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Development of Rapid Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to Burkholderia pseudomallei
research-article
Author(s):
Vichaya Suttisunhakul
a ,
Vanaporn Wuthiekanun
b ,
Paul J. Brett
c ,
Srisin Khusmith
a ,
Nicholas P. J. Day
b
,
d ,
Mary N. Burtnick
c ,
Direk Limmathurotsakul
b
,
e ,
Narisara Chantratita
a
,
b
,
Publication date (Electronic):
25 April 2016
Journal:
Journal of Clinical Microbiology
Publisher:
American Society for Microbiology
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Abstract
Burkholderia pseudomallei, the causative agent of melioidosis, is an environmental bacillus found in northeast Thailand. The mortality rate of melioidosis is ∼40%. An indirect hemagglutination assay (IHA) is used as a reference serodiagnostic test; however, it has low specificity in areas where the background seropositivity of healthy people is high. To improve assay specificity and reduce the time for diagnosis, four rapid enzyme-linked immunosorbent assays (ELISAs) were developed using two purified polysaccharide antigens (O-polysaccharide [OPS] and 6-deoxyheptan capsular polysaccharide [CPS]) and two crude antigens (whole-cell [WC] antigen and culture filtrate [CF] antigen) of B. pseudomallei. The ELISAs were evaluated using serum samples from 141 culture-confirmed melioidosis patients from Thailand along with 188 healthy donors from Thailand and 90 healthy donors from the United States as controls. The areas under receiver operator characteristic curves (AUROCC) using Thai controls were high for the OPS-ELISA (0.91), CF-ELISA (0.91), and WC-ELISA (0.90), while those of CPS-ELISA (0.84) and IHA (0.72) were lower. AUROCC values using U.S. controls were comparable to those of the Thai controls for all ELISAs except IHA (0.93). Using a cutoff optical density (OD) of 0.87, the OPS-ELISA had a sensitivity of 71.6% and a specificity of 95.7% for Thai controls; for U.S. controls, specificity was 96.7%. An additional 120 serum samples from tuberculosis, scrub typhus, or leptospirosis patients were evaluated in all ELISAs and resulted in comparable or higher specificities than using Thai healthy donors. Our findings suggest that antigen-specific ELISAs, particularly the OPS-ELISA, may be useful for serodiagnosis of melioidosis in areas where it is endemic and nonendemic.
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Most cited references31
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Defining the True Sensitivity of Culture for the Diagnosis of Melioidosis Using Bayesian Latent Class Models
Background Culture remains the diagnostic gold standard for many bacterial infections, and the method against which other tests are often evaluated. Specificity of culture is 100% if the pathogenic organism is not found in healthy subjects, but the sensitivity of culture is more difficult to determine and may be low. Here, we apply Bayesian latent class models (LCMs) to data from patients with a single Gram-negative bacterial infection and define the true sensitivity of culture together with the impact of misclassification by culture on the reported accuracy of alternative diagnostic tests. Methods/Principal Findings Data from published studies describing the application of five diagnostic tests (culture and four serological tests) to a patient cohort with suspected melioidosis were re-analysed using several Bayesian LCMs. Sensitivities, specificities, and positive and negative predictive values (PPVs and NPVs) were calculated. Of 320 patients with suspected melioidosis, 119 (37%) had culture confirmed melioidosis. Using the final model (Bayesian LCM with conditional dependence between serological tests), the sensitivity of culture was estimated to be 60.2%. Prediction accuracy of the final model was assessed using a classification tool to grade patients according to the likelihood of melioidosis, which indicated that an estimated disease prevalence of 61.6% was credible. Estimates of sensitivities, specificities, PPVs and NPVs of four serological tests were significantly different from previously published values in which culture was used as the gold standard. Conclusions/Significance Culture has low sensitivity and low NPV for the diagnosis of melioidosis and is an imperfect gold standard against which to evaluate alternative tests. Models should be used to support the evaluation of diagnostic tests with an imperfect gold standard. It is likely that the poor sensitivity/specificity of culture is not specific for melioidosis, but rather a generic problem for many bacterial and fungal infections.
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Development of a Prototype Lateral Flow Immunoassay (LFI) for the Rapid Diagnosis of Melioidosis
Raymond L. Houghton, Dana E. Reed, Mark A. Hubbard … (2014)
Introduction Burkholderia pseudomallei is an environmental Gram-negative bacillus and the cause of melioidosis. The clinical manifestations of melioidosis are broad and include disseminated disease with organ abscesses, severe sepsis, and mild infection of the skin and soft tissue [1]. Most patients have risk factors for infection, which include diabetes, heavy alcohol use, and chronic pulmonary or kidney disease [1]–[3]. The highest number of reported cases occurs in endemic regions of Thailand and Australia. Rising incidence rates have been recorded in northeast Thailand between 1997–2006, during which the average mortality rate was 42.6% [3]. In 2006, melioidosis and tuberculosis mortality rates in northeast Thailand were equivalent and second only to HIV/AIDS for infectious disease deaths [3]. In northern Australia the mortality rate over the last five years of the Darwin prospective melioidosis study was calculated at 9% [2]. The authors attributed the low mortality rate to early diagnosis and treatment, and access to and improvements in intensive care management [2]. Isolation of B. pseudomallei from clinical samples remains the “gold standard” against which other melioidosis diagnostics are compared [4]. Culture is routinely performed on multiple sample types (blood, urine, pus, sputum, etc.) and isolation of B. pseudomallei from any one of these cultures is diagnostic for melioidosis [5], [6]. However, recent modeling data has confirmed that culturing is an imperfect gold standard [7]. Furthermore, laboratory processing of positive samples takes 3–7 days [8]. This problem is compounded by the fact that many diagnostic laboratories may misidentify B. pseudomallei through lack of experience or validated diagnostic reagents [9]. Any delay in diagnostic confirmation is potentially important as B. pseudomallei requires therapy with ceftazidime or a carbapenem drug, which are not agents of choice for empirical therapeutic regimens. Taken together, these factors point to a clear need for a simple and rapid diagnostic test for accurate identification of B. pseudomallei directly on clinical samples or cultures. Prior to diagnostic test development we identified a number of potential B. pseudomallei diagnostic biomarkers by In vivo Microbial Antigen Discovery (InMAD) [10], [11] that are shed or secreted and may be targeted to diagnose acute disease. Capsular polysaccharide (CPS) proved to be the most encouraging target; this molecule is a polymer of 1,3-linked 2-O-acetyl-6-deoxy-β-d-manno-heptopyranose residues [12]. We confirmed CPS was present in melioidosis patient serum and urine samples by antigen-capture ELISA utilizing a CPS-specific monoclonal antibody (mAb 3C5) [10]. The current report describes the characterization of mAb 3C5, quantification of CPS within patient samples, and optimization of the Active Melioidosis Detect lateral flow immunoassay (AMD LFI) for the rapid diagnosis of melioidosis. Materials and Methods Bacterial cultures Bacterial isolates listed in Table 1 were cultured on trypticase soy agar containing 5% sheep blood. Escherichia coli and B. pseudomallei (strain Bp82) were cultured on Luria Bertani agar and brain heart infusion agar, respectively. Plates were incubated at 37°C for 18–24 h. All work with viable B. pseudomallei and Burkholderia mallei strains was conducted under BSL-3 containment. All other strains were grown under BSL-2 containment. 