Myxococcus xanthus is a model organism to study predation and competition. M. xanthus preys on a broad range of bacteria mediated by lytic enzymes, exopolysaccharides, Type-IV pilus-based motility, and specialized metabolites. Competition between M. xanthus and prey bacterial strains with various specialized metabolite profiles indicates a range of fitness, suggesting that specialized metabolites contribute to prey survival. To expand our understanding of how specialized metabolites affect predator–prey dynamics, we assessed interspecies interactions between M. xanthus and two strains of Bacillus cereus. While strain ATCC 14579 resisted predation, strain T was found to be highly sensitive to M. xanthus predation. The interaction between B. cereus ATCC 14579 and M. xanthus appears to be competitive, resulting in population loss for both predator and prey. Genome analysis revealed that ATCC 14579 belongs to a clade that possesses the biosynthetic gene cluster for production of thiocillins, whereas B. cereus strain T lacks those genes. Further, purified thiocillin protects B. cereus strains unable to produce this specialized metabolite, strengthening the finding that thiocillin protects against predation and contributes to the ecological fitness of B. cereus ATCC 14579. Lastly, strains that produce thiocillin appear to confer some level of protection to their own antibiotic by encoding an additional copy of the L11 ribosomal protein, a known target for thiopeptides. This work highlights the importance of specialized metabolites affecting predator–prey dynamics in soil microenvironments.The soil-dwelling delta-proteobacterium  From that initial screen, two B. cereus stains displayed differential susceptibility to predation: strain ATCC 14579 is resistant to predation whereas strain T is sensitive. We therefore hypothesized that strain ATCC 14579 may produce a specialized metabolite responsible for resistance to predation by M. xanthus.To expand our understanding of ormation  in 9 relB. cereus strains, which revealed that 14579 belongs to a clade possessing the thiocillin BGC while other close relatives do not. Thus, it appears that the thiocillin BGC was horizontally acquired from other bacteria, consistent with previous findings  for thiocillins, whereas the predation-resistant 14579 encodes all components for thiocillin biosynthesis. Thiocillins are ribosomally encoded, post-translationally modified, thiazolyl peptide antibiotics and are known to inhibit the interaction between the 23S rRNA and protein L11 within the 50S ribosomal subunit of Gram-positive bacteria . Thiocilfindings . Furtherfindings .M. xanthus predation, while the addition of purified thiocillin rescues these same mutants. Lastly, we observed that viability of M. xanthus was strongly affected during competition with 14579 cells in a thiocillin-dependent manner, strongly suggesting that the antimicrobial activity of thiocillins goes beyond Gram-positive bacteria, although the mechanism of action has not been determined. To our knowledge, thiocillin is the first specialized metabolite known to directly affect the fitness of M. xanthus. Overall, these findings show that thiocillin provides an advantage for B. cereus during competition with a naturally co-occurring predator, M. xanthus.Our predation assays here demonstrate that 14579 mutants lacking thiocillin precursor-encoding genes are sensitive to 22.1Myxococcus xanthus and Bacillus strains used in this study are listed in M. xanthus cultures were grown in liquid CYE media to mid-log phase before processing  to a final concentration of 2×109 cells/ml  were mixed at a ratio of 1:50 and 7 μL were spotted onto CFL starvation media  (The cells/ml . Predato mL H2O]  and incu mL H2O] . Molecul2.3All assays were monitored by microscopy using a Nikon SMZ10000 dissecting microscope. Images were taken using a QImaging Micropublisher CCD camera and processed with Qcapture software.2.4Bacillus cereus strains T and ATCC 14579 were sequenced at the University of Wisconsin Milwaukee School Freshwater Sciences Great Lakes Genomic Center using the Pacific Biosciences (PacBio) RSII SMRTcell system. Resulting reads were then assembled using Canu v.1.6  (B. cereus strains T and ATCC 14579 were annotated for open reading frames (ORFs) using Prodigal under default settings. Subsequent ORF calls were translated and given protein annotations using the PFAMs database  . FASTA fdatabase  under “sdatabase . FASTA fdatabase . To perf2.6B. cereus species relative to the presence of the thiocillin BGC, we examined 109 publicly available strains’ genomes (from the RefSeq database) including the original ATCC 14579 sequence (accession number AE016877.1). See http://tree.bio.ed.ac.uk/software/figtree/).To provide phylogenetic relationships between the 33.1M. xanthus can prey upon a large variety of bacteria, some strains are resistant to predation . Predation is visible as clearing of the prey colony relative to the buffer control, while resistance is indicated by a ring of cells at the edge of the original spot  were found to be sensitive to predation by M. xanthus, like strain T. Furthermore, we tested various mutant thiocillin strains  each of which lack antimicrobial activity against B. subtilis but retain the capacity to induce biofilm formation in B. subtilis  were determined on selective media that supported either the growth of B. cereus (LB) or M. xanthus (CYE with kanamycin). B. cereus strains were evaluated at 24 h when no growth of M. xanthus was detected. The results were normalized to the starting number of CFUs for each experiment (B. cereus mutants (∆tclE-H and T4V-producing strains) showed survival rates that were below 1%. In contrast, about 23% of the B. cereus ATCC 14579 cells survived, indicating that thiocillin provides a distinct advantage against predation by M. xanthus , consistent with previous reports indicating that only 4% of B. cereus strains possess thiocillin-like BGCs (B. cereus genomes lack the core tclA-tclX genes based on a BLAST-P cutoff (E < 10e-5) utilized by antiSMASH  as well as several strains that produce variant forms of thiocillin ; the assays reveal that only native thiocillin promotes survival of B. cereus in the presence of M. xanthus. We also show that purified thiocillin transiently protects non-thiocillin producing strains in a dose-dependent manner, including E. coli, from predation by M. xanthus. Together, these results indicate that the specialized metabolite, thiocillin, is important for protection from M. xanthus predation.Competition between different bacterial species is diverse and relies on many different strategies. In this study, we have focused on the use of specialized metabolites to influence predator–prey dynamics thought to occur in soil microenvironments. We show that Pseudomonas aeruginosa and Acinetobacter baumannii were found to be sensitive to thiostrepton (P. aeruginosa is sensitive to thiocillin and micrococcin (B. cereus ATCC 14579 significantly affects M. xanthus survival during predation assays, and that both predator and prey display losses in viability when thiocillin is present. In contrast, the antibiotic-null T4V variant of thiocillin did not protect B. cereus from predation and B. cereus cells producing the T4V thiocillin variant allowed M. xanthus predator cells to grow. To our knowledge, this is the first report of a bacterial specialized metabolite that negatively influences M. xanthus viability.Thiopeptides have not been widely reported to have antimicrobial activity against proteobacteria, although strepton , and P. rococcin . Here weB. cereus strains leads to the conclusion that a small clade of B. cereus acquired the thiocillin BGC, likely through a horizontal gain of function event, enabling this subset of strains to produce this specialized metabolite. PacBio sequencing revealed that B. cereus strain T lacks the BGC for thiocillin production while strain 14579 possesses it. Further analysis of publicly available B. cereus metagenomes revealed that 5 strains closely related to 14579 also possess the BGC and therefore likely can produce thiocillin whereas no other members of the broader B. cereus group possess genes related to thiocillin production. In support of our hypothesis, it is known that other genera, including Streptomyces, possess a similar BGC and are capable of thiocillin production. Because these bacteria are also soil inhabitants and live in similar ecological niches as Bacilli, horizontal gene transfer from Streptomyces species may account for the observed acquisition of this BGC within the Bacilli (Our analysis of the genomes of 109  Bacilli .rplK located near rpoA and rpoB. The identity and conservation of TclQ and TclT within the thiocillin-producing clade of 5 closely related strains of B. cereus is consistent with recent acquisition via horizontal gene transfer. We speculate that M. xanthus is inhibited by thiocillin at concentrations suitable to allow for inhibition of protein translation via L11 (M. xanthus cytoplasm and its mechanism of action are ongoing. Overall, this work demonstrates that thiocillin is an important contributor to survival of B. cereus ATCC 14579 during interspecies interactions with the predator M. xanthus. This specialized metabolite study extends our understanding of the chemical weaponry utilized between organisms known to interact with M. xanthus.It is known that thiocillin acts to inhibit L11 interactions with 23S rRNA to affect translation . Thus, t via L11 . DetaileThe datasets presented in this study are deposited in the NCBI database under accession numbers CP130491, CP138336, and CO138337.SM: Conceptualization, Formal analysis, Methodology, Visualization, Writing – original draft, Writing – review & editing, Investigation. OD: Methodology, Software, Visualization, Writing – review & editing. SA: Methodology, Software, Writing – review & editing. FS: Methodology, Software, Writing – review & editing. SK: Writing – review & editing. ES: Conceptualization, Supervision, Writing – review & editing. JK: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing.


