A selection process based on the robustness of anti-Listeria monocytogenes activity reveals two strains of Carnobacterium maltaromaticum with biopreservation properties in cheese.

Biopreservation is an approach consisting of using microorganisms as protective cultures and/or their metabolites to optimize the microbiological quality and shelf life of food by ensuring safety or reducing food waste. Biopreservation strain selection pipelines mainly focus on inhibition strength to identify strains of interest. However, in addition to inhibition strength, inhibition activity must be able to be expressed despite significant variations in food matrix properties. In this study, the anti-Listeria monocytogenes EGDelux properties of a collection of 77 Carnobacterium maltaromaticum strains were investigated by high throughput competition assays under varying conditions of co-culture inoculation level, time interval between inoculation with C. maltaromaticum and L. monocytogenes, pH, and NaCl, resulting in 1309 different combinations of C. maltaromaticum strains and culture conditions. This screening led to the selection of two candidate strains with potent and robust anti-L. monocytogenes activities. Deferred growth inhibition assays followed by halo measurements, and liquid co-culture followed by colony counting, revealed that these two strains exhibit a wide anti-Listeria spectrum. Challenge tests in Camembert and Saint-Nectaire cheese revealed both strains were able to inhibit a cocktail of five strains of L. monocytogenes with high potency and high reproducibility. These results highlight the importance of including the robustness criterion in addition to potency when designing a strain selection process for biopreservation applications.

Carnobacterium maltaromaticum as bioprotective culture against spoilage bacteria in ground meat and cooked ham.

This study assessed the bioprotective effect of Carnobacterium maltaromaticum (CM) against Pseudomonas fluorescens (PF) and Brochothrix thermosphacta (BT) in ground beef and sliced cooked ham stored in high- and low-oxygen-modified atmospheres (66/4/30% O2/N2/CO2 and 70/30% N2/CO2, respectively). Both meat products were inoculated with CM, PF, and BT individually or in combination and stored for 7 days (3 days at 4 °C + 4 days at 8 °C) for ground beef and 28 days (10 days at 4 °C + 18 days at 8 °C) for sliced cooked ham. Each food matrix was assigned to 6 treatments: NC (no bacterial inoculation, representing the indigenous bacteria of meat), CM, BT, PF, CM + BT, and CM + PF. Bacterial growth, pH, instrumental color, and headspace gas composition were assessed during storage. CM counts remained stable from inoculation and throughout the shelf-life. CM reduced the population of inoculated and indigenous spoilage bacteria, including BT, PF, and enterobacteria, and showed a negligible impact on the physicochemical quality parameters of the products. Furthermore, upon simulating the shelf-life of ground beef and cooked ham, a remarkable extension could be observed with CM. Therefore, CM could be exploited as a biopreservative in meat products to enhance quality and shelf-life.

Identification of novel Carnobacterium maltaromaticum strains in bone marrow samples of patients with acute myeloid leukemia using a metagenomic binning approach / Identificación de nuevas cepas de Carnobacterium maltaromaticum en muestras de médula ósea de pacientes con leucemia mieloide aguda mediante un enfoque de combinación metagenómica

The aim of this study aimed to examine the existence of a bacterial metagenome in the bone marrow of patients with acute myeloid leukemia (AML). We re-examined whole-genome sequencing data from the bone marrow samples of seven patients with AML, four of whom were remitted after treatment, for metagenomic analysis. After the removal of human reads, unmapped reads were used to profile the species-level composition. We used the metagenomic binning approach to confirm whether the identified taxon was a complete genome of known or novel strains. We observed a unique and novel microbial signature in which Carnobacterium maltaromaticum was the most abundant species in five patients with AML or remission. The complete genome of C. maltaromaticum “BMAML_KR01,” which was observed in all samples, was 100% complete with 8.5% contamination and closely clustered with C. maltaromaticum strains DSM20730 and SF668 based on single nucleotide polymorphism variations. We identified five unique proteins that could contribute to cancer progression and 104 virulent factor proteins in the BMAML_KR01 genome. To our knowledge, this is the first report of a new strain of C. maltaromaticum in patients with AML. The presence of C. maltaromaticum and its new strain in patients indicates an urgent need to validate the existence of this bacterium and evaluate its pathophysiological role.(AU)

Carnobacterium maltaromaticum boosts intestinal vitamin D production to suppress colorectal cancer in female mice.

