JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1996, p. 609–614 0095-1137/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 34, No. 3
Detection of Burkholderia pseudomallei DNA in Patients with Septicemic Melioidosis TARARAJ DHARAKUL,1* SIRIRURG SONGSIVILAI,1 SIRITHIP VIRIYACHITRA,1 VORAVICH LUANGWEDCHAKARN,1 BOONRAT TASSANEETRITAP,1 2 AND WIPADA CHAOWAGUL Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok,1 and Sappasitprasong Hospital, Ubonratchathani,2 Thailand Received 21 August 1995/Returned for modification 19 October 1995/Accepted 7 December 1995
Septicemic melioidosis is the most severe form of melioidosis, which is caused by Burkholderia pseudomallei. It is endemic in Southeast Asia and is the leading cause of death from community-acquired septicemia in northeast Thailand. A major factor that contributes to the high mortality is the delay in isolation and identification of the causative organism. More than half of the patients die within the first 2 days after hospital admission, before bacterial cultures become positive. The present study was undertaken to develop a rapid diagnostic method for identification of this organism. A nested PCR system that amplified a part of 16S rRNA gene that was highly specific to B. pseudomallei was developed. This system was able to detect as few as two bacteria present in the PCR. DNAs from all 30 clinical isolates of B. pseudomallei and none of the other bacteria tested were amplified. The described PCR system has been employed for the detection of the organism in clinical specimens, including buffy coat and pus from internal organs. The detection of B. pseudomallei in buffy coat specimens by PCR was shown to be comparable to the detection of bacteria from blood cultures in septicemic melioidosis cases. Melioidosis is an important infectious disease known to be endemic in Southeast Asia and the northern part of Australia. The causative agent, Burkholderia pseudomallei, is one of the most important causes of fatalities from community-acquired septicemia in northeastern Thailand (3). The highest mortality occurs in patients with the septicemic form of melioidosis, which is characterized by dissemination of the bacteria in the circulation and isolation of the bacteria from the blood and from various organs. Patients with septicemic melioidosis often deteriorate rapidly, and death often occurs within the first few days after hospitalization. Rapid diagnosis and prompt treatment with appropriate antibiotics can reduce the mortality by at least half (11). Current clinical practice in areas where melioidosis is endemic requires a combination of a high level of clinical suspicion and bacterial isolation by culture. This procedure, which remains the ‘‘gold standard’’ method for the laboratory diagnosis of melioidosis, generally takes 3 to 7 days, which means that in many cases the results are obtained after the patients have died. Therefore, improvement in the rapid laboratory diagnosis of melioidosis is urgently needed. Various methods, including those involving antibody and antigen detection as well as DNA detection, are currently being investigated (2, 5, 8). The present study employed a sensitive PCR amplification technique for detecting B. pseudomallei DNA in clinical specimens, especially buffy coat specimens, of acute melioidosis patients. This PCR system can specifically amplify B. pseudomallei DNA but did not amplify DNAs from any other bacteria tested. The assay was also sensitive and able to detect specific DNA from septicemic melioidosis patients while yielding negative results from patients not having melioidosis.
