Perspective

Molecular Population Genetic Analysis of Emerged Bacterial Pathogens: Selected Insights James M. Musser, M.D., Ph.D. Department of Pathology, Baylor College of Medicine, Houston, Texas, USA

Research in bacterial population genetics has increased in the last 10 years. Population genetic theory and tools and related strategies have been used to investigate bacterial pathogens that have contributed to recent episodes of temporal variation in disease fre- quency and severity. A common theme demonstrated by these analyses is that distinct bacterial clones are responsible for disease outbreaks and increases in infection frequency. Many of these clones are characterized by unique combinations of virulence genes or alleles of virulence genes. Because substantial interclonal variance exists in relative virulence, molecular population genetic studies have led to the concept that the unit of bacterial pathogenicity is the clone or cell line. Continued new insights into host–parasite interactions at the molecular level will be achieved by combining clonal analysis of bacterial pathogens with large-scale comparative sequencing of virulence genes.

To avert the threat of resurgent and new micro- students of the molecular evolutionary processes bial diseases, it is critical to gain insight into the in higher eukaryotic organisms. Bacteria were an molecular mechanisms contributing to temporal attractive group of experimental organisms be- variation in disease frequency and severity. Al- cause of their phenotypic diversity, short genera- though comprehensive, unambiguous under- tion times, haploid chromosomal genomes, and standing of the host and parasite factors accessory genetic elements. Hence, bacterial mediating these processes is not available for any population genetic research was originated by infectious agent, population genetic research in population geneticists interested in bacteria, the last 10 years has provided noteworthy new rather than bacterial geneticists or medical micro- information about the bacterial side of the equa- biologists interested in population genetics (1, 2). tion. This review will summarize the insights ac- In spite of its important implications for how the crued from population genetic analysis of bacteria field has developed over the last decade, this on- responsible for disease outbreaks or increases in togeny will not be discussed in detail here. How- infection frequency and severity. One of the pri- ever, the reader should recognize that bacterial mary themes emerging from this research is that population genetics is a discipline separate and distinct bacterial clones have been responsible for distinct from the study of the molecular epidemiol- several infection outbreaks (Table 1). Moreover, ogy of infectious agents. The research tools, meth- the distinct clones are frequently characterized by ods of data analysis, and general thought unique combinations of virulence genes or alleles of virulence genes. These observations have im- processes are very different from the typological portant implications for our understanding of in- thinking used by investigators of disease out- fectious diseases and the public health measures breaks or microbial pathogenesis (3-6). required to reduce their detrimental and poten- Early work on the clonal nature of bacterial tially devastating effect on society. pathogens was conducted largely with , through a framework supplied by serotyping of one or a few polymorphic surface antigens (7, 8). Population Genetics and Clonal Analysis of Only a few of the many possible O and H antigen Bacterial Pathogens: Basic Concepts serotypes were frequently associated with out- Population genetic study of bacterial pathogens breaks of infantile diarrhea in the United arose largely as an offshoot of research designed Kingdom and other countries, which suggested to address questions of longstanding interest to that isolates expressing these traits had special virulence properties (7-9). Because serotype Address for correspondence: James M. Musser, M.D., Ph.D., Department of Pathology, Baylor College of Medicine, One analysis of relatively few surface structures does Baylor Plaza, Houston, TX 77030 USA; fax 713-798-4595; not provide robust data for estimating overall

Vol. 2, No. 1 — January-March 1996 1 Emerging Infectious Diseases Perspective

Table 1. Representative Emerged Bacterial Pathogens Investigated with Molecular Population and other microbial Genetic Strategies pathogens provides a convenient strategy Organism Disease Reference for indexing overall Borrelia species Lyme disease 146-149,150,151 levels of chromosomal Escherichia coli O157:H7 hemorrhagic colitis; diversity in the sample hemolytic uremic syndrome 33-35 and for inferring ge- influenzae Invasive disease 156 netic relationships H. influenzae among strains. Be- biogroup aegyptius Brazilian purpuric fever 25,26 cause of the strong cor- Listeria monocytogenes Food-borne invasive disease 157, 158 relation of chromo- Mycobacterium tuberculosis Tuberculosis 91,105 somal divergence in- , 52-63,66,69-73 dexed by multilocus Salmonella species Food-borne illness 159-161 enzyme electro- Staphylococcus aureus Toxic shock syndrome; phoretic data and methicillin resistant strains 47,143,144 DNA-DNA hybridiza- Streptococcus pyogenes Invasive disease, tion studies (15-18), toxic-shock-like syndrome 82,83,85 several cryptic species S.pneumoniae Otitis, , sepsis, have been identified meningitis 125-131,136 once their existence 153-155 was initially discov- ered by population ge- netic analyses that levels of chromosomal diversity and relationships employed starch gel electrophoresis (16, 19-21). In among strains, the primary research tool used to most bacterial species, the number of allelic vari- examine the population genetics of emerging bac- ants is large, and it is unlikely that recombina- terial pathogens has been multilocus enzyme elec- tional processes would, by chance, frequently trophoresis (10, 11). This technique indexes allelic generate strains with the identical electromorph variation in sets of randomly selected structural profile. Hence, organisms with the same electro- genes located on the chromosome and provides a morph profile are generally thought to be similar basis for estimating overall levels of genotypic by descent, rather than by convergence through variation in populations, i.e., the sample of bacte- lateral gene flow. ria chosen for analysis. The key concept underly- Recently, convenient, rapid, and relatively inex- ing use of starch gel-based protein electrophoresis pensive large-scale DNA sequencing techniques in population genetics is that electromorphs (mo- have also been adopted by several laboratories. bility variants) of an enzyme can be directly Large-scale automated DNA sequencing has been equated with alleles of the corresponding struc- used to rapidly and unambiguously identify a tural gene. Moreover, electromorph profiles over a causative infectious agent and confirm or refute sample of different enzymes, therefore, correspond the identity of isolates recovered from temporally- to multilocus enzyme genotypes and are fre- linked patients thought to be involved in a disease quently referred to as electrophoretic types or ETs. outbreak. In addition, sequence-based studies The proteins analyzed are usually metabolic en- have been employed to define the nature and zymes expressed by virtually all isolates of a spe- extent of allelic variation in toxin and other viru- cies under the growth conditions used. The allelic lence factor genes and to rapidly identify muta- variation detected is unaltered by environmental tions associated with antimicrobial agent conditions such as culture conditions, laboratory resistance (22). storage, anatomic site of recovery, or specific clini- Unless noted, data on the population genetic cal disease. Allelic variation in these metabolic analysis of emerging bacterial pathogens summa- enzymes is selectively neutral, or nearly so, which rized in this article were generated by multilocus means that convergence to the same allele through enzyme electrophoresis, sometimes performed in adaptive evolution is unlikely (4, 12-14). As a con- concert with automated DNA sequencing. sequence, this approach to the study of bacterial

