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ISSN: 2476-2059
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DOI: 10.4172/2476-2059.1000113
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Research Article Open Access

Evaluation of Antimicrobial Resistance in Enterobacteriaceae and Coliforms

Isolated on Farm, Packaged and Loose Vegetables in Kentucky
Avinash M Tope*, Alexandra C Hitter and Shreya V Patel
Division of Food and Animal Science, College of Agriculture, Food Science and Sustainable Systems, Kentucky State University, Frankfort, KY 40601, USA
*Corresponding author: Avinash M Tope, Division of Food and Animal Science, College of Agriculture, Food Science, and Sustainable Systems, Kentucky State

University, 400 East Main St., Frankfort, KY 40601, USA, Tel: 502-597-6012; E-mail: [email protected]
Received date: 13 July, 2016; Accepted date: 10 August, 2016; Published date: 16 August, 2016
Copyright: © 2016 Tope AM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Fresh produce normally carry epiphytic microorganisms; however, it can be contaminated with pathogenic
bacteria. Categorized as ‘ready-to-eat’, most vegetables are consumed raw, thus, may present a food safety risk.
Over the last three decades, global consumption of fresh vegetables has increased considerably, and the market
has expanded by more than 20%. Concomitantly, the number of outbreaks involving fresh vegetables has increased
significantly. Enterobacteriaceae members are involved in most of the bacterial outbreaks linked to fresh produce.
There is a worldwide concern about the increased use of antimicrobials in agriculture. Antimicrobial resistant
bacteria enter the food chain from the farm, often due to the use of animal manure. In the current study, the
presence of entero-coliform bacteria and their antimicrobial susceptibilities in fresh vegetables sampled from small
farms to retail was evaluated. Samples of vegetables were (i) collected directly from small farms from central
Kentucky (n=59) and (ii) from four supermarkets in Frankfort, KY including loose and pre-packaged produce (n=72),
analyzed for isolation of entero-coliform species. Members of the Enterobacteriaceae were detected on 25% of farm
and 40% of retail produce, respectively. Approximately, 61% of the packaged produce and 19.4% of the loose
produce had entero-bacterial presence, respectively. Their resistance to fourteen common antimicrobials was tested
using Kirby-Bauer method. Approximately, 63% of isolates from farm and 70% of isolates from retail produce
displayed resistance to at least three antimicrobial agents, while 18% of the isolates from farm and 41% from retail
samples displayed resistance to at least ten antimicrobial agents. We conclude that ‘ready-to-eat’ fresh vegetables
can be a source of exposure to pathogens with multiple drug resistance (MDR), defined as resistance to at-least
three antimicrobial agents, leading to greater risks in immunocompromised individuals, and may serve as reservoirs
for resistance gene transfers in human colon.

Keywords: Enterobacteriaceae; Produce; Farm; Retail; Packaged; production, processing, distribution, and retail. Some of these products
Loose; Antimicrobial resistance; Multiple drug resistance have also gone through packaging, which can create an additional
chance for contamination to occur. In the United States, more than
Introduction 1500 different types of packagings are in use including items such as
bags, crates, hampers, baskets, cartons, bulk bins, and pallets [4].
Over the last three decades the global consumption of fresh
vegetables has increased significantly, thus the market segment for Classified as ‘ready-to-eat’ food, most fresh fruits and vegetables can
fresh produce has expanded by more than 20% [1]. A survey be and are consumed raw, without needing further processing, such as
conducted on American consumers’ choice of supermarkets revealed cooking, thereby can pose a food safety problem. Most fresh vegetables
that freshness of the produce and its availability across the year was the normally carry non-pathogenic epiphytic microorganisms, however,
single most deciding factor. In an effort to understand consumers’ contamination at the farming sites can arise because of various types of
attitudes toward produce packaging, the Produce Marketing soil treatments such as organic fertilizers, which include sewage sludge
Association, Yerecic Label and the Perishables Group conducted a and manure, use of contaminated irrigation water, as well as from the
three-part study. Almost 90% of the participants responded that the ability of pathogens to persist and proliferate in vegetables [3,5]. The
most important feature of packaged produce is its ability to preserve number of reported outbreaks involving fresh vegetables have
freshness and taste [2]. All this has led to a greater availability of “four increased significantly. The top five most concerning foodborne
range” produce, a term that refers to packaged, cleaned, possibly pathogens associated with recent outbreaks involving fresh fruits and
chopped and sometimes-mixed produce ready for consumption. vegetables were Listeria monocytogenes, Salmonella (non-typhoidal
Consumers also mentioned that they are attracted to packaged serotypes only), Escherichia coli (E.coli) O157:H7, E. coli non- O157
produce because of labels that contain information about the origin of STEC, and Campylobacter [6]. On average, Listeria monocytogenes
the produce, recipes and cooking ideas, nutritional information, and causes 1600 illnesses each year in the U.S. Of these illnesses, 1400
sell-by-date [3]. result in hospitalizations, Resulting in 250 deaths [7]. Since March
2016, the Centers for Disease Control and Prevention (CDC) has been
Fresh fruits and vegetables available at retail supermarkets, unlike collaborating with public health officials in several states and the US
produce at the farm have gone through the entire process of the food Food and Drug Administration (FDA) to investigate a multistate
supply chain. These products are vulnerable for potential outbreak of Listeria monocytogenes infections [8]. In March 2016, a
contamination at multiple avenues as they go through farm specific brand of pistachios were implicated in a Salmonella outbreak