10.1371/journal.pntd.0002727.t001 Table 1 Active Melioidosis Detect analytical reactivity and specificity. Bacterial isolate Strain name/DASH # Lateral Flow Result Burkholderia pseudomallei 7641; PHLS24; CDC2721620 Positive (+) Burkholderia pseudomallei Bp25; CDC2721628; 770429 Positive (+) Burkholderia pseudomallei CDC2721639; PHLS 66 Positive (+) Burkholderia pseudomallei K96243; NR 9320; CDC0022138 Positive (+) Burkholderia pseudomallei Bp92; CDC2721623 Positive (+) Burkholderia pseudomallei Thai 2 NE Human 88; PHLS 45 Positive (+) Burkholderia pseudomallei Bp104; CDC2721624 Positive (+) Burkholderia pseudomallei CDC2721635; PHLS 36 Positive (+) Burkholderia pseudomallei Bp73; Ln31348 Positive (+) Burkholderia pseudomallei PHLS 208 Positive (+) Burkholderia pseudomallei CDC2721102; F5013 Positive (+) Burkholderia pseudomallei BpG9709; CDC0032026 Positive (+) Burkholderia pseudomallei Sing Env 91; PHLS 19; CDC2721625 Positive (+) Burkholderia pseudomallei ATCC 23343; CDC2721676; NCTC 12939 Positive (+) Burkholderia pseudomallei Bp2889; SID2889 Positive (+) Burkholderia pseudomallei France Env 76; PHLS 33; CDC2721630; 7605 Positive (+) Burkholderia pseudomallei Bp68; CDC2721641 Positive (+) Burkholderia pseudomallei Indo 1 Monkey 90; PHLS 17; CDC2721619 Positive (+) Burkholderia pseudomallei Sing3 Human 88; PHLS 38; S6 Positive (+) Burkholderia pseudomallei 1106a; U1106a; CDC0022030 Positive (+) Burkholderia pseudomallei Bp53; CDC2721633; 307a Positive (+) Burkholderia pseudomallei Bp24; CDC2721620 Positive (+) Burkholderia pseudomallei BpG9313; CDC0032029 Positive (+) Burkholderia pseudomallei CDC2721162; B7210; B6195; 904-1111 Positive (+) Burkholderia pseudomallei CDC2721114; G6715 Positive (+) Burkholderia pseudomallei Thai NE Env 90; PHLS 216; CDC2721626 Positive (+) Burkholderia pseudomallei Bp H1406B; CDC0032028 Positive (+) Burkholderia pseudomallei F1394; 2002721096; 81A442 Positive (+) Burkholderia pseudomallei CDC2721123; H0929; 98-33; CDC0032024 Positive (+) Burkholderia pseudomallei Thai NE Human 99; PHLS 392 Positive (+) Burkholderia pseudomallei CDC1029240; H2001; 2001T-0229 Positive (+) Burkholderia pseudomallei CDC2721617; PHLS 5; NCTC 8016 Positive (+) Burkholderia pseudomallei Bp 14; CDC2721618 Positive (+) Burkholderia pseudomallei Bp H1442; CDC0032025 Positive (+) Burkholderia pseudomallei MSHR640; CDC8724880 Positive (+) Burkholderia pseudomallei Australian NT Human 1 97; 465a; CDC8724601 Positive (+) Burkholderia pseudomallei MSHR99; CDC8724881 Positive (+) Burkholderia pseudomallei MSHR362; CDC1756207 Positive (+) Burkholderia pseudomallei MSHR503; CDC8724890 Positive (+) Burkholderia pseudomallei #711; CDC2721675 Positive (+) Burkholderia pseudomallei PM19; CDC2734678; 620 Positive (+) Burkholderia pseudomallei MSHR296; CDC8724908 Positive (+) Burkholderia pseudomallei MSHR1200; CDC8724883 Positive (+) Burkholderia pseudomallei CDC2734694; PM40 Positive (+) Burkholderia pseudomallei PM26; CDC2734683 Positive (+) Burkholderia pseudomallei Malaysia5 Human; PHLS 75 Positive (+) Burkholderia pseudomallei MSHR1300; CDC8724901 Positive (+) Burkholderia pseudomallei PM115; CDC2734709 Positive (+) Burkholderia pseudomallei STW 424-1; CDC2721825 Positive (+) Burkholderia pseudomallei Bp40 Positive (+) Burkholderia pseudomallei MSHR365; CDC8724894 Positive (+) Burkholderia pseudomallei PM138; CDC2734661; SA923 Positive (+) Burkholderia pseudomallei Malaysia4 Human; PHLS 79 Positive (+) Burkholderia pseudomallei BpH1689; CDC0032024 Positive (+) Burkholderia pseudomallei CDC2721184 Positive (+) Burkholderia pseudomallei CDC2721634 Positive (+) Burkholderia pseudomallei CDC1756205 Positive (+) Burkholderia pseudomallei CDC8724905 Positive (+) Burkholderia pseudomallei CDC0022203 Positive (+) Burkholderia pseudomallei CDC2721637 Positive (+) Burkholderia pseudomallei CDC8724896; 1026b Positive (+) Burkholderia pseudomallei CDC8724889 Positive (+) Burkholderia pseudomallei CDC8724898 Positive (+) Burkholderia pseudomallei MSHR1655; 2002721686 (wcbR mutation) Negative (−) Burkholderia pseudomallei CDC8724899 Positive (+) Burkholderia pseudomallei CDC8724882 Positive (+) Burkholderia pseudomallei CDC8724900 Positive (+) Burkholderia pseudomallei CDC8724892 Positive (+) Burkholderia pseudomallei CDC8724893 Positive (+) Burkholderia pseudomallei CDC2721761 Positive (+) Burkholderia pseudomallei CDC8724885 Positive (+) Burkholderia pseudomallei CDC0022358 Positive (+) Burkholderia pseudomallei CDC8724877 Positive (+) Burkholderia pseudomallei CDC1756206 Positive (+) Burkholderia pseudomallei CDC8724895 Positive (+) Burkholderia pseudomallei CDC8724903 Positive (+) Burkholderia pseudomallei CDC8724878 Positive (+) Burkholderia mallei KC 238; Kweiyang #4; CDC2721277 Positive (+) Burkholderia mallei Kweiyang #1; CDC2734821 Positive (+) Burkholderia mallei KC1090; A188 Pasteur Institute; CDC2721278 Positive (+) Burkholderia mallei India 65-603; CDC0031066 Positive (+) Burkholderia mallei NCTC 10247; CDC2734315; Turkey 12 Positive (+) Burkholderia mallei Turkey 1; CDC0031065 Positive (+) Burkholderia mallei Turkey 5; CDC2734302 Positive (+) Burkholderia mallei NCTC 10260; CDC2734314; CDC2734301; Turkey 11; GB6; CCUG 19395 Positive (+) Burkholderia mallei Rob-DASH (2000031281); CDC0031304 Positive (+) Burkholderia mallei KC 234; 3873; China 7; CDC2721273 Positive (+) Burkholderia mallei KC 235; 3873-18; CDC2721274 Positive (+) Burkholderia mallei KC0248; CDC4017733 Positive (+) Burkholderia mallei KC 1091; A193 Pasteur Institute; CDC2721279 Positive (+) Burkholderia mallei KC 1092; CDC2721280; 52-236 Pasteur Institute Positive (+) Burkholderia mallei BURK011; CDC8724847; C2006251001 Positive (+) Burkholderia mallei GB9; CDC2734305; Strain 102; NCTC3708 Positive (+) Burkholderia mallei NCTC 3709 (Strain 106); CDC2724303; GB10 Positive (+) Burkholderia mallei Turkey 2; BURK063; CDC8724837 Positive (+) Burkholderia mallei Turkey 3; BURK064; CDC8724838 Positive (+) Burkholderia mallei Turkey 4; BURK065; CDC8724839 Positive (+) Burkholderia mallei Turkey 7; BURK068; CDC8724841 Positive (+) Burkholderia mallei CDC2734300; NCTC10247 Positive (+) Burkholderia mallei CDC2734301, NCTC10260 Positive (+) Burkholderia mallei CDC2734317; NCTC3709 Positive (+) Burkholderia mallei CDC2721275 Negative (−) Burkholderia mallei CDC2734299 Positive (+) Burkholderia mallei CDC2734311 Negative (−) Burkholderia mallei CDC0031063 Positive (+) Burkholderia mallei CDC0031064 Positive (+) Burkholderia mallei CDC2721276 Positive (+) Burkholderia mallei CDC2721648 Positive (+) Burkholderia mallei CDC2734312 Positive (+) Burkholderia mallei CDC2721280 Negative (−) Burkholderia thailandensis CDC3015869 (contains capsule operon) Positive (+) Burkholderia thailandensis CDC2721621 Negative (−) Burkholderia thailandensis CDC2721627 Negative (−) Burkholderia thailandensis CDC2721121 Negative (−) Burkholderia thailandensis CDC2721643 Negative (−) Burkholderia thailandensis CDC2721701 Negative (−) Burkholderia thailandensis CDC2721723 Negative (−) Burkholderia thailandensis CDC2721744 Negative (−) Burkholderia humptydooensis CDC2721687 Negative (−) Burkholderia oklahomensis CDC4002358 Negative (−) Burkholderia