Endemic and emerging infectious diseases caused by microbial pathogens pose a con-siderable threat to human and animal health worldwide1–5. At the molecular level, patho-gen evolution, transmission dynamics and genome plasticity, which account for many of the pathogenic properties of infectious agents, have been the focus of much research2,3,5,6. Functional and comparative genomics have improved both our understanding of micro-bial pathogenesis7,8 and what constitutes the border between commensal and pathogenic organisms9. It is becoming increasingly evi-dent that genome-sequence changes have an impact on the pathogenic potential and host tropism of many bacterial pathogens10.

With the availability of complete genome sequences for many strains of different bacte-rial pathogens, the development of genetic tools to screen for genome polymorphisms is being rigorously pursued. In this Opinion article, we discuss how genetic markers that are associated with bacterial genome fluidity can be developed and harnessed for diag-nostics, molecular epidemiology and vaccine production.

Mechanisms of genome alterationThere are three main mechanisms of large-scale genome alteration by which pathogens alter their genomes to evolve

into pathotypes — subgroups of strains that cause disease using common sets of virulence factors — that are adapted to specific host niches: gene acquisition by horizontal gene transfer (HGT); gene duplication followed by amplification and genome decay, which can occur through HGT; and DNA dele-tions, rearrangements and point mutations. These alterations create genetic variability that, upon selection, shapes the content of the genome in response to environmental conditions (FIG. 1).

Genome evolution is a continuous process that comprises long-term ‘macroevolution’, which leads to the development of new spe-cies or subspecies over millions of years, and short-term ‘microevolution’, which spans days or weeks and leads to the alteration of genes and traits in short time frames11. With respect to their susceptibility to these evolutionary processes, bacterial genomes comprise stable regions that form the core genome and variable regions that form the flexible gene pool12. The flexible component of the genome can accommodate and amel-iorate rearrangements owing to homologous recombination and the activities of phages, plasmids and transposons, and can also accommodate large mobile regions that are known as genomic islands (GEIs)13,14. It seems that some GEIs are acquired once

during the evolutionary lifetime of a lineage, and are subsequently subject to mutation to prevent further transmission and integra-tion. Pathogenicity islands (PAIs) — a sub-group of GEIs — were originally described in uropathogenic Escherichia coli (UPEC) as clusters of virulence genes that are absent in closely related strains or species15. There is substantial evidence indicating that HGT shapes the acquisition, integration and maintenance of PAIs in microbial genomes12,16,17.

Gene duplication and HGT are intercon-nected phenomena in which HGT enhances the propensity for gene amplification by augmenting the availability of multiple copies of identical DNA regions that can serve as targets for homologous recombina-tion18. Although a reversible phenomenon that does not result in permanent genetic change, amplification facilitates adaptation to changing environmental conditions18, which affects the fitness and immunogenic properties of many bacterial pathogens. In addition, short tandem DNA-sequence repeats that originate as a result of misalign-ment during replication can have an impact on gene regulation and protein production19.

Extensive genomic reduction is an additional evolutionary force that is active on a variable evolutionary timescale. One possible outcome of this process is the phe-nomenon of endosymbiosis, which occurs as the organism that is undergoing genomic reduction loses its metabolic and regula-tory versatility. Mechanisms for genomic ‘downsizing’ can permanently alter bacterial genotypes and result in adaptation to the environment through genome optimization. This can eventually result in a minimal genome that is sufficient to support only the basic metabolic activities of the pathogen or to allow successful exploitation of the host.

An interesting example of bacterial-genome optimization mediated by gene loss and increasing metabolic specialization is that of Yersinia pestis and its closest relative Yersinia pseudotuberculosis. In Y. pestis, 466 genes are inactivated or deleted compared with Y. pseudotuberculosis1,20,21. Y. pestis-specific phenotypes, such as cysteine requirement, the stimulatory effect of carbon dioxide on growth, the inability to

o p i n i o n

Genomic fluidity and pathogenic bacteria: applications in diagnostics, epidemiology and interventionNiyaz Ahmed, Ulrich Dobrindt, Jörg Hacker and Seyed E. Hasnain

Abstract | The increasing availability of DNA-sequence information for multiple pathogenic and non-pathogenic variants of individual bacterial species has indicated that both DNA acquisition and genome reduction have important roles in genome evolution. Such genomic fluidity, which is found in human pathogens such as Escherichia coli, Helicobacter pylori and Mycobacterium tuberculosis, has important consequences for the clinical management of the diseases that are caused by these pathogens and for the development of diagnostics and new molecular epidemiological methods.

