DETECTION OF SALMONELLA SPP. ISOLATED FROM READY-TO-EAT FOOD BY PCR AND CONVENTIONAL METHODS: A COMPARATIVE STUDY

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            DETECTION OF SALMONELLA SPP. ISOLATED FROM READY-TO-EAT FOOD BY PCR AND CONVENTIONAL METHODS: A COMPARATIVE STUDY

WALAA M. ALI; M.W. ABD AL-AZEEMand SERAGELDEEN SULTAN

Department of Microbiology, Fac. Vet. Med., South Valley Univ.

Received: 18 September 2017;       Accepted: 15 October 2017

INTRODUCTION

Ready-to-eat (RTE) food is processed foodstuffs which have gained popularity in recent times because they can be ingested without further thermal treatments (Rodriquez et al., 2010). Both fast-food restaurants and street vendors are offering RTE foods. The highly increasing demand for Ready-to-eat food can be illustrated by changing life style affecting food behavior; increasing number of women in work force and limited commitment to food preparation (Borch and Arinder, 2002 and Gudbjornsdottirm et al., 2004). In Egypt, the most ready - to – eat sandwiches sold in street vendors and fast food restaurants are Beef burger, kofta, El- Hawawshi and Liver (Kibda) sandwiches(Ayaz et al., 1985). During  the  last  two decades, Salmonella

Corresponding author: Dr. WALAA M. ALI

E-mail address:walaa_1286@yahoo.com

Present address: Department of Microbiology, Fac. Vet. Med., South Valley Univ.

was considered the most common food borne pathogen in the world due to its increasing incidence (Edwards and Ewing, 1972) and its association with consumption of ready-to-eat meat products. Worldwide, there are about 275 million humans had diarrheal diseases caused by Salmonella (Cabedo et al., 2008).Salmonellosis is one of the most common and widely distributed food-borne diseases and it is the second food-borne disease after campylobacteriosis in Europe (Botti et al., 2013; European Food Safety Authority, 2012). Typhoidal cases are stable with low numbers in developed countries, but non-typhoidal salmonellosis has increased worldwide. Typhoid fever usually causes mortality in 5 to 30% of typhoid-infected individual in the developing world. The World Health Organization (WHO) estimates 16 to 17 million cases occur annually, resulting in about 600,000 deaths. The mortality rates differ from region to region, but can be as high as 5 to 7% despite the use of appropriate antibiotic treatment. On the other hand, non-typhoidal cases account for 1.3 billion cases with 3 million deaths. In the United States, approximately 2 to 4 million cases of Salmonella gastroenteritis occur with about 500 deaths per year. A more accurate figure of salmonellosis is difficult to determine because normally only large outbreaks are investigated whereas sporadic cases are under-reported. (Portillo, 2000; Hanes, 2003; Hu and Kopecko, 2003).Traditional Salmonella detection methods are based on cultures using selective media and the characterization of suspicious colonies by biochemical and serological tests. These methods are generally time-consuming. Therefore, a rapid method is necessary for the identification of Salmonella isolates from clinical specimens(Alvarez et al., 2004).There has been a general move toward molecular methods of Salmonella detection and typing, which are based less on phenotypic features and more on stable genotypic characteristics (Arrach et al., 2008). PCR has become a potentially powerful alternative in microbiological diagnostics due to its simplicity, rapidity, reproducibility, and accuracy (Pickup et al., 2003).InvA gene could be used as specific marker gene for the rapid detection of Salmonella isolates from various biological samples irrespective of sample origin (Das et al., 2012).For these reasons, it was decided to carry out an investigation for PCR amplification assay as a rapid and sensitive alternative test for the detection of Salmonella spp. following isolation and identification by bacteriological and biochemical methods.

MATERIALS AND METHODS

1. Collection of samples:-

A total of one hundred Ready-to-eat sandwiches were collected from different fast-food restaurants and street venders with different sanitation levels. The collected sandwiches include beef burger, kofta, Hawawshy and liver (25 of each). Each collected sample was wrapped in a separate sterile plastic bag, sealed, labeled and kept in an ice box then transferred to the laboratory under complete aseptic conditions without undue delay and examined as quickly as possible. The collected samples were subjected to bacteriological examinations for the detection of Salmonellae (APHA, 2001).

2. Preparation of samples:-

At the laboratory the content (meat part) of each sandwich was aseptically and carefully removed then mixed well in a sterile mortar. Isolation and identification of salmonella in each one were detected.