Emerging Infectious Diseases 2 Vol. 2, No. 1 — January-March 1996 Perspective

Representative Insights spawned from a much larger base population of diverse nonpathogenic precursor clones. Accord- ing to this hypothesis, acquisition or loss of one or Brazilian Purpuric Fever more genes (or, perhaps, a shift in ecological niche) Brazilian purpuric fever (BPF), a serious inva- may produce a pathogenic form with the charac- sive disease of children, was first characterized in teristic viscerotropism for human conjunctivae. 1984 after an outbreak in Promissao, Sao Paulo Although population genetic analysis did not State, . Children with BPF have acute onset provide a simple reason for the BPF outbreak, the of fever and usually die within 48 h with dissemi- demonstration that the causative clone of bi- nated , vascular collapse, and hypotensive ogroup aegyptius was highly differentiated from shock (23). BPF is caused by Haemophilus influ- other phenotypically similar organisms provided enzae biogroup aegyptius, an organism associated an explanation for the unique infection manifesta- with sporadic or epidemic (24). Mul- tions and the unique group of characters associ- tilocus enzyme electrophoresis and other molecu- ated with the clone (28-30). Moreover, population lar techniques have demonstrated that isolates genetic analyses demonstrated distinct medical recovered from BPF patients represent a distinct correlates to isolates classified as biogroup aegyp- clone (25). tius. The results of numerous subsequent studies As a first step toward identifying the evolution- have confirmed that, as a population, H. influen- ary origin of this pathogenic H. influenzae bi- zae biogroup aegyptius strains vary in their behav- ogroup aegyptius clone, chromosomal variation ior, as one would expect of a genetically diverse set and genetic relationships were indexed among 17 of organisms. biogroup aegyptius isolates, and 2,209 encapsu- lated H. influenzae strains were recovered world- wide (26). Biogroup aegyptius isolates form three Escherichia coli O157:H7 distinct evolutionary lineages of the species H. Strains of E. coli expressing serotype O157:H7 influenzae. Isolates of the case clone are only very were recognized in the early 1980s as important distantly related to other isolates classified as causes of hemorrhagic colitis and hemolytic ure- biogroup aegyptius; that is, the case clone was no mic syndrome in North America (31). Disease usu- more related to other biogroup aegyptius isolates ally occurs after consumption of contaminated than are (for example) two H. influenzae isolates beef or other food. Several large outbreaks have selected at random from the species. The BPF case occurred, and more than 60 case clusters have clone was genetically allied with H. influenzae been reported in the (32). Because isolates expressing serotype c polysaccharide cap- several E. coli reference laboratories rarely iden- sule, a result that explains an earlier observation tified organisms expressing this serotype before (27) that BPF isolates, like serotype c strains, the early 1980s, reporting of these isolates has produce type 2 IgA1 protease, whereas other iso- increased dramatically. Because of the medical lates of biogroup aegyptius express type 1 IgA1 and economic importance of E. coli strains consid- protease. Thus, the population genetic evidence erable effort has been directed toward elucidating showed that biogroup aegyptius is polyphyletic genetic relationships among and between them as and that the BPF organism is a genetically distinct well as between them and other members of the clone unrelated to other isolates with the pheno- species; as a result extensive information is now typic criteria of biogroup aegyptius. available about clonal relationships among these The genetic diversity in the sample of all bi- important bacteria (33-35). ogroup aegyptius strains was approximately equal The observation that O157:H7 strains synthe- to that recorded for entire species of certain patho- size one or more Shiga-like toxins and lack the genic bacteria (16, 17). Therefore, the effective ability to rapidly ferment sorbitol initially sug- population size of aegyptius must be large; how- gested that strains of this serotype had shared a ever, this interpretation is difficult to reconcile recent common ancestor. To directly test this idea, with the observation that strains in the biogroup multilocus enzyme electrophoresis was used to are rare pathogens associated only with human assess genetic relatedness of 100 strains of E. coli disease. A possible explanation for the relatively serotypes recovered from patients with hemor- extensive genetic diversity among biogroup aegyp- rhagic colitis or hemolytic uremic syndrome (33). tius strains is that they represent cell lineages Although 25 distinct multilocus enzyme genotypes

