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Bacterial genomics in infectious disease and the clinical pathology laboratory.

Microbial pathogenesis researchers and infectious disease specialists have long sought to understand the molecular basis of human disease caused by bacteria. Although new data can be generated with current methodologies and paradigm shifts can occur with reevaluation of current knowledge, the most historic advancements have followed an introduction of new technologies. For example, Girolamo Fracastorao speculated about the nature of "contagion" as early as 1546, but the first written description of microorganisms did not occur until Antony van Leeuwenhoek fashioned the microscope 130 years later. (1) The microscope was then essential to the seminal studies of Louis Pasteur and Robert Koch. (1) Similarly, Oswald Avery discovered that DNA encoded heritable traits by using new extraction techniques to isolate nucleic acids from one strain of Pneumococcus and to transform them into another. (2) Today, "next-generation" sequencing platforms and bioinformatics tools are opening a new chapter in the history of infectious disease research and clinical pathology laboratory practice. (3) Herein, we will review clinically relevant discoveries generated from bacterial whole-genome sequencing, with an emphasis on human pathogens and laboratory medicine. The studies discussed in this article represent illustrative examples and are not intended to be a comprehensive review. A complete discussion of nextgeneration sequencing technologies, bioinformatics tools, and genomic medicine is also beyond the scope of this article, and we refer the reader to several excellent reviews. (4-7)

The first complete bacterial genome, Haemophilus influenzae strain Rd, was sequenced in 1995. (8) Although the H influenzae genome was relatively small, only 1830137 base pairs, the project took more than 1 year to complete and cost an estimated $1 million. (8) Two years later, DNA sequencing technology was advancing at a rate similar to Moore's law. Moore's law predicts a doubling of computational microprocessor power every 24 months. (9,10) Importantly, the cost for generating sequence data has also steadily decreased. It is now possible, depending on the instrument used, to sequence the complete genome of a bacterial strain in 1 day for much less than $1000. (11-13) Similarly, a single instrument can generate 100 or more bacterial genome sequences in less than 1 week. Assuming this trend continues, a $10 bacterial genome will be realistically obtained in the near future. Importantly, these low-cost, high-throughput sequencing platforms are readily available, enabling the pursuit of new investigations and novel applications that were previously inaccessible. Sequencing technologies provide pathologists, both in academic medical centers and community settings, a tremendous opportunity to perform infectious disease research, develop new clinical laboratory tests, and improve patient care.

Recently, the lay and scientific press have focused on the perceived slow pace of translational discoveries achieved through the human genome project. (3,14) In comparison, bacterial genomics has flourished because of technologic advancements stimulated by the human genome project. More than 1500 genomes from nearly 200 different species, including many significant human pathogens, have been deposited in publicly accessible databases (http://www.ncbi.nlm.nih.gov/genome?db= genome), and analysis of several thousand additional genomes is currently underway. Compared with humans and other model organisms, such as the worm Caenorhabditis elegans, the genome of a typical bacterium is several orders of magnitude smaller, greatly facilitating its investigation. On average, a bacterial genome contains 1 to 6 million base pairs that encode approximately 3000 genes. In comparison, the human genome contains 3.3 billion base pairs and encodes approximately 25 000 genes. Bacterial genomics is further simplified because the prokaryotes are haploid, having only a single allele for each coding sequence.

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The coupling of increased DNA sequencing capacity with decreased instrument and reagent cost and newly developed computational expertise has driven the intensive interrogation of the bacterial genomes into the forefront of modern infectious disease research. These advances are not projections for the future, but rather, they are mature technologies on the verge of routine clinical laboratory implementation. As such, many leading experts have strongly advocated the need for pathologists to become facile in genomics, (15,16) a third residency track in genomic pathology has been proposed, (17) and the American Board of Medical Specialties has certified clinical informatics as a new subspecialty (http://www.abms.org/ News_and_Events/Media_Newsroom/Releases/release_ Announcing_TwoNewSubspecialties_10312011.aspx, accessed January 10,2012). Importantly, recent achievements in bacterial genomics have transformed our understanding of virulence factors, host-pathogen interactions, and population genetics. Furthermore, they have opened new opportunities for the rational design of diagnostics, therapeutics, and vaccines that may significantly improve patient care.

