summary writing

ORIGINAL RESEARCH ARTICLE

Hygiene quality and presence of ESBL-producing Escherichia coli in raw food diets for dogs

Oskar Nilsson, DVM, BSc, PhD*

Department of Animal Health and Antimicrobial Strategies, National Veterinary Institute, Uppsala, Sweden

Background: Raw food diets are popular among some dog owners, even though there are concerns regarding

the infectious disease risk and public health implications. Hence, the two aims of this study were to investigate

the hygiene quality of raw food diets for dogs in the Swedish market and if Escherichia coli with transferable

resistance to extended spectrum cephalosporins (ESC) was present in such products.

Methods: Samples of raw food diets were suspended and further diluted in 0.9% saline. Appropriate dilutions

were 1) cultured on PetrifilmTMSEC to quantify the amount of E. coli in the samples and 2) mixed with

cefotaxime to a final concentration of 1 mg/L and cultured on PetrifilmTMSEC to quantify the amount of

ESC-resistant E. coli in the samples. Furthermore, undiluted suspensions were mixed 1:1 with double strength

MacConkey broth with cefotaxime, enriched overnight and finally cultured on MacConkey agar with

cefotaxime (1 mg/L). Suspected ESC-resistant E. coli were screened by PCR for genes encoding extended

spectrum beta lactamases and plasmid-mediated AmpC and their susceptibility to a panel of antimicrobials

was performed by broth microdilution using VetMIC GN-mo.

Results: Escherichia coli was isolated from all samples (n�39) and ESC-resistant E. coli was isolated from

nine samples (23%). All ESC-resistant E. coli were PCR-positive for the blaCMY-2 group and only one of them

was also resistant to a non-beta-lactam antibiotic.

Conclusion: The results of this study indicate that raw food diets could be a source of ESC-resistant E. coli to

dogs and highlight the need for maintaining good hygiene when handling these products to prevent infection.

Keywords: BARF; raw food diet; ESBL; AmpC; E. coli

*Correspondence to: Oskar Nilsson, Department of Animal Health and Antimicrobial Strategies, National

Veterinary Institute, SE-751 89 Uppsala, Sweden, Email: [email protected]

Received: 4 June 2015; Revised: 23 September 2015; Accepted: 25 September 2015; Published: 20 October 2015

R aw food diets are popular among some dog owners,

despite concerns regarding infectious disease risk

and public health (1, 2). The focus has mainly

been regarding the presence of Salmonella spp. as a

potential pathogen (3�7). Another concern would be

the presence of antibiotic-resistant bacteria, for example,

Enterobacteriaceae with transferable resistance to ex-

tended spectrum cephalosporins (ESC) due to extended

spectrum beta lactamases (ESBL) or plasmid-mediated

AmpC (pAmpC). Bacteria with such resistance are a

problem in human medicine and have been identified

in farm animals as well as in raw food diet products (3, 8).

In Sweden and Norway, ESBL and pAmpC are only

occasionally isolated from farm animals other than broilers

(9, 10). In Finland, the occurrence of ESBL and pAmpC

in poultry is lower than in many other countries (11).

As by-products from broiler slaughter are sometimes

used to manufacture raw food diet products, we identified

such diets as a potential source of ESBL and pAmpC to

dogs (12). It could also act as a source of antibiotic-

resistant microorganisms for people caring for the dogs

(13). The aim of this study was to investigate if Escherichia

coli with transferable resistance to ESC was present in

raw food diets for dogs in the Swedish market. In addition

the hygiene quality of the products was assessed by quan-

tification of E. coli present in the raw food diets.

Materials and methods Frozen samples of raw food diet products containing

poultry were purchased in shops in and around Uppsala,

Sweden, and from one Swedish internet shop, and stored

at �208C until analysed. Samples were thawed at 38C;

25 mL of saline (0.9%) was added to 25 g of feed and

treated for 30 sec at 230 rpm in a Stomacher (Stomacher

400 circulator, Seward, UK) before ]7 mL of liquid was

removed. Dilutions of 1:10 and 1:1,000 were prepared in

0.9% saline for quantitative isolation of E. coli and 1 mL of

each dilution was cultured on PetrifilmTMSEC (3M Health

infection ecology & epidemiology

T h e O n e H e a l t h J o u r n a l

�

Infection Ecology and Epidemiology 2015. # 2015 Oskar Nilsson. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Citation: Infection Ecology and Epidemiology 2015, 5: 28758 - http://dx.doi.org/10.3402/iee.v5.28758 (page number not for citation purpose)

Care, St Paul, USA) and incubated at 378C overnight.

