Core genome multilocus sequence typing (cgMLST) applicable to the monophyletic Klebsiella oxytoca species complex.

The environmental bacterium Klebsiella oxytoca displays an alarming increase of antibiotic-resistant strains that frequently cause outbreaks in intensive care units. Due to its prevalence in the environment and opportunistic presence in humans, molecular surveillance (including resistance marker screening) and high-resolution cluster analysis are of high relevance. Furthermore, K. oxytoca previously described in studies is rather a species complex (KoSC) than a single species comprising at least six closely related species that are not easily differentiated by standard typing methods. To reach a discriminatory power high enough to identify and resolve clusters within these species, whole genome sequencing is necessary. The resolution is achievable with core genome multilocus sequence typing (cgMLST) extending typing of a few housekeeping genes to thousands of core genome genes. CgMLST is highly standardized and provides a nomenclature enabling cross laboratory reproducibility and data exchange for routine diagnostics. Here, we established a cgMLST scheme not only capable of resolving the KoSC species but also producing reliable and consistent results for published outbreaks. Our cgMLST scheme consists of 2,536 core genome and 2,693 accessory genome targets, with a percentage of good cgMLST targets of 98.31% in 880 KoSC genomes downloaded from the National Center for Biotechnology Information (NCBI). We also validated resistance markers against known resistance gene patterns and successfully linked genetic results to phenotypically confirmed toxic strains carrying the til gene cluster. In conclusion, our novel cgMLST enables highly reproducible typing of four different clinically relevant species of the KoSC and thus facilitates molecular surveillance and cluster investigations.

Correction: GABenchToB: A Genome Assembly Benchmark Tuned on Bacteria and Benchtop Sequencers.

[This corrects the article DOI: 10.1371/journal.pone.0107014.].

Long-term changes in the subgingival microbiota in patients with stage III-IV periodontitis treated by mechanical therapy and adjunctive systemic antibiotics: A secondary analysis of a randomized controlled trial.

AIM: To explore whether adjunctive antibiotics can relevantly influence long-term microbiota changes in stage III-IV periodontitis patients. MATERIALS AND METHODS: This is a secondary analysis of a randomized clinical trial on periodontal therapy with adjunctive 500 mg amoxicillin and 400 mg metronidazole or placebo thrice daily for 7 days. Subgingival plaque samples were taken before and 2, 8, 14 and 26 months after mechanical therapy. The V4-hypervariable region of the 16S rRNA gene was sequenced with Illumina MiSeq 250 base pair paired-end reads. Changes at the ribosomal sequence variant (RSV) level, diversity and subgingival-microbial dysbiosis index (SMDI) were explored with a negative binomial regression model and non-parametric tests. RESULTS: Overall, 50.2% of all raw reads summed up to 72 RSVs (3.0%) that were generated from 163 stage III-IV periodontitis patients. Of those, 16 RSVs, including Porphyromonas gingivalis, Tannerella forsythia and Aggregatibacter actinomycetemcomitans, changed significantly over 26 months because of adjunctive systemic antibiotics. SMDI decreased significantly more in the antibiotic group at all timepoints, whereas the 2-month differences in alpha and beta diversity between groups were not significant at 8 and 14 months, respectively. CONCLUSIONS: Mechanical periodontal therapy with adjunctive antibiotics induced a relevant and long-term sustainable change towards an oral microbiome more associated with oral health.

Multicenter Preparedness Exercise Enables Rapid Development of Cluster-Specific PCR-Based Screening Assays from Bacterial Genomic Data.

PCR-based screening assays targeting strain-specific genetic markers allow the timely detection and specific differentiation of bacterial strains. Especially in situations where an infection cluster occurs, fast assay development is crucial for supporting targeted control measures. However, the turnaround times (TATs) for assay setup may be high due to insufficient knowledge about screening assay methods, workflows, and software tools. Here, two blind-coded and quality-controlled ring trials were performed in which five German laboratories established PCR-based screening assays from genomic data that specifically target selected bacterial clusters within two bacterial monospecies sample panels. While the first ring trial was conducted without a time limit to train the participants and assess assay feasibility, in the second ring trial, a challenging time limit of 2 weeks was set to force fast assay development as soon as genomic data were available. During both ring trials, we detected high interlaboratory variability regarding the screening assay methods and targets, the TATs for assay setup, and the number of screening assays. The participants designed between one and four assays per cluster that targeted cluster-specific unique genetic sequences, genes, or single nucleotide variants using conventional PCRs, high-resolution melting assays, or TaqMan PCRs. Assays were established within the 2-week time limit, with TATs ranging from 4 to 13 days. TaqMan probe delivery times strongly influenced TATs. In summary, we demonstrate that a specific exercise improved the preparedness to develop functional cluster-specific PCR-based screening assays from bacterial genomic data. Furthermore, the parallel development of several assays enhances assay availability.

