The first demonstration of conjugal plasmid transfer from Escherichia coli to Bartonella henselae is reported. Transconjugants bearing plasmids of incompatibility groups P (IncP) and Q (IncQ), expressing various resistance markers, were generated. Tn5 transposons delivered on suicide plasmids by conjugation showed transpositional insertion into random chromosomal sites.Bartonella (Rochalimaea) henselae is a newly recognized fastidious gram-negative bacillus (5, 14) that has been associated with a number of human diseases, including bacillary angiomatosis, parenchymal bacillary peliosis, cat scratch disease, and persisting bacteremia (for a review see reference 1). Bacterial virulence determinants involved in the pathogenesis of these manifestations may include factors that stimulate endothelial-cell migration and proliferation in vitro (6) and phasevariable type 4-like pili mediating adherence to and invasion of cultured epithelial cells (2). The development of tools for the genetic manipulation of B. henselae would aid the study of these and other potential virulence factors. To this end, we have established conjugal plasmid transfer from Escherichia coli to B. henselae and used this process to determine (i) functional origins of replication and antibiotic resistance genes useful for plasmid maintenance and (ii) appropriate transposon delivery systems based on suicide vectors useful for transposon mutagenesis in B. henselae.The bacterial strains and plasmids used in this study are depicted in Table 1. E. coli strains were grown at 37ЊC in Luria-Bertani broth supplemented with 1 mM diaminopimelic acid (DAP) and appropriate antibiotics. B. henselae strains were cultivated at 37ЊC and 5% CO 2 on Columbia agar supplemented with 5% defibrinated sheep blood (CSB agar) and appropriate antibiotics. Conditions for conjugal plasmid transfer were established by using E. coli ␤2155(pWB5) as the donor and B. henselae 49882Rif as a recipient. The IncP plasmid derivative pWB5 confers resistances to kanamycin (Km r ) and tetracycline (Tc r ) and is mobilized in trans by transfer functions of plasmid RK4 integrated in the chromosome of ␤2155. Growth of the dapA mutant ␤2155 is strictly dependent on exogenously supplied DAP; hence, the removal of DAP provides an efficient counterselection against this donor. The rifampin resistance (Rif r ) (80 g/ml) of recipient 49882Rif, a spontaneous Rif r mutant of B. henselae ATCC 49882, provides further means for counterselection against the E. coli donor.Matings were done by mixing phosphate-buffered salinewashed bacteria, corresponding to 0.1 ml of a stationary-phase broth culture of the donor with the bacterial content of a plate of the recipient, which had been grown for 3 days. The mixture, in approximately 0.1 ml of phosphate-buffered saline, was spotted onto a nitrocellulose filter disc (Millipore; 0.22-m pore size), placed on CSB agar containing 1 mM DAP, and incubated for 8 h at 37ЊC and 5% CO 2 . Bacteria were washed from the filter, and dilutions were plated on CSB agar containi...

During episodes of dental bacteremia, viridans group streptococci encounter platelets. Among these microorganisms, certain Streptococcus sanguis induce human and rabbit platelets to aggregate in vitro. In experimental rabbits, circulating streptococci induced platelets to aggregate, triggering the accumulation of platelets and fibrin into the heart valve vegetations of endocarditis. At necropsy, affected rabbit hearts showed ischemie areas. We therefore hypothesized that circulating S. sanguis might cause coronary thrombosis and signs of myocardial infarction (MI). Signs of MI were monitored in rabbits after infusion with platelet‐aggregating doses of 4 to 40 × 109 cells of S. sanguis 133‐79. Infusion resulted in dose‐dependent changes in electrocardiograms, blood pressure, heart rate, and cardiac contractility. These changes were consistent with the occurrence of MI. Platelets isolated from hyperlipidemic rabbits showed an accelerated in vitro aggregation response to strain 133‐79. Cultured from immunosuppressed children with septic shock and signs of disseminated intravascular coagulation, more than 60% of isolates of viridans streptococci induced platelet aggregation when tested in vitro. The data are consistent with a thrombogenic role for S. sanguis in human disease, contributing to the development of the vegetative lesion in infective endocarditis and a thrombotic mechanism to explain the additional contributed risk of periodontitis to MI. J Periodontol 1996;67:1138–1142.

A strain of Streptococcus sanguis that induced rabbit platelets to aggregate in vitro (Agg+ phenotype) was hypothesized to be a more virulent pathogen than an Aggstrain in experimental endocarditis in rabbits. A left ventricular catheter was implanted, and then an Agg+ or Aggstrain was inoculated intravenously. Vegetations formed on the aortic semilunar valves but were unaffected by the duration of implantation of the catheter. Vegetations enlarged by accumulating platelets and their mass increased directly with the duration of endocarditis. Inoculation of the Agg+ strain consistently caused endocarditis with significantly larger vegetations, a more severe clinical course (including febrile episodes, hematological changes, and signs of myocardial ischemia), more gross lesions in major organs, and greater mortality than inoculation with the Agg-strain, saline, or the Agg+ strain pretreated with monospecific rabbit immunoglobulin G or Fab fragments against its platelet aggregation-associated protein (PAAP; class II). In experimental endocarditis, PAAP expressed by Agg+ S. sanguis appeared to be an important virulence factor.INFECT. IMMUN. M.on July 10, 2020 by guest http://iai.asm.org/ Downloaded from

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