VFDB 2019: a comparative pathogenomic platform with an interactive web interface

In bacteria, the evolution of pathogenicity seems to be the result of the constant arrival of virulence factors (VFs) into the bacterial genome. However, the integration, retention, and/or expression of these factors may be the result of the interaction between the new arriving genes and the bacterial genomic background. To test this hypothesis, a phylogenetic analysis was done on a collection of 98 Escherichia coli/Shigella strains representing the pathogenic and commensal diversity of the species. The distribution of 17 VFs associated to the different E. coli pathovars was superimposed on the phylogenetic tree. Three major types of VFs can be recognized: (1) VFs that arrive and are expressed in different genetic backgrounds (such as VFs associated with the pathovars of mild chronic diarrhea: enteroaggregative, enteropathogenic, and diffusely-adhering E. coli), (2) VFs that arrive in different genetic backgrounds but are preferentially found, associated with a specific pathology, in only one particular background (such as VFs associated with extraintestinal diseases), and (3) VFs that require a particular genetic background for the arrival and expression of their virulence potential (such as VFs associated with pathovars typical of severe acute diarrhea: enterohemorragic, enterotoxigenic, and enteroinvasive E. coli strains). The possibility of a single arrival of VFs by chance, followed by a vertical transmission, was ruled out by comparing the evolutionary histories of some of these VFs to the strain phylogeny. These evidences suggest that important changes in the genome of E. coli have occurred during the diversification of the species, allowing the virulence factors associated with severe acute diarrhea to arrive in the population. Thus, the E. coli genome seems to be formed by an "ancestral" and a "derived" background, each one responsible for the acquisition and expression of different virulence factors.

ObjectiveCurrently, next generation sequencing (NGS) is widely used to decode potential novel or variant pathogens both in emergent outbreaks and in routine clinical practice. However, the efficient identification of novel or diverged pathogenomic compositions remains a big challenge. It is especially true for short DNA sequence fragments from NGS, since sequence similarity searching is vulnerable to false negatives or false positives, as is mismatching or matching with unrelated proteins. Therefore, this study aimed to establish a bioinformatics approach that can generate unique motif sequences for profiling searching, resulting in high specificity and sensitivity.ResultsIn this study, we introduced a Shortest Unique Representative Hidden Markov Model (HMM) approach to identify bacterial toxin, virulence factor (VF), and antimicrobial resistance (AR) in short sequence reads. We first construct unique representative domain sequences of toxin genes, VFs, and ARs to avoid potential false positives, and then to use HMM models to accurately identify potential toxin, VF, and AR fragments. The benchmark shows this approach can achieve relatively high specificity and sensitivity if the appropriate cutoff value is applied. Our approach can be used to recognize the protein sequences of known toxins and pathogens, identifies their common characteristics and then searches for similar sequences in other organisms.

Periodically disturbing biofilms reduces expression of quorum sensing-regulated virulence factors in Pseudomonas aeruginosa

Staphylococcus aureus uses quorum sensing and nutrient availability to control the expression of agr-regulated virulence factors. Quorum sensing is mediated by autoinducing peptide (AIP), which at a high concentration reduces expression of surface attachment proteins (coa, fnbpA) and increases expression of exotoxins (lukS) and proteases (splA). Nutrient availability can be sensed through the saeS/saeR system. Low nutrients increase expression of saeR, which augments expression of coa and fnbpA, distinct from the activity of AIP. The formation of spatial structure, such as biofilms, can alter quorum sensing and nutrient acquisition. In natural environments, biofilms encounter forces that may alter their spatial structure. These forces may impact quorum sensing and/or nutrient acquisition and thus affect the expression of agr-regulated virulence factors. However, this has not been studied. We show that periodically disturbing biofilms composed of S. aureus using a physical force affected the expression of agr-regulated virulence factors. In nutrient-poor environments, disturbance increased the expression of coa, fnbpA, lukS, and splA. Disturbance in a nutrient-rich environment at low or high disturbance amplitudes moderately reduced expression of coa and fnbpA but increased expression of lukS and splA. Interestingly, at an intermediate amplitude, the overall expression of agr-regulated virulence factors was the lowest; expression of lukS and splA remained unchanged relative to an undisturbed biofilm, while expression of coa and fnbpA significantly decreased. We hypothesize that these changes are a result of disturbance-driven changes in access to AIP and nutrients. Our results may allow the identification of environments where virulence is enhanced, or reduced, owing to a disturbance. IMPORTANCE Bacteria, such as Staphylococcus aureus, integrate signals from the environment to regulate genes encoding virulence factors. These signals include those produced by quorum-sensing systems and nutrient availability. We show that disturbing the spatial organization of S. aureus populations can lead to changes in the expression of virulence factors, likely by altering the ways in which S. aureus detects these signals. Our work may allow us to identify environments that increase or reduce the expression of virulence factors in S. aureus.

