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We investigated the general level of antibiotic resistance with further analysis of extended-spectrum beta-lactamase (ESBL) prevalence, as well as the population structure of E. coli in fecal flora of humans and Franklin's gulls (Leucophaeus pipixcan) in central parts of Chile. We found a surprisingly high carriage rate of ESBL-producing E. coli among the gulls 112/372 (30.1%) as compared to the human population 6/49 (12.2%.) Several of the E. coli sequence types (STs) identified in birds have previously been reported as Multi Drug Resistant (MDR) human pathogens including the ability to produce ESBLs. This means that not only commensal flora is shared between birds and humans but also STs with pathogenic potential. Given the migratory behavior of Franklin's gulls, they and other migratory species, may be a part of ESBL dissemination in the environment and over great geographic distances. Apart from keeping the antibiotic use low, breaking the transmission chains between the environment and humans must be a priority to hinder the dissemination of resistance.

With focus on environmental dissemination of antibiotic resistance among clinically relevant bacteria, such as the rising ESBL type of resistance among Escherichia coli, we investigated antibiotic resistance levels in wild birds in the Commander Islands and Kamchatka, Russia. Despite overall low resistance levels in randomly selected E. coli (one from each sample), we found multi-resistant ESBL-producing E. coli harbouring bla(CTX-M-14) and bla(CTX-M-15) using selective screening. Among these multi-resistant ESBL-producing E. coli we found one bla(CTX-M-15) harbouring strain belonging to the O25b-ST131 clone, recognized for its clonal disseminated worldwide as a human pathogen. The potential in acquiring resistant bacteria of human origin, especially highly pathogenic clones, as well as downstream consequences of that, should not be underestimated but further investigated.

Extended Spectrum beta-Lactamase (ESBL) producing Enterobacteriaceae started to appear in the 1980s, and have since emerged as some of the most significant hospital-acquired infections with Escherichia coli and Klebsiella being main players. More than 100 different ESBL types have been described, the most widespread being the CTX-M beta-lactamase enzymes (bla(CTX-M) genes). This study focuses on the zoonotic dissemination of ESBL bacteria, mainly CTX-M type, in the southern coastal region of France. We found that the level of general antibiotic resistance in single randomly selected E. coli isolates from wild Yellow-legged Gulls in France was high. Nearly half the isolates (47.1%) carried resistance to one or more antibiotics (in a panel of six antibiotics), and resistance to tetracycline, ampicillin and streptomycin was most widespread. In an ESBL selective screen, 9.4% of the gulls carried ESBL producing bacteria and notably, 6% of the gulls carried bacteria harboring CTX-M-1 group of ESBL enzymes, a recently introduced and yet the most common clinical CTX-M group in France. Multi locus sequence type and phylogenetic group designations were established for the ESBL isolates, revealing that birds and humans share E. coli populations. Several ESBL producing E. coli isolated from birds were identical to or clustered with isolates with human origin. Hence, wild birds pick up E. coli of human origin, and with human resistance traits, and may accordingly also act as an environmental reservoir and melting pot of bacterial resistance with a potential to re-infect human populations.

