Pseudomallei Isolates Research Articles

Molecular epidemiology of Burkholderia pseudomallei in Hainan Province of China based on O-antigen

BackgroundBurkholderia pseudomallei is a gram-negative bacterium widely found in Southeast Asia and northern Australia. This bacterium, which lacks an available vaccine, is the causative agent of melioidosis and has properties that potentially enable its exploitation as a bioweapon. MethodsPolymerase chain reaction assays targeting each of the lipopolysaccharide (LPS) genetic types were used to investigate genotype frequencies in B. pseudomallei populations. Silver staining, gas chromatography-mass spectrometry (GC-MS), and immunofluorescence were used to characterize LPS. ResultsIn our study, a total of 169 clinical B. pseudomallei isolates were collected from Hainan, China between 2004 and 2016. The results showed that LPS genotype A was the predominant type, comprising 91.1% of the samples, compared with only 8.9% of LPS genotype B. The majority of patients were male and were diagnosed with sepsis or pneumonia. Silver staining and GC-MS demonstrated that LPS genotypes A and B exhibited distinct phenotypes and molecular structures. Immunofluorescence tests showed there was no cross-reaction between LPS genotypes A and B. ConclusionThis is the first report on the molecular epidemiology of B. pseudomallei based on O-antigen in China. Tracking the regional distribution of different LPS genotypes offers significant insights relevant to the development and administration of LPS-based vaccines.

Meropenem-resistant Burkholderia pseudomallei: a concerning single case in Australia with no prior meropenem exposure

We report a case of cutaneous melioidosis in a 54-year-old male with a meropenem-resistant sub-population. He was empirically treated with episodic doxycycline and trimethoprim–sulfamethoxazole; however, the abscess re-accumulated. The patient had no prior exposure to meropenem. A sub-population of the isolate was meropenem resistant with an MIC >32 µg ml−1 and the identification was re-confirmed as Burkholderia pseudomallei. Whole-genome sequencing with ARDaP analysis only revealed a resistance determinant to doxycycline and did not reveal a resistance determinant to meropenem. Furthermore, no carbapenemases were detected through multiple bioinformatics tools. To date, this is the first reported case in Australia of a B. pseudomallei isolate resistant to meropenem without previous carbapenem exposure.

Use of Comparative Genomics To Resolve an Unusual Case of Aminoglycoside Susceptibility in the Melioidosis Pathogen Burkholderia pseudomallei in Bangladesh.

Melioidosis is an emerging tropical infectious disease with a rising global burden caused by the environmental bacterium Burkholderia pseudomallei. It is endemic in Southeast and South Asia, including Bangladesh. A rare aminoglycoside-susceptible B. pseudomallei isolate (Y2019) has recently been reported from a melioidosis patient in Dhaka, Bangladesh. To understand the geographical origins of Y2019, we subjected it and 10 other isolates from Bangladesh to whole-genome sequencing. In a phylogenetic tree with a global set of B. pseudomallei genomes, most Bangladeshi genomes clustered tightly within the Asian clade. In contrast, Y2019 was closely related to ST881 isolates from Sarawak, Malaysian Borneo, a gentamicin-sensitive sequence type, suggesting infection in Borneo. Y2019 also contained the same gentamicin sensitivity conferring nonsynonymous mutation in the drug efflux pump encoding the amrB gene. In the absence of a full travel history, whole-genome sequencing and bioinformatics tools have revealed the likely origin of this rare isolate.

Multi-locus sequence typing and genetic diversity of antibiotic-resistant genes and virulence-associated genes in Burkholderia pseudomallei: Insights from whole genome sequencing of animal and environmental isolates in Thailand

