Volume 8, Issue 5, May – 2023 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

Significance of Biofilm Formation by Enteric

Escherichia Coli Obtained from Fresh Produce in
Increased Antimicrobial Resistance and Persistence
Dr. Rekha Mehrotra, Dr. Vijay Veer Saharan
Associate Professor, Department of Microbiology Department of Microbiology,
Shaheed Rajguru College of Applied Sciences for Women, School of Life Sciences, Central University of Rajasthan,
University of Delhi, Vasundhara Enclave, New Delhi, India NH-8, Bandar Sindri, Ajmer, Rajasthan, India-305817

Dr. Preeti Verma, Assistant Professor,

Department of Microbiology
Shaheed Rajguru College of Applied Sciences for Women, University of Delhi, Vasundhara Enclave, New Delhi, India

Abstract:- The risk of enteric pathogen contamination I. INTRODUCTION

and growth on fresh produce is one of the main safety
concerns associated with the increasing number of With major advancements in various aspects of
foodborne outbreaks in recent years. Plants in general biological applications and technology, people around the
are not considered as host for enteric pathogens. world are still affected and falling ill from consumption of
However, the increased persistence of pathogenic and contaminated food with exerting heavy toll on both human
commensal E. coli in plants has led to the consideration health and nation’s economy. This is not only the case in
of these fresh produce as a secondary reservoir although developing countries with lack of proper sanitation and
factors associated in the increased persistence are not health but also in developed countries [1]. Human food
very clear. Biofilms formation by pathogenic E. coli and borne infections traditionally are acquired through the
other Enterobacter pose a serious threat to the safety of ingestion of foods of animal origin. Fresh fruits and
fresh produce as they can persist and colonize for long vegetables have emerged as new vehicles for the
periods of time in the food processing environment and transmission of diseases [2]. Leafy green vegetables
thus represent a source of recurrent contamination. (cabbage, spinach, lettuce, carrot and salad leaves of all
Concurrently, increased antibiotic resistance is equally a variety) have been identified as commodity group of highest
major concern with respect to biofilm formation as these concern in view of microbiological safety. Pathogenic E.
factors aids in the enhanced levels of virulence, coli, Salmonella sp. and Enteric bacteria are involved in
persistence and colonization of the pathogenic E. coli in food borne outbreaks causing gastroenteritis and chronic
fresh produce. In the present study the biofilm infection [3]. E. coli infection arising from contaminated
formation, antibiotic resistance and the multicellular food continues to be an immense problem with millions of
behavior by pathogenic/non-pathogenic E. coli strains cases occurring annually throughout the world [4]. In
derived from fresh produce has been investigated. The addition to the misery caused, financial loss is enormous.
formation of biofilm and its associated role with
antibiotic resistance has been addressed unveiling the Fresh vegetables are fundamental components of the
importance of factors leading to an increased host – human diet and there is considerable evidence of the health
microbe interaction. A total number of 33 E. coli strains and nutritional benefits associated with the consumption of
were isolated from ready- to- eat fresh produce. The fresh vegetables. This has led to significant rise in the
obtained results confirmed the ability to form biofilm by demand of fresh produce, changes in life styles and major
the E. coli strains obtained from fresh produce. Most of shifts in consumption trends [5]. Vegetables can become
the E. coli with increased number of cellulose/curli- contaminated with microorganisms capable of causing
fimbriae production was able to form stronger biofilm human diseases while still on the fields. The primary source
and showed a significant number of antibiotic of transmission of pathogenic E. coli to human is through
resistances. Overall, the present study suggests a contaminated foods consumption such as raw or
correlation between biofilm formation and antibiotic undercooked ground meat products, fresh produce and raw
resistance, implicating its important role in the food milk [6]. Cross contamination during preparations and poor
borne intoxications and increased virulence and its storage facilities along with contamination of water and
potential relevance for the management of food-borne other foods leads to increased infection.
illnesses linked with consumption of fresh produce.
In India, the burden of food-borne disease is unclear.
Keywords:- E. Coli, Fresh Produce, Biofilm, Curli- Most food-borne diseases go unreported, only few are
Fimbriae, Cellulose, Antimicrobial Resistance. reported by the media, usually those with high morbidity