For quantitative isolation of ESC-resistant E. coli, a 1 mL

aliquot of the undiluted suspension was mixed with 10 mL

of cefotaxime solution (0.1 mg/mL; Sigma Aldrich, China)

to create a final concentration of 1 mg/L before the mixture

was cultured on PetrifilmTMSEC as above. For qualitative

isolation of ESC-resistant E. coli, 5 mL of double strength

MacConkey broth (Lab M, Lancashire, UK) with cefo-

taxime (2 mg/L) was added to a 5 mL aliquot and in-

cubated at 378C overnight before 0.1 mL was streaked on

MacConkey agar (Difco, Hampshire, UK) with cefotax-

ime (1 mg/L) and incubated at 378C overnight.

Suspected E. coli colonies were confirmed by sub-

culture on horse blood agar (Oxoid, Basingstoke, UK) and

an indole test conducted. Furthermore, suspected ESC-

resistant E. coli were screened by PCR for genes encoding

ESBL and pAmpC, that is, the gene groups CTX-M-1,

CTX-M-2, CTX-M-8, CTX-M-9, CTX-M-25/26, MOX,

CIT, DHA, ACC, EBC, FOX, OXA-1, SHV, and TEM

(14). The number of E. coli or ESC-resistant E. coli on

each PetrifilmTMSEC was recorded.

Susceptibility testing of all ESC-resistant E. coli to a

panel of antimicrobials was performed by broth micro-

dilution using VetMIC GN-mo (SVA, Uppsala, Sweden)

according to standards of the CLSI (15). The E. coli

reference strains ATCC 25922 were used for quality

control and the results were interpreted according to

epidemiological cut-off values issued by EUCAST (www.

eucast.org, retrieved 2015-09-17; Table 1).

Results Altogether, 39 samples of eight different raw dog food

brands (1�11 samples/brand) were analysed. Depending on

the brand, the products contained by-products from animals

slaughtered in Finland (n�10), Norway (n�11), or Sweden

(n�18). Of the 39 samples, 22 contained only by-products

from poultry, whereas 17 contained by-products from

several animal species including poultry.

Escherichia coli was isolated from all 39 samples. In

34 (87%) of the samples, the amount of E. coli was higher

than 5�101 CFU/g, in 19 (49%) the amount was higher

than 5�102 CFU/g, and in 2 (5%) the amount was

higher than 5�104 CFU/g.

ESC-resistant E. coli was isolated from nine samples

(23%) and all isolates were PCR-positive for the blaCMY-2

group. The products with ESC-resistant E. coli originated

from Norway (n�3, 27%) or Sweden (n�6, 33%) and

contained either only poultry by-products (n�7, 32%)

or by-products from several animal species (n�2, 12%).

The number of colony forming units (CFU) of ESC-

resistant E. coli/gram could be calculated in five of the

nine samples, all of which were 510 CFU/g. All of these

samples were products that contained by-products exclu-

sively from poultry.

Only one of the ESC-resistant E. coli was also resistant

to a non-beta-lactam antibiotic (Table 1). That isolate

was also resistant to kanamycin.

Discussion It was not unexpected that ESC-resistant E. coli could be

isolated from raw food diet products containing poultry.

ESC-resistant E. coli bacteria is commonly isolated from

poultry in Europe and ESC-resistant Enterobacteriaceae

in raw food diet products has previously been described

(3, 8). Furthermore, studies have demonstrated an increased

risk of shedding of ESC-resistant Enterobacteriaceae

Table 1. Antibiogram of the nine Escherichia coli isolates with transferable resistance to extended spectrum cephalosporins isolated

from raw food diets for dogs

Resistance pattern

Isolate Am Ctx Caz Ci Nal Gm Sm Tc Ff Cs Su Trim Cm Km

1 64 �2 8 0.06 4 1 16 51 8 50.5 16 0.25 4 58

2 128 �2 8 0.06 2 1 8 51 54 50.5 32 0.25 52 58

3 128 �2 8 0.06 2 0.5 8 51 54 50.5 16 0.5 4 58

4 64 �2 4 0.06 2 1 8 51 54 1 58 0.25 4 58

5 64 �2 8 0.06 4 1 8 51 54 2 16 0.5 4 58

6 64 �2 16 0.06 2 1 8 51 54 2 16 0.5 4 58

7 �128 �2 �16 0.06 4 1 8 51 54 1 58 0.5 4 58

8 128 �2 8 0.06 2 1 8 2 8 1 16 0.5 4 58

9 64 �2 4 0.06 4 0.5 8 51 8 1 16 0.25 4 16

Bold and shaded figures indicate MIC above the epidemiological cut-off values issued by EUCAST (www.eucast.org, retrieved

2015-09-17).

