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Journal: Journal of clinical microbiology

183

Whole genome sequencing (WGS) is becoming available as a routine tool for clinical microbiology. If applied directly on clinical samples this could further reduce diagnostic time and thereby improve control and treatment. A major bottle-neck is the availability of fast and reliable bioinformatics tools. This study was conducted to evaluate the applicability of WGS directly on clinical samples and to develop easy-to-use bioinformatics tools for analysis of the sequencing data. Thirty-five random urine samples from patients with suspected urinary tract infections were examined using conventional microbiology, WGS of isolated bacteria and by directly sequencing on pellets from the urine. A rapid method for analyzing the sequence data was developed. Bacteria were cultivated from 19 samples, but only in pure culture from 17. WGS improved the identification of the cultivated bacteria and almost complete agreement was observed between phenotypic and predicted antimicrobial susceptibility. Complete agreement was observed between species identification, multi-locus-sequence typing and phylogenetic relationship for the Escherichia coli and Enterococcus faecalis isolates when comparing the results of WGS of cultured isolates and directly from the urine samples. Sequencing directly from the urine enabled bacterial identification in polymicrobic samples. Additional putative pathogenic strains were observed in some culture negative samples. WGS directly on clinical samples can provide clinically relevant information and drastically reduce diagnostic time. This may prove very useful, but the need for data analysis is still a hurdle to clinical implementation. To overcome this problem a publicly available bioinformatics tool was developed in this study.

Concepts: DNA, Bacteria, Molecular biology, Microbiology, Urinary tract infection, Urine, Escherichia coli, Biotechnology

154

The ability of Escherichia coli O157:H7 to induce cellular damage leading to disease in humans is related to numerous virulence factors, most notably stx gene encoding Shiga toxin (Stx), 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 (non-motile) 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 eae gene but lack of the bfpA gene, the stx-negative isolates were considered as atypical enteropathogenic E. coli (aEPEC). However, they had similar phenotypic characteristics as the Shiga toxin producing E. coli (STEC) isolates and belonged to the same sequence type ST11. 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 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, treatment strategy and prevention of transmission of these potentially pathogenic strains.

Concepts: DNA, Bacteria, Molecular biology, Genome, Escherichia coli, Plasmid, Escherichia coli O157:H7, Shiga toxin

153

Whole genome sequencing is becoming a leading technology in typing and epidemiology of microbial pathogens, but the increase in genomics information necessitates significant investment in bioinformatic resources and expertise, and currently used methodologies struggle with genetically heterogeneous bacteria such as the human gastric pathogen Helicobacter pylori. Here we demonstrate that the alignment-free analysis method Feature Frequency Profiling (FFP) can be used to rapidly construct phylogenetic trees of draft bacterial genome sequences on a standard desktop computer, and that the coupling with in silico genotyping methods gives useful information for comparative and clinical genomics and molecular epidemiology applications. FFP-phylogenetic trees of seven gastric Helicobacter species matched those obtained by analysis of 16S rDNA and ribosomal proteins, and FFP- and core genome single nucleotide polymorphism-based analysis of 63 H. pylori genomes showed again comparable phylogenetic clustering, consistent with genomotypes assigned using multi-locus sequence typing (MLST). Analysis of 377 H. pylori genomes highlighted conservation of genomotypes and linkage with phylogeographic characteristics, and predicted the presence of an incomplete or non-functional cag pathogenicity island in 18/276 genomes. In silico analysis of antibiotic susceptibility markers suggests that most H. pylori hspAmerind and hspEAsia isolates are predicted to carry the T2812C mutation conferring low level clarithromycin-resistance, while levels of metronidazole resistance were similar in all MLST-types. In conclusion, the use of FFP phylogenetic clustering and in silico genotyping allows determination of genome evolution and phylogeographic clustering, and can contribute to clinical microbiology by genomotyping for outbreak management, and the prediction of pathogenic potential and antibiotic susceptibility.

Concepts: DNA, Gene, Genetics, Bacteria, Microbiology, Genome, RNA, Helicobacter pylori

149

Matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) sample preparation methods including the direct, on-plate formic acid, and ethanol/formic acid tube extraction were evaluated for their ability to render highly pathogenic organisms non-viable and safe for handling in a Biosafety Level-2 laboratory. Of these, the tube extraction procedure was the most successful, with none of the tested strains surviving this sample preparation method. Tube extracts from several agents of bioterrorism and their near neighbors were analyzed in an eight laboratory study to examine the utility of the Bruker Biotyper and Vitek MS MALDI-TOF MS systems and their IVD, research use only, and Security-Relevant databases, as applicable, to accurately identify these agents. Forty-six distinct strains of Bacillus anthracis, Yersinia pestis, Francisella tularensis, Burkholderia mallei, Burkholderia pseudomallei, Clostridium botulinum, Brucella melitensis, Brucella abortus, Brucella suis, and Brucella canis were extracted and distributed to participating labs for analysis. A total of 35 near neighbor isolates were also analyzed.