oklahomensis CDC4021865 Negative (−) Burkholderia oklahomensis CDC4021866 Negative (−) Burkholderia vietnamiensis CDC2734483 Negative (−) Burkholderia pyrrocinia CDC2724646 Negative (−) Burkholderia caledonica CDC8724197 Negative (−) Burkholderia caribensis CDC8724200 Negative (−) Burkholderia ambifaria CDC8724201 Negative (−) Burkholderia anthina CDC8724199 Negative (−) Burkholderia cocovenenans CDC2734715 Negative (−) Burkholderia ferrariae CDC8724209 Negative (−) Burkholderia hydrophilia CDC2721759 Negative (−) Burkholderia fungorum CDC8724198 Negative (−) Burkholderia glathei CDC2734719 Negative (−) Burkholderia graminis CDC2734716 Negative (−) Burkholderia hospita CDC8724207 Negative (−) Burkholderia kururiensis CDC2734717 Negative (−) Burkholderia nodosa CDC8724205 Negative (−) Burkholderia phenazinium CDC2734718 Negative (−) Burkholderia phenoliruptrix CDC8724203 Negative (−) Burkholderia phymatum CDC8724208 Negative (−) Burkholderia phytofirmans CDC8724204 Negative (−) Burkholderia sacchari CDC8724202 Negative (−) Burkholderia silvatlantica CDC8724206 Negative (−) Burkholderia rhizoxinica CDC2734772 Negative (−) Burkholderia endofungorum CDC2734773 Negative (−) Burkholderia gladioli CDC3027208 Negative (−) Escherichia coli ATCC 25922 Negative (−) Pseudomonas aeruginosa * ATCC 27853 Negative (−) Streptococcus pneumoniae * ATCC 10015 Negative (−) Klebsiella pneumoniae * ATCC 13883 Negative (−) Staphylococcus aureus * ATCC 25923 Negative (−) Enterobacter cloacae * ATCC 23355 Negative (−) Providencia stuartii * ATCC 33672 Negative (−) *Indicates strains that were tested for reactivity against mAb 3C5 via western blot. Ethics section Clinical samples from patients with culture-positive melioidosis were obtained from sample archives (no identifiable private information supplied) at Mahidol-Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand and Menzies School of Health Research and Northern Territory Clinical School, Royal Darwin Hospital, Darwin, Northern Territory, Australia. Archived and de-identified melioidosis negative serum and urine samples were obtained from the University of Nevada School of Medicine, Reno, NV, USA. Quantitation of B. pseudomallei in urine samples B. pseudomallei was quantified in urine as previously described [6]. Briefly, 1 µl of urine was plated on Ashdown agar plates and incubated overnight at 37°C [13]. Colonies were counted and expressed as colony forming units (CFU)/ml (Table 1). The remaining urine was centrifuged at 3000 rpm for 5 min. The pellet was then plated on an Ashdown agar plate and incubated overnight. The lower limit of detection was 1 CFU/ml (1 colony from 1 µl) and the upper limit of detection was ≥106 CFU/ml (≥1000 colonies/1 µl). A positive B. pseudomallei liquid culture from urine samples that did not show growth on Ashdown agar plates was estimated to contain 1×105 448 MSID2 + 1×105 66 MSID5 + 1×105 187 MSID7 +
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The Genetic and Molecular Basis of O-Antigenic Diversity in Burkholderia pseudomallei Lipopolysaccharide
Apichai Tuanyok, Joshua Stone, Mark Mayo … (2012)
Introduction Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria, playing an important role in cell integrity and in signaling host innate immune response [1]. Structurally, LPS is composed of three major components: lipid A, the bacterial endotoxin that is embedded in the phospholipid bilayer of the outer membrane; core-oligosaccharide; and O-antigen. These three components are linked together as a part of the bacterial outer membrane. In a highly pathogenic bacterial species, such as Burkholderia pseudomallei, LPS has a major role in stimulating host innate immune response during infection [2]. B. pseudomallei LPS has been classified as a type II O-polysaccharide (O-PS) and is one of 4 different surface polysaccharides produced by this pathogen [3]. Previous studies have shown that B. pseudomallei LPS is required for serum resistance and virulence [4]. It has been well established in many bacterial diseases that overstimulation of the host cells by LPS can lead to the features of septic shock [5]. Likewise for B. pseudomallei, septicemia is a major cause of death. In Northeast Thailand especially in Ubon Ratchathani Province where melioidosis is highly endemic, the average incidence rate of melioidosis is 12.7 cases per 100,000 people per year with the average of 42.6% of mortality rate [6]. Cellular recognition of LPS by the innate immune system triggers the proinflammatory cytokines by host cells, which aids in the clearance of the pathogen. Previous studies have supported a potential role for B. pseudomallei LPS in protective immunity, with high concentrations of antibodies to LPS associated with survival in severe melioidosis [7], [8]. As a result, LPS has been used in vaccine development and provided protective immunity in a murine model of melioidosis [2]. In addition, it was demonstrated that LPS had an important role in bacterial virulence because the LPS mutant B. pseudomallei strain SRM117, which lacked the O-antigenic polysaccharide moiety was more susceptible to macrophage killing during the early phase of infection than its parental strain 1026b [9]. A previous study [10] identified LPS diversity based upon electrophoretic mobility with SDS-PAGE and detection using immunoblot analysis. This diversity included two serotypes (A and B) possessing different electrophoretic ladder profiles and a rough type that did not contain the ladder patterns; all were antigenically distinct [10]. Molecular structure of O-antigen serotype A or typical type has been described as the unbranched heteropolymers consisting of disaccharides repeats of -3)-β-D-glucopyranose-(1-3)-6-deoxy-α-L-talopyranose-(1- in which approx. 33% of the L-6dTalp residues bear 2-O-methyl and 4-O-acetyl substituents whereas the other L-6dTalp residues carry only 2-O-acetyl substituents [11]. We note that the structures are not known for any of the other B. pseudomallei O-antigens. B. thailandensis, a genetically related non-pathogenic species, has LPS that is cross-reactive to sera obtained from B. pseudomallei and B. mallei infections, and this has led to the development of a vaccine for melioidosis using LPS from B. thailandensis [12]. B. mallei, the causative agent of glanders, has O-antigen structure similar to those found in B. pseudomallei and B. thailandensis, except that it has different side-group modifications at the L-6dTalp residues which lack the acetylation at the O-4 position [13]. These structural differences are associated with the absence of oacA gene in B. mallei. oacA encodes for O-antigen acetylase A in B. thailandensis and its homolog in B. pseudomallei K96243 is identified as BPSL1936 [14]. B. pseudomallei genomes are very diverse due to horizontal gene transfer events [15], [16] and dynamic changes in repeated sequences [17]. This results in diverse phenotypic characteristics such as bacterial colony morphotypes [18], and importantly, may be implicated in the diverse clinical manifestations observed among melioidosis patients. The latter range from asymptomatic cases, to localized infections, to whole body sepsis, along with differential seroreactivities [19], all of which may be correlated with the great genomic diversity in this species [15], [17]. Nevertheless, the specific roles of genetic diversity in B. pseudomallei in differential clinical presentations of melioidosis requires further analysis, as clinical studies suggest host risk factors are the major determinant of disease severity [20]. Because LPS phenotypic diversity is important for serology and diagnostics, we investigated the genetic and molecular basis of differential LPS phenotypes in a large B. pseudomallei population. Bioinformatics, phenotypic characterization, as well as, population genetics approach were used in this study to better understand this important trait. Methods Comparative genomics of LPS biosynthesis genes Artemis and Artemis Comparison Tool (ACT) software [21] was used to display and compare multiple B. pseudomallei genomes. Genomes and nucleotide sequences used in this study are listed in Table 1. Mutations in O-antigen biosynthesis genes were identified using basic homologous gene based alignments. 