Antimicrobials included and cut-off values (mg/L) used are ampicillin (Am, �8), cefotaxime (Ctx, �0.25), ceftazidime (Caz, �0.5),

ciprofloxacin (Ci, �0.06), nalidixic acid (Nal, �16), gentamicin (Gm, �2), streptomycin (Sm, �16), tetracyclin (Tc, �8), florfenicol

(Ff, �16), coliston (Cs, �2), sulfamethoxazole (Su, �64), trimetoprim (Trim, �2), chloramphenicol (Cm, �16), kanamycin (Km, �8).

Oskar Nilsson

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Citation: Infection Ecology and Epidemiology 2015, 5: 28758 - http://dx.doi.org/10.3402/iee.v5.28758

among dogs fed raw food diets or raw meat in Canada

and the United Kingdom (16, 17). Together, this indicates

that raw food diets can also be a source of ESC-resistant

Enterobacteriaceae for dogs in Sweden. The risk probably

relates to feeding non-heat-treated animal products, re-

gardless of whether it is in the form of commercial diets

or not, as indicated by the previously reported increased

risk of shedding ESC-resistant E. coli in dogs fed raw meat

(17). In addition, the occurrence of ESC-resistant E. coli

in the products originating from Norway and Sweden in

this study is comparable to their prevalence in samples of

broiler meat in these two countries (9, 10).

Poultry from Finland, Sweden, or Norway was an

ingredient in all investigated products, and blaCMY-2 is the

dominant gene among ESC-resistant Enterobacteriaceae

from broilers in those countries (9�11). Therefore, it is

likely that most of the isolated ESC-resistant E. coli would

have a gene in the blaCMY-2 group. Although the isolates

were not sequenced in this study, one could speculate that

it will be the blaCMY-2 gene that confers resistance as

all ESC-resistant E. coli isolates from Swedish poultry

identified to date has carried blaCMY-2 (9). The resistance

pattern of the isolates is also similar to those which have

been described for isolates from poultry in Norway and

Sweden (9, 10).

The occurrence of E. coli in all products is comparable

to or higher than in previous studies (5, 18). The isolation

frequency of E. coli is comparable to that of broiler meat

95%), but considerably higher than that of pork (20%)

reported in the Swedish surveillance program Svarm (9).

Although E. coli could be isolated from all samples, it

should be noted that only two of the products contained

more E. coli than the maximum limit of coliform bacteria

(5�104 CFU/g) recommended by the Swedish Board of

Agriculture for animal feed materials of animal origin

(SJVFS 2011:40). Nineteen of the products contained

�5�102 CFU/g E. coli and would therefore have been

considered of unsatisfactory hygiene quality had they

been minced or mechanically separated meat intended for

human consumption (EC 2073/2005). According to this

regulation, a batch of minced or mechanically separated

meat is also classified as having unsatisfactory hygienic

quality if more than two of five samples contain between

5�101 and 5�102 CFU E. coli per gram. In this study,

15 of the 39 products tested contained that amount of

E. coli. However, as only one sample from each batch

was analysed, it is not possible to know if they had been

classified as having unsatisfactory hygiene quality or not

unless additional samples were analysed. The high inci-

dence of E. coli in these raw food diets highlights the

need for maintaining good hygiene when handling these

products to prevent infection.

Raw food diets for dogs are not intended for human

consumption. However, it is likely that they are handled in

kitchens where food for human consumption is also handled.

Thereby there is a possibility of cross-contamination and

a subsequent exposure of humans to bacteria from the

raw food diet products. The major concern would be

transient colonisation with ESC-resistant E. coli which

may allow gene transfer to E. coli adapted to the human

gut, resulting in prolonged colonisation with resistant

coliforms as a consequence (19). Thus, raw food diets

could constitute a source of ESC-resistant E. coli not

only for dogs but also for humans. However, at this time,

there are no indications of spread of ESC-resistant E. coli

from broiler meat to humans in Sweden (20). Hence, the

risk of spread from raw food diets to humans is probably

low, but this relies on good hygiene being maintained

when handling these products.

Acknowledgements

Kerstin Ekström, Annica Landén, and Mattias Myrenås are all

acknowledged for skilled technical assistance and Björn Bengtsson

is acknowledged for valuable discussions.

Conflict of interest and funding The study was funded by a grant from the Swedish Board

of Agriculture. The author has no conflict of interest to

declare.

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