Occurrence,Identification,and Antimicrobial Resistance Profiles of Extended-Spectrum and AmpC β-Lactamase-Producing Enterobacteriaceae from Fresh Vegetables Retailed in Gauteng Province,South Africa

Abstract

Extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase-producing Enterobacteriaceae are no longer restricted to the health care system, but represent increased risks related to environmental integrity and food safety. Fresh produce has been increasingly reported to constitute a reservoir of multidrug-resistant (MDR) potential human pathogenic Enterobacteriaceae. This study aimed to detect, identify, and characterize the antimicrobial resistance of ESBL/AmpC-producing Enterobacteriaceae isolates from fresh vegetables at point of sale. Vegetable samples (spinach, tomatoes, lettuce, cucumber, and green beans; n = 545) were purchased from retailers in Gauteng, the most densely populated province in South Africa. These included street vendors, trolley vendors, farmers' market stalls, and supermarket chain stores. Selective enrichment, plating onto chromogenic media, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmation of isolate identities showed that 17.4% (95/545) vegetable samples analyzed were contaminated with presumptive ESBL/AmpC-producing Enterobacteriaceae. Dominant species identified included Escherichia coli, Enterobacter cloacae, Enterobacter asburiae, and Klebsiella pneumoniae. Phenotypic antibiotic resistance analysis showed that 96.1% of 77 selected isolates were MDR, while resistance to aminoglycoside (94.8%), chloramphenicol (85.7%), and tetracycline (53.2%) antibiotic classes was most prevalent. Positive phenotypic analysis for ESBL production was shown in 61 (79.2%) of the 77 isolates, and AmpC production in 41.6% of the isolates. PCR and sequencing confirmed the presence of β-lactamase genes in 75.3% isolates from all vegetable types analyzed, mainly in E. coli, Enterobacter spp., and Serratia spp. isolates. CTX-M group 9 (32.8%) was the dominant ESBL type, while EBC (24.1%) was the most prevalent plasmidic type AmpC β-lactamase. Our findings document for the first time the presence of MDR ESBL/AmpC-producing Enterobacteriaceae in raw vegetables sold at selected retailers in Gauteng Province, South Africa.

Introduction

Extended-spectrum β-lactamase (ESBL)- and AmpC-producing Enterobacteriaceae have increased in occurrence globally in health care systems, agroecosystems, and fresh produce, due to the widespread use of broad-spectrum antibiotics (Ye et al., 2017). Dissemination of these antimicrobial-resistant microorganisms has been identified as one of the six main antibiotic resistance (AR)–related health risks globally (WHO, 2015). If infection by ESBL/AmpC-producing Enterobacteriaceae occurs, treatment options become limited as a result of expanded AR of the corresponding isolates (Freitag et al., 2018). Since ESBL/AmpC β-lactamases are capable of inactivating broad-spectrum penicillins and cephalosporins, their presence in Enterobacteriaceae is of clinical and epidemiological importance (Kolar et al., 2010). Clinically important ESBL-producing Enterobacteriaceae have been reported in different South African (SA) provinces (Eastern Cape [Vasaikar et al., 2017]; Western Cape [Peirano et al., 2011]; KwaZulu-Natal [Mahomed and Coovadia, 2014]; and Gauteng Province [Ehlers et al., 2009]). In 53 clinical isolates from Gauteng, ESBL gene prevalence was reported in 87% (Ehlers et al., 2009).

ESBLs, classified as Ambler class A enzymes, include TEM-, SHV- and CTX-M-type enzymes (Östholm, 2014; Ghafourian et al., 2015). More than 200 TEM and SHV variants have been documented, while 90 different enzymes within the CTX-M type have been described (Östholm, 2014). Class A enzymes hydrolyze ampicillin and extended-spectrum cephalosporins (Ghafourian et al., 2015). AmpC β-lactamases, classified as class C enzymes, are resistant to additional β-lactams, that is, cephamycins, and are not influenced negatively by class A enzyme inhibitors (Jacoby, 2009; Njage and Buys, 2017). Plasmid-mediated AmpC (pAmpC)-producing strains are distinguished from chromosomal AmpC since they are often not inducible (Mezzatesta et al., 2012). Six families of pAmpC-β-lactamases, including CIT, FOX, MOX, DHA, EBC, and ACC, have been described, with DHA, CMY (CIT family member), and FOX most commonly detected (Thomson, 2010). Co-occurrence of β-lactamase enzymes, especially AmpC β-lactamases and ESBLs, is common (Thomson, 2010).

