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.

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.

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.

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.

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.

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.

Efficient Hydrolysis of Gluten-Derived Celiac Disease-Triggering Immunogenic Peptides by a Bacterial Serine Protease from Burkholderia gladioli.

Celiac disease is an autoimmune disorder triggered by toxic peptides derived from incompletely digested glutens in the stomach. Peptidases that can digest the toxic peptides may formulate an oral enzyme therapy to improve the patients' health condition. Bga1903 is a serine endopeptidase secreted by Burkholderia gladioli. The preproprotein of Bga1903 consists of an N-terminal signal peptide, a propeptide region, and an enzymatic domain that belongs to the S8 subfamily. Bga1903 could be secreted into the culture medium when it was expressed in E. coli. The purified Bga1903 is capable of hydrolyzing the gluten-derived toxic peptides, such as the 33- and 26-mer peptides, with the preference for the peptide bonds at the carbonyl site of glutamine (P1 position). The kinetic assay of Bga1903 toward the chromogenic substrate Z-HPQ-pNA at 37 °C, pH 7.0, suggests that the values of Km and kcat are 0.44 ± 0.1 mM and 17.8 ± 0.4 s-1, respectively. The addition of Bga1903 in the wort during the fermentation step of beer could help in making gluten-free beer. In summary, Bga1903 is usable to reduce the gluten content in processed foods and represents a good candidate for protein engineering/modification aimed to efficiently digest the gluten at the gastric condition.

Kill and cure: genomic phylogeny and bioactivity of Burkholderia gladioli bacteria capable of pathogenic and beneficial lifestyles.

Burkholderia gladioli is a bacterium with a broad ecology spanning disease in humans, animals and plants, but also encompassing multiple beneficial interactions. It is a plant pathogen, a toxin-producing food-poisoning agent, and causes lung infections in people with cystic fibrosis (CF). Contrasting beneficial traits include antifungal production exploited by insects to protect their eggs, plant protective abilities and antibiotic biosynthesis. We explored the genomic diversity and specialized metabolic potential of 206 B. gladioli strains, phylogenomically defining 5 clades. Historical disease pathovars (pv.) B. gladioli pv. allicola and B. gladioli pv. cocovenenans were distinct, while B. gladioli pv. gladioli and B. gladioli pv. agaricicola were indistinguishable; soft-rot disease and CF infection were conserved across all pathovars. Biosynthetic gene clusters (BGCs) for toxoflavin, caryoynencin and enacyloxin were dispersed across B. gladioli, but bongkrekic acid and gladiolin production were clade-specific. Strikingly, 13 % of CF infection strains characterized were bongkrekic acid-positive, uniquely linking this food-poisoning toxin to this aspect of B. gladioli disease. Mapping the population biology and metabolite production of B. gladioli has shed light on its diverse ecology, and by demonstrating that the antibiotic trimethoprim suppresses bongkrekic acid production, a potential therapeutic strategy to minimize poisoning risk in CF has been identified.

Genomics-Driven Discovery of a Novel Glutarimide Antibiotic from Burkholderia gladioli Reveals an Unusual Polyketide Synthase Chain Release Mechanism.

A gene cluster encoding a cryptic trans-acyl transferase polyketide synthase (PKS) was identified in the genomes of Burkholderia gladioli BCC0238 and BCC1622, both isolated from the lungs of cystic fibrosis patients. Bioinfomatics analyses indicated the PKS assembles a novel member of the glutarimide class of antibiotics, hitherto only isolated from Streptomyces species. Screening of a range of growth parameters led to the identification of gladiostatin, the metabolic product of the PKS. NMR spectroscopic analysis revealed that gladiostatin, which has promising activity against several human cancer cell lines and inhibits tumor cell migration, contains an unusual 2-acyl-4-hydroxy-3-methylbutenolide in addition to the glutarimide pharmacophore. An AfsA-like domain at the C-terminus of the PKS was shown to catalyze condensation of 3-ketothioesters with dihydroxyacetone phosphate, thus indicating it plays a key role in polyketide chain release and butenolide formation.

Discovery and Biosynthesis of Bolagladins: Unusual Lipodepsipeptides from Burkholderia gladioli Clinical Isolates*.

Two Burkholderia gladioli strains isolated from the lungs of cystic fibrosis patients were found to produce unusual lipodepsipeptides containing a unique citrate-derived fatty acid and a rare dehydro-ß-alanine residue. The gene cluster responsible for their biosynthesis was identified by bioinformatics and insertional mutagenesis. In-frame deletions and enzyme activity assays were used to investigate the functions of several proteins encoded by the biosynthetic gene cluster, which was found in the genomes of about 45 % of B. gladioli isolates, suggesting that its metabolic products play an important role in the growth and/or survival of the species. The Chrome Azurol S assay indicated that these metabolites bind ferric iron, which suppresses their production when added to the growth medium. Moreover, a gene encoding a TonB-dependent ferric-siderophore receptor is adjacent to the biosynthetic genes, suggesting that these metabolites may function as siderophores in B. gladioli.

