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C-di-GMP signaling can directly influence bacterial behavior by affecting the functionality of c-di-GMP-binding proteins. In addition, c-di-GMP can exert a global effect on gene transcription or translation, for example, via riboswitches or by binding to transcription factors. In this study, we investigated the effects of changes in intracellular c-di-GMP levels on gene expression and protein production in the opportunistic pathogen Pseudomonas aeruginosa. We induced c-di-GMP production via an ectopically introduced diguanylate cyclase and recorded the transcriptional, translational as well as proteomic profile of the cells. We demonstrate that rising levels of c-di-GMP under growth conditions otherwise characterized by low c-di-GMP levels caused a switch to a non-motile, auto-aggregative P. aeruginosa phenotype. This phenotypic switch became apparent before any c-di-GMP-dependent role on transcription, translation, or protein abundance was observed. Our results suggest that rising global c-di-GMP pools first affects the motility phenotype of P. aeruginosa by altering protein functionality and only then global gene transcription.

Capsule polymers are crucial virulence factors of pathogenic bacteria and are used as antigens in glycoconjugate vaccine formulations. Some Gram-negative pathogens express poly(glycosylglycerol phosphate) capsule polymers that resemble Gram-positive wall teichoic acids and are synthesized by TagF-like capsule polymerases. So far, the biotechnological use of these enzymes for vaccine developmental studies was restricted by the unavailability of enantiopure CDP-glycerol, one of the donor substrates required for polymer assembly. Here, we use CTP:glycerol-phosphate cytidylyltransferases (GCTs) and TagF-like polymerases to synthesize the poly(glycosylglycerol phosphate) capsule polymer backbones of the porcine pathogen Actinobacillus pleuropneumoniae, serotypes 3 and 7 (App3 and App7). GCT activity was confirmed by high-performance liquid chromatography, and polymers were analyzed using comprehensive nuclear magnetic resonance studies. Solid-phase synthesis protocols were established to allow potential scale-up of polymer production. In addition, one-pot reactions exploiting glycerol-kinase allowed us to start the reaction from inexpensive, widely available substrates. Finally, this study highlights that multidomain TagF-like polymerases can be transformed by mutagenesis of active site residues into single-action transferases, which in turn can act in trans to build-up structurally new polymers. Overall, our protocols provide enantiopure, nature-identical capsule polymer backbones from App2, App3, App7, App9, and App11, Neisseria meningitidis serogroup H, and Bibersteinia trehalosi serotypes T3 and T15. IMPORTANCE Economic synthesis platforms for the production of animal vaccines could help reduce the overuse and misuse of antibiotics in animal husbandry, which contributes greatly to the increase of antibiotic resistance. Here, we describe a highly versatile, easy-to-use mix-and-match toolbox for the generation of glycerol-phosphate-containing capsule polymers that can serve as antigens in glycoconjugate vaccines against Actinobacillus pleuropneumoniae and Bibersteinia trehalosi, two pathogens causing considerable economic loss in the swine, sheep, and cattle industries. We have established scalable protocols for the exploitation of a versatile enzymatic cascade with modular architecture, starting with the preparative-scale production of enantiopure CDP-glycerol, a precursor for a multitude of bacterial surface structures. Thereby, our approach not only allows the synthesis of capsule polymers but might also be exploitable for the (chemo)enzymatic synthesis of other glycerol-phosphate-containing structures such as Gram-positive wall teichoic acids or lipoteichoic acids.

Syphilis is often called the great imitator because of its frequent atypical clinical manifestations that make the disease difficult to recognize. Because Treponema pallidum subsp. pallidum, the infectious agent of syphilis, is yet uncultivated in vitro, diagnosis is usually made using serology; however, in cases where serology is inconclusive or in patients with immunosuppression where these tests may be difficult to interpret, the availability of a molecular tool for direct diagnosis may be of pivotal importance. Here we present a fluorescence in situ hybridization (FISH) assay that simultaneously identifies and analyzes spatial distribution of T. pallidum in histological tissue sections. For this assay the species-specific FISH probe TPALL targeting the 16S rRNA of T. pallidum was designed in silico and evaluated using T. pallidum infected rabbit testicular tissue and a panel of non-syphilis spirochetes as positive and negative controls, respectively, before application to samples from four syphilis-patients. In a HIV positive patient, FISH showed the presence of T. pallidum in inguinal lymph node tissue. In a patient not suspected to suffer from syphilis but underwent surgery for phimosis, numerous T. pallidum cells were found in preputial tissue. In two cases with oral involvement, FISH was able to differentiate T. pallidum from oral treponemes and showed infection of the oral mucosa and tonsils, respectively. The TPALL FISH probe is now readily available for in situ identification of T. pallidum in selected clinical samples as well as T. pallidum research applications and animal models.

