Klebsiella pneumoniaeexhibiting a phenotypic hyper-splitting phenomenon including the formation of small colony variants

Abstract

In this study, we characterized aKlebsiella pneumoniaestrain in a patient with shrapnel hip injury, which resulted in multiple phenotypic changes, including the formation of a small colony variant (SCV) phenotype. Although already described since the 1960s, there is little knowledge about SCV phenotypes inEnterobacteriaceae. The formation of SCVs has been recognized as a bacterial strategy to evade host immune responses and compromise the efficacy of antimicrobial therapies, leading to persistent and recurrent courses of infections. In this case, 14 different, clonally identical resisto- and morpho-types were distinguished from the patient's urine and tissue samples. Whole genome sequencing revealed theK. pneumoniaehigh-risk clonal lineage belonging to sequence type 147. Subculturing the SCV colonies consistently resulted in the reappearance of the initial SCV phenotype and three stable normal-sized phenotypes with distinct morphological characteristics. Additionally, an increase in resistance was observed over time in isolates that shared the same colony appearance. Our findings highlight the complexity of bacterial behavior by revealing a case of phenotypic "hyper-splitting" in aK. pneumoniaeSCV and its potential clinical significance.

In-host evolution of classic to convergentKlebsiella pneumoniaesequence type 147 isolates and impact of associated capsular changes on different morphotypes

“…As described in the study of Doğan et al, all K. pneumoniae isolates belonged to ST147 with an O locus of O1/O2v1 and a capsule biosynthesis (KL) locus of KL64 for all isolates except 4D, which missed most of its K locus [11]. Interestingly, all isolates with the grey or g/d phenotype lacked at least one K locus gene (Supplementary table S1).…”

“…While short read data of all 14 isolates were obtained in the previous study [11], we now performed hybrid sequencing (Illumina and Nanopore) for isolates 5A-D. DNA was extracted using the MasterPure DNA Purification kit for Blood, v.2 (Lucigen, Middleton, WI, USA) according to the manufacturer’s instructions and quantified with the Qubit 4 fluorometer and the dsDNA HS Assay kit (Thermo Fisher Scientific, Waltham, MA,…”

“…Recently, we described K. pneumoniae isolates belonging to the international, high-risk clonal lineage of sequence type (ST)147 that showed different colony morphologies on blood agar (Tab. 1) [11]. The isolates were obtained at five different time points from urine and tissue of a patient with shrapnel shell injury.…”

“…The isolates were obtained at five different time points from urine and tissue of a patient with shrapnel shell injury. We identified four distinct phenotypes/morphologies for the isolates from the last two time-points, i.e., presenting as (i) white/whitish and (ii) grey colonies, respectively, with a smooth and shiny surface, (iii) grey and dry colonies (g/d), and (iv) small colony variants (SCVs), which were white and smooth [11]. When sub-cultivated on agar plates, the small colonies steadily split again into the four different morphotypes, a phenomenon that we subsequently termed “hyper-splitting” (Fig.…”

“…Here, beyond the superficial description [11], we characterized the underlying mechanisms of the morphotypes in detail and tracked the in-host evolution of these isolates. By combining genomics and transcriptomics with phenotyping, we also explored the hyper-splitting phenomenon and revealed associations between the phenotypes and virulence of this important international, high-risk clonal K. pneumoniae lineage.…”