Burkholderia pseudomallei is a Gram-negative bacillus and the etiological agent of melioidosis in humans and animals. The disease is highly endemic in northern Australia and Southeast Asia. Comprehensive genomic data are essential for understanding the bacteria's dissemination and genetic relationships among strains from different geographical regions. In this study, we conducted antimicrobial susceptibility testing and whole-genome sequencing of 54 B. pseudomallei isolates obtained from environmental and animal sources in southern Thailand between 2011 and 2018. Their genomics were determined of antibiotic-resistant genes (ARGs), virulence-associated genes, mobile genetic elements (MGEs), sequence types (STs), and single nucleotide polymorphisms (SNPs) to evaluate their epidemiological relatedness. Remarkably, all 54 isolates displayed sensitivity to antimicrobial agents typically used for melioidosis treatment. We identified nine distinct sequence types: ST392, ST51, ST409, ST508, ST376, ST1721, ST389, ST395, and ST289. Oxacillinase genes and the resistance nodulation family of efflux pumps (RND) were identified as contributors to antimicrobial resistance. Phylogenetic analysis demonstrated close genetic relations with other strains isolated from Southeast Asia. Furthermore, 172 virulence-associated genes were identified among the isolates, suggesting variations in clinical presentations. These findings underscore the importance of ongoing molecular genetic surveillance of B. pseudomallei for effective healthcare management and reducing melioidosis mortality.

A molecular epidemiological analysis of Burkholderia pseudomallei in southern Thailand.

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.

Genetic diversity, determinants, and dissemination of Burkholderia pseudomallei lineages implicated in melioidosis in Northeast Thailand

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.

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

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

Identification and mechanism determination of the efflux pump subunit amrB gene mutations linked to gentamicin susceptibility in clinical Burkholderia pseudomallei from Malaysian Borneo.

The bacterium Burkholderia pseudomallei is typically resistant to gentamicin but rare susceptible strains have been isolated in certain regions, such as Thailand and Sarawak, Malaysia. Recently, several amino acid substitutions have been reported in the amrB gene (a subunit of the amrAB-oprA efflux pump gene) that confer gentamicin susceptibility. However, information regarding the mechanism of the substitutions conferring the susceptibility is lacking. To understand the mechanism of amino acid substitution that confers susceptibility, this study identifies the corresponding mutations in clinical gentamicin-susceptible B. pseudomallei isolates from the Malaysian Borneo (n = 46; Sarawak: 5; Sabah: 41). Three phenotypically confirmed gentamicin-susceptible (GENs) strains from Sarawak, Malaysia, were screened for mutations in the amrB gene using gene sequences of gentamicin-resistant (GENr) strains (QEH 56, QEH 57, QEH20, and QEH26) and publicly available sequences (AF072887.1 and BX571965.1) as the comparator. The effect of missense mutations on the stability of the AmrB protein was determined by calculating the average energy change value (ΔΔG). Mutagenesis analysis identified a polymorphism-associated mutation, g.1056T > G, a possible susceptible-associated in-frame deletion, Delta V412, and a previously confirmed susceptible-associated amino acid substitution, T368R, in each of the three GENs isolates. The contribution of Delta V412 needs further confirmation by experimental mutagenesis analysis. The mechanism by which T368R confers susceptibility, as elucidated by in silico mutagenesis analysis using AmrB-modeled protein structures, is proposed to be due to the location of T368R in a highly conserved region, rather than destabilization of the AmrB protein structure.

Identification of Burkholderia cepacia strains that express a Burkholderia pseudomallei-like capsular polysaccharide.

Burkholderia pseudomallei and Burkholderia cepacia are Gram-negative, soil-dwelling bacteria that are found in a wide variety of environmental niches. While B. pseudomallei is the causative agent of melioidosis in humans and animals, members of the B. cepacia complex typically only cause disease in immunocompromised hosts. In this study, we report the identification of B. cepacia strains isolated from either patients or soil in Laos and Thailand that express a B. pseudomallei-like 6-deoxyheptan capsular polysaccharide (CPS). These B. cepacia strains were initially identified based on their positive reactivity in a latex agglutination assay that uses the CPS-specific monoclonal antibody (mAb) 4B11. Mass spectrometry and recA sequencing confirmed the identity of these isolates as B. cepacia (formerly genomovar I). Total carbohydrates extracted from B. cepacia cell pellets reacted with B. pseudomallei CPS-specific mAbs MCA147, 3C5, and 4C4, but did not react with the B. pseudomallei lipopolysaccharide-specific mAb Pp-PS-W. Whole genome sequencing of the B. cepacia isolates revealed the presence of genes demonstrating significant homology to those comprising the B. pseudomallei CPS biosynthetic gene cluster. Collectively, our results provide compelling evidence that B. cepacia strains expressing the same CPS as B. pseudomallei co-exist in the environment alongside B. pseudomallei. Since CPS is a target that is often used for presumptive identification of B. pseudomallei, it is possible that the occurrence of these unique B. cepacia strains may complicate the diagnosis of melioidosis.IMPORTANCEBurkholderia pseudomallei, the etiologic agent of melioidosis, is an important cause of morbidity and mortality in tropical and subtropical regions worldwide. The 6-deoxyheptan capsular polysaccharide (CPS) expressed by this bacterial pathogen is a promising target antigen that is useful for rapidly diagnosing melioidosis. Using assays incorporating CPS-specific monoclonal antibodies, we identified both clinical and environmental isolates of Burkholderia cepacia that express the same CPS antigen as B. pseudomallei. Because of this, it is important that staff working in melioidosis-endemic areas are aware that these strains co-exist in the same niches as B. pseudomallei and do not solely rely on CPS-based assays such as latex-agglutination, AMD Plus Rapid Tests, or immunofluorescence tests for the definitive identification of B. pseudomallei isolates.

