• View in gallery View in gallery

    Heterogeniety of Burkholderia pseudomallei lipo-polysacchaide (LPS). A, Representative silver-stained sodium do-decyl sulfate–polyacrylamide gel electrophoresis profiles of the three LPS types. Smooth type A and type B and rough type are shown in lanes 1, 2, and 3, respectively. B and C, Immunoblotting analysis of type A (lanes 1–4) and type B (lanes 5–9) LPS. The LPSs were probed with pooled serum from patients infected with the bacteria possessing LPS type A (B) and LPS type B (C). The numbers on the left represent molecular weight markers in kilodaltons (kD).

  • View in gallery

    Biofilm production by Burkholderia pseudomallei with different lipopolysaccharide (LPS) profiles. Representative isolates possessing type A, type B and rough type LPSs were cultured in biofilm-inducing medium and biofilm production was determined.15 Relative biofilm formation on the y axis represents the ratio of the optical density value at 630 nm of the test strain divided by that of the reference strain used in all experiments. The horizontal dotted lines indicate medians, and the boxes and error bars represent 25th–75th and 10th–90th percentiles, respectively. Differences between the three LPS groups were statistically significant by the Kruskal-Wallis test (type A versus type B; P = 0.0268; type A versus rough type; P = 0.0001; and type B versus rough type; P = 0.0264).

  • 1

    White NJ, 2003. Melioidosis. Lancet 361 :1715–1722.

  • 2

    Cheng AC, Currie BJ, 2005. Melioidosis: epidemiology, pathophysiology, and management. Clin Microbiol Rev 18 :383–416.

  • 3

    Anuntagool N, Naigowit P, Petkanchanapong V, Aramsri P, Panichakul T, Sirisinha S, 2000. Monoclonal antibody-based rapid identification of Burkholderia pseudomallei in blood culture fluid from patients with community-acquired septicaemia. J Med Microbiol 49 :1075–1078.

    • Search Google Scholar
    • Export Citation
  • 4

    Lowe P, Engler C, Norton R, 2002. Comparison of automated and nonautomated systems for identification of Burkholderia pseudomallei. J Clin Microbiol 40 :4625–4627.

    • Search Google Scholar
    • Export Citation
  • 5

    Petkanjanapong V, Naigowit P, Kondo E, Kanai K, 1992. Use of endotoxin antigens in enzyme-linked immunosorbent assay for the diagnosis of P. pseudomallei infections (melioidosis). Asian Pac J Allergy Immunol 10 :145–150.

    • Search Google Scholar
    • Export Citation
  • 6

    Sirisinha S, Anuntagool N, Dharakul T, Ekpo P, Wongrata-nacheewin S, Naigowit P, Petchclai B, Thamlikitkul V, Suputtamongkol Y, 2000. Recent developments in laboratory diagnosis of melioidosis. Acta Trop 74 :235–245.

    • Search Google Scholar
    • Export Citation
  • 7

    Tomaso H, Pitt TL, Landt O, Al Dahouk S, Scholz HC, Reisinger EC, Sprague LD, Rathmann I, Neubauer H, 2005. Rapid presumptive identification of Burkholderia pseudomallei with real-time PCR assays using fluorescent hybridization probes. Mol Cell Probes 19 :9–20.

    • Search Google Scholar
    • Export Citation
  • 8

    Wuthiekanun V, Anuntagool N, White NJ, Sirisinha S, 2002. Short report: a rapid method for the differentiation of Burkholderia pseudomallei and Burkholderia thailandensis. Am J Trop Med Hyg 66 :759–761.

    • Search Google Scholar
    • Export Citation
  • 9

    Woo PC, Lau SK, Woo GK, Fung AM, Ngan AH, Hui WT, Yuen KY, 2003. Seronegative bacteremic melioidosis caused by Burkholderia pseudomallei with ambiguous biochemical profile: clinical importance of accurate identification by 16S rRNA gene and groEL gene sequencing. J Clin Microbiol 41 :3973–3977.

    • Search Google Scholar
    • Export Citation
  • 10

    Anuntagool N, Aramsri P, Panichakul T, Wuthiekanun VR, Kinoshita R, White NJ, Sirisinha S, 2000. Antigenic heterogeneity of lipopolysaccharide among Burkholderia pseudomallei clinical isolates. Southeast Asian J Trop Med Public Health 31 (Suppl 1):146–152.

