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Abstract Background Glanders and melioidosis are contagious zoonotic diseases caused by Burkholderia mallei and B. pseudomallei, respectively. Bacterial isolation and polymerase chain reaction (PCR) have been used to detect these bacteria in animals suspected of infection; however, both methods require skilled experimental techniques and expensive equipment. These obstacles make it difficult to diagnose B. mallei and B. pseudomallei infections in areas where reagents and equipment are difficult to procure. To solve this problem, we developed an easy and ready-to-use dried-format diagnostic tool based on loop-mediated isothermal amplification (LAMP) method. Results The primer set targeting the internal transcribed spacer (ITS) region detected 10 genomic copies of B. mallei DNA and B. pseudomallei DNA using the conventional liquid LAMP method. This primer set did not detect any other Burkholderia species. Using this novel primer set, a dried-format in-house LAMP method with high sensitivity and specificity was developed. This method was used to test for the presence of B. mallei DNA in swabs collected from the nasal cavity and ulcerated skin of 19 B. mallei-infected horses and five uninfected horses and was compared with the real-time PCR method. These two tests showed 87.5% agreement for the positive samples and 100% agreement for the negative samples. This method detected all tested B. pseudomallei clinical isolates. Conclusions We established the first dry LAMP method for the detection of B. mallei and B. pseudomallei. This study provided a simple, rapid, cost-effective, and sensitive diagnostic tool for glanders and melioidosis.

BackgroundBurkholderia pseudomallei, causative agent of melioidosis, is a One Health concern as it is acquired directly from soil and water and causes disease in humans and agricultural and wild animals. We examined B. pseudomallei in soil and goats at a single farm in the Northern Territory of Australia where >30 goats acquired melioidosis over nine years.Methodology/principal findingsWe cultured 45 B. pseudomallei isolates from 35 goats and sampled soil in and around goat enclosures to isolate and detect B. pseudomallei and evaluate characteristics associated with its occurrence; 33 soil isolates were obtained from 1993-1994 and 116 in 2006. Ninety-two goat and soil isolates were sequenced; mice were challenged with six soil isolates to evaluate virulence. Sampling depth and total N/organic C correlated with B. pseudomallei presence. Twelve sequence types (STs) were identified. Most goat infections (74%) were ST617, some with high similarity to 2006 soil isolates, suggesting ST617 was successful at persisting in soil and infecting goats. ST260 and ST266 isolates were highly virulent in mice but other isolates produced low/intermediate virulence; three of these were ST326 isolates, the most common soil ST in 2006. Thus, virulent and non-virulent lineages can co-occur locally. Three genes associated with virulence were present in ST260 and ST266, absent in most ST326 isolates, and present or variably present in ST617.Conclusions/significanceAgricultural animals can influence B. pseudomallei abundance and diversity in local environments. This effect may persist, as B. pseudomallei was detected more often from soil collected inside and adjacent to goat enclosures years after most goats were removed. Following goat removal, the low virulence ST326, which was not isolated from soil when goats were present, became the predominant ST in soil by 2006. Although multiple diverse lineages of B. pseudomallei may exist in a given location, some may infect mammals more efficiently than others.

IntroductionBurkholderia pseudomallei, a Gram-negative bacterium inhabiting soil and fresh water, is the causative agent of melioidosis, a formidable disease in the tropics. The emergence of antibiotic resistance and the extended duration of treatment, up to 20 weeks, have posed significant challenges in combatting melioidosis. As an alternative approach, bacteriophage therapy is being explored.MethodsTo identify the most promising bacteriophage for future therapeutic applications, we designed a screening process to address the barrier of phage specificity due to the O-antigen receptor diversity. By using two biosafe strains, Bp82 (O-antigen type A) and 576mn (O-antigen type B), to represent the major serotype A and B, we screened 145 phage samples collected from soil and water in southern Thailand.ResultsTen of them demonstrated the ability to overcome differences in O-antigen types, yielding positive plaques formed on culture of both bacterial strains. Subsequently, we isolated 22 bacteriophages from these samples, one was adaptively mutated during the screening process, named ΦPK23V1, which had the ability to infect up to 83.3% (115/138) of tested B. pseudomallei strains, spanning both serogroups. Employing a panel of surface polysaccharide antigen mutant strains, we explored the role of capsular polysaccharide (CPS) and O-antigens as essential components for phage infection. All isolated phages were classified into the P2-like myophage group. Additionally, our research revealed a point mutation in the phage tail fiber gene (gpH), expanding the host range of ΦPK23V1, even in the absence of CPS and O-antigens.DiscussionHowever, it was evident that ΦPK23V1 is a lysogenic phage, which cannot be readily applied for therapeutic use. This discovery sheds light on the receptor binding domain of P2-like bacteriophages in B. pseudomallei. Collectively, our study has identified bacteriophages with a broad host range within B. pseudomallei strains, enhancing our understanding of phage–host interactions and offering insights into the role of the phage tail fiber gene in host cell entry.

