Exploring the characteristics of Burkholderia gladioli pathovar cocovenenans: Growth, bongkrekic acid production, and potential risks of food contamination in wet rice noodles and vermicelli.

This research investigated the presence of Burkholderia gladioli pathovar cocovenenans (BGC) in wet rice and starch products, Tremella, and Auricularia auricula in Guangzhou, China. It examined BGC growth and bongkrekic acid (BA) production in wet rice noodles and vermicelli with varying rice flour, edible starch ratios, and oil concentrations. A qualitative analysis of 482 samples revealed a detection rate of 0.62%, with three positive for BGC. Rice flour-based wet rice noodles had BA concentrations of 13.67 ± 0.64 mg/kg, 2.92 times higher than 100% corn starch samples (4.68 ± 0.54 mg/kg). Wet rice noodles with 4% soybean oil had a BA concentration of 31.72 ± 9.41 mg/kg, 5.74 times higher than those without soybean oil (5.53 ± 1.23 mg/kg). The BA concentration correlated positively (r = 0.707, P < 0.05) with BGC contamination levels. Low temperatures (4 °C and -18 °C) inhibited BGC growth and BA production, while higher storage temperatures (26 °C and 32 °C) promoted BGC proliferation and increased BA production. Reducing edible oil use and increasing edible starch can mitigate the risk of BGC-related food poisoning in wet rice noodles and vermicelli production. Further research is needed to find alternative oils that do not enhance BA production. Strengthening prevention and control measures is crucial across the entire production chain to address BGC contamination and BA production.

Wild mushrooms as potential reservoirs of plant pathogenic bacteria: a case study on Burkholderia gladioli.

Fruit bodies (sporocarps) of wild mushrooms growing in natural environments play a substantial role in the preservation of microbial communities, for example, clinical and food-poisoning bacteria. However, the role of wild mushrooms as natural reservoirs of plant pathogenic bacteria remains almost entirely unknown. Furthermore, bacterial transmission from a mushroom species to agricultural plants has rarely been recorded in the literature. In September 2021, a creamy-white Gram-negative bacterial strain was isolated from the sporocarp of Suillus luteus (slippery jack) growing in Bermuda grass (Cynodon dactylon) lawn in Southern Iran. A similar strain was isolated from the same fungus in the same area in September 2022. Both strains were identified as Burkholderia gladioli based on phenotypic features as well as phylogeny of 16S rRNA and three housekeeping genes. The strains were not only pathogenic on white button mushrooms (Agaricus bisporus) but also induced hypersensitive reaction (HR) on tobacco and common bean leaves and caused soft rot on a set of diverse plant species, that is, chili pepper, common bean pod, cucumber, eggplant, garlic, gladiolus, narcissus, onion, potato, spring onion, okra, kohlrabi, mango, and watermelon. Isolation of plant pathogenic B. gladioli strains from sporocarp of S. luteus in two consecutive years in the same area could be indicative of the role of this fungus in the preservation of the bacterium in the natural environment. B. gladioli associated with naturally growing S. luteus could potentially invade neighboring agricultural crops, for example, vegetables and ornamentals. The potential role of wild mushrooms as natural reservoirs of phytopathogenic bacteria is further discussed.IMPORTANCEThe bacterial genus Burkholderia contains biologically heterogeneous strains that can be isolated from diverse habitats, that is, soil, water, diseased plant material, and clinical specimens. In this study, two Gram-negative pectinolytic bacterial strains were isolated from the sporocarps of Suillus luteus in September 2021 and 2022. Molecular phylogenetic analyses revealed that both strains belonged to the complex species Burkholderia gladioli, while the pathovar status of the strains remained undetermined. Biological investigations accomplished with pathogenicity and host range assays showed that B. gladioli strains isolated from S. luteus in two consecutive years were pathogenic on a set of diverse plant species ranging from ornamentals to both monocotyledonous and dicotyledonous vegetables. Thus, B. gladioli could be considered an infectious pathogen capable of being transmitted from wild mushrooms to annual crops. Our results raise a hypothesis that wild mushrooms could be considered as potential reservoirs for phytopathogenic B. gladioli.

Unique Presentations of Burkholderia gladioli Infections in Several Strains of Immunocompromised Mice.

