Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 28, Num. 1, 2010, pp. 81-82

Indian Journal of Medical Microbiology, Vol. 28, No. 1, January-March, 2010, pp. 81-82

Correspondence

Important methodological considerations with respect to differentiation of CTX-M-15 and CTX-M-28 extended-spectrum beta-lactamases

*GA Menezes, MA Khan, JP Hays

Department of Microbiology (GAM), Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry - 605 006, India, Department of Medical Microbiology & Infectious Diseases (MAK,JPH), Erasmus MC, Rotterdam, The Netherlands

Correspondence Address: *Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry - 605 006, India, godfredmenezes@gmail.com

Date of Submission: 27-Feb-2009
Date of Acceptance: 16-May-2009

Code Number: mb10026

PMID: 20061778

DOI: 10.4103/0255-0857.58743

Dear Editor,

The acquisition and expression of beta-lactamases, by bacteria, is a major health concern in the treatment of infectious diseases, with the increase in carriage of extended-spectrum beta-lactamases (ESBLs) being of particular concern. CTX-M beta-lactamases constitute a family of these rapidly disseminating ESBL enzymes, having been identified in numerous countries within Africa, Asia, Europe, South America and the USA, also including India. ^[1],[2] Phylogenic studies have revealed five major groups of acquired CTX-M enzymes, CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25, with CTX-M-15 and CTX-M-28 (the subject of this letter) belonging to the CTX-M-1 group. ^[3] More specifically, CTX-M-15 has been frequently isolated from India, ^[4] whilst CTX-M-28 is much less common, only recently having been identified in India and Tunisia. ^[5],[6] Further, these two enzymes only differ by a single amino acid substitution present at the C-terminus of the protein (3'-end of the CTX-M gene), and it is usual to distinguish between these two CTX-M types (as well as other CTX-M types) using nucleotide sequencing (see GenBank accession numbers AY044436 and AJ549244).

In this respect, we would like to caution researchers to use at least one CTX-M flanking gene primer (3'- end) to obtain sequencing products that accurately differentiate between CTX-M-15 and CTX-M-28. In particular, if nucleotide polymorphisms exist in the PCR primers used to amplify CTX-M genes, and the resultant PCR products are subsequently sequenced, then these nucleotide polymorphisms will be present in any subsequent sequence data obtained from the original PCR product. For example, Achour et al. ^[5] used four CTX-M group-specific primer sets to screen for CTX-M genes, and then cloned and sequenced the resulting PCR products. However, the use of primers CTX-1F and CTX-1R generates a PCR product with 5'- and 3'- termini comprising CTX-M-28-specific sequences [Figure - 1]. Moreover, even if the gene present was actually CTX-M-15, subsequent PCR screening, cloning and sequencing would generate a CTX-M-28 sequence, due to the use of non-homologous primers. On the other hand, the use of CTX-M-15 specific primers and sequencing of subsequent PCR products will mis-identify any CTX-M-28 sequences [Figure - 1]. This may have been the case in publications by Moubareck et al. ^[6] and Weill et al. ^[7]

In fact, the mis-identification of CTX-M-15 and CTX-M-28 enzymes is due to the fact that the nucleotide sequences are very similar, with only two single nucleotide substitutions. Moreover, these sequence differences occur close to the 5'-end (position 21) and 3'-end (position 865) of the CTX-M gene, the 5'-end and 3'-end of genes usually being chosen as sites to design "specific" PCR screening primers [Figure - 1].

We, therefore, caution researchers against using CTX-M gene 3'-end PCR screening primers to generate PCR product that will be later used in sequencing reactions, especially in regions where CTX-M- 15 and CTX-M-28 are present.

References

1.	Tzouvelekis LS, Tzelepi E, Tassios PT, Legakis NJ. CTX-M-type beta-lactamases: An emerging group of extended spectrum enzymes. Int J Antimicrob Agents 2000;14:137-42.  Back to cited text no. 1
2.	Padmini BS, Raju AB, Mani KR. Detection of Entero bacteriaceae producing CTX-M extended spectrum beta-lactamases from a tertiary care hospital in south India. Indian J Med Microbiol 2008;26:163-6.  Back to cited text no. 2  [PUBMED]
3.	Bonnet R. Growing group of extended-spectrum beta- lactamases: The CTX-M enzymes. Antimicrob Agents Chemother 2004;48:1-14.  Back to cited text no. 3
4.	Muzaheed, Doi Y, Adams-Haduch JM, Endimiani A, Sidjabat HE, Gaddad SM, et al. High prevalence of CTX-M-15-producing Klebsiella pneumoniae among inpatients and outpatients with urinary tract infection in Southern India. J Antimicob Chemother 2008;61:1393-4.  Back to cited text no. 4
5.	Ben Achour N, Mercuri PS, Power P, Belhadj C, Ben Moussa M, Galleni M, et al. First detection of CTX-M-28 in a Tunisian hospital from a cefotaxime-resistant Klebsiella pneumoniae strain. Pathol Biol (Paris) 2008; Sep 30. [Epub ahead of print].  Back to cited text no. 5
6.	Moubareck C, Daoud Z, Hakimι NI, Hamzι M, Mangeney N, Matta H, et al. Countrywide spread of community- and hospital-acquired extended-spectrum beta-lactamase (CTX-M-15)-producing Enterobacteriaceae in Lebanon. J Clin Microbiol 2005;43:3309-13.  Back to cited text no. 6
7.	Weill, F-X, Perrier-Gros-Claude JD, Demartin M, Coignard S, Grimont PA. Characterization of extended-spectrum-beta-lactamase (CTX-M-15)-producing strains of Salmonella enterica isolated in France and Senegal. FEMS Microbiol Letters 2004;238:353-8.  Back to cited text no. 7

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