A metagenomic approach to unveil the association between fecal gut microbiota and short-chain fatty acids in diarrhea caused by diarrheagenic Escherichia coli in children.

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.

Transcription levels of hes and their involvement in the biofilm formation of Shiga toxin-producing Escherichia coli O91.

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.

Expression of hes, iha, and tpsA codified in locus of adhesion and autoaggregation and their involvement in the capability of shiga toxin-producing Escherichia coli strains to adhere to epithelial cells.

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.

A chimeric protein-based vaccine elicits a strong IgG antibody response and confers partial protection against Shiga toxin-producing Escherichia coli in mice.

Background: Shiga 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. Results: We 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. Conclusion: This 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.

Molecular and Genetic Characterization of Colicinogenic Escherichia coli Strains Active against Shiga Toxin-Producing Escherichia coli O157:H7.

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.

Vibrio cholerae, classification, pathogenesis, immune response, and trends in vaccine development.

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.

Microbiota Phenotype Promotes Anastomotic Leakage in a Model of Rats with Ischemic Colon Resection.

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.

Safety and Immunogenicity of a Chimeric Subunit Vaccine against Shiga Toxin-Producing Escherichia coli in Pregnant Cows.

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.

Enteropathy and gut dysbiosis as obstacles to achieve immune recovery in undetectable people with HIV: a clinical view of evidence, successes, and projections.

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.

Two centuries of vaccination: historical and conceptual approach and future perspectives.

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.

Bacteria from gut microbiota associated with diarrheal infections in children promote virulence of Shiga toxin-producing and enteroaggregative Escherichia coli pathotypes.

Background: Diarrheagenic E. coli (DEC) pathogenicity relies on the interaction of bacteria with the host's gut environment, which is regulated by the resident microbiota. Previously, we identified indicative bacterial species of gut microbiota in DEC-positive stool samples from children. Here, we evaluated the role of two indicative species, Citrobacter werkmanii (CW) and Escherichia albertii (EA), in the virulence of two DEC pathotypes, Shiga toxin-producing (STEC) and enteroaggregative (EAEC) Escherichia coli. Methods: We determined the effect of supernatants obtained from CW and EA cultures on the gene expression of STEC strain 86-24 and EAEC strain 042 by RNA-seq analysis. We evaluated IL-8 secretion from T84 cells infected with these DEC strains in the presence or absence of the supernatant from EA. The effect of the supernatant from EA on the growth and adherence of STEC and EAEC to cells was also evaluated. Finally, we studied the effect of the EA supernatant on the STEC-induced inflammation mediated by the long polar fimbriae (Lpf) in T84 cells and the expression of plasmid-encoded toxin (Pet) in EAEC. Results: RNA-seq analysis revealed that several virulence factors in STEC and EAEC were upregulated in the presence of supernatants from CW and EA. Interestingly, an increase in the secretion of IL-8 was observed in cells infected with STEC or EAEC in the presence of a supernatant from EA. Similar results were observed with the supernatants obtained from clinical strains of E. albertii. The supernatant from EA had no effect on the growth of STEC and EAEC, or on the ability of these DEC strains to adhere to cells. We found that Pet toxin in EAEC was upregulated in the presence of a supernatant from EA. In STEC, using mutant strains for Lpf fimbriae, our data suggested that these fimbriae might be participating in the increase in IL-8 induced by STEC in cells in the presence of a supernatant from EA. Conclusion: Supernatant obtained from an indicative species of DEC-positive diarrhea could modulate gene expression in STEC and EAEC, and IL-8 secretion induced by these bacteria. These data provide new insights into the effect of gut microbiota species in the pathogenicity of STEC and EAEC.

Characterization of Adherent-Invasive Escherichia coli (AIEC) Outer Membrane Proteins Provides Potential Molecular Markers to Screen Putative AIEC Strains.

Adherent-invasive E. coli (AIEC) is a pathotype associated with the etiopathogenesis of Crohn's disease (CD), albeit with an as-yet unclear role. The main pathogenic mechanisms described for AIEC are adherence to epithelial cells, invasion of epithelial cells, and survival and replication within macrophages. A few virulence factors have been described as participating directly in these phenotypes, most of which have been evaluated only in AIEC reference strains. To date, no molecular markers have been identified that can differentiate AIEC from other E. coli pathotypes, so these strains are currently identified based on the phenotypic characterization of their pathogenic mechanisms. The identification of putative AIEC molecular markers could be beneficial not only from the diagnostic point of view but could also help in better understanding the determinants of AIEC pathogenicity. The objective of this study was to identify molecular markers that contribute to the screening of AIEC strains. For this, we characterized outer membrane protein (OMP) profiles in a group of AIEC strains and compared them with the commensal E. coli HS strain. Notably, we found a set of OMPs that were present in the AIEC strains but absent in the HS strain. Moreover, we developed a PCR assay and performed phylogenomic analyses to determine the frequency and distribution of the genes coding for these OMPs in a larger collection of AIEC and other E. coli strains. As result, it was found that three genes (chuA, eefC, and fitA) are widely distributed and significantly correlated with AIEC strains, whereas they are infrequent in commensal and diarrheagenic E. coli strains (DEC). Additional studies are needed to validate these markers in diverse strain collections from different geographical regions, as well as investigate their possible role in AIEC pathogenicity.

