This survey aimed to find the prevalence and measure of Salmonella spp. , Salmonella Typhi and Salmonella Typhimurium in chopped fruits from peddler stables and hypermarkets in Malaysia. Analysis was carried out utilizing the most likely figure ( MPN ) – manifold polymerase concatenation reaction ( PCR ) method. The prevalence of Salmonella spp.
, Salmonella Typhi and Salmonella Typhimurium in 210 samples of chopped fruits examined were 23.3 % , 7.6 % and 3.8 % , severally with estimated measure changing from 0 to 19 MPN/g. This survey urged the authorization to look into the biosafety of chopped fruits in Malaysia.Salmonella spp.
are Gram-negative, facultatively anaerobiotic and bacillar bacteriums. So far there are more than 2500 serovars of Salmonella with typhoid febrility by Salmonella Typhi and stomach flu by Salmonella Typhimurium being the most dominant serovars in public wellness ( Namimatsu et al. , 2000 ; Park et al. , 2009 ) . Salmonella Typhi causes enteral febrility in worlds whereas Salmonella Typhimurium causes systemic disease in mice which closely resembles typhoid febrility in worlds ( Lin, Chiu, Chu, Huang, Lin, & A ; Ou, 2007 ) . In the United States, about 40,000 instances of salmonellosis are reported each twelvemonth which cause about 600 deceases yearly ( Albufera, Bhugaloo-Vial, Issack, & A ; Jaufeerally-Fakim, 2009 ) .
In Malaysia, it is hard to measure the position of salmonellosis due to the deficiency of elaborate epidemiological surveies by the public wellness and veterinary sector. Nevertheless, 171 salmonellosis patients with two deceases were reported due to ingestion of ready-to-eat nutrients in Kelantan in 2005 ( Tunung et al. , 2006 ) . The surveillance plan by Ministry of Health Malaysia ( Catching Disease Control Section, 2008 ) indicated that the incidence of typhoid febrility in Malaya from 1996 to 2006 was in the scope of 0.71 to 4.50 per 100,000 populations.Recently there is a crisp addition in the ingestion of fresh green goods worldwide due to their nutritionary benefits to worlds and alterations in diet.
The reported instances of foodborne eruptions associated with fresh green goodss have besides increased ( Abadias, Usall, Anguera, Solsona, & A ; Vinas, 2008 ) . Yaun, Sumner, Eifert, & A ; Marcy ( 2004 ) reported that the major eruptions associated with fresh green goodss have been linked with foodborne pathogens such as Salmonella spp. , Escherichia coli O157: H7, Listeria monocytogenes and Shigella spp. Although fresh green goodss are less often implicated as vehicles for salmonellosis, multistate eruptions of salmonellosis caused by apple, Citrullus vulgaris, boodle, melon, Mangifera indica and tomato ingestion have been reported ( Bordini, Ristori, Jakabi, & A ; Gelli, 2007 ) . To the best of our cognition, there is no published informations on the prevalence and figure of Salmonella spp. , Salmonella Typhi and Salmonella Typhimurium in chopped fruits in Malaysia.The gilded criterion for the sensing of Salmonella spp. from nutrient normally involves pre-enrichment, selective enrichment, isolation on selective agar media and verification of presumptive positive settlements utilizing biochemical and serological trials.
This conventional cultural method is really expensive and clip consuming ( Schonenbrucher, Mallinson, & A ; Bulte, 2008 ) . Consequently, polymerase concatenation reaction ( PCR ) -based methods for Salmonella spp. sensing has gained popularity. The manifold PCR applied in this survey, gives best consequences with figure of positive consequences similar to those obtained by conventional method. This farther reduces the labor, reagent cost and proving clip for multiple bacterial pathogens ( Cortez, Carvalho, Ikuno, Burger, & A ; Vidal-Martins, 2006 ) . However, PCR-based methods are limited to qualitative finding. Borowsky, Schmidt, & A ; Cardoso ( 2007 ) stated that the quantification of microorganisms nowadays in the nutrient samples is of import to measure the hazard to consumers. Therefore, the most likely figure ( MPN ) method which estimates the figure of micro-organism based on the chance is incorporated for the quantification of the PCR merchandises ( Mantynen, Niemela, Kaijalainen, Pirhonen, & A ; Lindstrom, 1997 ) .