J Food Microbiol Saf Hyg, an open access journal Volume 1 • Issue 2 • 1000113
ISSN:2476-2059
Citation: Tope AM, Hitter AC, Patel SV (2016) Evaluation of Antimicrobial Resistance in Enterobacteriaceae and Coliforms Isolated on Farm,
Packaged and Loose Vegetables in Kentucky. J Food Microbiol Saf Hyg 1: 113. doi: 10.4172/2476-2059.1000113

Page 2 of 7

[9]. There was a multistate outbreak of Shiga toxin-producing In the current study, following objectives were pursued. (1)
Escherichia coli O157 infections linked to Alfalfa sprouts produced by Determine the presence of Coliform and Enterobacteriaceae on
Jack & The Green Sprouts [10]. produce from farms and packaged and loose varieties of produce from
retail supermarkets as a risk factor for consumers; (2) Determine the
Enterobacteriaceae family of bacteria have a natural habitat in the
antimicrobial resistance profiles of Coliform and Enterobacteriaceae
digestive tract of warm-blooded animals. They are commonly found in
members isolated from farm and retail produce samples.
soils, plants, and in water [11] and can survive in soil depending on the
soil type, temperature and moisture content [12,13]. Many of these
bacteria found in fresh produce carry resistance factors to multiple Materials and Methods
antimicrobials, and thereby pose additional safety concerns for
consumers [14,15]. Sample collection
There is a worldwide concern about the increased prevalence of Farm samples: During May to September 2014, twenty small farms
antimicrobial resistance in bacteria. In 2011, 13,569,037 kg of were visited 1 to 3 times. Consenting small and limited- resource
antibiotics were used in production of food animals in the US. Of these farmers were contacted and recruited through a mailing list from
antibiotics, the FDA deemed only 8,255,697 kg as “medically Kentucky State University’s current outreach and extension programs,
important” [16]. In the conventional livestock industry, animals are such as the Small Farm Program, Third Thursday Thing, the Socially
usually maintained indoors in small and unsanitary living conditions, Disadvantaged Farmer Outreach Project and the Organic Association
fed antibiotics mixed in the feed, sometimes daily, as a “medically of Kentucky. Each farmer was given a survey that explored details
important” substitute to their living conditions [17]. The remaining regarding certification status, fertilization practices (such as the type(s)
amount of these antibiotics found a use mostly for promoting feed of manure or compost and/or chemicals, age of the manure or compost
efficiency, weight gain, and faster production rates. Thus, commercial and the time of application), the source of irrigation water,
animal husbandry, especially involving pig and cattle are the largest surrounding land use, handling practices during harvesting, post
users of antibiotics as growth promoters [18]. Antimicrobial resistant harvesting and handling practices such as washing, packaging and
bacteria enter the food chain from the farm environment, often storage.
through water runoff, animal manure, and spread to agricultural plants
During each visit, 1 to 2 samples of produce were randomly picked
[19]. The genes for antibacterial resistance have the potential for
from various locations on the field and immediately put into zip-lock
horizontal transfer to other related and non-related species, including
bags that were wiped with 70% alcohol. The sample size for small
the gastro-intestinal tracks of livestock, to the manure and can survive
vegetables and fruit was less than 100 grams. Samples from each were
even in composted animal waste.
assigned a code ID number, and were labeled with the date,
The nonselective and widespread use of antimicrobial agents in food immediately placed in a cooler, and transported to the laboratory
