In this study, our main observations were that K. pneumoniae fimH (i) has relatively low nucleotide diversity, (ii) moves horizontally across clonal groups (subspecies), (iii) contributes substantially to the diversification of K. pneumoniae clones, and (iv) has acquired a pathoadaptive mutation during endemic circulation of a large clone that causes nosocomial UTI.
At the population level,
fimH was analyzed in a group of 65
K. pneumoniae isolates of diverse origins. The
fimH locus could be amplified from almost 90% of the isolates, indicating the ubiquitous nature of type 1 fimbriae in
K. pneumoniae. The diversity of
fimH was compared to those of three other genes from the same isolates—
mdh,
fumC, and
tonB. Both
mdh and
fumC are housekeeping loci, commonly used for MLST (
4,
42,
47) because their variation is considered to be of a neutral nature due to high functional and structural conservation of the encoded proteins. The third locus,
tonB, has been used for MLST (
4,
42), although there is evidence in
E. coli that
tonB is under positive selection for structural variability (
2). The variability of
fimH is two- to threefold lower than that of the housekeeping and
tonB loci, primarily due to a lower level of silent changes. This trend might indicate the relatively late evolutionary acquisition of the type 1 fimbrial operon by
K. pneumoniae subspecies or homogenization of alleles due to selective sweeps, i.e., a periodic expansion of specific lineages across many habitats of the species. However, the
fimH diversity pattern is generally similar to that of other
K. pneumoniae genes, with a predominance of silent changes over replacement mutations. The
K. pneumoniae fimH nucleotide diversity of 1.8% ± 0.06% is similar to that reported previously for
E. coli, where nucleotide diversity is 1.64% ± 0.07%, and the dN/dS ratio is also significantly less than 1 (0.004/0.052 = 0.077) (
34).
The phylogeny of
fimH is less diverse and exhibits limited congruence with other loci, suggesting that
fimH is prone to relatively frequent horizontal transfer within or between clonal groups of
K. pneumoniae. Based on the level of between-group diversity, these clonal groups appear to represent distinct subspecies of
K. pneumoniae. Frequent horizontal transfer of
fimH has been reported for
E. coli and is thought to be driven by strong diversifying selection on
fimA (
47), which encodes the major subunit of type 1 fimbriae, a major surface antigen. In contrast, the housekeeping loci
mdh and
fumC were, in general, phylogenetically congruent and thus appeared less prone to horizontal transfer. However,
tonB exhibited signs of horizontal transfer, likely due to surface expression that subjects it to immune pressure for structural diversification.
The overall clonal diversity of
K. pneumoniae obtained here, based on four loci, is comparable to the diversity obtained previously with MLST based on seven loci that included six housekeeping genes (
rpoB,
gapA,
mdh,
pgi,
phoE, and
infB), as well as
tonB (
4). Under our scheme, 34 clones with unique MLST types were identified among 54 isolates, while the seven-locus scheme identified 40 unique MLST clones among 67 isolates. The comparable clonal diversity identified in our study, which used only four loci, shows the advantage of adding
fimH to the MLST scheme, the frequent horizontal transfer of which leads to the genetic diversification of
K. pneumoniae populations. The role of
fimH in clonal diversification is also evident in the analysis of clonal diversity of
K. pneumoniae isolates from different sources, where
fimH was a key locus in the diversification of several otherwise identical clones.
Among
K. pneumoniae isolates from different sources, environmental isolates were the most diverse, with all isolates exhibiting unique MLST profiles and with multiple loci contributing to diversity. This is likely due to the fact that the environment (water, soil, and plants) is the main natural reservoir of
K. pneumoniae, where clonal population diversity has been accumulating evolutionarily for a long time. The diversity of sepsis isolates tested was also high, in agreement with a previous study that grouped 43 sepsis isolates into 29 district sequence types by seven-gene MLST, suggesting that the ability to cause sepsis is a relatively common property of
K. pneumoniae isolates. Further, it has been demonstrated that isolates of environmental origin are equal in virulence to isolates of clinical origin (
39). This is supported by our observation that isolate Kp342, isolated from a plant and known to play a major role in nitrogen fixation (
7), exhibited 100% similarity in all four loci to that of sp3, a sepsis isolate (Fig. ).
In contrast, isolates from patients with liver abscesses are closely related. The earliest reports of a distinctive syndrome of community-acquired
K. pneumoniae septicemia with liver abscess came from Taiwan in the 1980s (
45). In 2007, it was suggested that isolates causing liver abscesses were members of a common clonal population, even though these strains were isolated on three different continents (
42). Furthermore, similar to our findings, several reports have demonstrated that isolates causing liver abscesses in the majority of cases are serotypes K1 and K2 (
8), representing only a small fraction of the 77 different serotypes known in
K. pneumoniae (
20). Still, the
fimH gene from one of the isolates tested here (3859) was sufficiently different from those of the other liver abscess isolates to indicate its horizontal transfer. Thus, liver abscess isolates of
K. pneumoniae have undergone some population diversification. It is possible either that the clone has been in circulation for a relatively long time or that pressure to exchange the type 1 fimbrial cluster is very high. Interestingly, isolates sp29 from a patient with sepsis and cas126 from the environment both displayed K1 serotypes. Both isolates belong to group A, according to all four genes, but are not phylogenetically linked to the eight liver isolates or to each other. Of the isolates in which all four genes were obtained, isolate 3861 was the only liver isolate of serotype K2, consistent with the absence of the
magA locus by PCR amplification.
