Screening of colistin-resistant bacteria in livestock animals from France

Abstract

Colistin is frequently used as a growth factor or treatment against infectious bacterial diseases in animals. The Veterinary Division of the European Medicines Agency (EMA) restricted colistin use as a second-line treatment to reduce colistin resistance. In 2020, 282 faecal samples were collected from chickens, cattle, sheep, goats, and pigs in the south of France. In order to track the emergence of mobilized colistin resistant (mcr) genes in pigs, 111 samples were re-collected in 2021 and included pig faeces, food, and water from the same location. All samples were cultured in a selective Lucie Bardet Jean-Marc Rolain (LBJMR) medium and colonies were identified using MALDI-TOF mass spectrometry and then antibiotic susceptibility tests were performed. PCR and Sanger sequencing were performed to screen for the presence of mcr genes. The selective culture revealed the presence of 397 bacteria corresponding to 35 different bacterial species including Gram-negative and Gram-positive. Pigs had the highest prevalence of colistin-resistant bacteria with an abundance of intrinsically colistin-resistant bacteria and from these samples one strain harbouring both mcr-1 and mcr-3 has been isolated. The second collection allowed us to identify 304 bacteria and revealed the spread of mcr-1 and mcr-3 in pigs. In the other samples, naturally, colistin-resistant bacteria were more frequent, nevertheless the mcr-1 variant was the most abundant gene found in chicken, sheep, and goat samples and one cattle sample was positive for the mcr-3 gene. Animals are potential reservoir of colistin-resistant bacteria which varies from one animal to another. Interventions and alternative options are required to reduce the emergence of colistin resistance and to avoid zoonotic transmissions.

Keywords: France; animals; colistin-resistant bacteria; mcr genes.

Figure 1

Geographical map showing the provenance of animal samples. The numbers in the bubbles represent the number of collected samples. The samples collected in 2020 are faecal samples only, while those collected in 2021 are from different origins. The localizations from where samples were collected are indicated by black stars.

Figure 2

Network screening analysis of colistin-resistant bacteria isolated from faecal samples of domestic animals in France using Cytoscape 3.9.0. A Isolated colistin-resistant bacteria from chicken. B From cattle; C From goats; D From sheep; E From pigs. Colistin-resistant bacteria are divided into two batches according to the Gram GNB and GPB. Bacteria carrying mcr genes are distinguished by blue zigzag arrows (edge). The number of edges for each bacterial species represents the number of isolated bacteria. The size of nodes also shows the variable number of isolated bacteria.

Figure 3

Screening of colistin-resistant bacteria isolated from collected samples of pigs in 2021. Edges in black represent faeces samples, red for food, and blue for water. Bacteria are devised into two groups according to the Gram, mcr genes cross-link with E. coli.

Figure 4

Hierarchical clustering analysis of antibiotic resistance phenotype of colistin-resistant bacteria isolated from domestic animals in France using MEV 4.9.0 software. The green colour refers to the sensitive phenotype of the bacteria to the antibiotic, and the red colour refer to resistance. amoxicillin (AMX), amoxicillin-clavulanic acid (AMC), cefepime (FEP), piperacillin/tazobactam (TPZ), cefalotin (KF), ceftriaxone (CRO), ertapenem (ETP), imipenem (IMP), fosfomycin (FF), nitrofurantoin (F), trimethoprim-sulfamethoxazole (SXT), amikacin (AK), ciprofloxacin (CIP), doxycycline (DO), colistin (CT), and gentamicin (GN).

Figure 5

Annotation heatmap of antibiotic resistance genes found in E. coli harbouring mcr genes isolated from faeces of pigs.