animal production systems remains to be the main cause for increased (Frankfort, KY) for analyses. Samples were kept in the refrigerator
resistance in pathogenic bacteria [20,21]. Most antimicrobial agents until analysis began. A total of 59 samples was collected from twenty
used in the food production system are the same antibiotics used in farms, which included conventional and organic (certified and non-
treating humans [22]. Since 1995, there has been a significant increase certified) farms.
in the use of fluoroquinolones in treating and preventing E. coli
Retail samples: During December 2014 to March 2015, three select
infections in chicken and turkeys prior to slaughter [23]. Over the past
varieties of vegetables (tomatoes, carrots and leafy greens), were
decade, there has been a 25% increase in fluoroquinolone resistance
collected at random, from 4 different grocery supermarkets, two of
for treating human E. coli infections [24]. Other top antibiotics used in
which were large chain supermarkets, and two were small
the pig and cattle industry include β-lactams, such as various
supermarkets in a lower income area from Frankfort, KY. Each
penicillins, and other classes of antibiotics such as macrolides, and
supermarket was visited thrice. From each supermarket, 50% of
tetracyclines.
produce samples were packaged and remaining 50% samples were
Bacteria with antimicrobial resistance are found in soil, and water from loose varieties. During each visit, packaged and loose vegetables
even in produce farms that do not use manure for fertilization. The of three varieties were picked from the shelf and immediately put into
likely source of the antimicrobial resistant bacteria in such farms zip-lock bags that were wiped with 70% alcohol. For supermarkets that
include water runoff, often from neighboring food animal farms. Many did not offer both packaged and loose varieties of carrots, potatoes
growers create buffer zones of unfarmed land to try to alleviate the risk were substituted. The sample size for the vegetables was less than 100
of water runoff from neighboring farms. However, information grams. Samples from each were assigned a code ID number, and were
regarding the effective location and size of the buffer zone required to labeled with the date, immediately placed in a cooler, and transported
minimize the risk of water runoff from such farms is yet unclear [25]. to the laboratory for analyses, which began within 24 h after collection.
Another potential source for developing antimicrobial resistance in A total of 72 produce samples was collected from four supermarkets
produce is through their traces in municipal water. Although water and each sample was tested in duplicate for detection of Coliforms and
treatment has been shown to remove most of the antibiotics, studies Enterobacteriaceae.
show that traces of certain classes of antibiotics do remain [26]. A
study conducted in Spain reported sulfonamides to be the most E. coli and Enterobacteriaceae identification
commonly detected antibiotics in sewage sludge and soil [27].
Using aseptic conditions, 10 grams of sample was then mixed with
There are a number of reports of antimicrobial resistance in bacteria 100 mL of lauryl sulfate tryptose broth (LST; Fisher Scientific,
found in meat products [28,29]. However, there are relatively fewer Hanover, IL), and pummeled in the 400 circulator machine for 5 min,
such reports on produce [30]. at 230 rotations per minute(RPM) (Seward Limited, London, UK). The
mixture was then placed in 9 mL of LST, in serial dilutions up to 10-4.

J Food Microbiol Saf Hyg, an open access journal Volume 1 • Issue 2 • 1000113
ISSN:2476-2059
Citation: Tope AM, Hitter AC, Patel SV (2016) Evaluation of Antimicrobial Resistance in Enterobacteriaceae and Coliforms Isolated on Farm,
Packaged and Loose Vegetables in Kentucky. J Food Microbiol Saf Hyg 1: 113. doi: 10.4172/2476-2059.1000113

