Biosolids for safe land application: does wastewater treatment plant size matters when considering antibiotics, pollutants, microbiome, mobile genetic elements and associated resistance genes?

Soil fertilization with wastewater treatment plant (WWTP) biosolids is associated with the introduction of resistance genes (RGs), mobile genetic elements (MGEs) and potentially selective pollutants (antibiotics, heavy metals, disinfectants) into soil. Not much data are available on the parallel analysis of biosolid pollutant contents, RG/MGE abundances and microbial community composition. In the present study, DNA extracted from biosolids taken at 12 WWTPs (two large-scale, six middle-scale and four small-scale plants) was used to determine the abundance of RGs and MGEs via quantitative real-time PCR and the bacterial and archaeal community composition was assessed by 16S rRNA gene amplicon sequencing. Concentrations of heavy metals, antibiotics, the biocides triclosan, triclocarban and quaternary ammonium compounds (QACs) were measured. Strong and significant correlations were revealed between several target genes and concentrations of Cu, Zn, triclosan, several antibiotics and QACs. Interestingly, the size of the sewage treatment plant (inhabitant equivalents) was negatively correlated with antibiotic concentrations, RGs and MGEs abundances and had little influence on the load of metals and QACs or the microbial community composition. Biosolids from WWTPs with anaerobic treatment and hospitals in their catchment area were associated with a higher abundance of potential opportunistic pathogens and higher concentrations of QACs.

Dairy manure as a potential source of crop nutrients and environmental contaminants.

Although animal manure is applied to agricultural fields for its nutrient value, it may also contain potential contaminants. To determine the variability in such contaminants as well as in valuable nutrients, nine uncomposted manure samples from Idaho dairies collected during 2.5 years were analyzed for macro- and micro-nutrients, hormones, phytoestrogens, antibiotics, veterinary drugs, antibiotic resistance genes, and genetic elements involved in the spread of antibiotic resistance. Total N ranged from 6.8 to 30.7 (C:N of 10 to 21), P from 2.4 to 9.0, and K from 10.2 to 47.7 g/kg manure. Zn (103 - 348 mg/kg) was more abundant than Cu (56 - 127 mg/kg) in all samples. Phytoestrogens were the most prevalent contaminants detected, with concentrations fluctuating over time, reflecting animal diets. This is the first study to document the presence of flunixin, a non-steroidal anti-inflammatory drug, in solid stacked manure from regular dairy operations. Monensin was the most frequently detected antibiotic. Progesterones and sulfonamides were regularly detected. We also investigated the relative abundance of several types of plasmids involved in the spread of antibiotic resistance in clinical settings. Plasmids belonging to the IncI, IncP, and IncQ1 incompatibility groups were found in almost all manure samples. IncQ1 plasmids, class 1 integrons, and sulfonamide resistance genes were the most widespread and abundant genetic element surveyed, emphasizing their potential role in the spread of antibiotic resistance. The benefits associated with amending agricultural soils with dairy manure must be carefully weighed against the potential negative consequences of any manure contaminants.

Salmonella persistence in soil depends on reciprocal interactions with indigenous microorganisms.

Fresh fruits and vegetables have numerous benefits to human health. Unfortunately, their consumption is increasingly associated with food-borne diseases, Salmonella enterica being their most frequent cause in Europe. Agricultural soils were postulated as reservoir of human pathogens, contributing to the contamination of crops during the growing period. Since the competition with the indigenous soil microbiota for colonization sites plays a major role in the success of invading species, we hypothesized that reduced diversity will enhance the chance of Salmonella to successfully establish in agricultural environments. We demonstrated that the abundance of Salmonella drastically decreased in soil with highly diverse indigenous prokaryotic community, while in soil with reduced prokaryotic diversity, Salmonella persisted for a long period. Furthermore, in communities with low diversity, Salmonella had an impact on the abundance of other taxa. The high physiological plasticity allows Salmonella to use agricultural soils as alternative habitat which might provide a route of animal/human infections. In addition, adjusted transcriptional profile with amino acid biosynthesis and the glyoxylate cycle most prominently regulated, suggests an adaptation to the soil environment. Our results underline the importance of the maintenance of diverse soil microbiome as a part of strategy aiming at reduced risk of food-borne salmonellosis outbreaks.

