Antibiotic Resistance Gene Content Research Articles

Co-localization of antibiotic resistance genes is widespread in the infant gut microbiome and associates with an immature gut microbial composition

BackgroundIn environmental bacteria, the selective advantage of antibiotic resistance genes (ARGs) can be increased through co-localization with genes such as other ARGs, biocide resistance genes, metal resistance genes, and virulence genes (VGs). The gut microbiome of infants has been shown to contain numerous ARGs, however, co-localization related to ARGs is unknown during early life despite frequent exposures to biocides and metals from an early age.ResultsWe conducted a comprehensive analysis of genetic co-localization of resistance genes in a cohort of 662 Danish children and examined the association between such co-localization and environmental factors as well as gut microbial maturation. Our study showed that co-localization of ARGs with other resistance and virulence genes is common in the early gut microbiome and is associated with gut bacteria that are indicative of low maturity. Statistical models showed that co-localization occurred mainly in the phylum Proteobacteria independent of high ARG content and contig length. We evaluated the stochasticity of co-localization occurrence using enrichment scores. The most common forms of co-localization involved tetracycline and fluoroquinolone resistance genes, and, on plasmids, co-localization predominantly occurred in the form of class 1 integrons. Antibiotic use caused a short-term increase in mobile ARGs, while non-mobile ARGs showed no significant change. Finally, we found that a high abundance of VGs was associated with low gut microbial maturity and that VGs showed even higher potential for mobility than ARGs.ConclusionsWe found that the phenomenon of co-localization between ARGs and other resistance and VGs was prevalent in the gut at the beginning of life. It reveals the diversity that sustains antibiotic resistance and therefore indirectly emphasizes the need to apply caution in the use of antimicrobial agents in clinical practice, animal husbandry, and daily life to mitigate the escalation of resistance.CR8ihftTez1HgYaufYhgHyVideo

A novel strategy for eliminating antibiotic resistance genes during fertilization of dewatered sludge by earthworms: Vermicomposting practice using Chinese herbal residues derived from Lianhua Qingwen as a bulking material

Vermicomposting is a sustainable sludge recycling technology that utilizes an eco-friendly composting using earthworms and microorganisms. However, a high abundance of antibiotic resistance genes (ARGs) remains in dewatered sludge that is not satisfactorily eliminated by vermicomposting. Chinese herbs have played a major role in curing many diseases in East Asia, leading to a large amount of Chinese herbal residues (CHRs) are difficult to dispose of. The present study investigated the feasibility of CHRs on the ARGs reduction in dewatered sludge during vermicomposting. The CHRs derived from Lianhua Qingwen were added separately to sludge with weight ratios of 0%, 10%, 30%, and 50%; sludge was then vermicomposted for 30 days. The results showed that co-vermicomposting of sludge and CHRs is a feasible strategy. The CHR treatments significantly (P < 0.05) decreased antibiotic concentration and bacterial population by 23.64%–49.68% and 42.58%–93.07%, respectively, compared to counterpart. Compared to the control, the CHR addition lowered the absolute abundances of macrolide, tetracycline, and sulfonamide ARGs by 42.69%–85.15%, 22.03%–75.24%, and 23.59%–90.66%, respectively. In addition, sludge containing 30% CHRs showed significant (P < 0.05) elimination of intⅠ-1 and tnpA-4 genes with abundance reductions of 71.40% and 52.33%, respectively, relative to the control. This study suggests that the CHRs can effectively reduce ARGs content in sludge by decreasing the bacterial population and horizontal gene transfer capacity during vermicomposting.