Salmonella spp., pathogenic Escherichia coli, and Shigella spp. have been implicated in foodborne disease outbreaks, while Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, and Enterobacter spp. are regarded as opportunistic human pathogenic bacteria (Baylis et al., 2011). The presence of ESBL/AmpC-producing Enterobacteriaceae on fresh produce has been studied worldwide (Kim et al., 2015; Nüesch-Inderbinen et al., 2015; Zurfluh et al., 2015).

Transfer of multidrug-resistant (MDR) Enterobacteriaceae onto fresh produce occurs through the use of contaminated irrigation water or during production via animal manure (van Hoek et al., 2015). Subsequent transfer to humans can happen through consumption of raw vegetables, potentially impacting consumer health negatively (Ye et al., 2017). Concomitantly AR genes can easily be transferred to commensal bacteria that typically colonize the human gut.

Fresh vegetables produced in SA are retailed nationally and to the South African Development Community (SADC) countries, Swaziland, the UK, Middle East, and Asian markets (DAFF, 2012a, b, 2016). Current knowledge regarding the occurrence of ESBL/AmpC-producing Enterobacteriaceae on fresh vegetables in SA is limited. The aim of this exploratory study was to detect, identify, and characterize the AR of ESBL- and AmpC-producing Enterobacteriaceae isolates from frequently consumed fresh vegetables from selected retailing sites in Gauteng, the most densely populated province in SA.

Materials and Methods

Sample collection

A total number of 545 vegetable samples was collected from 10 formal retailers, 10 street trading greengrocers, 10 mobile trolley vendors, and 13 vendors at two farmers' markets in Gauteng, SA, from September 2017 to May 2018 (Supplementary Fig. S1). In the informal markets, street traders typically display fresh produce on a table, underneath a shade covering, at the roadside, or they use mobile trolleys. The vegetable samples included, depending on availability, spinach (bunches, baby leaves, or minimally processed ready-to-eat [RTE] pillow packs; n = 200), tomatoes (n = 200), cucumbers (n = 45), lettuce (Iceberg lettuce heads or mixed salad leaf RTE pillow packs; n = 50), and green beans (n = 50 samples). All samples were transported in cooler boxes and stored at 4°C until further processing within 24 h.

Processing of fresh produce

At least three leaves from one spinach bunch and the inner leaves of three lettuce heads were used to prepare 50 g composite samples of each of the leafy vegetable samples. Each spinach or lettuce sample was aseptically cut into a sterile polyethylene strainer stomacher bag containing 200 mL buffered peptone water (BPW) (3M, Johannesburg, SA) in a 1:4 weight-to-volume ratio. A 150 g sample of tomatoes and cucumbers (composite of at least three tomatoes or cucumbers) and a 150 g sample of green beans were each placed into a sterile polyethylene stomacher bag containing 150 mL BPW in a 1:1 weight-to-volume ratio (Xu et al., 2015). Individual vegetable samples were blended for 5 min at 230 rpm in a Stomacher 400 circulator paddle blender (Seward Ltd., London, United Kingdom).

Isolation and identification of presumptive extended-spectrum and AmpC β-lactamase-producing Enterobacteriaceae

Each of the BPW–sample mixtures was incubated for 3–4 h at 37°C after which 1 mL of each sample was added to 9 mL Enterobacteriaceae enrichment broth (Oxoid, Johannesburg, SA) according to ISO 21528-1:2004 and incubated overnight at 30°C (Blaak et al., 2014). ESBL-producing microorganisms were detected by streaking 10 μL of each of the enriched samples onto ChromID ESBL agar plates (bioMérieux, Midrand, SA) and incubated overnight at 30°C (Blaak et al., 2014). All presumptive positive ESBL/AmpC-producing Enterobacteriaceae colonies based on colony color, including weakly colored colonies, on the chromogenic media were isolated and purified.

