Health Articles

 

Microsoft word - abdelnoorasb14p

Advanced Studies in Biology, Vol. 4, 2012, no. 8, 385 – 396
Identification of Virulence Genes among
Antibacterial-Resistant Escherichia coli
Isolated from Poultry
Fadi E. El-Rami , Elias A. Rahal , Fawwak T. Sleiman
and Alexander M. Abdelnoor *
1Department of Experimental Pathology, Immunology and Microbiology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon 2 Department of Animal and Veterinary Sciences, Faculty of Agricultural and Food Sciences, American University of Beirut, Beirut Lebanon *Address for Correspondence: Alexander M. Abdelnoor, Department of Microbiology & Immunology, Faculty of Medicine, American University of Beirut, P.O. Box 11-0236, Riad el-Solh, 11072020, Beirut Lebanon [email protected] Abstract
Food borne infections represent a global health hazard. The study was conducted to investigate the prevalence of virulence genes among Escherichia coli (E.coli) isolated from the cloacae of chickens that exhibited resistance to at least one of the commonly used antibacterial agents (ABA). Isolates were tested for antibacterial resistance using the Kirby-Bauer disk diffusion method. Ninety nine E.coli isolates expressed antibacterial resistance to at least one ABA and were tested for the presence of 13 virulence genes using PCR analysis. Nine enterotoxigenic E.coli (ETEC) and 5 enteropathogenic E.coli (EPEC) isolates were detected. All 9 ETEC-positive isolates were resistant to tetracycline (TE), 3 were resistant to sulfamethoxazole/trimethoprim (SXT) and 1 to ciprofloxacin (CIP). All 5 EPEC-positive isolates were resistant to TE, 4 were resistant to gentamicin (GN), 4 were resistant to SXT and 3 to CIP. These results are of public health concern since resistant isolates that harbor virulence genes could end up in food consumed by
386 Fadi E. El-Rami et al

humans. There did not appear to be an association between antibacterial resistance to
SXT, GN, or CIP and virulence traits.
Keywords: Virulence, antibacterial resistance, Escherichia coli, EPEC, ETEC

Introduction

Various disease outbreaks have been reported to be due to ingestion of food
contaminated with pathogenic Escherichia coli strains [12, 18, 20, 21]. In May 2011
in Germany there was an outbreak caused by E. coli strain O104:H4 that resulted in
810 cases of haemolytic uraemic syndrome and 39 deaths [9, 16].
The development and dissemination of antibacterial resistance (ABR) among
enteric bacteria isolated from poultry attracts attention due to its direct influence on
public health, through elevating the morbidity, mortality, and treatment costs of
infectious diseases [25].
E.coli represents a good candidate for the study of virulence and antibacterial
resistance. Although existing mostly as commensals, E.coli can become pathogenic
upon acquisition of virulence attributes, such as enterotoxins and adhesion or
invasion factors [6], and result in enteric/diarrhoeal disease, urinary tract infection,
and sepsis/meningitis [3]. Pathogenic E.coli are classified into 5 pathotypes based
on their virulence traits [14]: enterotoxigenic E. coli (ETEC), enteropathogenic E.
coli (EPEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), and
enteroaggregative E. coli (EAEC).
The aims of this study were to determine if E. coli isolated from cloacae of
chickens that were resistant to at least one ABA belonged to any one of the 5
pathotypes and if so, to see if there was an association between the pathotype and
ABR.

Materials and Methods

Specimens
Cloacae swabs were obtained from 103,000 layers housed by 6 farms (farms A,
B, D, E, I and J) and 36,000 broilers housed by 4 farms (farms C, F, G and H).
The average age of layers was 9.2 ± 7.7 months and that of broilers 1.5 ± 1.47
months.
Isolation and identification of E. coli
All specimens were cultured on MacConkey agar plates (BBL, Becton
Dickinson Microbiology Systems, Cockeysville, MD) and the suspected E.coli

Identification of virulence genes 387

colonies were sub-cultured on MacConkey agar plates. The final pure cultures were
identified using the API 20E kit (BioMerieux, Paris, France) and tested for
antimicrobial susceptibility.

Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was done using the Kirby-Bauer disk
diffusion method following the Clinical and Laboratory Standards Institute, CLSI
guidelines for inoculums' standardization, medium, and incubation conditions, and
E.coli ATCC 25922 used as a control. All isolates were tested for six antibacterial
agents (Oxoid, Basingstoke, UK): amoxicillin/clavulanic acid (AMC, 30 µg ml-1),
ceftriaxone (CRO, 30 µg ml-1), gentamicin (GN, 10 µg ml-1), tetracycline (TE, 30 µg
ml-1), ciprofloxacin (CIP, 5 µg ml-1), and sulfamethoxazole/ trimethoprim (SXT, 25
µg ml-1).

