Research Article Hiding in Fresh Fruits and Vegetables: … · 2020. 1. 11. · Research Article Hiding in Fresh Fruits and Vegetables: Opportunistic Pathogens May Cross Geographical

Research ArticleHiding in Fresh Fruits and Vegetables Opportunistic PathogensMay Cross Geographical Barriers

Zahra S Al-Kharousi1 Nejib Guizani1 Abdullah M Al-Sadi2

Ismail M Al-Bulushi1 and Baby Shaharoona3

1Department of Food Science amp Nutrition College of Agricultural and Marine Sciences Sultan Qaboos UniversityPO Box 34 Al-Khod 123 Muscat Oman2Department of Crop Sciences College of Agricultural and Marine Sciences Sultan Qaboos UniversityPO Box 34 Al-Khod 123 Muscat Oman3Department of Soils Water and Agricultural Engineering College of Agricultural and Marine Sciences Sultan Qaboos UniversityPO Box 34 Al-Khod 123 Muscat Oman

Correspondence should be addressed to Zahra S Al-Kharousi umohanedsqueduom

Received 30 September 2015 Accepted 24 January 2016

Academic Editor Susana Merino

Copyright copy 2016 Zahra S Al-Kharousi et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Different microbial groups of the microbiome of fresh produce can have diverse effects on human health This study was aimedat identifying some microbial communities of fresh produce by analyzing 105 samples of imported fresh fruits and vegetablesoriginated from different countries in the world including local samples (Oman) for aerobic plate count and the counts ofEnterobacteriaceae Enterococcus and Staphylococcus aureus The isolated bacteria were identified by molecular (PCR) andbiochemical methods (VITEK 2) Enterobacteriaceae occurred in 60 of fruits and 91 of vegetables Enterococcus was isolatedfrom 20 of fruits and 42 of vegetables E coli and S aureus were isolated from 22 and 7 of vegetables respectivelyNinety-seven bacteria comprising 21 species were similarly identified by VITEK 2 and PCR to species level E coli Klebsiellapneumoniae Enterococcus casseliflavus and Enterobacter cloacaewere themost abundant species many are known as opportunisticpathogens which may raise concern to improve the microbial quality of fresh produce Phylogenetic trees showed no relationshipbetween clustering of the isolates based on the 16S rRNA gene and the original countries of fresh produce Intercountry passage ofopportunistic pathogens in fresh produce cannot be ruled out which requires better management

1 Introduction

Being sources of high energy and rich in minerals vitaminsfibers and phenolics fruits and vegetables constitute animportant food group that has been linked to maintenanceof well-being of individuals [1] and to reduced incidence ofsome chronic diseases [2] In addition to the nutritional valueof fresh produce their diverse microbiomes can pass throughstomach to the gut where they establish specific associationswith the host resulting in various effects on human health [3]Recently interesting relationships have been found betweengut microbiota and obesity malnutrition cancer personalmotivation and decision-making in whichmicrobial balanceis critical for maintaining the healthy state [3 4]

On the other hand the increased consumption of fruitsand vegetables in recent years has been found to be accompa-nied by an increase in the number of human infections andoutbreaks [5] as these can serve as reservoirs for pathogensor opportunistic pathogens [3] Fruits and vegetables canbe contaminated with spoilage or pathogenic bacteria atany stage from production to consumption [6 7] Althoughtheir microflora is dominated by spoilage bacteria yeastsand molds fruits and vegetables can harbor pathogenicbacteria such as Salmonella Escherichia coli Bacillus cereusCampylobacter spp Yersinia enterocolitica Listeria monocy-togenes and Clostridium botulinum as well as some virusesand parasites [6] In Royal Hospital Oman May 2008 Bcereus caused a nosocomial outbreak with gastroenteritis

Hindawi Publishing CorporationInternational Journal of MicrobiologyVolume 2016 Article ID 4292417 14 pageshttpdxdoiorg10115520164292417

2 International Journal of Microbiology

and affected 58 individuals B cereus and its toxin werefound in different foods including vegetables [8] A moreserious outbreak MayndashJuly 2011 was caused by Shiga toxinproducing E coli O104H4 in Germany where 2987 cases ofgastroenteritis 855 cases of hemolytic-uremic syndrome and53 deaths were reported Fenugreek sprouts were found tobe contaminated with the causative agent [9] Bean sproutswere linked to two outbreaks in USA in 2014 one was causedby L monocytogenes in August while Salmonella Enteritidiscaused the other one just a month later L monocytogeneswas also involved in another outbreak that was linked tocaramel apples in October 2014 in USA [10] Opportunisticpathogens can cause life-threatening infections mainly inimmunocompromised people but they may have positiveeffects on the health of immunocompetent individuals bystimulating immune functions and priming the immunesystem continuously [3] Nonpathogenicmicrobes associatedwith fruits and vegetables may have various consequences onthe quality of the produce by affecting the rate of the foodspoilage Fruits and vegetables seemalso to be the sources thatdisseminate many microbes to food preparation areas [11]

In Oman large quantities of fruits and vegetables areimported nearly from all around the world to provide a year-round supply [12] to this countrywhich is located at the cross-roads of the exchange of cultivated plants [13] This studyaims at assessing the microbial load of some fresh fruits andvegetables imported into or grown in Oman and to identifythe isolated bacteria by biochemical and molecular methodswith emphasis on emergent opportunistic pathogens Thestudy will also investigate genetic relationships betweenbacteria isolated from fruits and vegetables originated fromdifferent countries in the world Findings from this study willprovide information on the sanitary conditions of fruits andvegetables produced and consumed in this part of the worldwhich will help in improving their quality and safety forconsumption To our knowledge this is the first report thatanalyzes microbial content of fresh produce in this countryand also relates the presence of particular opportunisticpathogens isolated from different local and imported samplesto particular effects on human health

2 Materials and Methods

21 Sample Collection Fruits and vegetables that are mostlyeaten rawwere selected for this studyThese contained 7 typesof fresh imported or locally produced fruits (banana Musaspp dates Phoenix dactylifera mango Mangifera indicapapaya Carica papaya pomegranate Punica granatumtomato Solanum lycopersicum and watermelon Citrulluslanatus) and 6 types of fresh imported or locally producedvegetables (cabbage Brassica oleracea capsicum Capsicumannuum carrot Daucus carota cucumber Cucumis sativuslettuce Lactuca sativa and radish Raphanus sativus) Sam-ples were purchased from local markets in Muscat or NizwaOman during the period of April to September 2014 Theimported fruits and vegetables originated from differentcountries and they were selected depending on their avail-ability in themarket during that period of timeThree samples

were obtained for each fruit or vegetable type originated fromthe same country The samples were collected aseptically andrefrigerated until analyzed within 24 hrs Table 1 representsthe origin of all samples

22 Microbiological Analysis of Fruits and Vegetables

221 Sample Preparation Aerobic Plate Count and Selec-tive Bacterial Counts Samples were analyzed in a safetycabinet (Purifier class II Labconco Kansas USA) and cutusing sterile scalpels Some fruits (banana mango papayapomegranate and watermelon) were peeled and the insideflesh was analyzed Twenty-five grams of the cut sample wasweighed in a sterile stomacher bag and mixed with 225mLof Maximum Recovery Diluent (MRD) and homogenizedfor 1min using a stomacher (Bagmixer 100 MiniMix Inter-science Bois Arpents France) Serial dilutions were preparedfrom the original homogenate in MRD Aerobic plate count(APC) was performed by spread plate method The plates ofstandard plate count agar (SPCA) were incubated at 35∘C for48 hrs [7]The count of Enterobacteriaceaewas performed onViolet Red Bile Glucose (VRBG) agar by pour plate methodand incubation at 35∘C for 24 hrs E coli was counted onTryptone Bile X-glucuronide (TBX) medium and the plateswere incubated at 2 temperatures 30 and 44∘C for 24 hrs[14] Staphylococcus aureuswas counted onBaird-Parker (BP)agar after incubation at 35∘C for 24 hrs [7] Enterococcus wascounted on Slanetz agar after incubation at 35∘C for 48 hrs[15] All microbiological media were from Oxoid Englandand all experiments were repeated three times

222 Identification of Bacteria For each selective medium(BPA VRBG TBX and Slanetz agar) one bacterial isolateshowing the typical morphology (details could be foundby searchingmanufacturerrsquos website (httpwwwoxoidcomUKblueindexaspc=UKamplang=EN)) was selected fromeach sample for identification Bacteria were preserved atminus80∘C using cryogenic vials with beads (Viabank UK)

(1) Identification of Bacteria by VITEK 2 Identification ofbacteria using biochemical tests was done using an automatedidentification equipment VITEK 2-compact 15 (bioMerieuxFrance) following instructions of the manufacturer In briefbacterial suspensions were prepared in sterile saline (045)and the density was adjusted to a McFarland standard of05ndash063 using VITEK 2 DensiCheck (bioMerieux France)GP and GN cards were used for Gram-positive and Gram-negative bacteria respectively

(2) Identification of Bacteria by Polymerase Chain Reaction(PCR) The second method was performed using polymerasechain reaction (PCR) targeting bacterial 16S rRNA [16]Briefly DNA was extracted using ldquofoodproof StarPrep TwoKitrdquo (Biotecon Diagnostics GmbH Potsdam Germany)The quality and quantity of DNA were checked usingNanoDropTM 2000 (Thermoscientific USA) PCR was doneby transferring 1 120583L of each primer (27F and 1492R DNAsequences 51015840-AGAGTTTGATCMTGGCTCAG-31015840 and 51015840-TACGGYTACCTTGTTACGACTT-31015840 resp) 22120583L of sterile