MATERIALS AND METHODS Bacterial strains. Thirty clinical isolates of B. pseudomallei were used in this study, of which 19 were obtained from melioidosis patients from Siriraj Hospital, Bangkok, Thailand. The other 11 isolates were obtained from two major hospitals in the northeastern part of Thailand; 4 were from Sappasitprasong Hospital, Ubonratchathani, and the other 7 were from Khonkaen Hospital, Khonkaen. Of these 30 isolates, 14, 4, 3, 7, and 2 were obtained from blood, lung, liver, soft tissue abscess, and urine, respectively. In addition, strains of Burkholderia cepacia, Pseudomonas putida, Pseudomonas aeruginosa, Haemophilus influenzae, Escherichia coli (ATCC 25922), Acinetobacter anitratus, Klebsiella pneumoniae, Enterobactor aerogenes, a Proteus sp., Serratia marcescens, Aeromonas hydrophila, Staphylococcus aureus, group A and group B streptococci, and group D enterococci were provided by the Department of Microbiology, Faculty of Medicine, Siriraj Hospital. Pseudomonas fluorescens type strain DMS2589 and Xanthomonas maltophilia type strain DMS904 were provided by the Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand. Collection and preparation of clinical specimens. Twenty-nine patients admitted to Sappasitprasong Hospital with suspected melioidosis were enrolled in this study. Twenty milliliters of blood was collected on the first day of admission. Ten milliliters of blood was injected into a blood culture tube. Buffy coat and plasma specimens were prepared from another 10 ml of heparinized blood. After centrifugation at 800 3 g for 10 min, the buffy coat was collected from the interface and pelleted by centrifugation at 6,000 3 g for 3 min. The buffy coat specimens were frozen at 2208C until use. In addition, liver pus was obtained after diagnostic percutaneous aspiration of a suspected case. Sputum was collected from two patients with suspected pulmonary melioidosis, by standard sputum collection procedures. The diagnosis of melioidosis was confirmed by isolation of B. pseudomallei from clinical specimens. Septicemic melioidosis was defined by positive isolation of the bacteria from blood cultures. Nineteen of the 29 suspected cases were bacteriologically confirmed melioidosis, of which 11 cases were septicemic melioidosis. The other eight cases were localized melioidosis. Primer design. Two sets of PCR amplification primers were designed from the variable region of the 16S rRNA gene of B. pseudomallei. Primers for secondround PCR were homologous or complementary to nucleotide sequences of B. pseudomallei and Burkholderia mallei (which have identical nucleotide sequences in this region) (12) but not to the sequences of other related species deposited in the GenBank database (National Center for Biotechnology Information release 88.0), including Burkholderia cepacia, Burkholderia gladioli, Burkholderia pickettii, and Burkholderia solanacearum, or to those of P. aeruginosa, X. maltophilia, and E. coli (Fig. 1). The amplified product from the first round of amplification is 717 bp in length, whereas the product from the second (nested) round is 397 bp in length. The nucleotide sequences of primers are shown in Table 1. Preparation of bacterial DNA for PCR. Bacterial DNAs were prepared by using a modified proteinase K digestion technique (9). Briefly, bacterial DNA (from 104 viable bacteria) was extracted in a total volume of 200 ml containing 10
* Corresponding author. Mailing address: Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, 2 Prannok Rd., Bangkok 10700, Thailand. Phone: 66-2-4197066. Fax: 66-24181636. Electronic mail address:
[email protected]. 609
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FIG. 1. Specificity of amplification primers. A comparison of nucleotide sequences, in the region corresponding to primers BS3L and BS4R, of the 16S rRNA genes of B. pseudomallei, B. mallei, B. cepacia, B. pickettii, B. gladioli, B. solanacearum, P. aeruginosa, X. maltophilia, and E. coli is shown. The sequences at the primer sites are underlined.
mM Tris-HCl (pH 7.8), 5 mM EDTA, 0.5% sodium dodecyl sulfate, 0.5% Tween 20, and 0.2 mg of proteinase K per ml. The tube was incubated at 568C for 2 h, and proteinase K was inactivated by heat denaturation at 958C for 10 min. Twenty microliters of the reaction mixture was then taken for PCR amplification. Preparation of DNA from clinical specimens. Clinical specimens, including buffy coat or pus (20 ml) or plasma (100 ml), were resuspended in 100 ml of TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA) and subjected to heat treatment at 1008C for 15 min. Cell debris was pelleted by centrifugation, and 20 ml of the supernatant was taken for DNA extraction by using the protocol described for DNA extraction from bacteria. PCR amplification. First-round PCR amplifications were carried out in a total volume of 100 ml containing 20 ml of DNA extract, 10 ml of 103 amplification buffer (containing 500 mM KCl, 100 mM Tris-HCl [pH 8.3], 15 mM MgCl2, and 0.01% [wt/vol] gelatin), 8 ml of deoxynucleoside triphosphates (2.5 mM each), 20
TABLE 1. Sequences of oligonucleotide primers for amplifying the B. pseudomallei 16S rRNA gene Primer
Outer U33 OL731 Inner BS3L BS4R a
Positionsa
Strand
Sequence (59339)
33–52 749–731
Sense Antisense
AAGTCGAACGGCAGCACGG TTTGCTCCCCACGCTTTCG
48–64 444–426
Sense Antisense
ACGGGCTTCGGCTGGTG CACTCCGGGTATTAGCCAG
According to reference 12.
