AAC
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Briñas, L.
Right arrow Articles by Torres, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Briñas, L.
Right arrow Articles by Torres, C.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, June 2003, p. 2056-2058, Vol. 47, No. 6
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.6.2056-2058.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Detection of CMY-2, CTX-M-14, and SHV-12 ß-Lactamases in Escherichia coli Fecal-Sample Isolates from Healthy Chickens

Laura Briñas,1 Miguel Angel Moreno,2 Myriam Zarazaga,1 Concepción Porrero,2 Yolanda Sáenz,1 María García,2 Lucas Dominguez,2 and Carmen Torres1*

Area de Bioquímica y Biología Molecular, Universidad de La Rioja, 26006 Logroño,1 Departamento de Patología Animal I, Facultad de Veterinaria, Universidad Complutense Madrid, Madrid, Spain2

Received 7 October 2002/ Returned for modification 20 December 2002/ Accepted 14 March 2003


   ABSTRACT
Top
 Abstract
Text
 References
 
Genes encoding the CMY-2, CTX-M-14, and SHV-12 ß-lactamases were detected in three of five Escherichia coli isolates from fecal samples from healthy chickens which showed resistance or diminished susceptibility to extended-spectrum cephalosporins. A -42 mutation at the promoter region of the ampC gene was detected in the other two isolates.


   TEXT
Top
 Abstract
 Text
References
 
Escherichia coli is an important pathogen of animals and humans, and it is also a common inhabitant of their intestinal tracts. An increase in the rate of ß-lactam resistance in E. coli isolates has been observed in the last few years, ß-lactamase synthesis being the main mechanism of resistance. Class A extended-spectrum ß-lactamases (ESBLs) (CTX-M or enzymes derived from the classic TEM-1 and SHV-1) and plasmidic class C ß-lactamases (FOX and CMY, among others) have been described for E. coli, and this fact is a significant cause of concern (5, 21). Several studies show the dissemination in Spain of ESBLs among human clinical isolates from hospitalized patients as well as from those in the community (4, 10; C. Sanchez, B. Padilla, C. Perez, E. Cercenado, M. Sanchez, J. Gomez, and E. Bouza, Abstr. 42nd Intersci. Conf. Antimicrob. Agents Chemother., abstr. K-1947, 2002). Very few studies on the characterization of ß-lactamases in E. coli isolates from sick animals (6, 7, 12, 27; T. Teshager, L. Dominguez, M. A. Moreno, Y. Sáenz, M. Zarazaga, C. Torres, and S. Cardeñosa, Letter, Antimicrob. Agents Chemother. 44:3483-3484, 2000) or from healthy animals (8) exist to date.

In this study, ß-lactamase characterization of five E. coli isolates recovered from fecal samples of healthy chickens which showed a cefotaxime and/or a ceftazidime MIC of >=4 µg/ml was carried out. These isolates belong to a collection of 120 recovered (between November 2000 and February 2001) at the slaughterhouse level from fecal samples of healthy chickens as part of the Spanish antimicrobial resistance surveillance program (17). MICs of 18 different antibiotics were determined for these isolates by the agar dilution method (18); the disk diffusion method was used for 3 additional antibiotics. Results are shown in Table 1. The ranges of the MICs of cefotaxime and ceftazidime were 0.25 to 128 µg/ml and 0.5 to 32 µg/ml, respectively. Three of the isolates showed resistance to nalidixic acid, ciprofloxacin, tetracycline, and trimethoprim-sulfamethoxazole, and two of them were also resistant to chloramphenicol, gentamicin, kanamycin, and tobramycin. All five isolates were susceptible to amikacin and fosfomycin.


