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Research ArticleBacteriological Contaminants of Some FreshVegetables Irrigated with Awetu River in Jimma TownSouthwestern Ethiopia

Desta Weldezgina1 and Diriba Muleta2

1Department of Biology College of Natural Sciences Jimma University Jimma Ethiopia2Institute of Biotechnology Addis Ababa University Addis Ababa Ethiopia

Correspondence should be addressed to Desta Weldezgina destaws2005gmailcom

Received 17 February 2016 Accepted 26 April 2016

Academic Editor Dario De Medici

Copyright copy 2016 D Weldezgina and D MuletaThis is an open access article distributed under theCreativeCommonsAttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

The main purposes of this study were to determine the bacteriological load and safety of some fresh vegetables irrigated withAwetu River in Jimma town southwestern Ethiopia Water and vegetable samples were collected from three different irrigationsites and analyzed for their bacteriological contaminants following standard procedures The maximum overall means of aerobicmesophilic bacteria Enterobacteriaceae aerobic spore formers staphylococci and total and fecal coliform counts were 806 710654 and 297 log CFU gminus1 and 1036 and 716MPN 100mLminus1 respectively The microflora of vegetable samples was dominated byBacillus species (327) followed by Enterobacteriaceae (25) and Micrococcus (16) Staphylococcus aureus and Salmonella sppwere detected in 240 and 207 of the samples respectively All the Staphylococcus aureus isolates were resistant to ampicillincefuroxime sodium and penicillin G (1000 each) All the Salmonella isolates were also resistant to tetracycline erythromycincefuroxime sodium and penicillin G (1000 each) The findings reveal that the river water used for irrigation in this study is apossible preharvest source of contamination to fresh vegetables which potentially constitutes a health risk to consumers

1 Introduction

Fresh and minimally processed vegetables and fruits providethe most important human diet that contains carbohydratesproteins vitamins minerals and fiber Lerici et al [1]reported that nutritional and other benefits of a regular intakeof fruits and vegetables are well documented internationallyTheir role in reducing the risk of lifestyle associated illnessessuch as heart disease diabetes and cancer has resultedin a further increase in desirability and consumption [2]For instance Food and Drug Administration (FDA) andWorld Health Organization (WHO) have recommended 5ndash9 servings of fruits and vegetables to be taken daily becausecorrect fresh produce intake alone could save 27million livesa year [3]

In contrast to their health benefits the consumption offresh fruits and vegetables has also been associated to riskfor consumers [4] Outbreaks of food infections associatedwith consumption of ready-to-eat vegetables have been

increasing [5] Garg et al [6] show that outbreaks of illnesscaused by bacteria viruses and parasites have been linkedepidemiologically to the consumption of a wide range ofvegetables and to a lesser extent of fruits Furthermore sur-veillance of vegetables has indicated that these foods canbe contaminated with various bacterial pathogens includingSalmonella spp Shigella spp Shiga toxigenic E coli (STEC)Listeria monocytogenes and Campylobacter spp [7]

Ijabadeniyi [2] reported that prepackaged fresh spinachwas recalled by the Food and Drug Administration (FDA)as a result of E coli outbreak in California USA The authoralso noted that fresh tomatoes consumed at restaurants in theUSA were also blamed for an outbreak of S typhimuriumIn addition there was an E coli O157H7 outbreak linked tolettuce from Taco Bell restaurants in northern USA [8]

The increase in outbreaks of foodborne illnesses due tofresh produce is a result of changes in dietary habits includinga higher per capital consumption of fresh or minimallyprocessed fruits and vegetables and the increased use of

Hindawi Publishing CorporationAdvances in BiologyVolume 2016 Article ID 1526764 11 pageshttpdxdoiorg10115520161526764

2 Advances in Biology

salad bars and meals eaten outside the home Ijabadeniyi[2] Changes in production and processing methods of dis-tribution consumption patterns and practices are otherfactors that have also contributed to increasing foodbornediseases due to raw consumed vegetables [9]

Ijabadeniyi [2] reported that other reasons given by theFood and Agriculture Organization (FAO) andWorld HealthOrganization (WHO) [10] for the increasing of foodborneinfectionpoisoning outbreaks are as follows microbial adap-tation increase in international trade and in susceptible pop-ulation and increase in worldwide travelling Furthermorechanges of a lifestyle of convenience consumer demandsregarding healthy food with no chemical preservatives andwith an extended shelf life and changes in human demo-graphics and behavior have also contributed to increase offoodborne infections

According to Suslow et al [11] the microbial quality ofirrigation water is critical because water contaminated withanimal or human wastes can introduce pathogens into veg-etable products during preharvest and postharvest activitiesvia direct or indirect contaminationThereforemicrobiologi-cal quality of irrigation water has a paramount importance tothe safety of fresh and minimally processed vegetables [12]Moreover Ibenyassine et al [13] reported that contaminatedirrigation water and surface runoff water may be the majorsources of pathogenic microorganisms that contaminatefruits and vegetables in fields Water from the river thatreceived both human and animal waste disposal poses ahealth risk due to contamination with all microorganismsof human and animal intestinal habitat such as Salmonellaand Listeria spp [14 15] Consequently the microbiologicalquality and safety of fresh vegetables is a significant concernto all stakeholders all over the world

Tambekar and Mundhada [16] reported that foodbornebacterial pathogens commonly detected in fresh vegetableswere E coli S aureus and Salmonella spp Aycicek et al[17] have also stated that plate count of aerobic mesophilicmicroorganisms found in food is one of the microbiologicalindicators for food quality in addition to the common indica-tors such as fecal coliform Information on themicrobial loadon fresh vegetables in Ethiopia has been studied by severalinvestigators [18ndash20]

According to Hailu [21] the use of rivers and hand-dug wells for various purposes is common in Jimma townThe author further noted that Awetu River is the primarysource of water for a range of activities such as recreationbathing washing clothes and household utensils small scaleagricultural irrigation car washing and other usesThe dete-rioration of the quality of Awetu River as a result of dischargeof municipal wastes and urban runoff has been well studiedHailu [21] For instance Deneke [22] investigated about thepollution profiles along Awetu River and found high loadof fecal coliforms According to Sofonias and Tsegaye [23]some of the water sources in Jimma town such as unprotectedsprings wells and Awetu River have greater chance to becontaminated via human and animal fecal matters andwastesdisposed from households hotels and small scale industriesThus these water sources could transmit diseases as a resultof contamination However a comprehensive investigation of

the quality and safety of vegetables irrigatedwithAwetu Riverincluding bacteriological quality and safety analyses is lack-ing Therefore this study was aimed at investigating the bac-teriological contaminants of some fresh vegetables irrigatedwith Awetu River in Jimma town southwestern Ethiopia

2 Materials and Methods

21 Description of the Study Area The study was conductedat Jimma town located 350 km southwest of Addis AbabaThe townrsquos geographical coordinates are 7∘411015840N latitude and36∘501015840E longitude The town is found with abundant meanannual rainfall between 1800 and 2300mmwhich makes thisregion one of the best watered Ethiopian highland areasconducive for agricultural production [24]

22 Study Design and Study Population Laboratory basedcross sectional study design was used The sampling siteswere three irrigation farms around Awetu River designatedas site A B and C along the river course at the same timeof the irrigation period The size of the farms is about 15 haand the average production of vegetables from these farmsis 30 kgha On the average the frequency of irrigation for thetarget vegetables is 3ndash5 daysweek depending on the humidityof the air at the farms

There are many farmers who are members of Microen-terprise Association around Awetu River They are activelyinvolved in irrigating their farms

Preliminary survey was made on the distribution andlocation of vegetable growers in the study area prior toresuming the actual sample collection The survey includedindividual residents and members of Microenterprise Asso-ciation of vegetable growers who regularly use Awetu Riverto irrigate their farms in Jimma town Purposive samplingwas made to gather information on Awetu River from a totalof 60 vegetable growers using structured questionnaire Thissurvey was performed in order to get general informationabout Awetu River including its pollution status and its effecton human health

23 Collection of Samples A total of 120 vegetable samples40 each from the three sites and 10 samples each of lettucecabbage tomato and carrot were randomly picked (15 kg)aseptically using sterilized scissors and cut into pieces Inaddition a total of 30 irrigation water samples (200mL each)were collected from three sites (10 samples each) The watersamples were collected from the depth of below 5 cm surfacewater using sterilized stopper glass bottles The sampleswere transported to Postgraduate and Research Laboratoryof Biology Department College of Natural Sciences JimmaUniversity using ice box The samples were processed forbacteriological analysis within 1ndash4 hrs of collection

24 Sample Preparation Mixed vegetable samples (unpro-cessed and large sized) were aseptically chopped into smallerpieces using a sterile stainless steel knife prior to weighing A25 g of subsample of each vegetable was aseptically weighedand vigorously shaken in 225mLof sterile 01 (wv) buffered

Advances in Biology 3

peptone water (Oxoid) for 3min separately to homogenizethe samples [25] In addition a tenfold serial dilution wasmade A 10mL of water sample was also mixed with 90mL ofpeptone water using vortex mixer Finally appropriate serialdilutions of the suspension were spread-plated on a suitablesolid media

