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Serologic responses to somatic O and colonization-factor antigens of enterotoxigenic Escherichia coli in travelers.

To improve the retrospective diagnoses of enterotoxigenic Escherichia coli (ETEC) as a cause of travelers' diarrhea, as well as to determine the presence of colonization-factor antigens in these infections, a study of serologic responses to antigens of ETEC was done. Paired sera from 60 United States students in Cholula, Puebla, Mexico, were analyzed for rises in titer of antibody to heat-labile toxin, eight somatic antigen O serogroups associated with ETEC, and two colonization-factor antigens, CFA/I and CFA/II. Only 9% had a response to O antigens, while 20% had responses to the colonization-factor antigens. Response to the colonization-factor antigens correlated significantly with response to the heat-labile toxin and with culture evidence of ETEC infection. Serologic studies confirmed that colonization-factor antigen has a role in naturally acquired cases of travelers' diarrhea and that it can be used as an additional determinant of infection with ETEC.

Animals

New surface-associated heat-labile colonization factor antigen (CFA/II) produced by enterotoxigenic Escherichia coli of serogroups O6 and O8.

Enterotoxigenic Escherichia coli (ETEC) belonging to serogroups O6 and O8 do not possess the H-10407-type colonization factor antigen (CFA/I). However, these frequently isolated ETEC were found to possess a second and distinct heat-labile surface-associated colonization factor antigen, termed CFA/II. Whereas CFA/I mediates mannose-resistant hemagglutination of human group A erythrocytes, CFA/II does not. CFA/II mediates mannose-resistant hemagglutination of bovine erythrocytes, and mannose-resistant hemagglutination is rapid only at reduced temperature (4 degrees C). Because CFA/II, like CFA/I, is spontaneously lost by many ETEC isolates in the laboratory, it was possible to produce specific anti-CFA/II serum by preparing antiserum against living cells of a prototype strain (PB-176) and adsorbing this serum with living and heat-treated cells of its CFA/II-negative derivative strain PB-176-P. This serum, which neutralized the colonization factor activity of CFA/II-positive strains in infant rabbits, was employed to confirm the presence of CFA/II on ETEC which exhibited mannose-resistant hemagglutination of bovine but not human erythrocytes. CFA/II, like CFA/I, mediates adherence of the bacteria to the mucosal surface of the small intestine, as demonstrated by indirect immunofluorescence. CFA/II appears to be an important virulence factor for humans since CFA/II-positive ETEC are frequently isolated from diarrhea cases, particularly travelers' diarrhea, in Mexico; these ETEC were not uncommon in a collection of isolates from Bangladesh. The O6:H16 strain of ETEC responsible for an outbreak of diarrhea in the United States was also shown to be CFA/II positive. CFA/I and CFA/II were never found on the same serotypes of ETEC, but 98% of the heat-stable and heat-labile enterotoxin-producing ETEC belonging to the frequently isolated serogroups O6, O8, O15, O25, O63, and O78 were positive for either CFA/I or CFA/II.

Animals

Genome-resolved analysis of colonization factor repertoires reveals ecological stratification in cervid gut microbiomes.

