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Biosolids that are applied to agricultural soil as an organic fertilizer are frequently contaminated with pharmaceutical residues that have persisted during wastewater treatment and partitioned into the organic phase. Macrolide antibiotics, which serve as a critically important human medicine, have been detected within biosolids. To determine the impacts of macrolide antibiotics on soil bacteria, every year for a decade, a series of replicated field plots received an application of a mixture of erythromycin, clarithromycin, and azithromycin at a realistic (0.1 mg kg soil(−1)) or an unrealistically high (10 mg kg soil(−1)) dose or were left untreated. The effects of repeated antibiotic exposure on the soil bacterial community, resistome, mobilome, and integron gene cassette content were evaluated by 16S rRNA and integron gene cassette amplicon sequencing, as well as whole-metagenome sequencing. At the unrealistically high dose, the overall diversity of the resistome and mobilome was altered, as 21 clinically important antibiotic resistance genes predicted to encode resistance to 10 different antibiotic drug classes were increased and 20 mobile genetic element variants (tnpA, intI1, tnpAN, and IS91) were increased. In contrast, at the realistic dose, no effect was observed on the overall diversity of the soil bacterial community, resistome, mobilome, or integron gene cassette-carrying genes. Overall, these results suggest that macrolide antibiotics entrained into soil at concentrations anticipated with biosolid applications would not result in major changes to these endpoints. IMPORTANCE Biosolids, produced from the treatment of sewage sludge, are rich in plant nutrients and are a valuable alternative to inorganic fertilizer when applied to agricultural soil. However, the use of biosolids in agriculture, which are frequently contaminated with pharmaceuticals, such as macrolide antibiotics, may pose a risk to human health by selecting for antibiotic resistance genes that could be transferred to plant-based food destined for human consumption. The consequences of long-term, repeated macrolide antibiotic exposure on the diversity of the soil bacterial community, resistome, and mobilome were evaluated. At unrealistically high concentrations, macrolide antibiotics alter the overall diversity of the resistome and mobilome, enriching for antibiotic resistance genes and mobile genetic elements of concern to human health. However, at realistic antibiotic concentrations, no effect on these endpoints was observed, suggesting that current biosolids land management practices are unlikely to pose a risk to human health due to macrolide antibiotic contamination alone.

Antibiotic resistance has emerged globally as one of the biggest threats to human and animal health. Although the excessive use of antibiotics is recognized as accelerating the selection for resistance, there is a growing body of evidence suggesting that natural environments are “hot spots” for the development of both ancient and contemporary resistance mechanisms. Given that pharmaceuticals can be entrained onto agricultural land through anthropogenic activities, this could be a potential driver for the emergence and dissemination of resistance in soil bacteria. Using functional metagenomics, we interrogated the “resistome” of bacterial communities found in a collection of Canadian agricultural soil, some of which had been receiving antibiotics widely used in human medicine (macrolides) or food animal production (sulfamethazine, chlortetracycline, and tylosin) for up to 16 years. Of the 34 new antibiotic resistance genes (ARGs) recovered, the majority were predicted to encode (multi)drug efflux systems, while a few share little to no homology with established resistance determinants. We characterized several novel gene products, including putative enzymes that can confer high-level resistance against aminoglycosides, sulfonamides, and broad range of beta-lactams, with respect to their resistance mechanisms and clinical significance. By coupling high-resolution proteomics analysis with functional metagenomics, we discovered an unusual peptide, PPP AZI 4 , encoded within an alternative open reading frame not predicted by bioinformatics tools. Expression of the proline-rich PPP AZI 4 can promote resistance against different macrolides but not other ribosome-targeting antibiotics, implicating a new macrolide-specific resistance mechanism that could be fundamentally linked to the evolutionary design of this peptide. IMPORTANCE Antibiotic resistance is a clinical phenomenon with an evolutionary link to the microbial pangenome. Genes and protogenes encoding specialized and potential resistance mechanisms are abundant in natural environments, but understanding of their identity and genomic context remains limited. Our discovery of several previously unknown antibiotic resistance genes from uncultured soil microorganisms indicates that soil is a significant reservoir of resistance determinants, which, once acquired and “repurposed” by pathogenic bacteria, can have serious impacts on therapeutic outcomes. This study provides valuable insights into the diversity and identity of resistance within the soil microbiome. The finding of a novel peptide-mediated resistance mechanism involving an unpredicted gene product also highlights the usefulness of integrating proteomics analysis into metagenomics-driven gene discovery.