View this table:
[in this window]
[in a new window]
  		TABLE 1. MIC values for five E. coli isolates recovered from healthy chickens with diminished susceptibility or resistance to cefotaxime and/or ceftazidime

 
The presence of genes encoding different types of ß-lactamases (TEM, SHV, OXA-1, CMY, CTX-M, FOX, and the mutation in the promoter of ampC) in the five isolates was studied. The detection of the blaTEM, blaSHV, and blaOXA-1 genes was carried out by PCR (Table 2). The blaTEM and blaSHV amplicons obtained were sequenced (both strands) using the same set of primers as for the PCRs. The DNA and the deduced amino acid sequences were compared with those previously described (GenBank database and G. Jacoby and K. Bush website [http://www.lahey.org/studies/webt.htm]). The nomenclature used for the blaTEM variants and promoters was as previously proposed (13, 15).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Primers and conditions used to amplify different ß-lactamase genes by the PCR technique

 
The detection of plasmidic class C ß-lactamases was carried out using specific primers for the amplification of blaCMY and blaFOX genes (Table 2). The complete sequence (approximately 1,800 bp) of the amplified fragment obtained with blaCMY primers was determined on both strands by applying a primer-walking strategy to obtain the whole blaCMY sequence as well as the upstream and downstream regions. The detection of plasmidic CTX-M-9-type and CTX-M-10-type (class A ß-lactamases) was also analyzed by PCR and by subsequently sequencing the amplicons (Table 2). Positive and negative controls were used in all reactions.

A positive blaTEM PCR result was obtained for three of the five E. coli isolates, and a positive blaSHV PCR result was obtained for an additional isolate (Table 3). Each of the three blaTEM amplicons was identified as a blaTEM-1b gene (the weak P3 promoter was identified in two of the isolates). The blaSHV amplicon was identified as a blaSHV-12 gene and was obtained in an E. coli isolate with an antibiotic resistance phenotype typical of an ESBL. The SHV-12 ß-lactamase had been previously found in human (19, 28) and animal E. coli clinical isolates (Teshager et al., letter, 2000) but never in healthy animal isolates.


View this table:
[in this window]
[in a new window]
  		TABLE 3. Plasmidic ß-lactamase genes and mutations in the promoter of the chromosomal ampC gene in five E. coli isolates recovered from healthy chickens with diminished susceptibility or resistance to cefotaxime and/or ceftazidime

 
A ß-lactamase CMY-2 was found in one E. coli isolate under study that also harbored a blaTEM-1b gene (Table 3). The sequence of the 1,800-bp amplicon, which included the blaCMY-2 gene, showed a genetic organization that enclosed (upstream of the blaCMY-2 gene) a partial region of the tnpA gene (encoding a transposase) and the right inverted repeat sequence of ISEcpI and (upstream of the blaCMY-2 gene) a partial region of an outer membrane lipoprotein gene. This genetic organization was similar to that previously described by Stapleton et al. (25; P. D. Stapleton, Abstr. InterSci. Conf. Antimicrob. Agents Chemother., abstr. 1457, 1999) in an E. coli plasmid carrying the blaCMY-4 gene and also to that found by Briñas et al. (7) in two E. coli isolates from sick animals (a chicken and a pig) which harbored the blaCMY-2 gene. The CMY-2 ß-lactamase has been previously detected in clinical bovine and porcine E. coli isolates (27) but never in healthy animals.

The only isolate (E. coli AOEC429432) for which cefotaxime had a MIC (128 µg/ml) higher than that of ceftazidime (0.5 µg/ml) gave a positive PCR result with the primers to detect blaCTX-M-9-like genes. This product was sequenced, and the deduced amino acid sequence revealed a CTX-M-14 ß-lactamase. This isolate also harbored a blaTEM-1b gene (Table 1). The nucleotide sequence of a 162-bp region upstream of the blaCTX-M-14 gene in this isolate was determined and analyzed with the BLASTN program at the National Center for Biotechnology Information. The sequence was 100% identical to the upstream region of the blaCTX-M-9 gene that was included in the In60 integron (23) (GenBank accession no. AF74129). Furthermore, our sequence included the 28-bp segment that had been proposed as a recognition site for the orf513 putative transposase (1). These results suggested that the blaCTX-M-14 gene in our isolate was located in an integron, as had been reported for other CTX-M ß-lactamases (1, 23). The CTX-M-14 ß-lactamase had been previously detected in human clinical E. coli isolates (4, 11, 16, 20, 24) but never before in animal isolates.

