Characterization of Clinically Relevant Strains of Extended-Spectrum β-Lactamase-Producing Klebsiella pneumoniae Occurring in Environmental Sources in a Rural Area of China by Using Whole-Genome Sequencing

Abstract

Klebsiella pneumoniae is a gram-negative, opportunistic pathogen, and a common cause of healthcare-associated infections such as pneumonia, septicemia, and urinary tract infection. The purpose of this study was to survey the occurrence of and characterize K. pneumoniae in different environmental sources in a rural area of Shandong province, China. Two hundred and thirty-one samples from different environmental sources in 12 villages were screened for extended-spectrum β-lactamase-(ESBL)-producing K. pneumoniae, and 14 (6%) samples were positive. All isolates were multidrug-resistant and a few of them belonged to clinically relevant strains which are known to cause hospital outbreaks worldwide. Serotypes, virulence genes, serum survival, and phagocytosis survival were analyzed and the results showed the presence of virulence factors associated with highly virulent clones and a high degree of phagocytosis survivability, indicating the potential virulence of these isolates. These results emphasize the need for further studies designed to elucidate the role of the environment in transmission and dissemination of ESBL-producing K. pneumoniae and the potential risk posed to human and environmental health.

Keywords: Klebsiella pneumoniae; environment; extended-spectrum β-lactamase; feces; multilocus sequence typing; pulsed-field gel electrophoresis; water; whole-genome sequencing.

Figure 1

Map showing village locations (A–L) and the surrounding area in a region of rural Shandong, China, where 231 environmental samples were collected for screening for ESBL-producing K. pneumoniae. White spots denote locations where samples were negative for ESBL-producing K. pneumoniae and red spots denote locations were samples were positive for ESBL-producing K. pneumoniae.

Figure 2

Genetic environment of CTX-M genes among K. pneumoniae isolated from environmental sources. Arrows represent direction of transcription. Red open reading frames (ORFs) denote the CTX-M genes, purple ORFs denote other antibiotic resistance genes, yellow ORFs denote genes related to mobile genetic elements, and gray ORFs denote other genes or genes of unknown function. (A) The genetic environments of bla_CTX−M−3 were similar in isolates KP2, KP4, KP7, KP9, and KP12. The two isolates KP8 and KP11 also shared a similar genetic environment surrounding bla_CTX−M−3. (B) Isolates KP1, KP3, KP5, and KP10 all carried bla_CTX−M−14 and shared the same genetic environment surrounding the gene, whereas isolates KP6 and KP13 had unique genetic environments surrounding bla_CTX−M−14. (C) The genetic environment of bla_{CTX−M−104} in the one isolate in the study which carried the gene.

Figure 3

Phylogeny of ESBL-producing K. pneumoniae isolated from environmental sources based on differences in SNPs. Red branches indicate isolates which were found related in the PFGE-analysis. The value represents the proportion of the scale of SNP differences.