Escherichia coli is currently unable to be reliably differentiated from Shigella species by routine matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. In the present study, a reliable and rapid identification method was established for Escherichia coli and Shigella species based on a short-term high-lactose culture using MALDI-TOF MS and artificial neural networks (ANN).Escherichia coli and Shigella species colonies, treated with (Condition 1)/without (Condition 2) a short-term culture with an in-house developed high-lactose fluid medium, were prepared for MALDI-TOF MS assays. The MS spectra were acquired in linear positive mode, with a mass range from 2000 to 12000 Da and were then compared to discover new biomarkers for identification. Finally, MS spectra data sets 1 and 2, extracted from the two conditions, were used for ANN training to investigate the benefit on bacterial classification produced by the new biomarkers.The Escherichia coli and Shigella species were summarized. Seven unreported MS peaks, with m/z 2330.745, m/z 2341.299, m/z 2371.581, m/z 2401.038, m/z 3794.851, m/z 3824.839 and m/z 3852.548, were discovered in only the spectra from the E. coli strains after a short-term high-lactose culture and were identified as belonging to acid shock protein. The prediction accuracies of the ANN models, based on data set 1 and 2, were 97.71±0.16% and 74.39±0.34% (n = 5), with an extremely remarkable difference (p < 0.001), and the areas under the curve of the receiver operating characteristic curve were 0.72 and 0.99, respectively.Twenty-seven characteristic MS peaks from the Escherichia coli from the Shigella species using MALDI-TOF MS and ANN.In summary, adding a short-term high-lactose culture approach before the analysis enabled a reliable and easy differentiation of  Escherichia coli (E. coli), the most common bacteria in clinical practice, is currently unable to be reliably differentiated from Shigella by routine MALDI-TOF MS analysis [E. coli and Shigella species are closely related and they both belong to the family Enterobacteriaceae, their MS spectra are very similar to each other. The MALDI-TOF MS identification results can be only reported as E. coli/Shigella species, which challenges the entity separation and rapid identification in epidemiology and clinical diseases[Matrix assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a fast and cost-effective method for bacterial identification, and it is used routinely in many laboratories and clinical testing organizations–3. Althoanalysis . Because diseases.E. coli and Shigella species are classified as separate species based on their biochemical characteristics and clinical relevance[E. coli and Shigella species to obtain accurate results. These approaches are classical and reliable, but may have a suboptimal diagnostic performance.relevance,7. AssesE. coli and Shigella species isolated from the clinical sample is urgently required for clinical diagnostics and public health. In this study, we present a novel short-term culture approach that is combined a MALDI-TOF MS analysis method to realize the accurate and reliable identification of E. coli and Shigella species.The accurate identification of Escherichia and Shigella species  at 35°C for 24 h to obtain fresh colonies. The strain colonies were inoculated into our in-house developed high-lactose fluid medium and were incubated at 35°C for 2 h . The colTM software   and an Autoflex maX MALDI-TOF/TOF system . The MS spectra were acquired in linear positive-ionization mode. Afterwards, the target MS peak was selected for the MS/MS analysis in a reflector mode. The MS/MS spectra were interpreted primarily with the FlexAnalysisany) See .The protein database searches were performed with MASCOT version 2.5  and BioTools 3.2 software  against the Swiss-Prot database for protein identification See .TM software R2015b  , a frequently used artificial neural network, was employed to recognize the classification of the target bacteria, and this was performed in MatlabUSA) See .E. coli strains obtained from the tryptic soy agar culture condition contained 27 fully conserved MS peaks, which were also shared with most of the E. coli strains. Analogously, the 27 MS peaks described above were also observed in the spectra of most of the Shigella strains. There was no significant differential peak between the spectra from the E. coli strains and the Shigella strains, leading an inability to identify them using MALDI-TOF MS  (Data sets < 0.001) . These rE. coli strains are part of the normal gut flora, whereas the Shigella species are considered to be pathogenic bacteria. The Shigella species has been separated from E. coli strains, as a requirement for clinical diagnostics, using biochemical methods and serological techniques, since the first Shigella species were discovered in 1898[E. coli and Shigella species are be considered to be part of the same phylogenetic continuum rather than clearly distinct species. The nuances, at the molecular level, between the E. coli and Shigella species lead to an indistinguishableness by routine sequencing of the 16S rRNA gene and MALDI-TOF MS-based identification[Most E. coli and Shigella species have a high degree of similarity. In our investigation, the E. coli and Shigella species shared 27 main characteristic MS peaks and had no significantly differentiated MS peaks. A routine MALDI-TOF MS analysis for the identification of E. coli and Shigella species is difficult. A recent study showed that a specialized automated system, based on an analysis of selective biomarker MS peaks in the spectra, using Biotyper software followed by an analysis with FlexAnalysis and ClinProTools software for the identification of E. coli and Shigella species, was better than traditional techniques, including an automated microbiology identification system and serotyping[E. coli and Shigella species and to rapidly distinguish the Shigella species from E. coli[E. coli and Shigella species is still difficult. To provide a reliable and efficient identification method for the E. coli and Shigella species for clinical diagnostic purposes, the biochemical method and the MALDI-TOF MS technique are combined. A novel high-lactose fluid medium is prepared on the optimized prescription. We added a short-term culture approach, using an in-house developed high-lactose fluid medium, before the routine MALDI-TOF MS assay, which enabled a reliable distinction between the E. coli and Shigella species. Seven MS peaks were newly observed in the spectra of the E. coli species only, which can be used as new biomarkers to distinguish E. coli and Shigella using MALDI-TOF MS.The MALDI-TOF MS technique is a highly cost-effective and time-efficient way to identify bacteria–11. Due erotyping. Paauw em E. coli. Howeverm E. coli. Even ifE. coli and Shigella strains. The proposed biomarkers are identified as fragments of acid shock protein, which makes the distinct mechanism clarified. Acid shock protein is encoded by the asr gene in E. coli, which is strongly induced by a high acid environment (pH < 5.0)[E. coli was under an acid shock condition. To our knowledge, E. coli strains can ferment lactose, by which organic acids are produced [E. coli species are stressed to produce many acid shock proteins to protect themselves against the low pH survival condition made by the lactose fermentation [E.coli group was 5.2±0.5, which support our hypothesis. Whereas the S. flexneri, S. sonnei, S. bogdii E. hermannii, Shimvellia blattae, E. fergusonii and E. albertii strains do not ferment lactose, while some S. dysenteriae strains ferment lactose slowly (delayed lactose fermentation), which leads to no significant change of pH, as well as the expression of acid shock protein when they survive in a high-lactose condition for a short time. Therefore, our method can be widely applied to differentiate all lactose-fermenting E. coli from Shigella species and other non-lactose-fermenting Escherichia species.The limited strains involved in our study and the unknown biomarkers may cause methodological limitations of its application. The distinct mechanism needs to be understood to determine whether the present approach successfully applies to any of the pH < 5.0). Acid shproduced . These oentation . After 21 was higher than that of data set 2, and BPNN model I performed better than BPNN model II. The E. coli and Shigella species were correctly classified, with 97.71±0.16% accuracy, using the BPNN model I. This encourages us to recommend this method for the identification of E. coli and Shigella species in the clinical laboratory. The short-term culture, combined with the MALDI-TOF MS assay, described here provides a novel bacteria identification strategy for allied bacteria groups, which are unable to be identified using the routine MALDI-TOF MS approach.The classification algorithm plays a critical role in the MALDI-TOF MS-based bacterial identification method–19. BPNNE. coli and Shigella using a MALDI-TOF MS. The new biomarkers increase the data separation, which significantly improves the classification efficiency and accuracy of artificial neural networks.In the present study, we provide a novel short-term culture approach, with optimized high-lactose fluid medium components, before the MS analysis in order to induce the expression of new biomarkers to distinguish S1 FigE. coli, Shigella and other Escherichia strains acquired from the linear positive mode, with a mass range from 2000 to 12000 Da. All the experimental strains were cultured on tryptic soy agar for 24 h followed by sample preparation and MALDI-TOF MS analysis.The MALDI-TOF MS spectra of the (TIF)Click here for additional data file.S2 FigE. coli, Shigella and other Escherichia strains acquired from the linear positive mode, with mass range from 2000 to 12000 Da. All the experimental strains were cultured on tryptic soy agar for 24 h, with an additional 2 h culture in the in-house developed high-lactose fluid medium followed by sample preparation and MALDI-TOF MS analysis. Newly discovered MS peaks, as identification biomarkers, are marked with the respective mass-to-charge ratio.The MALDI-TOF MS spectra of the (TIF)Click here for additional data file.S1 FileThe detailed methods of experiments in research work. The bacterial strains for experiments, culture conditions, sample preparation method, MALDI-TOF MS data acquisition method, protein identification method and artificial neural networks for bacterial identification were detailed.(DOCX)Click here for additional data file.


