lactamases escherichia coli strains isolated from market garden prod

Research ArticleIdentification of Extended-Spectrum 120573-LactamasesEscherichia coli Strains Isolated from Market GardenProducts and Irrigation Water in Benin

Wassiyath Mousseacute1 Haziz Sina1 Farid Baba-Moussa2 Pacocircme A Noumavo1

Nadegravege A Agbodjato1 Adolphe Adjanohoun3 and Lamine Baba-Moussa1

1Laboratoire de Biologie et de Typage Moleculaire en Microbiologie Faculte des Sciences et TechniquesUniversite drsquoAbomey-Calavi 05 BP 1604 Cotonou Benin2Laboratoire de Microbiologie et de Technologie Alimentaire FAST Universite drsquoAbomey-Calavi ISBA-Champ de foire01 BP 526 Cotonou Benin3Centre de Recherches Agricoles Sud Institut National des Recherches Agricoles du Benin Attogon BP 884 Cotonou Benin

Correspondence should be addressed to Lamine Baba-Moussa laminesaidyahoofr

Received 27 July 2015 Revised 19 October 2015 Accepted 12 November 2015

Academic Editor Frederick D Quinn

Copyright copy 2015 Wassiyath Mousse 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

The present study aimed at biochemical and molecular characterization of Escherichia coli strains isolated from horticulturalproducts and irrigation water of Cotonou The samples were collected from 12 market gardeners of 4 different sites Rapidrsquo E colimedium was used for identification of E coli strains and the antimicrobial susceptibility was performed by the agar disk diffusionmethodThe 120573-lactamases production was sought by the liquid acidimetric methodThe genes coding for 120573-lactamases and toxinswere identified by PCR method The results revealed that about 3495 of the analyzed samples were contaminated by E coliCabbages were the most contaminated by E coli (2826) in dry season All isolated strains were resistant to amoxicillin Thepenicillinase producing E coli carried blaTEM (6750) blaSHV (10) and blaCTX-M (2250) genes The study revealed that theresistance genes such as SLTI (3571) SLTII (3571) ETEC (715) and VTEC (2143) were carried Openly to the found resultsand considering the importance of horticultural products in Beninese food habits it is important to put several strategies aimingat a sanitary security by surveillance and sensitization of all the actors on the risks of some practices

1 Introduction

Fruits and leafy vegetables are interesting dietary sourceof nutrients micronutrients vitamins and fiber for humanbeings hence vital for health and fitness In most of Africantowns areas are often valorizing for market gardeningIndeed due to the strong urbanization of manyWest Africantowns the market gardening activity increases because itplays a vital role in the supply of populations with cheap andfresh products [1]This activity also provides employment formany low-income families generally living under conditionsof extreme poverty Among the commonly produced veg-etables we can cite tomatoes cabbage lettuce carrots great

nightshades green pepper basil parsley garden eggs greenbeans and leeks

In developing countries such as Benin gardeners useuntreated waste water and manure as fertilizers for theproduction of their crops This is a major contributing factorfor the outbreak of diseases In fact the practices used bythe market gardeners can foster a strong contamination ofvegetables by microorganisms some of which can be danger-ous for the consumer The differences in microbial profiles ofvarious vegetables result largely from unrelated factors suchas resident microflora in the soil application of nonresidentmicroflora via animal manures sewage or irrigation watertransportation and handling by individual retailers [2]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 286473 11 pageshttpdxdoiorg1011552015286473

2 BioMed Research International

Indeed several cases of typhoid fever have been associatedwith eating contaminated vegetables grown inor fertilizedwith contaminated soil or sewage [3]

Vegetables consumption is encouraged worldwide bygovernmental health agencies to protect against a rangeof illnesses such as cancers and cardiovascular diseasesHowever leafy greens consumed raw are increasingly beingidentified as important cause of human pathogens that weretraditionally associated with foods andor animal origin[4] Bacteria viruses and parasites on vegetables such asmarket garden products have been linked with illness [5]But bacterial pathogens continue to be amajor contributor toproduce associated food-borne illnesses Among those bac-teria Salmonella was the most commonly reported bacterialpathogen accounting for nearly half of the outbreaks due tobacteria [6] followed by E coli Thus it is established thatenterobacteria account for more than half of the outbreaksdue to bacteria To explain this high level of contaminationthe most frequently cited factors are water soil manure andanimals [7]

Among the enterobacteria E coli is a common part ofa normal microflora of man and most of the warm-bloodedanimals digestive tube Nevertheless some E coli strains arereported to be a leading cause of extraintestinal (meningitisurinary tract infections) andor intestinal pathologies [8]Thepathogenicity of those strains can be due not only to theirinvasion proportion but also to toxins production Enzymesdisplaying human cells cytopathic effect are part of such tox-ins [9] To fight the pathogenic E coli strains many antibioticmolecules are often used worldwide But due to the highmutation capability and the worse use of such antibioticsmany cases of E coli multiresistance strains are reportedIndeed since the advent of antibiotics manymicroorganismssuch as E coli have developed various strategies to thwart thepotential of action of these molecules [10] The most advisedantibiotic against pathogenic E coli strains is beta-lactamsThe most advised antibiotic against pathogenic E coli strainsis beta-lactams (cephemes monobactames carbapenemsetc) [11] The resistance to beta-lactams is thus the mainresistance mechanism among pathogenic E coli strains Thephenomenon is reported to be increased because of broadspectrum antibiotics uses in medicine and in agriculture [12]

Unfortunately in Cotonou (Benin) no data is availablein regard to the microbiological quality of market gardenproduct and the water used in its watering So this study isaimed at filling this gap by the molecular characterization ofextended-spectrum 120573-lactamases E coli strains isolated fromboth market garden products and irrigation water collectedin Cotonou

2 Material and Methods

21 Samples Collections for Market Garden Products Fourkinds of market garden products (lettuce cabbage greatnightshades and carrots)were collected from4 truck farmingsites (Figure 1) (Fidjrosse Jacquot Barrier Asecna Akpakparifle-range and ONIP Cadjehoun) in Cotonou Benin Thefour sites were selected after a prior investigation Per farmingsite three gardeners were randomly selected among those

who grow the four targeted products and two samples of eachproduct were collected per gardener The samples were col-lected both in dry season (January 2013-February 2013) andin rainy season (October 2013-November 2013) During thedry season a total of 82 samples (22 lettuces 22 cabbages 22great nightshades and 16 carrots) were collected because thecarrots samples that would be collected at ONIP Cadjehounwere not found During the rainy season a total of 84 samples(22 lettuces 16 cabbages 22 great nightshades and 22 carrots)were collected because the cabbages samples that would becollected at ONIP Cadjehoun were missed All the sampleswere collected in sterile Stomacher papers and then carried tolaboratory in icebox at about 4∘C

22 Samples Collections for Irrigation Water From the foursites listed above three kinds of irrigationwater (wells pondsand drilling) were collected for the irrigationThe water sam-ples were only collected in dry season (January 2013-February2013) Per farming site three gardeners were randomlyselected among those who grow the three targeted irrigationwater samples and two samples of each water were collectedper gardener Then a total of 22 irrigation water samples (8wells 6 ponds and 8 drilling sites) were collected because thepond water samples that would be collected at Akpakpa rifle-range were missed All the samples were collected in sterileStomacher papers and then carried to laboratory in icebox atabout 4∘C

