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Journal of Clinical Microbiology, August 2003, p. 3973-3977, Vol. 41, No. 8
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.8.3973-3977.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

CASE REPORT

Seronegative Bacteremic Melioidosis Caused by Burkholderia pseudomallei with Ambiguous Biochemical Profile: Clinical Importance of Accurate Identification by 16S rRNA Gene and groEL Gene Sequencing

Patrick C. Y. Woo,¹ Susanna K. P. Lau,¹ Gibson K. S. Woo,¹ Ami M. Y. Fung,¹ Antonio H. Y. Ngan,¹ Wai-ting Hui,¹ and Kwok-yung Yuen^1,2*

Department of Microbiology, The University of Hong Kong, University Pathology Building, Queen Mary Hospital,¹ HKU-Pasteur Research Centre, Hong Kong²

Received 21 January 2003/ Returned for modification 21 March 2003/ Accepted 28 April 2003

Top Abstract Case report References

 
An aerobic gram-negative bacterium was isolated from the blood and sputum of an 84-year-old, chair-bound nursing home resident with acute bacteremic pneumonia. Although the phenotypic characteristics suggested that the bacterium could be Burkholderia pseudomallei, the Vitek 1 system (GNI+), which can successfully identify 99% of B. pseudomallei strains, showed that the bacterium was "unidentified." Immunoglobulin G against the lipopolysaccharide (LPS) of B. pseudomallei, as detected by an LPS-based enzyme-linked immunosorbent assay with 95% sensitivity, was negative in both the acute-phase and convalescent-phase sera. Sequencing of the groEL gene showed that the isolate was B. pseudomallei. Proper identification of the bacterium in this study is crucial, since there would be a radical difference in the duration of antimicrobial therapy.

Top Abstract Case report References

 
An 84-year old Chinese woman was admitted to hospital because of fever, chills, and productive cough with yellow sputum for 3 days. She had known bronchiectasis as a result of old pulmonary tuberculosis. She was a nursing home resident and had been chair-bound for several years. On admission, her oral temperature was 40°C. Examination of the chest revealed reduced expansion and coarse crackles on the left side. The total white cell count was 19.6 x 10⁹/liter, with a neutrophil count of 18.0 x 10⁹/liter, a lymphocyte count of 0.6 x 10⁹/liter, and a monocyte count of 1.1 x 10⁹/liter. The hemoglobin level was 12.9 g/dl, and the platelet count was 249 x 10⁹/liter. The urea level in serum was 6.0 mmol/liter, the creatinine level was 96 µmol/liter, the albumin level was 34 g/liter, and globulin level was 50 g/liter. Liver enzymes were normal, and total bilirubin was 20 µmol/liter. The fasting serum glucose level was 21.8 mmol/liter. A chest radiograph revealed patchy infiltrates in the left lung. Sputum for Gram's smear and bacterial culture and blood cultures were performed. She was started on empirical intravenous amoxicillin-clavulanate.

On day 1 postincubation, the aerobic blood culture bottle turned positive with a gram-negative bacillus. The bacterium grew on blood agar, chocolate agar, and MacConkey agar as dry, wrinkled colonies of 1 mm in diameter after 48 h of incubation at 37°C in an aerobic environment, but no growth was detected in an anaerobic environment. The bacterium also grew at 42°C and was cytochrome oxidase positive, motile, and resistant to polymyxin B, suggestive of Burkholderia pseudomallei (11). However, the Vitek 1 system (GNI+) (bioMerieux Vitek), which can successfully identify 99% of B. pseudomallei strains (9), showed that it was "unidentified" in three separate experiments (Table 1). The bacterium was sensitive to ceftazidime, amoxicillin-clavulanate, and imipenem but resistant to cotrimoxazole, gentamicin, and amikacin. A Gram smear of the sputum showed numerous polymorphs, and a bacterium with the same biochemical profile was also recovered from the sputum culture. Amoxicillin-clavulanate was discontinued, and intravenous ceftazidime was commenced. Immunoglobulin G (IgG) antibodies against the lipopolysaccharide (LPS) of B. pseudomallei, determined using an in-house developed enzyme-linked immunosorbent assay (ELISA) based on a published protocol (12), was negative for both the acute-phase serum and the convalescent-phase serum obtained 2 weeks after admission.