Development and Evaluation of a Core Genome Multilocus Sequencing Typing (cgMLST) Scheme for Serratia marcescens Molecular Surveillance and Outbreak Investigations.

Serratia marcescens can cause a range of severe infections and contributes to nosocomial outbreaks. Although whole-genome sequencing (WGS)-based typing is the standard method for molecular surveillance and outbreak investigation, there is no standardized analytic scheme for S. marcescens core genome multilocus sequence typing (cgMLST). Here, the development and evaluation of a S. marcescens cgMLST scheme is reported with the goal of enabling a standardized methodology and typing nomenclature. Four hundred ninety-one high-quality S. marcescens WGS data sets were extracted from public databases and-using the genomic sequence of NCBI reference strain S. marcescens Db11 (NZ_HG326223.1) as a starting point-all Db11 genes present in ≥97% data sets used to create a cgMLST scheme. The novel scheme was evaluated using WGS data from 24 outbreak investigations (n = 175 isolates) distributed over three continents. Analysis of Db11 genes within the 491 data sets identified 2,692 target genes present in ≥97% of genomes (mean, 99.1%; median, 99.9%). These genes formed the novel cgMLST scheme, covering 47.8% of nucleotides in the Db11 genome. Analyzing 175 isolates from 24 outbreaks using the novel scheme gave comparable results to previous typing efforts for both general groupings and allelic distances within clusters. In summary, a novel cgMLST scheme for S. marcescens was developed and evaluated. The scheme and its associated nomenclature will improve standardization of typing efforts for molecular surveillance and outbreak investigation, allowing better understanding of S. marcescens genomic epidemiology and facilitating interlaboratory comparisons.

Gut microbiome and plasma metabolome changes in rats after oral gavage of nanoparticles: sensitive indicators of possible adverse health effects.

BACKGROUND: The oral uptake of nanoparticles is an important route of human exposure and requires solid models for hazard assessment. While the systemic availability is generally low, ingestion may not only affect gastrointestinal tissues but also intestinal microbes. The gut microbiota contributes essentially to human health, whereas gut microbial dysbiosis is known to promote several intestinal and extra-intestinal diseases. Gut microbiota-derived metabolites, which are found in the blood stream, serve as key molecular mediators of host metabolism and immunity. RESULTS: Gut microbiota and the plasma metabolome were analyzed in male Wistar rats receiving either SiO2 (1000 mg/kg body weight/day) or Ag nanoparticles (100 mg/kg body weight/day) during a 28-day oral gavage study. Comprehensive clinical, histopathological and hematological examinations showed no signs of nanoparticle-induced toxicity. In contrast, the gut microbiota was affected by both nanoparticles, with significant alterations at all analyzed taxonomical levels. Treatments with each of the nanoparticles led to an increased abundance of Prevotellaceae, a family with gut species known to be correlated with intestinal inflammation. Only in Ag nanoparticle-exposed animals, Akkermansia, a genus known for its protective impact on the intestinal barrier was depleted to hardly detectable levels. In SiO2 nanoparticles-treated animals, several genera were significantly reduced, including probiotics such as Enterococcus. From the analysis of 231 plasma metabolites, we found 18 metabolites to be significantly altered in Ag-or SiO2 nanoparticles-treated rats. For most of these metabolites, an association with gut microbiota has been reported previously. Strikingly, both nanoparticle-treatments led to a significant reduction of gut microbiota-derived indole-3-acetic acid in plasma. This ligand of the arylhydrocarbon receptor is critical for regulating immunity, stem cell maintenance, cellular differentiation and xenobiotic-metabolizing enzymes. CONCLUSIONS: The combined profiling of intestinal microbiome and plasma metabolome may serve as an early and sensitive indicator of gut microbiome changes induced by orally administered nanoparticles; this will help to recognize potential adverse effects of these changes to the host.

