Due to the spread of resistance, antibiotic exposure receives increasing attention. Ecological consequences for the different niches of individual microbiomes are, however, largely ignored. Here, we report the effects of widely used antibiotics (clindamycin, ciprofloxacin, amoxicillin, and minocycline) with different modes of action on the ecology of both the gut and the oral microbiomes in 66 healthy adults from the United Kingdom and Sweden in a two-center randomized placebo-controlled clinical trial. Feces and saliva were collected at baseline, immediately after exposure, and 1, 2, 4, and 12 months after administration of antibiotics or placebo. Sequences of 16S rRNA gene amplicons from all samples and metagenomic shotgun sequences from selected baseline and post-antibiotic-treatment sample pairs were analyzed. Additionally, metagenomic predictions based on 16S rRNA gene amplicon data were performed using PICRUSt. The salivary microbiome was found to be significantly more robust, whereas the antibiotics negatively affected the fecal microbiome: in particular, health-associated butyrate-producing species became strongly underrepresented. Additionally, exposure to different antibiotics enriched genes associated with antibiotic resistance. In conclusion, healthy individuals, exposed to a single antibiotic treatment, undergo considerable microbial shifts and enrichment in antibiotic resistance in their feces, while their salivary microbiome composition remains unexpectedly stable. The health-related consequences for the gut microbiome should increase the awareness of the individual risks involved with antibiotic use, especially in a (diseased) population with an already dysregulated microbiome. On the other hand, understanding the mechanisms behind the resilience of the oral microbiome toward ecological collapse might prove useful in combating microbial dysbiosis elsewhere in the body.

Escherichia coli is usually a non-pathogenic member of the human colonic flora. However, certain strains have acquired virulence factors and may cause a variety of infections in humans and in animals. There are three clinical syndromes caused by E. coli: (i) sepsis/meningitis; (ii) urinary tract infection and (iii) diarrhoea. Furthermore the E. coli causing diarrhoea is divided into different 'pathotypes' depending on the type of disease, i.e. (i) enterotoxigenic; (ii) enteropathogenic; (iii) enteroinvasive; (iv) enterohaemorrhagic; (v) enteroaggregative and (vi) diffusely adherent. The serotyping of E. coli based on the somatic (O), flagellar (H) and capsular polysaccharide antigens (K) is used in epidemiology. The different antigens may be unique for a particular serogroup or antigenic determinants may be shared, resulting in cross-reactions with other serogroups of E. coli or even with other members of the family Enterobacteriacea. To establish the uniqueness of a particular serogroup or to identify the presence of common epitopes, a database of the structures of O-antigenic polysaccharides has been created. The E. coli database (ECODAB) contains structures, nuclear magnetic resonance chemical shifts and to some extent cross-reactivity relationships. All fields are searchable. A ranking is produced based on similarity, which facilitates rapid identification of strains that are difficult to serotype (if known) based on classical agglutinating methods. In addition, results pertinent to the biosynthesis of the repeating units of O-antigens are discussed. The ECODAB is accessible to the scientific community at http://www.casper.organ.su.se/ECODAB/.

BackgroundThe brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines.Methodology/Principal FindingsTo examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies.Conclusions/SignificanceThe results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that rough vaccines are suitable for the control of brucellosis in endemic areas.

Diarrhea continues to be one of the most common causes of morbidity and mortality among infants and children in developing countries. Escherichia coli is an emerging agent among pathogens that cause diarrhea. The development of a highly applicable technique for the detection of different categories of diarrheagenic E. coli is important. We have used multiplex PCR by combining eight primer pairs specific for enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli, enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC). This facilitates the identification of five different categories of diarrheagenic E. coli from stool samples in a single reaction simultaneously. The prevalences of diarrheagenic E. coli were 22.5 and 12% in the diarrhea group and the control group, respectively. Among 587 fecal samples from Vietnamese children under 5 years of age with diarrhea, this technique identified 132 diarrheagenic E. coli strains. This included 68 samples (11.6%) with EAEC, 12 samples (2.0%) with EIEC, 39 samples (6.6%) with EPEC, and 13 samples (2.2%) with ETEC. Among the 249 age-matched controls, 30 samples were positive for diarrheagenic E. coli. The distribution was 18 samples (7.2%) with EAEC, 11 samples (4.4%) with EPEC, and 1 sample (0.4%) with ETEC.

The tailspike protein of Salmonella phage P22 is a viral adhesion protein with both receptor binding and destroying activities. It recognises the O-antigenic repeating units of cell surface lipopolysaccharide of serogroup A, B and D1 as receptor, but also inactivates its receptor by endoglycosidase (endorhamnosidase) activity. In the final step of bacteriophage P22 assembly six homotrimeric tailspike molecules are non-covalently attached to the DNA injection apparatus, mediated by their N-terminal, head-binding domains. We report the crystal structure of the head-binding domain of P22 tailspike protein at 2.3 A resolution, solved with a recombinant telluromethionine derivative and non-crystallographic symmetry averaging. The trimeric dome-like structure is formed by two perpendicular beta-sheets of five and three strands, respectively in each subunit and caps a three-helix bundle observed in the structure of the C-terminal receptor binding and cleaving fragment, reported here after full refinement at 1.56 A resolution. In the central part of the receptor binding fragment, three parallel beta-helices of 13 complete turns are associated side-by-side, while the three polypeptide strands merge into a single domain towards their C termini, with close interdigitation at the junction to the beta-helix part. Complex structures with receptor fragments from S. typhimurium, S. enteritidis and S. typhi253Ty determined at 1.8 A resolution are described in detail. Insertions into the beta-helix form the O-antigen binding groove, which also harbours the active site residues Asp392, Asp395 and Glu359. In the intact structure of the tailspike protein, head-binding and receptor-binding parts are probably linked by a flexible hinge whose function may be either to deal with shearing forces on the exposed, 150 A long tailspikes or to allow them to bend during the infection process.