Recent evidence suggests that the commensal microbes act as a barrier against invading pathogens and enteric infections are the consequences of multi-layered interactions among commensals, pathogens, and the host intestinal tissue. However, it remains unclear how perturbations of the gut microbiota compromise host infection resistance, especially through changes at species and metabolite levels.Bacteroides vulgatus, a dominant species of the Bacteroidetes phylum in mouse intestine, suppresses infection by Vibrio cholerae, an important human pathogen. Clindamycin (CL) is an antibiotic that selectively kills anaerobic bacteria, and accordingly Bacteroidetes are completely eradicated from CL-treated mouse intestines. The Bacteroidetes-depleted adult mice developed severe cholera-like symptoms, when infected with V. cholerae. Germ-free mice mono-associated with B. vulgatus became resistant to V. cholerae infection. Levels of V. cholerae growth-inhibitory metabolites including short-chain fatty acids plummeted upon CL treatment, while levels of compounds that enhance V. cholerae proliferation were elevated. Furthermore, the intestinal colonization process of V. cholerae was well-simulated in CL-treated adult mice.Here, we illustrate how B. vulgatus as an indigenous microbial species that can suppress intestinal infection. Our results also demonstrate that commensal-derived metabolites are a critical determinant for host resistance against V. cholerae infection, and that CL pretreatment of adult mice generates a simple yet useful model of cholera infection.Overall, we provide insights into how a symbiotic microbe and a pathogenic intruder interact inside host intestine. We identified  Vibrio cholerae is the causative agent of pandemic diarrheal disease, cholera. While cholera toxin (CT) and toxin-coregulated pilus (TCP) are known to be the major virulence determinants, its pathogenic mechanisms are starting to be understood as consequences of interaction with indigenous microbes, collectively termed gut microbiota [crobiota –4. A keycrobiota –7. This crobiota .Bacteroides species that reside in the human gut, such as Bacteroides fragilis and Bacteroides uniformis, utilise membrane attack complex/perforin toxins BSAP-1 and BSAP-2 for intraspecies antagonism [Microbiota-intrinsic factors such as interactions between the gut-residing species serve as the primary determinant of community composition. Such interactions include interbacterial niche competition  and secrtagonism . Microbitagonism .Shigella flexneri [Salmonella enterica [Clostridium difficile [Enterococcus [V. cholerae infection.Recent studies have demonstrated that antibiotic treatments alter the gut microbiota in humans and other mammals –15, and flexneri , Salmoneenterica , Clostriifficile , 19, andrococcus . The aborococcus –23. ThesIn this study, we uncover a unique microbiota-extrinsic treatment that induces severe cholera-like symptoms in adult mice, that are otherwise completely resistant. Furthermore, we show that a dramatic shift in metabolome production profile accounts for the compromised infection resistance in the host. This report highlights the importance of commensal-derived metabolites as a crucial determinant of host susceptibility to enteric infection.Bacteroides fragilis, a major species of the Bacteroidetes phylum [Colonization of enteric pathogens occurs depending on the composition of indigenous microbes inside the host intestine , 24, 25.s phylum , 30. On 8 CFU of the V. cholerae 7th pandemic strain, N16961, to monitor susceptibility to infection and infection-induced microbiota composition changes. In the control and SM-treated groups, microbiota composition did not change much following V. cholerae infection  or cecum was remarkably larger in CL-treated vs. control group. V. cholerae cells of > 109 CFU were grown per gram sample in SI and cecum, respectively. Likewise, ~ 109 CFU was also detected in fecal pellets shed from CL-treated mice  V. cholerae cells were almost completely eliminated inside the intestine of regular adult mice while (ii) the same number of inoculated cells was more highly proliferated in the CL-pretreated adult mouse intestine.Our results, shown in Fig. Staphylococcus aureus [Bacteroides spp. [V. cholerae infection, these previous reports also indicate that enhanced V. cholerae infectivity in adult mice might be attributed to CL-induced modulation of the host immune response. To address this issue, we conducted fecal microbiota transplantation (FMT) to germ-free (GF) mice and tested whether the GF mice with reconstituted intestinal microbiota develop differential resistance to V. cholerae infection. Fecal pellets freshly collected from PBS- or CL-treated mice were resuspended in PBS and transferred to GF mice via oral gavage. Following a 3-day transplant stabilization period, 5 × 108 N16961 cells were infected . Three distinct bacterial species were determined to belong to the Bacteroidetes phylum; ion Fig. . In respdsteinii  and a stdsteinii . As showion Fig. .Fig. 3SB. vulgatus was the most dominant species in the adult mouse intestine, we next assessed, under a more defined condition, the effect of B. vulgatus on altering host susceptibility to V. cholerae infection. To this end, we mono-associated GF mice with a suspension of B. vulgatus cells, grown anaerobically in vitro  CL-induced changes on metabolite level might have created an environment that strongly facilitates V. cholerae growth and pathogenesis.We next conducted a metabolome analysis to examine whether metabolomic profiles are altered in response to CL treatment and/or map Fig. . A tablemap Fig. . Such stV. cholerae growth is controlled by each of these major compounds. Of the 27 listed compounds, 17 were selected based on the availability and we monitored V. cholerae in vitro growth in M9 media supplemented with each metabolite. Growth curve experiments shown in Fig. V. cholerae growth. M9 media used in this particular set of growth experiments contain 0.4% glucose as primary carbon source, thereby enabling us to monitor whether the extraneously added metabolite promotes or inhibits V. cholerae growth. Seven metabolites shown in panels K~Q clearly inhibited V. cholerae growth. Importantly, these compounds were present in the largest quantities in regular SPF mouse intestines, but either absent or barely detectable in CL-treated mouse intestines  still prevented growth of V. cholerae  were ~ 0.29 and ~ 0.28 in the small intestine and cecum of CL-treated mice, respectively. In contrast, meaningful differences in colonization capabilities between two strains were not detected, when untreated adult mice were used , N-acetylglucosamine , and N-acetylneuraminic acid . Monosaccharides are produced as the result of mucin glycans being catabolized by the gut microbial species equipped with glycosidase genes. Bacteroides has evolved to express glycosidase genes which allow them to perform mucin degradation to full completion, and thus become well-adapted to thrive in the intestine [Clostridia produce pyruvate through glycolysis [Host-derived mucus glycan is a nutrient source utilized exclusively by the gut microbiota. Considering the microbial competition for nutritional resources in the intestinal tract and constant release of glycan into the lumen by epithelial cell turnover –62, the ntestine –63. The ycolysis , 64. Undycolysis .In this study, the dramatic changes in the cecal metabolomes of CL-treated mice strongly correlate with the compositional shift of the microbiota. Depletion of SCFAs and relatively high occurrence of amino sugars in the ceca of CL-treated mice indicate that catabolism of mucin glycan, a proliferation strategy for microbes belonging to the Bacteroidetes phylum, became inactive upon CL treatment.V. cholerae into the host intestine. When the pathogen enters the intestinal tract, the next strategy for its proliferation is to colonize the epithelial cells covered with a viscous layer of mucus. V. cholerae effectively responds to the mucus glycan and catabolize the polysaccharides to acquire competitive advantage in host intestine [V. cholerae motility [V. cholerae [VC1777-1779) attenuates colonization [V. cholerae needs to overcome a number of challenges such as acid stress, antimicrobial peptides, host immune response, and SCFAs [Utilization of the host glycan is also important for the invasion of ntestine –68. Glucnization . During nd SCFAs , 66, 68.nd SCFAs . In genend SCFAs .N-acetyl amino sugars and subsequent accumulation of SCFAs in the distal intestine are largely managed by specific gut microbiota  available for utilization by V. cholerae  genera.Our results suggest that catabolism of ota Fig. . Eliminarae Fig. . Of partntestine . In the B. vulgatus, that plays a significant role in inhibiting V. cholerae colonization in the adult mouse intestine. Moreover, we propose a simple antibiotic-treated mouse model to delineate V. cholerae infection, which might prove a useful tool for cholera vaccine research. We hope that the results presented in the present study will stimulate future investigations to better understand the in vivo probiotic function of B. vulgatus against V. cholerae infection and to devise better strategies for tackling this virulent and clinically important human pathogen.In conclusion, we identified a commensal bacterial species, Vibrio cholerae O1 serotype N16961 was used as a model pathogen in all experiments [V. cholerae from intestinal contents was thiosulfate-citrate-bile salt-sucrose (TCBS) medium. For enumeration of intestinal V. cholerae loads, mouse intestines were manually extracted from infected animals. The SIs, ceca, and colons were then homogenized in PBS (Sigma-Aldrich) and centrifuged at 1000 rpm for 1 min. The supernatants were serially diluted in PBS, and spotted on an LB agar plate containing streptomycin. B. vulgatus strains were isolated from C57BL/6 mouse intestines using Bacteroides Bile Esculin  agar plates. Subsequently, B. vulgatus strains were cultivated on Gifu Anaerobic Media (GAM) broth  in an anaerobic chamber maintained at 90% N2, 5% CO2, and 5% H2. All anaerobic culture media were deoxygenated for at least 24 h prior to usage. RAPD analysis was performed using six different B. vulgatus colonies recovered independently from two different CL-treated mice, following procedures described elsewhere [eriments . N16961 Experiments were performed using both SPF and GF mice in a C57BL/6 genetic background. SPF mice were obtained from Orient Bio , and GF mice were generated by the Yonsei University College of Medicine GF mouse facility. Mice were provided sterile water and autoclaved food. GF status was confirmed by negative microbial growth in mouse feces. GF mice were bred and housed in flexible-film isolators . When necessary, GF mice were transferred to isocages  for experimental grouping. All mouse experiments were conducted according to the guidelines provided by the Department of Animal Resources of Yonsei Biomedical Research Institute. The Committee on the Ethics of Animal Experiments at Yonsei University College of Medicine approved this study .For the antibiotic treatment presented in Fig. V. cholerae cells grown aerobically overnight at 37 °C were diluted 100-fold to inoculate fresh LB broth medium. The sub-cultured broth was incubated in a shaking incubator at 37 °C for 4 h. Finally, V. cholerae cells were centrifuged at 13,000 rpm for 10 min, and the resulting pellet was suspended in sterile PBS to the concentration of 1010 CFU per mL. For infection, 8- to 9-week-old mice were orally infected with 50 μl of V. cholerae cell suspension containing approximately 5 × 108 CFU of V. cholerae cells. The fluid accumulation ratio was calculated by using the equation (intestine weight) / . CT level was quantified by CT ELISA [V. cholerae cells in mouse feces, fecal pellets were physically disrupted to homogeneity, and fecal suspensions were serially diluted for CFU counting. Shortly after euthanasia, SI, cecum, and colon samples were manually extracted from each mouse and homogenized in 5 mL of PBS. The sample homogenates were centrifuged at low speed for remnant tissue removal, and the resulting supernatant was used for CFU counting.CT ELISA . To enumB. vulgatus mono-association, B. vulgatus was first seeded with thawed stock and passaged at least twice and less than 4 times for maximum viability. B. vulgatus cells were grown in GAM broth under anaerobic conditions. The cultured B. vulgatus cells were resuspended in PBS, and 108 CFU cells were delivered orogastrically to GF mice. Infant mouse infection was performed following procedures described previously [FMT was performed to transplant the antibiotic-treated gut microbiota into GF mice. SPF mice were treated either with CL or PBS as per the procedure described above, and fresh feces were collected 24 h post-antibiotic treatment. In each antibiotic-treated and control group, there were three mice, and two fecal pellets were collected from each mouse. The fecal pellets collected were pooled per group and suspended in 600 μl of PBS. The suspension was quick-centrifuged to remove the debris, and the resulting supernatants were orally administered. Each GF mouse received 50 μl of FMT solution by oral gavage. For eviously .lacZ-negative wild-type strain N16961 and lacZ-positive ΔtcpA were cultured as per the aforementioned V. cholerae culture protocol. Sub-cultured wild-type and ΔtcpA cells were centrifuged at 13,000 rpm for 2 min and then resuspended in PBS. Each strain was mixed at a 1:1 ratio, and 100 μl of the prepared mixture  was administered to 8- to 9-week-old female C57BL/6 mice (either untreated or CL-treated) by oral gavage. Each mouse was infected with 5 × 108 CFU of each strain.To proceed with the competitive index assay, The extraction method for bacterial DNA was performed using a PowerMax Soil DNA Isolation Kit (MO BIO). Each sequenced sample was prepared according to the Illumina 16S Metagenomic Sequencing Library protocols to amplify the V3 and V4 regions (519F-806R). The DNA quality was measured by PicoGreen and Nanodrop. Input gDNA (10 ng) was PCR amplified. The barcoded fusion primer sequences used for amplifications were as follows: 341F: 5′-CCTACGGGNGGCWGCAG, and 806R: 5′-GACTACHVGGGTATCTAATCC. The final purified product was then quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantification kits for Illumina Sequencing platforms) and qualified using a LabChip GX HT DNA High Sensitivity Kit . Paired-end (2 × 300 bp) sequencing was performed by Macrogen using the MiSeq™ platform . For species identification, sequenced reads were aligned using blast and NCBI 16S microbial database (version downloaded 2018.01.14). Taxonomy information was assigned only to OTUs that met both query coverage > 85% and identity percent > 85%. OTUs that did not meet these two criteria were left unassigned. In general, microbial species was identified with ~ 99% identity with a reference sequence.g, 4 °C, 60 min) for subsequent analysis with capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS).For extracting ionic metabolites, approximately 50 mg of cecal contents was dissolved in MilliQ water containing internal standards , Tsuruoka, Yamagata, Japan) at a ratio of 1:9 (w/v). After centrifugation, the supernatant was centrifugally filtered through a Millipore 5000-Da cutoff filter  to remove macromolecules  with commercial electrophoresis buffer  as the electrolyte. The spectrometer was scanned from m/z 50 to 1000 [m/z, peak area, and migration time (MT) for CE-TOFMS analysis [m/z values with the MTs determined by TOFMS. Areas of the annotated peaks were then normalized based on internal standard levels and sample amounts to obtain relative levels of each metabolite. Hierarchical cluster analysis (HCA) was performed using the proprietary software supplied by HMT, PeakStat.Metabolome analysis was conducted at HMT using the Basic Scan package with CE-TOFMS for ionic metabolites, based on the methods described previously , 75. Bri to 1000 . Peaks wanalysis . Signal N-acetylglucosamine (GluNAc), N-acetylneuraminic acid (NANA), glucaric acid, gluconolactone, mannosamine, and cholic acid. These metabolites were added at concentrations of 4, 1, 4, 10, 1, and 0.2 mM, respectively. N16961 growth was monitored by measuring OD600 values spectrophotometrically. To examine growth-inhibiting capabilities of the metabolites, N16961 was grown in M9 plus 0.4% glucose. However, M9 salt without glucose was used to assess the capabilities of the metabolites to promote the growth of V. cholerae.N16961 was grown in M9 media supplemented with 17 metabolites at 20 mM each, with the exception of t test was used to determine whether differences between groups were significant. A p value < 0.05 was considered to indicate statistical significance. All experiments were repeated for reproducibility.Data are expressed as mean ± standard error of the mean (SEM). Unpaired Student’s Additional file 1: Figure S1. Effects of a single-dose treatment of CL on host resistance to V. cholerae infection.Additional file 2: Figure S2. Random Amplified Polymorphic DNA (RAPD) analysis of B. vulgatus clones.Additional file 3: Figure S3. A V. cholerae ΔtcpA mutant is defective in colonizing the intestine of CL-treated adult mouse.


