Antimicrobial susceptibility and genetic relatedness of Salmonella serovars isolated from animal-derived dog treats in the USA

D. G. White1, A. Datta2, P. McDermott1, S. Friedman1, S. Qaiyumi, S. Ayers1, L. English1, S. McDermott1, D. D. Wagner1 and S. Zhao1,*

1 Division of Animal and Food Microbiology, Office of Research, Center for Veterinary Medicine and 2 Division of Field Science, Office of Regulatory Affairs, US Food and Drug Administration, Rockville, MD 20708, USA

Received 7 May 2003; returned 21 May 2003; revised 12 August 2003; accepted 13 August 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The objectives of this study were to determine the potential risk of dog treats in transmitting Salmonella to humans in the USA, and to characterize genetic relatedness and antimicrobial resistance among the isolates.

Methods: A total of 158 dog treats derived from pig ears and other animal parts were randomly collected nationwide and assayed for the presence of Salmonella. The Salmonella isolates were characterized using serotyping, pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing.

Results: Forty-one percent (65/158) of samples were positive for Salmonella. Eighty-four Salmonella isolates, comprising 24 serotypes, were recovered from the 65 positive samples. Fourteen samples were contaminated with more than one Salmonella serotype. PFGE analysis of 78 Salmonella isolates yielded 64 patterns. S. Infantis with PFGE patterns indistinguishable from those of strains identified in Canadian outbreaks in 1999 were recovered in several dog treat products. The majority of Salmonella isolates were susceptible to the antimicrobials tested; however, resistance was observed to tetracycline (26%), streptomycin (23%), sulfamethoxazole (19%), chloramphenicol (8%) and ampicillin (8%). Twenty-eight (36%) Salmonella isolates were resistant to at least one antimicrobial and 10 (13%) isolates displayed resistance to four or more antimicrobials. Two isolates were identified as S. Typhimurium DT104 with the characteristic penta-resistance phenotype (ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline). One S. Brandenburg isolate was resistant to eight antimicrobials. Seven Salmonella isolates also contained class I integrons encoding resistance genes to aminoglycosides, ß-lactam and streptothricin antimicrobials.

Conclusions: The study indicates that animal-derived dog treats in the USA could be a potential source of animal and human infections with Salmonella, including multidrug-resistant Salmonella strains.

Keywords: antimicrobial resistance, integrons, PFGE


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Salmonellosis in humans caused by non-typhoid Salmonella strains usually results in a self-limiting diarrhoea that does not warrant antimicrobial therapy. However, there are occasions when these infections can lead to life-threatening systemic infections that require effective chemotherapy.1 Of increasing concern is the worldwide emergence of multidrug-resistant phenotypes among Salmonella serotypes, in particular S. Typhimurium, and more recently S. Newport.2,3 S. Typhimurium DT104, resistant to at least five antimicrobials including ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline, has caused severe infections and deaths in animals and humans worldwide.4 More recently, a multi-state outbreak of multidrug-resistant S. Newport was reported by the US Centers for Disease Control and Prevention, which implicated raw or undercooked ground beef as the vehicle of transmission.3

In the summer of 1999, epidemiological investigations in Canada implicated dried pig ear dog treats as the source of S. Infantis outbreaks in humans,5 and phage typing and pulsed-field gel electrophoresis (PFGE) further established the link between human salmonellosis and pig ear-derived dog treats. One firm producing the dog treats implicated in the Canadian outbreak also distributes the same product throughout the USA. This prompted us to conduct a nationwide microbiological survey to investigate the prevalence of Salmonella in dog treats in the US market, and to characterize genetic relatedness and antimicrobial susceptibility phenotypes among isolates identified.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Salmonella isolation and identification

One hundred and fifty-eight dog treats (26 domestic and 132 imported products) were randomly collected in the USA by 16 district offices and seven regional laboratories of the US Food and Drug Administration (FDA). Salmonella were isolated and identified using methods described in the FDA Bacteriological Analytical Manual (http://www.cfsan.fda.gov/~ebam/bam-toc.html), followed by serotyping using Difco antisera (Becton Dickinson Microbiology System, Cockeysville, MD, USA). Phage typing was performed by the National Laboratories for Enteric Pathogens, Canadian Science Centre for Human and Animal Health, Manitoba, Canada.

PFGE

PFGE was performed to determine genomic DNA fingerprinting profiles of Salmonella according to methods described previously.6 The interpretation of the PFGE patterns was aided by use of Molecular Analyst Fingerprinting Plus Software, version 1.6 (Bio-Rad).