Development and Validation of a Burkholderia pseudomallei Core Genome Multilocus Sequence Typing Scheme To Facilitate Molecular Surveillance.

Burkholderia pseudomallei causes the severe disease melioidosis. Whole-genome sequencing (WGS)-based typing methods currently offer the highest resolution for molecular investigations of this genetically diverse pathogen. Still, its routine application in diagnostic laboratories is limited by the need for high computing power, bioinformatic skills, and variable bioinformatic approaches, with the latter affecting the results. We therefore aimed to establish and validate a WGS-based core genome multilocus sequence typing (cgMLST) scheme, applicable in routine diagnostic settings. A soft defined core genome was obtained by challenging the B. pseudomallei reference genome K96243 with 469 environmental and clinical genomes, resulting in 4,221 core and 1,351 accessory targets. The scheme was validated with 320 WGS data sets. We compared our novel typing scheme with single nucleotide polymorphism-based approaches investigating closely and distantly related strains. Finally, we applied our scheme for tracking the environmental source of a recent infection. The validation of the scheme detected >95% good cgMLST target genes in 98.4% of the genomes. Comparison with existing typing methods revealed very good concordance. Our scheme proved to be applicable to investigating not only closely related strains but also the global B. pseudomallei population structure. We successfully utilized our scheme to identify a sugarcane field as the presumable source of a recent melioidosis case. In summary, we developed a robust cgMLST scheme that integrates high resolution, maximized standardization, and fast analysis for the nonbioinformatician. Our typing scheme has the potential to serve as a routinely applicable classification system in B. pseudomallei molecular epidemiology.

Nitrate-rich diet alters the composition of the oral microbiota in periodontal recall patients.

BACKGROUND: This follow-up study evaluated microbiome changes in periodontal recall patients after consuming a nitrate-rich diet that led to a marked decrease of gingival inflammation. METHODS: Subgingival microbial samples of 37 patients suffering from gingival inflammation with reduced periodontium were taken before professional mechanical plaque removal (baseline) and subsequently after 2 weeks of regularly consuming a lettuce juice beverage (day 14) containing a daily dosage of 200 mg of nitrate (test group, n = 18) or being void of nitrate (placebo group, n = 19). Three hundred base pairs paired-end sequencing of the V3-V4 hypervariable region of the 16S rDNA was performed. RESULTS: At baseline, there were no significant differences about the bacterial diversity parameters between the groups (Mann-Whitney U test). After intervention in the test group, Rothia and Neisseria, including species reducing nitrate, increased significantly (negative binomial regression model). Alpha diversity decreased significantly from 115.69 ± 24.30 to 96.42 ± 24.82 aRSVs/sample (P = 0.04, Wilcoxon signed-rank test), accompanied by a significant change in beta diversity (P < 0.001, PERMANOVA). In the control group, however, no genus changed significantly, and alpha-, as well as beta-diversity did not change significantly. CONCLUSIONS: The decrease of gingival inflammation in periodontal recall patients induced by a nitrate-rich diet is accompanied by significant compositional changes within the subgingival microbiome.

Establishment and Evaluation of a Core Genome Multilocus Sequence Typing Scheme for Whole-Genome Sequence-Based Typing of Pseudomonas aeruginosa.

The environmental bacterium Pseudomonas aeruginosa, particularly multidrug-resistant clones, is often associated with nosocomial infections and outbreaks. Today, core genome multilocus sequence typing (cgMLST) is frequently applied to delineate sporadic cases from nosocomial transmissions. However, until recently, no cgMLST scheme for a standardized typing of P. aeruginosa was available. To establish a novel cgMLST scheme for P. aeruginosa, we initially determined the breadth of the P. aeruginosa population based on MLST data with a Bayesian approach (BAPS). Using genomic data of representative isolates for the whole population and all 12 serogroups, we extracted target genes and further refined them using a random data set of 1,000 P. aeruginosa genomes. Subsequently, we investigated reproducibility and discriminatory ability with repeatedly sequenced isolates and isolates from well-defined outbreak scenarios, respectively, and compared clustering applying two recently published cgMLST schemes. BAPS generated seven P. aeruginosa groups. To cover these and all serogroups, 15 reference strains were used to determine genes common in all strains. After refinement with the data set of 1,000 genomes, the cgMLST scheme consisted of 3,867 target genes, which are representative of the P. aeruginosa population and highly reproducible using biological replicates. We finally evaluated the scheme by reanalyzing two published outbreaks where the authors used single-nucleotide polymorphism (SNP) typing. In both cases, cgMLST was concordant with the previous SNP results and the results of the two other cgMLST schemes. In conclusion, the highly reproducible novel P. aeruginosa cgMLST scheme facilitates outbreak investigations due to the publicly available cgMLST nomenclature.