Role of Plasmids in Plant-Bacteria Interactions.

Plants are colonized by diverse microorganisms, which may positively or negatively influence the plant fitness. The positive impact includes nutrient acquisition, enhancement of resistance to biotic and abiotic stresses, both important factors for plant growth and survival, while plant pathogenic bacteria can cause diseases. Plant pathogens are adapted to negate or evade plant defense mechanisms, e.g. by the injection of effector proteins into the host cells or by avoiding the recognition by the host. Plasmids play an important role in the rapid bacterial adaptation to stresses and changing environmental conditions. In the plant environment, plasmids can further provide a selective advantage for the host bacteria, e.g. by carrying genes encoding metabolic pathways, metal and antibiotic resistances, or pathogenicity-related genes. However, we are only beginning to understand the role of mobile genetic elements and horizontal gene transfer for plant-associated bacteria. In this review, we aim to provide a short update on what is known about plasmids and horizontal gene transfer of plant associated bacteria and their role in plant-bacteria interactions. Furthermore, we discuss tools available to study the plant-associated mobilome, its transferability, and its bacterial hosts.

Shifts in abundance and diversity of mobile genetic elements after the introduction of diverse pesticides into an on-farm biopurification system over the course of a year.

Biopurification systems (BPS) are used on farms to control pollution by treating pesticide-contaminated water. It is assumed that mobile genetic elements (MGEs) carrying genes coding for enzymes involved in degradation might contribute to the degradation of pesticides. Therefore, the composition and shifts of MGEs, in particular, of IncP-1 plasmids carried by BPS bacterial communities exposed to various pesticides, were monitored over the course of an agricultural season. PCR amplification of total community DNA using primers targeting genes specific to different plasmid groups combined with Southern blot hybridization indicated a high abundance of plasmids belonging to IncP-1, IncP-7, IncP-9, IncQ, and IncW, while IncU and IncN plasmids were less abundant or not detected. Furthermore, the integrase genes of class 1 and 2 integrons (intI1, intI2) and genes encoding resistance to sulfonamides (sul1, sul2) and streptomycin (aadA) were detected and seasonality was revealed. Amplicon pyrosequencing of the IncP-1 trfA gene coding for the replication initiation protein revealed high IncP-1 plasmid diversity and an increase in the abundance of IncP-1ß and a decrease in the abundance of IncP-1ε over time. The data of the chemical analysis showed increasing concentrations of various pesticides over the course of the agricultural season. As an increase in the relative abundances of bacteria carrying IncP-1ß plasmids also occurred, this might point to a role of these plasmids in the degradation of many different pesticides.

Quantification of IncP-1 plasmid prevalence in environmental samples.

To study the role of broad-host-range IncP-1 plasmids in bacterial adaptability to irregular environmental challenges, a quantitative real-time PCR assay was developed that specifically detects the korB gene, which is conserved in all IncP-1 plasmids, in environmental samples. IncP-1 plasmid dynamics in a biopurification system for pesticide wastes were analyzed.

Increased abundance and transferability of resistance genes after field application of manure from sulfadiazine-treated pigs.

