An expanded database and analytical toolkit for identifying bacterial virulence factors and their associations with chronic diseases.

Virulence factor genes (VFGs) play pivotal roles in bacterial infections and have been identified within the human gut microbiota. However, their involvement in chronic diseases remains poorly understood. Here, we establish an expanded VFG database (VFDB 2.0) consisting of 62,332 nonredundant orthologues and alleles of VFGs using species-specific average nucleotide identity ( https://github.com/Wanting-Dong/MetaVF_toolkit/tree/main/databases ). We further develop the MetaVF toolkit, facilitating the precise identification of pathobiont-carried VFGs at the species level. A thorough characterization of VFGs for 5452 commensal isolates from healthy individuals reveals that only 11 of 301 species harbour these factors. Further analyses of VFGs within the gut microbiomes of nine chronic diseases reveal both common and disease-specific VFG features. Notably, in type 2 diabetes patients, long HiFi sequencing confirms that shared VF features are carried by pathobiont strains of Escherichia coli and Klebsiella pneumoniae. These findings underscore the critical importance of identifying and understanding VFGs in microbiome-associated diseases.

Phenotypic and genotypic characterization of Enterococcus faecalis and Enterococcus faecium isolated from fish, vegetables, and humans.

Enterococci, common hospital-acquired infections in immunocompromised patients, have garnered attention in clinical microbiology. To determine the clinical relevance of enterococci as food-borne pathogens, 116 fish, 90 vegetables, and 120 human diarrheal samples were tested for E. faecalis and E. faecium pathogenicity. Conventionally, 69 of 326 (21.17%) samples were positive for Enterococcus species, 52 (15.95%) of which were molecularly classified as E. faecalis and 13 (3.99%) as E. faecium. The E. faecalis contamination percentage of fresh fish (19.70%) was higher than frozen fish (4%). Cauliflower had the highest E. faecalis percentage (16.67%) when fish and vegetable samples didn't harbor the E. faecium atpA gene. 23.33% and 10.83% of participants' samples were molecularly confirmed as E. faecalis and E. faecium positive, respectively. E. faecalis isolates had all virulence genes, with gels being the most common (65.38%), while cylA and asa1 genes couldn't be detected in E. faecium isolates. E. faecalis showed the highest resistance against vancomycin and tetracycline (69.23%), whereas E. faecium extremely resisted tetracycline (76.92%) and erythromycin (69.23%) with the recognition of MDR among 44.2% of E. faecalis and 38.5% of E. faecium isolates. The great similarity of our isolates showed the clinical importance of food-borne antibiotic-resistant enterococci.

Streptococcus agalactiae isolated from clinical mastitis cases on large dairy farms in north China: phenotype, genotype of antimicrobial resistance and virulence genes.

Streptococcus agalactiae (Strep. agalactiae) is bovine mastitis pathogen and has thus became a matter of concern to dairy farms worldwide in terms of economic loss. The aims of this study were to (a) determine virulence genes, and (b) characterize the antimicrobial resistance (AMR) profiles and AMR genes and (c) figure out the relationship between AMR phenotypes and genotypes of Strep. agalactiae isolated from dairy cows in north China. A total of 20 virulence genes and 23 AMR genes of 140 isolates collected from 12 farms in six provinces were studied. The antimicrobial susceptibility of 10 veterinary commonly used antimicrobials were tested using the broth microdilution method. Results showed that all the isolates harbored the virulence genes lacIV, gapC, and dltA. The isolates that harbored the genes lacIII, fbsA, hylB, and cfb exhibited the high prevalence (99.29%), followed by isolates that harbored lacI (98.57%), bibA (97.86%), cylE (97.14%), lacII (92.14%), cspA (52.14%), pavA (25%), bca (2.14%), and scpB (0.71%). The fbsB, lmb, spbI, bac, and rib genes were not detected. The virulence patterns of B (fbsA_cfb_cylE_ hylB_bibA_cspA_ gapC_dltA_lacIII/IV) and C (fbsA_cfb_ bibA _ gapC_ dltA_lacIV) were dominant, accounting for 97.86% of the isolates. The following AMR genes were prevalent: pbp1A (97.14%), tet(M) (95.00%), lnu (A) (80.71%), erm (B) (75.00%), tet(O) (72.14%), blaZ (49.29%), tet(S) (29.29%), blaTEM (25.71%), erm (A) (17.14%), erm (C) (13.57%), tet (L) (10.71%), linB (2.86%), and erm (TR) (2.86%). The pbp2b, mecA1, mecC, lnu (D), erm (F/G/Q), and mef (A) genes were not detected. Eighty percent of the isolates harbored AMR genes and were highly resistant to tetracycline, followed by macrolides (10.71%), lincosamides (9.29%) and ß-lactams (4.29%). In conclusion, isolates only exhibited well correlation between tetracyclines resistance phenotype and genotype, and almost all isolates harbored intact combination of virulence genes.

