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summary writing for each research paper into 200 to 300 words. need to make sure no plagiarism and no AI.
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**files (5)**
– ## HygienequalityandpresenceofESBL-producingEscherichiacoliinrawfooddietsfordogs.pdf
– EstimationofEscherichiacoliinrawgroundbeef.pdf
– TheriskofsalmonellaesheddingbydogsfedSalmonella-contaminated.pdf
– Rawmeat-baseddietforpetsaneglectedsourceofhumanexposuretoSalmonellaandpathogenicEscherichiacoliclonescarryingmcr2019toJanuary2020.pdf
– MicrobiologicalQualityandPresenceofFoodbornePathogensinRawandExtrudedCanineDietsandCanineFecalSamples.pdf
**## HygienequalityandpresenceofESBL-producingEscherichiacoliinrawfooddietsfordogs.pdf**
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: Oskar.Nilsson@sva.se
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.
1
Citation: Infection Ecology and Epidemiology 2015, 5: 28758 – http://dx.doi.org/10.3402/iee.v5.28758
(page number not for citation purpose)
[http://creativecommons.org/licenses/by-nc/4.0/](http://creativecommons.org/licenses/by-nc/4.0/)
[http://www.infectionecologyandepidemiology.net/index.php/iee/article/view/28758](http://www.infectionecologyandepidemiology.net/index.php/iee/article/view/28758)
[http://dx.doi.org/10.3402/iee.v5.28758](http://dx.doi.org/10.3402/iee.v5.28758)
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
2
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Citation: Infection Ecology and Epidemiology 2015, 5: 28758 – http://dx.doi.org/10.3402/iee.v5.28758
[http://www.eucast.org](http://www.eucast.org/)
[http://www.eucast.org](http://www.eucast.org/)
[http://www.eucast.org](http://www.eucast.org/)
[http://www.infectionecologyandepidemiology.net/index.php/iee/article/view/28758](http://www.infectionecologyandepidemiology.net/index.php/iee/article/view/28758)
[http://dx.doi.org/10.3402/iee.v5.28758](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.
References
1. LeJeune JT, Hancock DD. Public health concerns associated
with feeding raw meat diets to dogs. J Am Vet Med Assoc 2001;
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2. Schlesinger DP, Joffe DJ. Raw food diets in companion animals:
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3. Finley R, Reid-Smith R, Ribble C, Popa M, Vandermeer M,
Aramini J. The occurrence and antimicrobial susceptibility of
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8. Seiffert SN, Hilty M, Perreten V, Endimiani A. Extended-
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antibiotics-/svarm-reports [cited 23 September 2015].
10. Mo SS, Norstrom M, Slettemeas JS, Lovland A, Urdahl AM,
Sunde M. Emergence of AmpC-producing Escherichia coli in
ESBL-producing E. coli in raw food diet products
Citation: Infection Ecology and Epidemiology 2015, 5: 28758 – http://dx.doi.org/10.3402/iee.v5.28758 3
(page number not for citation purpose)
[http://www.sva.se/en/antibiotics-/svarm-reports](http://www.sva.se/en/antibiotics-/svarm-reports)
[http://www.sva.se/en/antibiotics-/svarm-reports](http://www.sva.se/en/antibiotics-/svarm-reports)
[http://www.infectionecologyandepidemiology.net/index.php/iee/article/view/28758](http://www.infectionecologyandepidemiology.net/index.php/iee/article/view/28758)
[http://dx.doi.org/10.3402/iee.v5.28758](http://dx.doi.org/10.3402/iee.v5.28758)
the broiler production chain in a country with a low anti-
microbial usage profile. Vet Microbiol 2014; 171: 315�20.
11. Finnish Food Safety Authority. Finnish veterinary antimicro-
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agents. FINRES-Vet 2010�2012. Helsinki, Finland: Evira
publications; 2015.
12. Case LP, Daristotle L, Hayek MG, Raasch MR. Nutrient
content of pet food. In: Case LP, Daristotle L, Hayek MG,
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companion animal professionals. 3rd ed. Maryland Heights,
MO: Mosby; 2011, pp. 141�62.
13. Ewers C, Grobbel M, Bethe A, Wieler LH, Guenther S.
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bacteria in companion animals: action is clearly warranted!.
Berl Munch Tierarztl Wochenschr 2011; 124: 94�101.
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Antimicrob Chemother 2014; 69: 1497�500.
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mance standards for antimicrobial disc and dilution susceptibi-
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16. Lefebvre SL, Reid-Smith R, Boerlin P, Weese JS. Evaluation of
the risks of shedding Salmonellae and other potential pathogens
by therapy dogs fed raw diets in Ontario and Alberta. Zoonoses
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17. Schmidt VM, Pinchbeck GL, Nuttall T, McEwan N, Dawson S,
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31�40.