Periodic surveillance to find the prevalence and measure of Salmonella spp. in nutrients is of import to command human salmonellosis. As a effect, the aim of this survey was to find the prevalence and measure of Salmonella spp. , Salmonella Typhi and Salmonella Typhimurium in chopped fruits to measure the degree of consumer exposure to Salmonella. Another aim was to compare the happening of Salmonella spp. , Salmonella Typhi and Salmonella Typhimurium in chopped fruits from peddler stables and hypermarkets in a limited geographical location in Malaysia.
This survey will be the first biosafety appraisal of Salmonella spp. , Salmonella Typhi and Salmonella Typhimurium in chopped fruits in Malaysia which serve as an of import monitoring plan of Food Safety and Quality Division in the Ministry of Health Malaysia.
2.0 Materials and Methods
2.1 Sample aggregation
A sum of 210 samples of chopped fruits were purchased indiscriminately from 3 peddler stables at Seri Kembangan, hypermarket A at Kajang and hypermarket B at Kuala Lumpur. In this survey, seven types of fruits were analyzed ( Table 1 ) .
However, it was non possible to obtain all the samples at each trying location and on each trying juncture as this was dependent on their handiness during peculiar trying visit. All the samples were transported to the research lab in an ice box and examined within 2 hours after purchase.
2.2 Most likely figure ( MPN ) method
A 10 g of chopped fruit sample was aseptically weighed and transferred into a unfertile stomacher bag.
The sample was pummeled in a stomacher for 60 s with 90 milliliters of buffered peptone H2O ( BPW ; Merck, Darmstadt, Germany ) . A three-tube most likely figure ( MPN ) method was employed where 100 crease and 1000 fold dilutions of the stomacher fluids were prepared. Each dilution of the fluids was transferred into three tubings, with 1 milliliters in each tubing. All the tubings were incubated at 37°C for 24 h.
After incubation, MPN tubings were checked for turbidness where turbid tubings were subjected to DNA extraction followed by manifold PCR.
2.3 Plating method
The bacteriological method used for culturing Salmonella spp. , Salmonella Typhi and Salmonella Typhimurium was alterations of the methods of Rall, Rall, Aragon, & A ; Silva ( 2005 ) and Tunung et Al. ( 2006 ) . A one milliliter of pre-enriched BPW was transferred to 10 milliliters Selenite Cystine Broth ( SC ; Merck, Darmstadt, Germany ) and incubated at 37°C for 24 h.
A loopful of the broth civilization was so plated onto CHROMagar Salmonella ( CHROMagar Microbiology, Paris, France ) and Xylose Lysine Deoxycholate Agar ( XLD ; Eiken Chemical Co. , Tochigi, Japan ) . The home bases were incubated at 37°C for 24 H and the presumptive settlements obtained were purified by the run and isolation method utilizing the Tryptic Soy Agar ( TSA ; Merck, Darmstadt, Germany ) .
2.4 DNA extraction
Deoxyribonucleic acid was extracted from the turbid MPN tubings and presumably identified settlements from agar home bases utilizing a modified boiled cell method ( Chai et al. , 2007 ; Tang, Mohamad Ghazali, Saleha, Nishubuchi, & A ; Son, 2009 ) . A one milliliter part of each stock was centrifuged at 15,000 tens g for 3 min.
The pellet was suspended in 500 I?L sterile distilled H2O and vortexed smartly. The cell suspension was boiled for 10 proceedingss, cooled at -20EsC for 10 proceedingss, centrifuged once more at 15,000 tens g for 3 min. The supernatant was used as the DNA templet solution in the manifold PCR as described below.
DNA extraction from the presumably identified settlements from agar home base was the same except that the initial cell pelleting measure was omitted and a loopful of settlement was suspended in distilled H2O.
2.5 Multiplex PCR
Three sets of primers aiming a randomly selected-sequence of unknown map but is specific to Salmonella spp. , 23S rRNA cistron specific to Salmonella Typhi and fliC cistron specific to Salmonella Typhimurium were used in the manifold PCR check ( Table 2 ) . All oligonucleotide primers were synthesized by 1st BASE Laboratories, Malaysia.
