Clinical and Molecular Characterization of Human Burkholderia mallei Infection, Brazil.

We report a case of Burkholderia mallei causing glanders in a 73-year-old patient from the Northeast Region of Brazil. The patient was hospitalized with severe pneumonia. PCR and genomic sequencing confirmed B. mallei in pleural drainage. Genotyping revealed a novel genotype, emphasizing the need for genetic surveillance in zoonotic infections.

Tracking the footsteps of Burkholderia mallei: determination of the molecular differences and potential resistance genes.

Background/aim: Chemical biological radiological nuclear threats are at an important point in the agenda of world health today, as they can cause mass deaths. B. mallei attracts attention as a potential biological warfare agent due to its features such as multidrug resistance, a rapid transmission mechanism via aerosol, the absence of a complete treatment protocol for the infection it causes, and the absence of an approved vaccine for protection against the bacteria. B. mallei suspect samples must be studied by experienced personnel in biosafety level III laboratories. B mallei is a difficult and troublesome pathogen to diagnose and many unknowns about B. mallei today. Therefore, the aim of the study was to determine the molecular differences and potential resistance genes of B mallei strains. Materials and methods: Determination of the molecular differences and potential resistance genes of B mallei strains with new bioinformatics approaches by comparatively examining the data of 29 B mallei strains, 10 of which were isolated from Türkiye, on the genome list of the National Biotechnology Information Center (NCBI). Results: According to the genome annotations of the origins, the origin containing the highest number of CDS which is 5172 was found as the 11th strain obtained in Türkiye in 1949. The origin with the highest number of pseudogenes was determined as 23,344 (China 7) origin. Two hundred and eighty-five pseudogenes found in this strain were obtained from a knee effusion in Myanmar. According to chromosome 2 data, B. mallei strain was determined as the most similar strain to ATCC 23344, line 11 with NCTC 10229 strain, and SAVP1 strain was determined as the least similar strain. When the antimicrobial resistance gene markers of the isolates included in the study were examined, amrA and amrB, qacG ade, Burkholderia pseudomallei Omp38 were found to be carrying. Conclusion: In terms of public health, it was thought that the data obtained as a result of our study about B mallei, which is defined as a biological weapon, is very valuable for creating treatment protocols to be applied to possible epidemics in the future. In addition, the available genetic epidemiological data of these strains belonging to a category that is dangerous to work with in a laboratory environment were reviewed.

Protein Microarray-Guided Development of a Highly Sensitive and Specific Dipstick Assay for Glanders Serodiagnostics.

Burkholderia mallei, the causative agent of glanders, is a clonal descendant of Burkholderia pseudomallei, the causative agent of melioidosis, which has lost its environmental reservoir and has a restricted host range. Despite limitations in terms of sensitivity and specificity, complement fixation is still the official diagnostic test for glanders. Therefore, new tools are needed for diagnostics and to study the B. mallei epidemiology. We recently developed a highly sensitive serodiagnostic microarray test for human melioidosis based on the multiplex detection of B. pseudomallei proteins. In this study, we modified our array tests by using anti-horse IgG conjugate and tested sera from B. mallei-infected horses (n = 30), negative controls (n = 39), and horses infected with other pathogens (n = 14). Our array results show a sensitivity of 96.7% (confidence interval [CI] 85.5 to 99.6%) and a specificity of 100.0% (CI, 95.4 to 100.0%). The reactivity pattern of the positive sera on our array test allowed us to identify a set of 12 highly reactive proteins of interest for glanders diagnosis. The B. mallei variants of the three best protein candidates were selected for the development of a novel dipstick assay. Our point-of-care test detected glanders cases in less than 15 min with a sensitivity of 90.0% (CI, 75.7 to 97.1%) and a specificity of 100.0% (CI, 95.4 to 100.0%). The microarray and dipstick can easily be adopted for the diagnosis of both B. mallei and B. pseudomallei infections in different animals. Future studies will show whether multiplex serological testing has the potential to differentiate between these pathogens.

Characterization of immunoglobulin and cytokine responses in Burkholderia mallei infected equids.