WHOLE-GENOME SEQUENCING FACILITATES NEW DISCOVERIES IN MOLECULAR PATHOGENESIS RESEARCH

Perhaps the most productive line of bacterial genomics research has been the study of molecular pathogenesis. Many important discoveries bearing on host-pathogen interactions have been achieved. As a result, new opportunities for the design of vaccines, diagnostics, and therapies have been identified (Figure 1).

Mycobacterium tuberculosis (MTB) is a highly successful human pathogen, having infected approximately one-third of the world population. Hence, new knowledge that can be leveraged to design more effective vaccines or treatment regimens could confer a tremendous benefit to public health. To begin investigating MTB pathogenomics, Cole et al (18) sequenced the first MTB genome in 1998. That landmark study demonstrated that the MTB chromosome encodes approximately 250 genes involved in fatty acid metabolism, including a rich repertoire of polyketide biosynthetic pathways, which generate its characteristic acid-fast staining cell envelope. Researchers had not previously appreciated the depth to which the MTB genome was devoted to synthesizing the antiphagocytic cell envelope, confirming its importance as a virulence factor and a possible vaccine target. The genome information also provided new insight into the ability of MTB to adapt to environmental changes, such as transitioning between oxygen-rich airspaces and anaerobic granulomata. Those data had direct implications to therapy, suggesting that antituberculous agents could be designed to specifically target the different gene transcripts expressed during aerobic or anaerobic metabolism. More recently, whole-genome sequencing was used to compare multidrug-resistant (MDR) and extensively drug-resistant (XDR) MTB strains recovered in a single geographic region. (19) Results showed that the gene polymorphisms responsible for conferring resistance to rifampicin and pyrazinamide occurred at different loci in the MDR and XDR strains. That is, different mutations in the MDR and XDR MTB strains caused resistance to the same drugs, which suggests the XDR lineage did not directly evolve from the MDR lineage. Thus, surveillance efforts to contain MDR strains may be insufficient for preventing the emergence of XDR strains, and therefore, antibiotic stewardship programs should also be included in control strategies. Similarly, Ford et al (20) sequenced MTB strains recovered from cynomolgus macaques with active, latent, and reactivated disease. Those data demonstrated an unexpectedly similar MTB strain mutation rate during latency and active disease. That is, MTB strains were shown to accumulate mutations at a much higher rate during latent infection than was previously estimated by in vitro studies. Thus, MTB retains a high capacity to acquire the mutations that confer drug resistance during latency. This finding may explain why monotherapy for patients with latent MTB infection is a risk factor for selecting isoniazid-resistant strains. As a result, drug-resistance testing is now recommended for latent MTB infection, particularly in patients who are known to have high treatment failure rates, such as those who are positive for human immunodeficiency virus or who are otherwise immunocompromised.

As discussed below, Beres et al (21,22) have extensively studied the population genetic structure of serotype M3 group A Streptococcus (GAS, Streptococcus pyogenes) strains causing invasive infections in Ontario, Canada. More than 340 strains from this comprehensive, population-based collection have been genotyped. The availability of detailed patient information linked to each strain (ie, the disease phenotype caused by each strain) makes this pathogenomics approach quite powerful. An important finding in those studies has been the identification of nonrandom-strain genotype--disease phenotype associations. For example, one particular subclone was associated with significantly fewer human necrotizing fasciitis cases. (23) This observation led Olsen et al (24) to hypothesize that strains comprising this subclonal lineage lacked the molecular program needed to cause necrotizing fasciitis. Genome-wide comparison of the necrotizing fasciitis deficient subclone with closely related, fully virulent strains implicated a single nucleotide insertion in the disruption of a multiple gene virulence axis. That is, a single nucleotide change in an approximately 2 million base pair genome significantly decreased the necrotizing fasciitis capacity of this subclone. The result was decreased secreted protease activity of SpeB, a key virulence factor for tissue destruction and dissemination. Thus, if a new therapy was designed to specifically disrupt that virulence pathway, the bacterium could be rendered significantly less virulent. In a related line of investigation, Beres et al (21) also discovered that the gene-encoding regulator of protease B (ropB), the major positive regulator of SpeB, was the most highly polymorphic gene in the strain collection. Subsequent studies demonstrated that those polymorphisms showed a pattern of positive selection that decreases ropB regulatory function, disrupts SpeB-secreted protease activity, and alters strain virulence. (25,26) Taken together, these genomic data reveal 2 intersecting gene pathways that offer new targets for GAS vaccine design.