Correction to:British Journal of Cancer (2019) 120, 612–620; 10.1038/s41416-019-0389-6; published online 22 February 2019.The additional information of this manuscript originally stated that the authors declare no competing interests. This statement was incorrect, and should instead have stated the following:M.C.H. has the following competing interests to declare: Equity interest at Molecular MD; Consulting at Molecular MD, Blueprint Medicines, Deciphera Pharmaceuticals; Expert Testimony at Novartis; Licensed patent with royalty payments at Novartis. The remaining authors have no competing interests to declare.The authors apologise for any convenience this may have caused.


Linum are characterized by wide variability in size, morphology and number of chromosomes in karyotypes. Since such variability is determined mainly by the amount and composition of repeated sequences, we conducted a comparative study of the repeatomes of species from four sections forming a clade of blue-flowered flax. Based on the results of high-throughput genome sequencing performed in this study as well as available WGS data, bioinformatic analyses of repeated sequences from 12 flax samples were carried out using a graph-based clustering method.Members of different sections of the genus Linum (including the cultivated species L. usitatissimum) differed significantly from other studied species by a great number of satellite DNA families as well as their relative content in genomes.It was found that the genomes of closely related species, which have a similar karyotype structure, are also similar in the repeatome composition. In contrast, the repeatomes of karyologically distinct species differed significantly, and no similar tandem-organized repeats have been identified in their genomes. At the same time, many common mobile element families have been identified in genomes of all species, among them, Athila Ty3/gypsy LTR retrotransposon was the most abundant. The 30-chromosome members of the sect. The evolution of studied flax species was accompanied by waves of amplification of satellite DNAs and LTR retrotransposons. The observed inverse correlation between the total contents of dispersed repeats and satellite DNAs allowed to suggest a relationship between both classes of repeating sequences. Significant interspecific differences in satellite DNA sets indicated a high rate of evolution of this genomic fraction. The phylogenetic relationships between the investigated flax species, obtained by comparison of the repeatomes, agreed with the results of previous molecular phylogenetic studies.The online version of this article (10.1186/s12862-019-1375-6) contains supplementary material, which is available to authorized users. Repeated sequences are important components of eukaryotic genomes, and often constitute a significant part of plant genomes . They caLinum L. (Linacea) includes about 200 wild species. It’s assumed that the genus originated in Eurasia, and later it spreaded to Africa, Australia, North and South America. Recent molecular phylogenetic studies have shown that the genus Linum L. is not monophyletic but includes representatives of two sister clades: yellow-flowered and blue-flowered flax [Cathartolinum (Reichen b.) Griseb., Syllinum Griseb., Linopsis (Reichenb.) Engelmann and the genera Cliococca Bab., Hesperolinon (A. Gray) Small, Radiola Hill. and Sclerolinon C. M. Rogers. Blue-flowered flax is also subdivided into groups according to the botanical sections Stellerolinum Juz. ex Prob., Dasylinum (Planchon) Juz., Adenolinum (Reichenb.) Juz. (syn. Linum perenne L. group) and Linum L. The sect. Linum includes the essential cultural species L. usitatissimum. Many of the wild flax species are important medicinal or ornamental plants. Also, they are considered as potential donors of economically valuable traits to improve the cultivated species. In the last decade, studies of the genome of cultivated flax L. usitatissimum have been intensively conducted [Linum is still very limited. Many wild species have been studied using molecular-karyological approaches [Linum were carried out [Stellerolinum, L. stelleroides Planch. (2n = 20), formed the basal phylogenetic branch of the extant blue-flowered flax. After this branch, the phylogenetic branch, represented by modern species of the sect. Dasylinum , separated from the common trunk of the phylogenetic tree. Then, the phylogenetic branches corresponding to the sections Adenolinum  and Linum were separated from each other. Unlike species of most sections of blue-flowered flax, representatives of the sect. Linum were not a homogeneous group, and in turn, subdivided into several subgroups of karyologically distinct species. Particularly, L. narbonense L. (2n = 4x = 28), 16-chromosome species - L. grandiflorum Desf. and L. decumbens Desf., 30 chromosome species - L. usitatissimum L. and L. angustifolium Huds. and also high polyploid species from Australia and New Zealand ) represented individual subbranches inside the sect. Linum. In the present study, for better understanding of the organization and evolution of the flax genomes, we conducted low-depth sequencing and comparative repeatome study of the representatives of blue-flowered flax using NGS data.The genus red flax , 29. Besonducted –43, and onducted , 43. Howproaches –48. For proaches . Besidesried out , 49–54. ried out , 54 thatLinum were selected for genome sequencing. Three of them were obtained from the genebank of Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) : L. hirsutum subsp. hirsutum L. ), L. narbonense L. ) and L. usitatissimum L., var. Stormont cirrus ). The sample of L. perenne L. (per1) was collected from the natural population  by Dr. A.A. Svetlova, Komarov Botanical Institute RAS . The sample of L. stelleroides Planchon (ste) was kindly provided by Dr. L.N. Mironova, Botanic Garden Institute of the Far-Eastern Branch of the Russian Academy of Sciences .Five representatives of the genus L. leonii F.W.Schultz (accession number LIN 1672 (leo)), L. lewisii Pursh ), L. grandiflorum Desf. (accession number LIN 974 (gra)), L.decumbens Desf. (accession number LIN 1754 (dec)) and L. angustifolium Huds. (accession number LIN 1642 (ang)) were used.For genome size estimation, in addition to the above-listed samples, five species from IPK genebank: 0 C for night, fixation in ethanol:acetic acid fixative (3:1) at 2–40 C, hydrolysis at 500 C in 1 N HCl for 40 min, maceration with Cellulysin  and root tips squashing on the microscopic slides. About 50 prophase nuclei have been measured for each flax sample with Opton scanning microphotometer. Measurements were made in relation to to diploid rat hepatocytes containing 7.8 pg DNA per 2C nucleus [Hordeum vulgare var. Odesski 31 containing 22.6 pg DNA per 4C nucleus [The nuclear DNA content of the flax species was determined by comparative Feulgen photometry according to Boyko et al., . Briefly nucleus  and Hord nucleus .Flax seedlings were used for DNA extraction as described earlier . High-quL. leonii ), L. lewissii ), L. perenne ), L narbonense ) L.grandiflorum ), L. decumbens ) and L. angustifolium ).For the analysis of the repeatomes, raw paired-end reads obtained as a result of WGS of the five flax species carried out within the framework of this study and also raw paired-end reads of the WGS of wild flax species available in the European Nucleotide Archive (ENA) : L. leonTo identify and classify repetitive sequences in flax genomes, raw paired-end WGS reads were analyzed using the RepeatExplorer toolkit . For eacL. usitatissimum and L. bienne, tandem organization of the found putative satDNAs families were additionally examined by PCR amplification. For this purpose, primers were designed in opposite orientation to most conserved regions of monomers consensus sequences  optical map of L. usitatissimum cv. CDC Bethune [The characteristic ladder pattern of tandem repeats was checked after electrophoresis in a 2% agarose gel. The reliability of the PCR product was confirmed by Sanger sequencing. For  Bethune  availablFor phylogenetic tree construction based on repeatome compositions, the previously published approach has been used . ClusterStellerolinum, diploid species L. stelleroides, had the largest genome (1C = 1376 M bp), 39.47% of which was represented by mobile elements, and only 0.55% of satellite DNA. The genome of L. hirsutum from sect. Dasylinum (1C = 1066 M bp) contained 34.52% of TEs and 2.27% of satellite DNA. Genomes of diploid species of sect. Adenolinum were similar in size and repeatome composition. They had an average genome size 446 M bp, TEs - 29.57%, unclassified dispersed repeats - 4.46% and satellite DNA ~ 4.67%. Unlike previous sect. Linum united, several groups of species differed significantly in the structure of their karyotypes. We investigated representatives of three such karyo-groups, which were most widely represented in the European flora. It was found that, together with karyological differences, representatives of these three groups also differed significantly in the molecular organization of their genomes. Among them, the group represented by the autotetraploid species L. narbonense (2n = 4x = 28) had the largest genome size 1C = 1617 M bp (or 808 M bp per one subgenome). Dispersed repeats represented a significant portion of its genome (54.78%), and the proportion of satellite DNAs (0.87%) was smaller than in the representatives of the previous sections. The second group of sect. Linum constituted two karyologically similar diploid species, L. grandiflorum and L. decumbens (2n = 16). Their genomes were also very similar (1C ~ 480 Mbp), and contained ~ 25% of dispersed repeats and ~ 10% of satellite DNAs. The third group included karyologically similar allotetraploids (2n = 30): the cultivated species L. usitatissimum and its wild ancestor L. angustifolium. Both had the smallest genomes , ~ 18% of dispersed repeats and ~ 13% of satellite DNAs.The analysis of genome size and repeatomes showed that species from different sections differed significantly in genome size and repeatome composition Table . So, theL. hirsutum to 0.6% for the 30-chromosome species of sect. Linum. In L. stelleroides, together with Ty3-gypsy LRT-element Athila, Ty3-gypsy LRT-element Tekai also was widely represented (~ 16%), while the genome of L. narbonense contained considerable amount (~ 39%) of Ty3-gypsy LRT-element Ogre. In contrary, the genomes of other species contain minimal amounts of Ty3-gypsy LRT-element Ogre. Amplification of Ty3-gypsy LRT-element Tekai in L. stelleroides and Ty3-gypsy LRT-element Ogre in L. narbonense resulted in increasing of the genome sizes of these species.Comparison of the repeatomes of the studied species showed that they all contained spectra of TE similar in composition Table . In blueAdenolinum, 