Complete Genome Sequences of Two Methicillin-Resistant Staphylococcus haemolyticus Isolates of Multilocus Sequence Type 25, First Detected by Shotgun Metagenomics

The emergence of nosocomial infections by multidrug-resistantStaphylococcus haemolyticusisolates has been reported in several European countries. Here, we report the first two complete genome sequences ofS. haemolyticussequence type 25 (ST25) isolates 83131A and 83131B. Both isolates were isolated from the same clinical sample and were first identified through shotgun metagenomics

Complete Genome Sequences of Two Methicillin-Resistant Staphylococcus haemolyticus Isolates of Multilocus Sequence Type 25, First Detected by Shotgun Metagenomics

The emergence of nosocomial infections by multidrug-resistantStaphylococcus haemolyticusisolates has been reported in several European countries. Here, we report the first two complete genome sequences ofS. haemolyticussequence type 25 (ST25) isolates 83131A and 83131B. Both isolates were isolated from the same clinical sample and were first identified through shotgun metagenomics

Complete Coding Sequences of Five Dengue Virus Type 2 Clinical Isolates from Venezuela Obtained through Shotgun Metagenomics

Dengue is a disease endemic in Latin American countries, like Venezuela, and has become one of the most important public health problems. We report five complete coding sequences of dengue virus serotype 2 (DENV-2) isolated from DENV-infected patients in Venezuela. Phylogenetic analysis placed the isolates within the American/Asian genotype

Connections between Exoproteome Heterogeneity and Virulence in the Oral Pathogen Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterial pathogen associated with severe periodontitis and nonoral diseases. Clinical isolates of A. actinomycetemcomitans display a rough (R) colony phenotype with strong adherent properties. Upon prolonged culturing, nonadherent strains with a smooth (S) colony phenotype emerge. To date, most virulence studies on A. actinomycetemcomitans have been performed with S strains of A. actinomycetemcomitans, whereas the virulence of clinical R isolates has received relatively little attention. Since the extracellular proteome is the main bacterial reservoir of virulence factors, the present study was aimed at a comparative analysis of this subproteome fraction for a collection of R isolates and derivative S strains, in order to link particular proteins to the virulence of A. actinomycetemcomitans with serotype b. To assess the bacterial virulence, we applied different infection models based on larvae of the greater wax moth Galleria mellonella, a human salivary gland-derived epithelial cell line, and freshly isolated neutrophils from healthy human volunteers. A total number of 351 extracellular A. actinomycetemcomitans proteins was identified by mass spectrometry, with the S strains consistently showing more extracellular proteins than their parental R isolates. A total of 50 known extracellular virulence factors was identified, of which 15 were expressed by all investigated bacteria. Importantly, the comparison of differences in exoproteome composition and virulence highlights critical roles of 10 extracellular proteins in the different infection models. Together, our findings provide novel clues for understanding the virulence of A. actinomycetemcomitans and for development of potential preventive or therapeutic avenues to neutralize this important oral pathogen. IMPORTANCE Periodontitis is one of the most common inflammatory diseases worldwide, causing high morbidity and decreasing the quality of life of millions of people. The bacterial pathogen Aggregatibacter actinomycetemcomitans is strongly associated with aggressive forms of periodontitis. Moreover, it has been implicated in serious nonoral infections, including endocarditis and brain abscesses. Therefore, it is important to investigate how A. actinomycetemcomitans can cause disease. In the present study, we applied a mass spectrometry approach to make an inventory of the virulence factors secreted by different clinical A. actinomycetemcomitans isolates and derivative strains that emerged upon culturing. We subsequently correlated the secreted virulence factors to the pathogenicity of the investigated bacteria in different infection models. The results show that a limited number of extracellular virulence factors of A. actinomycetemcomitans have central roles in pathogenesis, indicating that they could be druggable targets to prevent or treat oral disease

Long-read sequencing-based in silico phage typing of vancomycin-resistant Enterococcus faecium

Abstract Background Vancomycin-resistant enterococci (VRE) are successful nosocomial pathogens able to cause hospital outbreaks. In the Netherlands, core-genome MLST (cgMLST) based on short-read sequencing is often used for molecular typing. Long-read sequencing is more rapid and provides useful information about the genome’s structural composition but lacks the precision required for SNP-based typing and cgMLST. Here we compared prophages among 50 complete E. faecium genomes belonging to different lineages to explore whether a phage signature would be usable for typing and identifying an outbreak caused by VRE. As a proof of principle, we investigated if long-read sequencing data would allow for identifying phage signatures and thereby outbreak-related isolates. Results Analysis of complete genome sequences of publicly available isolates showed variation in phage content among different lineages defined by MLST. We identified phage present in multiple STs as well as phages uniquely detected within a single lineage. Next, in silico phage typing was applied to twelve MinION sequenced isolates belonging to two different genetic backgrounds, namely ST117/CT24 and ST80/CT16. Genomic comparisons of the long-read-based assemblies allowed us to correctly identify isolates of the same complex type based on global genome architecture and specific phage signature similarity. Conclusions For rapid identification of related VRE isolates, phage content analysis in long-read sequencing data is possible. This allows software development for real-time typing analysis of long-read sequencing data, which will generate results within several hours. Future studies are required to assess the discriminatory power of this method in the investigation of ongoing outbreaks over a longer time period

