International Journal of Food Microbiology 172 (2014) 92–101

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Evaluation of a lytic bacteriophage, Φ st1, for biocontrol of Salmonella enterica serovar Typhimurium in chickens Chuan Loo Wong a, Chin Chin Sieo a,b,⁎, Wen Siang Tan a,b, Norhani Abdullah e,f, Mohd. Hair-Bejo c, Jalila Abu d, Yin Wan Ho b a

Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia d Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia e Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia f Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia b c

a r t i c l e

i n f o

Article history: Received 6 March 2013 Received in revised form 22 November 2013 Accepted 28 November 2013 Available online 7 December 2013 Keywords: Bacteriophage Phage therapy Salmonella Typhimurium Salmonellosis Chicken

a b s t r a c t In this study, a Salmonella Typhimurium lytic bacteriophage, Φ st1, which was isolated from chicken faecal material, was evaluated as a candidate for biocontrol of Salmonella in chickens. The morphology of Φ st1 showed strong resemblance to members of the Siphoviridae family. Φ st1 was observed to be a DNA phage with an estimated genome size of 121 kbp. It was found to be able to infect S. Typhimurium and S. Hadar, with a stronger lytic activity against the former. Subsequent characterisation of Φ st1 against S. Typhimurium showed that Φ st1 has a latent period of 40 min with an average burst size of 22 particles per infective centre. Approximately 86.1% of the phage adsorbed to the host cells within the initial 5 min of infection. At the optimum multiplicity of infection (MOI) (0.1), the highest reduction rate of S. Typhimurium (6.6 log10 CFU/ml) and increment in phage titre (3.8 log10 PFU/ml) was observed. Φ st1 produced adsorption rates of 88.4–92.2% at pH 7–9 and demonstrated the highest bacteria reduction (6.6 log10 CFU/ml) at pH 9. Φ st1 also showed an insignificant different (P N 0.05) reduction rate of host cells at 37 °C (6.4 log10 CFU/ml) and 42 °C (6.0 log10 CFU/ml). The in vivo study using Φ st1 showed that intracloacal inoculation of ~1012 PFU/ml of the phage in the chickens challenged with ~ 1010 CFU/ml of S. Typhimurium was able to reduce (P b 0.05) the S. Typhimurium more rapidly than the untreated group. The Salmonella count reduced to 2.9 log10 CFU/ml within 6 h of post-challenge and S. Typhimurium was not detected at and after 24 h of post-challenge. Reduction of Salmonella count in visceral organs was also observed at 6 h post-challenge. Approximately 1.6 log10 PFU/ml Φ st1 was found to persist in the caecal wall of the chicks at 72 h of post-challenge. The present study indicated that Φ st1 may serve as a potential biocontrol agent to reduce the Salmonella count in caecal content of chickens. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Salmonellosis, an infection induced by Salmonella, is one of the leading bacterial food-borne diseases affecting humans and animals worldwide. Chickens, which are one of the most consumed meat by human population, has been identified as the main reservoir of this zoonotic pathogen (Desin et al., 2013). In the United States, an estimated 1.4 million cases of food related illness caused by non-typhoidal Salmonella have been reported annually (Hootoon et al., 2011). This bacterium is normally transferred to humans via consumption of ⁎ Corresponding author at: Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Tel.: +60 3 89466702; fax: +60 3 89430913. E-mail addresses: [email protected] (C.L. Wong), [email protected] (C.C. Sieo), [email protected] (W.S. Tan), [email protected] (N. Abdullah), [email protected] (M. Hair-Bejo), [email protected] (J. Abu), [email protected] (Y.W. Ho). 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.11.034

contaminated chicken or egg products. The control of the spread of this bacterium from its primary source has proven to be challenging. Young chicks which are infected by serotype Typhimurium would normally not show any clinical symptoms. However, these chicks are able to shed the bacterium into the environment, leading to environmental contamination which promotes the spread of the organism within the flock (Hootoon et al., 2011). The current preventive strategies which include hygienic control in the farm, vaccination, supplementation with feed additives, such as antibiotics, prebiotics, probiotics and synbiotics, have not produced promising outcomes. Eventually, the live animals may be sent to slaughter contaminated. As suggested by USDA/FSIS (2008, 2010), the last phase of effort to control contamination can be made during transport of all animal species to the slaughtered processing plants. This is a major concern as incoming contaminated animals are associated with contamination of the abattoir environment in which animals on the same slaughter line using the same equipment and tools contribute to direct or indirect cross-contamination of carcasses

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during slaughter-dressing operations (Buncic, 2006). The search for new environmental-friendly intervention strategies is ongoing. In recent years, applications of bacteriophages to control bacterial pathogens have received new appraisal. They have also been identified as a prospective alternative biocontrol method for infections and contaminations by antimicrobial resistant pathogens (Hanlon, 2007). Bacteriophages, or phages, are natural predators of bacteria. They are abundant in the environment, with an estimated ratio of 10:1 with their bacterial counterparts (O'Flaherty et al., 2009). Phages are selfreplicating and self-limiting. Amplification occurs naturally as long as the bacterial host is present and they target only the specific host. In the case of phage therapy, virulent phages which eventually lead to lysis of the bacteria host are used. By far, applications of phages have been known to be safe and non-toxic. In fact, a number of phage products which are used as biosanitiation agents on ready-to-eat foods have been granted the “generally recognised as safe” (GRAS) status in the United States (Monk et al., 2010). The successful application of phages is highly dependent on the biological properties of the phages which will affect their performance in the biological system applied. Therefore, this current work was undertaken to characterise isolated Salmonella Typhimurium-specific phage in vitro, and to evaluate the efficiency of the phage in vivo (in chickens). The fundamental aim of this study is to use phage to minimise the microbial load on poultry in between the period of holding before slaughter to ante-mortem official inspection in the multistep sequence of poultry meat processing operations. Thus, intracloacal application of phage was employed to determine the ability of the phage to reduce the S. Typhimurium load in the caecum, which is the major colonisation site of Salmonella in chickens.