Genome sequencing was also used to investigate virulence determinants underlying the historic epidemic of bubonic plague known as "Black Death." (27) Genomes of contemporary Yersinia pestis strains were compared with those generated from mid-15th century archival materials. Those data revealed no unique gene content or mutations in the ancient strains that could explain their perceived high virulence. Thus, these findings are consistent with the hypothesis that factors other than the bacterial genotype, such as vector dynamics, living conditions, and host susceptibility, were responsible for the high mortality observed during Y pestis outbreaks. Importantly, the data validate public health efforts aimed at preventing vector-borne infectious diseases through environmental controls. Rather than targeting the causative bacterium, the most effective approach to controlling Y pestis is to eliminate food and shelter sources for rodents, to use insecticides proactively, and to promote health education.

A recently expanded area of genomics investigation is the discovery of small, noncoding RNAs (sRNAs) that may alter bacterial virulence. Small RNAs base pair with messenger RNAs (mRNA) to alter the translation or stability of the transcript. For example, approximately 100 sRNAs, many of which significantly alter the expression of virulence regulators or virulence factors, have been found in Escherichia coli. (28) Whole-genome sequencing studies recently identified sRNAs located within the Vibrio pathogenicity island that fine-tune the level and coordinate the timing of cholera virulence factor expression in vivo. (29) These sRNAs may be key to the high virulence associated with environmental outbreaks of E coli and Vibrio cholerae. A family of sRNAs was similarly shown to play an important role in regulating the expression of Legionella pneumophila virulence factors associated with adaption to an intracellular environment. (30) Genome-wide strategies have also identified putative sRNAs in nearly every human pathogen studied to date, including MTB, (31) GAS, (32) Yersinia pseudotuberculosis, (33) and Salmonella enterica. 34 Many investigators are now attempting to modulate these sRNAs as antimicrobial therapies. In principle, an exogenously administered sRNA could down-regulate virulence pathways, enhancing susceptibility of the pathogen to certain antibiotics or rendering it incapable of expressing virulence factors needed for human infection.

THE POPULATION GENETIC STRUCTURE OF BACTERIA GUIDES THE INVESTIGATION OF EPIDEMIOLOGY AND VIRULENCE

Antigen serotyping, multilocus sequence typing, and variable number of tandem repeat analysis are commonly used techniques for subclassifying isolates in public health laboratories for epidemiology studies. However, these are relatively low-resolution molecular techniques based on analysis of only one or a few gene targets. As a result, they have limited capacities for discriminating among closely related strains. In comparison, population genetic structures based on whole-genome sequence data have a much greater capacity to estimate genetic relationships. That is, population genomics allows investigators to study the fine population structure of strain collections, identify previously unrecognized genetic relationships, and infer the sequence of evolutionary events (Figure 2). These data have direct implications for epidemiologic investigations, forensics, virulence research, and vaccines.

An intensive genome-wide approach has been used to study the fine-structure molecular architecture of GAS. By comparing the whole-genome sequences of several different M-protein serotype GAS strains, Beres et al (35-37) confirmed that their core chromosomes were highly conserved. Unexpectedly, the major differences in gene content and allele diversity were contributed by exogenous, mobile genetic elements, such as prophage. (38,39) Thus, these genomics data demonstrated that many of the long-recognized differences in strain virulence, disease phenotype, and epidemic behavior of GAS strains from different serotypes are likely due to genes encoded on prophage, rather than on the core chromosome. (40) This finding suggests that a pan-serotype, anti-GAS strategy for new vaccines must target genes encoded on the core chromosome rather than on mobile genetic elements. Furthermore, Beres et al (21,23,41) have also examined the population genetic structure of a comprehensive collection of serotype M3 GAS strains isolated from patients with invasive infections in Ontario, Canada. Before the study, relatively little genetic diversity was thought to exist among strains of the same GAS serotype. However, at the whole-genome level, an unexpectedly complex population genetic structure was identified. (21) Importantly, a dynamic pattern of subclone emergence and decline was observed. Three distinct epidemic peaks were identified during the longitudinal study. By understanding the genetic events that lead to subclone emergence, investigators can predict and possibly prevent the next epidemic wave from occurring. Shea et al (22) extended this comparative pathogenomics approach to interrogate geographically and temporally matched GAS strains recovered from patients with pharyngitis. Results demonstrated that the pharyngitis strains have virtually identical population genetic structures as the invasive infection strains. Importantly, these data answered a crucial question in GAS pathogenesis research. The GAS strains causing oropharyngeal and invasive infections are derived from a common genetic pool. That is, invasive infection strains are immediately descended from pharyngitis strains, confirming that the human oropharynx is the primary reservoir for GAS. Thus, a vaccine that prevents GAS pharyngitis may also reduce GAS invasive infections by eliminating the oropharyngeal reservoir from which the strains causing invasive infections evolve.