International Journal of Microbiology 3

Table 1 Origin of fruits and vegetables and the number of positive samples for different microbial counts

Number Type of produce Originlowast APC Enterobacteriaceae S aureus Enterococcus E coli (30∘C) E coli (44∘C)Fruit (119899 = 60)

1 Banana Oman 3 3 0 0 0 0Philippine 3 2 0 0 0 0

2 Dates Oman 3 3 0 1 0 0Saudi Arabia 3 2 0 1 0 0

3 Mango Oman 3 3 0 1 0 0India Pakistan 3 3 1 1 0 0 0 0 0 0 0 0

4 Papaya Oman 3 3 0 3 0 0Thailand Philippine 3 3 3 3 0 0 0 0 0 0 0 0

5 Pomegranate Oman 1 0 0 0 0 0India Saudi Arabia 2 3 0 0 0 0 0 0 0 0 0 0

6 Tomato Oman 3 3 0 0 0 0Jordan Netherlands Syria 2 1 3 0 1 3 0 0 0 0 0 2 0 0 0 0 0 0

7 Watermelon Oman 3 1 0 1 0 0Egypt Iran 3 3 2 3 0 0 0 3 0 0 0 0

Vegetable (119899 = 45)

1 Cabbage Oman 3 3 0 3 3 3Netherlands 3 3 0 3 0 0

2 Carrot Oman 3 3 0 0 0 0Australia USA 3 3 3 3 0 0 0 0 0 0 0 0

3 Capsicum Oman 3 2 0 0 0 0Jordan UAE 3 3 3 2 0 0 0 0 0 0 0 0

4 Cucumber Oman 3 3 0 0 0 0UAE 3 3 0 1 0 0

5 Lettuce Oman 3 3 0 1 0 0Iran Jordan 3 3 3 1 0 0 3 3 0 1 0 2

6 Radish Oman 3 3 3 3 3 1China 3 3 0 3 1 1

APC aerobic plate count USA United States of America and UAE United Arab EmirateslowastThree samples were analyzed from each country

milliQ water and 1 120583L of the DNA sample to PCR reactiontubes containing PCR beads (puReTaq Ready-To-Go PCRbeads GE Healthcare UK) The thermal profile (Veriti 96-wellThermal cycler Applied Biosystems Singapore) for PCRreaction was as follows denaturation at 95∘C for 2minfollowed by 35 cycles of denaturation at 95∘C for 30 secannealing at 54∘C for 30 sec and extension at 72∘C for 1minThe final extension was at 72∘C for 10min and then kept at4∘C Aliquots of PCR products were checked by subjectingthem to 15 agarose gel electrophoresis and viewing them byGelDoc PCR products were sent for sequencing atMacrogen(Korea) using the same primers used for amplificationThe sequencing results were compared with those found atthe National Center for Biotechnology Information (NCBI)using BLAST search Then sequences of reference isolatesof each bacterial species were obtained from GenBank andneighbor joining trees were constructed for each bacterialgroupgenus using the Kimura 2 parameter evolutionarymodel (Mega 5) [17] Then bootstrap 50 majority-ruleconsensus trees were generated (1000 replications)

23 Statistical Methods Two-way analysis of variance(ANOVA) was used to determine if aerobic plate countsand Enterobacteriaceae counts varied significantly betweendifferent types of fruitsvegetables and between local andimported fruitsvegetables Data Disk 61 (Data DescriptionInc New York USA) was used to perform the statistical teststo identify any significant differences which were consideredas 119875 lt 005

3 Results

31 Bacterial Counts The origin and number of positivesamples of fruits and vegetables are shown in Table 1 whileFigure 1 shows the counts of different bacterial groups indifferent fruits and vegetables used in this study Aerobic platecount (APC) of local fruits (LF) (mean = 61 log CFU gminus1)was significantly greater than APC of imported fruits (IF)(mean = 50 log CFU gminus1) (ANOVA119875 = 00227 120572= 005) butAPC was not significantly different between local vegetables(LV) (mean = 63 logCFU gminus1) and imported vegetables (IV)


0

5

10

15

20

25

30

35

LF IF LV IV LF IF LV IV LF IF LV IV LF IF LV IV LF IF LV IV LF IF LV IV

Type of countcount

EnterococcusS aureusEnterobacteriaceae E coli (30∘C) E coli (44∘C)

RadishLettuceCucumberCapsicumCarrotCabbageWatermelon

TomatoPomegranatePapayaMangoDatesBanana

Aerobic plate

log(

mea

n+1

) (CF

U gminus

1)

Figure 1 Microbial counts (log (mean + 1) CFU gminus1) in local fruits (LF) imported fruits (IF) local vegetables (LV) and imported vegetables(IV)

(mean = 65 log CFU gminus1) (ANOVA 119875 = 02046 120572 = 005)APCdiffered significantly between the different types of fruits(ANOVA119875 = 00001120572=005) and vegetables (ANOVA119875 =00035 120572 = 005) Enterobacteriaceae and Enterococcus werefound in all groups that were tested LF IF LV and IVwhile Saureuswas found only in LV (radish)E coliwas isolated fromLV and IV but not from any fruit (Figure 1) Fifteen samples(714 119899 = 21) of the locally produced fruits were positivefor Enterobacteriaceae (mean = 49 log CFU gminus1) while abouthalf (538 119899 = 39) of the imported fruits were positivefor Enterobacteriaceae (mean = 42 log CFU gminus1) Seventeensamples (944 119899 = 18) of the local vegetables were positivefor Enterobacteriaceae (mean = 52 log CFU gminus1) while 24samples (889 119899 = 27) of the imported vegetables werepositive (mean = 53 logCFU gminus1) The count of Enterobac-teriaceae in LF was significantly greater than the count inIF (ANOVA 119875 = 00011 120572 = 005) but it did not differsignificantly between LV and IV (ANOVA 119875 = 02029120572 = 005) The count of Enterobacteriaceae was significantlydifferent from one type of fruit to another (ANOVA 119875 =00001120572= 005) as well as among different types of vegetables(ANOVA 119875 = 00001 120572 = 005)

At 30∘C E coli was isolated from 6 LV (3 cabbages and3 radishes mean = 38 logCFU gminus1) and from 2 IV samples(lettuce and radish mean = 19 log CFU gminus1) while at 44∘CE coli was isolated from 4 LV samples (3 cabbages and 1radish mean = 31 log CFU gminus1) and from 3 IV samples (2lettuces and 1 radish mean = 04 logCFU gminus1) Enterococ-cus was found in 6 LF (286 mean = 38 log CFU gminus1)

6 IF (154 mean = 26 logCFU gminus1) 7 LV (389 mean =42 log CFU gminus1) and 13 IV (481 mean = 38 logCFU gminus1)S aureuswas isolated only from 3 local radish samples (mean= 25 log CFU gminus1)

32 Identification of Bacteria Out of 130 bacteria (numberedfrom 1ndash130) that were isolated from fresh fruits and vegetables(except 4 reference strains numbers 127ndash130) a total of97 (746) isolates (21 species) were similarly identifiedby VITEK 2 and PCR to species level (Table 2) The mostencountered species was E coli (15 isolates) followed by Kleb-siella pneumoniae and Enterococcus casseliflavus (13 isolateseach) and then Enterobacter cloacae (12 isolates) Thirty-three bacteria were not similarly identified to species levelby VITEK 2 and PCR (Table 3) However to genus level 14isolates were not similarly identified by the 2 techniques Thedifferences in identification at genus level were only amongisolates of Enterobacteriaceae The 16S rRNA gene sequencesof bacteria numbers 1ndash130 were sequentially given accessionnumbers of KR265345-KR265474 by the GenBank

33 Phylogenetic Analysis Phylogenetic analysis showedgrouping of most bacterial isolates with the reference speciesobtained from NCBI except for few isolates from theEnterobacteriaceae K pneumoniae and R aquatilis madedistinct clear clusters while other isolates belonging toEnterobacteriaceae made several groups (Figure 2) E coliisolates grouped into two clusters (bootstrap values 66ndash86 Figure 3) However there was no relationship between


Table 2 Bacteria that were similarly identified to species level by VITEK 2 and PCR and their sources

Name of bacteria Number of isolates Rank SourceCitrobacter freundii 1 13 Tomato (Netherlands)Enterobacter amnigenus 4 7 Carrot (USA) lettuce (Oman) radish (China)Enterobacter asburiae 1 13 Carrot (Oman)

Enterobacter cloacae 12 4Cabbage carrot papaya radish watermelon (Oman)lettuce (Iran) papaya (Philippines) papaya (Thailand)tomato (Syria) watermelon (Egypt)

Enterobacter hormaechei 1 13 Tomato (Syria)Enterobacter ludwigii 3 8 Capsicum cucumber (UAE) tomato (Oman)

Enterococcus casseliflavus 13 2Cabbage (Netherlands) cucumber (UAE) dates (SaudiArabia) lettuce (Iran) radish (China) tomato ( Syria)watermelon (Iran) watermelon (Oman)

Enterococcus faecalis 7 5 Lettuce (Jordan) lettuce (Iran) radish mango papaya(Oman)

Enterococcus faecium 2 11 Cabbage (Oman) lettuce (Jordan)Enterococcus hirae 1 13 Papaya (Oman)Enterococcus raffinosus 1 13 Dates (Oman)

Escherichia coli 15 1 Cabbage radish (Oman) lettuce (Jordan) radish(China)

Klebsiella oxytoca 2 11 Radish (China) tomato (Oman)