25 Bacterial Counts A volume of 01mL aliquot of appro-priate dilution was spread-plated in duplicate on presolid-ified plates of Plate Count Agar (Oxoid) Violet Red BileGlucose Agar (Oxoid) and Mannitol Salt Agar (Oxoid) thatthen were incubated at optimum temperature and time forcounts of aerobic mesophilic bacteria Enterobacteriaceaeand staphylococci respectively Homogenized samples wereheated at 80∘C for 10 minutes in a water bath to count aerobicspore formers Thereafter a 01mL appropriate dilution wasspread-plated in duplicate on predried surfaces of PlateCount Agar (Oxoid) plates Inoculated plates were incubatedat 30∘Cndash32∘C for 24ndash48 hrs For microbial counts plateswith colonies between 30 and 300 were considered Totalcoliforms and fecal coliforms were enumerated by multipletube fermentation tests as described by APHA [26] Theresults were expressed as MPN 100mLminus1

26 Isolation and Characterization of Dominant MicrofloraAfter enumeration of aerobic mesophilic bacteria 10ndash20colonies with distinct morphological difference such as colorsize and shape were randomly picked from countable platesand aseptically transferred into a tube containing 5mL nutri-ent broth (Oxoid) and incubated at 30∘C for 24ndash48 hrs Thecultures were purified by repeated plating and pure cultureswere temporarily preserved on nutrient agar slants at 4∘Cfor a month for further work An overnight activated culturewas further characterized using the following standard testssuch as cell morphology gram staining motility bacte-rial endospore staining catalase cytochrome oxidase andOxidation Fermentation (OF) to differentiate into variousbacterial groups such as genus and family levels [27]

27 Isolation and Biochemical Identification of Some BacterialPathogens After counting staphylococci yellow colonieson Mannitol Salt Agar plates were aseptically picked andtransferred into 5mL nutrient broth and incubated at 37∘Cfor 24ndash36 hrs for further purification Then a loop of cul-ture from the nutrient broth was streaked on presolidifiedsurface of nutrient agar supplemented with 075 sodiumchloride and again incubated at 37∘C for 24ndash36 hrs so as toobtain distinct colonies Finally the distinct colonies werecharacterized using the established microbiological methodssuch as gram staining Gram positive cocci with clusteredarrangement under themicroscope were subjected to prelim-inary biochemical tests (the catalase and coagulase tests) forconformation

25 g or 25mL of each sample was aseptically transferredinto sterile flask containing 225mL buffered peptone water(BPW) then was homogenized for 5min and then wasincubated at 37∘C for 24 hrs for recovery and proliferationof cells Following the BPW enrichment a 1mL of culture

was transferred into 10mL of Rappaport Vassiliadis broth andwas incubated at 43∘C for 48 hrs A loop full of culture fromthe Rappaport Vassiliadis broth was streaked onto XyloseLysine Deoxycholate Agar (XLD) agar and incubated at 37∘Cfor 24 hrs Typical colonies that appeared red or colorlesswith no blackening were picked as presumptive Shigella andcolonies which had slightly transparent zone of reddish colorand a black center a pink-red zone surrounding the colonieswere considered as presumptive SalmonellaThe presumptiveSalmonella and Shigella colonies were further confirmedfollowing standard methods [28]

28 Antimicrobial Susceptibility Testing for S aureus andSalmonella spp The antimicrobial susceptibility testing forS aureus and Salmonella spp was determined according tomodified Kirby Bauer disc diffusion technique as describedby Clinical Laboratory Standard Institute (CLSI [29]) Thefollowing 9 drugs namely ampicillin (10 120583g) gentamycin(10 120583g) chloramphenicol (30120583g) tetracycline (30 120583g) ery-thromycin (15 120583g) Ttrimethoprim-sulfamethoxazole (co-tri-moxazole) (25 120583g) cefuroxime sodium (5 120583g) norfloxacin(10 120583g) and penicillin G (10 120583g) were used to determinethe antibiogram of the isolates The criteria used to selectthe antimicrobial agents tested in this study were based onavailability and frequency of prescription of the drugs forthe management of bacterial infection in Ethiopia Salmo-nella typhimurium (ATCC13311) and Staphylococcus aureus(ATCC25923) were used as reference strains for quality con-trol of the antibiotics used

29 Data Analysis Bacterial counts were calculated as colonyforming units per gram (CFU gminus1) and colony forming unitsper milliliter (CFUmLminus1) and converted into log

10

valuesThe statistical analysis was performed by one-way analysisof variance (ANOVA) followed by LSDrsquos Post Hoc MultipleComparison Test using statistical software (SPSS) packageversion 21 119901 lt 005 was considered statistically significant

3 Results

31 Sociodemographic Characteristics of the Study SubjectsMales accounted for 717 of the respondents (data notshown) The ages of vegetable growers ranged from 18 to 67years with average age of 35 years A large number of therespondents (483) were Muslims followed by Orthodox(333) Regarding theirmarital status 70weremarried but217 were unmarried About 55 of the respondents wereilliterate and the rest (45) attended elementary school (datanot shown)

32 Irrigation Conditions of Awetu River All the respondents(1000) used Awetu River for different purposes (Table 1)From all the respondents 983 used Awetu River for irri-gation 35 for washing their clothes and the rest 83 fortaking shower (Table 1) The majority of the respondents(967) irrigated tomato followed by potato (750) andcarrot (517) (Table 1)


Table 1 General information and irrigation conditions of AwetuRiver

CharacteristicsNumber of respondents

(119899 = 60)Frequency Percent ()

Using of Awetu River for any meansYes 600 1000No 00 00Application of Awetu RiverIrrigation 590 983Washing clothes 210 350Taking shower 50 83Types of vegetables irrigated with AwetuRiverTomato 580 967Potato 450 750Carrot 310 517Cabbage 230 383Lettuce 110 183Green pepper 60 100Onion 40 67Use of irrigated vegetablesFamily consumption 530 883Source of income 440 733Suspected diseases due to consumptionof the vegetablesTyphoid 100 667Diarrhea 70 467Anemia 20 133Suffering of Awetu River from anycontaminationYes 600 1000No 00 00Sources of contaminationWastes released from toilet 580 967Domestic waste 550 917Plastic and petrol washes 190 317Fecal matter 80 133

A significant number of the growers (883) used culti-vated vegetables for family consumption while 733 usedthem as their source of income (Table 1) The respondentscomplained of health problems due to consumption of thevegetables Accordingly some of the respondents suspectedtyphoid (667) others diarrhea (467) and the rest asso-ciated the issue with anemia (133) (Table 1)

Generally all the respondents (1000) believed thatAwetu River suffers from wastes released from Jimma townthat included toilet wastes (967) domestic wastes (917)plastic and petrol washes (3167) and fecal matter (133Table 1)

33 Bacterial Counts The mean bacterial counts(log CFUmLminus1 or gminus1) of aerobic mesophilic bacteria Enter-obacteriaceae aerobic spore formers and staphylococci atthe three sites were 858 742 575 and 264 logCFUmLminus1for water 694 609 524 and 297 for lettuce 806 710 654and 271 for carrot 741 624 571 and 275 for tomato 766670 589 and 276 logCFU gminus1 for cabbage respectively(Table 2) The mean microbial counts of different samples ofselected sites ranged from 255 to 942 log CFUmLminus1

The minimal and maximal mean counts of aerobicmesophilic bacteria Enterobacteriaceae aerobic spore form-ers and staphylococci obtained from different samples of thethree sites ranged from 592 to 942 50 to 836 430 to 704and 255 to 324 logCFU gminus1 or mLminus1 respectively (Table 2)Themean count of AMBwas the highest (942 logCFUmLminus1)in water samples of site C The count of Enterobacteriaceaewas also relatively higher (836 logCFUmLminus1) in water sam-ples of site C but the range of mean count for staphylococcirelatively in all samples was similar (Table 2) In additionMPN of total and fecal coliforms and their overall meanin vegetables also ranged from 8653 to 10360 and 5240to 7160MPN 100mLminus1 respectively However both weregt24000MPN 100mLminus1 in water samples (Table 2)

The mean counts (log CFUmLminus1 or gminus1) of AMB Enter-obacteriaceae and aerobic spore formers revealed statisticallysignificant (119901 lt 005) difference between all samples of thethree sites However there was no significant (119901 gt 005)difference between mean counts of total coliform and fecalcoliforms of all the samples collected from the three irrigationsites (Table 2) Likewise the mean counts of staphylococcidid not differ significantly (119901 gt 005) between the differentsite of irrigation farms for carrot and cabbage (Table 2) Meancounts of all bacterial groups differed significantly (119901 lt 005)between the different sample types with the highest meancount in carrot samples (data not shown)

34 Microflora Analysis Aerobic mesophilic bacterial floraof water samples collected from the three sites of thedownstream of Awetu River was dominated by Bacillusspp (353) Enterobacteriaceae (267) Micrococcus spp(120) Pseudomonas spp (83) Staphylococcus spp (67)Aeromonas spp (60) and Streptococcus spp (50) Sim-ilarly the most predominant generafamilies in lettuce car-rot tomato and cabbage samples were Bacillus spp (300340 317 and 323) Enterobacteriaceae (233 260 240and 250) Micrococcus spp (160 163 187 and 173)Pseudomonas spp (130 90 103 and 93) Staphylococcusspp (93 67 80 and 87) Aeromonas spp (60 50 57and 50) and Streptococcus spp (23 30 17 and 23)respectively (Figure 1) Generally the same dominant generawere isolated in both water and vegetable samples eventhough their distributions were relatively varied