Burkholderia mallei causes a highly fatal infectious disease in equines known as glanders. It is one of the OIE listed notifiable diseases, which entails strict control policy measures once B. mallei infection is confirmed in the susceptible hosts. Humans, especially equine handlers, veterinary professionals and laboratory workers are at greater risk to acquire the B. mallei infection directly through prolonged contact with glanderous equines, and indirectly through unprotected handling of B. mallei contaminated materials. Further, natural resistance of B. mallei to multiple antibiotics, aerosol transmission, lack of effective vaccine and treatment make this organism a potential agent of biological warfare. Results of experimental B. mallei infection in mouse and non-human primates and immunization with live attenuated B. mallei strains demonstrated that activation of early innate and adaptive immune responses play a critical role in controlling B. mallei infection. However, the immune response elicited by the primary hosts (equids) B. mallei infection is poorly understood. Therefore, we aimed to investigate immune responses in glanders affected horses (n = 23) and mules (n = 1). In this study, chronically infected equids showed strong humoral responses (IgM, IgG and IgA) specific to B. mallei type 6 secretory proteins such as Hcp1, TssA and TssB. The infected equids also elicited robust cellular responses characterized by significantly elevated levels of IFN-γ, TNF-α, IL-12, IL-17 and IL-6 in PBMCs. In addition, stimulation of equine PBMCs by Hcp1 resulted in the further elevation of these cytokines. Thus, the present study indicated that antibody response and T helper cell (Th) type 1-associated cytokines were the salient features of chronic B. mallei infection in horses. The immune responses also suggest further evaluation of these proteins as potential vaccine candidates.

First glanders cases detected in Nepal underscore the need for surveillance and border controls.

BACKGROUND: Glanders is a transmissible zoonotic disease caused by Burkholderia mallei that infects equids and humans. No glanders cases in equids were reported so far in Nepal. CASE PRESENTATION: Following suspected glanders in animals with clinical signs in different regions in Nepal, serum samples were tested by CFT, ELISA and Luminex® tests. Two horses and a mule tested positive for glanders by all tests, while two other equids only tested positive by ELISA and Luminex®. Analysis of swabs and pus samples by a PCR system targeting B. mallei confirmed the presence of the bacterium in the samples collected from the 3 equids that yielded positive results in all serological tests. Genotyping of the three PCR positive samples with a SNP-based method identified a genotype closely related to the B. mallei strains circulating in India. CONCLUSION: Confirmation of glanders cases underscores the need of implementing a surveillance program in Nepal and a strict control of the animal movement across the borders.

Molecular Typing of Burkholderia mallei Isolates from Equids with Glanders, India.

We collected 10 Burkholderia mallei isolates from equids in 9 districts in India during glanders outbreaks in 2013-2016. Multilocus variable-number tandem-repeat analysis showed 7 outbreak area-related genotypes. The study highlights the utility of this analysis for epidemiologically tracing of specific B. mallei isolates during outbreaks.

In Vitro Antibacterial Activity and In Vivo Efficacy of Sulbactam-Durlobactam against Pathogenic Burkholderia Species.

The Gram-negative bacterial genus Burkholderia includes several hard-to-treat human pathogens: two biothreat species, Burkholderia mallei (causing glanders) and B. pseudomallei (causing melioidosis), and the B. cepacia complex (BCC) and B. gladioli, which cause chronic lung infections in persons with cystic fibrosis. All Burkholderia spp. possess an Ambler class A Pen ß-lactamase, which confers resistance to ß-lactams. The ß-lactam-ß-lactamase inhibitor combination sulbactam-durlobactam (SUL-DUR) is in clinical development for the treatment of Acinetobacter infections. In this study, we evaluated SUL-DUR for in vitro and in vivo activity against Burkholderia clinical isolates. We measured MICs of SUL-DUR against BCC and B. gladioli (n = 150), B. mallei (n = 30), and B. pseudomallei (n = 28), studied the kinetics of inhibition of the PenA1 ß-lactamase from B. multivorans and the PenI ß-lactamase from B. pseudomallei by durlobactam, tested for blaPenA1 induction by SUL-DUR, and evaluated in vivo efficacy in a mouse model of melioidosis. SUL-DUR inhibited growth of 87.3% of the BCC and B. gladioli strains and 100% of the B. mallei and B. pseudomallei strains at 4/4 µg/ml. Durlobactam potently inhibited PenA1 and PenI with second-order rate constant for inactivation (k2/K) values of 3.9 × 106 M-1 s-1 and 2.6 × 103 M-1 s-1 and apparent Ki (Kiapp) of 15 nM and 241 nM, respectively, by forming highly stable covalent complexes. Neither sulbactam, durlobactam, nor SUL-DUR increased production of PenA1. SUL-DUR demonstrated activity in vivo in a murine melioidosis model. Taken together, these data suggest that SUL-DUR may be useful as a treatment for Burkholderia infections.

