In silico characterization of chromosomally integrated blaCTX-M genes among clinical Enterobacteriaceae in Africa: insights from whole-genome analysis

Abstract

Antimicrobial resistance (AMR) mediated by extended-spectrum β-lactamases (ESBLs) is a growing global concern, particularly among Enterobacteriaceae. The CTX-M-type ESBLs, encoded by the bla _CTX-M gene, are of significant public health importance due to their high prevalence and broad geographic distribution. Typically located on plasmids and often co-occurring with other AMR genes, bla _CTX-M contributes to multidrug resistance (MDR). However, increasing evidence suggests secondary chromosomal integration of bla _CTX-M, sometimes alongside other resistance determinants. The extent and implications of this mechanism remain poorly characterized, especially in Africa, where genomic surveillance is limited. In this study, we retrieved 295 chromosomal sequences of Enterobacteriaceae of African origin from the GenBank and performed in silico predictions of bla _CTX-M and other AMR genes. bla _CTX-M-carrying sequences were further characterized by in silico multilocus sequence typing and genome annotation. Chromosomal insertions were identified through alignment with reference genomes. Overall, 47 of 295 sequences (15.9%) harbored the bla _CTX-M gene, with the highest prevalence in Klebsiella pneumoniae (29/157, 18.5%), followed by Escherichia coli (13/72, 18.1%), Enterobacter spp. (4/38, 10.5%), and Shigella spp. (1/12, 8.3%). The most common allele was bla _CTX-M-15 (31/47, 66.0%), followed by bla _CTX-M-14 (12/47, 25.5%), bla _CTX-M-55 (3/47, 6.4%), and bla _CTX-M-27 (1/27, 3.7%). Co-occurrence of bla _CTX-M with additional AMR genes was frequently observed, with integration events often associated with mobile genetic elements such as ISEcp1 and IS26. Notably, strains from the same hospital setting were phylogenetically related and shared sequence types and AMR gene profiles, suggesting local clonal dissemination. These findings reveal a notable presence of chromosomally integrated bla _CTX-M among African Enterobacteriaceae, frequently in association with other resistance genes, thereby facilitating stable MDR propagation independent of plasmid maintenance. This evolutionary adaptation may have significant implications for the persistence and spread of MDR in clinical settings.

Keywords: Africa; Enterobacteriaceae; IS26; ISEcp1; blaCTX-M; chromosomal.

Figure 1

Genetic environment of bla_CTX-M in Shigella and E. coli chromosomes from Africa. From a total of 14 genomes, the ISEcp1 was observed upstream of bla_CTX-M in 11 strains, while wbuC existed downstream of bla_CTX-M in strains. CAC124 from Malawi exhibited multiple AMR genes on a composite transposon bracketed by directly oriented IS26 elements. Yellow; mobile genetic elements. Red; β-lactamase gene. Cyan; wbuC. Green; aminoglycoside resistance gene. Brown; chloramphenicol resistance gene.

Figure 2

Comparison analysis of CAC124 from Malawi with E. coli ST131 strains from various countries across the world. The IS26-flanked composite transposon in CAC124 was found in several E. coli ST131. Yellow; mobile genetic elements. Red; β-lactamase gene. Cyan; wbuC. Green; aminoglycoside resistance gene. Brown; chloramphenicol resistance gene.

Figure 3

MDR chromosomal insertions in E. cloacae strains from West Africa. (A) Strain EFN743 from Ghana harbored bla_CTX-M on a ~ 75 kbp IS26-flanked chromosomal insertion containing multiple AMR genes. The insertion was bracketed by putative 8 bp TSDs (represented by flags). (B) The insertion in strain EFN743 was similar to plasmid p23_A-OXA140 from Switzerland, though the gene arrangement was different. (C) Nigerian strain NN-BR118-1 carried bla_CTX-M on a ~ 13 kbp MDR insertion bounded by the int. gene on one end. This insertion was found in several sequences, though they lacked the int gene upstream, but harbored IS26 on the opposite end. Yellow; mobile genetic elements. Red; β-lactamase gene. Cyan; wbuC. Green; aminoglycoside resistance gene. Brown; chloramphenicol resistance gene. Pink; tetracycline resistance gene. Orange; folate pathway antagonist.

Figure 4

MDR chromosomal insertions in K. pneumoniae strains from West Africa. (A) Ghanaian strain MIN-106 possessed a 139,735 bp chromosomal insertion bounded by ISEcp1/bla_CTX-M-15/wbuC on one end. About 60,300 bp of this insertion was similar to plasmid p2247421-T20-ESBL_2, but the aligned regions did not include any AMR genes. (B) South African strain ST101:960186733 harbored multiple AMR genes on a 22,270 bp insertion bounded by ISEcp1/bla_CTX-M-15/wbuC on one end. The insertion was similar to the AMR gene cluster, which was observed in plasmids pB16KP0141–1 and pEc21617-310.

Figure 5

Core-SNP-based phylogenetic trees for E. coli (A) and K. pneumoniae (B). Clustering was seen among strains from the same geographic location. There was no obvious clustering based on the presence large bla_CTX-M-carrying MDR chromosomal insertions. Strains from the same country are indicated by the same color. Asterisks (*) represent the presence of large bla_CTX-M-carrying MDR chromosomal insertions.