Food Research International 39 (2006) 212–219

www.elsevier.com/locate/foodres

Frequency and antimicrobial resistance of enteric bacteriawith spoilage potential isolated from table eggs

A. Adesiyun a,¤, N. OYah a, N. Seepersadsingh a, S. Rodrigo a, V. Lashley b, L. Musai b

a School of Veterinary Medicine, Paraclinical Sciences Department, Faculty of Medical Sciences, University of the West Indies, St. Augustine Campus, Trinidad and Tobago

b Poultry Surveillance Unit, Ministry of Agriculture, Land and Marine Resources, Trinidad and Tobago

Received 21 July 2005; accepted 24 July 2005

Abstract

The prevalence of potential spoilage microorganisms on the shells and in the egg contents of table eggs sold in Trinidad was deter-mined. Table eggs samples were obtained from 23 poultry layer farms, 14 shopping malls and 102 supermarkets across the country.Each farm was visited twice approximately one month apart and 25 pooled eggs constituted a composite sample. Shopping mallswere each visited twice usually one month apart while supermarkets were each visited once over a 4-month period. For both mall andsupermarkets, six pooled eggs constituted a composite sample. Swabs of egg shells and pooled yolk and albumen (egg content) weretested for selected bacteria using standard methods. The resistance of bacteria to seven antimicrobial agents was detected using thedisc diVusion method. Of a total of 184 composite eggs (shells, yolk/albumen or both) sampled, 71 (38.6%) samples were positive forenteric microbes, other than E. coli, Salmonella, Campylobacter spp. and Listeria spp. Enterobacter spp. and Klebsiella spp. were iso-lated from 15 (8.2%) and 14 (7.6%), respectively, of pooled egg shells alone and from 6 (3.3%) and 3 (1.6%), respectively, of egg con-tent samples alone. Prevalence of enteric bacteria in egg contents was generally higher than found on egg shells with faeces/blood orcracks compared with those without, but the diVerences were not signiWcant (P > 0.05; X2). The microbial load of egg content was notsigniWcantly aVected by type of housing of laying birds, source of feeds, use of medicated feeds and temperatures at which eggs werekept at sale outlets. Of a total of 131 bacterial isolates tested, 125 (95.4%) exhibited resistance to one or more antimicrobial agentsand resistance was high to streptomycin (90.1%), tetracycline (51.9%) and kanamycin (30.5%). Failure to properly handle or heattable eggs sold in Trinidad poses a potential health hazard to consumers because of their poor microbial quality and high frequencyof resistance to antimicrobial agent. 2005 Elsevier Ltd. All rights reserved.

Keywords: Selected enteric bacteria; Antimicrobial resistance; Table eggs; Trinidad

1. Introduction

Table eggs are consumed worldwide in various dishesand are considered very nutritious and a cheap source ofprotein (Blumenthal, 1990; MAFF, 2000; Papadopoulouet al., 1997). The well known enteric pathogens particu-

* Corresponding author. Tel.: +1 868 645 2640x4237/4241; fax: +1868 645 7428.

E-mail address: abiodunadesiyun@hotmail.com (A. Adesiyun).

0963-9969/$ - see front matter 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.foodres.2005.07.008

larly Salmonella, Escherichia coli, Campylobacter spp.and Listeria spp. have been isolated from table eggs andtheir contents (Adesiyun et al., 2005b; Farber, Daley,& Coates, 1992; Hope, Baker, & Edel, 2002; Leasor &Foegeding, 1989; Shane, GiVord, & Yogasundram, 1986)and have been responsible for egg-borne epidemicsglobally (CDC, 1990; Danielsson, Mollby, & Brag,1979; Mazurek, Holbert, & Parrish, 2005; Rocourt,BenEmbarek, & Toyofuku, 2003; Todd, 1996). Othermembers of the family Enterobacteriaceae such as

A. Adesiyun et al. / Food Research International 39 (2006) 212–219 213

Pseudomonas spp., Citrobacter spp., Alcaligenes spp. andKlebsiella spp. have all been isolated from whole orcracked eggs with a potential to cause spoilage and enterthe food chain through table eggs causing infection inconsumers (Ibeh & Izuagbe, 1986; Jones, Musgrove, &Northcutt, 2004a; Musgrove, Jones, & Northcutt, 2004;Papadopoulou et al., 1997; Wadstrom & Ljungh, 1991).The prevalence of resistance to antimicrobial agentsamongst numerous bacteria isolated from eggs or theirwashings has been reported by others, emphasizing thepotential to cause therapeutic problems in consumers(Papadopoulou et al., 1997).

In Trinidad and Tobago, table eggs are used in sev-eral dishes and drinks but in 1992, an epidemic in mentalpatients which resulted in 10 mortalities was conWrmedto be egg nog-borne salmonellosis caused concern in thepopulation (Hyatali et al., 1992). Indar et al. (1998) dem-onstrated trans-ovarial transmission of Salmonella intable eggs sampled from poultry farms in Trinidad.Adesiyun et al. (2005a) reported the prevalence of anti-microbial residues in table eggs sold in the country,emphasizing the health risk they pose to consumers andin another report assessed the potential of table eggs inTrinidad serving as sources of Salmonella, E. coli, Cam-pylobacter spp. and Listeria spp. for consumers(Adesiyun et al., 2005b). To date, information is unavail-able on the presence of potential spoilage or infectiousmicroorganisms on or in table eggs nor is the eVect ofstorage conditions on the microbial load of eggs known.The frequency of resistance of these bacteria to com-monly used antimicrobial agents in the population hasalso not been reported.

