Advances in Environmental Biology

Advances in Environmental Biology, 9(22) Special 2015, Pages: 33-41

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Advances in Environmental Biology ISSN-1995-0756

EISSN-1998-1066

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Effect of some essential oils on verotoxin- producing Escherichia coli isolated from minced meat 1Nashwa

A. Ezzeldeen, 1Heidy Abo- El yazeed, 2Afaf Ali Amin, 2Hemat A. Khyralla and 3Mohamed A. Abdelmonem 1

Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt. Unit of Microbiology, Department of Food Safety, National Nutrition Institute, Cairo, Egypt . Department of Microbiology, Central Laboratory of Residue Analysis of Pesticides and Heavy Metals in Foods (QCAP), Dokki, Giza, Egypt. 2 3

ARTICLE INFO Article history: Received 5 August 2015 Accepted 20 September 2015 Available online 30 September 2015

Keywords: E. coli, verotoxin E. coli, essential oils

ABSTRACT Background: Escherichia coli are bacterial population of the gastro-intestinal tract of human and animals. They are pathogenic, enterotoxigenic, enteropathogenic, enteroinvasive, or enterohaemorrhagic according to the presence of specific virulence factors. Some isolates are shiga toxin producers, which are zoonotic agents causing serious diseases like diarrhoea, haemorrhagic colitis and haemolytic-uraemic syndrome. Shiga toxin–producing or verotoxin-producing E. coli are the most important recently emerged groups of foodborne pathogens. Objective: the objective of this work to study the prevalence of E. coli in minced meat through isolation and serotyping of recovered isolates and determine the effect of cinnamon, cumin and garlic oils on verotoxinproducing E. coli (VTEC) in minced meat. Results: Fifty minced meat samples in three Egyptian Governorates (Cairo, Giza and Qalubiya) were randomly collected, analyzed for isolation and serotyping of Escherichia coli. Forty four isolates (88%) revealed E .coli from the 50 minced meat samples. Further, thirty seven (84.1%) of E. coli isolates belonged to the following serotypes (O1, O26, O125, O126, O146, O166, O18, O114, O151, O157, O158, O27, O78, O148, O159, O168 and O8), while 7 (15.9%) were untypable with the available anti sera. E. coli isolates(37) screened for the detection of verotoxin-producing E. coli (VTEC). Result showed that all E. coli isolates gave negative results for VTEC (vt1, vt2 and eae) detection except O114, which had VTEC (vt1,vt2 and eae) with percentage of (2.7%). Effect of (cinnamon, cumin and garlic) oils on verotoxin-producing E. coli (VTEC) was determined, results showed that cumin and garlic oils were nondestructive for VTEC (vt1, vt2 and eae) genes, however VTEC completely destructed by treatment with cinnamon oil Conclusion: active component of the cinnamon oil, which was purified by (GC- MS) and identified as eugenol (1.72%), which had suppressive effect on VT production. Eugenol is useful for reducing the virulence of E. coli which produced VT.

© 2015 AENSI Publisher All rights reserved. To Cite This Article: To Cite This Article: Nashwa A. Ezzeldeen, Heidy Abo- El yazeed, Afaf Ali Amin, Hemat A. Khyralla and Mohamed A. Abdelmonem., Effect of some essential oils on verotoxin- producing Escherichia coli isolated from minced meat. Adv. Environ. Biol., 9(22), 33-41, 2015

INTRODUCTION Meat and meat products are highly prone to microbial contamination since they are rich in essential nutrients and perishable in nature [1]. Foodborne disease is any illness that results from ingestion of contaminated food. Food can contain pathogens or intoxications, or chemical agents that cause acute or chronic intoxications [2]. Family Enterobacteriaceae is the most challenging bacterial contaminant to raw and processed meat products worldwide. E. coli is the most predominant species in all food poisoning cases associated with meat. Contamination of raw meat is one of the main sources of foodborne illnesses [3]. Escherichia coli are bacterial population of the gastro-intestinal tract of human and animals [4] .They are commensals or pathogenic, enterotoxigenic, enteropathogenic, enteroinvasive, or enterohaemorrhagic according to the presence of specific virulence factors [5]. Some isolates are shiga-like toxin producers which are zoonotic agents causing serious diseases like diarrhoea, haemorrhagic colitis (HC) and haemolytic-uraemic syndrome (HUS). Shiga toxin– producing or verotoxin-producing E. coli (VTEC) are the most important recently emerged groups of foodborne Corresponding Author: Mohamed A. Abdelmonem, Department of Microbiology, Central Laboratory of Residue Analysis of Pesticides & Heavy Metals in Foods (QCAP), Dokki, Giza, Egypt. E-mail: [email protected]; 00201001471898.

