Ali O. Kiliç

Genetic diversity of vaginal lactobacilli from women in different countries based on 16S rRNA gene sequences

Aims: Lactobacilli are widely distributed in food and the environment, and some colonize the human body as commensal bacteria. The aim of this study was to determine the species of lactobacilli that colonize the vagina and compare them with those found in food and the environment.

Streptococcus gordonii utilizes several distinct gene functions to recruit Porphyromonas gingivalis into a mixed community

Dental plaque biofilm formation proceeds through a developmental pathway initiated by the attachment of pioneer organisms, such as Streptococcus gordonii, to tooth surfaces. Through a variety of synergistic interactions, pioneer organisms facilitate the colonization of later arrivals including Porphyromonas gingivalis, a potential periodontal pathogen. We have investigated genes of S. gordonii required to support a heterotypic biofilm community with P. gingivalis. By screening a plasmid integration library of S. gordonii, genes were identified that are crucial for the accumulation of planktonic P. gingivalis cells into a multispecies biofilm. These genes were further investigated by specific mutation and complementation analyses. The biofilm‐associated genes can be grouped into broad categories based on putative function as follows: (i) intercellular or intracellular signalling (cbe and spxB), (ii) cell wall integrity and maintenance of adhesive proteins (murE, msrA and atf), (iii) extracellular capsule biosynthesis (pgsA and atf), and (iv) physiology (gdhA, ccmA and ntpB). In addition, a gene for a hypothetical protein was identified. Biofilm visualization and quantification by confocal microscopy confirmed the role of these genes in the maturation of the multispecies community, including biofilm architectural development. The results suggest that S. gordonii governs the development of heterotypic oral biofilms through multiple genetic pathways.

Comparative study of vaginal Lactobacillus phages isolated from women in the United States and Turkey: Prevalence, morphology, host range, and DNA homology

ABSTRACT Lactobacilli play an important role in maintaining vaginal health. However, during bacterial vaginosis lactobacilli decrease for unknown reasons. Our preliminary study showed that phages could infect vaginal lactobacilli. Therefore, the aim of this study was to analyze the distribution, virulence, and types of vaginal Lactobacillusphages isolated from women of two countries: the United States and Turkey. A total of 209 vaginal lactobacilli were isolated from reproductive-aged women in the United States (n = 107) and Turkey (n = 102). By analysis of 16S rRNA gene sequence and by comparison of protein profiles, most lactobacilli were identified as L. crispatus, L. gasseri, andL. jensenii. After mitomycin C induction, 28% of American lactobacilli and 36% of Turkish lactobacilli released phages. A total of 67 phages were isolated and further characterized by their host range, electron microscopy, and DNA homology. All 67 phages were infective against lactobacilli from both collections. The host ranges of most phages were broad, including multiple Lactobacillusspecies. Even though the phages were all temperate, they were able to cause lytic infection in various strains. The electron micrographs of these phages showed a hexagon-shaped head and a long tail with or without a contractile tail sheath. Based on their morphology, these phages belonged to Bradleys phage groups A and B, and could be further classified into four morphotypes. All four types were found among American phages, but only three were found among Turkish isolates. DNA hybridization with labeled probes of the four types of phages revealed that additional genetic types existed within each morphotype among these phages. The phage genomic sizes ranged between 34 and 55 kb. Many of the lysogenic Lactobacillus strains released phages spontaneously at a high frequency of 10−3to 10−4 PFU/cell. In conclusion, lysogeny in vaginal lactobacilli is widely spread. Some lysogenic lactobacilli spontaneously release phages with a broad host range, which can be lytic against other vaginal lactobacilli regardless of their geographic origin.