Vol. 2, No. 1 — January-March 1996 3 Emerging Infectious Diseases Perspective were identified, cluster analysis found that Staphylococcus aureus Toxic Shock Syndrome O157:H7 isolates are closely related organisms. Toxic shock syndrome (TSS) was described in The results were interpreted to mean that 1978 (43) as a severe acute illness (characterized O157:H7 organisms recovered from epidemiologi- by high fever, erythematous rash, hypotension or cally unassociated North American outbreaks be- shock, multiorgan involvement, and desquama- long to a single geographically widespread tion of the skin) of young children associated with pathogenic clone with specific virulence properties infection with Staphylococcus aureus. Two years (33). Subsequent analysis of O157:H7 strains by later, it was recognized that TSS is a geographi- pulsed-field gel electrophoresis has supported this cally widespread disease affecting mainly young, idea (36). healthy, menstruating women, especially those us- To delineate clonal relationships among ing certain high absorbency tampons (44). Most O157:H7 organisms and other E. coli strains that vaginal isolates of S. aureus recovered from pa- cause hemorrhagic colitis and infantile diarrhea, tients with TSS produce a chromosomally encoded 1,300 isolates representing 16 serotypes from pa- toxin, designated as toxic shock syndrome toxin-1 tients with these diseases were studied by multilo- (TSST-1) (45). Evidence implicating TSST-1 as a cus enzyme electrophoresis and probing for genes major virulence factor in the pathogenesis of TSS encoding Shiga-like toxins (34). The O157:H7 has accumulated (46). Almost all strains recovered clone was closely related to a clone of O55:H7 from patients with menstrual TSS, which account strains that has a long history of worldwide asso- for approximately 90% of TSS cases, synthesize ciation with outbreaks of infantile diarrhea (34). TSST-1, whereas only 50%-60% of isolates from The data strongly suggested that the O157:H7 and cases of nonmenstrual TSS and 5%-25% of strains O55:H7 clones have recently radiated from a com- causing other diseases produce this protein. mon ancestral cell. The O157:H7 clone arose from Several questions of importance to both medical an O55:H7-like ancestor, perhaps through hori- bacteriology and evolutionary genetics were ad- zontal transfer and recombination events adding dressed in a study of 315 TSST-1-producing Shiga-like toxin genes and adhesion genes to an E. strains of S. aureus (47). It was discovered that the coli genome preadapted for causing diarrheal dis- organisms responsible for most cases of TSS with ease (34, 35). If, as the multilocus enzyme electro- a female urogenital focus are members of a single phoretic data indicate, O157:H7 and O55:H7 distinctive clone (designated as ET 41), a result organisms have shared a recent common ancestor, that explains the observation that isolates recov- it is likely that the close genetic affiliation would ered from patients with TSS share many traits be reflected at the nucleotide level. To test this (48, 49). The investigation also showed that TSST- notion, the gene (eae) (34) encoding intimin, a protein involved in bacterial attachment to entero- 1 is expressed by isolates of a great variety of cytes and subsequent effacement of the microvilli, clones representing virtually the full breadth of was sequenced from representative isolates of genotypic diversity in the species as a whole. In these two serotypes. The resulting sequence data addition, isolates of ET 41 represented 24% of a were consistent with the hypothesis that O157:H7 sample of TSST-1-producing strains recovered be- and O55:H7 organisms share a close genetic affin- fore 1978, which meant that the tst gene encoding ity and thereby provide a plausible explanation for the toxin neither evolved nor was acquired re- the observation that these bacteria cause similar cently by this species. The failure to recover iso- attaching and effacing lesions in cells grown in lates of ET 41 from non-human hosts effectively culture (38) and in animal models (39). Because eliminated the likelihood that animals are impor- conventional serotyping of E. coli does not provide tant in the transmission of this clone. a reliable basis for analyzing population structure Twenty-eight percent of isolates of S. aureus and can be grossly misleading as to genetic rela- cultured from the introitus, vagina, or cervix of tionships among isolates (40-42), many important unassociated healthy carriers or women with non- medical correlates of the population structure will TSS urogenital symptoms were ET 41 or closely not be recognized and understood fully until E. coli allied clones; no other single multilocus enzyme isolates are sorted out along clonal lineages. genotype accounted for more than 12% of normal vaginal isolates. These observations led to the hypothesis that isolates of ET 41 are more readily able to colonize the human vagina and, hence, are

Emerging Infectious Diseases 4 Vol. 2, No. 1 — January-March 1996 Perspective widely dispersed in an ecological niche of great Serogroup B ET-5 Complex Organisms consequence in TSS. Under this “adapted clone” Caugant et al. (52) demonstrated that an epi- hypothesis, isolates of ET 41 are responsible for demic of serogroup B that most vaginal cases because this clone has a special began in the 1970s in Norway and subsequently affinity for the cervicovaginal milieu, perhaps (but spread through much of Europe was caused by a not necessarily) as a consequence of variation in group of 22 very closely related clones, designated regulation of toxin- or other virulence-gene expres- as the ET-5 complex, that have no close genetic sion. In summary, data derived from clonal analy- relationship to other clone groups. Clones of this sis of TSST-1-producing S. aureus are consistent complex were traced intercontinentally to Chile with the notion that the “bloom” in TSS cases and , where they also caused contem- happened because of a change in the character of porary outbreaks of invasive disease. Clonal catemenial products (perhaps associated with de- analysis also showed that a severe epidemic of creasing levels of anti-TSST-1 antibody in human meningococcal disease in Cuba (characterized by populations), not because of a new S. aureus a high attack rate and incidence of septicemia) was strain. due to ET-5 complex organisms. The recovery of Two additional points are noteworthy regarding these same bacteria from outbreaks in Miami, population genetic analysis of S. aureus strains Florida, in 1980 and 1981, strongly suggested that producing TSST-1. First, if the gene encoding Cuban refugees imported the clones to Miami. TSST-1 were evolutionarily old, allelic variants Members of the ET-5 complex have seldom been differing in nucleotide and, perhaps, amino acid recognized as important pathogens in the United sequence would exist in natural populations. This States. However, ET-5 complex organisms were prediction was borne out by the identification of a responsible for a recent increase in meningococcal variant of TSST-1 associated with goat, sheep, and disease rates in Washington and Oregon (53). In occasionally bovine mastitis that is encoded by a addition, a serogroup B epidemic in greater Sao gene which differs from the “human” form by 14 Paulo, Brazil, was also caused by ET-5 complex nucleotides, resulting in 9 amino acid changes. members (54). The variant toxin retains mitogenic activity for Recently, clonal analysis has been used to study mouse splenocytes but differs significantly in serogroup B meningococcal isolates that caused other functions ascribed to TSST-1, including abil- invasive disease in The Netherlands between 1958 ity to induce a TSS-like disease in rabbits (50). and 1986 (55). Significant temporal variation in Second, if the rapid increase in TSS cases were the clonal composition of meningococcal popula- caused by a change in host character rather than tions was identified. Recent disease episodes were by the rapid spread of a single, new, hypervirulent caused predominantly by isolates of three clonal clone, subclonal heterogeneity would be present among isolates classified as ET 41. Examination lineages (designated I, III, and VI) that were not of RFLP patterns for the gene (coa) encoding co- represented in samples collected before 1975. In agulase has shown at least three distinct sub- addition, an epidemic in 1966-1967, and a hyper- clones of ET 41 (51). endemic disease wave in 1972 were caused mainly by two closely related clones (ET-11 and ET-17) Neisseria meningitidis expressing serotype 2b protein. Strong statistical Extensive work in the last 10 years has exam- deviation in the sex ratio was recorded for disease ined the molecular population genetics of Neisse- caused by clones of two lineages. Clones of lineage ria meningitidis, predominantly by clonal V were cultured far more frequently from female analysis, and more recently by DNA sequencing of than for male patients; whereas, clones of lineage putative virulence genes. This work suggests that IX were recovered from disease in male patients temporal variation in disease frequency and sever- approximately four times more often than aver- ity is usually associated with clonal replacement age. The cause(s) of these differences are unknown much like influenza epidemics are driven by anti- but warrant further investigation. genic shift. For most bacterial pathogens, few data are available regarding the frequency with which dis- tinctive clones are recovered in asymptomatic per- sons. Caugant et al. (56) studied the clonal composition of meningococcal isolates cultured