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DNA acquisitionby HGT (transformation, transduction and conjugation)

Genome reduction(deletions) Genome

optimization(rearrangementsand mutations)

Genomeoptimization(rearrangementsand mutations)

use hexose through the pentose–phosphate pathway, altered lipid A acylation, the gene-ral loss of adhesins and altered motility, can be explained by gene inactivation that con-tributes to the evolution of a highly virulent epidemic clone from a less-virulent, closely related progenitor21.

In addition, obligate symbiotic bacteria, as well as obligate intracellular bacterial pathogens, often undergo reductive genomic evolution through the generation of pseudo-genes and gene-deletion events22,23. Genomic reduction has been extensively studied in mycobacteria by monitoring the presence or absence of regions of difference (rD)24 in the context of the evolution of different clonal lineages of the tubercle bacilli (FIG. 2). rDs are genomic regions that have been deleted from the genomes of ancestral forms of myco-bacteria, thereby generating more successful forms (‘modern’ species). The ancestor of the Mycobacterium tuberculosis complex under-went a series of precisely timed genomic-deletion events to increase its fitness in new hosts. It is thought that rD1 is the cause of adaptation of M. tuberculosis to human hosts (that is, the creation of a ‘specialist’ mycobacterium), as the Mycobacterium avium complex, from which rD1 has been deleted, have remained ‘generalists’ (ReF. 25).

However, until the role of a given rD dele-tion in either virulence or host tropism is experimentally proven, its absence could simply be a marker that coincides with other genomic changes (for example, sin-gle-nucleotide polymorphisms (sNPs) or microdeletions) that are the actual cause of the functional adaptation.

Harnessing genomic fluidityE. coli. E. coli is an excellent representative of the full spectrum of pathogen genomic fluidity. As one of the model organisms that was initially used to analyse bacterial genome fluidity and its impact on the evolu-tion of pathogenic variants, the contribution of plasmid-mediated and phage-encoded virulence factors and PAIs has been fully described for E. coli16,26. Plasmids, phages and PAIs all play a crucial part in the evolution of different E. coli pathotypes (FIG. 3).

One main feature of the different intestinal E. coli pathotypes is the presence of pathotype-specific plasmids that often encode toxins. The characteristic protein toxins of enterotoxigenic, enteroaggrega-tive, enteroinvasive, enterohaemorrhagic and enteropathogenic E. coli (and also extraintestinal pathotypes) are plasmid-encoded27. Additionally, the coding regions

for identical or closely related protein toxins, such as alpha-haemolysin, cytotoxic necrotizing factor and autotransporter serine proteases, can be located on plasmids, as well as on chromosomal PAIs28. Furthermore, large so-called colicin plasmids seem to contain several gene clusters, including the salmochelin determinant, that can also be found within chromosomal PAIs in E. coli and closely related species.

A large fraction of the genomic dif-ferences between closely related E. coli strains can be accounted for by the activ-ity of phages. Approximately 40% of the enterohaemorrhagic E. coli (EHEC) O157-specific elements are present in 1 of the 18 cryptic prophages or in the only intact phage (933W), which also contains the virulence genes that are most characteristic of O157:H7 — those that encode the shiga toxin29. The fact that E. coli O157 prophages exhibit extensive structural and positional diversity suggests that prophage variation is one of the most important factors in generating genome diversity among O157 strains30,31.