Comparison of three non-human primate aerosol models for glanders, caused by Burkholderia mallei.

Burkholderia mallei is a gram-negative obligate animal pathogen that causes glanders, a highly contagious and potentially fatal disease of solipeds including horses, mules, and donkeys. Humans are also susceptible, and exposure can result in a wide range of clinical forms, i.e., subclinical infection, chronic forms with remission and exacerbation, or acute and potentially lethal septicemia and/or pneumonia. Due to intrinsic antibiotic resistance and the ability of the organisms to survive intracellularly, current treatment regimens are protracted and complicated; and no vaccine is available. As a consequence of these issues, and since B. mallei is infectious by the aerosol route, B. mallei is regarded as a major potential biothreat agent. To develop optimal medical countermeasures and diagnostic tests, well characterized animal models of human glanders are needed. The goal of this study was to perform a head-to-head comparison of models employing three commonly used nonhuman primate (NHP) species, the African green monkey (AGM), Rhesus macaque, and the Cynomolgus macaque. The natural history of infection and in vitro clinical, histopathological, immunochemical, and bacteriological parameters were examined. The AGMs were the most susceptible NHP to B. mallei; five of six expired within 14 days. Although none of the Rhesus or Cynomolgus macaques succumbed, the Rhesus monkeys exhibited abnormal signs and clinical findings associated with B. mallei infection; and the latter may be useful for modeling chronic B. mallei infection. Based on the disease progression observations, gross and histochemical pathology, and humoral and cellular immune response findings, the AGM appears to be the optimal model of acute, lethal glanders infection. AGM models of infection by B. pseudomallei, the etiologic agent of melioidosis, have been characterized recently. Thus, the selection of the AGM species provides the research community with a single NHP model for investigations on acute, severe, inhalational melioidosis and glanders.

Validation of three qPCR for the detection of Burkholderia mallei in equine tissue samples.

Burkholderia mallei is the causative agent of glanders, a zoonosis listed by the World Organization for Animal Health as of mandatory notification. In this work, a comparison of three qPCR protocols was made, two of them based on articles by other authors and one standardized in house, this last one aiming at a genomic region that does not exist in other species of the Burkholderia genus. All qPCRs showed high efficiency and good repeatability. However, reactions with Cq between 36 and 40 were considered suspicious and unreliable, requiring greater clinical criteria to analyze the results.

Methodological tools to study species of the genus Burkholderia.

Bacteria belonging to the Burkholderia genus are extremely versatile and diverse. They can be environmental isolates, opportunistic pathogens in cystic fibrosis, immunocompromised or chronic granulomatous disease patients, or cause disease in healthy people (e.g., Burkholderia pseudomallei) or animals (as in the case of Burkholderia mallei). Since the genus was separated from the Pseudomonas one in the 1990s, the methodological tools to study and characterize these bacteria are evolving fast. Here we reviewed the techniques used in the last few years to update the taxonomy of the genus, to study gene functions and regulations, to deepen the knowledge on the drug resistance which characterizes these bacteria, and to elucidate their mechanisms to establish infections. The availability of these tools significantly impacts the quality of research on Burkholderia and the choice of the most appropriated is fundamental for a precise characterization of the species of interest.Key points• Updated techniques to study the genus Burkholderia were reviewed.• Taxonomy, genomics, assays, and animal models were described.• A comprehensive overview on recent advances in Burkholderia studies was made.

16S rDNA and ITS Sequence Diversity of Burkholderia mallei Isolated from Glanders-Affected Horses and Mules in India (2013-2019).

