Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Daniels, J. B. Articles by Besser, T. E. Search for Related Content PubMed PubMed Citation Articles by Daniels, J. B. Articles by Besser, T. E. Agricola Articles by Daniels, J. B. Articles by Besser, T. E.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, December 2007, p. 8005-8011, Vol. 73, No. 24
0099-2240/07/$08.00+0     doi:10.1128/AEM.01325-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Molecular Epidemiology of bla_CMY-2 Plasmids Carried by Salmonella enterica and Escherichia coli Isolates from Cattle in the Pacific Northwest

Joshua B. Daniels, Douglas R. Call, and Thomas E. Besser^*

Department of Veterinary Microbiology and Pathology, Washington State University College of Veterinary Medicine, Pullman, Washington 99164

Received 13 June 2007/ Accepted 7 October 2007

Top ABSTRACT INTRODUCTION REFERENCES

 
Restriction analyses of bla_CMY-2-bearing plasmids and Salmonella and Escherichia coli hosts identified (i) shared highly similar plasmids in these species in rare cases, (ii) a clonal host-plasmid relationship in Salmonella enterica serotype Newport, and (iii) a very high diversity of strain types and plasmids among commensal E. coli isolates.

Top ABSTRACT INTRODUCTION REFERENCES

 
Nontyphoid Salmonella enterica subsp. enterica (NTS) causes approximately 36,000 confirmed cases of food-borne illness in the United States and an estimated 1.4 million unreported cases each year (9, 17, 25). A trend toward increasing antimicrobial resistance to broad-spectrum cephalosporins in some NTS serovars has raised concern because these drugs are regarded as first-line therapy in pediatric salmonellosis (11). The most recent surveillance data indicate that 0.6% of U.S. human NTS isolates are resistant to ceftriaxone and 3.4% are resistant to ceftiofur (a veterinary broad-spectrum cephalosporin) (7). The predominant mechanism of cephalosporin resistance in NTS in the United States is a cephamycinase encoded by plasmid-borne bla_CMY-2 genes (11, 27, 28, 30). bla_CMY-2, which likely originated from the chromosomal AmpC locus of Citrobacter freundii (1, 19), has been observed in plasmids in several species of the Enterobacteriacea (2-4, 15, 16, 18, 27, 29).

Commensal bacteria may serve as a reservoir of plasmid-borne antimicrobial resistance genes for pathogens, and there is evidence that plasmid transfer occurs readily between Escherichia coli and S. enterica. For example, a phylogenetic analysis of F plasmid-specific genes from reference collections of S. enterica and E. coli found several examples in which finO and traD sequence variants were shared between the two species (6). Furthermore, bla_CMY-2 Southern blot experiments with plasmids from E. coli and S. enterica have revealed similarities among isolates, suggesting that sequences (in addition to the bla_CMY-2 open reading frame) are shared among the plasmids harbored by these microbial genera (27, 28).

Cattle could represent an important niche for transfer of bla_CMY-2 plasmids between E. coli and NTS. Both bacterial genera inhabit the bovine gastrointestinal tract, and selection pressure favoring cephalosporin resistance is ubiquitous in some cattle production systems due to the frequent use of ceftiofur (13). For example, between 2001 and 2003, the percentage of ceftiofur-resistant NTS rose more rapidly among isolates from U.S. cattle than in those from human, chicken, turkey, and swine hosts (8). The predominance of bla_CMY-2--mediated cephalosporin resistance among NTS and E. coli isolates from cattle (20, 24, 30) led us to investigate the relationship between bla_CMY-2 plasmids and the two microbial genera. Assessing the diversity of host chromosomal and plasmid DNAs from commensal isolates of E. coli and clinical isolates of S. enterica permitted us to evaluate whether bla_CMY-2 dissemination in this ecological niche is clonal or due to epidemic plasmid spread and whether the nature of this process differed in a pathogen (S. enterica) and a potential reservoir of antimicrobial resistance genes (E. coli).

E. coli isolates from 46 animals originating in 14 herds and S. enterica isolates from 48 animals with salmonellosis originating in 47 herds were chosen to represent the bovine commensal flora and a major bovine pathogen, respectively. All isolates were obtained from cattle in Washington state or Idaho between 2001 and 2003. All isolates gave an amplicon of the appropriate length when tested by PCR using bla_CMY-2-specific primers described by Zhao et al. (30) (Table 1).