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Nashwa A. Ezzeldeen et al, 2015 Advances in Environmental Biology, 9(22) Special 2015, Pages: 33-41

pathogens [6]. These E. coli produce either one or two cytotoxins called Shiga toxins (stx1 and stx2) or verotoxins (vt1 and vt2) [7]. Verocytogenic E. coli including serotype O 157: H7 are among group causing chronic and potentially fatal illness, related to their ability to produce one or more toxins known as verotoxin or shiga - like toxin [8]. The STEC serogroups associated with human diseases are numerous which include O1, O2, O4, O5,O6, O22, O23, O26, O38, O45, O48, O50, O55,O73, O75, O91, O100, O103, O104, O105, O111, O113, O114, O115, O117, O118, O119, O121, O125, O126, O128, O132, O145, O153, O163, O165, and O166, as well as untypable isolates [9].Consumption of contaminated food with these STEC strains is the main cause of human infections [10]. Essential oils (Eos) called volatile or ethereal oils; are aromatic oily liquids obtained from plant material (flowers, buds, seeds, leaves, twigs, bark, herbs, wood, fruits and roots) [11]. Cinnamon essential oil has high antimicrobial properties, which formed clear zone when tested with Gram positive bacteria Bacillus subtilis and a Gram negative bacterium E. coli. Cinnamaldehyde contains high antibiotic quality since it is the main compound in cinnamon. Different amounts of specific compounds can affect the antimicrobial activity of EOs. For example, high concentrations of cinnamic aldehyde, eugenol or citral confer antimicrobial properties to Eos. Nowadays; every society requires more safe and high quality preservative-free foods with extended shelf life [12,13]. Food safety has received more attention due to increasing rate of new food-borne diseases. There are also new concerns about the application of herbal products with additional functional properties instead of synthetic chemical preservatives [14]. Among the natural antimicrobials, essential oils (EOs) have been widely investigated due to their anti -microbial (anti-fungal, anti -bacterial, anti-viral), anti oxidant, anti -mutagenic and anti -carcinogenic properties. MATERIALS AND METHODS Sampling: Fifty minced meat samples from different butcher's shops in three Governorates (Cairo, Giza and Qalyubiya), Egypt, were randomly collected. Sample processing: Samples were prepared according to the technique recommended by [15]. Bacteriological examination: Detection of E. coli according to Egyptian standards (E.S 1694:2005) 16 and (ISO 7251: 2005) 17 and serological profiling for serotyping of E. coli according to [18]. Screening of VTEC: VTEC screening was carried out after overnight incubation of the sample in Buffer peptone water at 37°C for 24 h, using Prep man ultra for extraction of DNA, TaqMan Environmental Master Mix, TaqMan E. coli 2011 O104:H4 Assay, Custom TaqMan VT2 (stx2) Assay, Custom TaqMan VT1 (stx1) Assay (Applied Bio system), Custom TaqMan O157 Assay and 7500 real time polymerase chain reaction (PCR) (Applied Bio system). The Master Mix Set-up was prepared as follow: 15 μL of 2X EMM 2.0 and 3 μL of 10X Target Assay Mix and 18 μL of total volume master mix per reaction. The dye settings for multiplex reaction were prepared as shown in Table 1 and the thermo cycler settings was carried out in 2 steps, enzyme activation and template denaturation step which occurred at 95°C /10 min and amplification step, which is repeated 45 times, including stage 1 at 95°C /15 min and stage 2 at 60°C /45 min. Positive reference strains included within the run and purchased from reference laboratory of E. coli in Rome Italy include E. coli O157 strain C210-03 genotype (eae+, VTx2+, VTx1+) and E. coli O104:H4 strain 11 2027 genotype (eae-, VTx2+, VTx1-). The procedure includes five main steps: 1.

