Specificities and Commonalities of Carbapenemase-Producing Escherichia coli Isolated in France from 2012 to 2015

Abstract

Carbapenemase-producing Escherichia coli (CP-Ec) represents a major public health threat with a risk of dissemination in the community as has occurred for lineages producing extended-spectrum β-lactamases. To characterize the extent of CP-Ec spread in France, isolates from screening and infection samples received at the French National Reference Center (F-NRC) laboratory for carbapenemase-producing Enterobacterales were investigated. A total of 691 CP-Ec isolates collected between 2012 and 2015 and 22 isolates collected before 2012 were fully sequenced. Analysis of their genome sequences revealed some disseminating multidrug-resistant (MDR) lineages frequently acquiring diverse carbapenemase genes mainly belonging to clonal complex 23 (CC23) (sequence type 410 [ST410]) and CC10 (ST10 and ST167) and sporadic isolates, including rare ST131 isolates (n = 17). However, the most represented sequence type (ST) was ST38 (n = 92) with four disseminated lineages carrying bla_OXA-48-like genes inserted in the chromosome. Globally, the most frequent carbapenemase gene (n = 457) was bla_OXA-48. It was also less frequently associated with MDR isolates being the only resistance gene in 119 isolates. Thus, outside the ST38 clades, its acquisition was frequently sporadic with no sign of dissemination, reflecting the circulation of the IncL plasmid pOXA-48 in France and its high frequency of conjugation. In contrast, bla_OXA-181 and bla_NDM genes were often associated with the evolution of MDR E. coli lineages characterized by mutations in ftsI and ompC. IMPORTANCE Carbapenemase-producing Escherichia coli (CP-Ec) might be difficult to detect, as MICs can be very low. However, their absolute number and their proportion among carbapenem-resistant Enterobacterales have been increasing, as reported by WHO and national surveillance programs. This suggests a still largely uncharacterized community spread of these isolates. Here, we have characterized the diversity and evolution of CP-Ec isolated in France before 2016. We show that carbapenemase genes are associated with a wide variety of E. coli genomic backgrounds and a small number of dominant phylogenetic lineages. In a significant proportion of CP-Ec, the most frequent carbapenemase gene bla_OXA-48, was detected in isolates lacking any other resistance gene, reflecting the dissemination of pOXA-48 plasmids, likely in the absence of any antibiotic pressure. In contrast, carbapenemase gene transfer may also occur in multidrug-resistant E. coli, ultimately giving rise to at-risk lineages encoding carbapenemases with a high potential of dissemination.

Keywords: drug resistance evolution; emergence; genomics; multidrug resistance; surveillance studies.

FIG 1

Origin of the isolates received by the F-NRC during the 2012–2015 period. (A) Absolute number of isolates per year from infection, screening, or unknown origins, differentiating between isolates that have or have not been sequenced. (B) Proportions of isolates from infection or from screening or unknown origins showing that isolates from infection origin represent the same proportions among total isolates that have been received by the F-NRC and isolates that have been sequenced.

FIG 2

Core genome phylogeny of CP-Ec isolates received by the F-NRC. ML phylogeny of the 713 CP-Ec isolates was built with RAxML (45) from 372,238 core SNPs after sequence alignment on MG1655-K12 (NC_000913.3) selected as the best reference. The genome sequence of Escherichia fergusonii strain ATCC 35469 (NC_011740.1) was used as an outgroup for the phylogenetic analysis. Genomes from Touchon et al. (40) were also incorporated into the analysis. Genomic features are indicated as in the figure key (left) from the inside to the outside circles: carbapenemases of the OXA, NDM, and other types, CTX-M ESBL, mutations in gyrA and parC QRDR region (FQ resistance), origin, main ST, phylogroups. Reference genomes from Touchon et al. (40) are indicated as Touchon. NA, not available.

FIG 3

Resistance gene content of the F-NRC CP-Ec isolates. (A) Box plot representation of the ARG content according to the phylogroup. The number of isolates belonging to each phylogroup is indicated within parentheses. The limits of the box indicate the lower and upper quartile. Outliers are indicated by points above the maximum value. (B) Relationships between the number of ARGs and the number of mutations in QRDRs. In the box plot representations, the median is indicated by an horizontal bar, and the mean is indicated by a × symbol.