PCR assays
A multiplex PCR method was performed to investigate the presence of virulence
genes. The primers used are listed in Table 1. E.coli isolates that exhibited resistance
to at least one ABA were studied. E.coli template DNA were obtained from pure
cultures of E.coli isolates grown overnight on MacConkey agar plates. Few colonies
were added to 0.5% Triton-X 100, boiled at 94 °C for 20 minutes, centrifuged, and
the supernatant was used as PCR template. Reference strains obtained from Centers
for Disease Control and Prevention (CDC, Atlanta, GA) were used as positive
controls as follows: H10407 (O78:H11) for ETEC, B170 (O111:NM) for EPEC
strains, 3591-78 (O75:NM) for EAEC, C4193-1 (O157:H7) for EHEC- particularly
O157;H7, and TD 213 (O124:NM) for EIEC.
Each PCR tube contained 50 µl of reaction mixture, composed of: Taq
polymerase (0.3 µl), 10x buffer (5 µl), dATP, dCTP, dGTP, dTTP (1.25 µl of each),
MgCl2 (3 µl), and a mixture of 26 primers (Table 1). All were purchased from
Thermofisher scientific, Germany. Six micro liter of E.coli template DNA was added
to the final PCR mixture.
The PCR cycling conditions adopted by the thermal cycler (Thermo Electron
corp., Milford, USA) were as follows: denaturation at 94 °C (35 cycles, 1.5 minutes
each), annealing at 55 °C (35 cycles, 1.5 minutes each), and strand elongation at
72 °C (1.5 minutes each). The PCR products were detected by electrophoresis (Owl
scientific plastics, USA) and visualized under UV light illumination (UVP Bio-
imaging systems, USA).

Results
Number of E. coli-resistant isolates
A total of 99 E. coli isolates were resistant to at least one antibacterial agent. The
majority of the isolates were resistant to TE (98.99%) followed by SXT (77.78%),

388 Fadi E. El-Rami et al

CIP (57.58%), GN (35.35%) and AMC (4.04%). None of the isolates were resistant
to CRO (Table 2).

E. coli-resistant isolates harboring virulence genes and their antibacterial
resistant patterns.
Of the 99 tested E.coli-resistant isolates, nine ETEC and 5 EPEC strains were
identified. All ETEC strains were located in layers farms, while 4 EPEC strains were
located in layers' farms and one EPEC isolate was located in broilers' farm (Figure
1and 2). No EAEC, EIEC, or EHEC isolates were found in any poultry farm studied.
All nine ETEC-positive isolates were resistant to TE. In addition, 3 were resistant
to SXT and 1 to CIP. Likewise, all 5 EPEC-positive isolates were resistant to TE. In
addition, 4 were resistant to SXT and 3 to CIP (Table 3).
Association between resistance and virulence genes

There didn't seem to be an association between resistance to GN, CIP or SXT and
the presence of a virulent gene. On the contrary, susceptible isolates having virulence
genes were more. In as much as TE was concerned, 14 isolates possessed a virulent
gene, Only 1 isolate was susceptible to TE and did not possess a virulent gene (Table
4).
Discussion

Food borne diseases are of utmost concern for public health authorities due to
their direct impact on consumer health. The feasible transfer of virulent and/or
antibacterial resistant organisms from food animals to humans due to direct contact
or food ingestion is well established [15]. It is conceivable that commensals, such as
E. coli, constitute a colonization barrier against pathogenic intruders, but if they
acquire virulence genes this would destroy their role as one of the first lines of
defense against pathogens [10]. Identifying the degree of antibacterial resistance as
well as virulence among the E.coli, as enteric commensals, of chickens draws a
better risk assessment image for future food borne infections related to poultry.
Antibacterial resistance is a global concern due to the uncontrolled increment,
ease of spread on mobile genetic elements, and its detrimental effect on public
health. The recorded resistance rates among chickens commensal E.coli against TE
(98.99%), SXT (77.78%), CIP (57.58%), and GN (35.35%) were remarkable and
alarming, due to the fact that these antibacterial agents are commonly used
therapeutically to treat human infections [15]. Moreover, some antibacterial agents
are administered to poultry as growth promoting factors and an earlier study

