OBJECTIVE: Blood-brain barrier (BBB) disorder is one of the early findings in cognitive impairments. We have recently found that Porphyromonas gingivalis bacteraemia can cause cognitive impairment and increased BBB permeability. This study aimed to find out the possible key virulence factors of P. gingivalis contributing to the pathological process.
METHODS: C57/BL6 mice were infected with P. gingivalis or gingipains or P. gingivalis lipopolysaccharide (P. gingivalis LPS group) by tail vein injection for 8 weeks. The cognitive behaviour changes in mice, the histopathological changes in the hippocampus and cerebral cortex, the alternations of BBB permeability, and the changes in Mfsd2a and Cav-1 levels were measured. The mechanisms of Ddx3x-induced regulation on Mfsd2a by arginine-specific gingipain A (RgpA) in BMECs were explored.
RESULTS: P. gingivalis and gingipains significantly promoted mice cognitive impairment, pathological changes in the hippocampus and cerebral cortex, increased BBB permeability, inhibited Mfsd2a expression and up-regulated Cav-1 expression. After RgpA stimulation, the permeability of the BBB model in vitro increased, and the Ddx3x/Mfsd2a/Cav-1 regulatory axis was activated.
CONCLUSIONS: Gingipains may be one of the key virulence factors of P. gingivalis to impair cognition and enhance BBB permeability by the Ddx3x/Mfsd2a/Cav-1 axis.

BACKGROUND: The most prevalent stomach infection in the world is caused by Helicobacter pylori (H. pylori). Several pathogenicity genes, including cagA, vacA, babA2, dupA, iceA, and oipA, are associated with an increased risk of gastrointestinal disease such as peptic ulcer and stomach cancer. This research aims to determine the prevalence of different H. pylori genotypes and correlate their risk in the development of gastrointestinal diseases in the Ecuadorian population.
METHODS: A cross-sectional research of 225 patients at the Calderón Hospital in Quito, Ecuador, was conducted. End point PCRs were run to determine the presence of 16S rRNA, cagA, vacA (m1), vacA (s1), babA2, dupA, iceA1, and oipA virulence genes. Chi-square test, odds ratios (OR) and 95% confidence intervals (CI) were utilized for the statistical analysis.
RESULTS: H. pylori infection was present in 62.7% of people. Peptic ulcers were seen in 22.2% and malignant lesions in 3.6% of patients. Genes oipA (93.6%), vacA (s1) (70.9%), and babA2 (70.2%) were the most prevalent. cagA/vacA (s1m1) and cagA/oipA (s1m1) combinations were found in 31.2% and 22.7% of the cases, respectively. Acute inflammation has a significant correlation with the genes cagA (OR = 4.96 95% CI: 1.1-22.41), babA2 (OR = 2.78 95% CI: 1.06-7.3), and the cagA/oipA combination (OR = 4.78, 95% CI: 1.06-21.62). Follicular hyperplasia was associated with iceA1 (OR = 3.13; 95% CI: 1.2-8.16), babA2 (OR = 2.56; 95% CI: 1.14-5.77), cagA (OR = 2.19; 95% CI: 1.06-4.52), and the cagA/oipA combination (OR = 2.32, 95% CI: 1.12-4.84). The vacA (m1) and vacA (s1m1) genes were associated with gastric intestinal metaplasia (OR = 2.71 95% CI: 1.17-6.29) (OR = 2.33 95% CI: 1.03-5.24). Finally, we showed that cagA/vacA (s1m1) gene combination increased the risk of duodenal ulcer development (OR = 2.89, 95% CI 1.10-7.58).
CONCLUSIONS: This study makes a significant contribution by offering genotypic information regarding H. pylori infection. The presence of several H. pylori genes was associated with the onset of gastrointestinal illness in the Ecuadorian population.