Industrial-scale aerobic composting of livestock manures with the addition of biochar: Variation of bacterial community and antibiotic resistance genes caused by various composting stages

The presence of large amounts of antibiotic resistance genes (ARGs) in livestock manures poses an impending, tough safety risk to ecosystems. To investigate more comprehensively the mechanisms of ARGs removal from industrial-scale composting of livestock manure based on biochar addition, we tracked the dynamics of bacterial community and ARGs at various stages of aerobic composting of livestock manures with 10% biochar. There were no significant effects of biochar on the bacterial community and the profiles of ARGs. During aerobic composting, the relative abundance of ARGs and mobile genetic elements (MGEs) showed overall trends of decreasing and then increasing. The key factor driving the dynamics of ARGs was bacterial community composition, and the potential hosts of ARGs were Caldicoprobacter, Tepidimicrobium, Ignatzschineria, Pseudogracilibacillus, Actinomadura, Flavobacterium and Planifilum. The retention of the thermophilic bacteria and the repopulation of the initial bacteria were the dominant reasons for the increase in ARGs at maturation stage. Additionally, among the MGEs, the relative abundance of transposon gene was substantially removed, while the integron genes remained at high relative abundance. Our results highlighted that the suitability of biochar addition to industrial-scale aerobic composting needs to be further explored and that effective measures are needed to prevent the increase of ARGs content on maturation stage.

The Sources and Potential Hosts Identification of Antibiotic Resistance Genes in the Yellow River, Revealed by Metagenomic Analysis.

The fate of antibiotic resistance genes (ARGs) has been revealed in various environmental media in recent years. Namely, the emergence of genes that resist colistin and carbapenems has attracted wide attention. However, the pollution condition of ARGs and sources in the Yellow River is still little understood, despite the river being the second longest in China. The present study determined the levels of ARG pollution in the Henan section of the Yellow River and evaluated the role of the aquaculture industry in the spread of ARGs. As revealed by the results, a total of 9 types of ARGs were detected in the sediments of the Yellow River, and the total ARG content in the Yellow River ranges from 7.27 to 245.45 RPKM. Sul1 and sul2 are the dominant ARGs, and the huge usage of sulfonamides, horizontal gene transfer, and wide bacteria host contribute to the prevalence of these two genes. The results of Spearman correlation analysis indicate that the breeding industry has little influence on ARGs in the Yellow River. Network analysis reveals that the opportunistic pathogen Pseudomonas is the potential host of sul1, tetG, and ANT(3′′)-IIa, which can pose a risk to human health.

Prevalence and Molecular Characterization of Extended-Spectrum β-Lactamase Producing Enterobacterales in Healthy Community Dogs in Israel.

Background: antimicrobial resistance is a global problem in human and veterinary medicine. We aimed to investigate the extended spectrum β-lactamase-producing Enterobacterales (ESBL-PE) gut colonization in healthy community dogs in Israel. Methods: Rectal swabs were sampled from 145 healthy dogs, enriched, plated on selective plates, sub-cultured to obtain pure cultures, and ESBL production was confirmed. Bacterial species and antibiotic susceptibility profiles were identified. WGS was performed on all of the ESBL-PE isolates and their resistomes were identified in silico. Owners’ questionnaires were collected for risk factor analysis. Results: ESBL-PE gut colonization rate was 6.2% (n = 9/145, 95% CI 2.9–11.5). Overall, ten isolates were detected (one dog had two isolates); the main species was Escherichia coli (eight isolates), belonging to diverse phylogenetic groups—B1, A and C. Two isolates were identified as Citrobacter braakii, and C. portucalensis. A phylogenetic analysis indicated that all of the isolates were genetically unrelated and sporadic. The isolates possessed diverse ESBL genes and antibiotic-resistance gene content, suggesting independent ESBL spread. In a multivariable risk factor analysis, coprophagia was identified as a risk factor for ESBL-PE gut colonization (p = 0.048, aOR = 4.408, 95% CI 1.014–19.169). Conclusions: healthy community dogs may be colonized with ESBL-PE MDR strains, some of which were previously reported in humans, that carry wide and diverse resistomes and may serve as a possible source for AMR.

Metagenomics Reveals the Diversity and Taxonomy of Carbohydrate-Active Enzymes and Antibiotic Resistance Genes in Suancai Bacterial Communities.