23 Samples Microbiological Analysis

231 Market Garden Products Once carried at the labo-ratory ten mL of each sample was aseptically poured intosterile bottles and diluted serially in distilled water up toa 10minus5 dilution Then 1mL of each tube dilutions 10minus4 and10minus5 was mixed with 15mL of Plate Count Agar (sim45∘C) andpoured in sterile Petri dishes After complete solidification asecond stratum (sim4mL) of the agar was added before beingincubated at 30∘C for 24 hours The colonies that grew werecounted per dish (30 to 300 colonies) and the values areexpressed as colony forming units (CFU)mL

232 Irrigation Water Once carried at the laboratory themicrobial analysis was done using the filter membranetechnique After having filtered the water samples through a045 120583m filter the filter was deposited on Plate Count Agarin Petri dish and incubated at 37∘C for 24 h The coloniesthat grew were counted per dish (30 to 300 colonies) and thevalues are expressed as colony forming units (CFU)mLusingthe formula cited above

24 Characterization and Identification of E coli Strains

241 Market Garden Products These strains were identifiedon Rapidrsquo E coli medium according to BRD 0701-0793standard Briefly 1mL of suspension of each tube dilutions10minus4 and 10minus5 has been paid aseptically in Petri dishes sterileThe melted and cooled (45∘C) Rapidrsquo E coli medium wasadded to the inoculum (sim15mL per box) Then the mixturewas homogenized before being left for solidification and

BioMed Research International 3

AkpakpaFidjross

Cadjehoun

Houeyiho

Cotonou

Cotonou 12

Cotonou 10

Cotonou 11

Cotonou 13

Cotonou 6Cotonou 3

Cotonou 2

Cotonou 1

Cotonou 9

Cotonou 5

Cotonou 7

Cotonou 8

Cotonou 4

Abom

ey-C

alav

i

N

S

W E

Agu gu s

6 03

Realisation LaCartoDGATFLASHUAC 2015Source IGN topographical background 1992

Market gardener siteDistrict capitalBorough border

Department borderPrincipal roadAtlantic Ocean

Niger

Nigeria Togo

Burkina Faso

Atlantic Ocean

6∘229984000998400998400N

6∘2599840030998400998400N

6∘229984000998400998400N

6∘2599840030998400998400N

2∘24

99840030

998400998400E 2∘28

9984000998400998400E

2∘24

99840030

998400998400E 2∘28

9984000998400998400E

(km)

S em e

e

e eSocirc-Ava

Figure 1 Overview of the garden sites

incubated at 44∘C for 24 h During the enumeration thepurple colonies with diameter le05mm are characteristicsof E coli producing beta-D-glucuronidase and beta-D-galactosidase and blue colonies of diameter le05mm arecharacteristics of E coli producing only 120573-D-galactosidaseThe number of seeds per gram of products analyzed is deter-mined by calculation taking into account the dilution factorThe research of E coli is completed by the indole productiontest [17]

242 Irrigation Water These germs are counted on themiddle of triphenyl tetrazolium chloride (TTC) Tergitol 7according to the standard BRD 0701-0793 A volume of100mL of water is filtered on the membrane filter this lastone is deposited on the middle of TTC that previously sankin Petri dishes after having beenmelted and cooled in a waterbath at 45∘C at a rate of 15mL per box The boxes are thenincubated in an oven at 44∘C for 24 h to 48 h The seeds of Ecoli appear in colonies of diameter le05mm and are yellowishwith yellow haloThe alleged settlements are seeded on 15mLof themedium EMB that sank in Petri dish after beingmeltedand cooled in a water bath at 45∘C The boxes are thenincubated at 44∘C for 24 h The purple colonies of diametergreaterle05mmare characteristics ofE coli on the agar EMB

25 Antibiotics Susceptibility Testing E coli Isolates Theantibiotics susceptibility pattern of the isolates was deter-mined using the disk diffusion method on Mueller-Hintonagar (Oxoid England) Inhibition zone diameter values wereinterpreted as recommended by the AntibiogramCommitteeof the French Society of Microbiology [11] The 13 antibi-otics (BioMerieux France) used in this study are amoxi-cillinclavulanic acid (2010 120583g) cefotaxime (30 120583g) ceftriax-one (30 120583g) amoxicillin (30 120583g) imipenem (10120583g) gentam-icin (10 120583g) tobramycin (10 120583g) amikacin (30120583g) kanamycin(30 120583g) nalidixic acid (30 120583g) ofloxacin (5 120583g) ciprofloxacin(5 120583g) and trimethoprimsulfamethoxazole (1252375 120583g)

26 Phenotypic Detection of the Penicillinase Theproductionof penicillinase by the isolated E coli strains was performedby tube acidimetric method [18] Benzyl-penicillin (600mg)was diluted in 400 120583L of distilled water before adding 300 120583Lof aqueous phenol red solution (1 wv) The pH of the pre-pared benzylpenicillin solution was then adjusted to 8 with1M NaOHThe final 1mL reaction volume was composed ofsuspension of two young isolated E coli colonies and about150 120583L of benzylpenicillin solution The E coli ATCC 25922strains were used as a control A yellow or orange colourwithin one hour at 37∘C indicates penicillinase activity


Table 1 Primers used to search genes in this study

Target genes Primers Primers sequences (51015840 rarr 31015840) Amplicon size (bp) Reference

E colimultirestant blaTEMOT-FOT-R

51015840-TTGGGTGCACGAGTGGGTTA-3101584051015840-TAATTGTTGCCGGGAAGCTA-31015840 467 [13]

E colimultirestant blaSHVSHV-FSHV-R

51015840-CGCCGGGTTATTCTTATTTGTCGC-3101584051015840-TCTTTCCGATGCCGCCGCCAGTCA-31015840 1017 [14]

E colimultirestant blaCTX-MCTX-FCTX-R

51015840-CGCTTTGCGATGTGCAG-3101584051015840-ACCGCGATATCGTTGGT-31015840 550 [13]

EHEC VT VT-FVT-R

51015840-GAGCGAAATAATTTATATGTG-3101584051015840-TGATGATGGCAATTCAGTAT-31015840 518 [15]

STEC SLTI (Stx1) SLTI-FSLTI-R

51015840-GAAGAGTCCGTGGGATTACG-3101584051015840-AGCGATGCAGCTATTAATAA-31015840 150 [16]

STEC SLTII (Stx2) SLTII-FSLTII-R

51015840-TTAACCACACCCACGGCAGT-3101584051015840-GCTCTGCATGCATCTCTGGT-31015840 255 [16]

ETEC LT LT-FLT-R

51015840-GCGACAAATTATACCGTGCT-3101584051015840-CCGAATTCTGTTATATATGT-31015840 315 [16]

VT verotoxin EHEC enterohemorrhagic E coli LT heat-labile enterotoxin ETEC enterotoxigenic E coli SLTI Shiga-like toxin I SLTII Shiga-like toxin IIand STEC Shiga toxin E coli

27 Phenotypic Detection of Extended-Spectrum Beta-Lacta-mase (ES120573L) The phenotypic detection of ESBL on the iso-lated E coli strains was performed by double disk synergy test[19 20] In this test the tested strains (106 bacteriamL) wereflooded onto Mueller-Hinton according to the AntibiogramCommittee of the French Society of Microbiology [11] Thetest was performed with amoxicillin + clavulanic acid discand the third-generation cephalosporins namely cefotaxime(30 120583g) and ceftriaxone (30 120583g) The amoxicillin + clavulanicacid disc was placed at the center of the inoculated Mueller-Hinton agar petri dish whereas the cefotaxime (30 120583g) andceftriaxone (30 120583g) discs were placed at both sides (about 15to 20mm) of the amoxicillin + clavulanic acid disc After 18 hincubation at 37∘C the enhancement of the zones of inhibi-tion of any of the cephalosporin discs towards the clavulanicacid disc confirms the strains as an ESBL producer [21]

