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In Malaysia, melioidosis is not a notifiable disease but has a high mortality rate, particularly in diabetic patients. Disseminated melioidosis can be fatal, with 16% to 37% of cases presenting with nonspecific symptoms, complicating early diagnosis. Some patients develop abscesses with or without bacteremia. We present a fatal case of disseminated melioidosis in a diabetic patient with vague initial symptoms. Remarkably, she exhibited low septic parameters, including a normal white blood cell count and low C-reactive protein levels, which masked the severity of the infection and delayed appropriate treatment. The absence of early radiological findings and specific laboratory tests further complicated the diagnostic process. This case underscores the necessity for heightened clinical awareness and the advancement of diagnostic tools, particularly in high-risk populations, to facilitate timely interventions and mitigate fatal outcomes. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei (B. pseudomallei) is a facultative intracellular parasitic pathogen with multiple immune escape mechanisms. Mitophagy is critical for mitochondrial quality control and function in various biological processes. We reported that B. pseudomallei infection induces mitophagy to promote its intracellular survival by decreasing mitochondrial reactive oxygen species (mtROS). Mechanically, B. pseudomallei infection leads to the rupture of host outer mitochondrial membrane (OMM) by DNM1L/DRP1 (dynamin 1-like). Furthermore, BipD, the type III secretion system (T3SS) needle tip protein of B. pseudomallei, hijacks the host KLHL9 (kelch-like 9)-KLHL13 (kelch-like 13)-CUL3 (cullin 3) E3 ubiquitin ligase complex to promote the K63-linked ubiquitination of IMMT/mitofilin (inner membrane protein, mitochondrial) at the K211 site. Then BipD-initiated mitophagy, via the conventional macroautophagy/autophagy pathway with the receptor SQSTM1 (sequestosome 1) involvement, decreases the mtROS production, which in turn facilitates the intracellular survival of B. pseudomallei. Here, our findings reveal an unexpected function of BipD and the KLHL9-KLHL13-CUL3 E3 ligase complex and suggest a novel mechanism used by bacterial pathogens that hijack host mitophagy for their survival. [ABSTRACT FROM AUTHOR]

Objective To express recombinant Burkholderia pseudomallei (B. pseudomallei) type Ⅲ secretion system BipD protein, prepare its polyclonal antibodies and verify their immunological traits. Methods The recombinant pET-28a-BipD plasmid was generated, and the pET-28a-BipD-carried E.coli BL21 (DE3) bacteria were induced with isopropyl-β-d-thiogalactoside (IPTG) to express recombinant BipD (rBipD) protein. The rBipD was obtained by affinity chromatography using His Trap column, then mixed with Fredrick's adjuvant to immunize BALB/c mice by intraperitoneal injection in order to obtain anti-rBipD polyclonal antibodies. The immunoreactivity of rBipD was detected by Western blot assay using rabbit anti-melioidosis serum and the serum from melioidosis patients. The immunogenicity of rBipD was evaluated using Western blotting and immunofluorescence staining. Finally, rBipD was used to establish an indirect ELISA to detect serum antibodies of clinical melioidosis patients. Results The recombinant plasmid pET-28a-BipD was successfully constructed and transformed into E.coli BL21 (DE3) to induce rBipD expression with IPTG treatment. The obtained rBipD had a relative molecular weight of 36×103 and a purity of 95.4%, and had good immunogenicity and immunoreactivity. It could induce the production of specific antibodies after immunizing mice, and mouse polyclonal antibodies against rBipD were prepared with the titer of 1∶512 000. rBipD of 5.0 μg/mL produced specific immune response with the serum of melioidosis patients, but had no specific reaction with the serum of tuberculosis patients, with statistical difference (P < 0.01). Conclusion rBipD with immunological activity is successfully prepared and purified, and its polyclonal antibodies are also developed, which provide a good tool for clinical immunological diagnosis and study of immune mechanism of B. pseudomallei infection.

Objective To analyze the mechanism that Hcp1 protein in type Ⅵ secretion system of Burkholderia pseudomallei (B. pseudomallei) mediates the formation of multinucleated giant cells (MNGCs) when host cells are infected by the bacterium. Methods The mutant strain (BPC006 Δhcp1) and complementation strain (BPC006 Δhcp1:: hcp1) were constructed by homologous recombination and plasmid complement technology, respectively. After RAW264.7 cells were infected with B. pseudomallei, the localization of Hcp1 in host cells was analyzed by immunofluorescence staining. The localization was further verified by cytoplasmic-membrane isolation in 293T cells after transfecting pCDNA4.1-Hcp1. The biological significance and effect of Hcp1 were explored by the anti-Hcp1 polyclonal antibody blocking and the formation of MNGC was detected by Giemsa staining. Results Western blotting showed that BPC006 Δhcp1 could not express Hcp1, while BPC006 Δhcp1:: hcp1 restored Hcp1 expression. The above results proved that the mutant and complement strains were successfully constructed. Both cellular immunofluorescence co-localization and cytoplasmic-membrane isolation experiments showed that Hcp1 localized to host cell membranes. Last but not least, compared with the control group, anti-Hcp1 polyclonal antibodies inhibited the formation of MNGC (P < 0.01). Conclusion Hcp1 protein in type Ⅵ secretion system of B. pseudomallei is able to translocate to the RAW264.7 cell membranes and plays an important role in the formation of MNGCs.

Abstract Melioidosis caused by Burkholderia pseudomallei (Bp) is a public health threat. Genomic-epidemiology research on this deadly disease is scarce. We investigated whole-genome sequences of Bp isolates in relation to environmental source and drug susceptibility. In total, 563 Bp isolates were collected from 11 Northeast Thai provinces during the period 2004–2021. Patients (n = 530 isolates), infected animals (n = 8), and environmental sources (n = 25) provided samples. Phylogenetic analysis revealed genetic diversity among the Bp isolates, including numerous well-supported clusters of varying sizes. Through in-depth analysis of 38 monophyletic clades (MCs), we found eleven associated with province of origin (p-value

Background: Melioidosis, an infectious disease caused by Burkholderia pseudomallei, is endemic in tropical countries. In nonendemic areas, the disease is rarely suspected because of varying clinical presentations and only a few attempts are made to isolate the pathogen. Many cases are left underdiagnosed or underreported in geographical areas where the disease is not endemic. This study aimed to analyze the clinical presentations, comorbidities, and antibiotic susceptibility patterns in patients with melioidosis in a tertiary care center. Materials and Methods: A retrospective study was done on culture-confirmed melioidosis patients admitted to a tertiary care center, from January 2015 to December 2022. Relevant information on clinical presentations, mortality rate, comorbidities, and antibiogram was collected from hospital medical records. Results: A total of 73 culture-confirmed cases of melioidosis were included in the study. Common clinical presentations were pneumonia (n = 35, 47.9%), septicemia (n = 13, 17.8%), and deep abscesses (n = 9, 12.3%). The mortality rate from melioidosis was 15.1% (n = 11). No significant difference was found in the mortality rate between pneumonia and septicemia groups (P = 0.716). Diabetes mellitus (DM) was the major comorbidity detected (n = 56, 76.7%). Isolates were susceptible to ceftazidime (n = 71, 97.3%), meropenem (n = 71, 97.3%), and co-trimoxazole (n = 60, 82.2%). Conclusions: Common clinical presentations of melioidosis in our setting were pneumonia and septicemia. DM was the major comorbidity. Nearly one in six patients died. Ceftazidime and meropenem were the effective antibiotics. These findings may help physicians to make an early microbiological diagnosis which is essential to reduce mortality.