Klebsiella pneumoniae 13 2Cabbage (Oman) capsicum (Jordan) banana(Philippines) dates (Oman) mango (Oman) papaya(Oman) watermelon (Iran)

Kluyvera intermedia 1 13 Cabbage (Netherlands)

Pantoea agglomerans 7 5Cabbage (Netherlands) carrot (Australia) carrot(USA) capsicum (UAE) lettuce (Jordan) radish(China) watermelon (Iran)

Rahnella aquatilis 3 8 Carrot (Australia) lettuce (Iran)Raoultella planticola 1 13 Cabbage (Netherlands)Serratia liquefaciens 1 13 Carrot (USA)Serratia marcescens 1 13 Tomato (Oman)Staphylococcus aureus 3 8 Radish (Oman)Reference strainsStaphylococcus aureus 1 ATCC 29213Staphylococcus aureus 1 ATCC 25923Escherichia coli 1 ATCC 25922Enterococcus faecalis 1 ATCC 29212Total 97

clustering of isolates and the countries or commoditiesfrom which they were obtained The 5 isolates (including 2reference strains) of S aureus also grouped together with avery high bootstrap support (100 Figure 4) Enterococcusspecies grouped into different clusters (50ndash100 bootstrapsupport Figure 5) There was also no relationship betweenclustering of isolates of the Enterococcus species and thecountries or commodities from which they were obtained(Figure 5)

4 Discussion

Most studies report the presence of pathogens in fruitsand vegetables but the counts are rarely documented [5]

APC can be used to monitor the hygienic quality of aproduct throughout processing and distribution [18] but itis difficult to be used to draw a conclusion on the safety ofa product [19] Nevertheless some researchers [20] founda strong association between APC and presence of E coliin beef carcasses where 88 of samples having APC ofge4 logCFUcm2 were positive for E coli while only 21 ofsamples having APC of lt2 logCFUcm2 were positive Inthis study E coli was isolated only from vegetable samples(from local cabbage and radish and from imported lettuceand radish) all withmeanAPCge 65 log CFU gminus1 (Figure 1) Itis possible that a certain association does exist between APCand a particular pathogen but it depends on the type of aproduct and needs a large number of samples to be tested


Table 3 Bacteria that were not similarly identified to species level by VITEK and PCR and their sources

Number of strains VITEK PCR Source20 Enterococcus faecium or E gallinarumlowast Enterococcus mundtii Cabbage (Oman)21 Enterococcus cecorum or Leuconostoc pseudomesenteroideslowast Enterococcus sulfureus Cabbage (Oman)27 Enterococcus cecorum or Leuconostoc pseudomesenteroideslowast Enterococcus sulfureus Lettuce (Jordan)31 Enterococcus faecium or E gallinarumlowast Enterococcus mundtii Lettuce (Iran)33 Enterococcus faecium or E gallinarumlowast Enterococcus mundtii Radish (Oman)36 Enterococcus faecium or E gallinarumlowast Enterococcus mundtii Radish (China)45 Enterococcus raffinosus Enterococcus gilvus Tomato (Syria)58 Klebsiella pneumoniae ssp pneumoniae Klebsiella oxytoca Carrot (Oman)65 Pantoea spp or Aeromonas sobrialowast Pantoea agglomerans Capsicum (Oman)66 Pantoea spp Pantoea agglomerans Capsicum (Oman)67 Pantoea spp Erwinia aphidicola Capsicum (Jordan)69 Pantoea spp Erwinia aphidicola Capsicum (Jordan)72 Ewingella americana or Pantoea spplowast Pantoea dispersa Cucumber (Oman)73 Pantoea spp Pantoea dispersa Cucumber (Oman)74 Pantoea spp Pantoea dispersa Cucumber (Oman)76 Pantoea spp Erwinia aphidicola Cucumber (UAE)77 Pantoea spp Erwinia rhapontici Cucumber (UAE)80 Leclercia adecarboxylata Enterobacter cancerogenus Lettuce (Oman)83 Pantoea spp Enterobacter cloacae Lettuce (Iran)85 Pantoea spp Pectobacterium carotovorum Radish (Oman)87 Pantoea spp Enterobacter cloacae Radish (Oman)91 Serratia ficaria Pantoea dispersa Banana (Oman)92 Pantoea spp Pantoea cypripedii Banana (Oman)98 Enterobacter cloacae complexlowastlowast Enterobacter oryzae Dates (Saudi Arabia)99 Pantoea spp Pantoea agglomerans Dates (Saudi Arabia)100 Pantoea spp Pseudocitrobacter faecalis Mango (Oman)103 Pantoea spp Pantoea dispersa Mango (India)104 Pantoea spp Pantoea dispersa Mango (Pakistan)108 Pantoea spp Pantoea eucrina Papaya (Thailand)109 Pantoea spp Pantoea dispersa Papaya (Thailand)112 Pantoea spp Escherichia hermannii Papaya (Philippines)113 Pantoea spp Enterobacter cloacae Papaya (Philippines)118 Pantoea spp Pantoea vagans Tomato (Syria)lowastLow discrimination organisms (same biopattern was produced by the tested strains) lowastlowastSlash line biopattern is the same for these organisms Enterobacterkobei E hormaechei E cloacae ssp cloacae E cloacae ssp dissolvens or E ludwigii

In the current study vegetables showed slightly largerAPCs than fruits (range of mean count = 41ndash70 and 11ndash67 logCFU gminus1 resp) Vegetables are usually grown closerto soil than fruits and thus might be contaminated easilyfrom soil [21] In addition some fruits that were observedto be physically contaminated with soil (watermelon) ordeposited with dust (papaya) showed high APC counts ofge6 logCFU gminus1 Leafy vegetableswere reported to give greaterAPC than nonleafy vegetables [7] The highest microbialcounts were obtained from the APC of leafy vegetablesimported lettuce (mean = 70 logCFU gminus1) followed by localcabbage (mean = 69 logCFU gminus1) This is 1 log less thanthe maximum APC (8 logCFU gminus1) that was reported incantaloupe spinach and lettuce in Saudi Arabia [7] but their

maximum Enterobacteriaceae count of 4 log CFU gminus1 (cab-bage and lettuce) was about 2 logs less than what was foundin the current study (papaya cabbage and radish) (Figure 1)Pomegranate was found to be the least contaminated fruithaving APC of 11 and 32 logCFU gminus1 (local and importedresp) with none of the other sought bacterial groups beingdetected Pomegranate juice [2] and extracts [22 23] wereshown to possess antibacterial activity but the low pH ofabout 35 of pomegranate [24] might also have contributedto this inhibitory activity

Clinically many members of the family of Enterobacte-riaceae are among the most potent and prevalent pathogens[25] indeed many of them have acquired resistance to mostantibiotics [21] Once ingested antibiotic resistance genes


74 Enterobacter amnigenus strain h-14 (KC1394341) 73 79-Enterobacter amnigenus lettuce Oman (KR265423)

78-Enterobacter amnigenus lettuce Oman (KR265422)93 90-Enterobacter amnigenus radish China (KR265434)

63-Enterobacter amnigenus carrot USA (KR265407)54-Kluyvera intermedia cabbage Netherlands (KR26539)

99 Kluyvera intermedia strain CD-10 (JF4965391) 80-Enterobacter cancerogenus lettuce Oman (KR265424)

99 Enterobacter cancerogenus isolate PSB20 (HQ2427331)98 53-Raoultella planticola cabbage Netherlands (KR265397)

Raoultella planticola strain ATCC33531 (NR_0249961)100-Pseudocitrobacter faecalis mango Oman (KR265444)

99 Pseudocitrobacter faecalis strain 52 CIT (KF0579421) 99 117-Citrobacter freundii tomato 2 Netherlands (KR265461)

Citrobacter freundii strain BAB-1679 (KF5351421)112-Enterobacter cloacae watermelon Egypt (KR265466) 116-Enterobacter ludwigii tomato Oman (KR265460)

66 52-Enterobacter cloacae cabbage Oman (KR265396)Enterobacter cloacae strain PYPB08 (JF3468951) 119-Enterobacter cloacae tomato Syria (KR265463) 57-Enterobacter cloacae carrot Oman (KR265401) 56-Enterobacter asburiae carrot Oman (KR265400) Enterobacter asburiae strain R2-343 (JQ6596231)71-Enterobacter ludwigii capsicum UAE (KR265415)

76 Enterobacter ludwigii strain ALS-2 (KJ6389891)56 115-Klebsiella oxytoca tomato Oman (KR265459)

58-Klebsiella oxytoca carrot Oman (KR265402)88-Klebsiella oxytoca radish China (KR265432)

99 Klebsiella oxytoca strain HP1 (AY6016791) 65 123-Enterobacter cloacae watermelon Egypt (KR265467)

75-Enterobacter ludwigii cucumber UAE (KR265419)60 121-Enterobacter cloacae watermelon Oman (KR265465)

69 107-Enterobacter cloacae papaya Oman (KR265451)52 111-Enterobacter cloacae papaya Philippines (KR265455)

110-Enterobacter cloacae papaya Thailand (KR265454)120-Enterobacter hormaechei tomato Syria (KR265464)

8054 Enterobacter hormaechei strain 22 BSL2 (KF2545941)99 85-Pectobacterium carotovorum radish Oman (KR265429)

Pectobacterium carotovorum ssp carotovorum strain BGZ-1 (KP2749031) 73 109-Pantoea dispersa papaya Thailand (KR265453)

64 104-Pantoea dispersa mango Pakistan (KR265448)80 103-Pantoea dispersa mango India (KR265447)

