Few recent works have studied isolation and ESBL detection in veterinary

Few recent works have studied isolation and ESBL detection in veterinary medicine (2, 16), and no data are available describing ESBL-mediated resistance in spp. isolated from bovine mastitis. In this research, the antimicrobial susceptibility and ESBL production were evaluated in isolated from animals with bovine clinical mastitis and selected for their resistance to ceftiofur, a broad-spectrum cephalosporin. Susceptibility to a range of antimicrobials, including those approved for both human and animal use (Table ?(Table1),1), was determined by disk diffusion assay according to Clinical and Laboratory Standards Institute interpretative criteria (3). ATCC 25922 and ATCC 25923 were used as quality control requirements. TABLE 1. Comparison of resistance profiles of the nine isolates From March 2008 to March 2009, 140 klebsiellae were isolated from milk samples, representing 26.6% of the total number of Gram-negative isolates obtained from bovine clinical mastitis. The isolates were subcultured on MacConkey agar supplemented with 8 g/ml of ceftiofur (5). Nine isolates, identified as isolates were screened against the panel in Table ?Table11 by the disk diffusion method and for ESBLs by the double-disk diffusion assay. A strain was considered an ESBL producer when it showed the expansion of an inhibition zone between a disk made up of amoxicillin-clavulanate (20 and 10 g) and disks made up of, respectively, ceftazidime (30 g) and cefotaxime (30 g) placed 25 mm apart (10). All isolates were tested by PCR targeting the Ambler class A -lactamase genes animal isolates (16), using previously published primer units (14, 15). Sequencing of amplified genes completed the study. The complete resistance profiles and the PCR results are summarized in Table ?Table1.1. Only isolate 205, identified as subsp. subsp. isolate in a case of bovine clinical mastitis. Isolate 205 also displayed a positive PCR result for genes and no spp. (11). The generally low level of resistance and the low prevalence of ESBL-producing strains (0.7%) should guarantee a good outcome of drug treatment for involved in mastitis, but antimicrobial resistance is not the only factor affecting therapy efficacy (1, 4). However, the described isolation must encourage that attention be paid to a phenomenon which could evolve and 133407-82-6 IC50 be enforced under the pressure of antimicrobial therapy, as the use of extended-spectrum cephalosporins in veterinary medicine may select ESBL producers (12). It is recommended to screen klebsiellae from cases of bovine mastitis in order to keep the prevalence of ESBL producers under control. Nucleotide sequence accession number. The sequence of the CTX-M class enzyme-encoding gene identified in our study was added to GenBank (accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”HM921043″,”term_id”:”302826889″,”term_text”:”HM921043″HM921043). Footnotes ?Published ahead of print on 18 August 2010. REFERENCES 1. Bradley, A. J., and M. J. Green. 2009. Factors affecting cure when treating bovine clinical mastitis with cephalosporin-based intramammary preparations. J. Dairy Sci. 92:1941-1953. [PMC free article] [PubMed] 2. Brisse, S., and E. van Duijkeren. 2005. Identification and antimicrobial susceptibility of 100 animal clinical isolates. Vet. Microbiol. 105:307-312. [PubMed] 3. Clinical and Laboratory Standards Institute. 2007. Performance standards for antimicrobial susceptibility testing; seventeenth informational supplement. CLSI document M100-S17. Clinical and Laboratory Standards Institute, Wayne, PA. 4. Constable, P. D., and D. E. Morin. 2003. Treatment of clinical mastitis using antimicrobial susceptibility profiles for treatment decisions. Vet. Clin. Food Anim. 19:139-155. [PubMed] 5. Donaldson, S. C., B. A. Straley, N. V. Hedge, A. A. Sawant, C. Debroy, and B. M. Jayarao. 2006. Molecular epidemiology of ceftiofur-resistant isolates from dairy calves. Appl. Environ. Microbiol. 72:3940-3948. [PMC free article] [PubMed] 6. Erskine, R. J., P. C. Bartlett, J. L. VanLente, and C. R. Phipps. 2002. Efficacy of systemic ceftiofur as a therapy for severe clinical mastitis in dairy cattle. J. Dairy Sci. 85:2571-2575. [PubMed] 7. Hillerton, J. E., and E. A. Berry. 2005. Treating mastitis in the cowa tradition or an archaism. J. Appl. Microbiol. 98:1250-1255. [PubMed] 8. Hogan, J., and K. L. Smith. 2003. Coliform mastitis. Vet. Res. 34:507-519. [PubMed] 9. Livermore, D. M. 1995. -Lactamases in laboratory and clinical resistance. Clin. Microbiol. Rev. 8:557-584. [PMC free article] [PubMed] 10. Livermore, D. M., and D. F. Brown. 2001. Detection of -lactamase-mediated resistance. J. Antimicrob. Chemother. 48(Suppl. 1):59-64. [PubMed] 11. Locatelli, C., I. Caronte, L. Scaccabarozzi, R. Migliavacca, L. Pagani, and P. Moroni. 2009. Extended-spectrum -lactamase production in E. coli strains isolated from clinical bovine mastitis. Vet. Res. Commun. 