2. Materials & methods 2.1. Salmonella cultures, media and growth conditions The Salmonella strain (S. Typhimurium 8720/06), obtained from the Veterinary Research Institute (VRI), Ipoh, Malaysia, was originally isolated from a local poultry farm. The culture was streaked on xylose– lysine–deoxycholate (XLD) selective agar (Merck KGaA, Darmstadt, Germany) and subsequently picked and grown in Luria Bertani (LB) broth (Difco, Kansas, USA) at 37 °C. The broth cultures were grown to mid-log phase (3-h-old, OD600 nm = 0.6) by incubation at 37 °C with shaking at 180 rpm. For long-term storage, S. Typhimurium in LB broth containing 20% (v/v) glycerol was prepared and stored at −80 °C.

2.2. Sample collection and bacteriophage isolation Samples of poultry excreta were collected from different poultry farms in Malaysia. A suspension of 10% (w/v) of chicken excreta was prepared in SM buffer (50 mM Tris–HCl [pH 7.5], 0.10 M NaCl, 8 mM MgSO4·7H2O and 0.01% gelatine [Sigma-Aldrich Co., Steinheim, Germany]) and enriched with a mid-log phase culture of S. Typhimurium to the ratio of 9:1 (v/v). The enrichment sample was incubated in an orbital shaker at 37 °C, 180 rpm for 24 h. The suspension was then centrifuged at 3000 × g for 5 min to remove debris. The supernatant was centrifuged again at 13,000 ×g for 10 min and the resulting supernatant was filtered through a 0.2 μm pore-size disposable syringe filter (Sartorius, Gottingen, Germany). The phages were then isolated using the agar overlay assay (Adams, 1959), in which 100 μl filtrate and 100 μl of mid-log phase S. Typhimurium were added to 3 ml of molten agar. The mixture was mixed and dispensed uniformly over the surface of solid LB agar (1.5%). The solidified agar plates were incubated for 24 h at 37 °C for plaque formation. Individual distinct plaques that appeared were differentiated on the basis of plaque morphology and agar overlay assay was repeated for these plaques for at least three times to obtain a homogenous plaque formation.

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2.3. Preparation of high titre phage lysates and purification A plaque was inoculated into 5 ml LB broth which had been seeded with 200 μl of mid-log phase S. Typhimurium. The mixture was incubated at 37 °C with agitation at 180 rpm until lysis was observed. This mixture was centrifuged, filter-sterilised and used for the preparation of high titre phage lysate. The filtered lysate was added to a 10 ml midlog phase host bacterial culture at the multiplicity of infection (MOI) of 1 and incubated at 37 °C with agitation at 180 rpm until the lysis of culture was observed. The lysed culture was clarified by centrifugation at 5600 × g for 15 min at 4 °C, decanted and filtered through 0.2 μm pore-size disposable syringe filters into sterile polypropylene tubes. The phage stock was titred using the agar overlay assay and stored at 4 °C. The high titre bacteriophage lysate was precipitated with 10% (w/v) polyethylene glycol (PEG) 8000 (Amresco, Solon, USA) and the pellet was resuspended with 10 ml of 1 M NaCl in TE buffer (10 mM Tris–HCl; pH 8.0 and 1 mM EDTA) using the method described in the Novagen's T7 Select® System Manual (2002). The partially purified phage suspension was layered onto the density gradients of caesium chloride (CsCl) (Amresco, Solon, USA). Purified phage particles were collected after centrifugation for 1 h at 209,700 × g in a Beckman SW41 rotor. The purified phage particle typically contained a titre around 1011 plaque forming units per ml (PFU/ml). 2.4. Phage morphology examination A drop of high titre purified phage stock was spotted onto a carboncoated copper grid and left at room temperature for 6 min. Negative staining with 2% (w/v) uranyl acetate for 10 min was performed on the sample before the grid was observed with a Philips HMG 400 transmission electron microscope (TEM) (Eindhoven, The Netherlands). The anti-Salmonella phage was classified using the guidelines of the International Committee on Taxonomy of Viruses (Fauquet et al., 2005). The phage size was determined from the average of three independent measurements. 2.5. Genomic characterisation The nucleic acid of the purified phage was extracted using the phenol extraction method described in the Novagen's T7 Select® System Manual (2002) with slight modifications. The nucleic acid, which was retained in the aqueous layer after being extracted twice with chloroform–isoamyl alcohol, was added with 1/4 volume of 3 M sodium acetate (pH 5.2) and precipitated with 2 volumes of absolute ethanol. After centrifugation at 12,000 × g for 10 min at 4 °C, the supernatant was discarded, the pellet was washed with 70% (v/v) of ice-cold ethanol, and then centrifuged again. The pellet of nucleic acid was air dried and resuspended in 50 μl of TE buffer. The extracted phage nucleic acid was analysed on a standard 0.7% (w/v) 1X Tris–acetate–EDTA (TAE) agarose gel. It was then digested with DNase Ι and RNase A according to the method described by Klieve and Gilbert (2005) to determine the type of nucleic acid. Its genome size was determined by pulse field gel electrophoresis (PFGE) (Hansen et al., 2007). 2.6. Physical and biological properties of phages 2.6.1. Lytic spectrum assay Quantitative determination was carried out to determine the degree of S. Typhimurium phage lytic activity on the other Salmonella pathogens. All the bacterial isolates used in this study are listed in Table 1. The lytic spectrum assay of each phage was determined by incubating the host culture until it reached mid-log phase (OD600 nm = 0.6). Exponential growing cultures were diluted serially from 10−1 to 10−6. For each dilution, aliquot of 0.3 ml was added with the same volume of phage lysates into a 1.5 ml microcentrifuge tube and the mixture was incubated immediately at 37 °C for 15 min. Control tubes consist a

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in an equal volume of LB broth at pH 7 and the set-up of the tubes was incubated at three different temperatures (25, 37 and 42 °C).