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Emergence of the USA300 community-acquired, methicillin-resistant Staphylococcus aureus (CA-MRSA) clone in the United States and throughout the world is a major public health concern. (42,43) Previous work has suggested that CA-MRSA infections are caused by a clonal group of organisms, but the conventional typing methods used in those studies lacked the resolving power needed to evaluate clonal evolution. Thus, until recently, an unresolved question in CA-MRSA research was whether a recent clonal expansion of a single ancestral strain or a simultaneous convergence of multiple strains toward a more virulent phenotype underlies the current epidemic. To unambiguously answer this question, Kennedy et al (44) compared the genome sequences of 10 CA-MRSA isolates recovered from diverse regions of the United States. Results demonstrated a single clonal lineage undergoing expansion and diversification. Importantly, these data suggest that the CA-MRSA clone will continue evolving under host-selective pressure, accumulating an increasingly diverse pool of derivatives. Thus, the emergence of an even higher virulence CA-MRSA clone is quite possible, further emphasizing the need for a preventative vaccine. Similarly, Harris et al (45) used whole-genome sequencing to construct a high-resolution map tracing the intercontinental spread and evolution of epidemic CAMRSA strains for more than 4 decades. Results demonstrated that although all CA-MRSA strains are closely related, there is a distinct phylogeny with geographically defined pockets of microevolution and subsequent dissemination. This information can be used to predict future evolutionary events that affect vaccine or drug design. That is, by understanding the mutations that are accumulating in CA-MRSA strains today, investigators can select vaccine and drug targets that are most likely to remain stable in the future. Harris et al (45) also used population genetics to trace person-to-person transmission of CAMRSA strains within a hospital. Importantly, if performed in real time, the genomics approach could identify unrecognized transmission chains for nosocomial CAMRSA infection, determine the point source, and precisely guide infection-control activities.

The MTB species may be one of the most ancient human pathogens. (46) However, until recently, hypotheses bearing on its origin, dissemination, and evolution could not be unequivocally tested, primarily because of the highly conserved nature of the MTB genome. (18,47-50) The low level of genetic diversity that exists among MTB strains renders phylogenetic analysis by multilocus sequence typing and spoligotyping generally uninformative. Thus, investigators have recently used a whole-genome approach to construct informative single-nucleotide polymorphism (SNP) panels for estimating genetic relationships among MTB strains. (51,52) Results identified multiple, deeply branching, phylogenetically distinct groups and subgroups. Importantly, strains within each of these divisions were nonrandomly associated with geography and ancient population migrations. Consistent with the prevailing hypothesis, the most ancestral cluster predominated in the Indian subcontinent, followed by East Asia, suggesting sites of species origin and initial spread before global dissemination. (51) This provides a temporal-geographic context for understanding the selective forces that first influenced MTB strain diversification. Given the unambiguous link between MTB dissemination and ancient human migration, these data also suggest that modern human population growth and global travel patterns could further exacerbate the ongoing epidemic. Furthermore, the finding that geographically disparate subgroups of MTB are evolving in isolation from others emphasizes the need to carefully select genetically stable vaccine targets that will provide coverage against all MTB subgroups. Similarly, by analyzing SNPs in a worldwide collection of Y pestis strains, investigators have estimated its origin in China, with subsequent evolution as geographic-specific lineages. (53) By studying the origins and subsequent evolution of these ancient pathogens, insight into their modern dissemination can be obtained. In addition, the emergence of new infectious diseases can be predicted. Future efforts to design therapies and vaccines must account for these data, including isolated pockets of strain evolution that may significantly affect medically relevant traits, such as virulence or transmissibility. (54)