3. Isolation of Salmonella spp.: (ISO-6579, 2002):

3.1. Pre-enrichment in Non- selective liquid media:

Twenty five grams of each prepared sample were taken, cut into small pieces using sterile forceps and scissors and blended for two minutes in sterile blender jar containing 225 ml of 0.1% sterile buffered peptone water (BPW) as a pre-enrichment broth which recommended by (Edel and Kamplmacher, 1973). Aseptically transfer each blended sample to 500 ml sterile wide-mouth, screw-caped jar and incubated at 37 °C for 18 ± 2 hrs.

3.2. Enrichment in selective liquid media:

0.1 ml of pre-enrichment culture was transferred into sterile tubes containing 10 ml of Rappaport Vassiliadis broth (RVS) and the tubes were then vortexed and incubated at   41.5°C ± 1for 24 hrs. (Rappaport et al., 1956 and Harvey and Price, 1981).

3.3. Selective plating:

Xylose lysine desoxychoclate agar (XLD) was used. A loopful of each incubated tube was streaked on (XLD) agar plates and incubated at 37°C for 24hrs. After incubation examine the plates for presence of typical colonies of salmonella. Typical colony of salmonella on XLD agar appears as pink colonies with or without black centers.

The suspected colonies were sub-cultured onto nutrient agar plate and incubated at 37°C for 24 hours. However, the purified suspected colonies were selected and streaked onto nutrient agar slope for further identification. The purified isolates were identified morphologically, biochemically and serologically.

4. Identification of Salmonella spp.:-

4.1. Morphological identification:

4.1.1. staining reaction: (Cruickshank et al., 1975):

Films of pure suspected cultures were stained with Gram's stain and examined microscopically under oil emersion lens. Salmonellae are 2-3 μm, pink to red Gram negative short bacilli with rounded end.

4.1.2. Motility test: (ICMSF, 1978):

Motility medium was inoculated by the stabbing technique to a depth of 5 mm and then incubated at 37°C for 24-48 hours. A circular growth from the line of stabbing represented a positive test. Salmonella spp. are motile except Salmonella pullorum and Salmonella gallinarum.

4.2. Biochemical identification (MacFaddin, 2000):

4.2.1. Hydrogen sulphide production test:

On Triple Sugar Iron (TSI) agar, isolated organisms were stabbed into the bottom of the butt with a needle, and then it was drawn over the slant, for production of a sufficient surface growth. The inoculated tubes were incubated at 37°C for 24-48 hours. Hydrogen sulphide (H2S) production was noted by blacking the medium, while acid formation is indicated by yellow color.

4.2.2. Citrate utilization test:

Slants and butts of Simmon ̓s citrate agar tubes were stabbed from pure cultures and incubated at 37°C for 48-96 hours. The development of blue colouration indicated utilization of citrate. Salmonella give positive result.

4.2.3. Urease test:

Christensen ̓s urea agar medium was inoculated with suspected isolates and incubated at 37°C for 24 hours. Development of pink colour denoted a hydrolysis of urea. Negative tubes were re-examined after further incubation for 24 hours. Salmonella give negative result.

4.2.4. Lysine iron agar:                 

Isolated organisms were stabbed into the bottom of the butt with a needle, and then it was drawn over the slant, for production of a sufficient surface growth. The inoculated tubes were incubated at 37°C for 24- 48 hours. Positive reaction was indicated by development of alkaline (purple) color in the slant and alkaline (purple) color in the butt with black coloration.  

5. Serological identification and serotyping of salmonella:

Isolates proved biochemically to be Salmonella microorganisms were subjected to serological identification according to Kauffman – White scheme (Kauffman, 1974).

6. Polymerase Chain Reaction (PCR) for detection of salmonella species:

6.1. DNA Extraction using QIA amp kit (Shah et al., 2009):

6.2. Amplification reaction for Salmonella-specific gene (invA) (Shanmugasamy et al., 2011):

The PCR cycling protocol (Thermal cycler) was applied as following:

  Initial denaturation at 94°C for 60 second.

  Denaturation at 94°C for 60 second.    

  Annealing at 64°C for 30 second.        For 35 cycles        

  Extension at 72°C for 30 second.

 Followed by a final extension at 72°C for 7 min.

6.3. Agarose Gel Electrophoresis and identification of PCR Products: According to (Surzycki, 2000).

RESULTS

Ι- Results of the conventional bacteriological methods:

A-Results of cultural examination:

Table 1: The Incidence of suspected salmonella strains among the different RTE samples

B-Results of biochemical identification:

Table 2: Results of biochemical identification for suspected isolated strains:

II-Results of serological identification:

Table 3: Results of serological test for identified biochemically isolated strains:

Table 4: Serotyping of Salmonella isolated and distribution of serotypes among different clinical samples:

Table 5: Serogroup and antigenic structures of serologically identified Salmonella strains isolated from the examined samples of RTE sandwiches:

III- Results of PCR:

A- Results of PCR assay:

Twenty eight strains that were isolated on XLD were taken for PCR assay (11 strains of these were biochemically positive and 17 strains were negative biochemically).