Melioidosis, a severe bacterial illness caused by Burkholderia pseudomallei, is prevalent in most parts of Thailand, including its southern region situated within the Malay Peninsula. Despite a lower reported incidence rate of melioidosis in the South compared to the Northeast, the mortality rate remains persistently high. This study aimed to better understand the epidemiology and investigate the presence of B. pseudomallei in the natural environment of southern Thailand. Using multi-locus sequence typing (MLST), we characterized B. pseudomallei isolates derived from human cases and compared them with previously reported sequence types (STs) from the same region. A total of 263 clinical isolates retrieved from 156 melioidosis patients between 2014 and 2020 were analyzed, revealing 72 distinct STs, with 25 (35%) matching STs from Finkelstein's environmental isolates collected in southern Thailand during 1964-1967. Notably, strains bearing STs 288, 84, 54, 289, and 46 were frequently found among patients. Additionally, we observed strain diversity with multiple STs in 13 of 59 patients, indicating exposure to various B. pseudomallei genotypes in the environmental sources of the infection. Environmental surveys were conducted in Songkhla Province to detect B. pseudomallei in soil and water samples where local patients lived. Of the 2737 soil samples from 208 locations and 244 water samples from diverse sources, 52 (25%) soil sampling locations and 63 (26%) water sources were cultured positive for B. pseudomallei. Positive soil samples were predominantly found in animal farming area and non-agricultural zones like mountains and grasslands, while water samples were frequently positive in waterfalls, streams, and surface runoffs, with only 9% of rice paddies testing positive. Collectively, a significant proportion of recent melioidosis cases in Songkhla Province can be attributed to known B. pseudomallei STs persisting in the environment for at least the past six decades. Further characterization of B. pseudomallei isolates from recent environment surveys is warranted. These findings illuminate the contemporary landscape of B. pseudomallei infections and their environmental prevalence in southern Thailand, contributing to the regional threat assessment in Thailand and Southeast Asia.

Abstract Background Burkholderia pseudomallei is the etiological agent of melioidosis and a CDC category B select agent with no available effective vaccine. Previous immunizations in mice have utilized the lipopolysaccharide (LPS) as a potential vaccine target because it is known as one of the most important antigenic epitopes in B. pseudomallei. Complicating this strategy are the four different B. pseudomallei LPS O-antigen types: A, B, B2, and rough. Sero-crossreactivity is common among O-antigens of Burkholderia species. Here, we identified the presence of multiple B. pseudomallei O-antigen types and sero-crossreactivity in its near-neighbor species. Results PCR screening of O-antigen biosynthesis genes, phenotypic characterization using SDS-PAGE, and immunoblot analysis showed that majority of B. mallei and B. thailandensis strains contained the typical O-antigen type A. In contrast, most of B. ubonensis and B. thailandensis-like strains expressed the atypical O-antigen types B and B2, respectively. Most B. oklahomensis strains expressed a distinct and non-seroreactive O-antigen type, except strain E0147 which expressed O-antigen type A. O-antigen type B2 was also detected in B. thailandensis 82172, B. ubonensis MSMB108, and Burkholderia sp. MSMB175. Interestingly, B. thailandensis-like MSMB43 contained a novel serotype B positive O-antigen. Conclusions This study expands the number of species which express B. pseudomallei O-antigen types. Further work is required to elucidate the full structures and how closely these are to the B. pseudomallei O-antigens, which will ultimately determine the efficacy of the near-neighbor B serotypes for vaccine development.

Abstract Background Phylogeographic reconstruction of some bacterial populations is hindered by low diversity coupled with high levels of lateral gene transfer. A comparison of recombination levels and diversity at seven housekeeping genes for eleven bacterial species, most of which are commonly cited as having high levels of lateral gene transfer shows that the relative contributions of homologous recombination versus mutation for Burkholderia pseudomallei is over two times higher than for Streptococcus pneumoniae and is thus the highest value yet reported in bacteria. Despite the potential for homologous recombination to increase diversity, B. pseudomallei exhibits a relative lack of diversity at these loci. In these situations, whole genome genotyping of orthologous shared single nucleotide polymorphism loci, discovered using next generation sequencing technologies, can provide very large data sets capable of estimating core phylogenetic relationships. We compared and searched 43 whole genome sequences of B. pseudomallei and its closest relatives for single nucleotide polymorphisms in orthologous shared regions to use in phylogenetic reconstruction. Results Bayesian phylogenetic analyses of >14,000 single nucleotide polymorphisms yielded completely resolved trees for these 43 strains with high levels of statistical support. These results enable a better understanding of a separate analysis of population differentiation among >1,700 B. pseudomallei isolates as defined by sequence data from seven housekeeping genes. We analyzed this larger data set for population structure and allele sharing that can be attributed to lateral gene transfer. Our results suggest that despite an almost panmictic population, we can detect two distinct populations of B. pseudomallei that conform to biogeographic patterns found in many plant and animal species. That is, separation along Wallace's Line, a biogeographic boundary between Southeast Asia and Australia. Conclusion We describe an Australian origin for B. pseudomallei, characterized by a single introduction event into Southeast Asia during a recent glacial period, and variable levels of lateral gene transfer within populations. These patterns provide insights into mechanisms of genetic diversification in B. pseudomallei and its closest relatives, and provide a framework for integrating the traditionally separate fields of population genetics and phylogenetics for other bacterial species with high levels of lateral gene transfer.

