IntroductionBrucella abortus is an intracellular pathogen that establishes chronic infections through immune evasion. Exosomes, a subtype of extracellular vesicles, mediate intercellular communication and can modulate host immune responses during infection. However, the proteomic composition and functional significance of exosomes from B. abortus-infected macrophages remain unclear.MethodsExosomes were isolated from RAW 264.7 macrophages infected or uninfected with B. abortus strain 2308, at 8 and 24 hours post-infection (hpi), using sequential centrifugation and immunoaffinity capture. Size and morphology were assessed by nanoparticle tracking analysis and transmission electron microscopy. Proteins were identified and quantified by label-free LC-MS/MS, followed by bioinformatic analyses for differential expression, functional enrichment, exclusive protein identification, and bacterial protein detection.ResultsExosomes from B. abortus-infected macrophages displayed distinct, time-dependent proteomic profiles. At 8 hpi, proteins involved in biosynthesis, energy metabolism, and endoplasmic reticulum processing were enriched, while lysosomal and antigen presentation components were reduced. At 24 hpi, enrichment shifted toward mitochondrial and redox regulation pathways, with sustained suppression of immune-related processes. Immune mediators (Csf3, Gsdmd, Ifi35) and retromer complex components were identified in a phase-specific manner. Sixty-six and twenty-four proteins were exclusive to infected exosomes at 8 and 24 hpi, respectively, reflecting a shift from metabolic/trafficking roles to immune regulation. Bacterial proteins GroEL and SodC were present at both time points, whereas Omp19, Omp2b, DnaK, and BAB1_0368 were restricted to early infection.ConclusionExosomes from B. abortus-infected macrophages exhibit dynamic proteomic remodeling that affects immune-related pathways, changes that may contribute to bacterial survival within the host. The presence of both host and bacterial-derived proteins within these vesicles suggests their potential relevance in brucellosis pathogenesis and highlights them as candidates worthy of further exploration as biomarkers or therapeutic targets.

Persister cells are a subpopulation of bacteria capable of surviving antibiotic treatments and are thought to contribute to disease chronicity and symptom relapse of chronic conditions. Crohn’s disease (CD) is a multifactorial chronic inflammatory condition of the gastrointestinal tract, and adherent-invasive Escherichia coli (AIEC) have emerged as a key contributor to its pathogenesis. AIEC can survive, replicate, and produce persister cells within macrophages; however, beyond the LF82 reference strain, little is known about the persistence phenotype and its variability among AIEC strains. In this study, the survival of two AIEC reference strains was analyzed following ciprofloxacin treatment, a fluoroquinolone antibiotic commonly used in CD therapy. In addition, four AIEC clinical isolates and two non-AIEC E. coli pathotypes were included for comparison. We investigated the roles of the resident antibiotic resistance plasmid, the stress response protein HtrA, and macrophage-induced persister formation. Our results revealed broad variability in persister cell formation among AIEC strains. Remarkably, the reference NRG857c strain exhibits a threateningly high-persistence phenotype, with persistence levels 200-fold higher than LF82 and certain clinical isolates. Neither the antibiotic resistance plasmid nor HtrA were required for this phenotype. Moreover, unlike LF82, NRG857c did not exhibit increased persistence following macrophage internalization. Overall, our findings demonstrate the presence of distinct persistence phenotypes among AIEC strains and identify NRG857c as a high-persistence variant. These observations underscore the need to consider bacterial persistence in the management of CD, particularly given the potential presence of AIEC strains with elevated persistence capabilities.

