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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 E nterobacteriaceae 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.

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 foodborne pathogens causing severe diseases. The ability of STEC to produce disease is associated with Shiga toxin (Stx) production. We investigated the occurrence of STEC on bovine and pork carcasses and walls of trucks where they were transported, and we characterized virulence genes and serotypes of STEC strains. We compared the whole genomic sequencing of a STEC O157:H7 strain isolated from a bovine carcass in this work and a STEC O157:H7 strain isolated from a child with HUS, both isolated in 2019. We studied the relationship between these isolates and others collected in the database. The results show a 40% of STEC and two different serogroups were identified (O130 and O157). STEC O157:H7 were isolated from bovine carcasses and harbored stx2, eae, ehxA, katP, espP, stcE, ECSP_0242/1773/2687/2870/2872/3286/3620 and were classified as lineage I/II. In STEC non-O157 isolates, three isolates were isolated from bovine carcasses and harbored the serogroup O130 and one strain isolated from pork carcasses was O-non-typeable. All STEC non-O157 harbored sxt1 gene. The analysis from the whole genome showed that both STEC O157:H7 strains belonged to the hypervirulent clade 8, ST11, phylogroup E, carried the allele tir 255 T > A T, and they were not clonal. The analysis of information allows us to conclude that the STEC strains circulate in pork and bovine carcasses arriving in transport. This situation represents a risk for the consumers and the need to implement an integrated STEC control in the food chain., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

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.

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.

Septic joint arthritis in the small joints of the hand can be caused by penetrating trauma, ruptured ganglion cysts, or open joint dislocations, among others. The use of external fixation for the treatment of this condition has been reported in the past as a means of temporary joint distraction, or for secondary fusion procedures. In the present article, the authors describe a surgical technique involving the use of a low-cost external fixator for the primary arthrodesis of infected distal interphalangeal joints of the hand. The external fixator is fabricated with simple materials, threaded Kirshner wires, bone cement, and an insulin syringe, which the authors have used to fuse the distal interphalangeal joint primarily when destroyed by septic arthritis., Competing Interests: Conflicts of Interest and Source of Funding: The authors report no conflicts of interest and no source of funding., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Escherichia coli , one of the most abundant bacterial species in the human gut microbiota, has developed a mutualistic relationship with its host, regulating immunological responses. In contrast, enterotoxigenic E. coli (ETEC), one of the main etiologic agents of diarrheal morbidity and mortality in children under the age of five in developing countries, has developed mechanisms to reduce the immune-activator effect to carry out a successful infection. Following infection, the host cell initiates the shutting-off of protein synthesis and stress granule (SG) assembly. This is mostly mediated by the phosphorylation of translation initiator factor 2α (eIF2α). We therefore evaluated the ability of a non-pathogenic E. coli strain ( E. coli HS) and an ETEC strain (ETEC 1766a) to induce stress granule assembly, even in response to exogenous stresses. In this work, we found that infection with E. coli HS or ETEC 1766a prevents SG assembly in Caco-2 cells treated with sodium arsenite (Ars) after infection. We also show that this effect occurs through an eIF2α phosphorylation (eIF2α-P)-dependent mechanism. Understanding how bacteria counters host stress responses will lay the groundwork for new therapeutic strategies to bolster host cell immune defenses against these pathogens.

Objective: To determine the dynamics of norovirus disease, a major cause of acute gastroenteritis (AGE), compared to other relevant etiologies, among families living in a lower middle income area., Study Design: Families with three or more members and with one or more healthy children <24 months of age were followed for 1-2 years to detect any AGE. Stool samples were tested for viral and bacterial pathogens and a questionnaire was completed for those with norovirus or rotavirus AGE., Results: Between April and June 2016, 110 families were enrolled, with 103 of them completing ≥12 months of follow-up. A total of 159 family AGE episodes were detected, mostly affecting one individual (92%). At least one pathogen was detected in 56% (94/169) of samples, of which 75/94 (80%) were sole infections. Norovirus was most common (n=26), followed closely by enteropathogenic Escherichia coli (EPEC) (n=25), rotavirus (n=24), and astrovirus (n=23). The annual incidence of family AGE was 0.77, and 0.12 for norovirus. Most norovirus AGE occurred in children <4 years old (96%). Only 13/159 (8%) index AGE cases resulted in a secondary case, of which four were associated with norovirus. The majority of norovirus strains were GII (85%), with a mild predominance of GII.4 (9/26; 35%); most norovirus isolates (69%) were recombinants., Conclusions: The family incidence of AGE in this lower middle income community was nearly one episode per year, mostly caused by viruses, specifically norovirus closely followed by rotavirus and astrovirus. Norovirus infections primarily affected children <4 years old and secondary cases were uncommon., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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.