Four strains of experimentally naïve mice (NOD. Cg-Prkdc scid Il2rg tm1Wjl /SzJ [NSG], NOD. Cg- Rag1 tm1Mom Il2rg tm1Wjl /SzJ [NRG], B6.129S(Cg)-Stat1 tm1Dlv/J [STAT1 -/-], and B6.129S7- Ifngr1 tm1Agt/J[IFNγR -/-] housed in a barrier facility developed unusual and seemingly unrelated clinical signs. Young NSG/NRG mice (n = 49, mean age = 4 ± 0.4 mo) exhibited nonspecific clinical signs of moderate-to-severe lethargy, hunched posture, decreased body condition, and pallor. In contrast to the NSG/NRG mice, the STAT1-/- and IFNγ R-/- mice (n = 5) developed large subcutaneous abscesses on the head and neck. These mice were euthanized, and samples were collected for culture. NSG/NRG mice had moderate-markedly enlarged livers (20 of 49, 40%) and spleens (17 of 49, 35%). The livers contained multiple, variably-sized, tan regions throughout all lobes. Histology revealed necrotizing hepatitis (13 of 17, 77%), splenic and hepatic extramedullary hematopoiesis (17 of 17, 100%), glomerular histiocytosis (6 of 17, 35%), and metritis (6 of 11, 55%) with perivascular inflammation, suggesting hematogenous spread Differentials for these lesions included mouse hepatitis virus, ectromelia virus, Pseudomonas aeruginosa, Salmonella spp., and Clostridium piliforme. Burkholderia gladioli was cultured from liver lesions and subcutaneous abscesses and confirmed with 16S ribosomal RNA sequencing. After completing systematic testing of the environment, failure of the water autoclave cycle was suspected as the cause of the outbreak. To address the situation, individually ventilated racks were sanitized and new breeders were purchased; these actions dramatically reduced B. gladioli infections. The current literature contains few reports of B. gladioli infections in immunocompromised mice, and its typical presentation is torticollis and rolling. B. gladioli infection is a potential differential for subcutaneous abscesses, hepatitis, and splenomegaly in immunocompromised mice. Careful monitoring of sterilization techniques is essential to prevent such infections in a barrier facility.

The Genomic-Driven Discovery of Glutarimide-Containing Derivatives from Burkholderia gladioli.

Glutarimide-containing polyketides exhibiting potent antitumor and antimicrobial activities were encoded via conserved module blocks in various strains that favor the genomic mining of these family compounds. The bioinformatic analysis of the genome of Burkholderia gladioli ATCC 10248 showed a silent trans-AT PKS biosynthetic gene cluster (BGC) on chromosome 2 (Chr2C8), which was predicted to produce new glutarimide-containing derivatives. Then, the silent polyketide synthase gene cluster was successfully activated via in situ promoter insertion and heterologous expression. As a result, seven glutarimide-containing analogs, including five new ones, gladiofungins D-H (3-7), and two known gladiofungin A/gladiostatin (1) and 2 (named gladiofungin C), were isolated from the fermentation of the activated mutant. Their structures were elucidated through the analysis of HR-ESI-MS and NMR spectroscopy. The structural diversities of gladiofungins may be due to the degradation of the butenolide group in gladiofungin A (1) during the fermentation and extraction process. Bioactivity screening showed that 2 and 4 had moderate anti-inflammatory activities. Thus, genome mining combined with promoter engineering and heterologous expression were proved to be effective strategies for the pathway-specific activation of the silent BGCs for the directional discovery of new natural products.

Heterologous Biosynthesis of Complex Bacterial Natural Products in Burkholderia gladioli.

Bacterial natural products (NPs) are an indispensable source of drugs and biopesticides. Heterologous expression is an essential method for discovering bacterial NPs and the efficient biosynthesis of valuable NPs, but the chassis for Gram-negative bacterial NPs remains inadequate. In this study, we built a Burkholderiales mutant Burkholderia gladioli Δgbn::attB by introducing an integrated site (attB) to inactivate the native gladiolin (gbn) biosynthetic gene cluster, which stabilizes large foreign gene clusters and reduces the native metabolite profile. The growth and successful heterologous production of high-value NPs such as phylogenetically close Burkholderiales-derived antitumor polyketides (PKs) rhizoxins, phylogenetically distant Gammaproteobacteria-derived anti-MRSA (methicillin-resistant Staphylococcus aureus) antibiotics WAP-8294As, and Deltaproteobacteria-derived antitumor PKs disorazols demonstrate that this strain is a potential chassis for Gram-negative bacterial NPs. We further improved the yields of WAP-8294As through promoter insertions and precursor pathway overexpression based on heterologous expression in this strain. This study provides a robust bacterial chassis for genome mining, efficient production, and molecular engineering of bacterial NPs.