PurposeThis study aimed to investigate differences in virulence factors between exoU and exoS lineages of Pseudomonas aeruginosa from microbial keratitis (MK).MethodInitially, the whole genome sequence (WGS) data of 20 exoU and 19 exoS P. aeruginosa keratitis isolates from India and Australia retrieved from NCBI genome database was assessed for the presence of different virulence genes using the Virulence Factors Database (VFDB). To confirm the VFDB results, a separate set of keratitis isolates consisting of 148 P. aeruginosa (36 from India and 112 from Australia) were screened by PCR for the presence of exoU, exoS, and the virulence genes found to be significantly different in the VFDB analysis. Flagellar length was measured by transmission electron microscopy (TEM) and phospholipase D (PLD) activity was determined by Amplex Red Phospholipase D Assay Kit.ResultsFrom 327 virulence-associated genes, the VFDB analysis identified significant differences in four virulence factor genes pilA, pldA, algP, and flaG between the exoU and exoS groups (all p < 0.05).When combining PCR and VFDB data from 187 keratitis isolates, pldA (83.6% vs. 31.8%, p < 0.01) and flaG (80% vs. 55.3%, p < 0.01) showed significantly higher prevalence in the exoU than the exoS. Similar trends were observed among Australian isolates while within the Indian isolates, only pldA (77.3% vs. 35.3%, p < 0.01) differed significantly. No association was found between flagellar length and flaG, but PLD activity correlated with the presence of the pldA.ConclusionThe pldA and flaG genes might be relevant virulence factors for the exoU group, potentially associated with the severity of MK.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10096-025-05294-3.

Staphylococcus aureus produces many virulence factors, including toxins, immune-modulatory factors, and exoenzymes. Previous studies involving the analysis of virulence expression were mainly performed by in vitro experiments using bacterial medium. However, when S. aureus infects a host, the bacterial growth conditions are quite different from those in a medium, which may be related to the different expression of virulence factors in the host. In this study, we investigated the expression of virulence factors in S. aureus grown in calf serum. The expression of many virulence factors, including hemolysins, enterotoxins, proteases, and iron acquisition factors, was significantly increased compared with that in bacterial medium. In addition, the expression of RNA III, a global regulon for virulence expression, was significantly increased. This effect was partially restored by the addition of 300 μM FeCl₃ into serum, suggesting that iron depletion is associated with the increased expression of virulence factors in serum. In chemically defined medium without iron, a similar effect was observed. In a mutant with agr inactivated grown in serum, the expression of RNA III, psm, and sec4 was not increased, while other factors were still induced in the mutant, suggesting that another regulatory factor(s) is involved. In addition, we found that serum albumin is a major factor for the capture of free iron to prevent the supply of iron to bacteria grown in serum. These results indicate that S. aureus expresses virulence factors in adaptation to the host environment.