Porphyromonas gingivalis were assessed over a 20-year period in the United States. Subgingival P. gingivalis was cultured pre-treatment from 2193 severe periodontitis patients during three time periods: 1999–2000 (936 patients), 2009–2010 (685 patients), and 2019–2020 (572 patients). The clinical isolates were tested for in vitro resistance to 4 mg/L for clindamycin and doxycycline, 8 mg/L for amoxicillin, and 16 mg/L for metronidazole, with a post hoc combination of data for metronidazole plus amoxicillin. Clindamycin-resistant P. gingivalis was significantly more prevalent in 2009–2010 (9.1% of patients) and 2019–2020  as compared to 1999–2000 (0.6%). P. gingivalis resistance to amoxicillin also significantly increased from 0.1% of patients in 1999–2000 to 1.3% in 2009–2010 and 2.8% (28-fold increase) in 2019–2020. P. gingivalis resistance to metronidazole, metronidazole plus amoxicillin, and doxycycline was low , and statistically unchanged, over the 20-year period. These findings are the first to reveal marked increases over 20 years in clindamycin-resistant and amoxicillin-resistant P. gingivalis in United States periodontitis patients. Increased antibiotic resistance of P. gingivalis and other periodontitis-associated bacteria threatens the efficacy of periodontal antimicrobial chemotherapy. Antibiotic resistance patterns of the major human periodontal pathogen  The World Health Organization and the United Nations have declared the rapid rise in antibiotic resistance in pathogenic bacteria as a global crisis threatening control of bacterial infections . The UniPorphyromonas gingivalis is a major periodontal disease bacterial pathogen [P. gingivalis is strongly associated with severe periodontitis [P. gingivalis has been linked to adverse disruption of the gut microbiome [pathogen . This Grpathogen , expresspathogen ,10,11,12pathogen . P. gingdontitis  and peridontitis  and may dontitis . Extraorcrobiome , developcrobiome , and thecrobiome ,24,25,26P. gingivalis from deep periodontal pockets, predisposing patients to impaired treatment outcomes [P. gingivalis-influenced systemic diseases [P. gingivalis persisting in their post-treatment subgingival microbiota [Conventional non-surgical and surgical periodontal therapy may fail to adequately remove outcomes ,29,30,31outcomes ,33,34,35diseases ,24,25,26crobiota ,42,43,44crobiota . P. gingivalis in the United States [Porphyromonas gingivalis clinical isolates in the United States prior to 2010 were rarely antibiotic-resistant [P. gingivalis resistant to clindamycin, amoxicillin, metronidazole, metronidazole plus amoxicillin, or doxycycline [Little is known about the present-day antibiotic susceptibility of periodontal d States ,47. Porpesistant ,48, withycycline . P. gingivalis in the United States has changed similarly to recent increases in the antibiotic resistance of other anaerobic bacteria at non-oral infection sites [P. gingivalis in United States periodontitis patients over a 20-year period.Because of a lack of longitudinal surveillance data, it is not known if the antibiotic resistance profile of subgingival on sites . To addrP. gingivalis, and diagnosed with severe periodontitis  [P. gingivalis in 1999–2000, 685 in 2009–2010, and 572 in 2019–2020, resulting in an overall total of 2193 study patients. The study patients were selected for three time periods over 20 years  from a retrospective search of consecutive de-identified laboratory records at the Oral Microbiology Testing Service (OMTS) Laboratory at Temple University School of Dentistry, Philadelphia, Pennsylvania. Patients were identified from the record search and included in the present study, forming 3 patient groups as their pre-treatment subgingival biofilms were consecutively evaluated by the OMTS Laboratory. The patients were all adults aged ≥ 35 years old, culture-positive for subgingival ontitis)  by perioEach patient clinically exhibited interproximal probing depths > 6 mm with bleeding on probing on ≥3 teeth, which strongly correlates  with severe periodontal attachment loss in adult patients . AvailabThe OMTS Laboratory was licensed during the 20-year study time period for high-complexity bacteriologic analysis and bacterial susceptibility testing by the Pennsylvania Department of Health and was federally certified by the United States Centers for Medicare and Medicaid Services to be in compliance with Clinical Laboratory Improvement Amendments (CLIA)-mandated proficiency testing, quality control, patient test management, personnel requirements, and quality assurance standards required of clinical laboratories engaged in diagnostic testing of human specimens in the United States . All labThis study was approved by the Temple University Human Subjects Institutional Review Board and was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2013. The Temple University Human Subjects Institutional Review Board reviewed the study protocol and judged it to be exempt from further ethical approval, since the retrospective analysis of de-identified laboratory data did not involve any patient contact, interaction, or intervention.P. gingivalis was isolated from pre-treatment subgingival biofilm samples from each study patient. The subgingival specimens were obtained by the patient’s diagnosing periodontists, following a standardized subgingival sampling protocol previously described [2, 10% H2, and 5% CO2. escribed . After iescribed . After tescribed . In brieescribed . Then, 0escribed  comprisiP. gingivalis was identified on EBBA primary isolation plates as circular, dome-shaped, dark-pigmented (brown to tan), raised surface colonies that lacked brick-red autofluorescence under long-wave ultraviolet light (365 nm wavelength) [P. gingivalis. The proportional recovery of P. gingivalis from each patient was calculated as the percentage of P. gingivalis colony-forming units relative to total subgingival anaerobic viable counts, as determined on nonselective EBBA primary isolation plates. The detection threshold for P. gingivalis on EBBA is estimated to be 1 colony per 500–1000 colonies  [elength)  but exhielength) . A subseelength) , with 10 counts) .P. gingivalis antibiotic resistance testing was performed using a direct plating method as previously described [r2 = 0.99) with the Clinical and Laboratory Standards Institute (CLSI)-approved agar dilution susceptibility assay for identifying antibiotic-resistant periodontal microorganisms [In vitro escribed , which crganisms . P. gingivalis and other putative periodontal bacterial pathogens in the OMTS Laboratory during the 20-year study period. After anaerobic incubation for 7 days, P. gingivalis isolates growing on antibiotic-supplemented media were considered resistant to the incorporated antibiotic concentration, as previously described [P. gingivalis isolates growing on antibiotic-supplemented media were subjected to MALDI-TOF mass spectrometry identification testing [P. gingivalis. Bacteroides thetaiotaomicron ATCC 29741, Clostridium perfringens ATCC 13124, and a multi-antibiotic-resistant clinical periodontal isolate of Fusobacterium nucleatum were used as positive and negative quality controls for all antibiotic resistance testing on drug-supplemented EBBA plates. In brief, 0.1 mL aliquots of subgingival biofilm dilutions for each patient were inoculated onto EBBA primary isolation plates supplemented with either amoxicillin at 8 mg/L, clindamycin at 4 mg/L, doxycycline at 4 mg/L, or metronidazole at 16 mg/L , which represent non-susceptible or resistant breakpoint drug concentrations for amoxicillin, clindamycin, and metronidazole against anaerobic bacteria as recommended by the CLSI , and forescribed . A subse testing  as part P. gingivalis clinical isolates exhibiting in vitro resistance to 4 mg/L clindamycin with the direct plating method [P. gingivalis strain were first prepared and adjusted to a 0.5 McFarland turbidity standard using sterile VMG I anaerobic dispersion solution and then streaked with sterile cotton-tipped swabs onto EBBA plates. After drying, predefined antibiotic gradient strips  containing clindamycin were applied onto the inoculated media surfaces. After 48 h of anaerobic incubation at 37 °C, the intersection between the border of P. gingivalis growth and the antibiotic gradient strip drug scale was read to determine in vitro minimum inhibitory concentration (MIC) values, following the manufacturer’s instructions. Clindamycin-resistant strains of P. gingivalis were identified using CLSI interpretative guidelines for clindamycin against anaerobic bacteria [B. thetaiotaomicron ATCC 29741 was used as a quality control strain in clindamycin gradient strip susceptibility testing.In a separate pilot study, 6 g method  were subg method . Pure cebacteria . B. thetP. gingivalis and the prevalence and subgingival proportions of antibiotic-resistant P. gingivalis. Means and standard deviation (SD) were calculated for continuous variables, and frequencies and percentages for categorical variables. Antibiotic resistance data were combined and analyzed post hoc from amoxicillin- and metronidazole-supplemented EBBA culture plates. This was based on previous studies demonstrating excellent agreement (98.5%) between periodontal pathogen antibiotic resistance patterns as determined from EBBA plates jointly supplemented with both amoxicillin and metronidazole, as compared to a post hoc combination of findings from EBBA plates individually supplemented with amoxicillin or metronidazole [P. gingivalis between the patient groups. Fisher’s exact test examined differences between the patient groups in the proportion of males, and the percentage of patients with antibiotic-resistant P. gingivalis in 2009–2010 and 2019–2020, as compared to 1999–2000, as well as between 2009 and 2010 and 2019 and 2020, for each of the tested antibiotics. A p-value ≤ 0.05 was required for statistical significance. The PC-based STATA/SE 16.1 for Windows  64-bit statistical software package was used in the data analysis.Descriptive analysis characterized the study patients and tabulated per patient the proportional cultivable recovery of nidazole . One-wayTest results were within expected ranges and outcomes for the three quality control bacterial strains subjected to in vitro amoxicillin, clindamycin, doxycycline, and metronidazole breakpoint resistance testing, as well as the quality control strain evaluated with clindamycin gradient strips.p-values > 0.05). The mean age of 1999–2000 patients was slightly but significantly lower, and the mean percentage of subgingival P. gingivalis in 2019–2020 patients was slightly but significantly lower than in the other two patient groups . No statistically significant gender differences were found between the three patient groups  but were not significantly different between patients evaluated in 2009–2010 and 2019–2020 (p = 0.922).In vitro resistance of 999–2000 . DiffereP. gingivalis to 8 mg/L amoxicillin was also rare in 1999–2000, with resistant isolates found in only one (0.1%) patient  and 2019–2020  as compared to 1999–2000 (p ≤ 0.002), but not between patients in 2009–2010 and 2019–2020 (p = 0.069). In vitro resistance of  patient . A signiP. gingivalis resistance, and no statistically significant temporal changes in drug resistance patterns, were found with metronidazole at 16 mg/L, the joint effects of