Page 3 of 7

One milliliter of the serial dilution of 10-3 was plated onto E. coli, and Statistical analysis
Enterobacteriaceae Petrifilm plates (3M Company, Maplewood, MN)
and 100 µL of the serial dilution of 10-3 was plated onto Eosin IBM SPSS statistical software (Chicago, IL) and Microsoft Excel
Methylene Blue (EMB; Fisher Scientific, IL) agar plates in duplicate, were used for statistical analyses. The Pearson Chi-Square was used to
and placed into the incubator at 37ºC for 48 h. Predominant analyze the differences between groups. Statistical significance was
Enterobacteriaceae isolates in the produce samples were identified defined at P<0.05.
using API 20E kit for biochemical characterization (Bio-Merieux Inc.,
Marcy I’Etoile, France). The API 20E testing strips were read and final Antimicrobial susceptibility
identification was made using API LAB PLUS computer software (Bio-
The antimicrobial susceptibility testing was performed by the
Merieux Inc., France).
standard CLSI (formerly known as NCCLS) using the Kirby Bauer disk
Analysis of E. coli O157:H7 was conducted for E. coli positive diffusion technique with Mueller-Hinton agar (Fisher Scientific, IL)
samples from the Petrifilm plates. The E. coli colonies were directly [31]. The antibiotics used in this study were amikacin (30 μg),
transferred from the Petrifilms into Nutrient broth (Remel, Lenexa, amoxicillin (30 μg), ampicillin (10 μg), cefoxitin (30 μg), ceftiofur (30
KS), and incubated at 37ºC, 24 h. One-hundred microliters of the μg), ceftriaxone (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg),
sample was plated onto CHROMagar O157 (CHROMagar gentamycin (10 μg), kanamycin (30 μg), nalidixic acid (30 μg),
Microbiology, Paris, France) agar, and placed into the incubator at streptomycin (10 μg), tetracycline (30 μg), and trimethoprim (5 μg).
37ºC, 48 h. E. coli O157:H7 was confirmed when characteristic mauve Bacterial cultures were grown in 5 mL of nutrient broth at 37ºC, 24 h.
colored colonies in the background of blue or steel gray colonies were Each overnight culture was spread evenly onto Mueller-Hinton agar
observed. and incubated at 37ºC, 48 h and the zones of inhibition were
measured.
Isolation of Salmonella spp.
Results
Colonies first identified through the API 20E biochemical testing kit
to be positive for Salmonella were placed into 5 mL of nutrient broth
and incubated at 37ºC, 24-48 h to stimulate growth. One milliliter of Farm samples
the nutrient broth cultures were transferred into 10 mL tetrathionate The occurrence of all Enterobacteriaceae including Coliforms
broth and incubated at 47ºC, 24 h for selective enrichment. isolated from farms and their antimicrobial resistance profiles is
Tetrathionate enrichment cultures (250 µL) were spread onto selective presented in Table 1. The most common strain identified from
Xylose Lysine Tergitol 4 agar (XLT4, Difco, Becton Dickinson, Sparks, Enterobacteriaceae was Pantoea spp. (27.3%). Other potentially
MD) and incubated at 37ºC, 24 h. The plates were evaluated for pathogenic isolates include Cronobacter sakazakii (18.2%), Serratia
colonies typical of Salmonella species after 24 h of incubation. The marcescens (18.2%), and Stenotrophomonas maltophilia (9.1%). While
colonies were also subjected to a second API biochemical testing for Cronobacter sakazakii and Serratia marcescens were resistant to nine
final confirmation. antibiotics, Pantoea spp isolates were resistant to thirteen of fourteen
antibiotics tested.

Bacteria % Detected Antibiotic Resistance

Kanamycin, Ciprofloxacin, Nalidixic acid, Chloramphenicol, Gentamycin, Ceftiofur, Amikacin, Ceftriaxone,

Cronobacter sakazakii 18.2 Trimethoprim.

Amoxicillin, Ceftiofur, Amikacin, Ceftriaxone, Nalidixic acid, Chloramphenicol, Gentamycin, Ampicillin,

Enterobacter cloacae 9.1 Tetracycline, Kanamycin, Trimethoprim.

Amoxicillin, Ceftiofur, Cefoxitin, Amikacin, Nalidixic acid, Chloramphenicol, Gentamycin, Ampicillin,

Pantoea spp. 27.3 Tetracycline, Ciprofloxacin, Streptomycin, Kanamycin, Trimethoprim

Proteus mirabilis 9.1 Ceftiofur, Ceftriaxone, Streptomycin, Trimethoprim.

Amoxicillin, Nalidixic acid, Ciprofloxacin, Gentamycin, Ampicillin, Tetracycline, Streptomycin, Kanamycin,

Serratia marcescens 18.2 Trimethoprim.

Serratia liquefacians 9.1 Ampicillin, Trimethoprim

Stenotrophomonas maltophilia 9.1 Gentamycin, Trimethoprim

Table 1: Occurrence (%) and Resistance pattern of Enterobacteriaceae isolated from farm produce.