Suancai, as a traditional fermented food in China with reputed health benefits, has piqued global attention for many years. In some circumstances, the microbial-driven fermentation may confer health (e.g., probiotics) or harm (e.g., antibiotic resistance genes) to the consumers. To better utilize beneficial traits, a deeper comprehension of the composition and functionality of the bacterial species harboring enzymes of catalytically active is required. On the other hand, ingestion of fermented food increases the likelihood of microbial antibiotic resistance genes (ARGs) spreading in the human gastrointestinal tract. Besides, the diversity and taxonomic origin of ARGs in suancai are little known. In our study, a metagenomic approach was employed to investigate distribution structures of CAZymes and ARGs in main bacterial species in suancai. Functional annotation using the CAZy database identified a total of 8796 CAZymes in metagenomic data. A total of 83 ARGs were detected against the CARD database. The most predominant ARG category is multidrug-resistant genes. The ARGs of antibiotic efflux mechanism are mostly in Proteobacteria. The resistance mechanism of ARGs in Firmicutes is primarily antibiotic inactivation, followed by antibiotic efflux. Due to the abundance of species with different ARGs, strict quality control including microbial species, particularly those with lots of ARGs, is vital for decreasing the risk of ARG absorption via consumption. Ultimately, we significantly widen the understanding of suancai microbiomes by using metagenomic sequencing to offer comprehensive information on the microbial functional potential (including CAZymes and ARGs content) of household suancai.

Genetic Diversity of Antimicrobial Resistance and Key Virulence Features in Two Extensively Drug-Resistant Acinetobacter baumannii Isolates.

In recent decades, Acinetobacter baumannii emerged as a major infective menace in healthcare settings due to scarce therapeutic options to treat infections. Therefore, undertaking genome comparison analyses of multi-resistant A. baumannii strains could aid the identification of key bacterial determinants to develop innovative anti-virulence approaches. Following genome sequencing, we performed a molecular characterization of key genes and genomic comparison of two A. baumannii strains, #36 and #150, with selected reference genomes. Despite a different antibiotic resistance gene content, the analyzed strains showed a very similar antibiogram profile. Interestingly, the lack of some important virulence determinants (i.e., bap, ata and omp33–36) did not abrogate their adhesive abilities to abiotic and biotic surfaces, as reported before; indeed, strains retained these capacities, although to a different extent, suggesting the presence of distinct vicarious genes. Conversely, secretion systems, lipopolysaccharide (LPS), capsule and iron acquisition systems were highly similar to A. baumannii reference strains. Overall, our analyses increased our knowledge on A. baumannii genomic content and organization as well as the genomic events occurring in nosocomial isolates to better fit into changing healthcare environments.

Genomic Analysis of Global Staphylococcus argenteus Strains Reveals Distinct Lineages With Differing Virulence and Antibiotic Resistance Gene Content.

Infections due to Staphylococcus argenteus have been increasingly reported worldwide and the microbe cannot be distinguished from Staphylococcus aureus by standard methods. Its complement of virulence determinants and antibiotic resistance genes remain unclear, and how far these are distinct from those produced by S. aureus remains undetermined. In order to address these uncertainties, we have collected 132 publicly available sequences from fourteen different countries, including the United Kingdom, between 2005 and 2018 to study the global genetic structure of the population. We have compared the genomes for antibiotic resistance genes, virulence determinants and mobile genetic elements such as phages, pathogenicity islands and presence of plasmid groups between different clades. 20% (n = 26) isolates were methicillin resistant harboring a mecA gene and 88% were penicillin resistant, harboring the blaZ gene. ST2250 was identified as the most frequent strain, but ST1223, which was the second largest group, contained a marginally larger number of virulence genes compared to the other STs. Novel S. argenteus pathogenicity islands were identified in our isolates harboring tsst-1, seb, sec3, ear, selk, selq toxin genes, as well as chromosomal clusters of enterotoxin and superantigen-like genes. Strain-specific type I modification systems were widespread which would limit interstrain transfer of genetic material. In addition, ST2250 possessed a CRISPR/Cas system, lacking in most other STs. S. argenteus possesses important genetic differences from S. aureus, as well as between different STs, with the potential to produce distinct clinical manifestations.