Evaluation of Klebsiella pneumoniae pathogenicity through holistic gene content analysis.

Klebsiella pneumoniae is a Gram-negative bacterium that causes both community- and healthcare-associated infections. Although various virulence factors and highly pathogenic phenotypes have been reported, the pathogenicity of K. pneumoniae is still not fully understood. In this study, we utilized whole-genome sequencing data of 168 clinical K. pneumoniae strains to assess pathogenicity. This work was based on the concept that the genetic composition of individual genomes (referred to as holistic gene content) of the strains may contribute to their pathogenicity. Holistic gene content analysis revealed two distinct groups of K. pneumoniae strains ('major group' and 'minor group'). The minor group included strains with known highly pathogenic clones (ST23, ST375, ST65 and ST86). The minor group had higher rates of capsular genotype K1 and presence of nine specific virulence genes (rmpA, iucA, iutA, irp2, fyuA, ybtS, iroN, allS and clbA) compared to the major group. Pathogenicity was assessed using Galleria mellonella larvae. Infection experiments revealed lower survival rates of larvae infected with strains from the minor group, indicating higher virulence. In addition, the minor group had a higher string test positivity rate than the major group. Holistic gene content analysis predicted possession of virulence genes, string test positivity and pathogenicity as observed in the G. mellonella infection model. Moreover, the findings suggested the presence of as yet unrecognized genomic elements that are either involved in the acquisition of virulence genes or associated with pathogenicity.

Evaluation of virulence determinants and cell surface properties associated with biofilm formation in methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrum beta-lactamase (ESBL) Escherichia coli from livestock and poultry origin.

Antibiotic resistance poses a persistent threat to modern medicine due to the emergence of novel antibiotic-resistant strains. Therefore, a timely understanding of antibiotic resistance and the virulence biology of pathogenic bacteria, particularly those of public health significance, is crucial for implementing effective mitigation strategies. This study aimed to investigate the virulence profiles of ten S. aureus isolates (NDa to NDj) and ten E. coli isolates (ND1 to ND10) originating from livestock and poultry, and to assess how various cell surface properties and biofilm formation abilities influence antibiotic resistance phenotypes. Antibiotic resistance profiling through phenotypic (AST) and genotypic methods (PCR) confirmed that NDa to NDe were methicillin-resistant S. aureus (MRSA) and ND1 to ND5 were extended-spectrum ß-lactamase (ESBL) producing E. coli isolates. Virulence properties such as hemolytic activity, coagulase activity, and nuclease activity were found to be independent of the antibiotic resistance phenotype in S. aureus. In contrast, biofilm formation phenotype was observed to influence antibiotic resistance phenotypes, with MRSA and ESBL E. coli isolates demonstrating higher biofilm formation potency. Chemical and enzymatic analysis of S. aureus and E. coli biofilms revealed proteins and polysaccharides as major components, followed by nucleic acids. Furthermore, cell surface properties such as auto-aggregation and hydrophobicity were notably higher in isolates with strong to medium biofilm-forming capabilities (ESBL and MRSA isolates), corroborated by genomic confirmation of various genes associated with biofilm, adhesion, and colonization. In conclusion, this study highlights that surface hydrophobicity and biofilm formation ability of MRSA (NDa to NDe) and ESBL E. coli (ND1 to ND5) isolates may influence antibiotic resistance phenotypes.

