutes, followed by 35 cycles of 94°C for 30 seconds, 58°C for 1 minute, and 72°C for 3 minutes, and lastly a final extension of 72°C for 10 minutes.Table 2
List of oligonucleotide primers used in pyrosequencing assays.
Organism
Target
Primer
Sequence (5′-3′)
Location (bp)a
B. mallei
fliP
BmfliP-F1b
ACGAACAGCGTGAGGAAGAG
2786291–2786310
IS407A
BmIS407A-R1
CTAGAAGCCCATTGGCCCTAT
2786443–2786423
Interface
Bm-S1
GGGGCAGGTCAACGA
2786417–2786403
Interface
Bm-S3
GGCAGGTCAACGAGC
2786415–2786401
B. mallei
fliP
BmfliP-F2b
CGAACAGCGTGAGGAAGAG
2786292–2786310
IS407A
BmIS407A-R1
CTAGAAGCCCATTGGCCCTAT
2786443–2786423
Interface
Bm-S3
GGCAGGTCAACGAGC
2786415–2786401
B. pseudomallei
fliP
BpfliP-F1
AGACGATGCTGCTGCTCAC
31112–31130
fliP
BpfliP-R1b
CCCGACGAGCACCTGATTC
31257–31239
fliP
BpfliP-R2b
GAACAGCGTGAGGAAGAGGG
31281–31262
fliP
BpfliP-S4
GCTGTCGTTCCTGCC
31134–31148
B. pseudomallei
fliP
BpfliP-F2b
GACGATGCTGCTGCTCAC
31113–31130
fliP
BpfliP-R3
AACAGCGTGAGGAAGAGGG
31280–31262
fliP
BpfliP-S5
AGCAGCGACAGCACG
31205–31191
Pyrosequencing primer sets forB. mallei and B. pseudomallei with respective target regions. Primers were designed using the design software supplied by the manufacturer.
adenotes location on the published sequences of either B. mallei GenBank accession number NC_006348 or B. pseudomallei GenBank accession number NC_009076.
bdenotes a biotinylated primer.
“Interface” denotes the sequence whereIS407A interrupts the fliP gene.
“F” denotes forward primer used for PCR.
“R” denotes reverse primer used for PCR.
“S” denotes the pyrosequencing primers.
The primers were first rehydrated in molecular grade water to bring them to a 100μM concentration for each primer. Forward and reverse primers were used at 1 μM concentration while the sequence primers were used at 100 μM.
The target sequence for the primer sets forB. mallei was: GCCTGCCGCAGCAGCGACAGCACGACGATGATCCGCGTGA, located at 2786360–2786399 bp in the sequence NCBI Genbank Accession #: NC_006348. The target sequence for the primer sets for B. pseudomallei was: GCGATGCTGCTGATGATGACGAGCTTCACGCGGATCATCA, located in the NCBI Genbank Accession #: NC_009076 at 31150–31189 bp.  PCR primer targets for both B. mallei and B. pseudomallei are all located on their respective chromosome 1. All primer coordinates were last verified on April 1, 2014.Figure 1
(a) Schematic ofBurkholderia mallei fliP gene withIS407A interruption that caused both gene inversion and loss of function. The large arrow within the block designates the orientation of the two parts of thefliP gene on the DNA strand. The location and direction of the various primers are noted by the small arrows. The pyrosequencing target inB. mallei is the interface between the insertion sequence element andfliP gene. (b) Schematic of theBurkholderia pseudomallei intact and functionalfliP gene. The large arrow within the block designates the orientation of the intact gene while the location and directions of the primers used are denoted by the small arrows. The pyrosequencing target inB. pseudomallei covers both sides of the point of thefliP gene that corresponds to where the insertion sequence is found inB. mallei.PCR amplicons were electrophoresed on a 1% agarose gel containing 15μg of ethidium bromide (final concentration of 0.2 μg/mL) in 1X TBE (44.5 mmol Tris-borate and 1 mmol EDTA, pH 8.3) for 60 min at 100 mV constant voltage. DNA bands were visualized with UV light and photographed using the GelDoc (Bio-Rad, Hercules, CA, USA).