The study was therefore conducted to determine theprevalence of selected enteric microbes on the shellsand in the egg contents of table eggs sold at outletsacross Trinidad as well as the antimicrobial resistanceof these bacteria. The study also investigated the possi-ble eVects of management practices at the poultry layerfarm level, as well as storage conditions at sale outlets,on the prevalence of these enteric pathogens in theeggs.

2. Materials and methods

Samples of table eggs were obtained from layer farmsand sale outlets (shopping mall and supermarkets).Farms rear layers which supply eggs to a majority of saleoutlets in Trinidad. Shopping malls are medium to largeshopping complexes with several shops including asupermarket. Table eggs are sold refrigerated in thesemall supermarkets. Supermarkets, which are in the large,medium and small categories, are outlets across thecountry selling essentially food items. They representsale outlets which include street vendors, kiosks, opentrucks/vehicles along highways and stores in neighbor-

hoods selling table eggs mostly at ambient or room tem-perature with a few at refrigeration temperature. Aquestionnaire was administered to obtain informationon location, farm size, number of layers and type ofhousing for laying birds, problem of rodents and free-Xying birds, type (medicated or non-medicated) andsource of feeds used and sale outlets for eggs. All farms,all shopping malls and selected supermarkets representa-tive of various regions of the island were sampled. Ateach farm, a total of 25 eggs were randomly collected ontwo occasions, approximately one month apart. At theshopping malls and supermarkets, six eggs were ran-domly sampled from diVerent producers available forsale during the visits. Shopping malls were sampledtwice, approximately one month apart, and the super-markets once. For farms, sterile crates were provided forthe collection of 25 eggs while at the shopping malls andsupermarkets, the eggs were purchased in the crates usedfor normal sales at each outlet. The temperatures (refrig-eration, ambient and air conditioned room temperature)at which eggs were kept at sale outlets were noted duringeach visit. Samples were transported to the laboratorywithin 2 h of collection. All eggs were processed in thelaboratory within 24 h of collection and when impracti-cable, they were stored overnight at the temperatures atwhich they were sold.

Overall, from 23 farms, a total of 46 composite eggssamples were processed while from 14 shopping malls, atotal of 31 composite eggs were processed. From the102 supermarket outlets studied, a total of 107 compos-ite eggs were processed. The eggs were processed as ear-lier described (Adesiyun et al., 2005b). BrieXy, wearingsterile gloves to handle egg samples from each source,for egg shells of either a pool of 6 (malls and supermar-kets) or 25 (farms), one sterile swab moistened in salinewas applied to the surface of each egg. The 6 or 25 swabsamples were submerged in 6 and 20 ml of saline,respectively, in universal bottles, the contents mixedwith VWR Mini- Vortexers (Henry Treemer, USA) andused to inoculate the appropriate enrichment broths ormedia. For yolk and albumen samples, a pool of 6 or 25eggs were submerged in 75% ethanol for 5 min afterwhich the pointed end of each egg was disinfected byXaming for 5–10 s with a Bunsen burner. A small holewas then made on the shell with a sterile scalpel bladeand the egg content (yolk and albumen) asepticallyemptied into a Stomacher bag. The content was blendedfor 30 s at normal speed in a Stomacher 400 (Seward,London, United Kingdom). The resulting mixture wasused to inoculate appropriate enrichment broths ormedia.

Swabs of egg shells (6 eggs from malls and supermar-kets and 25 egg from farms) and swabs of pooled eggcontents were used to inoculate MacConkey agar (DifcoLtd., Michigan, USA). The plates were incubated aerobi-cally overnight at 37 °C. Both lactose-fermenting and

214 A. Adesiyun et al. / Food Research International 39 (2006) 212–219

non-lactose-fermenting colonies were picked from Mac-Conkey plates, subcultured on blood agar, incubatedovernight at 37 °C to obtain pure colonies. The isolateswere identiWed using standard methods (Buchanan &Gibbons, 1974; Macfaddin, 2002).

The NCCLS (2002) procedure was used to determinethe antimicrobial resistance of isolates using the follow-ing antimicrobial agents (Difco, Michigan, USA) andconcentrations: kanamycin (30 mcg), streptomycin(10 mcg), nalidixic acid (30 mcg), gentamicin (10 mcg),tetracycline (30 mcg), sulphamethoxazole/trimethoprim(23.25/1.75 mcg) and enroXoxacin (5 mcg).

The prevalence and counts of microorganisms on eggshells and/or in egg content were compared for varioussources (farms, malls, supermarkets); and farm and saleoutlet practices such as type of housing on farms, sourceand type (medicated or non-medicated) of feed and tem-perature of storage at the sale outlet were also related tothe frequency of isolation of bacteria using the Statisti-cal Package for Social Sciences (SPSS) version 10. Allstatistical analyses were two-tailed using the �2 andFicher’s exact tests and interpreted at the 5% level of sig-niWcance.

3. Results

The prevalence of selected aerobic bacteria in tableeggs by sale outlets and location of isolation from tableeggs is shown in Table 1. Of the 139 outlets/farms tested,59 (42.4%) were positive for the enteric bacteria testedwith a range from 36.3% (37 of 102) for supermarket eggsamples to 69.6% (16 of 23) for eggs obtained directlyfrom farms. The diVerence was statistically signiWcant(P < 0.05; X2). Of a total of 184 composite eggs sampled,71 (38.6%) were positive for selected enteric bacteria butthe diVerence in frequency across sources was not signiW-cantly signiWcant (P > 0.05; X2). The bacteria weredetected at a higher frequency from the egg shells alone,

19.0% (35 of 184) than from egg contents alone, 13.0%(24 of 184). Eggs from farms had a signiWcantly(P < 0.05; X2) higher prevalence of enteric bacteria ontheir shells compared with eggs purchased from shop-ping malls and supermarkets.