Genomic approach to determine sources of neonatal Staphylococcus aureus infection from carriage in the Gambia.

Staphylococcus aureus is a major cause of neonatal infections in various anatomical sites, resulting in high morbidity and mortality in The Gambia. These clinical infections are often preceded by nasal carriage of S. aureus, a known risk factor. To determine whether potential sources of newborn S. aureus infections were from carriage, and to characterize S. aureus present in different anatomical sites (blood, ear, eye, umbilical cord, skin, pus, oropharynx, breast milk and vagina), we performed whole-genome sequencing of 172 isolates from clinical sites as well as from healthy and unhealthy carriage. A random selection of mothers (n = 90) and newborns (n = 42) participating in a clinical trial and testing positive for S. aureus were considered for this study. Sequence data were analyzed to determine S. aureus multilocus sequence types and selected antimicrobial and virulence gene profiles. Our findings revealed that in The Gambia, ST15 is the dominant sequence type associated with both carriage and clinical infection. In addition, S. aureus isolates causing clinical infection among neonates were genetically similar to those colonizing their oropharynx, and the different anatomical sites were not found to be uniquely colonized by S. aureus of a single genomic profile. Furthermore, while S. aureus associated with clinical infection had similar antimicrobial resistance gene profiles to carriage isolates, only hemolysin and adhesive factor virulence genes were significantly higher among clinical isolates. In conclusion, this study confirmed S. aureus oropharyngeal colonization among neonates as a potential source of clinical infection in The Gambia. Hence, interventions aiming to reduce neonatal clinical infections in The Gambia should consider decreasing oropharyngeal S. aureus carriage.Trial registration The trial was registered at ClinicalTrials.gov NCT03199547.

Proven anti-virulence therapies in combating methicillin- and vancomycin-resistant Staphylococcus aureus infections.

Introduction: Despite years of efforts to develop new antibiotics for eradicating multidrug-resistant (MDR) and multi-virulent Methicillin-Resistant Staphylococcus aureus (MRSA) and Vancomycin-Resistant Staphylococcus aureus (VRSA) infections, treatment failures and poor prognoses in most cases have been common. Therefore, there is an urgent need for new therapeutic approaches targeting virulence arrays. Our aim is to discover new anti-virulence therapies targeting MRSA and VRSA virulence arrays. Methodology: We employed phenotypic, molecular docking, and genetic studies to screen for anti-virulence activities among selected promising compounds: Coumarin, Simvastatin, and Ibuprofen. Results: We found that nearly all detected MRSA and VRSA strains exhibited MDR and multi-virulent profiles. The molecular docking results aligned with the phenotypic and genetic assessments of virulence production. Biofilm and hemolysin productions were inhibited, and all virulence genes were downregulated upon treatment with sub-minimum inhibitory concentration (sub-MIC) of these promising compounds. Ibuprofen was the most active compound, exhibiting the highest inhibition and downregulation of virulence gene products. Moreover, in vivo and histopathological studies confirmed these results. Interestingly, we observed a significant decrease in wound area and improvements in re-epithelialization and tissue organization in the Ibuprofen and antimicrobial treated group compared with the group treated with antimicrobial alone. These findings support the idea that a combination of Ibuprofen and antimicrobial drugs may offer a promising new therapy for MRSA and VRSA infections. Conclusion: We hope that our findings can be implemented in clinical practice to assist physicians in making the most suitable treatment decisions.

Genetic and phenotypic diversity of Flavobacterium psychrophilum isolates from Czech salmonid fish farms.