INTRODUCTION: Colonization factors (CFs) are important microbial traits associated with persistence and host adaptation in the gut, yet their large-scale organization in cervid gut microbiomes remains unclear. METHODS: A total of 3,311 non-redundant high-quality metagenome-assembled genomes (MAGs), derived from 688 cervid gut metagenomic samples across 15 publicly available projects and one in-house dataset, were analyzed. CF-associated genes were identified by comparison against the GHA CF database, and CF repertoires were characterized at genome, host-species, and gastrointestinal-segment levels. RESULTS: A total of 138,729 CF-associated genes spanning 71 CF families were identified. MAGs from Cervinae contained richer CF repertoires than those from Caprinae, and CF47 (Peptidase_C69), CF24_29 (QueH), and CF18 (Glycos_transf_2) were among the most prevalent families. CF repertoires were strongly structured by taxonomy, showed a moderate association with bacterial phylogenetic distance, and formed two recurrent genome-level configurations with distinct KEGG functional profiles. Integration of sample metadata further revealed differentiation of CF repertoires across host species and gastrointestinal segments, representing the major ecological dimensions examined in this study. Segment-associated CF variation was accompanied by redistribution of broader functional profiles, including enrichment of carbohydrate and lipid metabolism in the jejunum, membrane transport in the ileum, xenobiotics biodegradation in the cecum, and environmental adaptation in the rumen. DISCUSSION: These findings provide a genome-resolved view of CF repertoire organization in cervid gut microbiomes and demonstrate that colonization-associated functions are structured across microbial lineages and ecological contexts. This study highlights the importance of considering microbial taxonomy and host-associated environments when interpreting the distribution of CF repertoires in mammalian gut ecosystems.

Cervidae

Genomic insights into a diarrheal outbreak in Bangladesh reveal novel ETEC lineages and expansion of CS23 colonization factor.

Enterotoxigenic Escherichia coli (ETEC), a leading cause of diarrhea, is defined by heat-stable (ST) and/or heat-labile (LT) toxins and associated colonization factors (CFs). However, there is still a knowledge gap in understanding ETEC's evolution, particularly in endemic regions like Bangladesh. This study investigates the genomic attributes contributing to the rise of ETEC-associated diarrhea in Bangladesh during 2022-2023. Whole genome sequencing of 325 ETEC isolates (2022-2023), compared with historical strains (1980-2021), revealed significant evolutionary changes. Our findings showed a significant shift in ETEC toxin from LT to ST over the period 2013-2023. The most frequent virulence profile during this period was CFA/I + CS21 compared with previous years (1980-2021). The emergence of CS23-positive ETEC was reported for the first time in Bangladesh, which was considered a less common CF in previous studies. Notably, we report the four novel lineages "L26-L29" in this study through phylogenetic analysis, partly encompassing emergent CS23-positive ETEC strains. Additionally, the high prevalence of multi-drug-resistant ETEC strains and the presence of ESBL-CTX-M-resistant gene during 2022-2023 are a matter of great concern, underscoring the need for preventive measures. The switch of distinct toxin and CF combinations, the rapid emergence of CS23, ESBL-CTX-M resistance, and novel lineages may be the reason behind the increased number of ETEC diarrheal cases between 2022 and 2023. These findings highlight the rapid ETEC evolution that underscores the necessity of continued genomic surveillance to track ongoing changes.IMPORTANCEThis study expands on previous evidence, demonstrating a remarkable genomic diversity in ETEC strains from 2022 to 2023, particularly in virulence factors and AMR genes. The combined findings from these studies will be important for mitigating future diarrheal outbreaks by informing preventive measures, including future vaccine targets, and implementing antibiotic stewardship programs against ETEC infection. Importantly, this research underscores the necessity of continued genomic surveillance to track ongoing changes in ETEC. Such monitoring is essential for understanding the pathogen's evolving population structure, transmission dynamics, and resistance mechanisms.

Bangladesh

Detection and characterization of colonization factor of enterotoxigenic Escherichia coli isolated from adults with diarrhea.