Examining the activity of cefepime-taniborbactam against Burkholderia cepacia complex and Burkholderia gladioli isolated from cystic fibrosis patients in the United States.

The novel clinical-stage ß-lactam-ß-lactamase inhibitor combination, cefepime-taniborbactam, demonstrates promising activity toward many Gram-negative bacteria producing class A, B, C, and/or D ß-lactamases. We tested this combination against a panel of 150 Burkholderia cepacia complex (Bcc) and Burkholderia gladioli strains. The addition of taniborbactam to cefepime shifted cefepime minimum inhibitory concentrations toward the provisionally susceptible range in 59% of the isolates tested. Therefore, cefepime-taniborbactam possessed similar activity as first-line agents, ceftazidime and trimethoprim-sulfamethoxazole, supporting further development.

A prophage tail-like protein facilitates the endophytic growth of Burkholderia gladioli and mounting immunity in tomato.

A prophage tail-like protein (Bg_9562) of Burkholderia gladioli strain NGJ1 possesses broad-spectrum antifungal activity, and it is required for the bacterial ability to forage over fungi. Here, we analyzed whether heterologous overexpression of Bg_9562 or exogenous treatment with purified protein can impart disease tolerance in tomato. The physiological relevance of Bg_9562 during endophytic growth of NGJ1 was also investigated. Bg_9562 overexpressing lines demonstrate fungal and bacterial disease tolerance. They exhibit enhanced expression of defense genes and activation of mitogen-activated protein kinases. Treatment with Bg_9562 protein induces defense responses and imparts immunity in wild-type tomato. The defense-inducing ability lies within 18-51 aa region of Bg_9562 and is due to sequence homology with the bacterial flagellin epitope. Interaction studies suggest that Bg_9562 is perceived by FLAGELLIN-SENSING 2 homologs in tomato. The silencing of SlSERK3s (BAK1 homologs) prevents Bg_9562-triggered immunity. Moreover, type III secretion system-dependent translocation of Bg_9562 into host apoplast is important for elicitation of immune responses during colonization of NGJ1. Our study emphasizes that Bg_9562 is important for the endophytic growth of B. gladioli, while the plant perceives it as an indirect indicator of the presence of bacteria to mount immune responses. The findings have practical implications for controlling plant diseases.

Pan-genome analysis of the Burkholderia gladioli PV. Cocovenenans reveal the extent of variation in the toxigenic gene cluster.

Burkholderia gladioli has been reported as the pathogen responsible for cases of foodborne illness in many countries. The poisonous bongkrekic acid (BA) produced by B. gladioli was linked to a gene cluster absent in non-pathogenic strains. The whole genome sequence of eight bacteria strains, which were screened from the collected 175 raw food and environmental samples, were assembled and analyzed to detect a significant association of 19 protein-coding genes with the pathogenic status. Except for the common BA synthesis-related gene, several other genes, including the toxin-antitoxin genes, were also absent in the non-pathogenic strains. The bacteria strains with the BA gene cluster were found to form a single cluster in the analysis of all B. gladioli genome assemblies for the variants in the gene cluster. Divergence of this cluster was detected in the analysis for both the flanking sequences and those of the whole genome level, which indicates its complex origin. Genome recombination was found to cause a precise sequence deletion in the gene cluster region, which was found to be predominant in the non-pathogenic strains indicating the possible effect of horizontal gene transfer. Our study provided new information and resources for understanding the evolution and divergence of the B. gladioli species.

Nicotinic Acid Catabolism Modulates Bacterial Mycophagy in Burkholderia gladioli Strain NGJ1.