Table 2 shows the prevalence of the various entericbacteria on egg shells or in the egg content by source.The predominantly identiWed enteric aerobic bacteriafrom 184 composite eggs samples were Enterobacterspp., 23 (12.5%) and Klebsiella spp., 17 (9.2%) although atotal of 32 (17.4%) composite egg samples belonged tothe Enterobacteria group. Amongst the eggs positive forenteric bacteria, for egg shells, 30 (16.3%), 14 (7.6%), 2(1.1%) and 1 (0.5%) contained 1, 2, 3, and 4 microorgan-isms, respectively. For egg content samples, 25 (13.6%)were positive for one organism only, 9 (4.9%), 1 (0.5%)and 1 (0.5%) were contaminated by 2, 3 and 4 microor-ganisms, respectively. The diVerence in the prevalence ofthe various bacteria in composite Egg samples by sourcewas not statistically signiWcant (P > 0.05; X2).

Fifteen (8.2%) and 14 (7.6%) egg shells alone werepositive for Enterobacter spp. and Klebsiella spp., respec-tively, compared with 6 (3.3%) and 3 (1.6%), respectively,for egg contents only (Table 3). Overall, a total of 18(9.8%) egg shells alone and 11 (6.0%) of egg contentsalone, contained members of the Enterobacteria group.

The frequency of isolation of enteric bacteria fromegg shells with faeces or blood was 25.2% (36 of 143)compared with egg shells free of these materials, 26.8%(11 of 41) while for cracked egg shells, the frequency ofisolation of enteric bacteria was 39.1% (9 of 23) com-pared to a frequency of 32.3% (52 of 161) for intact eggs.The diVerences were not statistically signiWcant(P > 0.05; X2). For egg contents, 21.0% (30 of 143) eggswith faeces or blood on their egg shells contained entericbacteria while 17.1% (7 of 41) of eggs without faeces orblood on their shells were positive. Five (21.7%) of 23contents of eggs with cracked shells contained entericbacteria compared with 32 (19.9%) of 161 from eggs

Table 1Prevalence of enteric pathogens in table eggs by source

Prevalence with the same common letter (a,b,c) in the same column are not statistically signiWcantly diVerent (P > 0.05) but those with diVerent lettersare signiWcantly diVerent (P < 0.05).

a Enteric microbes other than Salmonella, E. coli and Campylobacter spp. and Listeria spp.b Pooled egg samples, 6 each from shopping malls and supermarkets and 25 each from farms.c Shell samples only were positive for selected enteric bacteria.d Egg content samples only were positive for selected enteric bacteria.e Both samples of egg shells and contents were positive for selected enteric bacteria.

Source Outlets/farms Composite eggs No. (%) of samples positivea for

No. of outletstested

No. (%) positivea forenteric bacteria

No. of composite eggsb

testedNo. (%) positivea forenteric bacteria

Shells onlyc Egg contentsonlyd

Egg shells/contentse

Farm 23 16 (69.6)a 46 21 (45.7)a 13 (28.3)a 7 (15.2)a 1 (2.2)a

Mall 14 6 (48.9)b 31 12 (38.7)a 6 (19.4)a 3 (9.7)a 3 (9.7)a

Supermarket 102 37 (36.3)a 107 38 (35.5)a 16 (15.0)a 14 (13.1)a 8 (7.5)a

Total 139 59 (42.4) 184 71 (38.6) 35 (19.0) 24 (13.0) 12 (6.5)a

A. Adesiyun et al. / Food Research International 39 (2006) 212–219 215

Tab

le 2

Pre

vale

nce

and

type

s of

ent

eric

mic

robe

s on

egg

she

lls a

nd in

egg

con

tent

by

sour

ce o

f eg

gs

aSh

ell,

cont

ent

or b

oth

posi

tive

for

the

sel

ecte

d m

icro

bes.

bE

nter

ic m

icro

bes

dete

cted

oth

er t

han

E.c

oli;

Sal

mon

ella

spp

. and

Cam

pylo

bact

er s

pp. a

nd L

iste

ria

spp.

cE

NT

: Ent

erob

acte

r sp

p., K

: Kle

bsie

lla s

pp.,

Pl :

Ple

siom

onas

spp

., S:

Ser

rati

a sp

p., P

: Pro

teus

spp

., C

T: C

itro

bact

er s

pp.,

Ps:

Pse

udom

onas

spp

., A

l: A

lcal

igen

es s

pp.,

CH

: Chr

omob

acte

r sp

p., A

: Aci

-ne

toba

cter

spp

., H

: Haf

nia

spp.

, EB

A: E

nter

obac

teri

a gr

oup.

dSi

xtee

n eg

gs w

ere

each

con

tam

inat

ed b

y on

e or

gani

sm.

eSe

ven

eggs

wer

e ea

ch c

onta

min

ated

by

one

orga

nism

.f

Tw

enty

egg

s w

ere

each

con

tam

inat

ed b

y on

e or

gani

sm.

gO

ne o

rgan

ism

isol

ated

fro

m b

oth

egg

shel

l and

con

tent

.

Out

let

No.

of c

ompo

site

eggs

tes

ted

No.

(%

)a of

eggs

pos

itiv

efo

r en

teri

c m

icro

besb

No.

(%

) of

egg

s po

siti

ve fo

r

EN

TK

PI

SP

CT

Ps

Al

CH

AH

EB

Ac

Far

m46

21 (

45.7

)d7

(15.

