First complete genome sequence of a Bacterial Panicle Blight causing pathogen, Burkholderia glumae, isolated from symptomatic rice grains from Bangladesh.

OBJECTIVES: Rice (Oryza sativa) is the most important food for more than two thirds of the world's population. Bangladesh is the third largest producer and consumer of rice globally. Recently, several symptoms of Bacterial Panicle Blight (BPB) in rice, including seedling blight, sheath rot, floret sterility, and spotted grains, have been detected in the country. In addition, the presence of the most prevalent and virulent causative agent of BPB, Burkholderia glumae, has been confirmed in rice displaying symptoms of the disease. BPB could become one of the next emerging diseases of rice in Bangladesh, and a complete genome of a B. glumae strain from the country will help clarify its origin and devise proper management systems to continue sustainable rice production. DATA DESCRIPTION: We report the first complete genome sequence of a B. glumae strain (BD_21g) isolated from symptomatic rice grains in Bangladesh (Natore District). The genome contains 2 chromosomes (1 and 2, with 3,417,499 and 3,855,283 bp, respectively) and 4 plasmids (1-4, with 123,248, 46,628, 88,744 and 53,064 bp, respectively).

Epidemiology and Clinical Features of Burkholderia Bacteremia at a Hospital in India.

Burkholderia spp. are opportunistic pathogens that cause infection in patients with disrupted immunity. The study intended to demonstrate the epidemiology and clinical features associated with Burkholderia spp. bacteremia. This retrospective study was performed to assess the clinical and laboratory characteristics of patients whose blood cultures were growing Burkholderia spp. and, based on their underlying comorbidities, were subjected to survival analysis from January 2022 to December 2022 at a university hospital in northern India. Three hundred patients with Burkholderia spp. bacteremia were included in this study conducted over 1 year. The mean age of the patients was 33.86 years with a male predominance of 56.67% (170/300, 56.67%). Underlying malignancies (207/300, 69.0%) were the most common clinical diagnosis, and catheter in situ (300/300, 100.0%) was the most common risk factor. Burkholderia cenocepacia (244/300, 81.33%) was the most common Burkholderia spp. isolated. All isolates were highly susceptible to minocycline. Kidney disease (P = 0.029), hypertension (P = 0.005), type 2 diabetes mellitus (P = 0.039), and respiratory disease (P <0.001) in patients were significantly associated with death owing to Burkholderia spp. bacteremia, whereas patients with malignancies (P <0.001) and undergoing treatment were significantly associated with a better outcome when the microorganism was susceptible to empirical antibiotics. The presence of indwelling devices, mechanical ventilation (P <0.001), and a hemodialysis catheter (P = 0.026) were statistically significant risk factors associated with poor outcomes.

Evaluating lignin degradation under limited oxygen conditions by bacterial isolates from forest soil.

Lignin, a heterogeneous aromatic polymer present in plant biomass, is intertwined with cellulose and hemicellulose fibrils, posing challenges to its effective utilization due to its phenolic nature and recalcitrance to degradation. In this study, three lignin utilizing bacteria, Klebsiella sp. LEA1, Pseudomonas sp. LEA2, and Burkholderia sp. LEA3, were isolated from deciduous forest soil samples in Nan province, Thailand. These isolates were capable of growing on alkali lignin and various lignin-associated monomers at 40 °C under microaerobic conditions. The presence of Cu2+ significantly enhanced guaiacol oxidation in Klebsiella sp. LEA1 and Pseudomonas sp. LEA2. Lignin-related monomers and intermediates such as 2,6-dimethoxyphenol, 4-vinyl guaiacol, 4-hydroxybenzoic acid, benzoic acid, catechol, and succinic acid were detected mostly during the late stage of incubation of Klebsiella sp. LEA1 and Pseudomonas sp. LEA2 in lignin minimal salt media via GC-MS analysis. The intermediates identified from Klebsiella sp. LEA1 degradation suggested that conversion and utilization occurred through the ß-ketoadipate (ortho-cleavage) pathway under limited oxygen conditions. The ability of these bacteria to thrive on alkaline lignin and produce various lignin-related intermediates under limited oxygen conditions suggests their potential utility in oxygen-limited processes and the production of renewable chemicals from plant biomass.

Burkholderia semiarida as Cause of Recurrent Pulmonary Infection in Immunocompetent Patient, China.