28 Detection of Resistance and Toxin Genes PolymeraseChain Reactions (PCR) were performed on total DNA of allconfirmed ESBL producer E coli to detect genes encodingmultidrug resistance (TEM SHV andCTX-M) and some vir-ulence factors namely VT (verotoxin) that encode enterohe-morrhagic E coli (VTEC) LT (heat-labile enterotoxin) whichencodes enterotoxigenic E coli (ETEC) and SLTI (Shiga-liketoxin I) and SLTII (Shiga-like toxin II) which encode Shigatoxin E coli (STEC) The DNA template was extracted bysuspending a loop of E coli colony in 500120583L sterile purewater and boiling for 10min at 95∘CThe suspensionwas thencentrifuged for 5min at 12000 rpm and 10120583L of the super-natant was used as target DNA DNA extracts were stored atminus20∘C until used

The primers for blaTEM blaSHV and blaCTX-M were usedfor multidrug resistance gene investigation by PCR amplifi-cation in 30 120583L containing for each 5 120583L of DNA 05 120583M ofeach primer (F and R) 15mM MgCl

2 250120583M dNTPs 1x

PCR buffer (Invitrogen) and 1U TaqDNApolymerase (Invit-rogen)The PCR program used for amplification consisted ofthe following (i) blaTEM (initial denaturation 94∘C for 5min

followed by 30 cycles 94∘C for 30 s 52∘C for 30 s and 72∘C for1min and a final elongation step 10min at 72∘C) (ii) blaSHV(initial denaturation was performed at 96∘C for 5min 30cycles of 96∘C for 15 s 50∘C for 15 s and 72∘C for 1min and afinal elongation step 10min at 72∘C) and (iii) blaCTX-M (initialdenaturation was performed at 95∘C for 5min 35 cycles of94∘C for 1min 54∘C for 1min and 72∘C for 2min and afinal elongation step 10min at 72∘C) Four genes encodingvirulence factors (VT SLTI SLTII and LT) were searchedin 25mL containing 75 120583L of DNA 125 120583L commercial 2xMaster Mix Polymerase (BioLabs) and 05 120583L of each primer(F andR) at 02 120583molLThePCRprogramused for amplifica-tion was 5min at 95∘C of initial denaturation followed by 40cycles of 45 s at 95∘C 45 s at 50∘C and 45 s at 72∘C and 10minat 72∘C for final extension One control positive for Stx2and LT was used The primers sequences and the expectedfragments are presented in Table 1

PCR products (10 120583L) were visualized after electrophore-sis at 150V for 30min on a 15 agarose gel containing ethid-ium bromide and visualized with UV transillumination A100 bp ladder standard was used as molecular weight ladder

29 Data Analysis The software Microsoft Office Excel 2010was used for processing of the data The software Epi Info6 version 604 cfr January 1999 has helped to make the testof Chi-squareThe test is considered statistically significant if119901 lt 005

3 Results

31 Environment of Market Gardening Sites The observationof gardening areas environment displays that the same sitesare cultivated by several market gardeners Among thosegardeners some of them live inside the sites in huts with theirfamily Considering themarket garden products we note thatonce collected they are sold to wholesaler but not directly tofinal consumers After harvesting the different garden prod-ucts are primarily washed with pond or residual waterobserved neighborhood of the gardening areas


Table 2 Total mesophilic count and E coli count of market gardenproducts and irrigation water collected in Cotonou

Samples FMT (CFUg) E coli (CFUg)Horticultural products

Lettuce 26 lowast 106 065 lowast 106

Carrot 25 lowast 106 044 lowast 106

Great nightshade 23 lowast 106 052 lowast 106

Cabbage 24 lowast 106 054 lowast 106

Water of wateringPool 15 lowast 106 0062 lowast 106

Well 25 lowast 106 083 lowast 106

Drilling 18 lowast 106 066 lowast 106

32 Total Mesophilic Microbial Flora (FMT) and E coliCount of Market Garden Products and Irrigation Water Themicrobiological analysis focused on the total aerobic countand E coli count from samples revealed a contaminationrate variation accordingly (Table 2) Considering the marketgarden products our data displays that the lettuce was thehighest microbial load (26lowast 106 CFUg) followed by the car-rots (25 lowast 106 CFUg) For the irrigation water we recordedthat the water of wells was the most contaminated one (25 lowast106) whereas the lowest colonies count was observed with thepool water (15lowast106 CFUg)The same trends were observedin the enumeration ofE coli (Table 2)Thus 065lowast106 CFUofE coliwere counted per gram of samples of lettuceThe waterof pool was the least contaminated samples tested (0062 lowast106 CFUg)

33 Identification of E coli Strains Among the 186 collectedsamples (market garden products and irrigation water) 65were contained by E coli strains It appears that the marketgarden products were most contaminated compared to theirrigation water (Figure 2(a)) The market garden productsglobal contamination patterns display high decreases fromdry season (7077) to the rainy season (20) (119901 lt 000001)

Considering the market garden products their E colicontamination rate varies not only according to the kind ofproducts (lettuce carrots cabbage and great nightshades)but also according to the seasons (Figure 1) Indeed our datashows that among the 46 E coli strains isolated during thedry season those of lettuce were the most contaminated ones(2609) followed successively by great nightshade (2391)cabbage (2826) and then carrots (2174) (Figure 2(b))The difference was not statistically significant (119901 gt 005)During the rainy season six E coli strains were isolatedfrom the 84 collected samples Three of the six (50)strains were isolated from great nightshade samples two(3333) were isolated from lettuce and one (1667) wasisolated from carrots

For the irrigation water out of the 22 collected samples13 of E coli strains were isolated Three (2308) strains wereisolated from the pool water samples whereas 5 (3846)strains were both isolated from drilling and wells water(Figure 2(c))

Focusing on the sampling site we denote a variationof contamination rate from a collection site to another(Figure 3) Thus it appears that market garden samplescollected at Fidjrosse Jacquot during the rainy season werethe most contaminated ones (119901 lt 005) During the rainyseason the sites of ONIP Cadjehoun and Akpakpa rifle-rangewere free of E coli contamination But during the dry seasonthe entire collection site harbors the E coli contamination atdifferent level (Figure 2(a)) Considering the irrigation waterFigure 2(b) shows that those collected at Barrier Asecna weremore contaminated (1016) than the ones collected in thethree other sites (119901 lt 005)

34 Susceptibility of Isolated E coli Strains to AntibioticsThe susceptibility of the 65 E coli strains isolated from oursamples varies from an antibiotic to another (119901 lt 005)Thus it was observed that all strains were resistant to amox-icillin Four of the tested antibiotics (amikacin ciprofloxacinimipenem and kanamycin) display a resistance rate under50 whereas the height remaining antibiotics (gentam-icin tobramycin nalidixic acid ofloxacin trimethoprimsul-famethoxazole amoxicillin clavulanic acid cefotaxime andceftriaxone) show their efficiency to less than 40 of thetested strains (Figure 4)

