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. 2023 Mar 23:14:1122691.
doi: 10.3389/fmicb.2023.1122691. eCollection 2023.

Species identification, antibiotic resistance, and virulence in Enterobacter cloacae complex clinical isolates from South Korea

Michidmaral Ganbold  1 , Jungyu Seo  1 , Yu Mi Wi  2 , Ki Tae Kwon  3 , Kwan Soo Ko  1
Affiliations

Species identification, antibiotic resistance, and virulence in Enterobacter cloacae complex clinical isolates from South Korea

Michidmaral Ganbold et al. Front Microbiol. .

Abstract

This study aimed to identify the species of Enterobacter cloacae complex (ECC) isolates and compare the genotype, antibiotic resistance, and virulence among them. A total of 183 ECC isolates were collected from patients in eight hospitals in South Korea. Based on partial sequences of hsp60 and phylogenetic analysis, all ECC isolates were identified as nine species and six subspecies. Enterobacter hormaechei was the predominant species (47.0%), followed by Enterobacter kobei, Enterobacter asburiae, Enterobacter ludiwigii, and Enterobacter roggenkampii. Multilocus sequence typing analysis revealed that dissemination was not limited to a few clones, but E. hormaechei subsp. xiangfangensis, E. hormaechei subsp. steigerwaltii, and E. ludwigii formed large clonal complexes. Antibiotic resistance rates were different between the ECC species. In particular, E. asburiae, E. kobei, E. roggenkampii, and E. cloacae isolates were highly resistant to colistin, whereas most E. hormaechei and E. ludwigii isolates were susceptible to colistin. Virulence was evaluated through serum bactericidal assay and the Galleria mellonella larvae infection model. Consistency in the results between the serum resistance and the G. mellonella larvae infection assay was observed. Serum bactericidal assay showed that E. hormaechei, E. kobei, and E. ludwigii were significantly more virulent than E. asburiae and E. roggenkampii. In this study, we identified the predominant ECC species in South Korea and observed the differences in antibiotic resistance and virulence between the species. Our findings suggest that correct species identification, as well as continuous monitoring is crucial in clinical settings.

Keywords: Enterobacter cloacae complex; Enterobacter hormaechei; antibiotic resistance; species identification; virulence.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Phylogenetic tree based on neighbor-joining method using hsp60 gene sequences of 183 clinical isolates and reference strains of Enterobacter cloacae complex (ECC). The sequences of the isolate and the reference strains were aligned using ClustalW option of mega 7.0 software program. A midpoint rooting option was applied to root the tree due to the absence of a reliable outgroup. The branch lengths are proportional to the changes in the nucleotides. Scale par indicates one substitution per 100 nucleotides.
Figure 2
Figure 2
Minimum spanning tree of 183 clinical isolates of Enterobacter cloacae complex (ECC), based on the allelic profiles of multilocus sequence typing (MLST). Species based on the partial hsp60 sequences are shown in different colors. Each node within the tree represents a single sequence type (ST), and the length of branches between each node represents the number of different alleles.
Figure 3
Figure 3
Results of serum bactericial assay among Enterobacter cloacae complex (ECC) species (A) and subspecies of Enterobacter hormaechei (B). Bacterial survival rates were determined after 3 h of incubation with normal human serum (NHS). Heat-inactivated serum (HIS) was used as a negative control. *p < 0.05; ***p < 0.001; ****p < 0.0001.
Figure 4
Figure 4
Results of Galleria mellonella larvae infection experiments. Survival of larvae infected with three isolates from each of the five Enterobacter cloacae complex (ECC) species, except Enterobacter roggenkampii. Enterobacter hormaechei subsp. xianfangensis (A), Enterobacter kobei (B), Enterobacter asburiae (C), Enterobacter ludwigii (D), and Enterobacter roggenkampii species (E). Three isolates from each species were selected for low (blue line), intermediate (black line), and high (red line) survival rates in the serum bactericidal assay. For E. roggenkampii, with no isolates showing high survival rate in serum bactericidal assay, two isolates were included. The percentages in parentheses represent the survival rates in the serum bactericidal assay. Five larvae were evaluated per isolate, and results were obtained from three independent experiments. p < 0.001; ****p < 0.0001.
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