Impact of template denaturation prior to whole genome amplification on gene detection in high GC-content species, Burkholderia mallei and B. pseudomallei.

OBJECTIVE: In this study, we sought to determine the types and prevalence of antimicrobial resistance determinants (ARDs) in Burkholderia spp. strains using the Antimicrobial Resistance Determinant Microarray (ARDM). RESULTS: Whole genome amplicons from 22 B. mallei (BM) and 37 B. pseudomallei (BP) isolates were tested for > 500 ARDs using ARDM v.3.1. ARDM detected the following Burkholderia spp.-derived genes, aac(6), blaBP/MBL-3, blaABPS, penA-BP, and qacE, in both BM and BP while blaBP/MBL-1, macB, blaOXA-42/43 and penA-BC were observed in BP only. The method of denaturing template for whole genome amplification greatly affected the numbers and types of genes detected by the ARDM. BlaTEM was detected in nearly a third of BM and BP amplicons derived from thermally, but not chemically denatured templates. BlaTEM results were confirmed by PCR, with 81% concordance between methods. Sequences from 414-nt PCR amplicons (13 preparations) were 100% identical to the Klebsiella pneumoniae reference gene. Although blaTEM sequences have been observed in B. glumae, B. cepacia, and other undefined Burkholderia strains, this is the first report of such sequences in BM/BP/B. thailandensis (BT) clade. These results highlight the importance of sample preparation in achieving adequate genome coverage in methods requiring untargeted amplification before analysis.

Molecular detection of Burkholderia mallei in different geographic regions of Brazil.

Glanders is a contagious disease of equids caused by the Gram-negative bacterium Burkholderia mallei. In Brazil, the disease is considered to be reemerging and has been expanding, with records of equids with positive serology in most of the federative units. However, there are few reports describing the genotypic detection of the agent. This study demonstrated the detection of B. mallei by species-specific PCR directly from tissues or from bacterial cultures, followed by amplicon sequencing in equids (equines, mules, and asinines) with positive serology for glanders in all five geographic regions of Brazil. The molecular evidence of B. mallei infection in serologically positive equids in this study expands the possibility of strain isolation and the conduction of epidemiological characterizations based on molecular information. The microbiological detection of B. mallei in cultures from nasal and palate swabs, even in equids without clinical manifestations, raises the possibility of environmental elimination of the agent.

A conserved active site PenA ß-lactamase Ambler motif specific for Burkholderia pseudomallei/B. mallei is likely responsible for intrinsic amoxicillin-clavulanic acid sensitivity and facilitates a simple diagnostic PCR assay for melioidosis.

Burkholderia pseudomallei is a soil- and water-dwelling Gram-negative bacterium that causes melioidosis in humans and animals. Amoxicillin-clavulanic acid (AMC) susceptibility has been hailed as an integral part of the screening algorithm for identification of B. pseudomallei, but the molecular basis for the inherent AMC susceptibility of this bacterium remains undefined. This study showed that B. pseudomallei (and the closely-related B. mallei) wild-type strains are the only Burkholderia spp. that contain a 70STSK73 PenA Ambler motif. This motif was present in >99.5% of 1820 analysed B. pseudomallei strains and 100% of 83 analysed B. mallei strains, and is proposed as the likely cause for their inherent AMC sensitivity. The authors developed a polymerase chain reaction (PCR) assay that specifically amplifies the penA70ST(S/F)K73-containing region from B. pseudomallei and B. mallei, but not from the remaining B. pseudomallei complex species or the 70STFK73 region from the closely-related penB of B. cepacia complex species. The abundance and purity of the 193-bp PCR fragment from putative B. pseudomallei isolates from clinical and environmental samples is likely sufficient for reliable confirmation of the presence of B. pseudomallei. The PCR assay is designed to be especially suited for use in resource-constrained areas. While not further explored in this study, the assay may allow diagnosis of putative B. mallei in culture isolates from animal and human samples.

Expression of virulence and antimicrobial related proteins in Burkholderia mallei and Burkholderia pseudomallei.