Enterobacter hormaechei, one of the species within the Enterobacter cloacae complex, is a relevant agent of healthcare-associated infections. In addition, it has gained relevance because isolates have shown the capacity to resist several antibiotics, particularly carbapenems. However, knowledge regarding colonization and virulence mechanisms of E. hormaechei has not progressed to the same extent as other Enterobacteriaceae species as Escherichia coli or Klebsiella pneumoniae. Here, we describe the presence and role of the type 3 fimbria, a chaperone-usher assembled fimbria, which was first described in Klebsiella spp., and which has been detected in other representatives of the Enterobacteriaceae family. Eight Chilean E. cloacae isolates were examined, and among them, four E. hormaechei isolates were found to produce the type 3 fimbria. These isolates were identified as E. hormaechei subsp. hoffmannii, one of the five subspecies known. A mutant E. hormaechei subsp. hoffmannii strain lacking the mrkA gene, encoding the major structural subunit, displayed a significantly reduced adherence capacity to a plastic surface and to Caco-2 cells, compared to the wild-type strain. This phenotype of reduced adherence capacity was not observed in the mutant strains complemented with the mrkA gene under the control of an inducible promoter. Therefore, these data suggest a role of the type 3 fimbria in the adherence capacity of E. hormaechei subsp. hoffmannii. A screening in E. hormaechei genomes contained in the NCBI RefSeq Assembly database indicated that the overall presence of the type 3 fimbria is uncommon (5.94–7.37%), although genes encoding the structure were detected in representatives of the five E. hormaechei subspecies. Exploration of complete genomes indicates that, in most of the cases, the mrkABCDF locus, encoding the type 3 fimbria, is located in plasmids. Furthermore, sequence types currently found in healthcare-associated infections were found to harbor genes encoding the type 3 fimbria, mainly ST145, ST78, ST118, ST168, ST66, ST93, and ST171. Thus, although the type 3 fimbria is not widespread among the species, it might be a determinant of fitness for a subset of E. hormaechei representatives.

Shiga toxin-producing Escherichia coli (STEC) poses a significant public health risk due to its zoonotic potential and association with severe human diseases, such as hemorrhagic colitis and hemolytic uremic syndrome. Ruminants are recognized as primary reservoirs for STEC, but swine also contribute to the epidemiology of this pathogen, highlighting the need for effective prevention strategies across species. Notably, a subgroup of STEC that produces Shiga toxin type 2e (Stx2e) causes edema disease (ED) in newborn piglets, economically affecting pig production. This study evaluates the immunogenicity of a chimeric protein-based vaccine candidate against STEC in pregnant sows and the subsequent transfer of immunity to their offspring. This vaccine candidate, which includes chimeric proteins displaying selected epitopes from the proteins Cah, OmpT, and Hes, was previously proven to be immunogenic in pregnant cows. Our analysis revealed a broad diversity of STEC serotypes within swine populations, with the cah and ompT genes being prevalent, validating them as suitable antigens for vaccine development. Although the hes gene was detected less frequently, the presence of at least one of these three genes in a significant proportion of STEC suggests the potential of this vaccine to target a wide range of strains. The vaccination of pregnant sows led to an increase in specific IgG and IgA antibodies against the chimeric proteins, indicating successful immunization. Additionally, our results demonstrated the effective passive transfer of maternal antibodies to piglets, providing them with immediate, albeit temporary, humoral immunity against STEC. These humoral responses demonstrate the immunogenicity of the vaccine candidate and are preliminary indicators of its potential efficacy. However, further research is needed to conclusively evaluate its impact on STEC colonization and shedding. This study highlights the potential of maternal vaccination to protect piglets from ED and contributes to the development of vaccination strategies to reduce the prevalence of STEC in various animal reservoirs.

Diarrheagenic Escherichia coli (DEC) is the main cause of diarrhea in children under five years old. The virulence of DEC is tightly regulated by environmental signals influenced by the gut microbiota and its metabolites. Short-chain fatty acids (SCFAs) are the main metabolic product of anaerobic fermentation in the gut, but their role in DEC diarrhea has not yet been established. In this study, we determine the levels of acetate, propionate, and butyrate in stool samples from children with diarrhea caused by DEC, and we identify bacteria from the fecal gut microbiota associated with the production of SCFAs. The microbiota and SCFAs levels in stool samples obtained from 40 children with diarrhea and 43 healthy children were determined by 16S rRNA gene sequencing and HPLC, respectively. Additionally, shotgun metagenomics was used to identify metagenome-assembled genomes (MAGs) in a subgroup of samples. The results showed significantly higher levels of all SCFAs tested in diarrheal samples than in healthy controls. The abundance of Streptococcus sp., Limosilactobacillus, Blautia, Escherichia, Bacteroides, Megamonas, and Roseburia was higher in the DEC group than in healthy individuals. Functional analysis of bacteria and their main metabolic pathways made it possible to identify species MAGs that could be responsible for the detected SCFAs levels in DEC-positive diarrhea. In conclusion, based on our results and published data, we suggest that SCFAs may be important in the crosstalk between the microbiota and DEC pathogens in the gut.