metronidazole at 16 mg/L plus amoxicillin at 8 mg/L, or doxycycline at 4 mg/L  in vitro t 4 mg/L . Only 1 P. gingivalis clinical isolates identified as resistant in vitro to 4 mg/L clindamycin with the direct plating method and then evaluated with gradient strip susceptibility testing were confirmed to be resistant to clindamycin, with all MIC values > 4 mg/L.All P. gingivalis resistant to breakpoint concentrations of clindamycin significantly increased by 15-fold (to 9.3% of patients) and resistant to amoxicillin by 28-fold (to 2.8% of patients) over a 20-year period. In comparison, no significant increases over 20 years were found with the initially low baseline levels of P. gingivalis resistance to metronidazole, metronidazole plus amoxicillin, and doxycycline. These findings are the first United States-specific data documenting 20-year temporal changes in the prevalence of antibiotic-resistant P. gingivalis in subgingival biofilms of periodontitis patients. With a global rise in antibiotic resistance documented for many bacterial species in the human microbiome ,64, it iP. gingivalis and other periodontal pathogens varies considerably between countries and geographic regions [P. gingivalis over time in the present study may stem from horizontal transfer and phenotypic expression of resistance genes for these antibiotics from other oral microorganisms [Importantly, the prevalence of antibiotic-resistant  regions ,6, which regions ,66 found regions , and mor regions . Since a regions ,69, the rganisms ,70, spurrganisms ,67.P. gingivalis to a 9.3% prevalence level in the United States is alarming and exceeded worldwide only by a 23.5% frequency of clindamycin-resistant P. gingivalis strains in Colombia in South America, where there is greater population exposure to antibiotic-containing over-the-counter products than in the United States [P. gingivalis is reported to be absent or low in the Netherlands, Germany, Switzerland, Japan, Spain, Italy, Turkey, Iran, and Brazil [erm antibiotic resistance genes [The increase in clindamycin-resistant d States ,72. Meand Brazil ,73. Consce genes . P. gingivalis-associated refractory cases of periodontitis and arresting progressive periodontal attachment loss [P. gingivalis clinical isolates [P. gingivalis is often part of the associated microbial etiology [P. gingivalis, as documented in the present study, these recommendations likely need to be re-considered and appropriately modified. In our opinion, empirical use of clindamycin in the treatment of P. gingivalis-associated periodontitis and periodontal abscesses in the United States should be minimized in the absence of P. gingivalis antibiotic susceptibility testing to limit further emergence of clindamycin-resistant P. gingivalis strains in the population. These findings have important clinical implications for United States periodontitis patients. Clinical treatment failures in periodontics have occurred when antibiotic therapy was given to patients where the targeted periodontal pathogens were resistant to the selected medication . Similarent loss . At the isolates , supportisolates ,38,42,45isolates , where Petiology . HoweverP. gingivalis in vitro resistance to amoxicillin increased to 2.8% of isolates in the United States over the 20 years under study. This compares to a much higher resistance rate (25%) to amoxicillin among P. gingivalis clinical isolates in Columbia [P. gingivalis resistance to amoxicillin elsewhere in the world [Columbia  and sligce rate 2% to amoxhe world ,73. P. gingivalis resistance to metronidazole and metronidazole plus amoxicillin in United States patients is consistent with global data [P. gingivalis metronidazole resistance rate (≥21.6%) detected in Columbia [P. gingivalis resistant in vitro to metronidazole, and none had P. gingivalis resistant to both metronidazole and amoxicillin, out of the 2193 P. gingivalis-positive periodontitis patients studied. The low-to-negligible level of metronidazole resistance among P. gingivalis and other anaerobic periodontal pathogens in United States periodontitis patients [The low-to-negligible levels of bal data ,6,73, exColumbia ,72. Onlypatients ,47,81 supatients ,42,43,44P. gingivalis in the United States  and that resistant to doxycycline by 37.7-fold (to 11.3% of patients) in United States periodontitis patients [The increased antibiotic resistance of as micra , anotheras micra . Over a patients . There iP. gingivalis was achieved using standardized and validated phenotypic criteria documented to have 100% concordance with definitive identification of P. gingivalis via MALDI-TOF mass spectrometry [P. gingivalis via molecular 16S rRNA gene sequencing analysis [P. gingivalis were merely due to laboratory error, changes in laboratory personnel, or examiner drift over time. Strengths of the present study include the relatively large number of severe periodontitis patients evaluated and the fairly long evaluation period of 20 years. Reliable identification of cultivable trometry , which ianalysis . The staanalysis , was empP. gingivalis than the other two groups (P. gingivalis in all patient groups averaged >10% of the cultivable subgingival microbiota, it is unlikely that these relatively small group differences impacted the present study results. More detailed demographic information for the study patients, such as their history of antibiotic and antidepressant drug use, was not available. The patients were not necessarily statistically representative of periodontitis patients throughout the United States. No clinical or radiographic evaluations of the study patients were made by calibrated examiners separate from the diagnostic information submitted by the participating private practice periodontists. MIC values of the test antibiotics against P. gingivalis were not determined, and carriage of antibiotic resistance genes by the P. gingivalis clinical isolates was not studied. It is important to note carriage of antibiotic resistance genes does not necessarily lead to phenotypic expression of bacterial antibiotic resistance since such genes in periodontal bacterial pathogens often remain silent and unexpressed [P. gingivalis resistance to clindamycin and amoxicillin in United States periodontitis patients. The present study data have a number of limitations. Different patients were evaluated at each of the study time periods, with no longitudinal cohort of patients evaluated. The patient groups differed, with one group having a slightly younger average patient age and another with <3% lower mean proportional levels of subgingival o groups . Howeverxpressed . AdditioP. gingivalis and may serve as part of a new antibiotic resistance baseline for dental antibiotic stewardship programs in the United States [Nevertheless, the present microbiological surveillance data better inform dental professionals in the United States about antibiotic resistance trends in the major human periodontal pathogen d States . AdditioP. gingivalis markedly increased in prevalence over a 20-year period in United States adults with severe periodontitis, whereas no significant temporal changes were found with low levels of P. gingivalis resistance to metronidazole, metronidazole plus amoxicillin, and doxycycline. These findings are the first to document the emergence of antibiotic-resistant periodontal P. gingivalis in the United States. They indicate a need for clinical caution when employing clindamycin or amoxicillin by itself in the treatment of United States periodontitis patients. Increased antibiotic resistance in P. gingivalis and other periodontitis-associated bacteria threatens the efficacy of periodontal antimicrobial chemotherapy. Systematic surveillance of the antibiotic susceptibility of target bacterial pathogens in periodontitis is necessary to detect changes in antibiotic resistance patterns, particularly when systemic/local antibiotic therapy is employed in clinical periodontal practice. The present study found clindamycin-resistant and amoxicillin-resistant


Helicobacter pylori. H. pylori induces chronic inflammation affecting the gastric epithelium, which can lead to DNA damage and the promotion of precancerous lesions. Disease manifestations associated with H. pylori are attributed to virulence factors with multiple activities, and its capacity to subvert host immunity. One of the most significant H. pylori virulence determinants is the cagPAI gene cluster, which encodes a type IV secretion system and the CagA toxin. This secretion system allows H. pylori to inject the CagA oncoprotein into host cells, causing multiple cellular perturbations. Despite the high prevalence of H. pylori infection, only a small percentage of affected individuals develop significant clinical outcomes, while most remain asymptomatic. Therefore, understanding how H. pylori triggers carcinogenesis and its immune evasion mechanisms is critical in preventing gastric cancer and mitigating the burden of this life-threatening disease. This review aims to provide an overview of our current understanding of H. pylori infection, its association with gastric cancer and other gastric diseases, and how it subverts the host immune system to establish persistent infection.Gastric cancer is a challenging public health concern worldwide and remains a leading cause of cancer-related mortality. The primary risk factor implicated in gastric cancer development is infection with  Gastric cancer (GC) remains among the deadliest cancers worldwide, taking close to one million lives annually. GC is the fourth leading cause of cancer-related deaths . AccordiH. pylori was initially described by Australian scientists Barry Marshall and Robin Warren in 1982 ) . H. pyloresponse ,47. Severesponse ,49. The nfection ,51, whicividuals ,53. The ividuals .H. pylori infection. IBD is an umbrella term for chronic relapsing–remitting digestive disorders, including Crohn’s Disease (CD) and ulcerative colitis (UC). H. pylori infection was regarded as a possible IBD risk factor due to similarities in the immunobiology of H. pylori infection and IBD. However, multiple studies have suggested an inverse association between H. pylori infection and the prevalence of IBD. There are essential epidemiological differences between H. pylori and IBD. H. pylori infection is more prevalent in developing countries than in developed countries. In contrast, the opposite is true for IBD, which is more prevalent in developed than developing countries. The prevalence of IBD is steadily increasing in developed countries, while rates of H. pylori infection are decreasing. IBD is less frequent among individuals who are H. pylori seropositive when compared to seronegative subjects [H. pylori infection [H. pylori infection regardless of age, ethnicity, and detection methods [H. pylori might exert a protective effect against IBD.IBD is another disease noted to have an inverse association with subjects . A meta-nfection . In that methods . These oH. pylori possesses a panel of virulence mechanisms that allow it to survive in the harshest environment of the body, which is the stomach (pH between 1.5 and 3.5) [H. pylori to promote GC include multiple virulence factors, described below. and 3.5) , colonizand 3.5) ,61,62,63H. pylori urease is a critical virulence factor for H. pylori. It is the most abundant protein expressed by H. pylori, representing 10–15% of total protein by weight. [H. pylori urease is a multimeric nickel-containing enzyme consisting of two subunits, α and β subunits, of 29.5 kDa and 66 kDa, respectively, which catalyzes the hydrolysis of urea into carbonic acid and ammonia. Six α and six β subunits aggregate to form a multimeric complex of 550 kDa. [H. pylori.  