## 2.4. Sanger Sequencing

The amplicons from the validation PCR and the initial pyrosequencing PCR reactions were purified using the Wizard DNA Purification Kit (Promega, Madison, WI, USA). Cycle sequencing was performed using the Beckman-Coulter Dye Terminator Cycle Sequencing (DTCS) Quick Start kit protocol (Beckman-Coulter, Fullerton, CA, USA). Cycle sequencing reactions with a final volume of 20μL consisted of 8 μL DTCS Quick Start master mix (Beckman-Coulter), 1.5 μL sequence primer at 25 pmol, and 8 μL of DNA. Cycle sequencing PCR was performed with the Biometra T3 Thermocycler (Biometra Inc.,) for 30 cycles under the following conditions: 96°C for 20 seconds, 50°C for 20 seconds, and 60°C for 4 minutes. The DNA produced from the cycle sequencing reaction was precipitated as per the Beckman-Coulter ethanol precipitation protocol. Finally, Sanger sequencing was performed on a Beckman-Coulter CEQ-8000 (Fullerton, CA) following the manufacturer’s directions. The resulting nucleotide sequences were compared to the public databases via NCBI nucleotide nBLAST alignment which confirmed or negated the sequences’ identity for eitherB. mallei orB. pseudomallei.


## 2.5. Pyrosequencing

Initial pyrosequencing PCR assays with the validation primers described above forB. mallei andB. pseudomalleiwere carried out in 50 μL reaction volumes. The amplicons were then used as template for the pyrosequencing assays using primers that nested within the amplicon sequence (Table 2 and Figure 1). One primer from each primer set carried a biotin tag at the 5′ end (Table 2). Different primer sets were used to examine if the target sequence could be produced consistently or if some primer locations were better than others. Finally, sepharose beads were used to capture the remaining biotinylated PCR product, after which the product-bead mixture was washed using a series of solutions that removed impurities and stripped the double-stranded DNA down to single-stranded DNA. This was done using the Pyromark vacuum prep worktable (Qiagen, Valencia, CA, USA). Specific pyrosequencing primers (Table 2) designed to target the regions of interest onB. mallei andB. pseudomallei were then annealed to the single-stranded DNA template before analysis on the Q96 ID Pyrosequencer (Qiagen). This procedure was performed as per the manufacturer’s instructions with each sample sequenced at least twice. The assay was conducted in the sequence analysis (SQA) mode and the pyrosequencing results were graded as pass or fail based on the test samples’ adherence to the predetermined qualifications as per the manufacturer’s directions. All strains were tested a minimum of two times to confirm results.


## 3. Results and Discussion

In the validation PCR assays, thefliP target regions ofB. mallei andB. pseudomallei were successfully amplified. ThefliP target gene region forB. mallei produced a 217 bp PCR product as predicted while the assay for theB. pseudomallei target region produced a 397 bp amplicon (data not shown). All tenB. mallei amplicons from the PCR assays when sequenced using the Sanger sequencing method had 100% nucleotide identity with the target sequence (B. mallei fliP gene and insertion sequence in GenBank accession NC_006348). When sequenced using the Sanger sequencing method, all ten amplicons produced from theB. pseudomallei DNA templates displayed 100% nucleotide identity with the published target gene (B. pseudomallei fliP gene sequence GenBank accession NC_009076).B. cepacia which possesses its own version of thefliP gene per published GenBank sequences have little to no nucleotide identity for eitherB. mallei orB. pseudomallei primers. As predicted none of theB. cepacia DNA templates nor the DNA from the six variousBurkholderia spp. produced amplicons when used in either of the two PCR assays. Additionally, the DNA templates from the remaining bacterial strains, including the seven bacterial collection strains which possess their own versions of thefliP genes, yielded negative results.When the DNA from the tenB. mallei strains were