View this table: [in this window] [in a new window]

TABLE 1. blaCMY-2 containing S. enterica and E. coli isolates

E. coli and S. enterica serotypes were determined by the Gastroenteric Disease Center (University Park, PA) and the National Veterinary Services Laboratory (Ames, IA), respectively. Isolates were assessed for pulsed-field gel electrophoresis (PFGE) type in accordance with PulseNet protocols (23). Plasmids were isolated by electroporation into E. coli DH10B and prepared for PstI restriction fragment length polymorphism (pRFLP) typing using previously described methods (12, 21). pRFLP typing and bla_CMY-2 Southern blotting were performed as described by Giles et al. (12) but using continuous voltage (7.2 V/cm for 1.5 h). Agar diffusion susceptibility testing was performed in accordance with CLSI standards (10). Gel images were analyzed using Bionumerics (Applied Maths, Belgium) with optimization and tolerance settings determined by the minimum values required to classify a standard plasmid (which was included with each gel) as indistinguishable from itself in an unweighted pair group method with arithmetic mean (UPGMA) analysis.

S. enterica included four serotypes: Newport (n = 35), Typhimurium (n = 5), Dublin (n = 7), and Muenster (n = 1); PFGE patterns were highly similar within serotypes (Fig. 1). Sixteen serotypes were identified among the 28 E. coli isolates that were typeable for both O and H antigens, but PFGE patterns were markedly more diverse than in the Salmonella spp.; each of the 46 isolates displayed a unique pattern (data not shown). The differences in serotype and PFGE diversities between the two genera may reflect the sources of the isolates: clinical versus commensal bacteria from healthy animals. Pathogenic Salmonella spp. have been described as inherently clonal (5, 14), whereas relatively little is known about the genetic diversity of nonpathogenic E. coli isolates from animal sources. Winokur et al. found diverse PFGE patterns among 55 bla_CMY-2 commensal E. coli isolates from clinical veterinary specimens (28). Taken together, these findings suggest that bla_CMY-2-bearing commensal E. coli isolates are not strongly clonal at the serotype and PFGE levels, regardless of the clinical status of the source.

View larger version (44K): [in this window] [in a new window]

FIG. 1. UPGMA dendrograms with negative images of ethidium bromide-stained gels from XbaI PFGE of blaCMY-2-positive S. enterica from cattle following normalization and analysis with Bionumerics software. The PFGE type designations correspond to those referred to in Table 1. Ser., serotype.

Forty pRFLP patterns were observed among the 94 isolates; however, repeatability analyses using a subset of 14 plasmids demonstrated that consistent self-grouping was observed only at the 90% similarity level. Thus, we considered plasmids with 90% similarity indistinguishable, resulting in the designation of 34 unique patterns. Four reference plasmids (A, B, C, and D), described by Giles et al. (12), were included as positive controls. Twelve pRFLP patterns were observed in more than one bacterial isolate, and two patterns were observed in both genera, consistent with exchange of some plasmids among commensal E. coli and S. enterica isolates (Fig. 2). Eighty-five plasmids had bla_CMY-2 Southern blot fragments identical to the A or C patterns previously described and were conserved within pRFLP types, consistent with horizontal-transfer activity (Fig. 3).

View larger version (44K): [in this window] [in a new window]

FIG. 2. UPGMA dendrogram with a negative image of an ethidium bromide-stained PstI plasmid RFLP gel after normalization in Bionumerics software. The bracketed strains indicate identical RFLP patterns from plasmids isolated from both E. coli and S. enterica. Plasmid type designations correspond to those in Table 1. Reference A, B, C, and D plasmids (12) were included as controls.

View larger version (32K): [in this window] [in a new window]

FIG. 3. (A) pRFLP of A, B, C, and D reference plasmids, followed by three examples of blaCMY-2 plasmids from S. enterica isolates from Washington state cattle. Lanes: 1, 8, and 12, 12-kb ladder; 2, \HindIII markers; 3, 4, 5, and 6, A, B, C, and D reference plasmids (12); 9, 10, and 11, pS7907, pS7909, and pS8129; 7, undigested reference plasmid D. (B) Southern hybridization of the gel from panel A with a full-length CMY-2 probe.

Each Salmonella serotype tended to be associated with a specific plasmid variant. Notably, for S. enterica serovar Newport, 26 of 35 isolates originated from 26 different herds but shared a single pRFLP type (type 18). Using the model of plasmid-bacterial-host associations proposed by Souza and Eguiarte (22), the relationship between S. enterica serovar Newport and its plasmids could be described as clonal, implying that bla_CMY-2 plasmids in this serotype were largely disseminated with an epidemic host bacterium. In contrast, the diversity of plasmids from E. coli was high, reflecting the high level of PFGE and serotype diversity observed between the isolates. The exceptions to this observation were five pRFLP types (10, 13, 16, 17) associated with multiple serologically distinguishable host strains, consistent with the idea of epidemic plasmids (22).

