Potential Virulence Research Articles

A pro-inflammatory diet is associated with growth and virulence of Escherichia coli in pediatric Crohn's disease.

Epidemiological studies have suggested an association between the inflammatory potential of dietary patterns and Crohn's disease (CD). However, relationships of these inflammatory dietary determinants with the microbiome remain largely unknown. In this cross-sectional study, we evaluate the association between the inflammatory potential of habitual diet, as assessed by the modified Children-Dietary Inflammatory Index (mC-DII), and the fecal microbiome and metabolome of children with CD in comparison to healthy children. A cross-sectional study including 51 children with CD between 6 and 18 years of age and 50 healthy controls was conducted. Dietary inflammatory potential was measured using the modified Children-Dietary Inflammatory Index (mC-DII) and diet quality assessed by the Healthy Eating Index (HEI)-2015 and alternate Mediterranean eating index (aMed). Microbiome was analysed using shotgun metagenomic sequencing and untargeted metabolomic analysis. A poor-quality, pro-inflammatory diet with similar mC-DII, HEI-2015 and aMed scores was found across healthy children and children with CD. In children with active disease, a pro-inflammatory diet was associated with decreased diversity, increased virulence potential and expansion of the Proteobacteria phylum dominated by Escherichia coli (E. coli) spp. Positive correlation between E. coli relative abundance and mC-DII was associated with a low intake of a cluster composed of fibers, vitamins and minerals with anti-inflammatory potential. A negative association between metabolites of fatty acid metabolism and HEI was found. In total, our results suggest that a pro-inflammatory diet may potentiate hallmarks of the inflammation-associated dysbiosis in CD and highlight the need for microbiome-targeted dietary interventions optimizing the anti-inflammatory potential of habitual diet in the management of pediatric CD.

Genetic diversity and virulence of Bacillus cereus group isolates from bloodstream infections.

Bacillus cereus catheter-related bloodstream infections (CRBSIs) are an increasing concern in Japanese hospitals. Although their clinical characteristics have been explored, the genetic relationships and virulence profiles of B. cereus isolates from CRBSIs remain understudied. Here, using advanced genomic techniques, we investigated the genetic diversity, phylogenetic relationships, and virulence profiles of B. cereus isolates from patients with bloodstream infections. We analyzed 28 B. cereus group strains isolated from blood samples at the University of Tokyo Hospital between 2005 and 2017 using whole-genome sequencing, core-genome single-nucleotide polymorphism (SNP) typing, and virulence gene profiling. Core-genome SNP analysis revealed significant genetic diversity among the isolates, suggesting multiple independent sources of infection. The isolates predominantly belonged to panC clades III and IV, with distinct virulence gene profiles. All panC clade III isolates contained hbl operon genes, whereas four isolates from clade IV harbored cereulide synthetase genes (cesABCD). One isolate possessed a capsule gene operon (capBCADE), a rare finding among clinical B. cereus strains. Biofilm formation ability was observed in 50% of catheter-related isolates, although this ability was not significantly different from that of the noncatheter-related isolates.IMPORTANCEThis study provides novel insights into the genetic diversity and virulence potential of B. cereus strains causing bloodstream infections in a Japanese hospital setting. These findings suggest diverse infection pathways and highlight the importance of continuous molecular epidemiological surveillance for effective infection control.

Determination of the Pathogenicity of the Variant UPM 1432/2019 IBDV in SPF Chicken Eggs and Chicken Fibroblast Cell Line

The infectious bursal disease virus (IBDV) is a nuisance to chicken productivity in the global poultry industry due to its high pathogenicity. In this study, the pathogenicity of the UPM 1432/2019 variant was evaluated using specific pathogen-free (SPF) chicken eggs and fibroblast cell lines. This viral strain was isolated from chickens displaying symptoms of watery diarrhoea and ruffled feathers. Following inoculation, the SPF chicken eggs, and cell lines were meticulously monitored for indicators of viral replication and the onset of cytopathic effects (CPE). The results showed that the virus could replicate in the SPF eggs and cell lines due to the presence of CPE in some cells. In addition, virus-induced weight loss in infected embryonated chicken eggs was evaluated. The UPM 1432/2019 and UPM 1219/2019 strains demonstrated similar mortality rates and CPE features. These findings indicate that the UPM 1432/2019 variant is pathogenic in SPF chicken eggs and fibroblast cell lines. Furthermore, the new UPM 1432/2019 variant caused IBD with pathogenic characteristics similar to the traditional UPM 1219/2019 variant. The emergence of the UPM 1432/2019 variant poses a risk to the economy and poultry production and is potentially resistant to the existing bursal disease vaccines. Consequently, the UPM 1432/2019 variant may significantly contribute to economic losses for chicken farm operators. Moreover, this study provides essential information on the potential virulence of the UPM 1432/2019 variant of IBDV and its potential impact on the poultry industry.