OBJECTIVES: Alterations in the composition of the microbiota in the intestine may promote development of celiac disease (CD). Using scanning electron microscopy (SEM) we previously demonstrated that rod-shaped bacteria were present on the epithelium of proximal small intestine in children with CD but not in controls. In this study we characterize the microbiota of proximal small intestine in children with CD and controls and identify CD-associated rod-shaped bacteria. METHODS: Proximal small intestine biopsies from 45 children with CD and 18 clinical controls were studied. Bacteria were identified by 16S rDNA sequencing in DNA extracted from biopsies washed with buffer containing dithiothreitol to enrich bacteria adhering to the epithelial lining, by culture-based methods and by SEM and transmission electron microscopy. RESULTS: The normal, mucosa-associated microbiota of proximal small intestine was limited. It was dominated by the genera Streptococcus and Neisseria, and also contained Veillonella, Gemella, Actinomyces, Rothia, and Haemophilus. The proximal small intestine microbiota in biopsies from CD patients collected during 2004-2007 differed only marginally from that of controls, and only one biopsy (4%) had rod-shaped bacteria by SEM (SEM+). In nine frozen SEM+ CD biopsies from the previous study, microbiotas were significantly enriched in Clostridium, Prevotella, and Actinomyces compared with SEM- biopsies. Bacteria of all three genera were isolated from children born during the Swedish CD epidemic. New Clostridium and Prevotella species and Actinomyces graevenitzii were tentatively identified. CONCLUSIONS: Rod-shaped bacteria, probably of the indicated species, constituted a significant fraction of the proximal small intestine microbiota in children born during the Swedish CD epidemic and may have been an important risk factor for CD contributing to the fourfold increase in disease incidence in children below 2 years of age during that time.

Salivary proline-rich proteins (PRPs) attach commensal Actinomyces and Streptococcus species to teeth. Here, gel filtration, mass spectrometry and Edman degradation were applied to show the release of a pentapeptide, RGRPQ, from PRP-1 upon proteolysis by Streptococcus gordonii. Moreover, synthetic RGRPQ and derivatives were used to investigate associated innate properties and responsible motifs. The RGRPQ peptide increased 2.5-fold the growth rate of S. gordonii via a Q-dependent sequence motif, and selectively stimulated oral colonization of this organism in a rat model in vivo. By contrast, growth of Streptococcus mutans, implicated in caries, was unaffected. While the entire RGRPQ sequence was required to block sucrose-induced pH-decrease by S. gordonii and S. mutans, the N-terminal Arg residue mediated pH-increase (i.e. ammonia production) by S. gordonii alone (which exhibits Arg catabolism to ammonia). Strains of commensal viridans streptococci exhibited PRP degradation and Arg catabolism, while cariogenic species did not. The RGRPQ peptide mediated via a differential Q-dependent sequence motif, adhesion inhibition and desorption of PRP-1-binding strains of A. naeslundii genospecies 2 (5 out of 10 strains) but not of S. gordonii (n=5). The inhibitable A. naeslundii strains alone displayed the same binding profile as S. gordonii to hybrid peptides terminating in RGRPQ or GQSPQ, derived from the middle or C-terminal segments of PRP-1. The present findings indicate the presence of a host-bacteria interaction where a host peptide released by bacterial proteolysis affects key properties in biofilm formation.

Salivary proline-rich proteins (PRPs) attach commensal Actinomyces and Streptococcus species to teeth. Here, gel filtration, mass spectrometry and Edman degradation were applied to show the release of a pentapeptide, RGRPQ, from PRP-1 upon proteolysis by Streptococcus gordond. Moreover, synthetic RGRPQ and derivatives were used to investigate associated innate properties and responsible motifs. The RGRPQ peptide increased 2.5-fold the growth rate of S. gordonii via a Q-dependent sequence motif and selectively stimulated oral colonization of this organism in a rat model in vivo. In contrast, the growth of Streptococcus mutans, implicated in caries, was not affected. While the entire RGRPQ sequence was required to block sucrose-induced pH-decrease by S. gordonii and S. mutans, the N-terminal Arg residue mediated the pH increase (i.e., ammonia production) by S. gordonii alone (which exhibits Arg catabolism to ammonia). Strains of commensal viridans streptococci exhibited PR-P degradation and Arg catabolism, whereas cariogenic species did not. The RGRPQ peptide mediated via a differential Q-dependent sequence motif, adhesion inhibition, and desorption of PRP-1-binding strains of A. naeslundii genospecies 2 (5 of 10 strains) but not of S. gordonii (n = 5). The inhibitable A. naeslundii strains alone displayed the same binding profile as S. gordond to hybrid peptides terminating in RGRPQ or GQSPQ, derived from the middle or C-terminal segments of PRP-1. The present findings indicate the presence of a host-bacterium interaction in which a host peptide released by bacterial proteolysis affects key properties in biofilm formation.