Brucella abortus is a facultative intracellular pathogen that causes a zoonosis called brucellosis. This disease leads to abortion and infertility in cattle, and diverse complications in humans. B. abortus is a successful intracellular bacterium that has developed the ability to evade the host's immune system and it replicates in professional and non-professional phagocytic cells, persisting in the different tissues, and organs of its hosts. It has been described that Brucella expresses a polar flagellum under certain conditions, but its function is still unknown. In this study we evaluated the role of the FlgJ, a protein, presumably a peptidoglycan hydrolase involved in flagellum formation and in the virulence of B. abortus strain 2308. B. abortus 2308 Δ flgJ mutant and complemented strains were constructed to study the function of the FlgJ protein in the context of the virulence of this pathogen in in vitro and in vivo assays. The results showed that the elimination of the flgJ gene delays the growth rate of B. abortus in culture, reduces its intracellular survival capacity in professional and non-professional phagocytic cells, rendering it unable to escape from the endocytic route and not reaching the endoplasmic reticulum. It also negatively affects their persistence in BALB/c mice. Functionally, the B. abortus 2308 flgJ gene restored motility to an E. coli flgJ mutant gene. Furthermore, it was discovered that the production of FlgJ protein is associated with the bacterial adherence by B. abortus . Therefore, although the specific function of the polar flagellum for Brucella is unknown, the data indicates that the flagellar flgJ gene and its product are required for full virulence of B. abortus 2308, since its deletion significantly reduces the fitness of this pathogen in vitro and in vivo ., (Copyright © 2020 Coloma-Rivero, Gómez, Alvarez, Saitz, del Canto, Céspedes, Vidal and Oñate.)

Shiga toxin-producing Escherichia coli (STEC) cause diarrhea and dysentery, which may progress to hemolytic uremic syndrome (HUS). Vaccination has been proposed as a preventive approach against STEC infection; however, there is no vaccine for humans and those used in animals reduce but do not eliminate the intestinal colonization of STEC. The OmpT, Cah and Hes proteins are widely distributed among clinical STEC strains and are recognized by serum IgG and IgA in patients with HUS. Here, we develop a vaccine formulation based on two chimeric antigens containing epitopes of OmpT, Cah and Hes proteins against STEC strains. Intramuscular and intranasal immunization of mice with these chimeric antigens elicited systemic and local long-lasting humoral responses. However, the class of antibodies generated was dependent on the adjuvant and the route of administration. Moreover, while intramuscular immunization with the combination of the chimeric antigens conferred protection against colonization by STEC O157:H7, the intranasal conferred protection against renal damage caused by STEC O91:H21. This preclinical study supports the potential use of this formulation based on recombinant chimeric proteins as a preventive strategy against STEC infections., Competing Interests: Competing interestsCurrently, an application for an international patent had been presented for the chimeric antigens developed and uses thereof (PCT/IB2019/054554). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© The Author(s) 2020.)

LEE-negative Shiga toxin-producing Escherichia coli (STEC) strains are important cause of infection in humans and they should be included in the public health surveillance systems. Some isolates have been associated with haemolytic uremic syndrome (HUS) but the mechanisms of pathogenicity are is a field continuos broadening of knowledge. The IrgA homologue adhesin (Iha), encoded by iha , is an adherence-conferring protein and also a siderophore receptor distributed among LEE-negative STEC strains. This study reports the presence of different subtypes of iha in LEE-negative STEC strains. We used genomic analyses to design PCR assays for detecting each of the different iha subtypes and also, all the subtypes simultaneously. LEE-negative STEC strains were designed and different localizations of this gene in STEC subgroups were examinated. Genomic analysis detected iha in a high percentage of LEE-negative STEC strains. These strains generally carried iha sequences similar to those harbored by the Locus of Adhesion and Autoaggregation (LAA) or by the plasmid pO113. Besides, almost half of the strains carried both subtypes. Similar results were observed by PCR, detecting iha LAA in 87% of the strains (117/135) and iha pO113 in 32% of strains (43/135). Thus, we designed PCR assays that allow rapid detection of iha subtypes harbored by LEE-negative strains. These results highlight the need to investigate the individual and orchestrated role of virulence genes that determine the STEC capacity of causing serious disease, which would allow for identification of target candidates to develop therapies against HUS., (© 2019 The Authors. Published by Elsevier Ltd.)

Background: Enterotoxigenic Escherichia coli (ETEC) cause significant diarrheal morbidity and mortality in children of resource-limited regions, warranting development of effective vaccine strategies. Genetic diversity of the ETEC pathovar has impeded development of broadly protective vaccines centered on the classical canonical antigens, the colonization factors and heat-labile toxin. Two non-canonical ETEC antigens, the EtpA adhesin, and the EatA mucinase are immunogenic in humans and protective in animal models. To foster rational vaccine design that complements existing strategies, we examined the distribution and molecular conservation of these antigens in a diverse population of ETEC isolates., Methods: Geographically diverse ETEC isolates (n = 1159) were interrogated by PCR, immunoblotting, and/or whole genome sequencing (n = 46) to examine antigen conservation. The most divergent proteins were purified and their core functions assessed in vitro., Results: EatA and EtpA or their coding sequences were present in 57.0% and 51.5% of the ETEC isolates overall, respectively; and were globally dispersed without significant regional differences in antigen distribution. These antigens also exhibited >93% amino acid sequence identity with even the most divergent proteins retaining the core adhesin and mucinase activity assigned to the prototype molecules., Conclusions: EtpA and EatA are well-conserved molecules in the ETEC pathovar, suggesting that they serve important roles in virulence and that they could be exploited for rational vaccine design., Competing Interests: The authors have declared that no competing interests exist.