Dynamics of integron structures across a wastewater network – Implications to resistance gene transfer

Class 1 and other integrons are common in wastewater networks, often being associated with antibiotic resistance genes (ARGs). However, the importance of different integron structures in ARG transfer within wastewater systems has only been implied, especially between community and hospital sources, among wastewater treatment plant compartments, and in receiving waters. This uncertainty is partly because current clinical class 1 integron qPCR assays (i.e., that target human-impacted structures, i.e., clintI1) poorly delineate clintI1 from non-impacted class 1 integron structures. They also say nothing about their ARG content. To fill these technical gaps, new real-time qPCR assays were developed for “impacted” class 1 structures (called aint1; i.e., anthropogenic class 1 integrons) and empty aint1 structures (i.e., carry no ARGs; called eaint1). The new assays and other integron assays then were used to examine integron dynamics across a wastewater network. 16S metagenomic sequencing also was performed to characterise associated microbiomes. aint1 abundances per bacterial cell were about 10 times greater in hospital wastewaters compared with other compartments, suggesting aint1 enrichment with ARGs in hospital sources. Conversely, the relative abundance of eaint1 structures were over double in recycled activated sludge compared with other compartments, except receiving waters (RAS; ∼30% of RAS class 1 structures did not carry ARGs). Microbiome analysis showed that human-associated bacterial taxa with mobile integrons also differed in RAS and river sediments. Further, class 1 integrons in RAS bacteria appear to have released ARGs, whereas hospital bacteria have accumulated ARGs. Results show that quantifying integron dynamics can help explain where ARG transfer occurs in wastewater networks, and should be considered in future studies on antibiotic resistance in the environment.

Effects of antibiotic duration on the intestinal microbiota and resistome: The PIRATE RESISTANCE project, a cohort study nested within a randomized trial.

BackgroundShortening antibiotic-treatment durations is a key recommendation of antibiotic-stewardship programmes, yet it is based on weak evidence. We investigated whether halving antibiotic courses would reduce antibiotic-resistance genes (ARG) in the intestinal microbiomes of patients treated for gram-negative bacteraemia.MethodsThis nested prospective cohort study included adult patients hospitalized at Geneva University Hospitals (Switzerland) participating in the PIRATE randomized trial assessing non-inferiority of shorter antibiotic courses (7 versus 14 days) for gram-negative bacteraemia (‘cases’) and, simultaneously, hospitalized patients with similar demography and comorbidity yet no antibiotic therapy (‘controls’). Stool was collected from case and control patients on days 7, 14, 30 and 90 after antibiotic initiation (day 1) and days 7 and 14 after admission, respectively, and analysed by whole-metagenome shotgun sequencing. The primary outcome was ARG abundance at day 30; secondary outcomes included microbiota-species composition and clustering over time.FindingsForty-five patients and 11 controls were included and evaluable; ARG analyses were conducted on the 29 per-protocol patients receiving 7 (±2) days or 14 (±3) days of antibiotic therapy. At day 30, ARGs were not detected at similar abundance in patients receiving 7 and 14 days (median counts/million [mCPM]: 96 versus [vs] 71; p=.38). By day 30, total ARG content between both groups was not significantly different from that of controls at D7 (362 and 370 mCPM vs 314 mCPM, p=.24 and 0.19). There were no significant differences amongst antibiotic-treated patients at any timepoint in bacterial diversity or clustering, but Shannon species diversity was significantly reduced compared to controls through day 14 (median 3.12 and 3.24 in the 7-day and 14-day groups vs 3.61 [controls]; p=.04 and 0.012). Patients treated for 14 days had reduced faecal phage content during and after therapy compared to other patient groups.InterpretationReducing antibiotic durations by half did not result in decreased abundance of ARGs in patients treated for gram-negative bacteraemia, nor did it improve microbiota species diversity.FundingThe study was funded by the University of Geneva's Louis-Jeantet Foundation (grant no. S04_12) and the Swiss National Science Foundation (NRP Smarter Healthcare, grant no. 407,440_167359).