Table 1 Salmonella strains used for lytic spectrum assay. Bacterial isolate

Source

Salmonella enterica serovar Typhimurium 8720/06 Salmonella enterica serovar Enteritidis 692/06 Salmonella enterica serovar Pullorum 8214/06 Salmonella enterica serovar Braenderup 9214/01 Salmonella enterica serovar Albany 234/02 Salmonella enterica serovar Corvallis 8677/04 Salmonella enterica serovar Hadar 1477/02 Salmonella enterica serovar Mbandaka 739/02 Salmonella enterica serovar Tennessee 1328/97

VRI VRI VRI VRI VRI VRI VRI VRI VRI

mixture of equal volume of exponential host culture and substitution of LB broth instead of phage lysates. Aliquot of 0.1 ml mixture was then spread plated on the appropriate medium and surviving bacteria colonies (CFU) were enumerated before and after incubation. 2.6.2. Phage adsorption rates and single-step growth kinetic A mid-log phase bacterial culture was infected with a phage suspension to a MOI ratio of 0.1. In the phage adsorption assay, the samples were allowed to adsorb for 5, 20, 30, 40 and 50 min at 37 °C, with shaking at 180 rpm. At the end of each adsorption period, the mixture was centrifuged at 7000 × g for 5 min at 4 °C. The supernatant was used for the determination of unabsorbed phage titre by the agar overlay assay. In order to determine the single-step growth kinetic of the phages, infected phages were obtained by centrifugation at 7000 ×g for 5 min at 4 °C after a short period of probable adsorption. The infected phages were resuspended in an equal volume of pre-warmed LB medium and were then incubated again at 37 °C with agitation. Samples were taken periodically and titrated to determine the titre of the phages. The phage titre was then plotted against time intervals. 2.6.3. Optimisation of phage infection Phage replication and lytic activity were determined in a mixture of phage-host at different MOI ratios. The mid-log phase bacterial culture was inoculated with dilutions of phage suspensions to give multiplicities of infection of approximately 0.1, 1, 10, 100 and 1000. Bacteria-free suspensions and phage-free suspensions were used as controls in all experiments. The populations of Salmonella and phages were enumerated at 30 min intervals for 8 h. For the enumeration of Salmonella, decimal dilutions of each suspension were spread on LB agar plates while the phages were enumerated using the agar overlay assay after dilutions of the filtrate were prepared in a SM buffer. The Salmonella and agar overlay plates were incubated at 37 °C for 24 h and 6 h, respectively. 2.6.4. Effects of pH and temperature on phage–host interactions A mid-log phase bacterial culture was pelleted and resuspended in an equal volume of LB broth adjusted in steps of 1 pH unit from pH 2 to 9. The resuspended culture was infected with a phage suspension to give a MOI ratio of 0.1 and incubated immediately at 37 °C, with agitation at 180 rpm. Initial phage and bacteria concentrations at 0 min were determined. The adsorption assays were conducted, and titration of unabsorbed phages was carried out at the time where the adsorption rate was maximum based on the results of the single-step kinetic growth curve. Phage-bacteria suspensions in LB media of different pH values were incubated at 37 °C, 180 rpm for 3.5 h during which the lysis of bacterial culture occurred. Phage-free suspensions and bacteria-free suspensions were used as controls in all experiments and were incubated under the same conditions as the phage-bacteria suspensions. Phage and/or bacteria titres at 3.5 h in phage-free suspensions, bacteria-free suspensions and mixture of phage-host suspensions were determined. The method described above to study the effects of pH was also used to determine the effects of incubation temperature on the phage–host interaction, except that the pelleted bacterial cells were resuspended

2.7. Chicken in vivo trial 2.7.1. Preparation of bacteria and phage inoculums S. Typhimurium 8720/06 (VRI) was grown overnight in LB broth at 37 °C, and 1% (v/v) of the overnight culture was transferred to fresh LB broth and incubated at 37 °C with shaking at 180 rpm until the culture reached the mid-log phase. The culture was centrifuged at 5000 ×g for 15 min at 4 °C. The cell pellet was washed 3 times with phosphatebuffer saline (PBS), after which it was resuspended in PBS to give an inoculum of ~1010 CFU/ml. Purified Φ st1 was used in this study and the titre of the phage was determined by the agar overlay assay prior to the in vivo experiment. 2.7.2. Experimental design Two hundred unsexed one-day-old White Leghorn chicks were obtained from a specific-pathogen-free (SPF) flock at the Veterinary Research Institute (VRI), Ipoh, Malaysia. The chicks were assigned randomly to 4 treatment groups of 50 chicks each. The chicks were fasted overnight before any inoculation was carried out. Subsequently, after inoculation of the materials, they were provided with water and antibiotic-free broiler ration ad libitum throughout the experimental period. The 4 treatment groups were Group I, in which the chicks were administered with 0.25 ml of PBS buffer only; Group II, in which the birds were administered with 0.25 ml of 1012 PFU/ml Φ st1 only; Group III, in which the birds were challenged with 0.25 ml of 1010 CFU/ml S. Typhimurium and followed by administration of 0.25 ml of 1012 PFU/ml Φ st1; and Group IV, in which the chicks were administered with 0.25 ml of 1010 CFU/ml S. Typhimurium only. All the materials were administered by intracloacal inoculation using a 1 ml graduated syringe attached to a moistened sterile 3.8 cm curve feeding needle. Prior to inoculation, 6 chicks from each group (samples at 0 h) were removed and euthanised by CO2 asphyxiation. The remaining chicks from Group 1 were inoculated with a PBS buffer, while Group 3 and Group 4 were challenged with S. Typhimurium. Then, six chicks were removed from each group (Groups 1, 2, 3 and 4) and kept isolated for 3 h before they were sacrificed (samples at 3 h). After 1 h of postchallenge with S. Typhimurium, the chicks from Group 2 and Group 3 were inoculated with Φ st1. Subsequent samplings were at 6, 12, 24, 48 and 72 h of post-challenge (PC) with S. Typhimurium. In each sampling, cloacal swabs were collected from each bird for detection of S. Typhimurium. The chicks were later euthanised by CO2 asphyxiation. All animal management and sampling procedures complied with the guidelines of the Guide for the Care and Use of Agriculture Animals in Agricultural Research and Teaching (Federation of Animal Science Societies, 2010). Necropsy of chicks was performed and sections of liver, heart and spleen were swabbed onto the XLD agar for qualitative assessments of the S. Typhimurium presence. All cloacal and viscera organ direct swabs were considered positive when ≥ 10 colonies of the target bacteria were recovered. The caeca of the chicks were aseptically removed and caecal content was weighed and diluted 1:10 in a PBS buffer. One millilitre of the diluted caecal content was transferred to a new microcentrifuge tube for recovery of the bacteriophage using the agar overlay assay. For determination of viable counts of S. Typhimurium, ten-fold serial dilutions of each diluted caecal sample in the PBS buffer were prepared, and an aliquot of 0.1 ml of each dilution was spread plated on the XLD agar. The plates were incubated for 24 h at 37 °C and the formation of colonies was enumerated. 2.8. Statistical analysis Variations between treatments on physical and biological properties of the phage were analysed using Student's t-test and univariate analysis of variance (ANOVA). Frequencies of Salmonella isolation from the