BACKGROUND: Burkholderia mallei and Burkholderia pseudomallei are both potential biological threat agents. Melioidosis caused by B. pseudomallei is endemic in Southeast Asia and Northern Australia, while glanders caused by B. mallei infections are rare. Here we studied the proteomes of different B. mallei and B. pseudomallei isolates to determine species specific characteristics. METHODS: The expressed proteins of 5 B. mallei and 6 B. pseudomallei strains were characterized using liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Subsequently, expression of potential resistance and virulence related characteristics were analyzed and compared. RESULTS: Proteome analysis can be used for the identification of B. mallei and B. pseudomallei. Both species were identified based on >60 discriminative peptides. Expression of proteins potentially involved in antimicrobial resistance, AmrAB-OprA, BpeAB-OprB, BpeEF-OprC, PenA as well as several other efflux pump related proteins and putative ß-lactamases was demonstrated. Despite, the fact that efflux pump BpeAB-OprB was expressed in all isolates, no clear correlation with an antimicrobial phenotype and the efflux-pump could be established. Also consistent with the phenotypes, no amino acid mutations in PenA known to result in ß-lactam resistance could be identified. In all studied isolates, the expression of virulence (related) factors Capsule-1 and T2SS was demonstrated. The expression of T6SS-1 was demonstrated in all 6 B. pseudomallei isolates and in 2 of the 5 B. mallei isolates. In all, except one B. pseudomallei isolate, poly-beta-1,6 N-acetyl-D-glucosamine export porin (Pga), important for biofilm formation, was detected, which were absent in the proteomes of B. mallei. Siderophores, iron binding proteins, malleobactin and malleilactone are possibly expressed in both species under standard laboratory growth conditions. Expression of multiple proteins from both the malleobactin and malleilactone polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) clusters was demonstrated in both species. All B. pseudomallei expressed at least seven of the nine proteins of the bactobolin synthase cluster (bactobolin, is a ribosome targeting antibiotic), while only in one B. mallei isolate expression of two proteins of this synthase cluster was identified. CONCLUSIONS: Analyzing the expressed proteomes revealed differences between B. mallei and B. pseudomallei but also between isolates from the same species. Proteome analysis can be used not only to identify B. mallei and B. pseudomallei but also to characterize the presence of important factors that putatively contribute to the pathogenesis of B. mallei and B. pseudomallei.

A Case Report of Burkholderia mallei Infection Leading to Pneumonia.

BACKGROUND: Glanders is a rare zoonotic disease caused by Burkholderia mallei. Humans can be infected by B. mallei, which causes cutaneous lymphadenitis and pneumonia, leading to sepsis and death in severe cases. CASE PRESENTATION: We report a case of a 60-year-old male who was diagnosed with glanders. The patient who had a history of diabetes presented with cough, expectoration, and fever. Computed tomography (CT) imaging showed B. mallei infection in the right upper lobe of the lung with mediastinal lymph node involvement and the lingual segment of the left lung. Moreover, the posterior basal segment of the lower lobe of both lungs had inflammation. Subsequently, B. mallei infection was confirmed by lymph node biopsy and bronchoalveolar lavage multiplex PCR-based targeted gene sequencing. After meropenem treatment, the patient was discharged, and CT imaging showed reduced absorption of pulmonary inflammatory lesions. CONCLUSIONS: Glanders is a rare disease that can cause skin infection, lymphadenitis, and pneumonia, and in severe cases, it can be life-threatening. The diagnosis of this disease mainly relies on microbiological culture and pathological biopsy. Diagnosis is also facilitated by multiplex PCRbased targeted gene sequencing. Glanders is treated with cephalosporins, carbapenems, and other sensitive antibiotics.

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.

Identification of Burkholderia mallei Isolates with Polymerase Chain Reaction-Restriction Fragment Length Polymorphism.

