Exploring ex vivo biofilm dynamics: consequences of low ampicillin concentrations on the human oral microbiome.

Prolonged exposure to antibiotics at low concentration can promote processes associated with bacterial biofilm formation, virulence and antibiotic resistance. This can be of high relevance in microbial communities like the oral microbiome, where commensals and pathogens share a common habitat and where the total abundance of antibiotic resistance genes surpasses the abundance in the gut. Here, we used an ex vivo model of human oral biofilms to investigate the impact of ampicillin on biofilm viability. The ecological impact on the microbiome and resistome was investigated using shotgun metagenomics. The results showed that low concentrations promoted significant shifts in microbial taxonomic profile and could enhance biofilm viability by up to 1 to 2-log. For the resistome, low concentrations had no significant impact on antibiotic resistance gene (ARG) diversity, while ARG abundance decreased by up to 84%. A positive correlation was observed between reduced microbial diversity and reduced ARG abundance. The WHO priority pathogens Streptococcus pneumoniae and Staphylococcus aureus were identified in some of the samples, but their abundance was not significantly altered by ampicillin. Most of the antibiotic resistance genes that increased in abundance in the ampicillin group were associated with streptococci, including Streptococcus mitis, a well-known potential donor of ARGs to S. pneumoniae. Overall, the results highlight the potential of using the model to further our understanding of ecological and evolutionary forces driving antimicrobial resistance in oral microbiomes.

Complete Genome Sequence of Streptococcus pneumoniae Serotype 19F Strain EF3030.

We report the complete genome sequence of Streptococcus pneumoniae EF3030, a serotype 19F isolate that colonizes the nasopharynx of mice while being mostly noninvasive. Such attributes make this strain highly attractive in pneumococcal carriage studies. The availability of its complete genomic sequence is likely to advance studies in the field.

Characterization of a Signaling System in Streptococcus mitis That Mediates Interspecies Communication with Streptococcus pneumoniae.

Streptococcus mitis is found in the oral cavity and nasopharynx and forms a significant portion of the human microbiome. In this study, in silico analyses indicated the presence of an Rgg regulator and short hydrophobic peptide (Rgg/SHP) cell-to-cell communication system in S. mitis Although Rgg presented greater similarity to a repressor in Streptococcus pyogenes, autoinducing assays and genetic mutation analysis revealed that in S. mitis Rgg acts as an activator. Transcriptome analysis showed that in addition to shp, the system regulates two other downstream genes, comprising a segment of a putative lantibiotic gene cluster that is in a conjugative element locus in different members of the mitis group. Close comparison to a similar lantibiotic gene cluster in Streptococcus pneumoniae indicated that S. mitis lacked the full set of genes. Despite the potential of SHP to trigger a futile cycle of autoinduction, growth was not significantly affected for the rgg mutant under normal or antibiotic stress conditions. The S. mitis SHP was, however, fully functional in promoting cross-species communication and increasing S. pneumoniae surface polysaccharide production, which in this species is regulated by Rgg/SHP. The activity of SHPs produced by both species was detected in cocultures using a S. mitis reporter strain. In competitive assays, a slight advantage was observed for the rgg mutants. We conclude that the Rgg/SHP system in S. mitis regulates the expression of its own shp and activates an Rgg/SHP system in S. pneumoniae that regulates surface polysaccharide synthesis. Fundamentally, cross-communication of such systems may have a role during multispecies interactions.IMPORTANCE Bacteria secrete signal molecules into the environment which are sensed by other cells when the density reaches a certain threshold. In this study, we describe a communication system in Streptococcus mitis, a commensal species from the oral cavity, which we also found in several species and strains of streptococci from the mitis group. Further, we show that this system can promote cross-communication with S. pneumoniae, a closely related major human pathogen. Importantly, we show that this cross-communication can take place during coculture. While the genes regulated in S. mitis are likely part of a futile cycle of activation, the target genes in S. pneumoniae are potentially involved in virulence. The understanding of such complex communication networks can provide important insights into the dynamics of bacterial communities.

Antibiotics alter the window of competence for natural transformation in streptococci.