The fimbriate colonization factor antigen (CEA) of Escherichia coli strain H-1047 was isolated and used to prepare anti-CFA antiserum. Enterotoxigenic E. coli (ETEC) isolated from 29 adults with diarrhea acquired in Mexico were examined for CFA by using this serum. Retrospectively, it was found that ETEC possessing the H-10407-type CFA were isolated from 25 (86%) of these diarrhea cases as compared with 2 of 11 (18%) from asymptomatic controls from whom ETEC had been isolated. CFA was found onE. coli of various serotypes, as demonstrated by bacterial agglutination by the anti-CFA serum. Heat treating the cells at 65 degress C for 1 h prevented the agglutination. CFA-positive strains did not react with anti-CFA serum when the cultures were grown at a low incubation temperature (18 degrees C). E. coli isolates identified serologically as CFA positive were shown to adhere to the intestinal villous surfaces of infant rabbits. By the indirect immunofluorescence technique, it was found that adhesion occurred preferentially in the upper 20 cm of the small intestine. Also, the ability or inability of various isolates to adhere to intestinal mucosa in vivo correlated with the presence or absence of fimbriae on the cells when grown in vitro. Agglutinability with anti-CFA serum, fimbriae, and adhesiveness were spontaneously lost by many isolates after laboratory passage in a manner previously described with E. coli H-10407. These observations suggest that the H-10407-type CFA plays a role in the virulence of ETEC possessing this antigen.

Adhesiveness

Hemagglutination of human group A erythrocytes by enterotoxigenic Escherichia coli isolated from adults with diarrhea: correlation with colonization factor.

Enterotoxigenic Escherichia coli (ETEC) of several different serotypes isolated from adults with diarrhea and known to possess the colonization factor antigen (CFA) were found to cause mannose-resistant hemagglutination (HA) of human group A erythrocytes. CFA-negative E. coli isolated during the same study did not possess the mannose-resistant hemagglutinin, although some non-ETEC, CFA-negative isolates did exhibit mannose-sensitive HA activity. The mannoseresistant hemagglutinin of ETEC was found to possess many characteristics previously associated with CFA, which is a surface-associated fimbriate heatlabile antigen, and the functionally and morphologically similar K88 and K99 antigens of animal-specific ETEC. Mannose-resistant HA and CFA titers were maximal when ETEC cells were grown on an agar medium (CFA agar) composed primarily of 1% Casamino Acids and 0.15% yeast extract, pH 7.4. Neither CFA nor HA were produced at a growth temperature of 18 degrees C; HA was completely inhibited by pretreatment of CFA-positive cells with the anti-CFA serum. The mannose-resistant hemagglutinin was lost spontaneously and simultaneously with CFA when clinical ETEC isolates were passaged on artificial medium in the laboratory, indicating plasmid control of both entities. The mannose-resistant hemagglutinin of ETEC was shown to be thermolabile, i.e., sensitive to heating at 65 degrees C, as was the CFA. Also, there was correlation between possession of CFA, as detected serologically and by demonstration of biological activity (adherence in the infant rabbit small intestine), presence of CFA-type fimbriae, and the ability of various E. coli isolates to cause mannose-resistant HA of human group A erythrocytes. These results indicate that the mannose-resistant HA of ETEC is another manifestation of CFA.

Agglutinins

Differences in serological responses and excretion patterns of volunteers challenged with enterotoxigenic Escherichia coli with and without the colonization factor antigen.

Double-blind studies were performed to compare the virulence of enterotoxigenic Escherichia coli with and without the fimbriate colonization factor antigen (CFA), using young healthy adults (mean age, 23 years) as volunteers. In the first study one group of volunteers ingested 1 X 10(6) E. coli H-10407, the CFA-positive strain, and another group ingested 1 X 10(6) E. coli H-10407-P, the CFA-negative spontaneous derivative of strain H-10407. The second study was similar except that the test strains were administered at a dose of 1 X 10(8) viable cells. Three parameters of infection were monitored: (i) diarrhea and associated symptoms; (ii) excretion pattern of test strains; and (iii) humoral antibody response to CFA, somatic antigen, and heat-labile enterotoxin. Significant signs of illness occurred only in six of seven volunteers who ingested E. coli H-10407 at a dose of 1 X 10(8). At both doses, E. coli H-10407-P appeared in the stool on day 1 postchallenge and disappeared by day 4. In contrast, strain H-10407 was persistently excreted from the first to the last day of the study. Also, only those volunteers in the H-10407 challenge groups (12 of 13 analyzed) responded with a fourfold antibody titer rise to CFA, somatic antigen, and/or heat-labile enterotoxin. No reversion of H-10407-P to H-10407 was detected.

