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Antimicrobial Agents and Chemotherapy, November 2008, p. 4198-4202, Vol. 52, No. 11
0066-4804/08/$08.00+0     doi:10.1128/AAC.00107-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Dominance of blaCTX-M within an Australian Extended-Spectrum β-Lactamase Gene Pool{triangledown}

Zhiyong Zong,1,3 Sally R. Partridge,1 Lee Thomas,2 and Jonathan R. Iredell1*

Centre for Infectious Diseases and Microbiology, University of Sydney,1 Institute for Clinical Pathology and Medical Research, Westmead Hospital, Sydney, New South Wales, Australia,2 Department of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, China3

Received 25 January 2008/ Returned for modification 13 April 2008/ Accepted 13 August 2008


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ABSTRACT
 
blaCTX-M genes, particularly blaCTX-M-15, are the dominant extended-spectrum β-lactamase (ESBL) genes among clinical isolates of Escherichia coli and Klebsiella pneumoniae in Sydney, Australia, where we also found one example of blaCTX-M-62, encoding a novel enzyme conferring ceftazidime resistance. ESBL genes were present in diverse community isolates and in a variety of associated conjugative plasmids.


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TEXT
 
The dominant mechanism of resistance to expanded-spectrum cephalosporins and monobactams among members of the family Enterobacteriaceae is the production of Ambler class A extended-spectrum β-lactamases (ESBLs), with more than 200 variants described (23). SHV-type ESBLs have been sporadically reported in Klebsiella pneumoniae isolates from Australia (24, 27), and a single isolate from Queensland, Australia was reported to carry a blaCTX-M-3-like gene in a study of ESBLs in invasive K. pneumoniae from 1996 to 1997 (24). SHV- and TEM-type ESBLs were dominant all over the world in members of the family Enterobacteriaceae during the 1990s (3, 5) but now appear less important than the widely distributed CTX-M enzymes (1, 5).

Reduced susceptibility (MIC ≥ 2 µg/ml) (Phoenix NMIC/ID-101 panel; Becton, Dickinson & Co., Franklin Lakes, NJ) to cefotaxime (CTX) and/or ceftazidime (CAZ) was observed in 206 of 9,946 Escherichia coli (2.1%) and 64 of 1,391 K. pneumoniae clinical isolates (4.6%) submitted to our laboratory from four regional hospitals and two associated community clinics in the western Sydney area of New South Wales, Australia, from March 2005 to May 2007. Of these, 81 randomly selected isolates (61 E. coli and 20 K. pneumoniae isolates) from different patients had been stored and were screened for blaCTX-M, blaSHV, and blaTEM genes by PCR (Table 1).


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  		TABLE 1. PCR primers used in this study

The majority (50 of 61 E. coli isolates and 10 of 20 K. pneumoniae isolates) yielded amplicons with blaCTX-M universal primers (18). Subsequent multiplex PCR (30) and analysis of sequences (ABI PRISM 3100 genetic analyzer; Applied Biosystems, Foster City, CA) revealed genes encoding CTX-M-3 (n = 4), CTX-M-15 (n = 33), and CTX-M-62 (n = 1) from the CTX-M-1 group and genes encoding CTX-M-9 (n = 3), CTX-M-14 (n = 17), CTX-M-24 (n = 2), and CTX-M-27 (n = 1) from the CTX-M-9 group (Table 2) (blaCTX-M-9 and blaCTX-M-14 coexisted in one isolate).


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  		TABLE 2. Isolates with ESBL genes

The CTX-M-3 enzymes identified in this study are encoded by the first reported and now widespread blaCTX-M-3 gene (e.g., GenBank accession no. Y10278) (here designated blaCTX-M-3a) (12), which is closely related to blaCTX-M-15. A novel variant of CTX-M-3 (Pro167Ser) has the CAZ resistance characteristic of this substitution (26) and was designated CTX-M-62. Ceftazidime resistance, but not cefotaxime resistance, was transferred to E. coli with blaCTX-M-62 on a conjugative plasmid, but the incompatibility group of the plasmid could not be determined. blaCTX-M-62 is a G509T variant of a blaCTX-M-3 gene, here designated blaCTX-M-3b (e.g., GenBank accession no. AB059404), previously reported from Asia, which differs from blaCTX-M-3a at 8 nucleotide positions (Table 3). Additional novel (silent) blaCTX-M variants were seen (Table 3): blaCTX-M-9b is a C109T variant of all previously deposited blaCTX-M-9 sequences (e.g., GenBank accession no. AF174129) and blaCTX-M-24 variants, including blaCTX-M-24a (e.g., GenBank accession no. AY143430) and the novel blaCTX-M-24e with AGG at codon 275. Using primers located in ISEcp1 (ISEcp1IR-F) and ISCR1 (CR1-F) combined with primers located in blaCTX-M-1 group genes (CTXM1-R) and blaCTX-M-9 group genes (CTXM9-R), blaCTXM-9 was found adjacent to ISCR1, while all other blaCTX-M genes were associated with ISEcp1, as expected.