The pyelonephritis-associated fimbria (P fimbria) is one of the most recognized adhesion determinants of extraintestinal pathogenic Escherichia coli strains (ExPECs). Twelve variants have been described for the gene encoding the P fimbria major structural subunit PapA and three variants for the gene encoding the adhesin subunit PapG. However, their distribution among the ExPEC diversity has not been comprehensively addressed. A complete landscape of that distribution might be valuable for delineating basic studies about the pathogenicity mechanisms of ExPECs and following up on the evolution of ExPEC lineages, particularly those most epidemiologically relevant. Therefore, we performed a massive descriptive study to detect the papA and papG variants along different E. coli genotypes represented by genomic sequences contained in the NCBI Assembly Refseq database. The most common papA variants were F11, F10, F48, F16, F12, and F7-2, which were found in significant association with the most relevant ExPEC genotypes, the phylogroups B2 and D, and the sequence types ST95, ST131, ST127, ST69, ST12, and ST73. On the other hand, the papGII variant was by far the most common followed by papGIII, and both were also found to have a significant association with common ExPEC genotypes. We noticed the presence of genomes, mainly belonging to the sequence type ST12, harboring two or three papA variants and two papG variants. Furthermore, the most common papA and papG variants were also detected in records representing strains isolated from humans and animals such as poultry, bovine, and dogs, supporting previous hypotheses of potential cross-transmission. Finally, we characterized a set of 17 genomes from Chilean uropathogenic E. coli strains and found that ST12 and ST73 were the predominant sequence types. Variants F7-1, F7-2, F8, F9, F11, F13, F14, F16, and F48 were detected for papA, and papGII and papGIII variants were detected for papG. Significant associations with the sequence types observed in the analysis of genomes contained in the NCBI Assembly Refseq database were also found in this collection in 16 of 19 cases for papA variants and 7 of 9 cases for the papG variants. This comprehensive characterization might support future basic studies about P fimbria-mediated ExPEC adherence and future typing or epidemiological studies to monitor the evolution of ExPECs producing P fimbria.

Shiga toxin-producing Escherichia coli (STEC) are recognized as being responsible for many cases of foodborne diseases worldwide. Cattle are the main reservoir of STEC, shedding the microorganisms in their feces. The serogroup STEC O91 has been associated with hemorrhagic colitis and hemolytic uremic syndrome. Locus of Adhesion and Autoaggregation (LAA) and its hes gene are related to the pathogenicity of STEC and the ability to form biofilms. Considering the frequent isolation of STEC O91, the biofilm-forming ability, and the possible role of hes in the pathogenicity of STEC, we propose to evaluate the ability of STEC to form biofilms and to evaluate the expression of hes before and after of biofilm formation. All strains were classified as strong biofilm-forming. The hes expression showed variability between strains before and after biofilm formation, and this may be due to other genes carried by each strain. This study is the first to report the relationship between biofilm formation, and hes expression and proposes that the analysis and diagnosis of LAA, especially hes as STEC O91 virulence factors, could elucidate these unknown mechanisms. Considering that there is no specific treatment for HUS, only supportive care, it is necessary to know the survival and virulence mechanisms of STEC O91.

Over the past two centuries, vaccines have been critical for the prevention of infectious diseases and are considered milestones in the medical and public health history. The World Health Organization estimates that vaccination currently prevents approximately 3.5–5 million deaths annually, attributed to diseases such as diphtheria, tetanus, pertussis, influenza, and measles. Vaccination has been instrumental in eradicating important pathogens, including the smallpox virus and wild poliovirus types 2 and 3. This narrative review offers a detailed journey through the history and advancements in vaccinology, tailored for healthcare workers. It traces pivotal milestones, beginning with the variolation practices in the early 17th century, the development of the first smallpox vaccine, and the continuous evolution and innovation in vaccine development up to the present day. We also briefly review immunological principles underlying vaccination, as well as the main vaccine types, with a special mention of the recently introduced mRNA vaccine technology. Additionally, we discuss the broad benefits of vaccines, including their role in reducing morbidity and mortality, and in fostering socioeconomic development in communities. Finally, we address the issue of vaccine hesitancy and discuss effective strategies to promote vaccine acceptance. Research, collaboration, and the widespread acceptance and use of vaccines are imperative for the continued success of vaccination programs in controlling and ultimately eradicating infectious diseases.