Isolate identities were determined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) (Bruker, Bremen, Germany) to species level as described by Standing et al. (2013). A single colony on nutrient agar was transferred to the MALDI-TOF polished steel target plate and further analyzed according to manufacturer's instructions (AOAC-OMA#2017.09), following calibration with the bacterial test standard. Non-Enterobacteriaceae isolates were not included in further analysis.

Antimicrobial susceptibility testing

A selection of 77 presumptive ESBL-producing Enterobacteriaceae isolates, representing all unique species per product type from each supplier, were selected for further analysis. The Kirby-Bauer disk diffusion technique was used to determine the resistance patterns of the isolates (Clinical Laboratory Standard Institute [CLSI], 2018). All isolates were screened for ESBL production by the double-disk synergy test (DDST) using cefotaxime 30 μg, ceftazidime 30 μg, and cefpodoxime 10 μg, alone or in combination with clavulanic acid 10 μg (Mast Diagnostics, Randburg, SA) (EUCAST, 2013). Zone diameters were compared with the CLSI and EUCAST criteria to determine if isolates were resistant, intermediate, or susceptible. Isolates showing resistance to cefoxitin and cefotaxime or ceftazidime were regarded as a phenotypic indicator of AmpC production (EUCAST, 2013). Production of ESBLs was confirmed using the cefepime ESBL disc set (Cefepime 30 μg, cefepime-clavulanic acid 30–10 μg) and AmpC production using the AmpC detection set (Mast Diagnostics) (EUCAST, 2013; CLSI, 2018). Additional antimicrobials tested for resistance or susceptibility of isolates included ampicillin 10 μg, amoxicillin-clavulanic acid 20/10 μg, amoxicillin 10 μg, trimethoprim-sulfamethoxazole 1.25/23.75 μg, imipenem 10 μg, neomycin 10 μg, tetracycline 30 μg, gentamycin 10 μg, chloramphenicol 10 μg (Mast Diagnostics) (CLSI, 2018). Isolates resistant to three or more antimicrobial classes were regarded as MDR. K. pneumoniae ATCC 700603, E. coli NCTC 13315, Enterobacter cloacae NCTC 1406, and E. coli ATCC 25922 were included as positive and negative controls as described by the manufacturer (Mast Diagnostics).

Characterization of β-lactamase genes

The presence of ESBL determinants (bla _TEM, bla _SHV, bla _CTX-M, bla _OXA) and pAmpC resistance genes (bla _ACC, bla _FOX, bla _MOX, bla _DHA, bla _CIT, bla _EBC) in the selected isolates was analyzed with PCR and sequencing. Single colonies of each presumptive ESBL-producing Enterobacteriaceae isolate were cultured aerobically under shaking conditions at 200 rpm in Tryptone soy broth (MERCK, Johannesburg, SA) for 24 h at 30°C. The cells were pelleted by centrifugation (12,500 g for 10 min), DNA was extracted using the Quick-gDNA Mini-Prep kit (Zymo Research, Irvine, CA), and the DNA concentration was determined using the Qubit dsDNA Broad Range Assay and a Qubit 2.0 fluorometer (Life Technologies, Johannesburg, SA). PCR was performed using the DreamTaq Green PCR Master Mix (ThermoFisher Scientific, Johannesburg, SA), specific primers, and thermocycling conditions for each of the genes as described in Supplementary Table S1.

PCR products were sequenced using BigDye Terminator v3.1 cycle sequencing on an ABI 3500XL sequencer in forward and reverse directions (InquabaBiotec, Johannesburg, SA). The sequences were edited with Chromas 2.6 and BioEdit sequence alignment editor software, and consensus sequences were subjected to BLAST nucleotide search analysis to identify the AR genes.