The mention Salmonella Typhi and Salmonella Typhimurium strains included as positive controls in each PCR assay were obtained from Institute for Medical Research, Malaysia.Multiplex PCR elaboration was carried out utilizing VeritiTM 96-Well Thermal Cycler ( Applied Biosystems, Foster City, CA, USA ) . We optimized the manifold PCR reaction conditions by a series of preliminary experiments so that the three independent PCR reactions can be performed in the same tubing ( informations non shown ) .
The optimized multiplex PCR reaction was performed in 50 AµL reaction mixtures incorporating 10 AµL of 5x PCR buffer, 0.2 millimeter of deoxynucleoside triphosphate mix, 0.2 I?M of ST11 and ST15 primers, 1.2 I?M for Fli15, Typ04, sty-1 and sty-2 primers, 2.5 millimeter MgCl2, 1.
5 U of Taq DNA polymerase and 4 I?L of DNA templet. All the stuffs used in the PCR were purchased from Vivantis Technologies, Selangor, Malaysia. The thermocycler conditions were initial denaturation at 94A°C for 2 min, followed by 35 rhythms of denaturation at 94A°C for 1 min, primer tempering at 53A°C for 1 min and extension at 72A°C for 1 min.
Last, a concluding extension at 72A°C for 7 min and indefinite keeping period at 4EsC was employed.For visual image of PCR merchandises, 5 I?L of PCR merchandises was loaded on 1.2 % agarose gel tally at 90 V for 40 min. The gel was so stained with ethidium bromide and viewed under UV ( UV ) visible radiation. A DNA-molecular ladder ( 100-bp ladder ) ( Vivantis Technologies, Selangor, Malaysia ) was included in each gel as molecular weight markers.
The mark cistrons specific to Salmonella spp. , Salmonella Typhi and Salmonella Typhimurium produced amplicons at 429 bp, 300 bp and 620 bp, severally. Fig. 1 shows the consequence of gel cataphoresis comparing assorted combinations of the PCR primer sets and verifying the multiplex PCR established for the current survey consists of three independent and specific PCR reactions.The prevalence of Salmonella spp. , Salmonella Typhi and Salmonella Typhimurium in 210 samples of chopped fruits examined were 23.
3 % , 7.6 % and 3.8 % , severally.
Coincident sensing of Salmonella spp. , Salmonella Typhi and Salmonella Typhimurium in 210 samples of chopped fruits utilizing manifold PCR are summarized in Table 3. It was shown that the prevalence of Salmonella spp. in entire chopped fruits from peddler stables ( 30 % ) were three times higher than those from hypermarkets ( 10 % ) with 140 and 70 samples examined, severally. Salmonella Typhi and Salmonella Typhimurium were detected in chopped fruits from peddler stables with prevalence for entire fruits being 11.4 % and 5.7 % , severally.
None of the samples from hypermarket was contaminated by Salmonella Typhi and Salmonella Typhimurium. Out of all the seven species of fruits examined, dragon fruits from peddler stables showed the highest prevalence of Salmonella spp. ( 75 % ) , Salmonella Typhi ( 40 % ) and Salmonella Typhimurium ( 25 % ) . On the other manus, papaia from hypermarkets showed zero taint of Salmonella spp. , Salmonella Typhi and Salmonella Typhimurium in all 10 samples examined.The estimated measure of Salmonella spp. , Salmonella Typhi and Salmonella Typhimurium in chopped fruits varied from 0 to 19 MPN/g ( Table 4 ) . The highest measure of Salmonella spp.
, Salmonella Typhi and Salmonella Typhimurium in chopped fruits from peddler stables and hypermarkets were 19 MPN/g. Most of the samples from peddler stables and hypermarkets showed a lower limit of 3 MPN/g and a upper limit of 19 MPN/g of Salmonella spp. The measure of Salmonella Typhi and Salmonella Typhimurium in chopped fruits from most locations were 0 MPN/g, but their presence in firedrake fruit from peddler stables reached a upper limit of 19 MPN/g.Table 5 demonstrates the difference between MPN-multiplex PCR and MPN-plating method in the sensitiveness in observing Salmonella spp.
