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Journal of Clinical Microbiology, October 2004, p. 4799-4802, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4799-4802.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

New Method for Laboratory Detection of AmpC ß-Lactamases in Escherichia coli and Klebsiella pneumoniae

K. Nasim,1 S. Elsayed,1,2 J. D. D. Pitout,1,2 J. Conly,1,3 D. L. Church,1,2,3 and D. B. Gregson1,2,3*

Department of Pathology and Laboratory Medicine,1 Department of Medicine, University of Calgary,3 Calgary Laboratory Services, Calgary, Alberta, Canada2

Received 6 November 2003/ Returned for modification 12 March 2004/ Accepted 26 April 2004


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ABSTRACT
 
A new cefoxitin-agar medium (CAM)-based assay was compared to the previously published modified three-dimensional (M3D) assay for the detection of AmpC production in Escherichia coli and Klebsiella pneumoniae. Clinical isolates of cefoxitin-resistant E. coli (n = 5) and K. pneumoniae (n = 7) and multiple control strains with and without AmpC enzymes were tested by both methods. The CAM method with 4 µg of cefoxitin/ml was equivalent to the M3D method for detecting AmpC production in E. coli and K. pneumoniae. This new method is easier to perform and interpret and allows for testing of multiple isolates on a single plate.


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TEXT
 
AmpC-mediated beta-lactam resistance (AmpC-R) in Escherichia and Klebsiella spp. is an emerging problem (28). High-level AmpC production is typically associated with in vitro resistance to all beta-lactam antibiotics except for carbapenems and cefepime. In addition, treatment failures with broad-spectrum cephalosporins have been documented (26, 36). These enzymes are not affected by available beta-lactamase inhibitors and, in association with the loss of outer membrane porins (OMP), can produce resistance to carbapenems (7, 8, 31). Genes for these beta-lactamases are found on the chromosomes of some members of the family Enterobacteriaceae (2, 19, 20, 25). Plasmid-mediated AmpC-R has arisen through the transfer of chromosomal genes for the inducible AmpC beta-lactamases onto plasmids (5, 9, 30, 34). Plasmids with these genes can spread among other members of the family Enterobacteriaceae (22, 35, 38), have been documented in many countries (4, 12, 14, 21), and can cause nosocomial outbreaks (7, 22).

Detecting AmpC-R is a challenge for laboratories (32). There is no National Committee for Clinical Laboratory Standards (NCCLS) guideline for its detection. We wished to address this issue by studying the use a cefoxitin agar medium (CAM), in comparison with a modified three-dimensional AmpC assay (M3D) for the detection of AmpC-R in clinical isolates of Escherichia coli and Klebsiella pneumoniae.

Isolates of E. coli containing MIR-1, FOX-1, and MOX-1; one K. pneumoniae strain known to be both AmpC and extended-spectrum-beta-lactamase (ESBL) positive; and E. coli ATCC 25922 were used as controls. Clinical isolates of E. coli, K. pneumoniae, and Enterobacter cloacae encountered at Calgary Laboratory Services and selected for study included 6 cefoxitin-sensitive ESBL-negative E. coli isolates, 8 AmpC-negative and ESBL-positive E. coli isolates, 55 cefoxitin-resistant and ESBL-negative E. coli isolates, 6 cefoxitin-resistant and ESBL-negative K. pneumoniae isolates, and 9 E. cloacae isolates showing in vitro susceptibility to extended-spectrum cephalosporins. Clinical isolates were identified using the Vitek automated microbial identification system (bioMerieux Inc., Durham, N.C.). Cefoxitin resistance was detected using the disk diffusion technique, and ESBL production was determined using double-disk diffusion methods, according to NCCLS guidelines (23, 24). Cefoxitin MICs were determined by the E-test method (AB Biodisk, Solna, Sweden) according to the manufacturer's recommendations.

The M3D assay was performed as described by Coudron et al. (13) and was used as the "gold standard" for detecting AmpC-R (Fig. 1). For the CAM assay, crude enzyme extracts were prepared by freezing and thawing cell pellets from centrifuged tryptic soy broth cultures as described previously (13). Mueller-Hinton agar with cefoxitin concentrations of 2, 4, 8, and 16 µg/ml was used. Plates were inoculated with E. coli ATCC 25922 to cover the entire surface. Circular wells with diameters of 5 mm were made in the agar and filled with 30 µl of extract from individual strains. Positive-control (E. coli with MOX-1) and negative-control (E. coli ATCC 11775) strains were included on each plate. Plates were incubated overnight aerobically at 35°C. A zone of growth around the periphery of a well was considered a positive CAM assay and evidence for the presence of an AmpC enzyme (Fig. 2).



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  		FIG. 1. M3D assay. AmpC-positive extracts distort the zone around the cefoxitin disk.