16- and 30-chromosome species of sect. Linum). Only one family of putative satellite repeat common to sect. Adenolinum and sect. Linum was identified using the Repeat Explorer. However, this putative satellite had low confidence, and PCR amplification did not confirm its tandem organization. For other karyologically different species, strictly specific sets of families of satellite DNAs were identified. At the same time, DNA sequences homologous to satellite repeats of one species were often found in genomes of other non-closely related species but in a low number of copies . In L. narbonense, only two families of satellite DNA were discovered which totally made up ~ 0.9% of the overall genome. In 16-chromosomal species of sect. Linum, two families of satellite DNAs common to both L. grandiflorum and L. decumbens were identified. One more family was found only in L. grandiflorum, and one was specific to L. decumbens. The highest number of families of satellite repeats (28 families) was found in 30-chromosomal species of the sect. Linum. In these species as well as in representatives of sect. Adenolinum, the amount of each satellite DNA family varied between L. usitatissimum and L. angustifolium.In total, 44 families of tandem-organized repeats were identified in the genomes of the studied species. Our results showed that the satellite DNA of blue-flower flax evolved much faster than dispersed repeats. Common families of satellite DNAs were only found within the three most closely related groups of species that had similar karyotypes  and 30-chromosome species (L.angustifolium and L. usitatissimum), respectively. The fourth cluster was represented by highly polyploid and closely related species from Australia and New Zealand  which were not examined in this study.The phylogenetic relationships of representatives of the genus  genomes , 49–54. stimated , 29, 53.Thus, the reconstructed here phylogeny of blue-flowered flax, which was based on the similarities and differences of the repeatomes, agreed with the results of molecular phylogenetic studies reported earlier.L. stelleroides, L. hirsutum and L. narbonense) had the largest chromosome sizes, whereas 30- chromosome species of the sect. Linum with the smallest chromosome sizes had the smallest genomes.As in the case of other angiosperms –66, genoL. stelleroides, L. hirsutum, and L. narbonense could be related to a significant amplification of certain mobile elements. The smallest genome sizes were found in 30-chromosomal species of sect. Linum (L. usitatissimum and L. angustifolium) which also contained the lowest amount of dispersed repeats . It should be noted that the content of dispersed repeats in L. usitatissimum, determined by using Repeat Explorer, was lower than that obtained earlier (24%) [Linum species [We revealed that all the studied genomes were characterized by similar sets of transposable elements. Among them, LTR type retroelements were the most widely represented. It was probably due to the common origin of those genomes. At the same time, differences were found both in the total content of dispersed repeats and in the content of individual classes of mobile elements in different species. Like other plants, in the studied species there is a direct relationship between the content of dispersed repeats in the genome and its size. Thus, the extremely large genome sizes in er (24%) . It coul species  could be species –69.It is known that satellite DNA families is often retained in related species during long evolutionary periods. In particular, in some plant groups, there are families of satellite DNAs specific to taxa of different rank up to families and orders –73. HoweLinum species was accompanied by waves of amplification of satellite DNAs and LTR retrotransposons. Those events together with polyploidization resulted in significant differences in genome size and karyotype structure of blue-flowered flax. The genomes of the studied flax species contained similar sets of mobile elements but differed in their amount. At the same time, comparison of satellite DNAs showed that most of the detected tandem repeats were species-specific (except for some very closely related species) which indicated the rapid and concerted evolution of this genome fraction. The phylogenetic relationships between the studied flax species, obtained by the estimation of the similarity of their repeatomes, agreed with the previous data based on other phylogenetic markers. A direct relationship between the sizes of the genomes and the total content of dispersed repeats and the inverse relationship between the sizes of the genomes and the total content and diversity of satellite DNAs were revealed. The lowest amount of dispersed repeats and highest content of satellite DNAs in the genomes of 30-chromosome flax from the sect. Linum was probably a result of their allotetraploid origin. Thus, our findings gave new useful information about Linum genome evolution and provided a valuable set of data that could be used in future investigation of Linum genome.The obtained results showed that the evolution of the blue-flowered Additional file 1:Putative satellite DNA sequences in genomes of blue-flowered flax. (DOCX 1037 kb)


Pleural involvement by non-tuberculous mycobacteria (NTM) in patients without distinct pulmonary disease is extremely rare. Vertebral osteomyelitis (VO) with or without pulmonary disease is also a rare clinical presentation of NTM infection, and pleural spread of NTM from VO has not been reported.Mycobacterium abscessus ssp. abscessus was detected in both the thoracic cavity and the paravertebral lesion. Both VO and the paravertebral abscess were improved by antimycobacterial treatment.A 63-year-old woman was admitted to our hospital with back pain persisting for 4 months and a 2-day history of fever and right chest pain. The patient was initially treated as right-sided empyema due to general bacteria. However, after removal of the chest tube, a previously overlooked paravertebral lesion was observed on CT. MRI confirmed VO at T7/8. VO of the thoracic spine due to non-tuberculous mycobacterial infection should be considered as a cause of pleuritis or empyema without pulmonary disease, especially in patients with back pain. Mycobacterium abscessus (M. abscessus) ssp. abscessus, a rapidly growing species of non-tuberculous mycobacteria (NTM), is well-known as a pathogen of the skin, soft tissues, bone, and lungs [M. avium-intracellulare complex (MAC), occasionally causes pleuritis or empyema, probably due to direct spread from pulmonary lesions [M. abscessus ssp. abscessus has not been reported [nd lungs –3. NTM,  lesions –9. Howevreported –13.M. abscessus complex [M. abscessus ssp. abscessus infection that presented as empyema without distinct pulmonary disease and was found to arise from VO.Vertebral osteomyelitis (VO) with or without pulmonary disease is also a very rare clinical presentation of NTM infection, including that caused by  complex –18. Appr complex . Accordi complex . However complex . Here weA 63-year-old woman was admitted to our hospital with back pain persisting for 4 months and a 2-day history of fever and right chest pain. On admission, her height and weight were 154 cm and 50 kg, respectively. She had no history of other diseases, including autoimmune disease, diabetes, bronchiectasis, old healed tuberculosis, trauma, or acupuncture. The patient had visited two other hospitals, where contusion of the thoracic spine had been diagnosed by MRI (two months before admission) and contrast CT (three weeks before admission) , treatment with imipenem/cilastatin (IPM/CS: 1 g/day i.v.), amikacin (AMK: 400 mg/day i.v.), and clarithromycin (CAM: 800 mg/day p.o.) was initiated based on a diagnosis of VO due to M. abscessus ssp. abscessus, with paravertebral abscess caused by direct spread. An antibiotic susceptibility test was performed with air-dried microplates containing serial dilutions of antimicrobial agents and modified Middlebrook 7H9 broth [Initial laboratory data included a white blood cell count of 7580/μl (85.0% neutrophils) and a C-reactive protein of 8.26 mg/dl. CT showed a right-sided pleural effusion Fig. c. Right dization , and wasbolletii , by multbolletii  and rpoBanalysis . Since tung Fig. f. M. absH9 broth , revealiH9 broth , 25.After continuation of treatment for three months, both the MRI-confirmed VO and the paravertebral abscess showed improvement Fig. c, f, i. M. abscessus complex pulmonary disease is generally of the nodular bronchiectatic type, so a subpleural caseous focus is an uncommon incipient lesion of M. abscessus complex infection compared to MAC infection [M. abscessus ssp. abscessus was not only isolated from the thoracic cavity, but also from the paravertebral abscess. The reason for non-recurrence of empyema was considered to be postoperative pleural adhesion induced by thoracoscopic curettage and subsequent drainage. Third, although vertebral biopsy was not performed, the paravertebral abscess and vertebral lesion both improved simultaneously with antimycobacterial treatment, suggesting that VO was also caused by M. abscessus ssp. abscessus infection. According to a review of 13 cases of pyogenic spondylitis with exudative pleural effusion, Staphylococcus aureus was the major pathogen [In patients with pleural involvement alone, the main pathway by which NTM reaches the thoracic cavity is thought to be direct discharge of an undetectable subpleural caseous focus into the pleural space . Howevernfection . In our pathogen . To the M. abscessus ssp. abscessus obtained in Japanese patients and a report on a patient successfully treated with this regimen [M. abscessus ssp. abscessus infection, but the patient preferred to stop medication when the lesion almost resolved on imaging. In recent years, it has been suggested that inducible and acquired resistance to CAM are the main causes of treatment-refractory M. abscessus ssp. abscessus pulmonary disease [M. abscessus spp. abscessus due to ribosomal methyl transferase gene erm(41). However, T/C polymorphism occurs at position 28 of erm(41) and C28 strains usually lose erm(41) function, resulting in susceptibility to macrolides [rrl gene region encoding the peptidyltransferase domain of the 23S rRNA [M. abscessus ssp. abscessus to CAM. However, the good clinical course of our patient makes it unlikely that acquired resistance to CAM developed during the treatment period.Her treatment  was selected on the basis of drug sensitivity data for  regimen , 28, 29. disease , 30–32. 23S rRNA , 32. BecDelayed diagnosis of spinal disease may lead to neurologic complications. Accordingly, VO of the thoracic spine due to NTM infection should be considered as a cause of pleuritis or empyema in patients without pulmonary disease, especially when back pain is present.


