Salivary microbiome and MRP-8/14 levels in children with gingivitis, healthy children, and their mothers.

BACKGROUND: Gingivitis is the most common form of periodontal disease among children and adolescents and is associated with disrupted host-microbiome homeostasis. Family is an important factor influencing the prevalence of gingivitis. In the present study, we investigated the salivary microbiome, oral hygiene habits, and the salivary level of myeloid-related protein (MRP)-8/14 in children aged 7-12 years with gingivitis, periodontally healthy children, and their mothers. METHODS: This study included 24 children with gingivitis (including four sibling pairs) and 22 periodontally healthy children (including two sibling pairs) and their mothers. The whole saliva was collected, DNA was extracted, the variable V3-V4 region of the eubacterial 16S ribosomal RNA gene was amplified, and sample library preparation was performed according to the Illumina protocol. The salivary levels of MRP-8/14 were analyzed by ELISA. RESULTS: Alpha diversity of the salivary microbiome was considerably higher in gingivitis children and mothers of gingivitis children compared to healthy children and their mothers, respectively. Significant differences in beta diversity between healthy and gingivitis children, healthy children and their mothers, and gingivitis children and their mothers were detected. Overall, the number of common core amplicon sequence variants between children and their own mothers was significantly higher than between children and other mothers. The salivary MRP-8/14 levels in children with gingivitis were significantly higher compared to healthy children; a similar tendency was also mentioned for mothers. CONCLUSION: Our study underlines the importance of family as an essential factor influencing oral health.

Effect of Antibiotic Eye Drops on the Nasal Microbiome in Healthy Subjects-A Pilot Study.

BACKGROUND: Antibiotic eye drops are frequently used in clinical practice. Due to the anatomical connection via the nasolacrimal duct, it seems possible that they have an influence on the nasal/pharyngeal microbiome. This was investigated by using two different commonly used antibiotic eye drops. METHODS: 20 subjects were randomized to four groups of five subjects receiving eye drops containing gentamicin, ciprofloxacin, or, as controls, unpreserved povidone or benzalkonium chloride-preserved povidone. Nasal and pharyngeal swabs were performed before and after the instillation period. Swabs were analyzed by Illumina next-generation sequencing (NGS)-based 16S rRNA analysis. Bacterial culture was performed on solid media, and bacterial isolates were identified to the species level by MALDI-TOF MS. Species-dependent antimicrobial susceptibility testing was performed using single isolates and pools of isolates. RESULTS: Bacterial richness in the nose increased numerically from 163 ± 30 to 243 ± 100 OTUs (gentamicin) and from 114 ± 17 to 144 ± 45 OTUs (ciprofloxacin). Phylogenetic diversity index (pd) of different bacterial strains in the nasal microbiome increased from 12.4 ± 1.0 to 16.9 ± 5.6 pd (gentamicin) and from 10.2 ± 1.4 to 11.8 ± 3.1 pd (ciprofloxacin). Unpreserved povidone eye drops resulted in minimal changes in bacterial counts. Preservative-containing povidone eye drops resulted in no change. A minor increase (1-2-fold) in the minimal inhibitory concentration (MIC) was observed in single streptococcal isolates. CONCLUSIONS: Antibiotic eye drops could affect the nasal microbiome. After an instillation period of seven days, an increase in the diversity and richness of bacterial strains in the nasal microbiome was observed.

Disturbances in microbial skin recolonization and cutaneous immune response following allogeneic stem cell transfer.

The composition of the gut microbiome influences the clinical course after allogeneic hematopoietic stem cell transplantation (HSCT), but little is known about the relevance of skin microorganisms. In a single-center, observational study, we recruited a cohort of 50 patients before undergoing conditioning treatment and took both stool and skin samples up to one year after HSCT. We could confirm intestinal dysbiosis following HSCT and report that the skin microbiome is likewise perturbed in HSCT-recipients. Overall bacterial colonization of the skin was decreased after conditioning. Particularly patients that developed acute skin graft-versus-host disease (aGVHD) presented with an overabundance of Staphylococcus spp. In addition, a loss in alpha diversity was indicative of aGVHD development already before disease onset and correlated with disease severity. Further, co-localization of CD45+ leukocytes and staphylococci was observed in the skin of aGVHD patients even before disease development and paralleled with upregulated genes required for antigen-presentation in mononuclear phagocytes. Overall, our data reveal disturbances of the skin microbiome as well as cutaneous immune response in HSCT recipients with changes associated with cutaneous aGVHD.