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cloacal and viscera organs were compared using Pearson's chi-square test. The significant differences among treatments obtained from the caecal contents of the birds were discriminated using Duncan's multiple range tests with significance set at P b 0.05. All data were analysed using SPSS software for Windows version 13 (SPSS Inc., Chicago, IL). 3. Results 3.1. Phage isolation and lytic spectrum assay A phage, Φ st1, was successfully isolated from the faecal materials obtained from a chicken farm. It produced clear plaques (0.5–1 mm in diameter) with no halo formation on the soft agar. Φ st1 not only could infect S. Typhimurium, but was also able to lyse Salmonella enterica serovar Hadar (S. Hadar). Under the experimental conditions, Φ st1 was able to reduce the host bacterial (S. Typhimurium) counts by 5–6 log10 CFU/ml (from 8.19 log10 CFU/ml to 2.43 log10 CFU/ml), and S. enterica serovar Hadar (S. Hadar) by 1–2 log10 CFU/ml. Φ st1 did not reveal any lytic activity against other Salmonella strains tested in this study (Fig. 1). As the lytic efficiency against S. Hadar was rather low, further assessment on this bacteria host was not performed. 3.2. Phage morphology examination Transmission electron microscopic examination of Φ st1 showed that it consisted of a hexagon nucleocapsid structure with a diameter of 67.43 ± 1.73 nm (head) and a long narrow tail of 172.69 ± 30.03 nm (Fig. 2). From its morphology, Φ st1 was presumptively identified as a member of the Siphoviridae family in the order of Caudovirales. 3.3. Phage genomic characterisation The analysis of nucleic acid type suggested that Φ st1 is a DNA phage as the genome was completely digested by DNase Ι, but refractory to the activities of RNase A. The estimated genome size of Φ st1 was approximately 121 kbp (Fig. 3). 3.4. Phage–host system 3.4.1. Latent period, burst size and adsorption rates The latent period for Φ st1 was observed to be 40 min. Within the latency period, Φ st1 produced an average burst size of 22 particles per infective centre (Fig. 4). The adsorption rate of Φ st1 was 86.1% within the first 5 min of infection, 96.1% at 20 min and reaching a maximum of 97.5% at 40 min (Fig. 5).

Fig. 2. Transmission electron micrograph of Φ st1 virion negatively stained with 2% uranyl acetate under 125 k magnification. Bar = 200 nm.

3.4.2. Phage replication assay The interactions and dynamics of phage–host populations were determined at the MOI ratios of 0.1, 1, 10, 100 and 1000. The results of the study showed that the highest reduction rate of host cells and increment in Φ st1 titre were observed at the lowest MOI ratio tested (MOI = 0.1). At this MOI ratio, Φ st1 titre was increased by 3.8 log10 PFU/ml (Fig. 6A) and the host bacteria (S. Typhimurium) was reduced by 6.6 log10 CFU/ml (Fig. 6B) at the end of the experimental period (8 h). However, at the MOI ratios of 1 and 10, Φ st1 titre was increased by only 2.0–2.5 log10 PFU/ml, while Salmonella-host decreased by 5.3–5.7 log10 CFU/ml. Compared to the lower MOI ratios, the MOI ratio of 100 displayed a slightly different pattern in the host cell reduction. A drastic reduction of 5.5 log10 CFU/ml of the host bacteria occurred during the initial 60 min of incubation, but phage production increased by only 2.2 log10 PFU/ml. At the MOI ratio of 1000, Φ st1 did not reduce any of the host bacteria throughout the incubation period despite the high concentration of phage input, and phage titre was observed to increase only by 1.1 log10 PFU/ml during this period. 3.4.3. Effect of pH on phage–host interactions Phage Φ st1 was mildly affected when incubated at pH 6–9 for 3 h, in which a reduction of phage titre ranging from 0.3 to 0.5 log10 PFU/ml was recorded. However, incubation of Φ st1 in pH 4 and 5 led to a decrease of phage titre of more than 1 log10 PFU/ml. At pH 2 and 3, 100% inactivation of Φ st1 was observed (data not shown). The adsorption rate of Φ st1 at different pH was also determined. At pH 2 and 3, adsorption of Φ st1 was not observed. Significantly higher adsorption rate

10 9 8

Log cfu/ml

7

*

6 5 4 3

*

2 1 0

Salmonella strains Fig. 1. Lytic activity of Φ st1 on Salmonella spp. Number of surviving Salmonella colonies (CFU) were enumerated before ( ) and after ( ) infected with Φ st1. * represents significant difference (P b 0.05) between the uninfected and phage infected culture.

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(kbp)

M

1

(bp)

M

1

242.5

2

2,322 2,027

2,322 2,027

194.0

1

9,416 6,557 4,361

9,416 6,557 4,361

339.5 291.0

M

23,130

23,130

485.0 436.5 388.0

(bp)

2

145.5 97.0

564

564

48.5

A

B

C

Fig. 3. (A) Uncut Φ st1 DNA genome determination using pulse field gel electrophoresis (PFGE). Lane M: λ-ladder PFG marker (New England Biolabs, #N0340S); Lane 1: Φ st1; (B) Phage nucleic acid type determination with DNase Ι. Lane M: λ DNA/Hind ΙΙΙ marker; Lane 1: undigested Φ st1 genome; Lane 2: Φ st1 genome digested with DNase Ι; (C) Phage nucleic acid type determination with RNase A. Lane M: λ DNA/Hind ΙΙΙ marker; Lane 1: undigested Φ st1 genome; Lane 2: Φ st1 genome digested with RNase A.

