The CTX-M b-lactamase pandemic Rafael Canto´n and Teresa M Coque In the past decade CTX-M enzymes have become the most prevalent extended-spectrum b-lactamases, both in nosocomial and in community settings. The insertion sequences (ISs) ISEcp1 and ISCR1 (formerly common region 1 [CR1] or orf513) appear to enable the mobilization of chromosomal b-lactamase Kluyvera species genes, which display high homology with blaCTX-Ms. These ISs are preferentially linked to specific genes: ISEcp1 to most blaCTX-Ms, and ISCR1 to blaCTX-M-2 or blaCTX-M-9. The blaCTX-M genes embedded in class 1 integrons bearing ISCR1 are associated with different Tn402-derivatives, and often with mercury Tn21-like transposons. The blaCTX-M genes linked to ISEcp1 are often located in multidrug resistance regions containing different transposons and ISs. These structures have been located in narrow and broad host-range plasmids belonging to the same incompatibility groups as those of early antibiotic resistance plasmids. These plasmids frequently carry aminoglycoside, tetracycline, sulfonamide or fluoroquinolone resistance genes [qnr and/or aac(60 )-Ib-cr], which would have facilitated the dissemination of blaCTX-M genes because of co-selection processes. In Escherichia coli, they are frequently carried in well-adapted phylogenetic groups with particular virulence-factor genotypes. Also, dissemination has been associated with different clones (CTX-M-9 or CTX-M-14 producers) or epidemic clones associated with specific enzymes such as CTX-M-15. All these events might have contributed to the current pandemic CTX-M b-lactamase scenario. Addresses Servicio de Microbiologı´a, Hospital Ramo´n y Cajal, 28034-Madrid, Spain Corresponding author: Canto´n, Rafael ([email protected])

Current Opinion in Microbiology 2006, 9:466–475 This review comes from a themed issue on Antimicrobials Edited by Fernando Baquero and Louis B Rice Available online 30th August 2006 1369-5274/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2006.08.011

Introduction The first plasmid-encoded b-lactamase that was able to destroy extended-spectrum b-lactam antibiotics was described in Germany in 1983. It was related to the production of a variant of the SHV-1 enzyme (for the origin of b-lactamase names see reference [1]), a broad Current Opinion in Microbiology 2006, 9:466–475

spectrum penicillinase found in Klebsiella pneumoniae. This report was followed by the description in France of variants of TEM-1 and TEM-2 enzymes with hydrolytic properties similar to SHV-1 derivatives. They were named as extended spectrum b-lactamases (ESBLs) in 1989 by Philippon et al. [2]. At the same time, a new family of ESBLs, the CTX-M, was detected in humans in Germany and Argentina (for epidemiological reviews see [3,4
]). These descriptions were preceded by the isolation in xlater recognized to be related to the CTX-M enzymes [3]. Since then, there has been a dramatic increase in the numbers of CTX-Ms enzymes and organisms producing them, and they have become the most prevalent ESBLs worldwide. This dominance has not only been observed in the nosocomial environment but also in the community setting [5,6
]. Moreover, CTX-M enzymes have been detected in pets, farm animals, products from the food chain and sewage [6
,7
,8,9]. The reasons for this dramatic increase are not yet well understood. Here, recent findings in this field are reviewed.

Geographic dissemination of CTX-M-producing isolates During the 1990s, TEM-ESBLs and SHV-ESBLs were dominant among ESBLs all over the world and CTX-Mproducing organisms were rarely recognized. Their presence was inferred in surveillance studies because of higher levels of resistance to cefotaxime than to ceftazidime, a characteristic that is usually present in all CTX-M-producing isolates [3,10
]. At that time, TEM-ESBLs and SHVESBLs were mainly associated with epidemic clones, and K. pneumoniae was the main carrier of the ESBL genes and a paradigm of the spread of the ESBLs in hospitals [4
]. This situation changed in the present century, somewhat earlier in South America than in Europe, and evolved accordingly with exponential-type dynamics. In addition, Escherichia coli was beginning to be recognized as the major source of ESBLs with a higher increase in the community than in the nosocomial setting [5,6
,7
]. The new epidemiological scenario included the increase in the number of different CTX-M enzymes and the recognition of multiple clones and genetic elements that carry blaCTX-M genes. The way by which CTX-M enzymes spread might follow an allodemic, rather than an epidemic pattern; this term reflecting that the increase of CTX-M enzymes had not been as a result of the dissemination of particular clones, but of the spread of both multiple specific clones and/or mobile genetic elements [11]. The suspicion that the increment of CTX-Ms in the hospital setting might be a consequence of the entrance of these enzymes from the community rather than the emergence and expansion in the nosocomial setting has been suggested in specific studies [6
,12
]. www.sciencedirect.com