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Volume 8, Issue 5, May – 2023 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
and/or occurring in urban areas [7, 8]. In the South-East BDAR (brown, dry and rough) indicating the expression of
Asia Region, WHO report shows nearly 150 million people curli-fimbriae only; d), SAW (smooth and white) indicating
fell ill with food borne diseases in 2010, which led to 175 no production of cellulose and curli-fimbriae. The distinct
000 deaths. Of these, 40% of food borne diseases burden colony morphotypes such as BDAR and RDAR have been
was among children under 5 years. reported in E. coli and Salmonella strains isolated from
clinical and animal sources [14, 15]. The BDAR and RDAR
Generally, it is considered that non-hosts morphotypes of Salmonella has been linked to increase their
environments such as plants do not support the colonization virulence or tolerance in long-term desiccation and nutrient
and survival of human enteric pathogens. However, depletion in biofilm [16]. However, limited information is
continuous increases in the microbial contamination of available on the role of production of curli fimbriae and
agricultural plants indicate that human enteric bacteria are cellulose and related colony morphotypes in agricultural
able to colonize and survive in fresh produce during plant-related E. coli and Salmonella.
cultivation and post-harvest processing [9, 10]. Further,
foodborne bacteria pathogens such as E. coli and Salmonella The present study focuses on the occurrence and
not only colonize or survive on the agricultural plant formation of biofilm as one of the approaches for
surface, but grow until the fresh produce is consumed by a colonization and persistence of enteric bacteria in fresh
new host. The most important strategies used by E. coli and produce with enhanced antimicrobial resistance.
Salmonella and other enteric bacteria during colonization or
survival in the agricultural plants is the formation of biofilm II. MATERIAL AND METHODS
[11]. A biofilm is an assembled and structural organization
of bacterial cells confined within a matrix of the EPS  Recovery and Isolation of E. Coli from Fresh Produce:
(extracellular polymeric substance) and which adhere to a The ready to eat fresh produce samples (n=170): leafy
living or inert surface. Several studies have demonstrated greens, carrot, radish, lettuce, cucumber and tomato were
that biofilm formation is significantly associated with E. brought to laboratory from various local markets of New
coli and Salmonella strains isolated from different sources Delhi (India) aseptically in sterile plastic containers. All
such as clinical and animal [12]. Biofilm formation samples were collected, labelled and transported to the
behaviors of many bacteria including E. coli is significantly laboratory aseptically. Bacteriological analysis of each
associated to enhance survival in natural environments, collected sample was carried out on the same day.
resistance to antimicrobial agents and during interaction
with hosts. Recently, few studies indicate that E. coli and For the recovery of E. coli from the collected samples,
Salmonella and other foodborne bacteria pathogens are able previously described methods were used with slight
to form biofilm on the surface of plants as well as in inner modifications. Presumptive identification of the produce
spaces of the plant tissues. It is also believed that bacterial isolated E. coli was done by conventional microbiological
cells confined in biofilm are more resistant to antibiotics and methods such as Gram’s staining and biochemical
stress conditions [13, 14]. Curli is a major component of characterization was performed to further characterize the
biofilm in many enteric bacteria including E. coli and are isolates such as IMViC, oxidase test and catalase test.
important for adherence to different biotic and abiotic Additionally, E. coli strains using molecular methods were
surfaces [15]. Thus, it is considered that biofilm formation screened and identified for the presence of uidA gene, Shiga
is a survival strategy of E. coli and Salmonella to withstand toxin (stx) and intimin gene (eae) by PCR assay. STEC
on the surfaces of the agricultural plant under unfavorable were identified using PCR primers targeting stx1, stx2, and
conditions. The production of curli fimbriae or thin eae genes, as described previously [17].
aggregative fimbriae and cellulose in E. coli was found to be
important components in the formation of extracellular  Biofilm Formation Assay:
polymeric matrix, which is essential during biofilm The biofilm development ability of the isolated E. coli
formation and persistence in various surfaces. Previous isolates was determined by using the microtiter plate method
studies suggest that production of curli fimbriae and as previously described [18]. The overnight growth of each
cellulose may be associated with the survival and bacterial strain with an OD600 of ⁓1.0 was diluted 1: 100 in
persistence of E. coli and Salmonella in the food fresh PBS and 200μl of the culture was added into the
environment. Evidences also suggest that production of triplicate well and incubated at 370C for 48 h. Culture was
cellulose and curli-fimbriae also offers protection to withdrawn, and each well was washed twice with sterile
bacterial cells against harsh environmental conditions such distilled water. The plates were air-dried, heat-fixed (60°C
as desiccation, osmotic shock, and UV radiation [13, 14]. for 2 hours), and stained with crystal violet (0.2%) for 10
Conversely, expression of cellulose and curli-fimbriae in E. min. The wells were washed twice to remove extra stain with
coli associated with their initial attachment in animal hosts distill water, air dried and solubilized in 200μl of 95%
or provides a physical barrier against the transmission of ethanol. Absorbance was obtained at OD570 . Wells
antimicrobial agents and compounds of the host immune containing only sterile media without bacterial inoculation
response. On the basis of the production of curli fimbriae were taken as blank or negative control.
and cellulose, E. coli displayed different colony
morphotypes; RDAR (red, dry and rough), indicating the
production of cellulose and curli; b), PDAR (pink, dry and
rough) indicating the production of cellulose only; c),