The virulence factor database (VFDB, http://www.mgc.ac.cn/VFs/) has served as a comprehensive repository of bacterial virulence factors (VFs) for >7 years. Bacterial virulence is an exciting and dynamic field, due to the availability of complete sequences of bacterial genomes and increasing sophisticated technologies for manipulating bacteria and bacterial genomes. The intricacy of virulence mechanisms offers a challenge, and there exists a clear need to decipher the ‘language’ used by VFs more effectively. In this article, we present the recent major updates of VFDB in an attempt to summarize some of the most important virulence mechanisms by comparing different compositions and organizations of VFs from various bacterial pathogens, identifying core components and phylogenetic clades and shedding new light on the forces that shape the evolutionary history of bacterial pathogenesis. In addition, the 2012 release of VFDB provides an improved user interface.

Pseudomonas aeruginosa (P. aeruginosa) possesses a plethora of virulence determinants, including the production of biofilm, pigments, exotoxins, proteases, flagella, and secretion systems. The aim of our present study was to establish the relationship between biofilm-forming capacity, the expression of some important virulence factors, and the multidrug-resistant (MDR) phenotype in P. aeruginosa. A total of three hundred and two (n = 302) isolates were included in this study. Antimicrobial susceptibility testing and phenotypic detection of resistance determinants were carried out; based on these results, isolates were grouped into distinct resistotypes and multiple antibiotic resistance (MAR) indices were calculated. The capacity of isolates to produce biofilm was assessed using a crystal violet microtiter-plate based method. Motility (swimming, swarming, and twitching) and pigment-production (pyoverdine and pyocyanin) were also measured. Pearson correlation coefficients (r) were calculated to determine for antimicrobial resistance, biofilm-formation, and expression of other virulence factors. Resistance rates were the highest for ceftazidime (56.95%; n = 172), levofloxacin (54.97%; n = 166), and ciprofloxacin (54.64%; n = 159), while lowest for colistin (1.66%; n = 5); 44.04% (n = 133) of isolates were classified as MDR. 19.87% (n = 60), 20.86% (n = 63) and 59.27% (n = 179) were classified as weak, moderate, and strong biofilm producers, respectively. With the exception of pyocyanin production (0.371 ± 0.193 vs. non-MDR: 0.319 ± 0.191; p = 0.018), MDR and non-MDR isolates did not show significant differences in expression of virulence factors. Additionally, no relevant correlations were seen between the rate of biofilm formation, pigment production, or motility. Data on interplay between the presence and mechanisms of drug resistance with those of biofilm formation and virulence is crucial to address chronic bacterial infections and to provide strategies for their management.

Klebsiella michiganensis is an opportunistic pathogen that causes an increasing number of serious infections. This study aimed to investigate the etiology of the severe clinical symptoms of sepsis in preterm infants and the characterization of K. michiganensis isolates. Whole-genome sequencing (WGS) was performed on three strains isolated from an infected preterm infant. Additionally, the genomic sequences of 534 K. michiganensis strains were obtained from the NCBI database. To gain deeper insights into these strains, we utilized the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG), and Pathogen Host Interactions (PHI) database annotation tools for comprehensive gene function analyses. Moreover, the multilocus sequence typing (MLST), EasyCGtree, and virulence factor database (VFDB) were employed to determine the sequence types (STs), construct phylogenetic trees, and identify potential virulence factors. Sequence analysis found that the three isolated strains had identical sequence characteristics and did not correspond to any of the known ST types. Virulence factor analysis revealed that the three strains harbored mrkABCDFHIJ, fimABCDEFGHIK, entABCDEFS, fepABCD, and capsule genes. These virulence factors are likely to play crucial roles in enhancing adhesion and metabolic capabilities, resisting phagocytosis (inducing immune cell damage), and ultimately contributing to prolonged bacteremia. The phylogenetic tree and comparative genomics of virulence factors showed the genetic and virulence factor diversity of the currently reported K. michiganensis strains. We identified a novel strain of K. michiganensis that exhibits high virulence and leads to severe septicemia phenotypes in preterm infants. Furthermore, comparative genomic analysis of previously reported K. michiganensis strains revealed the existence of three clades. This comprehensive analysis provides novel insights into the genetic relationships and virulence factor profiles of diverse strains of K. michiganensis. In future, it will be necessary to investigate the concept of the high virulence of K. michiganensis to determine the treatment method.