Burkholderia gladioli strain NGJ1 exhibits mycophagous activity on a broad range of fungi, including Rhizoctonia solani, a devastating plant pathogen. Here, we demonstrate that the nicotinic acid (NA) catabolic pathway in NGJ1 is required for mycophagy. NGJ1 is auxotrophic to NA and it potentially senses R. solani as a NA source. Mutation in the nicC and nicX genes involved in NA catabolism renders defects in mycophagy and the mutant bacteria are unable to utilize R. solani extract as the sole nutrient source. As supplementation of NA, but not FA (fumaric acid, the end product of NA catabolism) restores the mycophagous ability of ΔnicC/ΔnicX mutants, we anticipate that NA is not required as a carbon source for the bacterium during mycophagy. Notably, nicR, a MarR-type of transcriptional regulator that functions as a negative regulator of the NA catabolic pathway is upregulated in ΔnicC/ΔnicX mutant and upon NA supplementation the nicR expression is reduced to the basal level in both the mutants. The ΔnicR mutant produces excessive biofilm and is completely defective in swimming motility. On the other hand, ΔnicC/ΔnicX mutants are compromised in swimming motility as well as biofilm formation, potentially due to the upregulation of nicR. Our data suggest that a defect in NA catabolism alters the NA pool in the bacterium and upregulates nicR which in turn suppresses bacterial motility as well as biofilm formation, leading to mycophagy defects. IMPORTANCE Mycophagy is an important trait through which certain bacteria forage over fungal mycelia and utilize fungal biomass as a nutrient source to thrive in hostile environments. The present study emphasizes that nicotinic acid (NA) is important for bacterial motility and biofilm formation during mycophagy by Burkholderia gladioli strain NGJ1. Defects in NA catabolism potentially alter the cellular NA pool, upregulate the expression of nicR, a negative regulator of biofilm, and therefore suppress bacterial motility as well as biofilm formation, leading to mycophagy defects.

Selective Methylation by an ArsM S-Adenosylmethionine Methyltransferase from Burkholderia gladioli GSRB05 Enhances Antibiotic Production.

Arsenic methylation contributes to the formation and diversity of environmental organoarsenicals, an important process in the arsenic biogeochemical cycle. The arsM gene encoding an arsenite (As(III)) S-adenosylmethionine (SAM) methyltransferase is widely distributed in members of every kingdom. A number of ArsM enzymes have been shown to have different patterns of methylation. When incubated with inorganic As(III), Burkholderia gladioli GSRB05 has been shown to synthesize the organoarsenical antibiotic arsinothricin (AST) but does not produce either methylarsenate (MAs(V)) or dimethylarsenate (DMAs(V)). Here, we show that cells of B. gladioli GSRB05 synthesize DMAs(V) when cultured with either MAs(III) or MAs(V). Heterologous expression of the BgarsM gene in Escherichia coli conferred resistance to MAs(III) but not As(III). The cells methylate MAs(III) and the AST precursor, reduced trivalent hydroxyarsinothricin (R-AST-OH) but do not methylate inorganic As(III). Similar results were obtained with purified BgArsM. Compared with ArsM orthologs, BgArsM has an additional 37 amino acid residues in a linker region between domains. Deletion of the additional 37 residues restored As(III) methylation activity. Cells of E. coli co-expressing the BgarsL gene encoding the noncanonical radical SAM enzyme that catalyzes the synthesis of R-AST-OH together with the BgarsM gene produce much more of the antibiotic AST compared with E. coli cells co-expressing BgarsL together with the CrarsM gene from Chlamydomonas reinhardtii, which lacks the sequence for additional 37 residues. We propose that the presence of the insertion reduces the fitness of B. gladioli because it cannot detoxify inorganic arsenic but concomitantly confers an evolutionary advantage by increasing the ability to produce AST.

Outbreak of healthcare-associated bacteremia caused by Burkholderia gladioli due to contaminated multidose vials with saline solutions in three Croatian hospitals.

OBJECTIVES: Burkholderia gladioli has been associated with infections in patients with cystic fibrosis, chronic granulomatous disease, and other immunocompromising conditions. The aim of this study was to better depict the outbreak of healthcare-associated bacteremia caused by B. gladioli due to exposure to contaminated multidose vials with saline solutions. METHODS: An environmental and epidemiologic investigation was conducted by the Infection Prevention and Control Team (IPCT) to identify the source of the outbreak in three Croatian hospitals. RESULTS: During a 3-month period, 13 B. gladioli bacteremia episodes were identified in 10 patients in three Croatian hospitals. At the time of the outbreak, all three hospitals used saline products from the same manufacturer. Two 100-ml multidose vials with saline solutions and needleless dispensing pins were positive for B. gladioli. All 13 bacteremia isolates and two isolates from the saline showed the same antimicrobial susceptibility patterns and pulsed-field gel electrophoresis profile, demonstrating clonal relatedness. CONCLUSION: When an environmental pathogen causes an outbreak, contamination of intravenous products must be considered. Close communication between the local IPCT and the National Hospital Infection Control Advisory Committee is essential to conduct a prompt and thorough investigation and find the source of the outbreak.