Vol. 2, No. 1 — January-March 1996 5 Emerging Infectious Diseases Perspective from the nasopharnyx of healthy carriers in Nor- A group led by M. Achtman assembled 423 way and discovered that the frequency of recovery serogroup A meningococcal isolates, recovered pri- of clones (ET-5 complex and ET-37 complex) caus- marily from invasive episodes, and representing ing 80% of disease episodes were represented by organisms responsible for 23 epidemics or out- only 7% and 9%, respectively, of carrier isolates. breaks between 1915 and 1983. Thirty-four dis- This same study demonstrated that the clones tinctive clones were assigned to four complexes most commonly represented among carrier iso- representing groups of related clonal genotypes lates (19%) have never been recovered from pa- (61). Most epidemics were caused by a single clone, tients with invasive meningococcal infection. The and the same clone often was responsible for con- data reinforce the concept that bacterial clones current epidemics in contiguous countries. For vary dramatically in virulence potential. example, serogroup A clone I-1 caused a pandemic that began in North Africa and certain Mediterra- Serogroup C Disease nean countries in 1967 and spread throughout An increase of invasive disease due to serogroup West Africa in the subsequent 2 years; clone III-1 C N. meningitidis strains has been reported in has been responsible for disease outbreaks in Fin- several countries in recent years (57-60). Study of land, Brazil, Nepal, and . 121 isolates recovered from patients in Greater Recently Achtman’s group has extensively char- Sao Paulo, Brazil, between 1976 and 1990 identi- acterized more than 300 serogroup A isolates from fied a striking increase in isolates assigned to ET patients or carriers in one epidemic in The Gambia 11 complex (58). The percentage of invasive dis- in 1982-1983 and in 1984-1985 after an immuni- ease episodes caused by complex 11 organisms zation program at the end of 1983. Analysis of a increased from 8% in 1988 to 66% in 1990. Out- representative subgroup of 64 isolates showed breaks of serogroup C meningococci have also been that all were assigned to clone IV-1 (64, 65). Iso- recently reported from distinct regions of the lates of this clone were examined for subclonal United States (59) and (57, 60). Analysis variation with SDS-PAGE profiling, LPS profiling, of organisms collected from 13 U.S. outbreaks and genomic restriction endonuclease profiling, identified five distinct multilocus enzyme types, and rare variants were detected. Two cell-surface all very closely allied in overall chromosomal re- antigens (class 5 outer membrane protein and pili) latedness (59). Moreover, strains causing 4 of these were unusually variable, and the hypothesis was 13 outbreaks were identical in multilocus enzyme formulated that variation in the class 5 OMP type (designated ET-15) to organisms responsible occurs as a consequence of recombinational events for outbreaks in eastern Canada (60). Canadian affecting the translational reading frame. The investigators have reported (60) that ET-15 organ- role, if any, of this subclonal microheterogeneity in isms had a significantly higher case-fatality ratio serogroup A meningococcal epidemics is being as- than other invasive meningococcal disease iso- sessed. Clonal analysis has provided a framework lates, which may be due to a lower herd immunity that is being exploited to rationally select strains to the newly emerged clone. for further characterization by molecular and se- rologic techniques that may provide insight into Serogroup A Disease the forces driving a bacterial epidemic (66-68). Unlike other serogroups of Neisseria meningi- tidis, which are usually associated with endemic Clonal analysis also has demonstrated that se- disease, isolates expressing serogroup A capsular rogroup A isolates are a restricted phylogenetic polysaccharide are unusual in that they may cause subpopulation of the species N. meningitidis (69). large epidemics. For example, serogroup A organ- This result may mean that the genotype bestowing isms have been responsible for epidemics of inva- the epidemic phenotype has arisen a single time sive disease in Africa, China, Iran, Greece, and that it has not been successfully transferred Finland, Brazil, and Nepal (61). Major epidemics horizontally to unrelated phylogenetic lineages of every 5-10 years in the Sahel region of sub-Saha- the species. ran Africa have led to the description of a “menin- Moore et al. (70) employed clonal analysis to gitis belt” and to detailed studies by clonal analysis document the intercontinental spread of an epi- of the molecular epidemiology of serogroup A or- demic group A meningococcal clone complex by ganisms responsible for these and other outbreaks Muslim hajis pilgrims in 1987. Apparently this (62, 63). clone was carried from South Asia (Nepal and/or