Enterotoxigenic Escherichia coli (ETEC) is one of the most common diarrheal pathogens in the low- and middle-income regions of the world, however a systematic examination of the genomic content of isolates from Chile has not yet been undertaken. Whole genome sequencing and comparative analysis of a collection of 125 ETEC isolates from three geographic locations in Chile, allowed the interrogation of phylogenomic groups, sequence types and genes specific to isolates from the different geographic locations. A total of 80.8% (101/125) of the ETEC isolates were identified in E. coli phylogroup A, 15.2% (19/125) in phylogroup B, and 4.0% (5/125) in phylogroup E. The over-representation of genomes in phylogroup A was significantly different from other global ETEC genomic studies. The Chilean ETEC isolates could be further subdivided into sub-clades similar to previously defined global ETEC reference lineages that had conserved multi-locus sequence types and toxin profiles. Comparison of the gene content of the Chilean ETEC identified genes that were unique based on geographic location within Chile, phylogenomic classifications or sequence type. Completion of a limited number of genomes provided insight into the ETEC plasmid content, which is conserved in some phylogenomic groups and not conserved in others. These findings suggest that the Chilean ETEC isolates contain unique virulence factor combinations and genomic content compared to global reference ETEC isolates., Competing Interests: The authors have declared that no competing interests exist.

Enterotoxigenic Escherichia coli (ETEC) is a significant cause of childhood diarrhea and is a leading cause of traveler's diarrhea. ETEC strains encoding the heat-stable enterotoxin (ST) are more often associated with childhood diarrhea than ETEC strains that encode only the heat-labile enterotoxin (LT). Colonization factors (CFs) also have a demonstrated role in ETEC virulence, and two of the most prevalent CFs among ETEC that have caused diarrhea are colonization factor antigen I (CFA/I) and CS6. In the current report, we describe the genomes of 269 CS6- or CFA/I-encoding ST-only ETEC isolates that were associated with human diarrhea. While the CS6 and CFA/I ETEC were identified in at least 13 different ETEC genomic lineages, a majority (85%; 229/269) were identified in only six lineages. Complete genome sequencing of selected isolates demonstrated that a conserved plasmid contributed to the dissemination of CFA/I whereas at least five distinct plasmids were involved in the dissemination of ST and/or CS6. Additionally, there were differences in gene content between CFA/I and CS6 ETEC at the phylogroup and lineage levels and in association with their geographic location of isolation as well as lineage-related differences in ST production. Thus, we demonstrate that genomically diverse E. coli strains have acquired ST, as well as CFA/I or CS6, via one or more plasmids and that, in some cases, isolates of a particular lineage or geographic location have undergone additional modifications to their genome content. These findings will aid investigations of virulence and the development of improved diagnostics and vaccines against this important human diarrheal pathogen. IMPORTANCE Comparative genomics and functional characterization were used to analyze a global collection of CFA/I and CS6 ST-only ETEC isolates associated with human diarrhea, demonstrating differences in the genomic content of CFA/I and CS6 isolates related to CF type, lineage, and geographic location of isolation and also lineage-related differences in ST production. Complete genome sequencing of selected CFA/I and CS6 isolates enabled descriptions of a highly conserved ST-positive (ST + ) CFA/I plasmid and of at least five diverse ST and/or CS6 plasmids among the CS6 ETEC isolates. There is currently no approved vaccine for ST-only ETEC, or for any ETEC for that matter, and as such, the current report provides functional verification of ST and CF production and antimicrobial susceptibility testing and an in-depth genomic characterization of a collection of isolates that could serve as representatives of CFA/I- or CS6-encoding ST-only ETEC strains for future studies of ETEC pathogenesis, vaccine studies, and/or clinical trials.