The alternative sigma factors, rpoN1 and rpoN2 are required for mycophagous activity of Burkholderia gladioli strain NGJ1.

Bacteria utilize RpoN, an alternative sigma factor (σ54) to grow in diverse habitats, including nitrogen-limiting conditions. Here, we report that a rice-associated mycophagous bacterium Burkholderia gladioli strain NGJ1 encodes two paralogues of rpoN viz. rpoN1 and rpoN2. Both of them are upregulated during 24 h of mycophagous interaction with Rhizoctonia solani, a polyphagous fungal pathogen. Disruption of either one of rpoNs renders the mutant NGJ1 bacterium defective in mycophagy, whereas ectopic expression of respective rpoN genes restores mycophagy in the complementing strains. NGJ1 requires rpoN1 and rpoN2 for efficient biocontrol to prevent R. solani to establish disease in rice and tomato. Further, we have identified 17 genes having RpoN regulatory motif in NGJ1, majority of them encode potential type III secretion system (T3SS) effectors, nitrogen assimilation, and cellular transport-related functions. Several of these RpoN regulated genes as well as certain previously reported T3SS apparatus (hrcC and hrcN) and effector (Bg_9562 and endo-ß-1,3-glucanase) encoding genes are upregulated in NGJ1 but not in ΔrpoN1 or ΔrpoN2 mutant bacterium, during mycophagous interaction with R. solani. This highlights that RpoN1 and RpoN2 modulate T3SS, nitrogen assimilation as well as cellular transport systems in NGJ1 and thereby promote bacterial mycophagy.

Burkholderia gladioli E39CS3, an endophyte of Crocus sativus Linn., induces host resistance against corm-rot caused by Fusarium oxysporum.

AIM: To investigate the role of the leading saffron endophyte Burkholderia gladioli strain E39CS3 (BG-E39) in the inhibition of corm-rot and induced systemic resistance (ISR) in the host against the saffron specific pathogen, Fusarium oxysporum. METHODS AND RESULTS: We studied the interaction between BG-E39 and the corm-rot pathogen F. oxysporum in vitro and in vivo. BG-E39 strongly inhibited both the F. oxysporum strains and other saffron-specific and non-specific pathogens used in this study. Confrontation and microscopic analyses revealed that the endophyte possessed fungicidal activity against the pathogens and effectively induced cell death in the mycelia. The endophyte produced chitinases as well as ß-1,3-glucanase that may be involved in the pathogen cell wall degradation. BG-E39 did not cause corm-rot in Crocus sativus and the closely related plant, Gladiolus, thus establishing that it is non-pathogenic to these plants. The endophyte reduced corm-rot through antibiosis and enhanced the endogenous jasmonic acid (JA) levels and expression of JA-regulated and other plant defence genes. CONCLUSIONS: The bacterial endophyte BG-E39 provides resistance to the host plant against F. oxysporum corm-rot in nature. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study discovers the role of the saffron endophyte BG-E39 in providing resistance to the host against corm-rot. Therefore, this endophyte is a potential candidate for developing a microbial formulation for the biocontrol of the most common disease of C. sativus.

Solid-state fermentation production and characterization of an alkaline lipase from a newly isolated Burkholderia gladioli strain.

The newly isolated Burkholderia gladioli BRM58833 strain was shown to secrete an alkaline lipase highly active and stable in organic solvents. Lipase production was optimized through the cultivation of the strain by solid-state fermentation in wheat bran. The lipase extraction conditions were also optimized. The low-cost extract obtained has shown a high hydrolytic activity of 1096.7 ± 39.3 U·gds-1 (units per gram of dry solids) against pNPP and 374.2 ± 20.4 U·gds-1 against triolein. Proteomic analysis revealed the optimized extract is composed of two esterases and three true lipases, showing a preference for long-chain substrates. The highest activity was obtained at 50 °C and pH 9. However, the extract maintained more than 50% of its maximum activity between pH 8.0 and 10.0 and throughout the whole temperature range evaluated (32-70 °C). The enzymes were inhibited by SDS, EDTA, ZnSO4 and FeCl3 and activated by FeSO4, MgCl2 and BaCl2. The lipases conserved their activity when incubated in solvents as acetonitrile, diethyl ether, n-heptane n-hexane, toluene, methanol and t-butanol. The resistance of these lipases to solvents and expressive thermostability when compared to other lipases, reveal their potential both in hydrolysis reactions and in synthesis of esters.

