Prevalence of β-Lactam Drug-Resistance Genes in Escherichia coli Contaminating Ready-to-Eat Lettuce

Introduction

The increase and spread of antimicrobial drug-resistant bacterial strains has become a problem worldwide, and food may contribute as an important source of antibiotic resistance genes (ARGs) found in the human intestine. In particular, fresh vegetables carry saprophytic bacteria that harbor drug-resistance genes, which can enter the human gut when it is eaten uncooked (Raphael et al., 2011; Berman and Riley, 2013; Richter et al., 2019). Escherichia coli have been regarded as an indicator of contamination and health risk in foods (Paruch and Mæhlum, 2012). Some groups of E. coli can cause gastrointestinal infections as well as extraintestinal infections. In the United States, multistate outbreaks of gastroenteric illness caused by lettuce contaminated with E. coli O157:H7 have been widely reported (Glowacki et al., 2019). More recent studies have shown sequence types (STs) of extraintestinal pathogenic E. coli (ExPEC) shared by E. coli isolates from retail meat products and patients with community-acquired urinary tract infections (Yamaji et al., 2018; Borges et al., 2019).

In this study, we wished to discover if ExPEC and drug-resistant strains of ExPEC can be found on green leafy vegetables. Therefore, the purpose of this study was to determine the STs and ARG carriage of E. coli isolated from packaged ready-to-eat lettuce.

Materials and Methods

Ninety-one ready-to-eat lettuce packages were purchased from chain supermarkets located within a 11 kilometers vicinity of a university community in Northern California from October 2018 to July 2019. These lettuce products represented 12 brands and the main types included Boston (butterhead lettuce), iceberg (crisphead lettuce), and green/red leaf lettuce, which were distributed by four different California producers.

For each package, 25 g of lettuce were placed in a UV-pretreated polyethylene bag containing 50 mL of sterile phosphate-buffered saline (PBS; pH 7.4). After brief kneading by hand, the lettuce was incubated in PBS for 45 min at room temperature and washed three times. The combined rinsate (50 mL × 3) was then centrifuged at 12,000 g for 5 min at room temperature. The pellet was resuspended in 2 mL of PBS and preincubated in double strength MacConkey broth (Difco) at a 1:1 dilution at 35°C for 24 h. A separate 10 μL aliquot was then spread-plated on MacConkey agar (Difco) to isolate Gram-negative bacteria. E. coli was presumptively identified by lactose fermentation and indole (Sigma-Aldrich) tests.

Single colonies from tryptic soy agar plates were selected and inoculated into 2 mL tryptic soy broth and incubated in a shaking incubator for 15 h at 37°C. The aliquots of grown cultures were used to isolate DNA as described previously (Yamaji et al., 2018). The allelic number and the corresponding genotype number were designated by the curator of the multilocus sequence typing (MLST) based on the seven-gene scheme described at website https://enterobase.warwick.ac.uk/species/ecoli/allele_st_search

All isolates were assessed for susceptibility to ampicillin (AMP), cefoxitin (FOX), cefotaxime (CTX), cefuroxime (CXM), ceftriaxone (CRO), ampicillin/sulbactam (SAM), ceftazidime (CAZ), fosfomycin (FOS), gentamicin (CN), nitrofurantoin (NIT), trimethoprim-sulfamethoxazole (TMP-SMX), and nalidixic acid (NAL) by the standard disk diffusion assay, according to the Clinical and Laboratory Standards Institute-CLSI (CLSI, 2015). E. coli ATCC 25922 was used as reference strain in all tests.

The E. coli isolates were tested for the presence of bla _TEM, bla _SHV, bla _CTX-M, and bla _OXA β-lactamase genes by polymerase chain reaction (Dallenne et al., 2010). DNA sequences were visually inspected, aligned, and compared against sequences in GenBank with BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Statistical analysis was performed by Fisher's exact test (two-tailed) with significance set at p ≤ 0.05.

Results and Discussion

Of the 91 lettuce packages, 47 (52%) contained organic and 44 (48%) contained conventionally grown (nonorganic) lettuce. Thirty-four E. coli isolates were recovered from 18 (38%) organic and 7 (16%) conventionally grown lettuce (p = 0.01). The isolates were classified into 15 distinct STs. Ten isolates could not be assigned to a known ST (Table 1). The most common genotypes were ST8951 (16%) and ST5154 (12%). ST8951 has been previously identified in deciduous forest soil and turkey, including ground meat as well as clinical samples (https://enterobase.warwick.ac.uk/species/index/ecoli). The type of clinical sample is not described in the database. ST5154 has been reported from cattle, but not from any human infections (Ribeiro et al., 2016; https://enterobase.warwick.ac.uk/species/index/ecoli). Other genotypes, including ST1198, ST2625, ST2432, ST2819, ST4600, and ST5143, have been described as pathogens in human samples from Japan (Fukuoka), USA (Nashville and Tennessee), or Canada, China (Hangzhou and Shanghai), and Korea, as noted in the MLST database and previous studies (https://enterobase.warwick.ac.uk/species/index/ecoli).

ST1198 has been implicated in urinary tract infection and is a member of clonal complex 131 (Lee et al., 2010), which is one of the pandemic lineages of ExPEC (Riley, 2014). Foster et al. (2015) reported ST4600 as an enteropathogenic E. coli strain, isolated from a patient with diarrhea. The clinical sources of the other genotypes are not described.

Among 34 E. coli isolates, 26 (76%) were resistant to AMP, 17 (50%) to SAM, 8 (23%) to FOX, 7 (20%) to CXM, 1 (3%) to FOS, and 1 (3%) to NAL. Of the β-lactamase genes examined, bla _CTX-M was the most prevalent, found in 8 (23%) isolates, followed by bla _SHV, detected in 5 (15%) isolates, and bla _TEM detected in 4 (12%) isolates. The high frequency of E. coli strains carrying bla _CTX-M is surprising, considering that the most common β-lactam genes found in clinical strains of E. coli is bla _TEM and/or bla _SHV (Yamaji et al., 2018; Borges et al., 2019; Tarlton et al., 2019). The high frequency of CTX-M β-lactamase genes in ready-to-eat lettuce poses a public health concern, since it is the most common extended-spectrum beta-lactamases (ESBL) expressed by clinical strains of E. coli, which is becoming increasingly prevalent in extraintestinal infections worldwide (Yamaji et al., 2018; Borges et al., 2019; Richter et al., 2019; Tarlton et al., 2019).

To our surprise, in one strain (ST3222), we found a class A broad-spectrum β-lactamase SED-1 gene, bla _SED, reported by others to have been found in Citrobacter sedlakii in the bile fluid of a patient (Petrella et al., 2001). It suggests that some of these β-lactamase genes are horizontally transferred among saprophytic Gram-negative bacteria found in the environment, and that commensal E. coli from mammalian intestine contaminating lettuce could acquire them. Such genes can then enter the human intestine through ingestion of contaminated food product eaten uncooked.

Conclusions

We found that retail packaged lettuce purchased in Norther California frequently contains E. coli strains belonging to recognized ExPEC group, and that many of them carry ESBL genes. The magnitude of lettuce contributing to the spread of ExPECs and resistance genes into the human intestine may be underestimated.