35 Phenotypic Detection of the Penicillinase and ESBLsThe phenotypical investigation shows that 6769 of the 65isolated E coli strains produced penicillinase The 44 strainsproducing penicillinase were composed of all strains of Ecoli isolated from bothmarket garden products and irrigationwater (Figure 5) No strain of E coli isolated was producing120573-lactamase-producing plasmid to expanded-spectrum 120573-lactamases (also ESBLs)

36 Genomic Detection of the Penicillinase TheDNAof thosestrains was used to seek the presence of the blaTEM blaSHVand blaCTX-M genes The compilation of this investigation(Figure 5) displays that 6750 of the tested strains carriedthe blaTEM genes 10 carried the blaSHV and 2250 carriedthe blaCTX-M (Table 3) The distribution of the E coli strainsconsiderably varies according to the season (119901 lt 00005)

37 Detection of Toxins Genes The search of 4 toxins pro-duced by pathogenic E coli strains revealed that third oftested strains carried Shiga toxin 1 (3571) and Shiga toxin 2(3571) while 715 carried LT gene and 2143 carried VTgene (Table 4)

4 Discussion

The observation of the environment around the sites used forgardening revealed that several market gardeners live withtheir families on the sites There was not latrine in all theinvestigated sites Thus to relieve themselves they may havesome unhealthy behaviorsThus to support this idea Kifuani[22] found in a study led in Congo that the garden sites closeto human houses are much more exposed to the microor-ganisms (viruses protozoa nematodes etc) contaminationrisks of market garden products and their irrigation water


Table 3 Distribution of the penicillinase genes carried by strains of E coli according to the season

Dry season Rainy season TotalMarket garden products Water of watering Market garden products

blaTEM 4250 20 5 6750blaSHV 10 0 0 10blaCTX-M 20 0 250 2250Total 7250 20 750 100

7077

20

923

00

100

200

300

400

500

600

700

800

Market garden product Irrigation water

()

Dry seasonRainy season

(a)

26092174

23912826

3333

1667

5000

00000

100

200

300

400

500

600

Lettuce Carrot GreatNightshade

Cabbage

()


(b)

2308

3846 3846

00

100

200

300

400

500

Pool water Drilling water Well water

()

(c)

Figure 2 E coli contamination rates of collected samples according to the season and the kind of samples (a) Global contamination by Ecoli (b) Market garden products (c) Irrigation water


Table 4 Distribution of the toxins genes carried by strains of E coli according to the season


STEC (Stx1) 2143 0 1428 3571STEC (Stx2) 2143 0 1428 3571ETEC 715 0 0 715VTEC 0 1428 715 2143Total 50 1428 3571 100VT verotoxin VTEC enterohemorrhagic E coli LT heat-labile enterotoxin ETEC enterotoxigenic E coli Stx1 Shiga-like toxin I Stx2 Shiga-like toxin IIand STEC Shiga toxin E coli

1186 16942203 2711

000

1667

8333

000

100200300400500600700800900

Akp

akpa

rifl

e-ra

nge

Prop

ortio

n (

)

Sites of samples collection


Barr

ier

Ase

cna

ON

IP

Cadj

ehou

n

Jacq

uot

Fidj

ross

e

(a)


169

1016

508 508

000

200

400

600

800

1000

1200

Prop

ortio

n (

)

Akp

akpa

rifl

e-ra

nge

Barr

ier

Ase

cna

ON

IP

Cadj

ehou

n

Jacq

uot

Fidj

ross

e

(b)

Figure 3 E coli contamination rate of collected samples according to the site of samples collection (a)Market garden products (b) Irrigationwater

75

92

71

100

6065

23

4835

69

38

7160

CTX

AM

C

CRO

AM

X

GM TM AN K

CIP

OFX IPM NA

SXT

Resis

tanc

e (

)

Antibiotics

0

20

40

60

80

100

120

Figure 4 Resistance profile of the isolated E coli strains to 13antibiotics amoxicillin (AMX) amoxicillinclavulanic acid (AMC)cefotaxime (CTX) ceftriaxone (CRO) imipenem (IPM) gentam-icin (GM) tobramycin (TM) amikacin (AN) kanamycin (K)nalidixic acid (NA) ofloxacin (OFX) ciprofloxacin (CIP) andtrimethoprimsulfamethoxazole (SXT)

Indeed the microbiological quality focusing on the totalmesophilic flora of the garden product and irrigation watercollected in Cotonou is bad (Table 2) This high rate of

5682

2955

1364

00

100

200

300

400

500

600

Market garden products Irrigation water

Prop

ortio

n of

pro

duct

ion

()

Kind of samples


Figure 5 Phenotypical detection of penicillinase producer E colistrains

contamination by bacteria may be explained by the fact thatthe farming practices adopted by the market gardeners favorpermanent fecal contamination of irrigation water eitherdirectly (defecation) or indirectly through the streamingwater Such irrigation water is a reservoir of many cultures


pathogenicmicroorganisms such asE coli SalmonellaVibriocholerae and Shigella [23] Also the risk of contaminationincreases by the fact that most of the gardeners use chickendroppings as soil fertilizer [24]

However 3495 of the collected samples were con-taminated by E coli strains This proportion is lower thanthe 66 of E coli strains isolated from human infectionsreported in the United States by Griffin [25] Indeed itwas established that E coli strains were more frequentlyisolated from vegetables and fruits than Salmonella [26] Thepresence of Enterobacteriaceae may be due to poor hygieneconditions in which vegetables are usually grown [27] Thusthe inappropriate consumption of vegetables can be a sourceof water diseases (cholera bacillary dysentery etc) In dryseason the samples of market garden products are more con-taminated (7077) by E coli than those collected in the rainyseason (923) (119901 lt 000001) These results suggest that theirrigation water may play a major role in the microbiologicalquality of vegetables [28] Because in the rainy season gardenproduce was directly watered by rain the rain may clean themarket garden products and then increase the microbiologi-cal quality of leafy productsThese results are similar to thoseobtained by Koffi-Nevry et al [29] during their study on fecalcontamination of irrigation water

Among the samples of irrigation water those collectedfromwell (3846) and drilling (3846) weremore contam-inated than poolrsquos ones (2308) (Figure 2(c))This differencein proportion can be explained by the fact that the irrigationwater is contaminated by sewage therefore subject to thepresence of microorganisms [30] In addition the wells werethe openwells andmost well water is collected in tanks also inhalf-open tanks This observation was also made in Abidjanby Koffi-Nevry et al [29]

The E coli strains were isolated from all the sampledmarket garden products (lettuce carrot cabbage and greatnightshade) These results are similar to those reported onseveral market garden products such as cabbage carrots let-tuce tomato and pepper in Philippines [31] The presence ofE coli in these samples confirms the close link between food-borne infections associatedwith the consumption of crudities[32] Thus plants and leafy variety are universally suspectedas transmission way of food infections [3] Among the gardenproducts our results display that carrot samples were weaklycontaminated independently of the seasonThe low contami-nation rate of carrot samples can be explained by the fact thattheir edible parts are less exposed to direct contact of waterduring watering However this result seems contrary to thatobtained by Vital et al [31] when they found that the carrotwas most contaminated product In fact Vital et al [31] intheir study used samples of fresh produce from open airmarkets and supermarkets As vegetable leaves represent anappropriate surface of bacteria growth our data shows thatlettuce and great nightshade were highly contaminated byE coli strains both in dry and in rainy seasons Pathogenicbacteria are reported to survive on fresh vegetables for about30 days [33] We can then say that market garden leaves suchas lettuce are the most contaminated produce of gardens asalready reported in Cote drsquoIvoire [29] and in Senegal [30]