Wastewater treatment plants as a reservoir of integrase and antibiotic resistance genes - An epidemiological threat to workers and environment.

Conventional mechanical and biological wastewater treatment is unable to completely eliminate all pollutants, which can therefore enter surface water bodies together with treated wastewater. In addition, bioaerosols produced during wastewater treatment can pose a threat to the health of the wastewater treatment plant staff. In order to control the impact of a wastewater treatment plant (WWTP) on the surrounding environment, including its employees, samples of wastewater and water from a river which received treated wastewater were analysed in terms of their content of antibiotics and heavy metals, levels of selected physiochemical parameters, concentrations of antibiotic-resistance genes (ARGs) and genes of integrases. Furthermore, a quantitative analysis of ARGs in the metagenomic DNA from nasal and throat swabs collected from the WWPT employees was made. Both untreated and treated wastewater samples were dominated by genes of resistance to sulphonamides (sul1, sul2), MLS group of drugs (ermF, ermB) and beta-lactams (blaOXA). A significant increase in the quantities of ARGs and concentrations of antibiotics was observed in the river following the discharge of treated wastewater in comparison to their amounts in the river water upstream from the point of discharge. Moreover, a higher concentration of ARGs was detected in the DNA from swabs obtained from the wastewater treatment plant employees than from ones collected from the control group. Many statistically significant (p<0.05) correlations between the concentration of the gene of resistance to heavy metals cnrA versus ARGs, and between the ARGs content and the concentrations of heavy metals in both wastewater and river water samples were observed. The study has demonstrated that the mechanical and biological methods of wastewater treatment are not efficient and may affect the transmission of hazardous pollutants to the aquatic environment and to the atmospheric air. It has been shown that an activated sludge bioreactor can be a potential source of the presence of multi-drug resistant microorganisms in the air, which is a health risk to persons working in WWTPs. It has also been found that an environment polluted with heavy metals is where co-selection of antibiotic resistance may occur, in the development of which integrase genes play an essential role.

Significant effect of pH on tetracycline resistance genes reduction during sludge thermal hydrolysis treatment

Thermal hydrolysis (TH) treatment has been verified to effectively reduce antibiotic resistance genes (ARGs) in waste activated sludge. This study focused on the effect of TH parameters on ARGs reduction, and the optimal conditions basing on tetracycline (tet) resistance genes reduction rate in sludge phase were pH 3, temperature 160 °C and reaction time 2 h. The pH was found to play a critical role in tet genes reduction behavior. Additionally, the tet genes distributions in TH supernatant are considered as well because genes will release from solid to liquid phase as sludge cell breaking. The lowest genes content in TH supernatant was also observed at pH 3. Further investigation revealed that contents of DNA and organics released from sludge phase to liquid phase under pH 3 were less than that in neutral and alkaline condition; moreover, the organics in TH supernatant was found to protect DNA. Therefore, under the acidic condition, the genes had less releasing from the sludge phase and the DNA protection effect of organics was weaker, resulting in the lowest tet genes content in TH supernatant. Moreover, bacterial community structure after TH was closely related with the ARGs content. The bacterial on phylum and genus level showed various responses to TH pH values. Although the Firmicutes phylum and Escherichia-Shigella genus exhibited the stronger resistance and had higher accumulation in TH acidic condition, more possible total tet genes hosts were destroyed in acidic condition, causing less ARGs remaining in the sludge phase under pH 3.