Significant Short-Term Shifts in the Microbiomes of Smokers With Periodontitis After Periodontal Therapy With Amoxicillin & Metronidazole as Revealed by 16S rDNA Amplicon Next Generation Sequencing.

The aim of this follow-up study was, to compare the effects of mechanical periodontal therapy with or without adjunctive amoxicillin and metronidazole on the subgingival microbiome of smokers with periodontitis using 16S rDNA amplicon next generation sequencing. Fifty-four periodontitis patients that smoke received either non-surgical periodontal therapy with adjunctive amoxicillin and metronidazole (n = 27) or with placebos (n = 27). Subgingival plaque samples were taken before and two months after therapy. Bacterial genomic DNA was isolated and the V4 hypervariable region of the bacterial 16S rRNA genes was amplified. Up to 96 libraries were normalized and pooled for Illumina MiSeq paired-end sequencing with almost fully overlapping 250 base pairs reads. Exact ribosomal sequence variants (RSVs) were inferred with DADA2. Microbial diversity and changes on the genus and RSV level were analyzed with non-parametric tests and a negative binomial regression model, respectively. Before therapy, the demographic, clinical, and microbial parameters were not significantly different between the placebo and antibiotic groups. Two months after the therapy, clinical parameters improved and there was a significantly increased dissimilarity of microbiomes between the two groups. In the antibiotic group, there was a significant reduction of genera classified as Porphyromonas, Tannerella, and Treponema, and 22 other genera also decreased significantly, while Selenomonas, Capnocytophaga, Actinomycetes, and five other genera significantly increased. In the placebo group, however, there was not a significant decrease in periodontal pathogens after therapy and only five other genera decreased, while Veillonella and nine other genera increased. We conclude that in periodontitis patients who smoke, microbial shifts occurred two months after periodontal therapy with either antibiotics or placebo, but genera including periodontal pathogens decreased significantly only with adjunctive antibiotics.

No differences in microbiome changes between anti-adhesive and antibacterial ingredients in toothpastes during periodontal therapy.

AIM: This subgroup analysis of a 12-week randomized, double-blind, and two-center trial aimed to evaluate whether two different toothpaste formulations can differentially modulate the dental microbiome. MATERIAL AND METHODS: Forty one mild to moderate periodontitis patients used as an adjunct to periodontal treatment either a toothpaste with anti-adhesive zinc-substituted carbonated hydroxyapatite (HA) or with antimicrobial and anti-adhesive amine fluoride/stannous fluoride (AmF/SnF2 ) during a 12-week period. Plaque samples from buccal/lingual, interproximal, and subgingival sites were taken at baseline, 4 weeks after oral hygiene phase, and 8 weeks after periodontal therapy. Samples were analyzed with paired-end Illumina Miseq 16S rDNA sequencing. The differences and changes on community level (alpha and beta diversity) and on the level of single agglomerated ribosomal sequence variants (aRSV) were calculated with analysis of covariance (ANCOVA) and likelihood ratio test (LRT). RESULTS: Interproximal and subgingival sites harbored predominately Fusobacterium and Prevotella species associated with periodontitis, whereas buccal/lingual sites harbored mainly Streptococcus and Veillonella species associated with periodontal health. Alpha and beta diversity did not change noticeably differently between both toothpaste groups (P > 0.05, ANCOVA). Furthermore, none of the aRSVs showed a noticeably different change between the tested toothpastes during periodontal therapy (Padj . > 0.05, LRT). CONCLUSION: The use of a toothpaste containing anti-adhesive HA did not induce statistically noticeably different changes on microbial composition compared to an antimicrobial and anti-adhesive AmF/SnF2 formulation.

A Core Genome Multilocus Sequence Typing Scheme for Enterococcus faecalis.