Enrichment: Food sample (minced meat sample) was enriched by adding 25 g sample to 225 ml of enrichment broth (buffer peptone water) then incubated at 37°C for 24 h. 2.

Extraction of DNA: One ml of enrichment was transferred in micro centrifuge tube, centrifuged at maximum speed (15000rpm/3 min) to spin down the contents, and the supernatant removed. The pellets were re suspended in 100 μl of PrepMan® Ultra Sample preparation reagent and vortex to mix the contents. The tube was heated in a heat block at 95 to 100°C/10 min, and then centrifuged at 15000 rpm/3 min to spin down the contents. 10 μl of the supernatant (sample DNA) was transferred to a new tube containing 90 μl of water then vortex to mix the contents and then the sample DNA was ready for PCR. 3.

Preparing the sample for PCR:

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According to the number of samples, the premix solution of master mix and assay was calculated and added in external screw capped tube. Both the samples and negative controls require 15 μl of master mix and 3 μl assays. Premix solution (18 μl) was transferred into each well, gently pipette at the bottom of the well. 12 μl of unknown sample was transferred into each well, gently pipette to mix the solution. 12 μl of negative and positive control were transferred and the tubes closed. 4.

Preparation of PCR run: The samples runned on real-time PCR System, plate were loaded into the instrument, and then the cycle was adjusted as follow holding stage where the temperature was gradually raised to 95.0°C/10min followed by cycling stages which include 40 cycles; 15 min at 95.0°C and 1 h at 60°C, then the run started. 5.

Data analysis and documentation: Data analysis was carried out according to Flow-diagram of the screening procedure of VTEC according to EU RL (2011) [19] method. 25 g of sample was added to 225 ml BPW and enriched at 37°C, 18 to 24 h followed by extraction of DNA for screening and detection of the presence of Verotoxin genes. Negative result to vtx gene will be reported as absence of VTEC. Positive samples to vtx genes will undergo test for O104 and 0157 gene, followed by isolation onto MacConky agar or sorbitol MacConky agar, and then the isolated colonies were tested for vtx producing genes by real time PCR to confirm positive results by O104 and/or O157. Negative results indicate non VTEC producing O104 or O157 while positive VTEC, O104 and /or O157. The interpretation of results was carried out according to the analysis of verotxin where samples showing positive VT1 and /or VT2 are VTEC positive while samples showing negative VT1 and /or VT2 are VTEC negative. Application: The tested raw minced meat was treated with 3 natural essential oils (cinnamon , cumin and garlic) with concentration 500 µl / Kg minced meat according to International Organization for Standardization (ISO 7251:2005 and ISO 4831 :2006 ) 17, 20 of microbiology of food and animal feeding stuffs and preserved this tested raw minced meat in refrigerator at 4 o C for 3day , then screening for the presence or absence of VTEC in sample . Identification of the chemical composition of cinnamon essential oil using Gas Chromatography Mass Spectrometry (GC- MS) (Instrument type: Thermo scientific ISQ 120602) technique was used to identify the chemical component of cinnamon essential oil. Table 1: Dye settings for multiplex reaction. Parameter TaqMan® VT1 (stx1) Assay TaqMan® VT1 (stx2) Assay TaqMan® E. coli O104 Assay TaqMan® E. coli O157 Assay IPC, Internal positive control.

VT1 Probe Reporter = FAM™ Reporter = FAM™ Reporter = FAM™ Reporter = FAM™

IPC Reporter = VIC® Reporter = VIC® Reporter = VIC® Reporter = VIC®

RESULTS AND DISCUSSION Positive samples: Forty four samples (88%) out of 50 minced meat samples were positive for E. coli. Serotyping of Escherichia coli isolates: Serotyping of the 44 retrieved E. coli isolates indicated that 37(84.1% ) of them were typed (O1, O26, O125, O126, O146, O166, O18 , O114, O151 , O157, O158, O27, O78, O148, O159, O168 and O8 ) while the remaining 7 isolates (15.9%) were untypable with available antiserum used, as in Table (2). Table 2: Results of serotyping of E. coli isolates Polyvalent antiserum Monovalent antiserum Poly 1 O1 O26 Poly 2 O125 O126 O146 O166 Poly 3 O18 O114 O151 O157 O158 Poly 4 O27