Burkholderia semiarida was previously identified solely as a plant pathogen within the Burkholderia cepacia complex. We present a case in China involving recurrent pneumonia attributed to B. semiarida infection. Of note, the infection manifested in an immunocompetent patient with no associated primary diseases and endured for >3 years.

Burkholderia thailandensis Isolated from Infected Wound, Southwest China, 2022.

We report a clinical isolate of Burkholderia thailandensis 2022DZh obtained from a patient with an infected wound in southwest China. Genomic analysis indicates that this isolate clusters with B. thailandensis BPM, a human isolate from Chongqing, China. We recommend enhancing monitoring and surveillance for B. thailandensis infection in both humans and livestock.

Genetic Diversity and Virulence of Phytopathogenic Burkholderia glumae Strains Isolated from Rice Cultivars in Valleys of the High Jungle of Perú.

Burkholderia glumae causes bacterial leaf blight in rice, and its global spread has been exacerbated by climate change. To understand the genetic diversity and virulence of B. glumae strains isolated from rice cultivars in Perú, 47 isolates were obtained from infected rice fields, all belonging to B. glumae, and confirmed by recA and toxB sequences. The BOX-PCR typing group has 38 genomic profiles, and these turn into seven variable number tandem repeats (VNTR) haplotypes. There was no correlation between clustering and geographical origin. Nineteen strains were selected for phenotypic characterization and virulence, using both the maceration level of the onion bulb proxy and inoculation of seeds of two rice cultivars. Several strains produced pigments other than toxoflavin, which correlated with onion bulb maceration. In terms of virulence at the seed level, all strains produced inhibition at the root and coleoptile level, but the severity of symptoms varied significantly between strains, revealing significant differences in pathogenicity. There is no correlation between maceration and virulence scores, probably reflecting different virulence mechanisms depending on the host infection stage. This is the first study to evaluate the VNTR diversity and virulence of Peruvian strains of B. glumae in two commercial cultivars.

Genomic insights into an extensively drug-resistant and hypervirulent Burkholderia dolosa N149 isolate of a novel sequence type (ST2237) from a Vietnamese patient hospitalised for stroke.

OBJECTIVES: Burkholderia dolosa is a clinically important opportunistic pathogen in inpatients. Here we characterised an extensively drug-resistant and hypervirulent B. dolosa isolate from a patient hospitalised for stroke. METHODS: Resistance to 41 antibiotics was tested with the agar disc diffusion, minimum inhibitory concentration, or broth microdilution method. The complete genome was assembled using short-reads and long-reads and the hybrid de novo assembly method. Allelic profiles obtained by multilocus sequence typing were analysed using the PubMLST database. Antibiotic-resistance and virulence genes were predicted in silico using public databases and the 'baargin' workflow. B. dolosa N149 phylogenetic relationships with all available B. dolosa strains and Burkholderia cepacia complex strains were analysed using the pangenome obtained with Roary. RESULTS: B. dolosa N149 displayed extensive resistance to 31 antibiotics and intermediate resistance to 4 antibiotics. The complete genome included three circular chromosomes (6 338 630 bp in total) and one plasmid (167 591 bp). Genotypic analysis revealed various gene clusters (acr, amr, amp, emr, ade, bla and tet) associated with resistance to 35 antibiotic classes. The major intrinsic resistance mechanisms were multidrug efflux pump alterations, inactivation and reduced permeability of targeted antibiotics. Moreover, 91 virulence genes (encoding proteins involved in adherence, formation of capsule, biofilm and colony, motility, phagocytosis inhibition, secretion systems, protease secretion, transmission and quorum sensing) were identified. B. dolosa N149 was assigned to a novel sequence type (ST2237) and formed a mono-phylogenetic clade separated from other B. dolosa strains. CONCLUSIONS: This study provided insights into the antimicrobial resistance and virulence mechanisms of B. dolosa.

Understanding of the Site-Specific Microbial Patterns towards Accurate Identification for Patients with Diarrhea-Predominant Irritable Bowel Syndrome.

