Comparing genome-scale metabolic models of the non-resistant Enterococcus faecalis ATCC 19433 and the multi-resistant Enterococcus faecalis V583.

Enterococcus faecalis is a versatile lactic acid bacterium with a large variety of implications for humans. While some strains of this species are pathobionts being resistant against most of the common antibiotics, other strains are regarded as biological protectants or even probiotics. Accordingly, E. faecalis strains largely differ in the size and content of their accessory genome. In this study, we describe the genome-scale metabolic network reconstruction of E. faecalis ATCC 19433, a non-resistant human-associated strain. A comparison of the genome-scale metabolic model (GSM) of E. faecalis ATCC 19433 with a previously published GSM of the multi-resistant pathobiontic E. faecalis V583 reveals high similarities in the central metabolic abilities of these two human associated strains. This is reflected, e.g., in the identical amino acid auxotrophies. The ATCC 19433 strain, however, has a 14.1 % smaller genome than V583 and lacks the multiple antibiotic resistance genes and genes involved in capsule formation. Based on the measured metabolic fluxes at different growth rates, the energy demand at zero growth was calculated to be about 40 % lower for the ATCC 19433 strain compared to V583. Furthermore, the ATCC 19433 strain seems less prone to the depletion of amino acids utilizable for energy metabolism. This might hint at a lower overall energy demand of the ATCC 19433 strain as compared to V583.

Preparation and In Vitro Characterization of Lactococcus lactis-Loaded Alginate Particles as a Promising Delivery Tool for Periodontal Probiotic Therapy.

Probiotic microorganisms are used in a variety of food supplements and medical formulations to promote human health. In periodontal therapy, probiotics are mainly used in the form of gels, tablets or rinses that often tend to leak from the periodontal pocket, resulting in a strongly reduced therapeutic effect. In this pilot in vitro study, we present biodegradable alginate-based particles as an alternative, highly efficient system for a periodontal delivery of probiotic bacteria to the inflammation site. For this purpose, Lactococcus (L.) lactis was encapsulated using a standardized pump-controlled extrusion-dripping method. Time-dependent bacterial release in artificial saliva was investigated over 9 days. The effect of freeze drying was explored to ensure long-term storage of L. lactis-loaded particles. Additionally, the particles were bound to dentin surface using approved bioadhesives and subjected to shear stress in a hydrodynamic flow chamber that mimics the oral cavity in vitro. Thus, round particles within the range of 0.80-1.75 mm in radius could be produced, whereby the diameter of the dripping tip had the most significant impact on the size. Although both small and large particles demonstrated a similar release trend of L. lactis, the release rate was significantly higher in the former. Following lyophilization, particles could restore their original shape within 4 h in artificial saliva; thereby, the bacterial viability was not affected. The attachment strength to dentin intensified by an adhesive could resist forces between 10 and 25 N/m2. Full degradation of the particles was observed after 20 days in artificial saliva. Therefore, alginate particles display a valuable probiotic carrier for periodontal applications that have several crucial advantages over existing preparations: a highly stable form, prolonged continuous release of therapeutic bacteria, precise manufacturing according to required dimensions at the application site, strong attachment to the tooth with low risk of dislocation, high biocompatibility and biodegradability.

Liver cirrhosis and antibiotic therapy but not TIPS application leads to a shift of the intestinal bacterial communities: A controlled, prospective study.