Abstract Background Burkholderia pseudomallei is the etiologic agent of melioidosis, a significant cause of morbidity and mortality where this infection is endemic. Genomic differences among strains of B. pseudomallei are predicted to be one of the major causes of the diverse clinical manifestations observed among patients with melioidosis. The purpose of this study was to examine the role of genomic islands (GIs) as sources of genomic diversity in this species. Results We found that genomic islands (GIs) vary greatly among B. pseudomallei strains. We identified 71 distinct GIs from the genome sequences of five reference strains of B. pseudomallei: K96243, 1710b, 1106a, MSHR668, and MSHR305. The genomic positions of these GIs are not random, as many of them are associated with tRNA gene loci. In particular, the 3' end sequences of tRNA genes are predicted to be involved in the integration of GIs. We propose the term "tRNA-mediated site-specific recombination" (tRNA-SSR) for this mechanism. In addition, we provide a GI nomenclature that is based upon integration hotspots identified here or previously described. Conclusion Our data suggest that acquisition of GIs is one of the major sources of genomic diversity within B. pseudomallei and the molecular mechanisms that facilitate horizontally-acquired GIs are common across multiple strains of B. pseudomallei. The differential presence of the 71 GIs across multiple strains demonstrates the importance of these mobile elements for shaping the genetic composition of individual strains and populations within this bacterial species.

Abstract Background The ability of Burkholderia pseudomallei to survive in water likely contributes to its environmental persistence in endemic regions. To determine the physiological adaptations which allow B. pseudomallei to survive in aqueous environments, we performed microarray analyses of B. pseudomallei cultures transferred from Luria broth (LB) to distilled water. Findings Increased expression of a gene encoding for a putative membrane protein (BPSL0721) was confirmed using a lux-based transcriptional reporter system, and maximal expression was noted at approximately 6 hrs after shifting cells from LB to water. A BPSL0721 deficient mutant of B. pseudomallei was able to survive in water for at least 90 days indicating that although involved, BPSL0721 was not essential for survival. BPSL2961, a gene encoding a putative phosphatidylglycerol phosphatase (PGP), was also induced when cells were shifted to water. This gene is likely involved in cell membrane biosynthesis. We were unable to construct a PGP mutant suggesting that the gene is not only involved in survival in water but is essential for cell viability. We also examined mutants of polyhydroxybutyrate synthase (phbC), lipopolysaccharide (LPS) oligosaccharide and capsule synthesis, and these mutations did not affect survival in water. LPS mutants lacking outer core were found to lose viability in water by 200 days indicating that an intact LPS core provides an outer membrane architecture which allows prolonged survival in water. Conclusion The results from these studies suggest that B. pseudomallei survival in water is a complex process that requires an LPS molecule which contains an intact core region.

BackgroundBurkholderia pseudomallei possesses a diverse set of genes which encode a vast array of biological functions reflecting its clinical, ecological and phenotypic diversity. Strain variation is linked to geographic location as well as pattern of land uses. This soil-dwelling Gram-negative pathogen causes melioidosis, a tropical disease endemic in northern Australia and Southeast Asian regions including Bangladesh. Phylogeographic analyses of B. pseudomallei isolates by molecular typing techniques could be used to examine the diversity of this organism as well as to track melioidosis epidemics.MethodsIn this study, 22 B. pseudomallei isolates, of which 20 clinical and two soil isolates were analyzed, utilizing Real-time PCR assay and multilocus sequence typing (MLST). The sequences were then submitted to PubMLST database for analysis and construction of phylogenetic tree.FindingsA total of 12 different sequence types (STs) that includes four novel STs were identified for the first time. Strains having STs 1005, 1007 and 56 were the most widespread STs frequently isolated in Bangladesh. ST 1005, ST 56, ST 1007 and ST 211 have been detected not only in Bangladesh but are also present in many Southeast Asian countries.SignificanceST 1005 was detected in both soil and clinical samples of Gazipur. Most prevalent, ST 56 has been previously reported from Myanmar, Thailand, Cambodia and Vietnam, confirming the persistence of the genotype over the entire continent. Further large-scale study is necessary to find out the magnitude of the infection and its different reservoirs in the environment along with phylogeographic association.