Population genomics have also been used to study strain diversification, clonal evolution, and virulence of other important human pathogens, including Streptococcus pneumoniae, (55) Streptococcus agalactiae, (56) E coli, (57) and V cholerae. (58) Conversely, the study of Helicobacter pylori genomic lineages has been used to infer social relationships among ancient human populations and to resolve details of their migrations. (59) Emphasizing the importance of a genomic-based approach, each study discovered genetic relationships that were not previously detected by lower-resolution molecular techniques. Now, genomewide investigations of larger strain collections are needed to expand the scope of these instructive studies and to provide a genetic basis for designing diagnostic tests and antimicrobial therapies.

WHOLE-GENOME SEQUENCING IS CRUCIAL TO INVESTIGATING INFECTIOUS DISEASE OUTBREAKS

Outbreak investigations represent a specific application of population genomics, and full-genome analysis has become the preferred method for studying those events. Similar to evolutionary relationships inferred from genomic comparisons of geographically diverse strains, whole-genome sequencing of outbreak strains can precisely map their dissemination and clonal evolution. For example, Fittipaldi et al (60) recently sequenced the genomes of 601 serotype M59 GAS strains responsible for an ongoing epidemic of skin and soft tissue infections in Canada. Genome-wide analysis confirmed that M59 GAS strains recovered during the Canadian outbreak were clonally related by descent and genetically distinct from strains recovered in other geographic regions or times. Importantly, the extensive data set predicted precise patterns of geographic dissemination for subclonal lineages across Canada. Confirming the value of that approach, whole-genome sequencing results identified a previously unrecognized spread of the epidemic clone into 4 states of the United States. Ongoing analyses are now seeking to identify the genetic basis underlying the unusually high virulence of those strains.

To investigate a 3-year outbreak of MTB in British Columbia, Canada, Gardy et al (61) sequenced the genomes of 36 MTB strains with an identical variable number of tandem repeat pattern. Unexpectedly, the genomic data identified 2 genetically distinct lineages, revealing 2 concomitant outbreaks. Those data subsequently directed epidemiologists to map transmission events occurring within dynamic social networks among crack cocaine users in the community. In comparison, Truman et al (62) used a full-genome approach to demonstrate that epidemiologically unrelated leprosy cases in the southern United States were caused by the same unique strain of Mycobacterium leprae. That strain was also recovered from wild armadillos. Although causation was not proven, those data identified armadillos as the possible source of the zoonosis. As a result, public health officials have been advised to perform surveillance studies and to discourage direct contact with wild armadillos or consumption of their meat.

Foodborne outbreaks have also been investigated using whole-genome sequencing. The most common serotype of enterohemorrhagic E coli in North America is O157:H7. That lineage is distinguished from other serotypes by its highly homogenous (ie, clonal) population genetic structure. Eppinger et al (63) recently genotyped 231 isolates recovered during 3 concurrent foodborne outbreaks of human disease in the United States in the autumn of 2006. The resulting phylogenetic tree grouped the isolates into 3 discernable outbreak-associated clusters. That is, a genetically distinct subclone caused each outbreak, confirming that each outbreak was unrelated to the other two. If the whole-genome sequencing had been performed in real time, it could have directed the separate epidemiologic investigations. Genomics may have also determined each point source more quickly and identified additional patients who were not included in the original studies. That level of genetic diversity, although quite small compared with the genetic diversity observed within other Ecoliserotypes, provided new insight to the evolutionary dynamics and niche adaption of E coli O157:H7.