35 Prevalence of S aureus Shigella and Salmonella IsolatesThe total prevalence of S aureus and Salmonella was 240and 207 respectively (Table 3) The distribution of thesepathogens varied depending on the nature of vegetablesWithregard to sample types prevalence of S aureus was higher


Table 2 Bacterial counts from water and vegetable samples

Sample type Site Average log CFUmLminus1 or Gminus1plusmn SD Average MPN 100mLminus1 plusmn SDAMB Entero ASFs Staph Total coliform Fecal coliform

Water

A 765cplusmn 04 655

cplusmn 03 488

cplusmn 05 255

bplusmn 01 gt240000

aplusmn 0 gt 240000

aplusmn 0

B 869bplusmn 05 734

bplusmn 03 570

bplusmn 05 266

abplusmn 01 gt240000

aplusmn 0 gt240000

aplusmn 0

C 942aplusmn 01 836

aplusmn 01 665

aplusmn 05 271

aplusmn 01 gt240000

aplusmn 0 gt240000

aplusmn 0

Average 858 plusmn 03 742 plusmn 02 575 plusmn 05 264 plusmn 01 gt 240000 plusmn 0 gt 240000 plusmn 0

Lettuce

A 592cplusmn 05 500

cplusmn 0 430

cplusmn 01 277

bplusmn 02 84400

aplusmn 3305 52400

aplusmn 2024

B 690bplusmn 05 610

bplusmn 06 541

bplusmn 03 289

bplusmn 03 90800

aplusmn 3091 58800

aplusmn 2698

C 799aplusmn 05 717

aplusmn 04 602

aplusmn 05 324

aplusmn 04 97200

aplusmn 2698 65200

aplusmn 3091

Average 694 plusmn 05 609 plusmn 03 524 plusmn 03 297 plusmn 03 908 plusmn 3032 588 plusmn 2605

Carrot

A 705cplusmn 05 643

cplusmn 03 598

cplusmn 04 263

aplusmn 01 97200

aplusmn 2698 65200

aplusmn 3091

B 799bplusmn 05 708

bplusmn 04 659

bplusmn 04 274

aplusmn 01 103600

aplusmn 2024 71600

aplusmn 3305

C 915aplusmn 04 779

aplusmn 05 704

aplusmn 05 276

aplusmn 02 110000

aplusmn 0 78000

aplusmn 3373


Tomato

A 657cplusmn 04 538

cplusmn 04 512

cplusmn 04 262

cplusmn 01 78000

aplusmn 460 46000

aplusmn 0

B 753bplusmn 04 634

bplusmn 03 572

bplusmn 05 276

bplusmn 01 84400

aplusmn 3305 52400

aplusmn 2024

C 812aplusmn 04 699

aplusmn 05 629

aplusmn 01 288

aplusmn 01 97200

aplusmn 2698 58800

aplusmn 2698


Cabbage

A 682cplusmn 05 589

cplusmn 05 524

cplusmn 03 271

aplusmn 02 84400

aplusmn 3305 58800

aplusmn 2698

B 758bplusmn 04 681

bplusmn 05 581

bplusmn 05 273

aplusmn 02 90800

aplusmn 3091 65200

aplusmn 3091

C 857aplusmn 04 740

aplusmn 05 663

aplusmn 05 285

aplusmn 02 103600

aplusmn 2024 71600

aplusmn 3305


Mean values followed by different alphabets within a column of each sample types are significantly different using post hocmultiple comparisons test (119901 lt 005)where AMB stands for aerobic mesophilic bacteria ASFs aerobic spore formers Entero Enteriobacteriaceae and Staph staphylococci

00

50

100

150

200

250

300

350

400

Water Lettuce Carrot Tomato Cabbage

Perc

enta

ge (

)

Type of samples

BacillusEnterobacteriaceaeMicrococcusPseudomonas

StaphylococcusAeromonasStreptococcus

Figure 1 Distribution of dominant bacterial isolates fromwater andvegetable samples from irrigated farm land along the Awetu River

in lettuce (333) cabbage (267) and tomato (233)but relatively lower in water (200) and carrot samples(167)Higher frequency of Samonella spp was encounteredin cabbage (300) but it was lower in lettuce (133 Table 3)Shigella was not encountered in any of the samples analyzedThe prevalence of these all bacterial pathogens was not

significantly (119901 gt 005) different with respect to sample types(data not shown) However in terms of sample sites therewas significant (119901 lt 005) difference for S aureus but not forSalmonella spp (data not shown)

36 Antimicrobial Susceptibility Patterns of S aureus andSalmonella Isolates Staphylococcus aureus isolates weremost susceptible to chloramphenicol (1000) norfloxacin(9440) gentamycin (861) and erythromycin (806Table 4) Out of the tested drugs the highest resistancewas observed against ampicillin cefuroxime sodium andpenicillin G (1000 each)

All Salmonella isolates were also susceptible to chloram-phenicol norfloxacin gentamycin (1000 each) and co-trimoxazole (935) Out of the tested drugs the highestresistance was observed against tetracycline erythromycincefuroxime sodium penicillin G (1000 each) and ampi-cillin (903) (Table 4)

37 Multiple Drug Resistance of S aureus and SalmonellaIsolates Pattern of multiple drug resistance (MDR) amongS aureus isolates varied from three to seven antibiotics(Table 5) The highest MDR noted was AmpPenCefTeCot (1136 306) followed by AmpPenCef and AmpPenCefTe (736 194 each) The maximum MDR regis-tered was resistance to 7 antibiotics with the combinationAmpPenCefTeCotEryNorMDR to five antibiotics dom-inated the resistance patterns (1336 361 Table 5)


Table 3 Prevalence of S aureus and Salmonella isolates from water and vegetable samples

Sample type Sites Sample size Number of S aureuspositive samples ()

Number of Salmonellapositive samples ()

Water

A 10 0 0B 10 2 (20) 2 (20)C 10 4 (40) 5 (50)

Subtotal 30 6 (20) 7 (233)

Lettuce

A 10 3 (30) 1 (10)B 10 2 (20) 1 (10)C 10 5 (50) 2 (20)

Subtotal 30 10 (333) 4 (133)

Carrot

A 10 0 1 (10)B 10 1 (10) 2 (20)C 10 4 (40) 2 (20)

Subtotal 30 5 (167) 5 (167)

Tomato

A 10 1 (10) 1 (10)B 10 3 (30) 2 (20)C 10 3 (30) 3 (30)

Subtotal 30 7 (233) 6 (20)

Cabbage

A 10 1 (10) 2 (20)B 10 3 (30) 3 (30)C 10 4 (40) 4 (40)

Subtotal 30 8 (267) 9 (30)Total 150 36 (24) 31 (20)

Table 4 Antibiotic susceptibility of S aureus and Salmonella spp isolated from water and vegetable samples

Antimicrobial agent Disc content (120583g) S aureus (119899 = 36) Salmonella spp (119899 = 31)S R S R

Ampicillin 10 0 0 36 100 3 97 28 903Gentamycin 10 31 861 5 139 31 100 0 0Chloramphenicol 30 36 100 0 0 31 100 0 0Tetracycline 30 7 194 29 806 0 0 31 100Erythromycin 15 29 806 7 194 0 0 31 100Co-trimoxazole 25 18 50 18 50 29 935 2 65Cefuroxime sodium 5 0 0 36 100 0 0 31 100Penicillin 10 0 0 36 100 0 0 31 100Norfloxacin 10 34 944 2 56 31 100 0 0Where S stands for sensitive and R resistance

Pattern of multiple drug resistance (MDR) in Salmonellaisolates also varied from four to six antibiotics (Table 5) ThehighestMDRnotedwasAmpTeEryPenCef (2631 839)followed by TeEryPenCef (331 97) The maximumMDR registered was resistance to 6 antibiotics with thecombination of AmpTeEryPenCefCot (Table 5)

4 Discussion

In the current study all the respondents used Awetu Riverwithout any treatment for different purposes mainly forirrigation of vegetables thatmay be eaten raw Similarly Prabu

[30] reported that many people used Akaki River in AddisAbaba Ethiopia for different activities including irrigation ofvegetables that are served in the households of Addis Ababacity without any treatment

Some of the respondents complained of health problemssuch as typhoid due to consumption of vegetables irrigatedwith Awetu River Faruqui et al [31] demonstrated the prev-alence of typhoid epidemics in Santiago and Dakar Senegalwhich could be traced to fecal contamination ofwater or freshvegetables as noted in this studyMoreover in agreementwiththe current study Gerardi and Zimmerman [32] reportedClostridiumperfringes Staphylococcus aureus and someotherbacteria from wastewater


Table 5 Multiple drug resistance (MDR) of S aureus and Samonella spp isolated from water and vegetable samples

Isolates Number ofdrugs resisted Drugs resisted Resistant isolates

Subtotal number () Total Number ()

S aureus (36 isolates)

Three AmpCefPen 7 (194) 7 (194)Four AmpTeCefPen 7 (194) 7 (194)

Five AmpTeCotCefPen 11 (306) 13 (361)AmpGenTeCefPen 2 (56)

SixAmpTeEryCotCefPen 4 (111)

7 (194)AmpGenTeEryCefPen 1 (28)AmpGenTeCotCefPen 2 (56)

Seven AmpTeEryCotCefPenNor 2 (56) 2 (56)

