Emergence and transmission of the high-risk ST78 clone of OXA-48-producing Enterobacter hormaechei in a single hospital in Taiwan

Abstract

Carbapenem-resistant Enterobacter cloacae complex is a significant global healthcare threat, particularly carbapenemase-producing Enterobacter hormaechei (CPEH). From January 2017 to January 2021, twenty-two CPEH isolates from a regional teaching hospital in central Taiwan were identified with the carriage of carbapenemase genes bla_KPC-2, bla_IMP-8, and predominantly bla_OXA-48. Over 80% of these CPEH strains clustered into the high-risk ST78 lineage, carrying a bla_OXA-48 IncL plasmid (pOXA48-CREH), nearly identical to the endemic plasmid pOXA48-KP in ST11 Klebsiella pneumoniae. This OXA-48-producing ST78 lineage disseminated clonally from 2018 to 2021 and transferred pOXA48-CREH to ST66 and ST90 E. hormaechei. An IMP-8-producing ST78 strain harbouring a bla_IMP-8-carrying pIncHI2 plasmid appeared in 2018, and by late 2020, a KPC-2-producing ST78 strain was identified after acquiring a novel bla_KPC-2-carrying IncFII plasmid. These findings suggest that the high-risk ST78 lineage of E. hormaechei has emerged as the primary driver behind the transmission of CPEH. ST78 has not only acquired various carbapenemase-gene-carrying plasmids but has also facilitated the transfer of pOXA48-CREH to other lineages. Continuous genomic surveillance and targeted interventions are urgently needed to control the spread of emerging CPEH clones in hospital settings.

Keywords: Carbapenemase genes; Enterobacter hormaechei; OXA-48; ST78; mcr-9.1; plasmidome; resistome.

Graphical abstract

Figure 1.

Carbapenem-resistant E. hormaechei (CREH) isolates (n = 67) collected from January 2017 to January 2021 were clustered based on PFGE-XbaI profiles. For each non-duplicated isolate, features such as isolation time, location, specimen type, and colonization or infection with CREH are presented. In addition to antibiotic susceptibility testing results, the presence of specific carbapenemase genes (bla_OXA, bla_KPC, bla_IMP) or the mcr-9.1 gene, as well as the carriage of IncL- and IncHI2-type plasmid replicons, was detected by PCR and is indicated in black. The absence of these genetic loci is shown in light grey. Sequence types (STs) were determined by the MLST scheme for E. cloacae (http://pubmlst.org/ecloacae). Genotyping of bla_ACT was performed by sequencing PCR products amplified with specific primers (Table S1). Strains with one of the carbapenemase genes (bla_OXA, bla_KPC, bla_IMP) were classified as CPEH (n = 22) and highlighted with white text on a black background. Colour coding indicates antibiotic resistance: red for resistant, yellow for intermediate, and green for susceptible. Abbreviations for antibiotics are as follows: AN (amikacin), GM (gentamycin), AM (ampicillin), SAM (Ampicillin-Sulbactam), CZ (cefazolin), CMZ (cefmetazole), CTX (cefotaxime), CAZ (ceftazidime), CRO (ceftriaxone), FEP (cefepime), TZP (Piperacillin-Tazobactam), ETP (ertapenem), IMP (imipenem), MPM (meropenem), CL (colistin), CIP (ciprofloxacin), LVX (levofloxacin), TGC (tigecycline), MI (minocycline), SXT (Trimethoprim-Sulfamethoxazole).

Figure 2.

Emergence of carbapenemase-producing E. hormaechei (CPEH). (a) The time scale of clinical isolates of E. hormaechei carrying bla_IMP (blue), bla_OXA-48 (red), bla_KPC (green), and mcr-9.1 (purple) gene, detected by PCR. (b) The locations of patients with bla_OXA-48 or mcr-9.1-positive E. hormaechei colonization or infections are presented, including the intensive care unit (ICU), respiratory care centre (RCC), general ward, and outpatient department (OPD). (c) The type of specimens from which bla_OXA-48 or mcr-9.1-positive E. hormaechei were isolated included urine, sputum, blood, pus, and others.

Figure 3.