Identification of virulence genes 389

established an association between administration of TE and GN, and emergence of
resistance [8].
Virulence determinants of E. coli are other factors of concern. Out of the
thirteen virulence genes studied, 9 of 16 E.coli isolates in farm A possessed the
ETEC gene (Figure 1), four of 21 E.coli isolates in farm D possessed the EPEC gene
(Figure 2), and 1 of 9 E.coli isolates in farm F possessed the EPEC gene, reflecting a
high degree of clonality [26] which contributes to the vast dissemination of
pathogenicity traits according to the "clone concept" [13].
It is possible that an antibacterial resistant gene and a virulence gene are located
on the same transferable plasmid. A number of reports have attempted to establish an
association between resistance to an ABA and the presence of a virulent gene [1, 2,
5, 7, 19, 22]. However, our results do not indicate such an association with respect to
isolates resistant to SXT, CIP or GN. On the contrary, the number possessing
virulent genes was more in isolates that were susceptible to these ABA. In as much
as TE was concerned, there might be an association since virulence genes were
detected in resistant isolates and not in the one susceptible isolate. However, an
association could not be confirmed because only one isolate was susceptible to TE.
An interesting observation is that all ETEC and most EPEC isolates were
detected in layers, and only one EPEC isolate was identified among broilers. The
consequence of detecting pathogenic E. coli in healthy layers is deleterious, where
the continuous delivery of eggs and food products to humans will provide a
horizontal transfer of virulence and antibacterial resistance traits to human
populations [4, 15, 24].
In conclusion, the current study confirms the presence of virulent
antibacterial resistant E. coli, namely ETEC and EPEC strains in healthy chickens.
Such findings are indicative of a health hazard to humans since chicken and eggs are
commonly consumed foods.

References
[1] A.Franklin , R. Möllby ,Concurrent transfer and recombination between plasmids
encoding for heat-stable enterotoxin and drug resistance in porcine enterotoxigenic
Escherichia coli. Medical Microbiology and Immunology, 172 (1983), 137-147
[2] A.J. Mazaitis , R.Maas , W.K.Maas, Structure of a naturally occurring plasmid
with genes for enterotoxin production and drug resistance, Journal of Bacteriology,
145(1981),97-105
[3] A.M. Hammerum, O. Hueur , Human Health Hazards from Antimicrobial-
Resistant Escherichia coli of Animal Origin, Clinical Infectious Diseases,
48(2009)(7), 916-921
[4] B.Finlay,S. Falkow ,Common themes in microbial pathogenicity revisited.
Microbiology and Molecular Biology Review, 61(1997),136–169.

390 Fadi E. El-Rami et al

[5] C.L.Gyles , S. Palchaudhuri , W.K. Maas, Naturally occurring plasmid carrying
genes for enterotoxin production and drug resistance, Science, 198 (1977),198-199
[6] E.A. Garcia , Animal health and foodborne pathogens: enterohaemorrhagic
O157:H7 strains and other pathogenic Escherichia coli virotypes (EPEC, ETEC,
EIEC, EHEC), Polish Journal of Veterinary Sciences, 5(2002),103–115.
[7] F. Diarrassouba, M.S. Diarra, S. Bach, P. Delaquis , J. Pritchard ,E. Topp, B.J.,
Antibiotic resistance and virulence genes in commensal Escherichia coli and
Salmonella isolates from commercial broiler chicken farms. Journal of Food
Protection, 70(2007)(6),1316-27.
[8] F.E. El-Rami, F.T. Sleiman, A.M. Abdelnoor, Identification and Antibacterial
Resistance of Bacteria Isolated from Poultry, Polish Journal of Microbiology, In
Press (2012) .
[9] F.Scheutz , E.Møller Nielsen , J. Frimodt-Møller, N. Boisen, S. Morabito, R.
Tozzoli, J.P.Nataro,A. Caprioli, Characteristics of the enteroaggregative Shiga
toxin/verotoxin-producing Escherichia coli O104:H4 strain causing the outbreak of
haemolytic uraemic syndrome in Germany, May to June 2011. European
Surveillance, 16(2011)(24)
[10] H.Ochman, G. Lawrence , E.A.Groisman , Lateral gene transfer and the nature
of bacterial innovation, Nature, 405(2000), 299–304.
[11] J. Franke, S. Franke ,H. Schmidt, A.Schwarzkopf , L.H.Wieler ,G. Baljer , L.
Beutin, H. Karch H, Nucleotide sequence analysis of enteropathogenic Escherichia
coli (EPEC) adherence factor probe and development of PCR for rapid detection of
EPEC harboring virulence plasmids, Journal of Clinical Microbiology, 32(1994)(10),
2460-3.
[12] J.Meng , M.P.Doyle, Emerging and evolving microbial foodborne pathogens.
Bulletin de l'Institut Pasteur, 96 (1998)(3),151-163.
[13] J.M. Smith, N.H.Smith, M. O'Rourke, B.G. Spratt, How clonal are bacteria?
Proceedings of the National Academic of Sciences of the United States of America,
90(1993), 4384–4388.
[14] J.P.Nataro, J.B. Kaper, Diarrheagenic Escherichia coli, Clinical Microbiology
Reviews, 11(1998), 142–201.
[15] K.Mølbak , Spread of Resistant Bacteria and Resistance Genes from Animals to
Humans ? The Public Health Consequences. Journal of Veterinary Medicine, Series
B, 51(2004)(8-9), 364-369.
[16] M. Bielaszewska,A. Mellmann, W. Zhang, R. Köck ,A. Fruth, A. Bauwens,G.
Peters, H. Karch, Characterisation of the Escherichia coli strain associated with an
outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological
study.
doi:10.1016/S1473-3099(11)70165-7
[17] M.Vidal , E. Kruger , C. Durán, R. Lagos , M. Levine , V. Prado , C. Toro , R.
Vidal, Single multiplex PCR assay to identify simultaneously the six categories of