Of particular relevance to clinical laboratory medicine and public health, a panel of 1225 informative SNPs was designed for typing E coli O157:H7 isolates during outbreaks. Those sequences are publicly available, so clinical laboratories can develop diagnostic assays when future outbreaks are suspected. Similarly, Rasko et al (64) determined that a newly emerged clone of enteroaggregative E coli caused the outbreak of hemolytic-uremic syndrome associated with contaminated sprouts in Germany in 2011. Importantly, genomic data suggested that the high virulence of that strain was due to acquisition of a prophage encoding Shiga toxin 2, a distinct set of virulence factors and antibiotic-resistance genes. Thus, horizontal exchange of new genetic material allowed for the emergence of that highly virulent E coli strain. Similarly, a genome-wide approach also implicated mobile genetic elements as the source for the repertoire of new virulence factors expressed by the Listeria monocytogenes strain causing a recent outbreak of listeriosis in Canada. (65) By understanding the genetic basis of those outbreaks, investigators can validate diagnostic tests that detect the newly acquired virulence factors or design novel therapies that counteract them. In addition, future outbreaks can be anticipated, and therefore, contained, before becoming widespread.

Finally, the Amerithrax investigation into the US anthrax letter attacks during the autumn of 2001 demonstrated the value of whole-genome sequencing as a forensic tool. (66) Bacillus anthracis is a highly monomorphic species, so conventional low-resolution classification techniques cannot differentiate strains. To that end, the genome-wide investigation of evidentiary spores successfully validated a set of unique genetic markers for identifying the potential biologic source used in the mailings. Those markers were key to the Federal Bureau of Investigation response to the bioterrorism event. In comparison, Wright et al (67) used whole-genome sequencing in real time to demonstrate that a fatal, anthraxlike pulmonary infection in a patient from Texas was not related to bioterrorism. Rather, the genomics data proved that the Texas strain was closely related to other Bacillus cereus strains recovered in the region. However, it had naturally acquired a plasmid carrying the tripartite anthrax toxin genes, explaining the unusually severe clinical presentation of the patient. In either scenario, pathogenomics was crucial to guiding the coordinated response to a deliberate or natural event. This model strategy should be incorporated into the emergency response plan of all clinical laboratories with genome sequencing capability.

WHOLE-GENOME SEQUENCING GENERATES NEW INSIGHT TO INTRAHOST VARIATION, STRAIN VIRULENCE, AND THERAPEUTIC DESIGN

For decades, investigators have sought to understand the dynamics of host-pathogen interactions during the course of human infection. Whole-genome sequencing is now being used to identify the nucleotide-level genetic changes that occur in pathogenic bacteria in vivo. For example, patients with cystic fibrosis become chronically infected with Pseudomonas aeruginosa, leading to recurrent exacerbations and eventual, life-threatening pulmonary decline. However, the molecular pathways underlying the transition from acute infection to chronic carriage were unknown until Smith et al (68) sequenced the genomes of multiple P aeruginosa strains isolated from one patient over 8 years. Results identified an unexpected signature of genome-wide selection toward a less-virulent clone. That is, virulence factors and virulence regulators that are required to function during acute infection were selected against over time. Notably, gene pathways associated with surface antigen and exotoxin synthesis, secretion systems, motility, and multidrug efflux pumps were significantly altered. Those data are consistent with the idea that, as the infection persists, there may be a biologic advantage for a pathogen to evade host immunity by reducing its antigenic exposure. That finding explains, in part, the tenacious persistence of P aeruginosa infections in patients with cystic fibrosis and highlights the need to treat exacerbations with bactericidal rather than bacteriostatic agents. In contrast, whole-genome sequencing showed that intrahost genomic evolution of Burkholderia dolosa strains infecting patients with cystic fibrosis enhances their virulence characteristics, such as antibiotic resistance and membrane composition. (69) That evolutionary trend toward increased virulence parallels the deterioration of lung function associated with chronic B dolosa infection. Similarly, Kennemann et al (70) compared serial isolates of H pylori recovered from the stomachs of chronically infected patients. Results demonstrated a previously unrecognized pattern of genetic recombination and diversifying selection to increase the repertoire of H pylori adhesion proteins. Although these examples demonstrate contradictory themes of pathogen evolution toward an increased or decreased virulence phenotype, they have a common implication to translational research. Genomic changes that occur in vivo may also create new opportunities for therapy and diagnosis. That is, different molecular tools can be designed to specifically target the genotype of organisms present in acute or chronic infection.