Bacillus spp. are considered to be an eco-friendly and bio-safe alternative to traditional chemical fungicides/bactericides due to their intrinsic ability to induce native anti-stress pathways in plants. This review compiles information from multiple scientific databases  using the keywords “postharvest diseases”, “Bacillus”, “Bacillus subtilis”, “biocontrol”, “storage”, “losses”, and “fruits/vegetables”. To date, numerous examples of successful Bacillus spp. application in controlling various postharvest-emerged pathogens of different fruits/vegetables during handling, transportation, and storage have been described in the literature. The mechanism/s of such action is/are still largely unknown; however, it is suggested that they include: i) competition for space/nutrients with pathogens; ii) production of various bio-active substances with antibiotic activity and cell wall-degrading compounds; and iii) induction of systemic resistance. With that, Bacillus efficiency may depend on various factors including strain characteristics (epiphytes or endophytes), application methods (before or after harvest/storage), type of pathogens/hosts, etc. Endophytic B. subtilis-based products can be more effective because they colonize internal plant tissues and are less dependent on external environmental factors while protecting cells inside. Nevertheless, the mechanism/s of Bacillus action on harvested fruits/vegetables is largely unknown and requires further detailed investigations to fully realize their potential in agricultural/food industries.Postharvest diseases significantly reduce the shelf-life of harvested fruits/vegetables worldwide.  Fresh fruits and vegetables encounter disease between harvest and consumption, resulting in significant food waste and economic losses. According to the Food and Agriculture Organization  about 45Bacillus spp. viz. B. subtilis, is one of the most attractive agents for the development of natural plant protection products, as recommended by United States Food and Drug Administration. Bacillus spp. are generally recognized as safe microorganisms for application in the food industry. Bacillus spp. occupy the same niche as many pathogens and have the capacity to produce a wide range of bio-active substances with antibiotic activity. These substances induce various physiological features in host plant metabolism without causing adverse effects on the environment and human health [Bacillus spp. , produce endospores resistant to dynamic physical and chemical treatments, such as heat, desiccation, organic solvents, and UV irradiation, which therefore maintain their ability to trigger defense responses in host plants, even under unfavorable conditions [Bacillus-based biological products and serves as a potent bio-active component against pathogens [Bacillus strains on various plant species against a wide range of biotic  [Bacillus can be attributed to the synthesis of a wide range of biologically active compounds including antibiotics, siderophores, lipopeptides (LPs), enzymes, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, and exopolysaccharides. Furthermore, Bacillus spp. are known to affect regulation of phytohormone biosynthesis pathways, modulate ethylene levels in plants, and influence the emission of volatile organic compounds (VOCs) and the launch of host plants’ systemic resistance/tolerance [PGPB are a group of non-pathogenic beneficial bacteria that can directly and/or indirectly promote plant growth, disease resistance, and abiotic stress tolerance. They may live autonomously in the soil or colonize the rhizosphere, phyllosphere (epiphytes), and plants’ interior tissues (endophytes) ,10,11,12n health ,13. Furtnditions ,15,16. Tathogens ,19,20,21, pests) ,25,26,27, pests) ,28,29,30olerance ,32,33.Bacillus spp., including B. subtilis, for biocontrol of various postharvest diseases, in a wide range of fresh-cut fruit/vegetables during handling, transportation, and storage, has been frequently reported [B. subtilis to suppress the development of postharvest pathogens causing grey mold  has been demonstrated in strawberry, pear, apple, and tomato [Bacillus genus possess substantial potential to increase vegetables/fruit sets, quality, postharvest disease resistance, and tolerance under temperature fluctuations, mechanical injury associated with loading of product for transportation, unloading, packaging, and storage [Bacillus spp. in plant growth, development, and health under both normal and stressed conditions, their role in controlling postharvest disease, and the underlying mechanisms regulating fruits/vegetables storage quality remain largely unknown.The use of reported ,33,34,35d tomato ,40,41,42 storage . HoweverTrichoderma spp. for the control of rot (Botrytis) in strawberry [Lasiodiplodia theobromae) in stone fruits by B. subtilis [Bacillus spp., on postharvest physiology of various fruit/vegetables began to emerge, establishing them as enhancers of fresh fruit/vegetable resistance against a broad spectrum of postharvest diseases and unfavorable storage conditions associated with extending shelf life and maintaining nutritional qualities [Cucumis melo L.) fruit caused by Alternaria alternata was suppressed up to 77.2% when treated by B. subtilis EXWB1 prior to storage. In the presence of EXWB1 strain, A. alternata hyphae growth was hampered on melon fruit surfaces as well as wounded tissues. It was suggested that such a positive effect of B. subtilis EXWB1 on postharvest physiology of melon is connected to the ability of the EXWB1 strain to diminish the production of ethylene up to 72.3% and decrease the respiration rate of infected and non-infected melons by 26.1% and 71.9%, respectively [B. subtilis EXWB1. Likewise, it was revealed that treatment of fruit with EXWB1 contributes to maintenance of turgor pressure, titratable acidity in value close to the fresh fruit, increase of total sugar content up to 36.7%, and reduction of weight loss during storage [B. subtilis, B. pumilus, B. cereus, B. megaterium, and Agrobacterium radiobacter), treatment by strains of B. subtilis and A. radiobacter were most effective in controlling postharvest citrus fruit disease induced by Penicillium digitatum [B. pumilus and B. amyloliquefaciens effectively suppress the development of grey mold disease of pears and tomatoes caused by Botrytis cinerea [Bacillus strains  controlled grey mold of apple caused by Botrytis mali [B. subtilis AG1 against vine wood fungal pathogens of Phaeoacremonium aleophilum, Phaeomoniella chlamydospora, Verticillium dahliae, and Botryosphaeria rhodina has also been demonstrated [B. subtilis producing antibiotics and VOCs in suppressing the development of postharvest pathogens such as Rhizopus stolonifer (soft rot), Botrytis cinerea (gray mold), and Colletotrichum spp. in strawberry (Fragaria x ananassa) berries has been revealed [B. subtilis SK1-2 strain, exhibiting high antagonistic activity against Botryosphaeria dothidea, Diaporthe actinidiae, and Botrytis cinerea in in vitro culture, has been introduced as an effective antagonist for the bio-control of postharvest rot in kiwifruit [B. subtilis 9407 also exhibits strong antifungal activity against Botryosphaeria dothidea and significantly reduces the development of ring rot of apple fruit [B. subtilis of postharvest fungal rot caused by Aspergillus niger, Botryodiploidia theobromae, and Penicillium oxalicum has been reported in Dioscorea fruit [B. subtilis strain GA1 led to a reduced level of postharvest infection in apple caused by Botrytis cinerea during the first 5 d following pathogen inoculation and, notably, ~80% protection over the next 10 d [B. subtilis treatment on both fading of the litchi fruit during storage (30 d at 5 °C), and maintenance of proper quality  has been revealed. Interestingly, in all treatments by B. subtilis bacterial cells no change in fruit taste has been reported [B. subtilis CF-3 strain retained 65% of fruit quality after storage for 36 d at 10 °C, which was more than 30% higher in comparison to quality of non-treated controls [B. subtilis alleviated citrus fruit decay caused by Penicillium digitatum and P. italicum. It has been shown that B. subtilis H110 significantly reduced blue mold disease caused by P. expansum and black spot disease caused by Alternaria alternata on harvested apple pears (Pyrus bretschneideri Rehd.). It was suggested that mechanism by which B. subtilis H110 inhibits pathogens might be related to its ability to produce antagonistic protein and natural competition for nutrients and space [B. subtilis in the control of fungal rot in citrus [Monilinia fructicola infection in peaches and cherries has also been shown [B. subtilis strains APEC170 and Paenibacillus (Bacillus) polymyxa APEC136 reduced the symptoms of anthracnose of fruits caused by the fungal pathogens of Colletotrichum gloeosporioides and C. acutatum, as well as white rot caused by Botryosphaeria dothidea [B. subtilis SM21 effectively controlled rot caused by Rhizopus stolonifer and other pathogens including Monilinia fructicola, Cephalothecium, Rhizoctonia, and Alternaria in harvested peach fruit [B. subtilis and commercial wax Tag, enriched with different concentrations, significantly reduced the development of anthracnose, the avocado fruit rot complex (Dothiorella/Colletotrichum) during storage time [B. subtilis Ch-13 on induction of defense responses in potatoes against pathogens during both growing season and cold storage have indicated reduced colonization of bacterial antagonists on potato tuber surface during the storage period [B. subtilis-based microbial preparation increased the activity of peroxidase enzymes in potato tubers, formation of phytoalexin and ascorbic acid content by 2.4, 3.1, and 1.3 times, respectively. Treatment of banana fruit (Musa sp.) with the composition of bacterial strains  reduced the development of Fusarium oxysporum [Although the first reported microbial antagonist application was of rawberry , the firsubtilis . Later, ualities   into host plant tissues, before planting or during the vegetative phase, promoted plant (potato) growth, and protected plants from certain defects. These effects were maintained for a prolonged period leading to the better preservation of vegetables during storage [B. subtilis 10-4, applied alone or in combination with natural safe signaling molecules, such as salicylic acid (SA), promoted anti-stress activity in plants [B. subtilis 10-4 and SA were less infected by the pathogenic micromycetes Aspergillus, Pennicillium, and Alternaria with fully faded Cladosporium, Fusarium, and Mucor when compared to non-treated controls. Additionally, tubers treated with both B. subtilis 10-4 and B. subtilis 10-4 with SA retained antifungal activity even after a 30 d storage period compare to the non-treated controls, indicating the prolonged protective effect caused by B. subtilis treatment [B. subtilis together with SA is more effective in bio-controlling potato diseases during storage than B. subtilis alone [Bacillus spp.