Background: Enterotoxigenic Escherichia coli (ETEC) encoding heat-stable enterotoxin (ST) alone or with heat-labile enterotoxin (LT) cause moderate-to-severe diarrhea (MSD) in developing country children. The Global Enteric Multicenter Study (GEMS) identified ETEC encoding ST among the top four enteropathogens. Since the GEMS objective was to provide evidence to guide development and implementation of enteric vaccines and other interventions to diminish diarrheal disease morbidity and mortality, we examined colonization factor (CF) prevalence among ETEC isolates from children age <5 years with MSD and from matched controls in four African and three Asian sites. We also assessed strength of association of specific CFs with MSD., Methodology/principal Findings: MSD cases enrolled at healthcare facilities over three years and matched controls were tested in a standardized manner for many enteropathogens. To identify ETEC, three E. coli colonies per child were tested by polymerase chain reaction (PCR) to detect genes encoding LT, ST; confirmed ETEC were examined by PCR for major CFs (Colonization Factor Antigen I [CFA/I] or Coli Surface [CS] antigens CS1-CS6) and minor CFs (CS7, CS12, CS13, CS14, CS17, CS18, CS19, CS20, CS21, CS30). ETEC from 806 cases had a single toxin/CF profile in three tested strains per child. Major CFs, components of multiple ETEC vaccine candidates, were detected in 66.0% of LT/ST and ST-only cases and were associated with MSD versus matched controls by conditional logistic regression (p≤0.006); major CFs detected in only 25.0% of LT-only cases weren't associated with MSD. ETEC encoding exclusively CS14, identified among 19.9% of 291 ST-only and 1.5% of 259 LT/ST strains, were associated with MSD (p = 0.0011). No other minor CF exhibited prevalence ≥5% and significant association with MSD., Conclusions/significance: Major CF-based efficacious ETEC vaccines could potentially prevent up to 66% of pediatric MSD cases due to ST-encoding ETEC in developing countries; adding CS14 extends coverage to ~77%., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests. We note that the following authors are involved in the development of vaccines to prevent diarrhea caused by enterotoxigenic Escherichia coli: James P. Nataro: Has been named in patents for technology that may have relevance for ETEC vaccine technology; JPN is also a co-investigator on grants from non-profit agencies that support ETEC vaccine development. Halvor Sommerfelt: Has been named in patents for technology that may have relevance for ETEC vaccine technology including: INT Application Number PCT/IB2014/000267; INT Publication Number WO2014128555 A2 “ETEC Vaccine”. European Patent Application No. 14711297.3 United States Patent Application No. 14/769,342. China Patent Application No. 201480022329.6. Puntervoll P, Sommerfelt H, Clements J, Nataro JP, Zhang W, Taxt A. HS is also a co-investigator on grants from non-profit agencies that support ETEC vaccine development Ann-Mari Svennerholm: Has shares in the University of Göteborg spin-out biotech company Gotovax AB which is entitled to royalty from Scandinavian Biopharma on sales in travelers of the ETEC vaccine Etvax if it becomes a commercial product. A-MS is a co-inventor on ETEC vaccine patent application owned by Scandinavian Biopharma and has a research grant from the Swedish Foundation for Strategic Research for infection biology research. Myron M. Levine: Is a co-inventor of patents for ETEC vaccine technology including US patent #6,902,736 B2. “Isolation and characterization of the CSA operon (ETEC-CS4 pili) and methods of using same.” He is a member of the Scientific Advisory Board of the PaxVax Corporation. MML is also the recipient for grants relevant to ETEC vaccine development including the Enteric Center of Excellence for Translational Research (Enteric-CETR) “Immunoprophylactic Strategies to Control Emerging Enteric Infections” (NIAID U19AI109776; MML, PI)

Rotavirus G8P[8] infection has been common in Africa, but rare in the Americas. Among 23 rotavirus episodes observed during 18 months of surveillance of 100 families in Chile, 11 (48%) were identified as G8P[8]. Genotypes from these strains shared >99% identity with rotavirus sequences described in Asia, and may be misclassified as mixed G8/G12., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The coli surface antigen 26 (CS26) of enterotoxigenic Escherichia coli (ETEC) had been described as a putative adhesive pilus based on the partial sequence of the crsH gene, detected in isolates from children with diarrhea in Egypt. However, its production and activity as adherence determinant has not been experimentally addressed. The crsH was identified as a homolog of genes encoding structural subunits of ETEC colonization factors (CFs) CS12, CS18, and CS20. These CFs, along with the recently discovered CS30, belong to the γ 2 family of pili assembled by the chaperone-usher pathway (CU pili). Further, the complete CS26 locus, crsHBCDEFG , was described in an O141 ETEC strain (ETEC 100664) obtained from a diarrhea case in The Gambia, during the Global Enterics Multicenter Study. Here, we report that CS26 is a pilus of ∼10 nm in diameter, with the capacity to increase the cell adherence of the non-pathogenic strain E. coli DH10B. As for other related pili, production of CS26 seems to be regulated by phase variation. Deletion of crsHBCDEFG in ETEC 100664 significantly decreased its adherence capacity, which was recovered by in trans complementation. Furthermore, CrsH was cross-recognized by polyclonal antibodies directed against the major structural subunit of CS20, CsnA, as determined by Western blotting and immunogold labeling. ETEC CS26+ strains were found to harbor the heat-labile enterotoxin only, within three different sequence types of phylogroups A and B1, the latter suggesting acquisition through independent events of horizontal transfer. Overall, our results demonstrate that CS26 is an adhesive pilus of human ETEC. In addition, cross-reactivity with anti-CsnA antibodies indicate presence of common epitopes in γ 2 -CFs.