The CTX-M b-lactamase pandemic Canto´n and Coque 467

Moreover, a dramatic increase in prevalence of faecal carriers of CTX-M-producing organisms in the community has also been recognized [5,12
]. Although the CTX-M ESBLs are a heterogeneous family, the rapid spread of these enzymes is only comparable with the spread of the TEM-1 b-lactamase in the 1970s. Figure 1 shows the current situation of CTX-M ESBLs in different geographic areas. Nowadays, it is worth noting that an endemic situation is dominant in most European countries and in Asia and South America. The USA, but not Canada, still has sporadic reports of CTX-M-producing isolates [4
]. Some of the CTX-M enzymes are widely present in specific countries, such as CTX-M-9 and CTX-M-14 in Spain [13,14], CTX-M-1 in Italy [15] and CTX-M-2 in most South American countries, Japan and Israel [3,12
], whereas others such as CTX-M-15 have been detected worldwide [6,15,16
,17,18

].

Figure 2 illustrates the increase of ESBL-producing organisms in our institution — a 1200-bed university teaching hospital in northern Madrid (Spain) providing specialized health care for a population of about 500 000 habitants — since their first detection in 1989. Continuous monitoring of ESBL-producing organisms in our institution enabled us to observe the emergence of CTXM enzymes in 1991 in E. coli isolates with a shifting epidemiology of different ESBLs. We observed dominance of CTX-M-9 at the end of the 1990s, and the emergence of CTX-M-14 and CTX-M-1 group enzymes, including CTX-M-1, CTX-M-3, CTX-M-15 and CTXM-32, since 2001 (Figure 3). It is of note that in our institution in 2005, a total of 320 patients (65% of these were outpatients) were identified as being infected with ESBL-producing organisms, mainly causing urinary tract infections and E. coli being the most commonly species recovered (Canto´n et al., unpublished data).

Figure 1

In some countries, such as the USA, only sporadic reports of the isolation of CTX-M producing isolates have been published; however, in most European countries an endemic situation can be recognized. Different enzymes are not equally represented in all geographic areas: enzymes from the CTX-M-9 group are well represented in the countries surrounding the Mediterranean Sea and in the United Kingdom, CTX-M-2 has been mainly isolated in South America and Japan and CTX-M-15 is spread nearly worldwide. www.sciencedirect.com

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468 Antimicrobials

Figure 2

Graph of the isolation of ESBL-producing organisms at the Ramo´n y Cajal University Hospital in Madrid (Spain) since its first detection in 1989.

Figure 3

Shifting epidemiology of different ESBL-producing E. coli isolates from 1988 to 2002 at the Ramo´n y Cajal University Hospital in Madrid (see also the text for explanation). Current Opinion in Microbiology 2006, 9:466–475

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The CTX-M b-lactamase pandemic Canto´n and Coque 469

Origin of CTX-M enzymes Five different clusters of CTX-Ms (CTX-M-1, CTX-M2, CTX-M-8, CTX-M-9 and CTX-M-25) have been recognized on the basis of their amino acid sequences (http://www.lahey.org/studies/webt.stm). Table 1 shows different CTX-M enzymes within each cluster. Chromosomal b-lactamase genes from different Kluyvera species, particularly Kluyvera ascorbata and Kluyvera georgiana, have been identified as potential sources of specific blaCTX-M genes of CTX-M-1, CTX-M-9 and CTX-M-2 clusters. This is because of the high homology of blaCTXM genes with that of chromosomal K. georgiana KLUG-1 and K. ascorbata KLUA-1 b-lactamase genes [19,20]. In addition, it has been shown that neighbouring sequences of blaCTX-M-2, blaCTX-M-9, and blaCTX-M-10 genes exhibit high nucleotide identity with those surrounding of Kluyvera chromosomal b-lactamase genes [19,21,22].

The CR1 element has recently been renamed ISCR1 as it possesses the key motifs of the IS91-like element [24

,28]. It has also been suggested that this ISCR1 might participate in the gene mobilization process by a rolling-circle replication mechanism. Different bla genes, including blaCTX-M, blaCMY, blaDHA-1 and blaVEB, as well as other resistance genes, including qnrA, have been associated with ISCR1. The ISCR1 is embedded in the so-called class 1 integrons bearing ISCR1 [24

], which have complex modular structures (Figure 4) [13,23]. Similar sequences to ISCR1 have been found in Salmonella genomic islands (SGIs) and on conjugative transposons encoding sulfamethoxazole, trimethoprim and streptomycin resistance genes from Vibrio cholerae [24

]. These findings denote the sophisticated genetic rearrangement strategies that organisms create for mobilizing antibiotic resistance genes and the modular structure in which they are located.