OBJECTIVES: The gut-liver axis is discussed to play an important role in hepatic cirrhosis. Decompensated liver cirrhosis is associated with portal hypertension, which can lead to a variety of complications. Transjugular intrahepatic portosystemic shunt (TIPS) is an established treatment option for the complications of portal hypertension. In this study we focused on the effect of TIPS on intestinal microbial composition in cirrhotic patients. METHODS: Thirty patients with liver cirrhosis were compared to 18 healthy adults. Seventeen patients with cirrhosis and portal hypertension received a TIPS. Clinical characteristics, including age, sex, and liver function measured with a Child-Pugh score and model for end-stage liver disease score, were obtained. Intestinal microbial composition was assessed via 16S rRNA gene amplicon sequencing from stool probes before and after TIPS. RESULTS: TIPS led to a reduction of hepatic venous pressure gradient. However, TIPS did not cause a shift in the intestinal bacterial communities. Independent from the application of TIPS, antibiotic therapy was associated with a significant difference in the intestinal bacterial microbiota and also a reduced α-diversity. In addition, a significant difference was observed in the intestinal bacterial composition between patients with liver cirrhosis and healthy controls. CONCLUSION: The presence of liver cirrhosis and the use of antibiotic therapy, but not the application of TIPS, were associated with a significant shift of the intestinal bacterial communities, showing a high impact on the microbiota of patients with liver cirrhosis.

Synthetic mRNA delivered to human cells leads to expression of Cpl-1 bacteriophage-endolysin with activity against Streptococcus pneumoniae.

Endolysins are bacteriophage-encoded hydrolases that show high antibacterial activity and a narrow substrate spectrum. We hypothesize that an mRNA-based approach to endolysin therapy can overcome some challenges of conventional endolysin therapy, namely organ targeting and bioavailability. We show that synthetic mRNA applied to three human cell lines (HEK293T, A549, HepG2 cells) leads to expression and cytosolic accumulation of the Cpl-1 endolysin with activity against Streptococcus pneumoniae. Addition of a human lysozyme signal peptide sequence translocates the Cpl-1 to the endoplasmic reticulum leading to secretion (hlySP-sCpl-1). The pneumococcal killing effect of hlySP-sCpl-1 was enhanced by introduction of a point mutation to avoid N-linked-glycosylation. hlySP-sCpl-1N215D, collected from the culture supernatant of A549 cells 6 h post-transfection showed a significant killing effect and was active against nine pneumococcal strains. mRNA-based cytosolic Cpl-1 and secretory hlySP-sCpl-1N215D show potential for innovative treatment strategies against pneumococcal disease and, to our best knowledge, represent the first approach to mRNA-based endolysin therapy. We assume that many other bacterial pathogens could be targeted with this novel approach.

Analysis of FctB3 crystal structure and insight into its structural stabilization and pilin linkage mechanisms.

Streptococcus pyogenes harboring an FCT type 3 genomic region display pili composed of three types of pilins. In this study, the structure of the base pilin FctB from a serotype M3 strain (FctB3) was determined at 2.8 Å resolution. In accordance with the previously reported structure of FctB from a serotype T9 strain (FctB9), FctB3 was found to consist of an immunoglobulin-like domain and proline-rich tail region. Data obtained from structure comparison revealed main differences in the omega (Ω) loop structure and the proline-rich tail direction. In the Ω loop structure, a differential hydrogen bond network was observed, while the lysine residue responsible for linkage to growing pili was located at the same position in both structures, which indicated that switching of the hydrogen bond network in the Ω loop without changing the lysine position is advantageous for linkage to the backbone pilin FctA. The difference in direction of the proline-rich tail is potentially caused by a single residue located at the root of the proline-rich tail. Also, the FctB3 structure was found to be stabilized by intramolecular large hydrophobic interactions instead of an isopeptide bond. Comparisons of the FctB3 and FctA structures indicated that the FctA structure is more favorable for linkage to FctA. In addition, the heterodimer formation of FctB with Cpa or FctA was shown to be mediated by the putative chaperone SipA. Together, these findings provide an alternative FctB structure as well as insight into the interactions between pilin proteins.

Archaeome structure and function of the intestinal tract in healthy and H1N1 infected swine.