Emerging Infectious Diseases 6 Vol. 2, No. 1 — January-March 1996 Perspective

India) to Mecca, , where it was dis- association was present with isolates of the same seminated in epidemic form to other hajis (pil- clone, as well as those of distantly related phylo- grims) and to indigenous Saudis. The report of genetic lineages. The data were interpreted as invasive serogroup A meningococcal disease in strong circumstantial evidence that scarlet fever other Gulf nations and among hajis returning to toxin A itself, or, possibly, the product of a gene the United States, Europe ( and the United tightly linked to it, is a factor in the pathogenesis Kingdom), and Africa (Ethiopia, Sudan, and Chad) of TSLS. and the recovery of isolates of the same clone Because an increase in disease caused by complex (designated ET III-1) from persons in strains expressing the M1 serotype protein had these diverse geographic localities strongly sug- also been observed in England, Sweden, Norway, gested that an unusually virulent organism had , other European countries, and else- been rapidly dispersed intercontinentally. Spread where, we sought to determine if strains recovered of clone III-1 from Mecca to France by hajis was from these diverse localities were genetically al- independently confirmed by Riou et al. (71). This lied. Chromosomal diversity and relationships meningococcal clone also caused recent episodes of among 126 M1 strains from 13 countries on five invasive disease in Sweden (72) and Kenya (73). continents were analyzed by multilocus enzyme electrophoresis and restriction fragment profiling Streptococcus pyogenes Invasive Disease by pulsed-field gel electrophoresis (83). All isolates Severe invasive infections caused by S. pyo- were also examined for the speA gene by PCR, and genes have been reported with increased fre- to increase the possibility of identifying inter- quency in recent years in the United States (74, strain variation, strain subsets were examined by 75), Europe (76-78), and elsewhere (79, 80). These automated DNA sequencing for allelic polymor- include both soft tissue infections, such as celluli- phism in genes encoding M protein (emm), strep- tis, and deeper infections, including osteomyelitis, tococcal pyrogenic exotoxin A (speA), necrotizing fasciitis, and sepsis, many of which streptokinase (ska), pyrogenic exotoxin B (speB), have occurred in previously healthy persons. The and C5a peptidase (scp). Seven distinct emm1 observation that many patients have multiorgan alleles were identified that would express M pro- failure and other signs and symptoms mimicking teins differing at one or more amino acids in the staphylococcal toxic shock syndrome led to the N-terminus variable region. Although substantial characterization of a streptococcal “toxic-shock- levels of genetic diversity existed among M1 or- like syndrome” (TSLS) (81). Most S. pyogenes iso- ganisms, most invasive episodes were caused by lates recovered from such patients produce one or two subclones marked by distinctive multilocus more pyrogenic exotoxins with significant amino enzyme electrophoretic profile and PFGE restric- acid sequence homology and functional similarity tion fragment length polymorphism (RFLP) types. with several enterotoxins synthesized by S. One of these subclones (ET 1/RFLP pattern 1a) aureus (82). has the speA gene, and was recovered worldwide. To determine the genetic diversity and clonal Identity of speA, emm1, speB, and ska alleles in relationships among S. pyogenes isolates recov- virtually all isolates of ET 1/RFLP type 1a means ered from patients with TSLS or other invasive that these organisms have shared a common an- diseases in the United States, 108 organisms were cestor, and that global dispersion of this M1 sub- studied by multilocus enzyme electrophoresis and clone has occurred very recently. The occurrence analyzed for exotoxin A, B, and C synthesis (82). of the same emm and ska allele in strains that are The analysis showed that 33 distinctive clones well-differentiated in overall chromosomal char- were present among isolates comprising the sam- acter demonstrated that horizontal transfer and ple, but nearly half the disease episodes, including recombination play a fundamental role in diversi- more than two-thirds of the cases of TSLS, were fying natural populations of S. pyogenes. caused by strains of two related clones, designated The population genetic framework constructed ET-1 and ET-2 (82). The production of pyrogenic for S. pyogenes has been exploited to rationally exotoxin A (scarlet fever toxin, which is bacterio- choose strains for comparative molecular charac- phage-encoded), either alone or in combination terization of the gene (speA) encoding scarlet fever with other pyrogenic exotoxins, was associated toxin (84, 85). An analysis by Nelson et al. (84) with recovery in patients with TSLS. This identified four alleles of speA in natural

