Whole-Genome Sequencing-Based Species Classification, Multilocus Sequence Typing, and Antimicrobial Resistance Mechanism Analysis of the Enterobacter cloacae Complex in Southern China

Abstract

Members of the Enterobacter cloacae complex (ECC) are important opportunistic nosocomial pathogens that are associated with a great variety of infections. Due to limited data on the genome-based classification of species and investigation of resistance mechanisms, in this work, we collected 172 clinical ECC isolates between 2019 and 2020 from three hospitals in Zhejiang, China and performed a retrospective whole-genome sequencing to analyze their population structure and drug resistance mechanisms. Of the 172 ECC isolates, 160 belonged to 9 classified species, and 12 belonged to unclassified species based on ANI analysis. Most isolates belonged to E. hormaechei (45.14%) followed by E. kobei (13.71%), which contained 126 STs, including 62 novel STs, as determined by multilocus sequence typing (MLST) analysis. Pan-genome analysis of the two ECC species showed that they have an "open" tendency, which indicated that their Pan-genome increased considerably with the addition of new genomes. A total of 80 resistance genes associated with 11 antimicrobial agent categories were identified in the genomes of all the isolates. The most prevailing resistance genes (12/29, 41.38%) were related to β-lactams followed by aminoglycosides. A total of 247 β-lactamase genes were identified, of which the bla_ACT genes were the most dominant (145/247, 58.70%), followed by the bla_TEM genes (21/247, 8.50%). The inherent ACT type β-lactamase genes differed among different species. bla_ACT-2 and bla_ACT-3 were only present in E. asburiae, while bla_ACT-9, bla_ACT-12, and bla_ACT-6 exclusively appeared in E. kobei, E. ludwigii, and E. mori. Among the six carbapenemase-encoding genes (bla_NDM-1, bla_NDM-5, bla_IMP-1, bla_IMP-4, bla_IMP-26, and bla_KPC-2) identified, two (bla_NDM-1 and bla_IMP-1) were identified in an ST78 E. hormaechei isolate. Comparative genomic analysis of the carbapenemase gene-related sequences was performed, and the corresponding genetic structure of these resistance genes was analyzed. Genome-wide molecular characterization of the ECC population and resistance mechanism would offer valuable insights into the effective management of ECC infection in clinical settings. IMPORTANCE The presence and emergence of multiple species/subspecies of ECC have led to diversity and complications at the taxonomic level, which impedes our further understanding of the epidemiology and clinical significance of species/subspecies of ECC. Accurate identification of ECC species is extremely important. Also, it is of great importance to study the carbapenem-resistant genes in ECC and to further understand the mechanism of horizontal transfer of the resistance genes by analyzing the surrounding environment around the genes. The occurrence of ECC carrying two MBL genes also indicates that the selection pressure of bacteria is further increased, suggesting that we need to pay special attention to the emergence of such bacteria in the clinic.

Keywords: Enterobacter cloacae complex; MLST; Pan-genome; antimicrobial resistance mechanism; whole-genome sequencing-based species classification.

FIG 1

SNP phylogenetic tree of the genomes of 172 E. cloacae complex isolates. The species/subspecies, clades, and Hoffmann clusters are drawn in concentric circles.

FIG 2

Distributions of acquired β-lactamase genes in the 26 isolates. Blue and white squares represent the presence and absence of genes, respectively.

FIG 3

Genomic comparison of the plasmid pECC33-57 (red inner circle) with its close relatives. The ORFs of different gene functions are denoted by rectangles in various colors. Genes present in pECC33-57 but absent in the other plasmids are shown as blank spaces in the rings.

FIG 4

Schematic map of pECC33-49. The ORFs are colored based on gene function classification. The circles represent (from outside to inside) the predicted coding sequences (CDS); GC content; GC skew; and scale in kilobase pairs.