Fecal microbial community could not fully represent the intestinal microbial community. However, most studies analyzing diarrhea-dominant irritable bowel syndrome (IBS-D) were mainly based on fecal samples. We aimed to characterize the IBS-D microbial community patterns using samples at multiple intestinal sites. This study recruited 74 IBS-D patients and 20 healthy controls (HC). 22.34%, 8.51%, 14.89%, and 54.26% of them contributed to one, two, three, and four sites: duodenal mucosa (DM), duodenal lumen (DL), rectal mucosa (RM), and rectal lumen (RL) of intestinal samples, respectively. Then 16S rRNA gene analysis was performed on these 283 samples. The result showed that IBS-D microbial communities have specific patterns at each intestinal site differing from that of HC. Across hosts and sites, Bacillus, Burkholderia, and Faecalibacterium were the representative genera in duodenum of IBS-D, duodenum of HC, and rectum of HC, respectively. Samples from mucosa and lumen in rectum were highly distinguishable, regardless of IBS-D and HC. Additionally, IBS-D patients have lower microbial co-abundance network connectivity. Moreover, RM site-specific biomarker: Bacteroides used alone or together with Prevotella and Oscillospira in RM showed outstanding performance in IBS-D diagnosis. Furthermore, Bacteroides and Prevotella in RM were strongly related to the severity of abdominal pain, abdominal discomfort, and bloating in IBS-D patients. In summary, this study also confirmed fecal microbial community could not fully characterize intestinal microbial communities. Among these site-specific microbial communities, RM microbial community would be more applicable in the diagnosis of IBS-D. IMPORTANCE Microbial community varied from one site to another along the gastrointestinal tract, but current studies about intestinal microbial community in IBS-D were mainly based on fecal samples. Based on 283 intestinal samples collected from DM, DL, RM, and RL of HC and IBS-D, we found different intestinal sites had their site-specific microbial patterns in IBS-D. Notably, RM site-specific microbes Bacteroides, Prevotella, and Oscillospira could be used to discriminate IBS-D from HC accurately. Our findings could help clinicians realize the great potential of the intestinal microbial community in RM for better diagnosis of IBS-D patients.

The Impacts of Different Biological Treatments on the Transformation of Explosives Waste Contaminated Sludge.

The dinitrotoluene isomers 2,4 and 2,6-dinitrotoluene (DNT) represent highly toxic, mutagenic, and carcinogenic compounds used in explosive manufacturing and in commercial production of polyurethane foam. Bioremediation, the use of microbes to degrade residual DNT in industry wastewaters, represents a promising, low cost and environmentally friendly alternative technology to landfilling. In the present study, the effect of different bioremediation strategies on the degradation of DNT in a microcosm-based study was evaluated. Biostimulation of the indigenous microbial community with sulphur phosphate (2.3 g/kg sludge) enhanced DNT transformation (82% transformation, from 300 g/L at Day 0 to 55 g/L in week 6) compared to natural attenuation over the same period at 25 °C. The indigenous microbial activity was found to be capable of transforming the contaminant, with around 70% transformation of DNT occurring over the microcosm study. 16S rDNA sequence analysis revealed that while the original bacterial community was dominated by Gammaproteobacteria (30%), the addition of sulphur phosphate significantly increased the abundance of Betaproteobacteria by the end of the biostimulation treatment, with the bacterial community dominated by Burkholderia (46%) followed by Rhodanobacter, Acidovorax and Pseudomonas. In summary, the results suggest biostimulation as a treatment choice for the remediation of dinitrotoluenes and explosives waste.

Burkholderia species in human infections in Mexico: Identification of B. cepacia, B. contaminans, B. multivorans, B. vietnamiensis,B. pseudomallei and a new Burkholderia species.

BACKGROUND: Burkholderia sensu stricto is comprised mainly of opportunistic pathogens. This group is widely distributed in the environment but is especially important in clinical settings. In Mexico, few species have been correctly identified among patients, most often B. cepacia is described. METHODOLOGY/PRINCIPAL FINDINGS: In this study, approximately 90 strains identified as B. cepacia with the VITEK2 system were isolated from two medical centers in Mexico City and analyzed by MLSA, BOX-PCR and genome analysis. The initial identification of B. cepacia was confirmed for many strains, but B. contaminans, B. multivorans and B. vietnamiensis were also identified among clinical strains for the first time in hospitals in Mexico. Additionally, the presence of B. pseudomallei was confirmed, and a novel species within the B. cepacia complex was documented. Several strains misidentified as B. cepacia actually belong to the genera Pseudomonas, Stenotrophomonas and Providencia. CONCLUSIONS/SIGNIFICANCE: The presence of different Burkholderia species in Mexico was confirmed. Correct identification of Burkholderia species is important to provide accurate treatment for immunosuppressed patients.