Among enterococci, Enterococcus faecalis occurs ubiquitously, with the highest incidence of human and animal infections. The high genetic plasticity of E. faecalis complicates both molecular investigations and phylogenetic analyses. Whole-genome sequencing (WGS) enables unraveling of epidemiological linkages and putative transmission events between humans, animals, and food. Core genome multilocus sequence typing (cgMLST) aims to combine the discriminatory power of classical multilocus sequence typing (MLST) with the extensive genetic data obtained by WGS. By sequencing a representative collection of 146 E. faecalis strains isolated from hospital outbreaks, food, animals, and colonization of healthy human individuals, we established a novel cgMLST scheme with 1,972 gene targets within the Ridom SeqSphere+ software. To test the E. faecalis cgMLST scheme and assess the typing performance, different collections comprising environmental and bacteremia isolates, as well as all publicly available genome sequences from the NCBI and SRA databases, were analyzed. In more than 98.6% of the tested genomes, >95% good cgMLST target genes were detected (mean, 99.2% target genes). Our genotyping results not only corroborate the known epidemiological background of the isolates but exceed previous typing resolution. In conclusion, we have created a powerful typing scheme, hence providing an international standardized nomenclature that is suitable for surveillance approaches in various sectors, linking public health, veterinary public health, and food safety in a true One Health fashion.

Core Genome Multilocus Sequence Typing and Single Nucleotide Polymorphism Analysis in the Epidemiology of Brucella melitensis Infections.

The use of whole-genome sequencing (WGS) using next-generation sequencing (NGS) technology has become a widely accepted method for microbiology laboratories in the application of molecular typing for outbreak tracing and genomic epidemiology. Several studies demonstrated the usefulness of WGS data analysis through single-nucleotide polymorphism (SNP) calling from a reference sequence analysis for Brucella melitensis, whereas gene-by-gene comparison through core-genome multilocus sequence typing (cgMLST) has not been explored so far. The current study developed an allele-based cgMLST method and compared its performance to that of the genome-wide SNP approach and the traditional multilocus variable-number tandem repeat analysis (MLVA) on a defined sample collection. The data set was comprised of 37 epidemiologically linked animal cases of brucellosis as well as 71 isolates with unknown epidemiological status, composed of human and animal samples collected in Italy. The cgMLST scheme generated in this study contained 2,704 targets of the B. melitensis 16M reference genome. We established the potential criteria necessary for inclusion of an isolate into a brucellosis outbreak cluster to be ≤6 loci in the cgMLST and ≤7 in WGS SNP analysis. Higher phylogenetic distance resolution was achieved with cgMLST and SNP analysis than with MLVA, particularly for strains belonging to the same lineage, thereby allowing diverse and unrelated genotypes to be identified with greater confidence. The application of a cgMLST scheme to the characterization of B. melitensis strains provided insights into the epidemiology of this pathogen, and it is a candidate to be a benchmark tool for outbreak investigations in human and animal brucellosis.

Do we treat our patients or rather periodontal microbes with adjunctive antibiotics in periodontal therapy? A 16S rDNA microbial community analysis.

Empiric antibiotics are often used in combination with mechanical debridement to treat patients suffering from periodontitis and to eliminate disease-associated pathogens. Until now, only a few next generation sequencing 16S rDNA amplicon based publications with rather small sample sizes studied the effect of those interventions on the subgingival microbiome. Therefore, we studied subgingival samples of 89 patients with chronic periodontitis (solely non-smokers) before and two months after therapy. Forty-seven patients received mechanical periodontal therapy only, whereas 42 patients additionally received oral administered amoxicillin plus metronidazole (500 and 400 mg, respectively; 3x/day for 7 days). Samples were sequenced with Illumina MiSeq 300 base pairs paired end technology (V3 and V4 hypervariable regions of the 16S rDNA). Inter-group differences before and after therapy of clinical variables (percentage of sites with pocket depth ≥ 5mm, percentage of sites with bleeding on probing) and microbiome variables (diversity, richness, evenness, and dissimilarity) were calculated, a principal coordinate analysis (PCoA) was conducted, and differential abundance of agglomerated ribosomal sequence variants (aRSVs) classified on genus level was calculated using a negative binomial regression model. We found statistically noticeable decreased richness, and increased dissimilarity in the antibiotic, but not in the placebo group after therapy. The PCoA revealed a clear compositional separation of microbiomes after therapy in the antibiotic group, which could not be seen in the group receiving mechanical therapy only. This difference was even more pronounced on aRSV level. Here, adjunctive antibiotics were able to induce a microbiome shift by statistically noticeably reducing aRSVs belonging to genera containing disease-associated species, e.g., Porphyromonas, Tannerella, Treponema, and Aggregatibacter, and by noticeably increasing genera containing health-associated species. Mechanical therapy alone did not statistically noticeably affect any disease-associated taxa. Despite the difference in microbiome modulation both therapies improved the tested clinical parameters after two months. These results cast doubt on the relevance of the elimination and/or reduction of disease-associated taxa as a main goal of periodontal therapy.