–host plant–pathogen is one of the main barriers to the commercial development of preparations based on antagonistic bacteria and their compositions.Of particular interest for bio-control of postharvest diseases are endophytic strains of t plants ,57,58. F storage . In anotn plants ,61. Harvreatment . Interesis alone ,60. Perhis alone ,63. SA cBacillus spp. will play a pivotal role in determining the most effective application that maximally inhibits pathogen growth and function on a harvested host plant [Bacillus spp. suppresses postharvest pathogens are not yet fully understood, multiple mechanisms have been suggested including: (1) competition with pathogenic microflora for nutrients and suitable niches for colonization [Unraveling the mechanism/s of action of microbial antagonists including st plant . Althougnization ,33,64,72nization ,74,75,76nization ,77,78; anization ,77,79,80nization ,75,79,817–108 CFU/mL [Inhibition of rapid wound site colonization in fruits/vegetables triggered by antagonistic microorganisms is a crucial step in controlling postharvest decay . The eff8 CFU/mL . As simi8 CFU/mL ,57,58.Aureobasidium pullulans against Botrytis cinerea, Rhizopus stolonifer, Penicillium expansum, and Aspergillus niger, which infect table grapes and P. expansum and B. cinerea on apple fruit, direct physical interaction is not required for the antagonistic activity [Another significant contribution in bio-control of pathogens is competition for nutrients. In vitro studies demonstrated that bacterial inoculants take up nutrients faster than pathogens; this can lead to the inhibition of germination of pathogen spores at the wound site ,83. A fuactivity . In suchactivity ,85,86.Bacillus antibiotics are active against both gram-positive and gram-negative bacteria, as well as phytopathogenic fungi such as Aspergillus flavus, Alternaria solani, Fusarium oxysporum, Botryosphaeria ribis, Helminthosporium maydis, Phomopsis gossypii, and Colletotrichum gloeosporioides [Bacillus antibiotic substances break growing hyphae tips of Sclerotinia sclerotorum (the stimulant of sunflower white rot), A. alternata, Drechlera oryrae as soil fungi, and F. roseum, as well as Puccinia graminis  [B. subtilis has broad suppressive properties against more than 23 types of plant pathogens in vitro due to its ability to produce a broad range of antibiotics with a wide variety of structures and activities [Bacillus species may dedicate up to 8% of their genetic potential to the synthesis of a wide range of antimicrobial compounds, among which non-ribosomally synthesized LPs, lytic enzymes, and lantibiotics are suggested to be crucial for pathogen suppression [B. subtilis and B. amyloliquefaciens, ~5% and ~8% of the genomes are involved in the biosynthesis of wide range of antimicrobial compounds, respectively. In another study Ahmad and colleagues [B. subtilis (strain 330-2), about 10% were involved in antibiotic biosynthesis.Antibiotics are a heterogeneous group of low molecular weight organic compounds produced by bacteria, which suppress or diminish the growth and development of phytopathogenic microorganisms ,14,87. Aorioides . Bacilluls rust) . It has pression ,90,91,92lleagues  reportedBacillus spp. play pivotal roles in suppressing disease, giving them substantial status in the agriculture and biotechnology industries [Bacillus spp. cyclic LPs (CLPs) [Paenibacillus polymyxa was discovered in 1949 and categorized in the first group [B. subtilis (strain IAM1213) as the most effective producer of LPs [Bacillus, as well as actinomycetes and fungi. It was discovered that CLPs have a certain antibiotic selectivity. Therefore, if surfactin is mainly characterized by its antibacterial and antiviral properties, iturin and fungicin could be considered antifungal components [B. subtilis strains originating from private or public collections have been categorized as surfactin-producers [Fusarium oxysporum fungus development by B. subtilis GM5 due to the presence of surfactin and fengycin at spore germination, sprouting tube formation, and hyphae branching phases [B. subtilis ABS-S14, effective in bio-control of Penicillium digitatum-caused green mold in mandarin, produces all three CLPs. Research by Waewethongrak and co-workers [P. digitatum, while surfactins did not exhibit a direct effect. B. subtilis FZB24 producing iturin-like LPs were efficacious against various phytopathogenic fungi [B. subtilis, B. atrophaeus, and B. amyloliquefaciens producing iturin, fengycin, and surfactin possess high potential in the biotechnological industries due to their dynamic feature [Colletotrichum acutatum-induced anthracnose symptoms in tamarillo fruits were completely inhibited by iturin A and fengycin C, and reduced by 76% when treated with B. subtilis EA-CB0015 [B. subtilis inhibits the development of peach brown rot by producing iturin [B. subtilis strains was also observed in both individual activity and in the spectrum of LP level. Iturin, surfactin, and related compounds have been well known as the main components of the B. subtilis lipopeptide complex, whereas P. chimensis contains numerous homologs of bacillomycin and fenghin/plipastatin [ppsB is responsible for biosynthesis of fengycin, which represents the major antifungal compound produced by B. subtilis 9407 against Botryosphaeria dothidea. Biocontrol assays showed up to 50% reduction in ppsB gene expression compared with the wild-type strain, indicating the major role of fengycin in controlling apple ring rot disease caused by B. dothidea [Bacillus spp., B. amyloliquefaciens, and B. subtilis in particular, are also very efficient in obstructing different pathogens causing plant diseases [LPs synthesized by dustries ,95,96,97dustries . Dependidustries ,99, whics (CLPs) ,100,101.st group . In the r of LPs ,102. Sinmponents ,104. Theroducers . Researcg phases . Differeg phases . The antg phases ,107. Howg phases , while sg phases  and a nug phases ,109. Divg phases ,96,110. -workers  on the eic fungi . Previou feature ,114,115.g iturin . Neverthdiseases ,120,121.Bacillus spp. may vary according to the species and strain [B. subtilis is related to the control of Podosphaera fusca in cucurbits [B. subtilis strain ME488 suppress the Phytophthora blight in pepper and Fusarium wilt in cucumber [srfAA (surfactin), bacA (bacylisin), fenD (fengycin), ituC (iturin), spaS (subtilin), and bmyB (bacyllomicin) in 184 isolates of Bacillus spp. obtained from plant environments . According to different studies the ability of Bacillus spp. to suppress pathogens is associated with expression of the bmyB, fenD, ituC, srfAA, and srfAB genes [srfAA, bmyB, fenD, and bacA have been introduced as the most frequent antimicrobial peptide gene markers [Microbial antibiotics produced by d strain . It has ucurbits ; iturin,cucumber . Mora etcucumber  investigAB genes ,123,124. markers .Bacillus strains can produce varying amounts and types of LPs in response to the pathogens they encounter. [B. amyloliquefaciens significantly increased iturin and fengycin biosynthesis in the presence of such pathogens as Pythium aphanidermatum and F. oxysporum [Podosphaera fusca by B. subtilis strains UMAF6614, UMAF6616, UMAF6639, and UMAF8561 and was demonstrated for cucurbits [B. subtilis (strain GA1) on harvested apple fruit against grey mold caused by B. cinerea correlates with production of fengycins in infected apples. It was demonstrated that antibiotics gramicidin S and polymyxin B produced by Brevibacillus brevis and Paenibacillus polymyxa, respectively, controlled Botrytis cinerea (gray mold) of strawberry in both in vitro and in vivo [B. subtilis (strain fmbJ) inhibited Aspergillus flavus development in corn by producing antibiotics like bacillomycin D with identical amino acid sequences (Asn–Tyr–Asn–Pro–Glu–Ser–Thr) [Pseudomonas syringae (strains ESC-10 and ESC-11) controlled Penicillium digitatum (green mold) of lemon by producing an antibiotic syringomycin E. However, the production of syringomycin E was never detected on the fruit/vegetables, casting doubt on the role of the antibiosis in controlling of the postharvest diseases and suggesting the operation of an alternative mechanism independent of syringomycin biosynthesis [B. subtilis (strain 6051) forms a stable and extensive biofilm and secretes surfactin component to protect plants against Pseudomonas syringae [B. subtilis (strain RB14) plays a pivotal role in the suppression of diseases caused by Rhizoctonia solani in tomato. Bacilopeptins, belonging to iturin-group antifungal antibiotics produced by B. subtilis (strain FR-2) isolated from rhizosphere of garlic plant infected by Fusarium oxysporum [B. subtilis (strain CPA-8) in suppression of brown rot disease caused by Monilinia spp. in peaches governed by fengycin-like LPs. Likewise, CPA-8 also showed antifungal efficiency against other postharvest pathogens such as M. laxa, Penicillium digitatum, P. italicum, Botrytis cinerea, M. fructicola, and P. expansum [B. subtilis in biocontrol of postharvest diseases is mainly attributable to the production of iturin, surfactin, and gramicidin [Most counter. . For insxysporum . The proucurbits . Toure e in vivo . B. subtSer–Thr) . Bull etSer–Thr)  demonstrynthesis . During syringae . Asaka asyringae  found thxysporum . In addixysporum  demonstrexpansum . A numbeamicidin ,110,131.amicidin ,132. Howamicidin .3+ that is inaccessible to biological systems [3+ between PGPB and pathogenic microorganisms has been addressed as a main strategy for their function [Pseudomonads and possesses a strong antifungal effect. It was shown that pseudobacin produced by P. putida (strain WCS358), suppressed the development of Ralstonia solanacearum in eucalyptus, Erwinia carotovora in tobacco, and Botrytis cinerea in tomato [Biosynthesis of antimicrobial compounds may be indirectly stimulated by siderophores—low molecular weight peptides that possess a high affinity for Fe ions. Antimicrobial compounds are released by various microorganisms to dissolve and assimilate Fe systems . Bacterifunction . Pseudobn tomato . Systemin tomato . AntibioBacillus bacteria can be attributed to the synthesis of extracellular hydrolases such as chitinases and β1,3-glucanases capable of destroying the structural polysaccharides of the fungal cell wall (chitin and glucans) and lysing the hyphae of fungi [B. subtilis 739 showed that chitinase, chitosanase, β-1,3-glucanases, and proteases showed the most contribution to lysis of the native mycelium of various species of phytopathogenic fungi Alternaria alternata, Bipolaris sorokiniana, Fusarium culmorum, and Rhizoctonia solani [2O2, which in turn can also perform a signaling function by activating the genes regulating certain transcription factors or by interacting with other signaling components [2O2 [Alternative possible mechanisms of antagonistic function of of fungi ,136,137.of fungi . A reseaa solani . Chitinoa solani . In addia solani , enhancemponents ,141. Notnts [2O2 , which cnts [2O2 ,144.B. subtilis NSRS 89-24 have proved to be very active in degrading the fungal cell walls of rice blast and sheath blight pathogens [B. subtilis and postharvest pathogen F. oxysporum of yam (Dioscorea spp.) tubers by scanning electron microscopy. Their results indicated that fungal cell wall lysis by B. subtilis was due to extracellular chitinase production [B. subtilis APEC170 and Paenibacillus polymyxa APEC136 diminish the symptoms of anthracnose caused by Colletotrichum gloeosporioides and C. acutatum, and white rot caused by Botryosphaeria dothidea in harvested apples by inhibition of mycelial growth of these pathogens. This can be attributed to the increased production of chitinase, amylase, and protease by the APEC170 strain, as well as elevated levels of amylase and protease in the APEC136 strain [B. subtilis V26 enhance harvested tomato resistance (up to 79%) to diseases caused by Botrytis cinerea during storage. Similarly, biocontrol efficiency of B. subtilis is mainly attributed to the production of antifungal compounds, including chitinases and glucanases [Bacillus spp. play a pivotal role in their biocontrol activity against B. dothidea that cause diseases in harvested fruit.The lytic enzymes synthesized by antagonistic athogens . Swain eathogens  investigoduction . It has 6 strain  reporteducanases ,100,131.ucanases  also repBacillus spp. suppress the development of different diseases in harvested fruits/vegetables not only directly through the synthesis of metabolites with fungicidal activity, but also indirectly, through the launch of multiple defense response mechanisms  induced accumulation of ABA in plants and stimulated their growth under drought stress [flacca and sitiens and the rhizosphere bacterium Bacillus megaterium synthesizing ABA, it has been revealed that maintaining the normal