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 bla_CTX-M-1. From mixed salad and cilantro, IncF, IncI1, and IncP-1β 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.

High antibiotic releases from manufacturing facilities have been identified as a risk factor for antibiotic resistance development in bacterial pathogens. However, the role of antibiotic pollution in selection and transferability of antibiotic resistance genes (ARGs) is still limited. In this study, we analyzed effluents from azithromycin-synthesis and veterinary-drug formulation facilities as well as sediments from receiving river and creek taken at the effluent discharge sites, upstream and downstream of discharge. Culturing showed that the effluent discharge significantly increased the proportion of antibiotic resistant bacteria in exposed sediments compared to the upstream ones. Quantitative real-time PCR revealed that effluents from both industries contained high and similar relative abundances of resistance genes [sul1, sul2, qacE/qacEΔ1, tet(A)], class 1 integrons (intI1) and IncP-1 plasmids (korB). Consequently, these genes significantly increased in relative abundances in receiving sediments, with more pronounced effects being observed for river than for creek sediments due to lower background levels of the investigated genes in the river. In addition, effluent discharge considerably increased transfer frequencies of captured ARGs from exposed sediments into Escherichia coli CV601 recipient as shown by biparental mating experiments. Most plasmids exogenously captured from effluent and polluted sediments belonged to the broad host range IncP-1ε plasmid group, conferred multiple antibiotic resistance and harbored class 1 integrons. Discharge of pharmaceutical waste from antibiotic manufacturing sites thus poses a risk for development and dissemination of multi-resistant bacteria, including pathogens.

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 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.

Due to the high prevalence of colistin-resistant Enterobacteriaceae in poultry and pigs, process waters and wastewater from slaughterhouses were considered as a hotspot for isolates carrying plasmid-encoded, mobilizable colistin resistances (mcr genes). Thus, questions on the effectiveness of wastewater treatment in in-house and municipal wastewater treatment plants (WWTPs) as well as on the diversity of the prevailing isolates, plasmid types, and their transmissibility arise. Process waters and wastewater accruing in the delivery and unclean areas of two poultry and two pig slaughterhouses were screened for the presence of target colistin-resistant bacteria (i.e., Escherichia coli, Klebsiella spp., Enterobacter cloacae complex). In-house and municipal WWTPs (mWWTPs) including receiving waterbodies were investigated as well. Samples taken in the poultry slaughterhouses yielded the highest occurrence of target colistin-resistant Enterobacteriaceae (40.2%, 33/82), followed by mWWTPs (25.0%, 9/36) and pig slaughterhouses (14.9%, 10/67). Recovered isolates exhibited various resistance patterns. The resistance rates using epidemiological cut-off values were higher in comparison to those obtained with clinical breakpoints. Noteworthy, MCR-1-producing Klebsiella pneumoniae and E. coli were detected in scalding waters and preflooders of mWWTPs. A total of 70.8% (46/65) of E. coli and 20.6% (7/34) of K. pneumoniae isolates carried mcr-1 on a variety of transferable plasmids with incompatibility groups IncI1, IncHI2, IncX4, IncF, and IncI2 ranging between 30 and 360 kb. The analyzed isolates carrying mcr-1 on transferable plasmids (n = 53) exhibited a broad diversity, as they were assigned to 25 different XbaI profiles. Interestingly, in the majority of colistin-resistant mcr-negative E. coli and K. pneumoniae isolates non-synonymous polymorphisms in pmrAB were detected. Our findings demonstrated high occurrence of colistin-resistant E. coli and K. pneumoniae carrying mcr-1 on transferrable plasmids in poultry and pig slaughterhouses and indicate their dissemination into surface water.

Summary: 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.