Carnobacterium maltaromaticum was found to be specifically depleted in female patients with colorectal cancer (CRC). Administration of C. maltaromaticum reduces intestinal tumor formation in two murine CRC models in a female-specific manner. Estrogen increases the attachment and colonization of C. maltaromaticum via increasing the colonic expression of SLC3A2 that binds to DD-CPase of this bacterium. Metabolomic and transcriptomic profiling unveils the increased gut abundance of vitamin D-related metabolites and the mucosal activation of vitamin D receptor (VDR) signaling in C. maltaromaticum-gavaged mice in a gut microbiome- and VDR-dependent manner. In vitro fermentation system confirms the metabolic cross-feeding of C. maltaromaticum with Faecalibacterium prausnitzii to convert C. maltaromaticum-produced 7-dehydrocholesterol into vitamin D for activating the host VDR signaling. Overall, C. maltaromaticum colonizes the gut in an estrogen-dependent manner and acts along with other microbes to augment the intestinal vitamin D production to activate the host VDR for suppressing CRC.

Characterization of Bacteriophage cd2, a Siphophage Infecting Carnobacterium divergens and a Representative Species of a New Genus of Phage.

Carnobacterium divergens is frequently isolated from natural environments and is a predominant species found in refrigerated foods, particularly meat, seafood, and dairy. While there is substantial interest in using C. divergens as biopreservatives and/or probiotics, some strains are known to be fish pathogens, and the uncontrolled growth of C. divergens has been associated with food spoilage. Bacteriophages offer a selective approach to identify and control the growth of bacteria; however, to date, few phages targeting C. divergens have been reported. In this study, we characterize bacteriophage cd2, which we recently isolated from minced beef. A detailed host range study reveals that phage cd2 infects certain phylogenetic groups of C. divergens. This phage has a latent period of 60 min and a burst size of ~28 PFU/infected cell. The phage was found to be acid and heat sensitive, with a complete loss of phage activity when stored at pH 2 or heated to 60°C. Electron microscopy shows that phage cd2 is a siphophage, and while it shares the B3 morphotype with a unique cluster of Listeria and Enterococcus phages, a comparison of genomes reveals that phage cd2 comprises a new genus of phage, which we have termed as Carnodivirus. IMPORTANCE Currently, very little is known about phages that infect carnobacteria, an important genus of lactic acid bacteria with both beneficial and detrimental effects in the food and aquaculture industries. This report provides a detailed characterization of phage cd2, a novel siphophage that targets Carnobacterium divergens, and sets the groundwork for understanding the biology of these phages and their potential use in the detection and biocontrol of C. divergens isolates.

Membrane lipid composition of Carnobacterium maltaromaticum CNCM I-3298, a highly cryoresistant lactic bacterium.

The growing consumption of fermented products has led to an increasing demand for lactic acid bacteria (LAB), especially for LAB tolerant to freezing/thawing conditions. Carnobacterium maltaromaticum is a psychrotrophic and freeze-thawing resistant lactic acid bacterium. The membrane is the primary site of damage during the cryo-preservation process and requires modulation to improve cryoresistance. However, knowledge about the membrane structure of this LAB genus is limited. We presented here the first study of the membrane lipid composition of C. maltaromaticum CNCM I-3298 including the polar heads and the fatty acid compositions of each lipid family (neutral lipids, glycolipids, phospholipids). The strain CNCM I-3298 is principally composed of glycolipids (32%) and phospholipids (55%). About 95% of glycolipids are dihexaosyldiglycerides while less than 5% are monohexaosyldiglycerides. The disaccharide chain of dihexaosyldiglycerides is composed of α-Gal(1-2)-α-Glc chain, evidenced for the first time in a LAB strain other than Lactobacillus strains. Phosphatidylglycerol is the main phospholipid (94%). All polar lipids are exceptionally rich in C18:1 (from 70% to 80%). Regarding the fatty acid composition, C. maltaromaticum CNCM I-3298 is an atypical bacterium within the genus Carnobacterium due to its high C18:1 proportion but resemble the other Carnobacterium strains as they mostly do not contain cyclic fatty acids.