Salmonella spp (31 isolates)Four TeEryPenCef 3 (97) 3 (97)Five TeEryPenCefAmp 26 (839) 26 (839)Six TeEryPenCefAmpCot 2 (65) 2 (65)

Where Amp stands for ampicillin Pen penicillin Cef cefuroxime sodium Te tetracycline Cot co-trimoxazole Gen gentamycin Ery erythromycin andNor norfloxacin

The overall mean aerobic mesophilic count observed inthis study ranged from 694 to 806 logCFU gminus1 relativelyhigher than previous reports from Morocco Ibenyassine etal [33] but other studies reported a lower count that rangedfrom 2 to 6 logCFU gminus1 [17 34 35] Generally there is nospecification set for the permissible level of microbes for rawfood being served in Ethiopia However Hazard Analysis andCritical Control Points-Total QualityManagement (HACCP-TQM) Technical Guidelines lay down the microbial qualityfor raw foods where the food containing less than 4 4ndash669 669ndash769 and greater than 769 logCFU gminus1 (aerobicplate count) is rated as good average poor and spoiled foodrespectively [36]

According to this guideline specifically the mean countsof AMB in all food samples (this study)were 694 logCFU gminus1and above Hence they belong to the category of poor andspoiled food Aerobic organisms reflect the exposure of thesample to contamination and the existence of favorable con-ditions for multiplication of microorganisms [37] Ayciceket al [17] also stated that plate count of aerobic mesophilicmicroorganisms found in food is one of the microbiologicalindicators for food quality and most foods are regarded asharmful when they have large populations of aerobic meso-philic microorganisms even if the organisms are not knownto be pathogens [38]

The overall mean count of Enterobacteriaceae in thepresent study ranged from 609 to 710 log CFU gminus1 This ishigher than other studies conducted on lettuce and greenpepper 508 and 484 logCFU gminus1 respectively by Guchi andAshenafi [20] and Ibenyassine et al [33] in Ethiopia andMorocco respectively According to Gilbert et al [39] andguideline recommended for fresh fruit and vegetables inLondon overall mean counts (log CFU gminus1) of Enterobacte-riaceae in carrot (710) cabbage (670) tomato (624) andlettuce (609) revealed unsatisfactory level (ge4 logCFU gminus1)Guchi and Ashenafi [20] suggested that the high level of

Enterobactericeae in vegetables might indicate that the waterused for irrigation could be heavily contaminated with fecalmatter from sewerage effluent Although most of Enterobac-teriaceae are normal flora of vegetables Motarjemi et al [40]stated that high number clearly proves that poor hygienecould be a source of foodborne pathogens

In case of aerobic spore formers the overall mean countsranged from 524 to 654 logCFU gminus1 In all vegetables thecounts were higher compared to reports by Guchi andAshenafi [20] where the counts ranged between 347 and350 logCFU gminus1 in green pepper and lettuce respectivelyfrom Addis Ababa Ethiopia

In the present study the overall mean count of staph-ylococci from vegetable samples ranged from 271 to297 log CFU gminus1This is lower than themicrobiological stud-ies made on lettuce and green pepper from super mar-ket in Addis Ababa Ethiopia Guchi and Ashenafi [20]who reported 455 and 497 log CFU gminus1 respectively Highercounts of staphylococci from supermarketmay be due to skincontact and environmental contamination In the currentstudy although the counts of staphylococci were low healthrisk cannot be avoided since Erkan et al [41] reported thatcontamination of food stuffs during distribution and han-dling may allow bacterial growth and subsequent productionof toxins

The overall mean counts of total and fecal coliforms fromvegetable samples in the present study are relatively lowerthan Nipa et al [42] who reported gt1100MPN 100mLminus1from salad vegetables In addition the current finding is lowerthan the results obtained by Ashenafi [18] who reportedtotal coliform 15 times 103MPN 10 gminus1 and fecal coliform 37 times102MPN 10 gminus1 counts of raw consumed food like tomatoin Ethiopia A survey carried out on spring onions lettuceand cabbage cultivated with poor quality irrigation water inGhana also showed heavy contamination with fecal coliformbetween 40 times 103 and 93 times 108MPNgminus1 [43] The observed


difference in counts can be attributed in part to the degree oforiginal contamination storage conditions and the hygienicconditions of utensils and vegetables handlers

The total and fecal coliform counts fromwater samples inthe present study were gt2400MPN 100mLminus1 which is higherthan the WHO recommended standard [10] According tothe standard the fecal coliform level must not exceed 1000counts 100mLminus1 for the safe use of wastewater for irrigationof vegetables A relationship has been established betweencoliform levels and the incidence of pathogenic bacteria likeSalmonella [44]

Microbial groups that belong to seven genera wereisolated from the examined samples at varying percentageswith gram positive and gram negative flora accounting for595 and 405 respectively The gram positive cells wererepresented by bacteria from the genera Bacillus Micrococ-cus Staphylococcus and Streptococcus spp while the gramnegative microflora constituted members of Enterobacteri-aceae Pseudomonas spp and Aromonas spp This is partlysimilar to the previous reports by Guchi and Ashenafi [20] inlettuce and green pepper from Addis Ababa Ethiopia Thepredominant microflora of fresh vegetables in the presentstudy was generally Bacillus spp followed by members ofEnterobacteriaceae andMicrococcus spp

The predominance of Bacillus isolates (this study) amongthe gram positive bacteria is in agreement with Guchi andAshenafi [20] Enterobacteriaceae isolates were the dominantmicroflora among gram negative isolates unlike the previousreports on lettuce and green pepper from Ethiopia whichshowed the predominance of Pseudomonas isolates Guchiand Ashenafi [20] From water samples Ikpeme et al [45]also reported the predominant genera that include Bacillusspp (8651) followed by Pseudomonas spp (7123) andAeromonas spp (5258) from Nigeria similar to the presentstudy Kwashie [34] also reported dominant bacteria such asEnterobacteriaceae Bacillus spp Staphylococcus spp Pseu-domonas spp and Clostridium spp from soil samples irri-gated with wastewater This could be due to direct transferof bacterial cells from wastewater to soil which may even-tually be internalized into the vegetables indicating seriousconsumerrsquos health risk when consumed raw

The predominance of Bacillus spp was possibly due tothe presence of spores in the water and soil as well as otherenvironmental factors The survival of Bacillus depends onseveral factors such as nature of the organism resistance toa new physical environment and ability to form spores [46]High number of Bacillus spp could cause food poisoningIn addition endospores of Bacillus are more resistant thanthe vegetative cells to harsh weather conditions and evento antimicrobial treatments [47] Therefore the presence ofhigh percentage of Bacillus spp in fresh vegetables could haveconsumerrsquos health risk

The predominant Enterobacteriaceae in the present studyindicates that the water used for irrigation was heavilycontaminated with fecal matter and received sewerage fromdiverse sources In addition fresh fruit and vegetables oftencarry high levels of Enterobacteriaceae as part of their normalflora Gilbert et al [39] In the present study Micrococcusspp was among the dominant isolates due to its occurrence

in the wastewater and soil [48] This is in agreement withGuchi and Ashenafi [20] who reported thatMicrococcus sppare the second dominant microflora isolated from lettuce andgreen pepper in Addis Ababa EthiopiaMicrococcus spp arecommon environmental bacteria that could be introducedinto the fresh vegetables through cross-contamination forinstance from wastewater used by the growers during irri-gationMicrococcus is generally thought to be a saprotrophicor commensal organism though it can be an opportunisticpathogen particularly in hosts with compromised immunesystem such as HIV patients [49]

The prevalence of S aureus in the current study in allvegetables was lower than what is mentioned in the studyof Halablab et al [50] who reported higher prevalence of Saureus (515) fromLebanon In addition Ijabadeniyi [2] alsoreported 670 in broccoli and 330 in cauliflower in SouthAfricaThe presence of low S aureus (200) in the irrigationwater in this study was of lower results than those obtained byIkpeme et al [45] (25ndash33) from two rivers that are used forirrigation of vegetables in South Africa

Shigella was not isolated from any of the samples testedin the current study This is in line with Soriano et al [51]who reported (00) Shigella in all of the lettuces served inSpain University restaurants However Shigella was isolatedfrom 8 (125) samples of lettuces and 16 (250) samples ofgreen peppers by Guchi and Ashenafi [20] from supermarketof Addis Ababa Ethiopia

In the present study the prevalence of Salmonella sppin all vegetable samples was higher than in other reports byGuchi and Ashenafi [20] and Ijabadeniyi [2] who reported10 in lettuce and green peppers as well as 11 in broccoliand cauliflower respectively

In the present study all the bacterial pathogens isolatedfrom the surface water were also isolated from all vegetableseven if their prevalence was varied similar to the reportsby Ijabadeniyi [2] in South Africa Diverse prevalence ofpathogens in different samples shows the possibility of varia-tions in surface characteristics of the produce affecting path-ogen attachment and survival [52]

The antibiotic resistance patterns of S aureus isolates inthe current study showed low percentage of resistance tonorfloxacin (56) gentamycin (139) and erythromycin(194) This is partly similar to previous report by Donokoret al [53] from Ghana In the current study all S aureus iso-lates were resistant to penicillinG ampicillin and cefuroximesodium (1000 each) This finding is partly in agreementwith Sina et al [54]