Virulence factor discovery identifies associations between the Fic gene family and Fap2+ fusobacteria in colorectal cancer microbiomes.

Fusobacterium is a bacterium associated with colorectal cancer (CRC) tumorigenesis, progression, and metastasis. Fap2 is a fusobacteria-specific outer membrane galactose-binding lectin that mediates Fusobacterium adherence to and invasion of CRC tumors. Advances in omics analyses provide an opportunity to profile and identify microbial genomic features that correlate with the cancer-associated bacterial virulence factor Fap2. Here, we analyze genomes of Fusobacterium colon tumor isolates and find that a family of post-translational modification enzymes containing Fic domains is associated with Fap2 positivity in these strains. We demonstrate that Fic family genes expand with the presence of Fap2 in the fusobacterial pangenome. Through comparative genomic analysis, we find that Fap2+ Fusobacteriota are highly enriched with Fic gene families compared to other cancer-associated and human gut microbiome bacterial taxa. Using a global data set of CRC shotgun metagenomes, we show that fusobacterial Fic and Fap2 genes frequently co-occur in the fecal microbiomes of individuals with late-stage CRC. We further characterize specific Fic gene families harbored by Fap2+ Fusobacterium animalis genomes and detect recombination events and elements of horizontal gene transfer via synteny analysis of Fic gene loci. Exposure of a F. animalis strain to a colon adenocarcinoma cell line increases gene expression of fusobacterial Fic and virulence-associated adhesins. Finally, we demonstrate that Fic proteins are synthesized by F. animalis as Fic peptides are detectable in F. animalis monoculture supernatants. Taken together, our study uncovers Fic genes as potential virulence factors in Fap2+ fusobacterial genomes.IMPORTANCEAccumulating data support that bacterial members of the intra-tumoral microbiota critically influence colorectal cancer progression. Yet, relatively little is known about non-adhesin fusobacterial virulence factors that may influence carcinogenesis. Our genomic analysis and expression assays in fusobacteria identify Fic domain-containing genes, well-studied virulence factors in pathogenic bacteria, as potential fusobacterial virulence features. The Fic family proteins that we find are encoded by fusobacteria and expressed by Fusobacterium animalis merit future investigation to assess their roles in colorectal cancer development and progression.

Molecular and genetic analysis of methicillin-resistant Staphylococcus aureus (MRSA) in a tertiary care hospital in Saudi Arabia.

Methicillin-resistant Staphylococcus aureus (MRSA) continues to pose significant challenges in healthcare settings due to its multi-drug resistance (MDR) and virulence. This retrospective study examines the molecular and resistance profiles of MRSA isolates from a tertiary care hospital in Saudi Arabia, providing valuable insights into regional epidemiology. A total of 190 MRSA strains were analysed to assess antimicrobial susceptibility, genetic diversity, and virulence factors. Antimicrobial susceptibility testing was conducted according to CLSI guidelines, while molecular characterization involved spa typing, SCCmec typing, and DNA microarray analysis to determine clonal complexes (CCs), resistance genes, and virulence determinants. The isolates showed extensive resistance to beta-lactam antibiotics, with 78% classified as MDR. Notably, resistance to fusidic acid and ciprofloxacin was detected in 70% and 55% of isolates, respectively. The most prevalent clonal complexes-CC5, CC6, and CC22-comprised over 60% of the isolates and exhibited diverse spa types. The Panton-Valentine leukocidin (PVL) gene, linked to heightened virulence, was identified in approximately 20% of isolates, particularly within CC5, CC30, and CC80. Enterotoxin genes (sea and seb) and immune evasion genes (sak, chp, and scn) were also commonly detected, reflecting the isolates' capacity to adapt and persist within the hospital environment. These findings underscore the high burden of MDR MRSA with considerable genetic diversity and virulence potential. The study highlights the urgent need for strengthened molecular surveillance and targeted infection control measures to limit MRSA transmission and effectively manage infection risks in healthcare facilities.

