Genotypic Characterization of Clinical Klebsiella spp. Isolates Collected From Patients With Suspected Community-Onset Sepsis, Sweden

Abstract

Klebsiella is a genus of Gram-negative bacteria known to be opportunistic pathogens that may cause a variety of infections in humans. Highly drug-resistant Klebsiella species, especially K. pneumoniae, have emerged rapidly and are becoming a major concern in clinical management. Although K. pneumoniae is considered the most important pathogen within the genus, the true clinical significance of the other species is likely underrecognized due to the inability of conventional microbiological methods to distinguish between the species leading to high rates of misidentification. Bacterial whole-genome sequencing (WGS) enables precise species identification and characterization that other technologies do not allow. Herein, we have characterized the diversity and traits of Klebsiella spp. in community-onset infections by WGS of clinical isolates (n = 105) collected during a prospective sepsis study in Sweden. The sequencing revealed that 32 of the 82 isolates (39.0%) initially identified as K. pneumoniae with routine microbiological methods based on cultures followed by matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) had been misidentified. Of these, 23 were identified as Klebsiella variicola and nine as other members of the K. pneumoniae complex. Comparisons of the number of resistance genes showed that significantly fewer resistance genes were detected in Klebsiella oxytoca compared to K. pneumoniae and K. variicola (both values of p < 0.001). Moreover, a high proportion of the isolates within the K. pneumoniae complex were predicted to be genotypically multidrug-resistant (MDR; 79/84, 94.0%) in contrast to K. oxytoca (3/16, 18.8%) and Klebsiella michiganensis (0/4, 0.0%). All isolates predicted as genotypically MDR were found to harbor the combination of β-lactam, fosfomycin, and quinolone resistance markers. Multi-locus sequence typing (MLST) revealed a high diversity of sequence types among the Klebsiella spp. with ST14 (10.0%) and ST5429 (10.0%) as the most prevalent ones for K. pneumoniae, ST146 for K. variicola (12.0%), and ST176 for K. oxytoca (25.0%). In conclusion, the results from this study highlight the importance of using high-resolution genotypic methods for identification and characterization of clinical Klebsiella spp. isolates. Our findings indicate that infections caused by other members of the K. pneumoniae complex than K. pneumoniae are a more common clinical problem than previously described, mainly due to high rates of misidentifications.

Keywords: Illumina sequencing; Klebsiella; antimicrobial susceptibility; clinical microbiology; multidrug resistance; nanopore-based sequencing; whole-genome sequencing.

Figure 1

Overview of the results of the species identification. During the prospective sepsis study, species identification by MALDI-TOF MS (DB-4110) was performed for all collected bacterial isolates as part of the routine clinical practices. All isolates initially identified as Klebsiella spp. (n = 105) were subject to whole-genome sequencing (WGS). Genotypic species identification was performed by calculating the pairwise ANI on the assembled Illumina short-read data against reference genomes. The species identification by MALDI-TOF MS (DB-4110) and ANI agreed (solid line boxes) for 68 isolates, whereas the species were corrected based on the ANI results (dotted line boxes) for the remaining 37 isolates. ANI, average nucleotide identity; Km, K. michiganensis; Ko, K. oxytoca; Kpn, K. pneumoniae; Kqq, K. quasipneumoniae subsp. quasipneumoniae; Kqs, K. quasipneumoniae subsp. similipneumoniae; and Kqv, K. quasivariicola, Kv K. variicola.

Figure 2

Percentages of isolates per species carrying at least one resistance marker against at least one agent included in the current antibiotic class as predicted by ResFinder. At least one gene conferring resistance against β-lactam antibiotics was predicted for all Klebsiella isolates, and at least one quinolone resistance gene was predicted for all K. variicola, K. quasipneumoniae subsp. quasipneumoniae, and K. quasipneumoniae subsp. similipneumoniae. The single K. quasivariicola isolate was predicted resistant against β-lactam, fosfomycin, and quinolone, data not included in the figure. Km, K. michiganensis; Ko, K. oxytoca; Kpn, K. pneumoniae; Kqq, K. quasipneumoniae subsp. quasipneumoniae; Kqs, K. quasipneumoniae subsp. simili; and Kv K. variicola.