BACKGROUND: The salmonid pathogen Flavobacterium psychrophilum poses a significant economic threat to global aquaculture, yet our understanding of its genetic and phenotypic diversity remains incomplete across much of its geographic range. In this study, we characterise the genetic and phenotypic diversity of 70 isolates collected from rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta m. fario) from fish farms in the Czech Republic between 2012 and 2019 to compare their genomic content with all draft or complete genomes present in the NCBI database (n = 187). RESULTS: The Czech isolates underwent comprehensive evaluation, including multiplex PCR-based serotyping, genetic analysis, antimicrobial resistance testing, and assessment of selected virulence factors. Multiplex PCR serotyping revealed 43 isolates as Type 1, 23 as Type 2, with sporadic cases of Types 3 and 4. Multi-locus sequence typing unveiled 12 sequence types (ST), including seven newly described ones. Notably, 24 isolates were identified as ST329, a novel sequence type, while 22 were classified as the globally-distributed ST2. Phylogenetic analysis demonstrated clonal distribution of ST329 in the Czech Republic, with these isolates lacking a phage sequence in their genomes. Antimicrobial susceptibility testing revealed a high proportion of isolates classified as non-wild type with reduced susceptibility to oxolinic acid, oxytetracycline, flumequine, and enrofloxacin, while most isolates were classified as wild type for florfenicol, sulfamethoxazole-trimethoprim, and erythromycin. However, 31 isolates classified as wild type for florfenicol exhibited minimum inhibitory concentrations at the susceptibility breakpoint. CONCLUSION: The prevalence of the Czech F. psychrophilum serotypes has evolved over time, likely influenced by the introduction of new isolates through international trade. Thus, it is crucial to monitor F. psychrophilum clones within and across countries using advanced methods such as MLST, serotyping, and genome sequencing. Given the open nature of the pan-genome, further sequencing of strains promises exciting discoveries in F. psychrophilum genomics.

Virulence factors, antibiotic susceptibility and sequence type distribution of hospital-associated Clostridioides difficile isolates in Israel, 2020-2022.

Biofilm formation and toxin production are some of the virulence factors of Clostridioides difficile (C. difficile), which causes hospital-acquired C. difficile infection (HA-CDI). This work investigated the prevalence and distribution of different strains recovered from HA-CDI patients hospitalized in 4 medical centres across Israel, and characterized strains' virulence factors and antibiotic susceptibility. One-hundred and eighty-eight faecal samples were collected. C. difficile 's toxins were detected by the CerTest Clostridium difficile GDH + Toxin A + B combo card test kit. Toxin loci PaLoc and PaCdt were detected by whole-genome sequencing (WGS). Multi-locus sequence typing (MLST) was performed to classify strains. Biofilm production was assessed by crystal violet. Antibiotic susceptibility was determined using Etest. Fidaxomicin susceptibility was tested via agar dilution. Sequence type (ST) 42 was the most (13.8%) common strain. All strains harboured the 2 toxins genes; 6.9% had the binary toxin. Most isolates were susceptible to metronidazole (98.9%) and vancomycin (99.5%). Eleven (5.85%) isolates were fidaxomicin-resistant. Biofilm production capacity was associated with ST (p < 0.001). In conclusion, a broad variety of C. difficile strains circulate in Israel's medical centres. Further studies are needed to explore the differences and their contribution to HA-CDI epidemiology.

Long-term persistence of diverse clones shapes the transmission landscape of invasive Listeria monocytogenes.

