The fly route of extended-spectrum-β-lactamase-producing Enterobacteriaceae dissemination in a cattle farm: from the ecosystem to the molecular scale

Abstract

Introduction: This study aimed to understand the origin and to explain the maintenance of extended-spectrum β-lactamase (ESBL) Enterobacteriaceae isolated from food-producing animals in a third-generation cephalosporin (3GC)-free farm.

Methods: Culture and molecular approaches were used to test molecules other than 3GC such as antibiotics (tetracycline and oxytetracycline), antiparasitics (ivermectin, flumethrin, fenbendazol, and amitraz), heavy metal [arsenic, HNO₃, aluminum, HNO₃, cadmium (CdSO₄), zinc (ZnCl₂), copper (CuSO₄), iron (FeCl₃), and aluminum (Al₂SO₄)], and antioxidant (butylated hydroxytoluene) as sources of selective pressure. Whole-genome sequencing using short read (Illumina™) and long read (Nanopore™) technologies was performed on 34 genomes. In silico gene screening and comparative analyses were used to characterize the genetic determinants of resistance, their mobility, and the genomic relatedness among isolates.

Results: Our analysis unveiled a low diversity among the animal ESBL-producing strains. Notably, E. coli ST3268 was recurrently isolated from both flies (n = 9) and cattle (n = 5). These E. coli ST3268/bla _CTX-M-15/bla _TEM-1B have accumulated multiple plasmids and genes, thereby representing a reservoir of resistance and virulence factors. Our findings suggest that flies could act as effective mechanical vectors for antimicrobial gene transfer and are capable of transporting resistant bacteria across different environments and to multiple hosts, facilitating the spread of pathogenic traits. A significantly higher mean minimum inhibitory concentration of oxytetracycline (841.4 ± 323.5 mg/L vs. 36.0 ± 52.6 mg/L, p = 0.0022) in ESBL E. coli than in non-ESBL E. coli and bla _CTX-M-15 gene overexpression in oxytetracycline-treated vs. untreated ESBL E. coli (RQ_Oxy = 3.593, p = 0.024) confirmed oxytetracycline as a source of selective pressure in ESBL E. coli.

Discussion: The occurrence of ESBL E. coli in a farm without 3GC use is probably due to an as yet undefined human origin of Enterobacteriaceae bla _CTX-M-15 gene transmission to animals in close contact with cattle farm workers and the maintenance of the local ESBL E. coli reservoir by a high fly diversity and oxytetracycline selective pressure. These findings highlight the critical need for stringent vector control to mitigate antimicrobial resistance spread for preserving public health. Addressing this issue necessitates a multifaceted approach combining microbial genetics, vector ecology, and farm management practices.

Keywords: ESBL; Enterobacteriaceae; ST3268; oxytetracycline; selective pressure.

Figure 1

Core genome maximum likelihood phylogenetic tree of 23 E. coli isolates from farm number 13. Clusters ST3268 and ST155 represent groups of similar core genomes (0 to 25 single-nucleotide polymorphism difference). Hosts and antimicrobial susceptibility phenotypes are indicated by vertical colored stripes. The resistant phenotype is assigned to isolates that are resistant to at least one of the 17 antimicrobials tested. ST, sequenced type based on the Achtman MLST scheme (Wirth et al., 2006). Corresponding β-lactamase-associated resistance-coding genes are indicated by black squares and plasmids by black circles. IncF, plasmid incompatibility group F; RST, replicon typing system.

Figure 2

Schematic representation showing two combinations of the ultrastructural genetic background of the ST3268.1 ESBL E. coli subclusters (n = 7 isolates: BCA37F, -G, -H, -K, EC307, EC318, and EC338) and ST3268.2 (n = 2 isolates: BCA26.1 and EC347). Plasmids are shown as circles annotated for replicon, β-lactamase resistance genes, secretion system (T4S), toxin/antitoxin system (HigA/HigB), and phage-encoded protein genes. Supercoiled chromosomal DNA is schematically shown in red. a, truncated IncN_1_AY046276 replicon (247 bp). b, complete IncN_1_AY046276-pST3 replicon (512 bp).

Figure 3

Modulation of bla _CTX-M-15 gene expression under in vitro selective pressure. Relative quantification of bla _CTX-M-15 gene expression under oxytetracycline treatment in ESBL E. coli isolates. Error bars represent the standard deviation of at least two independent experiments. RQ, relative quantification. Error bars indicate the range between RQ min and RQ max. ** Statistically significant p-value < 0.05.

Figure 4

Core genome comparative analysis of 68 E. coli isolates from food-producing animals and flies. Maximum likelihood phylogenetic tree of 68 E. coli isolated from farms in Guadeloupe between 2018 and 2019. The farm number refers to our previous reference number (Gruel et al., 2021). Associated hosts and antimicrobial susceptibility phenotypes are indicated by vertical colored stripes. The resistant phenotype is assigned to isolates that are resistant to at least one of the 17 antimicrobials tested. Of these, 43 were ESBL producers. The colored clusters (ST115, ST3268, ST2015, ST2705, ST1630, and ST155) represent groups of similar core genomes (≤25 SNP). Only β-lactamase-encoding genes are indicated by black squares. Plasmid replicons are indicated by a black circle, and only the IncF RST was detailed. IncF, plasmid incompatibility group F; RST, replicon sequence typing.