In this study, the subspecies differentiation of 25 isolates of Burkholderia mallei was attempted based on their ribotype polymorphisms. The isolates were from human and equine infections that occurred at various times around the world. DNA samples from each isolate were digested separately with PstI and EcoRI enzymes and probed with an Escherichia coli-derived 18-mer rDNA sequence to identify diagnostic fragments. Seventeen distinct ribotypes were identified from the combined data obtained with the two restriction enzymes. The results demonstrate the general utility of ribotyping for the subspecies identification of B. mallei isolates.
In recent years, the scientific basis for the identification of microorganisms has undergone a shift in emphasis from the traditional reliance on biochemical and microscopic identification of phenotypic characteristics to techniques based on nucleotide sequence heterogeneities [1, for review]. Some of these techniques have been used to distinguish strains at the subspecies level, and thereby provide a sound basis for the epidemiological tracking of the likely source of an outbreak. These approaches typically rely on some variation of a DNA “fingerprint”; a unique or diagnostic hybridization pattern arising from the amplification or probing of repetitive sequences occurring in polymorphic regions of the genome.
Ribotyping is one such fingerprinting approach. Bacterial ribosomal RNA (rRNA) operons comprise a family of highly conserved genes, each of which is flanked by regions of DNA with much greater variability than that encoding the rRNA operons themselves. Restriction fragment length polymorphisms (RFLPs) arising from sequence differences in the flanking restriction sites, or from insertions, deletions or recombinations within the rDNA-containing fragments, can be identified by probing restriction-digested, size-fractionated and immobilized DNA fragments with labeled homologous DNA sequences. An advantage of ribotyping is that it enables genetic analysis of an organism without prior knowledge of its genomic DNA sequence. In addition, it can be a sensitive means to identify genetic heterogeneity in a readily interpretable pattern.
In the present work, the subspecies discrimination of 25 isolates of Burkholderia mallei was approached through polymorphisms identified by ribotyping, using PstI and EcoRI restriction enzymes. Ribotyping was previously used by others [2–8] to characterize isolates of the related organism Burkholderia pseudomallei, the causative agent of melioidosis, which is a significant public health problem in Southeast Asia and Northern Australia. A total of at least 22 different ribotypes were described from B. pseudomallei.
There are no previously reported B. mallei DNA polymorphisms known to us. We believed the previous success with B. pseudomallei suggested the utility of ribotyping for subspecies discrimination of B. mallei.
B. mallei is a Gram-negative rod-shaped obligate parasite that causes Glanders primarily in equines, but also in humans. Cats, dogs and many other mammals can be infected under experimental conditions, while hamsters  and mice  are the most common laboratory models with which to study B. mallei. Mortality is very high, there is no vaccine, and a chronic form of the disease sometimes develops that can exacerbate into the acute form even after many years . Glanders has disappeared from most regions of the world, leaving only enzootic foci in Asia and eastern Mediterranean countries, and sporadic human cases among those whose occupations involve direct contact with infected equines or work with the organism in laboratories. The organism has received increased attention recently because it was designated by the US Centers for Disease Control and Prevention as a Category B Bioterrorism Agent, (http://www.scchealth.org/docs/doche/bt/cats.html). Also, it has been reported recently that German saboteurs maliciously injected B. mallei into animals during World War I [12,13]. Other published reports include the construction of B. mallei strains containing multiple antibiotic resistance genes , a study of the correlation of antibiotic resistance with infectivity  and its alleged intentional release in Afghanistan . These reports suggest the importance of developing a reliable means for the forensic discrimination of various isolates of the organism, which was the objective of this work.
2 Materials and methods
2.1 Sources and growth of bacteria
Table 1 summarizes the available information on the strains used in this study. At the time of publication, arrangements were being made for safe deposit of these isolates with the American Type Culture Collection (http://www.atcc.org).
Dr. David Miller at USDA-APHIS. Separate isolates received from Etlik Veterinary Institute, Ankara, Turkey by Dr. Linda Schlater in 1984.
CA Gleiser Army Med. School
Ft. Detrick, then CDC
NCTC 10245, GB11, 10399, China 5, 2002721275
Lung and nose of horse
Naval Biological Lab, then CDC
2002721277, Kweiyang #4
Gleiser Army Med. Serv. Grad. School, then CDC
Human; from cord blood, nose, throat
2000031304, 2000031281, H1533
Lab infection (human). Srinivasan et al., 2001 .