Shiga toxin-producing Escherichia coli (STEC) are important foodborne pathogens that can cause severe disease. The ability to adhere to epithelial cells is an important virulence trait and pathogenicity islands (PAIs) play an important role. Recently, researchers identified a member of the Heat-resistant agglutinin family and characterized this antigen named Hemagglutinin from Shiga toxin-producing E. coli (Hes). More importantly, they showed that hes and other genes such as iha, pagC and agn43 were integrated in each of the four modules present in the new PAI named Locus of Adhesion and Autoaggregation (LAA) whose presence is associated with severe disease linked to with LEE-negatives STEC. The distribution of LAA among STEC strains isolates from different origins between 2000 and 2015 from cattle, the farm environment, and food and harboring diverse virulence was investigated. The STEC strains were characterized by PCR to detect three modules of LAA and agn43 (as marker of module IV), and phylogenetic groups were determined. LAA was found in 46% of LEE-negative STEC corresponding to serogroups O91, O174, O113, O171, O178, O130 and others. The presence of this PAI is associated with strains harboring stx2 (56%) and belonging to phylogroup B1 (91%). LAA is a novel pathogenicity island associated with strains isolated from Hemolytic Uremic Syndrome cases. Therefore, the results of this study contribute to a better understanding regarding the pathogenicity of this emergent subset of STEC strains harboring LAA as a predictor of virulence of LEE-negative STEC strains., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Purpose  The purpose of our study is to compare the intramedullary fixation of metacarpal fractures with cannulated headless screws and antegrade Kirschner wires in terms of final total active motion, grip strength, patient-related outcomes, need for casting, and return to work times. Methods  The authors performed a retrospective review of the hospital records. Thirty fractures were included in the study, 19 in the screw fixation group, and 11 in the Kirschner wire group. Grip strength, and total active motion, was measured at the latest follow-up for both the injured and contralateral hand. Pain was measured on the visual analog scale. Patients were requested to fill a Quick disabilities of the arm and hand score (DASH) questionnaire at the latest follow-up. Satisfaction was measured on a scale from 0 to 10. The time to return to work was quantified from the accident to the point when the patient was back to active duty. Postoperative casting time was also quantified. Results  The authors did not find any differences between the two groups in total active motion, grip strength, pain, satisfaction, or Quick DASH scores. We did find a difference in the return to work and casting times; these appeared to be shorter in the screw group. Conclusion  Due to the small number of cases, we have been unable to clearly conclude that there were any benefits in the application of one particular technique when compared with the other.

The treatment of extensive soft tissue defects in the thumb with dorsal metacarpal artery flaps has been previously reported in the literature. Island flaps from the dorsum of the index and long fingers have been the subject of many reports and studies. However, when the defect involves the whole thumb, a 360° circumferential defect, standard first or second dorsal metacarpal artery flaps are usually insufficient. There are fewer reports on the use of bilobed flaps for this application and we have found no reports on the use of bilobed racquet flaps or extended seagull flaps as treatment for this condition. We report the salvage of a thumb degloving injury with use of a bilobed racquet flap.

In March 2010, a massive outbreak of gastroenteritis started in the region of Antofagasta (northern Chile). The outbreak was mainly attributed to Norovirus genogroup II although ETEC strains were also isolated with high frequency from clinical samples. We review this outbreak and determined that ETEC was an underestimated etiologic agent., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Shiga Toxin-producing Escherichia coli (STEC) are a group of foodborne pathogens associated with diarrhea, dysentery, hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Shiga toxins are the major virulence factor of these pathogens, however adhesion and colonization to the human intestine is required for STEC pathogenesis. A subset of STEC strains carry the Locus of Enterocyte Effacement (LEE) pathogenicity island (PAI), which encodes genes that mediate the colonization of the human intestine. While LEE-positive STEC strains have traditionally been associated with human disease, the burden of disease caused by STEC strains that lacks LEE (LEE-negative) has increased recently in several countries; however, in the absence of LEE, the molecular pathogenic mechanisms by STEC strains are unknown. Here we report a 86-kb mosaic PAI composed of four modules that encode 80 genes, including novel and known virulence factors associated with adherence and autoaggregation. Therefore, we named this PAI as Locus of Adhesion and Autoaggregation (LAA). Phylogenomic analysis using whole-genome sequences of STEC strains available in the NCBI database indicates that LAA PAI is exclusively present in a subset of emerging LEE-negative STEC strains, including strains isolated from HC and HUS cases. We suggest that the acquisition of this PAI is a recent evolutionary event, which may contribute to the emergence of these STEC.

Adherent-invasive Escherichia coli (AIEC) strains are genetically variable and virulence factors for AIEC are non-specific. FimH is the most studied pathogenicity-related protein, and there have been few studies on other proteins, such as Serine Protease Autotransporters of Enterobacteriacea (SPATEs). The goal of this study is to characterize E. coli strains isolated from patients with Crohn's disease (CD) in Chile and Spain, and identify genetic differences between strains associated with virulence markers and clonality. We characterized virulence factors and genetic variability by pulse field electrophoresis (PFGE) in 50 E. coli strains isolated from Chilean and Spanish patients with CD, and also determined which of these strains presented an AIEC phenotype. Twenty-six E. coli strains from control patients were also included. PFGE patterns were heterogeneous and we also observed a highly diverse profile of virulence genes among all E. coli strains obtained from patients with CD, including those strains defined as AIEC. Two iron transporter genes chuA , and irp2 , were detected in various combinations in 68-84% of CD strains. We found that the most significant individual E. coli genetic marker associated with CD E. coli strains was chuA . In addition, patho-adaptative fimH mutations were absent in some of the highly adherent and invasive strains. The fimH adhesin, the iron transporter irp2 , and Class-2 SPATEs did not show a significant association with CD strains. The V27A fimH mutation was detected in the most CD strains. This study highlights the genetic variability of E. coli CD strains from two distinct geographic origins, most of them affiliated with the B2 or D E. coli phylogroups and also reveals that nearly 40% of Chilean and Spanish CD patients are colonized with E.coli with a characteristic AIEC phenotype.