To assess the safety of Bifidobacterium longum (B. longum) JDM301 based on complete genome sequences. The complete genome sequences of JDM301 were determined using the GS 20 system. Putative virulence factors, putative antibiotic resistance genes and genes encoding enzymes responsible for harmful metabolites were identified by blast with virulence factors database, antibiotic resistance genes database and genes associated with harmful metabolites in previous reports. Minimum inhibitory concentration of 16 common antimicrobial agents was evaluated by E-test. JDM301 was shown to contain 36 genes associated with antibiotic resistance, 5 enzymes related to harmful metabolites and 162 nonspecific virulence factors mainly associated with transcriptional regulation, adhesion, sugar and amino acid transport. B. longum JDM301 was intrinsically resistant to ciprofloxacin, amikacin, gentamicin and streptomycin and susceptible to vancomycin, amoxicillin, cephalothin, chloramphenicol, erythromycin, ampicillin, cefotaxime, rifampicin, imipenem and trimethoprim-sulphamethoxazol. JDM301 was moderately resistant to bacitracin, while an earlier study showed that bifidobacteria were susceptible to this antibiotic. A tetracycline resistance gene with the risk of transfer was found in JDM301, which needs to be experimentally validated. The safety assessment of JDM301 using information derived from complete bacterial genome will contribute to a wider and deeper insight into the safety of probiotic bacteria.

Bacterial pathogens continue to impose a major threat to public health worldwide in the 21st century. Intensified studies on bacterial pathogenesis have greatly expanded our knowledge about the mechanisms of the disease processes at the molecular level over the last decades. To facilitate future research, it becomes necessary to form a database collectively presenting the virulence factors (VFs) of various medical significant bacterial pathogens. The aim of virulence factor database (VFDB) (http://www.mgc.ac.cn/VFs/) is to provide such a source for scientists to rapidly access to current knowledge about VFs from various bacterial pathogens. VFDB is comprehensive and user-friendly. One can search VFDB by browsing each genus or by typing keywords. Furthermore, a BLAST search tool against all known VF-related genes is also available. VFDB provides a unified gateway to store, search, retrieve and update information about VFs from various bacterial pathogens.

Staphylococcus aureus is a Gram-positive bacterium that is present in the human microbiota. Nevertheless, these bacteria can be pathogenic to the humans. Due to the increasing occurrence of antibiotic-resistant S. aureus strains, new approaches to control this pathogen are necessary. The antimicrobial photodynamic inactivation (PDI) process is based in the combined use of light, oxygen, and an intermediary agent (a photosensitizer). These three components interact to generate cytotoxic reactive oxygen species that irreversibly damage vital constituents of the microbial cells and ultimately lead to cell death. Although PDI is being shown to be a promising alternative to the antibiotic approach for the inactivation of pathogenic microorganisms, information on effects of photosensitization on particular virulence factors is strikingly scarce. The objective of this work was to evaluate the effect of PDI on virulence factors of S. aureus and to assess the potential development of resistance of this bacterium as well as the recovery of the expression of the virulence factors after successive PDI cycles. For this, the photosensitizer 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin tetra-iodide (Tetra-Py+-Me) and six strains of S. aureus [one reference strain, one strain with one enterotoxin, two strains with three enterotoxins and two methicillin resistant strains (MRSA) – one with five enterotoxins and the other without enterotoxins] were used. The effect of photosensitization on catalase activity, beta hemolysis, lipases, thermonuclease, enterotoxins, coagulase production, and resistance/susceptibility to methicillin was tested. To assess the development of resistance after successive cycles of treatment, three strains of S. aureus (ATCC 6538, 2065 MA, and SA 3 MRSA) were used. The surviving colonies of a first cycle of PDI were collected from the solid medium and subjected to further nine consecutive cycles of PDI. The results indicate that the expression of some external virulence factors is affected by PDI and enterotoxin producing strains were more susceptible to PDI than non-toxigenic strains. The surviving bacteria did not develop resistance. PDI, contrarily to traditional antibiotics, inhibited the expression of virulence factors, efficiently inactivating either highly virulent strains and low virulent S. aureus strains, inactivating also antibiotic susceptible and resistant strains, without development of photoresistance after at least 10 consecutive cycles of treatment, and so this therapy may become a strong promising alternative to antibiotics to control pathogenic microorganisms.