(P b 0.05) was observed at pH 7–9 (88.4 to 92.2%). At pH 4, 5 and 6, Φ st1 adsorbed to the host cells at the rates of 52.3%, 62.6% and 74.8%, respectively (Fig. 7). The bacteria host, S. Typhimurium, also exhibited a lack of tolerance towards pH 2 and 3. The highest reduction rate (P b 0.05) of the host bacteria by Φ st1 was found to be at pH 8–9, in which the bacteria were reduced by approximately 6.6 log10 CFU/ml. Φ st1 reduced 3.9–6.0 log10 CFU/ml of the host bacteria at pH 5–7 and 0.7 log10 CFU/ml at pH 4. 3.4.4. Effects of temperature on phage–host interactions In general, Φ st1 was stable at the temperatures tested (25 °C, 37 °C and 42 °C), in which a reduction of only 0.1–0.5 log10 PFU/ml in the phage titre was observed after 3 h of incubation (data not shown). Although high survivability of Φ st1 was observed at these temperatures, the adsorption rate was significantly affected (Fig. 8). Φ st1 showed the highest and insignificantly different adsorption rates at 37 °C (97.4%) and 42 °C (96.5%). However, only 20.1% of Φ st1 adsorbed to the host bacteria at 25 °C. The analyses of the bacteria reduction rate by Φ st1 showed that the highest reduction rate was observed at 37 °C (6.4 log10 CFU/ml) and 42 °C (6.0 log10 CFU/ml), in which both

3.5. Chicken in vivo trial 3.5.1. Occurrence of Salmonella Typhimurium in cloacal and visceral organ swabs S. Typhimurium was not recovered above 10 CFU per sample in the cloaca and other visceral organs of the chicks from Group 1 (PBS only; unchallenged and non-treated) and Group 2 (unchallenged but treated with 1012 PFU/ml Φ st1) throughout the experimental period. However, higher occurrence of S. Typhimurium was observed in the chicks from Group 3 and Group 4 (Table 2). S. Typhimurium was detected in the cloaca of 83% of the chicks in Group 4 (challenged with 1010 CFU/ml S. Typhimurium but untreated with Φ st1) at 3 and 6 h after the chicks were challenged with the pathogen. Nonetheless, at 12 and 24 h of post challenge (PC), the chicks with S. Typhimurium in their cloaca decreased to 33%, and by 48 and 72 h, none of the chicks contained S. Typhimurium in their cloaca. At 3 h PC, S. Typhimurium was detected in the heart of a chick in Group 4, and at 6 h PC, S. Typhimurium was

120

9.00 8.00

100

7.00

% adsorbed phage

Phage titer (105 pfu/ml)

10.00

reduction rates were not significantly different (P b 0.05). Low temperature was found not conducive for bacteria lysis process as significant elimination of host was not observed at 25 °C (Fig. 8).

6.00 5.00 4.00 3.00 2.00 1.00 0.00

80 60 40 20 0

15 20 30 40 50 60 70 80 90 100 110 120 130

Time (min) Fig. 4. Single-step growth of S. Typhimurium phage in LB broth at 37 °C. Spontaneously released of Φ st1 ( ) at MOI ratio of 0.1.

-20

0

5

20

30

40

50

Time (min)

Fig. 5. Kinetics of adsorbed phages in dependency of the adsorption time at 37 °C. Bound phages of Φ st1 ( ) detected in the pellet after centrifugation at 5, 20, 30, 40 and 50 min after infection of S. Typhimurium host culture with Φ st1.

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the chicks from Group 3 (challenged with 1010 CFU/ml S. Typhimurium and treated with 1012 PFU/ml Φ st1), S. Typhimurium was not detected in the liver, heart and spleen of chicks from Group 3 at any of the sampling periods.

14 13 12

3.5.2. Enumeration of S. Typhimurium in caecal contents S. Typhimurium was not detected in the caeca of chicks from Group 1 (PBS only; unchallenged and non-treated) and Group 2 (unchallenged but treated with 1012 PFU/ml Φ st1). However, approximately 8.48 and 9.10 log10 CFU/ml of S. Typhimurium were found in the caecal contents of chicks in Group 3 (challenged with 1010 CFU/ml S. Typhimurium and treated with 1012 PFU/ml Φ st1) and Group 4 (challenged with 1010 CFU/ml S. Typhimurium only), respectively, at 3 h PC (Table 3). The number of caecal S. Typhimurium of the chicks in Group 4 was not significantly different at 3–12 h PC (6.82–9.10 log10 CFU/ml), but at 24 h PC, caecal S. Typhimurium decreased significantly (P b 0.05) to 4.58 log10 CFU/ml, and at 48 and 72 h PC, it was not detected. For the chicks in Group 3, the application of Φ st1 reduced the S. Typhimurium load significantly by 5.55 log10 CFU/ml (from 8.48 log10 CFU/ml to 2.93 log10 CFU/ml) at 6 h PC (Table 3). At 12 h PC, the number of caecal S. Typhimurium (2.61 log10 CFU/ml) was not significantly different from that at 6 h PC, but at 24, 48 and 72 h PC, S. Typhimurium was not detected.

11 10 9 8 7 6 0

30 60 90 120 150 180 210 240 300 360 420 480

Time (min)

S. Typhimurium titer (log10 cfu/ml)

B 10 9 8 7 6

3.5.3. Re-isolation of Φ st1 from caecal contents of chicks Φ st1 was not isolated from any caecal samples of chicks from Group 1 (PBS only; unchallenged and non-treated) and Group 4 (challenged with 1010 CFU/ml S. Typhimurium only). On the other hand, Φ st1 was detected in the caecal contents of chicks in Group 2 (unchallenged but treated with 1012 PFU/ml Φ st1) and Group 3 (challenged with 1010 CFU/ml S. Typhimurium and treated with 1012 PFU/ml Φ st1) (Table 4). The bacteriophage titres between these two groups were not significantly different throughout the sampling period. At 6 h PC, approximately 8.82 and 9.33 log10 PFU/ml of Φ st1 were isolated from the caecal contents of chicks in Group 2 and Group 3, respectively, and thereafter, the Φ st1 titre decreased progressively to 0.45 and 1.19 log10 PFU/ml (Group 2 and Group 3, respectively) at 72 h PC (Table 4).