Considering gardener sites the market garden samplescollected from Akpakpa rifle-range were more contaminatedwith E coli in the dry season Among the investigated sitesthis site was unprotected and therefore is open to any typeof movements including those of animals such as cattle forslaughtering in the same area In addition most of the hous-ing near the site does not have latrines so the site is currentlyused for defecation Thus the different manure (animal andhuman)may be the possible source of the high contaminationlevel observed through streaming water [23] In rainy seasonthe site closer to the beach Fidjrosse Jacquot was morecontaminated whereas the site of ONIP was the less contam-inated site independently of the season

The results of antibiotic susceptibility of E coli strainsshow that they were resistant to the majority of the 13 testedantibiotics at varying proportionsThus all the isolated E colistrains were resistant to amoxicillin and 92 of those strainswere resistant to the association of amoxicillin and clavulanicacid These proportions are slightly higher than the 952(amoxicillin) and 857 (amoxicillin and clavulanic acid)reported by Anago et al [34] in BeninThese rates are higherthan those obtained by some authors in developed countriesthey observed resistance to amoxicillin oscillating between42 [35] and 203 [36] The high difference observedbetween developing countries and the case of Benin may bedue to the fact that in developing countries the sale andadministration of antibiotics are regulated Thus the propor-tion observed in general in our study for this antibiotic wouldfind its explanation in a misuse of the antibiotic often sold onthe street without a medical prescription [37] This situationis observed not only in Benin but in most of the developingcountries such as Senegal [38] But we should also notethat selection pressure is exerted in both the medical fieldand agricultural field [39] For the other antibiotics such asceftriaxone higher resistance strains rates (71) comparedto those recorded in several studies conducted elsewhere inAfrica were observed [34 40] The strong resistance propor-tion of the tested germs to antibiotics observed in this studyalso confirms the increase of resistance in E coli

It was noticed that the majority of E coli (6769) strainsproduce penicillinase Among those strains 6750 10 and2250 harbor respectively blaTEM blaSHV and blaCTX-Mgenes The presence of genes blaTEM blaSHV and blaCTX-Mconfirms the resistance of E coli to the 120573-lactam antibioticsin this study Indeed the resistance to 120573-lactam antibioticssignificantly increases over the two decades and the presenceof blaTEM gene is due to the resistance to third-generationantibiotics that cause secretion of 120573-lactamases [41] Noneof the isolated strains produce expanded-spectrum 120573-lactamases (ESBLs) There were few strains isolated fromfood sector that produce ESBLs [42] But in clinical areahigh rates of resistance were reported all over the world [34]Originally the group CTX-M conferred for Enterobacteri-aceae a highest level of resistance for cefotaxime cefepimeaztreonam and ceftazidime [43] In specialty hospital at India[44] 14 of blaSHV and 50 of blaCTX-M were found by Ecoli ESBLs FromUniversity Hospital in Turkey blaCTX-M wascarried by 6097 of E coli ESBLs [45] These rates werehigher than the rate obtained in this study Thus origin of


sample collectionmay play an important role in the resistanceprofile of given strains Indeed clinical isolated strains aremore in contact with the antibiotics than the food isolated

The enterohemorrhagic Escherichia coli (EHEC STECand VTEC) are responsible for various infections rangingfrom watery diarrhea to hemorrhagic colitis which canprogress to hemolytic uremic syndrome in young childrenor thrombotic microangiopathy in adults [46] Moreoverstrains STEC and ETEC are responsible for severe diarrheain humans and animals [47] In this study the isolated E colistrainswere secreting Shiga toxin at levels of 3571 (Stx1) and3571 (Stx2) (Table 4) In the dry season STEC were foundto be Stx2 of 2143 and 2143 of Stx1 Stx2 is more virulentthan Stx1 [49] In a study by Nataro et al [47] on clinicalstrains 0 and 02 of E coli strains were respectively STECand ETEC Another study in Brazil on samples of infantdiarrhea observed rates (12) ETEC and (07) STEC [40]These types of strains are related to the production of choleratoxins [48] One study made in Brazil by Oliveira et al [49]showed that the prevalence of STEC ranged from 16 to515 0 to 50 and 467 to 733 respectively for beefcattle dairy cattle and goats depending on the farm Oliveiraet al [49] confirmed that the broad range of isolation ofSTEC observed in our study is probably associated with somefarm management practices such as the administration ofantibiotics inappropriate manipulations of diet and use ofnoncontaminated food and water A rate of 715 of isolatedE coli strains was secreting heat-labile enterotoxin therefore(ETEC) ETEC in contrast is pathogenic across all agegroups but it is most common among infants in developingcountries because immunity is acquired from repeated expo-sure [47] Irino et al [50] who conducted a study on dairycattle in Brazil assert that the presence of STEC (564 forStx1 and 406 for Stx2) may be due to food contaminationand failure water during handling by the farmer methodsThese different rates observed may be due to the diversityof the origin of strains and geographic variation [47] A rateof 833 of the strains was both STECVTEC This unusualcombination of virulence factors gives these strains a largepathogenicity [51] These various results obtained in thisstudy show that the consumption of garden produce raw orlittle cooked is very dangerous to humans

5 Conclusion

The food poisonings are a subject of growing concern in thefield of public health This study was designed to bring newknowledge to the biochemical and molecular characteristicsof E coli strains isolated from irrigation water and marketgarden products sold in Cotonou Benin

This study has allowed us to determine the biochemicalandmolecular characters of E coli strainsThus the microbi-ological quality of market garden products collected duringthe rainy season is better than those collected during thedry season The irrigation water plays an important role inthe contamination of market garden products Most of thestrains isolated produce penicillinase An annual inspectioncommittee is appropriate for the quality microbiology of themarket garden products and water of watering on the sites

Conflict of Interests

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

Acknowledgment

Thiswork was supported byGrant E4332-2F of InternationalFoundation of Science (IFS)

References

[1] O Cisse N F D Gueye and M Sy ldquoInstitutional andlegal aspects of urban agriculture in French-speaking WestAfrica from marginalization to legitimizationrdquo Environmentand Urbanization vol 17 no 2 pp 143ndash154 2005

[2] M O Ofor V C Okorie I I Ibeawuchi G O Ihejirika OP Obilo and S A Dialoke ldquoMicrobial contaminants in freshtomato wash water and food safety considerations in South-Eastern Nigeriardquo Life Sciences vol 1 pp 80ndash82 2009

[3] L R Beuchat ldquoVectors and conditions for preharvest contami-nation of fruits and vegetables with pathogens capable of caus-ing enteric diseasesrdquo British Food Journal vol 108 no 1 pp 38ndash53 2006

[4] C N Berger R K Shaw D J Brown et al ldquoInteraction ofSalmonella entericawith basil and other salad leavesrdquoThe ISMEJournal vol 3 no 2 pp 261ndash265 2009

[5] A M Sewell and J M Farber ldquoFoodborne outbreaks in Canadalinked to producerdquo Journal of Food Protection vol 64 no 11 pp1863ndash1877 2001

[6] S Sivapalasingam C R Friedman L Cohen and R V TauxeldquoFresh produce a growing cause of outbreaks of foodborneillness in the United States 1973 through 1997rdquo Journal of FoodProtection vol 67 no 10 pp 2342ndash2353 2004