Bovine mastitis is the most common disease affecting dairy cattle worldwide and it generates substantial losses for cattle breeders. One of the most common pathogens identified in infected milk samples is Staphylococcus aureus. Currently, there is no fast test for recognizing bacteria species on the market. The aim of this study was to bioinformatically and laboratory detect and characterize the fibronectin binding protein A (FnBPA) of S. aureus (SA) in milk samples obtained from cows diagnosed with mastitis. More than 90,000,000 amino acid sequences were subjected to bioinformatic detection in the search for a potential biomarker for bovine SA. The analysis of FnBPA included the detection of signal peptides and nonclassical proteins, antigenicity, and the prediction of epitopes. To confirm the presence of the fnbA gene in four SA isolates, amplification with specific primers was performed. FnBPA was detected by immunoblotting. The immunoreactivity and selectivity were performed with monoclonal anti-FnBPA antibodies and SA-negative serum. The bioinformatic analysis showed that FnBPA is a surface, conservative, immunoreactive, and species-specific protein with antigenic potential. Its presence was confirmed in all of the SA isolates we studied. Immunoblotting proved its immunoreactivity and specificity. Thus, it can be considered a potential biomarker in mastitis immunodiagnostics.

Streptococcus mutans is a major etiological agent for dental caries. We previously demonstrated that S. mutans strains expressing collagen-binding proteins (CBPs) were related to the pathogenesis of systemic diseases. However, their acquisition and colonization remain unknown. Here, we investigated the detection rates of CBP-positive S. mutans strains in children and their guardians to clarify the background for the acquisition and colonization in children. Saliva samples were collected from children and their mothers, and detection of S. mutans and collagen-binding genes (cnm, cbm) was performed by PCR after DNA extraction. The oral status of each child was examined, and their mothers were asked to complete a questionnaire. The isolation rate of Cnm-positive S. mutans was significantly higher in mothers than in children. Notably, the possession rates of CBP-positive strains in children were significantly higher in children whose mothers had CBP-positive strains than in children whose mothers did not have these strains. Furthermore, children with CBP-positive strains had a significantly shorter breastfeeding period than children without these strains. The present results suggest that nutritional feeding habits in infancy are one of the factors involved in the acquisition and colonization of CBP-positive S. mutans strains.

The outer membrane protein U (OmpU) is a conserved outer membrane protein in a variety of pathogenic Vibrio species and has been considered as a vital protective antigen for vaccine development. Vibrio mimicus (V. mimicus) is the pathogen causing ascites disease in aquatic animals. In this study, the prokaryotically expressed and purified His-tagged OmpU of V. mimicus (His-OmpU) was used as a subunit vaccine. The formalin inactivated V. mimicus, purified His tag (His-tag), and PBS were used as controls. The vaccinated yellow catfish were challenged with V. mimicus at 28 days post-vaccination, and the results showed that the His-OmpU and inactivated V. mimicus groups exhibited much higher survival rates than the His-tag and PBS groups. To fully understand the underlying mechanism, we detected the expression levels of several immune-related genes in the spleen of fish at 28 days post-vaccination and 24 h post-challenge. The results showed that most of the detected immune-related genes were significantly upregulated in His-OmpU and inactivated V. mimicus groups. In addition, we performed the serum bactericidal activity assay, and the results showed that the serum from His-OmpU and inactivated V. mimicus groups exhibited much stronger bactericidal activity against V. mimicus than those of His-tag and PBS groups. Finally, the serum agglutination antibody was detected, and the antibody could be detected in His-OmpU and inactivated V. mimicus groups with the antibody titers increasing along with the time post-vaccination, but not in His-tag or PBS group. Our data reveal that the recombinant OmpU elicits potent protective immune response and is an effective vaccine candidate against V. mimicus in yellow catfish.