FIG. 2. Ethidium bromide-stained agarose gel electrophoresis of the amplified 16S rRNA gene of B. pseudomallei from nine different clinical isolates (lanes 1 to 9). DNAs from all 30 isolates of B. pseudomallei DNA used in this study can be amplified.
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to the manufacturer’s protocol. Sequencing was carried out in both directions. The nucleotide sequences obtained were analyzed by using MacVecter software (International Biotechnologies, Inc.), and were compared with the sequences deposited in GenBank database release 88.0 (National Center for Biotechnology Information). Nucleotide sequence accession number. The partial sequence data for the 16S rRNA genes of four isolates of B. pseudomallei have been deposited in GenBank under accession numbers U29876 to U29879.
RESULTS
FIG. 3. Sensitivity of PCR amplification. Nested PCR for detection of the B. pseudomallei 16S rRNA gene was carried out with known amounts of bacteria as indicated.
pmol of each outer primer, and 2.5 U of Taq DNA polymerase (Perkin-Elmer). The tube was subjected to thermal cycling for 35 cycles, each consisting of 958C for 1 min (denaturation), 608C for 1 min (annealing), and 728C for 1 min (extension), and then subjected to final extension step for 10 min at 728C, in a DNA Thermal Cycler 480 (Perkin-Elmer). One-tenth of the product from the first round was taken to a new tube for the second-round PCR (nested PCR) with the inner primers in a 50-ml reaction mixture and subjected to PCR amplification for another 35 cycles with the same cycle profile. A pure culture of B. pseudomallei (103 bacteria per reaction) was used as the positive control and was included in the sample preparation step. TE buffer was used as a negative control in each of the three steps of the amplification process, including the sample preparation, first PCR, and nested PCR steps. The presence of amplified B. pseudomallei DNA product was analyzed on a 2% agarose gel stained with ethidium bromide and visualized under UV light. Strict laboratory precautions were carefully used to avoid contamination (6). The amplified products were verified by nucleotide sequence analysis. Each assay was carried out at least twice. The sensitivity of PCR detection was analyzed by using bacterial samples of known quantity (determined as CFU). Nucleotide sequencing. Nucleotide sequencing of the PCR-amplified products was performed in an automated nucleotide sequencer (Applied Biosystems model 373), using the PRISM dye terminator system (Perkin-Elmer), according
Detection of B. pseudomallei DNA by PCR. In the presence of B. pseudomallei DNA in the specimens, a band of 397-bp PCR-amplified products can be visualized in an ethidium bromide-stained agarose gel. In some cases with large amounts of bacterial DNA, three additional faint bands of 717, 702, and 412 bp, which are combined products of both rounds of PCR, can also be visualized. The fragments were confirmed to be parts of the 16S rRNA gene of B. pseudomallei by nucleotide sequencing (see below). These sets of primers can amplify DNAs from all clinical isolates (a total of 30 clinical isolates) of B. pseudomallei used in this study (Fig. 2). Sensitivity of nested PCR. The sensitivity of the nested PCR was determined by serial dilution of bacterial samples prior to DNA extraction. Diluted bacterial samples containing as few as two bacteria (equivalent to 15 fg of bacterial DNA) can be consistently amplified by nested PCR (Fig. 3). The sensitivity of the nested PCR was approximately 100-fold better than that of single-round amplification (data not shown). Specificity of nested PCR. PCR amplification was carried out with DNAs from panels of gram-negative and gram-positive bacteria, including B. cepacia, P. putida, P. aeruginosa, P. fluorescenes, X. maltophilia, E. coli, A. anitratus, K. pneumoniae, E. aerogenes, a Proteus sp., A. hydrophila, S. marcescens, H. influenzae, S. aureus, group A and group B streptococci, and group D enterococci. No amplified products were detectable, except in the cases of B. cepacia and X. maltophilia, for which a band of 717 bp can be seen. This DNA band is a result of the first-round amplification in which the outer set of primers can also amplify the B. cepacia and X. maltophilia 16S rRNA genes (confirmed by nucleotide sequencing [data not shown]). How-
FIG. 4. PCR amplification of DNAs from panels of gram-negative and gram-positive bacteria with primers specific to the 16S rRNA gene of B. pseudomallei. gr., group.
