Bacterial Supernatants Research Articles

Pheno- and genotyping in vitro dauer juvenile recovery in the nematode Heterorhabditis bacteriophora.

The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora is an effective biological-control agent of insect pests. The dauer juveniles (DJs) seek for, infect insects, and release cells of the carried symbiotic bacterium of the genus Photorhabdus. Inside the host, the DJs perceive signals from the insect's haemolymph that trigger the exit from the arrested stage and the further development to mature adults. This developmental step is called DJ recovery. In commercial production, a high and synchronous DJ recovery determines the success of liquid-culture mass production. To enhance the understanding about genetic components regulating DJ recovery, more than 160 mutant- and 25 wild type inbred lines (WT ILs) were characterized for DJ recovery induced by cell-free bacterial supernatant. The mutant lines exhibited a broader DJ recovery range than WT ILs (4.6-67.2% vs 1.6-35.7%). A subset of mutant lines presented high variability of virulence against mealworm (Tenebrio molitor) (from 22 to 78% mortality) and mean time survival under oxidative stress (70 mM H2O2; from 10 to 151 h). Genotyping by sequencing of 96 mutant lines resulted in more than 150 single nucleotide polymorphisms (SNPs), of which four results are strongly associated with the DJ recovery trait. The present results are the basis for future approaches in improving DJ recovery by breeding under in vitro liquid-culture mass production in H. bacteriophora. This generated platform of EMS-mutants is as well a versatile tool for the investigation of many further traits of interest in EPNs. KEYPOINTS: • Exposure to bacterial supernatants of Photorhabdus laumondii induces the recovery of Heterorhabditis bacteriophora dauer juveniles (DJs). Both, the bacteria and the nematode partner, influence this response. However, the complete identity of its regulators is not known. • We dissected the genetic component of DJ recovery regulation in H. bacteriophora nematodes by generating a large array of EMS mutant lines and characterizing their recovery pheno- and genotypes. • We determined sets of mutants with contrasting DJ recovery and genotyped a subset of the EMS-mutant lines via genotyping by sequencing (GBS) and identified SNPs with significant correlation to the recovery trait.

Dual production of polyhydroxyalkanoates and antibacterial/antiviral gold nanoparticles

Gold nanoparticles (AuNPs) have been explored for their use in medicine. Here, we report a sustainable, and cost-effective method to produce AuNPs using a bacterial strain such as Pseudomonas mendocina CH50 which is also known to be a polyhydroxyalkanoate (PHA) producer. A cell-free bacterial supernatant, which is typically discarded after PHA extraction, was used to produce spherical AuNPs of 3.5 ± 1.5 nm in size as determined by Transmission Electron Microscopy (TEM) analysis. The AuNPs/PHA composite coating demonstrated antibacterial activity against Staphylococcus aureus 6538P, and antiviral activity, with a 75% reduction in viral infectivity against SARS-CoV-2 pseudotype virus.

Isolation and Characterization of Enterococcus faecium from Fermented Korean Soybean Paste with Antibacterial Effects

In the present study, a total of eight Enterococcus faecium (OQ940301, OQ940302, OQ940303, OQ940304, OQ940305, OQ940308, OQ940309, and OQ940310) were isolated from soybean paste, a traditional Korean fermented food, and evaluated for their probiotic properties. The results showed that all the E. faecium strains survived in simulated human gastrointestinal conditions (4.1–5.59 log10 CFU/mL). In addition, the range of auto-aggregation was 5–25%, the hydrophobicity was around 94%, and it exhibited significant co-aggregation ability with Salmonella enterica and Staphylococcus aureus. However, all the isolates were shown to be resistant to Gentamycin. The bacterial cell-free supernatant showed antibacterial activity against S. enterica, Escherichia coli, Bacillus cereus, Listeria monocytogenes, and S. aureus. Furthermore, E. faecium exhibited potent anti-oxidant activity by scavenging 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) free radicals. In addition, safety was confirmed by evaluating the hemolytic activity in blood agar; none of the bacterial isolates showed hemolysis. These results demonstrated that E. faecium (OQ940301 and OQ940309) isolated from soybean paste showed a higher probiotic potential.