Although there was evidence of interspecies sharing of plasmids, the predominance of only two plasmid variants (A and C) in Salmonella isolates from an animal niche containing a plethora of E. coli-borne plasmid variants (presumably available for transfer to Salmonella) was conspicuous, suggesting that the major mechanisms of bla_CMY-2 dissemination differ between S. enterica and E. coli. This pattern is consistent with a recent study by Welch et al., who found greater diversity of plasmids among E. coli isolates than among the S. enterica isolates, using PCR primer sets representing 13 widely spaced loci from an entirely sequenced IncA/C bla_CMY-2 plasmid from S. enterica serovar Newport (26). Our observation that isolates limited to a solitary niche (cattle) are similarly diverse suggests a model of bla_CMY-2 dissemination in which insertions and deletions that occur during promiscuous plasmid sharing among E. coli isolates occasionally result in plasmids that are successful in a Salmonella host (such as pRFLP types 13 and 18). Also consistent with this model, conjugation experiments revealed that 40% of E. coli plasmids (versus 2% of Salmonella plasmids) were able to transfer or be mobilized to a Nal^r DH5 recipient. The subsequent success of a Salmonella host/plasmid clone, then, is likely modulated by diverse factors, including virulence, infectivity, and environmental persistence, as well as antimicrobial selection pressures. Factors that promote interspecies exchange of antimicrobial-resistance plasmids and enhance dissemination of S. enterica clones merit further study.

 
We thank Mike Kahn, Min-Su Kang, and Yubei Zhang for their thoughtful discussions and technical assistance. Paul Fey at the University of Nebraska provided the "A, B, C, and D" plasmids.

This work was partially funded by USDA-NRI Epidemiological Approaches to Food Safety grant 2005-01373, NIAID NIH contract N01-AI-30055, and the Agricultural Animal Health Program, WSU College of Veterinary Medicine, Pullman, WA.

 
* Corresponding author. Mailing address: Department of Veterinary Microbiology and Pathology, Washington State University College of Veterinary Medicine, P.O. Box 647040, Pullman, WA 99164-7040. Phone: (509) 335-6075. Fax: (509) 335-8529. E-mail: tbesser{at}vetmed.wsu.edu

Published ahead of print on 12 October 2007.

Top ABSTRACT INTRODUCTION REFERENCES

 

