ESBL-producing Enterobacteriaceae in Ready to Eat Vegetables

Pathogens producing extended-spectrum-β- lactamase (ESBL) such as the Enterobacteriaceae Escherichia coli and Klebsiella pneumoniae produce an enzyme that effectively render them resistant to beta-lactam antibiotics. These bacteria pose a risk to humans particularly in nosocomial environments, but also by contaminating foods such as ready-to-eat (RTE) vegetables.

After investigating the prevalence and characteristics of ESBL-producing E. coli and K. pneumoniae in RTE vegetables, Kim et al. determined that RTE vegetables, and sprouted seeds in particular, may play a role in spreading antimicrobial resistant bacteria and ESBL genes to humans.¹

From October 2012 to February 2013, this team of researchers collected 189 RTE vegetable samples from a South Korean market. The sprouted seed samples (n=91) contained the following types: alfalfa, broccoli, radish, rape, red cabbage, red kohlrabi, and red radish. Mixed salads (n=98) were composed of three to seven different types of the following vegetables: arugula, Batavia green, Batavia red, beet, bok choy, cabbage, carrot, cherry tomato, chicory, cucumber, kyona, leaf beet, lettuce, oak leaf, paprika, perilla, red beet, red bok choy, red cabbage, red chicory, red lettuce and romaine lettuce.

After collecting samples, the team enriched approximately 25g of each food sample in 225mL of Oxoid™ buffered peptone water (Thermo Fisher Scientific) for 24h at 37°C. Next, they streaked enriched samples onto Oxoid Brilliance ESBL agar (Thermo Fisher Scientific) for ESBL screening, followed by incubation at 37°C for 24h under aerobic conditions. From these cultures, the team transferred suspected E. coli colonies (blue or pink) or K. pneumoniae (green colony without a halo) onto Oxoid nutrient agar (Thermo Fisher Scientific) and incubated at 37°C for 24h. Following this, the team used Vitek2® (bioMérieux) to biochemically identify the cultures. They also used the Vitek AST-N224® (bioMérieux) card to investigate bacterial cultures for ESBL production, as well as the minimum inhibitory concentrations (MIC) of 16 antimicrobial agents.

The team found 10% of samples were contaminated with ESBL-producing E. coli and K. pneumoniae, with 94.7% of cases originating from sprouts. They also found a higher proportion of ESBL-positive isolates originated from sprouts (19.8%) compared with those from mixed salads (1.0%; p <0.05). This data adds weight to previous work that revealed sprouts were more likely to be contaminated compared with other vegetables such as carrots or salad greens.

Looking to antibiotic resistance, all isolates were resistant to cefotaxime, and many of the ESBL producers were also resistant to non-β-lactam antibiotics, including gentamicin, trimethoprim/sulfamethoxazole, and ciprofloxacin (73.7%, 63.2%, and 26.3% respectively).

As another aspect of this investigation, the team analyzed genes encoding β-lactamases, and were able to confirm and amplify the sequence of bla_CTX-M β- lactamase genes. They noted the insertion sequence ISEcp1 was upstream of all genes in the isolates examined in this study. Other sequences: orf477 and IS903 were detected downstream of the bla_CTX-M genes in all CTX-M-1 g and CTX-M-9 g isolates, respectively.

According to the authors, this is the first study to identify ESBL-producing E. coli or K. pneumoniae in RTE vegetables. They expect this work will lead to future studies involving food contamination or antibiotic resistance.

Learn more about microbiological examination of foods in our Food and Beverage Community

References:

1. Kim, H.S., et al. (2015) “Prevalence and characterization of extended-spectrum-β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in ready-to-eat vegetables.” International Journal of Food Microbiology,Aug 17;207:83-6. doi: 10.1016/j.ijfoodmicro.2015.04.049. Epub 2015 May 7.