Emergence of novel non-aggregative variants under negative frequency-dependent selection in Klebsiella variicola

Abstract

Klebsiella variicola is an emergent human pathogen causing diverse infections, some of which in the urinary tract. However, little is known about the evolution and maintenance of genetic diversity in this species, the molecular mechanisms and their population dynamics. Here, we characterized the emergence of a novel rdar-like (rough and dry) morphotype which is contingent both on the genetic background and the environment. We show that mutations in either the nitrogen assimilation control gene (nac) or the type III fimbriae regulator, mrkH, suffice to generate rdar-like colonies. These morphotypes are primarily selected for the reduced inter-cellular aggregation as a result of MrkH loss-of-function which reduces type 3 fimbriae expression. Additionally, these clones also display increased growth rate and reduced biofilm formation. Direct competitions between rdar and wild type clones show that mutations in mrkH provide large fitness advantages. In artificial urine, the morphotype is under strong negative frequency-dependent selection and can socially exploit wild type strains. An exhaustive search for mrkH mutants in public databases revealed that ca 8% of natural isolates analysed had a truncated mrkH gene many of which were due to insertions of IS elements, including a reported clinical isolate with rdar morphology. These strains were rarely hypermucoid and often isolated from human, mostly from urine and blood. The decreased aggregation of these mutants could have important clinical implications as we hypothesize that such clones could better disperse within the host allowing colonisation of other body sites and potentially leading to systemic infections.

Diverse polysaccharide production and biofilm formation abilities of clinical Klebsiella pneumoniae

“…This nding is important when considering the population dynamics suggesting that when removed from an environment where mucoviscosity or antibiotic resistance is not necessary, they may be outcompeted and less prone to spread a worrisome trait. When considering the mbriae allele variants we found that, in line with recent work 30,36 , insertion sequences in mrkH led to decreased bio lm formation, as all three isolates with this insertion displayed limited bio lm formation (Fig. 5).…”

“…2). However, similar to what was recently shown with Klebsiella variicola 30 , within the entire collection, the operon csgABCDEF was not present suggesting the phenotypes observed on congo red is due to another factor. In complementation of the congo red analysis, when grown on calco uor white agar and visualized under UV uorescence we saw diverse uorescence, indicating variability in cellulose production within the entire collection (Supplemental Figure S2).…”

“…Therefore, our work suggests that the isolates were adapted during or prior to the infection to the environment where it was isolated. This nding builds upon the previous work that revealed the structure of the environment largely impacts population evolution 30 .…”

“…Although the multi-drug resistant (MDR) group had quite a diversity of growth yields, this was comparably less diverse than the bio lm formation and mucoviscosity of this subset of isolates. Similar to antibiotic resistance, virulence factors such as mucoviscosity have been reported to be costly to the growth rate 30 . Although the overall correlation was not as signi cant (Fig.…”

“…It has recently been discovered that mutations within the gene encoding for the tip-adhesin of type III mbriae (mrkD) or within the switch of type I mbriae ( mH) impact bio lm formation in a capsule dependent manner 22 . Furthermore, a recent study revealed insertional inactivation of mrkH, encoding for a c-di-GMP transcriptional activator, resulted in decreased bio lm formation 30,36 . In addition, another chaperon-usher system tip adhesin EcpD has been shown to be important for adherence to epithelial cells 37 .…”