pyrosequenced withB. mallei pyrosequencing primers (Table 2) all ten tests were noted as “PASSED” and the resulting pyrograms were confirmed via NCBI nucleotide nBLAST alignment (Table 3). The NCBI nBLAST alignments confirmed that the pyrosequencing sequences matched with 100% nucleotide identity to publicly availablefliP sequences ofB. mallei. Different combinations of primer sequences (Table 2) were utilized to explore a larger target sequence and also resulted in 100% nucleotide identity no matter if the target was shifted slightly up or down stream (Figure 1). When the DNA from the tenB. mallei strains were pyrosequenced with theB. pseudomallei pyrosequencing primers (Table 2), all of the pyrograms were noted as “FAILED.” When the DNA from the tenB. pseudomallei strains were pyrosequenced using theB. pseudomallei sequencing primers (Table 2), all ten were noted as “PASSED” and when tested with theB. mallei pyrosequencing primers they were noted as “FAILED.” The resulting “PASSED” pyrograms were confirmed via NCBI nucleotide nBLAST and again matched with 100% nucleotide identity to publishedB. pseudomallei fliP sequences. The various primer sets that were designed and used (Table 2) generated sequences that shifted slightly up and down stream (Figure 1). The average pyrosequencing result obtained for theB. pseudomallei DNA templates was 44 nucleotides long (like theB. mallei results) and all had 100% nucleotide identity toB. pseudomallei GenBank sequences of thefliP gene and not forfliP gene sequences of otherBurkholderia species. Repeated testing gave the same results. As displayed in Table 3 the pyrosequencing results forB. mallei andB. pseudomallei show the highest percentage matches when applied to an NCBI BLAST search. Both assays demonstrated 100% sensitivity as they reliably and consistently identified the knownB. mallei orB pseudomallei DNA. This pyrosequencing method also demonstrated 100% specificity against the DNA samples of 15B. cepacia and six otherBurkholderia spp. All yielded negative PCR results and failed pyrosequencing readouts with both of theB. mallei andB. pseudomallei pyrosequencing primer sets (Table 4). All pyrosequencing nucleotide sequences will be submitted for entry into the NCBI databases.Table 3
Results of NCBI BLASTs of Sequences obtained by Pyrosequencing Assay.
Organism name
CBD Number
NCBI GenBank accession number
Burkholderia mallei
BB 0044
CP000548.1Burkholderia mallei
Burkholderia mallei
BB 0045
CP000548.1Burkholderia mallei
Burkholderia mallei
BB 0046
CP000548.1Burkholderia mallei
Burkholderia mallei
BB 0050
CP000548.1Burkholderia mallei
Burkholderia mallei
DD-675
CP000548.1Burkholderia mallei
Burkholderia mallei
DD-671
CP000548.1Burkholderia mallei
Burkholderia mallei
DD-372
CP000548.1Burkholderia mallei
Burkholderia mallei
DD-673
CP000548.1Burkholderia mallei
Burkholderia mallei
DD-677
CP000548.1Burkholderia mallei
Burkholderia mallei
DD-178
CP000548.1Burkholderia mallei
Burkholderia pseudomallei
BB 0047
CP006470.1Burkholderia pseudomallei
Burkholderia pseudomallei
BB 0048
CP006470.1Burkholderia pseudomallei
Burkholderia pseudomallei
BB 0049
CP003781.1Burkholderia pseudomallei
Burkholderia pseudomallei
BB 0051
CP006470.1Burkholderia pseudomallei
Burkholderia pseudomallei
BB 0052
CP003781.1Burkholderia pseudomallei
Burkholderia pseudomallei
BB 0053
CP006470.1Burkholderia pseudomallei
Burkholderia pseudomallei
BB 0054
CP006470.1Burkholderia pseudomallei
Burkholderia pseudomallei
BURK088
CP006470.1Burkholderia pseudomallei
Burkholderia pseudomallei
BURK099
CP006470.1Burkholderia pseudomallei
Burkholderia pseudomallei
NR-9320
CP006470.1Burkholderia pseudomallei
Outcome of pyrosequencing results compared against the NCBI database for alignment matches for eitherB. mallei or B. pseudomallei. Only the first match for each pyrosequence output is listed in the table. No sequences were obtained in any tests with B. cepacia or other bacterial strains and therefore no blast-searches were performed.Table 4
Results of PCR and pyrosequencing assays forB. mallei and B. pseudomallei.