Background: Methanogenic archaea represent a less investigated and likely underestimated part of the intestinal tract microbiome in swine. Aims/Methods: This study aims to elucidate the archaeome structure and function in the porcine intestinal tract of healthy and H1N1 infected swine. We performed multi-omics analysis consisting of 16S rRNA gene profiling, metatranscriptomics and metaproteomics. Results and discussion: We observed a significant increase from 0.48 to 4.50% of archaea in the intestinal tract microbiome along the ileum and colon, dominated by genera Methanobrevibacter and Methanosphaera. Furthermore, in feces of naïve and H1N1 infected swine, we observed significant but minor differences in the occurrence of archaeal phylotypes over the course of an infection experiment. Metatranscriptomic analysis of archaeal mRNAs revealed the major methanogenesis pathways of Methanobrevibacter and Methanosphaera to be hydrogenotrophic and methyl-reducing, respectively. Metaproteomics of archaeal peptides indicated some effects of the H1N1 infection on central metabolism of the gut archaea. Conclusions/Take home message: Finally, this study provides the first multi-omics analysis and high-resolution insights into the structure and function of the porcine intestinal tract archaeome during a non-lethal Influenza A virus infection of the respiratory tract, demonstrating significant alterations in archaeal community composition and central metabolic functions.

Faecalibacterium prausnitzii, Bacteroides faecis and Roseburia intestinalis attenuate clinical symptoms of experimental colitis by regulating Treg/Th17 cell balance and intestinal barrier integrity.

Ulcerative colitis (UC) is a severe inflammatory bowel disease (IBD) characterized by multifactorial complex disorders triggered by environmental factors, genetic susceptibility, and also gut microbial dysbiosis. Faecalibacterium prausnitzii, Bacteroides faecis, and Roseburia intestinalis are underrepresented species in UC patients, leading to the hypothesis that therapeutic application of those bacteria could ameliorate clinical symptoms and disease severity. Acute colitis was induced in mice by 3.5% DSS, and the commensal bacterial species were administered by oral gavage simultaneously with DSS treatment for up to 7 days. The signs of colonic inflammation, the intestinal barrier integrity, the proportion of regulatory T cells (Tregs), and the expression of pro-inflammatory and anti-inflammatory cytokines were quantified. The concentrations of SCFAs in feces were measured using Gas-liquid chromatography. The gut microbiome was analyzed in all treatment groups at the endpoint of the experiment. Results were benchmarked against a contemporary mesalazine treatment regime. We show that commensal species alone and in combination reduced disease activity index scores, inhibited colon shortening, strengthened the colonic epithelial barrier, and positively modulated tight junction protein expression. The expression level of pro-inflammatory cytokines was significantly reduced. Immune modulation occurred via inhibition of the loss of CD4 +CD25 +Treg cells in the spleen. Our study proofed that therapeutic application of F. prausnitzii, B. faecis, and R. intestinalis significantly ameliorated DSS-induced colitis at the level of clinical symptoms, histological inflammation, and immune status. Our data suggest that these positive effects are mediated by immune-modulatory pathways and influence on Treg/Th17 balance.

Pyrenebutyrate Enhances the Antibacterial Effect of Peptide-Coupled Antisense Peptide Nucleic Acids in Streptococcus pyogenes.

Antisense peptide nucleic acids (PNAs) inhibit bacterial growth in several infection models. Since PNAs are not spontaneously taken up by bacteria, they are often conjugated to carriers such as cell-penetrating peptides (CPPs) in order to improve translocation. Hydrophobic counterions such as pyrenebutyrate (PyB) have been shown to facilitate translocation of peptides over natural and artificial membranes. In this study, the capability of PyB to support translocation of CPP-coupled antisense PNAs into bacteria was investigated in Streptococcus pyogenes and Streptococcus pneumoniae. PyB enhanced the antimicrobial activity of CPP-conjugated antisense PNAs in S. pyogenes. The most significant effect of PyB was observed in combination with K8-conjugated anti-gyrA PNAs. In contrast, no significant effect of PyB on the antimicrobial activity of CPP-conjugated PNAs in S. pneumoniae was detected. Uptake of K8-FITC into S. pyogenes, Escherichia coli, and Klebsiella pneumoniae could be improved by pre-incubation with PyB, indicating that PyB supports the antimicrobial effect of CPP-antisense PNAs in S. pyogenes by facilitating the translocation of peptides across the bacterial membrane.