Streptococcus pneumoniae transformation occurs within a short competence window, during which the alternative sigma factor X (SigX) is activated to orchestrate the expression of genes allowing extracellular DNA uptake and recombination. Importantly, antibiotic stress promotes transcriptional changes that may affect more than 20% of the S. pneumoniae genome, including competence genes. These can be activated or repressed, depending on the antibiotic agent. For most antibiotics, however, it remains unknown whether transcriptional effects on competence translate into altered transformability. Here we investigate the effect of antibiotic subinhibitory concentrations on sigX expression using a luciferase reporter, and correlate for the first time with transformation kinetics. Induction of sigX expression by ciprofloxacin and novobiocin correlated with increased and prolonged transformability in S. pneumoniae. The prolonged effect of ciprofloxacin on competence and transformation was also observed in the streptococcal relatives Streptococcus mitis and Streptococcus mutans. In contrast, tetracycline and erythromycin, which induced S. pneumoniae sigX expression, had either an inhibitory or a nonsignificant effect on transformation, whereas streptomycin and the ß-lactam ampicillin, inhibited both sigX expression and transformation. Thus, the results show that antibiotics may vary in their effects on competence, ranging from inhibitory to stimulatory effects, and that responses affecting transcription of sigX do not always correlate with the transformation outcomes. Antibiotics that increase or decrease transformation are of particular clinical relevance, as they may alter the ability of S. pneumoniae to escape vaccines and antibiotics.

High-resolution profiles of the Streptococcus mitis CSP signaling pathway reveal core and strain-specific regulated genes.

BACKGROUND: In streptococci of the mitis group, competence for natural transformation is a transient physiological state triggered by competence stimulating peptides (CSPs). Although low transformation yields and the absence of a widespread functional competence system have been reported for Streptococcus mitis, recent studies revealed that, at least for some strains, high efficiencies can be achieved following optimization protocols. To gain a deeper insight into competence in this species, we used RNA-seq, to map the global CSP response of two transformable strains: the type strain NCTC12261T and SK321. RESULTS: All known genes induced by ComE in Streptococcus pneumoniae, including sigX, were upregulated in the two strains. Likewise, all sets of streptococcal SigX core genes involved in extracellular DNA uptake, recombination, and fratricide were upregulated. No significant differences in the set of induced genes were observed when the type strain was grown in rich or semi-defined media. Five upregulated operons unique to S. mitis with a SigX-box in the promoter region were identified, including two specific to SK321, and one specific to NCTC12261T. Two of the strain-specific operons coded for different bacteriocins. Deletion of the unique S. mitis sigX regulated genes had no effect on transformation. CONCLUSIONS: Overall, comparison of the global transcriptome in response to CSP shows the conservation of the ComE and SigX-core regulons in competent S. mitis isolates, as well as species and strain-specific genes. Although some S. mitis exhibit truncations in key competence genes, this study shows that in transformable strains, competence seems to depend on the same core genes previously identified in S. pneumoniae.

Increased oxygen exposure alters collagen expression and tissue architecture during ligature-induced periodontitis.

BACKGROUND AND OBJECTIVE: The aim of this study was to evaluate the effects of increased oxygen availability on gene expression and on collagen deposition/maturation in the periodontium following disease. MATERIAL AND METHODS: Male Wistar rats had ligatures placed around their molars to induce periodontal disease, and a subset of animals underwent hyperbaric oxygen (HBO) treatment for 2 h twice per day. At 15 and 28 d, tissue gene expression of COL1A1, transforming growth factor-ß1 and alkaline phosphatase was determined; other histological samples were stained with Picrosirius red to evaluate levels of collagen deposition, maturation and thickness. RESULTS: In animals that underwent HBO treatment, type I collagen expression was higher and collagen deposition, maturation and thickness were more robust. Reduced mRNA levels of transforming growth factor-beta1 and alkaline phosphatase in HBO-treated rats on day 28 suggested that a quicker resolution in both soft tissue and bone remodeling occurred following oxygen treatment. No differences in inflammation were observed between groups. CONCLUSIONS: The extracellular matrix regenerated more quickly in the HBO-treated group as evidenced by higher collagen expression, deposition and maturation.

Psychological distress and salivary secretory immunity.