Vol. 2, No. 1 — January-March 1996 7 Emerging Infectious Diseases Perspective populations, one of which (speA1) occurs in many epidemic, immigration from countries with high distinct clonal lineages and is, therefore, probably TB prevalence, and outbreaks in correctional in- evolutionarily old. The presence of identical exo- stitutions, nursing homes, shelters for the home- toxin A structural genes in diverse phylogenetic less, and other congregative environments have lineages means that the gene has been horizon- contributed to the resurgence (88). On a global tally distributed among clones, presumably by scale, one-third of the world’s population is in- bacteriophage-mediated transfer. Two other al- fected with this pathogen, and 8 million new TB leles (speA2 and speA3), characterized solely by cases occur each year. Moreover, nearly 3 million single nucleotide changes resulting in single people die annually of TB, making it the leading amino acid substitutions, were each identified in cause of death due to an infectious agent world- single clones (ET 1 and ET 2) that together have wide (88). Hence, there is a need to understand the caused most of TSLS episodes. The restriction of nature and extent of molecular variation in this speA2 and speA3 to single clonal lineages can be pathogen. interpreted as evidence that these two alleles are Although the population genetics of M. tubercu- evolutionarily younger than speA1. A fourth allele losis has not been examined by multilocus enzyme (speA4) also is present in a single phylogenetic electrophoresis, a recent study (91) analyzed DNA lineage and is 9% divergent from the other three sequence diversity in eight loci (192,875 nucleo- toxin alleles. The absence of synonymous (silent) tides) from unassociated isolates recovered in nucleotide changes in speA2 and speA3 is unusual North America and Europe. The data showed al- and suggests that the allelic variation is not selec- most a complete absence of coding sequence nu- tively neutral, which implies that the toxins are cleotide variation. To rule out the possibility that not functionally equivalent. Moreover, the muta- tions occur in a segment consisting of five amino restricted geographic sampling biased the data acids that are highly conserved in the aligned set, 350-bp fragments of genes encoding the beta sequences of staphylococcal enterotoxin A (SEA), subunit of RNA polymerase (rpoB), a 65-kilodalton staphylococcal enterotoxin B (SEB), SEC1, SEC3, heat shock protein (hsp65), the A subunit of DNA SED, SEE, and streptococcal pyrogenic exotoxin C gyrase (gyrA), an enzyme involved in aromatic (86). The segment of SPE A containing these vari- amino acid biosynthesis (aroA), RecA protein ations is immediately adjacent to a region contain- (recA), and a 1435-bp region of the gene (katG) ing cysteine residues involved in the formation of encoding a catalase-peroxidase enzyme important a disulfide loop believed to be required for mito- in isoniazid resistance (92-94) and host-parasite genicity of SPE A and other bacterial superan- interactions (95), were sequenced from one ran- tigens. Population genetic analysis then suggests domly selected isolate from each of seven countries that there are functional correlates of the allelic with well-differentiated human populations variation and that the alleles have been subject to (Switzerland, , Algeria, Somalia, Papua natural selection. Recent studies have shown that New Guinea, Vietnam, and Tibet). A virtual lack the ability of the SPEA2 and SPEA3 variants to of nucleotide variation was also found in these stimulate human peripheral blood mononuclear seven isolates, and sequencing of several genes cells exceeds that of SPEA1 (87). from many additional TB isolates has reinforced the concept of extremely restricted structural gene Mycobacterium tuberculosis polymorphism. The paucity of sequence variation Perhaps no bacterial infection in recent years was surprising for several reasons. First, paleo- has generated as much interest nationally as re- pathologic evidence suggests that humans got TB surgent tuberculosis (TB) (88). Largely because of as early as 3700 BC in Egypt and 2500-1500 BC the success of public health strategies, the inci- in Europe and also pre-Columbian North and dence of TB declined steadily in the United States South America (96). Moreover, M. tuberculosis since the early 1950s, and the disease was thought DNA recovered from lung lesions in a 1000-year- to be eradicable by the end of the first decade of old Peruvian mummy confirmed that the disease the 21st century (89). However, the yearly decline existed in the pre-Columbian New World (97). in TB incidence ended in 1984, and after several Second, as noted above, there is a very large global years of a plateau phase, resurged from 1988 pool of infected persons (88), and third, consider- through the present. An estimated 63,000 excess able chromosomal restriction fragment length cases occurred through 1993 (90). The HIV/AIDS polymorphism has been identified by probing with

Emerging Infectious Diseases 8 Vol. 2, No. 1 — January-March 1996 Perspective mobile elements such as IS6110 (98, 99). Based on suggested that many of these organisms were a population genetic interpretation of the data, it clonally related. More recent analysis using was posited that M. tuberculosis may be only IS6110 typing, several other molecular typing 15,000 to 20,000 years old, an age that dates strategies, and automated DNA sequencing to speciation and global dissemination to roughly the identify the exact nucleotide changes responsible same time as paleomigration into the New World. for resistance to isoniazid, rifampin, and strepto- The time frame is also consistent with speculation mycin has unambiguously demonstrated the ex- (100) that the agent of human TB arose from the istence of two abundant closely related subclones very closely related cattle pathogen M. bovis by (arbitrarily named W and W1, son of W) that have host specialization occurring since the domestica- clearly shared a recent common origin (105). Mul- tion of this animal some 8,000 - 10,000 years ago. tidrug resistance in these strains is due to sequen- Recent large-scale DNA sequencing results are tial accumulation of amino acid substitutions also consistent with an interpretation that M. conferring resistance to each drug alone, rather tuberculosis and M. bovis have shared a recent than a single-step molecular event, such as acqui- common ancestor. sition of a multidrug-resistance-conferring plas- These molecular population genetic findings mid. Progeny of these two subclones have now have considerable implications for M. tuberculosis spread well beyond the New York City borders. pathobiology research. First, the virtual absence The organisms have been isolated from patients of naturally occurring nucleotide substitutions in other New York communities (108), Atlanta, greatly increases the likelihood that missense mu- Miami, Denver, Las Vegas, and Paris, France tations identified in genes associated with resis- (109). Thus far, all patients documented to have tance to antimicrobial agents actually confer infection caused by W or W1 organisms can readily resistance rather than simply acting as conven- be epidemiologically connected with New York ient surrogate markers of resistance (93, 94, 101, City, that is, secondary, tertiary, or quaternary 102). Second, restricted allelic diversity means spread has not yet sufficiently obscured this im- that it is probable that only nominal amino acid portant epidemiologic thread. Dissemination of variation will occur in proteins of potential immu- these difficult to treat W and W1 organisms noprophylaxis, diagnostic, or virulence interest. throughout New York City and other cities dem- onstrates the devastating consequences of clonal Clones W and Son of W origin and spread of a bacterial pathogen. Because Commensurate with the rise of TB cases in the some persons now infected latently with W and United States was an increase in the number of W1 will later experience reactivation disease, dis- organisms resistant to one or more anti-TB medi- semination of W and W1 has adverse implications cations (103). This trend has been viewed with for TB control in the 21st century. great concern by public health authorities and clinicians, in part because no new first-line anti- Penicillin-Resistant Streptococcus pneumoniae TB agents have been introduced in several dec- The Gram-positive bacterial pathogen S. pneu- ades. Certain communities have contributed moniae is a major cause of illness and death world- disproportionately to the documented increase is wide (110, 112). In the United States, the organism resistant organisms, the most notable being New is responsible for more than 500,000 cases of pneu- York City (104), which has accounted for up to 60% monia, 55,000 episodes of bacteremia, 6,000 cases of all drug-resistant M. tuberculosis reported na- of meningitis, and 40,000 deaths each year (113). tionally in some surveys. Although strains with Until relatively recently, antibiotic resistance in S. several antimicrobial agent susceptibility pat- pneumoniae was rare, but it is now a global public terns have been identified, approximately 300 or- health problem (114-116). ganisms are invariably resistant to isoniazid, Resistance to penicillin in many organisms is streptomycin, rifampin, ethambutol, and variably due to the expression of altered high molecular- resistant to ethionamide, kanamycin, capreomy- weight penicillin-binding proteins (PBPs) that cin, and ciprofloxacin (105, 106). Early reports have reduced antibiotic affinity (117). Among re- based on IS6110 restriction fragment length poly- sistant strains, alterations in four (1A, 2X, 2A, and morphism typing (99), other molecular techniques 2B) of the five high molecular-weight PBPs ex- (98, 107), and classic epidemiologic investigations pressed by isolates of the species have been