Involvement of Streptococcus gordonii Beta-Glucoside Metabolism Systems in Adhesion, Biofilm Formation, and In Vivo Gene Expression

Streptococcus gordonii genes involved in beta-glucoside metabolism are induced in vivo on infected heart valves during experimental endocarditis and in vitro during biofilm formation on saliva-coated hydroxyapatite (sHA). To determine the roles of beta-glucoside metabolism systems in biofilm formation, the loci of these induced genes were analyzed. To confirm the function of genes in each locus, strains were constructed with gene inactivation, deletion, and/or reporter gene fusions. Four novel systems responsible for beta-glucoside metabolism were identified, including three phosphoenolpyruvate-dependent phosphotransferase systems (PTS) and a binding protein-dependent sugar uptake system for metabolizing multiple sugars, including beta-glucosides. Utilization of arbutin and esculin, aryl-beta-glucosides, was defective in some mutants. Esculin and oligochitosaccharides induced genes in one of the three beta-glucoside metabolism PTS and in four other genetic loci. Mutation of genes in any of the four systems affected in vitro adhesion to sHA, biofilm formation on plastic surfaces, and/or growth rate in liquid medium. Therefore, genes associated with beta-glucoside metabolism may regulate S. gordonii in vitro adhesion, biofilm formation, growth, and in vivo colonization.

Oral Streptococci and Cardiovascular Disease: Searching for the Platelet Aggregation-Associated Protein Gene and Mechanisms of Streptococcus sanguis-Induced Thrombosis

BACKGROUND Pathogenic mechanisms in infective endocarditis, disseminated intravascular coagulation, and cardiovascular events involve the aggregation of platelets into thrombi. Attendant infection by oral bacteria contributes to these diseases. We have been studying how certain oral streptococci induce platelet aggregation in vitro and in vivo. Streptococcus sanguis expresses a platelet aggregation-associated protein (PAAP), which contributes little to adhesion to platelets. When specific antibodies or peptides block PAAP, S. sanguis fails to induce platelet aggregation in vitro or in vivo. METHODS We used subtractive hybridization to identify the gene encoding for PAAP. RESULTS After subtraction of strain L50 (platelet aggregation-negative), four strain 133-79 specific sequences were characterized. Sequence agg4 encoded a putative collagen-binding protein (CbpA), which was predicted to contain two PAAP collagen-like octapeptide sequences. S. sanguis CbpA- mutants were constructed and tested for induction of platelet aggregation in vitro. Platelet aggregation was substantially inhibited when compared to the wild-type using platelet-rich plasma from the principal donor, but adhesion was unaffected. Other donor platelets responded normally to the CbpA- strain, suggesting additional mechanisms of response to S. sanguis. In contrast, CshA- and methionine sulfoxide reductase-negative (MsrA-) strains neither adhered nor induced platelet aggregation. CONCLUSIONS CbpA was suggested to contribute to site 2 interactions in our two-site model of platelet aggregation in response to S. sanguis. Platelet polymorphisms were suggested to contribute to the thrombogenic potential of S. sanguis.

Host-pathogen interactions in bacterial endocarditis : streptococcal virulence in the host

To identify streptococcal genes that are expressed during experimental endocarditis, we developed a promoter-less dual reporter gene-fusion (amy, cat) plasmid, pAK36. Chromosomal DNA from S. gordonii V288 was digested with Sau3Al. The resulting fragments were ligated into pAK36. Following transformation into S. gordonii, the library of random gene fusion clones was inoculated into a rabbit to induce experimental endocarditis. Chloramphenicol treatment effected positive selection. Upon euthanization of the rabbits, the valvular vegetations were excised in a sterile field. Surviving clones were isolated and screened in vitro for chloramphenicol sensitivity and negative amylase activity. From the 48 randomly picked, double-negative clones. DNA was isolated and analyzed by Southern hybridization with labeled pAK36 probe. Different insertion patterns were identified, suggesting that no fewer than 13 S. gordonii genes were induced. Therefore, S. gordonii genes are induced during experimental endocarditis, which may contribute to virulence.