5 4 3 2 1 0

0

30 60 90 120 150 180 210 240 300 360 420 480

Time (min) Fig. 6. Phage titre of Φ st1 (A) and infected host cell (S. Typhimurium) titre (B) at specific time intervals to give MOI ratios of 0.1(o), 1 (□), 10 (△), 100 (×), and 1000 (◊). Data are means ± standard deviations from three replicates of plate counts per MOI.

observed in 1/6 (17%), 2/6 (33%) and 1/6 (17%) of the liver, heart and spleen of this group of chicks, respectively. S. Typhimurium was not detected in the liver, heart and spleen of this group of chicks in subsequent samplings. The treatment of chicks with Φ st1 was found to reduce the presence of S. Typhimurium in the cloaca significantly. As observed in

4. Discussion The isolation of phages from environmental samples is possible especially from sources where the intended host is present (Higgins et al., 2007). In this study, Φ st1 against S. Typhimurium 8720/06 was

7

100

Phage adsorption rate (%)

90

6

80 5

70 60

4

50 3

40 30

2

20 1

10

Salmonella reduction (log10 cfu/ml)

Phage titer (log10 pfu/ml)

A

97

0

0 2

3

4

5

6

7

8

9

pH Fig. 7. Φ st1 adsorption rate ( ) and reduction in S. Typhimurium ( ) at different pH values. Data are means ± standard deviations from three replicates of plate counts.

C.L. Wong et al. / International Journal of Food Microbiology 172 (2014) 92–101

Phage adsorption rate (%)

120

7 6

100

5 80 4 60 3 40 2 20

1

0

Salmonella reduction (log10 cfu/ml)

98

0 25

37

42

Temperature (oC) Fig. 8. Φ st1 adsorption rate ( ) and reduction in S. Typhimurium ( ) at different temperature conditions. Data are means ± standard deviations from three replicates of plate counts.

successfully isolated from the poultry faecal materials of a local broiler farm. Φ st1 formed pin-sized plaques of b1 mm in diameter, which are similar to the size of plaques formed by the well-known Salmonella lytic bacteriophage, Felix 01 (O'Flynn et al., 2006; McLaughlin and King, 2008). The formation of the pin-sized plaques was probably due to delayed cell lysis caused by the small burst size of progeny phages (O'Flynn et al., 2006). The plaques formed by Φ st1 were however clear, indicating that Φ st1 is lytic (Yoon et al., 2007). One of the most important criteria to consider in the selection of a promising phage for effective phage therapy is the phage lytic efficiency. The screening of the lytic potential of phages in vitro provides a rigorous assessment of their lysis capabilities. Φ st1 is not only lytic towards its host cell (S. Typhimurium) but also slightly towards S. Hadar. Phages with narrow host range have also been widely isolated against other species of bacteria, for example, the lytic bacteriophages against Pediococcus sp. (Yoon et al., 2007), Ruminococcus albus AR67 (Klieve et al., 2004) and Pseudomonas plecoglossicida (Park et al., 2000). Phage P7 (Bigwood et al., 2008) and FGCSSa2 (Carey Smith et al., 2006) which are highly specific to S. Typhimurium have also been isolated. The degree of specificity of a phage depends on the attachment or adsorption of the phage cell to the receptor present on the bacterial cell during the initial stage of phage infection cycle (Parisien et al., 2008). The adsorption process often involves the highly specific binding of receptor-binding proteins (RBPs) in phages and the reversible binding of specific carbohydrate receptors exposed on the surface of bacterial

cell wall (Monteville et al., 1994). Therefore, there is a high possibility that the RBP presence on phages plays a major role in receptor specificity and hence in host cell recognition. Φ st1 isolated in this study is presumptively identified as a DNA phage from the Siphoviridae family. The 121 kbp genome size of Φ st1 is similar to that of siphophage T5 (Wang et al., 2005) (121 kbp) but is larger than those of some previously reported siphoviruses, which ranged from 40 to 50 kbp (Murphy et al., 1995; Pasharawipas et al., 2005). To date, the largest siphoviral genome reported is that of Bacillus phage SPBc2 with 134 kbp (Hatfull, 2008). Phages with large genome size of 200–300 kbp are predominantly the myovirus (Hill et al., 1989; Hansen et al., 2007; Seaman and Day, 2007). It has been suggested that large genome bacteriophages have higher level of fitness compared to small genome bacteriophages (Bull et al., 2004; Seaman and Day, 2007). The burst size of Φ st1 was 22 particles per cell, with an average latent period of 40 min. Salmonella-bacteriophage Felix 01 infection also demonstrated a small burst size of 14 particles per cell with a latent period of 60 min (O'Flynn et al., 2006). Similar small burst size and average latent period were also found in other bacteriophages such as Leuconostoc mesenteroides phage 1-A4 (24 particles per cell/20 min) (Mudgal et al., 2006), Pediococcus sp. phage ps05 (12 particles per cell/34 min) (Yoon et al., 2007), and Escherichia coli O157:H7 phage PPO1 (14 particles per cell/29 min) (Fischer et al., 2004). These burst sizes are considered small when compared to the other Salmonella-specific phages where burst sizes range from 100 to 200 particles per cell with

Table 2 The occurrence of S. Typhimurium in cloacal and other visceral organs of day-old chicks from treatment Group 3 (challenged and treated) and Group 4 (challenged and non-treated).a Treatment group

Organ swab

Hours of post-challenged (h) 0

3

6

12

24

48

72

1/6 (17)⁎ 0/6 (0) 0/6 (0) 0/6 (0) 5/6 (83)⁎

3/6 (50) 0/6 (0) 0/6 (0) 0/6 (0) 2/6 (33) 0/6 (0) 0/6 (0) 0/6 (0)

0/6 (0) 0/6 (0) 0/6 (0) 0/6 (0) 2/6 (33) 0/6 (0) 0/6 (0) 0/6 (0)

0/6 (0) 0/6 (0) 0/6 (0) 0/6 (0) 0/6 (0) 0/6 (0) 0/6 (0) 0/6 (0)