No. of isolates 4 2 1 3 2 4 3 1 2 1 2 2

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Poly 6

O78 O148 O159 O168 O8

2 3 1 1 3

From the 50 minced meat samples examined, 44 (88 %) isolates E. coli were recovered, E. coli prevalence rate to be from 85.65 – 100 % in fresh meat samples from abattoir and traditional open market each. Also this is in agreement with the study done in Trinidad where the isolation rate was 90% of E. coli from raw meat [21,22]. Serotyping of E. coli isolates was done, out of 44 isolates, 37 E. coli serovars isolated and 7 isolates were untypable with available antisera used. This serotyping of E. coli isolates was reported also that 15 strains 12 Escherichia coli serovars isolated from meat product samples belonged to the serovars: O166; O78; O126; O55; O26; O20; O25: K4; O114; O125: K70 ; O116 and the rest of the isolate strains were untypable [23]. Similar Escherichia coli serovars were isolated from meat products were recorded (O126; O26, O111; O126; O125 and O26 [24, 25]. The public health importance of isolated enteropathogenic serovars had been attributed to its enterotoxin, which is implicated in causing gastroenteritis, epidemic children diarrhea, and sporadic diarrhoea in children as well food poisoning [26]. Moreover three principle syndromes caused by enterohamorrhagic Escherichia coli have been linked to Escherichia coli O157: H7 which are hemorrhagic colitis with bloody stool, hemolytic uremic syndrome leading to renal failure in children; thermbutic thrombocytopenic purpura syndrome causing brain damage and high mortality rate [27]. In this study only one isolate E. coli O157 was detected from 50 minced meat samples with an incidence of 2 % [28]. Naturally, high frequency of E. coli in minced meat indicates high rate of contaminations during processing, E. coli presence in minced meat might have originated from animal tissues or contaminated tools used in slaughtering and related treatment or cutting process [29]. Results of Verocytotoxin detection among examine samples: Table (3) showed that all 37 E. coli isolates gave negative results for VTEC (vt1, vt2 and eae ) detection expect serotype O114 gave positive results for VTEC with percentage of ( 2.7%). Table 3: Screening of detection of VTEC in minced meat samples Serogroups Screening of VTEC VT1 VT2 O1 Negative Negative O26 Negative Negative O125 Negative Negative O126 Negative Negative O146 Negative Negative O166 Negative Negative O18 Negative Negative O114* Positive Positive O151 Negative Negative O157 Negative Negative O158 Negative Negative O27 Negative Negative O78 Negative Negative O148 Negative Negative O159 Negative Negative O168 Negative Negative O8 Negative Negative

eae Negative Negative Negative Negative Negative Negative Negative Positive Negative Negative Negative Negative Negative Negative Negative Negative Negative

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Photo 1: Showing the result of screening of minced meat samples for the detection of VTEC including internal control It is found that all minced meat samples were negative for VTEC ( Vt1, Vt2 and eae ) even E. coli O157 negative for verocytotoxin production indicate the non-toxigenic type of E. coli O157 but E. coli O114 is VTEC ( Vt1, Vt2 and eae ) positive as shown in Table 3 and Photo 1. There are two types of enteropathogenic E. coli (EPEC), typical, which possess the EPEC adherence factor (EAF) plasmid and atypical, which do not possess the EAF plasmid. Currently, the EPEC isolated in fatal illness, related to their ability to produce one or more industrialized countries are atypical while those from developing countries are typical. Interestingly [30]. During the past 23 year, a large number of human illness outbreaks have been traced worldwide to consumption of under cooked ground beef and other beef contaminated with shiga toxin-producing Escherichia coli (STEC). Although several routes exist for human infection with STEC, beef remains a main source 31. Infection with EPEC is a world-wide public health problem, related to consumption of contaminated ground beef 32. According to this study, E. coli O114 was enteropathogenic E. coli which is verocyto toxin-producing E. coli (VTEC). Results for the effect of three oils (cinnamon, cumin and garlic): The E. coli O114 strain was mixed with three oils (cinnamon, cumin and garlic) Photos from (2, 3 and 4)

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Photo(2) : Mixed with Cinnamon oil