Evaluation of an Accelerated Workflow for Surveillance of ESBL (CTX-M)-Producing Escherichia coli Using Amplicon-Based Next-Generation Sequencing and Automated Analysis

Outbreak management of extended spectrum β-lactamase (ESBL)-producing pathogens requires rapid and accurate diagnosis. However, conventional screening is slow and labor-intensive. The vast majority of the screened samples are negative and detection of non-outbreak-related resistant micro-organisms often complicates outbreak management. In a CTX-M-15-producing Escherichia coli outbreak, 149 fecal samples and rectal eSwabs were collected by a cross-sectional survey in a Dutch nursing home. Samples were processed by routine diagnostic methods. Retrospectively, ESBL-producing bacteria and resistance genes were detected directly from eSwab medium by an accelerated workflow without prior enrichment cultures by an amplicon-based next-generation sequencing (NGS) method, and culture. A total of 27 (18.1%) samples were positive in either test. Sensitivity for CTX-M detection was 96.3% for the phenotypic method and 85.2% for the NGS method, and the specificity was 100% for both methods, as confirmed by micro-array. This resulted in a positive predictive value (PPV) of 100% for both methods, and a negative predictive value (NPV) of 99.2% and 96.8% for the phenotypic method and the NGS method, respectively. Time to result was four days and 14 h for the phenotypic method and the NGS method, respectively. In conclusion, the sensitivity without enrichment shows promising results for further use of amplicon-based NGS for screening during outbreaks

Use of whole-genome sequencing to trace, control and characterize the regional expansion of extended-spectrum beta-lactamase producing ST15 Klebsiella pneumoniae

The study describes the transmission of a CTX-M-15-producing ST15 Klebsiella pneumoniae between patients treated in a single center and the subsequent inter-institutional spread by patient referral occurring between May 2012 and September 2013. A suspected epidemiological link between clinical K. pneumoniae isolates was supported by patient contact tracing and genomic phylogenetic analysis from May to November 2012. By May 2013, a patient treated in three institutions in two cities was involved in an expanding cluster caused by this high-risk clone (HiRiC) (local expansion, CTX-M-15 producing, and containing hypervirulence factors). A clone-specific multiplex PCR was developed for patient screening by which another patient was identified in September 2013. Genomic phylogenetic analysis including published ST15 genomes revealed a close homology with isolates previously found in the USA. Environmental contamination and lack of consistent patient screening were identified as being responsible for the clone dissemination. The investigation addresses the advantages of wholegenome sequencing in the early detection of HiRiC with a high propensity of nosocomial transmission and prolonged circulation in the regional patient population. Our study suggests the necessity for interinstitutional/regional collaboration for infection/outbreak management of K. pneumoniae HiRiCs

Low anti-staphylococcal IgG responses in granulomatosis with polyangiitis patients despite long-term Staphylococcus aureus exposure

Chronic nasal carriage of the bacterium Staphylococcus aureus in patients with the autoimmune disease granulomatosis with polyangiitis (GPA) is a risk factor for disease relapse. To date, it was neither known whether GPA patients show similar humoral immune responses to S. aureus as healthy carriers, nor whether specific S. aureus types are associated with GPA. Therefore, this study was aimed at assessing humoral immune responses of GPA patients against S. aureus antigens in relation to the genetic diversity of their nasal S. aureus isolates. A retrospective cohort study was conducted, including 85 GPA patients and 18 healthy controls (HC). Humoral immune responses against S. aureus were investigated by determining serum IgG levels against 59 S. aureus antigens. Unexpectedly, patient sera contained lower anti-staphylococcal IgG levels than sera from HC, regardless of the patients' treatment, while total IgG levels were similar or higher. Furthermore, 210 S. aureus isolates obtained from GPA patients were characterized by different typing approaches. This showed that the S. aureus population of GPA patients is highly diverse and mirrors the general S. aureus population. Our combined findings imply that GPA patients are less capable of mounting a potentially protective antibody response to S. aureus than healthy individuals

Critical steps in clinical shotgun metagenomics for the concomitant detection and typing of microbial pathogens

High throughput sequencing has been proposed as a one-stop solution for diagnostics and molecular typing directly from patient samples, allowing timely and appropriate implementation of measures for treatment, infection prevention and control. However, it is unclear how the variety of available methods impacts the end results. We applied shotgun metagenomics on diverse types of patient samples using three different methods to deplete human DNA prior to DNA extraction. Libraries were prepared and sequenced with Illumina chemistry. Data was analyzed using methods likely to be available in clinical microbiology laboratories using genomics. The results of microbial identification were compared to standard culture-based microbiological methods. On average, 75% of the reads corresponded to human DNA, being a major determinant in the analysis outcome. None of the kits was clearly superior suggesting that the initial ratio between host and microbial DNA or other sample characteristics were the major determinants of the proportion of microbial reads. Most pathogens identified by culture were also identified through metagenomics, but substantial differences were noted between the taxonomic classification tools. In two cases the high number of human reads resulted in insufficient sequencing depth of bacterial DNA for identification. In three samples, we could infer the probable multilocus sequence type of the most abundant species. The tools and databases used for taxonomic classification and antimicrobial resistance identification had a key impact on the results, recommending that efforts need to be aimed at standardization of the analysis methods if metagenomics is to be used routinely in clinical microbiology

Author Correction: Critical steps in clinical shotgun metagenomics for the concomitant detection and typing of microbial pathogens (vol 8, 13767, 2018)

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