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  		FIG. 2. CAM assay. AmpC-positive extracts produce a zone of growth around wells.

The M3D assay was negative with all AmpC-negative controls and positive with all known AmpC-positive controls and the nine E. cloacae clinical isolates (AmpC noninduced). Fifty-four of 55 E. coli strains and 1 of 6 Klebsiella pneumoniae strains were positive by the M3D method. The results of CAM with 4 µg of cefoxitin per ml were 100% concordant with those of the M3D method (Table 1). At higher and lower cefoxitin concentrations, the CAM method did not correlate as well with the M3D method. Cefoxitin MICs ranged between 16 and ≥256 µg/ml. No correlation was found between MICs and zone sizes by the CAM method (data not shown).


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  		TABLE 1. Summary results of AmpC detection assays

K. pneumoniae, E. coli, Salmonella spp., and Proteus mirabilis lack inducible AmpC enzymes (28). E. coli does carry an ampC gene but lacks the regulatory gene (ampR), leading to negligible enzyme production (18). In clinical isolates of E. coli, cephamycin resistance can be due to promoter or attenuator gene mutations (11, 15), the acquisition of plasmids with ampC genes (3, 12, 14, 16, 27, 29, 34, 35, 37, 39), and OMP changes (10). In our clinical strains of E. coli, only 1 of 55 showed no evidence of AmpC-R. In Klebsiella spp., interruption of a porin gene by insertion sequences has been described as a common type of mutation that causes increased cefoxitin resistance (17). In our study, only one of six cefoxitin-resistant K. pneumoniae strains was positive for AmpC enzymes by both the M3D and CAM methods, implying that cephamycin resistance in these isolates is due to OMP changes.

The CAM method is potentially useful for differentiating AmpC-R from other resistance mechanisms. In Klebsiella and Salmonella spp., both of which lack a chromosomal ampC gene, a positive test suggests plasmid-mediated AmpC resistance. In E. coli, once enzyme-mediated AmpC resistance has been detected, its plasmidic basis can be confirmed by transferring the plasmid to recipient bacterial strains via transconjugation or by detection of specific genes known to be transferred on plasmids (27).

Currently, detection of AmpC enzymes is a problem for clinical laboratories. Although lack of inhibition of activity against oxyimino-ß-lactams or cephamycins by beta-lactam inhibitors is indirect evidence for their presence, some AmpC enzymes have been shown to be susceptible to inhibition by tazobactam (1, 6). Inhibitors that are active against AmpC enzymes are not readily available. The CAM method with cefoxitin (4 µg/ml) is simpler to perform and easier to interpret than the M3D method. It allows for testing of up to five isolates and two controls on a single plate and is as sensitive as the M3D method. In addition, clinical isolates with multiple beta-lactamases have been described, resulting in difficulties in interpretation of MIC patterns (13, 33). The CAM method may prove useful in detecting the presence of AmpC-R in isolates that have multiple mechanisms of cephalosporin resistance. The control strain known to be both ESBL and AmpC positive was found to be positive for AmpC by this assay.

The CAM method with 4 µg of cefoxitin per ml is as sensitive and specific as the M3D method for AmpC detection in E. coli and K. pneumoniae. The new method is easier to perform and interpret and allows for the testing of multiple isolates on a single plate. Clinical and research laboratories can use this technique to confirm the presence of AmpC-mediated resistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Calgary Laboratory Services, #9, 3535 Research Rd., NW, Calgary, Alberta, Canada T2L 2K8. Phone: (403) 770-3309. Fax: (403) 770-3347. E-mail: dan.gregson{at}cls.ab.ca. Back