Urinary isolates of
K. pneumoniae also exhibited low diversity, i.e., they were highly clonal in nature. This may be due in part to the similar geographical origins of these isolates, with 20 of 21 collected from hospitals and foster care homes in Denmark. However, except for the
tonB locus, isolate C3091, collected in Washington, DC (
18), is grouped with UTI isolates from Denmark, suggesting that even geographically unlinked urinary isolates of
K. pneumoniae may be closely related. Also, the existence of the 13-isolate group, with identical MLST types isolated from patients in different locations, strongly suggests that this clone is involved in stable endemic circulation as a uropathogen among susceptible individuals in Denmark. Obtaining additional uropathogenic strains from more diverse geographic origins should provide a more definitive conclusion about the clonal diversity of uropathogenic
K. pneumoniae and the extent of endemic circulation of uropathogenic strains.
Considering the high clonal diversity and prevalence of K. pneumoniae isolates of environmental and sepsis origin, the clonal nature of the liver and uropathogenic isolates demonstrates the importance of certain clone-specific traits in K. pneumoniae pathogenicity. This allows future investigations to focus on comparative analysis of clinically distinctive clones in elucidating the molecular basis of K. pneumoniae pathogenesis.
Another interesting phenomenon observed in the endemic uropathogenic clone is
fimH diversification, which occurred in isolate cas665 through point mutation (S62A), rather than horizontal transfer. In
E. coli, the same mutation is proposed to be acquired under positive selection in uropathogenic isolates (
34), where A62 FimH variants demonstrate increased mannose-binding capability under static or low-shear conditions, increased urinary epithelium tropism, and enhanced urovirulence (
32). Indeed, when the mannose-binding capabilities of
K. pneumoniae FimH with and without mutation A62 were evaluated, the mutant variant exhibited dramatically increased binding, suggesting that S62A has been acquired in
K. pneumoniae under positive selection (S. G. Stahlhut et al., submitted for publication). To our knowledge, this is the first evidence of pathoadaptive diversification of uropathogenic bacteria that has been detected in the course of endemic circulation.
Aside from A62, no other mutations in the mature FimH peptide have been found in
K. pneumoniae that parallel the pathoadaptive mutations in FimH of
E. coli. In
E. coli FimH, about 50 different mutations have been characterized thus far, with over half occurring in hot-spot position
s, i.e., at specific amino acid residues (
34). Thus,
K. pneumoniae FimH appears to be under less positive selection for the acquisition of pathoadaptive changes. This is possibly due to the fact that
K. pneumoniae is primarily an environmental species that does not circulate continuously among the human population, in contrast to
E. coli. Thus, pathoadaptive mutations do not accumulate in significant numbers in
K. pneumoniae populations, especially considering the long-term instability of the pathoadaptive mutations due to their fitness trade-off in nonurinary habitats, where the gain of a functional advantage under novel conditions is accompanied by functionally detrimental effects in the original habitat. For FimH mutations, one such trade-off is increased sensitivity of the adhesin to inhibition by soluble mannosylated compounds (
32). Nevertheless, the occurrence of the S62A mutation in the course of the endemic circulation of a nosocomial uropathogenic clone of
K. pneumoniae strongly supports the importance of type 1 fimbriae in UTI caused by
K. pneumoniae, as has been shown for
E. coli.
Finally, it appears that the L(−12) deletion in the signal peptide of
K. pneumoniae FimH has been acquired on multiple occasions, suggesting an adaptive significance of the mutation. Although the mutation does not affect the structure or, implicitly, the function of FimH per se, it may affect the translocation of nascent FimH across the inner membrane. It has recently been demonstrated that signal peptide mutations are acquired under positive selection in
E. coli FimH and that they decrease the rate of protein translocation into the periplasm (
28). Because FimH initiates the biogenesis of type 1 fimbriae, decreased periplasmic FimH leads to the expression of fewer but longer fimbriae. It is thus conceivable that the L(−12) mutation may result in such morphological changes in fimbrial structure. If so, longer fimbriae could be adaptive for
K. pneumoniae strains by allowing the fimbrial tip (with FimH) to protrude through the thick capsule commonly produced by
K. pneumoniae isolates. It has been shown that capsular material can in fact interfere with fimbrial function (
29), making plausible the adaptive value of longer fimbriae. However, further studies are required to clarify the adaptive significance of the signal peptide mutation in
K. pneumoniae FimH. Also, though other within-species changes in
K. pneumoniae FimH do not overlap with the pathoadaptive changes observed in
E. coli FimH, their adaptive significance cannot be excluded.
Analysis of the genetic and structural diversity of the FimH adhesin of K. pneumoniae in the context of the population structure of the species allows certain insights into the physiological significance of the type 1 fimbriae. In particular, the high level of horizontal transfer of FimH that likely indicates the advantage of high structural variability of the major fimbrial subunit, FimA, shows that the type 1 fimbria of K. pneumoniae plays an important role in the colonization of habitats where such variability is essential. Whether the pressure comes from the adaptive or innate immunity of yet-unidentified host organisms or from phage escape remains to be determined. Also, the population analysis provided a framework in which to determine the rapid within-clone evolution of FimH in the endemic uropathogenic strain, indicating its functional significance in the latter subpopulation of K. pneumoniae. Thus, evolutionary tools can be successfully applied by microbiologists studying molecular mechanisms that underlie the ecology and pathogenesis of bacterial pathogens.
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