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Volume 8, Issue 5, May – 2023 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
 Cellulose and Curli Fimbriae Morphotype Screening:
The production of cellulose and curli-fimbria by all E.
coli strains was assessed using colony morphotypes on
congo red (CR) agar plates as described previously [13, 15].
Overnight grown fresh bacterial culture was inoculated on
CR agar plates prepared with LB agar without salt as a base
and supplemented with 40 μg/ml Congo red dye and 20
μg/ml of Coomassie brilliant blue and incubated at 25-28oC
for 72 hrs. Colony morphologies on CR plates were scored
as RDAR (Red Dry and Rough, indicates the expression of
both curli fimbriae and cellulose); BDAR (Brown Dry and
Rough, indicates the expression of curli-fimbriae only);
PDAR (Pink Dry and Rough, indicates the expression of
cellulose only); SAW (Smooth and White, indicates the no
expression of curli-fimbriae nor cellulose). The screening
experiment was performed in duplicates and at least 50-100
colonies per isolate was observed of each morphotype for
examination.

 Antimicrobial Susceptibility Testing: Fig 2 Percentage Recovery of E. Coli Colonize and Persist
Antimicrobial susceptibility of obtained E. coli in Vegetables/Fruits
isolates was determined using the Kirby-Bauer disc
diffusion method on MHA medium according to the The PCR analysis of the recovered enteric E. coli was
Clinical and Laboratory Standards Institute (CLSI 2012). performed for the confirmation of the isolated strains. The
The antimicrobial susceptibility was tested against seven diagnostic gene uidA encoding beta-glucuronidase in most
classes of drugs comprising of total 16 antibiotics. of the E. coli were used for analysis. Among the 36 picked
Reference strains E. coli ATCC 25922 was used as control. isolates of presumptive E. coli 83% of the isolates showed
the presence of the uid gene (Fig 3). The number of isolates
III. RESULTS obtained by genotypic analysis was less as compared to
phenotypic analysis. Hence 33 E. coli were identified using
 Recovery and Isolation of E. Coli from Fresh Produce: both conventional and molecular methods that were used for
To understand the factors associated with E. coli further analysis.
persistence on plants/fresh produce, a total of 33 (12.2%)
isolates of E. coli were recovered among the 170 fresh ready
to eat vegetable samples (Fig 1) Most isolates were obtained
from leafy greens (spinach and parsley) followed by lettuce,
carrot and cucumber (Fig 2).

Fig 1 E. Coli Growth on EMB Agar Showing Green Sheen:

(A) E. Coli Isolated From Vegetable (Cucumber) And Fig 3 PCR Amplification of ‘Uida’ (143bp) Genes from Six
(B) E. Coli 25922 Control Representatives E Coil Isolates

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Volume 8, Issue 5, May – 2023 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
 Biofilm Formation:  Prevalence of antibiotic resistance in Escherichia coli:
The elucidation of the biofilm formation was analyzed Among each class of antimicrobial drugs used in the
as described by Stepanović et al., 2000; the obtained data study, highest proportion of resistance was observed against
require the definition of the cut-off value that splits biofilm- quinolones (66.7%) followed by penicillin (36%) and
producing from non-biofilm-producing strains compared to aminoglycosides (28%). Carbapenem, macrolids and
negative control. According to the biofilm formation assays, cephalosporins showed moderate resistance 15%, 18% and
most of the produce-origin isolates were forming the strong 9% respectively. However, phenicol were 100% susceptible
biofilm. The OD570 for strong biofilm was observed to be in for the E. coli isolates. The drug resistance prevalence
the range of 0.408–0.854 for the E. coli isolates. The pattern of E. coli is represented, in Fig. 6. To summarize, E.
adherence ability of strains tested was classified into four coli, recovered isolates exhibited MDR pattern against
categories: strong biofilm producer, moderate biofilm majority of the critically and highly important antimicrobial
producer, weak biofilm producer, and no biofilm producer drugs (Fig. 7). It was observed that majority of the isolates
based upon the OD obtained. On the basis of the values showing MDR pattern were able to form strong biofilm.
defined, among the 33 isolates of E. coli, 55% (n = 18)
showed strong, 15% (n = 5) moderate, 18% (n = 6) weak
biofilm formation ability, while 12% (n = 4) were not able to
form biofilm. Statistically significant difference (P < 0.05)
was observed between biofilm producers categorized as
strong, moderate, weak or none (Fig. 4)

