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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.

In many jurisdictions sludge recovered from the sewage treatment process is a valued fertilizer for crop production. Pre-treatment of sewage sludge prior to land application offers the potential to abate enteric microorganisms that carry genes conferring resistance to antibiotics. Pre-treatment practices that accomplish this should have the desirable effect of reducing the risk of contamination of crops or adjacent water with antibiotic resistance genes carried in these materials. In the present study, we obtained municipal sludge that had been subjected to one of five treatments. There were, anaerobic-digestion or aerobic-digestion, in both instances with and without dewatering; and heat-treatment and pelletization. Each of the five types of biosolids was applied to an agricultural field at commercial rates, following which lettuce, carrots and radishes were planted. Based on qPCR, the estimated antibiotic gene loading rates were comparable with each of the five biosolids. However, the gene abundance in soil following application of the pelletized biosolids was anomalously lower than expected. Following application, the abundance of antibiotic resistance genes decreased in a generally coherent fashion, except sul1 which increased in abundance during the growing season in the soil fertilized with pelletized biosolids. Based on qPCR and high throughput sequencing evidence for transfer of antibiotic resistance genes from the biosolids to the vegetables at harvest was weak. Clostridia were more abundant in soils receiving any of the biosolids except the pelletized. Overall, the behavior of antibiotic resistance genes in soils receiving aerobically or anaerobically-digested biosolids was consistent and coherent with previous studies. However, dynamics of antibiotic resistance genes in soils receiving the heat treated pelletized biosolids were very different, and the underlying mechanisms merit investigation.

A previous soil metagenomics study recovered a novel cephalosporin resistance determinant, pbpTET A6, for which the exact resistance mechanism was unclear. This study used a three-dimensional structure-guided mutagenesis approach to demonstrate that PBPTET A6 is likely to be a class A penicillin-binding protein (PBP), and that its ability to confer cephalosporin resistance is directly linked to the functional integrity of its transpeptidase (TP) catalytic core. Screening of a library of PBPTET A6 variants carrying randomly introduced point mutations revealed additional residue modifications that compromised resistance, all of which were proximal to the TP active site except one which was found in a 29-amino-acid-long superstructure (α6-α7 loop) absent in other class A PBP homologues. Based on the site-specific mutagenesis results, it is hypothesized that residue arginine-400 plays an important role in limiting the access of certain cephalosporin compounds to the enzymatic core of the TP domain of PBPTET A6. Using a combination of adaptive evolution assays and whole-genome sequencing, the potential impact of PBPTET A6 on promoting the development of resistance in the clinically significant opportunistic pathogen Pseudomonas aeruginosa was investigated. Under the selective pressure of serial ceftazidime exposures, the pbpTET A6-expressing P. aeruginosa population readily evolved by excluding a ~400-kbp chromosomal element to acquire additional resistance against cephalosporins, suggesting that PBPTET A6 has a catalytic effect on facilitating antibiotic-resistance-associated genome adaptation. Overall, the soil environment contains genes conferring resistance to critically important antibiotics by cryptic mechanisms. Understanding what impact anthropogenic activities might have on the abundance and evolution of these genes should be a priority.

We examined the ability of composting to remove ARGs and enteric bacteria in litter obtained from broiler chickens fed with a diet supplemented with Bacitracin methylene disalicylate (BDM) (conventional chicken litter), or an antibiotic-free diet (raised without antibiotic (RWA) chicken litter). This was done by evaluating the litter before and after composting for the abundance of ten gene targets associated with antibiotic resistance or horizontal gene transfer, the composition of the bacterial communities, and the abundance of viable enteric bacteria. The abundance of gene targets was determined by qPCR and the microbial community composition of chicken litter determined by 16S rRNA gene amplicon sequencing. Enteric bacteria were enumerated by viable plate count. A majority of the gene targets were more abundant in conventional than in RWA litter. In both litter types, the absolute abundance of all of the target genes decreased after composting except sul1, intI1, incW and erm(F) that remained stable. Composting significantly reduced the abundance of enteric bacteria, including those carrying antibiotic resistance. The major difference in bacterial community composition between conventional and RWA litter was due to members affiliated to the genus Pseudomonas, which were 28% more abundant in conventional than in RWA litter. Composting favoured the presence of thermophilic bacteria, such as those affiliated with the genus Truepera, but decreased the abundance of those bacterial genera associated with cold-adapted species, such as Carnobacterium, Psychrobacter and Oceanisphaera. The present study shows that chicken litter from broilers fed with a diet supplemented with antibiotic has an increased abundance of some ARGs, even after composting. However, we can conclude that fertilization with composted litter represents a reduced risk of transmission of antibiotic resistance genes and enteric bacteria of poultry origin to soil and crops than will fertilization with raw litter.