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrhea worldwide. Among the 25 different ETEC adhesins, 22 are known as "colonization factors" (CFs), of which 17 are assembled by the chaperone-usher (CU) mechanism. Currently, there is no preventive therapy against ETEC, and CFs have been proposed as components for vaccine development. However, studies of diarrhea-causing ETEC strains worldwide indicate that between 15 and 50% of these are negative for known CFs, hindering the selection of the most widespread structures and suggesting that unknown adhesins remain to be identified. Here, we report the result of a comprehensive analysis of 35 draft genomes of ETEC strains which do not carry known adhesin genes; our goal was to find new CU pili loci. The phylogenetic profiles and serogroups of these strains were highly diverse, a majority of which produced only the heat-labile toxin. We identified 10 pili loci belonging to CU families β (1 locus), γ 2 (7 loci), κ (1 locus), and π (1 locus), all of which contained the required number of open reading frames (ORFs) to encode functional structures. Three loci were variants of previously-known clusters, three had been only-partially described, and four are novel loci. Intra-loci genetic variability identified would allow the synthesis of up to 14 different structures. Clusters of putative γ 2 -CU pili were most common (23 strains), followed by putative β-CU pili (12 strains), which have not yet been fully characterized. Overall, our findings significantly increase the number of ETEC adhesion genes associated with human infections.

The most common mechanism of trimethoprim (TMP)-resistance is the acquisition of dihydrofolate reductase enzyme resistant to this drug. Previous molecular characterization of TMP-genes resistance in Chilean isolates of Shigella sonnei searching for dfrA1 and dfrA8, showed solely the presence of dfrA8 (formerly dhfrIIIc). However, these genetic markers were absent in S. sonnei strains further isolated during an outbreak in 2009. To identify the TMP-resistance gene in these strains, a genomic DNA library from a TMP-resistant (TMP(R)) S. sonnei representative strain for the outbreak was used to clone, select and identify a TMP-resistance marker. The TMP(R) clone was sequenced by primer walking, identifying the presence of the dfrA14 gene in the sul2-strA'-dfrA14-'strA-strB gene arrangement, harbored in a native 6779-bp plasmid. The same plasmid was isolated by transforming with a ~4.2 MDa plasmid extracted from several TMP(R) S. sonnei strains into Escherichia coli. This plasmid, named pABC-3, was present only in dfrA14-positive strains and was homologous to a previously described pCERC-1, but different due to the absence of an 11-bp repetitive unit. The distribution of dfrA1, dfrA8, and dfrA14 TMP-resistance genes was determined in 126 TMP(R) S. sonnei isolates. Most of the strains (96%) carried only one of the three TMP-resistance genes assessed. Thus, all strains obtained during the 2009-outbreak harbored only dfrA14, whereas, dfrA8 was the most abundant gene marker before outbreak and, after the outbreak dfrA1 seems have appeared in circulating strains. According to PFGE, dfrA14-positive strains were clustered in a genetically related group including some dfrA1- and dfrA8-positive strains; meanwhile other genetic group included most of the dfrA8-positive strains. This distribution also correlated with the isolation period, showing a dynamics of trimethoprim genetic markers prevalent in Chilean S. sonnei strains. To our knowledge, dfrA14 gene associated to a small non-conjugative plasmid was detected for the first time in Shigella. Apparently, the strain causing the outbreak must have been introduced, changing drastically the genetic distribution of trimethoprim resistance in Chilean S. sonnei strains.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is the predominant causative agent of hemorrhagic colitis in humans and is the cause of haemolytic uraemic syndrome and other illnesses. Cattle have been implicated as the main reservoir of this organism. Here, we evaluated the immunogenicity and protective efficacy of a DNA vaccine encoding conserved sequences of truncated EHEC factor for adherence-1 (efa-1') in a mouse model. Intranasal administration of plasmid DNA carrying the efa-1' gene (pVAXefa-1') into C57BL/6 mice elicited both humoral and cellular immune responses. In animals immunized with pVAXefa-1', EHEC-secreted protein-specific IgM and IgG antibodies were detected in sera at day 45. Anti-EHEC-secreted protein sIgA was also detected in nasal and bronchoalveolar lavages. In addition, antigen-specific T-cell-proliferation, IL-10, and IFN-γ were observed upon re-stimulation with either heat-killed bacteria or EHEC-secreted proteins. Vaccinated animals were also protected against challenge with E. coli O157:H7 strain EDL933. These results suggest that DNA vaccine encoding efa-1' have therapeutic potential in interventions against EHEC infections. This approach could lead to a new strategy in the production of vaccines that prevent infections in cattle.

Background: Osteoarthritis is the most prevalent joint disease and a common cause of joint pain, functional loss, and disability. Conventional treatments demonstrate only modest clinical benefits without lesion reversal. Autologous mesenchymal stromal cell (MSC) treatments have shown feasibility, safety, and strong indications for clinical efficacy. We performed a randomized, active control trial to assess the feasibility and safety of treating osteoarthritis with allogeneic MSCs, and we obtain information regarding the efficacy of this treatment., Methods: We randomized 30 patients with chronic knee pain unresponsive to conservative treatments and showing radiological evidence of osteoarthritis into 2 groups of 15 patients. The test group was treated with allogeneic bone marrow MSCs by intra-articular injection of 40 × 10(6) cells. The control group received intra-articular hyaluronic acid (60 mg, single dose). Clinical outcomes were followed for 1 year and included evaluations of pain, disability, and quality of life. Articular cartilage quality was assessed by quantitative magnetic resonance imaging T2 mapping., Results: Feasibility and safety were confirmed and indications of clinical efficacy were identified. The MSC-treated patients displayed significant improvement in algofunctional indices versus the active controls treated with hyaluronic acid. Quantification of cartilage quality by T2 relaxation measurements showed a significant decrease in poor cartilage areas, with cartilage quality improvements in MSC-treated patients., Conclusions: Allogeneic MSC therapy may be a valid alternative for the treatment of chronic knee osteoarthritis that is more logistically convenient than autologous MSC treatment. The intervention is simple, does not require surgery, provides pain relief, and significantly improves cartilage quality.

