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
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Abstract |
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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
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Introduction |
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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.
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Materials and methods |
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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.
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Results |
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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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PFGE revealed 64 banding patterns among the 78 Salmonella isolates, which grouped into six clusters (AF) with 4865% 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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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.
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Discussion |
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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.
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Footnotes |
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References |
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