Genomic analysis of antibiotic-resistant Enterococcus spp. reveals novel enterococci strains and the spread of plasmid-borne Tet(M), Tet(L) and Erm(B) genes from chicken litter to agricultural soil in South Africa.

Manure from food animals exposed to antibiotics is often used as soil fertiliser, potentially releasing antibiotic-resistant bacteria (ARB) with diverse antibiotic-resistance genes (ARGs) into the soil. To determine the impact of chicken litter application on the soil resistome, Enterococcus spp. isolated from chicken litter and soil samples collected before and after the soil amendment were characterised, using whole-genome sequencing and bioinformatics tools. Nineteen Enterococcus spp. isolates from the three sources were sequenced on Illumina Miseq platform to ascertain the isolates' resistome, mobilome, virulome, clonality, and phylogenomic relationships. Multilocus sequence typing (MLST) analysis revealed eight novel sequence types (STs) (ST1700, ST1752, ST1753, ST1754, ST1755, ST1756, ST1004, and ST1006). The isolates harboured multiple resistance genes including those conferring resistance to inter alia macrolides-lincosamide-streptogramin (erm(B), lnu(B), lnu(G), lsaA, lsaE, eat(A), msr(C)), tetracycline (tet(M), tet(L), tet(S)), aminoglycosides (aac(6')-Ii, aac(6')-Iih, ant(6)-Ia, aph(3')-III, ant(9)-Ia), fluoroquinolones (efmA, and emeA), vancomycin (VanC {VanC-2, VanXY, VanXYC-3, VanXYC-4, VanRC}), and chloramphenicol (cat). The litter-amended soil harboured new ARB (particularly E. faecium) and ARGs (ant(6)-Ia, aac(6')-Ii, aph(3')-III), lnu(G), msr(C), and eat(A), efmA) that were not previously detected in the soil. The identified ARGs were associated with diverse mobile genetic elements (MGEs) such as insertion sequences (IS6, ISL3, IS256, IS30), transposons (Tn3 and Tn916) and plasmids (repUS43, repUS1, rep9b, and rep 22). Twenty-eight virulence genes encoding adherence/biofilm formation (ebpA, ebpB, ebpC), antiphagocytosis (elrA) and bacterial sex pheromones (Ccf10, cOB1, cad, and camE), were detected in the genomes of the isolates. Phylogenomic analysis revealed a close relationship between a few isolates from litter-amended soil and the chicken litter isolates. The differences in the ARG and ARB profiles in the soil before and after the litter amendment and their association with diverse MGEs indicate the mobilisation and transmission of ARGs and ARB from the litter to the soil.

Rethinking Manure Application: Increase in Multidrug-Resistant Enterococcus spp. in Agricultural Soil Following Chicken Litter Application.

The current study investigated the impact of chicken litter application on the abundance of multidrug-resistant Enterococcus spp. in agricultural soil. Soil samples were collected from five different strategic places on a sugarcane farm before and after manure application for four months. Chicken litter samples were also collected. Enterococci were enumerated using the Enterolert®/Quanti-Tray 2000® system and confirm and differentiated into species using real-time PCR. The antibiotic susceptibility profile of the isolates was determined using the disk diffusion method following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The overall mean bacterial count was significantly higher (p < 0.05) in manure-amended soil (3.87 × 107 MPN/g) than unamended soil (2.89 × 107 MPN/g). Eight hundred and thirty-five enterococci (680 from soil and 155 from litter) were isolated, with E. casseliflavus being the most prevalent species (469; 56.2%) and E. gallinarum being the least (16; 1.2%). Approximately 56% of all the isolates were resistant to at least one antibiotic tested, with the highest resistance observed against tetracycline (33%) and the lowest against chloramphenicol (0.1%); 17% of E. faecium were resistant to quinupristin-dalfopristin. Additionally, 27.9% (130/466) of the isolates were multidrug-resistant, with litter-amended soil harbouring more multidrug-resistant (MDR) isolates (67.7%; 88/130) than unamended soil (10.0%; 13/130). All isolates were susceptible to tigecycline, linezolid and gentamicin. About 7% of the isolates had a multiple antimicrobial resistance index > 0.2, indicative of high antibiotic exposure. Although organic fertilizers are regarded as eco-friendly compared to chemical fertilizers for improving soil fertility, the application of untreated animal manure could promote the accumulation of antibiotics and their residues and antibiotic-resistant bacteria in the soil, creating an environmental reservoir of antimicrobial resistance, with potential human and environmental health risks.