Genomics reveals the novel species placement of industrial contaminant isolates incorrectly identified as Burkholderia lata.

The Burkholderia cepacia complex (Bcc) is a closely related group of bacteria, composed of at least 20 different species, the accurate identification of which is essential in the context of infectious diseases. In industry, they can contaminate non-food products, including home and personal care products and cosmetics. The Bcc are problematic contaminants due to their ubiquitous presence and intrinsic antimicrobial resistance, which enables them to occasionally overcome preservation systems in non-sterile products. Burkholderia lata and Burkholderia contaminans are amongst the Bcc bacteria encountered most frequently as industrial contaminants, but their identification is not straightforward. Both species were historically established as a part of a group known collectively as taxon K, based upon analysis of the recA gene and multilocus sequence typing (MLST). Here, we deploy a straightforward genomics-based workflow for accurate Bcc classification using average nucleotide identity (ANI) and core-gene analysis. The workflow was used to examine a panel of 23 Burkholderia taxon K industrial strains, which, based on MLST, comprised 13 B. lata, 4 B. contaminans and 6 unclassified Bcc strains. Our genomic identification showed that the B. contaminans strains retained their classification, whilst the remaining strains were reclassified as Burkholderia aenigmatica sp. nov. Incorrect taxonomic identification of industrial contaminants is a problematic issue. Application and testing of our genomic workflow allowed the correct classification of 23 Bcc industrial strains, and also indicated that B. aenigmatica sp. nov. may have greater importance than B. lata as a contaminant species. Our study illustrates how the non-food manufacturing industry can harness whole-genome sequencing to better understand antimicrobial-resistant bacteria affecting their products.

Biological characteristics and salt-tolerant plant growth-promoting effects of an ACC deaminase-producing Burkholderia pyrrocinia strain isolated from the tea rhizosphere.

Plant growth-promoting rhizobacteria that produce 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase can promote plant growth and enhance abiotic stress tolerance. In this study, Burkholderia pyrrocinia strain P10, with an ACC deaminase activity of 33.01-µmol/h/mg protein, was isolated from the tea rhizosphere and identified based on morphological, biochemical, and molecular characteristics. In addition to its ACC deaminase activity at pH 5.0-9.0 and in response to 5% NaCl and 20% polyethylene glycol, strain P10 can also solubilize phosphorus compounds, produce indole-3-acetic acid, and secrete siderophores. Pot experiments revealed that strain P10 can significantly enhance peanut seedling growth under saline conditions (100- and 170-mmol/L NaCl). Specifically, it increased the fresh weight and root length of plants by 90.12% and 79.22%, respectively, compared with high-salt stress. These results provide new insights into the biological characteristics of Burkholderia pyrrocinia, which may be useful as a bio-fertilizer.

Characterization of zinc solubilization potential of arsenic tolerant Burkholderia spp. isolated from rice rhizospheric soil.

In this study, experiments were conducted to isolate, characterize, and evaluate rice rhizosphere bacteria for their arsenic (As) tolerance ability and zinc (Zn) solubilization potential in culture media and soil. Among 20 bacterial isolates recovered, six were found to solubilize inorganic Zn salt(s) efficiently under in vitro culture conditions. 16S rRNA gene sequence-based phylogenetic analysis indicated the affiliation of efficient Zn solubilizing bacteria (ZSB) to Burkholderia vietnamiensis and Burkholderia seminalis. Zinc solubilizing efficiency (ZSE) of the bacteria varied with the concentrations and types of Zn salts used in the experiments. Increasing trend in ZSE of the bacteria was noticed when the percentage of ZnO increased from 0.1 to 0.5 but the same decreased at 1.0%. Increased Zn solubilization was noticed when bacteria were incubated with lower concentration of Zn3(PO4)2 and ZnCO3. In general, Zn solubilization increased with increasing incubation time in lower volume medium, while some isolates failed to solubilize one or more tested Zn salts. However, enriched concentrated cells of the ZSB in glucose amended medium with 0.5% ZnO showed an increasing trend of Zn solubilization with time and were able to solubilize more than 300 mg/L Zn. This increased rate of Zn release by the ZSB was attributed to marked decline in pH that might be due to the enhanced gluconic acid production from glucose. As evident from the decreased ZSE of the bacteria in the presence of As(V) in particular, it seems arsenic imparts a negative effect on Zn solubilization. The ZSB were also able to increase the rate of Zn release in soil. A microcosm-based soil incubation study amending the enriched bacteria and 0.5% ZnO in soil showed an elevated level of both water-soluble and available Zn compared to un-inoculated control. During Zn solubilization in microcosms, viable cells in terms of colony-forming unit (CFU) declined by the same order of magnitude both in the presence and absence of ZnO that might be due to the nutrients limiting condition aroused during the incubation period rather than Zn toxicity. The bacteria in this study also exhibited plant growth promoting traits, such as growth in nitrogen-free medium, production of indole acetic acid (IAA), and solubilization of potassium and phosphate. Our findings suggested that Burkholderia spp. could be the potential candidates for enhancing Zn dissolution in the soil that might reduce the rate of inorganic Zn fertilization in agricultural soil.