[7] J C Heaton and K Jones ldquoMicrobial contamination of fruitand vegetables and the behaviour of enteropathogens in thephyllosphere a reviewrdquo Journal of Applied Microbiology vol104 no 3 pp 613ndash626 2008

[8] C Vernozy-Rozand J Flandrois V Livrelli et al ldquoEscherichiacoli entero-hemorragique (EHEC)O157H7rdquoAide-memoire 157Centre des medias Paris France 2001

[9] F A Clifton-Hadley ldquoDetection and diagnosis of Escherichiacoli O157 and other verocytotoxigenic E coli in animal faecesrdquoReviews in Medical Microbiology vol 11 no 1 pp 47ndash58 2000

[10] P De Wals Analyses Mutationnelles et Cinetiques de la 120573-Lactamase TEM-1 de Escherichia coli Vers une MeilleureComprehension du Phenomene de Resistance aux AntibiotiquesFaculte de Medecine 2007

[11] Comite de LrsquoAntibiogramme de la Societe Francaise le Microbi-ologie (CASFM) and C J Coordonnateur Soussy ldquoRecomman-dations 2012 Edition Janvier 2012rdquo p 56 2012

[12] D L Paterson and R A Bonomo ldquoExtended-spectrum 120573-lactamases a clinical updaterdquo Clinical Microbiology Reviewsvol 18 no 4 pp 657ndash686 2005

[13] J Gangoue-Pieboji B Bedenic S Koulla-Shiro et alldquoExtended-spectrum-120573-lactamase-producing Enterobacteria-ceae in Yaounde Cameroonrdquo Journal of Clinical Microbiologyvol 43 no 7 pp 3273ndash3277 2005

[14] M T Nuesch-Inderbinen H Hachler and F H KayserldquoDetection of genes coding for extended-spectrum SHV beta-lactamases in clinical isolates by a molecular genetic method


and comparison with the E testrdquo European Journal of ClinicalMicrobiology amp Infectious Diseases vol 15 no 5 pp 398ndash4021996

[15] K R S Aranda S H Fabbricotti U Fagundes-Neto and IC A Scaletsky ldquoSingle multiplex assay to identify simulta-neously enteropathogenic enteroaggregative enterotoxigenicenteroinvasive and Shiga toxin-producing Escherichia colistrains in Brazilian childrenrdquo FEMS Microbiology Letters vol267 no 2 pp 145ndash150 2007

[16] A Gassama-Sow P S SowM Gueye et al ldquoCharacterization ofpathogenic Escherichia coli in human immunodeficiency virusndashrelated diarrhea in Senegalrdquo Journal of Infectious Diseases vol189 no 1 pp 75ndash78 2004

[17] P Riegel M Archambaud D Clave andM Vergnaud Bacteriede Culture et drsquoIdentification Difficiles Editions BioMerieux2006

[18] E Koneman ldquoTest for determining inhibitoryrdquo in KonemanrsquosColor Atlas and Textbook of Diagnostic Microbiology LippincottWilliams ampWilkins 5th edition 2006

[19] V Jarlier M H Nicolas G Fournier and A PhilipponldquoExtended broad-spectrum 120573-lactamases conferring transfer-able resistance to newer beta-lactam agents in Enterobacteri-aceae hospital prevalence and susceptibility patternsrdquo Reviewsof Infectious Diseases vol 10 no 4 pp 867ndash878 1988

[20] K S Thomson and C C Sanders ldquoDetection of extended-spectrum 120573-lactamases in members of the family Enterobacte-riaceae comparison of the double-disk and three-dimensionaltestsrdquoAntimicrobial Agents and Chemotherapy vol 36 no 9 pp1877ndash1882 1992

[21] P Allouch R Labia P Pina and E Morin ldquoObservatoireshospitaliers de la sensibilite drsquoEscherichia coli et de Klebsiellapneumoniae a lrsquoassociation AmoxicillineAcide clavulanique en1994rdquoMedecine etMaladies Infectieuses vol 25 no 8-9 pp 934ndash939 1995

[22] K Kifuani Etude de la Qualite des Eaux des Effluents drsquoEauxUsees Domestiques UNIKIN Kinshasa Congo 2004

[23] R Koffi-Nevry B Assi-Clair M Koussemon A Wognin andN Coulibaly ldquoPotential Enterobacteria risk factors associatedwith contamination of lettuce (Lactuca sativa) grown in theperi-urban area of Abidjan (Cote drsquoIvoire)rdquo International Jour-nal of Biological and Chemical Sciences vol 5 no 1 pp 279ndash2902011

[24] S Petterson N Ashbolt and A Sharma ldquoMicrobial risks fromwaste water irrigation of salad crops a screening-level riskassessmentrdquo Journal of Food Science vol 75 no 5 pp M283ndashM290 2010

[25] P M Griffin ldquoEpidemiology of Shiga toxin-producing Escher-ichia coli infections in humans in the United Statesrdquo in Escher-ichia coli O157H7 and Other Shiga Toxi-Producing E coliStrains J B Kaper and A D OrsquoBrien Eds vol 5 pp 15ndash22American Society for Microbiology Washington DC USA1998

[26] ECSCF ldquoRisk profile on the microbiological contaminationof fruits and vegetables eaten rawrdquo Report of the EuropeanCommission Scientific Committee on Food ECSCF 2002

[27] P Amoah P Drechsel M Henseler and R C Abaidoo ldquoIrri-gated urban vegetable production in Ghana microbiologicalcontamination in farms and markets and associated consumerrisk groupsrdquo Journal of Water and Health vol 5 no 3 pp 455ndash466 2007

[28] SW Stine I Song C Y Choi andC PGerba ldquoEffect of relativehumidity on preharvest survival of bacterial and viral pathogens

on the surface of cantaloupe lettuce and bell peppersrdquo Journalof Food Protection vol 68 no 7 pp 1352ndash1358 2005

[29] R Koffi-Nevry J Assi-Clair E Assemand et al ldquoOrigine destemoins de contamination fecale de lrsquoeau drsquoarrosage de la laitue(lactuca sativa) cultivee dans la zone peri urbaine drsquoAbidjanrdquoJournal of Applied Biosciences vol 52 pp 3669ndash3675 2012

[30] M Ndiaye Impacts sanitaires des eaux drsquoarrosage de lrsquoagricultureurbaine de Dakar (Senegal) [These de Doctorat] Universite deGenese et de Lausanne 2009

[31] P G Vital K G B Dimasuay K W Widmer andW L RiveraldquoMicrobiological quality of fresh produce fromopen airmarketsand supermarkets in the Philippinesrdquo The Scientific WorldJournal vol 2014 Article ID 219534 7 pages 2014

[32] D A Rasko D R Webster J W Sahl et al ldquoOrigins of the Ecoli strain causing an outbreak of hemolytic-uremic syndromein GermanyrdquoTheNew England Journal of Medicine vol 365 no8 pp 709ndash717 2011

[33] M PescodWastewater Treatment and Use in Agriculture Foodand Agriculture Organization Rome Italy 1992

[34] E Anago L Ayi-Fanou C D Akpovi et al ldquoAntibiotic resis-tance and genotype of beta-lactamase producing Escherichiacoli in nosocomial infections in Cotonou Beninrdquo Annals ofClinical Microbiology and Antimicrobials vol 14 article 5 2015