Glanders is a highly contagious and fatal infection of equids caused by the bacteria known as Burkholderia mallei. It is one of the notifiable equine diseases and is still present in Asia, South America and Africa. In India, glanders re-emerged in 2006, and thereafter, increasing numbers of cases were reported in different regions of the country. Between 2013 and 2019, 39 B. mallei were isolated from glanders-affected horses (n = 30) and mules (n = 9) from seven states of India such as Uttar Pradesh, Haryana, Delhi, Himachal Pradesh, Gujarat, Maharashtra and Tamil Nadu. In this study, the phylogenetic relationships of these isolates were assessed by sequence analysis of 16S rDNA gene and ITS region. Purified PCR-amplified products of 16S rDNA gene and ITS region were sequenced, aligned and phylogenetic trees were constructed using MEGA 11 software. Additionally, B. mallei 16S rDNA (n = 36) and ITS (n = 18) sequences available in the GenBank were also included for analysis to determine the diversity of older B. mallei isolates with recent Indian isolates. Both the phylogeny showed that the majority of the recent isolates from India are closely related to each other, but are genetically diverse from older isolates that originated from India. Nucleotide substitutions were also observed in a single and double position in 12 recent and two old Indian isolates. The study also indicates that similar B. mallei strains were responsible for glanders outbreaks in different states (Uttar Pradesh- Himachal Pradesh and Uttar Pradesh- Haryana) and this is due to the migration of infected animals from one state to another state. This study implies that 16S rDNA and ITS region may be used for molecular characterization of B. mallei associated with glanders in resource-limited settings.

Validated Methods for Removing Select Agent Samples from Biosafety Level 3 Laboratories.

The Federal Select Agent Program dictates that all research entities in the United States must rigorously assess laboratory protocols to sterilize samples being removed from containment areas. We validated procedures using sterile filtration and methanol to remove the following select agents: Francisella tularensis, Burkholderia pseudomallei, B. mallei, Yersinia pestis, and Bacillus anthracis. We validated methanol treatment for B. pseudomallei. These validations reaffirm safety protocols that enable researchers to keep samples sufficiently intact when samples are transferred between laboratories.

Synthesis of the Oligosaccharides of Burkholderia pseudomallei and B. mallei Capsular Polysaccharide and Preliminary Immunological Studies of Their Protein Conjugates.

Efficient strategies were developed for the synthesis of 6-deoxy-d-manno-heptopyranose and its ß-(1 → 3)-linked oligomers as fragments of the common and major capsular polysaccharide, type I O-PS, of Burkholderia pseudomallei and Burkholderia mallei. The unusual heptose was synthesized from mannose, highlighted by the facile Wittig reaction and anti-Markovnikov hydroboration of the resultant olefin. The difficult ß-mannosidic linkage in the oligosaccharides was achieved in high stereoselectivity by H-bond-mediated aglycone delivery. All of the oligosaccharides were conjugated with the carrier protein CRM197. Preliminary immunological evaluations of the resultant glycoconjugates in mice verified their efficacy to elicit high titers of immunoglobulin G antibodies and robust T-cell-dependent immune responses. It was also found that the trisaccharide conjugates provoked the strongest immune responses, worthy of further in-depth study for vaccine development.

Pathological and Immunohistochemical Analyses of Naturally Occurring Equine Glanders Using an Anti-BpaB Antibody.

Glanders is caused by the gram-negative bacterium Burkholderia mallei. In this study, we investigated the histopathology and immunohistochemical localization of B. mallei in natural cases of equine glanders. Four horses showing clinical signs of nasal discharge and multiple cutaneous nodules or papulae in the hindlimbs and abdomen were reported in Mongolia. They tested positive for B. mallei infection on complement fixation, Rose Bengal agglutination, and mallein tests. Gross and histological lesions observed in these cases were similar to those previously reported in equine glanders. Immunohistochemistry using a monoclonal antibody to B. mallei BpaB showed localization of the bacterial antigen in the cytoplasm of neutrophils, macrophages, epithelioid cells, and multinucleated giant cells in the pyogranulomas and abscesses in target organs. Some alveolar type II cells and bronchiolar epithelial cells also contained the antigen. These results suggest that the anti-BpaB antibody is useful for identifying B. mallei-infected cell types in naturally infected horses.

Secondary metabolites from the Burkholderia pseudomallei complex: structure, ecology, and evolution.

