Novel chromosomal insertions of ISEcp1-blaCTX-M-15 and diverse antimicrobial resistance genes in Zambian clinical isolates of Enterobacter cloacae and Escherichia coli

Abstract

Background: The epidemiology of extended-spectrum β-lactamases (ESBLs) has undergone dramatic changes, with CTX-M-type enzymes prevailing over other types. bla_CTX-M genes, encoding CTX-M-type ESBLs, are usually found on plasmids, but chromosomal location is becoming common. Given that bla_CTX-M-harboring strains often exhibit multidrug resistance (MDR), it is important to investigate the association between chromosomally integrated bla_CTX-M and the presence of additional antimicrobial resistance (AMR) genes, and to identify other relevant genetic elements.

Methods: A total of 46 clinical isolates of cefotaxime-resistant Enterobacteriaceae (1 Enterobacter cloacae, 9 Klebsiella pneumoniae, and 36 Escherichia coli) from Zambia were subjected to whole-genome sequencing (WGS) using MiSeq and MinION. By reconstructing nearly complete genomes, bla_CTX-M genes were categorized as either chromosomal or plasmid-borne.

Results: WGS-based genotyping identified 58 AMR genes, including four bla_CTX-M alleles (i.e., bla_CTX-M-14, bla_CTX-M-15, bla_CTX-M-27, and bla_CTX-M-55). Hierarchical clustering using selected phenotypic and genotypic characteristics suggested clonal dissemination of bla_CTX-M genes. Out of 45 bla_CTX-M gene-carrying strains, 7 harbored the gene in their chromosome. In one E. cloacae and three E. coli strains, chromosomal bla_CTX-M-15 was located on insertions longer than 10 kb. These insertions were bounded by ISEcp1 at one end, exhibited a high degree of nucleotide sequence homology with previously reported plasmids, and carried multiple AMR genes that corresponded with phenotypic AMR profiles.

Conclusion: Our study revealed the co-occurrence of ISEcp1-bla_CTX-M-15 and multiple AMR genes on chromosomal insertions in E. cloacae and E. coli, suggesting that ISEcp1 may be responsible for the transposition of diverse AMR genes from plasmids to chromosomes. Stable retention of such insertions in chromosomes may facilitate the successful propagation of MDR clones among these Enterobacteriaceae species.

Keywords: AMR; Chromosomal insertion; Enterobacter cloacae; Escherichia coli; Extended spectrum β-lactamase; ISEcp1; Zambia; bla CTX-M.

Fig. 1

Phylogenetic analysis. Whole genome-based phylogenetic trees for 36 E. coli and 9 K. pneumoniae strains. a E. coli: a total of 12 STs were identified, including one novel type (marked with *). Four STs constituted 25/36 (69.4%) of E. coli strains, the most common being ST69 (9 strains), followed by ST131 (6 strains). b K. pneumoniae: a total of 3 STs were identified, with ST307 alone representing 6/9 (66.7%) strains

Fig. 2

AMR phenotypes, AMR genes and plasmid replicons. All but one strain displayed resistance to at least three antimicrobial classes. There was no phenotypic or genotypic resistance to imipenem, however, one strain (Zam_UTH_40) exhibited phenotypic resistance to colistin. A total of 12 AMR gene classes were identified. Within the β-lactamase gene class, the bla_CTX-M family showed the most diversity, with bla_CTX-M-15 being the most common variant. Most bla_CTX-M genes were located on plasmids, however, 7/45 (15.6%) strains harbored the genes on the chromosome. A total of 24 plasmid replicons were detected, with the most prevalent being IncFIB(AP001918)_1, which was present in 30/46 (65.2%) strains. Hierarchical clustering showed aggregation of strains of the same ST. Cefotaxime (CTX) is not shown here since all strains were selected using CTX. AMP; ampicillin. CHL; chloramphenicol. CIP; ciprofloxacin. CST; colistin. DOX; doxycycline. GEN; gentamicin. IPM; imipenem. NAL; nalidixic acid. NIT; nitrofurantoin