Effects of Antibiotic Duration on the Intestinal Microbiota and Resistome: The PIRATE RESISTANCE Project, a Cohort Study Nested Within a Randomized Trial

Background: Shortening antibiotic-treatment durations is a key recommendation of antibiotic-stewardship programmes, yet it is based on weak evidence. We investigated whether halving antibiotic courses would reduce antibiotic-resistance genes (ARG) in the intestinal microbiomes of patients treated for gram-negative bacteraemia. Methods: This nested prospective cohort study included adult patients hospitalized at Geneva University Hospitals (Switzerland) participating in the PIRATE randomized trial assessing non-inferiority of shorter antibiotic courses for gram-negative bacteraemia (‘cases’) and, simultaneously, hospitalized patients with similar demography and comorbidity yet no antibiotic therapy (‘controls’). Stool was collected from case and control patients on days 7, 14, 30 and 90 after antibiotic initiation (day 1) and days 7 and 14 after admission, respectively, and analysed by whole-metagenome shotgun sequencing. The primary outcome was ARG abundance at day 30; secondary outcomes included microbiota-species composition and clustering over time. Findings: Forty-five patients and 11 controls were included and evaluable; ARG analyses were conducted on the 29 per-protocol patients receiving 7 (±2) days or 14 (±3) days of antibiotic therapy. At day 30, ARGs were detected at similar abundance in patients receiving 7 and 14 days (median counts/million [mCPM]: 96 versus [vs] 71; p=.38). By D30, total ARG content in both groups was similar to that of controls at D7 (362 and 370 mCPM vs 314 mCPM, p=.24 and .19). There were no significant differences among antibiotic-treated patients at any timepoint in bacterial diversity or clustering, but Shannon species diversity was significantly reduced compared to controls through D14 (median 3.12 and 3.24 in the 7-day and 14-day groups vs 3.61 [controls]; p=.04 and .012). Fourteen-day antibiotic courses prompted a persistent trend toward reduced faecal phage content. Interpretation: Reducing antibiotic durations by half did not result in decreased abundance of ARGs in patients treated for gram-negative bacteraemia, nor did it improve microbiota species diversity. Funding: The study was funded by the University of Geneva’s Louis-Jeantet Foundation (grant no. S04_12) and the Swiss National Science Foundation (NRP Smarter Healthcare, grant no. 407440_167359). Declaration of Interests: All authors declare no competing interests. Ethics Approval Statement: The present study was approved by the Cantonal Ethics Committee of Geneva (no. 2017-00108); all participants provided written informed consent before enrolment.

Cross-border emergence of clonal lineages of ST38 Escherichia coli producing the OXA-48-like carbapenemase OXA-244 in Germany and Switzerland

BackgroundCarbapenemase-producing Gram-negative bacteria cause infections that are difficult to treat and represent a rising threat to healthcare systems worldwide. This study analysed isolates of Escherichia coli (E. coli), a species associated with nosocomial-acquired and community-acquired infections, from hospitals in Germany and Switzerland exhibiting a slight decrease in susceptibility to carbapenems. MethodsE. coli strains from Germany and Switzerland, obtained mainly in 2019, were first screened for carbapenemase genes by PCR and subsequently whole-genome-sequenced and analysed for their clonal relationship using multilocus sequence typing, single nucleotide polymorphisms, virulence and antibiotic-resistance gene content. ResultsThe analysis revealed the presence of extended β-lactamase (ESBL)-producing E. coli clones producing OXA-244, a point-mutation derivative of OXA-48, with a predominance of isolates exhibiting the sequence type (ST) ST38 in both Germany and Switzerland. These clustered exclusively into two distinct lineages: one encoding CTX-M-27, a recently emerged extended-spectrum β-lactamase, and the other CTX-M-14b. All OXA244/CTX-M-27 ST38 isolates harboured the Dr adhesin operon and a representative isolate exhibited a diffuse adherence (DAEC) phenotype and was invasive for Hela cells. ConclusionClonal lineages of ST38 are members of E. coli phylogenetic group D commonly associated with extra-intestinal infections. Their increased isolation in two different European countries indicates ongoing spread of ST38 ESBL-producing and OXA-244-producing E. coli clonal lineages. It is possible that members of the multidrug-resistant DEAC ExPEC group have expanded globally, but that this is currently underreported because of the inherent difficulty in detecting isolates expressing the OXA-244 allele.