72-Pantoea dispersa cucumber Oman (KR265416)

75 75

62

79 61 99

9783

8359

99

7099

6980

5672 69

55

84

568999

63

99

93

6092

73

6189

5499

84

59

9475

Pantoea dispersa strain F48 (KM0198461)73-Pantoea dispersa cucumber Oman (KR265417) 74-Pantoea dispersa cucumber Oman (KR265418) 108-Pantoea eucrina papaya Thailand (KR265452) Pantoea eucrina (HE6595141)91-Pantoea dispersa banana Oman (KR265435)92-Pantoea cypripedii banana Oman (KR265436)Pantoea cypripedii strain ATCC 29267 (NR_1188571)67-Erwinia aphidicola capsicum Jordan (KR265411) Erwinia aphidicola strain X 001 (NR_1047241) 76-Erwinia aphidicola cucumber UAE (KR265420) 69-Erwinia aphidicola capsicum Jordan (KR265413)

77-Erwinia rhapontici cucumber UAE (KR265421)Erwinia rhapontici strain ATCC 29283 (NR_1188581)65-Pantoea agglomerans capsicum Oman (KR265409) 66-Pantoea agglomerans capsicum Oman (KR265410)60-Pantoea agglomerans carrot Australia (KR265404)Pantoea vagans (HG4210101)118-Pantoea vagans tomato Syria (KR265462)81-Pantoea agglomerans lettuce Jordan (KR265425)125-Pantoea agglomerans watermelon Iran (KR265469) 64-Pantoea agglomerans carrot USA (KR265408)99-Pantoea agglomerans dates Saudi Arabia (KR265443) 70-Pantoea agglomerans capsicum UAE (KR265414)89-Pantoea agglomerans radish China (KR265433)Pantoea agglomerans strainzzx15 (KJ0094171) 55-Pantoea agglomerans cabbage Netherlands (KR265399) 98-Enterobacter oryzae dates Saudi Arabia (KR265442) Enterobacter oryzae strain R5-395 (JQ6597641)113-Enterobacter cloacae papaya Philippines (KR265457)87-Enterobacter cloacae radish Oman (KR265431)112-Escherichia hermannii papaya Philippines (KR265456)Escherichia hermannii strain Num 2 (KM0099901) 106-Klebsiella pneumoniae papaya Oman (KR265450)102-Klebsiella pneumoniae mango Oman (KR265446)94-Klebsiella pneumoniae banana Philippines (KR265438) 93-Klebsiella pneumoniae banana Philippines (KR265437) 97-Klebsiella pneumoniae dates Oman (KR265441)101-Klebsiella pneumoniae mango Oman (KR265445) 124-Klebsiella pneumoniae watermelon Iran (KR265468) 68-Klebsiella pneumoniae capsicum Jordan (KR265412) 126-Klebsiella pneumoniae watermelon Iran (KR265470) 96-Klebsiella pneumoniae dates Oman (KR265440)95-Klebsiella pneumoniae dates Oman (KR265439)51-Klebsiella pneumoniae cabbage Oman (KR265395)50-Klebsiella pneumoniae cabbage Oman (KR265394)Klebsiella pneumoniae strain NRC37 (KP2974431) 86-Enterobacter cloacae radish Oman (KR265430)

83-Enterobacter cloacae lettuce Iran (KR265427)84-Enterobacter cloacae lettuce Iran (KR265428)Serratia liquefaciens strain V4 (HQ3350011)62-Serratia liquefaciens carrot USA (KR265406) 59-Rahnella aquatilis carrot Australia (KR265403) 82-Rahnella aquatilis lettuce Iran (KR265426)61-Rahnella aquatilis carrot Australia (KR265405)Rahnella aquatilis strain HX2 (NR_0749211)Staphylococcus aureus ssp aureus strain FUA 2080 (JN1025651)

Figure 2 Neighbor joining tree based on 16S rRNA gene sequences of members of Enterobacteriaceae isolated from fresh produceStaphylococcus aureus (JN102565) was included as an outgroup KR-accession numbers correspond to gene sequences that belong to isolatesanalyzed in this study while others were obtained from NCBI database Bootstrap values above 50 are shown (1000 replications)


66

82

88

9-Escherichia coli cabbage Oman 30∘C (KR265353)7-Escherichia coli radish China 44∘C (KR265351)4-Escherichia coli lettuce Jordan 44∘C (KR265348)129-Escherichia coli ATCC 25922 (KR265473)8-Escherichia coli cabbage Oman 30∘C (KR265352)1-Escherichia coli cabbage Oman 44∘C (KR265345)

11-Escherichia coli lettuce Jordan 30∘C (KR265355)

Escherichia coli strain JCM 24006 (AB5485791)2-Escherichia coli cabbage Oman 44∘C (KR265346)Staphylococcus aureus ssp aureus strain FUA2080 (JN1025651)

10-Escherichia coli cabbage Oman 30∘C (KR265354)6-Escherichia coli radish Oman 44∘C (KR265350)15-Escherichia coli radish China 30∘C (KR265359)14-Escherichia coli radish Oman 30∘C (KR265358)13-Escherichia coli radish Oman 30∘C (KR265357)5-Escherichia coli lettuce Jordan 44∘C (KR265349)12-Escherichia coli radish Oman 30∘C (KR265356)3-Escherichia coli cabbage Oman 44∘C (KR265347)

Figure 3Neighbor joining tree based on 16S rRNAgene sequences ofE coli isolated from fresh produceE coli strain JCM24006 (AB548579)was included as a reference strain and Staphylococcus aureus (JN102565) was included as an outgroup KR-accession numbers correspond togene sequences that belong to isolates analyzed in this study while AB548579 and JN102565 were obtained from the NCBI database Bootstrapvalues above 50 are shown (1000 replications)

85

81

100

128-Staphylococcus aureus ATCC 25923 (KR265472)

16-Staphylococcus aureus radish Oman (KR265360)

17-Staphylococcus aureus radish Oman (KR265361)18-Staphylococcus aureus radish Oman (KR265362)

Enterococcus sulfureus ATCC 49903 (NR_1157661)

Staphylococcus aureus ssp aureus strain FUA2080 (JN1025651)


Figure 4 Neighbor joining tree based on 16S rRNA gene sequences of Staphylococcus aureus isolated from local radish Enterococcus sulfureusATCC 49903 (NR115766) was included as an outgroup KR-accession numbers correspond to gene sequences that belong to isolates analyzedin this study while others were obtained from NCBI database Bootstrap values above 50 are shown (1000 replications)

if present may be transferred to the normal flora of thehuman gut and thus possibly to the pathogenic bacteria[26] especially if these resistance determinants can persistin the human gut for as long as a year as was proposed byForslund and colleagues [27] Many outbreaks in associationwith consumption of fresh produce are due to bacteriabelonging to Enterobacteriaceae Outbreaks that were linkedto Salmonella Poona in cucumbers S Enteritidis in beansprouts E coli O121 in raw clover sprouts in 2014 and Ecoli O157H7 in ready-to-eat salads in 2013 are just someexamples [10]The normal flora of many fruits and vegetablescontains bacteria within the group of Enterobacteriaceae thatmight be present at high counts [28] Our results showedthat the counts of Enterobacteriaceae were comparable toAPCs (Figure 1) and only pomegranatewas free frombacteriabelonging to this family (Table 1) A total of 15 species ofEnterobacteriaceae were similarly identified by VITEK 2 andPCR (Table 2) In addition 11 other species within this groupwere identified by PCR but not by VITEK 2 (Table 3) The 3genera of Erwinia Pectobacterium and Pseudocitrobacter are

not included in the most updated version (701) of the VITEK2 identification database by GN card (isolates numbers 6769 76 77 85 and 100) and 6 other isolates could have beenmisidentified at genus level by VITEK 2 as compared to PCR(isolates numbers 80 83 87 91 112 and 113) though thesegenera are included inVITEK2databaseWithin this group 5species of the similarly identified genera byVITEK2 andPCRare not included in VITEK 2 latest version database includingPantoea dispersa (isolates numbers 72 73 74 103 104 and109) Pantoea cypripedii (isolate number 92) Enterobacteroryzae (isolate number 98) Pantoea eucrina (isolate number108) and Pantoea vagans (isolate number 118)

The genus of Klebsiella harbors opportunistic pathogenscapable of causing severe infections [29] Klebsiella pneumo-niae was one of the most dominant species in this study andwas isolated from local cabbage dates mango and papayaand from imported capsicum (Jordan) banana (Philippine)andwatermelon (Iran)Pantoea agglomerans (formerlyEnter-obacter agglomerans) andRahnella aquatilis are opportunisticpathogens as well [30] The genus of Enterobacter is one of


9350

80

6876

82 96

68

73

95

58 72

8482

100

55

10075

28-Enterococcus faecium lettuce Jordan (KR265372)Enterococcus faecium strain 132A (C4S1)