Burkholderia pseudomallei (Bp) causes the tropical disease melioidosis that afflicts an estimated 165,000 people each year. Bp is a facultative intracellular pathogen that transits through distinct intracellular stages including attachment to host cells, invasion through the endocytic pathway, escape from the endosome, replication in the cytoplasm, generation of protrusions towards neighboring cells, and host cell fusion allowing Bp infection to spread without exiting the intracellular environment. We have identified a TetR-like transcriptional regulator, BP1026B_II1561, that is up-regulated during the late stages of infection as Bp protrudes toward neighboring cells. We have characterized BP1026B_ II1561 and determined that it has a role in pathogenesis. A deletional mutant of BP1026B_II1561 is attenuated in RAW264.7 macrophage and BALB/c mouse models of infection. Using RNA-seq, we found that BP1026B_II1561 controls secondary metabolite biosynthesis, fatty acid degradation, and propanoate metabolism. In addition, we identified that BP1026B_II1561 directly controls expression of an outer membrane porin and genes in the shikimate biosynthetic pathway using ChIP-seq. Transposon mutants of genes within the BP1026B_ II1561 regulon show defects during intracellular replication in RAW264.7 cells confirming the role of this transcriptional regulator and the pathways it controls in pathogenesis. BP1026B_II1561 also up-regulates the majority of the enzymes in shikimate and tryptophan biosynthetic pathways, suggesting their importance for Bp physiology. To investigate this, we tested fluorinated analogs of anthranilate and tryptophan, intermediates and products of the shikimate and tryptophan biosynthetic pathways, respectively, and showed inhibition of Bp growth at nanomolar concentrations. The expression of these pathways by BP1026b_II1561 and during intracellular infection combined with the inhibition of Bp growth by fluorotryptophan/anthranilate highlights these pathways as potential targets for therapeutic intervention against melioidosis. In the present study, we have identified BP1026B_II1561 as a critical transcriptional regulator for Bp pathogenesis and partially characterized its role during host cell infection. [ABSTRACT FROM AUTHOR]

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]

Melioidosis caused by Burkholderia pseudomallei is an infectious disease with a high mortality rate. In acute melioidosis, sepsis is a major cause of death among patients. Once the bacterium enters the bloodstream, immune system dysregulation ensues, leading to cytokine storms. In contrast to B. pseudomallei, a closely related but non-virulent strain B. thailandensis has rarely been reported to cause cytokine storms or death in patients. However, the mechanisms in which the virulent B. pseudomallei causes sepsis are not fully elucidated. It is well-documented that monocytes play an essential role in cytokine production in the bloodstream. The present study, therefore, determined whether there is a difference in the innate immune response to B. pseudomallei and B. thailandensis during infection of primary human monocytes and THP-1 monocytic cells by investigating pyroptosis, an inflammatory death pathway known to play a pivotal role in sepsis. Our results showed that although both bacterial species exhibited a similar ability to invade human monocytes, only B. pseudomallei can significantly increase the release of cytosolic enzyme lactate dehydrogenase (LDH) as well as the increases in caspase-1 and gasdermin D activations in both cell types. The results were consistent with the significant increase in IL-1β and IL-18 production, key cytokines involved in pyroptosis. Interestingly, there was no significant difference in other cytokine secretion, such as IL-1RA, IL-10, IL-12p70, IL-15, IL-8, and IL-23 in cells infected by both bacterial species. Furthermore, we also demonstrated that ROS production played a crucial role in controlling pyroptosis activation during B. pseudomallei infection in primary human monocytes. These findings suggested that pyroptosis induced by B. pseudomallei in the human monocytes may contribute to the pathogenesis of sepsis in acute melioidosis patients. Author summary: Acute melioidosis, caused by B. pseudomallei infection, is considered one of the lethal infectious diseases reported in Southeast Asia and Northern Australia. Infection with this bacterium can lead to a severe systemic inflammatory response or sepsis, which is a major cause of death. The mortality rate of acute melioidosis may reach up to 50%. In contrast, very rare cases were reported from patients infected with B. thailandensis, a non-virulence strain. Although it is well-documented that these two bacteria share genetic similarities and can exhibit similarities in survival and replication inside most murine and human cells, there is very limited information on how only B. pseudomallei can cause sepsis in melioidosis patients, while B. thailandensis dose not. We established an infection model using primary human monocytes to demonstrate that only B. pseudomallei can induce pyroptosis, causing the release of key cytokines that are involved in sepsis. This information may contribute to understanding the mechanisms underlying pathogenesis in acute melioidosis and may guide future treatments. [ABSTRACT FROM AUTHOR]

Various nonribosomal peptide synthetases (NRPSs) create structural and functional diversity by incorporating α‐hydroxy acids into peptide backbones. Trigonic acid, an unusual cyclopropanol‐substituted hydroxy acid, is the source of the molecular warhead of malleicyprol, a critical virulence factor of human and animal pathogens of the Burkholderia pseudomallei (BP) group. The process of selecting and loading this building block remained enigmatic as the NRPS module designated for this task is incomplete. Using a combination of bioinformatics, mutational analyses, targeted metabolomics, and in vitro biochemical assays, we show that two trans‐acting enzymes are required to load this central building block onto the modular assembly line. An adenylation‐thiolation didomain enzyme (BurJ) activates trigonic acid, followed by the translocation of the enzyme‐bound α‐hydroxy acid thioester by an FkbH‐like protein with a mutated phosphatase domain (BurH). This specialized gateway is the first reported direct loading of an α‐hydroxy acid onto a bona fide NRPS module in bacteria and expands the synthetic biology toolbox for the site‐specific incorporation of non‐canonical building blocks. Moreover, insight into the biochemical basis of virulence factor biosynthesis can provide a foundation for developing enzyme inhibitors as anti‐virulence therapeutics against BP pathogen infections. [ABSTRACT FROM AUTHOR]

Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei, an intracellular pathogen with a high mortality rate and significant antibiotic resistance. The high mortality rate and resistance to antibiotics have drawn considerable attention from researchers studying melioidosis. This study evaluated the effects of various concentrations (75, 50, and 25 µg/mL) of promethazine hydrochloride (PTZ), a potent antihistamine, on biofilm formation and lipase activity after 24 h of exposure to B. thailandensis E264. A concentration-dependent decrease in both biofilm biomass and lipase activity was observed. RT-PCR analysis revealed that PTZ treatment not only made the biofilm structure loose but also reduced the expression of btaR1, btaR2, btaR3, and scmR. Single gene knockouts of quorum sensing (QS) receptor proteins (∆btaR1, ∆btaR2, and ∆btaR3) were successfully constructed. Deletion of btaR1 affected biofilm formation in B. thailandensis, while deletion of btaR2 and btaR3 led to reduced lipase activity. Molecular docking and biological performance results demonstrated that PTZ inhibits biofilm formation and lipase activity by suppressing the expression of QS-regulated genes. This study found that repositioning PTZ reduced biofilm formation in B. thailandensis E264, suggesting a potential new approach for combating melioidosis. Key Points: Repurposing promethazine hydrochloride to inhibit biofilms formation. Promethazine hydrochloride suppressed the expression of QS-regulated genes. The deletion of btaR1 affected the biofilm formation of B. thailandensis. [ABSTRACT FROM AUTHOR]

Melioidosis is a potentially severe disease caused by the gram-negative soil-dwelling bacterium called Burkholderia pseudomallei. The true breadth of the distribution of this tropical pathogen is starting to emerge with environmental and clinical isolates frequently characterized in new countries and regions. Even so, isolates, clinical cases, and genetic data from the continent of Africa remain scant. We previously confirmed the presence of B. pseudomallei in the environment of Ghana, unmasking a new area of endemicity for this pathogen. Here, we describe the genetic characteristics of isolates obtained from that environmental survey. Twenty-one isolates were subjected to whole genome sequencing and found to represent three discrete sequence types (ST), one of which was novel, and designated ST2058. Phylogenetic analysis places this novel isolate within a B. pseudomallei clade that includes genomes derived from the Americas, although it is closely related to a sub-clade that includes isolates from Africa. Importantly, phenotypic characterization demonstrates common features including API 20NE profiles and B. pseudomallei CPS to support existing diagnostics, and susceptibility to standard of care antibiotics often used in the clinical management of melioidosis. These findings add to our knowledge about the presence and distribution of B. pseudomallei in Africa and represent the first published genomes out of Ghana. [ABSTRACT FROM AUTHOR]