Figure 3

(A) Distribution of the number of resistance genes predicted by ResFinder per species. The sample size for each species was K. oxytoca (n = 16), K. michiganensis (n = 4), K. pneumoniae (n = 50), K. quasipneumoniae subsp. quasipneumoniae (n = 5), K. quasipneumoniae subsp. similipneumoniae (n = 3), K. variicola (n = 25), and K. quasivariicola (n = 1). Significant differences in the number of predicted resistance markers were observed between K. pneumoniae and K. oxytoca (p < 0.001), and between K. oxytoca and K. variicola (p < 0.001) using pairwise quasi-Poisson regression followed by adjustment of the resulting p-values by the Holm method. The single K. quasivariicola isolate was predicted resistant against β-lactam, fosfomycin, and quinolone, data not included in the figure. (B) Distribution of the number of resistance genes predicted by ResFinder per sample type. The sample size for each sample type was blood (n = 29), urine (n = 70), and nasopharynx (n = 4). No significant differences were detected between any sample types using pairwise Poisson regression (all values of p > 0.05). Km, K. michiganensis; Ko, K. oxytoca, Kpn, K. pneumoniae; Kqq, K. quasipneumoniae subsp. quasipneumoniae; Kqs, K. quasipneumoniae subsp. similipneumoniae; Kv, K. variicola; and NP, nasopharynx.

Figure 4

Distribution of sequence types (STs) for the three major groups of Klebsiella spp. in the current study. (A) K. oxytoca. “Other STs” include one isolate each of ST36, ST37, ST201, ST288, ST357, ST358, and ST359. “ND” refers to a single isolate for which the ST profile could not be determined. (B) K. pneumoniae. “Other STs” include one isolate each of ST11, ST20, ST30, ST37, ST39, ST187, ST215, ST220, ST294, ST309, ST381, ST485, ST462, ST678, ST685, ST788, ST870, ST872, ST966, ST1114, ST1948, ST3370, ST4069, ST4631, ST4725, and ST5442. (C) K. variicola. “Other STs” include one isolate each of ST149, ST261, ST280, ST281, ST282, ST283, ST284, ST285, ST286, ST287, and ST288. “ND” refers to a single isolate for which the ST profile could not be determined. MLST, multi-locus sequence typing; ND, not determined; and ST sequence type.

Figure 5

Percentage of isolates hosting each plasmid replicon type as predicted by PlasmidFinder. The total of 142 predicted plasmid replicons were distributed per species as follows: K. michiganensis n = 3, K. oxytoca n = 13, K. pneumoniae n = 84, K. quasipneumoniae subsp. quasipneumoniae n=3, K. quasipneumoniae subsp. similipneumoniae n = 2, K. variicola n = 36, and K. quasivariicola n = 1. The single K. quasivariicola isolate was predicted to host the plasmid replicon Col156, data not included in the figure. Km, K. michiganensis; Ko, K. oxytoca; Kpn, K. pneumoniae; Kqq, K. quasipneumoniae subsp. quasipneumoniae; Kqs, K. quasipneumoniae subsp. similipneumoniae; and Kv, K. variicola.

Figure 6

(A) Distribution of the number of plasmid replicons predicted by PlasmidFinder per species. The sample size for each species was K. oxytoca (n = 16), K. michiganensis (n = 4), K. pneumoniae (n = 50), K. quasipneumoniae subsp. quasipneumoniae (n = 5), K. quasipneumoniae subsp. similipneumoniae (n = 3), K. variicola (n = 25), and K. quasivariicola (n = 1, data not included in the figure). No significant differences in the number of predicted plasmid replicons were observed between any of the species using pairwise Poisson regression (all values of p > 0.05). (B) Distribution of the number of plasmid replicons predicted by PlasmidFinder per sample type. The sample size for each sample type was blood (n = 29), nasopharynx (n = 4), and urine (n = 70). Significant differences in the number of predicted plasmid replicons were observed between blood and nasopharynx (p = 0.01), and between urine and nasopharynx (p = 0.003), but not between blood and urine (p = 0.60) using pairwise Poisson regression followed by adjustment of the resulting p-values by the Holm method. Km, K. michiganensis; Ko, K. oxytoca; Kpn, K. pneumoniae; Kqq, K. quasipneumoniae subsp. quasipneumoniae; Kqs, K. quasipneumoniae subsp. similipneumoniae; Kv, K. variicola; and NP, nasopharynx.