Exposure of environmental bacteria to antibiotics may be increasing the global resistome. Antibiotic residues are entrained into agricultural soil through the application of animal and human wastes, and irrigation with reclaimed water. The impact of a mixture of three macrolide antibiotics on the abundance of selected genes associated with antibiotic resistance and genetic mobility were determined in a long-term field experiment undertaken in London, Canada. Replicated plots received annual applications of a mixture of erythromycin, clarithromycin and azithromycin every spring since 2010. Each antibiotic was added directly to the soil at a concentration of either 0.1 or 10 mg kg soil−1 and all plots were cropped to soybeans. By means of qPCR, no gene targets were enriched in soil exposed to the 0.1 mg kg soil−1 dose compared to untreated control. In contrast, the relative abundance of several gene targets including int1, sul2 and mphE increased significantly with the annual exposure to the 10 mg kg soil−1 dose. By means of high-throughput qPCR, numerous gene targets associated with resistance to aminoglycosides, sulfonamides, trimethoprim, streptomycin, quaternary ammonium chemicals as well as mobile genetic elements (tnpA, IS26 and IS6100) were detected in soil exposed to 10 mg kg soil−1, but not the lower dose. Overall, exposure of soil to macrolide antibiotics increased the relative abundance of numerous gene targets associated with resistance to macrolides and other antibiotics, and mobile genetic elements. This occurred at an exposure dose that is unrealistically high, but did not occur at the lower more realistic exposure dose.

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.

A screen for agents that potentiated the activity of paromomycin (PAR), a 4,5-linked aminoglycoside (AG), against wild-type Pseudomonas aeruginosa identified the RNA polymerase inhibitor rifampin (RIF). RIF potentiated additional 4,5-linked AGs, such as neomycin and ribostamycin, but not the clinically important 4,6-linked AGs amikacin and gentamicin. Potentiation was absent in a mutant lacking the AmgRS envelope stress response two-component system (TCS), which protects the organism from AG-generated membrane-damaging aberrant polypeptides and, thus, promotes AG resistance, an indication that RIF was acting via this TCS in potentiating 4,5-linked AG activity. Potentiation was also absent in a RIF-resistant RNA polymerase mutant, consistent with its potentiation of AG activity being dependent on RNA polymerase perturbation. PAR-inducible expression of the AmgRS-dependent genes htpX and yccA was reduced by RIF, suggesting that AG activation of this TCS was compromised by this agent. Still, RIF did not compromise the membrane-protective activity of AmgRS, an indication that it impacted some other function of this TCS. RIF potentiated the activities of 4,5-linked AGs against several AG-resistant clinical isolates, in two cases also potentiating the activity of the 4,6-linked AGs. These cases were, in one instance, explained by an observed AmgRS-dependent expression of the MexXY multidrug efflux system, which accommodates a range of AGs, with RIF targeting of AmgRS undermining mexXY expression and its promotion of resistance to 4,5- and 4,6-linked AGs. Given this link between AmgRS, MexXY expression, and pan-AG resistance in P. aeruginosa , RIF might be a useful adjuvant in the AG treatment of P. aeruginosa infections.

The ribosome-targeting antimicrobial, spectinomycin (SPC), strongly induced the mexXY genes of the MexXY-OprM multidrug efflux system in Pseudomonas aeruginosa and increased susceptibility to the polycationic antimicrobials polymyxin B and polymyxin E, concomitant with a decrease in expression of the polymyxin resistance-promoting lipopolysaccharide (LPS) modification loci, arnBCADTEF and PA4773-74. Consistent with the SPC-promoted reduction in arn and PA4773-74 expression being linked to mexXY , expression of these LPS modification loci was moderated in a mutant constitutively expressing mexXY and enhanced in a mutant lacking the efflux genes. Still, the SPC-mediated increase in polymyxin susceptibility was retained in mutants lacking arnB and/or PA4773-74, an indication that their reduced expression in SPC-treated cells does not explain the enhanced polymyxin susceptibility. That the polymyxin susceptibility of a mutant strain lacking mexXY was unaffected by SPC exposure, however, was an indication that the unknown polymyxin resistance ‘mechanism’ is also influenced by the MexXY status of the cell. In agreement with SPC and MexXY influencing polymyxin susceptibility as a result of changes in the LPS target of these agents, SPC treatment yielded a decline in common polysaccharide antigen (CPA) synthesis in wild-type P. aeruginosa but not in the Δ mexXY mutant. A mutant lacking CPA still showed the SPC-mediated decline in polymyxin MICs, however, indicating that the loss of CPA did not explain the SPC-mediated MexXY-dependent increase in polymyxin susceptibility. It is possible, therefore, that some additional change in LPS promoted by SPC-induced mexXY expression impacted CPA synthesis or its incorporation into LPS and that this was responsible for the observed changes in polymyxin susceptibility.