Virulence Factors Associated with Escherichia coli ...

Virulence Factors Associated with Escherichia coli Present in a Commercially Produced Competitive Exclusion Product Author(s): John J. Maurer, Charles L. Hofacre, Richard E. Wooley, Penelope Gibbs and Robrecht Froyman Source: Avian Diseases, Vol. 46, No. 3 (Jul. - Sep., 2002), pp. 704-707 Published by: American Association of Avian Pathologists Stable URL: http://www.jstor.org/stable/1592952 Accessed: 11-05-2018 14:38 UTC JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected]. Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://about.jstor.org/terms

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AVIAN DISEASES 46:704-707, 2002

Research NoteVirulence Factors Associated with Escherichia coli

Present in a Commercially Produced Competit Exclusion Product

John J. Maurer,A Charles L. Hofacre,A Richard E. Wooley,B Penelope Gi Robrecht Froymanc ADepartment of Avian Medicine BDepartment of Medical Microbiology and Parasitology College of Veterinary Medicine, The University of Georgia, Athens, GA 30602 CBayer AG, 51368 Leverkusen, Bayerwerk, Germany Received 19 October 2001

SUMMARY. In this study, we assessed the pathogenic potential of Escherichia coli associated with a commercial competitive exclusion (CE) product by examining the phenotypic

characteristics associated with E. coli virulent for humans and domestic animals. Most E. coli

isolates were capable of proliferating in iron-deplete chicken sera. Interestingly, none of the E. coli isolates from the commercial CE product contained the bacterial adhesin Tsh characteristic of avian pathogenic E. coli associated with airsacculitis and colisepticemia. In terms of virulence potential for humans, most E. coli isolates (78%) were sensitive to killing by 12.5% human sera. Because of their sensitivity to human sera, the E. coli in the CE product are not likely to cause a serious systemic infection in humans and, therefore, do not present a risk of causing septicemia in humans. Because these isolates also lack the gene tsh, they are also less likely to cause systemic disease or airsacculitis in poultry than pathogenic strains commonly isolated from diseased birds.

RESUMEN. Nota de Investigacidn-Factores de virulencia asociados con Escherichia coli presentes en un producto de exclusi6n competitiva producido comercialmente. Se investig6 el potencial pat6geno de Escherichia coli asociado con un producto comercial de exclusi6n competitiva, mediante el examen de las caracteristicas fenotipicas asociadas con el E. coli virulento para humanos y animales domesticos. La mayoria de las cepas de E. coli fueron capaces de proliferar en suero de polio sin presencia de hierro. Ninguno de los aislados de E. coli del producto de exclusi6n competitiva contenian la adhesina bacteriana Tsh, caracteristica de las cepas pat6genas de E. coli asociadas con aerosaculitis y colisepticemia. En terminos de la virulencia potencial para humanos, la mayoria (78%) de los aislados de E. coli fueron destruidos mediante el tratamiento con 12.5% de suero humano. Debido a esta

sensibilidad contra el suero humano, las cepas de E. coli presentes en el producto de exclusi6n competitiva posiblemente no causen una infecci6n sistemica en humanos y por lo tanto, no presentan un riesgo de septicemia en humanos. Debido a que estos aislados no tienen el gen tsh, existe menor posibilidad de que causen enfermedad sistemica o aerosaculitis en aves como la causada por las cepas pat6genas aisladas de aves enfermas. Key words: competitive exclusion, serum resistance, pathogenic E. coli, siderophore, tsh

Abbreviations: CAS = chrome azurol S; CE = competitive exclusion; PG = peptone

glucose

With public concern over food safety and cessing plants, there is considerable interest in federal mandate for meat processors to monitor affordable methods or products that reduce coliform and Salmonella levels in poultry-pro- contamination of food animals by human path704 This content downloaded from 198.137.18.158 on Fri, 11 May 2018 14:38:18 UTC All use subject to http://about.jstor.org/terms

Virulence factors of E. coli in CE product

ogens on the farm. Animals are most susceptible to colonization with Salmonella at an early age. By providing animals at birth with "good"

microflora present in competitive exclusion (CE) products, colonization of food animals by

Salmonella can be prevented (15). The CE cul-

705

PG broth alone in a 96-well sterile microtiter plate. The plate was incubated at 37 C for 5 hr, and optical

density was measured at a wavelength of 490 nm with a microtiter plate reader. A strain was considered sensitive if no growth was apparent in PG broth with chicken serum. Resistant E. coli had densities in sera

comparable with those observed for the no serum control. Avian E. coli V1 (19), Salmonella typhimuspecies of bacteria present provide the greatest rium SRI 1, and E. coli K12 laboratory strain HB101

tures that are undefined as to the number or

protection from Salmonella (16). However,were

included in this assay as the serum-resistant and

-sensitive controls. there is concern over the safety of CE products and whether administration may result in the Siderophore assay. Escherichia coli isolates were assayed for siderophore production by a coinadvertent contamination of food animals lormetric assay developed by Schwyn and Neilands with a human or animal pathogen present in (14). Bacteria were plated onto chrome azurol S the CE product. Because an undefined CE (CAS) agar and plates were incubated at 37 C for product ultimately consists of the gastrointes24 hr. Enterobactin-minus Salmonella strain LT-2 tinal microflora of healthy chickens (11), Eschenb-7 and an enterobactin-producing E. coli strain