Col. V.C. Micra, then USDA-APHIS
Iowa State University
Abbreviations: USAMRIID, United States Army Medical Research Institute of Infectious Diseases; USDA-APHIS, United States Department of Agriculture Animal and Plant Inspection Service; CDC, United States Center for Disease Control and Prevention. Strains GB4–GB12 were received from Dr. Dave Waag at USAMRIID, strains Turkey 1 through ISU were received from Dr. David Miller at USDA-APHIS, strains 273 through 304 were received from Dr. Tanja Popovic at CDC. Abbreviations are listed as they were recorded when the isolates were received by us. All the available information was included above, although it is not complete and cannot necessarily be verified independently.
2.2 DNA isolation
Isolates were streaked on Luria Broth (LB) plates supplemented with 4% glycerol and grown at 37 °C for 1–2 days. Individual colonies were inoculated into 5 ml LB + 4% glycerol liquid medium and grown at 37 °C for 1–3 days. Suspended cells (5 ml) were centrifuged at 5000g for 15 min and the resulting pellet was vigorously resuspended and washed in 4 ml TS buffer (0.05 M NaCl, 0.02 M Tris, pH 8). Vigorous resuspension was apparently critical to obtain digestible DNA and was presumably related to the removal of the polysaccharide capsule. Some isolates did not yield a clear interface between the pellet and the supernatant, which was rectified by increasing the TS volume to 25 ml. Cells were centrifuged and the pellets resuspended as before in 4 ml TS buffer. Following another centrifugation, cells were resuspended in 0.6 ml saline, to which 1.2 ml sucrose-RNase-lysozyme solution was added (a stock solution contained 2.0 ml of 1 mg/ml boiled RNase, 44 mg lysozyme, 8.6 gm RNase-free sucrose, and 19.0 ml TES4 buffer [0.05 M each of NaCl, ethylenediamine tetraacetic acid, and Tris, pH 8]). This suspension was incubated at 37 °C for 15 min, then at 55–60 °C for 3 min. To this solution was added (with gentle swirling) 0.6 ml 3.5% Sarkosyl (Sigma) in TES4, followed by a 20-min incubation at 55–60 °C. Pronase (Sigma) solution was prepared at 9 mg/ml in TES4 buffer and incubated at 37 °C for 60 min (autodigestion). An aliquot (0.25 ml) of this solution was added to the lysate followed by an overnight incubation at 37 °C. Two phenol/chloroform extractions were performed by adding 1 ml water, 2.5 ml water-saturated phenol and 1.25 ml chloroform to the lysate, shaking gently and incubating on ice for at least 30 min prior to centrifugation at 5000g at 4 °C for 15 min. Following the second extraction, the aqueous layer was removed and extracted with 1.25 ml chloroform only. Following centrifugation of the chloroform extract, the top (aqueous) layer was removed and 1.5 volumes of ice-cold isopropanol were added. This mixture was inverted gently to precipitate the DNA. Precipitated genomic DNA was removed with a bent glass pipet, washed in ice-cold 100% ethanol, dried briefly, and dissolved in 1 ml of TE buffer (10 mM Tris and 1 mM EDTA, pH 8.0) in a sterile tube. DNA concentrations were estimated based on comparisons with known standards in an agarose gel electrophoresis.
2.3 DNA analysis
DNA was digested with restriction enzymes according to the enzyme suppliers' recommendations. Restriction-digested DNA and 32P-labeled DNA molecular weight standards were size fractionated through a 20 cm long, 0.6% (w/v) agarose gel in 40 mM tris-acetate and 1 mM EDTA (TAE) at 30–60 V for 16–32 h, according to the fragment sizes to be resolved and visualized by autoradiography. Southern transfer of gels to nylon membranes was performed according to Sambrook et al. . Molecular weight standards (1–12 kb ladder from Invitrogen, Carlsbad, CA) and 1.5–48.5 kb Lambda DNA mono cut mix from New England Biolabs (Beverly, MA) were labeled with [32P]ATP. The 1–12 kb ladder standards were labeled in a 20 µl reaction using 5–10 pmol of 5′ DNA ends, phosphorylation exchange buffer, and 5 µl of 3000 Ci/mmol, and 10 mCi/ml gamma [32P]ATP. Reactions were incubated at 37 °C for 30 min, inactivated by heating to 65 °C for 10 min, precipitated with 2.2 µl of 3 M sodium acetate and 2.5 volumes cold 100% ethanol, stored at +40 °C for 60 min, centrifuged, and washed twice with cold 70% ethanol, dried and resuspended in TE buffer. For the Lambda standards, the same labeling was used following phosphatase treatment with 5 units of Antarctic Phosphatase (New England Biolabs) per µg of DNA according to the enzyme manufacturer's recommendations.