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  		TABLE 3. CTX-M gene variants

Four E. coli isolates yielded amplicons with blaSHV primers, all found to be the ESBL gene blaSHV-12. Sequencing of amplicons obtained from all 20 K. pneumoniae isolates suggested single blaSHV genes in 17 (3 blaSHV-1, 11 blaSHV-11, and 1 each of blaSHV-27, blaSHV-28, and blaSHV-109). SHV-109 is a novel variant most similar to SHV-61 (Thr268Met) and SHV-11 (Thr268Met with Leu10Arg in the signal peptide). The remaining three isolates appeared to have blaSHV-12 plus another blaSHV gene, with uncut and cut amplicons evident on electrophoresis after digestion with NheI (New England Biolabs, Ipswich, MA), which cuts at the position of a relevant sequence variation in blaSHV-12. Separate sequencing of purified uncut and cut bands revealed that one isolate had both blaSHV-12 and blaSHV-1 and that two isolates had both blaSHV-12 and blaSHV-11. Fifty-nine isolates also had blaTEM genes, all encoding (non-ESBL) TEM-1.

The 66 isolates (54 E. coli and 12 K. pneumoniae isolates) with blaCTX-M (n = 60) and/or blaSHV-12 (n = 7) (one isolate, JIE162, had both blaSHV-12 and blaCTX-M-15), were subjected to pulsed-field gel electrophoresis after XbaI (New England Biolabs) restriction of DNA purified from whole-cell extracts (14), and E. coli phylogenetic groups were assigned (7) (Table 2). Forty-eight unique strains were identified in this way among the 54 E. coli isolates, and 8 unique strains were identified from the 12 K. pneumoniae isolates.

Most (47/60) blaCTX-M genes transferred on conjugative plasmids to rifampin-resistant E. coli DH5{alpha}({Delta}lacZ) selected with rifampin (80 or 200 µg/ml) (Sigma, St. Louis, MO) plus ampicillin (80 µg/ml), CTX (2 µg/ml), or CAZ (2 µg/ml) by filter (29) and/or broth mating methods (9). Plasmid replicon typing of transconjugants as previously described (6), with an additional PCR for IncFII (22), revealed significant plasmid diversity (Table 2). Consistent with previous reports, multiple plasmid replicons were present in some transconjugants but IncF plasmids were numerically most important (13). All non-IncF amplicons and several IncF amplicons were sequenced, confirming the specificity of PCR typing. Several different HpaI (New England Biolabs) restriction patterns were observed among blaCTX-M-15 and blaCTX-M-14 plasmids (IncF and IncI1) extracted from transconjugants by alkaline lysis (28), but none matched the recently described epidemic IncFII plasmids in Europe (8) (not shown).

Three-quarters of the 66 isolates were not susceptible to gentamicin or tobramycin, and most were resistant to both (Table 2). Three-quarters were also resistant to trimethoprim-sulfamethoxazole. Aminoglycoside resistance was cotransferred with blaCTX-M-15 particularly. Although more than 60% of the original isolates were ciprofloxacin resistant, this phenotype was not transferred to transconjugants (Table 2). Variable associations of blaCTX-M-15 with genes conferring β-lactam and aminoglycoside resistance have been previously documented (2, 8), and further investigation is ongoing.

Nearly three-quarters of the 66 isolates with ESBL genes were recovered from urine. Two-thirds (35/54) of the E. coli isolates were from community-acquired infections, almost all of unique clonal type. K. pneumoniae isolates were more commonly (8/12) collected in the hospital setting and were less diverse (Table 2).

We detected no ESBL-type blaSHV, ESBL-type blaTEM, or blaCTX-M in 15 isolates. Despite having reduced susceptibility to CTX or CAZ (MIC ≥ 2 µg/ml), there was no zone enhancement to suggest an ESBL in any of these isolates by disk approximation test (15, 17), and none of the several less common ESBL genes were detected by PCR (Table 1). The majority (13/15) were cefoxitin resistant, and most carried either a plasmid-borne ampC gene (blaDHA or blaCMY-2-like; n = 7) or a metallo-β-lactamase gene (blaIMP-4; n = 3).

In summary, blaCTX-M genes are well-established in the general community here, and blaCTX-M-15 (and, to a lesser extent, blaCTX-M-14) is particularly dominant despite the presence of novel local variants. Our data indicate that these genes, including blaCTX-M-15, are associated with a variety of plasmid replicons and are present in a wide range of bacterial strains.

Nucleotide sequence accession numbers. The nucleotide sequences of blaCTX-M and blaSHV genes from representative isolates have been submitted to GenBank under accession nos. EU418908 to EU418920. The blaCTX-M-62 sequence is available under GenBank accession no. EF219134.


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ACKNOWLEDGMENTS
 
We are grateful to Glenys Conner, Peter Jelfs, Qinning Wang, and Matthew O'Sullivan for helpful advice and practical support.

Z.Z. was supported by an Endeavor International Postgraduate Student Scholarship from the Australian Government Department of Education, Science and Training. S.R.P. was supported by grants from the National Health and Medical Research Council of Australia.


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FOOTNOTES
 
* Corresponding author. Mailing address: CIDM, Level 3, ICPMR Building, Westmead Hospital, Westmead, NSW 2145, Australia. Phone: 61-2-9845-6255. Fax: 61-2-9891-5317. E-mail: jon.iredell{at}swahs.health.nsw.gov.au Back

{triangledown} Published ahead of print on 25 August 2008. Back


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Antimicrobial Agents and Chemotherapy, November 2008, p. 4198-4202, Vol. 52, No. 11
0066-4804/08/$08.00+0     doi:10.1128/AAC.00107-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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