[35] M Lemort S NeuvilleMMedus et al ldquoEvolution comparee dela sensibilite de souches de Escherichia coli isolees drsquoinfectionsurinaires de patients consultant aux urgences et de patientshospitalises en 2002 et 2004 a lrsquohopital Perpignanrdquo PathologieBiologie vol 54 no 8-9 pp 427ndash430 2006

[36] A Sotto C M De Boever P Fabbro-Peray A Gouby D Sirotand J Jourdan ldquoRisk factors for antibiotic-resistant Escherichiacoli isolated from hospitalized patients with urinary tractinfections a prospective studyrdquo Journal of ClinicalMicrobiologyvol 39 no 2 pp 438ndash444 2001

[37] D Guillemot and P Courvalin ldquoBetter control of antibioticresistancerdquo Clinical Infectious Diseases vol 33 no 4 pp 542ndash547 2001

[38] R Seck Resistance des souches drsquoEcherichia coli et Klebsiellapneumoniae isolees drsquoinfections urinaires [These de doctorat alrsquoUniversite Cheikh Anta Diop de Dakar] 2005

[39] H Allen J Donato K Hansen et al ldquoCall of the will antibioticresistance genes in natural environmentsrdquo Nature ReviewsMicrobiology vol 8 pp 251ndash259 2010

[40] J M Araujo G F Tabarelli K R S Aranda et al ldquoTypi-cal enteroaggregative and atypical enteropathogenic types ofEscherichia coli are the most prevalent diarrhea-associatedpathotypes among Brazilian childrenrdquo Journal of ClinicalMicro-biology vol 45 no 10 pp 3396ndash3399 2007

[41] S Nijssen A Florijn M J M Bonten F J Schmitz J Verhoefand A C Fluit ldquoBeta-lactam susceptibilities and prevalenceof ESBL-producing isolates among more than 5000 EuropeanEnterobacteriaceae isolatesrdquo International Journal of Antimicro-bial Agents vol 24 no 6 pp 585ndash591 2004

[42] R J Mesa V Blanc A R Blanch et al ldquoExtended-spectrum120573-lactamase-producingEnterobacteriaceae in different environ-ments (humans food animal farms and sewage)rdquo Journal ofAntimicrobial Chemotherapy vol 58 no 1 pp 211ndash215 2006

[43] N Lagha Etude de la resistance aux antibiotiques desenterobacteries productrices de 120573-lactamases a spectre etendu(BLSE) isolees de lrsquohopital de Laghouat [These de Doctorat]Universite Abou Bekr Belkaıd Tlemcen 2015


[44] S Jemima and S Verghese ldquoMultiplex PCR for bla(CTX-M)amp bla(SHV) in the extended spectrum beta lactamase (ESBL)producing gram-negative isolatesrdquo Indian Journal of MedicalResearch vol 128 no 3 pp 313ndash317 2008

[45] C Gulamber M Altindis R Kalayci et al ldquoMolecular charac-terization of nosocomial CTX-M type 120573-Lactamase producingEnterobacteriaceae fromUniversity Hospital in TurkeyrdquoAfricanJournal of Microbiology Research vol 6 pp 5552ndash5557 2012

[46] P Mariani-Kurkdjian and S Bonacorsi ldquoDiagnostic des infec-tions a Escherichia coli entero-hemorragiquesrdquo in Feuillets deBiologie Bacteriologie EHECSTEC vol 5 p 317 2014

[47] J P Nataro V Mai J Johnson et al ldquoDiarrheagenic Escherichiacoli infection in Baltimore Maryland and New Haven Con-necticutrdquoClinical Infectious Diseases vol 43 no 4 pp 402ndash4072006

[48] R Tozzoli A Caprioli S Cappannella V Michelacci M LMarziano and S Morabito ldquoProduction of the subtilase AB

5

cytotoxin by Shiga toxin-negative Escherichia colirdquo Journal ofClinical Microbiology vol 48 no 1 pp 178ndash183 2010

[49] M G Oliveira J R F Brito T A T Gomes et al ldquoDiversityof virulence profiles of Shiga toxin-producing Escherichia coliserotypes in food-producing animals in Brazilrdquo InternationalJournal of FoodMicrobiology vol 127 no 1-2 pp 139ndash146 2008

[50] K Irino M A M F Kato T M I Vaz et al ldquoSerotypes andvirulence markers of Shiga toxin-producing Escherichia coli(STEC) isolated from dairy cattle in Sao Paulo State BrazilrdquoVeterinary Microbiology vol 105 no 1 pp 29ndash36 2005

[51] F Scheutz E Moslashller Nielsen J Frimodt-Moslashller et al ldquoChar-acteristics of the enteroaggregative Shiga toxinverotoxinpro-ducing Escherichia coli O104H4 strain causing the outbreak ofhaemolytic uraemic syndrome in Germanyrdquo Euro Surveillancevol 16 p 19889 2011

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


Indeed several cases of typhoid fever have been associatedwith eating contaminated vegetables grown inor fertilizedwith contaminated soil or sewage [3]

Vegetables consumption is encouraged worldwide bygovernmental health agencies to protect against a rangeof illnesses such as cancers and cardiovascular diseasesHowever leafy greens consumed raw are increasingly beingidentified as important cause of human pathogens that weretraditionally associated with foods andor animal origin[4] Bacteria viruses and parasites on vegetables such asmarket garden products have been linked with illness [5]But bacterial pathogens continue to be amajor contributor toproduce associated food-borne illnesses Among those bac-teria Salmonella was the most commonly reported bacterialpathogen accounting for nearly half of the outbreaks due tobacteria [6] followed by E coli Thus it is established thatenterobacteria account for more than half of the outbreaksdue to bacteria To explain this high level of contaminationthe most frequently cited factors are water soil manure andanimals [7]

Among the enterobacteria E coli is a common part ofa normal microflora of man and most of the warm-bloodedanimals digestive tube Nevertheless some E coli strains arereported to be a leading cause of extraintestinal (meningitisurinary tract infections) andor intestinal pathologies [8]Thepathogenicity of those strains can be due not only to theirinvasion proportion but also to toxins production Enzymesdisplaying human cells cytopathic effect are part of such tox-ins [9] To fight the pathogenic E coli strains many antibioticmolecules are often used worldwide But due to the highmutation capability and the worse use of such antibioticsmany cases of E coli multiresistance strains are reportedIndeed since the advent of antibiotics manymicroorganismssuch as E coli have developed various strategies to thwart thepotential of action of these molecules [10] The most advisedantibiotic against pathogenic E coli strains is beta-lactamsThe most advised antibiotic against pathogenic E coli strainsis beta-lactams (cephemes monobactames carbapenemsetc) [11] The resistance to beta-lactams is thus the mainresistance mechanism among pathogenic E coli strains Thephenomenon is reported to be increased because of broadspectrum antibiotics uses in medicine and in agriculture [12]

Unfortunately in Cotonou (Benin) no data is availablein regard to the microbiological quality of market gardenproduct and the water used in its watering So this study isaimed at filling this gap by the molecular characterization ofextended-spectrum 120573-lactamases E coli strains isolated fromboth market garden products and irrigation water collectedin Cotonou