level of ABA in tomato plants is extremely necessary for the growth of the host in both normal and stress conditions [chanisms . These ibacillus ,153. Fort stress . Using tnditions . Differenditions . Recentlnditions . In pathnditions . In addinditions .Bacillus spp. produce low molecular weight VOCs , capable of easily propagating over long distances through diffusion in the air and pores in the soil. It has been established that exposure to VOCs can lead to a tissue-specific redistribution of endogenous auxins [B. subtilis UMAF6639 triggered in melon resistance to powdery mildew by activating JA- and SA-dependent protective reactions [Some strains of s auxins  and decrs auxins . In addis auxins . B. subteactions . Some oteactions .Almost all postharvest technologies manipulate the metabolism of the harvested product by inhibiting respiration rate of the product and the ethylene function as the key regulator of the ripening and senescence of fresh fruit and vegetables. Overproduction of ethylene leads to accelerated senescence and reduced shelf-life during postharvest storage ,161. To B. subtilis, by producing iturin and fungicin, induces the expression of phenylpropanoid metabolism genes in plants, thereby triggering the mechanisms of ISR [B. subtilis 168 producing surfactin and fengycin enhanced the resistance of tomato and bean plants to B. cinerea by activating the enzymes of the lipoxygenase pathway [B. amyloliquefaciens strain producing surfactin induced ISR in Brassica napus against Botrytis cinerea [Bacillus amyloliquefaciens FZB42 induced ISR in lettuce against Rhizoctonia solani, by the JA/ethylene-dependent signaling protective pathway through induction of PDF 1.2 gene expression [B. amyloliquefaciens FZB42 deficient in the synthesis of surfactin (CH1), as well as in the synthesis of surfactin, fengycin, and bacillomycin D (CH5) lost the ability to increase the resistance of lettuce to Rhizoctonia [B. subtilis strain BBG111 induces ISR in R. solani in the rhizosphere of rice due to the secretion of fengycin and surfactin, thereby causing a hypersensitive reaction and cell death. The immune responses develop over the JA/ethylene-, ABA- and auxin-dependent defense signaling pathways, which block the growth and development of the pathogen in the early stages of pathogenesis [B. amyloliquefaciens strain (FMB72) synthesizing 8.3 times more fengycin than the original strain of B. amyloliquefaciens ES-2-4 isolated from Scutellaria baicalensis Georgi. This strain possessed high biocidal activity against pathogens. Evidence from numerous studies indicate that Bacillus spp. elicit defense mechanisms in harvested fruits/vegetables and control of the postharvest decay through the accumulation of phytoalexins (scoparone and scopoletin) [B. subtilis (strain Ch-13) generate twice the protective response in potato tubers and increase the activity of peroxidase, ascorbic acid content, and the formation of phytoalexins in tubers during storage at 18 °C [Different studies have suggested that the production of antifungal compounds by microbial antagonists in host cells also contributes to the induction of defense mechanisms and biologically control harvested fruit/vegetable diseases. s of ISR . These d pathway , which d pathway . The B.  cinerea , as wellpression . It has pression . Howeverzoctonia . It has ogenesis . Future poletin) ,170. Comat 18 °C .2O2, after the onset of infection, and corresponds with the up-regulation of redox-sensitive transcription factors and PR genes. Thus, Pseudomonas putida LSW17S induced a rapid accumulation of transcription PR genes and production of H2O2 in tomato plants infected by P. syringae pv. tomato DC3000, which inhibited pathogen development [Rhizopus stolonifer on peach fruit treated with B. cereus AR156 and B. subtilis SM21 was associated with the generation of H2O2, and overexpression of chitinase genes, β1,3-glucanase, and phenylalanine–ammonium–lyase and the activity of their protein products [B. subtilis BSCBE4 and P. chlororaphis PA23 activated peroxidases and polyphenol oxidases catalyzing the final lignin biosynthesis reactions in pepper seedlings infected with Pythium aphanidermatum [B. subtilis (strain Bs16), Trichoderma viride (strains Tv1 and Tv13), Pseudomonas fluorescens (strains Pf1 and Py15) activate production of peroxidase, polyphenol oxidase, phenylalanine–ammonia–lyase and protect host plant tissues against various pathogens [Bacillus-induced defense responses in harvested fruit/vegetables against pathogens, the undelaying protective mechanisms still remain unclear and require more investigation.The ISR caused by endophytic bacteria is preserved in plants for a long time and effectively works against pathogens under storage conditions . It manielopment . The redproducts . B. subtdermatum . B. subtathogens . During athogens . NeverthThe effectiveness of potential microbial antagonists in suppressing pathogens in harvested fruits/vegetables depends on both characteristics of selected strain and application method. In general, microbial agents can be applied using pre- or post-harvest strategies .B. subtilis, reduces stress induced defects and positively affects crop yield during storage [Solanum tuberosum L.) tubers with endophytic B. subtilis strains 10-4 and 26D, positively affects plant growth, development, and yield of potato in the field, results in decreased infestation of tubers by pathogenic micromycetes Aspergillus, Pennicillium, and Alternaria and completely suppresses the development of Cladosporium, Fusarium, and Mucor, compared to non-inoculated control tubers both after harvesting and during storage [B. subtilis under field conditions results in colonization of the apple fruit surface by the microbial antagonist, which effectively controls the postharvest pathogens Penicillium expansum and Botrytis cinerea of apples [B. cinerea, Monilinia fructicola, Sclerotium rolfsii, and Geotrichum candidum [Bacillus spp. in the bio-control of postharvest avocado diseases, by use of the bacteria during vegetation, and in the pre-vegetation period before the laying of fruit for storage [Pathogens often infect fruit/vegetables in the field and live in plant tissues during vegetation season without causing any symptoms; however, these “hidden” infections may begin to develop during storage and become the major decay factor leading to significant food losses . Previou storage ,78. It h storage . This im storage ,177,178.f apples . In somecandidum ,180. Acccandidum  applicat storage ,181.Postharvest application of microbial antagonists has been introduced as a proper and practical method for control of harvested fruit and vegetables disease. According to this method, preparations containing microbial antagonists can be applied as postharvest sprays or as solutions in harvested fruit/vegetables  178,182182.Bacillus spp.  and ‘‘Bactril’’  contains B. nigrum 132, B. subtilis MBI600, and Bradyrhizobium japonicum [Bacillus spp. also can be considered one element, applied alongside other biological and physical methods, as part of an integrated vision of disease management. For example, mixtures of diverse antagonistic microorganisms with a broad spectrum of microbial activity and combination of different bio-controlling traits, leads to the control of two or more post-harvest diseases when applied under different environmental conditions [Bacillus spp.), biologically active compounds, and induced resistance, the introduction of more effective formulations, application methods and combinations with additional approaches for additive and/or synergistic effects, will not be unlikely [However, the application of one individual microbial antagonist cannot completely prevent all postharvest fruits/vegetables decay during storage. It is difficult to select an individual effective microbial strain with a broad spectrum of activity against wide range of pathogens ,182,183.aponicum . Bacillunditions . The effnditions . Integranditions ,137. GivB. subtilis strain B-3 (USA) [B. subtilis B-3 under commercial conditions for postharvest control of peach brown rot caused by Monilinia fructicola was conducted by Pusey et al. [B. subtilis added to each fruit was between 2 × 107 and 7 × 107 for B-3 from flask cultures, and 2 × 109 for B-3 from the fermenter culture. In a test at Byron, B. subtilis B-3 cultures, applied fresh or as a stored paste/powder, were equally effective as the chemical fungicide benomyl in control of brown rot. In a similar test at Clemson, stored forms of B-3 from both flask and fermenter cultures were as effective as benomyl [Bacillus spp. To date, several commercial microbial antagonists of the Bacillus genus, particularly B. subtilis, have been reported to control postharvest diseases in fruits/vegetables under laboratory and field conditions , has been formulated to control fungi of the Fusarium, Pythium, and Phytophthora genera on fruit and vegetables during storage [B. subtilis strains FZB24 and QST713 (Serenade and Rhio-plus) have already been commercially applied to control brown rot, powdery mildew, root rots, fire blight, and late blight on fruit/vegetables [B. subtilis strain QST 713 (Rhapsody) significantly reduced the development of postharvest rot in tomato [The first microorganism patented as a postharvest bio-controlling agent effective against brown rot of stone fruits was -3 (USA) . A piloty et al. , where t benomyl . In recenditions . For exa storage . Biocontgetables ,186. It n tomato .Bacillus spp. strains have a notable potential for being developed into a postharvest bio-controlling agent, which can be applied to different fruits/vegetables against a wide range of pathogens (Bacillus spp. C06 suppresses Monilinia fructicola (brown rot) incidence by 92% and reduced lesion diameter by 88% [Bacillus spp. T03-c reduced incidence and lesion diameter by 40% and 62%, respectively. Treatments with strains of MA-4, T03-c, and C06 significantly controlled brown rot by 91%, 100%, and 100%, respectively [B. amyloliquefaciens CPA8 have been developed as effective agents to control postharvest brown rot diseases in stone fruits. Gotor-Vila et al. [Monilinia spp. (more than 44.4%) in peaches, cherries, nectarines, plums, and apricots. However, one of the main factors restricting development and commercialization of postharvest bio-controlling products based on Bacillus spp. is the lack of knowledge about their multiple modes of actions in Bacillus spp.–hosts–pathogen interactions. Furthermore, success in the commercialization of prospective Bacillus spp. strains depends on the links between scientific organizations and industries as well. It follows that, even if microbial antagonists represent all the desirable characteristics, the commercial success requires economic and viable market demand.Different research carried out under laboratory conditions demonstrated that athogens . For exar by 88% . Bacilluectively . Two bioa et al.  demonstrBacillus spp. positively affect the postharvest physiology of fruits/vegetables by enhancing their resistance against different postharvest pathogens resulting in a prolonged storage period and extended “marketing” life, and preserving freshness and nutritional quality. Bacillus strains  may be developed as effective bio-control agents to decrease postharvest decay of fruits/vegetables. Developing these microbial antagonists as commercial agents for postharvest biological control is a strong prospect. Despite Bacillus spp. having a clear advantage as an eco-friendly strategy for preventing food losses during storage, the knowledge about the mode of action on postharvest physiology and preservation under pathogenic infection is limited and requires detailed investigation Therefore, understanding the mechanism/s of function for these bacteria will play an important role in the promotion of Bacillus-based products to support green technology in the agricultural and food industries.


Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technology (MALDI-TOF MS) is an innovative tool that has been shown to be effective for the identification of numerous arthropod groups including mosquitoes. A critical step in the implementation of MALDI-TOF MS identification is the creation of spectra databases (DB) for the species of interest. Mosquito legs were the body part most frequently used to create identification DB. However, legs are one of the most fragile mosquito compartments, which can put identification at risk. Here, we assessed whether mosquito thoraxes could also be used as a relevant body part for mosquito species identification using a MALDI-TOF MS biotyping strategy; we propose a double DB query strategy to reinforce identification success.Aedes aegypti BORA and Aedes albopictus Marseille, were dissected and analyzed by MALDI-TOF MS. Molecular identification using cox1 gene sequencing was also conducted on representative specimens to confirm their identification.Thoraxes and legs from 91 mosquito specimens belonging to seven mosquito species collected in six localities from Guadeloupe, and two laboratory strains, MS profiles obtained with both thoraxes and legs were highly compartment-specific, species-specific and species-reproducible, allowing high identification scores  when queried against the in-house MS reference spectra DB .Both thoraxes and legs could be used for a double DB query in order to reinforce the success and accuracy of MALDI-TOF MS identification.The online version of this article (10.1186/s13071-018-3157-1) contains supplementary material, which is available to authorized users. Culex pipiens form pipiens, Culex pipiens form molestus and hybrids)  and to the representativeness of cox1 gene diversity within a given species (i.e. Cx. quinquefasciatus). Indeed, genetic variations in the cox1 gene have been reported between specimens from the same mosquito species  collected in different provinces of Argentina, allowing the identification of different haplotypes [nad4 gene), it can influence the molecular identity rate obtained after DB query as observed here.To circumvent the limitations of morphological identification, molecular strategies based on DNA-barcoding using  applied , 33. Howplotypes . Even ifAe. aegypti by inclusion in the reference MS DB of MS spectra from laboratory-reared Ae. aegypti (Bora) specimens underlined the robustness of this approach.MS spectra variations between specimens from the same species with a different geographical origin was also observed. This phenomenon was reported for mosquitoes at the adult , 18 and Cx. atratus (s.l.) and D. magnus, cox1 sequences were not available in the GenBank DB, which resulted in failure to confirm the morphological identification. The lack of complete cox1 sequences in a public genomic DB frequently occurs, requiring the sequencing of other reference genes , or of several cox1 gene fragments [cox1 sequences queried against the BOLD DB allowed the identification of six out of seven species, confirming the morphological identification, including D. magnus specimens. Only specimens morphologically identified as Cx. atratus (s.l.) failed to be validated with cox1 sequence similarity lower than 97% following a query in BOLD. Nevertheless, as observed for Cx. quinquefasciatus in the GenBank DB, Cx. atratus (s.l.) cox1 sequences were available in the BOLD DB. However, it was not indicated to which member of the Cx. atratus complex corresponded the available cox1 sequences, i.e. Cx. atratus (s.s.) or Cx. atratus B [cox1 sequence similarity (< 89%) between Cx. atratus (s.l.) specimens from Guadeloupe and those available in the BOLD DB could suggest that the former belong to a different species of the Cx. atratus complex. The absence of another gene sequence target allowing to distinguish members of the Cx. atratus complex in the GenBank DB or the BOLD DB, did not allow us to definitively identify the specimens collected in Guadeloupe. However, the high homology of the cox1 sequences between specimens allowed us to consider them conspecific but as Cx. atratus (s.l.). The reproducibility of MS spectra, for each compartment, from specimens identified as Cx. atratus (s.l.), in addition to the elevated CCI obtained for both legs and thoraxes, were supplementary arguments to claim that these specimens could be considered as same species. The high efficiency of MALDI-TOF MS biotyping to distinct cryptic species has been repeatedly reported, notably distinguishing An. gambiae (s.s.) from An. coluzzi, corresponding to An. gambiae M and S molecular forms, respectively [Cx. atratus complex.For ragments , when potratus B . The lowectively , 20, 34.A critical step in the implementation of MALDI-TOF MS identification is the creation of MS reference spectra DB for the species of interest. The majority of the existing adult mosquito identification MS DB have been created using mosquito legs . HoweverAe. albopictus specimens revealed similar MS profiles between mixed compartments and thorax counterparts. In MS profiling, mainly abundant ionizable proteins are detectable. As proteins from legs were proportionally less abundant than those from the thorax, their protein quantities were insufficient to drastically change thorax MS profiles. Nevertheless, under the hypothesis that thorax MS profiles would be changed by the addition of legs for other species, it is probable that intensity of MS peaks corresponding to legs in the mixture could be variable according to the number of legs included. In such conditions, the reference MS spectra database from mixed legs and thoraxes would not be suitable to identify specimens which had lost several or all legs. Conversely, when legs were submitted alone, the MS profile was unchanged whatever the number of legs and only a decrease of the MS profile intensity was observed according to the number of legs used. For specimens with one or two legs remaining, the low ratio intensity of MS peaks/background can hinder peak detection, compromising their identification. The MS submission of a second compartment, such as the thorax, could then reveal specimen identity, especially for specimens which have lost several legs or for closely-related species.The comparison of MS profiles obtained with legs, thoraxes and a mix of legs and thoraxes from This new strategy consisting of submitting two body compartments per specimen to MALDI-TOF MS analysis, thereby producing distinct but species-specific MS spectra, should improve identification confidence. Moreover, the distinct topologies of the MSP dendrograms for each body part from paired specimens, pointed out that the proximity of MS spectra between mosquito species were different for legs and thoraxes. These MS pattern properties reinforce the species identification accuracy and reduce the risk of misidentification. The MS spectra specificity according to body part used, was also reported in others arthropods such as ticks  and it hIn addition to the standardization of sample preparation protocols , 20, 36,The present study points out MALDI-TOF biotyping strategy as an innovative and alternative tool for mosquito identification that could lead to a dramatic positive impact in the mosquito surveillance field worldwide. The double MS spectra DB query should reinforce identification quality and, in the case of missing legs, identification remains possible by the use of the thorax. To achieve this, the development of an international comprehensive and accessible mosquito spectra database is essential.Aedes aegypti (Bora strain) and Ae. albopictus  mosquitoes were raised in the laboratory using standard methods as previously described [Adult female mosquitoes, collected in the field or laboratory-reared, were used for this study. Field collection of mosquitoes was undertaken in 6 distinct sites from the Caribbean Island of Guadeloupe using BG sentinel traps Fig. . Collectescribed , 41. OnlAe. albopictus specimens were mixed prior to MALDI-TOF MS analysis.Legs and thoraxes from mosquitoes were processed as previously described . Brieflyn = 24) using the QIAamp DNA tissue extraction kit  according to the manufacturer’s instructions. Molecular identification of mosquito at the species level was performed by sequencing the PCR product of a fragment of the cytochrome c oxidase 1 gene (cox1) using the primers LCO1490 (forward) (5'-GGT CAA CAA ATC ATA AAG ATA TTG G-3') and HC02198 (reverse) (5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3') as previously described [http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the Barcode of Life Data Systems  to assig for 1 min was then performed and 1 μl of the supernatant of each sample was loaded on the MALDI-TOF steel target plate in quadruplicate . After air-drying, 1 μl of matrix solution composed of saturated α-cyano-4-hydroxycinnamic acid , 50% (v/v) acetonitrile, 2.5% (v/v) trifluoroacetic acid  and HPLC-grade water was added. To control matrix quality (i.e. absence of MS peaks due to matrix buffer impurities) and MALDI-TOF apparatus performance, matrix solution was loaded in duplicate onto each MALDI-TOF plate alone and with a bacterial test standard . Moreover, legs or thoraxes from two Ae. albopictus specimens reared at the laboratory and stored at -20 °C were included on each plate and were used as homogenization positive controls.Each compartment dissected was individually homogenized 3 × 1 min at 30 Hz using TissueLyser (Qiagen) and glass powder  in a homogenization buffer composed of a mix (50/50) of 70% (v/v) formic acid  and 50% (v/v) acetonitrile  for protein extraction according to the standardized automated setting described by Nebbak et al. . RespectProtein mass profiles were obtained using a Microflex LT MALDI-TOF Mass Spectrometer , with detection in the linear positive-ion mode at a laser frequency of 50 Hz within a mass range of 2–20 kDa. The setting parameters of the MALDI-TOF MS apparatus were identical to those previously used . The speMS spectra profiles were first controlled visually with flexAnalysis v.3.3 software . MS spectra were then exported to ClinProTools v.2.2 and MALDI-Biotyper v.3.0.  for data processing . MS spectra reproducibility was assessed by the comparison of the average spectral profiles  obtained from the four spots for each specimen according to body part with MALDI-Biotyper v.3.0 software . MS spectra reproducibility and specificity taking into account mosquito body part were demonstrated using clustering analyses and the composite correlation index (CCI) tool. In addition, ClinProTools software was used to identify discriminatory peaks among the 8 mosquito species for each body-part. Cluster analyses (MS dendrogram) were performed based on comparison of the MSP given by MALDI-Biotyper v.3.0. software and clustered according to protein mass profile . The CCI tool from MALDI-Biotyper v.3.0 software was also used to assess the spectral variations within and between each sample group, according to the body part, as previously described , 45. HigThe reference MS spectra were created using spectra from legs and thoraxes of two specimens per species collected in each site or reared at the laboratory using MALDI-Biotyper software v.3.0.  . MS specAdditional file 1:Table S1. Mass peak list distinguishing mosquito species using legs as biological material, based on the Genetic Algorithm model analysis of ClinProTools. The list includes unique species-specific mass peaks. Abbreviations: Da, Daltons; m/z, mass to charge ratio. (DOCX 16 kb)Additional file 2:Table S2. Mass peak list distinguishing mosquito species using thoraxes as biologic material, based on the Genetic Algorithm model analysis of ClinProTools. The list includes unique species-specific mass peaks. Abbreviations: Da, Daltons; m/z, mass to charge ratio. (DOCX 17 kb)Additional file 3:Figure S1. Resulting MS spectra for legs (red), thoraxes (green) and mix of legs and thoraxes (blue) from Ae. albopictus specimens. Five specimens per condition, loaded in quadruplicate, were tested. a Overlay of the mean MS profile per condition. b Gel view of the MS profiles per condition. An enlargement of the m/z window including the more intense MS peak from Ae. albopictus legs is presented as a MS spectra overlay (c), and gel view (d). (TIF 23971 kb)Additional file 4:Figure S2. Principal components analysis (PCA) from MS spectra for mosquito legs and thoraxes. PCA 3-dimensional (PCA1-PCA3) image from MS spectra of legs (red dots) and thoraxes (green dots) from Ae. aegypti (a), Cx. quinquefasciatus (b), Ae. taeniorynchus (c), P. cingulata (d), Cx. atratus (s.l.) (e), Cx. nigripalpus (f) and D. magnus (g). (TIF 44631 kb)Additional file 5:Figure S3. Geographical reproducibility of the MS spectra from legs  and thoraxes  included in the DB per mosquito species. LSVs obtained for Ae. aegypti , Cx. quinquefasciatus  and D. magnus , according to origins of the MS spectra from specimens of the same species included in the DB are shown. The dashed line represents the threshold value for relevant identification (LSVs > 1.8). Abbreviation: LSV, log-score value. (TIF 35156 kb)Additional file 6:Figure S4. Experimental design for mosquito identification using two distinct compartments by MALDI-TOF MS. The advantages of the creation of a MS reference database (DB) including mosquito legs and thoraxes are indicated. (PDF 95 kb)Additional file 7:Raw MS spectra from legs and thoraxes of mosquitoes included in the MS reference database. MS spectra were obtained using a Microflex LT MALDI-TOF Mass Spectrometer . (7Z 4576 kb)