For ribotyping experiments, genomic DNA was probed with a 32P-labeled 18-mer oligonucleotide. The oligomer was labeled using same method described above with the exception that the forward reaction buffer was substituted for the exchange buffer. The oligonucleotide sequence was derived from the Escherichia coli rDNA gene sequence gct cct agt acg aga gga . The same sequence had previously been used by us for detection of ribosomal RNA operons in many different species, and was chosen on this basis for its use in these experiments. Hybridization was conducted overnight at 37 °C in a solution containing 5×SSC (0.75 M NaCl and 0.075 M sodium citrate), 5×Denhardt's reagent and 0.5% (w/v) sodium dodecyl sulfate (SDS). Membranes were washed twice for 5 min each time in 2×SSC/0.1% (w/v) SDS at 50 °C. Autoradiography was performed using Kodak cassettes for 1–10 days using Kodak Biomax MR film exposed at +80 °C.
Autoradiography band assignment was performed visually by comparing results from several different gels run for varying times. The variation in run times was necessary, since shorter run times that retained all the fragments on the gel did not always yield sufficient separation, while longer runs would sometimes distort parts of the banding pattern and not be suitable for observation of the overall pattern. It was also sometimes observed that all band sizes did not always transfer with equal efficiency. The band size data in the tables are based on the information gleaned from all these gels, while the composite images shown illustrate the overall banding pattern but are not always the best example for the visualization of some bands from individual isolates. Minor bands were also sometimes observed, and some of these can be seen in figures. Typically, these minor bands would disappear at higher wash temperatures or with cleaner restriction digests.
Six restriction enzymes were initially tested for their ability to produce RFLPs from B. mallei DNA. Digested DNA from nine isolates (GB3–GB10 and GB12) was probed with a labeled 18-mer probe derived from E. coli rDNA. Results confirmed that the labeled oligonucleotide derived from E. coli bound to at least 2–3 distinct bands of the B. mallei DNA, depending on the isolate. The restriction enzymes BamH1, ClaI, HindIII and SmaI yielded few observable polymorphisms, whereas EcoR1 and Pst1 single-enzyme digests yielded highly polymorphic patterns. Consequently, EcoR1 and Pst1 were selected for further study with the complete panel of isolates. DNA was size-fractionated on agarose gels and hybridized fragments were sized by comparison with 32P-labeled commercial molecular weight standards in gels run for various times depending upon the size of the fragments to be resolved (typically 16–30 h at 60 V in a 20-cm gel). Southern-transferred DNA was probed with the rDNA oligomer, which yielded a polymorphic pattern of either 2 or 3 hybridized bands per isolate. Thirteen different EcoRI ribotypes were identified through the application of this method and were designated E-1 through E-13. Fig. 1 shows the EcoR1 ribotype patterns of all 25 isolates (image is a composite of five different exposures). Hybridization with the rDNA oligomer was repeated with PstI-digested and fractionated DNA, and 12 different PstI ribotypes were identified (designated P-1 through P-12). Fig. 2 shows the Pst1 ribotype patterns of all 25 isolates (image is a composite of two different exposures). Table 2 summarizes the observed band sizes of all the hybridized fragments with both restriction enzymes, as determined from electrophoretic size fractionations of various durations.
Autoradiogram of rDNA probing of EcoRI digests of B. mallei DNA. Lane 1: 1 kb markers, lane 2: GB3, lane 3: GB4, lane 4: GB5, lane 5: GB6, lane 6: GB7, lane 7: GB8, lane 8: GB9, lane 9: λ markers, lane 10: GB10, lane 11: GB12, lane 12: T2, lane 13: T4, lane 14: T6, lane 15: 1 kb markers, lane 16: T7, lane 17: T9, lane 18: 273, lane 19: 274, lane 20: 275, lane 21: 276, lane 22: λ markers, lane 23: 277, lane 24: 278, lane 25: 279, lane 26: 304, lane 27: 503, lane 28: 567, lane 29: ISU.
Autoradiogram of rDNA probing of PstI digests of B. mallei DNA. Lane 1: GB3, lane 2: GB4, lane 3: GB5, lane 4: GB6, lane 5: GB7, lane 6: GB8, lane 7: 1 kb markers, lane 8: GB9, lane 9: GB10, lane 10: GB12, lane 11: T2, lane 12: T4, lane 13: T6, lane 14: T7, lane 15: 1 kb markers, lane 16: T9, lane 17: 273, lane 18: 274, lane 19: 275, lane 20: 276, lane 21: 277, lane 22: λ markers, lane 23: 278, lane 24: 279, lane 25: 304, lane 26: 503, lane 27: 567, lane 28: ISU.