BACKGROUND: The foodborne bacterium Listeria monocytogenes (Lm) causes a range of diseases, from mild gastroenteritis to invasive infections that have high fatality rate in vulnerable individuals. Understanding the population genomic structure of invasive Lm is critical to informing public health interventions and infection control policies that will be most effective especially in local and regional communities. METHODS: We sequenced the whole draft genomes of 936 Lm isolates from human clinical samples obtained in a two-decade active surveillance program across 58 counties in New York State, USA. Samples came mostly from blood and cerebrospinal fluid. We characterized the phylogenetic relationships, population structure, antimicrobial resistance genes, virulence genes, and mobile genetic elements. RESULTS: The population is genetically heterogenous, consisting of lineages I-IV, 89 clonal complexes, 200 sequence types, and six known serogroups. In addition to intrinsic antimicrobial resistance genes (fosX, lin, norB, and sul), other resistance genes tetM, tetS, ermG, msrD, and mefA were sparsely distributed in the population. Within each lineage, we identified clusters of isolates with ≤ 20 single nucleotide polymorphisms in the core genome alignment. These clusters may represent isolates that share a most recent common ancestor, e.g., they are derived from the same contamination source or demonstrate evidence of transmission or outbreak. We identified 38 epidemiologically linked clusters of isolates, confirming eight previously reported disease outbreaks and the discovery of cryptic outbreaks and undetected chains of transmission, even in the rarely reported Lm lineage III (ST3171). The presence of animal-associated lineages III and IV may suggest a possible spillover of animal-restricted strains to humans. Many transmissible clones persisted over several years and traversed distant sites across the state. CONCLUSIONS: Our findings revealed the bacterial determinants of invasive listeriosis, driven mainly by the diversity of locally circulating lineages, intrinsic and mobile antimicrobial resistance and virulence genes, and persistence across geographical and temporal scales. Our findings will inform public health efforts to reduce the burden of invasive listeriosis, including the design of food safety measures, source traceback, and outbreak detection.

Clinical presentation and microbiological characteristics of community-acquired Staphylococcus aureus bacteraemia at a tertiary hospital in Costa Rica.

Introduction. Staphylococcus aureus is a leading agent in community-acquired bacteraemia (CAB) and has been linked to elevated mortality rates and methicillin resistance in Costa Rica.Gap statement and aim. To update and enhance previous data obtained in this country, we analysed the clinical manifestations of 54 S. aureus CAB cases in a tertiary hospital and delineated the sequence types (STs), virulome, and resistome of the implicated isolates.Methodology. Clinical information was retrieved from patient files. Antibiotic susceptibility profiles were obtained with disc diffusion and automated phenotypic tests. Genomic data were exploited to type the isolates and for detection of resistance and virulence genes.Results. Primary infections predominantly manifested as bone and joint infections, followed by skin and soft tissue infections. Alarmingly, 70% of patients continued to exhibit positive haemocultures beyond 48 h of treatment modification, with nearly a quarter requiring mechanical ventilation or developing septic shock. The 30-day mortality rate reached an alarming 40%. More than 60% of the patients were found to have received suboptimal or inappropriate antibiotic treatment, and there was an alarming tendency towards the overuse of third-generation cephalosporins as empirical treatment. Laboratory tests indicated elevated creatinine levels, leukocytosis, and bandaemia within the first 24 h of hospitalization. However, most showed improvement after 48 h. The isolates were categorized into 13 STs, with a predominance of representatives from the clonal complexes CC72 (ST72), CC8 (ST8), CC5 (ST5, ST6), and CC1 (ST188). Twenty-four isolates tested positive for mecA, with ST72 strains accounting for 20. In addition, we detected genes conferring acquired resistance to aminoglycosides, MLSB antibiotics, trimethoprim/sulfamethoxazole, and mutations for fluoroquinolone resistance in the isolate collection. Genes associated with biofilm formation, capsule synthesis, and exotoxin production were prevalent, in contrast to the infrequent detection of enterotoxins or exfoliative toxin genes.Conclusions. Our findings broaden our understanding of S. aureus infections in a largely understudied region and can enhance patient management and treatment strategies.

Genomic profiling of pan-drug resistant proteus mirabilis Isolates reveals antimicrobial resistance and virulence gene landscape.