1
1. Barlow, M., and B. G. Hall. 2002. Origin and evolution of the AmpC beta-lactamases of Citrobacter freundii. Antimicrob. Agents Chemother. 46:1190-1198.[Abstract/Free Full Text]
2
2. Bauernfeind, A., Y. Chong, and K. Lee. 1998. Plasmid-encoded AmpC beta-lactamases: how far have we gone 10 years after the discovery? Yonsei Med. J. 39:520-525.[Medline]
3
3. Bauernfeind, A., P. Hohl, I. Schneider, R. Jungwirth, and R. Frei. 1998. Escherichia coli producing a cephamycinase (CMY-2) from a patient from the Libyan-Tunisian border region. Clin. Microbiol. Infect. 4:168-170.[Medline]
4
4. Bauernfeind, A., I. Stemplinger, R. Jungwirth, and H. Giamarellou. 1996. Characterization of the plasmidic beta-lactamase CMY-2, which is responsible for cephamycin resistance. Antimicrob. Agents Chemother. 40:221-224.[Abstract]
5
5. Beltran, P., J. M. Musser, R. Helmuth, J. J. Farmer III, W. M. Frerichs, I. K. Wachsmuth, K. Ferris, A. C. McWhorter, J. G. Wells, A. Cravioto, et al. 1988. Toward a population genetic analysis of Salmonella: genetic diversity and relationships among strains of serotypes S. choleraesuis, S. derby, S. dublin, S. enteritidis, S. heidelberg, S. infantis, S. newport, and S. typhimurium. Proc. Natl. Acad. Sci. USA 85:7753-7757.[Abstract/Free Full Text]
6
6. Boyd, E. F., and D. L. Hartl. 1997. Recent horizontal transmission of plasmids between natural populations of Escherichia coli and Salmonella enterica. J. Bacteriol. 179:1622-1627.[Abstract/Free Full Text]
7
7. CDC. 2007. National Antimicrobial Resistance Monitoring System (NARMS): human isolates final report, 2004. U.S. Department of Health and Human Services, Atlanta, GA.
8
8. CDC. 2003. National Antimicrobial Resistance Monitoring System—Enteric Bacteria (NARMS): 2003 executive report. U.S. Department of Health and Human Services, Atlanta, GA.
9
9. CDC. 2005. Salmonella surveillance: annual summary, 2005. U.S. Department of Health and Human Services, Atlanta, GA.
10
10. CLSI. 2006. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. CLSI, Wayne, PA.
11
11. Fey, P. D., T. J. Safranek, M. E. Rupp, E. F. Dunne, E. Ribot, P. C. Iwen, P. A. Bradford, F. J. Angulo, and S. H. Hinrichs. 2000. Ceftriaxone-resistant Salmonella infection acquired by a child from cattle. N. Engl. J. Med. 342:1242-1249.[Abstract/Free Full Text]
12
12. Giles, W. P., A. K. Benson, M. E. Olson, R. W. Hutkins, J. M. Whichard, P. L. Winokur, and P. D. Fey. 2004. DNA sequence analysis of regions surrounding bla_CMY-2 from multiple Salmonella plasmid backbones. Antimicrob. Agents Chemother. 48:2845-2852.[Abstract/Free Full Text]
13
13. Hales, J. R. C. 2005. Antibacterials in the animal health industry: current markets and future prospects. PJB Publications, London, United Kingdom. http://www.pjbpubs.com/cms.asp?pageid=1897.
14
14. Hancock, D., T. E. Besser, J. Gay, D. Rice, M. Davis, and C. Gay. 2000. The global epidemiology of multiresistant Salmonella enterica serovar Typhimurium DT104, p. 217-243. In C. Brown and C. A. Bolin (ed.), Emerging diseases of animals. ASM Press, Washington, DC.
15
15. Huang, I. F., C. H. Chiu, M. H. Wang, C. Y. Wu, K. S. Hsieh, and C. C. Chiou. 2005. Outbreak of dysentery associated with ceftriaxone-resistant Shigella sonnei: first report of plasmid-mediated CMY-2-type AmpC beta-lactamase resistance in S. sonnei. J. Clin. Microbiol. 43:2608-2612.[Abstract/Free Full Text]
16
16. Koeck, J. L., G. Arlet, A. Philippon, S. Basmaciogullari, H. V. Thien, Y. Buisson, and J. D. Cavallo. 1997. A plasmid-mediated CMY-2 beta-lactamase from an Algerian clinical isolate of Salmonella Senftenberg. FEMS Microbiol. Lett. 152:255-260.[Medline]
17
17. Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro, P. M. Griffin, and R. V. Tauxe. 1999. Food-related illness and death in the United States. Emerg. Infect. Dis. 5:607-625.[Medline]
18
18. Odeh, R., S. Kelkar, A. M. Hujer, R. A. Bonomo, P. C. Schreckenberger, and J. P. Quinn. 2002. Broad resistance due to plasmid-mediated AmpC beta-lactamases in clinical isolates of Escherichia coli. Clin. Infect. Dis. 35:140-145.[CrossRef][Medline]
19
19. Philippon, A., G. Arlet, and G. A. Jacoby. 2002. Plasmid-determined AmpC-type beta-lactamases. Antimicrob. Agents Chemother. 46:1-11.[Free Full Text]
20
20. Pruiett, C. S. 2004. Identification and characterization of Escherichia coli with reduced susceptibility to ceftazidime from Washington and Idaho cattle herds. M.S. thesis. Washington State University, Pullman.
21
21. Rondon, M. R., S. J. Raffel, R. M. Goodman, and J. Handelsman. 1999. Toward functional genomics in bacteria: analysis of gene expression in Escherichia coli from a bacterial artificial chromosome library of Bacillus cereus. Proc. Natl. Acad. Sci. USA 96:6451-6455.[Abstract/Free Full Text]
22
22. Souza, V., and L. E. Eguiarte. 1997. Bacteria gone native vs. bacteria gone awry? Plasmidic transfer and bacterial evolution. Proc. Natl. Acad. Sci. USA 94:5501-5503.[Free Full Text]
23
23. Swaminathan, B., T. J. Barrett, S. B. Hunter, and R. V. Tauxe. 2001. PulseNet: the molecular subtyping network for foodborne bacterial disease surveillance, United States. Emerg. Infect. Dis. 7:382-389.[Medline]
24
24. Tragesser, L. A., T. E. Wittum, J. A. Funk, P. L. Winokur, and P. J. Rajala-Schultz. 2006. Association between ceftiofur use and isolation of Escherichia coli with reduced susceptibility to ceftriaxone from fecal samples of dairy cows. Am. J. Vet. Res. 67:1696-1700.[CrossRef][Medline]
25
25. Voetsch, A. C., T. J. Van Gilder, F. J. Angulo, M. M. Farley, S. Shallow, R. Marcus, P. R. Cieslak, V. C. Deneen, and R. V. Tauxe. 2004. FoodNet estimate of the burden of illness caused by nontyphoidal Salmonella infections in the United States. Clin. Infect. Dis. 38(Suppl. 3):S127-S134.[CrossRef][Medline]
26
26. Welch, T. J., W. F. Fricke, P. F. McDermott, D. G. White, M. L. Rosso, D. A. Rasko, M. K. Mammel, M. Eppinger, M. J. Rosovitz, D. Wagner, L. Rahalison, J. E. Leclerc, J. M. Hinshaw, L. E. Lindler, T. A. Cebula, E. Carniel, and J. Ravel. 2007. Multiple antimicrobial resistance in plague: an emerging public health risk. PLoS ONE 2:e309.[CrossRef]
27
27. Winokur, P. L., A. Brueggemann, D. L. DeSalvo, L. Hoffmann, M. D. Apley, E. K. Uhlenhopp, M. A. Pfaller, and G. V. Doern. 2000. Animal and human multidrug-resistant, cephalosporin-resistant Salmonella isolates expressing a plasmid-mediated CMY-2 AmpC beta-lactamase. Antimicrob. Agents Chemother. 44:2777-2783.[Abstract/Free Full Text]
28
28. Winokur, P. L., D. L. Vonstein, L. J. Hoffman, E. K. Uhlenhopp, and G. V. Doern. 2001. Evidence for transfer of CMY-2 AmpC beta-lactamase plasmids between Escherichia coli and Salmonella isolates from food animals and humans. Antimicrob. Agents Chemother. 45:2716-2722.[Abstract/Free Full Text]
29
29. Wu, S. W., K. Dornbusch, G. Kronvall, and M. Norgren. 1999. Characterization and nucleotide sequence of a Klebsiella oxytoca cryptic plasmid encoding a CMY-type beta-lactamase: confirmation that the plasmid-mediated cephamycinase originated from the Citrobacter freundii AmpC beta-lactamase. Antimicrob. Agents Chemother. 43:1350-1357.[Abstract/Free Full Text]
30
30. Zhao, S., D. G. White, P. F. McDermott, S. Friedman, L. English, S. Ayers, J. Meng, J. J. Maurer, R. Holland, and R. D. Walker. 2001. Identification and expression of cephamycinase bla_CMY genes in Escherichia coli and Salmonella isolates from food animals and ground meat. Antimicrob. Agents Chemother. 45:3647-3650.[Abstract/Free Full Text]

