lyng@icpmr.wsahs.nsw.gov.au

Tel:   (02) 9845-6255

CIDM – Public Health is a research group within the CIDM, which is one of the largest diagnostic and public health microbiology laboratories in Australia . Research in CIDM-PH is directed towards understanding of the epidemiology and pathogenesis of communicable diseases of public health importance, development of better methods of diagnosis and typing of micro-organisms, for surveillance and out break investigation and ultimately to prevention of disease and improvement in disease outcomes. This project is one of a number of current projects, using similar technology – multiplex PCR and reverse line blot assay – as a mean so “fingerprinting” bacterial strains to better understand the genetic basis of virulence and molecular epidemiology

Project:  Classification of pathogenic bacteria using multi locus gene sequencing (2009)

Background:  The 16S rRNA gene has been utilised for the identification of a large number of potential bacterial pathogens using ‘broad range’ primers. Although these amplification targets are highly conserved, limited homology between target genes and primers may preclude adequate gene amplification. 16S rRNA gene sequences from related bacteria may also be insufficiently polymorphic to permit their confident distinction. The rpoB gene, or the DNA-dependent RNA polymerase beta-subunit gene, has been proposed as a genome similarity predictor and as an alternative to 16S rRNA for phylogenetic and biodiversity studies. This gene is common to all bacteria, exists as a single copy in the genome and is a mosaic of conserved and variable sequence domains. It has been applied to a number of taxa, including the staphylococci, Enterobacteriaceae, Pseudomonas and Corynebacteria. This project tests the hypothesis that a multilocus sequence strategy may improve identification and classification of bacteria.

Aims:  To explore the effectiveness of using the sequencing of rpoB and 16S rRNA genes for the taxonomic and phylogenetic classification of clinically significant bacteria.

Methods:   Assay design and evaluation. The student will design universal primers and validate nucleic acid amplification methodology exploring DNA extraction, PCR and product purification and sequencing protocols. Selection of isolates. A total of 100 bacterial isolates stored in the Centre for Infectious Diseases and Microbiology (CIDM) Culture Collection and representing Streptococci and selection of nonfermentative and fermentative gram-negative bacteria will be used in the study. All of them are retrospective clinical isolates stored at –80C and passaged through BA plate prior to DNA extraction. ATCC strains will be used as controls. Analysis. Genomic sequences will be analysed and compared to those available in GenBank and genetic distances calculated using bioinformatics software packages. The relationships between complete sequences will be studied and discriminative partial sequences of the rpoB gene for different bacterial genera will be identified.

Significance:  The accurate and timely identification of clinically significant but fastidious bacteria is important for patient management and public health. The successful implementation of a ‘universal’ PCR for bacterial identification may lead to the development of new culture-independent methods of pathogen detection. Furthermore, these studies may lead to the identification of novel organisms and new associations of established species with human disease.

Selected Reference (Available on request)

• Adderson EE, et al. Identification of clinical coryneform bacterial isolates: Comparison of biochemical methods and sequence analysis of 16S rRNA and rpoB genes. J Clin Microbiol 2008;46:921-927.

• La Scola B, et al. Sequencing of the rpoB gene and flanking spacers for molecular identification of Acinetobacter species. J Clin Microbiol 2006;44:827-832.

• Mollet C, Drancourt M, Raoult D. rpoB sequence analysis as a novel basis for bacterial identification. Mol Microbiol 1997;26:1005-1011.

Study of genetic diversity of Campylobacter species based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs)

Background: Infection with Campylobacter spp. is the most frequently notified foodborne disease in Australia. Campylobacter infections have been notifiable in all Australian states except NSW for several years, because of its prevalence. The number of cases per year has continued to increase since the start of the notification. However, because infection is so common and the methods for subtyping Campylobacter spp. are unsatisfactory – not sufficiently discriminatory, too expensive and/or poorly reproducible – identification of outbreaks is difficult and therefore public health action is rarely possible.    

C. jejuni and C. coli are the two most common pathogens isolated from humans but their pathogenicity and epidemiology of infection are poorly understood, in part because of the lack of reliable typing methods. A robust, discriminatory typing method, which could be used routinely, would assist in rapid recognition of outbreaks and source identification, would improve understanding of global epidemiology, including risk factors and assist in disease control.

Clustered regularly interspaced short palindromic repeats (CRISPRs) arre distributed widely in bacteria and Archaea. They are the arrays of short direct repeats that are interspaced with non-repetitive variable sequences or spacers. It has been suggested that CRISPRs may be involved in replicon partitioning, DNA recombination and repair and resistance of host (bacterial) cell to foreign DNA.  The direct repeats in CRISPRs differ between species and spacer sequences are generally unique and similar to those of known phages and plasmids. The CRISPR region has been utilised for typing of several bacterial species, including Mycobacteria tuberculosis, by differentiation of spacers. Based on the information in GenBank, at least one CRISPR region has been found in C. jejuni strains, with variable numbers of spacers identified. The CRISPR region could be a potentially suitable target for differentiation of C. jejuni (and potentially C. coli) strains which could be used for routine strain typing and surveillance of campylobacteriosis.

Aims: To explore the genetic diversity of the CRISPR region in Campylobacter species by detection of polymorphism in direct repeats and spacers and to test its discriminatory power for epidemiological purposes.

Methods: Assay design. PCR primers will be designed to amplify the CRISPR region and to validate PCR amplification methodology. Based on the spacer sequences identified by sequencing of amplified CRISPR regions from representative clinical isolates and spacer sequences provided in GenBank, probes will be designed for reverse line blot hybridization, which will be the major technique used for the detection of spacer polymorphism.

Other molecular techniques will involve DNA extraction, PCR product purification and sequencing (including primer walking), and multiplex PCR.

Selectionof isolates. ~90 sporadic and 30-40 outbreak isolates of Campylobacter (including C. jejuni and C. coli) and some ATCC strains stored in the Centre for Infectious Diseases and Microbiology (CIDM) Culture Collection will be used in the study.

Analysis. Direct repeat and spacer sequences will be analysed and compared to those available in GenBank.  The genetic polymorphism among the isolates will be analysed by comparing their RLB patterns and genetic distances calculated using bioinformatics software (Bionumerics).

Significance: By conducting this project we can explore the genetic diversity presented in the CRISPR region and its possible role in pathogenicity, transmissibility, host specificity and evolution. A rapid, accurate strain typing method is important for the identification of outbreak-related strains and differentiation of epidemic from sporadic isolates, which will provide a reliable epidemiological tool for outbreak investigation. 

Contact: ProfLyn Gilbert, Centre for Infectious Diseases and Microbiology, ICPMR, Westmead Hospital (l.gilbert@usyd.edu.au).

Selected Reference (Available on request)

• Miller, M., P. Roche, K. et al. (2005). Australia’s notifiable diseases status, 2003 annual report of the National Notifiable Diseases Surveillance System. Commun. Dis. Intell. 29:1–61.

• O'Reilly LC. Inglis TJ. Unicomb L. Australian Campylobacter Subtyping Study Group. Australian multicentre comparison of subtyping methods for the investigation of Campylobacter infection. Epidemiology & Infection. 134:768-79, 2006.

• Bolotin, A., B. Quinquis, et al. (2005). "Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin." Microbiology 151(Pt 8): 2551-61.

• Kamerbeek, J., L. Schouls, et al. (1997). "Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology." J Clin Microbiol 35(4): 907-14.