In the present study high number of Salmonella spp wassusceptible to norfloxacin chloramphenicol and gentamycin(100 each) followed by co-trimoxazole (935) This resultis fairly in line with Akbarmehr [55] who reported thatSalmonella spp were highly susceptible to chloramphenicol(100) followed by gentamycin (9189) However isolatesof Salmonella spp exhibited resistance to tetracycline ery-thromycin penicillin G and cefuroxime sodium (100 each)followed by ampicillin (903) Cardoso et al [56] havealso reported 100 resistance of Salmonella enteritidis toboth tetracycline and erythromycin from Brazil The markedresistance of strains of Salmonella spp to ampicillin as shown


in the present study agrees with the findings of Ash et al [57]and Ikpeme et al [45] working on rivers in the United Statesand Nigeria respectively

In this study the two bacterial pathogens investigated (Saureus and Salmonella spp) showed high levels of multipledrug resistance This trend has also been reported especiallyin the developing world [58 59]

Generally the surface water pollution in this study mayhave originated from both human and animal sewage dis-posal by the informal settlement that lacks proper sanitationAccording to van Vuuren [60] lack of proper sanitationusually leads to disposal of both human and animal wastesin the wrong places including surface water Almost anyready-to-eat vegetables that have been contaminated withpathogens either from the environment or from human oranimal faeces or through storage processing and handlingcould potentially cause disease [4] As a result consumptionof these vegetables with elevated levels of bacterial pathogensmay lead to health disorders Thus regular monitoring ofmicrobial contamination of vegetables grown using wastewa-ter is necessary and consumption of contaminated vegetablesshould be avoided in order to reduce the health risk

5 Conclusion

The present study showed the potential hazard of freshvegetables collected from Jimma town which were irrigatedwith Awetu RiverThe hygienic quality of both water and veg-etable samples was poor since higher mean bacterial countswere recorded beyond the standard safe limits Microfloraof vegetable and water samples was dominated by Bacillusand Enterobacteriaceae The presence of high number ofpathogenic bacteria such as S aureus and Salmonella sppcould cause foodborne diseases Ampicillin penicillin G andcefuroxime sodium were the most resistant antimicrobialagents by S aureus and Salmonella spp However chloram-phenicol norfloxacin and gentamycin are the drug of choiceto be recommended for the treatment of both S aureusand Salmonella spp according to the in vitro assay of thepresent study Wastewater should be properly treated whenused for produce that may be eaten raw This safety measureshould be combinedwithGoodAgricultural Practices duringproduction of fresh vegetables It is important to thoroughlywash vegetables and dip them in food grade antibacterialchemicals for a good time to eliminate pathogens and sig-nificantly reduce the microbial load

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

The financial support of Jimma University is gratefully ac-knowledgedThe authors also would like to thank the farmersof the study area and all individuals that have rendered helpand support to this work

References

[1] C R Lerici M C Nicoli and M Anese ldquoThe ldquoweightrdquo givento food processing at the ldquoFood and Cancer Prevention IIIrdquosymposiumrdquo Italian Journal of Food Science vol 12 no 1 pp3ndash7 2000

[2] O A Ijabadeniyi Effect of irrigation water quality on themicrobiological safety of fresh vegetables [PhD thesis] PretoriaUniversity of Agricultural and Food Sciences JohannesburgSouth Africa 2010

[3] L M Johnston C L Moe D Moll and L A Jaykus ldquoTheepidemiology of produce associated outbreaks of foodbornediseaserdquo in Microbial Hazard Identification in Fresh Fruit andVegetables J James Ed John Wiley amp Sons New York NYUSA 2006

[4] 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

[5] L Pezzoli R Elson C L Little et al ldquoPacked with SalmonellaInvestigation of an intestinal outbreak of Salmonella infectionlinked to contamination of pre-packed basil in 2007rdquo FoodbornePathogens and Disease vol 5 no 5 pp 661ndash668 2008

[6] N Garg K L Garg and K G Mukerji Laboratory Manual ofFood Microbiology IK International Publishing House NewDelhi India 2010

[7] European Commission (EC) Risk Profile on the Microbiolog-ical Contamination of Fruits and Vegetables Eaten Raw Reportof the Scientific Committee on Food SCFCSFMHSURFFinal2002 httpeceuropaeufoodfsscscfout125 enpdf

[8] Centers For Disease Control And Prevention (CDC) ldquoFoodborne outbreak online databaserdquo 2015 httpwwwoutbreakda-tabasecomdetailstaco-bell-restaurants-lettuce-2006

[9] L R Beuchat and J-H Ryu ldquoProduce Handling and ProcessingPracticesrdquo Emerging Infectious Diseases vol 3 no 4 pp 459ndash465 1997

[10] World Health Organization (WHO) WHO Guidelines for theSafe Use of Wastewater Excreta and Greywater Wastewaterin Agriculture vol 2 World Health Organization GenevaSwitzerland 2006

[11] T V Suslow M P Oria L R Beuchat et al ldquoProductionpractices as risk factors in microbial food safety of fresh andfresh-cut producerdquo Comprehensive Reviews in Food Science andFood Safety vol 2 no 1 pp 38ndash77 2003

[12] E B Solomon C J Potenski and K R Matthews ldquoEffectof irrigation method on transmission to and persistence ofEscherichia coliO157H7 on lettucerdquo Journal of Food Protectionvol 65 no 4 pp 673ndash676 2002

[13] K Ibenyassine R AitMhand Y Karamoko N Cohen and MM Ennaji ldquoUse of repetitive DNA sequences to determine thepersistence of enteropathogenic Escherichia coli in vegetablesand in soil grown in fields treated with contaminated irrigationwaterrdquo Letters in Applied Microbiology vol 43 no 5 pp 528ndash533 2006

[14] M P Combarro M Gonzalez M Araujo A C Amezaga RA Sueiro and M J Garrido ldquoListeria species incidence andcharacterisation in a river receiving town sewage from a sewagetreatment plantrdquoWater Science andTechnology vol 35 no 11-12pp 201ndash204 1997

[15] D C Johnson C E Enriquez I L Pepper T L Davis C PGerba and J B Rose ldquoSurvival of Giardia Cryptosporidiumpoliovirus and Salmonella in marine watersrdquoWater Science andTechnology vol 35 no 11-12 pp 261ndash268 1997


[16] D H Tambekar and R H Mundhada ldquoBacteriological qualityof salad vegetables sold in Amravati city (India)rdquo Journal ofBiological Sciences vol 6 no 1 pp 28ndash30 2006

[17] H Aycicek B Sarimehmetoglu and S Cakiroglu ldquoAssessmentof the microbiological quality of meals sampled at the mealserving units of a military hospital in Ankara Turkeyrdquo FoodControl vol 15 no 5 pp 379ndash384 2004

[18] M Ashenafi ldquoMicrobial load incidence and antibiotic resis-tance of some disease-causing microorganisms on raw fooditems in consumed Ethiopiardquo MIRCEN Journal of AppliedMicrobiology and Biotechnology vol 5 no 3 pp 313ndash319 1989

[19] G Aberra T Frew T Asmamaw G Mulu and A SisayneshldquoA preliminary study of the microflora level of some fruits andvegetables pre- and post-preservationrdquo The Ethiopian Journalof Health Development vol 5 no 2 pp 57ndash65 1991

[20] B Guchi andMAshenafi ldquoMicrobial load prevalence and anti-biograms of salmonellaand shigella in lettuce and green pep-persrdquo Ethiopian Journal of Health Sciences vol 20 no 1 pp 43ndash47 2010

[21] D Hailu ldquoPollution status of Awetu stream as it crosses Jimmatown southwest of Ethiopia Post basic degree projectrdquo inAbstracts of All Public Health Faculty Graduate Student ResearchProjects Jimma University Jimma Ethiopia 1997

[22] I Deneke Assessment of drinking water quality and pollutionprofiles along Awetu stream (Jimma) [MS thesis] Addis AbabaUniversity Addis Ababa Ethiopia 2006

[23] K Sofonias and G Tsegaye ldquoMicrobial quality of Jimma watersupplyrdquo Ethiopian Journal of Education and Sciences vol 2 no1 p 23 2006

[24] AAlemuWTsegaye LGolassa andGAbebe ldquoUrbanmalariaand associated risk factors in Jimma town south-west EthiopiardquoMalaria Journal vol 10 pp 173ndash200 2011

[25] S Shalini Study on Microbiological Aspects of Fresh Fruit andVegetables (Including Green Leafy Vegetables) in and aroundNational Capital Region (NCR) Bhaskaracharya College ofApplied Sciences New Delhi India 2010

[26] American Public Health Association (APHA) Standard Meth-ods for the Examination of Water and Wastewater AmericanPublic Health Association (APHA)Washington DCUSA 21stedition 2005

[27] L John An introduction to bacterial identificationmdashgeneralprinciples 2001 httpwwwjlindquistnet

[28] T R Johnson and C L Case Laboratory Experiments in Micro-biology Pearson Education San Francisco Calif USA 8thedition 2007

[29] M CheesbroughDistrict Laboratory Practice in Tropical Coun-triesmdashPart 2 Cambridge University Press Cambridge UK2006

[30] P C Prabu ldquoImpact of heavy metal contamination of Akakiriver of Ethiopia on soil and metal toxicity on cultivated veg-etable cropsrdquo Electronic Journal of Environmental Agriculturaland Food Chemistry vol 8 no 9 pp 818ndash827 2009