Ribotypes and corresponding autoradiography bands from EcoR1 and Pst1 digests
Isolates of this ribotype
9.2, 8.6, 8.1
10.1, 9.2, 8.4
9.9, 8.6, 8.1
11.2, 10.2, 9.0
10.1, 9.8, 8.1
11.2, 10.2, 7.5
10.0, 9.2, 8.5
GB8, 273, 274, 304
12.0, 8.8, 8.4
10.1, 9.8, 8.1
15.0, 13.0, 8.8
11.4, 10.1, 10.0
12.0, 8.8, 6.6
T2, T4, T6, T7
10.1, 8.8, 8.4
10.1, 9.8, 8.1
9.5, 8.8, 8.4
11.4, 9.8, 8.1
21.0, 13.0, 8.8
10.0, 9.8, 8.4
12.0, 8.8, 6.6
10.9, 10.1, 8.4
11.2, 10.2, 8.8
10.1, 9.8, 8.1
11.2, 10.2, 8.8
10.1, 9.8, 8.1
Together, the digests with EcoRI and PstI enzymes yielded a total of 17 distinct ribotypes from 25 isolates (Table 2). Ribotypes 5 and 9 contained four isolates each; groups 3 and 8 contained two isolates each, while the remaining groups contained only a single isolate.
Godoy et al.  conducted Multi Locus Sequence Typing (MLST) on five isolates of B. mallei originally obtained from the National Collection of Type Cultures in London, England. No MLST-based sequence variation was observed among any of the B. mallei strains examined in that study and the authors concluded that the B. mallei isolates represented a clone within the B. pseudomallei species. Those B. mallei isolates are all included in this study and are designated here as GB4–GB6, GB10 and 275. They comprise four distinct ribotypes (1, 3, 7, and 8 in Table 2). All were distinguished from each other except GB5 and GB6, which together comprised Ribotype 3. The polymorphisms revealed through ribotyping and presented here do not specifically address the relationship of B. mallei to other closely-related organisms, though they do provide an effective forensic means by which most of these B. mallei isolates can be distinguished from each other.
Several other interesting observations emerge from the ribotyping results. The first involves the relationship between GB8 and 304. Isolate 304 was obtained from a laboratory worker who became infected with GB8 [20, and personal communication from Dr. Dave Waag]. The genotypic identity of GB8 and 304 and the clear discrimination of 304 from most of the rest of the isolates in this study serves as a general illustration of the utility of this genotyping scheme for the discrimination of potential sources of an infection or outbreak involving B. mallei.
On the other hand, although the relationship between GB8 and 304 is clear, it is not apparent from available information whether or not the genotypically indistinguishable isolates 273 and 274, have any relationship with GB8 or 304 (although 273 and 274 were themselves collected in the same country in the same year). There are also no documented historical parallels between the genotypically indistinguishable isolates GB12 and 275, or GB5 and GB6.
Interestingly, four isolates collected in Turkey (T2, T4, T6 and T7) are all Ribotype 9. Isolate T9, collected in the same country, has a different ribotype (with the PstI enzyme only). The similarity observed among four of the Turkey isolates is suggestive of a common origin of these isolates.
These data suggest a practical genotyping approach such as that illustrated in Fig. 3. Isolates would first be digested with EcoR1 and probed to determine their EcoR1 grouping. Isolates not adequately discriminated by their EcoR1 grouping would subsequently be digested with Pst1 to determine if they should be assigned the same or separate ribotype(s).
Hierarchical scheme for ribotype discrimination of B. mallei isolates. Isolates with indistinguishable EcoR1 groupings or indistinguishable ribotypes (Rt) are contained within the same box.
The same criteria used for discrimination of the 25 isolates from this study should be similarly useful for genotyping future isolates. If applied to more isolates and combined with more complete historical information, ribotyping may also elucidate the relationships among B. mallei strains with respect to geography and species. At a minimum, ribotyping is clearly a useful tool for the forensic discrimination of B. mallei isolates that might be encountered in an outbreak.
This work was funded by the US Army. We are grateful to Dr. David Waag (USAMRIID), Dr. David Miller (USDA-APHIS) and Dr. Tanja Popovic (CDC) for the Burkholderia mallei strains they provided to us for analysis, and to Dr. Diane Dutt and Dr. Vipin Rastogi for critical review of this manuscript.
(1999) Biological sabotage in World War I. In: Biological and Toxin Weapons: Research, Development and Use from the Middle Ages to 1945 (GeisslerE., MoonJ.E.v.C., Eds.), pp. 35–62. Oxford University Press, Oxford.
(2003) Multilocus sequence typing and evolutionary relationships among the causative agents of melioidosis and glanders, Burkholderia pseudomallei and Burkholderia mallei. J. Clin. Microbiol. 41 (5), 2068–2079.