Adolescent

Special O:K:H serotypes among enterotoxigenic E. coli strains from diarrhea in adults and children. Occurrence of the CF (colonization factor) antigen and of hemagglutinating abilities.

O:H serotypes previously found to be prevalent among a number of toxigenic strains from several geographic areas were examined for polysaccharide K antigen type. Members of each O:H serotype had the same type of K antigen and were found to be characterized by a certain fermentation pattern. Some O:H serotypes had no K antigen. The serofermentative types defined were: 06:K15:H16, 08:K40:H9, 015:H11, 025:K7:H42, 025:K98:H-, 078:H11, 078:H12, and 0149:H10. Some strains of the last-mentioned serotype, which were suspected of having caused a food-borne infection, had K88. This serotype belongs to the group of strains causing diarrhea in swine. The surface antigen (CF) described as a colonization factor [5] was demonstrated in 078:H-, 078:H11, and 078:H12 strains; but not in any strain of the other serotypes nor in any of 248 strains belonging to 078 but not isolated from cases of human diarrhea. Presence of the CF antigen was correlated with presence of a mannose-resistant ability to cause agglutination of human red cells. Behavior of the other serotypes as regards hemagglutinating abilities was examined and 025:K7:H42 strains were found to be very similar to the 078 strains in this respect.

ABO Blood-Group System

[Inhibition of Escherichia coli haemagglutinations by antibiotics].

Escherichia coli has been associated with a colonization factor antigen responsible for the bacterial adherence to the intestinal mucosa. This colonization factor is also responsible for haemagglutination. Among 32 antibiotics tested, only tetracyclines, chloramphenicol and nafcillin inhibit haemagglutination. Tetracycline hydrochloride, tetracycline phosphate and doxycycline are the most effective, followed by oxytetracycline, minocycline, chloramphenicol and nafcillin. Doxycline at a concentration at 0.8 mg/ml in buffered saline, chloramphenicol at 12.8 mg and nafcillin at 31 mg inhibit haemagglutination in 2 h. The inhibition of haemagglutination occurs both with E. coli susceptible and resistant to doxycycline. Pretreatment of crythrocytes with doxycycline does not prevent inhibition of haemagglutination where as pretreatment of E. coli does, suggesting that doxycycline acted on the bacteria. Once haemagglutination had occurred, doxycycline can reverse the haemagglutination, "unhooking" the bacteria from the erythrocytes. These data raise the possibility that some antibiotics may prevent adherence of E. coli to the intestinal mucosa, regardless of the susceptibility or resistance of the bacteria by a direct effect on the colonization factor antigen. Furthermore, some antibiotics may be able to detach the bacteria once adherence to the mucosa has occurred.

Anti-Bacterial Agents

Virulence factors of enterotoxigenic Escherichia coli.

Further evidence for the role of enterotoxigenic Escherichia coli as an etiologic agent of diarrhea is presented. A retrospective study of 71 cases of diarrhea in Mexican children demonstrated that greater than 40% of them harbored E. coli that produced heat-labile and/or heat-stable enterotoxin. The antigenic surface-associated colonization factor of E. coli strain H-10407 has been further characterized: this pilus-like antigen is produced under conditions of growth that repress the production of common pili of E. coli. The E. coli H-10407-type colonization factor pilus has been identified as one of the antigens possessed by a strain of E. coli that produced only heat-stable enterotoxin and that was responsible for an outbreak of pediatric diarrhea.

Antigens, Bacterial

Binding of lysozyme to common pili of Escherichia coli.