Knockdown of coagulation factor VII has potential anticoagulant and anti-inflammatory effects in a mouse model of group A streptococcal sepsis.

Infections originating from subcutaneous tissues are among the most common invasive infections caused by group A streptococcus (GAS) and associated with systemic coagulation activation. The role of intrinsic coagulation factors on GAS virulence has recently been determined, but the role of the extrinsic coagulation factor VII is unknown. Using a mouse model, in which GAS-sepsis emerges from a subcutaneous infection, we show that FVII is a negative acute phase protein. F7 knockdown using antisense oligonucleotides resulted in an attenuated systemic coagulation activation and inflammatory response in septic animals. The findings indicate FVII's ability to modify the host response.

Involvement of ribonuclease Y in pilus production by M49 Streptococcus pyogenes strain via modulation of messenger RNA level of transcriptional regulator.

Streptococcus pyogenes displays a wide variety of pili, which is largely dependent on serotype. A distinct subset of S. pyogenes strains that possess the Nra transcriptional regulator demonstrates thermoregulated pilus production. Findings obtained in the present study of an Nra-positive serotype M49 strain revealed involvement of conserved virulence factor A (CvfA), also referred to as ribonuclease Y (RNase Y), in virulence factor expression and pilus production, while a cvfA deletion strain showed reduced pilus production and adherence to human keratinocytes as compared with wild-type and revertant strains. Furthermore, transcript levels of pilus subunits and srtC2 genes were decreased by cvfA deletion, which was remarkable at 25°C. Likewise, both messenger RNA (mRNA) and protein levels of Nra were remarkably decreased by cvfA deletion. Whether the expression of other pilus-related regulators, including fasX and CovR, was subject to thermoregulation was also examined. While the mRNA level of fasX, which inhibits cpa and fctA translation, was decreased by cvfA deletion at both 37°C and 25°C, CovR mRNA and protein levels, as well as its phosphorylation level were not significantly changed, suggesting that neither fasX nor CovR is necessarily involved in thermosensitive pilus production. Phenotypic analysis of the mutant strains revealed that culture temperature and cvfA deletion had varied effects on streptolysin S and SpeB activities. Furthermore, bactericidal assay data showed that cvfA deletion decreased the rate of survival in human blood. Together, the present findings indicate that CvfA is involved in regulation of pilus production and virulence-related phenotypes of the serotype M49 strain of S. pyogenes.

The Gut Microbiome of an Indigenous Agropastoralist Population in a Remote Area of Colombia with High Rates of Gastrointestinal Infections and Dysbiosis.

An Indigenous agropastoralist population called the Wiwa from the Sierra Nevada de Santa Marta, in North-East Colombia, shows high rates of gastrointestinal infections. Chronic gut inflammatory processes and dysbiosis could be a reason, suggesting an influence or predisposing potential of the gut microbiome composition. The latter was analyzed by 16S rRNA gene amplicon next generation sequencing from stool samples. Results of the Wiwa population microbiomes were associated with available epidemiological and morphometric data and compared to control samples from a local urban population. Indeed, locational-, age-, and gender-specific differences in the Firmicutes/Bacteriodetes ratio, core microbiome, and overall genera-level microbiome composition were shown. Alpha- and ß-diversity separated the urban site from the Indigenous locations. Urban microbiomes were dominated by Bacteriodetes, whereas Indigenous samples revealed a four times higher abundance of Proteobacteria. Even differences among the two Indigenous villages were noted. PICRUSt analysis identified several enriched location-specific bacterial pathways. Moreover, on a general comparative scale and with a high predictive accuracy, we found Sutterella associated with the abundance of enterohemorrhagic Escherichia coli (EHEC), Faecalibacteria associated with enteropathogenic Escherichia coli (EPEC) and helminth species Hymenolepsis nana and Enterobius vermicularis. Parabacteroides, Prevotella, and Butyrivibrio are enriched in cases of salmonellosis, EPEC, and helminth infections. Presence of Dialister was associated with gastrointestinal symptoms, whereas Clostridia were exclusively found in children under the age of 5 years. Odoribacter and Parabacteroides were exclusively identified in the microbiomes of the urban population of Valledupar. In summary, dysbiotic alterations in the gut microbiome in the Indigenous population with frequent episodes of self-reported gastrointestinal infections were confirmed with epidemiological and pathogen-specific associations. Our data provide strong hints of microbiome alterations associated with the clinical conditions of the Indigenous population.