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TABLE 1. Biochemical profile and identification of the blood culture isolate by Vitek 1 system (GNI+)

Sequencing of the 16S rRNA genes of the isolates recovered from blood and sputum, using our published protocols (14, 15) and LPW81 5'-TGGCGAACGGGTGAGTAA-3' and LPW205 5'-CTTGTTACGACTTCACCC-3' (Gibco BRL, Rockville, Md.) as the PCR and sequencing primers, showed that they possessed the same nucleotide sequence. There was no base difference between the 16S rRNA gene sequence of the isolate and that of B. pseudomallei (GenBank accession no. AF093059), 12 (0.9%) base differences between the 16S rRNA gene sequence of the isolate and that of Burkholderia thailandensis (GenBank accession no. U91838), 21 (1.6%) base differences between the 16S rRNA gene sequence of the isolate and that of Burkholderia cepacia (GenBank accession no. AB051408), 23 (1.8%) base differences between the 16S rRNA gene sequence of the isolate and that of B. vietnamiensis (GenBank accession no. AF097534), and 57 (4.4%) base differences between the 16S rRNA gene sequence of the isolate and that of Burkholderia fungorum (in the Joint Genome Institute sequencing project) (Fig. 1).

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FIG. 1. Phylogenetic tree showing the relationship of the blood culture isolate to other Burkholderia species. The tree was inferred from 16S rRNA sequence data by the neighbor-joining method. A total of 1,308 nucleotide positions were included in the analysis. The tree was rooted with the sequence of B. pertussis. Bootstrap values were calculated from 1,000 trees. The scale bar indicates the number of substitutions per 100 bases using the Jukes-Cantor correction. Names and accession numbers are given as cited in the GenBank database.

Sequencing of the groEL genes of the isolates recovered from blood and sputum, using our published protocol (23) as well as LPW374 5'-AGAAGACATCATGGCCGT-3' and LPW377 5'-ATTACATGTCCATGCCCA-3' (Gibco BRL, Rockville, Md.), showed that they possessed the same nucleotide sequence. There were 0 to 5 (0 to 0.3%) base differences between the groEL gene sequence of the isolate and those of other B. pseudomallei strains we reported previously (23), 42 to 44 (2.6 to 2.7%) base differences between the groEL gene sequence of the isolate and those of other B. thailandensis strains that we reported previously (23), 77 (4.8%) base differences between the groEL gene sequence of the isolate and that of B. cepacia (GenBank accession no. AF104907), 78 (4.8%) base differences between the groEL gene sequence of the isolate and that of B. vietnamiensis (GenBank accession no. AF104908), and 170 (10.5%) base differences between the groEL gene sequence of the isolate and that of B. fungorum (in the Joint Genome Institute sequencing project), indicating that the isolate was a strain of B. pseudomallei (Fig. 2). A final diagnosis of melioidosis complicating diabetes mellitus and bronchiectasis was made. Ceftazidime was stopped after 2 weeks, and she was discharged with oral amoxicillin-clavulanate for another 20 weeks.

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FIG. 2. Phylogenetic tree showing the relationship of the blood culture isolate to other Burkholderia species. The tree was inferred from groEL sequence data by the neighbor-joining method. A total of 1,618 nucleotide positions were included in the analysis. The tree was rooted with the sequence of B. pertussis. Bootstrap values were calculated from 1,000 trees. The scale bar indicates the number of substitutions per 100 bases using the Jukes-Cantor correction. Names and accession numbers are given as cited in the GenBank database.

