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Antimicrob Agents Chemother. Feb 2007; 51(2): 796–799.
Published online Dec 4, 2006. doi:  10.1128/AAC.01070-06
PMCID: PMC1797763

Emergence and Dissemination of Enterobacteriaceae Isolates Producing CTX-M-1-Like Enzymes in Spain Are Associated with IncFII (CTX-M-15) and Broad-Host-Range (CTX-M-1, -3, and -32) Plasmids[down-pointing small open triangle]

Abstract

The spread of CTX-M-1-like enzymes in Spain is associated with particular plasmids of broad-host-range IncN (blaCTX-M-32, blaCTX-M-1), IncL/M (blaCTX-M-1), and IncA/C2 (blaCTX-M-3) or narrow-host-range IncFII (blaCTX-M-15). The identical genetic surroundings of blaCTX-M-32 and blaCTX-M-1 and their locations on related 40-kb IncN plasmids indicate the in vivo evolution of this element.

CTX-M enzymes are Ambler class A extended-spectrum β-lactamases, which are grouped in five lineages (http://www.lahey.org/studies/webt.htm). Enzymes within the CTX-M-1 cluster (CTX-M-1, -3, -10, -11, -12, -15, -22, -28, -29, -30, -32, and -33) are frequently detected in Europe and are involved in different epidemiological situations (2, 15, 16, 17, 26, 31, 40). Genes encoding CTX-M-1, CTX-M-3, and CTX-M-15 are disseminated in different continents through epidemic plasmids and/or particular epidemic strains (1, 4-6, 17, 19, 25, 26, 32, 41). A few CTX-M-32-producing strains have recently been recovered from both humans and farm animals in Spain, Italy, Greece, and Portugal, suggesting the existence of community reservoirs for CTX-M enzymes and ongoing widespread dissemination in the Mediterranean area (6, 11, 26, 30; L. Peixe et al., unpublished results). CTX-M-10 remains confined to Spain and France (22, 28). Other members of this cluster have scarcely been documented. Although Spain is one of the European countries with high prevalences of CTX-M-producing isolates, those corresponding to enzymes of the CTX-M-1 lineage have been reported only in specific studies (6, 11, 16, 30). In this work, we characterize at the molecular level all isolates producing CTX-M-1-like enzymes (other than the endemic CTX-M-10) (16, 28) since their first description in our institution in 2000.

Twenty-eight isolates (20 Escherichia coli and 8 Klebsiella pneumoniae isolates) recovered from 23 patients who attended the Ramón y Cajal Hospital in Madrid from 2000 to 2004 were studied (one isolate per patient and phenotype). Bacterial identification, susceptibility testing, and characterization of β-lactamases were performed as described previously (13, 14, 39, 39a). The presence of sequences linked to CTX-M genetic environments, such as ISEcp1, IS903, blaOXA-1, and aac(6′)-Ib-cr, was investigated by PCR and sequencing (25). Clonal relatedness was established by pulsed-field gel electrophoresis (21, 37) and identification of E. coli phylogenetic groups by a multiplex PCR assay (12). Conjugative transfer was tested by broth/filter mating experiments using E. coli K-12 strain BM21R (Nalr Rifr Lac+, plasmid-free) or two E. coli HB101 strains (Strr Lac and Kmr Lac) as recipients at a 1:10 donor/recipient ratio. Plasmid characterization included determination of plasmid size as described previously (3) and determination of incompatibility group by using a PCR-based replicon-typing assay, hybridization, sequencing, and comparison of restriction fragment length polymorphism (RFLP) patterns as described previously (10, 27, 35).

We identified E. coli isolates producing CTX-M-1 (nine isolates/three strains), CTX-M-3 (one isolate/one strain), CTX-M-15 (five isolates/three strains), and CTX-M-32 (five isolates/five strains) and K. pneumoniae isolates containing CTX-M-15 (eight isolates/three strains). Most E. coli isolates belonged to phylogroups associated with extraintestinal pathogenic E. coli (D [46.1%] and B2 [38.5%]), while groups linked to animal or human commensal strains were scarcely detected (A and B1 [7.7% each]). High clonal diversity was observed, although a CTX-M-15-producing K. pneumoniae strain was recovered from six patients located in different wards from 2002 to 2004 (Table (Table1)1) .