Detection and differentiation of Burkholderia species with pathogenic potential in environmental soil samples.

The Burkholderia pseudomallei phylogenetic cluster includes B. pseudomallei, B. mallei, B. thailandensis, B. oklahomensis, B. humptydooensis and B. singularis. Regarded as the only pathogenic members of this group, B. pseudomallei and B. mallei cause the diseases melioidosis and glanders, respectively. Additionally, variant strains of B. pseudomallei and B. thailandensis exist that include the geographically restricted B. pseudomallei that express a B. mallei-like BimA protein (BPBM), and B. thailandensis that express a B. pseudomallei-like capsular polysaccharide (BTCV). To establish a PCR-based assay for the detection of pathogenic Burkholderia species or their variants, five PCR primers were designed to amplify species-specific sequences within the bimA (Burkholderia intracellular motility A) gene. Our multiplex PCR assay could distinguish pathogenic B. pseudomallei and BPBM from the non-pathogenic B. thailandensis and the BTCV strains. A second singleplex PCR successfully discriminated the BTCV from B. thailandensis. Apart from B. humptydooensis, specificity testing against other Burkholderia spp., as well as other Gram-negative and Gram-positive bacteria produced a negative result. The detection limit of the multiplex PCR in soil samples artificially spiked with known quantities of B. pseudomallei and B. thailandensis were 5 and 6 CFU/g soil, respectively. Furthermore, comparison between standard bacterial culture and the multiplex PCR to detect B. pseudomallei from 34 soil samples, collected from an endemic area of melioidosis, showed high sensitivity and specificity. This robust, sensitive, and specific PCR assay will be a useful tool for epidemiological study of B. pseudomallei and closely related members with pathogenic potential in soil.

Exploring the value of MALDI-TOF MS for the detection of clonal outbreaks of Burkholderia contaminans.

BACKGROUND: Molecular genetics has risen in both output and affordability to become the gold standard in diagnosis, however it is not yet available for most routine clinical microbiology due to cost and the level of skill it requires. Matrix assisted laser desorption/ionisation - time of flight mass spectrometry (MALDI-TOF MS) approaches may be useful in bridging the gap between low-resolution phenotypic methods and bulky genotypic methods in the goal of epidemiological source-typing of microbes. Burkholderia has been shown to be identifiable at the subspecies level using MALDI-TOF MS. There have not yet been studies assessing the ability of MALDI-TOF MS to source-type Burkholderia contaminans isolates into epidemiologically relevant outbreak clusters. METHODS: 55 well-characterised B. contaminans isolates were used to create a panel for analysis of MALDI-TOF MS biomarker peaks and their relation to outbreak strains, location, source, patient, diagnosis and isolate genetics. Unsupervised clustering was performed and classification models were generated using biostatistical analysis software. RESULTS: B. contaminans spectra derived from MALDI-TOF MS were of sufficiently high resolution to identify 100% of isolates. Unsupervised clustering methods showed poor evidence of spectra clustering by all characteristics measured. Classification algorithms were discriminatory, with Genetic Algorithm models showing 100% recognition capability for all outbreaks, the pulsed-field gel electrophoresis (PFGE) typeability model, and 96.63% recognition for the location model. A consistent peak at m/z of approximately 6943 was identified in all non-typeable strains but in none of the typeable strains. CONCLUSIONS: MALDI-TOF MS successfully discriminates B. contaminans isolates into clonal, epidemiological clusters, and can recognise isolates non-typeable by PFGE. Further work should investigate this capability, and include peptide studies and genomic sequencing to identify individual proteins or genes responsible for this non-typeablity, particularly at the peak weight identified.