Antibiotic resistance gene reservoir in live poultry markets

The heavy use of antibiotics in farm animals contributes to the enrichment and spread of antibiotic resistance genes (ARGs) in "one-health" settings. Numerous ARGs have been identified in livestock-associated environments but not in Chinese live poultry markets (LPMs). We collected 753 poultry fecal samples from LPMs of 18 provinces and municipalities in China and sequenced the metagenomes of 130 samples. Bioinformatic tools were used to construct the gene catalog and analyze the ARG content. PCR amplification and Sanger sequencing were used to survey the distribution of mcr-1 gene in all 753 fecal samples. We found that a low number of genes but a high percentage of gene functions were shared among the poultry, human and pig gut gene catalogs. The poultry gut possessed 539 ARGs which were classified into 235 types. Both the ARG number and abundance were significantly higher in poultry than that in either pigs or humans. Fourteen ARG types were found present in all 130 samples, and tetracycline resistance (TcR) genes were the most abundant ARGs in both animals and humans. Moreover, 59.63% LPM samples harbored the colistin resistance gene mcr-1, and other mcr gene variants were also found. We demonstrated that the Chinese LPMs is a repository for ARGs, posing a high risk for ARG dissemination from food animals to humans under such a trade system, which has not been addressed before.

Effect of biochar amendment on the alleviation of antibiotic resistance in soil and phyllosphere of Brassica chinensis L.

The increasing prevalence of antibiotic resistance is a global threat to public health. Antibiotic resistance genes (ARGs) in soil have been demonstrated to be able to enter food chain. Strategies to mitigate the spread of antibiotic resistance from soil to crops are of great importance for food safety and human health. Soil amendment with biochar is a widely used approach to improve soil fertility. However, the impact of biochar on mitigating antibiotic resistance of soil and organically produced vegetables that are eaten raw is largely unknown. To gain insights into this impact, pot experiments planting Brassica chinensis L. in soil amended with biochar were conducted and antibiotic resistance genes (ARGs) were characterized by HT-qPCR targeting almost all major classes of ARGs and 10 mobile genetic elements (MGEs) marker genes. A total of 131 ARGs and 9 MGEs in soil and phyllosphere samples were identified. After biochar amendment, the abundance of ARGs was significantly decreased in non-planted soil. By comparison, no significant decrease of ARGs was found in rhizosphere and phyllosphere, indicating that vegetable planting affected antibiotic resistome in biochar-amended soil. To understand this effect, bacterial phylogeny structures within soil and phyllosphere were analyzed and found to correlate with their respective resistome, indicating that planting can influence the effect of biochar on soil antibiotic resistome by altering microbial community compositions. Structure equation models further revealed that a higher bacterial diversity corresponded to a decreased ARGs content. These results suggested that biochar amendment alone was not sufficient enough to alleviate ARGs level in planted soil and crops, instead, maintaining or increasing soil microbial diversity is potentially more useful in mitigating ARG spread and accumulation.

Influence of Soil Characteristics and Proximity to Antarctic Research Stations on Abundance of Antibiotic Resistance Genes in Soils.

Soil is an important environmental reservoir of antibiotic resistance genes (ARGs), which are increasingly recognized as environmental contaminants. Methods to assess the risks associated with the acquisition or transfer of resistance mechanisms are still underdeveloped. Quantification of background levels of antibiotic resistance genes and what alters those is a first step in understanding our environmental resistome. Toward this goal, 62 samples were collected over 3 years from soils near the 30-year old Gondwana Research Station and for 4 years before and during development of the new Jang Bogo Research Station, both at Terra Nova Bay in Antarctica. These sites reflect limited and more extensive human impact, respectively. A qPCR array with 384 primer sets targeting antibiotic resistance genes and mobile genetic elements (MGEs) was used to detect and quantify these genes. A total of 73 ARGs and MGEs encompassing eight major antibiotic resistance gene categories were detected, but most at very low levels. Antarctic soil appeared to be a common reservoir for seven ARGs since they were present in most samples (42%-88%). If the seven widespread genes were removed, there was a correlation between the relative abundance of MGEs and ARGs, more typical of contaminated sites. There was a relationship between ARG content and distance from both research stations, with a significant effect at the Jang Bogo Station especially when excluding the seven widespread genes; however, the relative abundance of ARGs did not increase over the 4 year period. Silt, clay, total organic carbon, and SiO2 were the top edaphic factors that correlated with ARG abundance. Overall, this study identifies that human activity and certain soil characteristics correlate with antibiotic resistance genes in these oligotrophic Antarctic soils and provides a baseline of ARGs and MGEs for future comparisons.