Negative results were obtained for the five E. coli isolates analyzed when the CTX-M-10-like, FOX, and OXA-1 sets of primers were used in the PCR assay.

The regulatory region of the chromosomal ampC gene was amplified by PCR (Table 2) with the five isolates and sequenced. The mutations in the promoter and attenuator region were studied by comparing the sequence with the same region in the E. coli K-12 ampC gene (14). No mutations were found in the two E. coli isolates which harbored the CMY-2 or CTX-M-14 ß-lactamases. Mutations at positions -18, -1, and +58 were detected in the E. coli isolate with the blaSHV-12 gene. Nevertheless, mutations at positions -42, -18, -1, and +58 were detected in the other two isolates (Table 1). Mutations at the -42 position were associated with hyperproduction of the chromosomal AmpC ß-lactamase and caused resistance to cefoxitin and a diminished susceptibility to extended-spectrum cephalosporins, as has been previously reported (9). The E. coli isolates which harbored blaCMY-2, blaCTX-M-14, or blaSHV-12 genes did not show a mutation at -42 position (Table 3).

To our knowledge, this is the first time that genes encoding SHV-12, CMY-2, and CTX-M-14 ß-lactamases have been detected in E. coli isolates from healthy animals. The use of extended-spectrum cephalosporins in chickens is very unusual, and the possibility of cross-selection with other antimicrobials used in poultry (such as sulfonamides and tetracyclines, among others) might explain this discovery and should be further analyzed in the future.


   ACKNOWLEDGMENTS
 
This work has been supported by grants from the Fondo de Investigaciones Sanitarias of Spain (FIS 01/973 and FIS 99/0938) and from the Ministerio de Agricultura, Pesca y Alimentacion of Spain.


   FOOTNOTES
 
* Corresponding author. Mailing address: Area de Bioquímica y Biología Molecular, Universidad de La Rioja, Madre de Dios, 51, 26006 Logroño, Spain. Phone: 34 941 299750. Fax: 34 941 299721. E-mail: carmen.torres{at}daa.unirioja.es. Back