, Salmonella Typhi and Salmonella Typhimurium in chopped fruits. Salmonella spp. were detected in 49 of 210 samples ( 23.3 % ) utilizing the MPN-multiplex PCR whereas merely in 20 samples ( 9.
5 % ) utilizing the MPN-plating method. The samples judged positive by the MPN-plating method were ever positive by the MPN-multiplex PCR method whereas the antonym was non true, bespeaking higher sensitiveness of the MPN-multiplex PCR method.
The three primer sets were used at the same time to guarantee the designation of Salmonella spp. , Salmonella Typhi and Salmonella Typhimurium in the same reaction tubing even in the presence of other related and non-related bacteriums. Salmonella Typhi and Salmonella Typhimurium were chosen because they are the most dominant serotypes among those isolated from the patients who presented enteral febrility and gastroenteritidis in worlds ( Namimatsu et al. , 2000 ) . In Malaysia, typhoid febrility is besides recognized as an endemic job which affects all age groups ( Thong, Cheong, Puthucheary, Koh, & A ; Pang, 1994 ) .
Our consequences back up these old observations.In this survey, MPN-PCR method was employed which allowed the sensing and quantification of Salmonella spp. , Salmonella Typhi and Salmonella Typhimurium in chopped fruits to be completed within 2 yearss. This was decidedly an advantage as compared with the MPN method coupled with conventional biochemical test-based trials which involved much work load, great sum of stuff and 7-10 yearss for complete designation ( Martin, Jofre, Garriga, Hugas, & A ; Aymerich, 2004 ) . If direct PCR was to be performed on nutrient samples, false positive consequences might hold occurred due to the dead cells ( Saroj, Shashidhar, Karani, & A ; Bandekar, 2008 ) . This job was solved by the incorporation of MPN method which allowed merely feasible cells to turn so as to complement manifold PCR method for the accurate sensing of Salmonella.
In Malaysia, many surveies on the incidence of Salmonella spp. from nutrient samples have been reported. These includes taint of Salmonella spp. in natural and ready-to-eat cooked nutrients ( Arumugaswamy, Rusul, Abdul Hamid, & A ; Cheah, 1995 ) , broilers ( Rusul, Khair, Son, Cheah, & A ; Yassin, 1996 ) , natural veggies ( Salleh et al. , 2003 ) and street nutrient and clinical samples ( Tunung et al. , 2006 ) .
This is the first prevalence survey describing coincident sensing of Salmonella spp. , Salmonella Typhi and Salmonella Typhimurium in chopped fruits in Malaysia. The consequence highlighted the concern of the presence of Salmonella spp. ( 23.3 % ) , Salmonella Typhi ( 7.6 % ) and Salmonella Typhimurium ( 3.8 % ) with bulk demoing the maximal measures ( 19 MPN/g ) in the chopped fruits.
Johannessen, Loncarevic, & A ; Kruse ( 2002 ) pointed out that Salmonella spp. had been isolated from fresh green goods, largely in low degrees. This was supported by Salleh et Al. ( 2003 ) who reported that the prevalence and taint degrees of Salmonella spp.
were lower in fresh veggies and fruits than in meat. However, the maximal figure of 19 MPN/g in 4 types of chopped fruits examined is considered high as fruits are normally eaten natural. Hence the hazard of geting salmonellosis becomes higher than devouring meat.
The prevalence of Salmonella spp. in chopped fruits from peddler stables was three times higher than those from hypermarkets ; Salmonella Typhi and Salmonella Typhimurium were detected merely from peddler stables. This might be attributed to the attitude and nutrient safety cognition of peddlers in Malaysia. Peddlers have been considered to be hapless, uneducated and lack grasp for safe nutrient handling. An informal survey by City Hall, known as Dewan Bandaraya Kuala Lumpur ( DBKL ) confirmed a deficiency of cognition of good nutrient handling and failure of peddlers to carry through the wellness demands ( Toh and Birchenough, 2000 ) . In fact, the taint of the fresh fruits may take topographic point while husbandmans grow them in Fieldss or groves, during reaping and station harvest home, processing and distribution. The usage of carnal manure as fertilisers, faecal taint by animate beings and employees, presence of domestic and wild animate beings, usage of contaminated H2O in irrigation and human use are known to be possible pre-harvest beginnings of the pathogens.
