CTX-M and plasmid-mediated AmpC-producing Enterobacteriaceae, Singapore.

To the Editor: In gram-negative bacteria, [beta]-lactamases are an important cause of antimicrobial resistance. In the 1990s, several new [beta]-lactamases, including CTX-M type and plasmid-mediated AmpC [beta]-lactamases, emerged.

CTX-M extended spectrum [beta]-lactamases (ESBLs) differ from those derived from TEM and SHV enzymes by their preferential hydrolysis of cefotaxime and ceftriaxone compared with ceftazidime. They also differ from an evolutionary standpoint and are more closely related to the chromosomal enzymes of Khoyvera species (1). These enzymes are increasingly described worldwide, particularly in South America, Europe, and East Asia.

AmpC enzymes confer resistance to oxyimino- and 7-[alpha]-methoxycephalosporins. They occur naturally in the chromosomes of bacteria such as Enterobacter, Citrobacter, Serratia, and Pseudomonas species. In the last decade, genes coding for AmpC [beta]-lactamases have made their way into plasmids and are increasingly detected in other species, notably Klebsiella and Escherichia coil (2).

We describe five strains of Enterobacteriaceae with unusual antimicrobial susceptibility patterns, which were isolated from patients in an 800-bed hospital in Singapore. K. pneumoniae EU17113, EU2673, and E. coli EU2657 were noted to be more susceptible to ceftazidime than ceftriaxone, whereas E. coli EU4855 and EB9505 were resistant to both cephalosporins. The National Committee for Clinical Laboratory Standards ESBL confirmatory test (3) was positive for strains EU17113, EU2673, and EU2657 but negative for strains EU4855 and EB9505. All strains were isolated from urine cultures except EB9505, which was isolated from blood culture. The isolates were identified by VITEK 2 (bioMerieux, Marcy l'Etoile, France) or Microbact 12E/A (Medvet Diagnostics, Thebarton, Australia).

The MICs by Etest (AB Biodisk, Solna, Sweden) and isoelectric points of [beta]-lactamases in crude extracts are shown in the Table. Polymerase chain reaction (PCR) for CTX-M genes was performed on strains EU17113, EU2673, and EU2657 by using primers CTX-1 and CTX-2 as described by Pai et al. (4). This yielded an [approximately equal to] 780-bp product with DNA extracts from strain EU17113 but not the others. The sequence of this product was identical to [bla.sub.CTX-M-9] as found in the GenBank database (accession no. AJ416345.1). PCR was repeated for strains EU2673 and EU2657 with a different primer set as described by Gniadkowski et al. (5). This produced [approximately equal to] 600-bp products which were identical to [bla.sub.CTX-M-11] and [bla.sub.CTX-M-15] (accession no. AJ310929.1 and AY463958.1) for EU2673, and [bla.sub.CTX-M-2], [bla.sub.CTX-M-20] and [bla.sub.Toho-1] (accession no. X92507.1, AJ416344.1, and D37830.1) for EU2657.

CTX-M ESBLs can be grouped into four clusters based on the similarity of their amino acid sequences: CTX-M-1 type (CTX-M-1, -3, -10, -11, -12, -15, -23, -28), CTX-M-2 type (CTX-M-2, -4, -5, -6, -7, -20, Toho1), CTX-M-9 type (CTX-M-9, -13, -14, -16, -17, -18, -19, -21, Toho-2), and CTX-M-8. Three of the four clusters of CTX-M-type enzymes are represented in our small sample of isolates. The only other report of CTX-M enzymes in Southeast Asia of which we are aware describes CTX-M-14 and -17 in Vietnam (6).

Diagnostic laboratories may fail to identify CTX-M-positive isolates as ESBL producers if ceftazidime resistance is used as the sole screening criterion, which is unlikely in Singapore because ceftriaxone is usually in the first-line panel for antimicrobial susceptibility testing of Enterobacteriaceae. In addition, ESBL screening is conducted in most laboratories by the Jarlier double disk diffusion method (7), with at least two different [beta]-lactam disks placed on either side of an amoxicillin-clavulanate disk. However, CTX-M producers may not be distinguished from other ESBL producers because once the ESBL test is positive, the organism is reported as resistant to all third-generation cephalosporins. The importance of the characteristic antimicrobial susceptibility pattern (cefotaxime- or ceftriaxone-resistant, ceftazidime-susceptible) may not be appreciated and may be lost in the edited report. We paid special attention to these isolates because we were evaluating a VITEK 2 at the time and noted the original susceptibility pattern while reviewing the machine reports.