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen that causes gastroenteritis and Hemolytic Uremic Syndrome. Cattle are the main animal reservoir, excreting the bacteria in their feces and contaminating the environment. In addition, meat can be contaminated by releasing the intestinal content during slaughtering. Here, we evaluated the safety and immunogenicity of a vaccine candidate against STEC that was formulated with two chimeric proteins (Chi1 and Chi2), which contain epitopes of the OmpT, Cah and Hes proteins. Thirty pregnant cows in their third trimester of gestation were included and distributed into six groups (n = 5 per group): four groups were administered intramuscularly with three doses of the formulation containing 40 µg or 100 µg of each protein plus the Quil-A or Montanide™ Gel adjuvants, while two control groups were administered with placebos. No local or systemic adverse effects were observed during the study, and hematological parameters and values of blood biochemical indicators were similar among all groups. Furthermore, all vaccine formulations triggered systemic anti-Chi1/Chi2 IgG antibody levels that were significantly higher than the control groups. However, specific IgA levels were generally low and without significant differences among groups. Notably, anti-Chi1/Chi2 IgG antibody levels in the serum of newborn calves fed with colostrum from their immunized dams were significantly higher compared to newborn calves fed with colostrum from control cows, suggesting a passive immunization through colostrum. These results demonstrate that this vaccine is safe and immunogenic when applied to pregnant cows during the third trimester of gestation.

BackgroundShiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen that causes gastrointestinal infections, ranging from acute diarrhea and dysentery to life-threatening diseases such as Hemolytic Uremic Syndrome. Currently, a vaccine to prevent STEC infection is an unmet medical need.ResultsWe developed a chimeric protein-based vaccine targeting seven virulence factors of STEC, including the Stx2B subunit, Tir, Intimin, EspA, Cah, OmpT, and AggA proteins. Immunization of mice with this vaccine candidate elicited significant humoral and cellular immune responses against STEC. High levels of specific IgG antibodies were found in the serum and feces of immunized mice. However, specific IgA antibodies were not detected in either serum or feces. Furthermore, a significantly higher percentage of antigen-specific CD4+ T cells producing IFN-γ, IL-4, and IL-17 was observed in the spleens of immunized mice. Notably, the immunized mice showed decreased shedding of STEC O157:H7 and STEC O91:H21 strains and were protected against weight loss during experimental infection. Additionally, infection with the STEC O91:H21 strain resulted in kidney damage in control unimmunized mice; however, the extent of damage was slightly lower in immunized mice. Our findings suggest that IgG antibodies induced by this vaccine candidate may have a role in inhibiting bacterial adhesion and complement-mediated killing.ConclusionThis study provides evidence that IgG responses are involved in the host defense against STEC. However, our results do not rule out that other classes of antibodies also participate in the protection against this pathogen. Additional work is needed to improve the protection conferred by our vaccine candidate and to elucidate the relevant immune responses that lead to complete protection against this pathogen.

Vibrio cholerae is the causative agent of cholera, a highly contagious diarrheal disease affecting millions worldwide each year. Cholera is a major public health problem, primarily in countries with poor sanitary conditions and regions affected by natural disasters, where access to safe drinking water is limited. In this narrative review, we aim to summarize the current understanding of the evolution of virulence and pathogenesis of V. cholerae as well as provide an overview of the immune response against this pathogen. We highlight that V. cholerae has a remarkable ability to adapt and evolve, which is a global concern because it increases the risk of cholera outbreaks and the spread of the disease to new regions, making its control even more challenging. Furthermore, we show that this pathogen expresses several virulence factors enabling it to efficiently colonize the human intestine and cause cholera. A cumulative body of work also shows that V. cholerae infection triggers an inflammatory response that influences the development of immune memory against cholera. Lastly, we reviewed the status of licensed cholera vaccines, those undergoing clinical evaluation, and recent progress in developing next-generation vaccines. This review offers a comprehensive view of V. cholerae and identifies knowledge gaps that must be addressed to develop more effective cholera vaccines.

