Tracking Down the Cause of Necrotizing Fasciitis in a Patient with Negative Cultures

In a patient suspected of lower abdominal necrotizing fasciitis cultures remained negative, possibly because broad spectrum antibiotics had already been given before tissue for culture was obtained. 16S-23S rDNANext Generation Sequencing showed that 99.7% of bacterial DNA was of Streptococcus pyogenes. 16S-23S rDNA can replace culture for identification of bacteria, also in polymicrobial infection

Development and validation of a reference data set for assigning Staphylococcus species based on next-generation sequencing of the 16S-23S rRNA region

Many members of the Staphylococcus genus are clinically relevant opportunistic pathogens that warrant accurate and rapid identification for targeted therapy. The aim of this study was to develop a careful assignment scheme for staphylococcal species based on next-generation sequencing (NGS) of the 16S-23S rRNA region. All reference staphylococcal strains were identified at the species level using Sanger sequencing of the 16S rRNA, sodA, tuf, and rpoB genes and NGS of the 16S-23S rRNA region. To broaden the database, an additional 100 staphylococcal strains, including 29 species, were identified by routine diagnostic methods, 16S rRNA Sanger sequencing and NGS of the 16S-23S rRNA region. The results enabled development of reference sequences encompassing the 16S-23S rRNA region for 50 species (including one newly proposed species) and 6 subspecies of the Staphylococcus genus. This study showed sodA and rpoB targets were the most discriminative but NGS of the 16S-23S rRNA region was more discriminative than tuf gene sequencing and much more discriminative than 16S rRNA gene sequencing. Almost all Staphylococcus species could be distinguished when the max score was 99.0% or higher and the sequence similarity between the best and second best species was equal to or >0.2% (min. 9 nucleotides). This study allowed development of reference sequences for 21 staphylococcal species and enrichment for 29 species for which sequences were publicly available. We confirmed the usefulness of NGS of the 16S-23S rRNA region by identifying the whole species content in 45 clinical samples and comparing the results to those obtained using routine diagnostic methods. Based on the developed reference database, all staphylococcal species can be reliably detected based on the 16S-23S rRNA sequences in samples composed of both single species and more complex polymicrobial communities. This study will be useful for introduction of a novel diagnostic tool, which undoubtedly is an improvement for reliable species identification in polymicrobial samples. The introduction of this new method is hindered by a lack of reference sequences for the 16S-23S rRNA region for many bacterial species. The results will allow identification of all Staphylococcus species, which are clinically relevant pathogens

Development of a reference data set for assigning Streptococcus and Enterococcus species based on next generation sequencing of the 16S-23S rRNA region

Background: Many members of Streptococcus and Enterococcus genera are clinically relevant opportunistic pathogens warranting accurate and rapid identification for targeted therapy. Currently, the developed method based on next generation sequencing (NGS) of the 16S-23S rRNA region proved to be a rapid, reliable and precise approach for species identification directly from polymicrobial and challenging clinical samples. The introduction of this new method to routine diagnostics is hindered by a lack of the reference sequences for the 16S-23S rRNA region for many bacterial species. The aim of this study was to develop a careful assignment for streptococcal and enterococcal species based on NGS of the 16S-23S rRNA region. Methods: Thirty two strains recovered from clinical samples and 19 reference strains representing 42 streptococcal species and nine enterococcal species were subjected to bacterial identification by four Sanger-based sequencing methods targeting the genes encoding (i) 16S rRNA, (ii) sodA, (iii) tuf and (iv) rpoB; and NGS of the 16S-23S rRNA region. Results: This study allowed obtainment and deposition of reference sequences of the 16S-23S rRNA region for 15 streptococcal and 3 enterococcal species followed by enrichment for 27 and 6 species, respectively, for which reference sequences were available in the databases. For Streptococcus, NGS of the 16S-23S rRNA region was as discriminative as Sanger sequencing of the tuf and rpoB genes allowing for an unambiguous identification of 93% of analyzed species. For Enterococcus, sodA, tuf and rpoB genes sequencing allowed for identification of all species, while the NGS-based method did not allow for identification of only one enterococcal species. For both genera, the sequence analysis of the 16S rRNA gene was endowed with a low identification potential and was inferior to that of other tested identification methods. Moreover, in case of phylogenetically related species the sequence analysis of only the intergenic spacer region was not sufficient enough to precisely identify Streptococcus strains at the species level. Conclusions: Based on the developed reference dataset, clinically relevant streptococcal and enterococcal species can now be reliably identified by 16S-23S rRNA sequences in samples. This study will be useful for introduction of a novel diagnostic tool, NGS of the 16S-23S rRNA region, which undoubtedly is an improvement for reliable culture-independent species identification directly from polymicrobially constituted clinical samples