The objective was to compare the antibacterial effects of adjunctive disinfection using diode laser and gaseous ozone compared to the medical dressings calcium hydroxide (Ca(OH)2) and chlorhexidine gel (CHX-Gel) onEnterococcus faecalisbiofilms in human root canals ex vivo. Root canals of 180 human extracted teeth were infected byE. faecalisand divided into 3 main groups (G): G1, control; G2, instrumentation and irrigation using 0.9% NaCl; G3, instrumentation and irrigation using 1% NaOCl. In each main group, the following treatments were applied: gaseous ozone, diode laser, and medical dressings of Ca(OH)2or CHX-Gel for 7 days (n=15). Reduction of colony forming units (CFUs) inside the root canal of planktons and frequencies of adherent bacteria after treatment were calculated. Bacterial reduction was significantly affected by the irrigation protocol (p<0.0005) and the disinfection method (p<0.0005), and a significant interaction between both factors could be observed (p<0.0005; ANOVA). In G3 (instrumentation using 1% NaOCl), no significant effect of disinfection methods could be demonstrated on planktonic bacteria (p=0.062; ANOVA) and frequencies of adherent bacteria (p>0.05; chi-square test). Instrumentation and irrigation using NaOCl combined with ozone or laser application resulted in comparable bacterial reduction onE. faecalisto the application of medical dressings.

The relevance of microorganisms in preterm birth is still under discussion. Using a diagnostic fluorescence in situ hybridization probe panel, we visualized Staphylococcus aureus and Streptococcus mitis group in two cases of acute chorioamnionitis. This technique provides spatial resolution and quantity of bacteria, clarifying the epidemiology and pathogenic pathways of acute chorioamnionitis.

During six years as German National Consultant Laboratory for Spirochetes we investigated 149 intestinal biopsies from 91 patients, which were histopathologically diagnosed with human intestinal spirochetosis (HIS), using fluorescence in situ hybridization (FISH) combined with 16S rRNA gene PCR and sequencing. Aim of this study was to complement histopathological findings with FISH and PCR for definite diagnosis and species identification of the causative pathogens. HIS is characterized by colonization of the colonic mucosa of the human distal intestinal tract by Brachyspira spp. Microbiological diagnosis of HIS is not performed, because of the fastidious nature and slow growth of Brachyspira spp. in culture. In clinical practice, diagnosis of HIS relies solely on histopathology without differentiation of the spirochetes. We used a previously described FISH probe to detect and identify Brachyspira spp. in histological gut biopsies. FISH allowed rapid visualization and identification of Brachyspira spp. in 77 patients. In most cases, the bright FISH signal already allowed rapid localization of Brachyspira spp. at 400× magnification. By sequencing, 53 cases could be assigned to the B. aalborgi lineage including “B. ibaraki” and “B. hominis”, and 23 cases to B. pilosicoli. One case showed mixed colonization. The cases reported here reaffirm all major HIS Brachyspira spp. clusters already described. However, the phylogenetic diversity seems to be even greater than previously reported. In 14 cases, we could not confirm HIS by either FISH or PCR, but found colonization of the epithelium by rods and cocci, indicating misdiagnosis by histopathology. FISH in combination with molecular identification by 16S rRNA gene sequencing has proved to be a valuable addition to histopathology. It provides definite diagnosis of HIS and allows insights into phylogeny and distribution of Brachyspira spp. HIS should be considered as a differential diagnosis in diarrhea of unknown origin, particularly in patients from risk groups (e.g. patients with colonic adenomas, inflammatory polyps, inflammatory bowel disease or HIV infection and in men who have sex with men).