10.1371/journal.pntd.0001453.t001 Table 1 LPS genotype identification and mutations in the four closely related Burkholderia species. Species Strain Source GenBank Accession Number LPS Genotype oacA B. pseudomallei K96243 Human NC_006350 A Intact B. pseudomallei 1026b Human AF064070 A Intact B. pseudomallei 1106a Human NC_009076 A Intact B. pseudomallei 1106b Human NZ_CM000774 A Intact B. pseudomallei 1710a Human NZ_CM000832 A Intact B. pseudomallei 1710b Human NC_007434 A Intact B. pseudomallei MSHR668 Human NC_009074 A Intact B. pseudomallei MSHR1655 Human NZ_CH899712 A* Frame-shift B. pseudomallei MSHR305 Human NZ_AAYX01000005 A Intact B. pseudomallei MSHR346 Human NC_012695 A Intact B. pseudomallei MSHR840 Human GU574442 B2 Intact B. pseudomallei MSHR139 Human HM852063 B2 Intact B. pseudomallei MSHR1950 Human HM852062 B2 Intact B. pseudomallei 112 Human NZ_ABBP01000549 A Frame-shift B. pseudomallei 14 Animal NZ_ABBJ01000798 A† Intact B. pseudomallei 406e Human NZ_CH899732 A Intact B. pseudomallei 576 Human NZ_ACCE01000003 B Intact B. pseudomallei 7894 Human NZ_ABBO01000695 A Intact B. pseudomallei 9 Human NZ_ABBL01000749 A Intact B. pseudomallei 91 Animal NZ_ABBK01000735 A Intact B. pseudomallei B7210 Human NZ_ABBN01000620 A‡ Intact B. pseudomallei BCC215 Human NZ_ABBR01000422 A Intact B. pseudomallei DM98 Human NZ_ABBI01002075 A Intact B. pseudomallei NCTC13177 Human NZ_ABBQ01000469 B Intact B. pseudomallei Pakistan 9 Human NZ_ACKA01000012 A Intact B. pseudomallei Pasteur 52237 Human NZ_CH899755 A Intact B. pseudomallei S13 Human NZ_CH899770 A Intact B. mallei ATCC23344 Human NC_006348 A Absent B. mallei NCTC10229 Animal NC_008836 A Absent B. mallei NCTC10247 Unknown NC_009080 A Absent B. mallei SAVP1 Animal NC_008785 A Absent B. mallei 2002721280 Unknown NZ_CH899691 A Absent B. mallei ATCC10399 Animal NZ_CH899681 A Absent B. mallei FMH Human NZ_DS264097 A Absent B. mallei GB horse4 Animal NZ_AAHO01000001 A Absent B. mallei JHU Human NZ_DS264109 A Absent B. mallei PRL-20 Animal NZ_AAZP01000025 A Absent B. thailandensis E264 Environment NC_007651 A Intact B. thailandensis Bt4 Environment NZ_ABBH01000548 A Intact B. thailandensis TXDOH Human NZ_ABBD01000533 A‡ 5′truncation B. oklahomensis C6786 Human NZ_ABBG01000257, NZ_ABBG01000258 N/A Intact B. oklahomensis EO147 Human NZ_ABBF01000376 A‡, ψ Intact Note: LPS genotypes and mutations were identified in the main O-antigen biosynthesis gene locus and the oacA homologs. Frame-shifted mutations of the O-antigen biosynthesis genes were found in their wbiI(*), wbiF(†),wbiE (‡), and wbiD (ψ) genes. We did not test the effects of wbiF mutant in B. pseudomallei 14, and wbiE mutant in B. pseudomallei B7210, due to unavailability of live bacterial cultures. The listed GenBank accession numbers are associated with the LPS genotype identification, not for oacA analysis. Details of the oacA mutations are demonstrated in Figure S1. PCR Analysis Multiplex-SYBR-Green PCR assays were designed to target 3 different LPS genotypes. Gene wbiE of B. pseudomallei K96243, gene BUC_3396 of B. pseudomallei 576, and gene BURP840_LPSb16 of B. pseudomallei MSHR840 were used as the PCR markers to investigate frequency of LPS genotypes A, B, and B2, respectively (Figures 1&2). PCR primers used in this study are as follows: wbiE_F, 5′-TCAAACCTATCCGCGTGTCGAAGT-3′; wbiE_R, 5′-TCGTCGTCAAGAAATCCCAGCCAT-3′; BUC3396_ F, 5′-AATCTTTTTCTGATTCCGTCC-3′; BUC3396_R, 5′ -ACCAGAAGACAAGGAGAAAGGCCA-3′; BURP840_LPSb16_F, 5′-AACCGGGTAGTTCGCGATTAC-3′; and BURP840_LPSb16_R, 5′-ATACGCCGGTGTAGAACAGTA-3′. The PCR assay was conducted in 10-µL reaction mixtures containing 1× SYBR-Green master mix (Applied Biosystems, USA), 0.3 µM of each PCR primer, and 0.1 to 1.0 ng of DNA template. Most tested DNA samples were made in collaborative laboratories in Thailand and Australia using various DNA extraction techniques. The reactions were performed on an ABI 7900HT Sequence Detection System (Applied Biosystems, USA) utilizing 40 cycles. Each cycle contained two steps: denaturation at 95°C for 15 s and annealing at 60°C for 30 s. The PCR products were further analyzed by melting them continuously from 60°C to 95°C to generate a dissociation curve. The melting temperatures of PCR amplicons for genes wbiE, BUC_3396, and BURP840_LPSb16 were constant at 87.0°C, 83°C, and 88.5°C, respectively. We used this assay to analyze DNA templates from 999 diverse B. pseudomallei strains isolated from clinical, animal, and environmental samples from Australia (n = 600), Thailand (n = 349), Malaysia (n = 27), Vietnam (n = 7), Papua New Guinea (n = 2), and unknown countries in Southeast Asia (n = 14), as well as 77 B. thailandensis strains, 2 B. thailandensis-like spp. strains, and 37 strains of unknown soil bacteria. 10.1371/journal.pntd.0001453.g001 Figure 1 Diversity of three biosynthesis gene clusters for the O-antigen moiety of the LPS in B. pseudomallei. The typical LPS ladder pattern (i.e serotype A) was associated with O-antigen biosynthesis gene cluster in K96243 (top) or LPS genotype A, whereas the atypical LPS ladder pattern (serotype B) found in approximately 13.8% of Australian strains was believed to be associated with a different O-antigen biosynthesis gene cluster or LPS genotype B observed in strain 576 (middle). Strain MSHR840 was identified as a variant serotype B strain, designated as LPS genotype B2, because many of its O-antigen biosynthesis genes (bottom) were similar to those found in strain 576. We note that genes encoding for key components of the O-antigens (e.g., wbiGHI, and rmlBAC), were conserved across these 3 different clusters. Note: * Target genes selected for PCR assays to represent each LPS genotype; GenBank accession number and nucleotide coordinates are indicated for each genome used in the analysis; gene apaH is shown in this figure as a flanking gene that is not involved in the O-antigen biosynthesis. 10.1371/journal.pntd.0001453.g002 Figure 2 Genotyping scheme and frequencies of three different LPS genotypes identified in B. pseudomallei populations. Multiplex SYBR-Green PCR assays were developed to target the presence of genes: wbiE, BUC_3396, and BURP840_LPSb16, which were the representatives of LPS genotypes A, B, and B2, respectively. PCR amplicons from these 3 gene targets were differentiated by melting dissociation (A); or sizing (B); lanes 1, 2, 3, and 4 are PCR products from strains K96243, 576, MSHR840, and non-DNA template control (NTC), respectively; and L, 1 kb-plus DNA ladder. We note that LPS genotype A was the most common LPS genotype, whereas a majority of the LPS genotype B was found in strains from Australia (approx.13.8%). Genotype B2 was found in strains from Australia and Papua New Guinea (PNG) only (C). DNA sequencing and analysis Whole genome sequencing was performed using 454 sequencing technology (Roche, USA) by US Army Edgewood Chemical Biological Center (ECBC), MD, USA. Artemis –based analysis and BLAST were used to annotate the O-antigen biosynthesis genes of B. pseudomallei strains MSHR840, MSHR139, and MSHR1950. DNA sequencing for wbiI and oacA genes was performed using ABI 3130×l Genetic Analyzer (Applied Biosystems, USA). LPS identification and characterization: Techniques for LPS extraction and SDS-PAGE analysis followed a previous study [10]. Immunoblot analysis was performed using sera from melioidosis patients with known infection with B. pseudomallei LPS genotype A or B strains as the primary antibodies. Horse radish peroxidase (HRP) – conjugated anti-human IgG was used as the secondary antibody in a standard immunoblot analysis. Monoclonal antibody 3D11, the B. mallei LPS-specific mAb (Research Diagnostics Inc., USA), was used as a primary antibody in the immunoblot analysis of the oacA mutant strains. Serum susceptibility tests Select B. pseudomallei strains were tested for growth, multiplication, and survival in the presence of 30% normal human serum (NHS) as previously described [4] with some modifications. Briefly, each B. pseudomallei strain was inoculated in a 2 mL of TSBDC media and incubated overnight at 37°C and 250 rpm in an orbital shaker. The overnight culture (100 µL) was used to inoculate 3 mL of TSBDC media and then incubated at the same conditions for 4 hr to reach mid exponential growth phase. Serum susceptibility tests were performed in 1.5 mL microfuge tubes containing 100 µL of bacterial culture, 300 µL of NHS (Lonza Inc., USA), and 600 µL PBS. The mixture was incubated at 37°C for 2 hr, and then the number of viable bacterial cells was determined using plate counting. B. pseudomallei 1026b and E. coli HB101 were used as positive and negative controls in this study, respectively. GenBank Accessions Nucleotide sequences and annotations of the O-antigen biosynthesis genes in B. pseudomallei strains MSHR840, MSHR139, and MSHR1950LPS were submitted to GenBank under accession nos. GU574442, HM852063, and HM852062, respectively. Results To better understand the diversity of genes responsible for the biosynthesis of O-antigen moiety of the LPS in B. pseudomallei, we first used a comparative analysis of all publicly available B. pseudomallei genomes to identify differences within LPS biosynthetic genes. Three different O-antigen biosynthesis gene categories, or genotypes, were identified. Secondly, we examined the genotype frequencies in B. pseudomallei populations using PCR assays targeting each of these genetic types. Thirdly, we correlated LPS genotypes with their differential phenotypes (serotypes). This led to our discovery of a natural mutation in an O-antigen biosynthesis gene in a clonal panel of B. pseudomallei strains isolated from a single human host. The adaptability of B. pseudomallei strains through LPS variation, even within a single human host, represents an important aspect of pathogen biology and a complication for melioidosis host response. Diversity of O-antigen biosynthesis genes in B. pseudomallei and its near-relative species We compared 27 B. pseudomallei, 10 B. mallei, 3 B. thailandensis, and 2 B. oklahomensis genomes (Table 1) to identify the LPS O-antigen biosynthesis genes. Assuming synteny and common genomic locations, along with known or predicted function, B. pseudomallei O-antigen biosynthesis genes were assigned to two major groups. Group A (LPS genotype A) was identical or very similar to the O-antigen biosynthesis operon observed in B. pseudomallei 1026b [4], whereas group B (LPS genotype B) was found in an atypical LPS strain 576 and also in the species type strain, NCTC13177. LPS genotype A was found in most B. pseudomallei and all B. mallei and B. thailandensis genomes examined. Surprisingly, the more distantly related B. oklahomensis strain EO147 also had LPS genotype A, which was different from the predicted O-antigen biosynthesis gene cluster in other B. oklahomensis strains (C6786, C7532, and C7533; data not shown). This may represent a lateral gene transfer event into EO147 and is deserving of additional study. Furthermore, regions within the two clusters had different levels of sequence conservation. Genes located at the ends of these two clusters (e.g., wbiGHI, and rmlBAC; Figure 1) had higher sequence similarity than most of the genes in the core of the clusters. Indeed, many of the cluster cores contain distinct gene composition. The conserved genes include those important for oligosaccharide synthesis and O-antigen biosynthesis [4]. LPS genotype frequencies were analyzed across a large strain collection using PCR-based assays. Multiplex-SYBR-Green PCR assays were designed to target a specific gene unique for each genotype. Gene wbiE (BPSL2676) of B. pseudomallei strain K96243 and gene BUC_3396 of strain 576 were used to represent the presence of LPS genotypes A and B, respectively (Figures 1&2). A total of 999 B. pseudomallei strains from different geographic locations and epidemiological origins (e.g., clinical, animal, and environmental strains) were tested for their LPS genotypes. We noted that 23 B. pseudomallei strains were collected from one melioidosis patient. We found that LPS genotype A was the most common genotype in both Australian and Southeast Asian strain populations (Figure 2). LPS genotype B was relatively rare in Southeast Asian strains (∼2.3%), but was found in 13.8% of Australian strains. Five strains from Australia and two strains from Papua New Guinea were non-typeable using these two PCR gene markers. Three of these strains, MSHR840, MSHR1950, and MSHR139 were further analyzed for O-antigen biosynthesis gene identification using whole genome sequencing. The O-antigen biosynthesis gene clusters from these strains were identified and annotated (GenBank accession nos. GU574442, HM852062, HM852063). Comparative genomics demonstrated that many genes in this new cluster were similar to those of the LPS genotype B genes of B. pseudomallei 576 and were distinct from the K96243 LPS genotype A genes. Hence, these newly identified O-antigen biosynthesis gene clusters represent a variant of the LPS genotype B and, consequentially, were designated as LPS genotype B2 (Table 1). Figure 1 shows the genomic comparison of these three different O-antigen biosynthesis gene clusters: A, B, and B2 (from B. pseudomallei strains K96243, 576, and MSHR840, respectively). We note that %G+C content of the core of these 3 different clusters is relatively low (∼59–60%) compared to the conserved parts of the O-antigen biosynthesis operon (∼68%). This supports the hypothesis that these genomic differences are due to genetic recombination e.g., horizontal gene transfer, which is common in B. pseudomallei [15], [16]. Comparative genomics of these three different clusters using homologous-based alignment are summarized in Table S1. Again, we note that genes wbiGHI, and rmlBAC are conserved among these three different clusters. Furthermore, gene BURP840_LPSb16 from strain MSHR840 was selected for use as a PCR marker to represent the LPS genotype B2. PCR genotype analysis (Figure 2) revealed that all seven of the previously non-typeable strains were positive for the LPS genotype B2. The LPS B2 genotype was found only in strains from Australia and Papua New Guinea. It is important to note that there is no known clonal relationship among these seven strains. The LPS B2 genotype genes were also found in a B. thailandensis-like spp. strain MSMB121, which was isolated in Australia (unpublished data). Complete LPS genotypic data are reported in Table S2. A novel LPS electrophoretic pattern - a type B variant LPS genotyping results were further examined by direct comparison to LPS electrophoretic phenotypes [10]. Due to the difficulty of international Select Agent transfer and BSL3 handling, we phenotyped only ∼ 24% of the isolates that were genotyped. We note that this is a limitation of our study. That said, all examined LPS A or B phenotypes were perfectly matched with their LPS A or B genotypes. In addition, 22 strains producing the rough LPS phenotype were all identified as LPS genotype A (Table S2). The genetic basis of the rough phenotype and its derivation from the A phenotype is known for only 16 of these strains (see below). SDS-PAGE revealed that LPS genotype B2 strains produced a distinct ladder pattern, though they were all detectable with type B sera using immunoblot hybridization. The B2 phenotype had a wider range of molecular weights (40–120 kDa) than the LPS types A and B. In total, three LPS banding patterns plus the rough LPS type (no ladder) can be detected (Figure 3). 