Retail Samples Three different types of loose and packaged vegetables (leafy greens,
tomatoes, and carrots) were collected during each experiment. These
Approximately 40.3% of the samples tested positive for presence of produce types were chosen based on a previous study in which higher
Enterobacteriaceae. Of the packaged produce samples, 61.1% tested contamination of E. coli was observed in produce samples that grew at
positive for Enterobacteriaceae, while only 19.4% of the loose produce or below the surface of the plant compared to produce samples that
samples tested positive for Enterobacteriaceae, and this was statistically grew above the surface [32]. Each vegetable was chosen as
significant (p ≤ 0.03) (Table 2).

J Food Microbiol Saf Hyg, an open access journal Volume 1 • Issue 2 • 1000113
ISSN:2476-2059
Citation: Tope AM, Hitter AC, Patel SV (2016) Evaluation of Antimicrobial Resistance in Enterobacteriaceae and Coliforms Isolated on Farm,
Packaged and Loose Vegetables in Kentucky. J Food Microbiol Saf Hyg 1: 113. doi: 10.4172/2476-2059.1000113

Page 4 of 7

representative of the placement, i.e. carrots as produce growing under presented in Table 3. As shown, the most common type of bacterial
the surface, leafy greens as produce growing at surface level, and strain (31%) was Stenotrophomonas maltophilia. Other potentially
tomatoes as produce growing above the surface level. As reported pathogenic isolates were Acinetobacter baumannii (3.4%), Serratia
earlier [32], most Enterobacteriaceae were detected (18.5%) on carrots, marcescens (17.2%), and Citrobacter freundii (3.4%). While
followed by tomatoes and leafy greens. Stenotrophomonas maltophilia strains were resistant to four
antibiotics, the remaining two isolates, Serratia marcescens and
Enterobacteriaceae Enterobacteriaceae Total Chrysobacterium indologenes were resistant to at least ten of the
Produce positive samplesª (%) positive samplesª samples
fourteen antibiotics tested.
Packaged 61.1 A 22 36 No E. coli was detected in any of the retail produce samples that
Loose 19.4 B 7 36 were tested. Only one sample tested positive for Salmonella spp. As
mentioned earlier, the most common isolate was Stenotrophomas
Total 40.3 29 72 maltophilia (31.0%). S. maltophila are gram-negative bacteria often
found in soil, water, and plants. They are opportunistic pathogens,
Table 2: Detection of Enterobacteriaceae in packaged and loose becoming increasingly more virulent, especially in hospitalized
produce samples from supermarkets *. ªPairs of data having different patients, and is associated with mortality rates of 14-69% in patients
letters (A and B) were significantly different (P<0.05). *Pearson Chi- with bacteremia [33].
Square test was performed (TS=12.9906; df=1, p- value <0.05)
Resistance patterns
%
Detecte
The prevalence of drug-resistant Enterobacteriaceae isolated from
Bacteria d Antibiotic Resistance farm produce is represented in Table 4. The results indicate that all the
Enterobacteriaceae isolates were resistant to trimethoprim (100%).
Amoxicillin, Ceftiofur, Amikacin, Cefoxitin,
Ceftriaxone, Nalidixic acid, Chloramphenicol, Antimicrobial Agent Concentration (µg) % Resistant % Susceptible
Acinetobacter Ciprofloxacin, Gentamycin, Ampicillin, Tetracycline,
baumannii 3.4 Streptomycin, Kanamycin, Trimethoprim. Amoxicillin 30 36.4 63.6

Amoxicillin, Ceftiofur, Cefoxitin, Ceftriaxone, Ceftiofur 30 36.4 63.6

Chryseobacteriu Nalidixic acid, Chloramphenicol, Ciprofloxacin,
m indologenes 10.3 Ampicillin, Tetracycline, Trimethoprim.
Cefoxitin 30 27.3 72.7
Proteus mirabilis 6.9 Ampicillin, Cefoxitin, Nalidixic acid, Trimethoprim.
Amikacin 30 36.4 63.6
Salmonella
Ceftriaxone 30 36.4 63.6
paratyphi A 3.4
Chloramphenicol 30 36.4 63.6
Amoxicillin, Ceftiofur, Amikacin, Cefoxitin,
Ceftriaxone, Chloramphenicol, Ciprofloxacin,
Kanamycin 30 54.5 45.5
Serratia Gentamycin, Ampicillin, Tetracycline, Kanamycin,
marcescens 17.2 Trimethoprim.
Ampicillin 10 72.7 27.3
Serratia Ciprofloxacin, Gentamycin, Kanamycin, Nalidixic
Gentamycin 10 36.4 63.6
liquefaciens 3.4 acid, Streptomycin, Trimethoprim
Ciprofloxacin 5 27.3 72.7
Stenotrophomon
as maltophilia 31.0 Ceftiofur, Amikacin, Ceftriaxone, Trimethoprim.
Streptomycin 10 36.4 63.6
Pasteurella
pneumotropica 3.4 Ampicillin, Kanamycin, Trimethoprim Nalidixic Acid 30 45.5 54.5