    • Search Google Scholar
    • Export Citation
  • 11

    Anuntagool N, Intachote P, Wuthiekanun V, White NJ, Sirisinha S, 1998. Lipopolysaccharide from nonvirulent Ara+ Burkholderia pseudomallei isolates is immunologically indistinguishable from lipopolysaccharide from virulent Ara-clinical isolates. Clin Diagn Lab Immunol 5 :225–229.

    • Search Google Scholar
    • Export Citation
  • 12

    Pitt TL, Aucken H, Dance DA, 1992. Homogeneity of lipopolysaccharide antigens in Pseudomonas pseudomallei. J Infect 25 :139–146.

  • 13

    Sermswan RW, Wongratanacheewin S, Trakulsomboon S, Thamlikitkul V, 2001. Ribotyping of Burkholderia pseudomallei from clinical and soil isolates in Thailand. Acta Trop 80 :237–244.

    • Search Google Scholar
    • Export Citation
  • 14

    Koonpaew S, Ubol MN, Sirisinha S, White NJ, Chaiyaroj SC, 2000. Genome fingerprinting by pulsed-field gel electrophoresis of isolates of Burkholderia pseudomallei from patients with melioidosis in Thailand. Acta Trop 74 :187–191.

    • Search Google Scholar
    • Export Citation
  • 15

    Taweechaisupapong S, Kaewpa C, Arunyanart C, Kanla P, Homchampa P, Sirisinha S, Proungvitaya T, Wongratanacheewin S, 2005. Virulence of Burkholderia pseudomallei does not correlate with biofilm formation. Microb Pathog 39 :77–85.

    • Search Google Scholar
    • Export Citation
  • 16

    Stepanovic S, Vukovic D, Dakic I, Savic B, Svabic-Vlahovic M, 2000. A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods 40 :175–179.

    • Search Google Scholar
    • Export Citation
  • 17

    DeShazer D, Brett PJ, Woods DE, 1998. The type II O-antigenic polysaccharide moiety of Burkholderia pseudomallei lipopolysaccharide is required for serum resistance and virulence. Mol Microbiol 30 :1081–1100.

    • Search Google Scholar
    • Export Citation
  • 18

    O’Toole G, Kaplan HB, Kolter R, 2000. Biofilm formation as microbial development. Annu Rev Microbiol 54 :49–79.

 
 
 

 

 
 
 

 

 

 

 

 

 

LIPOPOLYSACCHARIDE HETEROGENEITY AMONG BURKHOLDERIA PSEUDOMALLEI FROM DIFFERENT GEOGRAPHIC AND CLINICAL ORIGINS

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  • 1 Faculty of Tropical Medicine, and Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand; University of Oxford, Oxford, United Kingdom; Tropical and Emerging Infectious Diseases Division, Menzies School of Health Research, Charles Darwin University, Darwin, Northern Territory, Australia; Northern Territory Clinical School, Flinders University, Darwin, Northern Territory, Australia; Melioidosis Research Center, Departments of Biochemistry and Microbiology, Faculty of Medicine, and Department of Oral Diagnosis, Faculty of Dentistry, Khon Kaen University, Khon Kaen, Thailand

Heterogeneous patterns were obtained for lipopolysaccharide (LPS) from 1,327 Burkholderia pseudomallei isolates by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, silver staining, and immunoblot analysis. Two LPS serotypes (A and B) possessing different ladder profiles and a rough LPS without ladder appearances were identified. All three LPS types were antigenically distinct by immunoblotting. The predominant type A (97%) produced the lowest amount of biofilm. The two less common types (smooth type B and rough type) were found more in clinical than environmental isolates and more in Australian isolates than Thai isolates. These isolates were more often associated with relapse than with primary infection.

Melioidosis is a potentially fatal infectious disease endemic in southeast Asian countries and northern Australia.1,2 It has a broad clinical spectrum, ranging from a seropositive but asymptomatic condition to acute fatal septicemia. The causative agent is Burkholderia pseudomallei, a gram-negative bacillus found in soil and water in areas endemic for infection. Seropositive asymptomatic individuals may harbor the organisms for several years. Inappropriate or inadequate antibiotic treatment is associated with a high rate of relapse and no vaccine is currently available. Diagnosis remains a problem and a definitive diagnosis still relies on the isolation and identification of bacteria from clinical specimens. Attempts to develop reliable immunologic and molecular assays to replace the more time-consuming bacteriologic diagnosis have been made, but these new diagnostic methods remain unvali-dated.37 We reported a highly reliable latex agglutination test based on the use of monoclonal antibodies specific for a 200-kD exopolysaccharide antigen present only in B. pseudomallei.3,8 It was highly sensitive and specific for the detection of B. pseudomallei antigen in overnight broth hemocultures taken from patients suspected of having septicemic melioidosis.3