MetEx, a Metabolomics Explorer Application for Natural Product Discovery.

Advances in next-generation DNA sequencing technologies, bioinformatics, and mass spectrometry-based metabolite detection have ushered in a new era of natural product discovery. Microbial secondary metabolomes are complex, especially when otherwise silent biosynthetic genes are activated, and there is therefore a need for data analysis software to explore and map the resulting multidimensional datasets. To that end, we herein report the Metabolomics Explorer (MetEx), a publicly available web application for the analysis of parallel liquid chromatography-coupled mass spectrometry (LC-MS)-based metabolomics data. MetEx is a highly interactive application that facilitates visualization and analysis of complex metabolomics datasets, consisting of retention time, m/z, and MS intensity features, as a function of hundreds of conditions or elicitors. The software enables prioritization of leads from three-dimensional maps, extraction of two-dimensional slices from various higher order plots, organization of datasets by elicitor chemotypes, customizable library-based dereplication, and automatically scored lead selection. We describe the application of MetEx to the first UPLC-MS-guided high-throughput elicitor screen in which Burkholderia gladioli was challenged with 750 elicitors, and the resulting profiles were interrogated by UPLC-Qtof-MS and subsequently analyzed with the app. We demonstrate the utility of MetEx by reporting elicitors for several cryptic metabolite groups and by uncovering new natural products that remain to be characterized. MetEx is available at https://mo.princeton.edu/MetEx/.

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles.

Inferring the function of genes by manipulating their activity is an essential tool for understanding the genetic underpinnings of most biological processes. Advances in molecular microbiology have seen the emergence of diverse mutagenesis techniques for the manipulation of genes. Among them, transposon-insertion sequencing (Tn-seq) is a valuable tool to simultaneously assess the functionality of many candidate genes in an untargeted way. The technique has been key to identify molecular mechanisms for the colonization of eukaryotic hosts in several pathogenic microbes and a few beneficial symbionts. Here, Tn-seq is established as a method to identify colonization factors in a mutualistic Burkholderia gladioli symbiont of the beetle Lagria villosa. By conjugation, Tn5 transposon-mediated insertion of an antibiotic-resistance cassette is carried out at random genomic locations in B. gladioli. To identify the effect of gene disruptions on the ability of the bacteria to colonize the beetle host, the generated B. gladioli transposon-mutant library is inoculated on the beetle eggs, while a control is grown in vitro in a liquid culture medium. After allowing sufficient time for colonization, DNA is extracted from the in vivo and in vitro grown libraries. Following a DNA library preparation protocol, the DNA samples are prepared for transposon-insertion sequencing. DNA fragments that contain the transposon-insert edge and flanking bacterial DNA are selected, and the mutation sites are determined by sequencing away from the transposon-insert edge. Finally, by analyzing and comparing the frequencies of each mutant between the in vivo and in vitro libraries, the importance of specific symbiont genes during beetle colonization can be predicted.

Activity of Imipenem-Relebactam against Multidrug- and Extensively Drug-Resistant Burkholderia cepacia Complex and Burkholderia gladioli.

The Burkholderia cepacia complex (Bcc) and Burkholderia gladioli are opportunistic pathogens that most commonly infect persons with cystic fibrosis or compromised immune systems. Members of the Burkholderia genus are intrinsically multidrug resistant (MDR), possessing both a PenA carbapenemase and an AmpC ß-lactamase, rendering treatment of infections due to these species problematic. Here, we tested the ß-lactam-ß-lactamase inhibitor combination imipenem-relebactam against a panel of MDR Bcc and B. gladioli strains. The addition of relebactam to imipenem dramatically lowered the MICs for Bcc and B. gladioli: only 16% of isolates tested susceptible to imipenem, while 71.3% were susceptible to the imipenem-relebactam combination. While ceftazidime-avibactam remained the most potent combination drug against this panel of Bcc and B. gladioli strains, imipenem-relebactam was active against 71.4% of the ceftazidime-avibactam-resistant isolates. Relebactam demonstrated potent inactivation of Burkholderia multivorans PenA1, with an apparent Ki (Kiapp) value of 3.2 µM. Timed mass spectrometry revealed that PenA1 formed a very stable adduct with relebactam, without any detectable desulfation for as long as 24 h. Based on our results, imipenem-relebactam may represent an alternative salvage therapy for Bcc and B. gladioli infections, especially in cases where the isolates are resistant to ceftazidime-avibactam.