10.1371/journal.pntd.0001453.g003 Figure 3 Diversity of B. pseudomallei LPS banding patterns and their serological specificity. Panel A is silver strained SDS-PAGE of four different LPS phenotypes; panels B and C are immunoblotting analysis of the same LPS samples using sera from melioidosis patients with known infection by LPS genotype A, or B strains, respectively. Lanes 1–4 are typical (type A), atypical (type B), a novel atypical (type B variant or type B2), and rough LPS types, respectively; lane L is a pre-stained protein standard ladder. We note that the typical LPS was specifically seroreactive to the antibody from patient who was infected by LPS genotype A strain, whereas, the atypical LPS types (lanes 2 and 3) were seroreactive with the antibody from the LPS genotype B infected patient only. Rough LPS or no-banding LPS appearance (lane 4) was seronegative to both sera. Natural mutations in O-antigen genes and changes in bacterial phenotypes A frame-shift mutation observed in the O-antigen biosynthesis wbiI gene of B. pseudomallei strain MSHR1655 was correlated with its rough phenotype. This is one of nearly 100 strains that were isolated over 8 years from a patient with severe bronchiectasis associated with melioidosis. The mutation was an extra guanine inserted after nucleotide position 815 of the wbiI gene (Figure 4). The wbiI gene encodes an oligosaccharide epimerase/dehydratase and is conserved in all O-antigen biosynthesis gene clusters of B. pseudomallei. A mutation in this gene probably impacts on the synthesis of the O-antigen in this bacterial strain. There were 23 serial B. pseudomallei isolates observed from the chronically infected patient and the wbiI gene sequences were determined in all of them to detect frame shift mutations. The frame-shift mutation occurred in 16 isolates, all of which were collected on or after day 550 of the infection. The wild type sequence was present in the other seven isolates from earlier in the infection (Figure 4). Moreover, phenotypic characterization revealed that LPS samples extracted from the 16 wbiI mutated strains did not have the O-antigen ladder pattern (i.e the rough phenotype) based upon SDS-PAGE and silver straining (Figure 5A). Thus, it seems likely the frame-shift mutation in the wbiI gene blocks synthesis of the O-antigen. A recent study has reported that oacA gene, known to be involved in the acetylation at the O-4 position of the L-6dTalp residues of B. thailandensis O-antigen [14], is mutated in B. pseudomallei MSHR1655. Since MSHR1655 was isolated from the same patient above, we then sequenced the oacA gene in all of these clonal strains. We found that the oacA mutation occurred in the same 16 strains that had the wbiI mutation (Figure 4C). Additional study of the oacA gene in other whole genome sequenced strains determined that B. pseudomallei 112 and B. thailandensis TXDOH also had point mutation in their oacA genes (Table 1; Figure S1). To determine if the oacA gene plays only a single role in the side group modification of the L-6dTalp residues, or a dual role in combination with the synthesis of the O-antigen, both strains were tested for O-antigen production and immunogenic specificity. We found that B. pseudomallei 112 and B. thailandensis TXDOH expressed O-antigen type A ladder pattern and their O-antigen bands were strongly positive with the B. mallei LPS-specific mAb 3D11 (Figure 6) that recognized the lack of 4-O acetylation of the L-6dTalp residues [14]. This suggests the oacA gene in B. pseudomallei and B. thailandensis has a role in the acetylation at the O-4 position of the O-antigen L-6dTalp residues but is not involved in the synthesis of the O-antigen. Thus, we determined that the rough LPS phenotype observed in the 16 clonal chronic lung strains was due to the mutation of their wbiI gene, but not from the effect of the oacA mutation. In this study, we also identified six other independent rough LPS strains, but mutations did not occur in their wbiI or oacA genes. Searching for mutations in other genes of these strains warrants a follow up study to understand alternate mechanisms that generate the rough phenotype. 10.1371/journal.pntd.0001453.g004 Figure 4 Point mutations found in wbiI and oacA genes in clonal B. pseudomallei strains. These strains were collected chronologically from a single chronic lung patient who had severe bronchiectasis associated with melioidosis over almost 8 years. Panel A is the chronological order of these B. pseudomallei strains. Panel B demonstrates an extra base (“G”) that was found to cause frame-shift mutation in wbiI gene of all B. pseudomallei strains collected from day 550 onward. Panel C demonstrates the insertion of two extra bases “TC” in BPSL1936, the oacA homolog, in the same strains that had the wbiI mutation. Note: the wbiI gene of B. pseudomallei K96243 and oacA gene of B. thailandensis E264 [14] were used as comparisons. 10.1371/journal.pntd.0001453.g005 Figure 5 Differential LPS phenotypes and serum susceptibility of the chronic lung strains. Panel A demonstrates LPS phenotypes based upon SDS-PAGE analysis of select chronic lung strains; lanes 1–9, LPS samples from the chronic lung strains MSHR1043, MSHR1048, MSHR1218, MSHR1288, MSHR1290, MSHR1418, MSHR1459, MSHR1655, and MSHR3042, respectively; L, protein standard ladder. Panel B shows differential serum susceptibility in four select chronic lung B. pseudomallei strains grown in 30% of normal human serum (NHS); a well-known serum resistant B. pseudomallei strain 1026b, and a laboratory E. coli strain HB101 were used as the positive and negative controls in this study, respectively. We note that strains MSHR1655 and MSHR3042, the rough LPS strains that had mutation in their wbiI genes were unable to multiply in the presence of 30% NHS, whereas, the typical LPS strains MSHR1043 and MSHR1048 from the same patient were able to utilize the NHS as nutrients. 