As a result of the marked E. coli genome fluidity, the stepwise acquisition of ‘foreign’ DNA and loss or inactivation of DNA regions has resulted in different metabolic and pathogenic features that can distin-guish different strains and pathotypes. For example, the parallel gain and loss of mobile genetic elements, such as phages, virulence plasmids and the locus of enterocyte effacement PAI, in different E. coli lineages enabled the evolution of separate clones that belong to different E. coli pathotypes (enterohaemorrhagic and enteropathogenic E. coli) and are associated with specific disease symptoms32. similarly, enteroinva-sive E. coli and Shigella spp. evolved from commensal E. coli by the uptake of a large virulence plasmid and inactivation of the cad determinant that encodes a lysine decar-boxylase. This pathoadaptive mutation has also been termed a black hole33,34.

What are the potential consequences of our increasing knowledge of the genetic diversity and genome content of E. coli? The identification of novel GEI-encoded proteins that are involved in bacterial colonization and/or pathogenesis should aid in the devel-opment of new strategies to tackle E. coli infection35. Knowledge of the prevalence of genes among certain subgroups of E. coli should also facilitate specific preventive approaches; for example, development of a UPEC-specific vaccine36,37.

DNA polymorphisms that are a result of repeated DNA acquisition and gene loss can be useful for strain typing and

Figure 1 |mechanismsthatcontributetobacterialgenomeevolution.Genome plasticity results from DNA acquisition by horizontal gene transfer (HGT; for example, through the uptake of plasmids, phages and naked DNA) and genome reduction by DNA deletions, rearrangements and point muta-tions. The concerted action of DNA acquisition and gene loss results in a genome-optimization process that frequently occurs in response to certain growth conditions, including host infection or colonization.

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Common ancestor Mycobacterium leprae

Mycobacterium canettii

RD9

TbD1

RD8 RD7

RD12

RD14

Mycobacterium tuberculosis (ancestral)

M. tuberculosis

M. africanumMycobacteriumafricanum

Mycobacterium pinnipedii

Mycobacterium microti

Mycobacterium bovis

Mycobacterium caprae

M. bovis bacille Calmette–Guérin

Decay (pseudogenization)

RD10

RD13

RD4

RD1

RD2

RDsealRDMic

RDcan

risk assessment (that is, evaluation of the likelihood and severity of an infection that can be caused by a bacterial isolate). For example, the mutS–rpoS genomic region of E. coli isolates differs with phylogeny and pathotype38. GEIs and other mobile genetic elements can also be used as markers for diagnostic purposes to identify certain E. coli populations or subpopulations that have pathogenic potential and, in some cases, to predict their antibiotic resistance. The presence or absence of PAIs or GEIs can be confirmed without culturing the bacterium of interest by PCr and/or DNA sequencing. The detection of certain virulence-associated marker genes also facilitates risk assessment in routine diagnostics; for example, in assays to detect the shiga-toxin-encoding genes in certain intestinal pathogenic E. coli (shiga-toxin-expressing E. coli (sTEC)), as well as the K1-capsule determinant of E. coli isolates in newborn meningitis. Promising candidate virulence markers for subgroups of E. coli that lack common major virulence factors, such as extraintestinal pathogenic E. coli, can also be identified39–41. This removes the need to culture organisms in the laboratory and can prevent hazards that are related to the handling and transportation of live cultures.

Increased knowledge of genome fluid-ity, including information on the stability of PAIs or prophages, can be used to develop approaches that specifically atten-uate strains of pathogenic E. coli without affecting their viability. As such strains are also becoming increasingly antibiotic resistant, development of antimicrobial strategies that target bacterial virulence and reduce selective pressure for resist-ance is of substantial importance42. Data continue to accumulate regarding PAI-deletion frequencies in E. coli in response to different growth conditions and envi-ronmental stimuli, as well as information on the specificity of the P4-like phage integrases that are responsible for the chromosomal insertion and excision of PAIs43–45. Consequently, future preventive and therapeutic approaches could aim to selectively induce PAI deletion. It is tempt-ing to speculate whether an established bacterial infection could be treated by inducing virulence-gene deletion in the colonizing bacteria. Future attenuated vac-cines could also be developed by inducing PAI deletion, thereby offering prophylactic interventions.