19-Enterococcus faecium cabbage Oman (KR265363)43-Enterococcus hirae papaya Oman (KR265387)Enterococcus hirae (KF1835101)31-Enterococcus mundtii lettuce Iran (KR265375)Enterococcus mundtii (AB9047801)33-Enterococcus mundtii radish Oman (KR265377)20-Enterococcus mundtii cabbage Oman (KR265364)36-Enterococcus mundtii radish China (KR265380)45-Enterococcus gilvus tomato Syria (KR265389)Enterococcus gilvus (AB7424481)Enterococcus raffinosus (AB9325241)38-Enterococcus raffinosus dates Oman (KR265382)25-Enterococcus casseliflavus cucumber UAE (KR265369)44-Enterococcus casseliflavus tomato Syria (KR265388)47-Enterococcus casseliflavus watermelon Iran (KR265391)39-Enterococcus casseliflavus dates Saudi Arabia (KR265383)35-Enterococcus casseliflavus radish China (KR265379)46-Enterococcus casseliflavus watermelon Oman (KR265390)23-Enterococcus casseliflavus cabbage Netherlands (KR265367)22-Enterococcus casseliflavus cabbage Netherlands (KR265366)48-Enterococcus casseliflavus watermelon Iran (KR265392)30-Enterococcus casseliflavus lettuce 2 Iran (KR265374)Enterococcus casseliflavus strain CCFM8319 (KJ8038761)37-Enterococcus casseliflavus radish China (KR265381)24-Enterococcus casseliflavus cabbage Netherlands (KR265368)49-Enterococcus casseliflavus watermelon Iran (KR265393)130-Enterococcus faecalis ATCC 29212 (KR265474)29-Enterococcus faecalis lettuce 1 Iran (KR265373)Enterococcus faecalis strain P26-24 (KC1132051)34-Enterococcus faecalis radish Oman (KR265378)40-Enterococcus faecalis mango Oman (KR265384)42-Enterococcus faecalis papaya Oman (KR265386)41-Enterococcus faecalis papaya Oman (KR265385)26-Enterococcus faecalis lettuce Jordan (KR265370)32-Enterococcus faecalis radish Oman (KR265376)21-Enterococcus sulfureus cabbage Oman (KR265365)27-Enterococcus sulfureus lettuce Jordan (KR265371)Enterococcus sulfureus strain ATCC 49903 (NR_1157661)Staphylococcus aureus subsp aureus strain FUA2080 (JN1025651)

Figure 5 Neighbor joining tree based on sequencing 16S rRNA gene formembers ofEnterococcus isolated from fresh produce Staphylococcusaureus (JN102565) was included as an outgroup KR-accession numbers correspond to gene sequences that belong to isolates analyzed in thisstudy while others were obtained from NCBI database Bootstrap values above 50 are shown (1000 replications)

the largest genera in the family Enterobacteriaceae having atleast 19 species It also shows polyphyletic patterns when phy-logenetic trees are constructed based on 16S rRNA sequences[31] Many species within the genus of Enterobacter havebeen marked as emerging pathogens [30] Pseudocitrobacteris a new genus in the family of Enterobacteriaceae andPseudocitrobacter faecalis was isolated from a stool sample ofa patient and was found to possess NDM-1 carbapenemasewhich confers resistance to carbapenem antibiotics [32] In

this study one isolate was identified as P faecalis (from localmango) by PCR (Table 3) To our knowledge this is the firstreport of isolation of this species from fresh produce

Erwinia aphidicola was first isolated from the gut ofpea aphid by Harada and coworkers [33] In their studybiochemical tests showed that this species was the closestto Erwinia herbicola and Pantoea agglomerans but DNA-DNA hybridization separated it into a new species Later onthis species was recognized as an important plant pathogen


especially for beans [34] In this study the 3 isolates ofE aphidicola (from imported capsicum and cucumber)as identified by PCR were identified as Pantoea spp byVITEK 2 and this could be because of their similaritiesbased on biochemical reactions Rahnella aquatilis thoughis commonly present in vegetables [28] has been foundto be a primary pathogen and was isolated from bloodsputum urine and stool [35] In this study R aquatilis wasisolated from carrot (Australia) and lettuce (Iran) (Table 2)Likewise Serratia spp are often present in vegetables [28]but a species such as Serratia marcescens (isolated from localtomato in this study) has been recognized as an importantcause of nosocomial infections [36] Serratia liquefaciens wasalso found to cause bloodstream infection [37] Citrobacterfreundii isolated from tomato Netherlands in this studywas reported to cause nosocomial bacteremia [38] Raoultellaplanticola (isolated from cabbage Netherlands) was found tocause wound infection [39]

E coli is considered a better sanitary indicator thanEnterobacteriaceae [28] where its presence indicates recentfecal contamination [30] Unlike Osterblad and colleagues[40] who rarely found E coli in the examined 137 vegetablesamples E coli was the most encountered species in thisstudy properly due to its selective isolation on TBX mediumAccording to Public Health Leadership Society [41] freshproduce with E coli count of ge2 logCFU gminus1 is consideredunsatisfactory In this study local cabbage and radish andimported radish contained this unsatisfactory level (Figure 1)In the current study prevalence of E coli in local andimported vegetables (33 119899 = 18 and 15 119899 = 27 respor 22 in all vegetables) is greater than what was found byAbadias and coworkers [28] in whole vegetables (7 119899 = 28)The count of E coli isolated at 44∘Cwas less than the count at30∘C (Figure 1) indicating that for direct counting of E coli at44∘C on TBX it would be better to preincubate plates at 30∘Cin order to resuscitate any injured cells

S aureuswas foundonly in local radishwith amean countof 25 log CFU gminus1 It was reported that high levels of S aureusof gt4 logCFU gminus1 might indicate enterotoxin production[14] However other researchers [42] reported that S aureuswas attributed to be the cause of a food poisoning outbreak ina hotel in Japan in 2005 where pickled radish was one of thesources that S aureus was isolated from The counts of thispathogen in radish sashimi and frozen crab were 17 17 and20 logCFU gminus1 respectively but its enterotoxin was found ina vomit sample indicating that isolation of pathogens fromfoods is significant even at low levels as some of themmightproliferate in the food products if they are not kept at propertemperature (1ndash5∘C for fresh fruits and vegetables) [28]

Similar to previous findings [43] our results showed thatmore vegetable samples (LV = 389 IV = 481) harborEnterococcus than fruits (LF = 286 IF = 154) Enterococ-cus spp are involved in food intoxication and in spreadingantibiotic resistance through the food chain and they are aleading cause of nosocomial infections [15] where E faecalisand E faecium cause the majority of them [43] Enterococcuswas isolated from all types of the tested fruits and vegetables(local andor imported) except banana pomegranate carrot

and capsicum (Table 1) The highest counts of Enterococcuswere found in local cabbage followed by local papaya (49 and46 logCFU gminus1 resp Figure 1)The presence of Enterococcusindicates fecal contamination [30] that might not be recent asthey can persist for long time in the environment Howeverbesides gastrointestinal tract of animals and humans thisubiquitous organism can be found in soil and on plantsbut identification of species might help in discriminatingthe source of Enterococcus E faecalis and E faecium mightbe a better indicator of human fecal contamination thanEnterococcus casseliflavus and Enterococcus mundtii that aremore prevalent in environment [44] In this study E faecaliswas isolated from lettuce imported from Jordan and Iran andfrom radish mango and papaya produced in Oman whileE faecium was isolated from cabbage produced in Omanand from lettuce imported from Jordan In other studiesE faecalis was isolated from lettuce radish tomato applecucumber potato sprouts and carrot while E faecium wasisolated from dates lettuce tomato and soy product [15 43]

Similar to the result of others [43] E casseliflavuswas themost common species of Enterococcus (13 isolates Table 2)Three isolates of Enterococcus ludwigii (capsicum cucumber(UAE) tomato (Oman)) 1 isolate of Enterococcus hirae(papaya Oman) and 1 isolate of Enterococcus raffinosus(dates Oman) were found Three species within the genusof Enterococcus that were not similarly identified by VITEK2 and PCR are not included in the VITEK 2 latest versiondatabase including E mundtii (isolates numbers 20 31 33and 36) E sulfureus (isolates numbers 21 and 27) and Egilvus (isolate number 45) E hirae and E mundtii wereisolated previously from different fruits and vegetables [43]It was shown that genetic exchange of virulence determinantscan occur between food and clinical isolates of E faecium[45] In fact contaminated food might pose a risk and be asource for nosocomial infections [46]

Table 4 summarizes some bacteria that were previouslyreported to cause infections in human and that were isolatedin the present study from fresh produce Their sources couldbe environmental food or unknown Clearly these bacteriacan gain access to critical sites in the body and establishinfections Many of them are considered as opportunisticpathogens capable of establishing infections in immuno-compromised individuals However there are some sporadiccases where they can act as primary pathogens and thus causeinfections in immunocompetent healthy people Moreoverit is possible that opportunistic pathogens might emerge asimportant pathogens in future [3]Determining themicrobialquality of fresh produce served in hospitals would be ofparticular importance because translocation of pathogenicor opportunistic pathogens either to immunocompromisedpatients or to other environments in the hospitals might givethese bacteria andor their pathogenicity genetic determi-nants a better chance to be involved in the disease process

Phylogenetic analysis showed grouping of most bacterialisolateswith the reference strains obtained fromNCBI exceptfor few speciesmainly from the EnterobacteriaceaeMemberswithin Enterobacteriaceae have close genetic relationships


Table 4 Case reports for bacterial infections caused by bacteria listed in Table 2 as presented in the literature