2)g

3 (6

.5)

1 (2

.2)

3 (6

.5)

0 (0

.0)

0 (0

.0)

0 (0

.0)

0 (0

.0)

0 (0

.0)

1 (2

.2)

0 (0

.0)

11 (

23.9

)M

all

3112

(38

.7)e

4 (1

2.9)

g4

(12.

9)1

(3.2

)0

(0.0

)0

(0.0

)0

(0.0

)3

(9.7

)0

(0.0

)1

(3.2

)0

(0.0

)1

(3.2

)4

(12.

9)g

Supe

rmar

ket

107

38 (

35.5

)f12

(11.

2)g

10 (

9.3)

g6

(5.6

)4

(3.7

)6

(5.6

)5

(4.7

)g0

(0.0

)1

(0.9

)0

(0.0

)0

(0.0

)0

(0.0

)17

(15

.8)g

Tot

al18

471

(38

.6)

23 (1

2.5)

17 (

9.2)

8 (4

.2)

7 (3

.8)

6 (3

.3)

5 (2

.7)

3 (1

.6)

1 (0

.5)

1 (0

.5)

1 (0

.5)

1 (0

.5)

32 (

17.4

)

without cracked shells. The diVerences were not statisti-cally signiWcant (P > 0.05; X2).

Table 4 shows the prevalence and counts of totalaerobic bacteria in the egg contents of table eggs. Sev-enty-six (41.3%) of 184 composite eggs from all sourcescontained aerobic bacteria in their egg content and thelog10 mean log10 § SD total aerobic plate count (TAPC)was 6.0 § 7.7 cfu per ml. The diVerence in frequency ofaerobic bacteria in egg contents across sources was notstatistically signiWcant (P > 0.05; X2).

The log10 mean § log10 SD TAPC per ml for 50 eggssold at ambient or room temperatures was 3.56 § 2.46while for 26 eggs stored at refrigeration temperature itwas 3.81 § 2.65 but the diVerence was not statisticallysigniWcant (P > 0.05; X2). The type of housing for henlayers, problems with free-Xying bird and rodents, sourceof feeds and use of medicated feeds did not signiWcantly(P > 0.05; X2) aVect the microbial load of egg contentsfrom all sources studied.

The frequency of resistance amongst enteric bacteriatested according to the location of isolation on the egg isshown in Table 5. Overall, 125 (95.4%) of 131 isolatesstudied exhibited resistance to one or more antimicro-bial agents ranging from 80.0% (8 of 10) for Plesiomonasspp. to 100.0% found amongst isolates of Enterobacterspp., Serratia spp., Pseudomonas spp. and the Enterobac-teria group. The diVerence was not statistically signiW-cant (P > 0.05; X2). The frequency of resistance toantimicrobial agents amongst isolates recovered fromegg shells (97.3%) and egg content (94.7%) were similar(P > 0.05; X2). Considering the source of isolates, 32(94.1%) of 34, 22 (100.0%) of 22 and 71 (94.7%) of 75 iso-lates from layer farms, shopping malls and supermar-kets, respectively, were resistant to antimicrobial agents.

Regardless of the bacteria tested, resistance was high-est to streptomycin (90.1%) and lowest to sulphamethox-azole/trimethoprim (4.6%) as shown in Table 6 and thediVerence was statistically signiWcant (P < 0.05; X2). Atotal of 68 (51.9%) and 40 (30.5%) isolates exhibited resis-tance to tetracycline and kanamycin, respectively, while19.1%, 17.6% and 9.2% of the isolates were resistant tonalidixic acid, enroXoxacin and gentamicin, respectively.

4. Discussion

The fact that numerous genera of the family Entero-bacteriaceae were recovered from egg shells was notunexpected since it is known that freshly laid eggsbecome readily contaminated in their environments(Indar et al., 1998; Jones, Rives, & Carey, 1995). It hastherefore been suggested that laid eggs be removed fromthe nests as soon as practicable (Labaque, Navarro, &Martella, 2003). The Wnding of a signiWcantly higherprevalence of enteric bacteria on the shells of eggs sam-pled directly from the layer farms compared with shells

216 A. Adesiyun et al. / Food Research International 39 (2006) 212–219

of eggs from either the shopping malls or the supermar-kets may have resulted from the additional washings ofeggs carried out at the sale outlets to make them appear

clean and acceptable to the consumers. The practice ofwashing eggs with water containing detergents in Trini-dad is however not expected to remove all bacteria on

Table 3Frequency of isolation of enteric bacteria from egg shells and egg contents of table eggs

a Enteric microbes other than Salmonella, E. coli, Campylobacter and Listeria spp.b Of a total of 184 composite eggs samples each, of shell and egg content.c Eggs samples obtained from farms, shopping malls and supermarkets.d Enteric bacteria isolated from shells only.e Enteric bacteria isolated from egg content only.f Enteric bacteria isolated from shells and egg content of same composite egg.g Members of the Enterobacteria group other than the bacteria listed above and E. coli, Salmonella, Campylobacter spp. and Listeria spp.

Microorganisma No. (%)b of samplesc positive for

Egg shell onlyd Egg content onlye Egg shell/egg contentf

Enterobacter spp. 15 (8.2) 6 (3.3) 3 (1.6)Klebsiella spp. 14 (7.6) 3 (1.6) 1 (0.5)Citrobacter spp. 3 (1.6) 1 (0.5) 1 (0.5)Serratia spp. 3 (1.6) 3 (1.6) 0 (0.0)Proteus spp. 2 (1.1) 4 (2.2) 0 (0.0)Plesiomonas spp. 1 (0.5) 7 (3.8) 0 (0.0)Pseudomonas spp. 2 (1.1) 2 (1.1) 0 (0.0)Acinetobacter spp. 1 (0.5) 1 (0.5) 0 (0.0)Hafnia spp. 0 (0.0) 2 (1.1) 0 (0.0)Alcaligenes spp. 0 (0.0) 1 (0.5) 0 (0.0)Chromobacter spp. 0 (0.0) 1 (0.5) 0 (0.0)Enterobacteria groupg 18 (9.8) 11 (6.0) 3 (1.6)

Table 4Prevalence and counts of total aerobic bacteria in the contents of table eggs

a Grown on nutrient agar.