Melioidosis is an often-fatal neglected tropical disease caused by an environmental bacterium Burkholderia pseudomallei. However, our understanding of the disease-causing bacterial lineages, their dissemination, and adaptive mechanisms remains limited. To address this, we conduct a comprehensive genomic analysis of 1,391 B. pseudomallei isolates collected from nine hospitals in northeast Thailand between 2015 and 2018, and contemporaneous isolates from neighbouring countries, representing the most densely sampled collection to date. Our study identifies three dominant lineages, each with unique gene sets potentially enhancing bacterial fitness in the environment. We find that recombination drives lineage-specific gene flow. Transcriptome analyses of representative clinical isolates from each dominant lineage reveal increased expression of lineage-specific genes under environmental conditions in two out of three lineages. This underscores the potential importance of environmental persistence for these dominant lineages. The study also highlights the influence of environmental factors such as terrain slope, altitude, and river direction on the geographical dispersal of B. pseudomallei. Collectively, our findings suggest that environmental persistence may play a role in facilitating the spread of B. pseudomallei, and as a prerequisite for exposure and infection, thereby providing useful insights for informing melioidosis prevention and control strategies. An environmental bacterium, Burkholderia pseudomallei causes melioidosis, a fatal infection. A study from NE Thailand identifies that dominant lineages carry specific gene sets enhancing environmental persistence. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei , an environmental saprophyte, causes an infection that results in melioidosis. A high case-fatality rate is linked to it. Melioidosis is becoming an increasingly global infection. It can present a wide spectrum of manifestations and can affect any part of the human being. The head-and-neck region is a major site of melioidosis infection. Melioidosis in the head-and-neck region is a potentially life-threatening infection. Careful clinical correlation and proper evaluation are needed to establish an early diagnosis of melioidosis. Early diagnosis and prompt treatment are very required. A high index of suspicion in susceptible patients should prompt assessment for melioidosis. The awareness of melioidosis and its clinical manifestations with early diagnosis are needed to avoid failure of treatment which may result in high morbidity and mortality. As melioidosis in the head-and-neck region is not so common, there is very less information available. Therefore, a review article on melioidosis in the head-and-neck region is written with consideration of epidemiology, aetiopathology, clinical manifestations, diagnosis, treatment and prognosis. [ABSTRACT FROM AUTHOR]

We have continued crystal structural analyses of RND transporters, one of the main causes of antimicrobial resistance in Gram-negative bacteria. It has been more than twenty years since we first determined the structure of E. coli AcrB, and in 2023, we solved the crystal structure of its homolog, BpeB from Burkholderia pseudomallei. Does the impact of our work diminish as we continue to analyze the homologous proteins over and over again? No. We can gain deeper insights into the molecular mechanism of the RND transporter by gathering the knowledge accumulated so far. Even if it is not the first structure in the world, we can still find some significance in the work if we analyze it properly. [ABSTRACT FROM AUTHOR]

Background: Burkholderia pseudomallei, an environmental saprophyte bacterium, causes melioidosis in humans and animals. It was first discovered in Iran between 1967 and 1976 in small ruminants, equines, environments and humans. No subsequent studies have been conducted to determine the existence and prevalence of this pathogen in the country. Objectives: The present study aims to monitor the presence of B. pseudomallei in the ruminant population of the Golestan province of Iran, which largely depends on pastures. The ruminants can serve as sentinels to indicate the presence of the bacteria in the environment and its potential impact on human health in the One Health triad. Methods: Liver and lung abscesses from domestic sheep, cattle and goats in three industrial and three conventional slaughterhouses were sampled and analysed using 23S ribosomal DNA polymerase chain reaction (rDNA PCR) with primers CVMP 23‐1 and CVP‐23‐2 for B. pseudomallei, Burkholderia cepacia and Burkholderia vietnamiensis, as well as B. pseudomallei–specific TTS1 real‐time PCR, along with microbiological and biochemical assays. Results: Out of the 97 animals sampled, only 14 (15%) tested positive for 23S rDNA PCR. However, the follow‐up evaluation using TTS1 real‐time PCR and microbiological and biochemical assays did not confirm the presence of B. pseudomallei in the samples. Conclusions: Although B. pseudomallei was not detected in the current survey, conducting abattoir‐based surveillance of ruminants is a cost‐effective One Health approach to monitor pathogenic Burkholderia. Developing standards of clinical and laboratory good practices for Burkholderia infections is crucial for One Health surveillance. [ABSTRACT FROM AUTHOR]

Background: Burkholderia pseudomallei, the causative agent of melioidosis, is highly genetically recombinant, resulting in significant genomic diversity. Multiple virulence factors have been associated with specific disease presentations. To date, there are limited data relating to genomic diversity and virulence factors associated with melioidosis cases in North Queensland, Australia. Aim: To describe the genetic diversity of B. pseudomallei and identify virulence factors associated with clinical risk factors and patient outcomes. Methods: Whole genome sequencing of clinical isolates was performed and analysed with clinical data obtained from a retrospective melioidosis cohort study. Results: Fifty-nine distinct sequence types (STs) were identified from the 128 clinical isolates. Six STs comprised 64/128 (50%) isolates. Novel STs accounted for 38/59 (64%) STs, with ST TSV-13 as the most prevalent (n = 7), and were less likely to possess an LPS A genotype or YLF gene cluster (p < 0.001). These isolates were most likely to be found outside the inner city (aOR: 4.0, 95% CI: 1.7–9.0, p = 0.001). ST TSV-13 was associated with increased mortality (aOR: 6.1, 95% CI: 1.2–30.9, p = 0.03). Patients with a history of alcohol excess were less likely to be infected by fhaB3 (aOR 0.2, 95% CI: 0.1–0.7, p = 0.01) or YLF (aOR: 0.4, 95% CI: 0.2–0.9, p = 0.04) positive isolates. Conclusions: There are a significant number of novel sequence types in Townsville, Australia. An emerging novel ST appears to have an association with geographic location and mortality. Ongoing investigation is required to further understand the impact of this ST on the Townsville region. [ABSTRACT FROM AUTHOR]

Melioidosis is an emerging infection in India and is caused by a Gram-negative oxidase-positive bacilli, Burkholderia pseudomallei which is present in soil and surface water in the endemic region. Clinical manifestations are protean varying from asymptomatic infection, multiple skin abscesses, chronic pneumonia mimicking tuberculosis, and fulminant septic shock with abscesses in multiple internal organs. Isolation of bacilli from aspirated fluids remains the gold standard of diagnosis. Lack of microbiological services in many parts of the state, protean nature of clinical manifestations, lack of awareness among clinicians, and misidentification of the organism as Pseudomonas spp leads to delay in diagnosis and treatment. We have, retrospectively, analyzed 14 cases with culture-proven melioidosis diagnosed during 10 years. Case records of culture-positive patients were retrospectively analyzed for demographic data, clinical characteristics, management, complications, and clinical outcomes. Out of these, 13 cases were male. The most common age group affected was 50–59 years. The major occupation affected was manual laborer. Nonbacteremic melioidosis accounted for 12 cases. Concerning clinical presentations, intra-abdominal abscesses mainly in the liver and spleen accounted for seven cases. All the isolates were susceptible to ceftazidime, meropenem, and doxycycline, whereas 12 isolates were susceptible to chloramphenicol, 11 isolates were susceptible to trimethoprim–sulfamethoxazole combination, and 10 cases were susceptible to amoxicillin–clavulanic acid and ciprofloxacin. Ten cases recovered with ceftazidime administration along with surgical drainage of abscess and three cases recovered with meropenem administration and surgical drainage. One patient expired and was on piperacillin–tazobactam. Melioidosis is a disease of public health importance and early clinical and laboratory diagnosis is crucial in the effective management of melioidosis. [ABSTRACT FROM AUTHOR]

The reader will come to appreciate that: • Infection occurs through percutaneous inoculation or inhalation with contaminated soil and water. • Diagnosis of melioidosis can be difficult because of its diverse presentation. • Melioidosis is increasingly being diagnosed around the world. • Greater consideration for melioidosis in protracted or atypical pneumonia is warranted regardless of location. Melioidosis is a tropical infectious disease caused by the saprophytic gram-negative bacterium Burkholderia pseudomallei. Despite the infection being endemic in southeast Asia and northern Australia, the broad clinical presentations and diagnostic difficulties limit its early detection, particularly in children. Melioidosis more commonly affects the immunocompromised and adults. Melioidosis is increasingly being diagnosed around the world and whole-genome sequencing indicates that these cases are not linked with travel to endemic areas. Research has concentrated on the adult population with limited experience reported in the care of this uncommon, but potentially fatal condition in children presenting with bacteraemia and pneumonia. [ABSTRACT FROM AUTHOR]