Fig. 3

bla_CTX-M genes present on short chromosomal insertions in E. coli. a Zam_UTH_41. This E. coli ST8767 strain carried bla_CTX-M-14 on a 3,095 bp chromosomal insertion with ISEcp1 located 249 bp upstream of bla_CTX-M-14. Zam_UTH_26 (not shown) also had a similar genetic context for its insertion. b Zam_UTH_43. This E. coli ST131 O107:H5 strain harbored bla_CTX-M-15 on a 6,036 bp chromosomal insertion, with ISEcp1 located 255 bp upstream of bla_CTX-M-15. About 2.5 kb downstream of this insertion was another insertion that harbored genes conferring resistance to chloramphenicol (cat), β-lactams (bla_OXA-1), and aminoglycosides/quinolones (aac(6′)-Ib-cr5). F1, F2, F3, R1, R2, R3; primers used for confirmation of insertions

Fig. 4

bla_CTX-M genes present on large chromosomal insertions in E. coli. a Zam_UTH_18. This E. coli ST3580 strain possessed bla_CTX-M-15 on an 11,383 bp chromosomal insertion, which was very similar to plasmid pF609 (GenBank accession no. MK965545.1). bla_CTX-M-15 was closely associated with ISEcp1, which was located 255 bp upstream. The insertion also harbored the quinolone resistance gene qnrS1, located 4639 bp downstream of bla_CTX-M-15. The phenotypic AMR profile of this strain showed resistance to ampicillin and susceptibility to quinolones. b Zam_UTH_42. This E. coli ST648 strain harbored bla_CTX-M-15 on a 14,328 bp chromosomal insertion that was very similar to plasmid p13ARS_MMH0112-2 (GenBank accession no. LR697123.1). This insertion carried genes associated with resistance to aminoglycosides (aac(3)-IIa), aminoglycosides/quinolones (aac(6′)-Ib-cr5), β-lactams (bla_OXA-1, bla_TEM-1), and chloramphenicol (catB3). ISEcp1 was located 255 bp upstream of bla_CTX-M-15, however, unlike in other strains, ISEcp1 in this strain was truncated by IS1 and transposase. The phenotypic AMR profile of this strain was consistent with the AMR genotype for the insertion. Zam_UTH_47 (not shown) also had a similar genetic context for its insertion. F4, F5, F6, F7, R4, R5, R6, R7; primers used for confirmation of insertions. White; susceptible. Black; resistance phenotype in the absence of corresponding AMR gene. Red; β-lactam resistance. Brown; chloramphenicol resistance. Green; aminoglycoside and/or quinolone resistance

Fig. 5

bla_CTX-M gene on a large chromosomal insertion in E. cloacae. Zam_UTH_44. This E. cloacae ST316 strain carried bla_CTX-M-15 on a ~ 41 kb chromosomal insertion that exhibited nucleotide sequence homology with plasmid pCRENT-193_1 (GenBank accession no. CP024813.1). ISEcp1 was located 255 bp upstream of bla_CTX-M-15. The insertion also included diverse AMR genes encoding resistance to six antimicrobial classes, namely; aminoglycosides (aac(3)-IIa), quinolones (qnrB1), aminoglycosides/quinolones (aac(6′)-Ib-cr5), β-lactams (bla_OXA-1), trimethoprim (dfrA14), chloramphenicol (catB3), and tetracyclines (tet(A)). The phenotypic AMR profile of this strain was consistent with the AMR genotype of the insertion. F8, F9, R8, R9; primers used for confirmation of insertions. White; susceptible. Black; resistance phenotype in the absence of corresponding AMR gene. Red; β-lactam resistance. Brown; chloramphenicol resistance. Green; aminoglycoside and/or quinolone resistance. Pink; tetracycline resistance