Stress-induced impairments of mucosal immunity may increase susceptibility to infectious diseases. The present study investigated the association of perceived stress, depressive symptoms, and loneliness with salivary levels of secretory immunoglobulin A (S-IgA), the subclasses S-IgA1, S-IgA2, and their transporter molecule Secretory Component (SC). S-IgA/SC, IgA1/SC and IgA2/SC ratios were calculated to assess the differential effects of stress on immunoglobulin transport versus availability. This study involved 113 university students, in part selected on high scores on the UCLA Loneliness Scale and/or the Beck Depression Inventory. Stress levels were assessed using the Perceived Stress Scale. Unstimulated saliva was collected and analysed for total S-IgA and its subclasses, as well as SC and total salivary protein. Multiple linear regression analyses, adjusted for gender, age, health behaviours, and concentration effects (total protein) revealed that higher perceived stress was associated with lower levels of IgA1 but not IgA2. Perceived stress, loneliness and depressive symptoms were all associated with lower IgA1/SC ratios. Surprisingly, higher SC levels were associated with loneliness and depressive symptoms, indicative of enhanced transport activity, which explained a lower IgA1/SC ratio (loneliness and depression) and IgA2/SC ratio (depression). This is the first study to investigate the effects of protracted psychological stress across S-IgA subclasses and its transporter SC. Psychological stress was negatively associated with secretory immunity, specifically IgA1. The lower immunoglobulin/transporter ratio that was associated with higher loneliness and depression suggested a relative immunoglobulin depletion, whereby availability was not keeping up with enhanced transport demand.

Genome editing by natural genetic transformation in Streptococcus mutans.

Classical mutagenesis strategies using selective markers linked to designed mutations are powerful and widely applicable tools for targeted mutagenesis via natural genetic transformation in bacteria and archaea. However, the markers that confer power are also potentially problematic as they can be cumbersome, risk phenotypic effects of the inserted genes, and accumulate as unwanted genes during successive mutagenesis cycles. Alternative mutagenesis strategies use temporary plasmid or cassette insertions and can in principle achieve equally flexible mutation designs, but design of suitable counter-selected markers can be complex. All these drawbacks are eased by use of direct genome editing. Here we describe a strategy for directly editing the genome of S. mutans, which is applied to the widely studied reference strain UA159 (ATCC 700610) and has the advantage of extreme simplicity, requiring construction of only one synthetic donor amplicon and a single transformation step, followed by a simple PCR screen among a few dozen clones to identify the desired mutant. The donor amplicon carries the mutant sequence and extensive flanking segments of homology, which ensure efficient and precise integration by the recombination machinery specific to competent cells. The recipients are highly competent cells, in a state achieved by treatment with a synthetic competence pheromone.

Dental care providers' decision making regarding maintenance of compromised teeth and implant therapy indication: an analysis of gender and enrollment in teaching positions.

OBJECTIVES: The aim of this study was to evaluate Brazilian dentists' decision making regarding periodontally involved teeth and implant therapy indications as well as possible modifying factors such as gender, enrollment in teaching positions, and area of specialization. MATERIAL AND METHODS: This cross-sectional questionnaire-based study was conducted among Brazilian dentists between June and November 2012. The questionnaire comprised 27 questions divided into four different sections: socio-demographic characteristics, questions about their clinical practice toward implant therapy, decision making in four clinical cases, and agreement with different statements regarding endodontic, periodontal, and implant therapy. RESULTS: A total of 155 dentists answered the questionnaire with an average age of 35.5 years. Fifty-one percent were male, and 44.5% were involved in teaching positions. One hundred and thirty-six (87.7%) respondents had already pursued a post-graduation program or were currently involved in one. 33.5% placed dental implants, 42% provided implant maintenance care to their patients, while 30% performed treatment of peri-implant diseases themselves. Dental faculty and dentists who had not followed any further training program, preferred the use of regenerative therapies more frequently. As opposed, dentists not involved in teaching positions, and periodontists were more prone to select resective therapies. CONCLUSION: Results suggest that dentists' decision making did not follow the latest external evidence, which might be related to factors as professional expertise and patients' preferences. Considering the difficulties faced by professionals when handling complex cases, there is an urgent need to establish international protocols and guidelines to help the clinician identify this particular treatment option, which corresponds with their internal evidence and is based on good external evidence.