TABLE 1.
Clinical data on antimicrobial susceptibility, genetic environment, and plasmid characterization of Enterobacteriaceae isolates producing CTX-M-1-like enzymes in Madrid, Spain (2000 to 2004)a

ISEcp1 was located 46 and 49 bp upstream of blaCTX-M-3 and blaCTX-M-15, respectively, and 80 bp upstream of blaCTX-M-1 and blaCTX-M-32. CTX-M-32-producing isolates and one CTX-M-1-producing strain were associated with the presence of IS5 at the same position (downstream of ISEcp1 and 60 bp upstream of blaCTX-M). The presence of IS903 was not detected among transconjugants, while IS26 was found in most of the cases, probably due to the presence of this insertion sequence on known plasmids, such as IncFII, IncN, and IncL/M (see below) (7, 34, 36).

blaCTX-M-15 was located in closely related IncFII plasmids of various sizes (85 to 200 kb), transferability properties, and replicon contents (RepFII or RepFII-RepFIA). All FII replicons were identical to the epidemic CTX-M-15 plasmid pC15-1a (5) (GenBank accession no. AY458016), while all FIA replicons displayed 100% homology with the IncFII plasmid pRSB107 (GenBank accession no. AJ851089). In addition, all contained multiple antibiotic resistance determinants, including aac(6′)-Ib-cr and blaOXA-1. CTX-M-15-producing isolates from different European and Asian countries and Canada have previously been linked to epidemic IncFII plasmids carrying aac(6′)-Ib-cr and blaOXA-1 and various replicon contents, suggesting pandemic dissemination of CTX-M-15 by IncFII plasmid derivatives of a common element (5, 13a, 17, 20, 23-25). The diversity of replicons in our and other studies may be explained by the different recombinatorial events between IncFII plasmids with different variations in copA, which may alter their compatibility properties (13a, 17, 23, 29).

blaCTX-M-32 was located in a single 40-kb IncN plasmid recovered from different E. coli strains in our hospital since 2000. This gene was initially described in 2004 in the north part of Spain and has recently been detected among different species and hosts in Mediterranean countries, highlighting unnoticed dissemination and probable current widespread in Europe (6, 11, 26, 30, 33; L. Peixe et al., unpublished results). Otherwise, blaCTX-M-1 was associated with an IncL/M plasmid of 50 kb (isolates C75 and D63) or with an IncN plasmid of 40 kb. The RFLP pattern of this IncN plasmid (corresponding to one epidemic clone isolated from four patients at the intensive care cardio-pediatric unit over almost 1 year) was similar to that of the IncN plasmid containing blaCTX-M-32. CTX-M-32 differs from CTX-M-1 by a single Asp240-Gly substitution responsible for its increased level of resistance to ceftazidime (11). The identical genetic surroundings of both ISEcp1 and IS5-blaCTX-M might indicate in vivo evolution of plasmid under differential antibiotic selective pressure levels as described for blaCTX-M-3 and blaCTX-M-15 (1, 18). Finally, we detected blaCTX-M-3 on a 120-kb broad-host-range plasmid, IncA/C2, in one isolate from 2002 (38). This plasmid group seems to be widely spread since it has been linked to recent CTX-M-2- or CMY-producing isolates from different European and American countries after its first detection in the United States in 1997 (8, 9). CTX-M-3 is epidemic in Eastern Europe, where it has also been associated with a large conjugative plasmid, although an identification analysis of this plasmid has not yet been performed (1). Both CTX-M-1 and -3 have been recovered since from different Spanish hospitals in 2004 and 2005 (25a).

Although the evolution of particular CTX-M-1-like genes, such as blaCTX-M-3 and blaCTX-M-15 (1, 18) or blaCTX-M-1 and blaCTX-M-32 (this study), may contribute to the dissemination and diversity of these enzymes, the heterogeneous ISEcp1-bla regions detected for each member studied and the locations of genes on different plasmid platforms would suggest independent mobilization and dissemination events from a common ancestor.

In summary, we have described the emergence and spread of members of the CTX-M-1 cluster detected in our area at least since 2000 and associated with widely disseminated conjugative plasmids of broad (IncN, IncL/M, and IncA/C2) and narrow (IncFII) host ranges. Dissemination of IncN and IncA/C broad-host-range plasmids is of concern since it might contribute to the further spread of isolates producing CTX-M-1-like enzymes among other members of Enterobacteriaceae.

Acknowledgments

Ângela Novais was supported by a fellowship from the Ministerio de Ciencia y Tecnología of Spain (SAF 2003-09285). Raquel Moreira was a recipient of a “Leonardo da Vinci” fellowship funded by the APGEI (Associaçao Portuguesa de Gestao e Engenharia Industrial). This work was partially funded by research grants from the Ministerio de Ciencia y Tecnología of Spain (SAF 2003-09285), the Instituto de Salud Carlos III (FIS-PI040162), and the European Commission (grant SLHM-CT-2003-503335).

Footnotes

[down-pointing small open triangle]Published ahead of print on 4 December 2006.

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