2 Material and Methods

21 Samples Collections for Market Garden Products Fourkinds of market garden products (lettuce cabbage greatnightshades and carrots)were collected from4 truck farmingsites (Figure 1) (Fidjrosse Jacquot Barrier Asecna Akpakparifle-range and ONIP Cadjehoun) in Cotonou Benin Thefour sites were selected after a prior investigation Per farmingsite three gardeners were randomly selected among those

who grow the four targeted products and two samples of eachproduct were collected per gardener The samples were col-lected both in dry season (January 2013-February 2013) andin rainy season (October 2013-November 2013) During thedry season a total of 82 samples (22 lettuces 22 cabbages 22great nightshades and 16 carrots) were collected because thecarrots samples that would be collected at ONIP Cadjehounwere not found During the rainy season a total of 84 samples(22 lettuces 16 cabbages 22 great nightshades and 22 carrots)were collected because the cabbages samples that would becollected at ONIP Cadjehoun were missed All the sampleswere collected in sterile Stomacher papers and then carried tolaboratory in icebox at about 4∘C

22 Samples Collections for Irrigation Water From the foursites listed above three kinds of irrigationwater (wells pondsand drilling) were collected for the irrigationThe water sam-ples were only collected in dry season (January 2013-February2013) Per farming site three gardeners were randomlyselected among those who grow the three targeted irrigationwater samples and two samples of each water were collectedper gardener Then a total of 22 irrigation water samples (8wells 6 ponds and 8 drilling sites) were collected because thepond water samples that would be collected at Akpakpa rifle-range were missed All the samples were collected in sterileStomacher papers and then carried to laboratory in icebox atabout 4∘C

23 Samples Microbiological Analysis

231 Market Garden Products Once carried at the labo-ratory ten mL of each sample was aseptically poured intosterile bottles and diluted serially in distilled water up toa 10minus5 dilution Then 1mL of each tube dilutions 10minus4 and10minus5 was mixed with 15mL of Plate Count Agar (sim45∘C) andpoured in sterile Petri dishes After complete solidification asecond stratum (sim4mL) of the agar was added before beingincubated at 30∘C for 24 hours The colonies that grew werecounted per dish (30 to 300 colonies) and the values areexpressed as colony forming units (CFU)mL

232 Irrigation Water Once carried at the laboratory themicrobial analysis was done using the filter membranetechnique After having filtered the water samples through a045 120583m filter the filter was deposited on Plate Count Agarin Petri dish and incubated at 37∘C for 24 h The coloniesthat grew were counted per dish (30 to 300 colonies) and thevalues are expressed as colony forming units (CFU)mLusingthe formula cited above

24 Characterization and Identification of E coli Strains

241 Market Garden Products These strains were identifiedon Rapidrsquo E coli medium according to BRD 0701-0793standard Briefly 1mL of suspension of each tube dilutions10minus4 and 10minus5 has been paid aseptically in Petri dishes sterileThe melted and cooled (45∘C) Rapidrsquo E coli medium wasadded to the inoculum (sim15mL per box) Then the mixturewas homogenized before being left for solidification and


AkpakpaFidjross

Cadjehoun

Houeyiho

Cotonou

Cotonou 12

Cotonou 10

Cotonou 11

Cotonou 13

Cotonou 6Cotonou 3

Cotonou 2

Cotonou 1

Cotonou 9

Cotonou 5

Cotonou 7

Cotonou 8

Cotonou 4

Abom

ey-C

alav

i

N

S

W E

Agu gu s

6 03




Niger

Nigeria Togo

Burkina Faso

Atlantic Ocean

6∘229984000998400998400N

6∘2599840030998400998400N

6∘229984000998400998400N

6∘2599840030998400998400N

2∘24

99840030

998400998400E 2∘28

9984000998400998400E

2∘24

99840030

998400998400E 2∘28

9984000998400998400E

(km)

S em e

e

e eSocirc-Ava















EHEC VT VT-FVT-R






ETEC LT LT-FLT-R






2 250120583M dNTPs 1x





3 Results





















4 Discussion






7077

20

923

00

100

200

300

400

500

600

700

800


()


(a)

26092174

23912826

3333

1667

5000

00000

100

200

300

400

500

600


Cabbage

()


(b)

2308

3846 3846

00

100

200

300

400

500


()

(c)






1186 16942203 2711

000

1667

8333

000

100200300400500600700800900

Akp

akpa

rifl

e-ra

nge

Prop

ortio

n (

)



Barr

ier

Ase

cna

ON

IP

Cadj

ehou

n

Jacq

uot

Fidj

ross

e

(a)


169

1016

508 508

000

200

400

600

800

1000

1200

Prop

ortio

n (

)

Akp

akpa

rifl

e-ra

nge

Barr

ier

Ase

cna

ON

IP

Cadj

ehou

n

Jacq

uot

Fidj

ross

e

(b)


75

92

71

100

6065

23

4835

69

38

7160

CTX

AM

C

CRO

AM

X

GM TM AN K

CIP

OFX IPM NA

SXT

Resis

tanc

e (

)

Antibiotics

0

20

40

60

80

100

120



5682

2955

1364

00

100

200

300

400

500

600


Prop

ortio

n of

pro

duct

ion

()

Kind of samples















5 Conclusion





Acknowledgment


References





















































5












Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


AkpakpaFidjross

Cadjehoun

Houeyiho

Cotonou

Cotonou 12

Cotonou 10

Cotonou 11

Cotonou 13

Cotonou 6Cotonou 3

Cotonou 2

Cotonou 1

Cotonou 9

Cotonou 5

Cotonou 7

Cotonou 8

Cotonou 4

Abom

ey-C

alav

i

N

S

W E

Agu gu s

6 03




Niger

Nigeria Togo

Burkina Faso

Atlantic Ocean

6∘229984000998400998400N

6∘2599840030998400998400N

6∘229984000998400998400N

6∘2599840030998400998400N

2∘24

99840030

998400998400E 2∘28

9984000998400998400E

2∘24

99840030

998400998400E 2∘28

9984000998400998400E

(km)

S em e

e

e eSocirc-Ava















EHEC VT VT-FVT-R






ETEC LT LT-FLT-R






2 250120583M dNTPs 1x





3 Results





















4 Discussion






7077

20

923

00

100

200

300

400

500

600

700

800


()


(a)

26092174

23912826

3333

1667

5000

00000

100

200

300

400

500

600


Cabbage

()


(b)

2308

3846 3846

00

100

200

300

400

500


()

(c)






1186 16942203 2711

000

1667

8333

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169

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508 508

000

200

400

600

800

1000

1200

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75

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Resis

tanc

e (

)

Antibiotics

0

20

40

60

80

100

120



5682

2955

1364

00

100

200

300

400

500

600


Prop

ortio

n of

pro

duct

ion

()

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5 Conclusion





Acknowledgment


References





















































5












Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










EHEC VT VT-FVT-R






ETEC LT LT-FLT-R






2 250120583M dNTPs 1x





3 Results





















4 Discussion






7077

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100

200

300

400

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600

700

800


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(a)

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00000

100

200

300

400

500

600


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()


(b)

2308

3846 3846

00

100

200

300

400

500


()

(c)






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508 508

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200

400

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80

100

120



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100

200

300

400

500

600


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5 Conclusion





Acknowledgment


References





















































5












Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















4 Discussion






7077

20

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500

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400

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40

60

80

100

120



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100

200

300

400

500

600


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5 Conclusion





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5












Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





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400

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200

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500

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5 Conclusion





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5












Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





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500

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5 Conclusion





Acknowledgment


References





















































5












Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












5 Conclusion





Acknowledgment


References





















































5












Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







































5












Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

lactamases escherichia coli strains isolated from market garden prod

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