weight. . H. pylo550 kDa. . The relH. pylori urease can play a role in pathogenesis beyond its enzymatic action. Urease influences the host response by activating monocytes and polymorphonuclear leucocytes, leading to inflammation and damage to the epithelium [H. pylori to gastric epithelial cells via association with CD74 [H. pylori urease B was shown to bind CD74 on gastric epithelial cells and induce NF-κB activation and interleukin-8 (IL-8) production [ithelium . Approxiithelium ,67. The ithelium . This suithelium ,70, whicithelium ,72. Thisithelium . InteresH. pylori via the induction of hypoxia-induced factor-1α (HIF-1α) [H. pylori urease on HIF-1α involved TLR2 activation by urease B, recently found to bind TLR2 [An interesting recent report showed that urease may contribute to the carcinogenic potential of (HIF-1α) . HIF-1 i(HIF-1α) . A nonca(HIF-1α) , and boo(HIF-1α) . The invind TLR2 , and wasH. pylori to the gastric epithelium is a key step for colonizing the gastric mucosa. A bacterial adhesin that contributes to this process and determines colonization density, encoded by the babA2 gene, is the blood group antigen binding adhesin (BabA), which bind to Lewis B blood group antigens (Leb) on gastric epithelial cells [H. pylori strains into BabA high producers (BabA-H) with Leb binding activity, BabA low producers (BabA-L) without Leb binding activity, and BabA-negative strains (lacking the babA gene) [babA2 gene with lower levels of BabA expression appeared to be associated with the highest GC risk [Adhesins are bacterial cell-surface proteins contributing to the bacterial attachment to host cells. The adhesion of al cells ,81. One bA gene) . Studies GC risk . H. pylori outer inflammatory protein A (OipA), or HopH, is an outer membrane protein that behaves as an adhesin and stimulates IL-8 secretion. Yamaoka’s group showed that the functional status (on or off) of OipA is controlled by a slipped-strand mispairing mechanism that depends on the number of CT dinucleotide repeats in the 5′ region of the oipA gene [H. pylori strains with the oipA gene on “on” status are associated with greater colonization density, higher IL-8 production, and, consequently, higher neutrophil infiltration. Further, strains with the oipA gene on “on” status significantly associate with GC and PUD. HopQ is another outer membrane protein that behaves as an adhesin. HopQ binds to the human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) on host cells. HopQ alleles are usually found in H. pylori strains that contain the cag pathogenicity island  [H. pylori CagA oncoprotein into epithelial cells [The ipA gene . H. pylod below) . The binal cells , and is al cells ,87. H. pylori binding to gastric epithelial cells and enhancing colonization. They also induce the production of the inflammatory cytokines IL-6 and IL-8 [alpAB or alpA genes blocked or decreased the binding of H. pylori to epithelial cells [H. pylori to attach to gastric epithelial cells, the deletion of AlpAB alone reduced bacterial binding by 60–70%, and reduced H. pylori colonization of mice. The adherence-associated lipoproteins A and B (AlpA/AlpB) are outer membrane proteins participating in and IL-8 . Proof oal cells ,90. Moreal cells . DespitesabA gene, which is present in the H. pylori cag pathogenicity island (described below) [H. pylori primarily colonize [H. pylori adherence to the epithelium, a crucial step in establishing infection. In addition to sialic acid binding, SabA has been shown to interact with other host molecules, such as laminin [H. pylori to adhere to diverse types of gastric epithelial cells, and promote colonization in different stomach regions. Sialic acid-binding adhesin A (SabA) is another crucial virulence factor that facilitates adherence to the gastric epithelium and colonization of the gastric mucosa . SabA isd below) . SabA iscolonize ,94. The  laminin , fibrone laminin , and blob enhances the pathogenicity of H. pylori T4SS (discussed below) and triggers cellular proliferation and expression of proinflammatory cytokines, and oncogenic-related factors [Various allelic forms of these adhesins and OMPs, such as BabA, SabA, OipA, and HopQ, have been linked to GC. For instance, OipA activates epidermal growth factor receptor (EGFR), which sets off AKT and β-catenin cascades, and stimulates cellular proliferation and oncogenic transformation via LEF/TCF transcription factors. The translocation of β-catenin into the nucleus, and subsequent association with LEF/TCF transcription factors, are critical in Wnt signaling, which is triggered abnormally in several cancers . The bin factors . H. pylori cag pathogenicity island (cag PAI) is a region of about 40 kb DNA that encodes the cytotoxin-associated gene A (cagA), and approximately 27–30 additional genes that encode proteins that form a type IV secretion system (T4SS) [H. pylori. CagA is a 120–140 kDa protein whose expression by H. pylori represents the most potent risk factor for GC [H. pylori strains and are classified into four types based on the variation of flanking regions and orders of spacers. The four EPIYA motif types are EPIYA-A, -B, -C, and -D. CagA in Western strains of H. pylori contains EPIYA-A, EPIYA-B, and EPIYA-C motifs. In contrast, CagA from East Asian H. pylori strains have EPIYA-A, EPIYA-B, and EPIYA-D, but not the EPIYA-C motif [The m (T4SS) . The T4Sr for GC . The Cagr for GC ,100. Oncr for GC . The mag-C motif . Higashi-C motif . These m-C motif . The lev-C motif . Because-C motif , which mCagA has also been reported to impact epithelial cells in a tyrosine phosphorylation-independent manner. CagA has a conserved repeat motif, responsible for phosphorylation-independent activity (CRPIA: FPLKRHDKVDDLSKVG), in its C-terminal region, different from the EPIYA motifs. The CRPIA motif in nonphosphorylated CagA interacts with c-Met, the hepatocyte growth factor scatter factor receptor, which plays a role in the invasive growth of neoplastic cells. CagA binds c-Met, which then binds phospholipase Cγ (PLCγ) and turns on phosphatidylinositol 3-kinase/Akt signaling. This initiates β-catenin and NF-κB signaling, leading to proliferation and inflammation ,105. GrocagA− mutant strain developed these preneoplastic lesions [H. pylori cagA gene alone was transfected into cells have shown that its expression significantly affects the transfected cells. In one report, CagA transfected into human gastric epithelial cells revealed that CagA targets partitioning-defective 1 (PAR1). The CagA–PAR1 interaction causes junctional and polarity defects that release cells from growth-inhibitory signals and promote neoplasia [Support for the role of CagA as an oncoprotein was obtained from multiple observations. A study by Peek’s group that included Mongolian gerbils infected with the carcinogenic strain 7.13 resulted in gastric dysplasia and cancer in >50% of gerbils infected with the wild-type strain. In that study, none of the gerbils infected with the  lesions . Variouseoplasia . Using teoplasia . In thateoplasia . That ineoplasia . The miceoplasia . These oAnother bacterial product translocated by the T4SS into gastric epithelial cells is peptidoglycan, which is recognized by NOD1 (Nucleotide Binding Oligomerization Domain Containing 1), a cytoplasmic pattern recognition receptor ,113,114,H. pylori vacuolating toxin (VacA) is a major secreted virulence factor without known homologs in other bacterial species except for H. cetorum, a Helicobacter species found in the stomachs of marine mammals [H. pylori, and has pleiotropic effects on host cells. The pores induced by VacA in the membranes of the gastric epithelial lining lead to the leakage of small molecules and ions from the cells. This damage to the cells disrupts the epithelium’s barrier function, facilitating invasion and colonization of the stomach. VacA can also induce apoptosis in immune cells and gastric epithelial cells. This contributes to gastric inflammation and the progression of H. pylori-associated diseases. VacA also modulates the host immune response to H. pylori by suppressing the activation of specific immune cells and inhibiting the production of cytokines. This permits H. pylori to evade immune-mediated clearance and establish a persistent infection in the stomach.The  mammals . The VacH. pylori VacA binds to sphingomyelin on lipid rafts [VacA is first synthesized as a 140 kDa protoxin with an N-terminal signal peptide, a central region representing the toxin, and a C-terminus that mediates transport function. The central region (about 88 kDa), the mature virulent form of the toxin, is secreted after processing and is further cleaved into an A subunit and B subunit of 33 and 55 kDa, respectively. The p33 form was initially regarded as the pore-forming subunit, and the p55 form was ascribed to the cell binding function ,118. Howid rafts , and is id rafts . VacA isid rafts ,124. Vacid rafts , cytochrid rafts , and mitid rafts , ultimatH. pylori have the vacA gene, there is substantial diversity in the gene, which includes five heterogenic regions with considerable sequence differences. The first three regions identified correspond to the signal (s) sequence, middle (m), and intermediate (i) [vacA gene, were described [H. pylori strains with m1 VacA represent a higher risk factor for gastric ulcers [vacAs1i1m1 allelic combination is strongly associated with the existence of cagA [vacAs1m1 and cagA concurrently have a 4.8-fold greater risk of inducing preneoplastic lesions than other H. pylori strains [Even though all strains of iate (i) . There aescribed ,130. Theescribed . While sc ulcers , and Vacc ulcers ,134. Yam of cagA , and a r strains . The i r strains . The m r strains . The rolH. pylori’s ability to evade host immunity since it can act on several immune cells affecting innate and adaptive immunity. VacA impairs the functions of macrophages [++-calmodulin-dependent phosphatase calcineurin [H. pylori and highlight its important role in the pathogenesis of H. pylori-associated diseases.VacA is crucial in rophages ,140, eosrophages , mast cerophages , dendritrophages , and lymrophages ,145. Duerophages . Macrophrophages . VacA alH. pylori [napA gene expressed by most H. pylori strains. It is a 164-amino acid protein secreted by H. pylori and can be found in both the bacterial cell wall and the extracellular milieu. NAP’s three-dimensional structure was characterized by Zanotti et al. as having a quaternary structure consisting of a spherical dodecamer containing ∼17 kDa identical subunits with a four-helix bundle structure similar to bacterial ferritins [HP-NAP, or NapA, is another critical virulence factor of . pylori . HP-NAP erritins . HP-NAP H. pylori can cause DNA damage and mutations that lead to the initiation of cancer [H. pylori. Still, they can also cause collateral damage to the gastric mucosa, leading to inflammation and tissue injury [H. pylori-related diseases, including GC.Among the critical functions of HP-NAP is its ability to activate neutrophils to generate