Results

Identification of presumptive extended-spectrum and AmpC β-lactamase-producing Enterobacteriaceae isolates

Using MALDI-TOF analysis, 122 (28.2%) of the 432 presumptive extended-spectrum/AmpC β-lactamase-producing isolates obtained from the fresh vegetable samples were confirmed as Enterobacteriaceae belonging to 10 genera. The 310 non-Enterobacteriaceae isolates were predominantly identified as Pseudomonas spp. The Enterobacteriaceae isolates were identified as Enterobacter spp. (28.7%), including E. cloacae, Enterobacter asburiae, Enterobacter cowanii, and Enterobacter ludwigii; Serratia (18.9%), including predominantly Serratia fonticola; E. coli (18%); Klebsiella spp. (14.8%), including K. pneumoniae and Klebsiella oxytoca; Rahnella aquatilis (9%); Proteus spp. (4.9%), including Proteus penneri and Proteus mirabilis; Citrobacter spp. (2.5%), including Citrobacter farmeri and C. freundii; Kluyvera ascorbata (1.64%); Achromobacter xylosixidans (1.6%); and Raoultella ornithinolytica (0.8%). Presumptive ESBL/AmpC-producing Enterobacteriaceae were isolated from the vegetable types tested.

Phenotypic AR profiling

All the 77 selected presumptive ESBL-producing Enterobacteriaceae showed resistance to more than one antimicrobial agent, with 96.1% being MDR (resistant to ≥3 antimicrobial classes) (Fig. 1). Resistance to the aminoglycoside and chloramphenicol classes was dominant, observed in 94.8% and 85.7% of the isolates, respectively. All isolates with cephalosporin resistance (CTX30C, CAZ30C, CPD10C, or CPM30C) were further screened using DDST, after which 61/77 (79.2%) were tested positive for ESBL production (Fig. 1). All isolates that showed cefoxitin resistance (n = 46) were additionally screened with the AmpC detection set. From these 46 isolates, 32/77 (41.6%) were tested positive for AmpC production. This included 27 isolates showing resistance to cefoxitin, ceftazidime, and/or cefotaxime and additionally five isolates that showed cefoxitin resistance, but ceftazidime and/or cefotaxime susceptibility. All isolates displaying ESBL or AmpC phenotypes were further characterized for the identification of ESBL and/or AmpC resistance genes.

FIG. 1.

Summary of the species isolated from different fresh vegetables, indicating the phenotypic resistance profiles and the extended-spectrum β-lactamase/AmpC genetic determinants detected. The color code is given in the lower left corner of each section in grayscale: species identification (dark grey); isolate origin (black); phenotypic antimicrobial resistance—resistant (grey), intermediate resistant (light grey), or susceptible (white); genotypic determinants (black). AP10C, ampicillin; AUG30C, amoxicillin-clavulanic acid; A10C, amoxicillin; FOX30C, cefoxitin; CPM30C, cefepime; CPD10C, cefpodoxime; CPD10C/CLAV1C, cefpodoxime-clavulanic acid; CAZ30C, ceftazidime; CAZ/CLAV10C, ceftazidime-clavulanic acid; CTX30C, cefotaxime; CTX/CLAV10C, cefotaxime-clavulanic acid; TS25C, trimethoprim-sulfamethoxazole; IMI10C, imipenem; T30C, tetracycline; NE10C, neomycin; C10C, chloramphenicol.

Genotypic AR profiling

Genes encoding β-lactamases were detected in 58/77 (75.3%) isolates obtained from all vegetable types, mainly in E. coli (n = 20), Enterobacter spp. (n = 12), and Serratia spp. (n = 11) isolates. This included 37 (48%) broad-spectrum, 39 (51%) ESBL, and 20 (25.9%) AmpC genetic determinants (Fig. 1). The most frequently detected β-lactamase genes were bla _CTX-M (n = 28), followed by bla _SHV (n = 22), bla _TEM (n = 21), and bla _OXA (n = 5). ESBLs encoded by bla _CTX-M included CTX-M-14 (n = 15), CTX-M-15 (n = 6), CTX-M-27 (n = 4), and CTX-M-55 (n = 3); bla _TEM genes encoded TEM-3 (n = 3), while bla _SHV genes encoded SHV-18 (n = 6), SHV-28 (n = 1), and SHV-154 (n = 1). All the bla _OXA, 85.7% (n = 18) of the bla _TEM, and 63.6% (n = 14) of the bla _SHV sequences encoded broad-spectrum β-lactamases OXA-1, TEM-1, TEM-215, SHV-1, SHV-11, or SHV-26, respectively. Three isolates harbored more than one ESBL; one E. coli isolate carried the bla _TEM-3, bla _SHV-18, and bla _CTX-M-14 genes, and two isolates (E. coli and E. cowanii) carried the bla _TEM-3 gene in association with bla _CTX-M-14 and bla _SHV-18 genes, respectively. In 12 isolates (E. coli [n = 3]; Enterobacter spp. [n = 3]; Serratia spp. [n = 3]; R. aquatilis [n = 2]; and P. mirabilis [n = 1]), ESBL genes in association with broad-spectrum β-lactamases were detected (Fig. 1).