Impact of Pneumococcal and Viral Pneumonia on the Respiratory and Intestinal Tract Microbiomes of Mice.

With 2.56 million deaths worldwide annually, pneumonia is one of the leading causes of death. The most frequent causative pathogens are Streptococcus pneumoniae and influenza A virus. Lately, the interaction between the pathogens, the host, and its microbiome have gained more attention. The microbiome is known to promote the immune response toward pathogens; however, our knowledge on how infections affect the microbiome is still scarce. Here, the impact of colonization and infection with S. pneumoniae and influenza A virus on the structure and function of the respiratory and gastrointestinal microbiomes of mice was investigated. Using a meta-omics approach, we identified specific differences between the bacterial and viral infection. Pneumococcal colonization had minor effects on the taxonomic composition of the respiratory microbiome, while acute infections caused decreased microbial complexity. In contrast, richness was unaffected following H1N1 infection. Within the gastrointestinal microbiome, we found exclusive changes in structure and function, depending on the pathogen. While pneumococcal colonization had no effects on taxonomic composition of the gastrointestinal microbiome, increased abundance of Akkermansiaceae and Spirochaetaceae as well as decreased amounts of Clostridiaceae were exclusively found during invasive S. pneumoniae infection. The presence of Staphylococcaceae was specific for viral pneumonia. Investigation of the intestinal microbiomés functional composition revealed reduced expression of flagellin and rubrerythrin and increased levels of ATPase during pneumococcal infection, while increased amounts of acetyl coenzyme A (acetyl-CoA) acetyltransferase and enoyl-CoA transferase were unique after H1N1 infection. In conclusion, identification of specific taxonomic and functional profiles of the respiratory and gastrointestinal microbiome allowed the discrimination between bacterial and viral pneumonia. IMPORTANCE Pneumonia is one of the leading causes of death worldwide. Here, we compared the impact of bacterial- and viral-induced pneumonia on the respiratory and gastrointestinal microbiome. Using a meta-omics approach, we identified specific profiles that allow discrimination between bacterial and viral causative.

Dysbiosis and reduced small intestinal function are required to induce intestinal insufficiency in mice.

Extensive bowel resection can lead to short bowel syndrome and intestinal failure. Resection-induced dysbiosis may be related to the specific anatomic site of resection and influences the disease progression. Although patients with end-jejunostomy are at high risk for intestinal failure, preservation of the ileocecal valve and colon counteracts this risk. The present study investigated the role of the cecum in maintaining microbial homeostasis after different types of small bowel resection. Male C57BL6/J mice were anesthetized by intraperitoneal injection of ketamine-xylazine and received extended ileocecal resection (extended ICR), limited ileocecal resection (limited ICR), or mid-small bowel resection (SBR). Stool samples were collected before surgery and between postoperative days 2-7, for 16S rRNA gene sequencing. Only extended ICR, but neither limited ICR nor SBR, induced intestinal insufficiency. α-Diversity was reduced in both ICR variants but not after SBR. All resections resulted in an increase in Proteobacteria. Pathobionts, such as Clostridia, Shigella, and Enterococcus, increased after SBR while Muribaculaceae, Lactobacillus, and Lachnospiraceae decreased. Limited ICR resulted in an increase of members of the Clostridium sensu stricto group, Terrisporobacter and Enterococcus and a decrease of Muribaculaceae. The increase of Enterococcus was even more pronounced after extended ICR while Muribaculaceae and Akkermansia were dramatically reduced. Both ICR variants caused a decrease in steroid biosynthesis and glycosaminoglycan degradation-associated pathways, suggesting altered bile acid transformation and mucus utilization.NEW & NOTEWORTHY Resection-induced dysbiosis affects disease progression in patients with short bowel syndrome. Severe dysbiosis occurs after removal of the ileocecal valve, even in the absence of short bowel conditions, and is associated with the loss of Muribaculaceae and Akkermansia and an increase of Clostridium and Enterococcus. The preservation of the cecum should be considered in surgical therapy, and dysbiosis should be targeted based on its specific anatomical signature to improve postoperative bacterial colonization.