Mitophagy is critical for mitochondrial quality control and function to clear damaged mitochondria. Here, we found that Burkholderia pseudomallei maneuvered host mitophagy for its intracellular survival through the type III secretion system needle tip protein BipD. We identified BipD, interacting with BTB-containing proteins KLHL9 and KLHL13 by binding to the Back and Kelch domains, recruited NEDD8 family RING E3 ligase CUL3 in response to B. pseudomallei infection. Although evidently not involved in regulation of infectious diseases, KLHL9/KLHL13/CUL3 E3 ligase complex was essential for BipD-dependent ubiquitination of mitochondria in mouse macrophages. Mechanistically, we discovered the inner mitochondrial membrane IMMT via host ubiquitome profiling as a substrate of KLHL9/KLHL13/CUL3 complex. Notably, K63-linked ubiquitination of IMMT K211 was required for initiating host mitophagy, thereby reducing mitochondrial ROS production. Here, we show a unique mechanism used by bacterial pathogens that hijacks host mitophagy for their survival. The selective degradation of mitochondria by autophagy is essential for cell homeostasis. Here, Nan et al show that the effector protein BipD, secreted by Burkholderia pseudomallei, is able to modulate host mitophagy to promote its survival. [ABSTRACT FROM AUTHOR]

Objective To study the biological function of the possible efflux pump gene BPSL1972 in Burkholderia pseudomallei (Bp), so as to improve the drug resistance mechanism of Bp. Methods BPSL1972 gene knockout strain and its complement strain were constructed by homologous recombination and electric shock transformation, respectively. BPSL1972 protein expression vector was constructed by enzyme digestion and enzyme-linked method. The BPSL1972-encoded protein was expressed using a His-tag protein expression kit and the expressed proteins were verified by Western Blot. The changes in drug sensitivity, growth, biofilm formation, and cytotoxicity were compared by microbroth dilution assay, Kirby-Bauer assay, turbidimetric assay, tube assay, crystal violet staining, and CCK-8 kit. The physicochemical properties of BPSL1972 were predicted by Protscale and Protparam online websites, the signal peptide of BPSL1972 was predicted by SignalP 5.0 online website, and the transmembrane domain and gene function of BPSL1972 were predicted by Smart online website. The 3D structure of BPSL1972-encoded protein and its homology were analyzed through Phyre 2 online website. Results The predictions suggested that the BPSL1972-encoded protein is a class of neutral-stable hydrophobic protein with a lipoprotein signal peptide and a transmembrane region, classified under the RND efflux pump system with two lipoprotein outer membrane efflux proteins, potentially mediating macrolide resistance. Its 3D modeling results show high homology to VceC and fusaric acid efflux protein, possibly mediating the efflux of ion activators. Sequencing results confirmed the successful construction of both the deletion mutant strain and complement strain. However, there were no significant differences in biological characteristics between the deletion mutant strain and the wild-type strain, and there was also no significant difference in resistance to antibiotics such as cephalosporins, quinolones, carbapenems, aminoglycosides, tetracyclines, and surfactants such as sodium deoxycholate. Conclusion BPSL1972 may be an auxiliary gene of LysR family, and this auxiliary gene is secondary, thus resulting in no functional changes after its deletion. Further research is needed to investigate whether it has resistance to other antibiotics such as fusaric acid and its specific replacement or auxiliary function. [ABSTRACT FROM AUTHOR]

Melioidosis infecting a periurethral diverticulum and behaving as an abscess is a rare complication. A 76-year-old woman presented with pelvic pain, dysuria and urinary frequency. CT identified a large periurethral cystic collection and melioidosis was cultured in her urine. Cystoscopy revealed communication between urethra and diverticulum, requiring multiple transvaginal aspirations for re-accumulation and relapsing symptoms. No risk factors for melioidosis were identified, and was likely that isolated urinary infection is due to her urinary tract pathology. This challenging case with a rare pathogen highlights management and source control of melioidosis may need to adapt to anatomical variations promoting abscess reformation.

ABSTRACT Melioidosis is a serious infectious disease caused by the Gram-negative bacterium Burkholderia pseudomallei. Recently, Rab32-dependent immune vesicles emerge as a critical defense pathway to restrict the intracellular B. pseudomallei. However, B. pseudomallei can evade host immune vesicles and survive in the cytoplasm, although this mechanism is not well understood. In this study, we found Rab32-dependent vesicles could effectively combat B. pseudomallei infection, but not all intracellular B. pseudomallei were encapsulated in Rab32-positive vesicles. To explore how B. pseudomallei counteracted the Rab32-dependent defense pathway, transcriptomic profiling of B. pseudomallei was performed to characterize the response dynamics during infection. We found that the type III secretion system of B. pseudomallei was activated, and a variety of effector proteins were highly upregulated. Among them, BopE, BprD, and BipC were shown to interact with Rab32. Interestingly, BopE directly interacts with host Rab32, potentially suppressing Rab32 function by interfering with nucleotide exchange, which in turn restricts the recruitment of Rab32 to bacterial-containing vesicles. Knocking out of BopE can increase the proportion of Rab32-positive vesicles, suppressing the intracellular replication and virulence of B. pseudomallei. Collectively, our findings have demonstrated that BopE may be an important effector for B. pseudomallei to evade from the Rab32-dependent killing vesicles into the cytosol for survival and replication. Therefore, a deeper understanding of the interaction between BopE and the host Rab32-dependent restriction pathway may provide an effective therapeutic strategy for the elimination of intracellular B. pseudomallei.IMPORTANCEB. pseudomallei is facultative intracellular bacterium that has evolved numerous strategies to evade host immune vesicles and survive in the cytoplasm. Rab32-dependent vesicles are one of these immune vesicles, but the mechanism by which B. pseudomallei escape Rab32-dependent vesicles remains elusive. Here, we find B. pseudomallei infection leading the activation of the type III secretion system (T3SS-3) and increasing the expression of various effectors. Specifically, we identify that BopE, an effector secreted by T3SS-3, triggers vesicle escape to promote B. pseudomallei pathogenicity and survival. Mechanistically, BopE suppresses the activation of Rab32 by interfering with nucleotide exchange, ultimately triggering vesicle escape and intracellular survival. We also find knocking out the bopE gene can increase the proportion of Rab32-positive vesicles that trap B. pseudomallei, dampening the survival of B. pseudomallei both in vitro and in vivo. Taken together, our findings provide insights into the molecular mechanisms of pathogen effector-induced vesicle escape, indicating a potential melioidosis treatment via blocking B. pseudomallei BopE-host Rab32 interaction.

Therapies that modulate and appropriately direct the immune response are promising candidates for the treatment of infectious diseases. One such candidate therapeutic is DZ13, a short, synthetic, single-stranded DNA molecule. This molecule has enzymatic activity and can modulate the immune response by binding to and degrading the mRNA encoding a key immuno-regulatory molecule. Originally developed and entering clinical trials as an anti-cancer agent, DZ13 has also been evaluated as a treatment for viral infections, and has been shown to provide protection against infection with influenza virus in a mouse model of infection. In this work, we evaluated whether the immuno-modulatory properties of DZ13 could provide protection against the potential biothreat pathogen Burkholderia pseudomallei which causes the neglected tropical disease melioidosis. Treatment of mice infected with B. pseudomallei demonstrated that DZ13 did indeed provide excellent protection after only two post-exposure treatments. However, our data indicated that the enzymatic activity contained in DZ13 was not required for protection, with control oligonucleotide treatments lacking activity against the target mRNA equally as protective against B. pseudomallei. We have designed new sequences to study the mechanism of protection further. These novel sequences offer enhanced protection against infection, but are not directly anti-microbial and do not appear to be stimulating the immune system via TLR9 or other key innate immune sensors, despite containing CpG motifs. The molecular mechanism of these novel sequences remains to be elucidated, but the data highlights that these oligonucleotide-sensing pathways are attractive and relevant targets to modulate during bacterial and viral infections.

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 is a fatal infectious disease caused by Burkholderia pseudomallei. Its clinical manifestations are so varied that it has been called a great mimic. The primary melioidotic prostatic disease usually presents as an abscess of the prostate. This is the only documented case misdiagnosed as benign prostatic hyperplasia (BPH) because it does not show as a prostate abscess. The patient was a 66 years old male with a history of hypertension and alcoholism. Symptoms of difficulty urinating and fever had persisted for three days. Laboratory tests of the patient showed a marked increase in inflammatory markers. The first urine culture was performed to isolate Acinetobacter lwoffii, not Burkholderia pseudomallei. And CT showed prostatic hyperplasia with calcification. Thus, the preliminary diagnosis was BPH, urinary tract infection, and pulmonary infection. Cefmenoxime and Piperacillin were given for anti-infective treatment, but both of the effects were not satisfactory. Then, the second urine culture and multiple hemocultures were performed to isolate Burkholderia pseudomallei. The final diagnosis was primary melioidotic prostatic disease, BPH, pulmonary infection, bacteremia and multiple organ damage. The patient was treated with imipenem for two weeks and cured. Trimethoprim-sulfamethoxazole (TMP-SMX) was administered orally for twelve weeks after discharge. Therefore, clinicians need to have an understanding of primary melioidotic prostate disease to avoid misdiagnosis and mistreatment to improve the cure rate and survival rate of patients.