Litter Management Strategies and Their Impact on the Environmental and Respiratory Microbiome Might Influence Health in Poultry.

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.

Genetic features of verotoxigenic Escherichia coli O157:H7 isolated from clinical cases of Argentina and Chile.

We aimed to compare the genetic diversity existing in VTEC O157:H7 strains isolated from cases of human disease from Argentina and Chile. For it, 76 strains were studied in relation to the distribution of genes encoding virulence factors and subtyped by lineage-specific polymorphisms (LSPA-6), and phylogroups assignment. Our results show the almost exclusive circulation of VTEC O157:H7 isolates belonging to lineage I/II, associated with hypervirulent strains, and to the phylogroup E and, on the other hand, genetic diversity present among Argentinean and Chilean strains analyzed, mainly in relation to putative virulence determinants and nle profiles.

Landscapes and bacterial signatures of mucosa-associated intestinal microbiota in Chilean and Spanish patients with inflammatory bowel disease.

Inflammatory bowel diseases (IBDs), which include ulcerative colitis (UC) and Crohn's disease (CD), cause chronic inflammation of the gut, affecting millions of people worldwide. IBDs have been frequently associated with an alteration of the gut microbiota, termed dysbiosis, which is generally characterized by an increase in abundance of Proteobacteria such as Escherichia coli, and a decrease in abundance of Firmicutes such as Faecalibacterium prausnitzii (an indicator of a healthy colonic microbiota). The mechanisms behind the development of IBDs and dysbiosis are incompletely understood. Using samples from colonic biopsies, we studied the mucosa-associated intestinal microbiota in Chilean and Spanish patients with IBD. In agreement with previous studies, microbiome comparison between IBD patients and non-IBD controls indicated that dysbiosis in these patients is characterized by an increase of pro-inflammatory bacteria (mostly Proteobacteria) and a decrease of commensal beneficial bacteria (mostly Firmicutes). Notably, bacteria typically residing on the mucosa of healthy individuals were mostly obligate anaerobes, whereas in the inflamed mucosa an increase of facultative anaerobe and aerobic bacteria was observed. We also identify potential co-occurring and mutually exclusive interactions between bacteria associated with the healthy and inflamed mucosa, which appear to be determined by the oxygen availability and the type of respiration. Finally, we identified a panel of bacterial biomarkers that allow the discrimination between eubiosis from dysbiosis with a high diagnostic performance (96% accurately), which could be used for the development of non-invasive diagnostic methods. Thus, this study is a step forward towards understanding the landscapes and alterations of mucosa-associated intestinal microbiota in patients with IBDs.

Bacterial Gut Microbiota and Infections During Early Childhood.

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.

Gut Microbiota-Metabolome Changes in Children With Diarrhea by Diarrheagenic E. coli.

Background: Diarrheagenic Escherichia coli (DEC) strains are a main cause of diarrhea worldwide in children under 5 years old. DEC virulence is strongly regulated by environmental conditions and metabolites produced by the gut microbiota in the intestinal tract. In this study, we evaluated changes in gut microbiota-metabolome in children with or without diarrhea produced by DEC pathotypes. Goal: To determine gut microbiota composition and metabolome in stool samples obtained from healthy children and children with diarrhea positive for DEC pathotypes. Methods: We analyzed a total of 16 age-paired stool samples: 8 diarrheal samples positive for one DEC pathotype and 8 stool samples from healthy children. To identify the microbiota composition, we sequenced the V3-V4 region of the 16S rRNA and determined operational phylogenetic units (OPU). OPU were then used to predict metabolic pathways using the PICRUSt2 software. The presence of metabolites in stool samples was determined by LC-MS. A correlation analysis was performed with the main genera from each group and main metabolites. Bacteria associated with variance of main metabolites were identified using the MIMOSA2 software. Results: DEC and healthy groups showed a statistically different microbiota composition. A decrease in Firmicutes together with an increase in Bacteroidetes and Proteobacteria was found in the DEC group compared to the healthy group. Metabolic pathway predictions based on microbiota diversity showed that pathways involved in histidine and L-ornithine metabolism were significantly different between groups. A total of 88 metabolites detected by LC-MS were included in the metabolome analysis. We found higher levels of histamine and lower levels of ornithine in DEC samples than in the healthy group. Histamine and L-ornithine were associated with a specific microbiota species and the corresponding metabolic pathways. Conclusion: Stool samples from healthy children and children positive for DEC displayed a differential metabolome and microbiota composition. A strong correlation between a gut microbiota species and certain metabolites, such as histamine and L-ornithine, was found in the DEC group. This information might be useful to identify mechanisms and signaling molecules involved in the crosstalk between microbiota and DEC pathotypes.