Burkholderia mallei is the main cause of glanders as a dangerous contagious zoonosis disease that is mostly observed in single-hoofed animals, especially horses. Modern molecular techniques have been recently employed to improve epidemiology for identifying and searching for strains of this bacterium at different times and locations. Due to the unknown number of circulating strains and lack of preventive methods, glanders is still observed in the form of epidemics. The present study aimed to evaluate six field isolates plus two laboratory strains of Borkolderia mallei and Burkholderia pseudomallei using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. All the isolates and strains were microbially cultured in the glycerol nutrient and glycerol agar media. The individually grown colonies of the bacterium were used in the biochemical tests. The DNA of isolates was extracted by boiling, and the PCR-RFLP test was conducted on their genome. Finally, the bacterium was injected into guinea pigs to induce the Straus reaction. The biochemical assays (or bioassays) confirmed the isolates as Burkholderia mallei. The PCR-RFLP assay demonstrated a product for Burkholderia mallei with a length of 650 bp. Nevertheless, 250 and 400 bp were produced for Burkholderia pseudomallei. The swollen scrotum pointed to the occurrence of the Straus reaction. The PCR-RFLP is a proper differential diagnosis technique for B. mallei; moreover, it is a suitable method for differentiating between Burkholderia mallei and Burkholderia pseudomallei. This technique can detect Burkholderia mallei in a short time with high precision and sensitivity.

Pathogenic bacteria remodel central metabolic enzyme to build a cyclopropanol warhead.

Bacteria of the Burkholderia pseudomallei (BP) group pose a global health threat, causing the infectious diseases melioidosis, a common cause of pneumonia and sepsis, and glanders, a contagious zoonosis. A trait of BP bacteria is a conserved gene cluster coding for the biosynthesis of polyketides (malleicyprols) with a reactive cyclopropanol unit that is critical for virulence. Enzymes building this warhead represent ideal targets for antivirulence strategies but the biochemical basis of cyclopropanol formation is unknown. Here we describe the formation of the malleicyprol warhead. We show that BurG, an unusual NAD+-dependent member of the ketol-acid reductoisomerase family, constructs the strained cyclopropanol ring. Biochemical assays and a suite of eight crystal structures of native and mutated BurG with bound analogues and inhibitors provide snapshots of each step of the complex reaction mechanism, involving a concealed oxidoreduction and a C-S bond cleavage. Our findings illustrate a remarkable case of neofunctionalisation, where a biocatalyst from central metabolism has been evolutionarily repurposed for warhead production in pathogens.

Genetic diversity and spatial distribution of Burkholderia mallei by core genome-based multilocus sequence typing analysis.

Burkholderia mallei is the etiological agent of glanders, a highly contagious and often fatal disease in equids. Due to the high genetic clonality of B. mallei, high-resolution typing assays are necessary to differentiate between individual strains. Here we report on the development and validation of a robust and reproducible core genome-based Multi Locus Sequence Typing Assay (cgMLST) for B. mallei, which is based on 3328 gene targets and enables high-resolution typing at the strain level. The assay was validated using a set of 120 B. mallei genomes from public databases and 23 newly sequenced outbreak strains from in-house strain collections. In this cgMLST analysis, strains from different geographic regions were clearly distinguished by at least 70 allele differences, allowing spatial clustering while closely related and epidemiologically related strains were separated by only zero to three alleles. Neither the different sequencing technologies nor the assembly strategies had an influence on the cgMLST results. The developed cgMLST is highly robust, reproducible and can be used for outbreak investigations, source tracking and molecular characterization of new B. mallei isolates.

Molecular characterization of Burkholderia mallei strains isolated from horses in Brazil (2014-2017).

Glanders is an infectious zoonosis caused by Burkholderia (B.) mallei that mainly affects equids. The objective of this work was to provide additional knowledge on the diversity of the strains circulating in Brazil. Six Burkholderia mallei isolates obtained during necropsies of glanderous horses between 2014 and 2017 in two different states (Pernambuco and Alagoas) were analyzed by polymerase chain reaction-high-resolution melting (PCR-HRM). While four strains (9902 RSC, BM_campo 1, BM_campo 3 and UFAL2) clustered in the L3B2 branch, which already includes the Brazilian 16-2438_BM#8 strain, two strains (BM_campo 2.1 and BM_campo 2.2) clustered within the L3B3sB3 branch, which mostly includes older isolates, from Europe and the Middle East. Whole genome sequencing of two of these strains (UFAL2 and BM_campo 2.1), belonging to different branches, confirmed the HRM typing results and refined the links between the strains, including the description of the L3B3Sb3Gp1SbGp1 genotype, never reported so far for contemporary strains. These results suggest different glanders introduction events in Brazil, including a potential link with strains of European origin, related to colonization or trade.