Antimicrobial susceptibility determination

Salmonella isolates were assayed for susceptibility to 17 antimicrobials used by the National Antimicrobial Resistance Monitoring System (NARMS). MICs were determined by a broth microdilution method using the Sensititre system (Trek Diagnostic Systems, Westlake, OH, USA) and interpreted according to NCCLS criteria.7 Escherichia coli ATCC 25922 and 35218, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853 were used as quality control organisms in antimicrobial MIC determinations, according to NCCLS recommendations.

Identification of class I integrons and associated resistance genes

DNA templates, oligonucleotide primers and PCR conditions (including positive and negative controls) to detect the presence of class I integrons were as described previously.6 The PCR amplicons were purified using a PCR purification kit (Boehringer Mannheim, Indianapolis, IN, USA), and sequenced by BioServe Biotechnologies (Laurel, MD, USA). DNA sequences were analysed by searching the GenBank database using BLAST.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Prevalence of Salmonella in dog treats

Sixty-five (41%) of the 158 dog treat samples were contaminated with Salmonella, including eight (31%) domestic and 57 (43%) imported products. Eighty-four Salmonella isolates were recovered from the 65 positive samples. However, only 78 isolates were available for further analysis. A total of 24 serotypes were identified among the 78 isolates, including Anatum (15), Typhimurium (11), Infantis (eight), Derby (six), Ohio (six), Mbandaka (five), Bredeney (four), Worthington (three) and Newport (two), among others (Figure 1). Multiple serotypes of Salmonella were identified in 14 dog treats, including two serotypes from nine samples, and three serotypes from five samples. Five S. Typhimurium were phage-typed as DT104 (CVM 4193 and 4241), DT104a (CVM 4100 and 4198) or DT 17 (CVM 4194).



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Figure 1. Dendrogram of PFGE patterns of Salmonella recovered from animal-derived dog treats, and their association with serotype, source, integron and phage type. (a) Isolates with PFGE patterns indistinguishable from the Canadian outbreak strains; (b) isolates containing class I integrons; (c) S. Typhimurium DT104.

 
PFGE profiles

PFGE revealed 64 banding patterns among the 78 Salmonella isolates, which grouped into six clusters (A–F) with 48–65% pattern similarity (Figure 1). Overall, PFGE typing grouped the majority of isolates according to serotype; however, there were several interesting exceptions. One S. Derby isolate (CVM 4171) grouped into cluster B, which contained isolates of Newport, Muenchen, Infantis and Bredeney. The remaining S. Derby isolates grouped into cluster F. The PFGE patterns of two S. Muenchen isolates fell into two clusters (A and B) with 55% pattern similarity. Although four S. Bredeney isolates grouped into cluster B, one isolate (CVM 4157) was more closely related to S. Infantis isolates (74% pattern similarity) than the other three Bredeney isolates (68% pattern similarity). These four strains (S. Derby, CVM4171, S. Bredeney CVM 4175, and S. Muenchen CVM4244 and CVM4254), the PFGE patterns of which did not fall into their same serotype cluster, were re-analysed and confirmed several times by serotyping and PFGE.

Antimicrobial susceptibility profiles

All 78 Salmonella isolates were susceptible to amikacin, co-amoxiclav, cefoxitin, ceftiofur, ceftriaxone, ciprofloxacin and nalidixic acid. The most common resistance observed was to tetracycline (26%), followed by streptomycin (23%), sulfamethoxazole (19%), ampicillin and chloramphenicol (8%). Twenty-eight Salmonella (36%) were resistant to at least one antimicrobial, while 10 (13%) were resistant to four or more antimicrobials. Of the six isolates resistant to chloramphenicol, three were S. Typhimurium, two S. Derby and one S. Brandenburg. All chloramphenicol-resistant isolates also displayed decreased susceptibility to the fluorinated analogue florfenicol (>=16 mg/L), and the three S. Typhimurium strains possessed the putative phenicol efflux gene, flo (data not shown). Antimicrobial resistance phenotypes were strongly associated with particular serotypes (Table 1). In particular, S. Typhimurium and S. Derby isolates exhibited resistance to multiple antimicrobial agents more commonly than other serotypes. Two S. Typhimurium DT104 isolates displayed resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline, the typical penta-resistance pattern for S. Typhimurium DT104. Another S. Typhimurium isolate showed resistance to kanamycin in addition to the penta-resistance, but was untypeable by phage typing. One S. Brandenburg isolate from a Canadian sample exhibited resistance to eight antimicrobials, including ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline, gentamicin, apramycin and cefalothin. Interestingly, all eight S. Infantis isolates recovered from dog ear treats were susceptible to all tested antimicrobials.