Immune performance following antiretroviral therapy initiation varies among patients. Despite achieving viral undetectability, a subgroup of patients fails to restore CD4 + T-cell counts during follow-up, which exposes them to non-AIDS defining comorbidities and increased mortality. Unfortunately, its mechanisms are incompletely understood, and no specific treatment is available. In this review, we address some of the pathophysiological aspects of the poor immune response from a translational perspective, with emphasis in the interaction between gut microbiome, intestinal epithelial dysfunction, and immune system, and we also discuss some studies attempting to improve immune performance by intervening in this vicious cycle.

Abstract Shiga toxin-producing Escherichia coli (STEC) are recognized as responsible for many cases of foodborne diseases worldwide. Cattle are the main reservoir of STEC, shedding the microorganisms in their feces. The serogroup STEC O91 has been associated with hemorrhagic colitis and hemolytic uremic syndrome. Molecular data on which STEC O91 causes human disease still needs to be included. LAA and its hes gene are related to the pathogenicity of STEC and the ability to form biofilms. Considering the frequent isolation of STEC O91, the biofilm-forming ability, and the possible role of hes in the pathogenicity of STEC, we propose to analyze the ability of STEC to form biofilms and to evaluate the expression of hes before and after of biofilm formation. All strains were classified as strong biofilm-forming. The hes expression showed variability between strains before and after biofilm formation, and this may be due to other genes carried by each strain. This study is the first to report the relationship between biofilm formation, and hes expression and proposes that the analysis and diagnosis of LAA, especially hes as STEC O91 virulence factors, could elucidate these unknown mechanisms There is no treatment for HUS, so it is necessary to know the survival and virulence mechanisms of STEC O91.

Anastomotic leakage (AL) is a major cause of morbidity and mortality after colorectal surgery, but the mechanism behind this complication is still not fully understood. Despite the advances in surgical techniques and perioperative care, the complication rates have remained steady. Recently, it has been suggested that colon microbiota may be involved in the development of complications after colorectal surgery. The aim of this study was to evaluate the association of gut microbiota in the development of colorectal AL and their possible virulence strategies to better understand the phenomenon. Using 16S rRNA sequencing of samples collected on the day of surgery and the sixth day following surgery, we analyzed the changes in tissue-associated microbiota at anastomotic sites created in a model of rats with ischemic colon resection. We discovered a trend for lower microbial diversity in the AL group compared to non-leak anastomosis (NLA). There were no differences in relative abundance in the different types of microbial respiration between these groups and the high abundance of the facultative anaerobic Gemella palaticanis is a marker species that stands out as a distinctive feature.

The objective of this work was to molecularly and genotypically characterize and test the inhibitory activity of six colicinogenic Escherichia coli strains (ColEc) and their partially purified colicins against STEC O157:H7 isolated from clinical human cases. Inhibition tests demonstrated the activity of these strains and their colicins against STEC O157:H7. By PCR it was possible to detect colicins Ia, E7, and B and microcins M, H47, C7, and J25. By genome sequencing of two selected ColEc strains, it was possible to identify additional colicins such as E1 and Ib. No genes coding for stx1 and stx2 were detected after analyzing the genome sequence. The inhibitory activity of ColEc against STEC O157:H7 used as an indicator showed that colicins are potent growth inhibitors of E. coli O157:H7, being a potential alternative to reduce the presence of pathogens of public health relevance.