---

Applied and Environmental Microbiology, December 2007, p. 8005-8011, Vol. 73, No. 24
0099-2240/07/$08.00+0     doi:10.1128/AEM.01325-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Mataseje, L. F., Neumann, N., Crago, B., Baudry, P., Zhanel, G. G., Louie, M., Mulvey, M. R., and the ARO Water Study Group, (2009). Characterization of Cefoxitin-Resistant Escherichia coli Isolates from Recreational Beaches and Private Drinking Water in Canada between 2004 and 2006. Antimicrob. Agents Chemother. 53: 3126-3130 [Abstract] [Full Text]  
• Daniels, J. B., Call, D. R., Hancock, D., Sischo, W. M., Baker, K., Besser, T. E. (2009). Role of Ceftiofur in Selection and Dissemination of blaCMY-2-Mediated Cephalosporin Resistance in Salmonella enterica and Commensal Escherichia coli Isolates from Cattle. Appl. Environ. Microbiol. 75: 3648-3655 [Abstract] [Full Text]  
• Welch, T. J., Evenhuis, J., White, D. G., McDermott, P. F., Harbottle, H., Miller, R. A., Griffin, M., Wise, D. (2009). IncA/C Plasmid-Mediated Florfenicol Resistance in the Catfish Pathogen Edwardsiella ictaluri. Antimicrob. Agents Chemother. 53: 845-846 [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Daniels, J. B. Articles by Besser, T. E. Search for Related Content PubMed PubMed Citation Articles by Daniels, J. B. Articles by Besser, T. E. Agricola Articles by Daniels, J. B. Articles by Besser, T. E.