Streptococcus gordonii is a commensal bacterium that colonizes the hard and soft tissues present in the human mouth and nasopharynx. The cell wall-anchored polypeptides SspA and SspB expressed by S. gordonii mediate a wide range of interactions with host proteins and other bacteria. In this article we have determined the role of SspA and SspB proteins, which are members of the streptococcal antigen I/II (AgI/II) adhesin family, in S. gordonii adherence and internalization by epithelial cells. Wild-type S. gordonii DL1 expressing AgI/II polypeptides attached to and was internalized by HEp-2 cells, whereas an isogenic AgI/II(-) mutant was reduced in adherence and was not internalized. Association of S. gordonii DL1 with HEp-2 cells triggered protein tyrosine phosphorylation but no significant actin rearrangement. By contrast, Streptococcus pyogenes A40 showed 50-fold higher levels of internalization and this was associated with actin polymerization and interleukin-8 upregulation. Adherence and internalization of S. gordonii by HEp-2 cells involved beta1 integrin recognition but was not fibronectin-dependent. Recombinant SspA and SspB polypeptides bound to purified human alpha5beta1 integrin through sequences present within the NAV (N-terminal) region of AgI/II polypeptide. AgI/II polypeptides blocked interactions of S. gordonii and S. pyogenes with HEp-2 cells, and S. gordonii DL1 cells expressing AgI/II proteins inhibited adherence and internalization of S. pyogenes by HEp-2 cells. Conversely, S. gordonii AgI/II(-) mutant cells did not inhibit internalization of S. pyogenes. The results suggest that AgI/II proteins not only promote integrin-mediated internalization of oral commensal streptococci by host cells, but also potentially influence susceptibility of host tissues to more pathogenic bacteria.

Adhesion of bacteria is a key event in biofilm formation and is mediated by bacterial adhesins recognising host or bacterial partner receptors. In oral biofilm formation, primary Actinomyces and Streptococcus colonizers adhere to salivary pellicle proteins such as proline-rich proteins (PRPs) as well as to mucosal surfaces. Subsequently, Actinomyces and Streptococcus strains and other bacteria, such as Veillonella, Fusobacterium and Porphyromonas, adhere to each other. The nature of this community is highly important for the health or disease status, although specific pathogenic species may also have been implicated.

The aim of this thesis was to study key players in early oral colonisation, Actinomyces and Streptococcus species, and more specifically the nature of their adhesins and ligands. A further aim was to study the function of the salivary PRP proteins and an innate peptide derived thereof on bacterial adhesion, proliferation and regulation of pH, i.e. key factors in biofilm formation.

In paper I and II, adhesion, proliferation and pH affecting features of the RGRPQ (arginine-glycine-arginine-proline-glutamine) peptide, derived from PRP-1, were demonstrated. By use of an alanine-scan (I), motifs for adhesion inhibition and desorption of Actinomyces naeslundii, and proliferation stimulation, ammonia production and inhibition of sucrose induced pH drop by Streptococcus gordonii were indicated. The RGRPQ peptide also stimulated S. gordonii colonisation in vivo. In paper II, a more sophisticated quantitative structure-activity relationship (QSAR) study, using statistical molecular design (SMD) and multivariate modelling (partial least squares projections to latent structures, PLS), further narrowed down the RGRPQ peptide motifs. The R and Q amino acids were crucial for activity. For proliferation a hydrophobic and large size third position amino acid was crucial, while adhesion inhibition and desorption needed a small hydrophilic second position amino acid. All functions depended on a low polarity hydrophobic fourth position. Accordingly, activities could be optimized separately, with decreased function in the others.