reactive oxygen species (ROS)  and reacf cancer . HP-NAP f cancer ,152, whif cancer ,154,155.f cancer . Cytokine injury , which ae injury . Overall+ T helper (Th) cell responses to Th1 responses by inducing the production of IL-12 and IL-23 by neutrophils, monocytes, and dendritic cells [H. pylori infection. However, excessive production of IL-17 can also contribute to chronic inflammation and tissue damage. In addition to its influence on innate immune cells, HP-NAP affects the adaptive immune response. HP-NAP crosses the epithelial barrier and promotes the skewing of CD4ic cells ,159. ThoH. pylori’s survival and pathogenesis. H. pylori GGT (HpGGT) is a type I membrane protein anchored to the bacterial outer membrane. HpGGT is highly conserved and present in all strains [H. pylori cannot take up extracellular glutamine and glutathione directly, this enzyme allows H. pylori to use extracellular glutamine and glutathione as sources of glutamate for subsequent use in the tricarboxylic acid cycle [H. pylori in acidic conditions, as H. pylori ggt- isogenic mutant strains cannot colonize the gastric mucosa in animal models of infection, or do so less efficiently [H. pylori ggt- mutants could not be recovered from the stomachs of mice at 3–60 days postinfection [GGT is an enzyme that plays a crucial role in  strains . It invo strains . HpGGT h strains . Since Hid cycle . HpGGT iiciently ,164. A sH. pylori infection also affects mesenchymal stem cells (MSCs) by inducing their migration to the gastric mucosa, where they may contribute to GC development by differentiating into epithelial cells or assisting in angiogenesis [HpGGT has wide-ranging effects in gastric epithelial cells and plays a significant role in the pathogenesis of Hp-induced GC. HpGGT functions through multiple pathways to damage the gastric epithelial barrier. HpGGT induces epithelial cell apoptosis by a mitochondria-dependent pathway and reduces cell viability . HpGGT aogenesis . HpGGT wogenesis . Anotherogenesis , which iH. pylori infection. Another study showed that HpGGT induced microRNA-155 (miR-155) expression in both CCRF-CEM cells (a human T lymphoblast cell line) and primary human peripheral blood mononuclear cells [H. pylori-infected gastric mucosa [H. pylori colonization and infiltrating tumors [ggt- isogenic H. pylori mutant strains were reported to have lower Treg counts than wild-type-infected mice [HpGGT also affects immune cells directly. One of the earliest studies on the immune response effects of HpGGT showed that the enzyme could dampen T cell proliferation ,177. It ar cells . This rear cells , and reqc mucosa , promotig tumors . In suppted mice . These oH. pylori stealthily directs the expression of multiple receptors that influence T cell activity. The optimal balance between protective immunity and immune tolerance controls immune responses. T cell activity is determined by the combination of signals initiated by T cell receptor (TCR) recognition of antigen/MHC complexes and costimulatory molecules on APCs, divided into costimulatory and coinhibitory molecules , which inhibits effector T cells via PD-1 engagement on their surface, and fosters Treg cell development [H. pylori to promote B7-H1 expression by gastric epithelial cells is through the inhibition of miR-152 and miR-200b expression [H. pylori could induce the expression of B7-H1 by bone marrow-derived macrophages after binding to myosin heavy chain 9 (Myh9) on their surface [H. pylori-infected gastric epithelial cells simultaneously downregulate their expression of the costimulator B7-H2, thus, not only reducing effector T cell responses, but also altering T cell subset balances by increasing Tregs and decreasing Th17 cell numbers [H. pylori uses cagPAI to induce these effects on gastric epithelial cells, and activates the mTOR/p70 S6 kinase pathway to downregulate B7-H2 expression [H. pylori T4SS-mediated transfer of CagA and cell wall peptidoglycan (PG) fragment upregulated B7-H3 expression by gastric epithelial cells via activation of the p38MAPK signaling pathway [H. pylori effectively hijacks the regulated expression of coinhibitory molecules to avoid immunity, and may favor immune escape by developing cancer cells through these mechanisms. In addition to the various immune evasion mechanisms associated with the virulence factors described above, olecules . While telopment ,183,184.pression . They sh surface . The int numbers . This impression ,187. B7-pression ,189,190. pathway . Thus, HH. pylori’s properties since its initial identification as a human pathogen. While infection with H. pylori was initially linked with chronic gastritis and the development of peptic ulcer disease, research by multiple laboratories worldwide has established the bacterium as the most potent known risk factor for GC, one of the world’s deadliest cancers.Over the past 40 years, significant advances have been made in our comprehension of H. pylori infection, the high prevalence of the bacterium and the escalation of antibiotic resistance highlights the need for a protective vaccine. Though GC can be a devastating diagnosis, its bacterial etiology makes it a vaccine-preventable form of cancer. H. pylori has an extensive array of virulence factors that mediate pathogenesis and could represent potential vaccine targets to prevent the morbidity and mortality associated with the infection and GC. Nevertheless, vaccine development efforts have encountered various challenges. The variability among H. pylori strains, and the bacterium’s immune escape mechanisms, create significant obstacles to developing an effective vaccine. Clinical trials of candidate vaccines have yielded mixed or disappointing results.While antibiotic therapies are available to treat H. pylori vaccine is not currently available, continued research is promising and may eventually lead to an avenue to help decrease the burden of H. pylori-associated diseases. Research efforts into immune escape mechanisms used by H. pylori and GC, including the induced expression of novel immune checkpoint inhibitors, could yield candidate biomarkers to detect cancer progression in its early stages. In summary, H. pylori is the foremost risk factor for developing GC, and understanding how to override the bacterium’s immune evasion strategies is crucial for developing new approaches to treat and prevent the conditions associated with the infection.Although an


Phytophthora rubi is a primary causal agent of Phytophthora root rot and wilting of raspberry (Rubus idaeus L.) worldwide. The disease is a major concern for raspberry growers in Canada and USA. To date, no information is available on genomic diversity of P. rubi population from raspberry in Canada. Using a PCR-free library prep with dual-indexing for an Illumina HiSEQX running a 2x150 bp configuration, we generated whole genome sequence data of P. rubi isolates (n = 25) recovered during 2018 to 2020 from nine fields, four locations and four cultivars of raspberry growing areas of British Columbia, Canada. The assembled genome of 24 isolates of P. rubi averaged 8,541 scaffolds, 309× coverage, and 65,960,000 bp. We exploited single nucleotide polymorphisms (SNPs) obtained from whole genome sequence data to analyze the genome structure and genetic diversity of the P. rubi isolates. Low heterozygosity among the 72% of pathogen isolates and standardized index of association revealed that those isolates were clonal. Principal component analysis, discriminant analysis of principal component, and phylogenetic tree revealed that P. rubi isolates clustered with the raspberry specific cultivars. This study provides novel resources and insight into genome structure, genetic diversity, and reproductive biology of P rubi isolated from red raspberry. The availability of the P. rubi genomes also provides valuable resources for future comparative genomic and evolutionary studies for oomycetes pathogens. Phytophthora rubi, the primary causal agent of Phytophthora root rot and wilting (PRRW), is the most destructive pathogen of red raspberry (Rubus idaeus L.) in the Pacific Northwest regions of Canada  obtained from high throughput sequencing have recently been utilized to infer population structure, and its reproductive strategies in many species of Phytophthora such as P. capsici  generate genomic resources of P. rubi using whole genome sequencing, and ii) study the genome structure and genetic diversity of P. rubi isolates from raspberry.Although n Canada , no infon = 25) were collected over multiple years (2018 to 2020) from nine fields, and four cultivars of raspberry . After shearing, fragmented DNA was ligated with dual-indices using a KAPA Hyperprep PCR-free library kit (Roche) for Illumina according to the manufacturer’s instructions. Libraries were loaded and sequenced on an Illumina HiSEQX next-generation sequencing device running a 2 x 150 bp paired-end configuration at Admera Health LLC  according to the manufacturer’s directions. Raw paired-end sequence data for each sample are publicly available for download in NCBI BioProject accession number PRJNA856328.https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Paired-end reads were trimmed for the presence of adapters and low-quality sequences with BBDuk (https://jgi.doe.gov/data-and-tools/software-tools/bbtools/). To identify SNPs, paired-end reads were randomly subsampled to 50 million read pairs with reformat.sh (https://jgi.doe.gov/data-and-tools/software-tools/bbtools/) and then aligned to P. rubi reference genome . The processed reads were assembled using assemblers abyss-pe v2.3.5 (https://github.com/nextgenusfs/funannotate), with RNAseq data SRR10207404 . Statistical significance of r̄d test was calculated with 999 permutations of the random test. The r̄d values would be expected to be zero, if isolates are mating randomly (i.e. freely recombining)  . We carrmbining) . The advmbining)  distanceggplot2” . We excl. rubi isolates collected from diverse cultivars and locations were sequenced. The summary of total genome size, number of scaffolds, scaffold N50, total predicted genes, total predicted proteins, and percent repeat content of these isolates is shown in P. rubi genome assemblies ranged from 12.41 to 22.48%. In addition, BUSCO completeness scores ranged from 92.2 to 94.2%, with an average of 93.33%   Table 2.P rubi isolates corresponding to raspberry cultivars  can affect genotypic variability. Previous studies also reported the high genotypic diversity in clonal populations of P. infestans in Denmark  indicating that the P. rubi population from BC are predominantly clonal  on roots of six raspberry genotypes showed that the existence of three different races, suggesting a significant cultivar/isolate interaction  and accessions associated with the submission are Project: PRJEB62133 and Analyses: ERZ18306854.The datasets presented in this study can be found in online repositories. NCBI BioProject accession number is PRJNA856328. The variant data file (VCF) is available in the European Variation Archive (SS, RB, and KL conceived and designed the study. RB supervised the first author to conduct the experiment and drafting the manuscript. SS, RB, and KL curated the data. SS and ML performed all bioinformatics analyses. SS and RB wrote a first draft of the manuscript. All authors read and approved the submitted version.