BACKGROUND: Melioidosis is a disease caused by the bacterium Burkholderia pseudomallei, infecting humans and non-human primates (NHP) through contaminated soil or water. World-wide there are an estimated 165,000 human melioidosis cases each year, but recordings of NHP cases are sporadic. Clinical detection of melioidosis in humans is primarily by culturing B. pseudomallei, and there are no standardized detection protocols for NHP. NHP are an important animal model for melioidosis research including clinical trials and development of biodefense countermeasures. METHODOLOGY/PRINCIPLE FINDINGS: We evaluated the diagnostic potential of the multiple antigen serological assay, BurkPx, in NHP using two sera sets: (i) 115 B. pseudomallei-challenged serum samples from 80 NHP collected each week post-exposure (n = 52) and at euthanasia (n = 47), and (ii) 126 B. pseudomallei-naïve/negative serum samples. We observed early IgM antibody responses to carbohydrate antigens followed by IgG antibody recognition to multiple B. pseudomallei protein antigens during the second week of infection. B. pseudomallei negative serum samples had low to intermediate antibody cross reactivity to the antigens in this assay. Infection time was predicted as the determining factor in the variation of antibody responses, with 77.67% of variation explained by the first component of the principal component analysis. A multiple antigen model generated a binary prediction metric ([Formula: see text]), which when applied to all data resulted in 100% specificity and 63.48% sensitivity. Removal of week 1 B. pseudomallei challenged serum samples increased the sensitivity of the model to 95%. CONCLUSION/SIGNIFICANCE: We employed a previously standardized assay for humans, the BurkPx assay, and assessed its diagnostic potential for detection of B. pseudomallei exposure in NHP. The assay is expected to be useful for surveillance in NHP colonies, in investigations of suspected accidental releases or exposures, and for identifying vaccine correlates of protection.

Abstract Objective Burkholderia pseudomallei, the etiological cause of melioidosis, is a soil saprophyte endemic in South-East Asia, where it constitutes a public health concern of high-priority. Melioidosis cases are sporadically identified in nonendemic areas, usually associated with travelers or import of goods from endemic regions. Due to extensive intercontinental traveling and the anticipated climate change-associated alterations of the soil bacterial flora, there is an increasing concern for inadvertent establishment of novel endemic areas, which may expand the global burden of melioidosis. Rapid diagnosis, isolation and characterization of B. pseudomallei isolates is therefore of utmost importance particularly in non-endemic locations. Data description We report the genome sequences of two novel clinical isolates (MWH2021 and MST2022) of B. pseudomallei identified in distinct acute cases of melioidosis diagnosed in two individuals arriving to Israel from India and Thailand, respectively. The data includes preliminary genetic analysis of the genomes determining their phylogenetic classification in rapport to the genomes of 131 B. pseudomallei strains documented in the NCBI database. Inspection of the genomic data revealed the presence or absence of loci encoding for several documented virulence determinants involved in the molecular pathogenesis of melioidosis. Virulence analysis in murine models of acute or chronic melioidosis established that both strains belong to the highly virulent class of B. pseudomalleii.

In September 2021, a total of 25 patients diagnosed with COVID-19 developed acute melioidosis after (median 7 days) admission to a COVID-19 field hospital in Thailand. Eight nonpotable tap water samples and 6 soil samples were culture-positive for Burkholderia pseudomallei. Genomic analysis suggested contaminated tap water as the likely cause of illness.

We review the discovery of the tropical infectious disease melioidosis by Alfred Whitmore, a pathologist from England, and his assistant from India, C.S. Krishnaswami. We discuss how the subsequent disappearance of melioidosis from the medical literature of Burma holds parallels with the current neglect and under recognition of the disease. We urge global and national public health authorities to add melioidosis to existing neglected tropical diseases surveillance systems.

Abstract Background Melioidosis, caused by the category B biothreat agent Burkholderia pseudomallei, is a disease with a high mortality rate and requires an immediate culture-independent diagnosis for effective disease management. In this study, we developed a highly sensitive qPCR assay for specific detection of Burkholderia pseudomallei and melioidosis disease diagnosis based on a novel target sequence. Methods An extensive in-silico analysis was done to identify a novel and highly conserved sequence for developing a qPCR assay. The specificity of the developed assay was analyzed with 65 different bacterial cultures, and the analytical sensitivity of the assay was determined with the purified genomic DNA of B. pseudomallei. The applicability of the assay for B. pseudomallei detection in clinical and environmental matrices was evaluated by spiking B. pseudomallei cells in the blood, urine, soil, and water along with suitable internal controls. Results A novel 85-nucleotide-long sequence was identified using in-silico tools and employed for the development of the highly sensitive and specific quantitative real-time PCR assay S664. The assay S664 was found to be highly specific when evaluated with 65 different bacterial cultures related and non-related to B. pseudomallei. The assay was found to be highly sensitive, with a detection limit of 3 B. pseudomallei genome equivalent copies per qPCR reaction. The detection limit in clinical matrices was found to be 5 × 102 CFU/mL for both human blood and urine. In environmental matrices, the detection limit was found to be 5 × 101 CFU/mL of river water and 2 × 103 CFU/gm of paddy field soil. Conclusions The findings of the present study suggest that the developed assay S664 along with suitable internal controls has a huge diagnostic potential and can be successfully employed for specific, sensitive, and rapid molecular detection of B. pseudomallei in various clinical and environmental matrices.

Melioidosis, a potentially fatal disease caused by the bacterium Burkholderia pseudomallei continues to be neglected in the Indian Subcontinent despite bearing about 44% of the global burden. Diagnosis poses a significant challenge since the disease presents a wide range of symptoms and closely mimics tuberculosis and pneumonia both of which are endemic in India. Sophisticated diagnosis and treatment often become unaffordable for patients from rural or low-income backgrounds. We present five cases of melioidosis from a tertiary care hospital (Mahatma Gandhi Medical College and Research Institute ) in Pondicherry that exhibited predominantly high-grade fever, abdominal pain, and vomiting. Radiological imaging revealed abnormalities in the brain (1/5, 20%), lung (3/5, 60%), liver (2/5, 40%), spleen (2/5, 40%), kidney (2/5, 40%), and prostate gland (1/5, 20%). Burkholderia pseudomallei infection was confirmed through blood culture. Treatment with meropenem or ceftazidime was initiated immediately. Neuromelioidosis was confirmed in one patient. The clinical diagnoses for the remaining cases were as follows: septic shock, melioidosis with urosepsis, and refractory shock. Three patients required intensive care and of the five, one patient was discharged, one died, and three discontinued treatments against medical advice. In the case of the deceased patient, the clinical diagnosis encompassed refractory shock accompanied by lactic acidosis, melioidosis, and community-acquired pneumonia, which subsequently progressed to acute respiratory distress syndrome (ARDS). Notably, this patient presented with co-morbidities, notably type 2 diabetes mellitus. This exemplifies the difficulty faced by patients from low-income backgrounds which forces them to discontinue expensive treatment. The true burden of melioidosis in the Indian Subcontinent is uncertain as many cases remain undiagnosed. Unawareness of the disease, low index of suspicion among medical professionals, incorrect treatment, and discontinuation contribute to the disease burden. It is therefore imperative that melioidosis is brought to the attention of healthcare policymakers to determine the true burden of the disease by prioritizing nationwide surveillance and diagnosis.