Genetic Determinants and Biofilm Properties Useful in Estimation of UTI Pathogenicity of the Escherichia coli Strains Isolated from Free-Living Birds.

Background/Objectives: According to the One Health concept, wild birds can be indicators of ecosystem pollution and disease incidence. Escherichia coli strains are widespread worldwide, but there are still few reports on the association of human infections with a potential reservoir of highly pathogenic human strains in wild birds. Fecal E. coli with uropathogenic potential (UPEC) can be transmitted between birds and humans and may be a risk factor for urinary tract infections (UTIs). Results: The results showed that above 50% of the isolates were grouped as highly pathogenic, according to Clermont phylogroup classification. Such strains were found to be stronger biofilm producers, with a higher adherence of monocytes than low pathogenic. However, the highest cytotoxicity was observed for strains described as aquatic environmental. Convergence of the results of the analysis of monocyte activation by E. coli strains and the ability to form biofilm by individual phylogroups of the strains tested was demonstrated. Genetic determinants of the uropathogenicity of E. coli (UPEC) correlate with the evidence of strain pathogenicity during monocyte activation in in vitro assays. Methods: In this study, we assessed the virulence potential of environmental strains isolated from wild waterfowl using genetic analysis (Clermont phylogroup classification) and phenotypic methods, including analysis of the human monocyte response to biofilm formation. The estimation of the ability to form biofilms was tested using crystal violet, and the pathogenic potential of strains by monocyte activation assay including changes in morphology, adhesion and cytotoxicity. Conclusions: In conclusion, the virulence of E. coli strains isolated from free-living birds is significant, and they can be considered environmental reservoirs of pathogenic strains. According to our observations, they can be responsible for the dissemination of uropathogenic strains among humans.

Urban yellow-legged gull (Larus michahellis) and peri-urban Audouin's gull (Larus audouinii) as a source of Campylobacter and Salmonella of public health relevance.

Campylobacter spp. and Salmonella spp. are the leading cause of human enteric infections in the European Union. Some gull species act as reservoirs and play an important role in the epidemiology of these zoonotic agents. To gain insight into Campylobacter and Salmonella epidemiology we studied colonies of Audouin's gull (Larus audouinii) and yellow-legged gull (Larus michahellis) in Barcelona metropolitan area, Catalonia (north-eastern Spain). We assessed the occurrence, genetic diversity, virulence potential, and antimicrobial susceptibility of Campylobacter and Salmonella isolates recovered from gull faeces in different time periods within 2009-2018. The occurrence of Campylobacter was higher compared to Salmonella in both gull species. Also, the occurrence of both pathogens was significantly higher in Audouin's gull (45% for Campylobacter, 20% for Salmonella), than in yellow-legged gull (13% and 7%, respectively). All but one individual carried C. jejuni; the remaining positive yellow-legged gull carried C. lari. Salmonella serovar Typhimurium (including its monophasic variant) was the most frequent in both hosts followed by ser. Bredeney. Other serovars frequently associated with human salmonellosis (Infantis, London, Virchow) were only isolated from yellow-legged gulls. Multilocus Sequence Typing analyses showed that yellow-legged gull and not Audouin's gull carried several Campylobacter genotypes associated with human enteritis. Campylobacter isolates from both gull species revealed a high virulence potential, as opposed to Salmonella isolates which showed a lower prevalence of virulence-associated genes, particularly in Audouin's gull. Overall, a moderate to high frequency of antimicrobial resistance (including multidrug resistance) was found in both pathogens from both gull species. Campylobacter and Salmonella from yellow-legged gull showed a higher frequency of isolates resistant to antimicrobials of relevance in human medicine. Overall, our results highlight the potential public health threat associated with these gull species, particularly yellow-legged gull, in densely human populated areas.

Tracking the Prevalence of Antibiotic Resistance in Enterococcus Within the Spanish Poultry Industry: Insights from a One Health Approach.