Identification of novel Carnobacterium maltaromaticum strains in bone marrow samples of patients with acute myeloid leukemia using a metagenomic binning approach.

The aim of this study aimed to examine the existence of a bacterial metagenome in the bone marrow of patients with acute myeloid leukemia (AML). We re-examined whole-genome sequencing data from the bone marrow samples of seven patients with AML, four of whom were remitted after treatment, for metagenomic analysis. After the removal of human reads, unmapped reads were used to profile the species-level composition. We used the metagenomic binning approach to confirm whether the identified taxon was a complete genome of known or novel strains. We observed a unique and novel microbial signature in which Carnobacterium maltaromaticum was the most abundant species in five patients with AML or remission. The complete genome of C. maltaromaticum "BMAML_KR01," which was observed in all samples, was 100% complete with 8.5% contamination and closely clustered with C. maltaromaticum strains DSM20730 and SF668 based on single nucleotide polymorphism variations. We identified five unique proteins that could contribute to cancer progression and 104 virulent factor proteins in the BMAML_KR01 genome. To our knowledge, this is the first report of a new strain of C. maltaromaticum in patients with AML. The presence of C. maltaromaticum and its new strain in patients indicates an urgent need to validate the existence of this bacterium and evaluate its pathophysiological role.

Characteristics of key microorganisms and metabolites in irradiated marbled beef.

Insights into changes in microorganisms and metabolites in irradiated marbled beef may help elucidate the beneficial effects of irradiation on prolonging the shelf life of meat. In this study, 16S rRNA gene sequencing, ultra-high-performance liquid chromatography-tandem mass spectrometry, and Pearson's correlation analyses were conducted to detect key microorganisms, core metabolites, and potential correlation between the microbiome and metabolome in marbling beef. Microbiome analysis showed that irradiation effectively eradicated the spoilage bacterium Leuconostoc and reduced the proportions of Carnobacterium and Lactobacillus in marbled beef. Additionally, results of metabolomic analysis involving irradiated marbled beef revealed that metabolites with significant differences were mainly organic acids and their derivatives, lipids, and lipid-like molecules, including six core metabolites. Furthermore, a significant correlation between key bacteria and metabolites was observed. Carnobacterium, Lactobacillus, and Leuconostoc affected the accumulation of core metabolites in irradiated marbled beef by influencing amino acid and lipid metabolism. Characterization of the microbiota and metabolites, as well as clarification of their correlation, can contribute to a better understanding of the mechanisms whereby irradiation helps maintain meat quality.

Carnobacterium as a bioprotective and potential probiotic culture to improve food quality, food safety, and human health - a scoping review.

It is well-known that some bacteria can promote human and animal health. Bacteria of the genus Carnobacterium, while underexplored, have demonstrated significant probiotic and bioprotective potential. In this review, the recent scientific advances in this area are discussed. There are several requirements for a strain to be considered a probiotic or bioprotective agent, including the absence of antimicrobial resistance and the ability to colonize the gastrointestinal tract. Several researchers have reported such features in Carnobacterium bacteria, especially with regard to the production of antimicrobial substances. Research into animal production has advanced, especially in the aquaculture field, wherein inhibitory activity has been demonstrated against several important pathogens (for example Vibrio), and improvement in zootechnical indexes is evident. With respect to human health-related applications, research is still in the early stages. However, excellent in vitro results against pathogens, such as Candida albicans and Pseudomonas aeruginosa, have been reported. Carnobacterium bacteria have been assessed for a variety of applications in food, including direct application to the matrix and application to smart packaging, with proven effectiveness against Listeria monocytogenes. However, there is a lack of in vivo studies on Carnobacterium applications, which hinders its applications in various industries despite its high potential.