Identification of virulence genes 391

diarrheagenic Escherichia coli associated with enteric infections, Journal of Clinical
Microbiology 43(2005),5362–5365.
[18] National Institute of Health and Infectious Diseases Control Division, Ministry
of Health and Welfare of Japan, 1996 National Institute of Health and Infectious
Diseases Control Division, Ministry of Health and Welfare of Japan, Outbreak of
Enterohemorrhagic Escherichia coli O157:H7 Infection, Infectious Agents
Surveillance Report vol. 174 (1996), 180–181.
[19] N.M.Harnett, C.L.Gyles, Linkage of genes for heat-stable enterotoxin, drug
resistance, K99 antigen, and colicin in bovine and porcine strains of enterotoxigenic
Escherichia coli. American Journal Veterinary Research, 46(1985),428-433
[20] N.Pradel , V. Livrelli, C. De Champs, J.B. Palcoux, A. Reynaud , F. Scheutz ,J.
Sirot, B. Joly, C. Forestier , Prevalence and characterization of Shiga toxin-
producing Escherichia coli isolated from cattle, food, and children during a one-year
prospective study in France, Journal of Clinical Microbiology, 38(2000), 1023–1031.
[21] Ø. Olsvik, Y. Wasteson ,A. Lund, E. Hornes, Pathogenic Escherichia coli found
in food, International Journal of Food Microbiology, 12(1991)(1), 103-113.
[22]P.Boerlin, R. Travis, C.L. Gyles, Antimicrobial resistance and virulence genes of
Escherichia coli isolates from swine in Ontario, Applied and Environmental
Microbiology, 71 (2005)(11), 6753–6761.
[23] P.K. Fagan, M.A. Hornitzky, K.A. Bettelheim,S.P. Djordjevic, Detection of
shiga-like toxin (stx1 and stx2), intimin (eaeA), and enterohemorrhagic Escherichia
coli (EHEC) hemolysin (EHEC hlyA) genes in animal feces by multiplex PCR,
Applied Environmental Microbiology, 65(199), 868-872.
[24] R.D.Berg, The indigenous gastrointestinal microflora, Trends in Microbiology,
4(1996), 430-435.
[25] R.E.Warren , V.M. Ensor, P. O'Neill, Imported chicken meat as a potential
source of quinolone-resistant Escherichia coli producing extended-spectrum β-
lactamases in the UK. Journal of Antimicrobial Chemotherapy, 61(2008), 504-8.
[26] R.K.Selander, B.R. Levin , Genetic diversity and structure in Escherichia coli
populations. Science, 210(1980), 545–547.
[27]S.Stacy-Phipps , J.J.Mecca, J.B.Weiss, Multiplex PCR assay and simple
preparation method for stool specimens detect enterotoxigenic Escherichia coli DNA
during the course of infection. Journal of Clinical Microbiology, 33(1995),1054–
1059.
[28] T.A. Cebula, W.L. Payne, P. Feng, Simultaneous identification of strains of
Escherichia coli serotype O157:H7 and their Shiga-like toxin type by mismatch
amplification mutation assay-multiplex PCR, Journal of Clinical Microbiology, 33
(1995), 248-250

392 Fadi E. El-Rami et al

TABLE 1. Targets and primer sequences used in the detection of virulence genes of
Escherichia coli.