Burkholderia pseudomallei is generally considered to be a rather homogenous species, but colony variation occurs and variation in antibiotic susceptibility profiles and biochemical profiles using a number of commercial bacteriological identification systems have been reported.1,2,9 In addition to these phenotypic variations, we recently reported physicochemical and antigenic heterogeneity of the lipopolysaccharide (LPS) prepared from B. pseudomallei10,11 that could be responsible for false seronegativity in the patients with B. pseudomallei infection. A recently described case of seronegative bacteremic melioidosis caused by a B. pseudomallei strain possessing an ambiguous biochemical profile may have a similar explanation because the investigators based their conclusion on the LPS-based enzyme-linked immunosorbent assay system used for antibody detection.9

In our initial communication, we reported that 96% of the LPS from clinical isolates possessed a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) silver-staining ladder profile referred to as a typical ladder and approximately 3% exhibited different ladder characteristics referred to as an atypical ladder.10 The remaining 1% had no ladder detectable in the high molecular weight region but possessed a low molecular weight silver-staining band below the 29-kD marker that was designated as a no ladder LPS.10 All three LPS types were antigenically distinct, as evident from immunoblot reactivity against pooled sera from patients from whom the typical or atypical LPSs were isolated.10 To investigate the possible biologic significance of the different LPS phenotypes, we have now expanded our study to include environmental isolates as well as clinical isolates from patients with different clinical manifestations.

The LPSs were extracted from 1,327 isolates (Table 1) by proteinase K digestion and tested with pooled patient sera as previously described.11 In this large series of isolates, the proportion of isolates possessing different LPS patterns was similar to that in our initial report, i.e., 97:2:1 (Figure 1A and Table 1). Overall, 99% of the isolates possessed smooth type LPS exhibiting two different ladder profiles. These two LPS types were serologically distinct (Figure 1), most likely reflecting two different repeating polysaccharide side chains. We refer to the most common serotype (97%) as serotype A and the less abundant one (2%) as serotype B (corresponding to typical and atypical LPS, respectively, in the original report).10 The remaining 1% without a ladder profile and negative seroreactivity (tested against sera from patients from whom isolates with serotype A and type B LPSs were recovered) was considered to be a rough LPS (no ladder in the original report) and most likely represented only a lipid A core oligosaccharide. Our observation therefore differs from a previous report showing that B. pseudomallei isolates exhibited homogeneous SDS-PAGE patterns.12 The different LPS patterns of the isolates do not correlate with ribotypes13 or pulsed-field gel electrophoresis (PFGE) profiles.14 However, when quantitated for a capacity to produce biofilm,15,16 the three LPS types differed from one another (Figure 2). Those with a rough LPS (without ladder appearance) exhibited the highest biofilm-producing capacity and those with a smooth serotype A profile showed the lowest mean biofilm-producing capacity. Whether biofilm production is important in the pathogenesis of melioidosis is still unclear. However, we recently showed that it is most likely unrelated to the virulence of B. pseudomallei.15

Results of analyses of LPS data of B. pseudomallei isolates from different geographic regions and clinical manifestations are shown in Table 2. It is evident that a much higher proportion of the Australian isolates possessed the two less common LPS types (19.3%) in comparison with the Thai isolates (2.7%) (relative risk [RR] = 7.3, 95% confidence interval [CI] =3.7–14.1, P < 0.0001, by Fisher’s exact test). The LPSs of clinical isolates were more heterogeneous than the LPSs from environmental isolates. All environmental isolates had the smooth type LPS, and all except one belonged to serotype A. This atypical environmental isolate was obtained from soil on a goat farm where a B. pseudomallei outbreak had been reported. It exhibited the same PFGE profile as found in one of the isolates obtained from an infected goat. It is therefore possible that this environmental isolate was passed from an infected animal to the soil.