Abstract Background Melioidosis is a serious bacterial infection caused by Burkholderia pseudomallei, a gram-negative bacterium commonly found in soil and water. It can affect both humans and animals, and is endemic in regions such as Southeast Asia and Northern Australia. In recent years, there have been reports of an emergence of human melioidosis in other areas, including New Caledonia. Results During standard laboratory analysis in New Caledonia in 2021, a strain of B. pseudomallei was isolated from a goat. The strain was characterized using both MLST and WGS techniques and was found to cluster with previously described local human strains from the area. In parallel, several serological tests (CFT, ELISA, Luminex (Hcp1, GroEL, BPSS1840), arrays assay and a latex agglutination test) were performed on animals from the farm where the goat originated, and/or from three other neighboring farms. Using two commercial ELISA kits, seropositive animals were found only on the farm where the infected goat originated and tests based on recombinant proteins confirmed the usefulness of the Hcp1 protein for the diagnosis of melioidosis in animals. Conclusions Despite the regular reports of human cases, this is the first confirmed case of melioidosis in an animal in New Caledonia. These results confirm the presence of the bacterium in the region and highlight the importance of vigilance for both animal and human health. It is critical that all health partners, including breeders, veterinarians, and biologists, work together to monitor and prevent the spread of the disease.

The editorial "Genomics of pathogens and vectors" discusses the importance of genomics in understanding pathogenic organisms and their transmission dynamics. The use of high throughput genomic sequencing and bioinformatics has enabled precise identification of pathogens, tracking of mutations, and monitoring of antimicrobial resistance genes. The research highlights the role of genomics in advancing public health strategies, developing accurate diagnostics, and informing decision-making in the face of evolving pathogen transmission dynamics. The articles featured in the thematic issue demonstrate how genomics contributes to understanding pathogen dispersion, resistance to treatment, virulence, and diagnostics, emphasizing the urgent need for research and application of genomics to safeguard public health globally. [Extracted from the article]

This document is an editorial from the journal Frontiers in Genetics titled "Genomics of pathogens and vectors." It discusses the advancements in high throughput genomic sequencing and bioinformatics that have revolutionized our understanding of pathogenic organisms and their transmission dynamics. The use of genomics has enabled precise identification and tracking of pathogens based on mutational profiles, as well as the monitoring of antimicrobial resistance genes and virulence factors. The editorial highlights various studies that showcase how genomics and bioinformatics contribute to our knowledge of pathogen dispersion and transmission, resistance to treatment, virulence, pathogenicity, and diagnostics. The document emphasizes the importance of equitable access to these technologies for global health security and scientific growth. [Extracted from the article]

We report a case of liver abscess due to Burkholderia pseudomallei in a 45-year-old, non-diabetic patient from the Garhwal region of Uttarakhand. The patient reported chief complaints of abdominal pain and fever. Ultrasonography and contrast-enhanced computed tomography of the whole abdomen were suggestive of the left liver lobe abscess. Pus was aspirated from the abscess and sent for culture and sensitivity. Growth on the culture plate was identified as B. pseudomallei ; the patient was then treated with appropriate antibiotics and discharged in stable condition. Diagnosis of pseudomallei infection requires a high index of suspicion among clinicians as well as laboratory specialists. Early identification of organisms helped clinicians initiate appropriate management in our case. [ABSTRACT FROM AUTHOR]

Melioidosis, caused by Burkholderia pseudomallei , is a challenging infectious disease with global implications, primarily affecting Southeast Asia. We present the case of a 24-year-old male with a curious history of tobacco use, presenting with fevers, weight loss, and genitourinary symptoms. Diagnostic challenges arose as symptoms mimicked other diseases. Disseminated melioidosis was confirmed via Gram staining and positron emission tomography scan findings, emphasizing the disease's diverse clinical manifestations. Treatment with ceftazidime and cotrimoxazole led to prompt recovery. Notably, the patient's tobacco use linked to contaminated water highlights a unique transmission route. This case underscores the need for heightened awareness and preventive measures in endemic regions. [ABSTRACT FROM AUTHOR]

Background Melioidosis is caused by Burkholderia pseudomallei. Direct molecular detection from unamplified blood remains insensitive. Methods Three different extraction methods—QIAamp UCP Pathogen Mini Kit, High Pure PCR template and MagNA Pure Pathogen Universal—were trialled using spiked human ethylenediaminetetraacetic acid blood. A type III secretion system 1 (TTSS-1) polymerase chain reaction was used for detection. Results The QIAamp UCP Pathogen Mini Kit performed best, with a limit of detection of 1.5×102 cfu/ml. Conclusions It is planned to use the QIAamp UCP Pathogen Mini Kit to do a larger study on blood collected from patients with melioidosis. [ABSTRACT FROM AUTHOR]

Melioidosis is a bacterial infection caused by Burkholderia pseudomallei, that is common in tropical and subtropical countries including Southeast Asia and Northern Australia. The magnitude of undiagnosed and untreated melioidosis across the country remains unclear. Given its proximity to regions with high infection rates, Riau Province on Sumatera Island is anticipated to have endemic melioidosis. This study reports retrospectively collected data on 68 culture-confirmed melioidosis cases from two hospitals in Riau Province between January 1, 2009, and December 31, 2021, with full clinical data available on 41 cases. We also describe whole genome sequencing and genotypic analysis of six isolates of B. pseudomallei. The mean age of the melioidosis patients was 49.1 (SD 11.5) years, 85% were male and the most common risk factor was diabetes mellitus (78%). Pulmonary infection was the most common presentation (39%), and overall mortality was 41%. Lung as a focal infection (aOR: 6.43; 95% CI: 1.13–36.59, p = 0.036) and bacteremia (aOR: 15.21; 95% CI: 2.59–89.31, p = 0.003) were significantly associated with death. Multilocus sequence typing analysis conducted on six B.pseudomallei genomes identified three sequence types (STs), namely novel ST1794 (n = 3), ST46 (n = 2), and ST289 (n = 1). A phylogenetic tree of Riau B. pseudomallei whole genome sequences with a global dataset of genomes clearly distinguished the genomes of B. pseudomallei in Indonesia from the ancestral Australian clade and classified them within the Asian clade. This study expands the known presence of B. pseudomallei within Indonesia and confirms that Indonesian B. pseudomallei are genetically linked to those in the rest of Southeast Asia. It is anticipated that melioidosis will be found in other locations across Indonesia as laboratory capacities improve and standardized protocols for detecting and confirming suspected cases of melioidosis are more widely implemented. Author summary: Melioidosis is a disease caused by the bacterium Burkholderia pseudomallei. This disease can affect various organs such as the lungs, skin and soft tissues, central nervous system, internal organs, and others. The clinical manifestations vary, and the lack of laboratory examination support in some countries leads to underreporting of the disease. This also results in a varied death rate, ranging from 10–40% and even higher in some countries. The disease is endemic in the Southeast Asian region and northern Australia and is increasingly recognised in other tropical and sub-tropical locations globally. Indonesia has reported over 100 cases from at least 5 diverse regions. One province in Indonesia, Riau, located on the island of Sumatera bordering Malaysia, has been experiencing a relatively high number of melioidosis cases. This study reports melioidosis cases obtained from two referral hospitals in the province of Riau. The research explores the characteristics of patients, clinical manifestations, and outcomes in these cases. Additionally, the study analyzes factors influencing mortality from melioidosis in Riau. Phylogenetic analysis of six isolates of B. pseudomallei determined that B. pseudomallei in the province of Riau, Indonesia is genetically linked to those from neighbouring countries in Southeast Asia. [ABSTRACT FROM AUTHOR]

Objective: Burkholderia pseudomallei, the etiological cause of melioidosis, is a soil saprophyte endemic in South-East Asia, where it constitutes a public health concern of high-priority. Melioidosis cases are sporadically identified in nonendemic areas, usually associated with travelers or import of goods from endemic regions. Due to extensive intercontinental traveling and the anticipated climate change-associated alterations of the soil bacterial flora, there is an increasing concern for inadvertent establishment of novel endemic areas, which may expand the global burden of melioidosis. Rapid diagnosis, isolation and characterization of B. pseudomallei isolates is therefore of utmost importance particularly in non-endemic locations. Data description: We report the genome sequences of two novel clinical isolates (MWH2021 and MST2022) of B. pseudomallei identified in distinct acute cases of melioidosis diagnosed in two individuals arriving to Israel from India and Thailand, respectively. The data includes preliminary genetic analysis of the genomes determining their phylogenetic classification in rapport to the genomes of 131 B. pseudomallei strains documented in the NCBI database. Inspection of the genomic data revealed the presence or absence of loci encoding for several documented virulence determinants involved in the molecular pathogenesis of melioidosis. Virulence analysis in murine models of acute or chronic melioidosis established that both strains belong to the highly virulent class of B. pseudomalleii. [ABSTRACT FROM AUTHOR]