   REFERENCES
Top
 Abstract
 Text
 References
 

  1. Arduino, S. M., P. H. Roy, G. A. Jacoby, B. E. Orman, S. A. Pineiro, and D. Centron. 2002. BlaCTX-M-2 is located in an unusual class 1 integron (In35) which includes Orf513. Antimicrob. Agents Chemother. 46:2303-2306.[Abstract/Free Full Text]
  2. Bauernfeind, A., S. Wagner, R. Jungwirth, I. Schneider, and D. Meyer. 1997. A novel class C ß-lactamase (FOX-2) in Escherichia coli conferring resistance to cephamycins. Antimicrob. Agents Chemother. 41:2041-2046.[Abstract]
  3. Belaaouaj, A., C. Lapoumeroulie, M. M. Caniça, G. Vedel, P. Névot, R. Krishnamoorthy, and G. Paul. 1994. Nucleotide sequences of the genes coding for the TEM-like ß-lactamases IRT-1 and IRT-2 (formerly called TRI-1 and TRI-2). FEMS Microbiol. Lett. 120:75-80.[Medline]
  4. Bou, G., M. Cartelle, M. Tomas, D. Canle, F. Molina, R. Moure, J. M. Eiros, and A. Guerrero. 2002. Identification and broad dissemination of the CTX-M-14 ß-lactamase in different Escherichia coli strains in the northwest area of Spain. J. Clin. Microbiol. 40:4030-4036.[Abstract/Free Full Text]
  5. Bradford, P. A. 2001. Extended-spectrum ß-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin. Microbiol. Rev. 14:933-951.[Abstract/Free Full Text]
  6. Bradford, P. A., P. J. Petersen, I. M. Fingerman, and D. G. White. 1999. Characterization of extended-spectrum cephalosporin resistance in E. coli isolates associated with bovine calf diarrhoeal disease. J. Antimicrob. Chemother. 44:607-610.[Abstract/Free Full Text]
  7. Briñas, L., M. A. Moreno, T. Teshager, M. Zarazaga, Y. Sáenz, C. Porrero, L. Domínguez, and C. Torres. ß-lactamase characterization in Escherichia coli isolates with diminished susceptibility or resistance to extended-spectrum cephalosporins recovered from sick animals in Spain. Microb. Drug Resist., in press.
  8. Briñas, L., M. Zarazaga, Y. Sáenz, F. Ruiz-Larrea, and C. Torres. 2002. ß-Lactamase characterization in ampicillin-resistant Escherichia coli isolates obtained from foods, humans, and healthy animals. Antimicrob. Agents Chemother. 46:3156-3163.[Abstract/Free Full Text]
  9. Caroff, N., E. Espaze, I. Bérard, H. Richet, and A. Reynaud. 1999. Mutations in the ampC promoter of Escherichia coli isolates resistant to oxyiminocephalosporins without extended spectrum ß-lactamase production. FEMS Microbiol. Lett. 173:459-465.[Medline]
  10. Coque, T. M., A. Oliver, J. C. Pérez-Díaz, F. Baquero, and R. Cantón. 2002. Genes encoding TEM-4, SHV-2, and CTX-M-10 extended-spectrum ß-lactamases are carried by multiple Klebsiella pneumoniae clones in a single hospital (Madrid, 1989 to 2000). Antimicrob. Agents Chemother. 46:500-510.[Abstract/Free Full Text]
  11. Dutour, C., R. Bonnet, H. Marchandin, M. Boyer, C. Chanal, D. Sirot, and J. Sirot. 2002. CTX-M-1, CTX-M-3, and CTX-M-14 ß-lactamases from Enterobacteriaceae isolated in France. Antimicrob. Agents Chemother. 46:534-537.[Abstract/Free Full Text]
  12. Feria, C., E. Ferreira, J. D. Correia, J. Goncalves, and M. Canica. 2002. Patterns and mechanisms of resistance to ß-lactams and ß-lactamase inhibitors in uropathogenic Escherichia coli isolated from dogs in Portugal. J. Antimicrob. Chemother. 49:77-85.[Abstract/Free Full Text]
  13. Goussard, S., and P. Courvalin. 1999. Updated sequence information for TEM ß-lactamase genes. Antimicrob. Agents Chemother. 43:367-370.[Abstract/Free Full Text]
  14. Jaurin, B., and T. Grundström. 1981. ampC cephalosporinase of Escherichia coli K-12 has a different evolutionary origin from that of ß-lactamases of the penicillinase type. Proc. Natl. Acad. Sci. USA 78:4897-4901.[Abstract/Free Full Text]
  15. Leflon-Guibout, V., B. Heym, and M.-H. Nicholas-Chanoine. 2000. Updated sequence information proposed nomenclature for blaTEM genes and their promoters. Antimicrob. Agents Chemother. 44:3232-3234.[Abstract/Free Full Text]