Epidemiological information is key when interpreting whole genome sequence data - lessons learned from a large Legionella pneumophila outbreak in Warstein, Germany, 2013.

IntroductionWhole genome sequencing (WGS) is increasingly used in Legionnaires' disease (LD) outbreak investigations, owing to its higher resolution than sequence-based typing, the gold standard typing method for Legionella pneumophila, in the analysis of endemic strains. Recently, a gene-by-gene typing approach based on 1,521 core genes called core genome multilocus sequence typing (cgMLST) was described that enables a robust and standardised typing of L. pneumophila. Methods: We applied this cgMLST scheme to isolates obtained during the largest outbreak of LD reported so far in Germany. In this outbreak, the epidemic clone ST345 had been isolated from patients and four different environmental sources. In total 42 clinical and environmental isolates were retrospectively typed. Results: Epidemiologically unrelated ST345 isolates were clearly distinguishable from the epidemic clone. Remarkably, epidemic isolates split up into two distinct clusters, ST345-A and ST345-B, each respectively containing a mix of clinical and epidemiologically-related environmental samples. Discussion/conclusion: The outbreak was therefore likely caused by both variants of the single sequence type, which pre-existed in the environmental reservoirs. The two clusters differed by 40 alleles located in two neighbouring genomic regions of ca 42 and 26 kb. Additional analysis supported horizontal gene transfer of the two regions as responsible for the difference between the variants. Both regions comprise virulence genes and have previously been reported to be involved in recombination events. This corroborates the notion that genomic outbreak investigations should always take epidemiological information into consideration when making inferences. Overall, cgMLST proved helpful in disentangling the complex genomic epidemiology of the outbreak.

Development and evaluation of a core genome multilocus typing scheme for whole-genome sequence-based typing of Acinetobacter baumannii.

We have employed whole genome sequencing to define and evaluate a core genome multilocus sequence typing (cgMLST) scheme for Acinetobacter baumannii. To define a core genome we downloaded a total of 1,573 putative A. baumannii genomes from NCBI as well as representative isolates belonging to the eight previously described international A. baumannii clonal lineages. The core genome was then employed against a total of fifty-three carbapenem-resistant A. baumannii isolates that were previously typed by PFGE and linked to hospital outbreaks in eight German cities. We defined a core genome of 2,390 genes of which an average 98.4% were called successfully from 1,339 A. baumannii genomes, while Acinetobacter nosocomialis, Acinetobacter pittii, and Acinetobacter calcoaceticus resulted in 71.2%, 33.3%, and 23.2% good targets, respectively. When tested against the previously identified outbreak strains, we found good correlation between PFGE and cgMLST clustering, with 0-8 allelic differences within a pulsotype, and 40-2,166 differences between pulsotypes. The highest number of allelic differences was between the isolates representing the international clones. This typing scheme was highly discriminatory and identified separate A. baumannii outbreaks. Moreover, because a standardised cgMLST nomenclature is used, the system will allow inter-laboratory exchange of data.

High Interlaboratory Reproducibility and Accuracy of Next-Generation-Sequencing-Based Bacterial Genotyping in a Ring Trial.