AmpC resistance genes were detected in 18/58 (31%) isolates harboring β-lactamase genetic determinants (Fig. 1). In 17 isolates, only one pAmpC genetic determinant was detected; bla _MIR-20 (n = 4), bla _MIR-16 (n = 3), bla _ACT-58 (n = 2), and one isolate each carried bla _CMY-2, bla _MIR-14, bla _ACT-29, bla _ACT-10, bla _ACT-2, bla _EC, bla _CMY-161, or bla _CMY-87 respectively. Among these 17 isolates, five isolates (Enterobacter spp. [n = 2], E. coli [n = 1], R. aquatilis [n = 1], and S. fonticola [n = 1]) also harbored ESBL genetic determinants. One P. penneri isolate carried three AmpC genes (bla _ACT10, bla _DHA-18, and bla _CMY-49). The EBC family of the AmpC genetic determinants was the most dominant type.

Discussion

MDR ESBL/AmpC-producing Enterobacteriaceae were detected for the first time in raw vegetables retailed at selected sites in Gauteng Province, SA. Antibiotic-resistant opportunistic pathogens on fresh produce are a serious health concern that contributes toward the burden of AR in different environments, leading to increased risk of infection if colonization in humans occurs (Al-Kharousi et al., 2016). Enterobacteriaceae regarded as emerging bacterial threats include E. coli, K. pneumoniae, and Enterobacter spp. showing resistance to β-lactams and aminoglycosides (Fair and Tor, 2014).

Presumptive ESBL producers, predominantly E. coli, K. pneumoniae, E. cloacae, and E. asburiae, were detected in 17.4% of our vegetable samples analyzed. This is lower than the 25.4% reported by Zurfluh et al. (2015) for imported vegetables into Switzerland from the Dominican Republic, India, Thailand, and Vietnam, but higher than the 6% reported by Reuland et al. (2014) on retail vegetables in the Netherlands. Similar to Blaak et al. (2014), environmental ESBL-producing Enterobacteriaceae isolated from vegetables included S. fonticola and R. aquatilis.

Phenotypic confirmation of ESBL/AmpC production showed that 61 (79.9%) of the 77 analyzed Enterobacteriaceae isolates displayed an ESBL-producing phenotype and 41.6% an AmpC-producing phenotype, which is higher than results reported by van Hoek et al. (2015). Combined ESBL- and AmpC-producing phenotypes were also observed in 35% of the isolates. MDR phenotypes (resistance to ≥3 antimicrobial classes) were observed in 96.1% of our analyzed isolates. The most prevalent non-β-lactam resistance profiles showed resistance against aminoglycoside (94.8%), chloramphenicol (85.7%), and tetracycline (53.2%). This is higher than reports from similar studies that showed resistance to aminoglycosides (46.7–66.7%), chloramphenicol (33.3%) (Zurfluh et al., 2015; Ben Said et al., 2016), and tetracycline (46.7%) (Ben Said et al., 2016) in ESBL-producing Enterobacteriaceae.