Melioidosis is a serious human disease endemic in Southeast Asia, caused by a nonfermentative gram-negative bacterium, B. pseudomallei. B. pseudomallei is very different from the other nonfermentative gram-negative bacteria in terms of the spectrum of disease that it can cause. Illness can manifest as an acute, subacute, or chronic process. Moreover, the duration of treatment of melioidosis is very different from that of infections caused by other nonfermentative gram-negative bacteria. In melioidosis, prolonged antimicrobial treatment is mandatory, since a short course of antibiotics is associated with high disease relapse rates.

Although it is of crucial importance to make a correct diagnosis of melioidosis, this has always been difficult for both the clinicians and the laboratory scientists. In most clinical microbiology laboratories, this relies on isolation and accurate identification of the bacterium from clinical specimens such as blood, sputum, and pus. In some laboratories, serodiagnosis is also employed to complement bacterial culture. Although a lot of effort has been spent on the search for immunogenic proteins (17, 18), the most widely used serodiagnostic tests are still assays which detect antibodies against B. pseudomallei LPS (3, 12, 24, 25). For phenotypic identification of B. pseudomallei, this is often delayed because laboratory personnel are often unfamiliar with the bacterium, which may be overlooked as "Pseudomonas species" (8). Although it has recently been shown that the Vitek 1 system, a widely used automated bacterial identification system in clinical microbiology laboratories, is one of the best commercially available systems for phenotypic identification of B. pseudomallei (9), the identification of the remaining strains with ambiguous biochemical profiles has always been difficult.

Despite the success in using 16S rRNA gene sequencing for identifying most bacterial species (14-16, 19-22), there are "blind spots" within some major genera, in which 16S rRNA gene sequences are found not to be discriminative enough for the identification of certain species. In such circumstances, sequences of essential genes other than 16S rRNA, such as groEL, have been shown to be useful for the identification of some species that cannot be discriminated by 16S rRNA gene sequencing (6, 13). Since the difference between the 16S rRNA gene sequences of B. pseudomallei and that of B. thailandensis is less than 1%, 16S rRNA gene sequencing is not discriminative enough for identifying the two species confidently. Recently we have reported the use of groEL gene sequencing for successful identification of B. pseudomallei and discrimination between B. pseudomallei and B. thailandensis (23). In this report, we describe the clinical application and show the importance of applying such a technique in identifying a strain of B. pseudomallei with an ambiguous biochemical profile recovered from a patient without antibody response against the LPS of B. pseudomallei. Furthermore, it reinforced our previous observation that groEL gene sequencing is more discriminative than 16S rRNA gene sequencing in the identification of B. pseudomallei.

Proper identification of the bacterium in this study is crucial, since there would be a radical difference in the duration of antimicrobial therapy. If the bacterium was identified to be other nonfermentative gram-negative bacteria or other Burkholderia species, such as B. cepacia, a 2-week course of antibiotic would be sufficient. However, if it was B. pseudomallei (as in our case), antibiotics for six months would be the regimen of choice, since the relapse rate for a short course of antibiotics would be high (5). In the present case, the clinical picture of community-acquired pneumonia in the setting of diabetes mellitus was compatible with melioidosis. Moreover, the colonial morphology and simple conventional biochemical tests suggested that the isolate could be B. pseudomallei. However, identification of the bacterium using the Vitek 1 system showed it as "unidentified." In a recently published study, it was reported that only one out of 103 B. pseudomallei strains was unidentified with this automated bacterial identification system (9). According to this report, the probability that our isolate is B. pseudomallei is not high. Furthermore, both the acute and convalescent-phase sera obtained from our patient showed negative results in the ELISA, which has been shown to be 95.7% sensitive by a group in Thailand (12) and 95% sensitive by us (unpublished data) for culture-documented melioidosis. This further reduced the likelihood that our patient was suffering from melioidosis. The final diagnosis of melioidosis was confirmed only after 16S rRNA and groEL gene sequencing.