[31] N I Faruqui C A Scott and L Raschid-Sally ldquoConfrontingthe realities of wastewater use in irrigated agriculture lessonslearned and recommendationsrdquo inWastewater Use In IrrigatedAgriculture Confronting the Livelihood and Environmental Real-ities C A Scott N I Faruqui and L Raschid-Sally Eds pp173ndash185 CABI 2004

[32] M H Gerardi and M C Zimmerman Wastewater PathogensWastewater Microbiology Series Edited by MH Gerardi JohnWiley amp Sons Hoboken NJ USA 2005

[33] K Ibenyassine R A Mahand Y Karakomo B Anajjar MChouibani and M M Enanaji ldquoBacterial pathogens recoveredfrom vegetables irrigated by wastewater in Moroccordquo Journal ofEnvironmental Health vol 69 no 10 pp 47ndash51 2007

[34] K C KwashieMicrobial analysis of soil samples in a wastewaterirrigated vegetable production site case study at atonsu kumasi[MS thesis] Kwame Nkrumah University of Science andTechnology Kumasi Ghana 2009

[35] A S Angelidis E N Chronis D K Papageorgiou I I KazakisK C Arsenoglou and G A Stathopoulos ldquoNon-lactic acidcontaminating microbial flora in ready-to-eat foods a potentialfood-quality indexrdquoFoodMicrobiology vol 23 no 1 pp 95ndash1002006

[36] H Aycicek U Oguz and K Karci ldquoDetermination of totalaerobic and indicator bacteria on some raw eaten vegetablesfrom wholesalers in Ankara Turkeyrdquo International Journal ofHygiene and Environmental Health vol 209 no 2 pp 197ndash2012006

[37] G Tortora Microbiology Benjamin Publishing Co Inc NewYork NY USA 5th edition 1995

[38] R V Sudershan P Rao and K Polasa ldquoFood safety research inIndia a reviewrdquoAsian Journal of Food and Agro-Industry vol 2no 3 pp 412ndash433 2009

[39] R J Gilbert J de Louvois T Donovan et al ldquoGuidelines for themicrobiological quality of some ready-to-eat foods sampled atthe point of salerdquo Communicable Disease and Public Health vol3 no 3 pp 163ndash167 2000

[40] Y Motarjemi F Kaferstein G Moy and F Quevedo ldquoCon-taminated weaning food a major risk factor for diarrhoea andassociated malnutritionrdquo Bulletin of the World Health Organ-ization vol 71 no 1 pp 79ndash92 1993

[41] M E Erkan A Vural and T Ozekinci ldquoInvestigating the pres-ence of Staphylococcus aureus and Coagulase Negative Staph-ylococci (CNS) in some leafy green vegetablesrdquoResearch Journalof Biological Sciences vol 3 no 8 pp 930ndash933 2008

[42] MNNipaMMReazMMHasan et al ldquoPrevalence ofmultidrug resistant bacteria on raw salad vegetables sold in majormarkets of Chittagong City Bangladeshrdquo Middle-East Journalof Scientific Research vol 10 no 1 pp 70ndash77 2011

[43] P Amoah P Drechsel R C Abaidoo andW J Ntow ldquoPesticideand pathogen contamination of vegetables in Ghanarsquos urbanmarketsrdquo Archives of Environmental Contamination and Toxi-cology vol 50 no 1 pp 1ndash6 2006

[44] S M Goyal C P Gerba and J L Melnick ldquoOccurrence anddistribution of bacterial indicators and pathogens in canal com-munities along the Texas Coastrdquo Applied and EnvironmentalMicrobiology vol 34 no 2 pp 139ndash149 1977

[45] E Ikpeme J Nfongeh M E Eja L Etim and K Enyi-IdohldquoAntibiotic susceptibility profiles of enteric bacterial isolatesfrom dumpsite utisols and water sources in a rural communityin cross river staterdquo Nature and Science vol 9 no 5 pp 46ndash502011

[46] R E Godon ldquoThe genusBacillusrdquo inHandbook ofMicrobiologyA I Laskin and H A Lechevalier Eds vol 1-2 pp 319ndash336CRC Press Cleveland Ohio USA 1977

[47] Codex Alimantarius Code of Hygiene Practice for Fresh Fruitsand Vegetables Secretariateof the CODEX Alimetarius Com-mission Joint FAOWHO Food Standard programme Vial-edelle Terme di Caracalla Rome Italy 2007

[48] J Santamarıa and G A Toranzos ldquoEnteric pathogens and soila short reviewrdquo International Microbiology vol 6 no 1 pp 5ndash92003


[49] K J Smith R Neafie J Yeager and H G Skelton ldquoMicrococcusfolliculitis in HIV-1 diseaserdquo British Journal of Dermatology vol141 no 3 pp 558ndash561 1999

[50] M A Halablab I H Sheet and H M Holail ldquoMicrobiologicalquality of raw vegetables grown in Bekaa Valley LebanonrdquoAmerican Journal of Food Technology vol 6 no 2 pp 129ndash1392011

[51] J M Soriano H Rico J C Molto and J Manes ldquoAssessmentof the microbiological quality and wash treatments of lettuceserved in University restaurantsrdquo International Journal of FoodMicrobiology vol 58 no 1-2 pp 123ndash128 2000

[52] D O Ukuku C Liao and S V Gembeh ldquoAttachment of bac-terial human pathogens on fruit and vegetable surfaces Atlanta(Etats-Unis)rdquo Journal of Applied Microbiology vol 98 pp 380ndash396 2005

[53] E S Donkor T Nortey A Opitan N Dayie and M LAkyeh ldquoAntimicrobial susceptibility of Salmonella typhi andStaphylococcus aureus isolates and the effect of some media onsusceptibility testing resultsrdquoThe Internet Journal of Microbiol-ogy vol 4 no 2 pp 1ndash5 2008

[54] H Sina F Baba-Moussa A P Kayode et al ldquoCharacterizationof Staphylococcus aureus isolated from street foods toxin profileand prevalence of antibiotic resistancerdquo Journal of AppliedBiosciences vol 46 pp 3133ndash3143 2011

[55] J Akbarmehr ldquoAntimicrobial resistance in Salmonella isolatedfrom broiler chicken carcassesrdquo African Journal of MicrobiologyResearch vol 6 pp 1485ndash1488 2012

[56] M O Cardoso A R Ribeiro L R Dos Santos et al ldquoAntibi-otic resistance in Salmonella Enteritidis isolated from broilercarcassesrdquo Brazilian Journal of Microbiology vol 37 no 3 pp368ndash371 2006

[57] R J Ash B Mauck and M Morgan ldquoAntibiotic resistance ofgram negative bacteria in rivers United States of AmericardquoEmerging Infectious Diseases vol 8 no 7 pp 7ndash12 2002

[58] P I Umolu E N Okoli and I M Izomoh ldquoAntibiogram andbeta-lactamase production of Staphylococcus aureus isolatesfrom different human clinical specimens in Edo State NigeriardquoWest African Journal ofMedicine vol 21 no 2 pp 124ndash127 2002

[59] F Mills-Robertson S S Crupper M E Addy and P Men-sah ldquoAntibiotic resistance and genotyping of clinical groupB Salmonella isolated in Accra Ghanardquo Journal of AppliedMicrobiology vol 94 no 2 pp 289ndash294 2003

[60] L van Vuuren ldquoTime running out as Africa sprints towardsMDG deadlinerdquoTheWater Wheel vol 9 no 1 pp 25ndash27 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

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


salad bars and meals eaten outside the home Ijabadeniyi[2] Changes in production and processing methods of dis-tribution consumption patterns and practices are otherfactors that have also contributed to increasing foodbornediseases due to raw consumed vegetables [9]

Ijabadeniyi [2] reported that other reasons given by theFood and Agriculture Organization (FAO) andWorld HealthOrganization (WHO) [10] for the increasing of foodborneinfectionpoisoning outbreaks are as follows microbial adap-tation increase in international trade and in susceptible pop-ulation and increase in worldwide travelling Furthermorechanges of a lifestyle of convenience consumer demandsregarding healthy food with no chemical preservatives andwith an extended shelf life and changes in human demo-graphics and behavior have also contributed to increase offoodborne infections

According to Suslow et al [11] the microbial quality ofirrigation water is critical because water contaminated withanimal or human wastes can introduce pathogens into veg-etable products during preharvest and postharvest activitiesvia direct or indirect contaminationThereforemicrobiologi-cal quality of irrigation water has a paramount importance tothe safety of fresh and minimally processed vegetables [12]Moreover Ibenyassine et al [13] reported that contaminatedirrigation water and surface runoff water may be the majorsources of pathogenic microorganisms that contaminatefruits and vegetables in fields Water from the river thatreceived both human and animal waste disposal poses ahealth risk due to contamination with all microorganismsof human and animal intestinal habitat such as Salmonellaand Listeria spp [14 15] Consequently the microbiologicalquality and safety of fresh vegetables is a significant concernto all stakeholders all over the world

Tambekar and Mundhada [16] reported that foodbornebacterial pathogens commonly detected in fresh vegetableswere E coli S aureus and Salmonella spp Aycicek et al[17] have also stated that plate count of aerobic mesophilicmicroorganisms found in food is one of the microbiologicalindicators for food quality in addition to the common indica-tors such as fecal coliform Information on themicrobial loadon fresh vegetables in Ethiopia has been studied by severalinvestigators [18ndash20]