Vol. 2, No. 1 — January-March 1996 9 Emerging Infectious Diseases Perspective identified in resistant patient isolates. Research unknown. The frequency of use of beta-lactam has shown that two processes have contributed to antibiotics in Iceland and Sweden (a country the rise of these organisms. First, many distinct where resistant pneumococci are rare) in 1989 was susceptible strains are independently evolving to similar (133), and low compared to other industri- the resistance phenotype. Acquisition of penicillin alized countries (134). However, Iceland has a very binding protein gene segments from foreign do- high use of antimicrobial agents such as trimetho- nors, such as oral streptococci, is apparently a prim-sulfamethoxazole, metronidazol, and tetra- primary driving force (118). At the molecular level, cycline (133, 134), which means it is conceivable the result is generation of mosaic genes, and that selection for the multiresistant clone oc- thereby molecularly remodelled PBP proteins curred. A second possible factor contributing to the with decreased affinity for penicillin (119-123). clonal spread of the organisms is that 57% of Evidence shows that the Hex recombinational Iceland’s population of about 250,000 live in pathway (124) participates. Once a distinct drug- Reykjavik and its suburbs, where most of these resistant cell has been generated, progeny can be strains have been recovered. Moreover, almost transmitted locally and over intercontinental dis- 80% of Icelandic children 2 to 6 years of age in tances by person-to-person spread (125-130). Mul- Reykjavik attend day-care centers. Together these tilocus enzyme electrophoresis has been applied to factors may have provided a unique set of circum- analyze genetic relationships among penicillin-re- stances for introduction and rapid spread of the sistant strains of S. pneumoniae from global multiresistant clone. sources and to infer patterns of epidemiologic Investigating the molecular population genet- spread of these resistant organisms (125-131). ics of pneumococci has led to the realization that The importance of rapid local clonal spread of horizontal transfer and recombinational processes antibiotic-resistant S. pneumoniae is illustrated are also serving to generate variation in capsule by events in Iceland. Monitoring of antibiotic re- type and immunoglobulin A1 (IgA1) protease gene sistance patterns of pneumococci in Iceland alleles (135, 136). Coffey et al. (135) analyzed showed no detectable penicillin-resistant organ- European resistant strains expressing serotype 9 isms in 1983 to 1988. The first penicillin-resistant or 19 through a combined approach employing strain was recovered in December 1988 (132). The clonal analysis and RFLP profiling of genes encod- frequency of penicillin-resistant organisms rose ing PBP1A, PBP2B, and PBP2X. Analysis of a sharply over the next 3 years from 2.3% to 17% of resistant isolate synthesizing serotype 19 capsule all isolates in the first quarter of 1992 (132). Al- showed that it was identical in overall chromoso- most 70% of the resistant isolates expressed sero- mal character to a clone of organisms resistant to group 6 capsule polysaccharide and were also multiple antibiotics which expressed serotype resistant to tetracycline, , eryth- 23F, a result that was interpreted as evidence that romycin, and trimethoprim-sulfamethoxazole. To horizontal transfer of capsular biosynthesis genes test the hypothesis that these Icelandic isolates had occurred. More recently, Lomholt (136) has were clonally related, Soares et al. (128) examined shown that recombinational processes contribute 57 organisms for serotype, PBP pattern, pulsed- to allelic variation in the gene (iga) encoding IgA1 field chromosomal restriction endonuclease diges- protease. tion pattern, and multilocus enzyme electrophoretic genotype. All isolates were sero- type 6B and had closely similar or identical pat- Methicillin-Resistant Staphylococcus aureus terns for each of the molecular markers examined. Very soon after methicillin entered clinical use Surprisingly, the Icelandic organisms were indis- in the 1950s, strains of S. aureus resistant to this tinguishable from a subgroup of multiresistant antimicrobial agent were reported in the United serotype 6B pneumococci that occurs with high Kingdom (137). Within a few years, hospital out- incidence in Spain. The authors concluded that the breaks caused by methicillin-resistant S. aureus Spanish clone was imported to Iceland and noted (MRSA) occurred in Europe. MRSA were recog- that in recent years a favored vacation locality for nized as an important hospital infection control Icelandic families with young children had been problem in the United States in the mid-1970s, Spain. The factors responsible for the precipitous and these organisms have now achieved global spread of the clone in Iceland are largely distribution (138).