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REFERENCES
 
    1
  1. Babini, G. S., F. Danel, S. D. Munro, P. A. Micklesen, and D. M. Livermore. 1998. Unusual tazobactam-sensitive AmpC beta-lactamase from two Escherichia coli isolates. J. Antimicrob. Chemother. 41:115-118.[Abstract/Free Full Text]
    2. 2
  2. Barlow, M., and B. G. Hall. 2002. Origin and evolution of the AmpC ß-lactamases of Citrobacter freundii. Antimicrob. Agents Chemother. 46:1190-1198.[Abstract/Free Full Text]
    3. 3
  3. 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]
    4. 4
  4. 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]
    5. 5
  5. Bauernfeind, A., I. Stemplinger, R. Jungwirth, and H. Giamarellou. 1996. Characterization of the plasmidic ß-lactamase CMY-2, which is responsible for cephamycin resistance. Antimicrob. Agents Chemother. 40:221-224.[Abstract]
    6. 6
  6. Bonomo, R. A., J. Liu, Y. Chen, L. Ng, A. M. Hujer, and V. E. Anderson. 2001. Inactivation of CMY-2 beta-lactamase by tazobactam: initial mass spectroscopic characterization. Biochim. Biophys. Acta 1547:196-205.[CrossRef][Medline]
    7. 7
  7. Bradford, P. A., C. Urban, N. Mariano, S. J. Projan, J. J. Rahal, and K. Bush. 1997. Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC ß-lactamase, and the loss of an outer membrane protein. Antimicrob. Agents Chemother. 41:563-569.[Abstract]
    8. 8
  8. Cao, V. T., G. Arlet, B. M. Ericsson, A. Tammelin, P. Courvalin, and T. Lambert. 2000. Emergence of imipenem resistance in Klebsiella pneumoniae owing to combination of plasmid-mediated CMY-4 and permeability alteration. J. Antimicrob. Chemother. 46:895-900.[Abstract/Free Full Text]
    9. 9
  9. Carattoli, A., F. Tosini, W. P. Giles, M. E. Rupp, S. H. Hinrichs, F. J. Angulo, T. J. Barrett, and P. D. Fey. 2002. Characterization of plasmids carrying CMY-2 from expanded-spectrum cephalosporin-resistant Salmonella strains isolated in the United States between 1996 and 1998. Antimicrob. Agents Chemother. 46:1269-1272.[Abstract/Free Full Text]
    10. 10
  10. Chen, H. Y., and D. M. Livermore. 1993. Activity of cefepime and other beta-lactam antibiotics against permeability mutants of Escherichia coli and Klebsiella pneumoniae. J. Antimicrob. Chemother. 32(Suppl. B):63-74.[Abstract/Free Full Text]
    11. 11
  11. Clarke, B., M. Hiltz, H. Musgrave, and K. R. Forward. 2003. Cephamycin resistance in clinical isolates and laboratory-derived strains of Escherichia coli, Nova Scotia, Canada. Emerg. Infect. Dis. 9:1254-1259.[Medline]
    12. 12
  12. Coudron, P. E., N. D. Hanson, and M. W. Climo. 2003. Occurrence of extended-spectrum and AmpC ß-lactamases in bloodstream isolates of Klebsiella pneumoniae: isolates harbor plasmid-mediated FOX-5 and ACT-1 AmpC beta-lactamases. J. Clin. Microbiol. 41:772-777.[Abstract/Free Full Text]
    13. 13
  13. Coudron, P. E., E. S. Moland, and K. S. Thomson. 2000. Occurrence and detection of AmpC ß-lactamases among Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates at a veterans medical center. J. Clin. Microbiol. 38:1791-1796.[Abstract/Free Full Text]
    14. 14
  14. Doi, Y., N. Shibata, K. Shibayama, K. Kamachi, H. Kurokawa, K. Yokoyama, T. Yagi, and Y. Arakawa. 2002. Characterization of a novel plasmid-mediated cephalosporinase (CMY-9) and its genetic environment in an Escherichia coli clinical isolate. Antimicrob. Agents Chemother. 46:2427-2434.[Abstract/Free Full Text]
    15. 15
  15. Forward, K. R., B. M. Willey, D. E. Low, A. McGeer, M. A. Kapala, M. M. Kapala, and L. L. Burrows. 2001. Molecular mechanisms of cefoxitin resistance in Escherichia coli from the Toronto area hospitals. Diagn. Microbiol. Infect. Dis. 41:57-63.[CrossRef][Medline]
    16. 16
  16. Gazouli, M., L. S. Tzouvelekis, A. C. Vatopoulos, and E. Tzelepi. 1998. Transferable class C beta-lactamases in Escherichia coli strains isolated in Greek hospitals and characterization of two enzyme variants (LAT-3 and LAT-4) closely related to Citrobacter freundii AmpC beta-lactamase. J. Antimicrob. Chemother. 42:419-425.[Abstract/Free Full Text]
    17. 17
  17. Hernandez-Alles, S., V. J. Benedi, L. Martinez-Martinez, A. Pascual, A. Aguilar, J. M. Tomas, and S. Alberti. 1999. Development of resistance during antimicrobial therapy caused by insertion sequence interruption of porin genes. Antimicrob. Agents Chemother. 43:937-939.[Abstract/Free Full Text]
    18. 18
  18. Honore, N., M. H. Nicolas, and S. T. Cole. 1986. Inducible cephalosporinase production in clinical isolates of Enterobacter cloacae is controlled by a regulatory gene that has been deleted from Escherichia coli. EMBO J. 5:3709-3714.[Medline]