Genes expressing broad-spectrum β-lactamases, ESBLs, and/or AmpC β-lactamases were detected in 69.9% of our MDR isolates. Co-expression of ESBL and AmpC genes in environmental (van Hoek et al., 2015; Ye et al., 2017) and clinical (Tau et al., 2012; Kharat et al., 2017) Enterobacteriaceae isolates has also been reported. Globally the bla _CTX-M-type ESBL genes are predominant in Enterobacteriaceae, which was similar in our study, the majority being detected in E. coli isolates. bla _CTX-M-14 was the main genetic determinant detected from mostly E. coli and C. freundii isolates, which corresponds to results obtained from vegetable samples in Tunisia (Ben Said et al., 2016). Isolates harboring bla _CTX-M-15 included E. coli, E. cloacae, K. pneumoniae, R. aqualtilis, and S. fonticola and were second most prevalent in our study. bla _CTX-M-15 was the most prevalent gene detected in E. coli and K. pneumoniae isolates from fresh vegetables imported into Switzerland from India and the Dominican Republic (Zurfluh et al., 2015). This is in agreement with reports that bla _CTX-M-14 and bla _CTX-M-15 are predominant and have been associated with clinically relevant Enterobacteriaceae infections (Ehlers et al., 2009; Zurfluh et al., 2015).

In contrast to Njage and Buys (2014), who predominantly detected bla _{CTX-M Group 8/25}-positive E. coli isolates from lettuce in the North West Province (SA), no bla _{CTX-M Group 8/25} genes were detected in any of our E. coli isolates from the vegetable samples analyzed. The bla _CTX-M-15 (CTX-M group 1) and bla _CTX-M-14 (CTX-M group 9) genes detected in our environmental isolates, reported to be closely related to chromosomally encoded bla _FONA and bla _RAHN genes of S. fonticola and R. aquatilis, had no significant similarity in the GenBank database using NCBI BLAST based on total BLAST alignment scores. This contrasts results reported by Raphael et al. (2011) where sequences similar to bla _RAHN-2 and bla _FONA-5 were detected using bla _CTX-M primers.

In our study, five isolates, including E. coli, Enterobacter spp., R. aquatilis, S. fonticola, simultaneously harbored ESBL and AmpC genes. Environmental isolates are known to carry chromosomally encoded AmpC β-lactamases. However, Enterobacteriaceae harboring both chromosomal and pAmpC β-lactamases are increasingly reported to hydrolyze broad-spectrum cephalosporins more efficiently, resulting in adverse treatment options in clinical settings (Jacoby, 2009; Reuland et al., 2014).

The 18 isolates in which pAmpC resistance genes were detected predominantly included the EBC-type pAmpC β-lactamases (identified as bla _ACT/bla _MIR). This contrasts with two previous studies where bla _CIT, bla _DHA, or bla _ACC pAmpC β-lactamases were mostly detected in Enterobacteriaceae isolated from fresh produce and water samples (Njage and Buys, 2014; Ye et al., 2017). bla _ACT/MIR genes have been reported to be the dominant AmpC genetic determinants in Enterobacter spp., causing intra-abdominal infections (Khari et al., 2016), and were detected in seven of the Enterobacter spp. isolates in our study. The fact that fresh produce can serve as a reservoir of MDR ESBL/AmpC-producing Enterobacteriaceae, including their genetic determinants, constitutes a potential health risk to the consumer as resistance to antimicrobials frequently used to treat human infections was shown.

Conclusion

The results obtained from screening at these selected sites indicate that further investigation of different fresh produce types in Gauteng and other provinces in SA is necessary. Future studies should focus on the surveillance of production systems from farm to retail to identify potential sources of contamination that contribute to the presence and dissemination of antimicrobial-resistant microorganisms and their genetic determinants. Since AR is a worldwide problem, a global solution is required that integrates the contributions from government departments as well as from the scientific community.

Footnotes

Acknowledgments

The authors would like to acknowledge the financial assistance of the Water Research Commission in SA and the Department of Science and Technology–National Research Foundation (NRF), Centre of Excellence in Food Security. MALDI-TOF analysis was based on the research supported in part by the NRF (grant specific reference number (UID) 74426). Conclusions arrived at are those of the authors and are not necessarily to be attributed to the NRF. The authors would like to acknowledge Dr. Germán Villamizar-Rodríguez for guidance with genotypic characterization, and Ms. Zama Zulu for assistance with MALDI-TOF identification.

Disclosure Statement

No competing financial interests exist.

Supplementary Material

Supplementary Figure S1

Supplementary Table S1

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