Background/Objectives: Antimicrobial resistance (AMR) in Enterococcus species from poultry production represents a significant public health threat due to the potential transmission of AMR through the food chain. This study aimed to examine the relative prevalence, resistance patterns, and mannitol fermentation capacity of Enterococcus isolates from various poultry production systems in Spain over a seven-year period (2017-2023). Methods: A total of 215 Enterococcus isolates were analyzed. Phenotypic assessments were conducted to determine resistance rates and metabolic capacities, while genotypic characterization focused on detecting vancomycin-resistance genes (vanA, vanB, vanC, and vanD). Results: Enterococcus faecalis (62.3%) and Enterococcus faecium (29.77%) were the predominant species, primarily isolated from broilers (74.88%), with the highest frequency observed in one-week-old chicks (31.16%). High resistance rates to tetracyclines and streptogramins were identified, while resistance to vancomycin (0.47%) and tigecycline (3.03%) was low. Interestingly, a significant reduction in tetracyclines resistance was shown in this period for Enterococcus faecalis (from 100% to 70% (2017-2023) and Enterococcus faecium (from 100% to 40% (2018-2023)). Multidrug resistance (MDR) was detected in 26.98% of isolates. Mannitol fermentation tests revealed high metabolic capacity in Enterococcus faecalis (99.25%) and Enterococcus faecium (96.88%), associated with adaptability and virulence potential. Genotypic analysis showed a very low prevalence of vanB and vanC genes. Conclusions: These findings highlight the critical need for targeted surveillance and intervention strategies in poultry production to mitigate the risks posed by MDR Enterococcus to public health.

Plasmidome of Salmonella enterica serovar Infantis recovered from surface waters in a major agricultural region for leafy greens in California.

Non-typhoidal Salmonella enterica is a leading cause of gastrointestinal illnesses in the United States. Among the 2,600 different S. enterica serovars, Infantis has been significantly linked to human illnesses and is frequently recovered from broilers and chicken parts in the U.S. A key virulence determinant in serovar Infantis is the presence of the megaplasmid pESI, conferring multidrug resistance. To further characterize the virulence potential of this serovar, the present study identified the types of plasmids harbored by Infantis strains, recovered from surface waters adjacent to leafy greens farms in California. Sequencing analysis showed that each of the examined 12 Infantis strains had a large plasmid ranging in size from 78 kb to 125 kb. In addition, a second 4-kb plasmid was detected in two strains. Plasmid nucleotide queries did not identify the emerging megaplasmid pESI in the examined Infantis strains; however, the detected plasmids each had similarity to a plasmid sequence already cataloged in the nucleotide databases. Subsequent comparative analyses, based on gene presence or absence, divided the detected plasmids into five distinct clusters, and the phylogram revealed these Infantis plasmids were clustered based either on the plasmid conjugation system, IncI and IncF, or on the presence of plasmid phage genes. Assignment of the putative genes to functional categories revealed that the large plasmids contained genes implicated in cell cycle control and division, replication and recombination and defense mechanisms. Further analysis of the mobilome, including prophages and transposons, demonstrated the presence of genes implicated in the release of the bactericidal peptide microcin in the IncF plasmids and identified a Tn10 transposon conferring tetracycline resistance in one of the IncI1 plasmids. These findings indicated that the plasmids in the environmental S. enterica serovar Infantis strains from surface waters harbored a wide variety of genes associated with adaptation, survivability and antimicrobial resistance.

Comparative Genome Analysis of Canine Frederiksenia canicola Isolates.

Background/Objectives: The One Health approach is crucial for managing and controlling the spread of antimicrobial resistance. Frederiksenia canicola is a recently identified bacterial species that seems to be a component of the oral microbiota of dogs; however, its pathogenic nature is questionable. Methods: In this study, the antibacterial susceptibility of F. canicola isolates was determined using the disk diffusion and broth microdilution methods. Genome-wide comparative analyses were performed to identify the genetic factors driving virulence and antimicrobial drug resistance (e.g., virulence factors, antimicrobial resistance genes (ARGs) and prophage-related sequences). Results: Most of the F. canicola isolates lacked virulence-associated genes. F. canicola is likely resistant to clindamycin, lincomycin and neomycin, but susceptible to penicillin, erythromycin and enrofloxacin. Antimicrobial resistance genes were not found in the F. canicola genomes, but prophage-related sequences were identified, suggesting its potential in the transfer of genes associated with drug resistance between bacteria in the oral microbiome. Conclusions: F. canicola is presumably a commensal organism with low virulence potential, as evidenced by the absence of virulence-associated genes. As F. canicola can colonize a wide range of hosts, including humans, further investigation with a greater number of isolates is needed to better understand the role of F. canicola in disease development and the spread of drug resistance.