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.

Molecular epidemiology of Burkholderia mallei isolates from India (2015-2016): New SNP markers for strain tracing.

Glanders, caused by a bacterium called B. mallei, is primarily an infectious horse and human disease. Although its incidence is rare in developed countries, it is nonetheless prevalent in several geographical areas of the world. There is a lack of cost-effective, rapid and specific molecular typing tools for epidemiological tracing of glanders cases. We previously reported an SNP-based typing method that categorizes global B. mallei strains into three lineages (L1 to L3), as well as additional branches, sub-branches and groups. However, further discrimination of the Indian and Pakistani isolates within the L2B2sB2 sub-branch was not possible due to the lack of sufficient epidemiological markers. In this study, 10 B. mallei strains isolated from four states in India during 2015-2016 were whole genome sequenced; SNP analysis further confirmed their position in the L2B2sB2 branch. To better track the strains, four new markers targeting Indian or Pakistani strains, and specifically targeting sub-groups within the Indian strains, were identified. The new SNP markers were tested and validated on the 10 Indian isolates included in this study as well as on 6 contemporary B. mallei Pakistani strains. These rapid and discriminating typing tools will contribute to the epidemiological monitoring of B. mallei infections, particularly in South Asia and the Middle East, endemic regions of the disease.

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.

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.

A genetic variant of Burkholderia mallei detected in Kuwait: Consequences for the PCR diagnosis of glanders.

Glanders is a contagious zoonotic disease caused by Burkholderia mallei. Following the detection of glanders positive horses using the OIE complement fixation test, the tissues of two horses were analysed by PCR. While PCR systems targeting the Burkholderia pseudomallei complex gave positive signals, the species-specific PCR systems targeting B. mallei (fliP-IS407A) and B. pseudomallei (orf11)-the OIE recommended targets-resulted in negative signals. However, the presence of B. mallei in these tissues was confirmed with a recently described B. mallei-specific real-time PCR system and genotyping with MLST- and SNP-based methods, performed on the most positive tissue, identified a genotype closely related to B. mallei strains recently isolated in the Middle East. This study leads to recommendations regarding the use of PCR systems for the molecular diagnosis of glanders, especially in regions where the circulating B. mallei strains have not yet been fully genetically characterized.

Burkholderia collagen-like protein 8, Bucl8, is a unique outer membrane component of a putative tetrapartite efflux pump in Burkholderia pseudomallei and Burkholderia mallei.

Bacterial efflux pumps are an important pathogenicity trait because they extrude a variety of xenobiotics. Our laboratory previously identified in silico Burkholderia collagen-like protein 8 (Bucl8) in the hazardous pathogens Burkholderia pseudomallei and Burkholderia mallei. We hypothesize that Bucl8, which contains two predicted tandem outer membrane efflux pump domains, is a component of a putative efflux pump. Unique to Bucl8, as compared to other outer membrane proteins, is the presence of an extended extracellular region containing a collagen-like (CL) domain and a non-collagenous C-terminus (Ct). Molecular modeling and circular dichroism spectroscopy with a recombinant protein, corresponding to this extracellular CL-Ct portion of Bucl8, demonstrated that it adopts a collagen triple helix, whereas functional assays screening for Bucl8 ligands identified binding to fibrinogen. Bioinformatic analysis of the bucl8 gene locus revealed it resembles a classical efflux-pump operon. The bucl8 gene is co-localized with downstream fusCDE genes encoding fusaric acid (FA) resistance, and with an upstream gene, designated as fusR, encoding a LysR-type transcriptional regulator. Using reverse transcriptase (RT)-qPCR, we defined the boundaries and transcriptional organization of the fusR-bucl8-fusCDE operon. We found exogenous FA induced bucl8 transcription over 80-fold in B. pseudomallei, while deletion of the entire bucl8 locus decreased the minimum inhibitory concentration of FA 4-fold in its isogenic mutant. We furthermore showed that the putative Bucl8-associated pump expressed in the heterologous Escherichia coli host confers FA resistance. On the contrary, the Bucl8-associated pump did not confer resistance to a panel of clinically-relevant antimicrobials in Burkholderia and E. coli. We finally demonstrated that deletion of the bucl8-locus drastically affects the growth of the mutant in L-broth. We determined that Bucl8 is a component of a novel tetrapartite efflux pump, which confers FA resistance, fibrinogen binding, and optimal growth.