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FIG. 5. Alignment of nucleotide sequences of parts of the 16S rRNA genes of four isolates of B. pseudomallei (isolates U29876 to U29879) compared with published sequences for B. mallei, B. cepacia, B. gladioli, B. solanacearum, P. aeruginosa, X. maltophilia, and E. coli.
ever, this band can easily be distinguished by size from that of B. pseudomallei (Fig. 4). Nucleotide sequences. Nucleotide sequences were obtained from PCR-amplified products of four clinical isolates of B. pseudomallei. These sequences were identical to each other and almost identical to the nucleotide sequence of the gene encoding the 16S rRNA of B. mallei (Fig. 5). Yabuuchi et al. (12) reported that the nucleotide sequences of the genes encoding the 16S rRNAs of B. mallei and B. pseudomallei were identical. It should be noted that there is a discrepancy between the sequences obtained and the published sequence at position 172 (i.e., G in all four sequences obtained in this study and C in the sequences of B. mallei available from the GenBank database).
Detection of B. pseudomallei DNA in buffy coat specimens. This PCR system consistently amplified B. pseudomallei DNA from buffy coat, pus, and sputum specimens, but not from plasma specimens, from melioidosis patients (Fig. 6). The results of PCR amplifications and bacterial cultures are summarized in Table 2. Buffy coat and plasma specimens from 29 clinically suspected cases of melioidosis were tested; 19 of these cases were later confirmed to be melioidosis by bacterial cultures. Eleven of these 19 patients had blood cultures positive for B. pseudomallei during hospitalization. Of these 11 cases, 8 were positive for both PCR and blood cultures from the same specimens. One patient had a positive blood culture but was PCR negative. The other two patients had positive blood cultures from specimens taken at other times only and
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were also PCR negative (Table 2). Interestingly, one of eight patients with localized melioidosis had a positive PCR result from a buffy coat specimen, while the blood culture was negative. All 29 plasma samples were negative by PCR. B. pseudomallei DNA was also detected in liver pus from a patient with localized melioidosis and in the sputum of a patient with pulmonary melioidosis (Fig. 6). All of the buffy coat and plasma specimens from 10 patients who were culture negative for B. pseudomallei were PCR negative. DISCUSSION
FIG. 6. PCR amplification from clinical specimens. Lanes 2 to 9 are labeled with patient numbers (see Table 2).