According to Hailu [21] the use of rivers and hand-dug wells for various purposes is common in Jimma townThe author further noted that Awetu River is the primarysource of water for a range of activities such as recreationbathing washing clothes and household utensils small scaleagricultural irrigation car washing and other usesThe dete-rioration of the quality of Awetu River as a result of dischargeof municipal wastes and urban runoff has been well studiedHailu [21] For instance Deneke [22] investigated about thepollution profiles along Awetu River and found high loadof fecal coliforms According to Sofonias and Tsegaye [23]some of the water sources in Jimma town such as unprotectedsprings wells and Awetu River have greater chance to becontaminated via human and animal fecal matters andwastesdisposed from households hotels and small scale industriesThus these water sources could transmit diseases as a resultof contamination However a comprehensive investigation of

the quality and safety of vegetables irrigatedwithAwetu Riverincluding bacteriological quality and safety analyses is lack-ing Therefore this study was aimed at investigating the bac-teriological contaminants of some fresh vegetables irrigatedwith Awetu River in Jimma town southwestern Ethiopia

2 Materials and Methods

21 Description of the Study Area The study was conductedat Jimma town located 350 km southwest of Addis AbabaThe townrsquos geographical coordinates are 7∘411015840N latitude and36∘501015840E longitude The town is found with abundant meanannual rainfall between 1800 and 2300mmwhich makes thisregion one of the best watered Ethiopian highland areasconducive for agricultural production [24]

22 Study Design and Study Population Laboratory basedcross sectional study design was used The sampling siteswere three irrigation farms around Awetu River designatedas site A B and C along the river course at the same timeof the irrigation period The size of the farms is about 15 haand the average production of vegetables from these farmsis 30 kgha On the average the frequency of irrigation for thetarget vegetables is 3ndash5 daysweek depending on the humidityof the air at the farms

There are many farmers who are members of Microen-terprise Association around Awetu River They are activelyinvolved in irrigating their farms

Preliminary survey was made on the distribution andlocation of vegetable growers in the study area prior toresuming the actual sample collection The survey includedindividual residents and members of Microenterprise Asso-ciation of vegetable growers who regularly use Awetu Riverto irrigate their farms in Jimma town Purposive samplingwas made to gather information on Awetu River from a totalof 60 vegetable growers using structured questionnaire Thissurvey was performed in order to get general informationabout Awetu River including its pollution status and its effecton human health

23 Collection of Samples A total of 120 vegetable samples40 each from the three sites and 10 samples each of lettucecabbage tomato and carrot were randomly picked (15 kg)aseptically using sterilized scissors and cut into pieces Inaddition a total of 30 irrigation water samples (200mL each)were collected from three sites (10 samples each) The watersamples were collected from the depth of below 5 cm surfacewater using sterilized stopper glass bottles The sampleswere transported to Postgraduate and Research Laboratoryof Biology Department College of Natural Sciences JimmaUniversity using ice box The samples were processed forbacteriological analysis within 1ndash4 hrs of collection

24 Sample Preparation Mixed vegetable samples (unpro-cessed and large sized) were aseptically chopped into smallerpieces using a sterile stainless steel knife prior to weighing A25 g of subsample of each vegetable was aseptically weighedand vigorously shaken in 225mLof sterile 01 (wv) buffered










10


3 Results


















Water

A 765cplusmn 04 655

cplusmn 03 488

cplusmn 05 255

bplusmn 01 gt240000

aplusmn 0 gt 240000

aplusmn 0

B 869bplusmn 05 734

bplusmn 03 570

bplusmn 05 266


aplusmn 0 gt240000

aplusmn 0

C 942aplusmn 01 836

aplusmn 01 665

aplusmn 05 271

aplusmn 01 gt240000

aplusmn 0 gt240000

aplusmn 0


Lettuce

A 592cplusmn 05 500

cplusmn 0 430

cplusmn 01 277

bplusmn 02 84400

aplusmn 3305 52400

aplusmn 2024

B 690bplusmn 05 610

bplusmn 06 541

bplusmn 03 289

bplusmn 03 90800

aplusmn 3091 58800

aplusmn 2698

C 799aplusmn 05 717

aplusmn 04 602

aplusmn 05 324

aplusmn 04 97200

aplusmn 2698 65200

aplusmn 3091


Carrot

A 705cplusmn 05 643

cplusmn 03 598

cplusmn 04 263

aplusmn 01 97200

aplusmn 2698 65200

aplusmn 3091

B 799bplusmn 05 708

bplusmn 04 659

bplusmn 04 274

aplusmn 01 103600

aplusmn 2024 71600

aplusmn 3305

C 915aplusmn 04 779

aplusmn 05 704

aplusmn 05 276

aplusmn 02 110000

aplusmn 0 78000

aplusmn 3373


Tomato

A 657cplusmn 04 538

cplusmn 04 512

cplusmn 04 262

cplusmn 01 78000

aplusmn 460 46000

aplusmn 0

B 753bplusmn 04 634

bplusmn 03 572

bplusmn 05 276

bplusmn 01 84400

aplusmn 3305 52400

aplusmn 2024

C 812aplusmn 04 699

aplusmn 05 629

aplusmn 01 288

aplusmn 01 97200

aplusmn 2698 58800

aplusmn 2698


Cabbage

A 682cplusmn 05 589

cplusmn 05 524

cplusmn 03 271

aplusmn 02 84400

aplusmn 3305 58800

aplusmn 2698

B 758bplusmn 04 681

bplusmn 05 581

bplusmn 05 273

aplusmn 02 90800

aplusmn 3091 65200

aplusmn 3091

C 857aplusmn 04 740

aplusmn 05 663

aplusmn 05 285

aplusmn 02 103600

aplusmn 2024 71600

aplusmn 3305



00

50

100

150

200

250

300

350

400


Perc

enta

ge (

)

Type of samples













Water

A 10 0 0B 10 2 (20) 2 (20)C 10 4 (40) 5 (50)

Subtotal 30 6 (20) 7 (233)

Lettuce

A 10 3 (30) 1 (10)B 10 2 (20) 1 (10)C 10 5 (50) 2 (20)

Subtotal 30 10 (333) 4 (133)

Carrot

A 10 0 1 (10)B 10 1 (10) 2 (20)C 10 4 (40) 2 (20)

Subtotal 30 5 (167) 5 (167)

Tomato

A 10 1 (10) 1 (10)B 10 3 (30) 2 (20)C 10 3 (30) 3 (30)

Subtotal 30 7 (233) 6 (20)

Cabbage

A 10 1 (10) 2 (20)B 10 3 (30) 3 (30)C 10 4 (40) 4 (40)

Subtotal 30 8 (267) 9 (30)Total 150 36 (24) 31 (20)





4 Discussion









































5 Conclusion


Competing Interests


Acknowledgments


References






































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










10


3 Results


















Water

A 765cplusmn 04 655

cplusmn 03 488

cplusmn 05 255

bplusmn 01 gt240000

aplusmn 0 gt 240000

aplusmn 0

B 869bplusmn 05 734

bplusmn 03 570

bplusmn 05 266


aplusmn 0 gt240000

aplusmn 0

C 942aplusmn 01 836

aplusmn 01 665

aplusmn 05 271

aplusmn 01 gt240000

aplusmn 0 gt240000

aplusmn 0


Lettuce

A 592cplusmn 05 500

cplusmn 0 430

cplusmn 01 277

bplusmn 02 84400

aplusmn 3305 52400

aplusmn 2024

B 690bplusmn 05 610

bplusmn 06 541

bplusmn 03 289

bplusmn 03 90800

aplusmn 3091 58800

aplusmn 2698

C 799aplusmn 05 717

aplusmn 04 602

aplusmn 05 324

aplusmn 04 97200

aplusmn 2698 65200

aplusmn 3091


Carrot

A 705cplusmn 05 643

cplusmn 03 598

cplusmn 04 263

aplusmn 01 97200

aplusmn 2698 65200

aplusmn 3091

B 799bplusmn 05 708

bplusmn 04 659

bplusmn 04 274

aplusmn 01 103600

aplusmn 2024 71600

aplusmn 3305

C 915aplusmn 04 779

aplusmn 05 704

aplusmn 05 276

aplusmn 02 110000

aplusmn 0 78000

aplusmn 3373


Tomato

A 657cplusmn 04 538

cplusmn 04 512

cplusmn 04 262

cplusmn 01 78000

aplusmn 460 46000

aplusmn 0

B 753bplusmn 04 634

bplusmn 03 572

bplusmn 05 276

bplusmn 01 84400

aplusmn 3305 52400

aplusmn 2024

C 812aplusmn 04 699

aplusmn 05 629

aplusmn 01 288

aplusmn 01 97200

aplusmn 2698 58800

aplusmn 2698


Cabbage

A 682cplusmn 05 589

cplusmn 05 524

cplusmn 03 271

aplusmn 02 84400

aplusmn 3305 58800

aplusmn 2698

B 758bplusmn 04 681

bplusmn 05 581

bplusmn 05 273

aplusmn 02 90800

aplusmn 3091 65200

aplusmn 3091

C 857aplusmn 04 740

aplusmn 05 663

aplusmn 05 285

aplusmn 02 103600

aplusmn 2024 71600

aplusmn 3305



00

50

100

150

200

250

300

350

400


Perc

enta

ge (

)