0/6 (0) 0/6 (0) 0/6 (0) 0/6 (0) 0/6 (0) 0/6 (0) 0/6 (0) 0/6 (0)

(n positive/total; %)b Group 3

Group 4

Cloaca Liver Heart Spleen Cloaca Liver Heart Spleen

0/6 (0) 0/6 (0) 0/6 (0) 0/6 (0) 0/6 (0) 0/6 (0) 0/6 (0) 0/6 (0)

3/6 (50) 0/6 (0) 0/6 (0) 0/6 (0) 5/6 (83) 0/6 (0) 1/6 (17) 0/6 (0)

1/6 (17) 2/6 (33) 1/6 (17)

a Salmonella was not recovered above 10 CFU per sample in all organs of all chicks in Group 1 (unchallenged and non-treated) and Group 2 (unchallenged and treated). All treatments were administered by intracloacal application in 0.25 ml inoculum per chick. b Data are expressed as the number of positive chicks (n) from the total number of chicks sampled (6). The positive results taken into consideration were samples where ≥10 colonies of the target bacteria were recovered. Data within parentheses are percentages. ⁎ Indicates significant difference (P b 0.05) between the different treatment groups using Pearson chi-square test.

C.L. Wong et al. / International Journal of Food Microbiology 172 (2014) 92–101

99

Table 3 The enumeration of S. Typhimurium in the caecal contents of day-old chicks from treatment Group 3 (challenged and treated) and Group 4 (challenged and non-treated).x Treatment group

Hours of post-challenged (h) 0

3

6

12

24

48

72

2.93 ± 1.32bcA 8.99 ± 0.23aB

2.61 ± 1.65bcA 6.82 ± 1.39aB

0.00bA 4.58 ± 1.50bB

0.00b 0.00c

0.00b 0.00c

y

Average colonisation factor Group 3 Group 4

0.00b 0.00c

8.48 ± 0.37a 9.10 ± 0.23a

x

Salmonella was not detected in all organs of all chicks in Group 1 (unchallenged and non-treated) and Group 2 (unchallenged and treated). All treatments were administered by intracloacal application in 0.25 ml inoculum per chick. Data are mean values ± SEM of chicks. y Mean of log10 number of S. Typhimurium per ml of caeca content. a–c Values within rows with different superscripts are significantly different (P b 0.05). A–B Values within columns with different superscripts are significantly different (P b 0.05).

phage multiplication would also be observed (Goode et al., 2003; Huff et al., 2006). This is also known as the “lysis from without” phenomenon (Hudson et al., 2006). According to Kasman et al. (2002), replication of phages is not necessary in in vivo studies as long as high doses of phages were applied to the target cells as early as the infection occurs. Φ st1 was inactivated within 3 h of incubation at pH 2 and 3 but was relatively stable at pH 4–9. Bacteriophages of Aeromonas hydrophila were also unable to survive at pH 3 for 1 h at 22 °C (Chow and Rouf, 1983). According to Muller-Merbach et al. (2007), extreme pH conditions would alter the saccharidic side chains present on the bacterial host, and thus, would damage their function to act as receptor molecules. These altered polysaccharidic side chains would eventually impair phage adsorption. However, Salmonella-phages, such as st104a and Felix 01, were found to survive the acidic condition of pH 2.5 (porcine gastric juice) for up to 2 h (O'Flynn et al., 2006). Φ st1 also demonstrated a high host cell reduction (~ 6 log10 CFU/ml) at 37 and 42 °C. This could be an advantage as these temperatures are within the body temperature range of humans and animals, thus allowing the phages to react effectively in vivo. In salmonellosis, the majority of infections and transmissions in commercial broiler farms are primarily via faecal–oral route. Hence, it is important to generate an effective administration route for Salmonella colonisation in the intestinal tract in order to study the biology of Salmonella infection in chicks and also to evaluate a potential therapeutic product against Salmonella colonisation (Brito et al., 1995; Cox et al., 1996). Various possible methods of administration of this pathogen have been demonstrated with the majority of Salmonella infections performed through oral inoculation (Brito et al., 1995; Toro et al., 2005; Atterbury et al., 2007). However, in the present in vivo study, intracloacal inoculation of S. Typhimurium was performed to ensure establishment of the target bacteria at the caecal wall lining. This inoculation method eliminates the passage through the acidic conditions of the crop and gizzard (pH ~3–5) during transit to gastrointestinal tract, which would eliminate some of the bacteria (Cox et al., 1972) and reduce the level of establishment at the target site (caecum).

similar or shorter latent period (Carey Smith et al., 2006; McLaughlin et al., 2006). Variations in latent period and burst size of different phage isolates could be due to the differences in medium, host cell, pH and temperature (Hadas et al., 1997; You et al., 2001; Guttman et al., 2005; Muller-Merbach et al., 2007). Understanding the interaction of a phage and its host cell is of paramount importance in practical phage therapy (Payne and Jansen, 2001; Cairns et al., 2009). In phage–host interaction, there is a threshold termed ‘proliferation/replication threshold’, whereby a minimum concentration of a bacterial population (~104 CFU/ml) must be present in order to sustain phage proliferation (Kasman et al., 2002). Likewise, there is also an inundation threshold for the phage population, in which a minimum phage concentration is needed to reduce the bacterial population. Hence, the effect of the ratio of phage to bacteria concentration is important in determining the efficiency of therapeutic phage application. The ratio of infectious virions to cells in a culture is commonly defined as multiplicity of infection (MOI). In the present study, the MOI ratio of 0.1 showed the highest reduction rate of host cell count (6.6 log10 CFU/ml) and increment in phage titre (3.8 log10 PFU/ml) after 8 h. At high MOI ratios of 100, although new viral progenies were produced less efficiently (2.2 log10 PFU/ml) in the whole infection process, it reduced the host cells rapidly (5.5 log10 CFU/ml) within 60 min (Fig. 6). Theoretically, a low MOI ratio is advantageous for large scale production and commercialisation of phage products, as it would reduce the cost of preparation, purification and application of phage products. Several in vivo studies have used low MOI to test the therapeutic efficiencies of phages (Raya et al., 2006; Toro et al., 2005; Barrow et al., 1998). In this approach, phages are allowed to auto-replicate in the biological system during the process of therapy. Bigwood et al. (2008), Atterbury et al. (2007) and O'Flynn et al. (2004) suggested that in the in vivo therapeutic application and food biocontrol approach, an efficient phage treatment regime would be the rapid elimination of host cells in a short period of time using high MOI ratios. Given the relatively high phage titre in comparison to the lower numbers of host cells, the chances of phage to bind to the targeted bacteria would be increased but lysis of cells without