Multiplex PCR for plasmid-mediated AmpC genes was performed on strains EU4855 and EB9505 by using the method described by Perez-Perez and Hanson (8). Amplified products of [approximately equal to] 460 bp were produced which, when sequenced, were identical to the gene sequences encoding C-1 molecular subgroup plasmid-mediated AmpC enzymes CMY-2 (accession no. X91840.1), and LAT-3 (accession no. U77414.1) (9). This family of enzymes is thought to be derived from the chromosomal [beta]-lactamase of C. freundii and includes some of the most widely distributed plasmid-mediated AmpC [beta]-lactamases. CMY-2 has been recently found in E. coli strains from Malaysia, which shares a common border with Singapore (10).

Although identifying these enzymes has little impact on managing a patient, recognizing CTX-M and plasmid-mediated AmpC enzymes affects antimicrobial drug-resistant surveillance because important new mechanisms of extended-spectrum cephalosporin resistance are represented.

The following sequences have been submitted to GenBank: EU17113 (accession no. AY517474), EU2673 (accession no. AY517476), EU2657 (accession no. AY517475), EU4855 (accession no. AY517473), and EB9505 (accession no. AY514304).

Table. Characteristics of isolates containing CTX-M and
plasmid-mediated AmpC [beta]-lactamases

                           MIC ([micro]g/mL) (a)

Isolate      Species      CAZ    FEP   CRO    AZT         pl (b)

EU17113   K. pneumoniae     4      4    128      2     7.2, 7.5, 8.0
EU2673    K. pneumoniae   128    128   >256   >256    5.8, 7.2, 7.5,
                                                            9.0
EU2657       E. coli       16    128   >256    64     5.1, 6.3, 7.2,
                                                         7.5, 8.0
EU4855       E. coli      >256     4   >256    64           8.8
EB9505       E. coli       128     1    256     8        5.1, 8.7

Isolate   Sequenced [beta]-lactamase

EU17113          CTX-M-9 type
EU2673          CTX-M-15 type
EU2657           CTX-M-2 type
EU4855            CMY-2 type
EB9505            CMY-2 type

(a) K. pneumoniae, Klebsiella pneumoniae; E. coli, Escherichia coli;
CAZ, ceftazidime; FEP, cefepime; CRO, ceftriaxone; AZT, aztreonam.

(b) Isoelectric points of [beta]-lactamases.

Acknowledgements

We thank Lily Ng, Ong Lan Huay, Lin Xiuhua, and Yeap Yee Chin Yvonne for technical assistance.

The VITEK 2 was provided by SPD Scientific Pte Ltd, Singapore.

Tse Hsien Koh, * Grace Chee Yeng Wang, * Li-Hwei Sng, * Zhao Yi, * and Tse Yuen Kohl ([dagger])

* Singapore General Hospital, Singapore; and ([dagger])Ngee Ann Polytechnic, Singapore

References

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(2.) Philippon A, Arlet G, Jacoby GA. Plasmid-determined AmpC-type beta-lactamases. Antimicrob Agents Chemother. 2002;46: 1-11.

(3.) National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disk susceptibility tests. 8th ed. Wayne (PA): The Committee; 2003.

(4.) Pai H, Choi EH, Lee HJ, Hang JY, Jacoby GA. Identification of CTX-M-14 extended-spectrum beta-lactamase in clinical isolates of Shigella sonnei, Escherichia coli, and Klebsiella pneumoniae in Korea. J Clin Microbial 2001;39:3747-9.

(5.) Gniadkowski M, Schneider I, Palucha A, Jongwirth R, Mikiewicz B, Bauernfeind A. Cefotaxime-resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX-M-3 cefotaxime-hydrolyzing beta-lactamase that is closely related to the CTX-M-1/MEN-1 enzyme. Antimicrob Agents Chemother. 1998;42:827-32.

(6.) Cao V, Lambert T, Nhu DQ, Loan HK, Hoang NK, Arlet G, et al. Distribution of extended-spectrum beta-lactamases in clinical isolates of Enterobacteriaceae in Vietnam. Antimicrob Agents Chemother. 2002;46:3739-43.

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(8.) Perez-Perez FJ, Hanson ND. Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol. 2002;40:2153-62.

(9.) Walther-Rasmussen J, Hoiby N. Plasmid-borne AmpC beta-lactamases. Can J Microbiol. 2002;48:479-93.

(10.) Palasubramaniam S, Subramaniam G. Ho SE, Sekaran M, Parasakthi N. Plasmid encoded CMY and AmpC beta-lactamase mediated resistance to extended-spectrum beta-lactams in Klebsiella pneumoniae and E. coli In: Abstracts of the 4th International Symposium on Antimicrobial Agents and Resistance; Seoul, Korea; July 16-18, 2003; abstract GN 10, Seoul, Korea: Asian-Pacific Research Foundation for Infectious Diseases; 2003.

Address for correspondence: Tse Hsicn Koh, Department of Pathology, Singapore General Hospital, 1 Hospital Drive, 169608, Singapore; fax: [phone]; email: [e-mail]