Burkholderia vietnamiensis causing a non-lactational breast abscess in a non-cystic fibrosis patient in Tamil Nadu, India.

Burkholderia cepacia complex is a Gram-negative opportunistic pathogen usually found in people with an immunocompromised condition such as cystic fibrosis (CF). In a tropical country like India, this organism has been associated with a number of hospital-acquired infections including sepsis. We present here a report of a case of Burkholderia vietnamiensis causing a non-lactational breast abscess in a non-CF patient. The pathogen was identified as B. cepacia using Vitek system and matrix-assisted laser desorption ionisation-time of flight. This was confirmed by polymerase chain reaction (PCR) using recA genus-specific gene and sequencing of the PCR amplicons. recA-restriction fragment length polymorphism and recA gene sequencing revealed that the isolate is B. vietnamiensis. This is the first description of B. vietnamiensis isolated from a clinical case from India.

Design of a new multiplex PCR assay for rice pathogenic bacteria detection and its application to infer disease incidence and detect co-infection in rice fields in Burkina Faso.

Crop diseases are responsible for considerable yield losses worldwide and particularly in sub-Saharan Africa. To implement efficient disease control measures, detection of the pathogens and understanding pathogen spatio-temporal dynamics is crucial and requires the use of molecular detection tools, especially to distinguish different pathogens causing more or less similar symptoms. We report here the design a new molecular diagnostic tool able to simultaneously detect five bacterial taxa causing important diseases on rice in Africa: (1) Pseudomonas fuscovaginae, (2) Xanthomonas oryzae, (3) Burkholderia glumae and Burkholderia gladioli, (4) Sphingomonas and (5) Pantoea species. This new detection tool consists of a multiplex PCR, which is cost effective and easily applicable. Validation of the method is presented through its application on a global collection of bacterial strains. Moreover, sensitivity assessment for the detection of all five bacteria is reported to be at 0.5 ng DNA by µl. As a proof of concept, we applied the new molecular detection method to a set of 256 rice leaves collected from 16 fields in two irrigated areas in western Burkina Faso. Our results show high levels of Sphingomonas spp. (up to 100% of tested samples in one field), with significant variation in the incidence between the two sampled sites. Xanthomonas oryzae incidence levels were mostly congruent with bacterial leaf streak (BLS) and bacterial leaf blight (BLB) symptom observations in the field. Low levels of Pantoea spp. were found while none of the 256 analysed samples was positive for Burkholderia or Pseudomonas fuscovaginae. Finally, many samples (up to 37.5% in one studied field) were positive for more than one bacterium (co-infection). Documenting co-infection levels are important because of their drastic consequences on epidemiology, evolution of pathogen populations and yield losses. The newly designed multiplex PCR for multiple bacterial pathogens of rice is a significant improvement for disease monitoring in the field, thus contributing to efficient disease control and food safety.

Custom Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometric Database for Identification of Environmental Isolates of the Genus Burkholderia and Related Genera.

Success of discovery programs for microbial natural products is dependent on quick and concise discrimination between isolates from diverse environments. However, laboratory isolation and identification of priority genera using current 16S rRNA PCR-based methods are both challenging and time-consuming. An emerging strategy for rapid isolate discrimination is protein fingerprinting via matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Using our in-house environmental isolate repository, we have created a main spectral (MSP) library for the Bruker Biotyper MALDI mass spectrometer that contains 95 entries, including Burkholderia, Caballeronia, Paraburkholderia, and other environmentally related genera. The library creation required the acquisition of over 2,250 mass spectra, which were manually reviewed for quality control and consolidated into a single reference library using a commercial software platform. We tested the effectiveness of the reference library by analyzing 49 environmental isolate strains using two different sample preparation methods. Overall, this approach correctly identified all strains to the genus level provided that suitable reference spectra were present in the MSP library. In this study, we present a fast, accurate method for taxonomic assignment of environmentally derived bacteria from the order Burkholderiales, providing a valuable alternative to traditional PCR-based methods. The MSP library described in the manuscript is available for use.IMPORTANCE The Gram-negative proteobacterial order Burkholderiales has emerged as a promising source of novel natural products in recent years. This order includes the genus Burkholderia and the newly defined genera Caballeronia and Paraburkholderia However, development of this resource has been hampered by difficulties with rapid and selective isolation of Burkholderiales strains from the environment. Environmental metagenome sequencing has revealed that the potential for natural products is not evenly distributed throughout the microbial world. Thus, large but targeted microbial isolate libraries are needed to effectively explore the chemical potential of natural products. To study these organisms efficiently, methods to quickly identify isolates to the genus level are required. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is already used in clinical settings to reliably identify unknown bacterial pathogens. We have adapted similar methodology using the MALDI Biotyper instrument to rapidly identify environmental isolates of Burkholderia, Caballeronia, and Paraburkholderia for downstream natural product discovery.