Antimicrobial Activity of Peptide-Coupled Antisense Peptide Nucleic Acids in Streptococcus pneumoniae.

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and is responsible for multiple other infectious diseases, such as meningitis and otitis media, in children. Resistance to penicillins, macrolides, and fluoroquinolones is increasing and, since the introduction of pneumococcal conjugate vaccines (PCVs), vaccine serotypes have been replaced by non-vaccine serotypes. Antisense peptide nucleic acids (PNAs) have been shown to reduce the growth of several pathogenic bacteria in various infection models. PNAs are frequently coupled to cell-penetrating peptides (CPPs) to improve spontaneous cellular PNA uptake. In this study, different CPPs were investigated for their capability to support translocation of antisense PNAs into S. pneumoniae. HIV-1 TAT- and (RXR)4XB-coupled antisense PNAs efficiently reduced the viability of S. pneumoniae strains TIGR4 and D39 in vitro. Two essential genes, gyrA and rpoB, were used as targets for antisense PNAs. Overall, the antimicrobial activity of anti-gyrA PNAs was higher than that of anti-rpoB PNAs. Target gene transcription levels in S. pneumoniae were reduced following antisense PNA treatment. The effect of HIV-1 TAT- and (RXR)4XB-anti-gyrA PNAs on pneumococcal survival was also studied in vivo using an insect infection model. Treatment increased the survival of infected Galleria mellonella larvae. Our results represent a proof of principle and may provide a basis for the development of efficient antisense molecules for treatment of S. pneumoniae infections. IMPORTANCE Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and is responsible for the deaths of up to 2 million children each year. Antibiotic resistance and strain replacement by non-vaccine serotypes are growing problems. For this reason, S. pneumoniae has been added to the WHO "global priority list" of antibiotic-resistant bacteria for which novel antimicrobials are most urgently needed. In this study, we investigated whether CPP-coupled antisense PNAs show antibacterial activity in S. pneumoniae. We demonstrated that HIV-1 TAT- and (RXR)4XB-coupled antisense PNAs were able to kill S. pneumoniae in vitro. The specificity of the antimicrobial effect was verified by reduced target gene transcription levels in S. pneumoniae. Moreover, CPP-antisense PNA treatment increased the survival rate of infected Galleria mellonella larvae in vivo. Based on these results, we believe that efficient antisense PNAs can be developed for the treatment of S. pneumoniae infections.

Metagenomic Sequencing for the Diagnosis of Plasmodium spp. with Different Levels of Parasitemia in EDTA Blood of Malaria Patients-A Proof-of-Principle Assessment.

Molecular diagnostic approaches are increasingly included in the diagnostic workup and even in the primary diagnosis of malaria in non-endemic settings, where it is difficult to maintain skillful microscopic malaria detection due to the rarity of the disease. Pathogen-specific nucleic acid amplification, however, bears the risk of overlooking other pathogens associated with febrile illness in returnees from the tropics. Here, we assessed the discriminatory potential of metagenomic sequencing for the identification of different Plasmodium species with various parasitemia in EDTA blood of malaria patients. Overall, the proportion of Plasmodium spp.-specific sequence reads in the assessed samples showed a robust positive correlation with parasitemia (Spearman r = 0.7307, p = 0.0001) and a robust negative correlation with cycle threshold (Ct) values of genus-specific real-time PCR (Spearman r = -0.8626, p ≤ 0.0001). Depending on the applied bioinformatic algorithm, discrimination on species level was successful in 50% (11/22) to 63.6% (14/22) instances. Limiting factors for the discrimination on species level were very low parasitemia, species-depending lacking availability of reliable reference genomes, and mixed infections with high variance of the proportion of the infecting species. In summary, metagenomic sequencing as performed in this study is suitable for the detection of malaria in human blood samples, but the diagnostic detection limit for a reliable discrimination on species level remains higher than for competing diagnostic approaches like microscopy and PCR.