OBJECTIVES:Shiga toxin-producing Escherichia coli strains LAA-positive are important cause of human infection. The capability to adhere to epithelial cells is a key virulence trait, and genes codified in LAA pathogenicity island could be involved in the adhesion during the pathogenesis of LAA-positive STEC strains. Thus, our objectives were to compare hes-negative and hes-positive STEC strains in their adherence capability to epithelial cells (HEp-2) and to evaluate the expression levels of the hes, iha, and tpsA in the bacteria adhered and non-adhered to HEp-2 cells. These genes are encoded in LAA, and are virulence factors that participate in adhesion and autoaggregation.RESULTS:We could not observe differences between the adhesion of strains but also in the expression level of of hes, iha, and tpsA. Genes encoded in LAA contribute to the adhesion phenotype though the expression of STEC adhesins is a coordinated event that depends not only the strain but also on the environment as well as its genetic background. Therefore, the results of this study suggest that LAA ,the most prevalent PAI among LEE-negative STEC strains, plays a role in pathogenesis.

Gut microbiota composition during the first years of life is variable, dynamic and influenced by both prenatal and postnatal factors, such as maternal antibiotics administered during labor, delivery mode, maternal diet, breastfeeding, and/or antibiotic consumption during infancy. Furthermore, the microbiota displays bidirectional interactions with infectious agents, either through direct microbiota-microorganism interactions or indirectly through various stimuli of the host immune system. Here we review these interactions during childhood until 5 years of life, focusing on bacterial microbiota, the most common gastrointestinal and respiratory infections and two well characterized gastrointestinal diseases related to dysbiosis (necrotizing enterocolitis and Clostridioides difficile infection). To date, most peer-reviewed studies on the bacterial microbiota in childhood have been cross-sectional and have reported patterns of gut dysbiosis during infections as compared to healthy controls; prospective studies suggest that most children progressively return to a “healthy microbiota status” following infection. Animal models and/or studies focusing on specific preventive and therapeutic interventions, such as probiotic administration and fecal transplantation, support the role of the bacterial gut microbiota in modulating both enteric and respiratory infections. A more in depth understanding of the mechanisms involved in the establishment and maintenance of the early bacterial microbiota, focusing on specific components of the microbiota-immunity-infectious agent axis is necessary in order to better define potential preventive or therapeutic tools against significant infections in children.

Aerial and respiratory tract-associated bacterial diversity has been scarcely studied in broiler production systems. This study examined the relationship between the environmental air and birds’ respiratory microbiome, considering a longitudinal sampling. Total viable bacteria and coliforms in the air were quantified, and the 16S rRNA gene was sequenced from tracheal and air samples obtained through a novelty protocol. Air results showed a decrease in coliforms over time. However, at week 3, we reported an increase in coliforms (from 143 to 474 CFUc/m3) associated with litter management. Additionally, 16S rRNA gene results indicated a distinctive air microbial community, associated primarily with Bacillota phylum particularly of the Bacilli class (>58%), under all conditions. Tracheal results indicated a predominance of Escherichia coli/Shigella at the beginning of the productive cycle, shifting toward the middle and end of the cycle to Gallibacterium. However, at week 3, the dominance of Escherichia coli/Shigella (>99.5%) associated with litter aeration by tumbling stood out. Tracheal and air samples displayed a statistically different community structure, but shared differentially abundant features through time: Enterococcus, Gallibacterium, and Romboutsia ilealis. These results indicate the impact of production management protocols on the birds’ respiratory system that should be considered a breakpoint in poultry farm health.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is the most prevalent serotype associated with severe diseases worldwide. Biofilms by STEC O157:H7 constitute a high risk to public health and the food industry since they allow cross-contamination of surfaces and the consequent transmission to humans. This study aimed to detect the presence of adhesins genotypically and determine the ability to form biofilm and the curli expression in a collection of 30 O157:H7 strains from healthy cattle and human cases. The efa1, iha, fimCD, ehaA, lpfA1-3, and lpfA2-2 genes were detected in all strains; cah was frequently detected in strains isolated from humans (16/20), and agn43 was the least prevalent gene (3/30). All strains could form a biofilm, although those isolated from cattle were the most biofilm-formers. The curli-negative phenotype was the most prevalent phenotype observed at 37 °C and room temperature. The association between curli production and biofilm formation could not be determined, but the highest proportion of curli-positive strains at room temperature were strong biofilm-formers. These results highlight the possibility of the persistence of STEC O157:H7 in environmental conditions and food processing facilities, increasing the risk of contamination or infection.