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Updated: Nov 5, 2025
Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS
Published on: July 11, 2016
Genotypic Characterization of Clinical Klebsiella spp. Isolates Collected From Patients With Suspected
Patricia Saxenborn1, John Baxter1, Andreas Tilevik1
1Systems Biology Research Centre, School of Bioscience, University of Skövde, Skövde, 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.
Insights
Whole-genome sequencing revealed frequent misidentification of Klebsiella pneumoniae, with other species like Klebsiella variicola often implicated in infections. This highlights the need for advanced methods to accurately diagnose Klebsiella infections and manage drug resistance.
Area of Science:
- Clinical microbiology
- Bacterial genomics
- Infectious diseases
Background:
- Klebsiella species are opportunistic pathogens causing diverse human infections.
- Highly drug-resistant Klebsiella pneumoniae poses a significant clinical challenge.
- Conventional methods often misidentify Klebsiella species, underrecognizing clinical significance.
Purpose of the Study:
- To characterize the diversity and traits of Klebsiella species in community-onset infections using whole-genome sequencing (WGS).
- To assess the accuracy of conventional microbiological methods in identifying Klebsiella species.
- To investigate antimicrobial resistance profiles and genotypic diversity within clinical Klebsiella isolates.
Main Methods:
- Whole-genome sequencing (WGS) of 105 clinical Klebsiella isolates from a prospective sepsis study in Sweden.
- Comparison of WGS results with initial identification by routine microbiological methods (culture and MALDI-TOF MS).
- Analysis of antimicrobial resistance genes, prediction of multidrug-resistance (MDR), and multi-locus sequence typing (MLST).
Main Results:
- 39.0% of isolates initially identified as K. pneumoniae were misidentified by conventional methods; 23 were K. variicola, and nine were other K. pneumoniae complex members.
- Significantly fewer resistance genes were found in K. oxytoca compared to K. pneumoniae and K. variicola (p < 0.001).
- High genotypic multidrug-resistance (MDR) was predicted in K. pneumoniae complex (94.0%) and K. oxytoca (18.8%), often involving beta-lactam, fosfomycin, and quinolone resistance markers.
Conclusions:
- High-resolution genotypic methods like WGS are crucial for accurate identification and characterization of clinical Klebsiella isolates.
- Infections caused by Klebsiella species other than K. pneumoniae are more common than previously recognized due to misidentification.
- Understanding the true species distribution and resistance profiles is essential for effective clinical management of Klebsiella infections.