Organism name (number)
B. mallei Assays
B. pseudomallei Assays
PCR
Pyrosequence
PCR
Pyrosequence
B. mallei (10)
+
PASS
−
FAIL
B. pseudomallei (10)
−
FAIL
+
PASS
B. cepacia (15)
−
FAIL
−
FAIL
Burkholderia species (8)
−
FAIL
−
FAIL
Other Bacteria (17)
Gram negative strains (10)
−
FAIL
−
FAIL
Gram positive strains (7)
−
FAIL
−
FAIL
“+” denotes a positive result or amplicon was produced.
“−” denotes a negative results.
“PASS” denotes the pyrosequencing assay produced a reliable sequence.
“Fail” denotes the pyrosequencing assay produced no reliable sequence or no sequence at all.Reliable detection and differentiation ofB. mallei andB. pseudomallei continue to be challenging because of the difficulty in developing a consistently sensitive, selective, and accurate assay [25]. Current molecular techniques such as ribotyping, and multilocus sequence typing are employed for detection and differentiation, but these methods can be labor intensive and challenging for laboratories and their usefulness is questionable in distinguishing differences betweenB. mallei andB. pseudomallei [19, 28]. Here we present a new method for detection and differentiation based on single gene target pyrosequencing. Pyrosequencing is alternative sequencing method, which provides real-time read-out, that is, highly suitable for sequencing short stretches of DNA [36]. Other key advantages of pyrosequencing include simple frequency data and the ability to generate sequence signals immediately downstream of the primer. Additionally, both sample preparation and single-strand DNA processing are relatively rapid [37]. Furthermore, pyrosequencing eliminates the need for labeled primers, dNTPs, or gel electrophoresis. We chose pyrosequencing as our target platform because it requires the least amount of sample manipulation, while still providing real-time read-out results that are highly suitable for sequencing short stretches of DNA (≤40 nucleotides). However, the target sequences and the assays described in this paper can be adapted to standard and real-time PCR in those laboratories that do not possess a pyrosequencer. In our experience pyrosequencing is cost-effective in testing samples when compared to qPCR, standard PCR, and dideoxynucleotide chain-terminating sequencing methods. Cost analysis was not in the scope of our paper but could be addressed by other researchers.Clinically, when infection is suspected and before any treatment options can be explored proper pathogen identification is essential. This ensures that both the proper treatment and safety protocols are followed to protect both patient and healthcare practitioner. We proposed an effective method of detection and positive identification ofB. mallei andB. pseudomallei based upon the overall genomic plasticity and multipleIS elements separatingB. malleifromB. pseudomallei [32, 38]. TheIS407A insertion that causes thefliP gene truncation, inversion, and subsequent loss of flagella functionality inB. malleiprovided the target for reliable identification of both species. Our pyrosequencing assays targeted a short nucleotide sequence that encompassed both the invertedfliP sequence as well as theIS407A sequence. As this sequence is unique toB. mallei it was our assertion that this sequence should provide 100% identification ofB. mallei. Examination of DNA from ten uniqueB. mallei isolates validated this claim. The fully functional, nontruncated, noninvertedfliP gene ofB. pseudomalleialso provided a reliable target for identification. We focused on the region of the gene where theIS407A insertion sequence apparently was introduced to create theB. mallei fliP/IS407A interface.TheBurkholderia species contain several genes includingfliP that contribute to the biosynthesis and function of the flagellar organelle [39]. While thefliP gene is present throughout the variousBurkholderia species, minor sequence differences enabled us to employ specific targeting.B. thailandensisis genetically and phenotypically the closest neighbor to bothB. mallei andB. pseudomallei[40]. However,B. thailandensis is avirulent towards humans and animals and therefore only three were examined in this study.B. cepacia, (a common opportunistic pathogen) is also genetically related toB. mallei andB. pseudomalleias well as familiar to clinicians and laboratorians in the United States. In addition,B. cepacia carries a version of thefliP gene and has been mistaken forB. pseudomallei[19]. Thus we added moreB. cepaciaisolates to our examination of theB. pseudomalleiassay.B. cepacia does not contain anyIS element in itsfliP gene and thus we predicted that none of the DNA from these isolates would generate positive results with theB. malleiprimers. The results validated our prediction. TheB. cepacia DNA also produced negative results in the