This study characterized 89 isolates based on their clinical, geographic, genotypic, and phenotypic characteristics. It was noted that all the four recognized phylogroups, were represented with Klebsiella pneumoniae subsp. pneumoniae isolates predominating, consistent with findings from other studies (42–44). Some differences were observed in the characteristics of the phylogroups. For example, Klebsiella variicola subsp. variicola isolates are typically linked to bloodstream infections and UTIs (45,46); however, those identified in this study were mainly associated with SSTIs (Figure 1) and were largely antibiotic susceptible (Figure 4). In previous studies, Klebsiella quasipneumoniae subsp. similipneumoniae isolates were linked to nosocomial infections such as UTIs (47,48). Yet, in this study, one isolate, kkp034, caused a community-acquired SSTI, potentially indicating a broader distribution of this phylogroup in Kenya as found in other countries, such as sewage in Brazil (49) and a turtle in China (50).

The lineages identified in this study were highly diverse, and most of them were local strains that have not been described in other countries. Globally disseminated high-risk lineages such as ST14, ST15, ST307, and ST607, were identified (Figure 3). These high-risk lineages are bacterial pathogens that easily acquire and disseminate antimicrobial resistance (51). For example, ST14, ST15, and ST147 have been linked to the spread of carbapenemase resistance genes in many countries (52,53). In this study, the two extensively drug resistant (XDR) ST147 strains carried a bla_OXA-181 (kkp001) and a bla_NDM gene (kkp003), respectively, while the MDR ST14/15 strains carried several ESBL genes. Notably, kkp018 (ST15) harbored a mobile colistin-resistant gene, mcr-8, and an AmpC betalactamase gene, blaDHA (Figure 4). ST307 and ST607 are emerging strains linked to ESBL infections (Long, et al., 2017), and they were observed in six MDR strains isolated from SSTIs and UTIs (Figure 1 and 5). ST17 is a regional strain that has been implicated in outbreaks in Kilifi (6), Mwanza (54), and Kilimanjaro (55). This study identified three MDR and ESBL positive ST17 strains, isolated from a community-acquired SSTI, a nosocomial UTI, and a hospital environmental swab (Figures 1 and 5). High-risk clones have also been linked to specific serotypes, e.g., ST607-K25, responsible for a nosocomial outbreak at a neonatal intensive care unit in a hospital in France (56). Significantly, this study identified three MDR and ESBL-positive ST607-K25 clones associated with SSTIs (Figures 5).

The presence of these high-risk strains in Kenya indicate the significant clinical and public health threat they pose. However we noticed that this threat was highest in Kisumu and Nairobi, the two largest cities in the country, were most of the global lineages (79%, 11/14) were identified. These cities are travel hubs with large referral hospitals serving patients from a wide geographical area locally and global travelers. The diverse patient population could explain the concentration of local, regional and global lineages, the great strain diversity, and novel alleles in isolates from the two counties compared to Kisii, Kilifi, and Kericho counties.

Plasmids are the main vehicle for AMR gene transmission and in this study we found that a majority of the AMR genes were carried in plasmids, particularly IncFIB(K) and IncFII(pKP91) (Table 2), which are common among Enterobacterales. The predominant plasmid replicon types belonged to the diverse Incompatibility (Inc) family (57), whose host range is mainly limited to Enterobacterales (58) known to have large multi-replicon plasmids (59). The multi-replicon IncF plasmids contain the FII, FIA, and/or FIB replicons (Table 2 and S2) which account for their high abundance (Figure 2). Unsurprisingly, antibiotic susceptible KP isolates (kkp012, kkp030, kkp102, and kkp0112) contained few or no plasmids and were ESBL negative and non-MDR. The MDR kkp070 was exceptional because although it possessed no plasmid replicons, it had several resistance genes integrated into a genetic island in the chromosome. These integration events were not uncommon, as plasmid-associated AMR genes were detected in the chromosome of several isolates: kkp005 and kkp006 carried a bla_CTX-M-15 gene, kkp109 had a dfrA14 trimethoprim resistant gene, and kkp001 possessed several AMR genes (aac(6’)-Ib9, bla_CTX-M-15, arr-2, and rmtF). The integration of AMR genes into the chromosome is alarming because the resistance is transferred clonally, becomes part of the core genome, and increases the spread and prevalence of non-susceptible KP lineages.