Carnobacterium Species Capable of Growth at Pressures Ranging Over 5 Orders of Magnitude, from the Surface of Mars (103 Pa) to Deep Oceans (107 Pa) in the Solar System.

Several permanently cold solar system bodies are being investigated with regard to their potential habitability, including Mars and icy moons. In such locations, microbial life would have to cope with low temperatures and both high and low pressures, ranging from ∼102 to 103 Pa on the surface of Mars to upward of ∼108-109 Pa in the subsurface oceans of icy moons. The bacterial genus Carnobacterium consists of species that were previously shown to be capable of growth in the absence of oxygen at low temperatures and at either low pressure or high pressure, but to date the entire pressure range of the genus has not been explored. In the present study, we subjected 14 Carnobacterium strains representing 11 species to cultivation in a complex liquid medium under anaerobic conditions at 2°C and at a range of pressures spanning 5 orders of magnitude, from 103 to 107 Pa. Eleven of the 14 strains showed measurable growth rates at all pressures tested, representing the first demonstration of terrestrial life forms capable of growth under such a wide range of pressures. These findings expand the physical boundaries of the capabilities of life to occur in extreme extraterrestrial environments.

First isolation of Carnobacterium maltaromaticum from farmed Rainbow Trout in Virginia.

OBJECTIVE: Carnobacterium maltaromaticum is considered an emerging pathogen of salmonids in the United States and around the world. METHODS: Bacterial cultures obtained from the posterior kidney and skin of moribund Rainbow Trout Oncorhynchus mykiss from a commercial aquaculture facility in Virginia, USA, grew C. maltaromaticum, which was confirmed by additional phenotypic and molecular characterization. RESULT: A presumptive diagnosis based on the clinical signs, necropsy observations, histopathology, and bacterial cultures was bacterial septicemia due to C. maltaromaticum. CONCLUSION: This represents the first documentation of C. maltaromaticum in Rainbow Trout from Virginia.

Characterization of novel bacteriocin produced by bacteriocinogenic Carnobacterium maltaromaticum isolated from raw milk.

This study was designed to assess newly isolated bacteriocin-producing strain as potential food preservative. A bacteriocin producing lactic acid bacterium, named Carnobacterium maltaromatium KCA018, was screened from raw milk using deferred and spot-on-the-lawn assays. The crude cell free supernatant (CFS) was purified to obtain proteinaceous bacteriocin by ammonium sulfate precipitation (assigned as bacteriocin KCA) and tested for bacteriocin production, physical stability, antimicrobial activity, and bacteriocin-encoding gene detection. The growth curves of C. maltaromatium KCA018 reached late exponential phase after 15 h of incubation at 25 °C and 30 °C (Fig. 2). The maximum production of bacteriocin KCA was reached after 12 h of incubation at 25 °C, showing the antimicrobial activity of more than 3000 AU/ml against Listeria monocytogenes. The purified bacteriocin KCA was stable up to 67 °C for 30 min of exposure and between pH 4 and 7, showing more than 6000 AU/ml. The antibacterial activity of bacteriocin KCA was lost in the presence of pronase, proteinase K, and trypsin. Purified bacteriocin KCA showed higher antibacterial activity against Gram-positive bacteria than against Gram-negative bacteria. The CFS and purified bacteriocin KCA effectively inhibited the growth of L. monocytogenes ATCC 1911, E. faecalis ATCC 19433, and E. feacium ATCC 11576. The molecular weight of purified bacteriocin KCA was estimated at approximately 5 kDa. The positive amplification was observed for pisA and cbnBM1 with approximately between 100 and 200 bp. The newly identified bacteriocin can be a promising preservative for application in food.