The Exoproteome and Surfaceome of Toxigenic Corynebacterium diphtheriae 1737 and Its Response to Iron Restriction and Growth on Human Hemoglobin.

Toxin-producing Corynebacterium diphtheriae strains are the etiological agents of the severe upper respiratory disease, diphtheria. A global phylogenetic analysis revealed that biotype gravis is particularly lethal as it produces diphtheria toxin and a range of other virulence factors, particularly when it encounters low levels of iron at sites of infection. To gain insight into how it colonizes its host, we have identified iron-dependent changes in the exoproteome and surfaceome of C. diphtheriae strain 1737 using a combination of whole-cell fractionation, intact cell surface proteolysis, and quantitative proteomics. In total, we identified 1414 of the predicted 2265 proteins (62%) encoded by its reference genome. For each protein, we quantified its degree of secretion and surface exposure, revealing that exoproteases and hydrolases predominate in the exoproteome, while the surfaceome is enriched with adhesins, particularly DIP2093. Our analysis provides insight into how components in the heme-acquisition system are positioned, showing pronounced surface exposure of the strain-specific ChtA/ChtC paralogues and high secretion of the species-conserved heme-binding HtaA protein, suggesting it functions as a hemophore. Profiling the response of the exoproteome and surfaceome after microbial exposure to human hemoglobin and iron limitation reveals potential virulence factors that may be expressed at sites of infection. Data are available via ProteomeXchange with identifier PXD051674.

Serotype diversity of Actinobacillus pleuropneumoniae detected by real-time PCR in clinical and subclinical samples from Spanish pig farms during 2017–2022

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a challenging respiratory disease for the global swine industry. Variations in the serotypes associated with clinical disease have been observed in different regions worldwide. This study aimed to provide an updated epidemiological assessment of A. pleuropneumoniae serotypes in Spain, incorporating bacterial characterization through serotyping and toxinotyping. Serotypes 9/11, 2, 4, 5, 17, and 13 were frequently identified in diseased animals. Furthermore, qPCR of lung samples from an outbreak, even when samples were pooled, emerged as a robust diagnostic tool, enabling the rapid detection of A. pleuropneumoniae and their serotypes without the need for microbiological isolation. This technology also facilitates serotype monitoring of apparently healthy herds through the testing of oral fluids. The study revealed the frequent simultaneous presence of diverse serotypes within a farm. Serotypes 1, 7, 10, 12, 18, and 19 were frequently found in subclinically infected animals but were rarely detected in acute pleuropneumonia outbreaks in the current study. These results provide valuable information for interpreting the potential virulence of the different serotypes in Spain. However, other predisposing factors and the immune status of the herds such as type of vaccines used when appropriate, should be carefully considered before drawing definitive conclusions. Nevertheless, the study offers valuable insights that underscore the necessity for detailed regional data to contribute toward a comprehensive understanding of the disease dynamics and toward formulating effective control measures for porcine pleuropneumonia.

Genomic Diversity and Virulence Factors of Clostridium perfringens Isolated from Healthy and Necrotic Enteritis-Affected Broiler Chicken Farms in Quebec Province.