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Table 1. Antimicrobial resistance profiles of Salmonella serovars isolated from animal-derived dog treatsa
 
Presence of class I integrons

Seven of the 78 Salmonella isolates carried class I integrons, including three S. Typhimurium, three S. Derby and one S. Brandenburg. The three S. Typhimurium isolates possessed two integrons of 1.0 and 1.2 kb, three S. Derby carried a 1 kb integron, and one S. Brandenburg isolate carried a 650 bp integron. The 1 kb integron identified in both S. Typhimurium and S. Derby isolates contained the aadA2 gene encoding resistance to streptomycin, whereas the 1.2 kb integron identified in S. Typhimurium isolates contained the blaPSE-1 gene encoding resistance to ampicillin. Interestingly, the 650 bp integron identified in S. Brandenburg contained the sat-1 gene, which confers resistance to the streptothricin antimicrobial, nourseothricin.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our findings indicate that animal-derived dog treats on the US market were commonly contaminated with Salmonella. Of the 24 Salmonella serotypes recovered, several serotypes, including Typhimurium, Newport, Heidelberg, Muenchen, Montevideo, Infantis, Mbandaka and Agona, were among the top 15 serotypes causing human salmonellosis in the USA.8 Other Salmonella serotypes, such as Anatum, Derby, Ohio and Worthington, have also been associated with human salmonellosis cases.8 For some serotypes (S. Anatum, S. Infantis, S. Worthington and S. Typhimurium), indistinguishable PFGE patterns were found in different samples from different geographical locations. Of particular public health significance, several PFGE patterns of S. Infantis recovered from domestic and imported dog treats were indistinguishable from the PFGE patterns of S. Infantis implicated in a Canadian outbreak.5

An increased incidence in the occurrence of multidrug-resistant Salmonella has been widely reported in the past decade, presumably attributed to the extensive use of antimicrobial agents in human and veterinary medicine.2 A high percentage of Salmonella isolates in our study were resistant to tetracycline, streptomycin and sulphonamides. This is not surprising, as these antimicrobials are widely used in swine and other animal production environments for treatment and prevention of disease and for growth promotion. Resistant phenotypes also appear to be associated with particular serotypes, which is consistent with other published findings.9 Class 1 integrons were identified in S. Typhimurium, S. Derby and S. Brandenburg, all of which were isolated from pig ear-derived dog treats. The integron of S. Brandenburg carried an unusual sat-1 gene encoding resistance to streptothricin, a drug that has not been used in the USA. This suggests that resistance to this drug developed elsewhere, perhaps in Germany, where the majority of reports of streptothricin resistance have originated. In addition, we previously identified the same aadA and pse-1 genes on class I integrons reported here in Salmonella isolated from imported seafood products.10 These examples may represent instances of resistance gene dissemination on an international scale.

In summary, animal-derived dog treats in the USA were often found to be contaminated with a variety of Salmonella serotypes, including antimicrobial-resistant variants. These findings indicate a potential public health threat posed by these products and highlight the importance of implementing preventive measures to reduce Salmonella contamination.


    Footnotes
 
* Corresponding author. Tel: +1-301-827-8139; Fax: +1-301-827-8127; E-mail: szhao{at}cvm.fda.gov Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Helms, M., Vastrup, P., Gerner-Smidt, P. et al. (2002). Excess mortality associated with antimicrobial drug-resistant Salmonella typhimurium. Emerging Infectious Diseases 8, 490–5.[ISI][Medline]

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4 . Glynn, M. K., Bopp, C., Dewitt, W. et al. (1998). Emergence of multidrug-resistant Salmonella enterica serotype typhimurium DT104 infections in the United States. New England Journal of Medicine 338, 1333–8.[Abstract/Free Full Text]

5 . Clark, C., Cunningham, J., Ahmed, R. et al. (2001). Characterization of Salmonella associated with pig ear dog treats in Canada. Journal of Clinical Microbiology 39, 3962–8.[Abstract/Free Full Text]

6 . Zhao, S., White, D. G., Ge, B. et al. (2001). Identification and characterization of integron-mediated antibiotic resistance among Shiga toxin-producing Escherichia coli isolates. Applied and Environmental Microbiology 67, 1558–64.[Abstract/Free Full Text]

7 . National Committee for Clinical Laboratory Standards. (2001). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals: Approved Standard M31-A. NCCLS, Wayne, PA, USA.

8 . Olsen, S. J., Bishop, R., Brenner, F. W. et al. (2001). The changing epidemiology of Salmonella: trends in serotypes isolated from humans in the United States, 1987–1997. Journal of Infectious Diseases 183, 753–61.[CrossRef][ISI][Medline]

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10 . Zhao, S., Datta, A. R., Ayers, S. et al. (2003). Antimicrobial-resistant Salmonella serovars isolated from imported food. International Journal of Food Microbiology 84, 87–92.[ISI][Medline]