18. Strohmeyer RA, Morley PS, Hyatt DR, Dargatz DA, Scorza
AV, Lappin MR. Evaluation of bacterial and protozoal con-
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Butaye P, et al. In situ ESBL conjugation from avian to human
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20. Borjesson S, Jernberg C, Brolund A, Edquist P, Finn M, Landen
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E. coli from Swedish broilers and association with human
clinical isolates. Clin Microbiol Infect 2013; 19: E309�11.
Oskar Nilsson
4
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[http://www.infectionecologyandepidemiology.net/index.php/iee/article/view/28758](http://www.infectionecologyandepidemiology.net/index.php/iee/article/view/28758)
[http://dx.doi.org/10.3402/iee.v5.28758](http://dx.doi.org/10.3402/iee.v5.28758)
**EstimationofEscherichiacoliinrawgroundbeef.pdf**
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**## HygienequalityandpresenceofESBL-producingEscherichiacoliinrawfooddietsfordogs.pdf**
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: Oskar.Nilsson@sva.se
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.
1
Citation: Infection Ecology and Epidemiology 2015, 5: 28758 – http://dx.doi.org/10.3402/iee.v5.28758
(page number not for citation purpose)
[http://creativecommons.org/licenses/by-nc/4.0/](http://creativecommons.org/licenses/by-nc/4.0/)
[http://www.infectionecologyandepidemiology.net/index.php/iee/article/view/28758](http://www.infectionecologyandepidemiology.net/index.php/iee/article/view/28758)
[http://dx.doi.org/10.3402/iee.v5.28758](http://dx.doi.org/10.3402/iee.v5.28758)
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
2
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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.
References
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with feeding raw meat diets to dogs. J Am Vet Med Assoc 2001;
219: 1222�5.
2. Schlesinger DP, Joffe DJ. Raw food diets in companion animals:
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3. Finley R, Reid-Smith R, Ribble C, Popa M, Vandermeer M,
Aramini J. The occurrence and antimicrobial susceptibility of
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food diets in three Canadian cities. Zoonoses Public Health
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4. Mehlenbacher S, Churchill J, Olsen KE, Bender JB. Availability,
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5. Weese JS, Rousseau J, Arroyo L. Bacteriological evaluation
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6. Chengappa MM, Staats J, Oberst RD, Gabbert NH, McVey S.
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7. Joffe DJ, Schlesinger DP. Preliminary assessment of the risk
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2002; 43: 441�2.
8. Seiffert SN, Hilty M, Perreten V, Endimiani A. Extended-
spectrum cephalosporin-resistant Gram-negative organisms in
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9. SVARM. Swedish Veterinary Antimicrobial Resistance Mon-
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antibiotics-/svarm-reports [cited 23 September 2015].
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Sunde M. Emergence of AmpC-producing Escherichia coli in
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Citation: Infection Ecology and Epidemiology 2015, 5: 28758 – http://dx.doi.org/10.3402/iee.v5.28758 3
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[http://www.infectionecologyandepidemiology.net/index.php/iee/article/view/28758](http://www.infectionecologyandepidemiology.net/index.php/iee/article/view/28758)
[http://dx.doi.org/10.3402/iee.v5.28758](http://dx.doi.org/10.3402/iee.v5.28758)
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content of pet food. In: Case LP, Daristotle L, Hayek MG,
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13. Ewers C, Grobbel M, Bethe A, Wieler LH, Guenther S.
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mance standards for antimicrobial disc and dilution susceptibi-
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16. Lefebvre SL, Reid-Smith R, Boerlin P, Weese JS. Evaluation of
the risks of shedding Salmonellae and other potential pathogens
by therapy dogs fed raw diets in Ontario and Alberta. Zoonoses
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17. Schmidt VM, Pinchbeck GL, Nuttall T, McEwan N, Dawson S,
Williams NJ. Antimicrobial resistance risk factors and char-
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31�40.
18. Strohmeyer RA, Morley PS, Hyatt DR, Dargatz DA, Scorza
AV, Lappin MR. Evaluation of bacterial and protozoal con-
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19. Smet A, Rasschaert G, Martel A, Persoons D, Dewulf J,
Butaye P, et al. In situ ESBL conjugation from avian to human
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20. Borjesson S, Jernberg C, Brolund A, Edquist P, Finn M, Landen
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**EstimationofEscherichiacoliinrawgroundbeef.pdf**
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**TheriskofsalmonellaesheddingbydogsfedSalmonella-contaminated.pdf**
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**Rawmeat-baseddietforpetsaneglectedsourceofhumanexposuretoSalmonellaandpathogenicEscherichiacoliclonescarryingmcr2019toJanuary2020.pdf**
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**MicrobiologicalQualityandPresenceofFoodbornePathogensinRawandExtrudedCanineDietsandCanineFecalSamples.pdf**
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**## HygienequalityandpresenceofESBL-producingEscherichiacoliinrawfooddietsfordogs.pdf**
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: Oskar.Nilsson@sva.se
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.
1
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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
2
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Citation: Infection Ecology and Epidemiology 2015, 5: 28758 – http://dx.doi.org/10.3402/iee.v5.28758
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[http://www.eucast.org](http://www.eucast.org/)
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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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