Sclerotinia sclerotiorum (Lib.) de Bary is a highly destructive fungal pathogen that seriously damages the yield and quality of Brassica napus worldwide. The complex interaction between the B. napus and S. sclerotiorum system has presented significant challenges in researching rapeseed defense strategies. Here, we focus on the infection process of S. sclerotiorum, the defense mechanisms of rapeseed, and recent research progress in this system. The response of rapeseed to S. sclerotiorum is multifaceted; this review aims to provide a theoretical basis for rapeseed defense strategies. Brassica napus) is an allopolyploidy (AACC) resulting from the natural hybridization of Brassica rapa (AA) and Brassica oleracea (CC) (Rapeseed (cea (CC) . It is acea (CC) . Rapeseecea (CC) . Winter cea (CC) . Rapeseecea (CC) . Rapeseecea (CC) .Sclerotinia sclerotiorum (Lib.) de Bary is the ascomycetes’ highly destructive fungal pathogen. It is widespread and infects over 700 plant species, including many economically important crops like rapeseed, sunflower (Helianthus annuus), peanut (Arachis hypogaea), soybean (Glycine max), garden lettuce (Lactuca sativa), and sugar beet (Beta vulgaris) , and it induces a hypersensitive response (HR) in plant cells by secreting OA into plant tissues, ultimately leading to programmed cell death (PCD)  . Sclerotre cases . S. sclet tissue . Ascospot tissue . Previouth (PCD) . No vari methods .S. sclerotiorum. Developments in various tools, techniques, and expanded research on S. sclerotiorum have generated valuable insights into defense strategies for rapeseed. Here, we introduce the infection process of S. sclerotiorum, the defense strategies of rapeseed, recent research on the identification of defense-related genes in rapeseed, and the resistance strategies of transgenic rapeseed. The B. napus–S. sclerotiorum system has a complex regulatory network, and this review aims to provide a reference for future studies on S. sclerotiorum resistance strategies in rapeseed.In recent years, the increasing global demand for rapeseed has led to intensified cultivation practices, increasing the urgency for effective strategies against S. sclerotiorum is a typical necrotrophic pathogen, and recent studies have revealed that it undergoes a transient biotrophic phase after initially colonizing plant tissues before transitioning into a necrotrophic pathogen  can inactivate plant polygalacturonase-inhibiting proteins (PGIPs), promoting the dissociation of polygalacturonases (PG)-PGIP and enhancing necrotrophic virulence (PLANT DEFENSIN1.2 (PDF1.2) in A. thaliana, affecting the plant defense response  and 24 hpi, respectively. The expression changes of SA–JA crosstalk genes such as BnWRKY70, BnNPR1, BnMPK4, and BnEDS1 suggest the importance of the orderly expression of SA and JA pathways  and effector-triggered immunity (ETI). PTI relies on transmembrane pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs), while ETI involves the use of proteins encoded by intracellular resistance (R) genes. These mechanisms contribute to plant defense together . The invm system . Severalrotiorum . Variousrotiorum . Studiesathogens . These sathogens . SA and pathways . Additio defense . The horB. napus–S. sclerotiorum interaction provides a comprehensive understanding of the molecular events during S. sclerotiorum infection and the defense strategies in rapeseed. Illumina sequencing was conducted at various time points following S. sclerotiorum infection, leading to the categorization of differentially expressed genes (DEGs) into several major groups, including hydrolytic enzymes, secondary metabolites, detoxification processes, signaling pathways, developmental processes, secreted effectors, OA regulation, and the production of ROS. The results revealed a noteworthy increase in the expression of genes related to nucleic acid binding during the brief biotrophic phase (12–24 hpi), coinciding with the upregulation of genes such as the pathogen-induced cutinase A gene SsCuta and lipid degradation-related genes SS1G_09557, SS1G_01953, and SS1G_11930; these lipolytic enzymes also seem to be involved in cuticle penetration facilitated by the mechanical pressure applied by infection cushions during the same period (SsACP1), polyketide synthase (SsPKS), and non-ribosomal peptide synthase (SsNRPS), and genes encoding subtilisin-like serine proteases  can degrade cell wall glycoproteins and may play an essential role in fungal customization  and eburicol 14 alpha-demethylase (CYP51 and SS1G_04805) were upregulated in the early stages of infection , and Ss12040 (ITC hydrolases gene). SsQDO deletion lines showed decreased pathogenicity and increased sensitivity to flavonols. Ss12040, a homolog of SaxA from Pseudomonas syringae, was essential for pathogenicity on ITC-defended plants. Together, these genes constitute the defense network of S. sclerotiorum against the antitoxins produced by rapeseed. Furthermore, salicylate hydroxylase and specific extracellular effectors, such as SsLysM and SsPINE1, are very important in inhibiting plant defense pathways  was intricate, with most TFs in families such as WRKY, MYB, NAC, and others being downregulated. The critical gene ICS1 in SA biosynthesis showed an initial increase in expression followed by a decrease during the infection period  domain protein (JAZ) , ERF, and ORA59 increased, and the activation of the JA/ET pathway resulted in elevated expression of genes responsible for chitinase and pathogenesis-related (PR) genes such as PDF1.2, PR2, PR3, and PR4. This underlines the significance of the JA/ET pathway in defense against S. sclerotiorum. Additionally, the upregulation of PGIPs serves to inhibit PG in the cell wall, preventing cell wall degradation  signaling pathway during the early stages of n period . Conversver time . Howeverin (JAZ) . JAZ inhed genes . The ET ed genes . Followiradation . Long norapeseed .A. thaliana  plants were more susceptible. Importantly, BnMPK3 and BnMPK6 confer resistance to rapeseed by positively regulating critical genes of the ET pathway, BnACS and BnEIN3  W box region in promoters to inhibit or activate downstream gene expression . WRKY33 amalexin . In rape control . Conversam genes . WRKY70 rotiorum . Plant mnd ORA59 . In raperotiorum . Heat shrotiorum . In raperotiorum . Since Hrotiorum .BnNPR1 significantly decreased following S. sclerotiorum infection. BnNPR1-RNAi plants showed increased sensitivity to S. sclerotiorum, resulting in the accumulation of ROS and the suppression of the SA pathway. However, the expression of JA/ET pathway-related genes increased, and this increased sensitivity may be attributed to the early stage role of the SA pathway , is important for plant systemic acquired resistance (SAR). NPR1 is responsible for promoting the expression of downstream defense genes . In rape pathway .Triticum aestivum and are typically characterized by a cupin domain. Various GLP isoforms exhibit diverse enzymatic activities, including OXO (true germins), superoxide dismutase (SOD), polyphenol oxidase (PPO), and ADP glucose pyrophosphatase/phosphodiesterase (AGPPase)  . In rapenfection .2+ is very important in HR and PCD, and they are closely associated with ROS in various plant immune responses . Six of these BnCNGC genes (four in group I and two in group IV-A) were strongly induced by SA and S. sclerotiorum. Susceptible B. oleracea displayed increased calcium signaling during S. sclerotiorum infection, which might be linked to cell death  effects. Recent research has identified several quantitative trait loci (QTLs) associated with S. sclerotiorum resistance in rapeseed, with some of these QTLs showing links to flowering time (FT) and yield  and leucine-rich repeat (LRR) domain  domain . Li et aS. sclerotiorum; genetic engineering is an effective way to improve rapeseed resistance. Since the degradation of rapeseed cell walls is significant for S. sclerotiorum colonization, a promising strategy is to impede pathogen colonization as the first line of plant defense. Overexpressing PGIPs typically inhibits pathogen-produced PG and affects pathogen colonization , guaiacyl (G), and syringyl (S) units. It serves to reinforce the mechanical strength of the cell wall and plays a positive role in supporting plant growth and development, and has various functions in mitigating biotic and abiotic stresses . Ferulatrotiorum . Strengtradation .A. thaliana  is a key regulator of floral organ abscission  in rapeseed also showed increased tolerance to S. sclerotiorum  are a class of small cationic peptides known for their wide-ranging antibacterial effects, achieved through mechanisms such as the inhibition of protein transport, cell membrane penetration, and binding to DNA/RNA . In raperotiorum . Similarrotiorum .S. sclerotiorum, has been found to exert an influence on the subcellular localization of QCR8 (the subunit of the cytochrome b-c1 complex) in Nicotiana benthamiana, consequently impairing its biological functions  in rapeseed was shown to be induced by S. sclerotiorum; 5′-deletions and promoter activity analysis showed that a 189-bp region was essential for S. sclerotiorum to induce responses, and the promoter D7pBnGH17, which connects this region to the core promoter region, was induced after S. sclerotiorum infection, but it was less active under normal growth conditions  improved the resistance to S. sclerotiorum and delayed disease, no strategy of complete immunity to S. sclerotiorum has been reported. Second, the current studies mainly stay in the laboratory stage and lack field experiments, which will be limited to molecular breeding and application. The relationship between yield, quality, and disease resistance should also be balanced. Third, in addition to the search for R genes, the search and development of S genes are equally important; inactivation of the S genes usually induces long and broad-spectrum resistance with little adverse effect on crop growth and yield (In recent years, significant progress in studies of the nd yield . In addind yield , and mayR-SC: Writing – original draft. J-YW: Writing – review & editing. RS: Writing – review & editing. X-LT: Writing – review & editing.


Perinatally HIV-infected infants can be infected with a drug-resistant virus or select for drug resistance by exposure to sub-therapeutic levels of maternal antiretroviral drugs present in breastmilk or from sub-therapeutic infant prophylaxis. We report a case of dolutegravir resistance detected in a treatment-naive perinatally HIV-infected infant whose mother was receiving tenofovir/lamivudine/dolutegravir. This case was detected during a national survey of HIV drug resistance in Haiti amongst infants testing positive for HIV through the national early infant diagnosis program between April 2020 and March 2021. This unique case underscores the need for prompt management of high viral loads in pregnant and breastfeeding women and supports HIV drug resistance surveillance efforts targeted at antiretroviral therapy-naive infants born to mothers in low-and middle-income countries. The rapid decrease in plasma levels of HIV RNA in women receiving dolutegravir (DTG)-based antiretroviral therapy (ART), coupled with high levels of viral load suppression, is associated with potential reduction in mother-to-child-transmission and raises hopes for eliminating mother-to-child transmission of HIV –5. HowevHIV drug resistance (HIVDR) in perinatally HIV-infected infants may occur because of infection with drug-resistant virus, exposure to sub-therapeutic levels of antiretroviral drugs in breastmilk or during infant postnatal prophylaxis (PNP) –8. PriorRecently, WHO recommended using DTG-containing ART for children aged at least 4 weeks and weighing at least 3 kg ; thus, DFollowing, WHO survey guidance , Haiti cDuring the survey period, 5185 infants were born to HIV-infected mothers; 276 (5.3%) of the infants tested positive for HIV. Of these, 143 (52%) had remnant specimens available for HIVDR testing of which 121 (85%) and 105 (73%) yielded a successful quality-assured genotype for protease/reverse transcriptase (PR/RT) and integrase regions, respectively. The mean age of the infants was 8.2 months [95% confidence interval (CI) 7.3–9.2], and the majority  had experienced exposure to antiretroviral drugs for PMTCT. Most mothers had received DTG-based ART , and most infants  had been breastfed or were breastfeeding at the time of specimen collection.The overall prevalence of resistance to EFV/NVP was 50.9% 95% CI 41.7–60.0). The prevalence of any NRTI resistance was 11.2% (95% CI 6.6–18.5) and was driven by ABC and 3TC resistance , 92% of which was because of the M184V mutation alone which confers low-level resistance to ABC (Fig. .7–60.0. In this national survey of HIVDR in ART-naive infants in Haiti, the prevalence of INSTI resistance was very low in this population, occurring in only one of 58 infants whose mothers were receiving DTG-based ART. Nonetheless, the presence of DTG resistance in this one infant merits attention and signals a need for further surveillance in this population as DTG-based ART is rolled out globally because of limited available treatment options and the need for lifelong treatment.Pharmacokinetic studies have shown that DTG can be passed through the placenta and breastmilk in sub-therapeutic levels raising concerns for selection of drug resistance ,13. HoweThe high prevalence of NRTI and NNRTI resistance is consistent with findings of similar studies –11 and uA key limitation of this analysis is the inability to ascertain if the infant was infected with a DTG-resistant virus or if the resistance was selected because of ingesting sub-therapeutic levels of DTG in breastmilk. It is, however, worth noting that, as the infant was not receiving a nonnucleoside reverse transcriptase inhibitor containing PNP, it is likely that the observed resistance may at least be attributed to infection with a resistant virus. Overall, well designed studies are needed to determine the mechanism of DTG resistance in perinatally infected infants and prevention strategies.In conclusion, we report the first case of detected DTG resistance in an ART-naive HIV-infected infant identified from a nationally representative survey in Haiti. While the near absence of INSTI resistance is highly reassuring, close monitoring of infants initiating ART is required to ensure successful treatment outcomes. Strengthening routine HIVDR surveillance will also help detect any possible increase in the prevalence of DTG resistance in this population and provide information to guide paediatric HIV treatment in countries using a public-health approach.Authors’ contributions: K.F. was the principal investigator. M.R.J., G.D.S., J.D.V.O., I.J., J.B., E.P., N.D. and H.G., were involved in study design and data collection. M.R.J., A.G., O.S. and S.C.I. conceived the sub-study. S.C.I. performed the statistical analysis. S.C.I., M.R.J. and A.G. wrote the manuscript. All authors provided valuable input to interpretation of the data and critically reviewed the paper for important intellectual content. All authors reviewed and approved the final version of the manuscript.There are no conflicts of interest.