Bacterial secondary metabolites play important roles in promoting survival, though few have been carefully studied in their natural context. Numerous gene clusters code for secondary metabolites in the genomes of members of the Bptm group, made up of three closely related species with distinctly different lifestyles: the opportunistic pathogen Burkholderia pseudomallei, the non-pathogenic saprophyte Burkholderia thailandensis, and the host-adapted pathogen Burkholderia mallei. Several biosynthetic gene clusters are conserved across two or all three species, and this provides an opportunity to understand how the corresponding secondary metabolites contribute to survival in different contexts in nature. In this review, we discuss three secondary metabolites from the Bptm group: bactobolin, malleilactone (and malleicyprol), and the 4-hydroxy-3-methyl-2-alkylquinolines, providing an overview of each of their biosynthetic pathways and insight into their potential ecological roles. Results of studies on these secondary metabolites provide a window into how secondary metabolites contribute to bacterial survival in different environments, from host infections to polymicrobial soil communities.

Lipopolysaccharides from Different Burkholderia Species with Different Lipid A Structures Induce Toll-Like Receptor 4 Activation and React with Melioidosis Patient Sera.

Lipopolysaccharides (LPSs) of Gram-negative bacteria comprise lipid A, core, and O-polysaccharide (OPS) components. Studies have demonstrated that LPSs isolated from the pathogenic species Burkholderia pseudomallei and Burkholderia mallei and from less-pathogenic species, such as Burkholderia thailandensis, are potent immune stimulators. The LPS structure of B. pseudomallei, the causative agent of melioidosis, is highly conserved in isolates from Thailand; however, the LPSs isolated from other, related species have not been characterized to enable understanding of their immune recognition and antigenicities. Here, we describe the structural and immunological characteristics of the LPSs isolated from eight Burkholderia species and compare those for B. pseudomallei to those for the other seven species. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), gas chromatography (GC), SDS-PAGE, Toll-like receptor 4 (TLR4) stimulation, and immunoblot analysis were performed on these Burkholderia species. MALDI-TOF profiles demonstrated that Burkholderia lipid A contains predominantly penta-acylated species modified with 4-amino-4-deoxy-arabinose residues at both terminal phosphate groups. The lipid A could be differentiated based on mass differences at m/z 1,511, 1,642, 1,773, and 1,926 and on fatty acid composition. LPSs of all species induced TLR4-dependent NF-κB responses; however, while SDS-PAGE analysis showed similar LPS ladder patterns for B. pseudomallei, B. thailandensis, and B. mallei, these patterns differed from those of other Burkholderia species. Interestingly, immunoblot analysis demonstrated that melioidosis patient sera cross-reacted with OPSs of other Burkholderia species. These findings can be used to better understand the characteristics of LPS in Burkholderia species, and they have implications for serological diagnostics based on the detection of antibodies to OPS.

Melioidosis patient serum-reactive synthetic tetrasaccharides bearing the predominant epitopes of Burkholderia pseudomallei and Burkholderia mallei O-antigens.

Melioidosis and glanders, respectively caused by the Gram-negative bacteria Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm), are considered as urgent public health issues in developing countries and potential bioterrorism agents. Bp and Bm lipopolysaccharides (LPS) have been identified as attractive vaccine candidates for the development of prophylactic measures against melioidosis and glanders. Bp and Bm express structurally similar LPSs wherein the O-antigen (OAg) portion consists of a heteropolymer whose repeating unit is a disaccharide composed of d-glucose and 6-deoxy-l-talose residues, the latter being diversely acetylated and methylated. Herein we report the synthesis of two tetrasaccharides mimicking the main substitution epitopes of Bp and Bm LPS OAgs. The assembly of the tetrasaccharides was achieved using a sequential glycosylation strategy while relying on the late-stage epimerization of the inner rhamnose into a 6-deoxy-l-talose residue. We show that these synthetic compounds strongly react with culture-confirmed Thai melioidosis patient serum and closely mimic the antigenicity of native Bp OAg. Our results suggest that these tetrasaccharides could be suitable candidates for the development of vaccines and/or diagnostic tools against melioidosis and glanders.

A novel selective medium for the isolation of Burkholderia mallei from equine specimens.