The station crop taint beginnings include contaminated rinsing H2O or ice, human use, animate beings, contaminated equipment or transit vehicles, cross-contamination and high storage temperatures ( Beuchat, 2002 ; Johannessen, Loncarevic, & A ; Kruse, 2002 ; Bordini, Ristori, Jakabi, & A ; Gelli, 2007 ) . As a consequence, it can be concluded that there is a broad taint path which consequences in insecure fresh green goods unsuitable for ingestion without heat intervention.From our observation during sampling, the peddlers were found to be less hygienic in the readying of chopped fruits as compared to animal trainers in hypermarkets. The peddlers seldom wear baseball mitts in contrast to animal trainers in hypermarket who usually wear baseball mitts during handling of chopped fruits ; although have oning baseball mitts, does non ever warrant safe nutrient unless baseball mitts are often changed. Hawkers normally pack the chopped fruits in plastic bags without sealing and utilize crushed ice to chill the chopped fruits during show.
On the other manus, the chopped fruits in hypermarkets are sealed utilizing polystyrene packaging and displayed in hair-raiser. Besides, peddlers usually wash fruits, custodies, cutting board and knives in the pails with H2O. Viswanathan and Kaur ( 2001 ) pointed out that the running H2O is rarely available at the seller ‘s base, nutrients are inefficaciously protected from flies and therefore the sanitation of street peddling operations required much concerns. They besides emphasized that the sellers might pollute the fresh green goods during sliting, chopping and manus commixture. Apart from that, the sloughing of Salmonella spp. by human bearers during handling of nutrient can be recognized as another beginning of taint ( Hatha and Lakshmanaperumalsamy, 1997 ) .This survey showed the MPN-multiplex PCR method was considered to be more successful in observing Salmonella spp.
, Salmonella Typhi and Salmonella Typhimurium than MPN-plating method ( Table 5 ) . This was consistent with the findings by Mahon, Murphy, Jones, & A ; Barrow ( 1994 ) who compared the manifold PCR technique and standard bacteriological technique for the sensing of Salmonella on the poulet tegument. Out of the 10 positive samples detected by manifold PCR, merely 5 of them were positive by civilization. They concluded that PCR may hold advantages over standard civilization methods for the sensing of Salmonella in domestic fowl samples. Busse ( 1995 ) stressed that the procedure of insulating Salmonella from nutrient is sometime prone to failure as they can be lost during enrichment even a contaminated part has been drawn. Apart from that, Salmonella spp.
can come in feasible but nonculturable province ( VBNC ) under unfavorable status which contributes to difficulty in culturing though many different conventional civilization media and enrichment governments have been proposed for isolation of Salmonella from nutrient and environmental samples ( Way, Josephson, Pillai, Abbaszadegan, Gerba, & A ; Pepper, 1993 ) . Smith, Newton, Harwood, & A ; Barer ( 2002 ) besides claimed that many major pathogens, including Salmonella enterica, are able to come in a VBNC province where they can non be recovered by standard civilization methods but retain viability and can revive under favorable conditions.In decision, this survey demonstrated distribution of Salmonella spp. , Salmonella Typhi and Salmonella Typhimurium in chopped fruits in Malaysia with peddler stables demoing well higher taint frequence than hypermarkets.
Therefore, sliced fruits in Malaysia pose a wellness hazard to consumers. It is of import to transport out the hazard appraisal of Salmonella in fresh green goods and set up control steps that can guarantee the original centripetal quality and nutritionary values of the fresh fruits.
This survey was supported by Science Fund ( project no. 545260 ) from Ministry of Science, Technology and Innovation, Malaysia and in portion by a Grant-in-Aid for Science Research ( KAKENHI 19101010 ) from the Japan Society for the Promotion of Sciences.
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210Primer appellationTargetPrimer sequence 5 ‘ to 3 ‘Length ( bp )Amplicon size ( bp )