16S rRNA and Secondary Gene Target Sequencing on Pure Cultures

16S rRNA gene PCR*, sequencing, and phylogenetic analysis. Secondary gene target sequencing and phylogenetic analysis is used complementarily with 16S gene sequencing. The choice of specific secondary gene target is dependent on 16S gene sequencing results.

• Legionellosis

Pure cultures of isolate implicated in clinical illness or environmental isolates.

Slants or plates of any suitable media and swabs in transport media are all acceptable. Samples submitted for isolation and/or identification of strict anaerobes must be submitted under oxygen-free conditions. Pre-reduced anaerobically sterilized (PRAS)/Cary and Blair transport medium or PRAS Cooked Meat broth are acceptable.

Store and ship samples at room temperature.

Shipping of specimens shall be done by a TDG certified individual in accordance with TDG regulations. For additional information regarding classification of specimens for the purposes of shipping, consult either Part 2 Appendix 3 of the TDG Regulations or section 3.6.2 of the IATA Dangerous Goods Regulations as applicable.

Clinical illness with symptoms suggestive of bacterial infection

Completed Special Bacteriology requisition form detailing all patient information and relevant clinical information.  If possible, attach lab results that have already been done at local or provincial laboratories.

All patient and strain history must be included. Enteric bacteria, Mycobacteria, Aerobic Actinomycetes, common nosocomial agents (MRSA, VRE), Neisseria, Eukaryotes, and Risk Group 3 bacteria are NOT accepted by Special Bacteriology. Please note that duplicate bacterial isolates that have been isolated from the same patient, source, and identified as the same organism by the sender, will be rejected unless additional information is provided to warrant testing. Acceptance and/or rejection of the additional sample(s) is at the discretion of the Special Bacteriology lab.   Contact lab for further information.  Please refer to the Guide to Services for test services offered by the NML.

16S gene and secondary gene target (if applicable) sequencing results are compared against publically-available databases (GenBank) and known type strains.

15 calendar days for the 16S/secondary gene target final report. Report may be delayed in cases where staff or resources are limited, or for poor or slow growing organisms.  In cases where further testing is required, the final report may be delayed and status of request will be forwarded in a preliminary report. Final report is sent once all testing is complete.

1. Clinical and Laboratory Standards Institute.  2018. Interpretive Criteria for Identification of bacteria and Fungi by DNA Target Sequencing; Approved Guideline MM18-Ed2. Volume 38, Number 15. Wayne, PA.
2. Fry, N. K., S. Warwick, N. A. Saunders, and T.M. Embley. 1991. The use of 16S ribosomal RNA analyses to investigate the phylogeny of the family Legionellaceae. J. Gen. Microbiol. 137:1215-1222.
3. Khamis, A, Raoult, D, La Scola, B. 2004. rpoB Gene Sequencing for Identification of Corynebacterium Species. JCM 42: 3925-3931
4. Kolbert CP, Rys PN, Hopkins M, Lynch DT, Germer JJ, O’Sullivan CE, et al. 16S ribosomal DNA sequence analysis for identification of bacteria in a clinical microbiology laboratory.  In: Persing DH, Tenover FD, Versalovic J, Tang Y-W, Unger ER, Relman DA, White TJ, eds. Molecular microbiology: diagnostic principles and practice. Washington, DC: ASM Press 2004. p 361–77.
5. Ratcliff RM, Lanser JA, Manning PA, Heuzenroeder MW.  1998.  Sequence-based classification scheme for the genus Legionella targeting the mip gene.  J Clin Microbiol. 36(6):1560-7.
6. Yamamoto, S, PJM Bouvet, and S Harayama.  1999.  Phylogenetic structures of the genus Acinetobacter based on gyrB sequences: comparison with the grouping by DNA-DNA hybridization.  International Journal of Systematic Bacteriology.  49: 87-95.  

National case definition: Legionnaires' disease and Pontiac fever (Legionellosis)

Legionella