A systematic review and meta-analysis of the global prevalence and relationships among Burkholderia pseudomallei sequence types isolated from humans, animals, and the environment.

Burkholderia pseudomallei, a highly pathogenic bacterium responsible for melioidosis, exhibits ecological ubiquity and thrives within soil and water reservoirs, posing significant infection risks to humans and animals through direct contact. The aim of this study was to elucidate the genetic diversity and prevalence patterns of B. pseudomallei sequence types (STs) across a global spectrum and to understand the relationships between strains isolated from different sources. We performed a systematic review and meta-analysis in this study. Extensive research was carried out across three comprehensive databases, including PubMed, Scopus, and ScienceDirect with data collected from 1924 to 2023. A total of 40 carefully selected articles contributed 2737 B. pseudomallei isolates attributed to 729 distinct STs and were incorporated into the systematic review. Among these, ST46 emerged as the most prominent, featuring in 35% of the articles and demonstrating a dominant prevalence, particularly within Southeast Asia. Moreover, ST51 consistently appeared across human, animal, and environmental studies. Subsequently, we performed a meta-analysis, focusing on nine specific STs: ST46, ST51, ST54, ST70, ST84, ST109, ST289, ST325, and ST376. Surprisingly, no statistically significant differences in their pooled prevalence proportions were observed across these compartments for ST46, ST70, ST289, ST325, and ST376 (all p > 0.69). Conversely, the remaining STs, including ST51, ST54, ST84, and ST109, displayed notable variations in their prevalence among the three domains (all p < 0.04). Notably, the pooled prevalence of ST51 in animals and environmental samples surpassed that found in human isolates (p < 0.01). To the best of our knowledge, this study is the first systematic review and meta-analysis to investigate the intricate relationships between STs and their sources and contributes significantly to our understanding of B. pseudomallei diversity within the One Health framework.

Phylogeographic characterization of Burkholderia pseudomallei isolated from Bangladesh.

Burkholderia 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. In 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. A 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. ST 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.

In Vitro Susceptibility of Burkholderia pseudomallei Isolates to Cefiderocol and Ceftazidime/Avibactam from Odisha, India.

Introduction and Objectives The availability of a limited arsenal of antibacterial agents effective against Burkholderia pseudomallei, the causative agent of melioidosis, together with sporadic reports of emergence of resistance necessitates an evaluation of in vitro activity of new antimicrobials against clinical B. pseudomallei isolates. Cefiderocol (CFDC), a novel siderophore cephalosporin, and ceftazidime-avibactam (CZA), a new β lactam combination agent, have shown promising results for the treatment of difficult-to-treat Gram-negative bacilli infections with limited treatment options. This study was conducted to determine the in vitro activity of CFDC and CZA against a contemporary collection of 60 B. pseudomallei clinical isolates. Materials and Methods Minimum inhibitory concentrations (MIC) of CFDC and CZA were determined by broth microdilution and E-test, respectively. The performance of disk diffusion was also evaluated for CFDC. Results All B. pseudomallei isolates were susceptible to CFDC and CZA with MIC range of 0.125 to 2 mg/L and 0.19 to 1 mg/L, respectively. Zone diameters for CFDC ranged from 31 to 40 mm. Conclusion CFDC and CZA exhibited excellent in vitro activity against 60 B. pseudomallei isolates. Further pharmacokinetic-pharmacodynamics studies and clinical trials are needed to prove the clinical efficacy of CFDC and CZA in the treatment of melioidosis.