Exploring the Bioprotective Potential of Halophilic Bacteria against Major Postharvest Fungal Pathogens of Citrus Fruit Penicillium digitatum and Penicillium italicum

Citrus fruits are vulnerable to green mold (caused by Penicillium digitatum) and blue mold (caused by Penicillium italicum) during storage, posing significant challenges to the industry. Therefore, biological control utilizing antagonistic bacteria has emerged as a dependable strategy for managing postharvest diseases. In this study, halophilic bacterial isolates were carefully selected from diverse saline ecosystems, including the Dead Sea, the Agadir Sea, the Rabat Sea, saline soil, and water of the Amassine Oued in Taounate, based on rigorous in vitro and in vivo antagonism bioassays. Out of 21 bacteria from different saline environments, 10 were chosen for further characterization based on the 16S rDNA gene. Notably, the EAM1 isolate demonstrated exceptional inhibitory effects, reaching a 90% inhibition rate against P. digitatum, while the ER2 isolate closely followed with an 89% inhibition rate against P. italicum. Furthermore, in bacterial supernatant experiments, six bacterial isolates effectively curbed the growth of P. digitatum, and three demonstrated efficacy against P. italicum development. In an in vivo trial spanning ten days of incubation, three highly effective isolates against P. digitatum displayed zero severity, and two of these isolates also demonstrated zero severity against P. italicum. Interestingly, a comparison of bacterial filtrates revealed that all isolates exhibited a severity level of over 50% against the pathogen causing green rot (P. digitatum), while the severity was lower than 50% for the supernatants of the two isolates used against P. italicum. In conclusion, this study highlights the promising role of halophilic bacteria, specifically Bacillus amyloliquefaciens EAM1 and B. amyloliquefaciens ER2, in controlling postharvest fruit pathogens. The findings shed light on the potential of utilizing these bioprotective agents to address the challenges posed by green and blue citrus molds, providing valuable insights for the citrus industry.

Regulation of serotonin production by specific microbes from piglet gut

BackgroundSerotonin is an important signaling molecule that regulates secretory and sensory functions in the gut. Gut microbiota has been demonstrated to affect serotonin synthesis in rodent models. However, how gut microbes regulate intestinal serotonin production in piglets remains vague. To investigate the relationship between microbiota and serotonin specifically in the colon, microbial composition and serotonin concentration were analyzed in ileum-cannulated piglets subjected to antibiotic infusion from the ileum when comparing with saline infusion. Microbes that correlated positively with serotonin production were isolated from piglet colon and were further used to investigate the regulation mechanisms on serotonin production in IPEC-J2 and a putative enterochromaffin cell line RIN-14B cells.ResultsAntibiotic infusion increased quantities of Lactobacillus amylovorus (LA) that positively correlated with increased serotonin concentrations in the colon, while no effects observed for Limosilactobacillus reuteri (LR). To understand how microbes regulate serotonin, representative strains of LA, LR, and Streptococcus alactolyticus (SA, enriched in feces from prior observation) were selected for cell culture studies. Compared to the control group, LA, LR and SA supernatants significantly up-regulated tryptophan hydroxylase 1 (TPH1) expression and promoted serotonin production in IPEC-J2 cells, while in RIN-14B cells only LA exerted similar action. To investigate potential mechanisms mediated by microbe-derived molecules, microbial metabolites including lactate, acetate, glutamine, and γ-aminobutyric acid were selected for cell treatment based on computational and metabolite profiling in bacterial supernatant. Among these metabolites, acetate upregulated the expression of free fatty acid receptor 3 and TPH1 while downregulated indoleamine 2,3-dioxygenase 1. Similar effects were also recapitulated when treating the cells with AR420626, an agonist targeting free fatty acid receptor 3.ConclusionsOverall, these results suggest that Lactobacillus amylovorus showed a positive correlation with serotonin production in the pig gut and exhibited a remarkable ability to regulate serotonin production in cell cultures. These findings provide evidence that microbial metabolites mediate the dialogue between microbes and host, which reveals a potential approach using microbial manipulation to regulate intestinal serotonin biosynthesis.

Aqueous Extracts of Fermented Macrofungi Cultivated in Oilseed Cakes as a Carbon Source for Probiotic Bacteria and Potential Antibacterial Activity.