Pseudomonas Tetracycline 30 54.5 45.5

luteola 3.4 Ceftriaxone, Nalidixic acid, Trimethoprim.
Trimethoprim 5 100 0
Citrobacter
braakii 3.4 DR1 ≥ 1 100

Burkholderia MDR2 ≥ 3 63.6

cepacia 3.4
MDR3 ≥ 10 18.2
Citrobacter
freundii 3.4
Table 4: Prevalence of drug-resistant Enterobacteriaceae (%) from
Pantoea spp. 6.9 produce sampled from farms. 1Drug resistance to one or more
antimicrobials. 2Microbial drug resistance to 3 or more antimicrobials.
3Microbial drug resistance to 10 or more antimicrobials.
Table 3: Occurrence (%) Enterobacteriaceae from supermarket samples
and their resistance profile.
All Enterobacteriaceae isolates from farms and supermarkets were
The occurrence of individual Enterobacteriaceae member from the resistant to at least one antibiotic. Overall, 63.6% of the isolates
retail supermarkets along with their antimicrobial resistance profile is displayed MDR to at least five antibiotics, whereas 18.2% of the isolates

J Food Microbiol Saf Hyg, an open access journal Volume 1 • Issue 2 • 1000113
ISSN:2476-2059
Citation: Tope AM, Hitter AC, Patel SV (2016) Evaluation of Antimicrobial Resistance in Enterobacteriaceae and Coliforms Isolated on Farm,
Packaged and Loose Vegetables in Kentucky. J Food Microbiol Saf Hyg 1: 113. doi: 10.4172/2476-2059.1000113

Page 5 of 7

displayed MDR to at least-ten antibiotics. No statistically significant times the oxygen level is below 1%, even though the FDA recommends
differences were observed in the MDR profiles of bacterial isolates the oxygen level reach 1-3% to maintain safety and quality of the
from organically and conventionally raised produce. produce [36].
The prevalence of drug-resistant Enterobacteriaceae isolated from The results of this study indicate that packaged produce was more
retail produce is represented in Table 5. Generally, the results indicate contaminated with Enterobacteriaceae than loose varieties. In
that the Enterobacteriaceae tested were resistant to nalidixic acid addition, since a certain packaging type (MAP) affects the oxygen level
(86.2%) and trimethoprim (75.9%). Approximately 70% of the isolates in produce affecting the overall quality, a vigorous evaluation of
displayed MDR to at least five antibiotics, whereas 41.4% of the isolates efficacy of various packaging types is required.
displayed MDR to at least ten antibiotics.
In efforts to lower the chances of foodborne outbreaks from produce
The intent of the present study was to provide some assessment on the FDA offers tips on how to handle fresh fruits and vegetables safely.
the Enterobacteriaceae count, vis-à-vis, and the microbiological quality Although washing raw produce by the consumer is encouraged, the
of packaged and loose produce available at retail grocery supermarkets FDA mentions that there is no need to wash the produce prior to
and offer a comparative evaluation with produce on the farm. Based consumption if the package indicates that the produce is pre-washed.
largely on unfounded reports that loose produce poses a greater risk of [5]. However, in 2010 Consumer Reports published a report on tests
foodborne illness than packaged produce [34], however, there are few performed on 208 bags of “pre-washed” greens from 16 different
scientific reports on the microbiological analysis of packaged and loose brands of salad greens. In that study, while the top foodborne
retail produce [35]. pathogens such as E coli O157:H7 and Salmonella were not found, the
study reported that 39% of the packaged greens exceeded safe levels for
Antimicrobial Agent Concentration (µg) % Resistant % Susceptible coliform bacteria, and 23% had exceeded safe levels for Enterococcus
Amoxicillin 30 44.8 51.1
bacteria, which are the two indicators of poor sanitation methods and
fecal contamination [37,38].
Ceftiofur 30 48.3 51.7
Overall, all Enterobacteriaceae members isolated from both farm
Cefoxitin 30 51.7 48.3 and retail produce samples were resistant to trimethoprim. A one-year
study evaluating impact of production environment on the prevalence
Amikacin 30 55.2 44.8 of antimicrobial resistance reported isolating E.coli, 4.5% of which
Ceftriaxone 30 58.6 41.4 were resistant to multiple antibiotics, including trimethoprim and
sulfamethoxazole [39]. In another study from Jamaica, antimicrobial
Chloramphenicol 30 37.9 62.1 resistance of Pseudomonas aeruginosa isolated from fresh produce
from retail and supermarkets reported isolating strains, 5% of which
Kanamycin 30 55.2 44.8
were resistant to at-least three antibiotics, including trimethoprim and
Ampicillin 10 58.6 41.4 sulfamethoxazole [40].