Pemphigus is a chronic autoimmune blistering disease. Pemphigus blisters can damage the natural skin barrier and increase the risk of life-threatening conditions. Colonization of pemphigus wounds with methicillin-resistant Staphylococcus aureus (MRSA) prolongs wound healing and increases mortality rate. Assessing MRSA prevalence, types, and toxin and adhesion genes can facilitate the detection of MRSA strains which cause infections, selection of appropriate treatments, and healing of pemphigus wounds. This study aimed to determine the SCCmec, the direct repeat unit (dru) types (dts), and the toxin, MSCRAMM, and biofilm genes of MRSA strains isolated from pemphigus wounds.
In this cross-sectional study, 118 S. aureus isolates were gathered from 118 patients with pemphigus. MRSA detection was performed using the mecA gene. Using the polymerase chain reaction method, all MRSA isolates were assessed for the presence of the sea, seb, sec, tst, eta, pvl, hla, hlb, MSCRAMM, and ica genes. Typing and subtyping were performed through respectively SCCmec typing and dru typing methods. The Bionumerics software was used for analyzing the data and drawing the minimum spanning tree.
From 118 S. aureus isolates, 51 were MRSA. SCCmec typing revealed the prevalence of SCCmec II with a prevalence of 64.7% (33 out of 51 isolates) and SCCmec III with a prevalence of 35.3% (18 out of 51 isolates). Dru typing indicated seven dts, namely dts 10a, 10g, 10m, 13i, 8h, 8i, and 9ca in two main clusters. The dt9ca was a new dru type and was registered in the dru-typing database (www.dru-typing.org). The prevalence rates of the hla, sea, and sec genes in MRSA isolates were respectively 54.9%, 27.4%, and 1.9%, while the hlb, seb, eta, and pvl genes were not detected at all. Only one MRSA with SCCmec III and dt10a carried the tst encoding gene. MSCRAMM gene analysis revealed the high prevalence of the eno (31.3%) and the fib (21.5%) genes. The prevalence rates of the icaA and icaD biofilm formation genes were 3.9% and 5.8%, respectively. There were no significant differences between the two detected SCCmec types and between the two detected dts clusters respecting the prevalence of the encoding genes of virulence factors and MSCRAMMs.
The toxin genes hla and sea are prevalent among MRSA strains with SCCmec II and III isolated from pemphigus wounds. The most prevalent dts are dt10a and dt10g among MRSA with SCCmec III and dt8h and dt8i among MRSA with SCCmec II.

BACKGROUND: Little is known on significance, diversity and characteristics of gut E. coli in goats despite their importance as food animals globally. We characterized the temporal dynamics in diversity of E. coli in fecal samples from a cohort of goat kids and adult meat goats on pasture over a one-year period. Isolates were characterized based on phylogenetic grouping, virulence genes; shiga toxins 1 and 2 (Stx1&Stx2) (STEC), intimin (eaeA), hemolysin (hly) and select important sero-groups (026, 045, 0103, 0126 and 0146) using molecular methods.
RESULTS: A total of 516 E. coli isolates were screened. Prevalence of virulence genes and STEC was 65 and 56% respectively. Prevalence of virulence genes and STEC was significantly higher in goat kids less than six months (76% /66%) than adults (48% /28%). Isolates with virulence profiles of two or more genes were also higher in young goat kids (50%) than adults (20%). Entero-pathogenic E. coli (EPEC-eaeA gene only) were mostly from pre-weaned goat kids while hly gene only isolates were significantly higher in adults. The stx1, stx2 and hly genes peaked around weaning (60, 63 and 52%) respectively. Goats kids were mostly hosts to group D (59%) while adults older than one year had B1 (75%) isolates. Group D isolates were most abundant at weaning (64%) and diarrhea samples (74%). Group B2 isolates overall (6%) were mostly detected around weaning (63%) while A isolates were 4% overall. Twenty-four isolates belonged to sero-groups 026, 0103 and 0146 with 70% of the isolates detected around weaning. Nineteen of these isolates were STEC with most harboring the stx1/stx2/hly/eae (25%) profile. Most belonged to O26 sero-group (75%) and phylogroup D (75%).
CONCLUSIONS: To our knowledge this is the first study to highlight longitudinal age related differences in E. coli phylogenetic diversity, abundance of virulence genes and select important sero-groups in goats. Differences detected suggest a possible role of age and weaning stress in influencing E. coli diversity in the gut of goats. The findings are relevant to both animal and public health to advise on further studies on caprine E. coli isolates as animal and human pathogens.