The O-antigenic repeating units of lipopolysaccharides from Salmonella serogroups A, B, and DI serve as receptors for the phage P22 tailspike protein, which also has receptor destroying endoglycosidase (endorhamnosidase) activity, integrating the functions of both hemagglutinin and neuraminidase in influenza virus. Crystal structures of the tailspike protein in complex with oligosaccharides, comprising two 0-antigenic repeating units from Sabnonella typhiumurium, SalmoneUa enteritidis, and Salmonella typhi 253Ty were determined at 1.8 A resolution. The active-site topology with Asp-392, Asp-395, and Glu-359 as catalytic residues was identified. Kinetics of binding and cleavage suggest a role of the receptor destroying endorhamnosidase activity primarily for detachment of newly assembled phages.The infection of Salmonella by phage P22 starts with the recognition of the 0-antigenic repeating units of the cell surface lipopolysaccharide by the homotrimeric tailspike protein (TSP), which is present in six copies on the attachment apparatus (1, 2). Phages that use lipopolysaccharide as receptors are faced with the chemical diversity of the 0-antigenic repeats, which differ in carbohydrate composition and stereochemistry of the 0-glycosidic linkage. In addition, microheterogeneity conceming the number of repeating units and additional modifications as acetylation or glucosylation known as form variation is an important feature of lipopolysaccharide (3-5). P22 has adapted to Salmonella serotypes A, B, and Dl ( Fig. 1), sharing a common trisaccharide repeating unit a-Dmannose-(1,4)-a-L-rhamnose-(1,3)-a-D-galactose for the 0-antigen but differing in their branching carbohydrate moieties, a 3,6-dideoxyhexose a-(1,3)-linked to D-mannose. Dideoxyhexoses are paratose (serogroup A), abequose (serotype B), or tyvelose (serotype D1) and reflect the correlation of serotype classification and chemical structure of the 0-antigenic repeats of Salmonella (6, 7). In addition, P22 tolerates the 0-antigen 122, which shows an a-(1,4)-linked D-glucose at D-galactose as in Salmonella typhi 253Ty (8). In the phage-host interaction, the dideoxyhexose can be viewed as a wobble position that allows for some flexibility in a specific interaction.Receptor destroying enzymatic activities are well known for viruses that use carbohydrates as receptors. Influenza A and B virus and paramyxovirus have neuraminidases releasing terminal N-acetylmuraminic acid from glycoproteins and glycolipids (9), whereas influenza virus C and some coronaviruses that recognize an 0-acetylated sialic acid epitope have a sialate 9-0-acetylesterase, removing an acetyl group (10, 11). Endoglycosidase or acetylesterase activities have also been demonstrated for a large number of phages acting on encapsulated Gram-negative bacteria like Escherichia coli, Salmonella, or Klebsiella (12). TSP possesses receptor destroying endorhamnosidase activity cleaving the a(1,3)-O-glycosidic bond between rhamnose and galactose of the 0-antigenic repeats (13,14). To elucidate the s...

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Bacteriophage P22 binds to its cell surface receptor, the repetitive O-antigen structure in Salmonella lipopolysaccharide, by its six homotrimeric tailspikes. Receptor binding by soluble tailspikes and the receptor-inactivating endorhamnosidase activity of the tailspike protein were studied using octa- and dodecasaccharides comprising two and three O-antigen repeats of Salmonella enteritidis and Salmonella typhimurium lipopolysaccharides. Wild-type tailspike protein and three mutants (D392N, D395N, and E359Q) with defective endorhamnosidase activity were used. Oligosaccharide binding to all three subunits, measured by a tryptophan fluorescence quench or by fluorescence depolarization of a coumarin label attached to the reducing end of the dodecasaccharide, occurs independently. At 10 degrees C, the binding affinities of all four proteins to oligosaccharides from both bacterial strains are identical within experimental error, and the binding constants for octa- and dodecasaccharides are 1 x 10(6) M(-1) and 2 x 10(6) M(-1), proving that two O-antigen repeats are sufficient for lipopolysaccharide recognition by the tailspike. Equilibration with the oligosaccharides occurs rapidly, but the endorhamnosidase produces only one cleavage every 100 s at 10 degrees C or about 2 min(-1) at the bacterial growth temperature. Thus, movement of virions in the lipopolysaccharide layer before DNA injection may involve the release and rebinding of individual tailspikes rather than hydrolysis of the O-antigen.