Background: Glanders and melioidosis are contagious zoonotic diseases caused by Burkholderia mallei and B. pseudomallei, respectively. Bacterial isolation and polymerase chain reaction (PCR) have been used to detect these bacteria in animals suspected of infection; however, both methods require skilled experimental techniques and expensive equipment. These obstacles make it difficult to diagnose B. mallei and B. pseudomallei infections in areas where reagents and equipment are difficult to procure. To solve this problem, we developed an easy and ready-to-use dried-format diagnostic tool based on loop-mediated isothermal amplification (LAMP) method. Results: The primer set targeting the internal transcribed spacer (ITS) region detected 10 genomic copies of B. mallei DNA and B. pseudomallei DNA using the conventional liquid LAMP method. This primer set did not detect any other Burkholderia species. Using this novel primer set, a dried-format in-house LAMP method with high sensitivity and specificity was developed. This method was used to test for the presence of B. mallei DNA in swabs collected from the nasal cavity and ulcerated skin of 19 B. mallei-infected horses and five uninfected horses and was compared with the real-time PCR method. These two tests showed 87.5% agreement for the positive samples and 100% agreement for the negative samples. This method detected all tested B. pseudomallei clinical isolates. Conclusions: We established the first dry LAMP method for the detection of B. mallei and B. pseudomallei. This study provided a simple, rapid, cost-effective, and sensitive diagnostic tool for glanders and melioidosis. [ABSTRACT FROM AUTHOR]

Background: Burkholderia pseudomallei, causative agent of melioidosis, is a One Health concern as it is acquired directly from soil and water and causes disease in humans and agricultural and wild animals. We examined B. pseudomallei in soil and goats at a single farm in the Northern Territory of Australia where >30 goats acquired melioidosis over nine years. Methodology/Principal findings: We cultured 45 B. pseudomallei isolates from 35 goats and sampled soil in and around goat enclosures to isolate and detect B. pseudomallei and evaluate characteristics associated with its occurrence; 33 soil isolates were obtained from 1993–1994 and 116 in 2006. Ninety-two goat and soil isolates were sequenced; mice were challenged with six soil isolates to evaluate virulence. Sampling depth and total N/organic C correlated with B. pseudomallei presence. Twelve sequence types (STs) were identified. Most goat infections (74%) were ST617, some with high similarity to 2006 soil isolates, suggesting ST617 was successful at persisting in soil and infecting goats. ST260 and ST266 isolates were highly virulent in mice but other isolates produced low/intermediate virulence; three of these were ST326 isolates, the most common soil ST in 2006. Thus, virulent and non-virulent lineages can co-occur locally. Three genes associated with virulence were present in ST260 and ST266, absent in most ST326 isolates, and present or variably present in ST617. Conclusions/Significance: Agricultural animals can influence B. pseudomallei abundance and diversity in local environments. This effect may persist, as B. pseudomallei was detected more often from soil collected inside and adjacent to goat enclosures years after most goats were removed. Following goat removal, the low virulence ST326, which was not isolated from soil when goats were present, became the predominant ST in soil by 2006. Although multiple diverse lineages of B. pseudomallei may exist in a given location, some may infect mammals more efficiently than others. Author summary: We studied genomic diversity in the environmental bacterial pathogen, Burkholderia pseudomallei, which caused the disease melioidosis in goats at a small farm in northern Australia. By comparing genomes from 92 B. pseudomallei isolates from goats and soil, we discovered 12 diverse lineages at this location, three of which infected the majority of goats and were cultured from the soil of goat enclosures. We also conducted a mouse challenge experiment using six isolates from this farm and determined that two soil isolates displayed much greater virulence than the others. Finally, we investigated soil factors associated with B. pseudomallei occurrence and found 1) most B. pseudomallei isolates were sampled from irrigated soils, and 2) B. pseudomallei was much more likely to be encountered at soil depths of 30 cm than 10 cm. The presence of this pathogen in the soil severely impacted maintenance of the goat herd due to high mortality and emphasizes its One Health implications for other livestock operations in tropical countries where B. pseudomallei is endemic. Our findings provide insight into the importance of more fully understanding soil habitats that promote B. pseudomallei presence, thereby increasing the risk of melioidosis to humans and agricultural animals. [ABSTRACT FROM AUTHOR]