Septicemic melioidosis represents the most severe clinical aspect of this disease. More than half of the mortality occurs within the first 48 h of admission (3). A delay in diagnosis is one of the important factors contributing to death (4). Standard bacterial culture requires approximately 3 to 5 days for the identification of the causative organism (7). Improved rapid laboratory diagnosis of B. pseudomallei would be extremely beneficial in cases of severe melioidosis. The detection of antibody to B. pseudomallei is of limited use in areas where B. pseudomallei is endemic, since the majority of the population have already been exposed to this organism. Detection of the rRNA gene by PCR has several advantages in terms of both sensitivity and specificity because of the multiple copies of
TABLE 2. Summary of clinical data for patients used in this studya Condition and patient
Septicemic melioidosis 1 2 3 4 5 6 7 8 9 10 11 Localized melioidosis 12 13 14 15 16 17 18 19 20 Nonmelioidosis C01 C02 C03 C04 C05 C06 C07 C08 C09 C10
Diagnosis
Septicemic Septicemic Septicemic Septicemic Septicemic Septicemic Septicemic Septicemic Septicemic Septicemic Septicemic
melioidosis melioidosis melioidosis melioidosis melioidosis melioidosis melioidosis melioidosis melioidosis melioidosis melioidosis
Blood culture result
PCR result for B. pseudomallei from buffy coat
Other bacterial culture(s) positive for B. pseudomallei
B. pseudomallei B. pseudomallei B. pseudomallei B. pseudomallei B. pseudomallei B. pseudomallei B. pseudomallei B. pseudomallei B. pseudomallei NGb NG
1 1 1 1 1 1 1 1 2 2 2
Sputum, cerebrospinal fluid Liver pus, urine Sputum Throat swab, urine Foot pus Tracheal aspirate, urine Sputum, throat swab Sputum, urine Leg pus Other blood culture Other blood culture, liver pus
Pulmonary melioidosis Pulmonary melioidosis Pulmonary melioidosis Pulmonary melioidosis Pulmonary melioidosis Melioidosis of urinary tract Melioid liver abscess Melioid splenic abscess Melioid liver abscess
NG NG NG NG NG NG NG NG NG
Pneumonia Pneumonia Pneumonia Pneumonia Liver abscess Amebic liver abscess Splenic abscess E. coli septicemia AIDS Diabetes mellitus with chronic renal failure
NG NG NG NG NG NG NG E. coli NG NG
1 2 2 2 2 2 2 2 NDc
Sputum Sputum Pleural fluid Sputum Sputum Urine Liver pus Splenic pus Liver pus
2 2 2 2 2 2 2 2 2 2
a Blood cultures and PCR amplification were carried out with the same blood specimens. Results of bacterial cultures for these patients who had B. pseudomallei recovered from other sites, as well as results of blood cultures taken at different times, are also shown. b NG, no growth. c ND, not done.
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targeted DNA sequences and the conservation of sequences in the same species (1). In the present study, a PCR amplification method was developed for the detection of B. pseudomallei DNA in clinical specimens. Leukocytes from the buffy coat were used primarily as the main source of specimen because of the superiority of buffy coat to plasma or serum for the detection of B. pseudomallei DNA. Nested PCR was used initially because of its high sensitivity and less complicated procedure compared with one-step PCR and hybridization, which are not practical for rapid diagnosis in laboratories in areas of endemicity. This nested PCR assay is at least 100-fold more sensitive than the previously reported detection of the 23S rRNA gene of B. pseudomallei by PCR and hybridization (8) and has been proven useful in clinical situations. False-positive results can be avoided by careful laboratory precautions (6). The nested PCR method developed was shown to be highly sensitive, being able to detect as few as two organisms present in the reaction. This PCR system was specific to B. pseudomallei; it amplified DNAs from all 30 clinical isolates from various parts of Thailand and did not amplify DNAs from any of the other bacteria tested. After first-round amplification with the outer pair of primers, in the presence of a large amount of bacterial DNA, only the rRNA genes of B. pseudomallei, B. cepacia, and X. maltophilia were amplified (a band of 717 bp). However, after the second round of PCR with the inner primer pair, only B. pseudomallei DNA was amplified (a unique band of 397 bp). B. mallei was not available