Bacteria Specimen of isolation Country RefCitrobacter freundii Blood urine USA South Africa [38 47]Enterobacter amnigenus Eye USA [48]Enterobacter asburiae Blood South Africa [47]Enterobacter cloacae Stool USA [49]Enterobacter hormaechei Bloodtrachea nasopharynxrectum USA [50]Enterobacter ludwigii Bone India [51]Enterococcus casseliflavus Cerebrospinal fluid Italy [52]Enterococcus faecalis Blood South Africa [47]Enterococcus faecium Cerebrospinal fluid USA [53]Enterococcus hirae Blood France [54]Enterococcus raffinosus Pelvic hematoma Germany [55]Escherichia coli Stool USA [49]Klebsiella oxytoca Stool Austria [56]Klebsiella pneumoniae Urineliver trachea USA South Africa [47 57]Pantoea agglomerans Blood Italy [58]Rahnella aquatilis Blood Italy [58]Raoultella planticola Peritoneal fluid soft tissue wound Brazil Ireland Saudi Arabia [39 59 60]Serratia liquefaciens Blood USA [37]Serratia marcescens Eye USA [61]Staphylococcus aureus Blood Italy [62]

and species often give polyphyletic groups in trees con-structed using 16S rRNA sequences [63ndash65] The 5 isolatesof S aureus were grouped together with a very high boot-strap support (100 Figure 4) Likewise it was shown thatsequencing of 16S rRNAgene could be accurately used to gen-erate phylogenetic relationships of the genus Staphylococcus[66] Sequencing of 16S rRNA gene gave good discriminationof the genus Enterococcus into 8 clusters namely E faeciumE hirae E mundtii E gilvus E raffinosus E casseliflavus Efaecalis and E sulfureus (Figure 5 bootstrap percentages 50ndash100) Similar discrimination of the genus Enterococcus waspreviously shown by sequencing 16S rRNA and rpoA genes[67]

Although bacteria can contaminate fresh produce atany stage from farm to fork in the current study somesamples were peeled andmanywere obtained in their originalpackages whichmay suggest contamination from the originalcountries No relationship was found between clustering ofthe isolates based on the 16S rRNA gene and the countriesor commodities from which they were obtained Thus cir-culation of these bacterial species among different countriescannot be ruled out and this may raise concerns aboutthe role played by quarantine in limiting spread of humanand plant pathogens via agricultural commodities Howevermore epidemiological studies should be done specifically foreach species taking into account increasing the number ofisolates and further characterizing the strains in order toestablish some relationships between the different genotypes

In conclusion this study analyzed for the first time themicrobial load of some fresh fruits and vegetables in Omanat the point of retail sale The presence of high counts ofAPC andEnterobacteriaceae aswell as fecal bacteria indicates

that the hygienic conditions of fruits and vegetables in Omanare not satisfactory and should be improved Understandingthe complex microbial ecosystem that is unique for eachproduce in a given country would be important to establishfully controlled systems for fruits and vegetables that canbe applied during harvest production distribution andmarketing aimed at controlling spoilage and pathogenicmicrobes The presence of S aureus possible pathogenicenterococci and E coli as well as other opportunistic andemerging pathogens in the examined fresh fruits and veg-etables requires further investigation of virulence determi-nants which might be important in future for tracking theirpathogenicity evolution

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors thank Sultan Qaboos University for providingfinancial support for this study

References

[1] V Prasanna T N Prabha and R N Tharanathan ldquoFruitripening phenomenamdashan overviewrdquo Critical Reviews in FoodScience and Nutrition vol 47 no 1 pp 1ndash19 2007

[2] Y-L Lee T Cesario Y Wang E Shanbrom and L ThruppldquoAntibacterial activity of vegetables and juicesrdquo Nutrition vol19 no 11-12 pp 994ndash996 2003


[3] G Berg A Erlacher K Smalla and R Krause ldquoVegetablemicrobiomes is there a connection among opportunistic infec-tions human health and our rsquogut feelingrsquordquo Microbial Biotech-nology vol 7 no 6 pp 487ndash495 2014

[4] C A Lozupone J I Stombaugh J I Gordon J K Jansson andR Knight ldquoDiversity stability and resilience of the human gutmicrobiotardquo Nature vol 489 no 7415 pp 220ndash230 2012

[5] A N Olaimat and R A Holley ldquoFactors influencing themicrobial safety of fresh produce a reviewrdquo Food Microbiologyvol 32 no 1 pp 1ndash19 2012

[6] L R Beuchat ldquoEcological factors influencing survival andgrowth of human pathogens on raw fruits and vegetablesrdquoMicrobes and Infection vol 4 no 4 pp 413ndash423 2002

[7] S A Hassan A D Altalhi Y A Gherbawy and B A El-Deeb ldquoBacterial load of fresh vegetables and their resistanceto the currently used antibiotics in Saudi Arabiardquo FoodbornePathogens and Disease vol 8 no 9 pp 1011ndash1018 2011

[8] S S Al-Abri A K Al-Jardani M S Al-Hosni P J Kurup S Al-Busaidi and N J Beeching ldquoA hospital acquired outbreak ofBacillus cereus gastroenteritis Omanrdquo Journal of Infection andPublic Health vol 4 no 4 pp 180ndash186 2011

[9] S Hauswaldt M Nitschke F Sayk W Solbach and J K-M Knobloch ldquoLessons learned from outbreaks of Shiga toxinproducing Escherichia colirdquo Current Infectious Disease Reportsvol 15 no 1 pp 4ndash9 2013

[10] Centers for Disease Control and Prevention (CDC) 2014httpwwwcdcgovoutbreaks

[11] J W Leff and N Fierer ldquoBacterial communities associated withthe surfaces of fresh fruits and vegetablesrdquo PLoSONE vol 8 no3 Article ID e59310 pp 1ndash9 2013

[12] L U Opara F A Al-Said and A Al-Abri ldquoAssessment of whatthe consumer values in fresh fruit quality case study of OmanrdquoNew Zealand Journal of Crop and Horticultural Science vol 35no 2 pp 235ndash243 2007

[13] K Hammer J Gebauer S Al Khanjari and A Buerkert ldquoOmanat the cross-roads of inter-regional exchange of cultivatedplantsrdquo Genetic Resources and Crop Evolution vol 56 no 4 pp547ndash560 2009

[14] K Nguz J Shindano S Samapundo and A HuyghebaertldquoMicrobiological evaluation of fresh-cut organic vegetablesproduced in Zambiardquo Food Control vol 16 no 7 pp 623ndash6282005

[15] H Abriouel N B Omar A C Molinos et al ldquoComparativeanalysis of genetic diversity and incidence of virulence factorsand antibiotic resistance among enterococcal populations fromraw fruit and vegetable foods water and soil and clinicalsamplesrdquo International Journal of Food Microbiology vol 123no 1-2 pp 38ndash49 2008

[16] S N Al-Bahry M A Al-Zadjali I Y Mahmoud and A EElshafie ldquoBiomonitoring marine habitats in reference to antibi-otic resistant bacteria and ampicillin resistance determinantsfrom oviductal fluid of the nesting green sea turtle Cheloniamydasrdquo Chemosphere vol 87 no 11 pp 1308ndash1315 2012

[17] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[18] D J Reasoner and E E Geldreich ldquoA new medium for theenumeration and subculture of bacteria from potable waterrdquoApplied and Environmental Microbiology vol 49 no 1 pp 1ndash71985

[19] M J Allen S C Edberg and D J Reasoner ldquoHeterotrophicplate count bacteriamdashwhat is their significance in drinkingwaterrdquo International Journal of Food Microbiology vol 92 no3 pp 265ndash274 2004

[20] G R Siragusa W J Dorsa C N Cutter G L Bennett J EKeen and M Koohmaraie ldquoThe incidence of Escherichia colion beef carcasses and its association with aerobic mesophilicplate count categories during the slaughter processrdquo Journal ofFood Protection vol 61 no 10 pp 1269ndash1274 1998

[21] R Ruimy A Brisabois C Bernede et al ldquoOrganic and conven-tional fruits and vegetables contain equivalent counts of Gram-negative bacteria expressing resistance to antibacterial agentsrdquoEnvironmental Microbiology vol 12 no 3 pp 608ndash615 2010

[22] N S Al-Zoreky ldquoAntimicrobial activity of pomegranate (Punicagranatum L) fruit peelsrdquo International Journal of Food Microbi-ology vol 134 no 3 pp 244ndash248 2009

[23] S M S Menezes L N Cordeiro and G S B Viana ldquoPunicagranatum (pomegranate) extract is active against dental plaquerdquoJournal of Herbal Pharmacotherapy vol 6 no 2 pp 79ndash922006

[24] S A Al-Maiman and D Ahmad ldquoChanges in physical andchemical properties during pomegranate (Punica granatum L)fruit maturationrdquo Food Chemistry vol 76 no 4 pp 437ndash4412002

[25] P H Nhung K Ohkusu N Mishima et al ldquoPhylogeny andspecies identification of the family Enterobacteriaceae based ondnaJ sequencesrdquo Diagnostic Microbiology amp Infectious Diseasevol 58 no 2 pp 153ndash161 2007

[26] SMathur and R Singh ldquoAntibiotic resistance in food lactic acidbacteriamdasha reviewrdquo International Journal of Food Microbiologyvol 105 no 3 pp 281ndash295 2005

[27] K Forslund S Sunagawa J R Kultima et al ldquoCountry-specificantibiotic use practices impact the human gut resistomerdquoGenome Research vol 23 no 7 pp 1163ndash1169 2013

[28] M Abadias J Usall M Anguera C Solsona and I VinasldquoMicrobiological quality of fresh minimally-processed fruitand vegetables and sprouts from retail establishmentsrdquo Interna-tional Journal of FoodMicrobiology vol 123 no 1-2 pp 121ndash1292008

[29] D Jonas B Spitzmuller F D Daschner J Verhoef and SBrisse ldquoDiscrimination of Klebsiella pneumoniae and Klebsiellaoxytoca phylogenetic groups and other Klebsiella species byuse of amplified fragment length polymorphismrdquo Research inMicrobiology vol 155 no 1 pp 17ndash23 2004