Deciphering Additional Roles for the EF-Tu, l-Asparaginase II and OmpT Proteins of Shiga Toxin-Producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) causes outbreaks and sporadic cases of gastroenteritis. STEC O157:H7 is the most clinically relevant serotype in the world. The major virulence determinants of STEC O157:H7 are the Shiga toxins and the locus of enterocyte effacement. However, several accessory virulence factors, mainly outer membrane proteins (OMPs) that interact with the host cells may contribute to the virulence of this pathogen. Previously, the elongation factor thermo unstable (EF-Tu), l-asparaginase II and OmpT proteins were identified as antigens in OMP extracts of STEC. The known subcellular location of EF-Tu and l-asparaginase II are the cytoplasm and periplasm, respectively. Therefore, we investigate whether these two proteins may localize on the surface of STEC and, if so, what roles they have at this site. On the other hand, the OmpT protein, a well characterized protease, has been described as participating in the adhesion of extraintestinal pathogenic E. coli strains. Thus, we investigate whether OmpT has this role in STEC. Our results show that the EF-Tu and l-asparaginase II are secreted by O157:H7 and may also localize on the surface of this bacterium. EF-Tu was identified in outer membrane vesicles (OMVs), suggesting it as a possible export mechanism for this protein. Notably, we found that l-asparaginase II secreted by O157:H7 inhibits T-lymphocyte proliferation, but the role of EF-Tu at the surface of this bacterium remains to be elucidated. In the case of OmpT, we show its participation in the adhesion of O157:H7 to human epithelial cells. Thus, this study extends the knowledge of the pathogenic mechanisms of STEC.

Repertoire and Diversity of Toxin - Antitoxin Systems of Crohn's Disease-Associated Adherent-Invasive Escherichia coli. New Insight of T his Emergent E. coli Pathotype.

Adherent-invasive Escherichia coli (AIEC) corresponds to an E. coli pathovar proposed as a possible agent trigger associated to Crohn's disease. It is characterized for its capacity to adhere and to invade epithelial cells, and to survive and replicate inside macrophages. Mechanisms that allow intestinal epithelium colonization, and host factors that favor AIEC persistence have been partly elucidated. However, bacterial factors involved in AIEC persistence are currently unknown. Toxin-antitoxin (TA) systems are recognized elements involved in bacterial persistence, in addition to have a role in stabilization of mobile genetic elements and stress response. The aim of this study was to elucidate the repertoire and diversity of TA systems in the reference AIEC NRG857c strain and to compare it with AIEC strains whose genomes are available at databases. In addition, toxin expression levels under in vitro stress conditions found by AIEC through the intestine and within the macrophage were measured. Our results revealed that NRG857c encodes at least 33 putative TA systems belonging to types I, II, IV, and V, distributed around all the chromosome, and some in close proximity to genomic islands. A TA toxin repertoire marker of the pathotype was not found and the repertoire of 33 TA toxin genes described here was exclusive of the reference strains, NRG857c and LF82. Most toxin genes were upregulated in the presence of bile salts and acidic pH, as well as within the macrophage. However, different transcriptional responses were detected between reference strains (NRG857c and HM605), recalling the high diversity associated to this pathotype. To our knowledge this is the first analysis of TA systems associated to AIEC and it has revealed new insight associated to this emergent E. coli pathotype.

Distribution of Locus of Adhesion and Autoaggregation and hes Gene in STEC Strains from Countries of Latin America.

Shiga toxin-producing Escherichia coli (STEC) are zoonotic food pathogens associated with foodborne diarrheal illness, hemorrhagic colitis, and complications such as hemolytic uremic syndrome (HUS). The ability to adhere to epithelial cells is an important virulence trait, and pathogenicity islands (PAIs) play an important role on it. Some STEC carrying a PAI named locus of enterocyte effacement (LEE-positive) have been frequently associated to HUS; however, STEC that do not carry LEE (LEE-negative) have also been associated with this outcome. The burden of disease caused by LEE-negative STEC has increased recently in several countries like Argentina, Chile, and Paraguay. A new PAI -the Locus of Adhesion and Autoagregation (LAA)-has been associated to severe disease in humans. In this study, we aimed to analyze the distribution of LAA and its possible predictor, the gene hes, in LEE-negative STEC strains isolated from Chile and Paraguay from different sources. The presence of the different LAA modules and hes were detected by PCR. LAA was found in 41.6% and 41.0% of strains isolated from Chile and Paraguay, respectively. Strains were isolated from diverse origins and belonged to several serogroups including O91, O103, and O113. The hes gene was detected in 50% of the isolates from Paraguay and Chile. Therefore, the detection of LAA and hes in STEC could complement current genetic evaluation schemes, allowing to classify LEE negative STEC strains as LAA-positive or LAA-negative STEC strains.