Concepts: Bacteria, Mass spectrometry, Brucellosis, Burkholderia mallei, Burkholderia, Brucella, Burkholderia pseudomallei

148

Infection with Leishmania donovani is typically asymptomatic, but a significant number of individuals may progress to visceral leishmaniasis (VL), a deadly disease that threatens 200 million people in endemic areas. While diagnosis of acute VL has been simplified by the use of cost-effective confirmatory serological tests, similar standardized tools are not widely available for detecting asymptomatic infection which can be 4-20 times more prevalent than active disease. A simple and accurate serological test capable of detecting asymptomatic L. donovani infection will be useful for surveillance programs targeting VL control and elimination. To address this unmet need, we evaluated recombinant antigens for their ability to detect serum antibodies in 104 asymptomatic L. donovani infected individuals (qualified as positive for L. donovani-specific antibodies by direct agglutination test; DAT) from the VL hyperendemic Mymensingh district of Bangladesh. The novel proteins rKR95 and rTR18 possessed the greatest potential and detected 69% of DAT positive individuals, with rKR95 being more robust in reactivity. Agreement in results with individuals with high DAT responses, who are more likely to progress to VL disease, was 74%. When considered along with rK39, a gold standard antigen used to confirm clinical diagnosis of VL but which is now becoming widely used for surveillance, rKR95 and rTR18 conferred a sensitivity of 84% based on a theoretical combined estimate. Our data indicate that incorporating rKR95 and rTR18 with rK39 in serological tests amenable to rapid or high-throughput screening could enable simple and accurate detection of asymptomatic infection. Such tests will be important tools to measure L. donovani infection rates, a primary goal in surveillance and a critical measurement with which to assess elimination programs.

Concepts: Immune system, Antibody, Epidemiology, Leishmaniasis, Visceral leishmaniasis, Leishmania, Serology, Direct agglutination test

132

The subject of laboratory error was examined during a symposium at the ASM Microbe 2016 conference in Boston.….

Concepts: Subject, Object, Subject-object problem

45

Vibrio parahaemolyticus is the leading seafood-transmitted bacterial pathogen worldwide where it causes gastroenteritis and rarely lethal septicemia.….

Concepts: Immune system, Bacteria, Microbiology, United States, North America, Foodborne illness, Vibrio, Vibrio parahaemolyticus

42

Routine full characterization of Mycobacterium tuberculosis (TB) is culture-based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near point of care.We demonstrate a low-cost DNA extraction method for TB WGS direct from patient samples. We initially evaluated the method using the Illumina MiSeq sequencer (40 smear-positive respiratory samples, obtained after routine clinical testing, and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction was obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. Using an Illumina MiSeq/MiniSeq the workflow from patient sample to results can be completed in 44/16 hours at a reagent cost of £96/£198 per sample.We then employed a non-specific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis BCG strain (BCG), and to combined culture-negative sputum DNA and BCG DNA. For flowcell version R9.4, the estimated turnaround time from patient to identification of BCG, detection of pyrazinamide resistance, and phylogenetic placement was 7.5 hours, with full susceptibility results 5 hours later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of the MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis in direct samples.

Concepts: DNA, Molecular biology, Tuberculosis, Full genome sequencing, Mycobacterium, Mycobacterium tuberculosis, Mycobacterium bovis, Pyrazinamide

41

Our previous study showed that bacterial genomes can be identified using 16S rRNA sequencing in urine specimens of both symptomatic and asymptomatic patients who are culture negative using standard urine culture protocols. In the present study, we used a modified culture protocol that included plating larger volumes of urine, incubation under varied atmospheric conditions and prolonged incubation times to demonstrate that many of the organisms identified in urine by 16S rRNA gene sequencing are, in fact, cultivable using an expanded quantitative urine culture (EQUC) protocol. Sixty-five urine specimens (from 41 patients with overactive bladder and 24 controls) were examined using both the standard and EQUC culture techniques. Fifty-two of the 65 urine samples (80%) grew bacterial species using EQUC, while the majority of these [48/52 (92%)] were reported as no growth at 10(3) colony forming units (CFU)/mL by the clinical microbiology laboratory using the standard urine culture protocol. Thirty-five different genera and 85 different species were identified by EQUC. The most prevalent genera isolated were Lactobacillus (15%), followed by Corynebacterium (14.2%), Streptococcus (11.9%), Actinomyces (6.9%) and Staphylococcus (6.9%). Other genera commonly isolated include Aerococcus, Gardnerella, Bifidobacterium, and Actinobaculum. Our current study demonstrates that urine contains communities of living bacteria that comprise a resident female urine microbiota.

Concepts: Gene, Archaea, Bacteria, Gut flora, Biology, Organism, Ribosomal RNA, 16S ribosomal RNA

39

Human noroviruses are a major cause of viral gastroenteritis, with an estimated 3 million cases per year in the UK. Human noroviruses (HuNoVs) have recently been isolated from pet dogs in Europe (Summa et al, 2012), raising concerns about potential zoonotic infections. With 31% UK households owning a dog, this could prove an important transmission route. To examine this risk, canine tissues were studied for their ability to bind to HuNoV in vitro. In addition, canine stool samples were analysed for the presence of viral nucleic acid and canine serum samples were tested for the presence of anti-HuNoV antibodies. Results showed that seven different genotypes of HuNoV VLPs can bind to canine gastrointestinal tissue, suggesting that infection is at least theoretically possible. Although HuNoV RNA was not identified in stool samples from 248 dogs, serological evidence of previous exposure to HuNoV was obtained in 43/325 canine serum samples. Remarkably, canine seroprevalence to different HuNoV genotypes mirrored the seroprevalence in the human population. Though entry and replication within cells has not been demonstrated, the canine serological data indicates that dogs produce an immune response to HuNoV, implying productive infection. In conclusion this study reveals zoonotic implications for HuNoV, and to elucidate the significance of this finding, further epidemiological and molecular investigations will be essential.

Concepts: Immune system, Antibody, Virus, Europe, Diarrhea, Dog, Gastroenteritis, Norovirus