Inventory of Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae in France as Assessed by a Multicenter Study

Abstract

The objective of this study was to perform an inventory of the extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae isolates responsible for infections in French hospitals and to assess the mechanisms associated with ESBL diffusion. A total of 200 nonredundant ESBL-producing Enterobacteriaceae strains isolated from clinical samples were collected during a multicenter study performed in 18 representative French hospitals. Antibiotic resistance genes were identified by PCR and sequencing experiments. The clonal relatedness between isolates was investigated by the use of the DiversiLab system. ESBL-encoding plasmids were compared by PCR-based replicon typing and plasmid multilocus sequence typing. CTX-M-15, CTX-M-1, CTX-M-14, and SHV-12 were the most prevalent ESBLs (8% to 46.5%). The three CTX-M-type EBSLs were significantly observed in Escherichia coli (37.1%, 24.2%, and 21.8%, respectively), and CTX-M-15 was the predominant ESBL in Klebsiella pneumoniae (81.1%). SHV-12 was associated with ESBL-encoding Enterobacter cloacae strains (37.9%). qnrB, aac(6')-Ib-cr, and aac(3)-II genes were the main plasmid-mediated resistance genes, with prevalences ranging between 19.5% and 45% according to the ESBL results. Molecular typing did not identify wide clonal diffusion. Plasmid analysis suggested the diffusion of low numbers of ESBL-encoding plasmids, especially in K. pneumoniae and E. cloacae However, the ESBL-encoding genes were observed in different plasmid replicons according to the bacterial species. The prevalences of ESBL subtypes differ according to the Enterobacteriaceae species. Plasmid spread is a key determinant of this epidemiology, and the link observed between the ESBL-encoding plasmids and the bacterial host explains the differences observed in the Enterobacteriaceae species.

Keywords: AAC(3)-II; AAC(6′)-Ib; ESBL; plasmid; plasmid-mediated quinolone resistance; plasmid-mediated resistance.

FIG 1

Epidemiological context of ESBL-producing Enterobacteriaceae in 18 representative French hospitals in 2012 (n = 50,378 Enterobacteriaceae). (a) Distribution of bacterial species in the 200 ESBL-producing Enterobacteriaceae isolates from clinical samples. C. freundii, Citrobacter freundii; P. mirabilis, Proteus mirabilis; C. braakii, Citrobacter braakii; E. aerogenes, Enterobacter aerogenes; E. hermannii, Escherichia hermannii. (b) Annual prevalence of ESBL-producing Enterobacteriaceae (the arrows indicate the extreme values and the horizontal dashes the mean values). (c) The clinical samples from which the 200 ESBL-encoding Enterobacteriaceae were isolated. (d) Hospital units corresponding to patients infected by the 200 ESBL-encoding Enterobacteriaceae strains.

FIG 2

The diversity of ESBLs in Enterobacteriaceae isolated from clinical samples in 18 French hospitals (n = 200). (a) Prevalence of ESBLs in Enterobacteriaceae. (b) Distribution of ESBLs in E. coli (n = 124), K. pneumoniae (n = 37), and E. cloacae (n = 29).

FIG 3

Percentage of ESBL-encoding isolates harboring plasmid-mediated genes encoding resistance to gentamicin, amikacin, and fluoroquinolones. (a) Distribution of proteins encoded by resistance genes in ESBL-producing Enterobacteriaceae. (b) Distribution of proteins encoded by aac(6′)-Ib-cr, aac(3)-II, qnrB, qnrA1, and qnrS1 in CTX-M-15 (n = 93)-, CTX-M-1 (n = 31)-, CTX-M-14 (n = 30)-, and SHV-12 (n = 16)-producing Enterobacteriaceae.

FIG 4

(a and b) Multidimensional scaling analysis (MDS) scatter plots derived from DiversiLab data from the ESBL-producing E. coli strain showing ESBL types (a) and the E. coli phylogroup (b). The scale indicates the dissimilarity between strains for the x and y axes. (c and d) Dendrogram of K. pneumoniae (c) and E. cloacae (d) strains. The vertical dashed line indicates the criterion for clonality (≥95% similarity). ST, sequence type.

FIG 5

Percentage of ESBL-producing isolates harboring virulence factors for the E. coli (n = 124) (a) and K. pneumoniae (n = 37) (b) species. (c and d) Percentages of ESBL-producing isolates harboring 0 to 11 virulence factors for the different E. coli phylogroups (c) and the ST131 E. coli sequence type (d).