Lipopolysaccharides (LPS) are cell wall components from Gram-negative bacteria and are composed of three covalently linked regions: the O-antigen, the core oligosaccharide, and the lipid A moiety, which carries most of their endotoxic activity. The objective of this study was to isolate and compare the lipid A structures from ruminal LPS derived from total mixed ration (TMR)- and pasture-fed cows, by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Ruminal bacteria were collected from two rumen-cannulated Holstein cows; one fed a TMR (60:40, forage–concentrate) and the other pasture fed. The representativeness of each sample was validated by comparing the rumen microbiome from the cows in our study to the core rumen microbiome from the previous literature. Lipopolysaccharides from each respective sample were extracted with a phenol–water extraction procedure and purified via ultracentrifugation. To isolate lipid A from the core and O-antigen, pure ruminal LPS samples were hydrolyzed with acetic acid. Lipid A derived from the TMR-fed cow potentially exhibited a tetra-acylated structure, whereas lipid A derived from the pasture-fed cow potentially exhibited a penta-acylated lipid A structure. Both samples were quantified using limulus amebocyte lysate (LAL) assay and exhibited low endotoxic activity, consistent with the MALDI-TOF MS observations. Results indicate that the lipid A acylation pattern differs between diets, and that ruminal bacteria express solely under-acylated lipid A structures contrary to hexa-acylated lipid A, typically expressed by bacteria such as E. coli.

Melioidosis, caused by Burkholderia pseudomallei, is a notifiable disease associated with a high mortality rate in Thailand. The disease is highly endemic in northeast Thailand, while its prevalence in other parts of the country is poorly documented. This study aimed at improving the surveillance system for melioidosis in southern Thailand, where the disease was believed to be underreported. Two adjacent southern provinces, Songkhla and Phatthalung, were selected as the model provinces to study melioidosis. There were 473 individuals diagnosed with culture-confirmed melioidosis by clinical microbiology laboratories at four tertiary care hospitals in both provinces from January 2014 to December 2020. The median age was 54 years (IQR 41.5–64), 284 (60%) of the patients were adults ≥50 years of age, and 337 (71.2%) were male. We retrospectively analyzed 455 patients treated at either Songklanarind Hospital, Hatyai Hospital, Songkhla Provincial Hospital, or Phatthalung Provincial Hospital, of whom 181 (39.8%) patients died. The median duration from admission to death was five days (IQR 2–17). Of the 455 patients, 272 (57.5%) had at least one clinical risk factor, and 188 (39.8%) had diabetes. Two major clinical manifestations, bacteremia and pneumonia, occurred in 274 (58.1%) and 166 (35.2%) patients, respectively. In most cases, 298 (75%) out of 395 local patients were associated with rainfall. Over the seven years of the study, the average annual incidence was 2.87 cases per 100,000 population (95% CI, 2.10 to 3.64). This study has confirmed that these two provinces of southern Thailand are endemic to melioidosis; even though the incidence rate is much lower than that of the Northeast, the mortality rate is comparably high.

The current study was initiated when our specific-pathogen-free laboratory toms developed unexpectedly high levels of cross-reactive antibodies to human SARS-CoV-2 (SCoV2) receptor binding domain (RBD) upon mating with feline coronavirus (FCoV)-positive queens. Multi-sequence alignment analyses of SCoV2 Wuhan RBD and four strains each from FCoV serotypes 1 and 2 (FCoV1 and FCoV2) demonstrated an amino acid sequence identity of 11.5% and a similarity of 31.8% with FCoV1 RBD (12.2% identity and 36.5% similarity for FCoV2 RBD). The sera from toms and queens cross-reacted with SCoV2 RBD and reacted with FCoV1 RBD and FCoV2 spike-2, nucleocapsid, and membrane proteins, but not with FCoV2 RBD. Thus, the queens and toms were infected with FCoV1. Additionally, the plasma from six FCoV2-inoculated cats reacted with FCoV2 and SCoV2 RBDs, but not with FCoV1 RBD. Hence, the sera from both FCoV1-infected cats and FCoV2-infected cats developed cross-reactive antibodies to SCoV2 RBD. Furthermore, eight group-housed laboratory cats had a range of serum cross-reactivity to SCoV2 RBD even 15 months later. Such cross-reactivity was also observed in FCoV1-positive group-housed pet cats. The SCoV2 RBD at a high non-toxic dose and FCoV2 RBD at a 60–400-fold lower dose blocked the in vitro FCoV2 infection, demonstrating their close structural conformations essential as vaccine immunogens. Remarkably, such cross-reactivity was also detected by the peripheral blood mononuclear cells of FCoV1-infected cats. The broad cross-reactivity between human and feline RBDs provides essential insights into developing a pan-CoV vaccine.