E. coli genome fluidity could also be exploited by using attenuated E. coli strains as probiotics. Examples of naturally occurring attenuated variants that are

derived from a virulent extraintestinal pathogenic ancestor include E. coli strain Nissle 1917 and asymptomatic bacteriuria (ABU) isolates. E. coli strain Nissle 1917 and ABU strain 83972 have been shown to be closely related to UPEC isolates from symptomatic urinary-tract infections. These attenuated strains have evolved by reductive genome evolution, which included deletion of a PAI (and other genomic-DNA regions) and virulence-gene inactivation owing to point muta-tion9,46–48. Interestingly, both Nissle 1917 and ABU strain 83972 are used as probiot-ics and are able to out-compete pathogenic bacteria under certain conditions49–51.

Genome fluidity that is manifested by the loss and transfer of shiga-toxin-gene (stx)-containing phages during the course of an EHEC infection could result in dif-ferent E. coli pathotypes (EHEC versus atypical enteropathogenic E. coli), and thus has clinical, diagnostic and epide-miological consequences52. shiga toxin is considered to be the crucial factor in the development of haemolytic uraemic syndrome, a leading cause of acute renal failure in children. recently, it has been shown that loss of the stx-carrying phages by EHEC O157:H7 does occur during human infection53. As stx detection is

routinely used to screen for EHEC, stx-negative variants of E. coli (which are still able to cause diarrhoea in humans) are not detected. This is crucial from a public-health perspective, as it hampers appropriate clinical management and epidemiological investigations54.

Helicobacter pylori. H. pylori is an impor-tant human pathogen that has evolved by aggregating different GEIs in its chromo-some, including GEIs that encode three major type Iv secretion systems55–57. The first secretion system identified comprises 29 genes that are located on the cag PAI58. Because of its geographically conserved insertion, deletion and substitution patterns, the cag PAI has recently been used as a marker of human migration59. sequence polymorphisms in the form of insertion and deletion (indel) changes within an unstable region between the right junction of the cag PAI and the gene that encodes glutamate racemase (glr)60 have been widely used as a signature for strain identification. Based on the instabil-ity of the cagA right junction, several inde-pendent molecular-epidemiology studies have been carried out that involve the gen-otyping of hundreds of strains and isolates from different continents to decipher the

Figure 2 |currentevolutionaryscenarioforpathogenicmycobacteria.extensive genomic downsizing has been a dominant trend in the evolution of the fittest forms of mycobacteria with vari-ous host tropisms. Polymorphisms in genomic regions of differences (rDs), which are indicated by differently coloured boxes, have recently become the most accepted method for the identifi-cation of tubercle bacilli24,25. Figure adapted, with permission, from ReF. 24 (2002) National Academy of Sciences.

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LEE

LEE

Black hole

PhageStxφ

Plasmids PAls

Invasion plasmidpEAFpENT

Commensal E. coliEnterotoxigenic E. coli

Enteroinvasive E. coli andShigella spp.

Uropathogenic E. coli

Enteropathogenic E. coli

LEE

Enterohaemorrhagic E. coli

geographical partitioning of H. pylori with reference to gene-pool characteristics and their impact on transmission patterns in individual countries58,59,61–65. For example, the impact of human migration in Peru and India on the survival and persistence of H. pylori has recently been reviewed using core-genome haplotyping through the analysis of multilocus sequence types and cag-PAI genotypes59,66. H. pylori can import DNA from strains from differ-ent populations during co-infection65. However, it seems that the cag PAI and the region that surrounds it was exchanged by the Amerindian strains in Peru59, where the human population underwent major changes in recent history with the arrival of European conquerors and settlers. The European H. pylori strains harboured an intact, functional cag PAI, and the ‘endogenous’ H. pylori in Peru could have been out-competed by the newly arrived cag-PAI-positive strains59.