Pueraria montana chloroplast genome variation architecture and phylogeny, we investigated the chloroplast variation architecture of 104 P. montana accessions from across China. P. montana’s chloroplast genome showed high diversity levels, with 1674 variations, including 1118 single nucleotide polymorphisms and 556 indels. The intergenic spacers, psbZ-trnS and ccsA-ndhD, are the two mutation hotspot regions in the P. montana chloroplast genome. Phylogenetic analysis based on the chloroplast genome dataset supported four P. montana clades. P. montana variations were conserved among and within clades, which showed high gene flow levels. Most P. montana clades were estimated to have diverged at 3.82–5.17 million years ago. Moreover, the East Asian summer monsoon and South Asian summer monsoon may have accelerated population divergence. Our results show that chloroplast genome sequences were highly variable and can be used as molecular markers to assess genetic variation and relationships in P. montana.Despite having a generally conserved structure, chloroplast genome data have been helpful for plant population genetics and evolution research. To mine  Chloroplasts are maternally inherited in most angiosperms and have their own genome. The chloroplast genome is circular, ranging from 107 to 218 kb among the photosynthetic plant species : 11.42–13.22 Ma) in the middle Miocene and 7.04 Ma (95% HPD: 5.72–8.72 Ma) in the late Miocene, respectively . ChloropThe four subclades identified in the phylogeny and PCA analyses were used to compare intra-clade divergence. The mean π of the four subclades were calculated as 0.00038, 0.00040, 0.00010, and 0.00055, respectively A,B. Cladthomsonii accession from GenBank. Four clades and the entire collection showed relatively high divergences. An analysis of molecular variance (AMOVA) suggested that both variations contributed to inter- and intra-clade differences (Genetic distances (Fst) between the four subclades were 0.319–0.716, indicating high genetic differences among them A. Clade ferences C.Arnebia euchroma and Arnebia guttata ) were calculated using the whole chloroplast genome dataset to assess genetic diversity among the different clades. The network of all samples was constructed. Haplotype data were analyzed with DnaSP version 6 , and a mP. montana accessions from different subclades and nine Glycininae species as the outgroups. According to the average values of Egan et al. [We used the chloroplast genome to estimate the divergence times of the different subclades. This dataset included 16 n et al.  in a calThe divergence time analyses were performed using BEAST 2 . We chosP. montana’s genetic diversity, identifying genome-wide variability and contributing to a better understanding of the divergence history among its populations. The phylogenetic, PCA, and structure analyses showed genetic differentiation and divergence among P. montana populations. Chloroplast genome sequences showed high variability and provided novel insights into evolutionary history.This study used whole chloroplast genomes to examine


Paspalum vaginatum Sw.) caused by Microdochium paspali seriously impacts the landscape value of turf and poses a challenge to the maintenance and management of golf courses. Little is known about the genome of M. paspali or the potential genes underlying pathogenicity. In this study, we present a high-quality genome assembly of M. paspali with 14 contigs using the Nanopore and Illumina platform. The M. paspali genome is roughly 37.32 Mb in size and contains 10,365 putative protein-coding genes. These encompass a total of 3,830 pathogen-host interactions (PHI) genes, 481 carbohydrate-active enzymes (CAZymes) coding genes, 105 effectors, and 50 secondary metabolite biosynthetic gene clusters (SMGCs) predicted to be associated with pathogenicity. Comparative genomic analysis suggests M. paspali has 672 species-specific genes (SSGs) compared to two previously sequenced non-pathogenic Microdochium species, including 24 species-specific gene clusters (SSGCs). Comparative transcriptomic analyses reveal that 739 PHIs, 198 CAZymes, 40 effectors, 21 SMGCs, 213 SSGs, and 4 SSGCs were significantly up-regulated during the process of infection. In conclusion, the study enriches the genomic resources of Microdochium species and provides a valuable resource to characterize the pathogenic mechanisms of M. paspali.The sparse leaf patch of seashore paspalum ( It is distributed worldwide, especially in Asia and Europe  (M. maydis can cause tar-spot disease of maize (Zea mays) (M. poae was reported to cause leaf blight on Kentucky bluegrass (Poa pratensis) and creeping bentgrass (Agrostis stolonifera) in North China  , M. maydea mays) , and recth China . Some Mid barley .Microdochium species mainly focused on the identification and biological characterization of the pathogenic or endophytic fungi, but there is a lack of genome-scale studies. In the NCBI database, only a few gene sequences commonly used for fungal classifications have been recorded for Microdochium species, and the genomes of only two endophytes, M. bolleyi  proteins, carbohydrate-active enzymes (CAZymes), secreted proteins and effector proteins, species-specific genes (SSGs). The species-specific secondary metabolite biosynthetic gene clusters (SMGCs) were further analyzed by comparative genomics with the non-pathogenic fungi, M. bolleyi and M. trichocladiopsis. In addition, comparative transcriptomics analysis were performed to identify the putative disease-related factors during the infection of M. paspali on seashore paspalum. This study enriches the genome resources of Microdochium species and provides a scientific basis for further characterization of the mechanisms of pathogenicity and pathogen-host interactions.In this study, we sequenced the genome of 2.2.1.M. paspali strain I16 was preserved at the College of Agro-grassland Science, Nanjing Agricultural University. It was isolated from symptomatic leaves of seashore paspalum  at the research station of the Jurong Turf Institute. The strain, stored in glycerol (20%), was inoculated on potato dextrose agar (PDA) medium, and incubated for 3 d at 25°C in the dark. Thereafter, actively growing mycelial plugs were transferred to fresh PDA at 25°C for 7 d, and subsequently the mycelia were collected. Highly purified total genomic DNA was extracted according to a modified CTAB method  library was constructed following the manufacturer’s protocols of a Ligation Sequencing Kit SQK-LSK109 and a Native Barcoding Expansion Pack EXP-NBD104/114 . The prepared ONT library was loaded onto flow cells for single molecule sequencing on a PromethION device (Oxford Nanopore Technologies). Illumina sequencing was conducted on an Illumina NovaSeq  after the genomic DNA was randomly fragmented by Covaris M220 Focused Ultra-sonicator device . The library for Illumina sequencing was constructed following the steps of DNA fragment end repair, 3′ A-tailing, adapter ligation, purification, and PCR amplification. The raw sequencing data were filtered to obtain clean reads, by removing reads containing adapters and ploy-N and low-quality reads with more than 50% of the bases having Q-scores ≤10 in the whole read. The clean reads were analyzed by Genomescope v1.0 (k = 19) to estimate the genome size, heterozygosity, and duplication rate before assembly. An initial splicing result was obtained by performing genome error correction and splicing with NECAT software , Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups of Proteins (COG), Nonredundant (Nr), Pathway, and Reference Sequence (Refseq) with BLASTP    to predie <1e-5) , and thee <1e-5)  and Intee <1e-5) .2.4.Microdochium bolleyi (accession no. GCA_001566295.1) and Microdochium trichocladiopsis (accession no. GCA_020744255.1) were selected as the reference genomes of homologous species for M. paspali. The genomes of Fusarium graminearum (accession no. GCA_900044135.1), Fusarium poae (accession no. GCA_019609905.1), Fusarium equiseti (accession no. GCA_910393935.1), Fusarium solani (accession no. GCA_020744495.1), Fusarium oxysporum (accession no. GCA_013085055.1), Fusarium verticillioides (accession no. GCA_000149555.1), Clarireedia jacksonii (accession no. GCA_026225895.1), Magnaporthiopsis poae (accession no. GCA_000193285.1), Botrytis cinerea (accession no. GCA_000143535.4), Sclerotinia sclerotiorum (accession no. GCA_001857865.1), and Truncatella angustata (accession no. GCA_020726525.1), were selected for comparisons with the genomic data of M. paspali. Ortholog clustering analysis was performed using OrthoFinder v2.3.12 , and these genes were integrated with the results of unique orthologous groups and classified as SSGs of M. paspali. Gene sequences in the genomes of the three Microdochium species were compared using DIAMOND v0.9.29     to idente <1e-5)  was furt2.5.M. paspali and seashore paspalum, both fungal mycelia and plant leaf tissues were collected after inoculation. Specifically, ten actively growing mycelial plugs (5 mm in diameter) of M. paspali were put into potato dextrose broth (PDB) medium and grown for 7 days. Mycelia were collected and rinsed with sterile distilled water (SDW) and a portion was stored directly in centrifuge tubes in liquid nitrogen. This served as the control (0 h of inoculation) with the rest being used to make a mycelial suspension by adding SDW and pulverizing with a grinder. The mycelial suspension was evenly sprayed on healthy seashore paspalum cultivar “Sea Spray” until water drops fell off the leaves. The plants were then transferred to a sterilized glass box and placed in a greenhouse at 25°C. Inoculated plants were watered daily to maintain high humidity for infection. It was observed that the mycelium of M. paspali began to grow gradually within 0–12 h after infection, and there were no visible symptoms on the leaves. At 24 h post infection, the water-soaked leaves could be observed, and at 36 h the symptoms of the leaves began to worsen and chlorosis was observed. At 48 h post infection the mycelium of M. paspali spread all over the leaves, which appeared to be uniformly blighted and showed obvious black lesions. Therefore, we collected leaf tissues at 6 h, 12 h, 24 h, 36 h, and 48 h after inoculation  was applied to measure the expression level of a gene or transcript by StringTie  and RNA concentrations and purity were measured using the NanoDrop 2000 . RNA integrity was assessed using the RNA Nano 6,000 Assay Kit and the Agilent Bioanalyzer 2,100 system . RNA-seq was performed using the Illumina NovaSeq6000 platform and the 150 bp paired-end chemistries. Raw reads were processed through in-house perl scripts to obtain clean reads by removing reads containing adapters and ploy-N and low-quality reads with more than 50% of the bases having Q-scores ≤10 in the whole read. Subsequently, Hisat2 v2.2.1  was usedtringTie  employintringTie  providedtringTie .2.6.E-value <1e-5) based on the PHI v4.14 database  (Microdochium and comparisons with known clusters using antiSMASH v6.0 (Homologs of known PHI genes were annotated using the BLASTP (database . The prege >35%) . IdentifASH v6.0  at defauM. paspali were identified according to