Plant biomass colonized by macrofungi can contain molecules with bioactive properties with applications to human/animal health. This work aimed to verify antibacterial activities from aqueous extracts from oil seed cakes of Jatropha curcas (JSC) and cottonseed (CSC), fermented by macrofungi for probiotic bacteria cultivation. Coriolopsis sp., Tyromyces sp., Panus lecomtei, and Pleurotus pulmonarius were cultivated in solid and submerged media. The aqueous extract of unfermented JSC was more efficient than glucose for the growth of all probiotic bacteria. Extracts from four macrofungi fermented in CSC favored Lactobacillus acidophilus growth. In solid fermentation, macrofungi extracts cultivated in JSC favored Bifidobacterium lactis growth. All fungi extracts showed more significant growth than carbohydrates among the four probiotic bacteria evaluated. Regarding antimicrobial activities, no fungal extract or bacterial supernatant showed a more significant inhibition halo for enteropathogenic bacteria than ampicillin (control). Extracts from P. lecomtei and Coriolopsis sp. in CSC showed inhibition halos for Salmonella enterica. Supernatants from L. acidophilus, B. lactis, and Lactobacillus rhamnosus resulted in more significant inhibition of Staphylococcus aureus than the control, which indicates possible antimicrobial activity. Unfermented JSC supernatant showed better results for bacterial growth, while supernatants and aqueous extracts from CSC fermentation can be used for probiotic bacteria culture.

Antipathogenic Efficacy of Biogenic Silver Nanoparticles and Antibiofilm Activities Against Multi-drug-Resistant ESKAPE Pathogens.

The silver nanoparticles (AgNPs) were produced by employing a biogenic loom and tested for antipathogenic assets against multi-drug-resistant (MDR) ESKAPE bacteria. Biogenically synthesized AgNPs were characterized adopting various high-throughput techniques such as UHRTEM, SEM with EDX, DLS, TGA-DTA, and XRD and spectroscopic analysis showed polydispersion of nanoparticles. In this context, AgNPs with the attribute of spherical-shaped nanoparticles with an average size of 26nm were successfully synthesized utilizing bacterial supernatant. The antipathogenic activities of AgNPs were assessed against 11 strains of MDR ESKAPE bacteria including Enterococcus faecium; methicillin-resistant Staphylococcus aureus; Klebsiella pneumonia; Acinetobacter baumannii; Pseudomonas aeruginosa; Enterobacter aerogenes; and Enterobacter species. The exposure of biogenic AgNPs in a well diffusion assay showed all the growth inhibitions of ESKAPE bacteria at 200μg/ml after 18h of incubation. Growth kinetics demonstrated maximum killing at 60μg/ml after 4h of completion. The highest biofilm depletions were found at 100μg/ml in adhesion assay. Live/dead assays showed effective killing of the ESKAPE bacteria at 10μg/ml in pre-existing biofilms. The effective inhibitory concentrations of AgNPs were investigated ranging from 10 to 200μg/ml. The selected pathogens found sensitive to AgNPs are statistically significant (P < 0.05) than that of cefotaxime/AgNO3. Consequently, a broad spectrum of antimicrobial potentials of AgNPs can be alternative to conventional antimicrobial agents for future medicine.

Anti-tumor effects of Bacteroides fragilis and Bifidobacterium bifidum culture supernatants on mouse breast cancer

Breast cancer is one of the most important causes of cancer related morbidity and mortality in the world. Breast contains several bacterial species performing specialized functions. Bacteroides and Bifidobacterium, as microbial flora, play pivotal role against breast cancer development in vivo and in vitro. In this study we evaluated the anti-proliferative and anti-tumor effects of Bacteroides fragilis (F.S), Bifidobacterium bifidum (B.S) and Bifidobacterium bifidum + Bacteroides fragilis supernatants (B.F.S) in in vivo and in vitro model of 4 T1 cell line breast cancer. Bacterial supernatants assessed in both model. Our results indicated that the relative tumor volume significantly increased in the mice receiving intratumoral PBS. Three out of the 4 mice were cleared from the tumor thoroughly in 10–25 days after administration of B.F.S. mice receiving intratumoral B.F.S significantly secreted higher interferon γ and lower IL-10 compared with the other groups. Overall, our findings indicated that intratumoral administration of B.F.S effectively inhibited the growth of breast tumors through induction of necrosis and suppressing proliferation and angiogenesis.