The relatedness of cagA-gene sequences from Indians and Europeans was exam-ined to test the hypothesis of gene flow in India through Indo–Aryans and the arrival of Neolithic practices and languages from

the Fertile Crescent66. The cag PAI of the Indian strains was found to be completely evolved (that is, an intact PAI without any indels), and was probably acquired from a European source well before the arrival of H. pylori in India66. These observations support the notion that anciently acquired genomic landmarks could be used as markers in studying pathogen biology at the interface of human anthropology.

The CagA antigen, one of the principal virulence factors of H. pylori, is encoded on the cag PAI and is a universally accepted virulence marker that is linked to gastric and peptic ulcer disease and gastric cancer67–69. H. pylori strains are often determined to be carcinogenic or ulcerogenic based on the composition of their cag PAI; for example, using the pres-ence or absence of a particular motif (the EPIyA motif; type C or D) within the cagA gene. EPIyA motifs are crucially involved in the phosphorylation of CagA, which is linked to the severity of atrophic gastritis and gastric carcinoma in patients who are infected with CagA-positive strains of H. pylori67. Therefore, several diagnostic and phenotypic studies have been based

on this phosphorylation event70–72, and many molecular diagnostic methods target genes or gene products that are encoded by the cag PAI, including CagA70,73,74.

Mycobacteria. Genomic deletions repre-sent an important mechanism of genetic variability in mycobacteria75. Analysis of such deletions at the population level and comparison with reference strains is a useful method for identifying genes that could be associated with transmissibility and virulence. Other methods that detect small changes in the genome, using a range of molecular markers, are also important in the context of assessing the most predominant strain in an epidemiological setting76–79.

reductive genomic evolution markers have now been validated for the lineage identification of certain pathogens, including members of the M. tuberculosis complex. Members of this complex, such as M. tuberculosis, Mycobacterium africanum or Mycobacterium bovis, can be differenti-ated by the presence or absence of certain rDs (FIG. 2), which represent markers of unidirectional deletion events (once a

Figure 3 |contributionofhorizontalacquisitionofmobilegeneticelementstotheevolutionofEscherichia colipathotypes.The uptake of mobile genetic elements (phages, virulence plasmids and pathogenic-ity islands), as well as the loss of chromosomal-DNA regions in different E. coli lineages, has enabled the evolution of separate clones, which

belong to different E. coli pathotypes and are associated with specific disease symptoms. Lee, locus of enterocyte effacement; PAi, pathogenic-ity island; peAF, enteropathogenic E. coli adhesion-factor plasmid; peNT, enterotoxin-encoding plasmids; Stx, Shiga-toxin-encoding bacteriophage.

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region is lost it cannot be regained80,81), and by certain sNPs82–85. These rDs24 or sNPs82, together with minisatellite and spoligotype markers, permit identifica-tion of ancestral or modern forms of M. tuberculosis2,86.

As discussed above, the evidence sug-gests that successive genome deletions in pathogenic mycobacteria have led to an increasingly defined host preference. For example, genomic deletions that are specific to M. tuberculosis (TbD1) might have ren-dered the organism more human-specific and aggressive in terms of outbreak poten-tial, compared with its ancestors. similarly, a specific genomic deletion could have

contributed to the greater pathogenic suc-cess of Mycobacterium pinnipedii in seals compared with other hosts76,87. several dele-tions in the M. bovis genome are suggested to be the cause of the successful attenuation of virulence in M. bovis bacille Calmette–Guérin (BCG) vaccine strains24. The deletion marker rD1 is always absent in attenuated strains such as M. bovis BCG24, and has been shown to be a powerful virulence-linked marker that carries virulence-associated genes, such as those that encode the EsAT6 and CFP10 antigens (discussed below)88–90.

