r.lan@unsw.edu.au

Molecular evolution of bacterial pathogens

We employ molecular and bioinformatic approaches to investigate the evolution of bacterial pathogens and emergence of pathogenic clones.

Project 1 Molecular evolution and single nucleotide polymorphismtyping of Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium is a common cause of salmonellosis among humans and domestic animals worldwide. Effective surveillance and control of Typhimurium pathogens require sensitive and accurate subtyping of strains to determine the potential sources of infection.

We have used a novel method of mutation detection to show that there is a low but detectable level of mutational variation within the Typhimurium clone. Mutational changes, which accumulate over time, would reflect true genetic relationships of isolates. This project is to develop a typing scheme using single nucleotide polymorphisms (SNPs). A SNP (‘snip’) is a variation at an individual nucleotide level where a single base differs between individual sequences. SNPs will be identified from genome sequence comparisons and selected for typing of worldwide isolates. The project will involve analysis of genome sequences, development of novel real-time PCR assays and sequencing to identify SNPs that are informative for molecular typing of Typhimurium. The data will be used to determine the relationships of the worldwide isolates to understand how Typhimurium evolves.

• Hu, H., R. Lan, and P. R. Reeves. 2006. Adaptation of multilocus sequencing to study variation within a major clone: evolutionary relationships of phage types in Salmonella enterica serovar Typhimurium. Genetics 172: 1-8.

• McClelland, M. et al. 2001 Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 413:852-856.

Project 2 Molecular phage typing of Salmonella enterica serovar Typhimurium

The Anderson phage-typing scheme is commonly used in epidemiological surveillance of Typhimurium infections. It is based on combinations of resistance or degree of sensitivity of Typhimurium isolates to a series of specific bacteriophages. However, a molecular based method is highly desirable.

This project is to obtain molecular markers and develop a molecular typing scheme to replace phage typing. We have obtained 22 markers correlated with phage typing. The markers used can unambiguously differentiate 8 of the 33 the phage types studied, showing great promise for development of a molecular phage typing scheme. This project will obtain more markers to further develop the typing system. The two typing schemes to be developed will complement each other for both long and short term molecular epidemiological surveillance of Typhimurium infections. The project involves cloning, sequencing, and bioinformatic analysis.

• Hu, H., R. Lan, and P. R. Reeves. 2002. Fluorescent Amplified Fragment Length Polymorphism Analysis of Salmonella enterica Serovar Typhimurium Reveals Phage-Type- Specific Markers and Potential for Microarray Typing. J Clin Microbiol 40:3406-3415.

• Lan, R., Stevenson, G., Donohoe, K., Ward, L.R., Reeves, P.R., 2006. Molecular markers with potential to replace phage typing for Salmonella enterica serovar Typhimurium. J. Microbiol. Methods In press.

Project 3 Molecular evolution of Shiga toxin producing E. coli

E. coli is a highly variable species. Most strains are commensal in the large intestine of mammals or birds, but a small proportion are pathogenic, and fall into discrete pathovars, each with a particular mode of pathogenesis, of which at least 8 are now recognised. They include enteropathogenic enterohaemorrhagic, enterotoxigenic, enteroaggregative and enteroinvasive E. coli (EPEC, EHEC, ETEC, EAEC and EIEC). A range of serotypes, based on both O and H antigens, is found in each pathovar. Most serotypes are clones and the variety of serotypes gives a good indication of the diversity of pathogenic forms. However the serogroup, based on O antigen only, often does not reflect clonal relatedness.

We have in collaboration with 3 other laboratories developed a multilocus sequence typing (MLST) scheme and is being used to study E. coli population structure and evolution of the pathogenic forms. MLST is a method based on sequencing of 7 house keeping genes from each strain and use the variation revealed to determine the relationships and study global epidemiology. We have used MLST to study the origins of Shigella and EIEC. In this project we will extend the study to Shiga toxin producing E. coli (STEC) to identify clones or clonal complexes with potential to cause haemorrhagic uraemic syndrome and determine the relationship of STEC to other E. coli. The project involves PCR, sequencing, and bioinformatic analysis.