Melioidosis, a severe bacterial illness caused by Burkholderia pseudomallei, is prevalent in most parts of Thailand, including its southern region situated within the Malay Peninsula. Despite a lower reported incidence rate of melioidosis in the South compared to the Northeast, the mortality rate remains persistently high. This study aimed to better understand the epidemiology and investigate the presence of B. pseudomallei in the natural environment of southern Thailand. Using multi-locus sequence typing (MLST), we characterized B. pseudomallei isolates derived from human cases and compared them with previously reported sequence types (STs) from the same region. A total of 263 clinical isolates retrieved from 156 melioidosis patients between 2014 and 2020 were analyzed, revealing 72 distinct STs, with 25 (35%) matching STs from Finkelstein's environmental isolates collected in southern Thailand during 1964–1967. Notably, strains bearing STs 288, 84, 54, 289, and 46 were frequently found among patients. Additionally, we observed strain diversity with multiple STs in 13 of 59 patients, indicating exposure to various B. pseudomallei genotypes in the environmental sources of the infection. Environmental surveys were conducted in Songkhla Province to detect B. pseudomallei in soil and water samples where local patients lived. Of the 2737 soil samples from 208 locations and 244 water samples from diverse sources, 52 (25%) soil sampling locations and 63 (26%) water sources were cultured positive for B. pseudomallei. Positive soil samples were predominantly found in animal farming area and non-agricultural zones like mountains and grasslands, while water samples were frequently positive in waterfalls, streams, and surface runoffs, with only 9% of rice paddies testing positive. Collectively, a significant proportion of recent melioidosis cases in Songkhla Province can be attributed to known B. pseudomallei STs persisting in the environment for at least the past six decades. Further characterization of B. pseudomallei isolates from recent environment surveys is warranted. These findings illuminate the contemporary landscape of B. pseudomallei infections and their environmental prevalence in southern Thailand, contributing to the regional threat assessment in Thailand and Southeast Asia. Author summary: This study conducted a genetic analysis of Burkholderia pseudomallei, the bacterium causing a deadly tropical disease, from 156 patients in southern Thailand during 2014–2020. It found various genotypes, some of which were consistent with the genotypes found in the environment dating back to the 1960s. Extensive testing of soil and water in the region revealed contamination with B. pseudomallei. Areas such as animal farms, mountains, grasslands, and natural water sources e.g., streams and waterfalls, were mostly affected. This finding confirmed that some B. pseudomallei genotypes have had a long-standing presence in the environment, contributing to recent human cases. This study not only enhance our understanding of the spread of melioidosis throughout Southeast Asia but also highlights the importance of ongoing research and monitoring of B. pseudomallei in natural environments to address potential risks associated with the bacterium's persistence. [ABSTRACT FROM AUTHOR]

Abstract 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

BackgroundBurkholderia pseudomallei is an environmental bacterium that causes melioidosis. A facultative intracellular pathogen, B. pseudomallei can induce multinucleated giant cells (MNGCs) leading to plaque formation in vitro. B. pseudomallei can switch colony morphotypes under stress conditions. In addition, different isolates have been reported to have varying virulence in vivo, but genomic evolution and the relationship with plaque formation is poorly understood.Methodology/principle findingsTo gain insights into genetic underpinnings of virulence of B. pseudomallei, we screened plaque formation of 52 clinical isolates and 11 environmental isolates as well as 4 isogenic morphotype isolates of B. pseudomallei strains K96243 (types II and III) and 153 (types II and III) from Thailand in A549 and HeLa cells. All isolates except one environmental strain (A4) and K96243 morphotype II were able to induce plaque formation in both cell lines. Intracellular growth assay and confocal microscopy analyses demonstrated that the two plaque-forming-defective isolates were also impaired in intracellular replication, actin polymerization and MNGC formation in infected cells. Whole genome sequencing analysis and PCR revealed that both isolates had a large genomic loss on the same region in chromosome 2, which included Bim cluster, T3SS-3 and T6SS-5 genes.Conclusions/significanceOur plaque screening and genomic studies revealed evidence of impairment in plaque formation in environmental isolates of B. pseudomallei that is associated with large genomic loss of genes important for intracellular multiplication and MNGC formation. These findings suggest that the genomic and phenotypic differences of environmental isolates may be associated with clinical infection.

