Advances in Clinical Sample Preparation for Identification and Characterization of Bacterial Pathogens Using Metagenomics

Whole genome sequencing (WGS) plays an increasing role in communicable disease control through high-resolution outbreak tracing, laboratory surveillance and diagnostics. However, WGS has traditionally relied on microbial culture in order to obtain pathogen specific DNA for sequencing. This has severely limited the application of whole genome sequencing on pathogens with fastidious culturing requirements. In addition, the widespread adoption of culture-independent diagnostic tests has reduced availability of cultured isolates for confirmatory testing and surveillance. These recent developments have created demand for the implementation of techniques enabling direct sequencing of microbial genomes in clinical samples without having to culture an isolate. However, sequencing of specific organisms from clinical samples can be affected by high levels of contaminating DNA from the host and other commensal microorganisms. Several methods have been introduced for selective lysis of host cells and/or separate specific organisms from a clinical sample. This review examines the different approaches for sample preparation that have been used in diagnostic and public health laboratories for metagenomic sequencing

The Association of Mycobacterium avium subsp. paratuberculosis with Inflammatory Bowel Disease.

The association of Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) with Crohn's disease is a controversial issue. M. paratuberculosis is detected by amplifying the IS900 gene, as microbial culture is unreliable from humans. We determined the presence of M. paratuberculosis in patients with Crohn's disease (CD) (n = 22), ulcerative colitis (UC) (n = 20), aphthous ulcers (n = 21) and controls (n = 42) using PCR assays validated on bovine tissue. Culture from human tissue was also performed. M. paratuberculosis prevalence in the CD and UC groups was compared to the prevalence in age and sex matched non-inflammatory bowel disease controls. Patients and controls were determined to be M. paratuberculosis positive if all three PCR assays were positive. A significant association was found between M. paratuberculosis and Crohn's disease (p = 0.02) that was not related to age, gender, place of birth, smoking or alcohol intake. No significant association was detected between M. paratuberculosis and UC or aphthous ulcers; however, one M. paratuberculosis isolate was successfully cultured from a patient with UC. We report the resistance of this isolate to ethambutol, rifampin, clofazamine and streptomycin. Interestingly this isolate could not only survive but could grow slowly at 5°C. We demonstrate a significant association between M. paratuberculosis and CD using multiple pre-validated PCR assays and that M. paratuberculosis can be isolated from patients with UC

Genome-wide comparison of Corynebacterium diphtheriae isolates from Australia identifies differences in the Pan-genomes between respiratory and cutaneous strains

Abstract Background Corynebacterium diphtheriae is the main etiological agent of diphtheria, a global disease causing life-threatening infections, particularly in infants and children. Vaccination with diphtheria toxoid protects against infection with potent toxin producing strains. However a growing number of apparently non-toxigenic but potentially invasive C. diphtheriae strains are identified in countries with low prevalence of diphtheria, raising key questions about genomic structures and population dynamics of the species. This study examined genomic diversity among 48 C. diphtheriae isolates collected in Australia over a 12-year period using whole genome sequencing. Phylogeny was determined using SNP-based mapping and genome wide analysis. Results C. diphtheriae sequence type (ST) 32, a non-toxigenic clone with evidence of enhanced virulence that has been also circulating in Europe, appears to be endemic in Australia. Isolates from temporospatially related patients displayed the same ST and similarity in their core genomes. The genome-wide analysis highlighted a role of pilins, adhesion factors and iron utilization in infections caused by non-toxigenic strains. Conclusions The genomic diversity of toxigenic and non-toxigenic strains of C. diphtheriae in Australia suggests multiple sources of infection and colonisation. Genomic surveillance of co-circulating toxigenic and non-toxigenic C. diphtheriae offer new insights into the evolution and virulence of pathogenic clones and can inform targeted public health actions and policy. The genomes presented in this investigation will contribute to the global surveillance of C. diphtheriae both for the monitoring of antibiotic resistance genes and virulent strains such as those belonging to ST32

Comparative genomics between human and animal associated subspecies of the Mycobacterium avium complex : a basis for pathogenicity

Background: A human isolate of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis 43525) was sequenced and compared genomically to other mycobacterial pathogens. M. paratuberculosis 43525 was recently isolated from a patient with ulcerative colitis and belongs to the M. avium complex, a group known to infect both humans and animals. While M. paratuberculosis is a known pathogen of livestock, there are only 20 human isolates from the last 20 years, therefore we took the opportunity to perform a whole genome comparison between human and animal mycobacterial pathogens. We also compared virulence determinants such as the mycobactin cluster, PE/PPE genes and mammalian cell entry (mce) operons between MAC subspecies that infect animals and those that infect humans. M. tuberculosis was also included in these analyses given its predominant role as a human pathogen. Results: This genome comparison showed the PE/PPE profile of M. paratuberculosis 43525 to be largely the same as other M. paratuberculosis isolates, except that it had one PPE and one PE_PGRS protein that are only present in human MAC strains and M. tuberculosis. PE/PPE proteins that were unique to M. paratuberculosis 43525, M. avium subsp. hominissuis and a caprine M. paratuberculosis isolate, were also identified. In addition, the mycobactin cluster differed between human and animal isolates and a unique mce operon flanked by two mycobactin genes, mbtA and mbtJ, was identified in all available M. paratuberculosis genomes. Conclusions: Despite the whole genome comparison placing M. paratuberculosis 43525 as closely related to bovine M. paratuberculosis, key virulence factors were similar to human mycobacterial pathogens. This study highlights key factors of mycobacterial pathogenesis in humans and forms the basis for future functional studies.11 page(s

Schematic of genomic rearrangements and the IS<i>1311</i> locations in the K10 (Type II) (top), S397 (Type III) (middle) and the Telford (Type I) (bottom) closed reference genomes.

Analysis conducted using MAUVE genome alignment tool: blocks of the same colour indicate homologous genomic regions; lines connect these regions between the different reference strains. Coloured arrows indicate the IS1311 loci; arrows with a grey outline indicate a locus that contains the C-strain specific SNP. The colour of the arrow heads indicates analogous loci, as shown in Table 4.</p

Comparison of MIC values between ATCC19698 and isolate 43525.

<p>Comparison of MIC values between ATCC19698 and isolate 43525.</p

SNPs present in IS1311 used to differentiate between subspecies of M. avium and strains of MAP.

SNPs present in IS1311 used to differentiate between subspecies of M. avium and strains of MAP.</p