Analysis of antifungal resistance genes in Candida albicans and Candida glabrata using next generation sequencing.

INTRODUCTION/OBJECTIVES: An increase in antifungal resistant Candida strains has been reported in recent years. The aim of this study was to detect mutations in resistance genes of azole-resistant, echinocandin-resistant or multi-resistant strains using next generation sequencing technology, which allows the analysis of multiple resistance mechanisms in a high throughput setting. METHODS: Forty clinical Candida isolates (16 C. albicans and 24 C. glabrata strains) with MICs for azoles and echinocandins above the clinical EUCAST breakpoint were examined. The genes ERG11, ERG3, TAC1 and GSC1 (FKS1) in C. albicans, as well as ERG11, CgPDR1, FKS1 and FKS2 in C. glabrata were sequenced. RESULTS: Fifty-four different missense mutations were identified, 13 of which have not been reported before. All nine echinocandin-resistant Candida isolates showed mutations in the hot spot (HS) regions of FKS1, FKS2 or GSC1. In ERG3 two homozygous premature stop codons were identified in two highly azole-resistant and moderately echinocandin-resistant C. albicans strains. Seven point mutations in ERG11 were determined in azole-resistant C. albicans whereas in azole-resistant C. glabrata, no ERG11 mutations were detected. In 10 out of 13 azole-resistant C. glabrata, 12 different potential gain-of-function mutations in the transcription factor CgPDR1 were verified, which are associated with an overexpression of the efflux pumps CDR1/2. CONCLUSION: This study showed that next generation sequencing allows the thorough investigation of a large number of isolates more cost efficient and faster than conventional Sanger sequencing. Targeting different resistance genes and a large sample size of highly resistant strains allows a better determination of the relevance of the different mutations, and to differentiate between causal mutations and polymorphisms.

Emergence of a dalbavancin induced glycopeptide/lipoglycopeptide non-susceptible Staphylococcus aureus during treatment of a cardiac device-related endocarditis.

In the present study, we demonstrated the emergence of dalbavancin non-susceptible and teicoplanin-resistant Staphylococcus aureus small colony variants which were selected in vivo through long-term treatment with dalbavancin. A 36-year-old man presented with a cardiac device-related S. aureus endocarditis and received long-term therapy with dalbavancin. Consecutively, two glycopeptide/lipoglycopeptide susceptible and two non-susceptible S. aureus isolates were obtained from blood cultures and the explanted pacemaker wire. The isolates were characterized by: standard typing methods, antimicrobial susceptibility testing, auxotrophic profiling, proliferation assays, scanning and transmission electron microscopy, as well as whole genome sequencing. The isolated SCVs demonstrated a vancomycin-susceptible but dalbavancin non-susceptible and teicoplanin-resistant phenotype whereof the respective MICs of the last isolate were 16- and 84-fold higher than the susceptible strains. All four strains were indistinguishable or at least closely related by standard typing methods (spa, MLST, and PFGE), and whole genome sequencing revealed only eight sequence variants. A consecutive increase in cell wall thickness (up to 2.1-fold), an impaired cell separation with incomplete or multiple cross walls and significantly reduced growth rates were observed in the present study. Therefore, the mutations in pbp2 and the DHH domain of GdpP were identified as the most probable candidates due to their implication in the biosynthesis and metabolism of the staphylococcal cell wall. For the first time, we demonstrated in vivo induced dalbavancin non-susceptible/teicoplanin resistant, but vancomycin and daptomycin susceptible S. aureus SCVs without lipopeptide or glycopeptide pretreatment, thus, indicating the emergence of a novel lipoglycopeptide resistance mechanism.

Neutralization of antimicrobial substances in new BacT/Alert FA and FN Plus blood culture bottles.

Time to detection (TTD) in automated blood culture systems is delayed for sensitive microorganisms in the presence of antimicrobial substances and has been associated with worse outcomes for sepsis patients on inadequate empirical therapy. While resin addition removes antimicrobial substances to various degrees from blood culture media, media formulations and the blend of resins may influence performance. The BacT/Alert 3D system (bioMérieux) was investigated using the new resin-containing medium types FA Plus (aerobic) and FN Plus (anaerobic). TTD was compared between control and test bottles containing relevant bacteria or Candida albicans, with and without defined concentrations of antimicrobials. Failure of neutralization was defined as a negative blood culture on day 3. In general, growth delay was nonlinear, concentration dependent, bottle type specific, and reciprocally associated with MICs. Substance-specific serum drug concentrations corresponding to a predefined, clinically relevant 3-h delay of TTD were calculated. Where appropriate, a time interval allowing for drug elimination below this critical level was obtained by pharmacokinetic modeling. Clarithromycin, clindamycin, gentamicin, linezolid, tigecycline, vancomycin, and fluconazole were neutralized. For ciprofloxacin and piperacillin-tazobactam, which were only incompletely neutralized in combination with the most sensitive test strains, a maximum waiting time for blood draw of 1 h was determined based on pharmacokinetics. One or more test strains did not grow in bottles containing either amoxicillin-clavulanate, cefepime, cefotaxime, meropenem, or metronidazole, and we thus recommend particular caution in timing of blood draws if patients have been pretreated with these agents.