Although acute melioidosis is the most common outcome of Burkholderia pseudomallei infection, we have documented a case, P314, where disease severity lessened with time, and the pathogen evolved towards a commensal relationship with the host. In the current study, we used whole-genome sequencing to monitor this long-term symbiotic relationship to better understand B. pseudomallei persistence in P314's sputum despite intensive initial therapeutic regimens. We collected and sequenced 118 B. pseudomallei isolates from P314's airways over a >16-year period, and also sampled the patient's home environment, recovering six closely related B. pseudomallei isolates from the household water system. Using comparative genomics, we identified 126 SNPs in the core genome of the 124 isolates or 162 SNPs/indels when the accessory genome was included. The core SNPs were used to construct a phylogenetic tree, which demonstrated a close relationship between environmental and clinical isolates and detailed within-host evolutionary patterns. The phylogeny had little homoplasy, consistent with a strictly clonal mode of genetic inheritance. Repeated sampling revealed evidence of genetic diversification, but frequent extinctions left only one successful lineage through the first four years and two lineages after that. Overall, the evolution of this population is nonadaptive and best explained by genetic drift. However, some genetic and phenotypic changes are consistent with in situ adaptation. Using a mouse model, P314 isolates caused greatly reduced morbidity and mortality compared to the environmental isolates. Additionally, potentially adaptive phenotypes emerged and included differences in the O-antigen, capsular polysaccharide, motility, and colony morphology. The >13-year co-existence of two long-lived lineages presents interesting hypotheses that can be tested in future studies to provide additional insights into selective pressures, niche differentiation, and microbial adaptation. This unusual melioidosis case presents a rare example of the evolutionary progression towards commensalism by a highly virulent pathogen within a single human host.

Burkholderia pseudomallei is a Gram-negative bacterium and the causative agent of melioidosis in humans and animals in the tropics. The clinical manifestations of melioidosis are diverse, ranging from localized infections to whole-body sepsis. The effective serological method is crucial for the point-of-care diagnosis of melioidosis. The aim of this study was to develop indirect immunofluorescence assay (IFA)-based methods for detecting immunoglobulin G (IgG) antibodies in melioidosis patients. These methods use whole-cell antigens made from recombinant E. coli strains that express major B. pseudomallei antigens, including TssM, OmpH, AhpC, BimA, and Hcp1. A total of 271 serum samples from culture-confirmed melioidosis patients (n = 81), patients with other known infections (n = 70), and healthy donors (n = 120) were tested. Our study showed that the recombinant TssM strain had the highest performance, with 92.6% sensitivity, 100% specificity, 100% positive predictive value, 96.9% negative predictive value, 97.8% efficiency, 97.0% accuracy, and no cross-reactivity. The method agreement analysis based on k efficiency calculations showed that all five IFA methods perfectly agreed with the standard culturing method, while the traditional indirect hemagglutination (IHA) method moderately agreed with the culture. In summary, our investigations showed that the TssM-IFA method could be used for melioidosis diagnosis.

Burkholderia pseudomallei, the Gram-negative bacterium which causes melioidosis, is a threat to human and a wide range of animal species. There is an increased concern of melioidosis in Indonesian primate facilities, especially following case reports of fatal melioidosis in captive macaques and orangutans. Our preliminary serosurveillance of immunoglobulin G (IgG) to B. pseudomallei lipopolysaccharide showed that a significant number of captive and wild macaques in the western part of Java, Indonesia, have been exposed to B. pseudomallei. To better characterize the humoral immune response in those animals, a panel of assays were conducted on the same blood plasma specimens that were taken from 182 cynomolgus macaques (M. fascicularis) and 88 pig-tailed macaques (M. nemestrina) reared in captive enclosures and wild habitats in the western part of Java, Indonesia. The enzyme-linked immunosorbent assays (ELISAs) in this study were conducted to detect IgG against B. pseudomallei proteins; alkyl hydroperoxide reductase subunit C (AhpC), hemolysin-coregulated protein (Hcp1), and putative outer membrane porin protein (OmpH). The performances of those immunoassays were compared to ELISA against B. pseudomallei LPS, which has been conducted previously. Seropositivity to at least one assay was 76.4% (139/182) and 13.6% (12/88) in cynomolgus macaques and pig-tailed macaques, respectively. Analysis of demographic factors showed that species and primate facility were significant factors. Cynomolgus macaques had higher probability of exposure to B. pseudomallei. Moreover, macaques in Jonggol facility also had higher probability, compared to macaques in other facilities. There were no statistical associations between seropositivity with other demographic factors such as sex, age group, and habitat type. There were strong positive correlations between the absorbance results of AhpC, HcpI, and OmpH assays, but not with LPS assay. Our analysis suggested that Hcp1 assay would complement LPS assay in melioidosis serosurveillance in macaques.