  16. Ma, L., Y. Ishii, F. Y. Chang, K. Yamaguchi, M. Ho, and L. K. Siu. 2002. CTX-M-14, a plasmid-mediated CTX-M type extended-spectrum ß-lactamase isolated from Escherichia coli. Antimicrob. Agents Chemother. 46:1985-1988.[Abstract/Free Full Text]
  17. Moreno, M. A., L. Dominguez, T. Teshager, I. A. Herrero, and M. C. Porrero. 2000. Antibiotic resistance monitoring: the Spanish programme. The VAV Network. Red de Vigilancia de Resistencias Antibioticas en Bacterias de Origen Veterinario. Int. J. Antimicrob. Agents 14:285-290.[CrossRef][Medline]
  18. National Committee for Clinical Laboratory Standards. 2000. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. NCCLS document M7-A5. National Committee for Clinical Laboratory Standards, Wayne, Pa.
  19. Nüesch-Inderbinen, M. T., F. H. Kayser, and H. Hachler. 1997. Survey and molecular genetics of SHV ß-lactamases in Enterobacteriaceae in Switzerland: two novel enzymes, SHV-11 and SHV-12. Antimicrob. Agents Chemother. 41:943-949.[Abstract]
  20. Pai, H., E.-H. Choi, H.-J. Lee, J. Y. Hong, and G. A. Jacoby. 2001. Identification of CTX-M-14 extended-spectrum ß-lactamase in clinical isolates of Shigella sonnei, Escherichia coli, and Klebsiella pneumoniae in Korea. J. Clin. Microbiol. 39:3747-3749.[Abstract/Free Full Text]
  21. Philippon, A., G. Arlet, and G. A. Jacoby. 2002. Plasmid-determined AmpC-type ß-lactamases. Antimicrob. Agents Chemother. 46:1-11.[Free Full Text]
  22. Pitout, J. D., K. S. Thomson, N. D. Hanson, A. F. Ehrhardt, E. S. Moland, and C. C. Sanders. 1998. ß-Lactamases responsible for resistance to expanded-spectrum cephalosporins in Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis isolates recovered in South Africa. Antimicrob. Agents Chemother. 42:1350-1354.[Abstract/Free Full Text]
  23. Sabaté, M., F. Navarro, E. Miró, S. Campoy, B. Mirelis, J. Barbé, and G. Prats. 2002. Novel complex sul1-type integron in Escherichia coli carrying blaCTX-M-9. Antimicrob. Agents Chemother. 46:2656-2661.[Abstract/Free Full Text]
  24. Saladin, M., V. T. B. Cao, T. Lambert, J. L. Donay, J. L. Herrmann, Z. Ould-Hocine, C. Verdet, F. Delisle, A. Philippon, and G. Arlet. 2002. Diversity of CTX-M ß-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals. FEMS Microb. Lett. 209:161-168.[Medline]
  25. Stapleton, P. D., K. P. Shannon, and G. L. French. 1999. Carbapenem resistance in Escherichia coli associated with plasmid-determined CMY-4 ß-lactamase production and loss of an outer membrane protein. Antimicrob. Agents Chemother. 43:1206-1210.[Abstract/Free Full Text]
  26. Steward, C. D., J. K. Rasheed, S. K. Hubert, J. W. Biddle, P. M. Raney, G. J. Anderson, P. P. Williams, K. L. Britain, A. Oliver, J. E. McGowan, Jr., and F. C. Tenover. 2001. Characterization of clinical isolates of Klebsiella pneumoniae from 19 laboratories using the National Committee for Clinical Laboratory Standards extended-spectrum ß-lactamases detection methods. J. Antimicrob. Agents 39:2864-2872.
  27. Winokur, P. L., D. L. Vonstein, L. J. Hoffman, E. K. Uhlenhopp, and G. V. Doern. 2001. Evidence for transfer of CMY-2 AmpC ß-lactamase plasmids between Escherichia coli and Salmonella isolates from food animals and humans. Antimicrob. Agents Chemother. 45:2716-2722.[Abstract/Free Full Text]
  28. Yan, J.-J., W.-C. Ko, S.-H. Tsai, H.-M. Wu, Y.-T. Jin, and J.-J. Wu. 2000. Dissemination of CTX-M-3 and CMY-2 ß-lactamases among clinical isolates of Escherichia coli in southern Taiwan. J. Clin. Microbiol. 38:4320-4325.[Abstract/Free Full Text]

Antimicrobial Agents and Chemotherapy, June 2003, p. 2056-2058, Vol. 47, No. 6
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.6.2056-2058.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Briñas, L.
Right arrow Articles by Torres, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Briñas, L.
Right arrow Articles by Torres, C.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Clin. Vaccine Immunol. Clin. Microbiol. Rev.
J. Clin. Microbiol. ALL ASM JOURNALS