Procoagulant Activity of Blood and Microvesicles Is Disturbed by Pneumococcal Pneumolysin, Which Interacts with Coagulation Factors.

The coagulation and contact systems are parts of the innate immune system as they prevent bleeding and dissemination of pathogens and also contribute to microbial killing by inflammatory reactions and the release of antimicrobial peptides. Here, we investigated the influence of Streptococcus pneumoniae on the coagulation and contact system. S. pneumoniae (pneumococci), but no other investigated streptococcal species, impairs coagulation of blood by autolysis and release of pneumolysin. Defective blood coagulation results from the lysis of tissue factor-producing mononuclear cells and their procoagulant microvesicles, which are the main trigger for blood coagulation during sepsis. In addition, pneumolysin binds coagulation and contact system factors, but this does not result in activation. Thus, pneumococci modulate activation of the coagulation system by releasing pneumolysin, which could potentiate lung injury during pneumonia.

The role of bacterial corrosion on recolonization of titanium implant surfaces: An in vitro study.

BACKGROUND: Inflammation triggered by bacterial biofilms in the surrounding tissue is a major etiological factor for peri-implantitis and subsequent implant failure. However, little is known about the direct effects of bacterial corrosion and recolonization on implant failure PURPOSE: To investigate the influence of oral commensals on bacterial corrosion and recolonization of titanium surfaces. MATERIALS AND METHODS: Streptococcus sanguinis (S. sanguinis) and Porphyromonas gingivalis (P. gingivalis), which are key bacteria in oral biofilm formation, were cultured on commercially pure titanium and titanium-aluminum-vanadium (Ti6Al4V) plates in artificial saliva/brain heart infusion medium under aerobic or anaerobic conditions. Biofilm formation was examined after 7 and 21 days by crystal violet and live/dead staining. Titanium ions released into culture supernatants were analyzed over a period of 21 days by atomic absorption spectrometry. Visual changes in surface morphology were investigated using scanning electron microscopy. Biofilm formation on sterilized, biocorroded, and recolonized implant surfaces was determined by crystal violet staining. RESULTS: S. sanguinis and P. gingivalis formed stable biofilms on the titanium samples. Bacterial corrosion led to a significant increase in titanium ion release from these titanium plates (p < 0.01), which was significantly higher under aerobic conditions on pure titanium (p ≤ 0.001). No obvious morphological surface changes, such as pitting and discoloration, were detected in the titanium samples. During early biofilm formation, the addition of titanium ions significantly decreased the number of live cells. In contrast, a significant effect on biofilm mass was only detected with P. gingivalis. Bacterial corrosion had no influence on bacterial recolonization following sterilization of titanium and Ti6Al4V surfaces. CONCLUSION: Bacterial corrosion differs between oral commensal bacteria and leads to increased titanium ion release from titanium plates. The titanium ion release did not influence biofilm formation or bacterial recolonization under in vitro conditions.

The Non-phosphorylating Glyceraldehyde-3-Phosphate Dehydrogenase GapN Is a Potential New Drug Target in Streptococcus pyogenes.