Characterization of a cryptic plasmid from Lactobacillus fermentum KC5b and its use for constructing a stable Lactobacillus cloning vector

Lactobacillus fermentum KC5b, a strain originally isolated from the human vagina, contains a cryptic plasmid pKC5b. The sequence and genetic organization of the 4392-bp plasmid were determined. It contains two convergently oriented replicons, which are homologous to each other and to the stable replicon of the Enterococcus faecium plasmid pMBB1. The two replicons of pKC5b were used either individually or together to construct Lactobacillus-Escherichia coli shuttle plasmids. Only the plasmid pSP1 that carried both replicons transformed lactobacilli, suggesting a complementary function between the two replicons. Since the replicons had a high homology to those of other plasmids that replicate via a theta-like mechanism and no detectable single-stranded intermediates were found for the plasmid, it is possible that pKC5b may replicate via a theta-like mechanism. The new shuttle plasmid pSP1 has been transformed and stably maintained in several Lactobacillus strains. As an initial application, pSP1 was used to clone the S-layer protein gene (slpA) of Lactobacillus acidophilus ATCC 4356 into a heterologous vaginal Lactobacillus strain and achieved surface-bound expression of the protein.

Analysis of lactobacillus products for phages and bacteriocins that inhibit vaginal lactobacilli.

Objective: Bacterial vaginosis is associated with an unexplained loss of vaginal lactobacilli. Previously, we have identified certain vaginal lactobacilli-released phages that can inhibit in vitro other vaginal lactobacilli. However, there is no apparent route for phages to be transmitted among women. The purpose of this study was to identify whether certain Lactobacillus products commonly used by women release phages or bacteriocins that can inhibit vaginal lactobacilli. Methods: From 26 Lactobacillus products (2 acidophilus milks, 20 yogurts, 3 Lactobacillus pills, and 1 vaginal douche mix), lactobacilli were isolated with Rogosa SL agar (Difco, Detroit, MI). From these lactobacilli, phages and bacteriocins were induced with mitomycin C and tested against a collection of vaginal Lactobacillus strains. Results: From the 26 products, 43 Lactobacillus strains were isolated. Strains from 11 yogurts released phages, among which 7 inhibited vaginal lactobacilli. Eleven strains released bacteriocins that inhibited vaginal lactobacilli. While about one-half of the vaginal strains were lysed by bacteriocins, less than 20% were lysed by phages. Conclusions: Some vaginal lactobacilli were inhibited in vitro by phages or bacteriocins released from Lactobacillus products used by women, implying that vaginal lactobacilli may be reduced naturally due to phages or bacteriocins from the environment.

Horizontol dissemination of TEM- and SHV-typr beta-lactamase genes-carrying resistance plasmids amongst clonical isolates of Enterobacteriaceae.

The extended-spectrum β-lactamase (ESBL)-producing bacteria have been isolated at increasing frequency worldwide. Expression of ESBL is often associated with multidrug resistance and dissemination by resistance plasmids. During a two-month period in 2000, 133 clinical isolates of enterobacterial strains were randomly collected from outpatients and inpatients at a university hospital in Turkey. The ESBL producing strains were determined by double-disk synergy (DDS) testing. Twenty ESBL producing strains (15%) including Escherichia coli (n = 9), Klebsiella pneumoniae (n = 7), Klebsiella oxytoca (n = 2) and Enterobacter aerogenes (n = 2) were detected and further analyzed for their resistance transfer features, plasmid profile and nature of the resistance genes. Plasmid transfer assays were performed using broth mating techniques. TEM- and SHV- genes were analyzed by polymerase chain reaction (PCR) and hybridization using specific probes. EcoRI restriction enzyme analyses of R plasmids were used in the detection of epidemic plasmids. Fourteen plasmid profiles (A, B1, B2, C1, and C2 to L) were obtained with EcoRI restriction enzyme analysis. Most of these plasmids were detected to carry both TEM- and SHV-derived genes by PCR, and confirmed by localizing each gene by hybridization assay. Epidemiological evidence indicated that there was an apparent horizontal dissemination of conjugative R plasmids among multidrug-resistant enterobacterial genera and species in this hospital.