Burkholderia pseudomallei is the environmental bacillus that causes melioidosis; a disease clinically significant in Australia and Southeast Asia but emerging in tropical and sub-tropical regions around the globe. Previous studies have placed the ancestral population of the organism in Australia with a single lineage disseminated to Southeast Asia. We have previously characterized B. pseudomallei isolates from New Guinea and the Torres Strait archipelago; remote regions that share paleogeographic ties with Australia. These studies identified regional biogeographical boundaries. In this study, we utilize whole-genome sequencing to reconstruct ancient evolutionary relationships and ascertain correlations between paleogeography and present-day distribution of this bacterium in Australasia. Our results indicate that B. pseudomallei from New Guinea fall into a single clade within the Australian population. Furthermore, clades from New Guinea are region-specific; an observation possibly linked to limited recent anthropogenic influence in comparison to mainland Australia and Southeast Asia. Isolates from the Torres Strait archipelago were distinct yet scattered among those from mainland Australia. These results provide evidence that the New Guinean and Torres Strait lineages may be remnants of an ancient portion of the Australian population. Rising sea levels isolated New Guinea and the Torres Strait Islands from each other and the Australian mainland, and may have allowed long-term isolated evolution of these lineages, providing support for a theory of microbial biogeography congruent with that of macro flora and fauna. Moreover, these findings indicate that contemporary microbial biogeography theories should consider recent and ongoing impacts of globalisation and human activity.

Introduction Melioidosis is a complex bacterial disease discovered just over 100 years ago in Rangoon, Burma (now Myanmar) in south-east Asia. (1) Transmission occurs mostly in tropical and subtropical regions [...]

Burkholderia pseudomallei (Bp) causes the disease melioidosis. The main cause of mortality in this disease is septic shock triggered by the host responding to lipopolysaccharide (LPS) components of the Gram-negative outer membrane. Bp LPS is thought to be a weak inducer of the host immune system. LPS from several strains of Bp were purified and their ability to induce the inflammatory mediators TNF-α and iNOS in murine macrophages at low concentrations was investigated. Innate and adaptive immunity qPCR arrays were used to profile expression patterns of 84 gene targets in response to the different LPS types. Additional qPCR validation confirmed large differences in macrophage response. LPS from a high-virulence serotype B strain 576a and a virulent rough central nervous system tropic strain MSHR435 greatly induced the innate immune response indicating that the immunopathogenesis of these strains is different than in infections with strains similar to the prototype strain 1026b. The accumulation of autophagic vesicles was also increased in macrophages challenged with highly immunogenic Bp LPS. Gene induction and concomitant cytokine secretion profiles of human PBMCs in response to the various LPS were also investigated. MALDI-TOF/TOF was used to probe the lipid A portions of the LPS, indicating substantial structural differences that likely play a role in host response to LPS. These findings add to the evolving knowledge of host-response to bacterial LPS, which can be used to better understand septic shock in melioidosis patients and in the rational design of vaccines.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a potentially life-threatening infection that can affect humans and a wide variety of animals in the tropics. In December 2017, a swine melioidosis case was discovered during a meat inspection at a privately-owned slaughterhouse in Nakhon Si Thammarat Province in southern Thailand. The infection, which continued for several months, caused a dispute about where the disease began. An environmental investigation into two farms—both involved in raising the first infected pig—ensued. Through genetic analysis, the investigation revealed that a contaminated water supply at one farm was the probable source of infection. The three local sequence types identified in the investigation were types 51, 298 and 392.

This communication presents a successful story of an attempt to treat and manage a case of canine melioidosis, a severe tropical disease caused by Burkholderia pseudomallei. A 10-year-old dog was trapped with barbed wires, causing an infected wound around its neck and back, which was later diagnosed as severe melioidosis. The dog was treated based on a modified human protocol. Intravenous meropenem injections (20 mg/kg twice daily) were given for 14 days to prevent death from sepsis prior to treatment with oral sulfamethoxazole-trimethoprim (25 mg/kg twice daily) for 20 weeks to eliminate the bacteria. Canine melioidosis is an unusual infection in dogs, even in Thailand where melioidosis is highly endemic. This successful case management was solely based on proper diagnosis and appropriate treatments.

ABSTRACT Whole-genome sequence (WGS) data are commonly used to design diagnostic targets for the identification of bacterial pathogens. To do this effectively, genomics databases must be comprehensive to identify the strict core genome that is specific to the target pathogen. As additional genomes are analyzed, the core genome size is reduced and there is erosion of the target-specific regions due to commonality with related species, potentially resulting in the identification of false positives and/or false negatives. IMPORTANCE A comparative analysis of 1,130 Burkholderia genomes identified unique markers for many named species, including the human pathogens B. pseudomallei and B. mallei. Due to core genome reduction and signature erosion, only 38 targets specific to B. pseudomallei/mallei were identified. By using only public genomes, a larger number of markers were identified, due to undersampling, and this larger number represents the potential for false positives. This analysis has implications for the design of diagnostics for other species where the genomic space of the target and/or closely related species is not well defined.