[Effect of the method of measurement on the interpretation of renal clearance]. / Der Einfluss der Messmethode auf die Bedeutung der Nierenclearance.

The concept of renal clearance measured during steady-state conditions includes the effect of filtration and secretion as well as urine flow. On the other hand, renal clearance, as used in nuclear medicine, only describes the effect of filtration and secretion. A way for determining steady-state renal clearance with a bolus injection technique is described. Steady-state renal clearance reflects the efficiency of filtration and secretion, urine flow, and the steady-state chemical potentials in the kidneys.

[A new concept for detailed measuring of the excretory function of the kidney]. / Ein neues Konzept zur detaillierten Messung der Ausscheidungsfunktion der Nieren.

A new method to investigate renal function in detail without use of catheters is demonstrated. This method combines the advantages of two techniques, the physiological clearance measurement technique and the isotope nephrogram technique. The function of each kidney is quantitated in terms of the velocity of renal blood flow, the velocity of filtration and secretion and the velocity of urine flow to the bladder. The new concept is illustrated by two clinical examples.

[Radionuclide determination of renal clearance equivalent to physiological clearance measurements]. / Nuklearmedizinische Bestimmung der Nierenclearance äquivalent zur physiologischen Clearancemessung.

The concept of renal clearance in nuclear medicine describes the tubular secretion and glomerular filtration of a substance being removed from the blood plasma. The concept of clearance as used physiologically is rather wider and includes consideration of the outflow of the substances remained back in the kidneys. The present paper discusses clearance measurements as seen from a thermo-dynamic point of view, in which isotope clearances become equivalent with the physiological concept of clearance. In addition, it is possible to quantify each single step of the excretory function of each kidney separately.

[Direct and non-invasive determination of cardiac volume]. / Direkte und nicht-invasive Bestimmung des Herzzeitvolumens.

An improved isotope method for direct and non-invasive determination of heart volume, ejection volume and volume/time is described. By taking account of the reduction in the radio-active marker during the procedure, very good correlation of the results with those of cine ventriculography carried out at the same time can be expected.

[A new nuclear medicine method for studying heart function without ECG-trigger and without the effect of body background radiation]. / Eine neue nuklearmedizinische Methode zur Untersuchung der Herzfunktion ohne EKG-Trigger und ohne Einfluss der Körperhintergrundstrahlung.

The intensity fluctuations of the radiation from a beating heart filled with radioactively labeled blood is analysed in such a way that the stroke volume and thus the cardiac output can be determined. The method is independent of any background radiation and irregularities of the heart rate. Further features are the speed of the method and the immediate imaging of the kinetics of the heart wall.

Noise analysis and relaxation experiments of transport of hydrophobic anions across lipid membranes at equilibrium and nonequilibrium.

Under equilibrium and nonequilibrium steady-state conditions, the spectral intensity of current noise SJ(f) generated by the transport of hydrophobic anions across lipid bilayer membranes was investigated. The experimental results were compared with different reaction models. SJ(f) showed a characteristic increase proportional to f2 between frequency-independent tails at low and high frequencies. This gradient was found to be independent of applied voltage which indicates the contribution of a single voltage-dependent reaction step of ion translocation across the membrane. From the shape of SJ(f) at low frequencies the rate constant of ion desorption from the membrane into the aqueous phase could be estimated. Unambiguous evidence for the application of a general model, which includes the coupling of slow ion diffusion in the aqueous phase to ion adsorption/desorption at the membrane interface, could not be obtained from the low-frequency shape of SJ(f). The shot noise of this ion transport determines the amplitude of SJ(f) at high frequencies which decreases with increasing voltage applied. Analysis of voltage-jump current-relaxation experiments and of current noise carried out on one membrane yielded significant differences of the derived ion partition coefficient. This deviation is qualitatively described on the basis of incomplete reaction steps.

[Results of computed tomography of the lumbar vertebral discs (author's transl)]. / Ergebnisse der computertomographischen Diagnostik an der lumbalen Bandscheibe.

As shown in 150 patients, myelography and computed tomography (CT) lead to identical results in the investigation of vertebral disc lesions of the lower lumbar spine. In less severe disc changes, where myelography is not indicated, CT findings largely correspond with clinical findings. CT investigations are risk free, without side effects, painless, fast and economical.