The adhesion of pathogenic bacteria to host cells and the extracellular matrix (ECM) is considered an important step in the pathogenesis of microorganisms. It has been described that Leptospira spp. bind to multiple receptors on host cells and to the ECM to initiate infection. Most studies of Leptospira adherence described until now have focused on the in vitro attachment of recombinant L. interrogans proteins to ECM components. These putative adhesins may be involved in the colonization of the host, contributing to the bacterial invasion process. Certainly, in vitro cell adhesion studies have contributed to the elucidation of leptospiral pathogenesis mechanisms. Here, we describe a cell adhesion assay that can be used for studying the interactions between putative leptospiral adhesins and host components.

Invasins and intimins, members of virulence-related adhesin family which is involved in attachment and adherence to epithelial cells during infection, are found in various pathogens. These pathogens can attach to enterocytes and lead to the formation of a pedestal-like structure. Invasins and intimins belong to type Ve secretion systems, and the N-terminal β-barrel domain acts as a translocation pore to secrete the C-terminal passenger domain. However, the relationship between invasins/intimins and type III secretion system (T3SS) has been poorly studied. Based on the transposon insertion mutant library of Edwardsiella piscicida, we got a transposon insertion mutant with significant T3SS defect and identified the mutated gene ETAE_0323 (named inV later). This gene encoded a protein with 2359 amino acid residues and was predicted to be an invasin. To study the relationship between InV and T3SS, strains with N-terminus or C-terminus deleted InV fragments were made. However, none of them was able to copy the phenotype of the transposon insertion mutant previously identified. The localization of InV in ΔT3SS strain was not significantly different from WT, suggesting that the T3SS defect in the transposon insertion mutant was likely to be caused by polar effect. Nevertheless, depletion of inV still showed dramatic internalization and virulence defect in HeLa cell and zebrafish model, respectively, suggesting InV as a virulence related protein.

Bovines are the primary reservoir of enterohemorrhagic Escherichia coli (EHEC) O157:H7 and the main source of its transmission to humans. Here, we present a one-year longitudinal study of fecal shedding of E. coli O157. E. coli O157 obtained from recto-anal mucosal samples were characterized by multiplex PCR. The E. coli O157 prevalence ranged from 0.84% in July to 15.25% in November. The confinement within pens resulted in prevalence of 11%. Most animals (61.86%; 75/118) shed E. coli O157 at least in one sampling occasion. Of the positive animals, 82.19%, 16.44%, and 1.37% were stx positive on one, two and three sampling occasions, respectively. All the E. coli O157 isolated strains carried the genes eae and rfbO157, whereas 11%, 33% and 56% contained stx1, stx2 and stx1/stx2, respectively. The stx1/stx2 and stx2 types were significantly higher during the grazing and finishing periods, respectively, in comparison with the rearing and grazing periods. The presence of stx2a subtype was evident in four isolates, whereas stx2c was present in at least seven. However, both subtypes were present simultaneously in two isolates. The stx1/stx2c, stx1/stx2d and stx1/stx2NT genotypes occurred in 24, 2 and 15 isolates, respectively. The simultaneous occurrence of stx1 and stx2c significantly increased during grazing. Some cases of within-pen and between-pen transmission occurred throughout the study. Contagion levels during in-field grazing were higher than during permanent confinement in the pens. Thus, the individual patterns of shedding varied depending on the proportion of animals shedding the bacteria within pens and the time of shedding.