Given that the population structure of members of the M. tuberculosis complex is highly clonal, and that HGT between strains

does not occur or is rare, rDs can serve as unidirectional markers for differentiating between closely related species, such as M. bovis, M. africanum and M. tuberculosis, and for gaining deeper insights into phe-notypic traits that have relevance to patho-genicity or vaccine efficacy91–93. In addition to deletion markers88 and conserved genes94, regions of the M. tuberculosis genome that cause strand slippage during replication, which results in strain diversity and gene-function redundancies, also have diagnostic applications95,96.

short-tandem repeats in the form of minisatellites97 and microsatellites98 have robust clinical applications in the manage-

Table 1 | Exploitation of bacterial genome fluidity for diagnostic and health-care applications

molecularmarkerorassay geneticormolecularchange Applicationtohealthcare

escherichia coli

virulence genes (for example, stx and kpsK1)

Presence or absence of the locus; expression of functional virulence factors

Diagnostic markers and antigens; markers of infection

Genomic islands, plasmids and phages

Presence or absence of the locus; expression of functional virulence factors

Diagnostic markers and antigens; markers of infection

Black holes Presence or absence of the locus; expression of functional virulence factors

Diagnostic markers and antigens; markers of infection

core-genome MLST evolution of housekeeping genes Lineage identification

SNPs Base substitution in candidate genes Screening for antimicrobial resistance, molecular epidemiology and evolution

Helicobacter pylori

indel of cag right junction Small-scale insertions and deletions at the right junction of the cag PAi

Markers of human migration and H. pylori lineage identification

cagA Presence or absence of the locus; expression of functional toxin

Diagnostic markers and antigens; markers of invasive gastric inflammation

cagA ePiYA motif Strain-specific tyrosine phosphorylation of cagA Geographical markers of gastric-cancer predisposition

Plasticity-region cluster Presence or absence of loci Putative virulence genes; possible interventional targets

core-genome MLST evolution of housekeeping genes Human-migration studies; lineage identification

Mycobacteria

rDs reductive evolution Diagnostic and genotyping applications for epidemiology; vaccine candidates (rD1)

Drs indel in Dr region Spoligotyping for epidemiology; strain identification

iS6110 rFLPs Mobile-element instability Outbreak investigation; strain identification

DUs Tandem duplication Quality control of BcG vaccines

SNPs Base substitution in candidate genes Screening for antimicrobial resistance, molecular epidemiology and evolution

MirUs (minisatellites) Tandem-repeat tract expansion or shrinkage Large-scale, high-throughput analysis that can monitor spread and transmission dynamics

Microsatellites replication errors Molecular epidemiology; strain identification

Pe–PPe protein family variable-repeat motifs and antigenic variation Diagnostic markers and antigens; putative virulence markers

erp protein family Plasticity of nucleotide-repeat motifs Diagnostic antigens and virulence markers; vaccine candidates (rD1)

BcG, Mycobacterium bovis bacille calmette–Guérin; Dr, direct repeat; DU, genome-duplication marker; MirU, mycobacterial repetitive interspersed unit; MLST, multilocus sequence typing; PAi, pathogenicity island; rD, region of difference; rFLP, restriction-fragment length polymorphism; SNP, single-nucleotide polymorphism.

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ment of tuberculosis outbreaks. shrinkage and expansion of these repeats, as well as their gain and loss, creates sufficient allelic diversity97 to allow the individualization of strains, thereby allowing investiga-tors to track the movement of clones during tuberculosis epidemics. Dozens of mycobacterial interspersed repetitive unit (MIrU) loci have now been tested in different laboratories, and independent databases exist for strain comparisons (for example, MIrU–vNTrplus; see Further information). MIrU-based markers2,97, together with broad classification markers, such as Is6110 restriction-fragment length polymorphisms99 and the spacer types100 in the direct repeat regions, are widely used in mycobacterial epidemiological studies2,101 (TABLe 1).