Matrix-assisted laser desorption/ionization time-of-flight MS for the accurate identification of Burkholderia cepacia complex and Burkholderia gladioli in the clinical microbiology laboratory.

Introduction. Burkholderia cepacia complex (Bcc) bacteria, currently consisting of 23 closely related species, and Burkholderia gladioli, can cause serious and difficult-to-treat infections in people with cystic fibrosis. Identifying Burkholderia bacteria to the species level is considered important for understanding epidemiology and infection control, and predicting clinical outcomes. Matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF) is a rapid method recently introduced in clinical laboratories for bacterial species-level identification. However, reports on the ability of MALDI-TOF to accurately identify Bcc to the species level are mixed.Aim. The aim of this project was to evaluate the accuracy of MALDI-TOF using the Biotyper and VITEK MS systems in identifying isolates from 22 different Bcc species and B. gladioli compared to recA gene sequencing, which is considered the current gold standard for Bcc.Methodology. To capture maximum intra-species variation, phylogenetic trees were constructed from concatenated multi-locus sequence typing alleles and clustered with a novel k-medoids approach. One hundred isolates representing 22 Bcc species, plus B. gladioli, were assessed for bacterial identifications using the two MALDI-TOF systems.Results. At the genus level, 100 and 97.0 % of isolates were confidently identified as Burkholderia by the Biotyper and VITEK MS systems, respectively; moreover, 26.0 and 67.0 % of the isolates were correctly identified to the species level, respectively. In many, but not all, cases of species misidentification or failed identification, a representative library for that species was lacking.Conclusion. Currently available MALDI-TOF systems frequently do not accurately identify Bcc bacteria to the species level.

Major Aspects of Burkholderia gladioli and Burkholderia cepacia Infections in Children.

BACKGROUND: Burkholderia cepacia complex is an aerobic, non-spore-forming, catalase-positive, nonfermentative, Gram-negative bacterium common in environment. It is a serious pathogen especially for patients with cystic fibrosis (CF). But pathogenicity of Burkholderia is not limited to patients with CF. Herein, we aimed to reveal clinical patterns and outcomes of Burkholderia infections in pediatric patients in our hospital and also antimicrobial susceptibility of the isolated strain. METHODS: This retrospective study was conducted in Ankara Hematology Oncology Children's Training and Research Hospital. Patients with isolates of Burkholderia spp. between January 6, 2013, and January 12, 2018, were included in the study. RESULTS: Burkholderia spp. was isolated from 55 patients. 94.6% of these patients had underlying diseases and had prior hospitalization within a year. Burkholderia gladioli grew in 15 patients' samples (27.3%); 38 patients grew B. cepacia (69.1%). None of the patients that B. gladioli was isolated was diagnosed as CF;. all had nosocomial infections. B. gladioli seemed to be more susceptible to aminoglycosides, piperacillin-tazobactam, carbapenems and ciprofloxacin than B. cepacia (P = 0.00), whereas B. cepacia seemed to be more susceptible to ceftazidime than B. gladioli (P = 0.032). In addition, B. cepacia was more susceptible to trimethoprim-sulfamethoxazole and levofloxacin than B. gladioli, but this difference was not statistically significant (P = 0.76). CONCLUSIONS: The incidence of nosocomial infections caused by Burkholderia spp. is rare especially in pediatric literature. In our study, nosocomial Burkholderia infections occurred mostly in intensive care unit patients. The surveillance of Burkholderia infections is still very important, and the clinicians should be aware of changing epidemiology and increasing resistance of the microorganism. Besides, there are no internationally agreed minimal inhibitory concentration breakpoints and disk-diffusion test thresholds for susceptibility testing for Burkholderia. Thus, the methods which were used for antibiotic susceptibility testing in our center might cause uncertainty about the results and internationally agreed minimal inhibitory concentration breakpoints and disk-diffusion test thresholds for susceptibility testing for Burkholderia is still a gap to fill for the current literature.

Impact of Plant Growth Promoting Rhizobacteria in the Orchestration of Lycopersicon esculentum Mill. Resistance to Plant Parasitic Nematodes: A Metabolomic Approach to Evaluate Defense Responses Under Field Conditions.

The present study deals with biological control of Meloidogyne incognita in 45-days old Lycopersicon esculentum, inoculated with Pseudomonas aeruginosa(M1) and Burkholderia gladioli (M2). The improved plant growth and biomass of nematode infested Plant growth promoting rhizobacteria (PGPR) inoculated plants was observed. Remarkable reduction in the numbers of second stage juvenile (J2s), root galls was recorded after treatment of microbes relative to experimental controls. Moreover, the lowered activities of oxidative stress markers (H2O2 (hydrogen peroxide), O2- (superoxide anion), malondialdehyde (MDA)) was estimated in plants after rhizobacterial supplementation. Higher activities of enzymatic (SOD (Superoxide dismutase), POD (Guaiacol peroxidase), CAT (Catalase), GPOX (Glutathione peroxidase), APOX (Ascorbate peroxidase), GST (Glutathione-S-transferase), GR (Glutathione reductase), DHAR (Dehydroascorbate reductase), PPO (Polyphenol oxidase)) and non-enzymatic (glutathione, ascorbic acid, tocopherol) antioxidants were further determined in nematode infected plants following the addition of bacterial strains. The upregulation of photosynthetic activities were depicted by evaluating plant pigments and gas exchange attributes. An increase in the levels of phenolic compounds (total phenols, flavonoids, anthocyanins), osmoprotectants (total osmolytes, carbohydrates, reducing sugars, trehalose, proline, glycine betaine, free amino acids) and organic acids (fumaric, succinic, citric, malic acid) were reflected in infected plants, showing further enhancement after application of biocontrol agents. The study revealed the understanding of plant metabolism, along with the initiative to commercially exploit the biocontrol agents as an alternative to chemical nematicides in infected fields for sustainable agriculture.