Community-acquired bacteria frequently detected by means of quantitative polymerase chain reaction in nosocomial early-onset ventilator-associated pneumonia.

OBJECTIVE: To test whether real-time polymerase chain reaction allows for rapid quantitative detection of Streptococcus pneumoniae, Chlamydia pneumoniae, Mycoplasma pneumoniae, and Legionella pneumophila in bronchoalveolar lavage fluids and to determine the prevalence of these pathogens in nosocomial ventilator-associated pneumonia. DESIGN: Prospective epidemiologic study applying a new molecular biology-based diagnostic tool during a 27-month period. SETTING: Three medical intensive care units of a tertiary care university hospital. PATIENTS: One hundred patients suffering from nosocomial ventilator-associated pneumonia, hospitalized for > or =14 days, intubated for reasons other than pneumonia, and mechanically ventilated for >48 hrs. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: S. pneumoniae, M. pneumoniae, and C. pneumoniae were detected in bronchoalveolar lavage fluids of 100 patients in 20 (20%), three (3%), and two (2%) cases, respectively. There of 17 (71%) revealed no growth or no significant growth by conventional culture. In one patient, S. pneumoniae and M. pneumoniae were detected simultaneously. Corresponding colony-forming units/mL were partly up to 10 CFU/mL with Gram stainings showing signs of acute inflammation in 80%. A significant temporary correlation between the number of days on ventilator, development of nosocomial pneumonia, and the frequency of detection of these pathogens was found for day 4. CONCLUSIONS: S. pneumoniae, M. pneumoniae, and C. pneumoniae should be considered as causative agents in critically ill patients who develop early-onset nosocomial ventilator-associated pneumonia. Thus, empirical antimicrobial regimens should cover S. pneumoniae, Chlamydia, and Mycoplasma alike. Quantitative polymerase chain reaction is a fast diagnostic tool allowing for detection of these bacteria within 3 hrs in pretreated patients.

No evidence of involvement of Chlamydia pneumoniae in severe cerebrovascular atherosclerosis by means of quantitative real-time polymerase chain reaction.

BACKGROUND AND PURPOSE: All studies reporting high numbers of Chlamydia pneumoniae DNA positives in stroke patients published to date have used polymerase chain reaction (PCR) techniques highly prone to generate false-positive results. The aim of this study was to analyze the prevalence of C. pneumoniae DNA in plaques of the carotid artery as well as in peripheral blood by means of a new, closed, real-time PCR system. METHODS: Carotid endarterectomy specimens and preoperative peripheral blood mononuclear cells (PBMC) of 75 individuals with severe cerebrovascular atherosclerosis were analyzed by means of a C. pneumoniae-specific quantitative ompA-based real-time PCR TaqMan system. Plaques were also cultured onto HEp-2 cells. Before the surgical intervention, C. pneumoniae-specific IgM, IgG, and IgA as well as C-reactive protein (CRP) levels were determined. RESULTS: 89% of all patients studied had C. pneumoniae-specific antibodies, but the pathogen was not detected in a single carotid atheroma by real-time PCR and cell culture. However, C. pneumoniae DNA was detected in 4 PBMC samples (5.3%) at very low levels (<1 inclusion/6 mL EDTA blood). No statistical significance was found between symptomatic/asymptomatic patients, C. pneumoniae PCR, results and CRP values after correction for multiplicity-of-test adjustment. CONCLUSIONS: By means of a closed, highly sensitive, and specific real-time PCR, C. pneumoniae was not detected in cerebrovascular atherosclerosis. PCR on PBMC was not predictive for endovascular chlamydia infection and most likely stem from previous C. pneumoniae respiratory tract infection in individual cases.

Comparison of a new quantitative ompA-based real-Time PCR TaqMan assay for detection of Chlamydia pneumoniae DNA in respiratory specimens with four conventional PCR assays.