WHOLE-GENOME SEQUENCING UNCOVERS NEW ANTIBIOTIC-RESISTANCE MECHANISMS

Antibiotic-resistant strains are a major cause of concern among specialists in infectious disease. Next-generation sequencing techniques have recently been used to identify the genetic basis of new antibiotic-resistance mechanisms. For example, Boyle-Vavra et al (71) compared the whole-genome sequences of MRSA strains isolated before and after daptomycin treatment failure. Of the 2 genetic differences identified among the strains, one was a SNP in the multiple peptide resistance factor (mprF). The same point mutation had previously been identified in a genome-wide survey of daptomycin-resistant MRSA strains obtained by in vitro passage, but it had been not yet been confirmed to occur in vivo. (72) A similar strategy discovered 2 additional gene mutations conferring daptomycin resistance after treatment failure of a vancomycin-resistant Enterococci faecalis infection. (73) Whole-genome sequencing has also provided new insight into the genomic plasticity that underlies the ability of S pneumoniae strains to readily acquire linezolid, fluoroquinolone, rifampicin, and macrolide resistance.74-76 Taken together, these data provide a framework for designing new drug derivatives or combination therapies that retain efficacy against resistant strains. They also provide a tool for predicting how resistance mechanisms will develop against future antimicrobial agents. Thus, new compounds that are less likely to be affected by acquired-resistance mechanisms can be designed.

MOLECULAR DIAGNOSTIC TESTS ARE DEVELOPED USING WHOLE-GENOME SEQUENCE DATA

Whole-genome sequencing of pathogens will soon become a standard tool in the diagnostic laboratory. Although 16S sequencing is accepted as the gold standard for identifying unknown bacteria, in many cases, it lacks discriminatory power needed to unambiguously classify some groups of organisms. (77) In comparison, genomics can precisely identify the species or subspecies of an unknown isolate. (67,78) That is, by comparing the genomes of closely related organisms that are not readily distinguished by biochemical phenotyping or 16S sequencing, their differences at the gene content level or even the gene allele level can be determined. Whole-genome sequence information can also guide molecular diagnostic test development by identifying appropriate nucleic acid targets for detection. That information can then be used to select species-specific sequences. Bannantine et al (79,80) used that strategy for Mycobacterium avium subsp paratuberculosis, the etiologic agent of Johne disease. Comparative genomics confirmed that Mycobacterium avium subsp paratuberculosis, Mycobacterium avium subsp avium, and Mycobacterium tuberculosis are closely related. However, 21 genes unique to the former subspecies were identified. (79,80) ISMap02 was subsequently selected as a putative target sequence for the design of a highly sensitive, real-time polymerase chain reaction test. That molecular diagnostic assay is now used to rapidly identify and cull infected cattle. (81) ISMap02 was selected, in part, because the sequence data showed it to be present at 6 copies per genome. Similarly, wholegenome sequence data have identified unique, multicopy, gene targets that increased the sensitivity of molecular diagnostic tests developed for other human pathogens, including Brucella spp, (82) Coxiella burnetii, (83) Tropheryma whipplei, (84,85) and Neisseria meningitidis. (86) Genomics has also proven useful for identifying organisms that have escaped detection by established molecular diagnostic tests. For example, whole-genome sequencing determined that the new Swedish variant of Chlamydia trachomatis has a large deletion in the gene region targeted by conventional assays. Consequently, because of its ensuing false-negative laboratory test results, many infections went untreated, and the organism rapidly disseminated. (87) Furthermore, analysis of the T whipplei genome elucidated the metabolic defects it must overcome to grow in a cell-free culture medium. (88) Researchers should now apply wholegenome sequencing strategies to additional scenarios in the clinical laboratory, such as the identification of bacterial species that cannot be cultured with conventional techniques. (89-91)