Since LPS has been implicated as one of the virulence factors for a number of gram-negative bacteria, including B. pseudomallei,17 we investigated possible associations between the LPS types and various clinical parameters of the patients from whom these isolates were obtained (Table 2). There was no association between the LPS type and disease severity (e.g., fatal versus non-fatal, and septicemic versus localized), clinical manifestations (neurologic versus non-neurologic), or underlying risk factors (diabetic versus non-diabetic). The two less abundant LPS patterns were found more in isolates obtained from patients with relapse melioidosis than from those with primary infection (RR = 6.6, 95% CI = 2.2–19.6, P = 0.012, by Fisher’s exact test). This suggests that the bacteria with these less common LPSs survive in the host better than those with the more common LPS type. Since each LPS type is antigenically distinct, antigenic polymorphism among the B. pseudomallei LPS may allow the bacteria to evade host immunity.

We identified one B. pseudomallei isolate from a primary and relapse infection that had the same PFGE pattern and ribotype but different a LPS type (patient 4; Table 3). However, the role of specific immunity in the control of melioidosis remains uncertain. These uncommon LPS types were not associated with a survival advantage ex vivo because bacteria with different LPS phenotypes were equally resistant to killing and equally able to replicate in 30% normal human serum (data not presented).

Variability in LPS has been observed to exhibit different degrees of surface colonization.18 The interrelationship between LPS and biofilm synthesis and the possible involvement in host adhesion and host persistence have been previously reported in Pseudomonas aeruginosa and P. fluorescens.18 We reported herein that the B. pseudomallei isolates with a rough LPS appeared to have a higher capacity to produce biofilm (Figure 2). Although it is logical to predict that such a combination may allow the organism to evade host defenses and therefore survive better inside the host, our most recent report15 that the biofilm by itself is probably unrelated to the virulence of B. pseudomallei. The involvement of LPS in the pathogenesis of melioidosis remains inconclusive. Further biochemical characterization and structural elucidation of the different O-polysaccharide side chains and the lipid A of B. pseudomallei LPS should provide additional insights into the possible role of LPS in pathogenesis, diagnosis, and vaccine development.

Table 1

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis silver-stained profile of Burkhoderia pseudomallei lipopolysaccharide (LPS)

LPS ladder pattern
OriginNo. of isolatesType A (%)Type B (%)Rough type (%)
* NTCC strains 1688, 4845, 4846, 6700, 7131, 7383, 8016, and 8707 were used for comparison.
Human
    Thailand1,0561,028 (97)22 (2)6 (1)
    Australia3728 (76)6 (16)3 (8)
    China42 (50)1 (25)1 (25)
    Cambodia22 (100)00
    Bangladesh11 (100)00
Animals
    Australia1111 (100)00
    Hong Kong1919 (100)00
    Malaysia1111 (100)00
Soil
    Australia32 (67)1 (33)0
    Thailand127127 (100)00
    Laos4747 (100)00
Water
    Australia11 (100)00
NTCC*88 (100)00
    Total1,3271,287 (97)30 (2)10 (1)
Table 2

Relationship of lipopolysaccharide (LPS) types with different geographic, clinical, and laboratory parameters*

LPS status
VariableNo.Smooth type A (%)Smooth type B (%)Rough type (%)
* Parameters in bold in the variable column are significantly different from one another (P < 0.05).
† Data from Thai patients only.
‡ Data from Australian patients only.
§ Indirect hemagglutination test (IHA) commonly used for antibody detection in many areas endemic for infection. A cut-off dilution for positive sera in the present study is 1:160.
Thailand1,0561,028 (97.3)22 (2.1)6 (0.6)
Australia5242 (80.8)7 (13.5)3 (5.8)
Clinical1,1521,110 (96.4)29 (2.5)10 (0.9)
Environmental178177 (99.4)1 (0.6)0
Primary338324 (95.9)11 (3.4)3 (0.9)
Relapse118 (72.7)2 (18.2)1 (9.1)
Fatal173161 (93.1)9 (5.2)3 (1.7)
Non-fatal213199 (93.4)10 (4.7)4 (1.8)
Septicemic208193 (92.8)11 (5.3)4 (1.9)
Non-septicemic179167 (93.3)9 (5.0)3 (1.7)
Diabetic177166 (93.8)8 (4.5)3 (1.7)
Non-diabetic203189 (93.1)10 (4.9)4 (2.0)
Neurologic‡1611 (68.8)3 (18.8)2 (12.5)
Non-neurologic‡2117 (80.9)3 (14.3)1 (4.8)
IHA§
    Positive9884 (85.7)7 (7.1)7 (7.1)
    Negative4743 (91.5)4 (8.5)0
Table 3

Heterogeneity of lipopolysaccharides (LPSs) isolated from the same individual patients

Clinical specimens
PatientInfectionTypeDate obtainedLPS typeRibotype
1PrimaryHemoculture9/6/89B17
Urine9/6/89A17
2PrimaryHemoculture7/22/96A4
Parotid pus7/17/96A4
ReinfectedParotid pus6/24/97B8
3PrimaryHemoculture11/14/98B81
Urine11/15/98Rough26
4PrimarySputum10/3/98A23
RelapseHemoculture2000B23
Figure 1.
Figure 1.
Figure 1.