Identification of the Biosynthetic Gene Cluster for the Organoarsenical Antibiotic Arsinothricin.

The soil bacterium Burkholderia gladioli GSRB05 produces the natural compound arsinothricin [2-amino-4-(hydroxymethylarsinoyl) butanoate] (AST), which has been demonstrated to be a broad-spectrum antibiotic. To identify the genes responsible for AST biosynthesis, a draft genome sequence of B. gladioli GSRB05 was constructed. Three genes, arsQML, in an arsenic resistance operon were found to be a biosynthetic gene cluster responsible for synthesis of AST and its precursor, hydroxyarsinothricin [2-amino-4-(dihydroxyarsinoyl) butanoate] (AST-OH). The arsL gene product is a noncanonical radical S-adenosylmethionine (SAM) enzyme that is predicted to transfer the 3-amino-3-carboxypropyl (ACP) group from SAM to the arsenic atom in inorganic arsenite, forming AST-OH, which is methylated by the arsM gene product, a SAM methyltransferase, to produce AST. Finally, the arsQ gene product is an efflux permease that extrudes AST from the cells, a common final step in antibiotic-producing bacteria. Elucidation of the biosynthetic gene cluster for this novel arsenic-containing antibiotic adds an important new tool for continuation of the antibiotic era. IMPORTANCE Antimicrobial resistance is an emerging global public health crisis, calling for urgent development of novel potent antibiotics. We propose that arsinothricin and related arsenic-containing compounds may be the progenitors of a new class of antibiotics to extend our antibiotic era. Here, we report identification of the biosynthetic gene cluster for arsinothricin and demonstrate that only three genes, two of which are novel, are required for the biosynthesis and transport of arsinothricin, in contrast to the phosphonate counterpart, phosphinothricin, which requires over 20 genes. Our discoveries will provide insight for the development of more effective organoarsenical antibiotics and illustrate the previously unknown complexity of the arsenic biogeochemical cycle, as well as bring new perspective to environmental arsenic biochemistry.

Amelioration of Chlorpyrifos-Induced Toxicity in Brassica juncea L. by Combination of 24-Epibrassinolide and Plant-Growth-Promoting Rhizobacteria.

Pervasive use of chlorpyrifos (CP), an organophosphorus pesticide, has been proven to be fatal for plant growth, especially at higher concentrations. CP poisoning leads to growth inhibition, chlorosis, browning of roots and lipid and protein degradation, along with membrane dysfunction and nuclear damage. Plants form a linking bridge between the underground and above-ground communities to escape from the unfavourable conditions. Association with beneficial rhizobacteria promotes the growth and development of the plants. Plant hormones are crucial regulators of basically every aspect of plant development. The growing significance of plant hormones in mediating plant-microbe interactions in stress recovery in plants has been extensively highlighted. Hence, the goal of the current study was to investigate the effect of 24-epibrassinolide (EBL) and PGPRs (Pseudomonas aeruginosa (Ma), Burkholderia gladioli (Mb)) on growth and the antioxidative defence system of CP-stressed Brassica juncea L. seedlings. CP toxicity reduced the germination potential, hypocotyl and radicle development and vigour index, which was maximally recuperated after priming with EBL and Mb. CP-exposed seedlings showed higher levels of superoxide anion (O2-), hydrogen peroxide (H2O2), lipid peroxidation and electrolyte leakage (EL) and a lower level of nitric oxide (NO). In-vivo visualisation of CP-stressed seedlings using a light and fluorescent microscope also revealed the increase in O2-, H2O2 and lipid peroxidation, and decreased NO levels. The combination of EBL and PGPRs reduced the reactive oxygen species (ROS) and malondialdehyde (MDA) contents and improved the NO level. In CP-stressed seedlings, increased gene expression of defence enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APOX), glutathione peroxidase (GPOX), dehydroascorbate reductase (DHAR) and glutathione reductase (GPOX) was seen, with the exception of catalase (CAT) on supplementation with EBL and PGPRs. The activity of nitrate reductase (NR) was likewise shown to increase after treatment with EBL and PGPRs. The results obtained from the present study substantiate sufficient evidence regarding the positive association of EBL and PGPRs in amelioration of CP-induced oxidative stress in Brassica juncea seedlings by strengthening the antioxidative defence machinery.