Acquisition of an IncL-type pOXA48 plasmid in E. hormaechei Isolates. (a) BRIG comparison of the bla_OXA-48-carrying IncL plasmid, named pOXA48-CREH, in 11 clinical isolates collected in this study, aligned against pOXA48-CREH of 18CRE35 (CP090194.1). (b) Alignment of pOXA48-CREH with the endemic plasmid pOXA48-KP (CP040036.1) found in ST11_KL64 K. pneumoniae. The grey-shaded connection indicates an inversion between two copies of IS1999, causing IS1 (yellow) duplication on pOXA48-CREH.

Figure 4.

Phylogenomic relatedness, plasmidome, and resistome of representative carbapenemase-producing E. hormaechei strains. A phylogenetic tree was constructed based on cgSNP analysis of representative E. hormaechei strains (n = 14) using E. hormaechei Eh1 (GCA_009834325.1) as the reference. The distance between nodes is presented as the substitution rate per site. The plasmid Inc-type and the carriage of antimicrobial resistance (AMR) genes were determined by PlasmidFinder and ResFinder from the Centre for Genomic Epidemiology (http://www.genomicepidemiology.org/). The absence of the indicated AMR gene is shown in light grey.

Figure 5.

Comparison of pIncFIB-CREH. (a) Alignment of pIncFIB plasmids in representative E. hormaechei ST78, ST90, and ST66 strains. (b) Alignment of the class I integron-flanked AMR cassettes on the pIncFIB plasmids identified in representative strains in this study against the Tn3-flanked AMR cassette on the pIncFIB plasmid (CP034755.1) of the ST78 reference strain Eh1.

Figure 6.

Comparison of AMR cassettes on pIncHI2-CREH. (a) Alignment of the region containing major AMR cassettes of the pIncHI2 plasmids in representative E. hormaechei ST90, ST78, and ST66 strains and the corresponding region of the closely related plasmid pEC-IMPQ (EU855788.1). (b) Alignment of the large IncHI2-IncC hybrid plasmid (450,336 bp) identified in CRECL35 (ST90) against the IncC plasmid (CP129795.1; 184,336 bp) in K. pneumoniae 2020C07-229 (SAMN36281330). (c-e) Detailed presentation of other AMR cassettes on the pIncHI2-IncC hybrid plasmid in CRECL35. (f) Insertion of bla_MOX-6 into the chromosome of CRECL35 (ST90).

Figure 7.

Acquisition of a bla_KPC-2-carrying pIncFII and a multi-drug-resistance pIncC plasmid in ST78 E. hormaechei strain CRECL55. (a) Mauve alignment of the pIncFII plasmid in E. hormaechei CRECL55 (ST78) with pEC974-3 (CP021843.1) identified in E. coli strain EC974 (SAMN07192703). (b) Mauve alignment of the pIncC plasmid in CRECL55 with the closely-related plasmid pIncC-L117 (CP040034.1) identified in K. pneumoniae KPC160117 (SAMN11246288).

Figure 8.

Emergence and transmission of carbapenemase-producing E hormaechei during 2017-2020. The index OXA-48-producing E. hormaechei strain emerged in May 2017, potentially transferring the bla_OXA-48-carrying IncL plasmid, likely acquired from ST11 K. pneumoniae, to an ST66 E. hormaechei isolate. Clonal expansion of this OXA-48-producing ST78 lineage has occurred since 2018. In 2019, an ST90 E. hormaechei strain acquired pOXA48-CREH, probably from the OXA-48-producing ST78 lineage, and disseminated on a small scale. Through acquiring a novel bla_KPC-2-carrying pIncFII plasmid and a variant of the pIncC plasmid, a KPC-2-producing ST78 E. hormaechei was identified at the end of 2020. Echoing the capability of ST78 E. hormaechei in acquiring various types of drug-resistance plasmids, in this high-risk lineage, an IMP-8-producing E. hormaechei was initially found in 2018 after harbouring a bla_IMP-8-pIncHI2 plasmid. Collectively, ST78 E. hormaechei emerged as the primary driving force behind the transmission of carbapenemase genes in this hospital setting.