Enterotoxigenic Escherichia coli (ETEC), a leading cause of acute diarrhea, colonizes the intestine by means of adhesins. However, 15 to 50% of clinical isolates are negative for known adhesins, making it difficult to identify antigens for broad-coverage vaccines. The ETEC strain 1766a, obtained from a child with watery diarrhea in Chile, harbors the colonization factor CS23 but is negative for other known adhesins. One clone, derived from an ETEC 1766a genomic library (clone G10), did not produce CS23 yet was capable of adhering to Caco-2 cells. The goal of this study was to identify the gene responsible for this capacity. Random transposon-based mutagenesis allowed the identification of a 4,110-bp gene that codes for a homologue of the temperature-sensitive hemagglutinin (Tsh) autotransporter described in avian E. coli strains (97% identity, 90% coverage) and that is called TleA (Tsh-like ETEC autotransporter) herein. An isogenic ETEC 1766a strain with a tleA mutation showed an adhesion level similar to that of the wild-type strain, suggesting that the gene does not direct attachment to Caco-2 cells. However, expression of tleA conferred the capacity for adherence to nonadherent E. coli HB101. This effect coincided with the detection of TleA on the surface of nonpermeabilized bacteria, while, conversely, ETEC 1766a seems to secrete most of the produced autotransporter to the medium. On the other hand, TleA was capable of degrading bovine submaxillary mucin and leukocyte surface glycoproteins CD45 and P-selectin glycoprotein ligand 1 (PSGL-1). These results suggest that TleA promotes colonization of the intestinal epithelium and that it may modulate the host immune response., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Serogroup W Neisseria meningitidis was the main cause of invasive meningococcal disease in Chile during 2012. The case-fatality rate for this disease was higher than in previous years. Genotyping of meningococci isolated from case-patients identified the hypervirulent lineage W:P1.5,2:ST-11, which contained allele 22 of the fHbp gene.

Efforts to develop vaccines for prevention of acute diarrhea have been going on for more than 40 y with partial success. The myriad of pathogens, more than 20, that have been identified as a cause of acute diarrhea throughout the years pose a significant challenge for selecting and further developing the most relevant vaccine candidates. Based on pathogen distribution as identified in epidemiological studies performed mostly in low-resource countries, rotavirus, Cryptosporidium, Shigella, diarrheogenic E. coli and V. cholerae are predominant, and thus the main targets for vaccine development and implementation. Vaccination against norovirus is most relevant in middle/high-income countries and possibly in resource-deprived countries, pending a more precise characterization of disease impact. Only a few licensed vaccines are currently available, of which rotavirus vaccines have been the most outstanding in demonstrating a significant impact in a short time period. This is a comprehensive review, divided into 2 articles, of nearly 50 vaccine candidates against the most relevant viral and bacterial pathogens that cause acute gastroenteritis. In order to facilitate reading, sections for each pathogen are organized as follows: i) a discussion of the main epidemiological and pathogenic features; and ii) a discussion of vaccines based on their stage of development, moving from current licensed vaccines to vaccines in advanced stage of development (in phase IIb or III trials) to vaccines in early stages of clinical development (in phase I/II) or preclinical development in animal models. In this first article we discuss rotavirus, norovirus and Vibrio cholerae. In the following article we will discuss Shigella, Salmonella (non-typhoidal), diarrheogenic E. coli (enterotoxigenic and enterohemorragic), and Campylobacter jejuni.

In Part II we discuss the following bacterial pathogens: Shigella, Salmonella (non-typhoidal), diarrheogenic E. coli (enterotoxigenic and enterohemorragic) and Campylobacter jejuni. In contrast to the enteric viruses and Vibrio cholerae discussed in Part I of this series, for the bacterial pathogens described here there is only one licensed vaccine, developed primarily for Vibrio cholerae and which provides moderate protection against enterotoxigenic E. coli (ETEC) (Dukoral(®)), as well as a few additional candidates in advanced stages of development for ETEC and one candidate for Shigella spp. Numerous vaccine candidates in earlier stages of development are discussed.