Gentamycin 10 51.7 48.3 The Enterobacteriaceae isolated from farm produce displayed
greater resistance to ampicillin (72.7%) than the Enterobacteriaceae
Ciprofloxacin 5 55.2 44.8 isolated from supermarket produce (58.6%). For the farm produce
samples, the most common isolate was Pantoea spp. (27.3%),
Streptomycin 10 41.4 58.6
commonly found in soil and plants. As observed in the previous
Naladixic Acid 30 86.2 13.8 studies, most of the Pantoea spp. isolated in the current study was
resistant to aminoglycosides as well as to ampicillin [41,42]. The
Tetracycline 30 58.6 41.4
Enterobacteriaceae isolated from supermarket produce displayed
Trimethoprim 5 75.9 24.1 greater resistance to nalidixic acid (86.2%) compared to the resistance
(45.5%) found in Enterobacteriaceae from farm produce. The most
DR1 ≥1 100 common isolate from the supermarket produce was
MDR2 ≥ 3 70
Stenotrophomonas maltophilia (31%), commonly found in water, soil,
and plant roots. Stenotrophomonas maltophilia, commonly found to
MDR3 ≥ 10 41.4 be resistant to a number of quinolones, including nalidixic acid [43].
All the Acinetobacter baumannii, Cronobacter sakazakii, and
Table 5: Prevalence of drug-resistant Enterobacteriaceae (%) from Salmonella spp. isolates showed MDR. The MDR strains have arisen in
produce sampled from supermarkets. 1Drug resistance to one or more Enterobacteriaceae, and this is a concern because of their potential for
antimicrobials. 2Microbial drug resistance to 3 or more antimicrobials. widespread complications in management of infected patients [44].
3Microbial drug resistance to 10 or more antimicrobials. Studies show the use of antibiotics in agriculture can drive the
extensive transmission of antimicrobial resistant bacteria [45,46].
Among the sampled packaged produce, various members of All of the Enterobacteriaceae isolated from farm and supermarket
Enterobacteriaceae were detected in 61.1% of the sample compared to produce displayed resistance to at least one of the 14 antibiotics tested.
9.4% of the loose varieties of the same produce. Packaging adds However, only 18.2% of the Enterobacteriaceae isolated from farm
another element to the food production chain, allowing for another produce displayed MDR to ten antibiotics, compared to 41.4% of the
avenue for contamination in the entire food production process. One Enterobacteriaceae isolated from supermarket produce. Steps involved
of the most common types of packaging is Modified Atmosphere in processing and packaging of the produce may have contributed
Packaging (MAP), as it can increase the shelf life of the product. MAP toward this factor. A study conducted in North Carolina analyzed fresh
changes the air level of the produce inside the packaging, but often

J Food Microbiol Saf Hyg, an open access journal Volume 1 • Issue 2 • 1000113
ISSN:2476-2059
Citation: Tope AM, Hitter AC, Patel SV (2016) Evaluation of Antimicrobial Resistance in Enterobacteriaceae and Coliforms Isolated on Farm,
Packaged and Loose Vegetables in Kentucky. J Food Microbiol Saf Hyg 1: 113. doi: 10.4172/2476-2059.1000113

Page 6 of 7

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Citation: Tope AM, Hitter AC, Patel SV (2016) Evaluation of Antimicrobial Resistance in Enterobacteriaceae and Coliforms Isolated on Farm,
Packaged and Loose Vegetables in Kentucky. J Food Microbiol Saf Hyg 1: 113. doi: 10.4172/2476-2059.1000113

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ISSN:2476-2059