    19. 19
  19. Korfmann, G., C. C. Sanders, and E. S. Moland. 1991. Altered phenotypes associated with ampD mutations in Enterobacter cloacae. Antimicrob. Agents Chemother. 35:358-364.[Abstract/Free Full Text]
    20. 20
  20. Lindberg, F., and S. Normark. 1986. Contribution of chromosomal beta-lactamases to beta-lactam resistance in enterobacteria. Rev. Infect. Dis. 8(Suppl. 3):S292-S304.
    21. 21
  21. Manchanda, V., and N. P. Singh. 2003. Occurrence and detection of AmpC beta-lactamases among Gram-negative clinical isolates using a modified three-dimensional test at Guru Tegh Bahadur Hospital, Delhi, India. J. Antimicrob. Chemother. 51:415-418.[Abstract/Free Full Text]
    22. 22
  22. Nadjar, D., M. Rouveau, C. Verdet, L. Donay, J. Herrmann, P. H. Lagrange, A. Philippon, and G. Arlet. 2000. Outbreak of Klebsiella pneumoniae producing transferable AmpC-type beta-lactamase (ACC-1) originating from Hafnia alvei. FEMS Microbiol. Lett. 187:35-40.[Medline]
    23. 23
  23. National Committee for Clinical Laboratory Standards. 2000. Performance standards for antimicrobial disk susceptibility tests. Approved standard, 7th ed. NCCLS document M2-A7. National Committee for Laboratory Standards, Wayne, Pa.
    24. 24
  24. National Committee for Clinical Laboratory Standards. 2002. Performance standards for antimicrobial susceptibility testing. Twelfth informational supplement. NCCLS document M100-S12. National Committee for Laboratory Standards, Wayne, Pa.
    25. 25
  25. Normark, S., S. Lindquist, and F. Lindberg. 1986. Chromosomal beta-lactam resistance in enterobacteria. Scand. J. Infect. Dis. Suppl. 49:38-45.[Medline]
    26. 26
  26. 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]
    27. 27
  27. Perez-Perez, F. J., and N. D. Hanson. 2002. Detection of plasmid-mediated AmpC ß-lactamase genes in clinical isolates by using multiplex PCR. J. Clin. Microbiol. 40:2153-2162.[Abstract/Free Full Text]
    28. 28
  28. Philippon, A., G. Arlet, and G. A. Jacoby. 2002. Plasmid-determined AmpC-type ß-lactamases. Antimicrob. Agents Chemother. 46:1-11.[Free Full Text]
    29. 29
  29. Pornull, K. J., G. Rodrigo, and K. Dornbusch. 1994. Production of a plasmid mediated AmpC-like beta-lactamase by a Klebsiella pneumoniae septicaemia isolate. J. Antimicrob. Chemother. 34:943-954.[Abstract/Free Full Text]
    30. 30
  30. Queenan, A. M., S. Jenkins, and K. Bush. 2001. Cloning and biochemical characterization of FOX-5, an AmpC-type plasmid-encoded ß-lactamase from a New York City Klebsiella pneumoniae clinical isolate. Antimicrob. Agents Chemother. 45:3189-3194.[Abstract/Free Full Text]
    31. 31
  31. 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]
    32. 32
  32. Subha, A., V. R. Devi, and S. Ananthan. 2003. AmpC beta-lactamase producing multidrug resistant strains of Klebsiella spp. and Escherichia coli isolated from children under five in Chennai. Indian J. Med. Res. 117:13-18.[Medline]
    33. 33
  33. Tzouvelekis, L. S., A. C. Vatopoulos, G. Katsanis, and E. Tzelepi. 1999. Rare case of failure by an automated system to detect extended-spectrum ß-lactamase in a cephalosporin-resistant Klebsiella pneumoniae isolate. J. Clin. Microbiol. 37:2388.[Free Full Text]
    34. 34
  34. Walther-Rasmussen, J., and N. Hoiby. 2002. Plasmid-borne AmpC beta-lactamases. Can. J. Microbiol. 48:479-493.[CrossRef][Medline]
    35. 35
  35. 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]
    36. 36
  36. Wong-Beringer, A., J. Hindler, M. Loeloff, A. M. Queenan, N. Lee, D. A. Pegues, J. P. Quinn, and K. Bush. 2002. Molecular correlation for the treatment outcomes in bloodstream infections caused by Escherichia coli and Klebsiella pneumoniae with reduced susceptibility to ceftazidime. Clin. Infect. Dis. 34:135-146.[CrossRef][Medline]
    37. 37
  37. 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 ß-lactamase: confirmation that the plasmid-mediated cephamycinase originated from the Citrobacter freundii AmpC ß-lactamase. Antimicrob. Agents Chemother. 43:1350-1357.[Abstract/Free Full Text]
    38. 38
  38. Yan, J. J., C. H. Chiu, W. C. Ko, C. L. Chuang, and J. J. Wu. 2002. Ceftriaxone-resistant Salmonella enterica serovar Hadar: evidence for interspecies transfer of blaCMY-2 in a Taiwanese university hospital. J. Formos. Med. Assoc. 101:665-668.[Medline]
    39. 39
  39. 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]

Journal of Clinical Microbiology, October 2004, p. 4799-4802, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4799-4802.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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