Shiga-toxin producing Escherichia coli (STEC) is the etiologic agent of acute diarrhea, dysentery, and hemolytic-uremic syndrome (HUS). There is no approved vaccine for STEC infection in humans, and antibiotic use is contraindicated, as it promotes Shiga toxin production. In order to identify STEC-associated antigens and immunogenic proteins, outer membrane proteins (OMPs) were extracted from STEC O26:H11, O103, O113:H21, and O157:H7 strains, and commensal E. coli strain HS was used as a control. SDS-PAGE, two-dimensional-PAGE analysis, Western blot assays using sera from pediatric HUS patients and controls, and matrix-assisted laser desorption ionization-tandem time of flight analyses were used to identify 12 immunogenic OMPs, some of which were not reactive with control sera. Importantly, seven of these proteins have not been previously reported to be immunogenic in STEC strains. Among these seven proteins, OmpT and Cah displayed IgG and IgA reactivity with sera from HUS patients. Genes encoding these two proteins were present in a majority of STEC strains. Knowledge of the antigens produced during infection of the host and the immune response to those antigens will be important for future vaccine development., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea, mainly in developing countries. Although there are 25 different ETEC adhesins described in strains affecting humans, between 15% and 50% of the clinical isolates from different geographical regions are negative for these adhesins, suggesting that additional unidentified adhesion determinants might be present. Here, we report the discovery of Coli Surface Antigen 23 (CS23), a novel adhesin expressed by an ETEC serogroup O4 strain (ETEC 1766a), which was negative for the previously known ETEC adhesins, albeit it has the ability to adhere to Caco-2 cells. CS23 is encoded by an 8.8-kb locus which contains 9 open reading frames (ORFs), 7 of them sharing significant identity with genes required for assembly of K88-related fimbriae. This gene locus, named aal (adhesion-associated locus), is required for the adhesion ability of ETEC 1766a and was able to confer this adhesive phenotype to a nonadherent E. coli HB101 strain. The CS23 major structural subunit, AalE, shares limited identity with known pilin proteins, and it is more closely related to the CS13 pilin protein CshE, carried by human ETEC strains. Our data indicate that CS23 is a new member of the diverse adhesin repertoire used by ETEC strains.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea. Three adhesins (Tia, TibA, EtpA), an iron acquisition system (Irp1, Irp2, and FyuA), a GTPase (LeoA), and an autotransporter (EatA) are ETEC virulence-related proteins that, in contrast to the classical virulence factors (enterotoxins and fimbrial colonization factors) have not heretofore been targets in characterizing isolates from epidemiological studies. Here, we determined the occurrence of these nonclassical virulence genes in 103 ETEC isolates from Chilean children with diarrhea and described their association with O serogroups and classical virulence determinants. Because tia, leoA, irp2, and fyuA are harbored by pathogenicity islands inserted into the selC and asnT tRNA genes (tDNAs), we analyzed the regions flanking these loci. Ten additional tDNAs were also screened to identify hot spots for genetic insertions. Associations between the most frequent serogroups and classical colonization factor (CF)-toxin profiles included O6/LT-STh/CS1-CS3-CS21 (i.e., O6 serogroup, heat-labile [LT] and human heat-stable [STh] enterotoxins, and CFs CS1, -3 and -21), O6/LT-STh/CS2-CS3-CS21, and O104-O127/STh/CFAI-CS21. The eatA and etpA genes were detected in more than 70% of the collection, including diverse serogroups and virulence profiles. Sixteen percent of the ETEC strains were negative for classical and nonclassical adhesins, suggesting the presence of unknown determinants of adhesion. The leuX, thrW, and asnT tDNAs were disrupted in more than 65% of strains, suggesting they are hot spots for the insertion of mobile elements. Sequences similar to integrase genes were identified next to the thrW, asnT, pheV, and selC tDNAs. We propose that the eatA and etpA genes should be included in characterizations of ETEC isolates in future epidemiological studies to determine their prevalence in other geographical regions. Sequencing of tDNA-associated genetic insertions might identify new ETEC virulence determinants.

Context: The natural process of luteolysis and luteal regression is induced by withdrawal of gonadotropin support., Objective: The objectives of this study were: 1) to compare the functional changes and apoptotic features of natural human luteal regression and induced luteal regression; 2) to define the ultrastructural characteristics of the corpus luteum at the time of natural luteal regression and induced luteal regression; and 3) to examine the effect of human chorionic gonadotropin (hCG) on the steroidogenic response and apoptotic markers within the regressing corpus luteum., Design: Twenty-three women with normal menstrual cycles undergoing tubal ligation donated corpus luteum at specific stages in the luteal phase. Some women received a GnRH antagonist prior to collection of corpus luteum, others received an injection of hCG with or without prior treatment with a GnRH antagonist., Main Outcome Measure: Main outcome measures were plasma hormone levels and analysis of excised luteal tissue for markers of apoptosis, histology, and ultrastructure., Results: The progesterone and estradiol levels, corpus luteum DNA, and protein contents in induced luteal regression resembled those of natural luteal regression. hCG treatment raised progesterone and estradiol in both natural luteal regression and induced luteal regression. The increase in apoptosis detected in induced luteal regression by cytochrome c in the cytosol, activated caspase-3, and nuclear DNA fragmentation, was similar to that observed in natural luteal regression. The antiapoptotic protein Bcl-2 was significantly lower during natural luteal regression. The proapoptotic proteins Bax and Bak were at a constant level. Apoptotic and nonapoptotic death of luteal cells was observed in natural luteal regression and induced luteal regression at the ultrastructural level. hCG prevented apoptotic cell death, but not autophagy., Conclusion: The low number of apoptotic cells disclosed and the frequent autophagocytic suggest that multiple mechanisms are involved in cell death at luteal regression. hCG restores steroidogenic function and restrains the apoptotic process, but not autophagy.