The PE–PPE gene families of M. tuberculosis constitute approximately 10% of the coding capacity of its genome, and are probably expanding through gene duplication and amplification. Importantly, members of these families are functionally linked to the EsAT6 (esx) gene cluster, which encodes a system for the secretion of members of the EsAT6 family of potent T-cell antigens102,103. It has recently been sug-gested that the esx-gene cluster facilitates the presentation of important antigens, including members of the PE–PPE protein families, to the host immune system. The preponderance of repetitive tracts within these gene families could contribute to the antigenic variation that is displayed by M. tuberculosis. It has also been speculated that the EsAT6 and CFP10 loci, coupled with their associated PE–PPE genes, might constitute a putative immuno-genicity island89,102,104,105. Two independent members of the PE–PPE families (rv2430c106 and rv2608 (ReF. 95)) have been shown to elicit a strong host humoral immune response that allows the specific detection of active M. tuberculosis infection in some patients. The PE–PPE family genes are non-randomly organized in the M. tuberculosis genome, and follow a defined pattern that has a role not only in function but also in pathogenesis107. The predicted cell-surface localization of the PE–PPE proteins also sug-gests a role for these proteins in host–patho-gen interactions. Moreover, in addition to rD1, other rDs that are deleted in the BCG strains have been shown to encode genes from the PE–PPE families. Finally, a com-parative genomic analysis of laboratory and clinical isolates of M. tuberculosis revealed the presence of sequence polymorphisms within the PE–PPE genes, a finding that is also suggestive of an important role for these

proteins in pathogenesis and immunity108.The M. tuberculosis Erp family of

exported proteins encode variable-repeat motifs within the central region of the protein109, and variations in the repeat length between the different mycobacterial species have potential uses in diagnostics. Homologues of the erp gene are present in Mycobacterium leprae, Mycobacterium smegmatis, M. avium, Mycobacterium marinum, Mycobacterium xenopi and the extracellular toxin-producing Mycobacterium ulcerans. Interestingly, the Erp protein has been reported to be asso-ciated with mycobacterial pathogenesis110, and deletion mutants of the erp gene can stimulate an immune response against mycobacteria. In another study, it was sug-gested that initial bacterial growth and the consequent histological outcome of infec-tion are conditioned by both the nature and number of Erp repeats103.

Nilsson et al.111 observed in Salmonella enterica serovar Typhimurium that extensive genomic deletions can occur over a short evolutionary timescale — possibly within weeks — leading to a reduction in microbial metabolic and regulatory versatility. Another process of in vitro genome evolution was observed for mycobacteria by analysis of suc-cessive generations of the original BCG vac-cine strain, which evolved frequently through various genomic-deletion and tandem-duplication events91. Consequently, because they are frequently sub-cultured, present-day BCG vaccine strains rapidly lose their protec-tive efficiency. Conversely, older versions of the BCG vaccine strain are considered to be superior in providing protection against tuberculosis compared with more commonly used variants. Genome instability in these bacteria can result in a variable antigenic repertoire that differs from M. tuberculosis or M. bovis and therefore seriously impacts on the protective efficacy of vaccine strains91. In addition, duplication and amplification of certain genetic elements could lead to the emergence of novel functions in BCG strains that could affect their antigenic repertoire and subsequent immunological proper-ties91. Molecular markers that are based on genomic deletions and tandem duplications could therefore be employed to validate older versions of the BCG vaccine strain. In addi-tion, these markers could be useful in quality validation and the genetically controlled pro-duction of BCG strains. Genome-duplication (DU) markers, for example DU1 and DU2 (ReF. 91), constitute potential candidates for the evaluation of protective efficacy of dif-ferent BCG strains. The potential uses of

molecular methods in routine quality control and identification of BCG vaccine strains are being discussed112,113.

ConclusionsIn the study of bacterial genome fluidity, many questions remain, including: how is bacterial genome fluidity regulated; what environmental stimuli are responsible for this fluidity; what is the in vivo relevance of bacterial genome fluidity; and how can bacterial genome fluidity be exploited for the generation and selection of optimally adapted microorganisms? Answering these questions will not only increase our knowledge of the mechanisms that are involved in the evolution and adaptation of bacterial pathogens, but will also have an impact on the development of accurate diagnostics and timely therapeutic interventions against infectious diseases. As the magnitude of bacterial genome fluidity becomes clear, the key research goals are evident: first, the exploitation of these data to design, improve and adapt appropriate diagnostics; second, the expansion of our knowledge of the underlying biology of each pathogen; and, finally, the exploitation of bacterial genome dynamics to develop new approaches to combat infectious diseases.