Avian necrotic enteritis due to the Gram-positive bacterium Clostridium perfringens has re-emerged following the ban on antibiotic growth promoters in many poultry producing countries. The limited number of previous studies has left important gaps in our understanding of the genetic diversity and virulence traits of the pathogen. To address these knowledge gaps, in this study, we sequenced the genomes of 41 Clostridium perfringens isolates recovered from commercial broiler chicken flocks in Quebec, Canada, including isolates from healthy birds and those affected by necrotic enteritis. We sought to understand the pangenome diversity and interrogated the genomes for key virulence factors involved in necrotic enteritis pathogenesis. On average, the genomes had a GC content of 28% and contained 3206 coding sequences. A variable presence of toxins, degradative hydrolytic enzymes, and collagen-binding proteins was also found. Through pangenome analysis, we revealed a total of 10,223 genes, 652 (6.4%) of which formed the core genome. Additionally, we identified 17 different plasmids, 12 antibiotic resistance genes, and nine prophage regions. Overall, our results demonstrated a relatively high genetic diversity among chicken Clostridium perfringens isolates collected from the same geographical location, offering new insights into potential virulence mechanisms and adaptation of the pathogen within poultry populations.

Candida albicans recovered from persistent candidemia exhibit enhanced virulence traits.

Candida albicans catheter-related candidemia is largely driven by microbial adhesion and biofilm formation on central venous catheters. Cells that disperse from these biofilms can enter the bloodstream, spread to distant organs, and sustain the cycle of infection. In this study, we investigated the virulence potential of C. albicans isolates obtained from the blood of catheterized patients experiencing persistent candidemia, comparing them to isolates that were cleared from the bloodstream early in the infection. Our results show that isolates persisting in the bloodstream for four days or longer, despite antifungal treatment, exhibited enhanced adherence, filamentation, and biofilm formation in vitro, along with increased expression of key virulence-related genes. Notably, cells dispersed from second-generation biofilms formed by these persistent isolates displayed even more pronounced pathogenic characteristics, including improved immune evasion. Furthermore, in vivo experiments using Galleria mellonella revealed that persistent isolates were significantly more virulent than their non-persistent counterparts.

Deciphering pathogenicity and virulence of the first Staphylococcus debuckii isolate from diabetic foot osteomyelitis.

This study identifies Staphylococcus debuckii as a new coagulase-negative staphylococcal species isolated from diabetic foot osteomyelitis (DFOM) and provides an in-depth analysis of its pathogenic and virulence profile, as well as demonstrating its potential to cause infection. The S. debuckii NSD001 strain was examined for its planktonic growth, biofilm production, and phagocytosis rates in murine macrophages compared to S. aureus NSA739. Additionally, persistence and replication within human osteoblasts were investigated, while the zebrafish embryo model was employed to assess virulence. Genomic sequencing and bioinformatic analysis were also conducted to identify genes associated with virulent potential. S. debuckii NSD001 exhibited robust planktonic growth and significant biofilm production, highlighting its capacity to initiate and maintain an infection, and demonstrated similar rates of phagocytosis as S. aureus NSA739 in murine macrophages, suggesting a mechanism for evading initial host defenses. The strain persisted and replicated within human osteoblasts, indicative of a strategy for intracellular survival and facilitation of chronic osteomyelitis. The zebrafish embryo model revealed a slower, yet fatal, virulence profile for S. debuckii NSD001 compared to the rapid lethality induced by S. aureus NSA739. Genomic sequencing and bioinformatic analysis uncovered various genes corroborating its virulence. S. debuckii NSD001 poses a significant concern in DFOM due to its ability to form biofilms and survive within host cells, presenting challenges for current treatment strategies. This underscores the need for updated clinical protocols and increased awareness among healthcare professionals to effectively manage infections caused by this emerging pathogen.

Variations in Kojic Acid Production and Corn Infection Among Aspergillus flavus Isolates Suggest a Potential Role as a Virulence Factor.

Kojic acid is a secondary metabolite with strong chelating and antioxidant properties produced by Aspergillus flavus and A. oryzae. Although antioxidants and chelators are important virulence factors for plant pathogens, the ecological role of kojic acid remains unclear. We previously observed a greater gene expression of antioxidants, especially kojic acid, by non-aflatoxigenic A. flavus when co-cultured with aflatoxigenic A. flavus. Aflatoxin production was also reduced. In this study, we investigated kojic acid production in 22 A. flavus isolates from Louisiana and compared them to four common A. flavus strains in liquid medium and on corn kernels. Corn kernel infection was assessed by quantifying the maize beta tubulin DNA content of the kernels using drop digital PCR (ddPCR). Maize beta tubulin DNA content decreased with increased corn kernel infection. Greater kojic acid production by A. flavus isolates coincided with greater levels of corn kernel infection. All isolates produced 60 and 700 times more kojic acid than aflatoxin and cyclopiazonic acid (a known virulence factor), respectively, which varied among sclerotial size categories. A. flavus strains with small sclerotia, which were rarely isolated from corn, produced the least kojic acid and infected corn kernels the least, while medium and large sclerotia strains-mainly isolated from corn-produced the most kojic acid and were more infectious. Non-aflatoxigenic isolates from Louisiana produced the most kojic acid. These results suggest that kojic acid is a potential virulence factor and may increase the pathogenic success of medium and large sclerotia-producing A. flavus, which could ultimately lead to more effective A. flavus biocontrol strains. Further studies are required to determine the effects that kojic acid has on the redox environment during corn infection and how the altered redox environment decreases aflatoxin production.