[30] J M T Hamilton-Miller and S Shah ldquoIdentity and antibioticsusceptibility of enterobacterial flora of salad vegetablesrdquo Inter-national Journal of Antimicrobial Agents vol 18 no 1 pp 81ndash832001

[31] C Brady I Cleenwerck S Venter T Coutinho and P DeVos ldquoTaxonomic evaluation of the genus Enterobacter basedon multilocus sequence analysis (MLSA) Proposal to reclassifyE nimipressuralis and E amnigenus into Lelliottia gen novas Lelliottia nimipressuralis comb nov and Lelliottia amnigenacomb nov respectively E gergoviae and E pyrinus intoPluralibacter gen nov as Pluralibacter gergoviae comb novand Pluralibacter pyrinus comb nov respectively E cowanii EradicincitansE oryzae andE arachidis intoKosakonia gen novasKosakonia cowanii comb novKosakonia radicincitans combnov Kosakonia oryzae comb nov and Kosakonia arachidiscomb nov respectively and E turicensis E helveticus andE pulveris into Cronobacter as Cronobacter zurichensis nom


nov Cronobacter helveticus comb nov and Cronobacter pul-veris comb nov respectively and emended description of thegenera Enterobacter and Cronobacterrdquo Systematic and AppliedMicrobiology vol 36 no 5 pp 309ndash319 2013

[32] P Kampfer S P Glaeser M W Raza S A Abbasi andJ D Perry ldquoPseudocitrobacter gen nov a novel genus ofthe Enterobacteriaceae with two new species Pseudocitrobacterfaecalis sp nov and Pseudocitrobacter anthropi sp nov isolatedfrom fecal samples from hospitalized patients in PakistanrdquoSystematic and Applied Microbiology vol 37 no 1 pp 17ndash222014

[33] H Harada H Oyaizu Y Kosako and H Ishikawa ldquoErwiniaaphidicola a new species isolated from pea aphid Acyrtho-siphon pisumrdquo Journal of General and Applied Microbiology vol43 no 6 pp 349ndash354 1997

[34] F Marın M Santos F Carretero J A Yau and F DianezldquoErwinia aphidicola isolated from commercial bean seeds(Phaseolus vulgaris)rdquoPhytoparasitica vol 39 no 5 pp 483ndash4892011

[35] K Tash ldquoRahnella aquatilis bacteremia from a suspectedurinary sourcerdquo Journal of Clinical Microbiology vol 43 no 5pp 2526ndash2528 2005

[36] A Hejazi and F R Falkiner ldquoSerratia marcescensrdquo Journal ofMedical Microbiology vol 46 no 11 pp 903ndash912 1997

[37] L A Grohskopf V R Roth D R Feikin et al ldquoSerratialiquefaciens bloodstream infections from contamination ofepoetin alfa at a hemodialysis centerrdquoThe New England Journalof Medicine vol 344 no 20 pp 1491ndash1497 2001

[38] V Drelichman and J D Band ldquoBacteremias due to Citrobacterdiversus and Citrobacter freundii incidence risk factors andclinical outcomerdquo Archives of Internal Medicine vol 145 no 10pp 1808ndash1810 1985

[39] B Nada and M Areej ldquoRaoultella planticola a central venousline exit site infectionrdquo Journal of Taibah University MedicalSciences vol 9 no 2 pp 158ndash160 2014

[40] M Osterblad O Pensala M Peterzens H Heleniusc and PHuovinen ldquoAntimicrobial susceptibility of Enterobacteriaceaeisolated from vegetablesrdquo Journal of Antimicrobial Chemother-apy vol 43 no 4 pp 503ndash509 1999

[41] R J Gilbert J de Louvois T Donovan et al ldquoGuidelines forthe microbiological quality of some ready-to-eat foods sampledat the point of sale PHLS Advisory Committee for Food andDairy Productsrdquo Communicable Disease and Public Health vol3 no 3 pp 163ndash167 2000

[42] S Kuramoto H Kodama K Yamada et al ldquoFood poisoningattributable to Staphylococcus aureus deficient in all of thestaphylococcal enterotxoin gene so far reportedrdquo JapaneseJournal of Infectious Diseases vol 59 no 5 p 347 2006

[43] L L McGowan C R Jackson J B Barrett L M Hiott andP J Fedorkacray ldquoPrevalence and antimicrobial resistance ofenterococci isolated from retail fruits vegetables and meatsrdquoJournal of Food Protection vol 69 no 12 pp 2976ndash2982 2006

[44] A B Boehm and L M Sassoubre ldquoEnterococci as indicatorsof environmental fecal contaminationrdquo in Enterococci FromCommensals to Leading Causes of Drug Resistant Infections MS Gilmore D B Clewell Y Ike et al Eds Massachusetts Eyeand Ear Infirmary Boston Mass USA 2014

[45] T J Eaton andM J Gasson ldquoMolecular screening of Enterococ-cus virulence determinants and potential for genetic exchangebetween food and medical isolatesrdquo Applied and EnvironmentalMicrobiology vol 67 no 4 pp 1628ndash1635 2001

[46] T G Emori and R P Gaynes ldquoAn overview of nosocomialinfections including the role of the microbiology laboratoryrdquoClinical Microbiology Reviews vol 6 no 4 pp 428ndash442 1993

[47] K Gqunta J van Wyk P Ekermans C Bamford C Moodleyand S Govender ldquoFirst report of an IMI-2 carbapenemase-producing Enterobacter asburiae clinical isolate in SouthAfricardquo Southern African Journal of Infectious Diseases vol 30no 1 pp 34ndash35 2015

[48] C Westerfeld G N Papaliodis I Behlau M L Durand andL Sobrin ldquoEnterobacter amnigenus endophthalmitisrdquo RetinalCases amp Brief Reports vol 3 no 4 pp 409ndash411 2009

[49] A W Paton and J C Paton ldquoEnterobacter cloacae producinga Shiga-like toxin II-related cytotoxin associated with a case ofhemolytic-uremic syndromerdquo Journal of Clinical Microbiologyvol 34 no 2 pp 463ndash465 1996

[50] P N Wenger J I Tokars P Brennan et al ldquoAn outbreakof Enterobacter hormaechei infection and colonization in anintensive care nurseryrdquo Clinical Infectious Diseases vol 24 no6 pp 1243ndash1244 1997

[51] A Khajuria A K Praharaj N Grover and M Kumar ldquoFirstreport of an Enterobacter ludwigii isolate coharboring NDM-1 and OXA-48 carbapenemasesrdquo Antimicrobial Agents andChemotherapy vol 57 no 10 pp 5189ndash5190 2013

[52] C Iaria G Stassi G B Costa R di Leo A Toscano andA Cascio ldquoEnterococcal meningitis caused by Enterococcuscasseliflavus First case reportrdquo BMC Infectious Diseases vol 5article 3 2005

[53] C Zeana C J Kubin P Della-Latta and S M HammerldquoVancomycin-resistant Enterococcus faecium meningitis suc-cessfully managed with linezolid Case report and review of theliteraturerdquo Clinical Infectious Diseases vol 33 no 4 pp 477ndash482 2001

[54] C Poyart T Lambert PMorand et al ldquoNative valve endocardi-tis due to Enterococcus hiraerdquo Journal of Clinical Microbiologyvol 40 no 7 pp 2689ndash2690 2002

[55] B S Freyaldenhoven G Schlieper R Lutticken andR R Rein-ert ldquoEnterococcus raffinosus infection in an immunosuppressedpatient case report and review of the literaturerdquo Journal ofInfection vol 51 no 3 pp e121ndashe124 2005

[56] C Hogenauer C Langner E Beubler et al ldquoKlebsiella oxytocaas a causative organism of antibiotic-associated hemorrhagiccolitisrdquo The New England Journal of Medicine vol 355 no 23pp 2418ndash2426 2006

[57] A Elemam J Rahimian and W Mandell ldquoInfection withpanresistant Klebsiella pneumoniae a report of 2 cases and abrief review of the literaturerdquo Clinical Infectious Diseases vol49 no 2 pp 271ndash274 2009

[58] M C Liberto G Matera R Puccio T Lo Russo E Colosimoand E Foca ldquoSix cases of sepsis caused by Pantoea agglomeransin a teaching hospitalrdquo New Microbiologica vol 32 no 1 pp119ndash123 2009

[59] M S Alves L W Riley and B M Moreira ldquoA case of severepancreatitis complicated by Raoultella planticola infectionrdquoJournal of Medical Microbiology vol 56 no 5 pp 696ndash6982007

[60] K Orsquo Connell J Kelly and U NiRiain ldquoA rare case of soft-tissue infection caused by Raoultella planticolardquo Case Reports inMedicine vol 2010 Article ID 134086 2 pages 2010

[61] R A Equi and W R Green ldquoEndogenous Serratia marcescensendophthalmitis with dark hypopyon a case report and reviewrdquoSurvey of Ophthalmology vol 46 no 3 pp 259ndash268 2001


[62] C Tascini A Di Paolo M Polillo et al ldquoCase report ofa successful treatment of methicillin-resistant Staphylococcusaureus (MRSA) bacteremia and MRSAvancomycin-resistantEnterococcus faecium cholecystitis by daptomycinrdquo Antimicro-bial Agents and Chemotherapy vol 55 no 5 pp 2458ndash24592011

[63] P H Nhung K Ohkusu N Mishima et al ldquoPhylogeny andspecies identification of the family Enterobacteriaceae basedon dnaJ sequencesrdquo Diagnostic Microbiology and InfectiousDisease vol 58 no 2 pp 153ndash161 2007