Is Shiga Toxin-Negative Escherichia coli O157:H7 Enteropathogenic or Enterohemorrhagic Escherichia coli? Comprehensive Molecular Analysis Using Whole-Genome Sequencing

The ability of Escherichia coli O157:H7 to induce cellular damage leading to disease in humans is related to numerous virulence factors, most notably the stx gene, encoding Shiga toxin (Stx) and carried by a bacteriophage. Loss of the Stx-encoding bacteriophage may occur during infection or culturing of the strain. Here, we collected stx-positive and stx-negative variants of E. coli O157:H7/NM (nonmotile) isolates from patients with gastrointestinal complaints. Isolates were characterized by whole-genome sequencing (WGS), and their virulence properties and phylogenetic relationship were determined. Because of the presence of the cae gene but lack of the bfpA gene, the stx-negative isolates were considered atypical enteropathogenic E. coli (aEPEC). However, they had phenotypic characteristics similar to those of the Shiga toxin-producing E. coli (STEC) isolates and belonged to the same sequence type, STI1. Furthermore, EPEC and STEC isolates shared similar virulence genes, the locus of enterocyte effacement region, and plasmids. Core genome phylogenetic analysis using a gene-by-gene typing approach showed that the sorbitol-fermenting (SF) stx-negative isolates clustered together with an SF STEC isolate and that one non-sorbitol-fermenting (NSF) stx-negative isolate clustered together with NSF STEC isolates. Therefore, these stx-negative isolates were thought either to have lost the Stx phage or to be a progenitor of STEC O157: H7/NM. As detection of STEC infections is often based solely on the identification of the presence of stx genes, these may be misdiagnosed in routine laboratories. Therefore, an improved diagnostic approach is required to manage identification, strategies for treatment, and prevention of transmission of these potentially pathogenic strains

Reprint of "Application of next generation sequencing in clinical microbiology and infection prevention"

Current molecular diagnostics of human pathogens provide limited information that is often not sufficient for outbreak and transmission investigation. Next generation sequencing (NGS) determines the DNA sequence of a complete bacterial genome in a single sequence run, and from these data, information on resistance and virulence, as well as information for typing is obtained, useful for outbreak investigation. The obtained genome data can be further used for the development of an outbreak-specific screening test. In this review, a general introduction to NGS is presented, including the library preparation and the major characteristics of the most common NGS platforms, such as the MiSeq (Illumina) and the Ion PGM (TM) (ThermoFisher). An overview of the software used for NGS data analyses used at the medical microbiology diagnostic laboratory in the University Medical Center Groningen in The Netherlands is given. Furthermore, applications of NGS in the clinical setting are described, such as outbreak management, molecular case finding, characterization and surveillance of pathogens, rapid identification of bacteria using the 16S-23S rRNA region, taxonomy, metagenomics approaches on clinical samples, and the determination of the transmission of zoonotic micro-organisms from animals to humans. Finally, we share our vision on the use of NGS in personalised microbiology in the near future, pointing out specific requirements

Improved Detection of Five Major Gastrointestinal Pathogens by Use of a Molecular Screening Approach▿ §

The detection of bacterial and parasitic gastrointestinal pathogens through culture and microscopy is laborious and time-consuming. We evaluated a molecular screening approach (MSA) for the detection of five major enteric pathogens: Salmonella enterica, Campylobacter jejuni, Giardia lamblia, Shiga toxin-producing Escherichia coli (STEC), and Shigella spp./enteroinvasive E. coli (EIEC), for use in the daily practice of a clinical microbiology laboratory. The MSA consists of prescreening of stool specimens with two real-time multiplex PCR (mPCR) assays, which give results within a single working day, followed by guided culture/microscopy of the positive or mPCR-inhibited samples. In the present 2-year overview, 28,185 stool specimens were included. The MSA was applied to 13,974 stool samples (49.6%), whereas 14,211 samples were tested by conventional methods only (50.4%). The MSA significantly increased the total detection rate compared to that of conventional methods (19.2% versus 6.4%). The detection of all included pathogens, with the exception of S. enterica, significantly improved. MSA detection frequencies were as follows: C. jejuni, 8.1%; G. lamblia, 4.7%; S. enterica, 3.0%; STEC, 1.9%; and Shigella spp./EIEC, 1.4%. The guided culture/microscopy was positive in 76.8%, 58.1%, 88.9%, 16.8%, and 18.1% of mPCR-positive specimens, respectively. Of all mPCRs, only 1.8% was inhibited. Other findings were that detection of mixed infections was increased (0.9% versus 0.02%) and threshold cycle (CT) values for MSA guided culture/microscopy-positive samples were significantly lower than those for guided culture/microscopy-negative samples. In conclusion, an MSA for detection of gastrointestinal pathogens resulted in markedly improved detection rates and a substantial decrease in time to reporting of (preliminary) results