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.

First record of Burkholderia mallei Turkey 10 strain originating from glanderous horses from Brazil.

Burkholderia (B.) mallei is the causative agent of glanders in Equidae. This study describes the first record of the Turkey 10 strain of B. mallei in glanderous horses in Northeastern of Brazil. This description should contribute to the future actions of diagnosis, control, and eradication of this disease in Brazil.

Evaluation of Burkholderia mallei ΔtonB Δhcp1 (CLH001) as a live attenuated vaccine in murine models of glanders and melioidosis.

BACKGROUND: Glanders caused by Burkholderia mallei is a re-emerging zoonotic disease affecting solipeds and humans. Furthermore, B. mallei is genetically related to B. pseudomallei, which is the causative agent of melioidosis. Both facultative intracellular bacteria are classified as tier 1 select biothreat agents. Our previous study with a B. mallei ΔtonB Δhcp1 (CLH001) live-attenuated vaccine demonstrated that it is attenuated, safe and protective against B. mallei wild-type strains in the susceptible BALB/c mouse model. METHODOLOGY/PRINCIPAL FINDING: In our current work, we evaluated the protective efficacy of CLH001 against glanders and melioidosis in the more disease-resistant C57BL/6 mouse strain. The humoral as well as cellular immune responses were also examined. We found that CLH001-immunized mice showed 100% survival against intranasal and aerosol challenge with B. mallei ATCC 23344. Moreover, this vaccine also afforded significant cross-protection against B. pseudomallei K96243, with low level bacterial burden detected in organs. Immunization with a prime and boost regimen of CLH001 induced significantly greater levels of total and subclasses of IgG, and generated antigen-specific splenocyte production of IFN-γ and IL-17A. Interestingly, protection induced by CLH001 is primarily dependent on humoral immunity, while CD4+ and CD8+ T cells played a less critical protective role. CONCLUSIONS/SIGNIFICANCE: Our data indicate that CLH001 serves as an effective live attenuated vaccine to prevent glanders and melioidosis. The quantity and quality of antibody responses as well as improving cell-mediated immune responses following vaccination need to be further investigated prior to advancement to preclinical studies.

A real-time loop mediated isothermal amplification assay for molecular detection of Burkholderia mallei, the aetiological agent of a zoonotic and re-emerging disease glanders.

Burkholderia mallei, a potential biological warfare agent, is the causative agent of an infectious, fatal, and zoonotic disease, called glanders. Accurate and early diagnosis of glanders is important to control the disease lethality and infection spread. Molecular detection of B. mallei is considered strenuous because B. mallei is a subtractive genomic clone of B. pseudomallei. The present study was aimed at development of a real-time LAMP assay for detection of B. mallei. The LAMP assay was highly sensitive and could detect ≥250 fg of genomic DNA of B. mallei and ≥100 copies of recombinant plasmid containing target DNA sequence. In artificially spiked blood and water samples, it could detect ≥2.1 × 103 and ≥4.73 × 102 CFU/mL of B. mallei, respectively. The assay was highly specific for B. mallei as none of the other bacteria used in the study tested positive. The reported LAMP assay being simple and rapid can be a viable alternative to PCR-based glanders diagnostic assays in glanders endemic regions with resource-limited settings.