Cronobacter spp. can cause systemic infections, such as meningitis, sepsis, and necrotizing enterocolitis, in immunocompromised patients, especially neonates. Although some virulence factors have been reported previously, the pathogenesis of Cronobacter remains unclear. In this study, we compared genome sequences from different Cronobacter species, sequence types, and sources, with the virulence genes in the virulence factor database. The results showed that Cronobacter has species specificity for these virulence genes. Additionally, two gene clusters, including sfp encoding fimbriae and hly encoding hemolysin, were discovered. Through cell adhesion, cytotoxicity, and hemolysis assays, we found that the isolates possessing the two gene clusters had higher cytotoxicity and stronger hemolysis capacity than those of other isolates in this study. Moreover, analysis of type VI secretion system (T6SS) cluster and putative fimbria gene clusters of Cronobacter revealed that T6SS have species specificity and isolates with high cytotoxicity possessed more complete T6SS cluster construction than that of the rest. In conclusion, the two novel gene clusters and T6SS cluster were involved in the mechanism underlying the cytotoxicity of Cronobacter.

In the process of host–pathogen interactions, bacterial pathogens always employ some special genes, e.g., virulence factors (VFs) to interact with host and cause damage or diseases to host. A number of VFs have been identified in bacterial pathogens that confer upon bacterial pathogens the ability to cause various types of damage or diseases. However, it has been clarified that some of the identified VFs are also encoded in the genomes of nonpathogenic bacteria, and this finding gives rise to considerable controversy about the definition of virulence factor. Here 1988 virulence factors of 51 sequenced pathogenic bacterial genomes from the virulence factor database (VFDB) were collected, and an orthologous comparison to a non-pathogenic bacteria protein database was conducted using the reciprocal-best-BLAST-hits approach. Six hundred and twenty pathogen-specific VFs and 1368 common VFs (present in both pathogens and nonpathogens) were identified, which account for 31.19% and 68.81% of the total VFs, respectively. The distribution of pathogen-specific VFs and common VFs in pathogenicity islands (PAIs) was systematically investigated, and pathogen-specific VFs were more likely to be located in PAIs than common VFs. The function of the two classes of VFs were also analyzed and compared in depth. Our results indicated that most but not all T3SS proteins are pathogen-specific. T3SS effector proteins tended to be distributed in pathogen-specific VFs, whereas T3SS translocation proteins, apparatus proteins, and chaperones were inclined to be distributed in common VFs. We also observed that exotoxins were located in both pathogen-specific and common VFs. In addition, the architecture of the two classes of VFs was compared, and the results indicated that common VFs had a higher domain number and lower domain coverage value, revealed that common VFs tend to be more complex and less compact proteins.

The vast majority of the species of the genus Candida spp.is commensal in humans; however, some are opportunistic pathogens that can cause infection, called candidosis. Among the different types of candidosis, we highlight the vulvovaginal (VVC) which can occur in two main clinical variants: chronic (cVVC) and episodic or sporadic. The incidence of cVVC has been worrying the scientific community, promoting the research on genotypic and phenotypic causes of its occurrence. We summarize important findings on factors that favor chronic vulvovaginal candidosis with respect to molecular epidemiology and the expression of various virulence factors, while clarifying the terminology involving these infections. The aim of this review was to gather research that linked virulence factors to VVC and its persistence and recurrence, using two databases (Pubmed and Google Scholar). Predisposing factors in women for the occurrence of cVVC and some studies that refer new preventive and alternative therapies were also included, where appropriate. Several studies have been shedding light on the increasing number of persistence and recurrences of VVC. The expression of virulence factors has been related to both chronic forms of VVC and antifungal resistance. Other studies report mutations occurring in the genome of Candida spp. during the infection phase which may be important indications for new therapies. The introduction of preventive therapies and new therapies has revealed great importance and is also highlighted here.