33(Suppl. 1):S141-S144. [PubMed] 12. Madec, J.-Y., C. Lazizzera, P. Chatre, D. Meunier, S. Martin, G. Lepage, M.-F. Mnard, P. Lebreton, and T. Raumbaud. 2008. Prevalence 133407-82-6 IC50 of fecal carriage of acquired expanded-spectrum cephalosporin resistance in strains from cattle in France. J. Clin. Microbiol. 46:1566-1567. [PMC free article] [PubMed] 13. Munoz, M. A., F. L. Welcome, Y. H. Schukken, and R. N. Zadoks. 2007. Molecular epidemiology of two mastitis outbreak on a dairy farm in New York State. J. Clin. Microbiol. 45:3964-3971. [PMC free article] [PubMed] 14. Pagani, L., R. Migliavacca, L. Pallecchi, C. Matti, E. Giacobone, G. Amicosante, E. Romero, and G. M. Rossolini. 2002. Emerging extended-spectrum -lactamases in from a hospital in northern Italy. J. Clin. Microbiol. 41:4264-4269. [PMC free article] [PubMed] 16. Vo An, T. T., E. van Duijkeren, A. C. Fluit, and W. Gaastra. 2007. Characteristics of extended-spectrum cephalosporin-resistant and isolates from horses. Vet. Microbiol. 124:248-255. [PubMed]. ESBL detection in veterinary medicine (2, 16), and no data are available describing ESBL-mediated resistance in spp. isolated from bovine mastitis. In this research, the antimicrobial susceptibility and ESBL production were evaluated in isolated from animals with bovine clinical mastitis and selected for their resistance to ceftiofur, a broad-spectrum cephalosporin. Susceptibility to a range of antimicrobials, including those approved for both human and animal use (Table ?(Table1),1), was determined by disk diffusion assay according to Clinical and Laboratory Standards Institute interpretative criteria (3). ATCC 25922 and ATCC 25923 were used as Pax1 quality control standards. TABLE 1. Comparison of resistance profiles of the nine isolates From March 2008 to March 2009, 140 klebsiellae were isolated from milk samples, representing 26.6% of the total number of Gram-negative isolates obtained from bovine clinical mastitis. The isolates were subcultured on MacConkey agar supplemented with 8 g/ml of ceftiofur (5). Nine isolates, identified as isolates were screened against the panel in Table ?Table11 by the disk diffusion method and for ESBLs by the double-disk diffusion assay. A strain was considered an ESBL producer when it showed the expansion of an inhibition zone between a disk containing amoxicillin-clavulanate (20 and 10 g) and disks containing, respectively, ceftazidime (30 g) and cefotaxime (30 g) placed 25 mm apart (10). All isolates were tested by PCR targeting the Ambler class A -lactamase genes animal isolates (16), using previously published primer sets (14, 15). Sequencing of amplified genes completed the study. The complete resistance profiles and the PCR results are summarized in Table ?Table1.1. Only isolate 205, identified as subsp. subsp. isolate in a case of bovine clinical mastitis. Isolate 205 also displayed a positive PCR result for genes and no spp. (11). The generally low level of resistance and the low prevalence of ESBL-producing strains (0.7%) should guarantee a good outcome of drug treatment for involved in mastitis, but antimicrobial resistance is not the 133407-82-6 IC50 only factor affecting therapy efficacy (1, 4). However, the described isolation must encourage that attention be paid to a phenomenon which could evolve and be enforced under the pressure of antimicrobial therapy, as the use of extended-spectrum cephalosporins in veterinary medicine may select ESBL producers (12). It is recommended to 133407-82-6 IC50 screen klebsiellae from cases of bovine mastitis in order to keep the prevalence of ESBL producers under control. Nucleotide sequence accession number. The sequence of the CTX-M class enzyme-encoding gene identified in our study was added to GenBank (accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”HM921043″,”term_id”:”302826889″,”term_text”:”HM921043″HM921043). Footnotes ?Published ahead of print on 18 August 2010. REFERENCES 1. Bradley, A. J., and M. J. Green. 2009. Factors affecting cure when treating bovine clinical mastitis with cephalosporin-based intramammary preparations. J. Dairy Sci. 92:1941-1953. [PMC free article] [PubMed] 2. Brisse, S., and E. van Duijkeren. 2005. Identification and antimicrobial susceptibility of 100 animal clinical isolates. Vet. Microbiol. 105:307-312. [PubMed] 3. Clinical and Laboratory Standards Institute. 2007. Performance standards for antimicrobial susceptibility testing; seventeenth informational supplement. CLSI document M100-S17. Clinical and Laboratory Standards Institute, Wayne, PA. 4. Constable, P. D., and D. E. Morin. 2003. Treatment of clinical mastitis using antimicrobial susceptibility profiles for treatment decisions. Vet. Clin. Food Anim. 19:139-155. [PubMed] 5. Donaldson, 133407-82-6 IC50 S. C., B. A. Straley, N. V. Hedge, A. A. Sawant, C. Debroy, and B. M. Jayarao. 2006. Molecular epidemiology of ceftiofur-resistant isolates from dairy.

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