Spreading manure containing antibiotics in agriculture is assumed to stimulate the dissemination of antibiotic resistance in soil bacterial populations. Plant roots influencing the soil environment and its microflora by exudation of growth substrates might considerably increase this effect. In this study, the effects of manure from pigs treated with sulfadiazine (SDZ), here called SDZ manure, on the abundance and transferability of sulfonamide resistance genes sul1 and sul2 in the rhizosphere of maize and grass were compared to the effects in bulk soil in a field experiment. In plots that repeatedly received SDZ manure, a significantly higher abundance of both sul genes was detected compared to that in plots where manure from untreated pigs was applied. Significantly lower abundances of sul genes relative to bacterial ribosomal genes were encountered in the rhizosphere than in bulk soil. However, in contrast to results for bulk soil, the sul gene abundance in the SDZ manure-treated rhizosphere constantly deviated from control treatments over a period of 6 weeks after manuring, suggesting ongoing antibiotic selection over this period. Transferability of sulfonamide resistance was analyzed by capturing resistance plasmids from soil communities into Escherichia coli. Increased rates of plasmid capture were observed in samples from SDZ manure-treated bulk soil and the rhizosphere of maize and grass. More than 97% of the captured plasmids belonged to the LowGC type (having low G+C content), giving further evidence for their important contribution to the environmental spread of antibiotic resistance. In conclusion, differences between bulk soil and rhizosphere need to be considered when assessing the risks associated with the spreading of antibiotic resistance.

Salmonella enterica relies on carbon metabolism to adapt to agricultural environments.

Salmonella enterica, a foodborne and human pathogen, is a constant threat to human health. Agricultural environments, for example, soil and plants, can be ecological niches and vectors for Salmonella transmission. Salmonella persistence in such environments increases the risk for consumers. Therefore, it is necessary to investigate the mechanisms used by Salmonella to adapt to agricultural environments. We assessed the adaptation strategy of S. enterica serovar Typhimurium strain 14028s to agricultural-relevant situations by analyzing the abundance of intermediates in glycolysis and the tricarboxylic acid pathway in tested environments (diluvial sand soil suspension and leaf-based media from tomato and lettuce), as well as in bacterial cells grown in such conditions. By reanalyzing the transcriptome data of Salmonella grown in those environments and using an independent RT-qPCR approach for verification, several genes were identified as important for persistence in root or leaf tissues, including the pyruvate dehydrogenase subunit E1 encoding gene aceE. In vivo persistence assay in tomato leaves confirmed the crucial role of aceE. A mutant in another tomato leaf persistence-related gene, aceB, encoding malate synthase A, displayed opposite persistence features. By comparing the metabolites and gene expression of the wild-type strain and its aceB mutant, fumarate accumulation was discovered as a potential way to replenish the effects of the aceB mutation. Our research interprets the mechanism of S. enterica adaptation to agriculture by adapting its carbon metabolism to the carbon sources available in the environment. These insights may assist in the development of strategies aimed at diminishing Salmonella persistence in food production systems.

Influence of sewage sludge stabilization method on microbial community and the abundance of antibiotic resistance genes.

Municipal sewage sludge (MSS) and other biosolids are of high interest for agriculture. These nutrient-rich organic materials can potentially serve as organic fertilizers. Besides an increase of organic matter in soil, other positive effects were shown after their application. Especially the positive influence on circular economy increased the attention paid to management of MSS in recent years. Unfortunately, the use of sewage sludge has some drawbacks. Biosolids are frequently polluted with heavy metals, xenobiotic organic compounds and industrial chemicals, which may be hazardous for the environment and humans. Here, we investigated the influence of stabilization method and the size of wastewater treatment plant on the structure of microbial communities as well as the abundance of antibiotic resistance genes (ARG) and mobile genetic elements (MGE). All tested ARG and MGE were detectable in almost all of the samples. Interestingly, the presence of MGE as well as particular heavy metals correlated positively with the presence of several ARG. We conclude that the distribution of ARG and MGE in biosolids originated from municipal wastewater treatment plants, cannot be explained by the size of the facility or the applied stabilization method. Moreover, we postulate that the presence of pollutants and long-term impacts should be assessed prior to a possible use of sewage sludge as fertilizer.

Linking low-level stable isotope fractionation to expression of the cytochrome P450 monooxygenase-encoding ethB gene for elucidation of methyl tert-butyl ether biodegradation in aerated treatment pond systems.