BACKGROUND: Burkholderia mallei is a Gram-negative bacterium that causes glanders, a zoonotic disease, especially in equine populations (e.g. horses, donkeys, and mules). B. mallei usually grows slowly on most culture media, and this property makes it difficult to isolate from clinical specimens. One of the problems is that B. mallei is easily overgrown by other bacteria, especially in animal specimens collected from non-sterile sites. The aim of this study was to develop a new selective agar for the laboratory diagnosis of glanders. We formulated a new agar, named BM agar, to enrich B. mallei growth, but inhibit the growth of other bacteria and fungi based on their antimicrobial profiles. We compared the growth of B. mallei on BM with Xie's and PC agars, the two previously described selective agars for B. mallei. RESULTS: BM agar could sufficiently grow almost all of the tested B. mallei strains within 72 h: only one out of the 38 strains grew scantly after 72 h of incubation. BM agar was further tested with other Burkholderia species and various bacterial species commonly found in the nasal cavities and on the skin of horses. We have found that other Burkholderia species including B. pseudomallei and B. thailandensis can grow on BM agar, but non-Burkholderia species cannot. Furthermore, the specificities of the three selective agars were tested with or without spiking B. mallei culture into clinical specimens of non-sterile sites collected from healthy horses. The results showed that BM agar inhibited growths of fungi and other bacterial species better than PC and Xie's agars. We have also found that growth of B. mallei on BM agar was equivalent to that on 5% horse blood agar and was significantly greater than those on the other two agars (P < 0.05). CONCLUSIONS: We believe that BM agar can be used to efficiently isolate B. mallei from mixed samples such as those typically collected from horses and other contaminated environments.

A Machine Learning-Based Raman Spectroscopic Assay for the Identification of Burkholderia mallei and Related Species.

Burkholderia (B.) mallei, the causative agent of glanders, and B. pseudomallei, the causative agent of melioidosis in humans and animals, are genetically closely related. The high infectious potential of both organisms, their serological cross-reactivity, and similar clinical symptoms in human and animals make the differentiation from each other and other Burkholderia species challenging. The increased resistance against many antibiotics implies the need for fast and robust identification methods. The use of Raman microspectroscopy in microbial diagnostic has the potential for rapid and reliable identification. Single bacterial cells are directly probed and a broad range of phenotypic information is recorded, which is subsequently analyzed by machine learning methods. Burkholderia were handled under biosafety level 1 (BSL 1) conditions after heat inactivation. The clusters of the spectral phenotypes and the diagnostic relevance of the Burkholderia spp. were considered for an advanced hierarchical machine learning approach. The strain panel for training involved 12 B. mallei, 13 B. pseudomallei and 11 other Burkholderia spp. type strains. The combination of top- and sub-level classifier identified the mallei-complex with high sensitivities (>95%). The reliable identification of unknown B. mallei and B. pseudomallei strains highlighted the robustness of the machine learning-based Raman spectroscopic assay.

Development of Rose Bengal test against mallein test for rapid diagnosis of equine glanders.

BACKGROUND AND AIM: Burkholderia mallei, the etiologic agent of the disease known as glanders. Clinical and bacteriological diagnosis of glanders is difficult in the early stages of the disease. Currently, mallein (allergic hypersensitivity test) is used for the diagnosis of glanders. The mallein test requires an experienced laboratory person and lasts 48 h. Therefore, in order to quickly diagnose the disease, especially in areas (such as the borders of the country) that cannot be kept animals, new methods should be used to identify the disease. The Rose Bengal is a serological diagnostic test and has been recommended by the World Organization for Animal Health (OIE). In this study, the Rose Bengal test (RBT) was evaluated for the diagnosis of equine glanders, and its diagnostic was compared with mallein test. MATERIALS AND METHODS: Sera from 70 naturally infected culture-positive horses, 3 equines that were sensitized by injecting antigen and 110 healthy equines were tested. Specificity and sensitivity of RBT and mallein test when testing culture-positive equines were calculated. RESULTS: Diagnosis of glanders with both methods yield the same results, but Rose Bengal test is much faster than mallein test for diagnosis of equine glanders. CONCLUSION: By comparative RBT with mallein test, it can be considered, RBT test has been used for rapid detection of glanders with features such as, ease of use and can be applicable without specialized equipment and trained personnel. Because the RBT is simpler and rapid to perform, the inclusion of the test as a supplementary test for the diagnosis of glanders in field conditions is recommended.