Molecular and epidemiological analysis of a Burkholderia cepacia sepsis outbreak from a tertiary care hospital in Bangladesh.

BACKGROUND: Burkholderia cepacia complex (Bcc) is a group of serious pathogens in cystic fibrosis patients and causes life threatening infections in immunocompromised patients. Species within the Bcc are widely distributed within the environment, can survive in the presence of disinfectants and antiseptics, and are inherently multidrug resistant (MDR). METHODS: Dhaka Medical College Hospital (DMCH) patients with a B. cepacia positive blood culture between 20 October 2016 to 23rd September 2017 were considered as outbreak cases. Blood stream infections (BSIs) were detected using BacT/ALERT 3D at DMCH. B. cepacia was isolated on chromogenic UTI media followed by MALDI-TOF. Minimum inhibitory concentration (MIC) of clinically relevant antibiotics was determined by agar dilution. Whole genome sequencing was performed on an Illumina MiSeq platform. Patients' demographic and clinical data were collected. Patients' clinical history and genomic data of the outbreak strains were merged to investigate possible outbreaks. Ninety-one B. cepacia genomes were downloaded from 'Burkholderia Genome Database' and the genomic background of the global strains were compared with our outbreak strains. RESULTS: Among 236 BSIs, 6.35% (15/236) were B. cepacia. Outbreak cases were confined to the burn critical care unit and, to a lesser extent, the paediatrics department. There was a continuum of overlapping cases at DMCH between 23 October 2016 to 30 August 2017. Core genome SNPs showed that the outbreak strains were confined to a single clade, corresponded to a common clone (ST1578). The strains were shown to be MDR and associated with a mortality of 31% excluding discharge against medical advice. MIC profiles of the strains suggested that antibiotics deployed as empirical therapy were invariably inappropriate. The genetic background of the outbreak strains was very similar; however, a few variations were found regarding the presence of virulence genes. Compared to global strains from the Burkholderia Genome Database, the Bangladeshi strains were genetically distinct. CONCLUSIONS: Environmental surveillance is required to investigate the aetiology and mode of transmission of the B. cepacia outbreak. Systematic management of nosocomial outbreaks, particularly in resource limited regions, will mitigate transmission and will improve patients' outcomes.

Reciprocal antibiotic collateral sensitivity in Burkholderia multivorans.

Antibiotic collateral sensitivity (CS) occurs when a bacterium that acquires resistance to a treatment drug exhibits decreased resistance to a different drug. Here we identify reciprocal CS networks and candidate genes in Burkholderia multivorans. Burkholderia multivorans was evolved to become resistant to each of six antibiotics. The antibiogram of the evolved strain was compared with the immediate parental strain to determine CS and cross-resistance. The evolution process was continued for each resistant strain. CS interactions were observed in 170 of 279 evolved strains. CS patterns grouped into two clusters based on the treatment drug being a ß-lactam antibiotic or not. Reciprocal pairs of CS antibiotics arose in ≥25% of all evolved strains. A total of 68 evolved strains were subjected to whole-genome sequencing and the resulting mutation patterns were correlated with antibiograms. Analysis revealed there was no single gene responsible for CS and that CS seen in B. multivorans is likely due to a combination of specific and non-specific mutations. The frequency of reciprocal CS, and the degree to which resistance changed, suggests a long-term treatment strategy; when resistance to one drug occurs, switch to use of the other member of the reciprocal pair. This switching could theoretically be continued indefinitely, allowing life-long treatment of chronic infections with just two antibiotics.