Salmonella specific PCR with primers for InvA gene were performed. The results of PCR assay on DNA obtained from the yielded cultures are given in Photo (1) and Photo (2).

Table 6: Results of PCR assay for isolated Salmonella:

Photo (1): Agarose gel electrophoresis of PCR of invA gene (284 bp) for identification and characterization of (11 biochemically positive Salmonella species).

Lane M: 100 bp ladder as molecular size DNA marker.

Lane 1: Control positive Salmonellae for invA gene.

Lane 2: Control negative (No DNA).

Lanes 3, 9 & 12: Positive S. Enteritidis strains for invA gene.

Lane 4: Positive S. Virchow strain for invA gene.

Lanes 5, 7, 10 & 13: Positive S. Typhimurium strains for invA gene.

Lane 11: Positive S. Haifa strain for invA gene.

Lanes 6 & 8: Negative Salmonella species.

Photo (2): Agarose gel electrophoresis of PCR of invA gene (284 bp) for identification and characterization of (17 biochemically negative Salmonella species).

Lane M: 100 bp ladder as molecular size DNA marker.

Lane 1: Control positive Salmonellae for invA gene.

Lane 2: Control negative (No DNA).

Lanes 3, 4, 5&6: Positive Salmonella strains for invA gene.

Lanes7,8,9,10,11,12&13:Negative Salmonella species.

B- Comparison between PCR and biochemical reactions for detection of Salmonella:

Table 7: Comparison between PCR and biochemical reactions for detection of Salmonella:

C- Comparison between conventional bacteriological examination, biochemical tests, serological tests and PCR test for detection of Salmonella:

Table 8: Comparison between conventional methods, biochemical tests, serological tests and PCR test for Salmonella isolates:

DISCUSSION

Food borne illness associated with the consumption of ready to eat foods has been reported all over the world(FAO, 1988; Garcia et al., 2004; Chumber et al., 2007; Ghosh et al., 2007).Ready to eat food has been implicated in cases of food poisoning or gastroenteritis in human beings (Eley, 1996).

From the results obtained in Table (1)it was evident that Twenty-eight strains of Salmonella spp were isolated from 100 ready-to-eat meat products with a total percentage of (28%) using XLD agar.

In our study XLD had high efficiency in primary isolation of salmonella from clinical samples. This may be attributed to additional material found in XLD agar e.g. Xylose and Lysine. Xylose is fermented by most enteric organisms except Shigella Spp and Providencia Spp. Lysine is added to identify Salmonella. As xylose is exhausted, Salmonella Spp. organisms decarboxylate lysine causing alkaline conditions which give the colonies red color. Our results agreed with Isenberg, (2004)who reported the high efficiency of XLD agar in primary isolation of Shigella and Salmonella Spp.

From the results obtained inTable (1)it was cleared that although proper cooking temperature sufficient to kill the organism, its presence in ready-to-eat meat and meat products reflect: highly contaminated raw materials, insufficient heating during cooking and post cooking contamination most probably from contaminated workers hands or through using contaminated utensils. In the same time the cross contamination between raw and ready prepared foods is considered as a main source for post cooking contamination (National Academy of Science, 1985).

The obtained results in Table (2)declared that biochemical reactions could detect only 11strains out of 28 Salmonella isolates, with a percentage of (39.3%) as following 2 beef burger samples (7.1%), 6 kofta samples (21.4%), 1 hawawshy sample (3.6%) and 2 liver samples (7.1%). There were 17 (60.7%) negative cases: 5 from beef burger, 4 from kofta, 4 from hawawshy and 4 from liver samples.

The negative results obtained by biochemical reactions may be attributed to the weak metabolic activity or weakly reactive organisms leading to some false-negative reactions, these data were supported by the findings of Elmer et al. (1997).

Serological tests were done after biochemical reaction, since biochemical reactions identified the isolates as salmonella spp. that needs further identification to determine its species. This is in general agreement with the FDA (1995, 2001) that recommended serological tests being conducted on isolates that were retained as presumptive Salmonella after urease testing.  

The serological identification were performed for the only 11 biochemically +Ve isolates according to  Kauffman – White scheme  for the determination of Somatic (O) and flagellar (H) antigens using Salmonella antiserum.