erichia coli is expected to be present in AN this 194 were included as negative and positive con-

product because the organism is a constituent, trols, respectively (15). Siderophore-producing E. although minor part, of the animals' microflora coli produce an orange halo around colonies grown on to CAS agar, whereas nonproducers appear blue (18). This organism varies in its potential on this medium. cause disease, existing as commensal, opportunScreening of E. coli isolates for the avian ist, or pathogen in its animal host (2,9). Disease pathogenic E. coli hemagglutinin gene, tsb. outcome is ultimately dictated by both the orPresence or absence of tsh among E. coli was deterganism's genetic potential and the predisposimined by Southern analysis with a digoxigenin-lation of the animal health as defined by its gebeled DNA probe generated from the amplification

netics, environment, and physiologic state of the tsh gene by polymerase chain reaction (10). (1,3). It is therefore not uncommon to find Colonies were patched onto nylon membranes and

pathogenic strains harbored by a healthy animal processed for Southern hybridizations as described by host (1,3,7). Without characterization of E.Sambrook coli et al. (13). The DNA:DNA hybridizations and washes were done at 68 C. Hybridizing DNA component of CE product, poultry producers fragments were detected with anti-DIG antibody-alrun the risk of introducing not only a potential kaline phosphatase conjugate (Boehringer-Mannhuman pathogen but a strain virulent for heim, healthy birds as well. In this study, we assessed Indianapolis, IN). Avian E. coli 97-2716 and laboratory strain E. coli K12 LE392 served as positive

the pathogenic potential of E. coli associated and negative controls, respectively, for DNA:DNA with the CE product AVIGUARD. hybridizations for tsh (10).

MATERIALS AND METHODS

RESULTS AND DISCUSSION

Escherichia coli isolates. We isolated E. coli

Several phenotypic characteristics wer from a commercial CE product, AVIGUARD?

amined to determine whether the compe (Bayer, Bayerwerk, Germany), by reconstituting exclusion product AVIGUARD? could r the product in sterile water and plating the rehy-

drated product onto MacConkey agar. An indole pathogenic E. coli colonizing chickens wi

and oxidase test was performed on lactose-positive innocuous E. coli population that is inc colonies. Sixty isolates were propagated forof further surviving and proliferating in human

study. Serum resistance. We assessed the resistance of

For bacteria to become septicemic in a

malian or avian host, they must be capab E. coli to chicken and human serum by the microtiter growing in an iron-limiting environmen

method described by Lee et al. (8). Briefly, bacteria Many bacteria acquire "sequestered" ir were grown as a standing overnight culture at 37 C

their immediate environment by prod in peptone glucose (PG) broth. The cultures were hydroxamate and catechol siderophores, bactin and enterobactin. Fifty-six of 58 ul1 of 25% chicken or human serum in PG broth isolates or (96%) were capable of producing

diluted 1:1000 in PG broth. One hundred microliters of the diluted culture was used to inoculate 100


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J. J. Maurer et al.

rophores. Two isolates did not grow on the

caps the lipopolysaccharide of gram-negative

bacteria (17). Many commensal E. coli are typeable with regard to the O-antigen (4), therefore ed for clinical and commensal E. coli isolated it is not surprising to find that they are also from broiler chickens. However, this difference serum resistant. Despite the relative resistance was small, with 90% and 75%, respectively, of of these E. coli isolates to killing by chicken the virulent E. coli and normal intestinal E. coli complement, most isolates (78%) were sensitive being positive for siderophores (17). This mark- to human sera. er alone is not sufficient to determine virulence Although some of the E. coli associated with because iron in an oxygenic environment is nat- the CE product were serum resistant, they urally limiting to most bacteria, pathogenic or lacked the signature adhesin gene tsh, which is otherwise, and microorganisms respond by pro- believed to be important in the ability of the

minimal medium used for this assay. Differences in siderophore production have been report-

ducing iron-scavenging compounds like side- organism to colonize its avian host and cause

rophores (4).

airsacculitis or colibacillosis (12). However, oth-

In addition to being able to scavenge iron er virulence factors, like eaeA and tir, may be away from transferrin and lactoferin in the present that may contribute to other poultry

blood, bacteria must be able to survive in the diseases (5). The E. coli associated with this CE presence of bacteriocidal agents in serum, most product are also less likely to cause septicemia notably complement. We examined the ability in humans because of their sensitivity to human of these E. coli isolates to survive and proliferate serum. This step is the first in determining the in chicken sera. Sixty percent of E. coli isolates relative safety of CE product for humans and were resistant to 12.5% chicken sera. Another animals by characterizing genes or factors as13% of these isolates exhibited an intermediate sociated with the resident bacteria. resistance to the serum, whereas only 27% of avian E. coli isolates were sensitive. Wooley et

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