Early diagnosis is essential for the successful management of Burkholderia pseudomallei infection, but it cannot be achieved by the current gold standard culture technique. Therefore, this study aimed to develop a lateral flow immunoassay (LFIA) targeting B. pseudomallei capsular polysaccharide. The development was performed by varying nitrocellulose membrane reaction pads and chase buffers. The prototype LFIA is composed of Unisart CN95 and chase buffer containing tris-base, casein, and Surfactant 10G. The assay showed no cross-reactivity with E. coli, S. aureus, P. aeruginosa, and P. acne. The limit of detections (LODs) of the prototype LFIA was 107 and 106 CFU/mL B. pseudomallei in hemoculture medium and artificial urine, respectively. These LODs suggest that this prototype can detect melioidosis from positive hemoculture bottles but not straight from urine. Additionally, these LODs are still inferior compared to Active Melioidosis Detect (AMDTM). Overall, this prototype holds the potential to be used clinically with hemoculture bottles. However, further improvements should be considered, especially for use with urine samples. [ABSTRACT FROM AUTHOR]

Background: Melioidosis, a tropical infectious disease caused by Burkholderia pseudomallei, is epidemic in most region in Southeast Asia with high case fatality. However, there is scanty information regarding the disease's epidemiological pattern, demographics, and underlying risk factors. Method: This 5-year retrospective study of 185 confirmed cases which were taken from the Negeri Sembilan Melioidosis Registry between 2018 and 2022. We aim to describe the incidence, mortality rate, case fatality, relationship with meteorology, and factors that influence mortality in this central region of Peninsular Malaysia. Results: Incidence rate (IR) of melioidosis in Negeri Sembilan is varied at 1.9 to 5.1 with mean of 3.1 in 100,000 population per year. IR varied between districts in the state from zero to 22.01 in 100,000 population per year. Mortality rate were ranged from 0.17 to 0.74 cases with mean of 0.44 cases in 100,000 population per year. The case fatality rate of this state scattered from 8.70% to 16.67%. There were no significant linear associations between cases and deaths with monthly rainfall and humidity. The mean age of patients was 52.8 years, predominated with age around 41–60 years old. Males (77.8%) predominated, and the majority of cases were Malays (88.9%) and had exposed to soil related activities (74.6%). Mortality from melioidosis was more likely in Bumiputera and non-Malaysians (p<0.05). Patients who had at least one comorbidity were at a higher risk of death from melioidosis (p<0.05). Diabetes mellitus was found in 41.1% of all identified cases, making it a major underlying risk factor for both developing and dying from melioidosis (aOR:19.32, 95%CI:1.91–195.59, p<0.05). Hypertension and mortality status in melioidosis are also significantly correlated (aOR: 7.75, 95% CI: 2.26–26.61, p<0.05). Conclusion: The epidemiological patterns of cases reported from Negeri Sembilan are consistent for the most part from previous studies in other states in Malaysia and global with regard to its incidence, case fatality, demographic and predisposing chronic diseases. Diabetes mellitus and hypertension were significantly linked to increased mortality among all determinants. Author summary: Melioidosis is an environmentally acquired infection caused by Burkholderia pseudomallei, with the global distribution of this bacteria is comparatively restricted to temperate zones. The details on epidemiological pattern, demographics, and underlying risk factors of the disease, however, are still scarce. This study examines a 5-year retrospective analysis of 185 confirmed cases that were obtained between 2018 and 2022 from the Negeri Sembilan Melioidosis Registry. In this central region of Peninsular Malaysia, the authors detailed the incidence, death rate, case fatality, correlation with meteorology, and factors influencing mortality. Findings indicated that the state had a 3.1 in 100,000 population yearly mean incidence rate of melioidosis, with a case fatality rate ranging from 8.70% to 16.67%. The incidences vary throughout districts. Nevertheless, no significant relationships were found between monthly humidity and rainfall and the number of cases and fatalities. The main factors found to be associated with a higher mortality rate included bumiputra ethnicities, underlying comorbidities and diabetes mellitus. Remarkably, there was also a correlation found between the mortality status in melioidosis with hypertension. Despite a few limitations, these findings could contribute to improving healthcare professionals' knowledge of the illness and for better prevention care. [ABSTRACT FROM AUTHOR]

In contrast to "frank" pathogens, like Salmonella entrocolitica, Shigella dysenteriae, and Vibrio cholerae, that always have a probability of disease, "opportunistic" pathogens are organisms that cause an infectious disease in a host with a weakened immune system and rarely in a healthy host. Historically, drinking water treatment has focused on control of frank pathogens, particularly those from human or animal sources (like Giardia lamblia, Cryptosporidium parvum, or Hepatitis A virus), but in recent years outbreaks from drinking water have increasingly been due to opportunistic pathogens. Characteristics of opportunistic pathogens that make them problematic for water treatment include: (1) they are normally present in aquatic environments, (2) they grow in biofilms that protect the bacteria from disinfectants, and (3) under appropriate conditions in drinking water systems (e.g., warm water, stagnation, low disinfectant levels, etc.), these bacteria can amplify to levels that can pose a public health risk. The three most common opportunistic pathogens in drinking water systems are Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa. This report focuses on these organisms to provide information on their public health risk, occurrence in drinking water systems, susceptibility to various disinfectants, and other operational practices (like flushing and cleaning of pipes and storage tanks). In addition, information is provided on a group of nine other opportunistic pathogens that are less commonly found in drinking water systems, including Aeromonas hydrophila, Klebsiella pneumoniae, Serratia marcescens, Burkholderia pseudomallei, Acinetobacter baumannii, Stenotrophomonas maltophilia, Arcobacter butzleri, and several free-living amoebae including Naegleria fowleri and species of Acanthamoeba. The public health risk for these microbes in drinking water is still unclear, but in most cases, efforts to manage Legionella, mycobacteria, and Pseudomonas risks will also be effective for these other opportunistic pathogens. The approach to managing opportunistic pathogens in drinking water supplies focuses on controlling the growth of these organisms. Many of these microbes are normal inhabitants in biofilms in water, so the attention is less on eliminating these organisms from entering the system and more on managing their occurrence and concentrations in the pipe network. With anticipated warming trends associated with climate change, the factors that drive the growth of opportunistic pathogens in drinking water systems will likely increase. It is important, therefore, to evaluate treatment barriers and management activities for control of opportunistic pathogen risks. Controls for primary treatment, particularly for turbidity management and disinfection, should be reviewed to ensure adequacy for opportunistic pathogen control. However, the major focus for the utility's opportunistic pathogen risk reduction plan is the management of biological activity and biofilms in the distribution system. Factors that influence the growth of microbes (primarily in biofilms) in the distribution system include, temperature, disinfectant type and concentration, nutrient levels (measured as AOC or BDOC), stagnation, flushing of pipes and cleaning of storage tank sediments, and corrosion control. Pressure management and distribution system integrity are also important to the microbial quality of water but are related more to the intrusion of contaminants into the distribution system rather than directly related to microbial growth. Summarizing the identified risk from drinking water, the availability and quality of disinfection data for treatment, and guidelines or standards for control showed that adequate information is best available for management of L. pneumophila. For L. pneumophila, the risk for this organism has been clearly established from drinking water, cases have increased worldwide, and it is one of the most identified causes of drinking water outbreaks. Water management best practices (e.g., maintenance of a disinfectant residual throughout the distribution system, flushing and cleaning of sediments in pipelines and storage tanks, among others) have been shown to be effective for control of L. pneumophila in water supplies. In addition, there are well documented management guidelines available for the control of the organism in drinking water distribution systems. By comparison, management of risks for Mycobacteria from water are less clear than for L. pneumophila. Treatment of M. avium is difficult due to its resistance to disinfection, the tendency to form clumps, and attachment to surfaces in biofilms. Additionally, there are no guidelines for management of M. avium in drinking water, and one risk assessment study suggested a low risk of infection. The role of tap water in the transmission of the other opportunistic pathogens is less clear and, in many cases, actions to manage L. pneumophila (e.g., maintenance of a disinfectant residual, flushing, cleaning of storage tanks, etc.) will also be beneficial in helping to manage these organisms as well. [ABSTRACT FROM AUTHOR]