Effect of the combination of superchilling and super-chilled storage on shelf-life and bacterial community dynamics of beef during long-term storage.

This study investigated the effect of superchilling (-30 °C until the core temperature achieved -3 °C, and - 1 °C until 24 h, SC) on shelf-life and bacterial community dynamics of beef loins, with a typical very fast chilling (-30 °C until the core temperature achieved 0 °C, and - 1 °C until 24 h, VFC) and conventional chilling (0- 4 °C for 24 h, CC) as controls. The super-chilled storage (-1 °C) was adopted after each chilling procedure, and physicochemical traits and microbiological quality were evaluated during a long-term storage. No remarkable adverse impact on meat color and lipid oxidation were observed in SC treatment. The bacterial composition results showed that Carnobacterium spp. were the main bacteria in SC treatment in the late storage period (63- 84 days). The loss of Lactobacillus spp., due to the "ultra-low temperature" during the superchilling, might be the reason that the SC did not result in a longer shelf-life compared with CC samples.

Carnobacterium maltaromaticum associated with meningoencephalitis and otitis in stranded common thresher sharks (Alopias vulpinus).

Juvenile common thresher sharks (Alopias vulpinus) have been recently stranding along the California coastline. Using Illumina sequencing of the bacterial 16S rRNA gene along with necropsy, cytological, bacteriological, and histological techniques, we screened microbial communities and described lesions characterizing affected sharks with the purpose of identifying potential pathogen sources and pathologic processes. Histopathological assessment of moribund sharks revealed severe meningoencephalitis, as previously described in stranded salmon sharks (Lamna ditropis), along with inflammation of the inner ear and subcutaneous tissues surrounding the endolymphatic ducts. Furthermore, inflamed areas were characterized by the prevalence of Carnobacterium maltaromaticum, suggesting this bacterium as a potential pathogen that gains access to the inner ear through the endolymphatic ducts, with subsequent spread into the brain. The absence or low abundance of this bacterium in the spiral valve in both healthy and infected sharks suggests that Carnobacterium is not a commensal member of their digestive communities and the spiral valve is unlikely to be the source of the pathogen. Furthermore, phylogenetic analysis suggests that C. maltaromaticum strains isolated from diseased sharks have minimal genetic variation and differ from other strains originating from food or diseased teleosts. While a C. maltaromaticum-like organism has previously been associated with meningoencephalitis in salmon shark strandings, this is the first study to report common thresher shark strandings associated with C. maltaromaticum, involving the endolymphatic ducts as portals of entry to the brain.

Characterization of bacterial communities in Coregonus peled fillets during chilled storage and interactions between selected bacterial strains.

AIM: Coregonus peled fillets were used as a model to evaluate the dominant bacterial growth of chilled fish during storage after shipping and interactions of selected bacterial strains. METHODS AND RESULTS: Coregonus peled fillets were transported by air and land in ice boxes about 48 h from aquatic products company in Xinjiang, China, to the laboratory located in Dalian, China. Both culture-dependent (plate counts on nonselective media) based on 16S rRNA gene sequencing and culture-independent (Illumina-MiSeq high-throughput sequencing) methods were used. To detect interactions among bacterial populations from chilled fish, the influence of 18 test strains on the growth of 12 indicator isolates was measured by a drop assay and in liquid culture medium broth. The results showed that bacterial counts exceeded 7.0 log CFU/g following storage for 4 days at 4 °C. When the bacterial counts exceeded 8.5 log CFU/g after 12 days, the predominant micro-organisms were Aeromonas, Pseudomonas, Carnobacterium, Psychrobacter and Shewanella, as measured by the culture-independent method. All test strains showed inhibiting effects on the growth of other strains in liquid culture. Pseudomonas isolates showed antibacterial activity for approximately 60% of the indicator strains on nutritional agar plates. The majority of test isolates enhancing indicator strain growth were the strains isolated on day 0. CONCLUSIONS: High-throughput sequencing approach gives whole picture of bacterial communities in chilled C. peled fillets during storage, while growth interferences between selected bacterial strains illustrate the complexity of microbial interactions. SIGNIFICANCE AND IMPACT OF THE STUDY: We determined the bacterial communities and growth interferences in chilled Coregonus peled after shipping and these are the first data concerning microbiota in C. peled using a culture-independent analysis. The present study will be useful for manufacture and preservation of C. peled products by providing with valuable information regarding microbiological spoilage of C. peled.