Symbiosis of Carpenter Bees with Uncharacterized Lactic Acid Bacteria Showing NAD Auxotrophy.

Eusocial bees (such as honey bees and bumble bees) harbor core gut microbiomes that are transmitted through social interaction between nestmates. Carpenter bees are not eusocial; however, recent microbiome analyses found that Xylocopa species harbor distinctive core gut microbiomes. In this study, we analyzed the gut microbiomes of three Xylocopa species in Japan between 2016 and 2021 by V1 to V2 region-based 16S rDNA amplicon sequencing, and 14 candidate novel species were detected based on the full-length 16S rRNA gene sequences. All Xylocopa species harbor core gut microbiomes consisting of primarily lactic acid bacteria (LAB) that were phylogenetically distant from known species. Although they were difficult to cultivate, two LAB species from two different Xylocopa species were isolated by supplementing bacterial culture supernatants. Both genomes exhibited an average LAB genome size with a large set of genes for carbohydrate utilization but lacked genes to synthesize an essential coenzyme NAD, which is unique among known insect symbionts. Our findings of phylogenetically distinct core LAB of NAD auxotrophy reflected the evolution of Xylocopa-restricted bacteria retention and maintenance through vertical transmission of microbes during solitary life. We propose five candidate novel species belonging to the families Lactobacillaceae and Bifidobacteriaceae, including a novel genus, and their potential functions in carbohydrate utilization. IMPORTANCE Recent investigations found unique microbiomes in carpenter bees, but the description of individual microbes, including isolation and genomics, remains largely unknown. Here, we found that the Japanese Xylocopa species also harbor core gut microbiomes. Although most of them were difficult to isolate a pure colony, we successfully isolated several strains. We performed whole-genome sequencing of the isolated candidate novel species and found that the two Lactobacillaceae strains belonging to the Xylocopa-specific novel LAB clade lack the genes for synthesizing NAD, a coenzyme central to metabolism in all living organisms. Here, we propose a novel genus for the two LAB species based on very low 16S rRNA gene sequence similarities and genotypic characters.

Exoproducts of the Most Common Achromobacter Species in Cystic Fibrosis Evoke Similar Inflammatory Responses In Vitro.

Achromobacter is a genus of Gram-negative rods, which can cause persistent airway infections in people with cystic fibrosis (CF). The knowledge about virulence and clinical implications of Achromobacter is still limited, and it is not fully established whether Achromobacter infections contribute to disease progression or if it is a marker of poor lung function. The most commonly reported Achromobacter species in CF is A. xylosoxidans. While other Achromobacter spp. are also identified in CF airways, the currently used Matrix-Assisted Laser Desorption/Ionization Time Of Flight Mass Spectrometry (MALDI-TOF MS) method in routine diagnostics cannot distinguish between species. Differences in virulence between Achromobacter species have consequently not been well studied. In this study, we compare phenotypes and proinflammatory properties of A. xylosoxidans, A. dolens, A. insuavis, and A. ruhlandii using in vitro models. Bacterial supernatants were used to stimulate CF bronchial epithelial cells and whole blood from healthy individuals. Supernatants from the well-characterized CF-pathogen Pseudomonas aeruginosa were included for comparison. Inflammatory mediators were analyzed with ELISA and leukocyte activation was assessed using flow cytometry. The four Achromobacter species differed in morphology seen in scanning electron microscopy (SEM), but there were no observed differences in swimming motility or biofilm formation. Exoproducts from all Achromobacter species except A. insuavis caused significant IL-6 and IL-8 secretion from CF lung epithelium. The cytokine release was equivalent or stronger than the response induced by P. aeruginosa. All Achromobacter species activated neutrophils and monocytes ex vivo in a lipopolysaccharide (LPS)-independent manner. Our results indicate that exoproducts of the four included Achromobacter species do not differ consistently in causing inflammatory responses, but they are equally or even more capable of inducing inflammation compared with the classical CF pathogen P. aeruginosa. IMPORTANCE Achromobacter xylosoxidans is an emerging pathogen among people with cystic fibrosis (CF). Current routine diagnostic methods are often unable to distinguish A. xylosoxidans from other Achromobacter species, and the clinical relevance of different species is still unknown. In this work, we show that four different Achromobacter species relevant to CF evoke similar inflammatory responses from airway epithelium and leukocytes in vitro, but they are all equally or even more proinflammatory compared to the classic CF-pathogen Pseudomonas aeruginosa. The results suggest that Achromobacter species are important airway pathogens in CF, and that all Achromobacter species are relevant to treat.