"Switching Partners": Piperacillin-Avibactam Is a Highly Potent Combination against Multidrug-Resistant Burkholderia cepacia Complex and Burkholderia gladioli Cystic Fibrosis Isolates.

In persons with cystic fibrosis (CF), airway infection with Burkholderia cepacia complex (Bcc) species or Burkholderia gladioli presents a significant challenge due to inherent resistance to multiple antibiotics. Two chromosomally encoded inducible ß-lactamases, a Pen-like class A and AmpC are produced in Bcc and B. gladioli Previously, ceftazidime-avibactam demonstrated significant potency against Bcc and B. gladioli isolated from the sputum of individuals with CF; however, 10% of the isolates tested resistant to ceftazidime-avibactam. Here, we describe an alternative antibiotic combination to overcome ceftazidime-avibactam resistance. Antimicrobial susceptibility testing was performed on Bcc and B. gladioli clinical and control isolates. Biochemical analysis was conducted on purified PenA1 and AmpC1 ß-lactamases from Burkholderia multivorans ATCC 17616. Analytic isoelectric focusing and immunoblotting were conducted on cellular extracts of B. multivorans induced by various ß-lactams or ß-lactam-ß-lactamase inhibitor combinations. Combinations of piperacillin-avibactam, as well as piperacillin-tazobactam plus ceftazidime-avibactam (the clinically available counterpart), were tested against a panel of ceftazidime-avibactam nonsusceptible Bcc and B. gladioli The piperacillin-avibactam and piperacillin-tazobactam-ceftazidime-avibactam combinations restored susceptibility to 99% of the isolates tested. Avibactam is a potent inhibitor of PenA1 (apparent inhibitory constant [Kiapp] = 0.5 µM), while piperacillin was found to inhibit AmpC1 (Kiapp = 2.6 µM). Moreover, piperacillin, tazobactam, ceftazidime, and avibactam, as well as combinations thereof, did not induce expression of blapenA1 and blaampC1 in the B. multivorans ATCC 17616 background. When ceftazidime-avibactam is combined with piperacillin-tazobactam, the susceptibility of Bcc and B. gladioli to ceftazidime and piperacillin is restored in vitro Both the lack of blapenA1 induction and potent inactivation of PenA1 by avibactam likely provide the major contributions toward susceptibility. With in vivo validation, piperacillin-tazobactam-ceftazidime-avibactam may represent salvage therapy for individuals with CF and highly drug-resistant Bcc and B. gladioli infections.

Supplementation with plant growth promoting rhizobacteria (PGPR) alleviates cadmium toxicity in Solanum lycopersicum by modulating the expression of secondary metabolites.

The current study evaluated the synergistic role of Plant growth promoting rhizobacteria (PGPR), Pseudomonas aeruginosa and Burkholderia gladioli on different physiological, biochemical and molecular activities of 10-days old Solanum lycopersicum seedlings under Cd stress. Cd toxicity altered the levels of phenolic compounds (total phenols (30.2%), flavonoids (92.7%), anthocyanin (59.5%), polyphenols (368.7%)), osmolytes (total osmolytes (10.3%), total carbohydrates (94%), reducing sugars (64.5%), trehalose (112.5%), glycine betaine (59%), proline (54.8%), and free amino acids (63%)), and organic acids in S. lycopersicum seedlings. Inoculation of P. aeruginosa and B. gladioli alleviated Cd-induced toxicity, which was manifested through enhanced phenolic compound levels and osmolytes. Additionally, the levels of low molecular weight organic acids (fumaric acid, malic acid, succinic acid, and citric acid) were also elevated. The expression of genes encoding enzymes for phenols and organic acid metabolism were also studied to be modulated that included CHS (chalcone synthase; 138.4%), PAL (phenylalanine ammonia lyase; 206.7%), CS (citrate synthase; 61.3%), SUCLG1 (succinyl Co-A ligase; 33.6%), SDH (succinate dehydrogenase; 23.2%), FH (fumarate hydratase; 12.4%), and MS (malate synthase; 41.2%) and found to be upregulated in seedlings inoculated independently with P. aeruginosa and B. gladioli. The results provide insights into the role of micro-organisms in alleviating Cd-induced physiological damage by altering levels of different metabolites.