Proteus mirabilis is a gram-negative pathogen that caused significant opportunistic infections. In this study we aimed to identify antimicrobial resistance (AMR) genes and virulence determinants in two pan-drug resistant isolate "Bacteria_11" and "Bacteria_27" using whole genome sequencing. Proteus mirabilis "Bacteria_11" and "Bacteria_27" were isolated from two different hospitalized patients in Egypt. Antimicrobial susceptibility determined using Vitek 2 system, then whole genome sequencing (WGS) using MinION nanopore sequencing was done. Antimicrobial resistant genes and virulence determinants were identified using ResFinder, CADR AMR database, Abricate tool and VF analyzer were used respectively. Multiple sequence alignment was performed using MAFFT and FastTree, respectively. All genes were present within bacterial chromosome and no plasmid was detected. "Bacteria_11" and "Bacteria_27" had sizes of approximately 4,128,657 bp and 4,120,646 bp respectively, with GC content of 39.15% and 39.09%. "Bacteria_11" and "Bacteria_27" harbored 43 and 42 antimicrobial resistance genes respectively with different resistance mechanisms, and up to 55 and 59 virulence genes respectively. Different resistance mechanisms were identified: antibiotic inactivation, antibiotic efflux, antibiotic target replacement, and antibiotic target change. We identified several genes associated with aminoglycoside resistance, sulfonamide resistance. trimethoprim resistance tetracycline resistance proteins. Also, those responsible for chloramphenicol resistance. For beta-lactam resistance, only blaVEB and blaCMY-2 genes were detected. Genome analysis revealed several virulence factors contribution in isolates pathogenicity and bacterial adaptation. As well as numerous typical secretion systems (TSSs) were present in the two isolates, including T6SS and T3SS. Whole genome sequencing of both isolates identify their genetic context of antimicrobial resistant genes and virulence determinants. This genomic analysis offers detailed representation of resistant mechanisms. Also, it clarifies P. mirabilis ability to acquire resistance and highlights the emergence of extensive drug resistant (XDR) and pan-drug resistant (PDR) strains. This may help in choosing the most appropriate antibiotic treatment and limiting broad spectrum antibiotic use.

The coral pathogen Vibrio coralliilyticus uses a T6SS to secrete a group of novel anti-eukaryotic effectors that contribute to virulence.

Vibrio coralliilyticus is a pathogen of coral and shellfish, leading to devastating economic and ecological consequences worldwide. Although rising ocean temperatures correlate with increased V. coralliilyticus pathogenicity, the specific molecular mechanisms and determinants contributing to virulence remain poorly understood. Here, we systematically analyzed the type VI secretion system (T6SS), a contact-dependent toxin delivery apparatus, in V. coralliilyticus. We identified 2 omnipresent T6SSs that are activated at temperatures in which V. coralliilyticus becomes virulent; T6SS1 is an antibacterial system mediating interbacterial competition, whereas T6SS2 mediates anti-eukaryotic toxicity and contributes to mortality during infection of an aquatic model organism, Artemia salina. Using comparative proteomics, we identified the T6SS1 and T6SS2 toxin arsenals of 3 V. coralliilyticus strains with distinct disease etiologies. Remarkably, T6SS2 secretes at least 9 novel anti-eukaryotic toxins comprising core and accessory repertoires. We propose that T6SSs differently contribute to V. coralliilyticus's virulence: T6SS2 plays a direct role by targeting the host, while T6SS1 plays an indirect role by eliminating competitors.

Adaptations to cationic biocide exposure differentially influence virulence factors and pathogenicity in Pseudomonas aeruginosa.

Cationic biocides (CBs), which include quaternary ammonium compounds (QACs), are employed to mitigate the spread of infectious bacteria, but resistance to such surface disinfectants is rising. CB exposure can have profound phenotypic implications that extend beyond allowing microorganisms to persist on surfaces. Pseudomonas aeruginosa is a deadly bacterial pathogen that is intrinsically tolerant to a wide variety of antimicrobials and is commonly spread in healthcare settings. In this study, we pursued resistance selection assays to the QAC benzalkonium chloride and quaternary phosphonium compound P6P-10,10 to assess the phenotypic effects of CB exposure in P. aeruginosa PAO1 and four genetically diverse, drug-resistant clinical isolates. In particular, we sought to examine how CB exposure affects defensive strategies and the virulence-associated "offensive" strategies in P. aeruginosa. We demonstrated that development of resistance to BAC is associated with increased production of virulence-associated pigments and alginate as well as pellicle formation. In an in vivo infection model, CB-resistant PAO1 exhibited a decreased level of virulence compared to wild type, potentially due to an observed fitness cost in these strains. Taken together, these results illustrate the significant consequence CB resistance exerts on the virulence-associated phenotypes of P. aeruginosa.