Selected references (available upon request)

• Salim, A., R. Lan, and P. R. Reeves. 2005. Relationships of pathogenic clones of Vibrio cholerae. Emerging Infectious Diseases 11:1758-1760.

• Dziejman, M., E. Balon, D. Boyd, C. M. Fraser, J. F. Heidelberg, and J. J. Mekalanos. 2002. Comparative genomic analysis of Vibrio cholerae: genes that correlate with cholera endemic and pandemic disease. Proc Natl Acad Sci USA 99:1556-1561.

Project 4 Molecular evolution and typing of Bordetella pertussis

Pertussis, commonly known as whooping cough, is an acute respiratory disease caused by Bordetella pertussis. It is estimated that 20–40 million cases and 400,000 deaths occur annually worldwide. Disease is most severe in infants and young children but often mild in adults. However adults are an important reservoir of infection.

In most countries there was a 90% reduction in the incidence of pertussis in the 2 decades after vaccination was introduced in the 1940s. However, increases in pertussis infection rates, observed across the globe in the 1990s, have led to it being now regarded as a re-emerging infectious disease. One of the key factors contributing to the increased incidence of pertussis is believed to be antigenic shifts in circulating strains escaping from vaccine-induced immunity.

This project will develop an integrated approach for identification of variants of genes encoding antigens represented in the acellular pertussis vaccine. The method can then be used for surveillance of circulating pertussis strains. The project involves real time PCR, sequencing, and bioinformatic analysis.

Selected references (available upon request)

• Mooi, F.R., van Loo, I.H., King, A.J., 2001. Adaptation of Bordetella pertussis to vaccination: a cause for its reemergence? Emerg Inf Dis 7, 526-528.

• Muyldermans, G., Pierard, D., Hoebrekx, N., Advani, R., Van Amersfoorth, S., De Schutter, I., Soetens, O., Eeckhout, L., Malfroot, A., Lauwers, S., 2004. Simple algorithm for identification of Bordetella pertussis pertactin gene variants. J Clin  Microbiol 42, 1614-1619.

• Poynten, M., McIntyre, P.B., Mooi, F.R., Heuvelman, K.J., Gilbert, G.L., 2004. Temporal trends in circulating Bordetella pertussis strains in Australia. Epidemiol & Inf 132, 185-193.

Project 5 Single nucleotide polymorphism (SNP) typing of Bordetella pertussis

Bordetella pertussis is genetically homogenous. Conventional typing methods such as multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE) have shown only limited discrimination in B. pertussis. Mutational changes, which accumulate over time, would reflect true genetic relationships of isolates. Recently, single nucleotide polymorphisms (SNPs) have been used for typing of genetically homogeneous pathogens such as Y. pestis and E. coli O157. This method has not been used for the typing of B. pertussis. This project is to develop a typing scheme for B. pertussis SNPs. Currently, we are identifying SNPs in B. pertussis genomes using comparative genomic sequencing. The SNPs identified will be used as molecular markers to type Australian and worldwide B. pertussis isolates to understand the evolution of B. pertussis. The project involves genome comparisons, real-time PCR, sequencing and bioinformatic analysis.

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Selected references (available upon request)

• Halperin, S. A., 2007. The Control of Pertussis — 2007 and Beyond. New England Journal of Medicine. 356:110-113

• Mooi, F.R., van Loo, I.H., King, A.J., 2001. Adaptation of Bordetella pertussis to vaccination: a cause for its reemergence? Emerg Inf Dis 7, 526-528.

• Zhang, W. et al., 2006. Probing genomic diversity and evolution of Escherichia coli O157 by single nucleotide polymorphisms. Genome Research 16:757-767.