The strict human pathogen Streptococcus pyogenes causes infections of varying severity, ranging from self-limiting suppurative infections to life-threatening diseases like necrotizing fasciitis or streptococcal toxic shock syndrome. Here, we show that the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase GapN is an essential enzyme for S. pyogenes. GapN converts glyceraldehyde 3-phosphate into 3-phosphoglycerate coupled to the reduction of NADP to NADPH. The knock-down of gapN by antisense peptide nucleic acids (asPNA) significantly reduces viable bacterial counts of S. pyogenes laboratory and macrolide-resistant clinical strains in vitro. As S. pyogenes lacks the oxidative part of the pentose phosphate pathway, GapN appears to be the major NADPH source for the bacterium. Accordingly, other streptococci that carry a complete pentose phosphate pathway are not prone to asPNA-based gapN knock-down. Determination of the crystal structure of the S. pyogenes GapN apo-enzyme revealed an unusual cis-peptide in proximity to the catalytic binding site. Furthermore, using a structural modeling approach, we correctly predicted competitive inhibition of S. pyogenes GapN by erythrose 4-phosphate, indicating that our structural model can be used for in silico screening of specific GapN inhibitors. In conclusion, the data provided here reveal that GapN is a potential target for antimicrobial substances that selectively kill S. pyogenes and other streptococci that lack the oxidative part of the pentose phosphate pathway.

All Driven by Energy Demand? Integrative Comparison of Metabolism of Enterococcus faecalis Wildtype and a Glutamine Synthase Mutant.

Lactic acid bacteria (LAB) play a significant role in biotechnology, e.g., food industry and also in human health. Many LAB genera have developed a multidrug resistance in the past few years, causing a serious problem in controlling hospital germs worldwide. Enterococcus faecalis accounts for a large part of the human infections caused by LABs. Therefore, studying its adaptive metabolism under various environmental conditions is particularly important to promote the development of new therapeutic approaches. In this study, we investigated the effect of glutamine auxotrophy (ΔglnA mutant) on metabolic and proteomic adaptations of E. faecalis in response to a changing pH in its environment. Changing pH values are part of the organism's natural environment in the human body and play a role in the food industry. We compared the results with those of the wildtype. Using a genome-scale metabolic model constrained by metabolic and proteomic data, our integrative method allows us to understand the bigger picture of the adaptation strategies of this bacterium. The study showed that energy demand is the decisive factor in adapting to a new environmental pH. The energy demand of the mutant was higher at all conditions. It has been reported that ΔglnA mutants of bacteria are energetically less effective. With the aid of our data and model we are able to explain this phenomenon as a consequence of a failure to regulate glutamine uptake and the costs for the import of glutamine and the export of ammonium. Methodologically, it became apparent that taking into account the nonspecificity of amino acid transporters is important for reproducing metabolic changes with genome-scale models because it affects energy balance. IMPORTANCE The integration of new pH-dependent experimental data on metabolic uptake and release fluxes, as well as of proteome data with a genome-scale computational model of a glutamine synthetase mutant of E. faecalis is used and compared with those of the wildtype to understand why glutamine auxotrophy results in a less efficient metabolism and how-in comparison with the wildtype-the glutamine synthetase knockout impacts metabolic adjustments during acidification or simply exposure to lower pH. We show that forced glutamine auxotrophy causes more energy demand and that this is likely due to a disregulated glutamine uptake. Proteome changes during acidification observed for the mutant resemble those of the wildtype with the exception of glycolysis-related genes, as the mutant is already energetically stressed at a higher pH and the respective proteome changes were in effect.

The versatile role of the contact system in cardiovascular disease, inflammation, sepsis and cancer.

The human contact system consists of plasma proteins, which - after contact to foreign surfaces - are bound to them, thereby activating the zymogens of the system into enzymes. This activation mechanism gave the system its name - contact system. It is considered as a procoagulant and proinflammatory response mechanism, as activation finally leads to the generation of fibrin and bradykinin. To date, no physiological processes have been described that are mediated by contact activation. However, contact system factors play a pathophysiological role in numerous diseases, such as cardiovascular diseases, arthritis, colitis, sepsis, and cancer. Contact system factors are therefore an interesting target for new therapeutic options in different clinical conditions.