10.1371/journal.pntd.0001453.g006 Figure 6 Phenotypic effects of the oacA mutation in B. pseudomallei 112 and B. thailandensis TXDOH revealed by immunoblot analysis. LPS samples from B. pseudomallei K96243 and 112, B. mallei ATCC23344, and B. thailandensis E264 and TXDOH, Lanes 1–5, respectively, hybridized against serotype A patient's serum (panel A), and B. mallei LPS-specific mAb 3D11 (panel B). As predicted, LPS samples from B. pseudomallei 112 (lane 2) and B. thailandensis TXDOH (lane 5) were strongly positive to the mAb 3D11 due to the mutation of their oacA genes. Lane L is a pre-stained protein standard ladder. Because LPS is essential for outer membrane integrity and serum resistance, four B. pseudomallei strains from this chronic lung patient were further tested in serum bactericidal assays. Two of the wbiI mutant strains that expressed the rough LPS phenotype (MSHR1655 and MSHR3042) were unable to grow in the presence of 30% normal human serum (NHS). In contrast, two early infection isolates from the same patient expressing the typical LPS A phenotype (MSHR1043 and MSHR1048) were able to resist the inhibitory human serum effect and grow (Figure 5B). Furthermore, we also confirmed that the LPS genotype B2 strains were killed in growth media containing 30% NHS, whereas the LPS genotype B strains were resistant (Figure S2). We believe that this finding of serum susceptibility in LPS genotype B2 is important and deserves further investigation. Discussion Two major (A, B) and one minor (B2) LPS genotypes exist unequally in B. pseudomallei populations Despite the fact that genes responsible for the O-antigen biosynthesis in B. pseudomallei 1026b were identified many years ago [4], diversity of these genes across multiple B. pseudomallei strains has not been well studied until now. Advances in genome sequencing and comparative genomics have provided insights into the complexity and diversity of B. pseudomallei genomes. B. pseudomallei genomic studies can now strive for correlations between genomic diversity and differential phenotypes; perhaps the clinical outcomes of individual strains of B. pseudomallei may be predicted using basic genomic analysis. In our current study, we were able to establish a correlation between differential LPS phenotypes and diversity of O-antigen biosynthesis genes or known as LPS genotypes. Three different major LPS genotypes have been identified so far. LPS genotype A was designated to the strains that contained the O-antigen biosynthesis genes that were identical or very similar to those found in a reference strain 1026b [4], whereas the LPS genotype B category is represented by the atypical LPS strain 576. Finally, LPS B2 genotype was identified as a variant of the LPS genotype B because many of its O-antigen biosynthesis genes were similar to those of LPS genotype B, and both groups were serotype B positive. LPS genotype A was the most common genotype in both geographic locations: Southeast Asia and Australia where it accounted for 97.7% and 85.3% of the populations, respectively. Interestingly, the frequency of LPS genotype B was relatively high (approx. 13.8%) in Australian strains, whereas they accounted for only 2.3% of the strains from Southeast Asia. LPS genotype B2 was found in only 7 strains, 5 of which were from Australia, and the other 2 strains were from Papua New Guinea. In addition, LPS genotype B2 was also found in a member of B. thailandensis-like species which was recently discovered in Australia [22]. This would suggest that the LPS genotype B2 genes in B. pseudomallei may be acquired by horizontal gene transfer from a common soil bacterial species in Australia, or vice versa. Comparative genomics and phenotypic characterization of this LPS genotype B2 in B. pseudomallei and its near-relative species warrants further investigation. Because the LPS genotypes B and B2 were frequently found in Australia but not in Southeast Asia, it is possible that this finding may be due to different therapies used for clinical cases in these 2 endemic locations. We have investigated this and found that the majority of these isolates were obtained before any exposure to antibiotics or treatment therapy. In addition, some of the LPS genotype B strains were collected from soil in Australia, and 2 strains of the LPS genotype B2 were found in animal cases. This confirms that the occurrence of LPS types B and B2 in Australia is not associated with the exposure to antibiotics or treatment therapy. Although, we phenotyped only 24% of the isolates that were genotyped, most tested strains were perfectly matched between their genotypes and phenotypes, except those 16 rough LPS genotype A strains from a single chronic case that had mutations in their wbiI genes (Figure 4). In this current study, we were unable to identify the genetic basis or mutations in 6 independent LPS genotype A strains that did not produce the O-antigen (Table S2). Because the typical LPS was also found in B. thailandensis, the use of anti-LPS antibody based latex agglutination for the identification of B. pseudomallei in environmental specimens was not successful in an early study [23]. B. thailandensis LPS has also been shown to cross-react with rabbit and mouse sera obtained from inoculation with B. pseudomallei or B. mallei suggesting that LPS molecules from B. thailandensis, a non-pathogenic bacterium, may be useful in ongoing efforts to develop novel vaccines and/or diagnostic reagents [24]. This has brought to our attention whether low-grade B. thailandensis infections might naturally provide protection against melioidosis. Although the O-antigen biosynthesis genes in B. pseudomallei and B. thailandensis are similar, a recent study by a Singaporean group has revealed that lipid A components of the LPS from both B. pseudomallei and B. thailandensis must be different; the murine and human macrophages produced lower levels of tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-10 in response to B. pseudomallei LPS than in response to B. thailandensis LPS in vitro [25]. In our current study, the typical LPS was also found in B. oklahomensis strain EO147, formerly known as an American B. pseudomallei strain [26], suggesting that the typical LPS is widely spread in multiple Burkholderia species. This group includes highly pathogenic species such as B. pseudomallei and B. mallei, but also non-pathogenic species: B. thailandensis, B. thailandensis-like species, and B. oklahomensis. The evolution of LPS diversity across these closely related species is likely a function of differential selection and horizontal transfer of genetic elements. This diversity could play a role in frequency and distribution of disease in humans. However, without understanding molecular structures of these O-antigen types, it is difficult to access the phenotypic effects of this genetic diversity. Structural analysis of the O-antigen types B and B2 deserves further investigations. In addition, we have found that the LPS genotype B2 strains were sensitive to 30% NHS, whereas the LPS type B strains were resistant (Figure S2). This finding demonstrates a level of phenotypic differences between these two serologically related groups. We believe that the consequences for case treatment associated with these differential serum susceptibilities also warrant further investigations. The rough LPS phenotype – Adaptation to survival and persistence in a host? A previous study has shown that the two less common LPS phenotypes (smooth type B and rough type) were more prevalent in clinical than environmental isolates and more prevalent in Australian isolates than Thai isolates [10]. In our current study, LPS genotype B was found in both clinical and environmental strains from Australia, whereas the rough LPS was still found only in clinical strains. Based on our description of the molecular basis for LPS phenotypes, it is unlikely that B. pseudomallei will readily switch its LPS phenotype from A to B, or vice versa, as has been suggested previously [10]. The gene compositions of LPS genotypes A and B are very different and a simple switching mechanism is difficult to envision. In addition, we have found that at least some rough LPS strains have mutations in their O-antigen biosynthesis genes. These include 16 clonally related isolates from a single chronic lung infected patient (Table S2). All of these strains were identified as LPS genotype A with mutations in their O-antigen biosynthesis genes. Using Tn5-OT182 mutagenesis, DeShazer and colleagues identified at least seven genes in the O-antigen biosynthesis operon of B. pseudomallei 1026b that were responsible for O-antigen biosynthesis and serum resistance; these included rmlB, rmlD, wbiA, wbiC, wbiE, wbiG, and wbiI [4]. In our current study, we found point mutations in wbiI and oacA genes of B. pseudomallei