assays with theB. pseudomallei primers even though theB. cepacia fliP gene is fully functional. Alignment comparison betweenB. pseudomallei (NCBI GenBank Accession #:NC_009076) andB. cepacia fliP gene resulted in up to 88% alignment matches (NCBI sequence alignment). Disruption of thefliP gene givesB. mallei its distinct nonmotile phenotype, while the differences betweenB. cepacia andB. pseudomallei fliP genes provide genotypic targeting while still retaining their motility phenotype. While theB. cepacia fliP is fully functional, the sensitivity of pyrosequencing enables the exploitation of even a 12% difference because the oligonucleotides used in our pyrosequencing assay had low homology with theB. cepacia fliP sequence. It is only through sequencing that one can definitively differentiate betweenB. pseudomalleiandB cepacia. Our detection assays can enable the laboratory to identify an isolate as eitherB. mallei or B. pseudomallei with the confidence of eliminating false positives withB. cepacia. If positive for either select agent, the laboratory can immediately send the isolate to a Laboratory Reference Network (LRN) laboratory where it can be further examined and the CDC contacted if necessary. AlthoughB. cepacia is not a focus of LRN laboratories, it is still a serious concern for the clinical laboratory and clinicians. Therefore a new pyrosequencing assay to reliably and consistently identifyB. cepaciais being explored by our laboratory.To date, we have not yet identified in the literature anyB. mallei strains that do not have theIS407A element disrupting thefliP gene. Historically, this interruption with the subsequent rearrangement of the gene partial sequences’ orientation and loss of genetic material has been one feature that distinguishes theB. malleispecies fromB. pseudomallei. Additionally, the element seems to be stable inB. mallei strains although it can theoretically move. Researchers have suggested thatB. malleievolved from a sole strain ofB. pseudomalleiand that the genome is “closed” and in “an evolutionary bottleneck in the mammalian host” that offered no opportunity for new genes [41]. It would be very interesting to see if in the future anyB. mallei isolates have lost theIS element adjacent to the partialfliP gene sequence. If and when that occurs new assays can be designed using the partialfliP sequence and whatever DNA sequence remains after the exit of theIS element.With new detection assays there is always a concern that since many are developed using pure culture they are susceptible to variable results when challenged with mixed cultures. Clinical samples are routinely cultured in nutrient rich media to identify pathogens and by custom and necessity to isolate each potential pathogen. Our study is designed for usage in clinical applications after the organism has been isolated from a clinical sample such as blood, skin, or deep tissue wound and the organism is not yet identified. So the fact that our assay was developed using pure cultures should have no negative impact on future applications. However, in the future, we plan to examine the possibility of direct detection straight from a clinical sample without using culturing and to test mixtures of bacteria, samples, and specimens such as swamp and irrigation waters, wounds, and sputum to explore if our assays can be utilized without the necessity of culturing to isolate the potential pathogen.Although PCR based assays are prized for their high sensitivity this can also be a potential drawback. One of the primary drawbacks of PCR assay is “carry-over” products resulting from subsequent PCR runs which then become contaminants producing false positives [42, 43]. Pyrosequencing requires no initial PCR amplification or subsequent post PCR manipulation. The target sample DNA can be utilized directly as the template. Here we performed PCR only as an experimental validation step in the beginning of the study and later switched to extracted genomic DNA as template. The ability of pyrosequencing to identify short nucleotide sequences gives our assays specificity vital to the successful identification and differentiation of the twoBurkholderia species. It is important to note that this assay is a starting point which will require further validation with the necessary cost analysis. In particular as bothB. mallei andB. pseudomallei are Tier 1 designees access to multiple strains of DNA is strictly limited. Thus we could not test more isolates as would be required by the CDC. However, we are confident that this work is a starting point that focuses on a single gene as a target and can be useful to other researchers whether in the clinical or basic research laboratory.



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*Source: 109583-2014-10-02.xml*