The obtained results in Table (3)declared that serological tests could detect only 9 strains out of 11 Salmonella isolates, with a percentage of (81.8%) as following 1 beef burger sample (9.1%), 5 kofta samples (45.4%), 1 hawawshy sample (9.1%) and 2 liver samples (18.2%). There were 2 (18.2%) negative cases: 1 from beef burger and 1 from kofta samples.

From the results of Table (4)we found that different serotypes have been isolated from different clinical samples. one strain of Salmonella typhimurium was isolated from beef burger samples, one strain of Salmonella typhimurium, two strains of Salmonella enteritidis, one strain of Salmonella virchow, one strain of Salmonella haifa were isolated from kofta samples, one strain of Salmonella typhimurium was isolated from hawawshy samples and one strain of Salmonella typhimurium and one strain of Salmonella enteritidis were isolated from liver samples.

From obtained results inTable (4) it was obvious that Salmonella Typhimurium was the most common serotype isolated from different clinical samples followed by Salmonella enteritidis. This results were agreed withtheWHO (1988)that reported that Salmonella typhimurium occurs more, and was more widely distributed than any other serovars, this organism cause severe outbreaks of salmonellosis in all kinds of animals and was frequently the cause of both sporadic cases and outbreaks of gastroenteritis in man all over the world. Also these results were agreed with Herikstad et al. (2002)who stated thatSalmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis are the most frequently isolated serovar from food- borne outbreaks throughout the world and with Zhao et al. (2002) who stated that the most common type of Salmonella are typhimurium and enteritidis;they have been causing illness for more than 100 years.

Salmonella enterica serovar virchow (S. virchow) is recognized as one of the more common, invasive Salmonella serotypes(Messer et al., 1997; Willocks et al., 1996)and accounted for 46% of septicaemia cases caused by Salmonella species (Ashdown and Ryan, 1990).

In addition to bacteremia, S. virchow is capable of causing a range of extra-intestinal infections, which may be associated with serious morbidity and even mortality as Meningitis. (Ashdown and Ryan 1990; Messer et al., 1997).

Kaibu et al. (2005)reported that a case of a 78 years old male who died of food intoxication in Nagasaki 2004 due to Salmonella Haifa. 

From the results obtained in Table (5)Salmonella isolates were characterized antigenically and serologically to determine their serogroup and antigenic structure, the isolates were located in 3 serogroups with characteristic antigenic features based on O and H antigen.

PCR amplification assay is a new tool for molecular biology; it is so sensitive that a single DNA molecule can be amplified. Presence of salmonella in intensive livestock production presents explicit public health risks in addition to food industry losses. PCR provides a rapid means to monitor specific microorganisms in a variety of samples. This assay is an epidemiologically useful tool to distinguish salmonella spp.

Amplification of DNA sequences unique to an organism using the PCR improves both the speed of detection and the level of sensitivity at which organisms can be detected (Buffone et al., 1991; Ramamurthy et al., 1993) and has been increasingly used to identify several bacterial species from food and clinical samples (Stone et al., 1994). Another advantage is that PCR is not dependent on the utilization of a substrate or the expression of antigens, thereby circumventing phenotypic variations in biochemical patterns and lack of detectable antigens (Hoorfar et al., 1999).

Lampel et al. (2000) and Ferretti et al. (2001)proposed a rapid method with primers for invA gene, which allowed the detection of Salmonella serotypes within a maximum of 12 hours in many clinical samples. In this study, Salmonellagenomic DNA was extracted from cultures of the local isolates by using a simple, rapid and reliable protocol based on the boiling.

From the obtained results in Table (6)it is cleared that invasion gene operon, invA was detected in only 13 Salmonella isolates, representing multiple serotypes, out of 28 clinical isolates. This agreed with Das et al. (2012)who reported that invA gene could be used as specific marker gene for the rapid detection of Salmonella isolates from various biological samples irrespective of sample origin.

The chromosomally located invA gene contains sequences unique to Salmonella is found in all known serovars and has been established as an international standard suitable PCR for rapid, less expensive, and sensitive detection of this genus (Rahn et al., 1992).

The obtained result in this study showed that 13strains of Salmonella Spp. were isolated from 100 ready-to-eat meat products with a total percentage of (13%) using XLD agar. Salmonella was recovered in meat products by many investigators such as Abd El - Aziz (1987) (10%), Ahmed (1988) (8%), Siriken et al. (2006) (7%), El-Mossalami et al. (1989) (6%), El-Mossalami (2003) (5%), Torky (2004) (5%), El-Sherif and El-Mossalami (1998) (3.3%)and Mosupye and Holy (1999) (2%). 