Source No. of composite egg samples tested Aerobic bacteria

No. (%) positivea Log10 mean + log10 SD (cfu/ml)

Farm 46 16 (34.8) 2.8 § 2.8Mall 31 12 (38.7) 7.4 § 7.8Supermarket 107 48 (44.9) 8.2 § 8.8

Total 184 76 (41.3) 6.0 § 7.7

Table 5Frequency of resistance to antimicrobial agents amongst bacteria from egg shell and yolk/albumen

a A mixture of yolk and albumen.b Resistant to one or more antimicrobial agents.c Included Acinetobacter (2 isolates), Chromobacter (2 isolates), Hafnia (2 isolates) and Alcaligenes (1 isolate).

Type of bacteria No. of isolatestested

No. (%) of isolatesresistantb

Location of isolation from eggs

Shell Contenta

No. of isolatestested

No. (%) of isolatesresistantb

No. of isolatestested

No. (%) of isolatesresistantb

Enterobacter spp. 30 30 (100.0) 19 19 (100.0) 11 11 (100.0)Klebsiella spp. 19 18 (94.7) 15 14 (93.3) 4 4 (100.0)Plesiomonas spp. 10 8 (80.0) 1 1 (100.0) 9 7 (77.8)Serratia spp. 8 8 (100.0) 4 4 (100.0) 4 4 (100.0)Proteus spp. 7 6 (85.7) 3 3 (100.0) 4 3 (75.0)Citrobacter spp. 6 5 (83.3) 4 4 (100.0) 1 1 (100.0)Pseudomonas spp. 4 4 (100.0) 2 2 (100.0) 2 2 (100.0)Enterobacteria group 40 40 (100.0) 24 24 (100.0) 16 16 (100.0)Othersc 7 6 (85.7) 1 0 (0.0) 6 6 (100.0)

Total 131 125 (95.4) 73 71 (97.3) 57 54 (94.7)

A. Adesiyun et al. / Food Research International 39 (2006) 212–219 217

egg shells compared to other recommended procedures(Chavez, Knape, & Coufal, 2002; Himathongkham,Riemann, & Ernst, 1999; James, Lechevalier, & Ketter-ingham, 2002). Reports exist on higher prevalence andcounts of bacteria on unwashed egg shells than thosewashed (Jones et al., 2004a; Labaque et al., 2003). It hasbeen documented that the storage of eggs at the sale out-lets, depending on storage conditions, particularly thetemperature and duration, may aVect the microbial loadof both egg shells or content but not the prevalence ofbacteria (Humphrey, Whitehead, & Gawler, 1991; Jones,Curtis, & Anderson, 2004b; Schoeni, Glass, & McDer-mott, 1995).

The isolation of these bacteria from egg contentsalone may suggest vertical transmission as reported forSalmonella (Humphrey, 1994; Thorne, 1991; Todd, 1996)but being a prevalence study, the possibility of prior pen-etration of egg shells to contaminate contents cannot beignored. It has however been suggested that verticaltransmission with resultant contaminated eggs is rarewith certain microorganisms like Campylobacter jejuni(Sahin, Kobalka, & Zhang, 2003).

The spectrum of enteric bacteria found in the currentstudy, other than the known common egg-associatedpathogens such as Salmonella, Listeria spp. and Cam-pylobacter spp. (Adesiyun et al., 2005b; Farber et al.,1992; Hope et al., 2002; Leasor & Foegeding, 1989;Shane et al., 1986), agrees with published reports whereEnterobacter spp., Klebsiella spp., Citrobacter spp., Pro-teus spp., and Alcaligenes spp. have been recovered fromeggs or their wash water elsewhere (Ibeh & Izuagbe,1986; Jones et al., 2004a; Moats, 1980; Musgrove et al.,2004; Papadopoulou et al., 1997; Soriano, Rico, &Molto, 2000). The Wndings indicate that these bacteriamay be playing an important role in the spoilage of eggscoupled with the fact that some of these have caused

infection in humans (Danielsson et al., 1979; Donnelly,Srivastava, & Oyejide, 2002; Guerrant, Moore, & Kirsh-cenfeld, 1975; Klipstein & Engert, 1977; Wadstrom &Ljungh, 1991). Dishes containing raw or improperlycooked contaminated eggs pose health risk to consumersas they could contain enteric pathogens (Hayes, Nylen,& Smith, 1999; Lievonen, Havulinna, & Maiala, 2004).

Also of public health and economic signiWcance wasthe Wnding that 11 enteric bacteria were isolated fromegg contents, albeit at diVerent frequencies. These bacte-ria, in high counts, may cause spoilage of eggs whilesome have the potential to cause infection in consumers(Thorne, 1991). Papadopoulou et al. (1997) had isolatedEnterobacter cloacae, E. coli, Pseudomonas spp. and Cit-robacter spp. from table eggs in Greece, as found in thepresent study. Ibeh and Izuagbe (1986) also reportedthat Klebsiella spp., S. aureus and Alcaligenes spp. werethe predominant bacteria isolated from cracked eggs inNigeria. The enteric bacteria detected in this study, par-ticularly Proteus spp., Citrobacter spp., Enterobacterspp., Klebsiella spp., Aeromonas spp. and Plesiomonasspp. are causal agents for diarrhoea (Danielsson et al.,1979), while Alcaligenes spp. and Pseudomonas spp.cause egg spoilage (Donnelly et al., 2002; Moats, 1980).

