Researchers from a German institute have expanded knowledge about produce harboring antibiotic resistance genes that often escape traditional molecular detection methods.

These antibiotic resistance genes might evade cultivation-independent detection, but could still be transferred to human pathogens, according to a team from the Julius Kühn Institut.

Fresh fruits and vegetables are increasingly recognized as a source of pathogenic bacteria, antibiotic-resistant bacteria, and antibiotic resistance genes. The study in the journal mBio explored methods to characterize the transferable resistome – the collection of antibiotic resistance genes present in bacteria – associated with produce.

Mixed salad, arugula, and cilantro bought from supermarkets in Germany were analyzed by cultivation- and DNA-based methods. Before and after a nonselecive enrichment step, tetracycline (TET)-resistant E. coli were isolated and plasmids conferring TET resistance were captured by exogenous plasmid isolation. TET-resistant E. coli isolated from arugula and cilantro carried IncF, IncI1, IncN, IncHI1, IncU, and IncX1 plasmids. From mixed salad and cilantro, IncF, IncI1, and IncP-1β plasmids were captured.

Although initially low in abundance, TET-resistant E. coli were isolated from all purchased produce samples after nonselecive enrichment. TET-resistant E. coli isolates were mostly isolated from cilantro followed by mixed ready-to-eat salad and arugula.

According to the researchers, their findings suggest produce might be a hot spot for contamination with E. coli carrying multi-drug resistance plasmids that occur at low abundance.

Direct detection of IncI and IncF plasmids in total community DNA (TC-DNA) failed, but they became detectable in DNA extracted from enrichment cultures. That confirms cultivation-independent DNA-based methods are not always sufficiently sensitive to detect the transferable resistome in the rare microbiome, according to the researchers.

The team found that IncF and IncI plasmids were the most prevalent types in E. coli isolates from produce. The study demonstrated that multi-drug resistance plasmids present in produce-associated bacteria were transferable to sensitive E. coli recipients, a process that could occur in the human gut.

A total of 24 samples from different locally produced or imported produce was analyzed. The mixed salad and arugula were purchased from local supermarkets in Braunschweig in June and September 2016 and cilantro was obtained from supermarkets in Braunschweig and Magdeburg in May 2017.

Resistance- and virulence-associated traits of E. coli isolates are almost exclusively found on IncF group plasmids. However, no real-time PCR (RT-PCR) systems that allow the cultivation-independent detection and quantification of these plasmids in total community DNA are available.

Almost all E. coli isolates were resistant to antibiotics from at least one class and two isolates were resistant to eight classes; tetracyclines, penicillins, third generation cephalosporins, fluoroquinolones, aminoglycosides, sulfonamides, phenicols, and trimethoprim.

The team found three extended-spectrum beta-lactamase (ESBL) producing E. coli which were isolated from two cilantro samples.

Plasmids from seven different Inc groups were found in the 63 E. coli isolates.

The study showed that bacteria associated with produce can carry various plasmids that might represent an important link between the environmental and human gut microbiomes.

Researchers said produce-associated bacteria should be considered an important route of disseminating transferable antibiotic resistances, which might be relevant for patients under antibiotic treatment.

“In conclusion, this study showed that produce that we eat might contain bacteria such as E. coli carrying transferable multidrug resistance plasmids. Although E. coli numbers are typically low, our nonselective enrichments showed that proliferation can easily occur,” they added.