Chlamydia pneumoniae, an important respiratory pathogen, is difficult to culture, and detection rates by conventional PCRs vary considerably. A new quantitative ompA-based real-time PCR assay based on TaqMan technology for detection of C. pneumoniae in respiratory samples is described, and its performance in terms of sensitivity and reproducibility is compared with those of four published conventional PCRs (one single-step PCR targeting a cloned PstI fragment; two nested PCRs, one targeting the 16S rRNA gene followed by hybridization and the other targeting the ompA gene; and a touchdown enzyme time-release [TETR] PCR also targeting the 16S rRNA gene). Both ompA-based PCRs showed the best analytical sensitivity. All five assays could detect even lower target levels from spiked sputum, with the 16S rRNA assays performing better than the ompA-based nested PCR (10(-6) inclusion-forming units [IFU] were detected in four of four and two of four replicates by the 16S rRNA TETR PCR and the 16S rRNA nested PCR, respectively). In general, the ompA-based real-time protocol produced the most consistent positive results for all replicates tested down to 10(-6) IFU. Eight of 45 patient sputum specimens (18%) were C. pneumoniae DNA positive in at least one of four replicates tested by at least one assay. Without taking into consideration the analytical sensitivity or the reproducibility of the test results, the numbers of C. pneumoniae DNA-positive sputum specimens (n = 8) were four, three, two, two, and one for the 16S rRNA TETR assay, the PstI-based single-step PCR, the ompA-based real-time PCR, the ompA-based nested touchdown PCR, and the 16S rRNA-based nested PCR, respectively. However, the overall rate of concordance of positive results was low. Only one cell culture-positive sputum specimen was positive by four of five assays (14 of 16 replicates; mean cycle threshold value, 25; 10(8) particles/ml of sputum). Thirty-seven specimens were C. pneumoniae negative by all five assays for all replicates tested, as were all negative controls (n = 65 to 100 per testing panel). No PCR inhibitors were detected by real-time PCR or by the 16S rRNA-based nested assay. We confirm that the analytical sensitivity of an assay for the detection of C. pneumoniae does not necessarily predict its ability to detect its target in sputum. A quantitative, fast, and easy-to-handle diagnostic approach such as the ompA-based real-time TaqMan PCR described here might improve the detection of C. pneumoniae in respiratory samples.

Reliability of nested PCR for detection of Chlamydia pneumoniae DNA in atheromas: results from a multicenter study applying standardized protocols.

The present multicenter study was designed to find explanations for the discrepancies in the reported rates of detection of Chlamydia pneumoniae DNA in endarterectomy specimens. Coded identical sets of (i) a C. pneumoniae DNA dilution series (panel 1; n = 10), (ii) spiked control tissue specimens (panel 2; n = 10 specimens, including 5 negative controls), and (iii) endarterectomy specimens (panel 3; 15 atheromas, 5 negative controls) were analyzed at four laboratories by three standardized DNA extraction methods in each laboratory and a nested touchdown PCR protocol targeting the ompA gene of C. pneumoniae. Panel 1 samples were correctly identified as positive to levels of 0.3 inclusion-forming units (IFU)/PCR mixture (100%) and 0.03 IFU/PCR mixture (50%). All negative controls were correctly reported as negative. Panel 2 samples were identified as C. pneumoniae positive to levels of 0.01 IFU/PCR mixture (100%) and 0.005 IFU/PCR mixture (91%), independent of the DNA extraction method used, and only one false-positive result was reported. For panel 3 samples, 5 of 240 (2%) analyses (in which DNA extractions and PCR were performed at the same laboratory) were positive; the positive specimens were from three endarterectomy specimens and two negative controls. After exchange of DNA extracts between laboratories, 13 of 15 atheroma samples were C. pneumoniae DNA positive in at least 1 of a series of 48 analyses per atheroma sample; however, the overall positivity rate did not exceed 5% (33 of 720 analyses) and therefore was lower than that for the negative controls (8%; 19 of 240 analyses). Not a single positive result could be achieved when all panel 3 extracts (n = 240 analyses) were reamplified by a 16S rRNA PCR followed by hybridization with a C. pneumoniae-specific probe. Statistical analyses demonstrated that positive results did not occur in an independent and random fashion and could most likely be explained by amplicon carryover at the nested PCR level as well as amplicon introduction during DNA extraction, but not by the patterns of distribution of very low target levels or a certain DNA extraction protocol. The results of studies by nested PCR for detection of the prevalence of C. pneumoniae will always be questionable.