WHOLE-GENOME SEQUENCING IDENTIFIES NEW PATHOGENS THAT CAUSE HUMAN DISEASE

The potential of bacterial genomics as a tool for new pathogen discovery has yet to be fully realized. Approximately 90% of the human microbiome cannot be cultivated. (92) These uncultivable organisms may represent the biologic equivalent of "dark matter" in the human microbiome, representing a new frontier in clinical microbiology and bacterial pathogenesis. Alterations in the human microbiome are associated with different ailments, such as Crohn disease; however, a single, causative pathogen has yet to be implicated. (89) As metagenomics becomes better established and the human microbiome becomes better defined, the role of microbial communities in human disease will be better understood. New bacterial species will surely be identified, and new diagnostic assays that detect those organisms will be needed. Demonstrating proof of this concept, Salzberg et al (93) recently discovered 3 new endosymbiont species of the genus Wolbachia that were unintentionally interrogated when the genomes of their fruit fly hosts were sequenced. Another unexpected Wolbachia sp strain was identified during bioinformatic analysis of the Culex sp mosquito genome. (94) Although technically challenging to deconvolute sequence data from mixed samples, there is incredible potential to use this strategy for discovering new human pathogens in clinical samples. For example, Gaynor et al (95) sequenced the DNA library generated from a nasopharyngeal aspirate collected from a 3-year-old with culture-negative pneumonia. As expected, most of the sequence reads mapped to the human genome, but 6 unknown sequences with distant homology to BK and JC viruses were also identified. As a result, a new polyomavirus was identified and subsequently recovered from symptomatic children in Australia and the United States. (96) Similarly, Palacios et al (97) used unbiased high-throughput sequencing to identify a previously unknown Old World arenavirus in specimens collected from 3 recipients of visceral organ transplants from a single donor, who each died of a febrile illness. These genome-based techniques should now be applied to highly important clinical scenarios, such as culture-negative sepsis or pneumonia, in which a bacterial infection is strongly suspected, but no known pathogens are recovered. (98,99) Given the recent emphasis placed on sepsis as a measure of health care quality, it represents a prime opportunity for genomics-based discovery. (100)

FUTURE CHALLENGES TO IMPLEMENTING WHOLE-GENOME SEQUENCING OF BACTERIA IN THE CLINICAL LABORATORY

Next-generation sequencing instruments are capable of economically generating tremendous amounts of bacterial genomics data. Today, there are unprecedented opportunities for infectious disease researchers to test hypotheses that were previously intractable. Much has been learned, and many new investigations are underway. Wholegenome sequencing is now the preferred method to study bacterial virulence, investigate outbreaks, and characterize new organisms. Species assignment of isolates that cannot be otherwise identified and real-time molecular epidemiology of nosocomial infections are also opportunities to affect patient care. Furthermore, well-curated databases containing bacterial strain genotype-patient disease phenotype information are being developed for clinical decision support. As more strains are sequenced and new discoveries are made, those relationships will need to be continuously reevaluated. However, the data analysis bottleneck must be overcome before bacterial genome sequencing can be fully embraced in a clinical laboratory setting. One technologist can sequence several strains in a single day, but a team of physician-scientists may require a week or more to complete the data analysis. To that end, new bioinformatics tools must be developed to rapidly extract the most meaningful genetic information from the complex data sets. Bioinformatics expertise must also be developed beyond the confines of major bacterial genomics centers. As our ability to generate genome sequence data continues to rapidly expand and these bioinformatics tools translate to routine clinical use, pathologists will be expected to interpret the results for our clinical colleagues and patients. Many leaders in our field have acknowledged the need to train the next generation of genomic pathologists, (15) even suggesting a residency track in genomic pathology. (17) Finally, to be effectively implemented in clinical pathology laboratories, new regulatory guidelines and reimbursement models must be developed to support the efforts of pathologists.

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Randall J. Olsen, MD, PhD; S. Wesley Long, MD, PhD; James M. Musser, MD, PhD

Accepted for publication January 24, 2012.

From the Department of Pathology and Genomic Medicine, The Methodist Hospital System, Houston, Texas; and the Center for Molecular and Translational Human Infectious Diseases Research, The Methodist Hospital Research Institute, Houston.

This article was published as an Early Online Release March 22, 2012.

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Randall J. Olsen, MD, PhD, Department of Pathology and Genomic Medicine, The Methodist Hospital System, 6565 Fannin St, R6-114, Houston, TX 77030 (e-mail: RJOlsen@tmhs.org).
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Author:Olsen, Randall J.; Long, S. Wesley; Musser, James M.
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Date:Nov 1, 2012
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