Multidimensional compound-specific stable isotope analysis (CSIA) was applied in combination with RNA-based molecular tools to characterize methyl tertiary (tert-) butyl ether (MTBE) degradation mechanisms occurring in biofilms in an aerated treatment pond used for remediation of MTBE-contaminated groundwater. The main pathway for MTBE oxidation was elucidated by linking the low-level stable isotope fractionation (mean carbon isotopic enrichment factor [ε(C)] of -0.37‰ ± 0.05‰ and no significant hydrogen isotopic enrichment factor [ε(H)]) observed in microcosm experiments to expression of the ethB gene encoding a cytochrome P450 monooxygenase able to catalyze the oxidation of MTBE in biofilm samples both from the microcosms and directly from the ponds. 16S rRNA-specific primers revealed the presence of a sequence 100% identical to that of Methylibium petroleiphilum PM1, a well-characterized MTBE degrader. However, neither expression of the mdpA genes encoding the alkane hydroxylase-like enzyme responsible for MTBE oxidation in this strain nor the related MTBE isotope fractionation pattern produced by PM1 could be detected, suggesting that this enzyme was not active in this system. Additionally, observed low inverse fractionation of carbon (ε(C) of +0.11‰ ± 0.03‰) and low fractionation of hydrogen (ε(H) of -5‰ ± 1‰) in laboratory experiments simulating MTBE stripping from an open surface water body suggest that the application of CSIA in field investigations to detect biodegradation may lead to false-negative results when volatilization effects coincide with the activity of low-fractionating enzymes. As shown in this study, complementary examination of expression of specific catabolic genes can be used as additional direct evidence for microbial degradation activity and may overcome this problem.

Inhibition of nitrification by low oxygen concentrations in an aerated treatment pond system with biofilm promoting mats.

Inhibition of nitrification in the presence of low oxygen concentrations (below 1.2 mg/L) and temperature dependency at oxygen saturation levels were observed in an aerated treatment pond system with biofilm promoting mats in two parallel ponds for remediation of ammonium, methyl tertiary butyl ether (MTBE), and benzene-contaminated groundwater. Within the first 18 months, at an average oxygen concentration of 0.7 +/- 0.5 mg/L along the ponds, no significant decrease of ammonium or significant formation of nitrification products were observed. After increasing the aeration to oxygen saturation levels, the ammonium removal increased up to a maximum of 27%, with concomitant formation of nitrite and nitrate (up to 26 and 0.6 mM). The subsequent reduction of aeration in one pond to the previous level resulted in a definitive stop of nitrification, while, in the other pond, nitrification was well-correlated with the water temperature, reaching up to 45% ammonium removal.

Detection, Isolation, and Characterization of Plasmids in the Environment.

Plasmids play a major role in the bacterial adaptation to changing and stressful environmental conditions caused by antibiotics, heavy metals, and disinfectants. However, the investigation of the ecology and diversity of environmental plasmids is challenging due to their typically low abundance in soil bacterial communities and the low cultivability of their hosts. Here we discuss the potentials and limitations of cultivation-dependent and cultivation-independent approaches for detecting and quantifying plasmids in total community DNA from environmental samples. Protocols for PCR-based detection of plasmid-specific sequences in total community DNA are presented. Furthermore, protocols to obtain and characterize plasmids either from isolates (endogenous plasmid isolation) or by capturing into a recipient strain by biparental and triparental mating will be provided.

Composted Sewage Sludge Influences the Microbiome and Persistence of Human Pathogens in Soil.

Composted sewage sludge (CSS) gained attention as a potential fertilizer in agriculture. Application of CSS increases soil microbial activity and microbial biomass, however, it can also lead to increased chemical and microbiological risks. In this study, we performed microcosm experiments to assess how CSS reshapes the microbial community of diluvial sand (DS) soil. Further, we assessed the potential of CSS to increase the persistence of human pathogens in DS soil and the colonization of Chinese cabbage (Brassica rapa L. subsp. pekinensis (Lour.) Hanelt). The results revealed that CSS substantially altered the prokaryotic community composition. Moreover, addition of CSS increased the persistence of Salmonella enterica serovar Typhimurium strain 14028s and S. enterica serovar Senftenberg in DS soil. However, the enhanced persistence in soil had no impact on the colonization rate of B. rapa grown on soil inoculated with Salmonella. We detected Salmonella in leaves of 1.9% to 3.6% of plants. Addition of CSS had no impact on the plant colonization rate. The use of sewage sludge composts is an interesting option. However, safety measures should be applied in order to avoid contamination of crop plants by human pathogens.