FIG 4

Carbapenemase gene content of the F-NRC CP-Ec isolates. (A) Relationship between the carbapenemase allele and the ARG content. In the box plot representation, the median is indicated by an horizontal bar and the mean by a × symbol. The limits of the box indicate the lower and upper quartile. Outliers are indicated by points above the maximum value. (B) Number of isolates carrying a specific carbapenemase allele according to the number of QRDR mutations in their genomes. (C) Per-year evolution of the percentage of isolates carrying the different carbapenemase alleles in the 2012–2015 period. The year of isolation is indicated as in the figure key (right). Given their small number (n = 22), strains isolated before 2012 are not indicated.

FIG 5

Features of the main STs associated with CP-Ec isolates in France. At the top of the figure, for each ST, the phylogroup, the total number of isolates, and the number of isolates associated with urinary tract infections are indicated. Intermediate (Inter) and minor ST indicate ST represented by four to six isolates and one to three isolates, respectively. (A) Proportion of the isolates according to the carbapenemase allele in each ST. (B) Box plot representation of the ARGs content according to the ST. (C) Proportion of isolates according to the number of mutations in QRDR. (D) Proportion of isolates with a specific PBP3 allele. PBP3 characterized by a YRIP, YRIK, or YRIN insertion between positions 333 and 334, the YRIN insertion is generally associated with a A413V mutation (YRIN-L) and less frequently also with a E349K mutation (YRIN-K-L) (10). (E) Proportion of isolates carrying specific mutations in ompC: R195L, G138D or acquisition of an ST38-like ompC allele by recombination (10).

FIG 6

Core genome phylogeny of ST38 isolates. ML Phylogeny was based on genome sequences of 92 CP-Ec from the F-NRC and 464 genome sequences retrieved from EnteroBase and from the NCBI database, including 331 carrying a carbapenemase gene. A core genome (2,900,000-nt) alignment of the de novo assemblies on the sequence of GCA_005886035.1 used as a reference was performed by using Parsnp (47); ML phylogeny was built with RAxML (45) from 6,170 core SNPs after removing recombined regions with Gubbins (48). The genome sequence of CNRC6O47 (ST963) was used as an outgroup for the phylogenetic analysis. F-NRC isolates are indicated by red triangles (inner circle). Other genomic features are indicated as indicated in the figure key (left) from the inside to the outside circles: carbapenemases of the OXA, NDM, and other types; absence of any plasmid of any Inc type as identified by using PlasmidFinder (14); ESBL of the CTX-M types; number of mutations in gyrA and parC QRDRs (FQ resistance); number of ARGs; mutations in ftsI, ompC, and ompF; and geographical origin. The four OXA-48-like clades (G1, G2, G3, and G4) clustering most French isolates are colored in blue, red, violet, and green. A fifth clade (G5) corresponding to a possible outbreak of VIM-4 isolates in east of Paris is colored in brown.

FIG 7

bla_OXA-48 integration sites of ST38 CP-Ec clades 1, 2, 3, and 4. The genome sequences of GCA_005886035.1 (CP040390.1), CNR65D6, GCA_004759025.1 (CP038505.1), and CNR85I8 were chosen as representative genomes of clusters 1, 2, 3, and 4, respectively, and aligned, by using BlastN, against the sequence of GCA_001900295.1 (CP010116.1), an ST38 isolate that does not carry a carbapenemase gene and whose genome sequence was complete. Gene annotations were derived from strain MG1655 (NC_000913.3). The regions surrounding the bla_OXA-48 insertion sites in each clade were more precisely aligned against the corresponding sequences of CP010116.1 and of representative genomes of the other clades by using Easyfig (52). bla_OXA-48 is represented by a brown arrow, cointegrated genes of pOXA-48 origin are indicated by light green arrows, and the IS1 transposase is indicated by a green arrow. Sequences used to perform the BLAST analysis on Illumina contigs are underlined in red. It is interesting to note that, in the G3 clade, bla_OXA-48 integration occurs in a genomic island inserted at tRNASec. Another genomic island is present in G1-type strains but does not carry bla_OXA-48.