Study of Copper Resistant Pseudomonas Strains Isolated from Soil in Sarcheshmeh Copper Mine, in Iran and Investigation of the Mechanisms Involved in Copper Resistance

Heavy metal contaminated sites are the potential source of metal resistance microorganisms. The major focus of this study is to isolate and identify copper ions resistant Pseudomonas from the soil samples collected from mining and refining sites of Sarcheshmeh copper mine in the Kerman Province of Iran and investigate on possible mechanisms for copper resistance. Isolates were selected based on high level of copper ions resistance. The screened isolate was identified as Pseudomonas putida Cu-2 by morphological, biochemical and 16S rDNA gene sequencing analysis. A high degree of copper ions resistance associated with multiple antibiotic resistances was also detected in the screened isolate. The growth pattern of screened isolate with all the studied Cu concentrations was similar to that of control (without Cu ions) indicating that Cu ions would not affect the growth of isolated strain. A decrease in the amount of exopolysaccharides (EPS) was detected after Cu ions – P. Putida Cu-2 culture supernatant interaction. The Fourier transform infrared spectroscopy (FT-IR) peaks for the EPS extracted from the bacterial culture supernatant and the interacted Cu ions were nearly similar. The EPS covering of the Cu ions was established by FT-IR. The study of bacterial EPS covered Cu ions with E. coli PTCC 1338 and S. aureus PTCC 1113 exhibited less toxicity compared to uncoated Cu ions. The presence of copA gene that encodes for the multi-copper oxidase was investigated in the selected strain. The present study suggests that the capping of Cu ions by bacterially produced EPS and the presence of copA gene serve as the probable mechanisms of copper resistance.

Exploring bacterial cell-free supernatants, unfiltered ferments and crude bacteria uses of Xenorhabdus and Photorhabdus (Morganellaceae) for controlling Botrytis cinerea (Helotiales: Sclerotiniaceae)

The pathogen Botrytis cinerea (Helotiales: Sclerotiniaceae) is a wound necrotrophic fungus that causes significant losses in fruits and vegetables worldwide. The entomopathogenic nematode (EPN) symbiotic bacteria, Xenorhabdus spp. and Photorhabdus spp., are well-known associated biological control agents that produce a diverse range of natural antifungal compounds. This study aimed to evaluate the efficacy of different control strategies against B. cinerea using: (i) EPN symbiotic bacterial cell-free supernatants, (ii) unfiltered ferments and (iii) crude bacteria isolates. The antifungal efficacy of X. bovienii, X. nematophila, X. kozodoii and P. laumondii subsp. laumondii cell-free supernatants obtained after the bacterial fermentations were tested in vitro at two different concentrations (10% and 20%). Furthermore, the antifungal effect of X. nematophila and P. laumondii unfiltered ferments were tested in vitro, and their dissuasive effect was also tested over tomato leaves. Finally, the antifungal capacity of the crude X. nematophila and P. laumondii isolate was tested comparing their effect with the fungicide effect of the commercial Bacillus amyloliquefaciens (Serenade® ASO fungicide). The results showed that employing the bacterial cell-free supernatants or the unfiltered ferments could have different antifungal efficacy against this pathogen. Applying X. nematophila cell-free supernatant and unfiltered ferments at 20% concentration resulted in the highest inhibition effect compared with the control (distilled water), 82% and 100%, respectively. Furthermore, P. laumondii-isolate can control the growth of Botrytis in in vitro conditions, showing no significant differences with the efficacy of Bacillus amyloliquefaciens in a four-day experiment. Overall, this study builds on a better understanding of the effects of these novel biocontrol agents against B. cinerea and helps to develop an innovative formulation of these bacterial products as an efficient biocontrol tool.