for this study; however, it has been documented that B. mallei is genetically identical to B. pseudomallei at the level of the nucleotide sequences of the 16S rRNA genes (12), and therefore this PCR protocol should amplify B. mallei DNA as well. However, this is not considered a problem in clinical situations, since glanders caused by B. mallei is a disease of animals and is epidemiologically different from melioidosis. Only sporadic cases of glanders occur in animals in Asia, Africa, and South America (10). This PCR amplification system has been proven useful in clinical situations, in which the PCR results were obtained before bacterial cultures became positive. With the developed PCR system, eight of nine buffy coat specimens from blood culture-positive patients with septicemic melioidosis were PCR positive. In addition, one of eight blood culture-negative patients with localized melioidosis also had B. pseudomallei in the buffy coat which was detectable by PCR. Therefore, the PCR system developed appears to be almost as sensitive as the standard bacterial culture system for the detection of B. pseudomallei. PCR amplification can be performed directly from clinical samples which may contain small numbers of bacteria. This
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technique has proven valuable in identifying bacterial pathogens that have resisted detection and identification by traditional microbiological methods (1). The PCR method described here allows identification of B. pseudomallei directly from buffy coat, sputum, and pus from internal organs and probably from other types of clinical specimens. The test could be completed within one work day. Therefore, this procedure would be a valuable laboratory diagnostic tool in medical centers that have facilities for PCR. ACKNOWLEDGMENTS This work was supported by grants from the National Science and Technology Development Agency of Thailand and the Anandhamahidol Foundation to T. Dharakul and S. Songsivilai. We acknowledge the Department of Microbiology, Faculty of Medicine, Siriraj Hospital, the Microbiology Laboratory of Khonkaen Hospital, and the Department of Medical Sciences, Ministry of Public Health, Thailand, for providing bacterial strains. REFERENCES 1. Anderson, B. 1994. Broad range polymerase chain reaction for detection and identification of bacteria. J. Fla. Med. Assoc. 81:835–837. 2. Anuntagool, N., P. Rugdech, and S. Sirisinha. 1993. Identification of specific antigens of Pseudomonas pseudomallei and evaluation of their efficacies for diagnosis of melioidosis. J. Clin. Microbiol. 31:1232–1236. 3. Chaowagul, W., N. J. White, D. A. B. Dance, Y. Wattanagoon, P. Naigowit, T. M. E. Davis, S. Looareesuwan, and N. Pitakwatchara. 1989. Melioidosis: a major cause of community-acquired septicemia in Northeastern Thailand. J. Infect. Dis. 159:890–899. 4. Dance, D. A. B. 1991. Melioidosis: the tips of the iceberg? Clin. Microbiol. Rev. 4:52–60. 5. Desakorn, V., M. D. Smith, V. Wuthiekanun, D. A. B. Dance, H. Aucken, P. Suntharasamai, A. Rajchanuwong, and N. J. White. 1994. Detection of Pseudomonas pseudomallei antigen in urine for the diagnosis of melioidosis. Am. J. Trop. Med. Hyg. 51:627–633. 6. Kitchin, P. A., Z. Szotyori, C. Fromholc, and N. Almond. 1990. Avoidances of false positives. Nature (London) 344:201. 7. Leelarasamee, A., and S. Bovornkitti. 1989. Melioidosis: review and update. Rev. Infect. Dis. 11:413–425. 8. Lew, A. E., and P. M. Desmarchelier. 1994. Detection of Pseudomonas pseudomallei by PCR and hybridization. J. Clin. Microbiol. 32:1326–1332. 9. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Habor Laboratory Press, Cold Spring Habor, N.Y. 10. Sanford, J. P. 1985. Pseudomonas species (including melioidosis and glanders), p. 1250–1254. In G. L. Mandell, R. G. Douglas, and J. E. Bennett (ed.), Principles and practice of infectious diseases, 2nd ed. John Wiley & Sons, Inc., New York. 11. White, N. J., D. A. B. Dance, W. Chaowagul, Y. Wattanagoon, V. Wuthiekanun, and N. Pitakwatchara. 1989. Halving of mortality of severe melioidosis by ceftazidime. Lancet ii:697–700. 12. Yabuuchi, E., Y. Kosako, H. Oyaizu, I. Yano, H. Hotta, Y. Hashimoto, T. Ezaki, and M. Arakawa. 1992. Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov. Microbiol. Immunol. 36:1251–1275.