Niyaz Ahmed is at the Pathogen Evolution Laboratory, Center for DNA Fingerprinting and Diagnostics,

Nacharam, Hyderabad 500 076, India

Ulrich Dobrindt and Jörg Hacker are at the University of Würzburg, Institute for Molecular Biology of Infectious

Diseases, Röntgenring 11, 97070 Würzburg, Germany.

Jörg Hacker is also at the Robert-Koch Institut, Nordufer 20, 13353 Berlin, Germany.

Seyed E. Hasnain is at the University of Hyderabad, Hyderabad, 500 046, India, The Institute of Life

Sciences, HCU Campus, Hyderabad, 500 046, India and the Jawaharlal Nehru Centre for Advanced

Scientific Research, Jakkur, Bangalore 560 064, India.

N.A. and U.D. contributed equally to this work. Correspondence to J.H. and S.E.H.

e-mails: j.hacker@mail.uni-wuerzburg.de; vc@uohyd.ernet.in

doi:10.1038/nrmicro1889 Published online 8 April 2008.

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DATABASESentrez Genome Project: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genomeprjEscherichia coli | Helicobacter pylori | Mycobacterium avium | Mycobacterium bovis | Mycobacterium leprae | Mycobacterium marinum | Mycobacterium smegmatis | Mycobacterium tuberculosis | Mycobacterium ulcerans | Yersinia pestis | Yersinia pseudotuberculosis

FURTHER inFoRMATionJörg Hacker and Ulrich Dobrindt’s homepage: http://www.infektionsforschung.uni-wuerzburg.de/Niyaz Ahmed’s homepage: http://www.pathogen-evolution.orgseyed e. Hasnain’s homepage: http://www.isogem.org/hasnain.htmlinstitute Pasteur (colibri database): http://genolist.pasteur.fr/colibri/institute Pasteur (PyloriGene database): http://genolist.pasteur.fr/PyloriGene/J. craig venter institute (comprehensive microbial resource): http://www.jcvi.org/cms/research/projects/cmr/MirU–vNtrplus : http://www.miru-vntrplus.orgWellcome trust sanger institute (pathogen sequencing unit): http://www.sanger.ac.uk/Projects/Pathogens/

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113. Shin, J., Wood, D., Robertson, J., Minor, P. & Peden, K. WHO informal consultation on the application of molecular methods to assure the quality, safety and efficacy of vaccines, Geneva, Switzerland, 7–8 April 2005. Biologicals 35, 63–71 (2007).

AcknowledgementsResearch in the laboratories of N.A. and S.E.H. was supported by several grants from the Department of Biotechnology, Ministry of Science & Technology, Government of India. Research in the laboratories of U.D. and J.H. was supported by grants from the German Research Foundation (SFB479, TP A1), the European Community (European virtual institute for functional genomics of bacterial pathogens; CEE LSHB-CT-2005-512,061) and the Bavarian Research Foundation.

o p i n i o n

Searching for the cause of Kawasaki disease — cytoplasmic inclusion bodies provide new insightAnne H. Rowley, Susan C. Baker, Jan M. Orenstein and Stanford T. Shulman

Abstract | Kawasaki disease (KD) has emerged as the most common cause of acquired heart disease in children in the developed world. The cause of KD remains unknown, although an as-yet unidentified infectious agent might be responsible. By determining the causative agent, we can improve diagnosis, therapy and prevention of KD. recently, identification of an antigen-driven igA response that was directed at cytoplasmic inclusion bodies in KD tissues has provided new insights that could unlock the mysteries of KD.

Kawasaki disease (KD) is an acute child-hood illness that usually affects previously healthy infants and children. The disease is manifested by a high spiking fever and, in classic cases, four of five additional features: rash; red eyes; red lips and mouth; swollen and red hands and feet;

and swollen glands in the neck. These symptoms resolve spontaneously within 1–3 weeks, or sooner after treatment with intravenous gammaglobulin and aspirin. However, inflammation of medium-sized arteries throughout the body, particularly of the coronary arteries, can occur during

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