YjbH contributes to Staphylococcus aureus skin pathology and immune response through Agr-mediated α-toxin regulation.

Staphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs) with Methicillin-Resistant S. aureus (MRSA) strains being a major contributor in both community and hospital settings. S. aureus relies on metabolic diversity and a large repertoire of virulence factors to cause disease. This includes α-hemolysin (Hla), an integral player in tissue damage found in various models, including SSTIs. Previously, we identified a role for the Spx adapter protein, YjbH, in the regulation of several virulence factors and as an inhibitor of pathogenesis in a sepsis model. In this study, we found that YjbH is critical for tissue damage during SSTI, and its absence leads to decreased proinflammatory chemokines and cytokines in the skin. We identified no contribution of YjbI, encoded on the same transcript as YjbH. Using a combination of reporters and quantitative hemolysis assays, we demonstrated that YjbH impacts Hla expression and activity both in vitro and in vivo. Additionally, expression of Hla from a non-native promoter reversed the tissue damage phenotype of the ΔyjbIH mutant. Lastly, we identified reduced Agr activity as the likely cause for reduced Hla production in the ΔyjbH mutant. This work continues to define the importance of YjbH in the pathogenesis of S. aureus infection as well as identify a new pathway important for Hla production.

Genomic Dissection of an Enteroaggregative Escherichia coli Strain Isolated from Bacteremia Reveals Insights into Its Hybrid Pathogenic Potential.

Escherichia coli is a frequent pathogen isolated from bloodstream infections. This study aimed to characterize the genetic features of EC092, an E. coli strain isolated from bacteremia that harbors enteroaggregative E. coli (EAEC) genetic markers, indicating its hybrid pathogenic potential. Whole-genome sequencing showed that EC092 belongs to phylogroup B1, ST278, and serotype O165:H4. Genes encoding virulence factors such as fimbriae, toxins, iron-uptake systems, autotransporter proteins (Pet, Pic, Sat, and SepA), and secretion systems were detected, as well as EAEC virulence genes (aggR, aatA, aaiC, and aap). EC092 was found to be closely related to the other EAEC prototype strains and highly similar in terms of virulence to three EAEC strains isolated from diarrhea. The genomic neighborhood of pet, pic, sat, sepA, and the EAEC virulence genes of EC092 and its three genetically related fecal EAEC strains showed an identical genomic organization and nucleotide sequences. Also, EC092 produced and secreted Pet, Pic, Sat, and SepA in the culture supernatant and resisted the bactericidal activity of normal human serum. Our results demonstrate that the strain EC092, isolated from bacteremia, is a hybrid pathogenic extraintestinal E. coli (ExPEC)/EAEC with virulence features that could mediate both extraintestinal and intestinal infections.

Rli51 Attenuates Transcription of the Listeria Pathogenicity Island 1 Gene mpl and Functions as a Trans-Acting sRNA in Intracellular Bacteria.

Listeria pathogenicity island 1 (LIPI-1) is a genetic region containing a cluster of genes essential for virulence of the bacterial pathogen Listeria monocytogenes. Main virulence factors in LIPI-1 include long 5' untranslated regions (5'UTRs), among which is Rli51, a small RNA (sRNA) in the 5'UTR of the Zn-metalloprotease-coding mpl. So far, Rli51 function and molecular mechanisms have remained obscure. Here, we show that Rli51 exhibits a dual mechanism of regulation, functioning as a cis- and as a trans-acting sRNA. Under nutrient-rich conditions, rli51-mpl transcription is prematurely terminated, releasing a short 121-nucleotide-long sRNA. Rli51 is predicted to function as a transcription attenuator that can fold into either a terminator or a thermodynamically more stable antiterminator. We show that the sRNA Rli21/RliI binds to a single-stranded RNA loop in Rli51, which is essential to mediate premature transcription termination, suggesting that sRNA binding could stabilize the terminator fold. During intracellular infection, rli51 transcription is increased, which generates a higher abundance of the short Rli51 sRNA and allows for transcriptional read-through into mpl. Comparative intracellular bacterial transcriptomics in rli51-null mutants and the wild-type reference strain EGD-e suggests that Rli51 upregulates iron-scavenging proteins and downregulates virulence factors from LIPI-1. MS2 affinity purification confirmed that Rli51 binds transcripts of the heme-binding protein Lmo2186 and Lmo0937 in vivo. These results prove that Rli51 functions as a trans-acting sRNA in intracellular bacteria. Our research shows a growth condition-dependent mechanism of regulation for Rli51, preventing unintended mpl transcription in extracellular bacteria and regulating genes important for virulence in intracellular bacteria.