Heterogeniety of Burkholderia pseudomallei lipo-polysacchaide (LPS). A, Representative silver-stained sodium do-decyl sulfate–polyacrylamide gel electrophoresis profiles of the three LPS types. Smooth type A and type B and rough type are shown in lanes 1, 2, and 3, respectively. B and C, Immunoblotting analysis of type A (lanes 1–4) and type B (lanes 5–9) LPS. The LPSs were probed with pooled serum from patients infected with the bacteria possessing LPS type A (B) and LPS type B (C). The numbers on the left represent molecular weight markers in kilodaltons (kD).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 74, 3; 10.4269/ajtmh.2006.74.348

Figure 2.
Figure 2.

Biofilm production by Burkholderia pseudomallei with different lipopolysaccharide (LPS) profiles. Representative isolates possessing type A, type B and rough type LPSs were cultured in biofilm-inducing medium and biofilm production was determined.15 Relative biofilm formation on the y axis represents the ratio of the optical density value at 630 nm of the test strain divided by that of the reference strain used in all experiments. The horizontal dotted lines indicate medians, and the boxes and error bars represent 25th–75th and 10th–90th percentiles, respectively. Differences between the three LPS groups were statistically significant by the Kruskal-Wallis test (type A versus type B; P = 0.0268; type A versus rough type; P = 0.0001; and type B versus rough type; P = 0.0264).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 74, 3; 10.4269/ajtmh.2006.74.348

*

Address correspondence to Narisara Anuntagool, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand. E-mail: narisara@tropmedres.ac

Authors’ addresses: Narisara Anuntagool, Vanaporn Wuthiekanun, and Nicholas J. White, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand, Telephone: 66-2-354-1389, Fax: 66-2-354-9169, E-mail: narisara@tropmedres.ac. Bart J. Currie, P.O. Box 41096 Casuarina, Northern Territory 0811, Australia, Telephone: 61-8-8922-8056, Fax: 61-8-8927-5287, E-mail: bart@menzies.edu.au. Rasana W. Sermswan, Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. Surasakdi Wongratanacheewin, Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand, Telephone: 66-4-336-3515, Fax: 66-4-334-8375, E-mail: sura_wng@kku.ac.th. Suwimol Taweechaisupapong, Deparment of Oral Diagnosis, Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002, Thailand. Sansanee C. Chaiyaroj and Stitaya Sirisinha, Deparment of Microbiology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand, Telephone: 66-2-201-5675, Fax: 66-2-644-5411, E-mail: scssr@mahidol.ac.th.

Acknowledgments: We thank Mark Mayo and Daniel Gal (Menzies School of Health Research, Darwin, Northern Territory, Australia) for assistance with isolate processing and some of the PFGE data mentioned. We also thank Prof. Nicholas PJ Day and Kasia Stepniewska (Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand) for their assistance in statistical analysis.

Financial support: This work was supported by grants RDG 4530209 and PDF/48/2544 from the Thailand Research Fund (Bangkok, Thailand) and the Wellcome Trust-Mahidol University-Oxford Tropical Medicine Research Programme funded by the Wellcome Trust of Great Britain, and by a project grant from the Australian National Health and Medical Research Council.

REFERENCES

  • 1

    White NJ, 2003. Melioidosis. Lancet 361 :1715–1722.

  • 2

    Cheng AC, Currie BJ, 2005. Melioidosis: epidemiology, pathophysiology, and management. Clin Microbiol Rev 18 :383–416.

  • 3

    Anuntagool N, Naigowit P, Petkanchanapong V, Aramsri P, Panichakul T, Sirisinha S, 2000. Monoclonal antibody-based rapid identification of Burkholderia pseudomallei in blood culture fluid from patients with community-acquired septicaemia. J Med Microbiol 49 :1075–1078.

    • Search Google Scholar
    • Export Citation
  • 4

    Lowe P, Engler C, Norton R, 2002. Comparison of automated and nonautomated systems for identification of Burkholderia pseudomallei. J Clin Microbiol 40 :4625–4627.