Although the col plasmid family were the second most dominant group, the majority of col-like plasmids did not contain any resistance or virulence genes, except for the abundant colE1 type, Col (pHAD28), which typically carries qnrS1 and other AMR genes (58) as observed in kkp0e21 (Table 2). In addition, col plasmids benefit KP because they produce bacteriocins lethal to rival bacteria. One of the other plasmid families identified was the pK1433 (kkp056) and is associated with the blaKPC2 gene (60), which provides a mechanism for carrying and spreading KPC-type genes. The isolates with plasmids carrying a bla_CTX-M gene also carried bla_OXA, bla_SHV, or bla_TEM genes, implying that these resistance genes are transferred together (e.g., ESBL-positive kkp081 and kkp059 isolates possessed bla_CTX-M-15, bla_SHV-134, and bla_TEM181 genes). The most intriguing isolates were the ESBL-positive isolates (kkp015, kkp070, and kkp107) which only contained the bla_SHV penicillinase gene. We hypothesize that the ESBL phenotypes in these isolates may have been due to alternative resistance mechanisms such as up-regulation of MDR efflux pumps. The expression of genes encoding MDR efflux pumps is noteworthy because their activity induces resistance of different antibiotic classes non-specifically and can cause phenotypic and genotypic discordance.

Examination of the virulence factors among the KP phylogroups highlighted one main difference. K. quasipneumoniae subsp similipneumoniae did not demonstrate the Type VI protein secretion system found in the other phylogroups (Table S5); instead, it possessed the Type II secretion as previously noted (61). Siderophores were the most significant virulence genes detected. The ubiquitous enterobactin scavenges iron from host cells; however, their activity is neutralized by human lipocalin-2 protein (62). In response, KP uses the more virulent yersiniabactin to bind iron and other heavy metals like copper to avoid metal toxicity and phagocytosis using reactive oxygen species (63). According to Holt, et al. (2015), acquiring yersiniabactin is usually the first step in accumulating more potent siderophores to make them more invasive. Yersiniabactin genes were found among the K. pneumoniae subsp pneumoniae isolates causing various clinical infections and from diverse geographical locations. Most of them were MDR and ESBL positive, making their infections persistent and antibiotic-resistant and likely contributing to their dominance over the other phylogroups.

Classical hypervirulent KP (hvKP) isolates are generally antibiotic susceptible (64), and they are characterized by having rmpA/A2 and magA virulence factors as well as the K2 capsule type (53). This study identified one potentially hypervirulent isolate (kkp043), which carried rmpA and rmpA2 genes. In addition, it also carried genes for aerobactin and salmochelin that were carried on a repB_KLEB_VIR plasmid (Table 2). A trade-off between virulence and antibiotic resistance was demonstrated in this potentially hypervirulent isolate (kkp043) as it was non-MDR and ESBL-negative and had only one other plasmid bearing the IncFIA(HI1) replicon, dfrA5, and sul1 genes (Table 2). Additional examples were the two isolates (kkp012 and kkp045) with magA genes which were ESBL-negative and non-MDR. Furthermore, non-MDR K2 strains possessed the ybt operon, which the MDR K2 strains lacked (Figure 5). Despite this, there is growing concern about the rise of MDR hvKP globally (39,65–68), which cause severe infections with few treatment options (69) emphasizing the significance of monitoring virulence characteristics of KP in Kenya.

The study had several limitations. The first was that the two sequencing technologies utilized had different strengths and weaknesses, which could introduce bias in the genomic analysis. The draft genomes from the long reads had fewer contigs (<=12) (Table S1) and were more contiguous, but they had less depth/coverage, while the short reads produced less contiguous draft genomes with greater depth (>40) (Table S1). More contiguity enabled better plasmids reconstruction, while sufficient depth enabled better assignment of multilocus sequence types. The second limitation was that susceptibility tests were carried out on the Vitek2^® platform using a limited number of antibiotics and were not verified using another phenotypic method. Nevertheless, there was good concordance between the phenotypic and genotypic results. Finally, in vivo tests were not conducted to confirm the virulence gene activity, so the virulence results are only predicted.