Phenotypic heterogeneity of capsule production across opportunistic pathogens

Phenotypic heterogeneity allows bacteria to adapt fast to changing environments. Extracellular capsules are well-known virulence factors, but also increase the cell adaptability and prevalence under hostile conditions. To limit their cost, some species regulate capsule production by genetic phase variation. Here, we demonstrated that phenotypic heterogeneity is a major mechanism controlling capsule production in <i>Klebsiella</i> and <i>Acinetobacter</i> species. We designed a method to agnostically measure heterogeneity and show that 71% of <i>Klebsiella pneumoniae</i> strains can be heterogeneous. This is mostly associated with <i>K. pneumoniae</i> strains that do not encode <i>rmp</i>, a genetic determinant of hypervirulence. Capsule serotype exchanges across several genetic backgrounds revealed that heterogeneity depends on specific genome-capsule locus interactions. Importantly, we showed that heterogeneity provides a fitness advantage especially in conditions where the capsule is costly, as estimated by comparing non-heterogeneous and heterogeneous strains during competition with their non-capsulated variants. Finally, heterogeneity impacts phage adsorption patterns, and could thus alter the rate of horizontal gene transfer events. This unsuspected heterogeneity may help understand the transition from commensalism to pathogenesis and can have important implications in virulence, environmental survival and evolution of some ESKAPE pathogens.IMPORTANCEThe polysaccharidic capsule is present in ~50% of species across the bacterial phylogeny, including all ESKAPE microorganisms, the six most significant multidrug-resistant (MDR) nosocomial pathogens. It is also an important virulence factor and a major target for both phage therapy and the development of vaccines. Here, we reveal that in two major genera of ESKAPE pathogens, <i>Klebsiella</i> spp. and <i>Acinetobacter</i> spp., capsule production within clonal populations is heterogeneous, leading to mixed populations of hyper-, hypo-, and intermediate-capsulated cells. Such heterogeneity responds to different environmental cues, including changes in nutrient availability and spatial structure. We show that this plasticity, known to enable faster, more efficient adaptation to environmental changes, limits capsule costs and could explain <i>Klebsiella</i> and <i>Acinetobacter</i> resilience. Finally, capsule heterogeneity can play a major role in bacterial evolution, as a driver of horizontal gene transfer, and in treatment failure. Thus, it should be taken into account in the design of prophylactic strategies and antimicrobial therapy.