Plant Species-Dependent Increased Abundance and Diversity of IncP-1 Plasmids in the Rhizosphere: New Insights Into Their Role and Ecology.

IncP-1 plasmids, first isolated from clinical specimens (R751, RP4), are recognized as important vectors spreading antibiotic resistance genes. The abundance of IncP-1 plasmids in the environment, previously reported, suggested a correlation with anthropogenic pollution. Unexpectedly, qPCR-based detection of IncP-1 plasmids revealed also an increased relative abundance of IncP-1 plasmids in total community DNA from the rhizosphere of lettuce and tomato plants grown in non-polluted soil along with plant age. Here we report the successful isolation of IncP-1 plasmids by exploiting their ability to mobilize plasmid pSM1890. IncP-1 plasmids were captured from the rhizosphere but not from bulk soil, and a high diversity was revealed by sequencing 14 different plasmids that were assigned to IncP-1ß, δ, and ε subgroups. Although backbone genes were highly conserved and mobile elements or remnants as Tn501, IS1071, Tn402, or class 1 integron were carried by 13 of the sequenced IncP-1 plasmids, no antibiotic resistance genes were found. Instead, seven plasmids had a mer operon with Tn501-like transposon and five plasmids contained putative metabolic gene clusters linked to these mobile elements. In-depth sequence comparisons with previously known plasmids indicate that the IncP-1 plasmids captured from the rhizosphere are archetypes of those found in clinical isolates. Our findings that IncP-1 plasmids do not always carry accessory genes in unpolluted rhizospheres are important to understand the ecology and role of the IncP-1 plasmids in the natural environment.

Whole Genome Sequencing of Escherichia coli From Store-Bought Produce.

The role of agriculture in the transfer of drug resistant pathogens to humans is widely debated and poorly understood. Escherichia coli is a valuable indicator organism for contamination and carriage of antimicrobial resistance (AMR) in foods. Whilst whole genome sequences for E. coli from animals and associated meats are common, sequences from produce are scarce. Produce may acquire drug resistant E. coli from animal manure fertilizers, contaminated irrigation water and wildlife, particularly birds. Whole genome sequencing was used to characterize 120 tetracycline (TET) resistant E. coli from store-bought, ready-to-eat cilantro, arugula and mixed salad from two German cities. E. coli were recovered on the day of purchase and after 7 days of refrigeration. Cilantro was far more frequently contaminated with TET-resistant E. coli providing 102 (85%) sequenced strains. Phylogroup B1 dominated the collection (n = 84, 70%) with multi-locus sequence types B1-ST6186 (n = 37, 31%), C-ST165 (n = 17, 14%), B1-ST58 (n = 14, 12%), B1-ST641 (n = 8, 7%), and C-ST88 (n = 5, 4%) frequently identified. Notably, seven strains of diverse sequence type (ST) carried genetic indicators of ColV virulence plasmid carriage. A number of previously identified and novel integrons associated with insertion elements including IS26 were also identified. Storage may affect the lineages of E. coli isolated, however further studies are needed. Our study indicates produce predominantly carry E. coli with a commensal phylogroup and a variety of AMR and virulence-associated traits. Genomic surveillance of bacteria that contaminate produce should be a matter of public health importance in order to develop a holistic understanding of the environmental dimensions of AMR.

Salmonella Establishment in Agricultural Soil and Colonization of Crop Plants Depend on Soil Type and Plant Species.