It is also relevant to note that 41.3% of the 184 pooledcomposite egg contents were contaminated with aerobicbacteria, having log10 mean TAPC per ml of 2.8. It wasno surprise that the contents of eggs sampled directlyfrom farms had a considerably lower prevalence (34.8%)and log10 mean TAPC per ml of 2.8 compared with eggsfrom supermarkets, 44.9% and 8.2%, respectively. This isbecause enteric bacteria have been demonstrated to becapable of multiplying in egg content at ambient temper-ature (Humphrey, 1994; Messens, Duboccage, & Gri-jspeerdt, 2004) and even at refrigeration temperature(Kim, Emery, & Rinke, 1989). It has also been reported

Table 6Frequency of resistance of bacterial isolates from egg shell and content to eight antimicrobial agents

a From egg shell and contents.b K, kanamycin; S, streptomycin; NA, nalidixic acid; CN, gentamicin; TE, tetracycline; SXT, trimethoprim/sulphamethoxazole; ENR, enroXoxa-

cin.

Type of bacteria detected No. of isolates testeda No. (%) of isolates resistant to

Sb TE K NA ENR CN SXT

Enterobacter spp. 30 29 (96.7) 16 (53.3) 11 (36.7) 3 (10.0) 4 (13.3) 1 (3.3) 1 (3.3)Klebsiella spp. 19 18 (94.7) 1 (5.3) 7 (36.8) 1 (5.3) 0 (0.0) 3 (15.8) 0 (0.0)Plesiomonas sp. 10 6 (60.0) 5 (50.0) 1 (10.0) 2 (20.0) 1 (10.0) 0 (0.0) 0 (0.0)Serratia spp. 8 8 (100.0) 6 (75.0) 6 (75.0) 2 (25.0) 3 (37.5) 0 (0.0) 0 (0.0)Proteus spp. 7 5 (71.4) 1 (14.3) 1 (14.3) 1 (14.3) 1 (14.3) 0 (0.0) 0 (0.0)Citrobacter spp. 6 5 (83.3) 3 (50.0) 1 (16.7) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)Pseudomonas spp. 4 2 (50.0) 4 (100.0) 1 (25.0) 3 (75.0) 3 (75.0) 2 (50.0) 1 (25.0)Acinetobacter spp. 2 1 (50.0) 1 (50.0) 0 (0.0) 1 (50.0) 0 (0.0) 0 (0.0) 0 (0.0)Chromobacter spp. 2 2 (100.0) 0 (0.0) 0 (0.0) 2 (100.0) 2 (100.0) 0 (0.0) 0 (0.0)Hafnia spp. 2 2 (100.0) 2 (100.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)Alcaligenes spp. 1 1 (100.0) 1 (100.0) 0 (0.0) 1 (100.0) 1 (100.0) 0 (0.0) 1 (100.0)Enterobacteria group 40 39 (97.5) 28 (70.0) 12 (30.0) 9 (22.5) 8 (20.0) 6 (15.0) 3 (7.5)

Total 131 118 (90.1) 68 (51.9) 40 (30.5) 25 (19.1) 23 (17.6) 12 (9.2) 6 (4.6)

218 A. Adesiyun et al. / Food Research International 39 (2006) 212–219

that the Wnal microbial load of egg contents depends ontemperature and length of storage (Humphrey, 1994).The fact that eggs at sale outlets, particularly at super-markets, are kept for days or even weeks at ambient tem-peratures or at refrigeration temperatures (open andclosed refrigerators) during which multiplication of thecontaminants in the egg content can occur, therebyincreasing their population and increasing their spoilageor health risk potential, cannot be ignored.

It was rather a surprise Wnding that cracks in eggs didnot signiWcantly aVect the prevalence of all aerobicenteric bacteria assayed for in either the egg shells or eggcontents. The lack of statistically signiWcant diVerence inthe frequency of contamination of cracked anduncracked eggs may be due to the high level of contami-nation found in uncracked eggs. Cracked eggs have beenreported to be important sources of contamination ofegg contents (CDC, 1990; Ibeh & Izuagbe, 1986; Todd,1996). A multitude of factors including a failure to detectdirt on eggs visually and vertical transmission may havebeen responsible for the Wndings.

It is of therapeutic relevance that regardless of thetype of bacteria and the source (farm, malls or super-markets) or location on the egg (shell or content), resis-tance was generally high (80.0–100.0%) to antimicrobialagents some of which are used in treating human dis-eases thereby posing health hazard to consumers(Papadopoulou et al., 1997). The rather high prevalenceof resistance exhibited to antimicrobial agents by the eggisolates of enteric bacteria is however expected since ithas been reported that 80.7% of the layer farmers inTrinidad used medicated feeds containing nitrofuran-toin, tylosin and chlortetracycline while only 26.1%claimed adherence to withdrawal period after use (Ades-iyun et al., 2005a). Furthermore, the table eggs fromwhich these enteric bacteria were isolated were reportedto have a prevalence of antimicrobial agents of 13.0% of184 composite eggs, comprising a 6.5% prevalence forsulfonamides, 3.8% for macrolides, 2.7% for tetracyclineand 0.0% for beta-lactams (Adesiyun et al., 2005a). Itwas therefore not a surprise that relatively high fre-quency of resistance was exhibited by the enteric bacte-ria to streptomycin and tetracycline.