[64] F Rezzonico T H Smits E Montesinos J E Frey and BDuffy ldquoGenotypic comparison of Pantoea agglomerans plantand clinical strainsrdquo BMCMicrobiology vol 9 article 204 2009

[65] C Sproer U Mendrock J Swiderski E Lang and E Stacke-brandt ldquoThe phylogenetic position of Serratia Buttiauxella andsome other genera of the family Enterobacteriaceaerdquo Interna-tional Journal of Systematic Bacteriology vol 49 no 4 pp 1433ndash1438 1999

[66] T Takahashi I Satoh and N Kikuchi ldquoPhylogenetic relation-ships of 38 taxa of the genus Staphylococcus based on 16S rRNAgene sequence analysisrdquo International Journal of SystematicBacteriology vol 49 no 2 pp 725ndash728 1999

[67] S M Naser F L Thompson B Hoste et al ldquoApplication ofmultilocus sequence analysis (MLSA) for rapid identification ofEnterococcus species based on rpoA and pheS genesrdquoMicrobiol-ogy vol 151 no 7 pp 2141ndash2150 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


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International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


and affected 58 individuals B cereus and its toxin werefound in different foods including vegetables [8] A moreserious outbreak MayndashJuly 2011 was caused by Shiga toxinproducing E coli O104H4 in Germany where 2987 cases ofgastroenteritis 855 cases of hemolytic-uremic syndrome and53 deaths were reported Fenugreek sprouts were found tobe contaminated with the causative agent [9] Bean sproutswere linked to two outbreaks in USA in 2014 one was causedby L monocytogenes in August while Salmonella Enteritidiscaused the other one just a month later L monocytogeneswas also involved in another outbreak that was linked tocaramel apples in October 2014 in USA [10] Opportunisticpathogens can cause life-threatening infections mainly inimmunocompromised people but they may have positiveeffects on the health of immunocompetent individuals bystimulating immune functions and priming the immunesystem continuously [3] Nonpathogenicmicrobes associatedwith fruits and vegetables may have various consequences onthe quality of the produce by affecting the rate of the foodspoilage Fruits and vegetables seemalso to be the sources thatdisseminate many microbes to food preparation areas [11]

In Oman large quantities of fruits and vegetables areimported nearly from all around the world to provide a year-round supply [12] to this countrywhich is located at the cross-roads of the exchange of cultivated plants [13] This studyaims at assessing the microbial load of some fresh fruits andvegetables imported into or grown in Oman and to identifythe isolated bacteria by biochemical and molecular methodswith emphasis on emergent opportunistic pathogens Thestudy will also investigate genetic relationships betweenbacteria isolated from fruits and vegetables originated fromdifferent countries in the world Findings from this study willprovide information on the sanitary conditions of fruits andvegetables produced and consumed in this part of the worldwhich will help in improving their quality and safety forconsumption To our knowledge this is the first report thatanalyzes microbial content of fresh produce in this countryand also relates the presence of particular opportunisticpathogens isolated from different local and imported samplesto particular effects on human health

2 Materials and Methods

21 Sample Collection Fruits and vegetables that are mostlyeaten rawwere selected for this studyThese contained 7 typesof fresh imported or locally produced fruits (banana Musaspp dates Phoenix dactylifera mango Mangifera indicapapaya Carica papaya pomegranate Punica granatumtomato Solanum lycopersicum and watermelon Citrulluslanatus) and 6 types of fresh imported or locally producedvegetables (cabbage Brassica oleracea capsicum Capsicumannuum carrot Daucus carota cucumber Cucumis sativuslettuce Lactuca sativa and radish Raphanus sativus) Sam-ples were purchased from local markets in Muscat or NizwaOman during the period of April to September 2014 Theimported fruits and vegetables originated from differentcountries and they were selected depending on their avail-ability in themarket during that period of timeThree samples

were obtained for each fruit or vegetable type originated fromthe same country The samples were collected aseptically andrefrigerated until analyzed within 24 hrs Table 1 representsthe origin of all samples

22 Microbiological Analysis of Fruits and Vegetables

221 Sample Preparation Aerobic Plate Count and Selec-tive Bacterial Counts Samples were analyzed in a safetycabinet (Purifier class II Labconco Kansas USA) and cutusing sterile scalpels Some fruits (banana mango papayapomegranate and watermelon) were peeled and the insideflesh was analyzed Twenty-five grams of the cut sample wasweighed in a sterile stomacher bag and mixed with 225mLof Maximum Recovery Diluent (MRD) and homogenizedfor 1min using a stomacher (Bagmixer 100 MiniMix Inter-science Bois Arpents France) Serial dilutions were preparedfrom the original homogenate in MRD Aerobic plate count(APC) was performed by spread plate method The plates ofstandard plate count agar (SPCA) were incubated at 35∘C for48 hrs [7]The count of Enterobacteriaceaewas performed onViolet Red Bile Glucose (VRBG) agar by pour plate methodand incubation at 35∘C for 24 hrs E coli was counted onTryptone Bile X-glucuronide (TBX) medium and the plateswere incubated at 2 temperatures 30 and 44∘C for 24 hrs[14] Staphylococcus aureuswas counted onBaird-Parker (BP)agar after incubation at 35∘C for 24 hrs [7] Enterococcus wascounted on Slanetz agar after incubation at 35∘C for 48 hrs[15] All microbiological media were from Oxoid Englandand all experiments were repeated three times

222 Identification of Bacteria For each selective medium(BPA VRBG TBX and Slanetz agar) one bacterial isolateshowing the typical morphology (details could be foundby searchingmanufacturerrsquos website (httpwwwoxoidcomUKblueindexaspc=UKamplang=EN)) was selected fromeach sample for identification Bacteria were preserved atminus80∘C using cryogenic vials with beads (Viabank UK)

(1) Identification of Bacteria by VITEK 2 Identification ofbacteria using biochemical tests was done using an automatedidentification equipment VITEK 2-compact 15 (bioMerieuxFrance) following instructions of the manufacturer In briefbacterial suspensions were prepared in sterile saline (045)and the density was adjusted to a McFarland standard of05ndash063 using VITEK 2 DensiCheck (bioMerieux France)GP and GN cards were used for Gram-positive and Gram-negative bacteria respectively

(2) Identification of Bacteria by Polymerase Chain Reaction(PCR) The second method was performed using polymerasechain reaction (PCR) targeting bacterial 16S rRNA [16]Briefly DNA was extracted using ldquofoodproof StarPrep TwoKitrdquo (Biotecon Diagnostics GmbH Potsdam Germany)The quality and quantity of DNA were checked usingNanoDropTM 2000 (Thermoscientific USA) PCR was doneby transferring 1 120583L of each primer (27F and 1492R DNAsequences 51015840-AGAGTTTGATCMTGGCTCAG-31015840 and 51015840-TACGGYTACCTTGTTACGACTT-31015840 resp) 22120583L of sterile











Vegetable (119899 = 45)










3 Results



0

5

10

15

20

25

30

35


Type of countcount




Aerobic plate

log(

mea

n+1

) (CF

U gminus

1)






















4 Discussion





























75 75

62

79 61 99

9783

8359

99

7099

6980

5672 69

55

84

568999

63

99

93

6092

73

6189

5499

84

59

9475






66

82

88






85

81

100












9350

80

6876

82 96

68

73

95

58 72

8482

100

55

10075

























Acknowledgment


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











Vegetable (119899 = 45)










3 Results



0

5

10

15

20

25

30

35


Type of countcount




Aerobic plate

log(

mea

n+1

) (CF

U gminus

1)






















4 Discussion





























75 75

62

79 61 99

9783

8359

99

7099

6980

5672 69

55

84

568999

63

99

93

6092

73

6189

5499

84

59

9475






66

82

88






85

81

100












9350

80

6876

82 96

68

73

95

58 72

8482

100

55

10075

























Acknowledgment


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

5

10

15

20

25

30

35


Type of countcount




Aerobic plate

log(

mea

n+1

) (CF

U gminus

1)






















4 Discussion





























75 75

62

79 61 99

9783

8359

99

7099

6980

5672 69

55

84

568999

63

99

93

6092

73

6189

5499

84

59

9475






66

82

88






85

81

100












9350

80

6876

82 96

68

73

95

58 72

8482

100

55

10075

























Acknowledgment


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















4 Discussion





























75 75

62

79 61 99

9783

8359

99

7099

6980

5672 69

55

84

568999

63

99

93

6092

73

6189

5499

84

59

9475






66

82

88






85

81

100












9350

80

6876

82 96

68

73

95

58 72

8482

100

55

10075

























Acknowledgment


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























75 75

62

79 61 99

9783

8359

99

7099

6980

5672 69

55

84

568999

63

99

93

6092

73

6189

5499

84

59

9475






66

82

88






85

81

100












9350

80

6876

82 96

68

73

95

58 72

8482

100

55

10075

























Acknowledgment


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















75 75

62

79 61 99

9783

8359

99

7099

6980

5672 69

55

84

568999

63

99

93

6092

73

6189

5499

84

59

9475






66

82

88






85

81

100












9350

80

6876

82 96

68

73

95

58 72

8482

100

55

10075

























Acknowledgment


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


66

82

88






85

81

100












9350

80

6876

82 96

68

73

95

58 72

8482

100

55

10075

























Acknowledgment


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


9350

80

6876

82 96

68

73

95

58 72

8482

100

55

10075

























Acknowledgment


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















Acknowledgment


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










Acknowledgment


References















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Hiding in Fresh Fruits and Vegetables: … · 2020. 1. 11. · Research Article Hiding in Fresh Fruits and Vegetables: Opportunistic Pathogens May Cross Geographical