Colistin resistance genes (mer-1 to mer-5) in agricultural soil fertilized with animal manure in Northern Italy

Fertilization with animal manure is a common agricultural practice in Italy. Waste products from intensive animal farming, such as cattle, swine and poultry farms, are the most frequently utilized. However, manure may contain residues of antimicrobials used during rearing that may have a negative impact on the environment, including the dissemination of antimicrobial resistance genes [1, 2]. Colistin is an antimicrobial commonly used in animals, and recent studies showed that resistance can be induced by plasmid-mediated mcr genes [3]. The presence of antimicrobials, such as colistin, in animal manure used as fertilizer of agricultural soil may promote the dissemination of mcr genes into the environment. This study aimed at investigating the presence of colistin residues and mer-1 to mcer-5 genes in agricultural soil before and after fertilization with manure/slurry from intensive animal (i.e. dairy, swine and chicken) farms in Northern Italy. A total of 84 samples (1 sample of manure/slurry per farm; 1 sample of soil before fertilization, TO, per farm; and 1 sample of soil about 30 days after fertilization, T30, per farm) were collected from dairy (n = 11), swine (n = 10) and chicken (n = 7) farms. All samples were analysed by HPLC- MS/MS and by SYBR® Green Real-Time PCR for the detection of colistin and mer-1 to mer-5 genes, respectively. None of the samples contained colistin residues, whereas all the mcr genes were found. In swine farms, all 5 mer genes (i.e. 5 slurry, 2 TO and 3 T30 samples for mer-1; 3 slurry samples for mer-2; 3 slurry, 2 TO and 2 T30 samples for mer-3; 2 slurry and 1 T30 samples for mcr-4; 1 slurry and 1 T30 samples for mcr-5) were detected. In chicken and dairy farms, mer- 1 (i.e. 3 manure samples in dairy,-and 3 TO and 1 T30 samples in chicken), mcr-3 (i.e. 2 manure in dairy samples, and 1 manure and 1 TO samples in chicken), mcr-4 (i.e. 1 manure sample in dairy) and mcr-S (i.e. 1 TO sample in dairy) genes were found. Overall, mcr-1 was the prevalent gene (20%), followed by mcer-3 (13%), mer-4 (4.8%), mer-5 (3.6%), and mer-2 (3.5%). This study showed that all the mcr genes were present in all types of animal (i.e. dairy, swine and chicken) manure and related fertilized soils regardless of the presence of colistin residues. Furthermore, a higher prevalence and diversity of mer genes was found in swine manure and related fertilized soils compared to those from dairy and chicken farms. In conclusion, the presence of plasmid- mediated mcr genes both in manure and in agricultural soil is concerning for environmental and public health and further research is needed to understand their ecology into the environment.