The fact that the frequency of resistance to antimicro-bial agents amongst the enteric bacteria did not signiW-

cantly vary across outlets (farms, malls andsupermarkets) may be a reXection of a common source,i.e., the farms, which supply the sale outlets and this maysuggest common practice on these farms concerning theuse of antimicrobial agents. It is known that all layerfarms in Trinidad receive their medicated feeds from onesource. It has been reported that storage condition andduration will not aVect the prevalence of diVerent typesof bacteria on egg shells or in egg contents but the rate ofmultiplication and the microbial load (Humphrey et al.,1991; James et al., 2002; Schoeni et al., 1995). It has been

established that resistance of bacteria to antimicrobialagents is genetically controlled (plasmids or chromo-somes) (Shears, Suliman, & Hart, 1988; Washington,1979; Yang, Park, & Seo, 2001).

Although the enteric bacteria recovered from tableeggs in the current study are not the common establishedpathogens, it was concluded that the prevalence andresistance exhibited by the enteric bacteria recoveredfrom table eggs in Trinidad may pose a health hazard toconsumers where the consumption of diets or drinkscontaining improperly cooked or raw eggs exists. Thereis a need to educate the population on good sanitarypractices in handling eggs and consumption of onlyproperly cooked eggs or egg products.

Acknowledgements

The assistance of the staV of the Poultry SurveillanceUnit of the Ministry of Agriculture, Land and MarineResources is appreciated for sample collection. We aregrateful to Gerard Ramirez, Shakti Dookeran, AweedaNewaj-Fyzul and Anil Pooran for their technical sup-port. We thank Bernadette Abrams for typing the manu-script.

References

Adesiyun, A. A., OYah, N. V., Seepersadsingh, N., Rodrigo, Lashley, &Georges (2005a). Prevalence of antimicrobial residues in table eggsin Trinidad. Journal of Food Protection, 68, 1501–1505.

Adesiyun, A. A., OYah, N. V., Seepersadsingh, N., Rodrigo, Lashley, &Musai. (2005b). Microbial health risk posed by table eggs in Trini-dad. Epidemiology and Infection (in press).

Blumenthal, D. (1990). From the chicken to the egg. FDA Con-sumer(April), 7–10.

Buchanan, R. E., & Gibbons, N. E. (Eds.). (1974). Bergeys’ manual ofdeterminative bacteriology (8th ed.). Baltimore, USA: Williams andWilkins Company.

Centers for Disease Control (CDC), Update: (1990). Salmonella enteri-tidis infections and grade A shell eggs, United States, 1989.MMWR 38, 877–880.

Chavez, C., Knape, K. D., Coufal, C. D., et al. (2002). Reduction of eggshell aerobic plate counts by ultraviolet irradiation. Poultery Sci-ence, 81, 1132–1135.

Danielsson, M. L., Mollby, R., Brag, H., et al. (1979). Enterotoxigenicenteric organisms in Swedish food. Journal of Hygiene (Cam-bridge), 83, 33–40.

Donnelly, T. M., Srivastava, K. K., Oyejide, A., et al. (2002). Oozingostrich egg: omphalitis caused by Enterobacteriaceae. Lab Animal(NY), 31, 23–24.

Farber, J. M., Daley, E., & Coates, F. (1992). Presence of Listeria spp.in whole eggs and wash water samples from Ontario and Quebec.Food Research and International, 25, 143–145.

Guerrant, R. L., Moore, R. A., Kirshcenfeld, P. M., et al. (1975). Role oftoxigenic and invasive bacteria in acute diarrhea of childhood. NewEngland Journal of Medicine, 293, 567–573.

Hayes, S., Nylen, G., Smith, R., et al. (1999). Undercooked hens’ eggsremain a risk factor for sporadic Salmonella enteritidis infection.Communicable Disease and Public Health, 2, 66–67.

A. Adesiyun et al. / Food Research International 39 (2006) 212–219 219

Himathongkham, S., Riemann, H., & Ernst, R. (1999). EYcacy of disin-fection of shell eggs externally contaminated with Salmonella ente-ritidis: implications for egg testing. International Journal of FoodMicrobiology, 49, 161–167.

Hope, B. K., Baker, R., Edel, E. D., et al. (2002). An overview of the S.enteritidis risk assessment for shell eggs and egg products. RiskAnalysis, 22, 203–218.

Humphrey, T. J. (1994). Contamination of egg shell and contents withSalmonella enteritidis: a review. International Journal of FoodMicrobiology, 21, 31–40.

Humphrey, T. J., Whitehead, A., Gawler, A. H., et al. (1991). Numberof Salmonella enteritidis in the contents of naturally contaminatedhens’ eggs. Epidemiology and Infection, 106, 489–496.

Hyatali, I., Quamina, E., Mohess, C., & et al. (1992). Report of the ill-ness and resultant deaths at St. Ann’s hospital, Port of Spain, Trin-idad, Government Printery.

Ibeh, I. N., & Izuagbe, Y. S. (1986). An analysis of the microXora ofbroken eggs used in confectionery products in Nigeria and theoccurrence of enterotoxigenic Gram-negative bacteria. Interna-tional Journal of Food Microbiology, 3, 71–77.

Indar, L., Baccus-Taylor, G., Commissiong, E., et al. (1998). Salmonel-losis in Trinidad: evidence for transovarian transmission of Salmo-nella in farm eggs. West Indian Medical Journal, 47, 50–53.

James, C., Lechevalier, V., & Ketteringham, L. (2002). Surface pasteur-ization of shell eggs. Journal of Food Engineering, 53, 193–197.