Development of duplex qPCR targeting Carnobacterium maltaromaticum and Vagococcus salmoninarum.

In November 2018, Vagococcus salmoninarum was identified as the causative agent of a chronic coldwater streptococcosis epizootic in broodstock brook trout (Salvelinus fontinalis) at the Iron River National Fish Hatchery in Wisconsin, USA. By February 2019, the epizootic spread to adjacent raceways containing broodstock lake trout (Salvelinus namaycush), whereby fish were found to be coinfected with Carnobacterium maltaromaticum and V. salmoninarum. To differentiate these two pathogens and determine the primary cause of the lake trout morbidity, a quantitative real-time PCR (qPCR) was developed targeting the C. maltaromaticum phenylalanyl-tRNA synthase alpha subunit (pheS) gene. The qPCR was combined with a V. salmoninarum qPCR, creating a duplex qPCR assay that simultaneously quantitates C. maltaromaticum and V. salmoninarum concentrations in individual lake trout tissues, and screens presumptive isolates from hatchery inspections and wild fish from national fish hatchery source waters throughout the Great Lakes basin. Vagococcus salmoninarum and C. maltaromaticum were co-detected in broodstock brook trout from two tribal hatcheries and C. maltaromaticum was present in wild fish in source waters of several national fish hatcheries. This study provides a powerful new tool to differentiate and diagnose two emerging Gram-positive bacterial pathogens.

Resistance of biofilm- and pellicle-embedded strains of Escherichia coli encoding the transmissible locus of stress tolerance (tLST) to oxidative sanitation chemicals.

Biofilm formation in food processing plants reduces the efficacy of sanitation. The presence of transmissible locus of stress tolerance (tLST) also enhances resistance of planktonic cells of Escherichia coli to sanitation chemicals but the role of tLST in resistance of biofilm-embedded cells remains unclear. This study investigated the link of tLST to biofilm formation and its contribution to resistance of biofilm-embedded E. coli to sanitation. Biofilms were formed as single-strain and as dual-strain biofilms in association with E. coli, Aeromonas australensis or Carnobacterium maltaromaticum. Biofilms on stainless steel were compared to floating biofilms formed at the air-liquid interface (pellicles). The resistance of biofilm-embedded tLST positive strains of E. coli to chlorine, hydrogen peroxide, and peroxyacetic acid was higher than the resistance of tLST negative strains. Higher biofilm density as measured by crystal violet staining was observed in tLST-positive strains of E. coli when compared to tLST negative strains. Biofilm density positively correlated to resistance to disinfectants. The use of confocal laser scanning microscopy detected more compact structure of pellicles compared to solid surface-attached biofilms, resulting in higher chlorine resistance despite the absence of tLST in strains of E. coli. Collectively, the findings of this study elucidated the impact of tLST in strains of E. coli on biofilm formation and sanitizer resistance. These findings may inform the development of improved sanitization protocols for food facilities.

Carnobacterium inhibens isolated in blood culture of an immunocompromised, metastatic cancer patient: a case report and literature review.