Today, next-generation whole-genome sequencing (WGS) is increasingly used to determine the genetic relationships of bacteria on a nearly whole-genome level for infection control purposes and molecular surveillance. Here, we conducted a multicenter ring trial comprising five laboratories to determine the reproducibility and accuracy of WGS-based typing. The participating laboratories sequenced 20 blind-coded Staphylococcus aureus DNA samples using 250-bp paired-end chemistry for library preparation in a single sequencing run on an Illumina MiSeq sequencer. The run acceptance criteria were sequencing outputs >5.6 Gb and Q30 read quality scores of >75%. Subsequently, spa typing, multilocus sequence typing (MLST), ribosomal MLST, and core genome MLST (cgMLST) were performed by the participants. Moreover, discrepancies in cgMLST target sequences in comparisons with the included and also published sequence of the quality control strain ATCC 25923 were resolved using Sanger sequencing. All five laboratories fulfilled the run acceptance criteria in a single sequencing run without any repetition. Of the 400 total possible typing results, 394 of the reported spa types, sequence types (STs), ribosomal STs (rSTs), and cgMLST cluster types were correct and identical among all laboratories; only six typing results were missing. An analysis of cgMLST allelic profiles corroborated this high reproducibility; only 3 of 183,927 (0.0016%) cgMLST allele calls were wrong. Sanger sequencing confirmed all 12 discrepancies of the ring trial results in comparison with the published sequence of ATCC 25923. In summary, this ring trial demonstrated the high reproducibility and accuracy of current next-generation sequencing-based bacterial typing for molecular surveillance when done with nearly completely locked-down methods.

Real-Time Genome Sequencing of Resistant Bacteria Provides Precision Infection Control in an Institutional Setting.

The increasing prevalence of multidrug-resistant (MDR) bacteria is a serious global challenge. Here, we studied prospectively whether bacterial whole-genome sequencing (WGS) for real-time MDR surveillance is technical feasible, returns actionable results, and is cost-beneficial. WGS was applied to all MDR isolates of four species (methicillin-resistant Staphylococcus aureus [MRSA], vancomycin-resistant Enterococcus faecium, MDR Escherichia coli, and MDR Pseudomonas aeruginosa) at the University Hospital Muenster, Muenster, Germany, a tertiary care hospital with 1,450 beds, during two 6-month intervals. Turnaround times (TAT) were measured, and total costs for sequencing per isolate were calculated. After cancelling prior policies of preemptive isolation of patients harboring certain Gram-negative MDR bacteria in risk areas, the second interval was conducted. During interval I, 645 bacterial isolates were sequenced. From culture, TATs ranged from 4.4 to 5.3 days, and costs were €202.49 per isolate. During interval II, 550 bacterial isolates were sequenced. Hospital-wide transmission rates of the two most common species (MRSA and MDR E. coli) were low during interval I (5.8% and 2.3%, respectively) and interval II (4.3% and 5.0%, respectively). Cancellation of isolation of patients infected with non-pan-resistant MDR E. coli in risk wards did not increase transmission. Comparing sequencing costs with avoided costs mostly due to fewer blocked beds during interval II, we saved in excess of €200,000. Real-time microbial WGS in our institution was feasible, produced precise actionable results, helped us to monitor transmission rates that remained low following a modification in isolation procedures, and ultimately saved costs.

Impact of the Daily Use of a Microcrystal Hydroxyapatite Dentifrice on De Novo Plaque Formation and Clinical/Microbiological Parameters of Periodontal Health. A Randomized Trial.

AIM: This 12-week prospective, randomized, double-blind, two-center trial evaluated the impact of a microcrystalline zinc hydroxyapatite (mHA) dentifrice on plaque formation rate (PFR) in chronic periodontitis patients. We hypothesized that mHA precipitates cause delayed plaque development when compared to a fluoridated control (AmF/SnF2), and therefore would improve periodontal health. MATERIAL & METHODS: At baseline and after 4 and 12 weeks, PFR and other clinical and microbiological parameters were recorded. Seventy periodontitis patients received a mHA or AmF/SnF2 dentifrice as daily oral care without hygiene instructions. Four weeks after baseline, participants received full mouth debridement and continued using the dentifrices for another 8 weeks. RESULTS: Primary outcome PFR did not change statistically significantly from baseline to weeks 4 and 12, neither in mHA (n = 33; 51.7±17.2% vs. 48.5±16.65% vs. 48.4±19.9%) nor in AmF/SnF2-group (n = 34; 52.3±17.5% vs. 52.5±21.3% vs. 46.1±21.8%). Secondary clinical parameters such as plaque control record, gingival index, bleeding on probing, and pocket probing depth improved, but between-group differences were not statistically significant. Microbiological analyses showed similar slight decreases in colony-forming units in both groups. CONCLUSION: In patients with mild-to-moderate periodontitis, periodontal therapy and use of a mHA-or AmF/SnF2 dentifrice without instructions induced comparable improvements in periodontal health but did not significantly reduce the PFR. TRIAL REGISTRATION: ClincalTrials.gov NCT02697539.