Photo(3) : Mixed with Garlic oil

Photo (4) : Mixed with Cumin oil

Screening the effect of VTEC (E. coli O114) isolate after treatment with natural essential oils (cinnamon, cumin and garlic ) with concentration 500 µl / Kg minced meat according to (ISO 7251:2005 ) [17]. Photos (3,4) Showed that, VTEC ( VT1, VT2 and eae) are still present in minced meat and not destruction after treatment with cumin oil and garlic oil, however VTEC are completely destructed after treatment with cinnamon oil which showed in Photo(2). EOs act to inhibit the growth of bacterial cells and also inhibit the production of toxic bacterial metabolites. Most EOs have a more powerful effect on Gram-positive bacteria than Gram-negative species, and this effect is most likely due to differences in the cell membrane33.Many bacterial pathogens, including EHEC, have a conserved membrane histidine sensor kinase (QseC) to activate the expression of virulence factors including VT2 [34]. Inhibitors of the QseC-mediated activation, which is referred to as a quorum sensing system, are likely useful for antivirulence therapy [35]. Recently, a library of 150000 small organic molecules to identify inhibitors of the QseC-mediated activation in EHEC, and found that 1 compound, N-phenyl-4-{[(phenylamino) thioxomethyl] amino}-benzenesulfonamide, inhibited the QseCmediated activation of virulence genes including the VT2 gene without inhibiting the growth of EHEC O157 [36]. However, they did not examine its effect on the expression of the VT1 gene. Many EHEC O157 strains have both VT1 and VT 2 genes in the genome of prophage of λ bacteriophage [37]. The VT1 and VT2 genes may be regulated by the QseC-mediated activation. One of the possible mechanisms for the suppression of EHEC O157 by eugenol is inhibition of the QseC-mediated activation of VT genes by binding to QseC or membranes near QseC. Since many of the spices reduced the amounts of extracellular VTs, other essential oils in the spices may also bind to membranes and inhibit the QseC-mediated activation to some extent. Chemical compositions of cinnamon essential oil: An active component of the cinnamon oil, which purified by (GC- MS) and identified as eugenol and other component showed in Table (4) and Figure (1). Eugenol (1.72%) component which present only in cinnamon oil not found in cumin and garlic oils. Peak identification performed by comparing the relative retention time of each peak with those of standard compounds. Also the essential oils mixed with their major component and injected into GLC to verify the peak identify. Peak area measured by triangulation and the percentage of each

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essential oil component was calculated as of the summation of peak area [38,39]. When we examined the suppressive effect of eugenol on VT production by E. coli , the amounts of both intracellular and extracellular VTs were found to decrease with an increase in eugenol concentration. Eugenol is useful for reducing the virulence of E. coli, which produced VT [40]. Table (4) and Figure (1) demonstrated the percentage of the components of cinnamon essential oil. Data prevailed 15 components. The major components were cinnamic aldehyde (72.87%), cinnamic acid (8.88%), cinnemyl acetate (2.83%), eugonal (1.72%), linalool (1.61%), caryophellene (1.44%), P-cymene (1.03%), limonene (1.00). The total components presented (93.93%), while the unknown ones were (6.07%).The main compounds identified in cinnamon essential oil, was cinnamic aldehyde (87.7%) followed by α-pinene (7.98%) and β - pinene (4.23%) [41]. Table 4: Chemical composition of cinnamon essential oil by using (GC-Ms). Chemical Peak Retention Area % Constituents No. time 2 4.040 0.64  - pinine 4 5.345 0.20 - pinine



Limonene Alpha terpined Myrcene P-cymene Benzaldehyde Cinnamic aldehyde Phenel ethyl alcohol Eugonal Cinnamyle acetate Linalool Caryophellene Comphene Cinnamic acid unknown

3 18 6 11 12 28 2.4 32 31 14 15 13 36 -

7.577 25.121 6.505 9.32 17.291 37.76 32.79 41.58 41.255 14.446 21.395 4.655 50.280 -

1.00 0.34 0.45 1.03 0.33 72.87 0.35 1.72 2.83 1.61 1.44 0.24 8.88 6.07

Fig. 1: Gas Chromatography Mass (GC-Ms) for chemical components of cinnamon essential oil.

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