Jones, D. R., Curtis, P. R., Anderson, K. E., et al. (2004b). Microbialcontamination in inoculated shell eggs. II. EVects of layer strainand egg storage. Poultry Science, 83, 95–100.

Jones, D. R., Musgrove, M. T., & Northcutt, J. K. (2004a). Variation inexternal and internal microbial populations in shell eggs duringextended storage. Journal of Food Protection, 67, 2657–2660.

Jones, F. T., Rives, D. V., & Carey, J. B. (1995). Salmonella contamina-tion in commercial eggs and an egg production facility. Poultry Sci-ence, 74, 753–757.

Kim, C. J., Emery, D. A., Rinke, H., et al. (1989). EVect of time and tem-perature on growth of Salmonella enteritidis in experimentally inoc-ulated eggs. Avian Diseases, 33, 735–742.

Klipstein, F. A., & Engert, R. F. (1977). Immunological relationshipsbetween cholera toxin and the heat labile and heat stable enterotox-ins of coliform bacteria. Infection Immunology, 18, 110–117.

Labaque, M. C., Navarro, J. L., & Martella, M. B. (2003). Microbialcontaminant of artiWcially contaminated Greater Rhea (RheaAmericana) eggs. Poultry Science, 44, 355–358.

Leasor, S. B., & Foegeding, P. M. (1989). Listeria species in commerciallybroken raw liquid whole egg. Journal of Food Protection, 52, 777–780.

Lievonen, S., Havulinna, A. S., & Maiala, R. (2004). Egg consumptionpatterns and Salmonella risk in Finland. Journal of Food Protec-tion, 67, 2416–2423.

Macfaddin, J. F. (2002). Biochemical tests for identiWcation of medicalbacteria. New York: Williams and Wilkins.

Mazurek, J., Holbert, L., Parrish, M. K., et al. (2005). Raw eggs-lessonslearned from an outbreak of Salmonella serotype enteritidis infec-tion associated with meringue pie. Journal of Public Health andManagement and Practice, 11, 201–207.

Messens, W., Duboccage, L., Grijspeerdt, K., et al. (2004). Growth ofSalmonella serovars in hens’ egg albumen as aVected by storageprior to inoculation. Food Microbiology, 21, 25–32.

Ministry of Agriculture, Fisheries and Food (MAAF) (2000). Eggs andpoultry meat frequently asked question. Available from URL http://www.maV.-gov.uk/foodrin/poultry/epfaq.htm.

Moats, W. A. (1980). ClassiWcation of bacteria from commercial eggwashers and washed and unwashed eggs. Applied EnvironmentalMicrobiology, 29, 179–185.

Musgrove, M. T., Jones, D. R., Northcutt, J. K., et al. (2004). IdentiWca-tion of Enterobacteriaceae from washed and unwashed commercialshell eggs. Journal of Food Protection, 67, 2613–2616.

National Committee for Clinical Laboratory Standards (NCCLS).(2002). Performance standards for antimicrobial disc and dilutionsusceptibility for bacteria isolated from animals; Approved Stan-dards, (2nd ed., Vol. 22). pp. 1–52.

Papadopoulou, C., Dimitriou, D., Levidiotou, S., et al. (1997). Bacterialstrains isolated from eggs and their resistance to currently usedantibiotics: is there a health hazard for consumers?. ComparativeImmunology Microbiology and Infectious Diseases, 20, 35–40.

Rocourt, J., BenEmbarek, P., Toyofuku, H., et al. (2003). Quantitativerisk assessment of Listeria monocytogenes in ready-to-eat foods:the FAO/WHO approach. FEMS Immunology and Medical Micro-biology, 35, 263–267.

Sahin, O., Kobalka, P., & Zhang, Q. (2003). Detection and survival ofCampylobacter in chicken eggs. Jouranl of Applied Microbiology,95, 1070–1079.

Schoeni, J. L., Glass, K. A., McDermott, J. L., et al. (1995). Growth andpenetration of Salmonella enteritidis, Salmonella heidelberg andSalmonella typhimurium in eggs. International Journal of FoodMicrobiology, 24, 385–396.

Shane, S. M., GiVord, D. H., & Yogasundram, K. (1986). Campylobac-ter jejuni contamination of eggs. Veterinary Research Communica-tions, 10, 487–492.

Shears, P., Suliman, G., & Hart, C. A. (1988). Occurrence of multipleantibiotic resistance and R-plasmids in Enterobacteriaceae isolatedfrom children in Sudan. Epidemiology and Infection, 100, 73–81.

Soriano, J. M., Rico, H., Molto, J. C., et al. (2000). Microbial evaluationof Spanish potato omelette and cooked meat samples in universityrestaurants. Journal of Food Protection, 63, 1273–1276.

Thorne, G. M. (1991). Salmonella: The chicken and the eggs. ClinicalMicrobiology Newsletter, 13, 65–68.

Todd, E. C. D. (1996). Risk assessment of use of cracked eggs in Can-ada. International Journal of Food Microbiology, 30, 125–143.

Wadstrom, T., & Ljungh, A. (1991). Aeromonas and Plesiomonas asfood and waterborne pathogens. International Journal of FoodMicrobiology, 12, 303–312.

Washington, J. A. (1979). The eVects and signiWcance of sub-minimalinhibitory concentrations of antibiotics. Reviews of Infectious Dis-eases, 1, 781–786.

Yang, S. J., Park, K. Y., Seo, K. S., et al. (2001). Multidrug-resistanceSalmonella typhimurium and Salmonella enteritidis identiWed bymultiplex PCR from animals. Journal of Veterinary Science, 2, 181–188.