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Intrinsic methicillin resistance is due to the studies (147), 16S rRNA gene sequencing (148), expression of an altered penicillin-binding protein genomic fingerprinting by arbitrarily primed po- (PBP) termed PBP 2a (139) that is encoded by the lymerase chain reaction (149), and other chromosomal mec gene (140, 141). Evidence has techniques (Valsangiacomo C, Balmelli T, Pif- been presented that mec originated as a conse- faretti J-C, pers. comm.). Moreover, different geno- quence of a recombinational event fusing about species of B. burgdorferi have been associated with 300 bp of a staphylococcal beta-lactamase gene distinct clinical manifestations of Lyme borreliosis and a segment of a gene encoding a PBP from an (150-152). unknown donor bacterium, perhaps E. coli (142). Although population genetics has only been Multilocus enzyme electrophoresis and other applied to the study of for molecular population genetic techniques were approximately a decade, considerable insight has used to determine the extent of mec distribution been gained into the molecular mechanisms of among phylogenetic lineages of the species and temporal variation in disease frequency and sever- genetic relationships among MRSA strains circu- ity, host adaptation of clonal lineages, and the lating in various geographic regions at different relationship of disease severity and naturally oc- times (143, 144). The mec gene is harbored by curring bacterial clones. The work cited in this many divergent phylogenetic lineages repre- review represents only a small part of the contri- senting a large portion of the breadth of chromo- bution of molecular population genetic investiga- somal diversity in the species S. aureus. On the tions to an understanding of temporal variation in basis of additional evidence, it was proposed that disease frequency and severity, microbial patho- multiple episodes of horizontal transfer and re- genicity, and evolution of virulence genes. For combination have contributed to the spread of the example, contributions have also been made from mec resistance determinant in natural popula- studies of Vibrio cholerae (153-155), encapsulated tions. The identification of a single multilocus H. influenzae (156), L. monocytogenes (157-158), enzyme genotype among MRSA organisms recov- Salmonella spp. (159-161), and other pathogens ered in the , Denmark, Switzer- (162). land, Egypt, and Uganda, soon after the Changes in human behavior, simple processes widespread introduction of methicillin into clini- of microbial evolution, and increasing resistance cal use in the 1960s, meant that MRSA isolates to antimicrobial agents will continue to supply recovered from those localities at that time were mankind with new infectious disease challenges progeny of a single ancestral cell that had probably and, therefore, motivation for molecular popula- acquired the mec determinant recently. The mul- tion genetics studies. The genomes of two bacterial tilocus enzyme electrophoretic data demonstrat- pathogens have now been sequenced (163-164), ing association of mec with highly divergent and it is likely that the genomes of most major members of the S. aureus species effectively ruled human and veterinary viral and bacterial patho- out the idea (145) that all extant MRSA are lineal gens will be sequenced in their entirety in the next descendants of a single clone, and that mec was decade. Hence, the trend toward molecular dissec- acquired just once by methicillin-sensitive clones tion of microbial populations by large-scale DNA of this pathogen. sequencing will accelerate. DNA sequence-based and conventional molecular population genetic studies are cost-effective and should be encour- Borrelia Species Associated with Lyme Disease aged in the fight to limit the detrimental impact Molecular population genetic strategies have of infectious agents on human, animal, and plant also been used to delineate accurate phylogenetic health. relationships among emerging organisms. For ex- ample, Boerlin et al. (146) studied 50 isolates classified as Borrelia burgdorferi by multilocus Acknowledgments enzyme electrophoresis and identified three dis- D. Meyer assisted in preparing the manuscript. tinct genetic clusters that were well differentiated Research in the author’s laboratory is supported by Public from one another in overall chromosomal charac- Health Service Grants AI-33119, AI-37004, and DA-09238, ter. The investigators proposed that each cluster and by awards from the Texas Advanced Technology Program and the Moran Foundation. (The author is an represented a genospecies, and this idea was sub- Established Investigator of the American Heart sequently supported by DNA-DNA reassociation Association.)

Vol. 2, No. 1 — January-March 1996 11 Emerging Infectious Diseases Perspective

Dr. Musser is a member of the Section of Molecular 16. Selander RK, McKinney RM, Whittam TS, Bibb WF, Pathobiology, Department of Pathology, and associate Brenner DJ, Nolte FS, et al. Genetic structure of professor of pathology, and microbiology and populations of . J Bacteriol immunology, Baylor College of Medicine, Houston, 1985;163:1021-37. Texas. He is also associate director, Clinical 17. Musser JM, Hewlett EL, Peppler MS, Selander RK. Genetic diversity and relationships in populations of Microbiology Laboratory, and medical director, Bordetella spp. J. Bacteriol 1986;166:230-7. Molecular Diagnostic Laboratory, The Methodist 18. Gilmour MN, Whittam TS, Kilian M, Selander RK. Hospital. He has had a long-standing interest in Genetic relationships among the oral streptococci. J elucidating the molecular forces responsible for Bacteriol 1987:5247- 57. temporal variation in disease frequency and severity. 19. 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Proc Natl Acad Sci USA 1983;80:1751- 28. Carlone GM, Gorelkin L, Gheesling LL, et al. Poten- 5. tial virulence- associated factors in Brazilian purpuric 14. Dykhuizen DE, de Framond J, Hartl DL. Selective fever. J Clin Microbiol 1989;27:609-14. neutrality of glucose-6-phosphate dehydrogenase al- 29. Rubin LG. Phase-variable expression of the 145-kDa lozymes in Escherichia coli. Mol Biol Evol 1984;1:162- surface protein of Brazilian purpuric fever case-clone 70. strains of Haemophilus influenzae biogroup aegyp- 15. Ochman H, Whittam TS, Caugant DA, Selander RK. tius. J Infect Dis 1995;171:713-7. Enzyme polymorphism and genetic structure in Es- cherichia coli and Shigella. J Gen Microbiol 1983;129:2715-26.

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