In paper III and IV, focus was on the bacterial adhesins and their binding epitopes. The genes for FimA major subunit proteins of type-2 fimbriae were sequenced from A. naeslundii genospecies 1 and 2 and Actinomyces odontolyticus, each with unique carbohydrate binding specificities (III). Three major subtypes of FimA proteins were found that correlated with binding specificity, including a novel fimA gene in A. odontolyticus. All subtypes contained a pilin, LPXTG and E box motif. In paper IV, multiple PRP binding patterns for Actinomyces and Streptococcus strains were mapped using a hybrid peptide construct. The two most deviating binding groups deviated in type-1 fimbriae mediated binding to milk and saliva protein ligands.

In conclusion, differences in bacterial adhesins and their ability to utilise salivary proteins may render bacteria tropism for different niches. Peptides derived from protein receptors, such as RGRPQ, may be important modulators of biofilm formation, giving commensal bacteria a competitive edge in the bacterial community.

Salivary proline-rich proteins (PRPs) attach commensal Actinomyces and Streptococcus species to teeth. Here, gel filtration, mass spectrometry and Edman degradation were applied to show the release of a pentapeptide, RGRPQ, from PRP-1 upon proteolysis by Streptococcus gordonii. Moreover, synthetic RGRPQ and derivatives were used to investigate associated innate properties and responsible motifs. The RGRPQ peptide increased 2.5-fold the growth rate of S. gordonii via a Q-dependent sequence motif and selectively stimulated oral colonization of this organism in a rat model in vivo. In contrast, the growth of Streptococcus mutans, implicated in caries, was not affected. While the entire RGRPQ sequence was required to block sucrose-induced pH-decrease by S. gordonii and S. mutans, the N-terminal Arg residue mediated the pH increase (i.e., ammonia production) by S. gordonii alone (which exhibits Arg catabolism to ammonia). Strains of commensal viridans streptococci exhibited PRP degradation and Arg catabolism, whereas cariogenic species did not. The RGRPQ peptide mediated via a differential Q-dependent sequence motif, adhesion inhibition, and desorption of PRP-1-binding strains of A. naeslundii genospecies 2 (5 of 10 strains) but not of S. gordonii (n=5). The inhibitable A. naeslundii strains alone displayed the same binding profile as S. gordonii to hybrid peptides terminating in RGRPQ or GQSPQ, derived from the middle or C-terminal segments of PRP-1. The present findings indicate the presence of a host-bacterium interaction in which a host peptide released by bacterial proteolysis affects key properties in biofilm formation.

Oral commensal Streptococcus gordonii proteolytically cleave the salivary PRP-1 polypeptide into an RGRPQ innate peptide. The Arg and Gln termini are crucial for RGRPQ-mediated ammonia production and proliferation by S. gordonii SK12 and adhesion inhibition and desorption by Actinomyces naeslundii T14V, respectively. Here we have applied (i) a multivariate approach using RGRPQ-related peptides varied at amino acids 2, 3, and 4 simultaneously and (ii) size and N- and C-terminal modifications of RGRPQ to generate structure activity information. While the N-terminal arginine motif mediated ammonia production independent of peptide size, other responses required more or less full-length peptide motifs. The motifs for adhesion inhibition and desorption were the same. The adhesion and proliferation motifs required similarly a hydrophobic/low polarity amino acid 4 but differentially a hydrophilic or hydrophobic character of amino acids 2/3, respectively; polar peptides with small/hydrophilic and hydrophilic amino acids 2 and 3, respectively, had high adhesion inhibition/desorption activity, and lipophilic peptides with large/hydrophobic amino acids 2 and 3 had high proliferation activity. Accordingly, while RIWWQ had increased proliferation but abolished adhesion/desorption activity, peptides designed with hydrophilic amino acids 2 and 3 were predicted to behave in the opposite way. Moreover, a RGRPQ mimetic for all three responses should mimic small hydrophilic, large nitrogen-containing, and hydrophobic/low polarity amino acids 2, 3, and 4, respectively. Peptides fulfilling these criteria were 1-1.6-fold improved in all three responses. Thus, both mimetics and peptides with differential proliferation and adhesion activities may be generated for evaluation in biofilm models.