Pathotype Target
product size (bp) GCA CAC GGA GCT CCT CAG TC TCC TTC ATC CTT TCA ATG GCT TT stII- F AAA GGA GAG CTT CGT CAC ATT TT stII- R AAT GTC CGT CTT GCG TTA GGA C CTC GGC ACG TTT TAA TAG TCT GG GTG GAG AGC TGA AGT TTC TCT GC AGC TCA GGC AAT GAA ACT TTG AC TGG GCT TGA TAT TCC GAT AAG TC CAC AGG CAA CTG AAA TAA GTC TGG ATT CCC ATG ATG TCA AGC ACT TC GAA CGT TGG TTA ATG TGG GGT AA TAT TCA CCG GTC GGT TAT CAG T eae- F TCA ATG CAG TTC CGT TAT CAG TT eae- R GTA AAG TCC GTT ACC CCA ACC TG CAG GGT AAA AGA AAG ATG ATA A TAT GGG GAC CAT GTA TTA TCA GGA AGT CAA ATT CAT GGG GGT AT bfp- R GGA ATC AGA CGC AGA CTG GTA GT uidA- F GCG AAA ACT GTG GAA CTG GG TGA TGC TCC ATA ACT TCC TG eae- F TCA ATG CAG TTC CGT TAT CAG TT eae- R GTA AAG TCC GTT ACC CCA ACC TG ACG ATG TGG TTT ATT CTG GA CTT CAC GTC ACC ATA CAT AT stx1-F CAG TTA ATG TGG TGG CGA AGG stx1-R CAC CAG ACA ATG TAA CCG CTG stx2-F ATC CTA TTC CCG GGA GTT TAC G stx2-R GCG TCA TCG TAT ACA CAG GAG C Abbreviations :enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), and enteroaggregative E. coli (EAEC)
Identification of virulence genes 393
TABLE 2. Source and type of birds, and number of E.coli isolates from each source
resistant to each antibacterial agent tested.
Farm Type Resistant Number of isolates resistant to Abbreviations: AMC: amoxicillin/clavulanic acid; GN: gentamicin; TE: tetracycline; CIP: ciprofloxacin; SXT: sulfamethaxazole/trimethoprim; CRO: ceftriaxone. * Resistant E.coli isolates to at least one ABA.
394 Fadi E. El-Rami et al

TABLE 3. Resistance profiles of pathogenic Escherichia coli isolated from poultry.

Isolate Pathotype Isolate ABA
Antibacterial Resistance Profile source exposure* A11 ETEC Layer TE A12 ETEC Layer TE *ABA exposure = antibacterial agent used as a food growth promoting factor.
TE = tetracycline. GN = Gentamicin. AMC= amoxicillin/clavulanic acid; CIP=
ciprofloxacin; SXT= sulfamethaxazole/trimethoprim; CRO= ceftriaxone.
R = resistant. ETEC = enterotoxigenic E. coli, EPEC = enteropathogenic E. coli.
TABLE 4. Resistance and susceptibility profiles of virulent and avirulent
Escherichia coli isolated from poultry.
ABA Number
Resistant and Susceptible Resistant and Susceptible and possess did not virulent gene* virulent gene TE 14 0 84 1 *Virulent gene for EPEC or ETEC. Abbreviations : ABA = antibacterial agent. TE = tetracycline. GN = Gentamicin. CIP= ciprofloxacin; SXT= sulfamethaxazole/trimethoprim. ETEC = enterotoxigenic E. coli, EPEC = enteropathogenic E. coli. Total number of pathogenic E.coli = 14. Total number of isolated E.coli =99.
Identification of virulence genes 395


Figure 1. Gel image of ETEC isolates (A: Lanes A1, 2, 3, 4, 5, 7, 8, 11) and
reference strains ( B: EPEC, EAEC, ETEC, EIEC, O157;H7). Lane L: 100
bp ladder. Abbreviations: enterotoxigenic E. coli (ETEC), enteropathogenic E.
coli (EPEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli
(EHEC), enteroaggregative E. coli (EAEC).

396 Fadi E. El-Rami et al


Figure 2. Gel image of EPEC isolates (A: Lanes D12, 13, 14, 16) and
reference strains ( B: EPEC, EAEC, ETEC, EIEC, O157;H7). Lane L: 100
bp ladder. Abbreviations: enterotoxigenic E. coli (ETEC), enteropathogenic E.
coli (EPEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli
(EHEC), enteroaggregative E. coli (EAEC).

Received: April, 2012

Source: http://modul.mercubuana.ac.id/files/openjournal/Journal%20Of%20Visual/learner/abdelnoorASB5-8-2012.pdf