Exploring Cefiderocol Resistance Mechanisms in Burkholderia pseudomallei.

Cefiderocol is a siderophore cephalosporin designed mainly for treatment of infections caused by β-lactam and multidrug-resistant Gram-negative bacteria. Burkholderia pseudomallei clinical isolates are usually highly cefiderocol susceptible, with in vitro resistance found in a few isolates. Resistance in clinical B. pseudomallei isolates from Australia is caused by a hitherto uncharacterized mechanism. We show that, like in other Gram-negatives, the PiuA outer membrane receptor plays a major role in cefiderocol nonsusceptibility in isolates from Malaysia.

Workflow for Identification of Burkholderia pseudomallei Clinical Isolates in Myanmar.

Burkholderia pseudomallei, the highly infectious and causative organism of melioidosis, was first identified in Myanmar in 1911. B. pseudomallei was identified in Myanmar because of its genetic relatedness to Burkholderia species. In this study, we identified two isolates of Burkholderia cenocepacia, two Acinetobacter baumannii complexes, and 18 clinical isolates of B. pseudomallei using Vitek 2. These isolates were first screened using a latex agglutination test, which showed positive results in 20 of the 22 isolates. All isolates were cultured on Ashdown՚s agar and further tested using molecular methods. Specific PCR for type III secretion system (TTSs) gene clusters indicated 19 B. pseudomallei isolates out of 22 isolates. Furthermore, 16S rRNA and recA gene sequencing were used as the gold standard methods and yielded the same results. RapID NF Plus detected 16 B. pseudomallei out of 22 isolates. Vitek 2 and RapID NF Plus should be considered key tools in the diagnosis of melioidosis and surveillance of B. pseudomallei in Myanmar; however, accurate identification must be confirmed by TTS1 PCR. This study evaluated the presumptive workflow for the investigation of B. pseudomallei infections using different methods and options, in line with the available equipment.

DNase I and chitosan enhance efficacy of ceftazidime to eradicate Burkholderia pseudomallei biofilm cells

Biofilm-associated Burkholderia pseudomallei infection contributes to antibiotic resistance and relapse of melioidosis. Burkholderia pseudomallei biofilm matrix contains extracellular DNA (eDNA) that is crucial for biofilm establishment. However, the contribution of eDNA to antibiotic resistance by B. pseudomallei remains unclear. In this study, we first demonstrated in vitro that DNase I with the administration of ceftazidime (CAZ) at 24 h considerably inhibited the 2-day biofilm formation and reduced the number of viable biofilm cells of clinical B. pseudomallei isolates compared to biofilm treated with CAZ alone. A 3–4 log reduction in numbers of viable cells embedded in the 2-day biofilm was observed when CAZ was combined with DNase I. Confocal laser-scanning microscope visualization emphasized the competence of DNase I followed by CAZ supplementation to significantly limit B. pseudomallei biofilm development and to eradicate viable embedded B. pseudomallei biofilm cells. Furthermore, DNase I supplemented with chitosan (CS) linked with CAZ (CS/CAZ) significantly eradicated shedding planktonic and biofilm cells. These findings indicated that DNase I effectively degraded eDNA leading to biofilm inhibition and dispersion, subsequently allowing CAZ and CS/CAZ to eradicate both shedding planktonic and embedded biofilm cells. These findings provide efficient strategies to interrupt biofilm formation and improve antibiotic susceptibility of biofilm-associated infections.

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

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

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

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

New genetic markers for &lt;i&gt;Burkholderia pseudomallei&lt;/i&gt; strains typing

Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease in humans and animals that may be asymptomatic long-term and quickly develop to pneumonia and septicemia in immunocompromised state and due to predisposing factors. Melioidosis is endemic in countries with tropical and subtropical climates, where B. pseudomallei is a part of the soil and water microbiota of stagnant water bodies, as well as the plant rhizosphere. The recording of imported melioidosis cases in countries with temperate climate zone, along with the continued threat of using this pathogen as a bioterrorism agent, indicate the relevance of research aimed at developing modern methods for its diagnosing and typing. Current research in the intraspecific differentiation of pathogenic strains of infectious diseases tends to use two or more types of molecular markers. A promising direction for the B. pseudomallei strains genotyping is based on using a combination of VNTR loci, which provides a high discriminating ability of the method of multi-locus variable tandem repeat number analysis (MLVA), and slowly evolving single nucleotide polymorphisms (SNPs). The aim of this work was to identify new VNTR and SNP loci suitable for use as genetic markers in molecular typing of the melioidosis causative agent. 20 strains of B. pseudomallei from the collection of the Volgograd Plague Control Research Institute and whole genome sequences of 85 B. pseudomallei strains from the GenBank NCBI database were analyzed. While typing melioidosis causative agent strains from the GenBank NCBI database for 4 VNTR loci, 74 genotypes were identified, of which 64 were unique (HunterGaston index 0.997). It was found that the high allelic polymorphism of VNTR loci limited a potential to determine the geographical and phylogenetic relationships of B. pseudomallei isolates by using the MLVA-4 scheme, and the identified null alleles increased the risk of homoplasia. In this regard, the MLVA-4 scheme was supplemented with a VNTR marker in the BPSS1974 locus encoding a collagen-like protein as well as with SNP markers in the genes of lipid A biosynthesis lauroyl acyltransferase, RNA polymerase sigma factor RpoH, and glutamine amidotransferase. For amplification of the select loci, primers and probes were designed as part of the developed scheme, which were tested in the typing of B. pseudomallei collection strains. According to the results of a cluster analysis for 105 strains of the melioidosis causative agent, an integrated approach turned out to be optimal, allowing differentiation of strains within SNP groups based on VNTR profiles. The methodological approach developed for B. pseudomallei genetic typing, based on a comprehensive analysis of 4 SNP- and 5 MLVA-markers in our modification, allowed to differentiate strains according to the geographical regions of their origin and establish the clonal origin of isolates upon revealing cases of melioidosis.

Genomics and structural insight into the masking of gentamicin-resistance in clinical Burkholderia pseudomallei strain VB29710 from India

The present study reported a rare gentamicin-susceptible β-lactamase (PenA, OXA-57) expressing clinical Burkholderia pseudomallei isolate VB29710 from India. Whole-genome sequencing and structural analyses revealed the insertion of R962 and L963 into AmrB, the transmembrane-protein of the AmrAB-OprA efflux-pump that affected aminoglycoside-efflux through local alterations in backbone conformation.

Genomic diversity of resistant and virulent factors of Burkholderia pseudomallei clinical strains recovered from Guangdong using whole genome sequencing.

Burkholderia pseudomallei (B. pseudomallei) is a highly infectious agent and causes melioidosis, in both humans and animals, which is endemic in Southeast Asia and Northern Australia. This study aims to determine the molecular epidemiology, resistant determinants, and genomic diversity of the clinical isolates of B. pseudomallei to further elucidate the phylogenetic and evolutionary relationship of the strains with those in other endemic regions. In this study, we obtained eight clinical B. pseudomallei isolates from Guangdong province from 2018 to 2019. All the isolates were sequenced using the Illumina NovaSeq platform. The draft genomes of B. pseudomallei were further used to find antibiotic-resistant genes (ARGs), virulence factors, and gene mutations. Multilocus sequence typing (MLST) and single nucleotide polymorphism (SNP) analysis were performed to characterize the diversity and epidemiology of the strains. All isolates were susceptible to antibiotics commonly used for melioidosis treatment. Class D beta-lactamases genes OXA-57 and OXA-59, as well as various mutation factors such as amrA, amrB, omp38, gyrA, and ceoB were identified. MLST analysis of the B. pseudomallei strains identified eight different sequence types (STs): ST1774, ST1775, ST271, ST562, ST46, ST830, ST1325, and ST10. Phylogenetic analysis found that the strains used in this study showed high genetic diversity. We also report 165 virulence factors among B. pseudomallei strains responsible for different neurological disorders, pneumonia, skin lesions, and abscesses. All strains recovered in this study were susceptible to commonly used antibiotics. However, high genetic diversity exists among the isolates. The surveillance, diagnosis, and clinical features of melioidosis varied in different geographical locations. These regional differences in the clinical manifestations have implications for the practical management of the disease. The present study reports the identification of different mutation and virulence factors among B. pseudomallei strains responsible for different neurological disorders, pneumonia, skin lesions, and abscesses.