multiple software analyses. The protein sequences of all genes were analyzed using SignalP v5.0  by K-mer analysis  and M. bolleyi (38.84 Mb)  .M. paspali with an average gene length of 1703 bp and an average of 2.71 exons per gene, while no gene was predicted on contig 14. The total length of genes were 17.66 Mb, accounting for 47.32% of the genome , Long interspersed nuclear elements (LINE), short interspersed nuclear elements (SINE), and DNA element) were 0.51%%  were detected in all samples  were defined as species-specific gene clusters (SSGCs), and there were 24 SSGCs identified in M. paspali genome (Only 285 SSGs were annotated in the NR database and most (218 genes) had unknown functions. GO analysis showed that the functions of SSGs were classified into sixteen terms, of which twelve SSGs were significantly enriched in term of pathogenesis . Eighty- paspali , S11. SSi genome .The expression of most SSGs was significantly altered after infection, with two hundred and thirteen SSGs being up-regulated at least at one stage after infection . Among t3.4.M. paspali and other fungi was compared with the genomes of two non-pathogenic Microdochium species, M. bolleyi and M. trichocladiopsis, and 12 other representative pathogenic fungi. The phylogenetic tree was constructed according to sequence alignment of 3,024 single-copy orthologous proteins, which suggested that M. paspali had a close evolutionary relationship with other Microdochium species , SMGCs, and SSGs were mainly concentrated at the edges of contigs, and there were also more up-regulated genes during infection in these pathogenic factor-enriched regions  held more densely distributed SSGs and pathogenic factors than other contigs  were up-regulated during infection, suggesting that a high number of SSGs responded to the process of M. paspali-seashore paspalum interaction, and might play important roles in the pathogenicity of M. paspali. It was worth noting that the three short contigs  of M. paspali contained more dense SSGs and pathogenicity-related genes  than other contigs  and all four up-regulated SSGs encoding CAZymes belonged to GHs . The observed result suggestes GHs may be widely involved in the interaction between M. paspali and seashore paspalum. Existing studies indicate GHs decomposed the cell wall during the infection of plant pathogenic fungi, helping them to invade and colonize, and also play a toxic role by inducing the immune response of the host plants . However, the four SSGs have not been functionally annotated and their function needs to be further analyzed. Among the up-regulated effectors, six  were consistently up-regulated at 6–48 h after infection, which might also be the targets of interest to understand the pathogenic mechanism of M. paspali. In the Pfam database, g2155 is annotated as phospholipase, which can aid pathogen invasion by disrupting components of the host cell membrane, and widely exists in a variety of pathogenic fungi and bacteria . It contained thirty-one genes, and its core biosynthetic gene g3022 was a SSG of M. paspali, which was predicted to be responsible for the production of tenuazonic acid (TeA). TeA is a type of tetramic acidic  toxin that is generally considered to be a potential virulence factor (Alternaria tenuis (Phoma sorghina (a pathogen of sorghum) and Magnaporthe oryzae (a pathogen of rice) were also reported to produce TeA , respectively. The core biosynthetic genes of the two gene clusters, MGG_07803 and g3022, were both predicted to be the fungal non-ribosomal peptide synthetase and polyketide synthase (NRPS-PKS) hybrid enzyme. Transcriptome analysis revealed that fourteen genes  in SMGC21 were up-regulated after infection. Therefore, it is hypothesized that M. paspali has the potential to produce TeA, which may play an important role in the pathogenicity of M. paspali. Interestingly, we also found that four SSGCs  significantly up-regulated during infection were all partially or fully distributed on different SMGCs , suggesting that these SSGCs might be involved in the biosynthesis of certain secondary metabolic genes (SMs) in M. paspali. Whether the biosynthetic SMs have toxic effects on plants and what types of SMs they are will be an important direction for our future research.During the complex processes of interaction with host plants, pathogenic fungi can produce a variety of different secondary metabolites, which affect the symptom expression and disease progression of plant hosts, and are also important virulence factors . Bioinfoe factor , which ce factor . TeA wasa tenuis . The pladuce TeA . The biour genes . Through5.M. paspali was sequenced for the first time. The results provide a valuable reference for genome assemblies of other related pathogenic fungi in the genus of Microdochium. In addition, comparative genomics and transcriptomics analyses identified large numbers of pathogenicity-related genes in M. paspali. These provide an important baseline for further characterization of pathogenic and evolutionary mechanisms and may assist in developing effective management strategies to control M. paspali on turfgrass.In this study, the whole genome of https://www.ncbi.nlm.nih.gov/genbank/, JAVBIV000000000; https://www.ncbi.nlm.nih.gov/, PRJNA1007419.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: PJ: Data curation, Software, Writing – original draft. YK: Writing – review & editing. ZZ: Writing – review & editing. HZ: Writing – review & editing. YD: Writing – review & editing. KL: Writing – review & editing. ZY: Writing – review & editing, Conceptualization. JH: Conceptualization, Writing – review & editing, Methodology, Project administration. YZ: Resources, Formal analysis, Writing – review & editing.This study was supported by Jiangsu Institute of Botany Talent Fund (JIBTF202212).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Phytobacter diazotrophicus is an Enterobacterales species that was originally identified as a plant growth-promoting, Gram-negative bacterium. Recently, this species has been recognized as relevant to opportunistic human and nosocomial infections in clinical settings. Its frequent misidentification as other Enterobacterales species from clinical examination occasionally causes a delay in the identification of nosocomial outbreaks. Here, we report the emergence of New Delhi metallo-β-lactamase (NDM)-producing P. diazotrophicus isolated from hospitalized pediatric patients and hospital environments in Tokyo, Japan. In our case, these isolates were found during an investigation of carbapenem-resistant Enterobacterales in relation to nosocomial infections. Whole-genome sequencing is useful for overcoming the difficulty of species identification. Furthermore, we found that blaNDM-1 was carried by an IncA/C2 plasmid (approximately 170 kbp), which was transferrable from the clinical isolates to the recipient strain Escherichia coli J53. Our study demonstrated that P. diazotrophicus behaves as a carrier of blaNDM-harboring plasmids, potentially disseminating resistance to carbapenems among Enterobacterales.Phytobacter diazotrophicus, an Enterobacterales species that has recently been found to be relevant to clinical settings, is often misidentified as other bacteria in clinical laboratories. Here, we found NDM-producing P. diazotrophicus in hospitalized pediatric patients and their environment in Tokyo, Japan. Given that the isolates carried blaNDM-1-harboring transferrable plasmids, the influence of such bacteria could be greater with the mediation of horizontal transfer of carbapenem resistance. Our findings suggest that P. diazotrophicus should be recognized as an NDM-carrier, for which more attention should be paid in clinical settings.Early detection of nosocomial outbreaks is important to minimize the spread of bacteria. When an outbreak is caused by multidrug-resistant bacteria such as carbapenem-resistant Enterobacterales, a delay in findings makes it difficult to control it because such bacteria often spread not only among human patients but also in hospital environments.  The ID 32 E Api Kit  indicated that one of these was Pantoea spp. Due to the necessity of another approach for identification, 16S rRNA sequencing using a MicroSEQ 500 16S rDNA Sequencing Kit  was performed, and a BLAST search for these sequences on the NCBI website using the megablast algorithm made another candidate, P. diazotrophicus  indicated that these four isolates were possibly rophicus .https://www.bioinformatics.babraham.ac.uk/projects/trim_galore) and assembled using Spades v.3.12.0  as a definitive analysis. A next-generation sequencing (NGS) library was prepared from genomic DNA using the Nextera XT DNA Library Preparation Kit  to obtain 2 × 300 bp paired-end short reads on the MiSeq platform (Illumina). Quality trimming was performed using Trim Galore v.0.6.7  was updated in our lab in April 2022. As a result, Phytobacter ursingii was hit with all four isolates with high scores: 2.22, 2.06, 2.11, and 1.99, for TA9730, TA9734, TA9759, and TA9832, respectively. The species identification is less accurate with the MALDI system than with WGS, but it is useful for the recognition of the P. diazotrophicus outbreak because all four isolates are determined to be identical species.After the current study concluded, we conducted a more thorough investigation of the isolates in April 2023. First, we tested Type Strain Genome Server, another genome-based species identification tool . The confication . Given trophicus , 21, thehttps://github.com/rrwick/Porechop), these long reads were assembled with trimmed paired-end short reads using Unicycler v.0.4.8 (https://dfast.ddbj.nig.ac.jp). Gene markers for plasmid replicon type and antimicrobial resistance (AMR)-related genes were identified using PlasmidFinder  with a library prepared using the Native Barcoding Kit (Oxford Nanopore Technologies). After quality trimming using NanoFilt v.0.1.0  and adap v.0.4.8 . Genes widFinder  and ResFidFinder , respectP. diazotrophicus isolates carried IncFIB(K) and IncA/C2 plasmids  was identical to the other three plasmids, except for the lack of a fosfomycin-resistant gene (fosA) , suggesting that the common backbone of these plasmids contributes to the dissemination among Enterobacterales, even though the β-lactamase genes carried are diverse.Four plasmids , and mule (fosA) ; Fig. 2,Escherichia coli J53 was tested using the mating method (−3 and 4.1 × 10−3 for pTMTA97302 and pTMTA97342, respectively.The conjugative transfer of pTMTA97302 and pTMTA97342 from clinical isolates to g method . The trag method , was appP. diazotrophicus as shown by the previous reports which mentioned frequent misidentification of this species (P. diazotrophicus is an opportunistic pathogen (blaKPC and blaIMP-6 (P. diazotrophicus because our isolates carried a blaNDM-1-harboring plasmid, which could spread carbapenem resistance via the horizontal transfer of plasmids in clinical settings. Considering the potential as a carrier of antimicrobial resistance genes, P. diazotrophicus should be more recognized as a clinically relevant pathogen.In summary, our initial identification using general methods had failed to identify  species , 10, andpathogen . Notably