Comparative Genomics and Pathogenicity Analysis of Three Fungal Isolates Causing Barnyard Grass Blast.

Barnyard grass is one of the most serious rice weeds, often growing near paddy fields and therefore potentially serving as a bridging host for the rice blast fungus. In this study, we isolated three fungal strains from diseased barnyard grass leaves in a rice field. Using a pathogenicity assay, we confirmed that they were capable of causing blast symptoms on barnyard grass and rice leaves to various extents. Based on morphology characterization and genome sequence analyses, we confirmed that these three strains were Epicoccum sorghinum (SCAU-1), Pyricularia grisea (SCAU-2), and Exserohilum rostratum (SCAU-6). The established Avirulence (Avr) genes Avr-Pia, Avr-Pita2, and ACE1 were detected by PCR amplification in SCAU-2, but not in SCAU-1 or SCAU-6. Furthermore, the whole-genome sequence analysis helped to reveal the genetic variations and potential virulence factors relating to the host specificity of these three fungal pathogens. Based on the evolutionary analysis of single-copy orthologous proteins, we found that the genes encoding glycoside hydrolases, carbohydrate esterases, oxidoreductase, and multidrug transporters in SCAU-1 and SCAU-6 were expanded, while expansion in SCAU-2 was mainly related to carbohydrate esterases. In summary, our study provides clues to understand the pathogenic mechanisms of fungal isolates from barnyard grass with the potential to cause rice blast.

Characterization of Shigella flexneri serotype 6 strains from geographically diverse low- and middle-income countries.

Shigella flexneri serotype 6 (Sf6) is one of the most common serotypes recovered from surveillance studies of moderate to severe diarrhea. Despite the clinical significance of Sf6, this serotype is understudied. In this work, we have performed both serotype-specific genomic and phenotypic comparisons of Sf6 isolates to one another and non-S. flexneri serotypes. Comparative genomic analyses identified significant nucleotide homology between Sf6 strains (n = 325), despite a broad range of collection timeframes and geographic locations. We identified Sf6 specific factors, including a potential novel Shigella virulence factor (type II secretion system). Additionally, we identified established Shigella virulence genes (ospG) and metabolic genes (rutABCDEFGR) that were absent in Sf6 strains while present in the majority of 728 non-Sf6 strains. Complete sequencing of 11 clinical Sf6 strains, demonstrated that the Sf6 virulence plasmid (pINV) is ~38 kb smaller than the average non-Sf6 pINV (~228 kb). Comparisons of S. flexneri species level antibiotic susceptibility highlighted that clinical Sf6 isolates from Africa in the Global Enteric Multicenter Study (GEMS) and Vaccine Impact on Diarrhea in Africa (VIDA) study demonstrated geographic, serotype-specific susceptibility pattern. Phenotypic analyses of Sf6 identified reduced intracellular invasion and cytokine induction from HT-29 cells, as well as reduced Ipa protein effector secretion, compared with S. flexneri serotype 2a strain 2457T. Together these data highlight conserved and unique serotype-specific genotypic and phenotypic features for Sf6. This level of conservation has not been noted for other S. flexneri serotypes and is promising for vaccine and diagnostic assays to provide global Sf6-specific coverage.IMPORTANCEShigellosis is an ongoing global public health crisis with >270 million annual episodes among all age groups; however, the greatest disease burden is among children in low- and middle-income countries (LMIC). The lack of a licensed Shigella vaccine and the observed rise in antimicrobial-resistant Shigella spp. highlights the urgency for effective preventative and interventional strategies. The inclusion of S. flexneri serotype 6 (Sf6) is a necessary component of a multivalent vaccine strategies based on its clinical and epidemiological importance. Given the genomic diversity of Sf6 compared with other S. flexneri serotypes and Sf6 unique O-antigen core structure, serotype-specific characterization of Sf6 is a critical step to inform Shigella-directed vaccine and alternative therapeutic designs. Herein, we identified conserved genomic content among a large collection of temporally and geographically diverse Sf6 clinical isolates and characterized genotypic and phenotypic properties that separate Sf6 from non-Sf6 S. flexneri serotypes.