Common pili from Escherichia coli were found to bind hen egg white lysozyme. The binding was highly dependent on ionic strength, and the maximum binding occurred near an ionic strength of 0.02. The pili were aggregated by lysozyme, and this process could be followed by optical turbidity, electron microscopy, and coprecipitation. Near the maximum saturation of binding, one lysozyme molecule was bound by two pilus protein subunits. Electron micrographs of this aggregate indicated that they were paracrystalline structures. Piliated bacteria were more readily agglutinated by lysozyme than were nonpiliated bacteria. Since lysozyme is considered to be an antibacterial humoral factor and since pili are considered to be a colonization factor, the binding of lysozyme may represent an important bacterium-host interaction

Bacterial Proteins

Colonization of porcine small intestine by Escherichia coli: colonization and adhesion factors of pig enteropathogens that lack K88.

The colonizing and adhesive attributes of enterotoxigenic acapsular and/or nonpiliated mutants from K88-negative enteropathogenic Escherichia coli strains were compared with their capsulated and piliated parents (parents were piliated when grown in vitro and in vivo). Acapsular, nonpiliated mutants from three different colonizing strains of enteropathogenic E. coli lost their ability to colonize the ileum of newborn pigs. Acapsular, piliated and capsular, nonpiliated mutants were derived from one of the parental strains (987), and both mutants lacked the ability to colonize the ileum of pigs. The only mutants available from a fourth strain (431) were acapsular and piliated, and they colonized as well as their parents. These data indicate that both capsule and pili are involved in colonization by strain 987. In contrast, capsule is not required for colonization by strain 431, but pili may be.

Animals

Hemagglutination by Escherichia coli in septicemia and urinary tract infections.

The agglutination of erythrocytes from various animal species by Escherichia coli was studied. The 405 strains of E. coli were isolated from urine in patients with urinary tract infections, from blood in septicemic patients, or from feces in persons without intestinal or urinary disorders. In urinary tract infections, d-mannose-resistant agglutination (MRHA) of human erythrocytes was the most common finding (23% of the strains). The highest frequency of mannose-sensitive hemagglutination (MSHA) attributed to type I (common type) pili occurred with guinea pig erythrocytes (11.5%). Of the 78 E. coli strains isolated from blood cultures, 11 (14%) produced MRHA of human erythrocytes and only one gave MSHA. In the stool cultures, only 1 of 170 E. coli strains was MSHA reacting, whereas 28 strains (16.5%) showed MRHA of human erythrocytes. No MRHA strain reacted with antiserum against colonization factor antigen (CFA)/I of pilus nature in enterotoxigenic human E. coli strains (O78:H12). MRHA of bovine erythrocytes, reputedly typical of enterotoxigenic E. coli of serogroups O6 and O8, was shown by only two strains, neither of which agglutinated with CFA/II antiserum. The most common hemagglutinating pattern of E. coli from urine and blood thus was MRHA for human erythrocytes. This agglutination may have been caused by pili or other surface properties of one or more serotypes. These may represent a new class of colonization-promoting antigens (adhesins).

Animals

Genome-wide association analysis reveals specialization to hosts and niches in multiple species of the Lactobacillaceae.

The Lactobacillaceae inhabit diverse environments, but the extent of their habitat adaptation remains unclear and the colonization factors unknown. First, we applied multiple machine learning models to determine if we can distinguish strains of the same species isolated from two different habitats based on their gene content. Surprisingly, we show that no species is differentially adapted to the oral cavity versus the human gut, or food versus the human gut, while only Lactobacillus crispatus showed specialization to the human urogenital system versus human gut. We then asked which species of Lactobacillaceae are habitat-specialized and how they could be identified. Using multiple lifestyle predictors incorporated in logistic regression models, we found that Limosilactobacillus reuteri, Ligilactobacillus ruminis, L. salivarius, L. crispatus, and L. mucosae displayed the highest degrees of host specialization. Applying our microbial genome-wide association study tool, aurora, to these species identified genes encoding adhesins and bacteriocins as the strongest and most common adaptation factors. This work establishes a generalizable framework for identifying novel species-habitat pairs with strong evidence of specialization and for uncovering the genomic features underlying within-species host and habitat adaptation.

Humans