Table 4 The re-isolation of Φ st1 from caecal contents of day-old chicks from treatment Group 2 (unchallenged and treated) and Group 3 (challenged and treated).x Treatment group

Hours of post-challenged (h) 0

3

6

12

24

48

72

9.33 ± 0.43a 8.82 ± 0.32a

6.97 ± 0.53b 7.55 ± 0.67ad

6.12 ± 0.31b 6.03 ± 0.24b

2.39 ± 0.77c 1.55 ± 0.57c

0.45 ± 0.45d 1.19 ± 0.77ce

Total PFU/ml recoveredy,z Group 2 Group 3 x

0.00d 0.00e

0.00d 0.00e

The total count of Φ st was not detected in the caecal contents of all chicks in Group 1 (unchallenged and non-treated) and Group 4 (challenged and non-treated). All treatments were administered by intracloacal application in 0.25 ml inoculum per chick. Data are mean values ± SEM of chicks. y Mean of log10 number of Φ st1 per ml of caeca content. z Values within columns were not significantly different (P N 0.05). a–e Values within rows with different superscripts are significantly different (P b 0.05).

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C.L. Wong et al. / International Journal of Food Microbiology 172 (2014) 92–101

Similarly, phage treatment can be applied in a number of ways for treatment of infected chicks. Phages have been inoculated orally into Salmonella infected chicks (Toro et al., 2005; Atterbury et al., 2007; Higgins et al., 2007) and campylobacteriosis infected chicks (Wagenaar et al., 2005; Loc Carrillo et al., 2005). Intramuscular administration (Barrow et al., 1998; Huff et al., 2006) and phages delivered through aerosol spray and drinking water have also been carried out (Borie et al., 2009). Vent lip application of phages is another possible method of administration to reduce pathogens, such as Salmonella, which favour colonisation of the lower gastrointestinal tract (Corrier et al., 1994; Miyamoto et al., 2000; Andreatti Filho et al., 2007; Sivula et al., 2008). Although intracloacal application is not commonly used in the treatment of diseases in animals, this route of administration could be a plausible alternative for phages which are sensitive to acidic conditions. This would lead to elimination and inhibition of pathogen through fast and direct contact of the phage with the bacteria host. In this case, this approach would directly reduce the Salmonella counts in the faecal droppings of the chickens which will otherwise be horizontally transferred to the other birds or the environment. The persistence of phages in vivo is important for complete elimination of the pathogen. Otherwise, regrowth of the pathogen would occur. In the present study, 1012 PFU/ml of Φ st1 was inoculated intracloacally, and approximately 6 h after post-challenge, 8.8 and 9.3 log10 PFU/ml of Φ st1 were present in the chicks of Group 2 (unchallenged but treated with 1012 PFU/ml Φ st1) and Group 3 (challenged with 1010 CFU/ml S. Typhimurium and treated with 1012 PFU/ml Φ st1), respectively. Although an increment of Φ st1 count which indicated the multiplication of phages in vivo liberated from the lysis of the target cells was not detected especially in the chicks from Group 3, the pathogen was rapidly eliminated from the chicks (within 12 h as compared to within 24 h without the treatment of Φ st1). Rapid elimination of pathogens is essential to control the spread of pathogens, especially in the case of Salmonella infection, where the primary route of transmission is via faecal–oral route. Massive bacterial multiplication within the gut and rapid tissue invasion following ingestion of Salmonella contaminated source would result in the death of chickens, in which mortality rates vary enormously from 10 to 80% in severe outbreaks (Snoeyenbos, 1991; Atterbury et al., 2007). In addition, the intermittent shedding/ excretion of Salmonella in the faeces by infected chicks would create a large reservoir of Salmonella proliferation in the environment and become a massive source of infection in animals. Contaminations may occur during handlings of the animals and this would ultimately lead to a high carcass contamination rate in processing plants (Poppe et al., 1998). The absence of increment in the phage titre as observed in the chicks of Group 3 could be due to “lysis from without”. In many of the studies, lysis from without would only occur when a high ratio of phage to host (multiplicity of infection: MOI) was used (Bigwood et al., 2008). Most of the successful bacteriophage therapies in animal models have used high MOI although bacteriophages could be administered at small doses given by their exponential growth attributes in host cells (Berchieri, 1991; Fiorentin et al., 2005; Atterbury et al., 2007). According to Huff et al. (2006), an effective bacteriophage treatment would be to ensure that a sufficiently large number of bacteriophages are present when bacterial infection is systemic. Atterbury et al. (2007) demonstrated that a 3 log CFU/ml reduction in S. Typhimurium by high titre Φ10 was observed after 8 h. In the present study, a single administration of high titre Φ st1 was found to successfully reduce ~6 log10 CFU/ml of S. Typhimurium rapidly within 6 h of postchallenge. Results of the present study indicated the possibility of biocontrol of S. Typhimurium by Φ st1 via intracloacal inoculation. This substantial reduction could potentially reduce the risk of product contamination at slaughter. Conflict of interest statement The authors declare that there are no conflicts of interest.

Acknowledgements This study was financially supported by the Ministry of Science, Technology and Innovation of Malaysia under the Science Fund Programme. We wish to thank the Veterinary Research Institute, Ipoh, for providing the pathogenic bacterial isolates and Professor Son Radu, Universiti Putra Malaysia for the Campylobacter strains used in the bacteriophage typing scheme in this study. We would also like to acknowledge Dr. Ooi Chee Hong for his kind assistance in the chicken faecal sampling and Dr. Chai Lay Ching for her guidance in the PFGE analyses. Appreciation is also expressed to the undergraduate students of the Faculty of Biotechnology and Biomolecular Sciences and the internship students from UCSI University, Kuala Lumpur.

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