Highly-conserved regulatory activity of the ANR family in the virulence of diarrheagenic bacteria through interaction with master and global regulators

ANR (AraC negative regulators) are a novel class of small regulatory proteins commonly found in enteric pathogens. Aar (AggR-activated regulator), the best-characterized member of the ANR family, regulates the master transcriptional regulator of virulence AggR and the global regulator HNS in enteroaggregative Escherichia coli (EAEC) by protein–protein interactions. On the other hand, Rnr (RegA-negative regulator) is an ANR homolog identified in attaching and effacing (AE) pathogens, including Citrobacter rodentium and enteropathogenic Escherichia coli (EPEC), sharing only 25% identity with Aar. We previously found that C. rodentium lacking Rnr exhibits prolonged shedding and increased gut colonization in mice compared to the parental strain. To gain mechanistic insights into this phenomenon, we characterized the regulatory role of Rnr in the virulence of prototype EPEC strain E2348/69 by genetic, biochemical, and human organoid-based approaches. Accordingly, RNA-seq analysis revealed more than 500 genes differentially regulated by Rnr, including the type-3 secretion system (T3SS). The abundance of EspA and EspB in whole cells and bacterial supernatants confirmed the negative regulatory activity of Rnr on T3SS effectors. We found that besides HNS and Ler, twenty-six other transcriptional regulators were also under Rnr control. Most importantly, the deletion of aar in EAEC or rnr in EPEC increases the adherence of these pathogens to human intestinal organoids. In contrast, the overexpression of ANR drastically reduces bacterial adherence and the formation of AE lesions in the intestine. Our study suggests a conserved regulatory mechanism and a central role of ANR in modulating intestinal colonization by these enteropathogens despite the fact that EAEC and EPEC evolved with utterly different virulence programs.

Pseudophomins A–D Produced from Pseudomonas sp. HN8-3 Using an OSMAC Approach and Their Roles in Biocontrol of Phytophthora capsici in Cucumbers

In this study, two new cyclic lipopeptides (CLPs) pseudophomins C (3) and D (4) and two known CLPs pseudophomins A (1) and B (2) were produced and characterized from the bacterial supernatant of Pseudomonas sp. HN8-3 by an OSMAC (one strain-many compounds) approach. OSMAC is a strategy that involves feeding of a single microorganism with divergent substrates to stimulate the production of new secondary metabolites. These pseudophomins were purified and identified via chromatographic methods, droplet collapse assay, genome mining, spectroscopic and spectrometric analyses, and single-crystal X-ray diffraction (XRD). Moreover, bioactivity tests showed that pseudophomins could lyse the zoospores of Phytophthora capsici in vitro, and coapplication of pseudophomins with zoospores of P. capsici further reduced the incidence of P. capsici on cucumber leaves. Collectively, these results indicated that pseudophomins have the potential to be developed as biopesticides for controlling P. capsici in cucumber.

Microassay validation for bacterial IAA estimation as a new fine-tuned PGPR screening assay

The detection and quantification of Indole -3 Acetic Acid (IAA) produced by Plant growth promoting rhizobacteria (PGPR) rely on a standard well-documented assay, which remains time-consuming, laborious, and costly. These drawbacks led to sway interest to economic and reliable assays. The aim of this work is to validate and standardize a fast, reliable, and cost-effective microassay to quantify IAA produced by bacteria with an easy microplate method. In order to validate the accuracy of the IAA microplate assay, bacterial samples from different genera were assayed using two methods: the conventional IAA estimation assay and the IAA micro- assay. The microassay shows a prominent reduction in used bacterial supernatant volume as well as Salkowski reagent volume of about 92.5%. It is considerably cheaper than the conventional one of around 56%. The newly performed microplate assay is 23 times faster. The result of IAA quantitative analysis for 13 bacterial strains showed that Bacillus muralis and Bacillus toyonensis produced the highest IAA concentration (23.64±0.003μg/ml and 23.35±0.006μg/ml, respectively). The obtained data from both methods were highly correlated with an R-value of 0.979. The microassay offers the ability to read the optical density of all samples simultaneously since used volumes of bacterial supernatants and Salkowski reagent were minimized to place the mixture in 96-well microplates, which reduces greatly required labor. Furthermore, the application of the IAA micro-plate assay reduces drastically the reagent waste and toxicity hazard of Salkowski reagent in the environment, thus, we can classify it as eco-friendly respecting the Green Chemistry concept according to Environmental Protection Agency (EPA). The IAA microassay is a, reliable, rapid and cost-effective and eco-friendly method to screen plant growth promoting potential of more than 23 bacterial strains by microplate. It could be an alternative for the conventional IAA assay as a routine research tool.