Type of samples













Water

A 10 0 0B 10 2 (20) 2 (20)C 10 4 (40) 5 (50)

Subtotal 30 6 (20) 7 (233)

Lettuce

A 10 3 (30) 1 (10)B 10 2 (20) 1 (10)C 10 5 (50) 2 (20)

Subtotal 30 10 (333) 4 (133)

Carrot

A 10 0 1 (10)B 10 1 (10) 2 (20)C 10 4 (40) 2 (20)

Subtotal 30 5 (167) 5 (167)

Tomato

A 10 1 (10) 1 (10)B 10 3 (30) 2 (20)C 10 3 (30) 3 (30)

Subtotal 30 7 (233) 6 (20)

Cabbage

A 10 1 (10) 2 (20)B 10 3 (30) 3 (30)C 10 4 (40) 4 (40)

Subtotal 30 8 (267) 9 (30)Total 150 36 (24) 31 (20)





4 Discussion









































5 Conclusion


Competing Interests


Acknowledgments


References






































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















Water

A 765cplusmn 04 655

cplusmn 03 488

cplusmn 05 255

bplusmn 01 gt240000

aplusmn 0 gt 240000

aplusmn 0

B 869bplusmn 05 734

bplusmn 03 570

bplusmn 05 266


aplusmn 0 gt240000

aplusmn 0

C 942aplusmn 01 836

aplusmn 01 665

aplusmn 05 271

aplusmn 01 gt240000

aplusmn 0 gt240000

aplusmn 0


Lettuce

A 592cplusmn 05 500

cplusmn 0 430

cplusmn 01 277

bplusmn 02 84400

aplusmn 3305 52400

aplusmn 2024

B 690bplusmn 05 610

bplusmn 06 541

bplusmn 03 289

bplusmn 03 90800

aplusmn 3091 58800

aplusmn 2698

C 799aplusmn 05 717

aplusmn 04 602

aplusmn 05 324

aplusmn 04 97200

aplusmn 2698 65200

aplusmn 3091


Carrot

A 705cplusmn 05 643

cplusmn 03 598

cplusmn 04 263

aplusmn 01 97200

aplusmn 2698 65200

aplusmn 3091

B 799bplusmn 05 708

bplusmn 04 659

bplusmn 04 274

aplusmn 01 103600

aplusmn 2024 71600

aplusmn 3305

C 915aplusmn 04 779

aplusmn 05 704

aplusmn 05 276

aplusmn 02 110000

aplusmn 0 78000

aplusmn 3373


Tomato

A 657cplusmn 04 538

cplusmn 04 512

cplusmn 04 262

cplusmn 01 78000

aplusmn 460 46000

aplusmn 0

B 753bplusmn 04 634

bplusmn 03 572

bplusmn 05 276

bplusmn 01 84400

aplusmn 3305 52400

aplusmn 2024

C 812aplusmn 04 699

aplusmn 05 629

aplusmn 01 288

aplusmn 01 97200

aplusmn 2698 58800

aplusmn 2698


Cabbage

A 682cplusmn 05 589

cplusmn 05 524

cplusmn 03 271

aplusmn 02 84400

aplusmn 3305 58800

aplusmn 2698

B 758bplusmn 04 681

bplusmn 05 581

bplusmn 05 273

aplusmn 02 90800

aplusmn 3091 65200

aplusmn 3091

C 857aplusmn 04 740

aplusmn 05 663

aplusmn 05 285

aplusmn 02 103600

aplusmn 2024 71600

aplusmn 3305



00

50

100

150

200

250

300

350

400


Perc

enta

ge (

)

Type of samples













Water

A 10 0 0B 10 2 (20) 2 (20)C 10 4 (40) 5 (50)

Subtotal 30 6 (20) 7 (233)

Lettuce

A 10 3 (30) 1 (10)B 10 2 (20) 1 (10)C 10 5 (50) 2 (20)

Subtotal 30 10 (333) 4 (133)

Carrot

A 10 0 1 (10)B 10 1 (10) 2 (20)C 10 4 (40) 2 (20)

Subtotal 30 5 (167) 5 (167)

Tomato

A 10 1 (10) 1 (10)B 10 3 (30) 2 (20)C 10 3 (30) 3 (30)

Subtotal 30 7 (233) 6 (20)

Cabbage

A 10 1 (10) 2 (20)B 10 3 (30) 3 (30)C 10 4 (40) 4 (40)

Subtotal 30 8 (267) 9 (30)Total 150 36 (24) 31 (20)





4 Discussion









































5 Conclusion


Competing Interests


Acknowledgments


References






































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Water

A 765cplusmn 04 655

cplusmn 03 488

cplusmn 05 255

bplusmn 01 gt240000

aplusmn 0 gt 240000

aplusmn 0

B 869bplusmn 05 734

bplusmn 03 570

bplusmn 05 266


aplusmn 0 gt240000

aplusmn 0

C 942aplusmn 01 836

aplusmn 01 665

aplusmn 05 271

aplusmn 01 gt240000

aplusmn 0 gt240000

aplusmn 0


Lettuce

A 592cplusmn 05 500

cplusmn 0 430

cplusmn 01 277

bplusmn 02 84400

aplusmn 3305 52400

aplusmn 2024

B 690bplusmn 05 610

bplusmn 06 541

bplusmn 03 289

bplusmn 03 90800

aplusmn 3091 58800

aplusmn 2698

C 799aplusmn 05 717

aplusmn 04 602

aplusmn 05 324

aplusmn 04 97200

aplusmn 2698 65200

aplusmn 3091


Carrot

A 705cplusmn 05 643

cplusmn 03 598

cplusmn 04 263

aplusmn 01 97200

aplusmn 2698 65200

aplusmn 3091

B 799bplusmn 05 708

bplusmn 04 659

bplusmn 04 274

aplusmn 01 103600

aplusmn 2024 71600

aplusmn 3305

C 915aplusmn 04 779

aplusmn 05 704

aplusmn 05 276

aplusmn 02 110000

aplusmn 0 78000

aplusmn 3373


Tomato

A 657cplusmn 04 538

cplusmn 04 512

cplusmn 04 262

cplusmn 01 78000

aplusmn 460 46000

aplusmn 0

B 753bplusmn 04 634

bplusmn 03 572

bplusmn 05 276

bplusmn 01 84400

aplusmn 3305 52400

aplusmn 2024

C 812aplusmn 04 699

aplusmn 05 629

aplusmn 01 288

aplusmn 01 97200

aplusmn 2698 58800

aplusmn 2698


Cabbage

A 682cplusmn 05 589

cplusmn 05 524

cplusmn 03 271

aplusmn 02 84400

aplusmn 3305 58800

aplusmn 2698

B 758bplusmn 04 681

bplusmn 05 581

bplusmn 05 273

aplusmn 02 90800

aplusmn 3091 65200

aplusmn 3091

C 857aplusmn 04 740

aplusmn 05 663

aplusmn 05 285

aplusmn 02 103600

aplusmn 2024 71600

aplusmn 3305



00

50

100

150

200

250

300

350

400


Perc

enta

ge (

)

Type of samples













Water

A 10 0 0B 10 2 (20) 2 (20)C 10 4 (40) 5 (50)

Subtotal 30 6 (20) 7 (233)

Lettuce

A 10 3 (30) 1 (10)B 10 2 (20) 1 (10)C 10 5 (50) 2 (20)

Subtotal 30 10 (333) 4 (133)

Carrot

A 10 0 1 (10)B 10 1 (10) 2 (20)C 10 4 (40) 2 (20)

Subtotal 30 5 (167) 5 (167)

Tomato

A 10 1 (10) 1 (10)B 10 3 (30) 2 (20)C 10 3 (30) 3 (30)

Subtotal 30 7 (233) 6 (20)

Cabbage

A 10 1 (10) 2 (20)B 10 3 (30) 3 (30)C 10 4 (40) 4 (40)

Subtotal 30 8 (267) 9 (30)Total 150 36 (24) 31 (20)





4 Discussion









































5 Conclusion


Competing Interests


Acknowledgments


References






































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Water

A 10 0 0B 10 2 (20) 2 (20)C 10 4 (40) 5 (50)

Subtotal 30 6 (20) 7 (233)

Lettuce

A 10 3 (30) 1 (10)B 10 2 (20) 1 (10)C 10 5 (50) 2 (20)

Subtotal 30 10 (333) 4 (133)

Carrot

A 10 0 1 (10)B 10 1 (10) 2 (20)C 10 4 (40) 2 (20)

Subtotal 30 5 (167) 5 (167)

Tomato

A 10 1 (10) 1 (10)B 10 3 (30) 2 (20)C 10 3 (30) 3 (30)

Subtotal 30 7 (233) 6 (20)

Cabbage

A 10 1 (10) 2 (20)B 10 3 (30) 3 (30)C 10 4 (40) 4 (40)

Subtotal 30 8 (267) 9 (30)Total 150 36 (24) 31 (20)





4 Discussion









































5 Conclusion


Competing Interests


Acknowledgments


References






































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






































5 Conclusion


Competing Interests


Acknowledgments


References






































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















5 Conclusion


Competing Interests


Acknowledgments


References






































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





5 Conclusion


Competing Interests


Acknowledgments


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

Documents

New Research Article Bacteriological Contaminants of Some Fresh …downloads.hindawi.com/archive/2016/1526764.pdf · 2019. 7. 30. · possible preharvest source of contamination to