    • Search Google Scholar
    • Export Citation
  • 5

    Petkanjanapong V, Naigowit P, Kondo E, Kanai K, 1992. Use of endotoxin antigens in enzyme-linked immunosorbent assay for the diagnosis of P. pseudomallei infections (melioidosis). Asian Pac J Allergy Immunol 10 :145–150.

    • Search Google Scholar
    • Export Citation
  • 6

    Sirisinha S, Anuntagool N, Dharakul T, Ekpo P, Wongrata-nacheewin S, Naigowit P, Petchclai B, Thamlikitkul V, Suputtamongkol Y, 2000. Recent developments in laboratory diagnosis of melioidosis. Acta Trop 74 :235–245.

    • Search Google Scholar
    • Export Citation
  • 7

    Tomaso H, Pitt TL, Landt O, Al Dahouk S, Scholz HC, Reisinger EC, Sprague LD, Rathmann I, Neubauer H, 2005. Rapid presumptive identification of Burkholderia pseudomallei with real-time PCR assays using fluorescent hybridization probes. Mol Cell Probes 19 :9–20.

    • Search Google Scholar
    • Export Citation
  • 8

    Wuthiekanun V, Anuntagool N, White NJ, Sirisinha S, 2002. Short report: a rapid method for the differentiation of Burkholderia pseudomallei and Burkholderia thailandensis. Am J Trop Med Hyg 66 :759–761.

    • Search Google Scholar
    • Export Citation
  • 9

    Woo PC, Lau SK, Woo GK, Fung AM, Ngan AH, Hui WT, Yuen KY, 2003. Seronegative bacteremic melioidosis caused by Burkholderia pseudomallei with ambiguous biochemical profile: clinical importance of accurate identification by 16S rRNA gene and groEL gene sequencing. J Clin Microbiol 41 :3973–3977.

    • Search Google Scholar
    • Export Citation
  • 10

    Anuntagool N, Aramsri P, Panichakul T, Wuthiekanun VR, Kinoshita R, White NJ, Sirisinha S, 2000. Antigenic heterogeneity of lipopolysaccharide among Burkholderia pseudomallei clinical isolates. Southeast Asian J Trop Med Public Health 31 (Suppl 1):146–152.

    • Search Google Scholar
    • Export Citation
  • 11

    Anuntagool N, Intachote P, Wuthiekanun V, White NJ, Sirisinha S, 1998. Lipopolysaccharide from nonvirulent Ara+ Burkholderia pseudomallei isolates is immunologically indistinguishable from lipopolysaccharide from virulent Ara-clinical isolates. Clin Diagn Lab Immunol 5 :225–229.

    • Search Google Scholar
    • Export Citation
  • 12

    Pitt TL, Aucken H, Dance DA, 1992. Homogeneity of lipopolysaccharide antigens in Pseudomonas pseudomallei. J Infect 25 :139–146.

  • 13

    Sermswan RW, Wongratanacheewin S, Trakulsomboon S, Thamlikitkul V, 2001. Ribotyping of Burkholderia pseudomallei from clinical and soil isolates in Thailand. Acta Trop 80 :237–244.

    • Search Google Scholar
    • Export Citation
  • 14

    Koonpaew S, Ubol MN, Sirisinha S, White NJ, Chaiyaroj SC, 2000. Genome fingerprinting by pulsed-field gel electrophoresis of isolates of Burkholderia pseudomallei from patients with melioidosis in Thailand. Acta Trop 74 :187–191.

    • Search Google Scholar
    • Export Citation
  • 15

    Taweechaisupapong S, Kaewpa C, Arunyanart C, Kanla P, Homchampa P, Sirisinha S, Proungvitaya T, Wongratanacheewin S, 2005. Virulence of Burkholderia pseudomallei does not correlate with biofilm formation. Microb Pathog 39 :77–85.

    • Search Google Scholar
    • Export Citation
  • 16

    Stepanovic S, Vukovic D, Dakic I, Savic B, Svabic-Vlahovic M, 2000. A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods 40 :175–179.

    • Search Google Scholar
    • Export Citation
  • 17

    DeShazer D, Brett PJ, Woods DE, 1998. The type II O-antigenic polysaccharide moiety of Burkholderia pseudomallei lipopolysaccharide is required for serum resistance and virulence. Mol Microbiol 30 :1081–1100.

    • Search Google Scholar
    • Export Citation
  • 18

    O’Toole G, Kaplan HB, Kolter R, 2000. Biofilm formation as microbial development. Annu Rev Microbiol 54 :49–79.

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