The absence of antibody response to the LPS of B. pseudomallei could be due to the antigenic heterogeneity of LPS among B. pseudomallei isolates. Recently it has been reported that out of 739 B. pseudomallei isolates recovered from patients and animals of different geographical regions, the LPS extracted from 711 (96.2%) revealed identical typical ladder patterns, whereas those extracted from 21 (2.8%) and 7 (0.9%) showed atypical or no ladder patterns (1). Furthermore, it was shown that there were no immunological cross-reactivity between typical and atypical LPS, as demonstrated by Western blot assays using sera obtained from melioidosis patients from whom the typical and atypical LPS were obtained (1). This observation is in line with the 95% sensitivity of the LPS-based ELISA. We speculate that the LPS of the B. pseudomallei isolated from our patient probably possessed the atypical ladder pattern or did not possess any ladder pattern, leading to the apparent negative antibody response. LPS from B. pseudomallei with LPS of other types should be included to improve the sensitivity of LPS-based antibody detection systems.

The source of the B. pseudomallei infection in our patient remains elusive. B. pseudomallei is a natural saprophyte that can be isolated from soil, stagnant streams, rice paddies, and ponds, which have been quoted as the major natural reservoirs of the bacterium (8). However, our patient, being a nursing home resident and having been chair bound for several years, had no recent contact with these usual sources of B. pseudomallei. Since the incubation period of melioidosis can be more than 20 years (10), one possible cause of the bacteremic pneumonia would be reactivation of a latent focus as a result of poor diabetic control. On the other hand, it is also possible that our patient acquired the infection via contact with a contaminated water supply of the nursing home. It was reported recently that the source of a cluster of melioidosis cases was linked to the community water supply (4). In this outbreak, typing by pulsed-field gel electrophoresis revealed that the B. pseudomallei strains isolated from six of the nine patients showed restriction digestion patterns identical to that for a strain isolated from the community water supply but not for soil isolates (4). This is in line with the evidence that B. pseudomallei bacteria, similar to Legionella pneumophila bacteria, have the ability to survive as endosymbionts in free-living amebae (7), and water supply has been quoted as a major reservoir and source of legionellosis (2). Further studies should be carried out to ascertain the importance of water supplies as reservoirs of B. pseudomallei, which would have important implications for infection control measures that have to be taken.

Nucleotide sequence accession number. The 16S rRNA gene and groEL gene sequences of the B. pseudomallei strain in the present study were submitted to GenBank and given accession numbers AY198339 and AY198338, respectively.

 
This work is partly supported by the University Development Fund, University Research Grant Council, and the Committee for Research and Conference Grant, The University of Hong Kong.

 
* Corresponding author. Mailing address: Department of Microbiology, The University of Hong Kong, University Pathology Building, Queen Mary Hospital, Hong Kong. Phone: (852) 28554892. Fax: (852) 28551241. E-mail: hkumicro{at}hkucc.hku.hk.

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Journal of Clinical Microbiology, August 2003, p. 3973-3977, Vol. 41, No. 8
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.8.3973-3977.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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• Inglis, T. J. J., Sagripanti, J.-L. (2006). Environmental Factors That Affect the Survival and Persistence of Burkholderia pseudomallei. Appl. Environ. Microbiol. 72: 6865-6875 [Full Text]  
• ANUNTAGOOL, N., WUTHIEKANUN, V., WHITE, N. J., CURRIE, B. J., SERMSWAN, R. W., WONGRATANACHEEWIN, S., TAWEECHAISUPAPONG, S., CHAIYAROJ, S. C., SIRISINHA, S. (2006). LIPOPOLYSACCHARIDE HETEROGENEITY AMONG BURKHOLDERIA PSEUDOMALLEI FROM DIFFERENT GEOGRAPHIC AND CLINICAL ORIGINS.. Am J Trop Med Hyg 74: 348-352 [Abstract] [Full Text]  
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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Woo, P. C. Y. Articles by Yuen, K.-y. Search for Related Content PubMed PubMed Citation Articles by Woo, P. C. Y. Articles by Yuen, K.-y.