Survival of Antibiotic Resistant Bacteria and Horizontal Gene Transfer Control Antibiotic Resistance Gene Content in Anaerobic Digesters.

Understanding fate of antibiotic resistant bacteria (ARB) vs. their antibiotic resistance genes (ARGs) during wastewater sludge treatment is critical in order to reduce the spread of antibiotic resistance through process optimization. Here, we spiked high concentrations of tetracycline-resistant bacteria, isolated from mesophilic (Iso M1-1—a Pseudomonas sp.) and thermophilic (Iso T10—a Bacillus sp.) anaerobic digested sludge, into batch digesters and monitored their fate by plate counts and quantitative polymerase chain reaction (QPCR) of their corresponding tetracycline ARGs. In batch studies, spiked ARB plate counts returned to baseline (thermophilic) or 1-log above baseline (mesophilic) while levels of the ARG present in the spiked isolate [tet(G)] remained high in mesophilic batch reactors. To compare results under semi-continuous flow conditions with natural influent variation, tet(O), tet(W), and sul1 ARGs, along with the intI1 integrase gene, were monitored over a 9-month period in the raw feed sludge and effluent sludge of lab-scale thermophilic and mesophilic anaerobic digesters. sul1 and intI1 in mesophilic and thermophilic digesters correlated positively (Spearman rho = 0.457–0.829, P < 0.05) with the raw feed sludge. There was no correlation in tet(O) or tet(W) ratios in raw sludge and mesophilic digested sludge or thermophilic digested sludge (Spearman rho = 0.130–0.486, P = 0.075–0.612). However, in the thermophilic digester, the tet(O) and tet(W) ratios remained consistently low over the entire monitoring period. We conclude that the influent sludge microbial composition can influence the ARG content of a digester, apparently as a result of differential survival or death of ARBs or horizontal gene transfer of genes between raw sludge ARBs and the digester microbial community. Notably, mesophilic digestion was more susceptible to ARG intrusion than thermophilic digestion, which may be attributed to a higher rate of ARB survival and/or horizontal gene transfer between raw sludge bacteria and the digester microbial community.

Bacterial phylogeny structures soil resistomes across habitats.

SummaryAncient and diverse antibiotic resistance genes (ARGs) have previously been identified from soil1–3, including genes identical to those in human pathogens4. Despite the apparent overlap between soil and clinical resistomes4–6, factors influencing ARG composition in soil and their movement between genomes and habitats remain largely unknown3. General metagenome functions often correlate with the underlying structure of bacterial communities7–12. However, ARGs are hypothesized to be highly mobile4,5,13, prompting speculation that resistomes may not correlate with phylogenetic signatures or ecological divisions13,14. To investigate these relationships, we performed functional metagenomic selections for resistance to 18 antibiotics from 18 agricultural and grassland soils. The 2895 ARGs we discovered were predominantly novel, and represent all major resistance mechanisms15. We demonstrate that distinct soil types harbor distinct resistomes, and that nitrogen fertilizer amendments strongly influenced soil ARG content. Resistome composition also correlated with microbial phylogenetic and taxonomic structure, both across and within soil types. Consistent with this strong correlation, mobility elements syntenic with ARGs were rare in soil compared to sequenced pathogens, suggesting that ARGs in the soil may not transfer between bacteria as readily as is observed in the clinic. Together, our results indicate that bacterial community composition is the primary determinant of soil ARG content, challenging previous hypotheses that horizontal gene transfer effectively decouples resistomes from phylogeny13,14.