LysR-Type Transcriptional Regulator Contributes to Pseudomonas cannabina pv. alisalensis Virulence by Regulating Type Three Secretion System

Pseudomonas cannabina pv. alisalensis (Pcal) causes bacterial blight on cabbage. In a previous study, we screened for reduced virulence using Tn5 transposon mutants and identified a LysR-type transcriptional regulator (LTTR) as a potential virulence factor in Pcal. However, the role of LTTR in Pcal virulence has not been thoroughly investigated. In this study, we demonstrated that the Pcal NN14 mutant (with Tn5 insertion in the LTTR-encoding gene) showed reduced disease symptoms and bacterial populations in cabbage, indicating that LTTR contributes to Pcal virulence. RNA-seq analysis identified 39 LTTR-dependent genes. Genes associated with 13 of the type three secretion system (T3SS), two of flagellar apparatus, ABC transporters, and transcription factors were expressed at lower levels in the NN14 mutant compared to the wild type. Conversely, tssH and hcp, type six secretion system (T6SS)-related genes, showed higher expression in NN14. Furthermore, these differences in gene expression were observed in minimal medium, but not in nutrient-rich medium, suggesting that LTTR acts as a global regulator responsive to nutrient conditions. Additionally, LTTR activated the expression of T3SS-related genes during Pcal infection. We also demonstrated that NN14 showed a reduced ability to induce hypersensitive reaction (HR) cell death in non-host plants. Collectively, these results suggest that LTTR contributes to Pcal virulence by regulating T3SS in response to environmental changes.

Comparative Genome Analysis of Piscine Vibrio vulnificus: Virulence-Associated Metabolic Pathways.

Vibriosis caused by Vibrio vulnificus is a major problem in aquatic animals, particularly brown marble groupers (Epinephelus fuscoguttatus). V. vulnificus biotype I has recently been isolated and classified into subgroups SUKU_G1, SUKU_G2, and SUKU_G3 according to the different types of virulence genes. In a previous study, we have shown that biotype I V. vulnificus strains were classified into three subgroups according to the different types of virulence genes, which exhibited different phenotypes in terms of growth rate and virulence. To gain insight into the different genetic features revealed by the potential virulence mechanisms of V. vulnificus in relation to a spectrum of pathogenesis, comparative genomic analyses of three biotype I V. vulnificus strains belonging to different subgroups (SUKU_G1, SUKU_G2, and SUKU_G3) were performed. The V. vulnificus genome is composed of two circular chromosomes with average sizes of 3 Mbp and 1.7 Mbp that are evolutionarily related based on the analysis of orthologous genes. A comparative genome analysis of V. vulnificus revealed 5200 coding sequences, of which 3887 represented the core genome and the remaining 1313 constituted the dispensable genome. The most virulent isolate (SUKU_G1) carries unique enzymes that are important for lipopolysaccharide (LPS) and capsular polysaccharide (CPS) synthesis, as well as flagellar glycosylation, and harbors another type of repeat in toxin (RTX) and bacterial defense mechanisms. The less virulent isolate (SUKU_G2) shares enzymes related to CPS biosynthesis or flagellar glycosylation, while the avirulent isolate (SUKU_G3) and a less virulent isolate (SUKU_G2) share enzymes related to the production of rare sugars. Interestingly, the isolates from the three subgroups containing specific CMP-N-acetylneuraminate-producing enzymes that are correlated with their growth abilities. Collectively, these observations provide an understanding of the molecular mechanisms underlying disease pathogenesis and support the development of strategies for bacterial disease prevention and control.