Salmonella spp. was isolated from 2 beef burger samples with a percentage (8%). This obtained results was higher than those recorded by Usama, Maha (2009) (2.5%), Mohamed, Enas (2011) (5%), Soliman, 1988 (5%), El-Mossalami (1989) (6%) and Fathi and Thabet (2001) (6%). But the results in this study were lower than those obtained by Badrie et al. (2003) who found that (18.5%) of beef burger samples were positive for salmonella, Fathi et al. (1994) who isolate salmonella from (15.8%) of beef burger samples, also Abd El Fath, Rabab (2015) who isolated Salmonella from (13.33%) of examined samples of beef burger and Abou-zaid et al. (2001) who isolated Salmonella from (11.6%) of ready-to-eat beef burger. Soliman et al. (2002) detected Salmonella in (10%) of ready-to-eat beef burger. In contrast Duitschaever et al. (1977); Karim, (1977); El-sherbeeny et al. (1985); Youssef et al., (1999); Ebraheem, (2001); Chung et al. (2003); El-Mossalami, (2003) and Zaki, (2003) failed to isolate Salmonella spp. from RTE beef burger. 

Salmonella spp. was isolated from 5 Kofta samples with a percentage of (20%). The obtained result was higher than those recorded byHassan, Shereen (2009) (13.33%), Al-kour (2001) (12%), Soliman, 1988 (10%),Shaltout et al. (2013) (8%),El-Mossalami (2003) (5%), Usama, Maha (2009) (2.5%). Also the obtained results were lower than those obtained by Hassanin et al. (2014) (33.3%) and Abd El Fath, Rabab (2015) (26.67%).

On the otherside, some investigators failed to detect salmonella in kofta samples asHassan (1986); Tolba (1994) Hussein (1996); Ebraheem, (2001); Küplül et al. (2003); Abd El-Aziz, Doaa (2004); Al-Mutairi (2011) and Mohamed, Enas (2011).  

In the examined hawawshy samples, Salmonella spp. was isolated from 3 samples (12%) as present in table (6). These results were lower than those obtained by Hassanin et al. (2014) which isolate Salmonella from (40%) of examined samples. Also our results for hawawshy disagree with those reported by Ebraheem, 2001; El-Mossalami, 2003 and Ismail-Soad (2006). They recorded negative results of Salmonella spp. in the examined hawawshy sandwiches.

Salmonella spp. was isolated from 3 Liver samples with a percentage of (12%).This datawere higher than those obtained by Abd-El-Malek (2014)whoisolates Salmonella typhimurium from 7% of examined samples. On the contrary Abou, (1995); Ebraheem, (2001) and Büyükyِrük, (2014) failed to isolate salmonella from the examined samples.

The obtained results in Table (7)declared comparison between the results of PCR and those of biochemical reactions for detection of Salmonella. It was found that PCR could detect 13 samples as Salmonella isolates that include (9 biochemically positive and 4 biochemically negative). While biochemical reactions could detect 11 samples as Salmonella isolates and when examined by PCR, it excluded 2 samples as non-Salmonella isolates. So, we found that PCR is more specific than conventional method, because the conventional methods for Salmonella spp. have very poor specificity, andthere were numerous false- positiveresults Dusch and Altwegg (1995); Perez et al. (2003).

Oliveira et al. (2003)reported that PCR assay using the invA primers specific for Salmonella spp. considerably decreased the number of false-negative results which commonly occur in diagnostic laboratories.

The obtained results in Table (8) declared comparison between the results of PCR and those of biochemical reactions, serological reactions and cultural methods for detection of Salmonella. It was found that PCR assay were more superior to cultural methods and biochemical test for isolation of Salmonella. While PCR assay had the same results of serological test, for the strains that were biochemically positive, so the PCR assay were used to confirm the serological results.

Theseresults agreed withSallam et al. (2014) who mentioned that the higher prevalence of Salmonella positive samples that were obtained by PCR in the present study in comparison with that of cultural conventional method indicated that PCR is more sensitive and reliable than the phenotypic based culture method.

From our study, we can conclude that the identification of Salmonella should not be based on the conventional bacteriological methods only, but also on molecular methods.

The culture techniques are universally recognized as the standard methods for the detection of bacterial pathogens, such as salmonella in food stuffs. These techniques generally take longer time and are less sensitive compared to PCR based methods.

In conclusion, identification of Salmonella and screening of invA gene through PCR based procedures can have major benefit in public health specifically for rapid diagnosis, epidemiological investigations, ideal vaccine, development of treatment, and prophylactic strategies for salmonellosis.

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