In this study, we provide evidence that closely related M. tuberculosis strains can diversify sufficiently to lead to recognizably altered phenotypes, which appear to be associated with epidemiologic characteristics. The process of evolution/adaptation of M. tuberculosis in heterogeneous host populations is complicated by the pathogen’s obligate intracellular lifestyle, requiring coexistence with the host to survive. Thus, strain-specific changes in the bacterial population potentially involve a combination of pathogen characteristics, properties of the host, and environmental factors. Here, we show that subtle genetic changes in strain H gave rise to BE, a strain with altered phenotypic properties in both tissue culture and guinea pig infection models. Interestingly, the phenotypes appear to correlate with our empirical epidemiologic observation(s).
We show that the strain groups C and H are closely related to BE, H6, and C28 by descent and share key molecular signatures ( and ) [19
]. Plasticity in the DR region, typically driven by homologous recombination between DRs, transposition of IS6110
, and/or slippage during DNA replication, has been used to detail microevolutionary events [27
]. Interestingly, some unrelated M. tuberculosis
lineages and M. tuberculosis
complex members, such as M. bovis
], share the DVR25 duplication. It is possible, albeit unlikely, that the duplication events arose independently (convergence) or from random lineage-specific loss of the second copy of DVR25. Thus, the use of a combination of molecular markers with varying clock speeds can shed light on the order of events and group and distinguish closely related strains of M. tuberculosis
Although the distribution of the 5 variants in our population may be anecdotal, it likely reflects the consequences of microevolutionary events modifying bacterial properties and/or host-pathogen interactions. Faster comparative M. tuberculosis
growth, in vitro or in vivo, has been used as a proxy for virulence or bacterial fitness [34
]. However, the correlation between growth rates and epidemiologic success is unclear [35
]. One study examining growth and cytokine patterns among a large group of phylogenetically related strains did not find consistent profiles except within large clusters [39
]. We found that strains that have caused extensive disease (C and H) grew significantly slower in human monocytes compared with other strains (). Our intracellular studies suggest that the faster growing strains elicited significantly more TNF-α and IL-1β than these slower growing strains (). Previous studies have suggested that the cytokine-inducing capacity of M. tuberculosis
is a property of bacillary load, which is determined by growth kinetics [35
]. However, our results with heat-killed bacilli suggest that the differential cytokine profiles induced by these strains were independent of bacillary load. Previous studies have shown that early innate immune response is driven by intrinsic bacterial properties, independent of replication [15
]. In one report using human macrophage infections, overall levels of cytokine production appeared to track with phylogenetic lineages, although with some intralineage variation [17
]. A recent study suggested that innate immune response and virulence, largely consistent within lineages, appears associated with distinct cell envelope lipid [41
]. In contrast, a study by Wang et al [42
] showed considerable variation among a diverse group of Beijing strains but did not find a distinct cytokine profile associated with successful sublineages.
Proteomic analysis of BE, H6, and C28 grown in broth cultures identified several differences in protein levels [43
]. Strains H6 and C28 were found to produce higher levels of proteins involved in virulence, detoxification, metabolic pathways, and adaptation, and lower levels of proteins involved in cell wall and cell processes. Additionally, BfrB and Cfp29, B- and T-cell antigens, respectively [44
], were more abundant in H6 and C28 than in BE. The differential abundance of proteins may partly explain the subtle differentials in growth rates and cytokine induction in monocytes. These data suggest that, during the course of microevolution, M. tuberculosis
can alter proteomic profiles, potentially affecting biomedically relevant properties.
Guinea pigs are relatively susceptible to low-dose aerosol infection and have been used extensively to examine virulence and pathogenesis of M. tuberculosis
]. Animals infected with strains BE, H6, or C28 had a significantly shorter median survival than those infected with C or H. In guinea pigs, survival is often proportional to levels of inflammation but not necessarily to bacillary burden and/or progressive pathology [46
]. In our study, the strains that induced higher levels of proinflammatory cytokines in vitro were associated with more inflammation in guinea pigs, as indicated by lung pathology, as well as more rapid mortality. In contrast, CDC1551 infection in mice has been associated with significantly longer survival than other M. tuberculosis
strains, despite inducing high levels of proinflammatory cytokines [37
The clinical strains in this report represent one of the largest groups of clustered strains circulating in the NYC region. In the early 1990s, the C strain was associated with an outbreak in a large homeless shelter in Manhattan and widespread dissemination in NYC, including 20% of all drug-susceptible tuberculosis cases reported in a large urban NYC hospital [48
]. The H strain and, to a lesser extent, BE have caused large clusters in the region for the last 15 years. Similarly, the most frequent drug-susceptible strain in San Francisco is closely related to H [33
]. The high prevalence of our strains among US-born patients was largely consistent with previous reports [19
Our study has demonstrated that natural variation, with detectable phenotypic changes, among clinical isolates may alter epidemiologic patterns in populations. Although our epidemiologic observation may be subject to confounding, it is possible that infection with M. tuberculosis
strains that are good inducers of proinflammatory cytokines are controlled more efficiently in healthy individuals with an effective innate response. This may explain the limited number of secondary cases associated with the outbreak of immunogenic CDC1551 strain [36
]. However, the spread of such strains may be unhindered in immunocompromised populations. Consistent with our findings, a separate study comparing tuberculosis cases infected with strains C and BE showed that BE cases were significantly associated with more respiratory AFB smear-positive status, HIV coinfection, and other traditional tuberculosis risk factors [50
]. In contrast, M. tuberculosis
strains that elicit a less protective innate immune response, such as the slower growing C and H strains, may more effectively cause active disease and promote spread in the general population. Thus, host factors can facilitate the selective spread of otherwise less epidemiologically fit organisms.
Numerous studies have highlighted large strain clusters and variants that have caused extensive disease. Reasons for spread have largely been attributed to individual- and ecological-level risk factors. Population-based studies are poised to identify variants and are capable of indicating M. tuberculosis microevolutionary processes within an epidemiologic context. Studies exacting the nature of other polymorphisms inherent in bacterial populations and their consequences by methods such as whole-genome sequencing are warranted. Our studies highlight the utility of examining strains that have, over the course of diversification, altered their epidemiology. Identifying such variants may be useful to study the natural evolution of virulence, pathogenesis, and bacterial fitness.