isolates that were collected from a chronic lung patient (Figure 4). We hypothesize that the frame-shift mutation in the wbiI genes blocks O-antigen biosynthesis in all mutant strains, but not from the effect of the oacA mutation. This is because we observed the oacA mutations in B. pseudomallei 112 and B. thailandensis TXDOH that had normal O-antigen biosynthesis gene cluster (Table 1 and Figure S1). Our study has demonstrated that these two oacA mutant strains expressed O-antigens identical to those found in B. mallei due to lack of the 4-O acetylation of the L-6dTalp residues of the O-antigen. The lack of the 4-O acetylation of the L-6dTalp residues has recently been described in the oacA knock-out mutant B. thailandensis ZT0715 and a wild-type B. mallei ATCC23344 [14]. We have demonstrated that these wbiI mutant strains produced rough LPS and were sensitive to normal human serum suggesting that the wbiI gene encoding for epimerase, or dehydratase, was essential for the biosynthesis of B. pseudomallei O-antigen. Although loss of the O-antigen might compromise serum survival it might also be adaptive in particular niches. B. pseudomallei survival or persistence in the host might be enhanced without the surface presentation of the O-antigenic moiety of the LPS, as it would not be recognized by host immune systems and would, therefore, avoid being killed by antibodies. The O-antigenic polysaccharide of B. pseudomallei modulates the host cell response, which in turn controls the intracellular fate of B. pseudomallei inside macrophage. This was concluded from the observation that the O-antigen mutant B. pseudomallei strain SRM117 was more susceptible to macrophage killing during the early phase of infection than the parental wild-type strain 1026b [27]. This was also confirmed by the same group when they demonstrated the importance of intracellular killing by the human polymorphonuclear cells (PMNs), macrophages (Mϕs), and susceptibility to killing by 30% normal human serum [28]. LPS and CPS (capsular polysaccharide) have been used as subunits in immunizing BALB/c mice against B. pseudomallei infection [2]. Mice vaccinated with LPS developed predominantly IgM and IgG3 responses, whereas the mice vaccinated with the CPS developed a predominantly IgG2b response. Furthermore, immunization with the LPS provided an optimal protective response, and the immunized mice challenged by the aerosol route showed a small increase in the mean time to death compared with the unvaccinated controls [2]. Previously, it was shown that B. pseudomallei LPS from strain 1026b signaled through Toll-like receptor (TLR) 2 and not through TLR4 [29]. This was observed in the TLR2 knock-out mutant mice that displayed a markedly improved host defense, but it was not observed in TLR4 knock-out mice [29]. In contrast, a study in HEK293 cells demonstrated that heat-killed B. pseudomallei strains K96243 or BP-1 activated TLR2 and TLR4, and in the presence of MD-2, LPS and lipid A from BP-1 are TLR4 ligands [30]. We note that B. pseudomallei 1026b and K96243 expressed the typical O-antigen type A, but the O-antigen type of BP-1 was not reported in that study. Although there was no report of association between the LPS types and disease severity (e.g., fatal versus non-fatal, and septicemia versus localized), clinical manifestations (neurologic versus non-neurologic), or underlying risk factors (diabetic versus non-diabetic) observed in a previous study [10], full phenotypic characterization including virulence in animal models, innate immune response, etc of these different LPS types warrants further investigations given the LPS diversity that we have described. Supporting Information Figure S1 Point mutations found in gene BPSL1936 ( oacA homolog) of B. pseudomallei MSHR1655 and 112, and B. thailandensis TXDOH. These point mutations (panel A): in strain MSHR1655, the mutation was associated with 2 extra bases, “TC”, inserted right after nucleotide no. 298 of this gene; in strain 112, it was associated with a deletion of one base, “T”, at nucleotide no. 112; and in B. thailandensis TXDOH, it was associated with the 5′ truncation mutation. Amino acid sequence analysis (panel B) has demonstrated that the point mutations in MSHR1655 and 112 potentially caused frame-shift mutations in their BPSL1936 genes, and then split the gene into 2 separated open reading frames (ORFs). Two known amino acid motifs, VXXFFXXSG and WXLXXEXXXY, were present in both ORFs of MSHR1655, whereas only the latter motif was present in strain 112. We noted that both amino acid motifs were absent in B. thailandensis TXDOH. (PPT) Click here for additional data file. Figure S2 O-antigen types and differential serum susceptibility. O-antigen type A and B strains including B. pseudomallei 1026b, NCTC13178, and NCTC13179, MSHR367b, MSHR98, respectively, were resistant to 30% normal human serum (NHS); whereas the O-antigen type B2 strains : B. pseudomallei MSHR840, MSHR454, and MSHR1950, and B. thailandensis-like sp. strain MSMB121 were sensitive to the 30% NHS. A rough O-antigen type strain MSHR3042, a member of the chronic lung strains (see text), was also sensitive. We noted that B. thailandensis E264 was able to survive, but unable to multiple in the presence of 30% NHS. E. coli HB101 was used as a control serum sensitive strain. (PPT) Click here for additional data file. Table S1 Comparison of LPS genotype A, B, and B2 gene clusters. (DOC) Click here for additional data file. Table S2 List of bacterial strains used in this study and their LPS genotyping PCR results. (XLS) Click here for additional data file.
Author and article information
Contributors
G. A. Land: Role: Editor
Journal
Journal ID (nlm-ta): J Clin Microbiol
Journal ID (iso-abbrev): J. Clin. Microbiol
Journal ID (hwp): jcm
Journal ID (pmc): jcm
Journal ID (publisher-id): JCM
Title:
Journal of Clinical Microbiology
Publisher:
American Society for Microbiology
(1752 N St., N.W., Washington, DC
)
ISSN
(Print):
0095-1137
ISSN
(Electronic):
1098-660X
Publication date
(Electronic preprint):
24
February
2016
Publication date
(Electronic):
25
April
2016
Publication date
(Print):
May
2016
Publication date PMC-release: 25
April
2016
Volume: 54
Issue: 5
Pages: 1259-1268
Affiliations
[a
]Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University,
Bangkok, Thailand
[b
]Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol
University, Bangkok, Thailand
Author notes
Address correspondence to Narisara Chantratita,
narisara@
123456tropmedres.ac
.
Citation Suttisunhakul V, Wuthiekanun V, Brett PJ, Khusmith S, Day NPJ, Burtnick MN, Limmathurotsakul D, Chantratita N. 2016. Development of rapid enzyme-linked immunosorbent assays for detection of antibodies to Burkholderia pseudomallei. J Clin Microbiol 54:1259–1268. doi: 10.1128/JCM.02856-15.
Article
Publisher ID: 02856-15DOI: 10.1128/JCM.02856-15
PMC ID: 4844749
PubMed ID: 26912754
SO-VID: e6bcbfe3-8376-4003-805f-82b626e25397
Copyright © Copyright © 2016 Suttisunhakul et al.
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This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
History
Date
received
: 3
November
2015
Date
revision requested
: 9
December
2015
Date
accepted
: 6
February
2016
Page count
Figures: 3,
Tables: 4,
Equations: 0,
References: 41,
Pages: 10,
Words: 7840
Funding
Funded by: The Wellcome Trust
Award ID: 087769/Z/08/Z
Award Recipient
: Narisara Chantratita
Funded by: Faculty of Tropical Medicine, Mahidol University
http://dx.doi.org/10.13039/501100006439
Award Recipient
: Vichaya Suttisunhakul
Categories
Subject:
Immunoassays
ScienceOpen disciplines: Microbiology & Virology
Data availability:
ScienceOpen disciplines: Microbiology & Virology