Oral inoculation of Fusobacterium nucleatum exacerbates ulcerative colitis via the secretion of virulence adhesin FadA.

Fusobacterium nucleatum (F. nucleatum), an anaerobic resident of the oral cavity, is increasingly recognized as a contributing factor to ulcerative colitis (UC). The adhesive properties of F. nucleatum are mediated by its key virulence protein, FadA adhesin. However, further investigations are needed to understand the pathogenic mechanisms of this oral pathogen in UC. The present study aimed to explore the role of the FadA adhesin in the colonization and invasion of oral F. nucleatum in dextran sulphate sodium (DSS)-induced colitis mice via molecular techniques. In this study, we found that oral inoculation of F. nucleatum strain carrying the FadA adhesin further exacerbated DSS-induced colitis, leading to elevated alveolar bone loss, disease severity, and mortality. Additionally, CDH1 gene knockout mice treated with DSS presented increases in body weight and alveolar bone density, as well as a reduction in disease severity. Furthermore, FadA adhesin adhered to its mucosal receptor E-cadherin, leading to the phosphorylation of ß-catenin and the degradation of IκBα, the activation of the NF-κB signalling pathway and the upregulation of downstream cytokines. In conclusion, this research revealed that oral inoculation with F. nucleatum facilitates experimental colitis via the secretion of the virulence adhesin FadA. Targeting the oral pathogen F. nucleatum and its virulence factor FadA may represent a promising therapeutic approach for a portion of UC patients.

Characterization of Helicobacter pylori iceA and babA2 virulence genes in dyspeptic patients at a teaching hospital in Ghana.

Introduction: Helicobacter pylori (H. pylori) infection is endemic in Africa. It is a major aetiological factor in the development of peptic ulcer disease and distal gastric cancers. Existing data shows that clinical outcomes are dependent on the virulence of the infecting strain, host´s susceptibility, and environmental factors. In Ghana, a previous study showed that the majority of symptomatic individuals harboured cagA and vacA virulent strains. The main objective of this study was to characterize and assess the significance of other virulence factors, specifically iceA and babA2 in Ghana. Methods: H. pylori iceA and babA2 genes were investigated in dyspeptic patients at the Korle Bu Teaching Hospital (KBTH), Accra, Ghana. The study employed a cross-sectional design consecutively recruiting patients with upper gastrointestinal symptoms for endoscopy. Nucleic acid was extracted from gastric biopsies using a commercial kit (QIAGEN DNeasy tissue kit). H. pylori babA2 and iceA genes were amplified using extracted deoxyribonucleic acid (DNA) and primers by polymerase chain reaction (PCR). Results: majority, (71.1%), of the study participants, were H. pylori positive when tested with urease-campylobacter-like organism (CLO). In total, 46 H. pylori urease CLO-positive samples were randomly analyzed by PCR for iceA, of which, 12 (26%) and 7 (15%) were found to have iceA1 and iceA2 respectively. Of the CLO-positive samples, 9 were randomly analysed for babA2 by PCR. Three samples were babA2 positive and 6 were babA2 negative. Conclusion: in Ghana, although H. pylori is endemic, iceA prevalence is rather low and probably exerts a limited effect on bacterial virulence. Further evaluation would be required, not only to determine association with other virulence factors but more importantly, inter-relationships with wider host and environmental factors that impact on disease pathogenesis.