Chitinase A, a tightly regulated virulence factor of Salmonella enterica serovar Typhimurium, is actively secreted by a Type 10 Secretion System.

As a facultative intracellular pathogen, Salmonella enterica serovar Typhimurium is one of the leading causes of food-borne diseases in humans. With the ingestion of fecal contaminated food or water, S. Typhimurium reaches the intestine. Here, the pathogen efficiently invades intestinal epithelial cells of the mucosal epithelium by the use of multiple virulence factors. Recently, chitinases have been described as emerging virulence factors of S. Typhimurium that contribute to the attachment and invasion of the intestinal epithelium, prevent immune activation, and modulate the host glycome. Here we find that the deletion of chiA leads to diminished adhesion and invasion of polarized intestinal epithelial cells (IEC) compared to wild-type S. Typhimurium. Interestingly, no apparent impact on interaction was detected when using non-polarized IEC or HeLa epithelial cells. In concordance, we demonstrate that chiA gene and ChiA protein expression was solely induced when bacteria gain contact with polarized IEC. The induction of chiA transcripts needs the specific activity of transcriptional regulator ChiR, which is co-localized with chiA in the chitinase operon. Moreover, we established that after chiA is induced, a major portion of the bacterial population expresses chiA, analyzed by flow cytometry. Once expressed, we found ChiA in the bacterial supernatants using Western blot analyses. ChiA secretion was completely abolished when accessory genes within the chitinase operon encoding for a holin and a peptidoglycan hydrolase were deleted. Holins, peptidoglycan hydrolases, and large extracellular enzymes in close proximity have been described as components of the bacterial holin/peptidoglycan hydrolase-dependent protein secretion system or Type 10 Secretion System. Overall, our results confirm that chitinase A is an important virulence factor, tightly regulated by ChiR, that promotes adhesion and invasion upon contact with polarized IEC and is likely secreted by a Type 10 Secretion System (T10SS).

Inhalable vaccine of bacterial culture supernatant extract mediates protection against fatal pulmonary anthrax

ABSTRACT Pulmonary anthrax is the most fatal clinical form of anthrax and currently available injectable vaccines do not provide adequate protection against it. Hence, next-generation vaccines that effectively induce immunity against pulmonary anthrax are urgently needed. In the present study, we prepared an attenuated and low protease activity Bacillus anthracis strain A16R-5.1 by deleting five of its extracellular protease activity-associated genes and its lef gene through the CRISPR-Cas9 genome editing system. This mutant strain was then used to formulate a lethal toxin (LeTx)-free culture supernatant extract (CSE) anthrax vaccine, of which half was protective antigen (PA). We generated liquid, powder, and powder reconstituted formulations that could be delivered by aerosolized intratracheal inoculation. All of them induced strong humoral, cellular, and mucosal immune responses. The vaccines also produced LeTx neutralizing antibodies and conferred full protection against the lethal aerosol challenges of B. anthracis Pasteur II spores in mice. Compared to the recombinant PA vaccine, the CSE anthrax vaccine with equal PA content provided superior immunoprotection against pulmonary anthrax. The preceding results suggest that the CSE anthrax vaccine developed herein is suitable and scalable for use in inhalational immunization against pulmonary anthrax.

The deterrent ability of Xenorhabdus nematophila and Photorhabdus laumondii compounds as a potential novel tool for Lobesia botrana (Lepidoptera: Tortricidae) management