Screening of a transposon insertion mutant library of Pseudomonas aeruginosa for increased susceptibility to paromomycin identified a number of genes whose disruption enhanced susceptibility of this organism to multiple aminoglycosides, including tobramycin, amikacin, and gentamicin. These included genes associated with lipid biosynthesis or metabolism ( lptA , faoA ), phosphate uptake ( pstB ), and two-component regulators ( amgRS , PA2797-PA2798) and a gene of unknown function (PA0392). Deletion mutants lacking these showed enhanced panaminoglycoside susceptibility that was reversed by the cloned genes, confirming their contribution to intrinsic panaminoglycoside resistance. None of these mutants showed increased aminoglycoside permeation of the cell envelope, indicating that increased susceptibility was not related to enhanced aminoglycoside uptake owing to a reduced envelope barrier function. Several mutants ( pstB , faoA , PA0392, amgR ) did, however, show increased cytoplasmic membrane depolarization relative to wild type following gentamicin exposure, consistent with the membranes of these mutants being more prone to perturbation, likely by gentamicin-generated mistranslated polypeptides. Mutants lacking any two of these resistance genes in various combinations invariably showed increased aminoglycoside susceptibility relative to single-deletion mutants, confirming their independent contribution to resistance and highlighting the complexity of the intrinsic aminoglycoside resistome in P. aeruginosa . Deletion of these genes also compromised the high-level panaminoglycoside resistance of clinical isolates, emphasizing their important contribution to acquired resistance.

Pan-aminoglycoside-resistant Pseudomonas aeruginosa mutants expressing the mexXY components of the aminoglycoside-accommodating MexXY-OprM multidrug efflux system but lacking mutations in the mexZ gene encoding a repressor of this efflux system and in the mexXY promoter have been reported (S. Fraud and K. Poole, Antimicrob. Agents Chemother. 55:1068–1074, 2011). Genome sequencing of one of these mutants, K2966, revealed the presence of a mutation within the predicted promoter region of the rplU-rpmA operon encoding ribosomal proteins L21 and L27, consistent with an observed 2-fold decrease in expression of this operon in the mutant relative to wild-type P. aeruginosa PAO1. Moreover, correction of the mutation restored rplU-rpmA expression and, significantly, reversed the elevated mexXY expression and pan-aminoglycoside resistance of the mutant. Reduced rplU-rpmA expression was also observed in a second mexXY -expressing pan-aminoglycoside-resistant mutant, K2968, which, however, lacked a mutation in the rplU-rpmA promoter region. Restoration of rplU-rpmA expression in the K2968 mutant following chromosomal integration of the rplU-rpmA operon derived from wild-type P. aeruginosa failed, however, to reverse the elevated mexXY expression and pan-aminoglycoside resistance of this mutant, although it did so for K2966, suggesting that the mutation impacting rplU-rpmA expression in K2968 also impacts other mexXY -related genes. Increased mexXY expression owing to reduced rplU-rpmA expression in K2966 and K2968 was dependent on PA5471, whose expression was also elevated in these mutants. Thus, mutational disruption of ribosome function, by limiting expression of ribosomal constituents, promotes recruitment of mexXY and does so via PA5471, reminiscent of mexXY induction by ribosome-disrupting antimicrobial agents. Interestingly, reduced rplU-rpmA expression was also observed in a mexXY -expressing pan-aminoglycoside-resistant clinical isolate, suggesting that ribosome-perturbing mutations have clinical relevance in the recruitment of the MexXY-OprM aminoglycoside resistance determinant.