Background The autotransporter protein Burkholderia intracellular motility A (BimA) facilitates the entry of Burkholderia pseudomallei into the central nervous system (CNS) in mouse models of melioidosis. Its role in the pathogenesis of human cases of CNS melioidosis is incompletely defined. Methods Consecutive culture-confirmed cases of melioidosis at 2 sites in tropical Australia after 1989 were reviewed. Demographic, clinical, and radiological data of the patients with CNS melioidosis were recorded. The bimA allele (bimA Bm or bimA Bp ) of the B. pseudomallei isolated from each patient was determined. Results Of the 1587 cases diagnosed at the 2 sites during the study period, 52 (3.3%) had confirmed CNS melioidosis: 20 (38.5%) had a brain abscess, 18 (34.6%) had encephalomyelitis, 4 (7.7%) had isolated meningitis, and 10 (19.2%) had extra-meningeal disease. Among the 52 patients, there were 8 (15.4%) deaths; 17/44 (38.6%) survivors had residual disability. The bimA allele was characterized in 47/52; 17/47 (36.2%) had the bimA Bm allele and 30 (63.8%) had the bim A Bp allele. Patients with a bimA Bm variant were more likely to have a predominantly neurological presentation (odds ratio [OR]: 5.60; 95% confidence interval: 1.52–20.61; P  = .01), to have brainstem involvement (OR: 7.33; 1.92–27.95; P  = .004), and to have encephalomyelitis (OR: 4.69; 1.30–16.95; P  = .02). Patients with a bimA Bm variant were more likely to die or have residual disability (OR: 4.88; 1.28–18.57; P  = .01). Conclusions The bimA allele of B. pseudomallei has a significant impact on the clinical presentation and outcome of patients with CNS melioidosis. [ABSTRACT FROM AUTHOR]

Bacteriophages (phages), viruses that infect bacteria, are found in abundance not only in the environment but also in the human body. The use of phages for the diagnosis of melioidosis, a tropical infectious disease caused by Burkholderia pseudomallei, is emerging as a promising novel approach, but our understanding of conditions under which Burkholderia prophages can be induced remains limited. Here, we first demonstrated the isolation of Burkholderia phages from the hemocultures of melioidosis patients. The B. pseudomallei-positive hemoculture bottles were filtered to remove bacteria, and then phages were isolated and purified by spot and double agar overlay plaque assays. Forty blood samples (hemoculture-confirmed melioidosis) were tested, and phages were found in 30% of the samples. Transmission electron microscopy and genome analysis of the isolated phages, vB_HM387 and vB_ HM795, showed that both phages are Myoviruses. These two phages were stable at a pH of 5-7 and temperatures of 25-37°C, suggesting their ability to survive in human blood. The genome sizes of vB_HM387 and vB_HM795 are 36.3 and 44.0 kb, respectively. A phylogenetic analysis indicated that vB_HM387 has homologs, but vB_HM795 is a novel Myovirus, suggesting the heterogeneity of Burkholderia phages in melioidosis patients. The key finding that Burkholderia phages could be isolated from the blood of melioidosis patients highlights the potential application of phage-based assays by detecting phages in blood as a pathogen-derived biomarker of infection. [ABSTRACT FROM AUTHOR]

Background: Melioidosis, caused by the category B biothreat agent Burkholderia pseudomallei, is a disease with a high mortality rate and requires an immediate culture-independent diagnosis for effective disease management. In this study, we developed a highly sensitive qPCR assay for specific detection of Burkholderia pseudomallei and melioidosis disease diagnosis based on a novel target sequence. Methods: An extensive in-silico analysis was done to identify a novel and highly conserved sequence for developing a qPCR assay. The specificity of the developed assay was analyzed with 65 different bacterial cultures, and the analytical sensitivity of the assay was determined with the purified genomic DNA of B. pseudomallei. The applicability of the assay for B. pseudomallei detection in clinical and environmental matrices was evaluated by spiking B. pseudomallei cells in the blood, urine, soil, and water along with suitable internal controls. Results: A novel 85-nucleotide-long sequence was identified using in-silico tools and employed for the development of the highly sensitive and specific quantitative real-time PCR assay S664. The assay S664 was found to be highly specific when evaluated with 65 different bacterial cultures related and non-related to B. pseudomallei. The assay was found to be highly sensitive, with a detection limit of 3 B. pseudomallei genome equivalent copies per qPCR reaction. The detection limit in clinical matrices was found to be 5 × 102 CFU/mL for both human blood and urine. In environmental matrices, the detection limit was found to be 5 × 101 CFU/mL of river water and 2 × 103 CFU/gm of paddy field soil. Conclusions: The findings of the present study suggest that the developed assay S664 along with suitable internal controls has a huge diagnostic potential and can be successfully employed for specific, sensitive, and rapid molecular detection of B. pseudomallei in various clinical and environmental matrices. [ABSTRACT FROM AUTHOR]

Introduction: Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a disease endemic in many tropical countries globally. Clinical presentation is highly variable, ranging from asymptomatic to fatal septicemia, and thus the outcome of infection can depend on the host immune responses. The aims of this study were to firstly, characterize the macrophage immune response to B. pseudomallei and secondly, to determine whether the immune response was modified in the presence of novel inhibitors targeting the virulence factor, the macrophage infectivity potentiator (Mip) protein. We hypothesized that inhibition of Mip in B. pseudomallei would disarm the bacteria and result in a host beneficial immune response. Methods: Murine macrophage J774A.1 cells were infected with B. pseudomallei K96243 in the presence of small-molecule inhibitors targeting the Mip protein. RNA-sequencing was performed on infected cells four hours post-infection. Secreted cytokines and lactose dehydrogenase were measured in cell culture supernatants 24 hours post-infection. Viable, intracellular B. pseudomallei in macrophages were also enumerated 24 hours post-infection. Results: Global transcriptional profiling of macrophages infected with B. pseudomallei by RNA-seq demonstrated upregulation of immune-associated genes, in particular a significant enrichment of genes in the TNF signaling pathway. Treatment of B. pseudomallei-infected macrophages with the Mip inhibitor, AN_CH_37 resulted in a 5.3-fold reduction of il1b when compared to cells treated with DMSO, which the inhibitors were solubilized in. A statistically significant reduction in IL-1β levels in culture supernatants was seen 24 hours post-infection with AN_CH_37, as well as other pro-inflammatory cytokines, namely IL-6 and TNF-α. Treatment with AN_CH_37 also reduced the survival of B. pseudomallei in macrophages after 24 hours which was accompanied by a significant reduction in B. pseudomallei-induced cytotoxicity as determined by lactate dehydrogenase release. Discussion: These data highlight the potential to utilize Mip inhibitors in reducing potentially harmful pro-inflammatory responses resulting from B. pseudomallei infection in macrophages. This could be of significance since overstimulation of pro-inflammatory responses can result in immunopathology, tissue damage and septic shock. [ABSTRACT FROM AUTHOR]

Background: Melioidosis is an emerging infectious disease caused by the Gram-negative bacterium Burkholderia pseudomallei, which can lead to a range of clinical manifestations including acute septicaemia and chronic localized infections. Osteomyelitis is an uncommon but well-recognized complication of melioidosis, with the calcaneus being a relatively rare site of involvement. Case Description: The authors present a case series of three patients with calcaneal osteomyelitis caused by Burkholderia pseudomallei, infection. All three patients presented with non-specific symptoms like fever and weight loss, followed by localized ankle/foot pain and swelling. Diagnosis was made based on imaging findings (radiographs and MRI) and isolation of Burkholderia pseudomallei, from blood or pus cultures. Treatment involved a combination of initial intravenous antibiotic therapy (meropenem or ceftazidime) for 4-6 weeks, followed by oral trimethoprimsulfamethoxazole for 6 months. One patient required surgical drainage of a plantar abscess. All patients had a favorable clinical outcome with a resolution of osteomyelitis on follow-up imaging. Conclusion: Calcaneal osteomyelitis is a rare but potentially serious complication of melioidosis. Early recognition and prompt initiation of appropriate antibiotic therapy are crucial for favorable outcomes. Clinicians should maintain a high index of suspicion for osteomyelitis in patients with melioidosis, especially in patients from Endemic areas as well as in patients with risk factors like diabetes. Close follow-up and monitoring for potential relapse or reactivation are essential, particularly in immunocompromised individuals. [ABSTRACT FROM AUTHOR]