The grapevine moth, Lobesia botrana (Lepidoptera: Tortricidae), is a critical pest for vineyards and causes significant economic losses in wine-growing areas worldwide. Identifying and developing novel semiochemical cues (e.g. volatile bacterial compounds) which modify the ovipositional and trophic behaviour of L. botrana in vineyard fields could be a novel control alternative in viticulture. Xenorhabdus spp. and Photorhabdus spp. are becoming one of the best-studied bacterial species due to their potential interest in producing toxins and deterrent factors. In this study, we investigated the effect of the deterrent compounds produced by Xenorhabdus nematophila and Photorhabdus laumondii on the ovipositional moth behaviour and the larval feeding preference of L. botrana. Along with the in-vitro bioassays performed, we screened the potential use of 3 d cell-free bacterial supernatants and 3 and 5 d unfiltered bacterial ferments. In addition, we tested two application systems: (i) contact application of the bacterial compounds and (ii) volatile bacterial compounds application. Our findings indicate that the deterrent effectiveness varied with bacterial species, the use of bacterial cell-free supernatants or unfiltered fermentation product, and the culture times. Grapes soaked in the 3 d X. nematophila and P. laumondii ferments had ∼ 55% and ∼ 95% fewer eggs laid than the control, respectively. Likewise, the volatile compounds emitted by the 5 d P. laumondii fermentations resulted in ∼ 100% avoidance of L. botrana ovipositional activity for three days. Furthermore, both bacterial fermentation products have larval feeding deterrent effects (∼65% of the larva chose the control grapes), and they significantly reduced the severity of damage caused by third instar larva in treated grapes. This study provides insightful information about a novel bacteria-based tool which can be used as an eco-friendly and economical alternative in both organic and integrated control of L. botrana in vineyard.

Legionella pneumophila and Free-Living Nematodes: Environmental Co-Occurrence and Trophic Link.

Free-living nematodes harbor and disseminate various soil-borne bacterial pathogens. Whether they function as vectors or environmental reservoirs for the aquatic L. pneumophila, the causative agent of Legionnaires' disease, is unknown. A survey screening of biofilms of natural (swimming lakes) and technical (cooling towers) water habitats in Germany revealed that nematodes can act as potential reservoirs, vectors or grazers of L. pneumophila in cooling towers. Consequently, the nematode species Plectus similis and L. pneumophila were isolated from the same cooling tower biofilm and taken into a monoxenic culture. Using pharyngeal pumping assays, potential feeding relationships between P. similis and different L. pneumophila strains and mutants were examined and compared with Plectus sp., a species isolated from a L. pneumophila-positive thermal source biofilm. The assays showed that bacterial suspensions and supernatants of the L. pneumophila cooling tower isolate KV02 decreased pumping rate and feeding activity in nematodes. However, assays investigating the hypothesized negative impact of Legionella's major secretory protein ProA on pumping rate revealed opposite effects on nematodes, which points to a species-specific response to ProA. To extend the food chain by a further trophic level, Acanthamoebae castellanii infected with L. pneumphila KV02 were offered to nematodes. The pumping rates of P. similis increased when fed with L. pneumophila-infected A. castellanii, while Plectus sp. pumping rates were similar when fed either infected or non-infected A. castellanii. This study revealed that cooling towers are the main water bodies where L. pneumophila and free-living nematodes coexist and is the first step in elucidating the trophic links between coexisting taxa from that habitat. Investigating the Legionella-nematode-amoebae interactions underlined the importance of amoebae as reservoirs and transmission vehicles of the pathogen for nematode predators.

Killed fungal pathogen triggers antifungal metabolites in Alcaligenes faecalis for plant defense

Sclerotium rolfsii is a broad host necrotrophic pathogen causing serious damages in crop yields. Apart from chemical fungicides being used to control this pathogen, no bio-fungicides have been reported till date. In this study, we have tried to utilize heat killed S. rolfsii hyphae for triggering biosynthesis of antifungal compounds in the endophytic bacterium Alcaligenes faecalis BHU 12. The endophytic bacterial cell free supernatant (CFS) obtained by growing BHU 12 in presence of freeze-crushed and autoclaved S. rolfsii hyphae caused prominent S. rolfsii hyphal degeneration and inhibition of sclerotial germination under in vitro conditions. This result was further corroborated under in planta conditions wherein spraying of the CFS at the point of infection inhibited further pathogen ingress. This observation was justified by the presence of gallic and shikimic acid in the CFS which served as antifungal agent and augmentor of plant defense system respectively. Infected plants sprayed with the CFS were found to display a prominent increase in phenylalanine ammonia lyase activity and a corresponding increase in total phenolics. In this context, our results described the possible alternative of using endophytic bacterial metabolite compounds as biofungicides. A simultaneous augmentation in seed germination upon treatment with the CFS suggests a possibility of using endophytic bacterial supernatants as biocontrol and biofertilizer alternative instead of whole bacterial cell since indigenous soil microbiota combined with cumbersome inoculation techniques prevents successful colonization of endophytic microbes in planta.