BACKGROUND: Carnobacterium species are lactic acid-producing Gram-positive bacteria that have been approved by the US Food and Drug Administration and Health Canada for use as a food bio-preservative. The use of live bacteria as a food additive and its potential risk of infections in immunocompromised patients are not well understood. CASE PRESENTATION: An 81-year-old male with a history of metastatic prostate cancer on androgen deprivation therapy and chronic steroids presented to our hospital with a 2-week history of productive cough, dyspnea, altered mentation, and fever. Extensive computed tomography imaging revealed multifocal pneumonia without other foci of infection. He was diagnosed with pneumonia and empirically treated with ceftriaxone and vancomycin. Blood cultures from admission later returned positive for Carnobacterium inhibens. He achieved clinical recovery with step-down to oral amoxicillin/clavulanic acid for a total 7-day course of antibiotics. CONCLUSIONS: This is the fourth reported case of bacteremia with Carnobacterium spp. isolated from humans. This case highlights the need to better understand the pathogenicity and disease spectrum of bacteria used in the food industry for bio-preservation, especially in immunocompromised patients.

Investigation of the microbiota associated with ungerminated and germinated Norwegian barley cultivars with focus on lactic acid bacteria.

The microbial community of ungerminated and germinated barley grains from three different cultivars grown at four different locations in Norway was investigated by culture dependent and culture independent methods. Lactic acid bacteria (LAB) was focused in this study and was isolated from germinated barley. The number of LAB ranged between 2.8 and 4.6 log cfu/g in ungerminated grains and between 4.9 and 6.3 log cfu/g in germinated grains. In total 66 out of 190 isolates were Gram+, catalase-negative and presumptive LAB. The LAB isolates were by 16S rRNA sequencing identified to be Carnobacterium maltaromaticum (6), Lactococcus lactis (2), Enterococcus sp. (1) and Leuconostoc sp. (57). Germination significantly influenced the bacterial composition. Regarding the different cultivars and growth places no significant difference in bacterial composition was seen. The most abundant bacterial genus was Pantoea (18.5% of the total sequences), followed by Rhizobium (10.1%) and Sphingomonas (9.9%). Fungal composition was significantly influenced by the germination process and the cultivation place, but no significant difference in fungal composition was detected between the 3 cultivars. The most abundant fungal genera were Cryptococcus (43.8% of all the sequences), Cladosporium (8.2%), Pyrenophora (7.4%) and Vagicola (6.3%). This study revealed knowledge of barley grain associated microbes of Norwegian barley that can be useful to control the malt quality. Germination affected both bacterial and fungal microbiota composition. No difference in bacterial microbiota composition was seen regarding cultivars and cultivation place, however, the fungal microbiota composition was significantly influenced by the cultivation place. Differences in fungal community of ungerminated and germinated barley samples of different geographical locations were more pronounced than differences in bacterial communities.

Gut microbiota mediate Plutella xylostella susceptibility to Bt Cry1Ac protoxin is associated with host immune response.

Insect gut microbiotas have a variety of physiological functions for host growth, development, and immunity. Bacillus thuringiensis (Bt) is known to kill insect pests by releasing insecticidal protoxins, which are activated in the insect midgut. However, the interplay among Bt infection, host immunity, and gut microbiota are still unclear. Here we show that Bt Cry1Ac protoxin interacts with the gut microbiota to accelerate the mortality of P. xylostella larvae. Cry1Ac protoxin was found to cause a dynamic change in the midgut and hemocoel microbiota of P. xylostella, with a significant increase in bacterial load and a significant reduction in bacterial diversity. In turn, loss of gut microbiota significantly decreased the Bt susceptibility of P. xylostella larvae. The introduction of three gut bacterial isolates Enterococcus mundtii (PxG1), Carnobacterium maltaromaticum (PxCG2), and Acinetobacter guillouiae (PxCG3) restored sensitivity to Bt Cry1Ac protoxin. We also found that Cry1Ac protoxin and native gut microbiota can trigger host midgut immune response, which involves the up-regulation of expression of Toll and IMD pathway genes and most antimicrobial peptide genes, respectively. Our findings further shed light on the interplay between insect gut microbiota and host immunity under the Bt toxin killing pressure, and this may provide insights for improving the management of Bt resistance and lead to new strategies for biological control of insect pests.