Fig 6 Percentage Prevalence of Antimicrobial Resistance of

E. Coli Against Clinically Important Antibiotic Classes
Fig 4 Biofilm Formation Ability of Vegitable/Fruit-Origin
Escherichia Coil Isolates (N=33), Strong Biofilm Producer
(N=18; 55%), Moderate Biofilm Producer (5; 15%),
Weak Biofilm Producer (6; 18%), and No-Biofilm
Producer (4;12%)

 Cellulose and Curli Fimbriae Morphotype Screening:

The E. coli isolates screened for cellulose and curli
fimbriae showed distinct colony morphotypes distribution.
The cellulose and curli fimbriae occurrence are exhibited as
RDAR, PDAR, BDAR and SAW. The distribution of E. coli
showing multicellular behaviors was observed as RDAR (n
= 16), PDAR (n = 8), BDAR (n = 7) and SAW (n = 2) as
depicted in Fig 5.

Fig 7 Percentage Resistant E. Coli Isolates Against

Individual Antibiotic

IV. DISCUSSION

Survival of human enteric pathogens in plants and

formation of biofilm has led to increase in the number of
food borne illnesses. The main question arises is how these
human pathogens invade the fresh produce and after
invading not only survive inside the plant, vegetable or fruits
also behaves as a carrier of the particular pathogens to
Fig 5 RDAR (red, dry and rough), PDAR (pink, dry and human. In the present study, we observed the survival
rough), BDAR (brown, dry and rough), SAW (smooth and strategies adopted by the human enteric pathogens to
white). Each bar represents no of E. coli isolates for overcome the environmental adverse conditions to colonize
particular colony morphotype and persist in the plant’s habitat. The isolated E. coli

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Volume 8, Issue 5, May – 2023 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
displayed multicellular behavior showing RDAR, BDAR, V. CONCLUSION
PDAR morphotype, which indicates the
expression/production of both or either of cellulose or curli- In conclusion, the present study suggests that fresh
fimbriae [12, 13]. Mechanisms involved in surface produce (leafy greens, ready-to-eat produce) are potential
attachment of human pathogens to plants are biofilm reservoirs or sources of multidrug-resistant E. coli strains.
formation, fimbriae, extracellular polysaccharide and Further, this study reports the prevalence and pattern of
presence of flagella. Curli fimbriae and cellulose are the correlation of Biofilm formation and MDR in E. coli
extracellular polysaccharide supposed to be linked with originating from fresh produce. The major limitation of the
biofilm formation, majorly found to be determinant of cell- present study is that the analysis relied on phenotypic
cell interactions and adherence to biotic and abiotic surfaces. resistance data. Therefore, we did not have the opportunity
The occurrence of high number of RDAR morphotype in our to account for the multiple genetic determinants that
studies signifies the importance of cell adhesion to the plant underlie drug resistance. Despite this limitation, our study
surface. This is in agreement with the previous studies which represents an important contribution to providing a large
showed that cellulose and thin aggregative fimbriae are data set to identify E. coli, in particular to characterize the
important for E. coli interaction with plants [19, 20]. The level and dynamics of biofilm formation and antimicrobial
distinct colony morphotypes such as RDAR and BDAR have resistance in these pathogens present in leafy greens/fresh
also been reported in E. coli strains isolated from clinical and produce.
animal sources. Studies have shown increased survivability
of the E. coli possessing RDAR morphotype. Interestingly, ACKNOWLEDGMENT
E. coli isolates in our collection majorly displayed RDAR
and BDAR morphotypes, which suggests of their survival in The authors would like to thank the management of
the plant environment with virulence potential. A similar Shaheed Rajguru College of Applied Science for Women,
significant finding has been reported in E. coli isolated from University of Delhi, for providing facilities for carrying out
human gastrointestinal tract [12, 13]. Moreover, it has also the present study.
been shown that on plant surface the biofilm formation is
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