Human pathogenic bacteria, such as Salmonella enterica, are able to colonize crop plants. So far, not much is known about biotic and abiotic factors influencing this colonization in field soil. This understanding, however, is imperative for the provision of safe fresh produce to the consumer. In this study, we investigated the effects of soil type, organic fertilization, plant species and the way of Salmonella entry into the plant production system, on the survival of S. enterica in soil as well as the colonization of plants. The selected S. enterica serovar Typhimurium strain 14028s, S. Typhimurium strain LT2 and S. Senftenberg were able to persist in soil for several weeks. Salmonella's persistence in soil was prolonged in loamy, if compared to sandy soil, and when applied together with organic fertilizer. The leaves of lettuce and corn salad were colonized by S. enterica providing evidence for internalization from the soil via the root. Colonization rates were affected by soil type, plant species and S. enterica strain. Overall, S. enterica was detected in leaves of 0.5-0.9% of the plants, while lettuce was more frequently colonized than corn salad. Plants grown in sandy soil were more often colonized than plants grown in loamy soil. After spray inoculation, S. enterica could be detected on and in leaves for several weeks by cultivation-depending methods, confirmed by confocal microscopy using GFP-labeled S. Typhimurium 14028s. On the one hand, transcriptome data from S. Typhimurium 14028s assessed in response to lettuce medium or lettuce root exudates showed an upregulation of genes associated with biofilm formation and virulence. On the other hand, lettuce inoculated with S. Typhimurium 14028s showed a strong upregulation of genes associated with plant immune response and genes related to stress response. In summary, these results showed that organic fertilizers can increase the persistence of Salmonella in soil and that soil type and plant species play a crucial role in the interactions between human pathogens and crop plants. This understanding is therefore a starting point for new strategies to provide safe food for the consumer.

Salmonella adapts to plants and their environment during colonization of tomatoes.

Humans and animals are considered typical hosts for Salmonella, however, also plants can be colonized. Tomatoes were linked to salmonellosis outbreaks already on several occasions. The aim of this study was, therefore, to establish a comprehensive view on the interaction between Salmonella enterica and tomatoes, and to test the hypothesis that colonization of plants is an interactive process. We assessed the persistence of Salmonella in agricultural soil, the colonization pattern in and on tomatoes, as well as the reciprocal responses of tomatoes to different Salmonella strains and Salmonella to root exudates and tomato-related media. This study revealed that Salmonella can persist in the soil and inside the tomato plant. Additionally, we show that Salmonella strains have particular colonization pattern, although the persistence inside the plant differs between the tested strains. Furthermore, the transcriptome response of tomato showed an up-regulation of several defense-related genes. Salmonella transcriptome analysis in response to the plant-based media showed differentially regulated genes related to amino acid and fatty acid synthesis and stress response, while the response to root exudates revealed regulation of the glyoxylate cycle. Our results indicate that both organisms actively engage in the interaction and that Salmonella adapts to the plant environment.

Soil amendment with sewage sludge affects soil prokaryotic community composition, mobilome and resistome.

Municipal sewage sludge (MSS) is often directly applied to fields despite concerns regarding the spread of harmful microbes and associated resistance genes (RGs). In this four month microcosm study, the dynamics of prokaryotic communities in agricultural soil and changes in mobile genetic elements and RGs following amendment with stabilized MSS were investigated. TaqMan-based quantitative real-time (q)PCR showed that RG prevalence was high when compared to untreated soil and genes for class 1 integrons (intI1), streptomycin RGs (aadA, strA) and tetracycline RG (tet(W)) were detectable for the duration of the four month study. High-throughput qPCR revealed an enhanced prevalence of aminoglycoside RGs (aacC, aadE), macrolide lincosamide-streptogramin B RGs (ermB, ermF) and tetracycline RGs (tet(L), tet(M), tet(X)). Illumina MiSeq sequencing of 16S rRNA gene fragments amplified from total community DNA revealed significant changes in the prokaryotic community composition both at phylum and genus levels, with lower richness and evenness after MSS amendment followed by gradual recovery after 119 days. Conjugative plasmids captured from MSS using exogenous isolation belonged predominantly to the IncP-1 plasmid group. Our results provide new insights into short- and medium-term effects of MSS amendment on soil prokaryotic communities, including the mobilome and resistome.

