A sampling and metagenomic sequencing-based methodology for monitoring antimicrobial resistance in swine herds.

OBJECTIVES: Reliable methods for monitoring antimicrobial resistance (AMR) in livestock and other reservoirs are essential to understand the trends, transmission and importance of agricultural resistance. Quantification of AMR is mostly done using culture-based techniques, but metagenomic read mapping shows promise for quantitative resistance monitoring. METHODS: We evaluated the ability of: (i) MIC determination for Escherichia coli; (ii) cfu counting of E. coli; (iii) cfu counting of aerobic bacteria; and (iv) metagenomic shotgun sequencing to predict expected tetracycline resistance based on known antimicrobial consumption in 10 Danish integrated slaughter pig herds. In addition, we evaluated whether fresh or manure floor samples constitute suitable proxies for intestinal sampling, using cfu counting, qPCR and metagenomic shotgun sequencing. RESULTS: Metagenomic read-mapping outperformed cultivation-based techniques in terms of predicting expected tetracycline resistance based on antimicrobial consumption. Our metagenomic approach had sufficient resolution to detect antimicrobial-induced changes to individual resistance gene abundances. Pen floor manure samples were found to represent rectal samples well when analysed using metagenomics, as they contain the same DNA with the exception of a few contaminating taxa that proliferate in the extraintestinal environment. CONCLUSIONS: We present a workflow, from sampling to interpretation, showing how resistance monitoring can be carried out in swine herds using a metagenomic approach. We propose metagenomic sequencing should be part of routine livestock resistance monitoring programmes and potentially of integrated One Health monitoring in all reservoirs.

Abundance and diversity of the faecal resistome in slaughter pigs and broilers in nine European countries.

Antimicrobial resistance (AMR) in bacteria and associated human morbidity and mortality is increasing. The use of antimicrobials in livestock selects for AMR that can subsequently be transferred to humans. This flow of AMR between reservoirs demands surveillance in livestock and in humans. We quantified and characterized the acquired resistance gene pools (resistomes) of 181 pig and 178 poultry farms from nine European countries, sequencing more than 5,000 Gb of DNA using shotgun metagenomics. We quantified acquired AMR using the ResFinder database and a second database constructed for this study, consisting of AMR genes identified through screening environmental DNA. The pig and poultry resistomes were very different in abundance and composition. There was a significant country effect on the resistomes, more so in pigs than in poultry. We found higher AMR loads in pigs, whereas poultry resistomes were more diverse. We detected several recently described, critical AMR genes, including mcr-1 and optrA, the abundance of which differed both between host species and between countries. We found that the total acquired AMR level was associated with the overall country-specific antimicrobial usage in livestock and that countries with comparable usage patterns had similar resistomes. However, functionally determined AMR genes were not associated with total drug use.

Author Correction: Abundance and diversity of the faecal resistome in slaughter pigs and broilers in nine European countries.

In the version of this Article originally published, the surname of author Oksana Lukjancenko was spelt incorrectly as 'Lukjacenko'. This has now been corrected.

Aminobacter MSH1-Mineralisation of BAM in Sand-Filters Depends on Biological Diversity.

BAM (2,6-dichlorobenzamide) is a metabolite of the pesticide dichlobenil. Naturally occurring bacteria that can utilize BAM are rare. Often the compound cannot be degraded before it reaches the groundwater and therefore it poses a serious threat to drinking water supplies. The bacterial strain Aminobacter MSH1 is a BAM degrader and therefore a potential candidate to be amended to sand filters in waterworks to remediate BAM polluted drinking water. A common problem in bioremediation is that bacteria artificially introduced into new diverse environments often thrive poorly, which is even more unfortunate because biologically diverse environments may ensure a more complete decomposition. To test the bioaugmentative potential of MSH1, we used a serial dilution approach to construct microcosms with different biological diversity. Subsequently, we amended Aminobacter MSH1 to the microcosms in two final concentrations; i.e. 10(5) cells mL(-1) and 10(7) cells mL(-1). We anticipated that BAM degradation would be most efficient at "intermediate diversities" as low diversity would counteract decomposition because of incomplete decomposition of metabolites and high diversity would be detrimental because of eradication of Aminobacter MSH1. This hypothesis was only confirmed when Aminobacter MSH1 was amended in concentrations of 10(5) cells mL(-1). Our findings suggest that Aminobacter MSH1 is a very promising bioremediator at several diversity levels.

Fungal-bacterial consortia increase diuron degradation in water-unsaturated systems.

Bioremediation of pesticide-polluted soil may be more efficient using mixed fungal-bacterial cultures rather than the individual strains alone. This may be due to cooperative catabolism, where the first organism transforms the pollutant to products which are then used by the second organism. In addition, fungal hyphae may function as transport vectors for bacteria, thereby facilitating a more effective spreading of degrader organisms in the soil. A more rapid mineralization of the phenylurea herbicide diuron was found in sand with added microbial consortia consisting of both degrading bacteria and fungi. Facilitated transport of bacteria by fungal hyphae was demonstrated using a system where herbicide-spiked sand was separated from the consortium by a layer of sterile glass beads. Several fungal-bacterial consortia were investigated by combining different diuron-degrading bacteria (Sphingomonas sp. SRS2, Variovorax sp. SRS16, and Arthrobacter globiformis D47) and fungi (Mortierella sp. LEJ702 and LEJ703). The fastest mineralization of (14)C-labeled diuron was seen in the consortium consisting of Mortierella LEJ702, Variovorax SRS16, and A. globiformis D47, as measured by evolved (14)CO2. In addition, the production of diuron metabolites by this consortium was minimal. Analyses of 16S rDNA suggested that bacteria were transported more efficiently by LEJ702 than by LEJ703. Finally, it was determined that the fungal growth differed for LEJ702 and LEJ703 in the three-member consortia. This study demonstrates new possibilities for applying efficient fungal-bacterial consortia for bioremediation of polluted soil.

Fungal hyphae stimulate bacterial degradation of 2,6-dichlorobenzamide (BAM).

Introduction of specific degrading microorganisms into polluted soil or aquifers is a promising remediation technology provided that the organisms survive and spread in the environment. We suggest that consortia, rather than single strains, may be better suited to overcome these challenges. Here we introduced a fungal-bacterial consortium consisting of Mortierella sp. LEJ702 and the 2,6-dichlorobenzamide (BAM)-degrading Aminobacter sp. MSH1 into small sand columns. A more rapid mineralisation of BAM was obtained by the consortium compared to MSH1 alone especially at lower moisture contents. Results from quantitative real-time polymerase chain reaction (qPCR) demonstrated better spreading of Aminobacter when Mortierella was present suggesting that fungal hyphae may stimulate bacterial dispersal. Extraction and analysis of BAM indicated that translocation of the compound was also affected by the fungal hyphae in the sand. This suggests that fungal-bacterial consortia are promising for successful bioremediation of pesticide contamination.