Manure and Doxycycline Affect the Bacterial Community and Its Resistome in Lettuce Rhizosphere and Bulk Soil.

Manure application to agricultural soil introduces antibiotic residues and increases the abundance of antibiotic-resistant bacteria (ARB) carrying antibiotic resistance genes (ARGs), often located on mobile genetic elements (MGEs). The rhizosphere is regarded as a hotspot of microbial activity and gene transfer, which can alter and prolong the effects of organic fertilizers containing antibiotics. However, not much is known about the influence of plants on the effects of doxycycline applied to soil via manure. In this study, the effects of manure spiked with or without doxycycline on the prokaryotic community composition as well as on the relative abundance of ARGs and MGEs in lettuce rhizosphere and bulk soil were investigated by means of a polyphasic cultivation-independent approach. Samples were taken 42 days after manure application, and total community DNA was extracted. Besides a pronounced manure effect, doxycycline spiking caused an additional enrichment of ARGs and MGEs. High-throughput quantitative PCR revealed an increase in tetracycline, aminoglycoside, and macrolide-lincosamide-streptogramin B (MLSB) resistance genes associated with the application of manure spiked with doxycycline. This effect was unexpectedly lower in the rhizosphere than in bulk soil, suggesting a faster dissipation of the antibiotic and a more resilient prokaryotic community in the rhizosphere. Interestingly, the tetracycline resistance gene tetA(P) was highly enriched in manure-treated bulk soil and rhizosphere, with highest values observed in doxycycline-treated bulk soil, concurring with an enrichment of Clostridia. Thus, the gene tetA(P) might be a suitable marker of soil contamination by ARB, ARGs, and antibiotics of manure origin. These findings illustrate that the effects of manure and doxycycline on ARGs and MGEs differ between rhizosphere and bulk soil, which needs to be considered when assessing risks for human health connected to the spread of ARGs in the environment.

The Transferable Resistome of Produce.

Produce is increasingly recognized as a reservoir of human pathogens and transferable antibiotic resistance genes. This study aimed to explore methods to characterize the transferable resistome of bacteria associated with produce. Mixed salad, arugula, and cilantro purchased from supermarkets in Germany were analyzed by means of cultivation- and DNA-based methods. Before and after a nonselective enrichment step, tetracycline (TET)-resistant Escherichia coli were isolated and plasmids conferring TET resistance were captured by exogenous plasmid isolation. TET-resistant E. coli isolates, transconjugants, and total community DNA (TC-DNA) from the microbial fraction detached from leaves or after enrichment were analyzed for the presence of resistance genes, class 1 integrons, and various plasmids by real-time PCR and PCR-Southern blot hybridization. Real-time PCR primers were developed for IncI and IncF plasmids. TET-resistant E. coli isolated from arugula and cilantro carried IncF, IncI1, IncN, IncHI1, IncU, and IncX1 plasmids. Three isolates from cilantro were positive for IncN plasmids and blaCTX-M-1 From mixed salad and cilantro, IncF, IncI1, and IncP-1ß plasmids were captured exogenously. Importantly, whereas direct detection of IncI and IncF plasmids in TC-DNA failed, these plasmids became detectable in DNA extracted from enrichment cultures. This confirms that cultivation-independent DNA-based methods are not always sufficiently sensitive to detect the transferable resistome in the rare microbiome. In summary, this study showed that an impressive diversity of self-transmissible multiple resistance plasmids was detected in bacteria associated with produce that is consumed raw, and exogenous capturing into E. coli suggests that they could transfer to gut bacteria as well.IMPORTANCE Produce is one of the most popular food commodities. Unfortunately, leafy greens can be a reservoir of transferable antibiotic resistance genes. We found that IncF and IncI plasmids were the most prevalent plasmid types in E. coli isolates from produce. This study highlights the importance of the rare microbiome associated with produce as a source of antibiotic resistance genes that might escape cultivation-independent detection, yet may be transferred to human pathogens or commensals.