Mobilization and expression of blaCTX-M genes Different genetic elements might be involved in the mobilization of blaCTX-M genes. These elements include: the ISEcp1-like insertion sequences (IS) associated with most genes within CTX-M-1, CTX-M-2 and CTX-M-9 clusters; the CR1 (common region 1, formerly orf513) element, a putative transposase found to be linked to blaCTX-M-2 and blaCTX-M-9 genes [13,22,23,24

]; and phage-related sequences, which are only identified in the surroundings of the blaCTX-M-10 gene in Spain [25]. A recent in vitro study demonstrated the mobilization of blaCTX-M-2 gene from its progenitor K. ascorbata as a result of the participation of the ISEcp1B gene [26]. This element belongs to the IS1380 family. A relevant characteristic of these ISs is their ability to mobilize by a oneended transposition mechanism. Sequence comparison reveals that ISEcp1B might recognize a wide range of DNA sequences, which might be used to mobilize structurally unrelated genes, including blaCTX-Ms. Thus, there is the potential that a transposition mechanism participates in the blaCTX-M gene mobilization [27

].

It is of note that different IS elements have been linked to different blaCTX-M genes within a specific CTX-M cluster. As an example, blaCTX-M-9 is associated with class 1 integrons containing ISCR1, whereas blaCTX-M-14 is mainly associated with ISEcp1. Also, some specific blaCTX-M genes such us blaCTX-M-10 might be associated with both ISEcp1 and phage-related sequences [3,23,26,27

,29]. These facts would demonstrate the availability of these sequences in nature and the possibility of triggering different recombinatorial adaptive processes. Similar to other ISs, the ISEcp1-like family has been shown to act as a promoter region for high-level expression of different CTX-M enzymes, including CTX-M-14, CTX-M-18, CTX-M-17 and CTX-M-19 [3,27

]. Other plasmid-mediated b-lactamases genes, such as blaCMYtype genes or genes encoding 16S rRNA methylases (rtmC), might also be associated, in their expression and mobilization, with ISEcp1 [30,31]. Furthermore, it has also been recently recognized that the ISCR1

Table 1 Different CTX-M clusters and origin of blaCTX-M. CTX-M cluster

Year (enzyme, country) Enzymes

Origin a

a

CTX-M-1

CTX-M-2

CTX-M-8

CTX-M-9

CTX-M-25

1989 (CTX-M-1, Germany) CTX-M-1, -3, -10, -11, -12, -15, -22, -23 -29, -30, -32, -33, -28, -36, -54, UOE-1

1986 (FEC-1, Japan)

1996 (CTX-M-8, Brazil) CTX-M-40

1994 (CTX-M-9, Spain)

2000 (CTX-M-25, Canada) CTX-M, -26, -25, -39, -41

K. ascorbata

K. ascorbata

CTX-M-2, -4, -6, -7, -20, -31, -44 (previously TOHO-1), FEC-1

K. georgiana

CTX-M-9, -13, -14, -16, -17, -18, -19, -24, -27, -45 (previously TOHO-2), -46, -47, -48, -49, -50, K. georgiana

ND

Year of first isolation or description (first enzyme described and country of isolation); CTX-M-14 and CTX-M-18 are identical; ND: not defined.

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470 Antimicrobials

Figure 4

Modular schematic structure of the backbone of class 1 integrons containing blaCTX-M genes, on the basis of available information from sequences of In117, InV117 (blaCTX-M-2) and In60 variants (blaCTX-M-9) [13,29,33]. Each integron includes the conserved regions 50 CS and 30 CS flanking a variable number of gene cassettes, followed by ISCR1, blaCTX-M genes, sequences showing high homology with Kluyvera genomes (represented as open boxes because ORFs of different lengths and orientations have been described in blaCTX-M-2 and blaCTX-M-9 integrons), and a second copy of the 30 CS, designated 30 CS2, followed by a truncated version of Tn402-tni module. Sequences upstream and downstream integron correspond to the tnp module and mer operon of Tn21-like transposons, respectively. Vertical bars indicate terminal repeats of the integrons (white) or Tn21-transposons (black). Light grey box indicates the presence of IS1326 and/or IS1353 within Tn402. White arrows with adjacent circles (59-base element) symbolize a gene cassette. The variable region might contain different gene cassette arrays. Similarly coloured arrows represent related modules.

elements provide a putative promoter region for the expression of unrelated antibiotic resistance genes such as blaCTX-M, qnrA and dfrA10 [32].

Dissemination of blaCTX-M genes blaCTX-M genes and transposable elements

Recent studies have analyzed the complete genetic context of specific blaCTX-M genes providing insights about their dissemination in both nosocomial and community settings. The association of bla genes with transposons of Tn21 family might have influenced the spread of those located in class 1 integrons bearing ISCR1, particularly those coding for metallo-b-lactamases or specific cefotaximases such as CTX-M-2 and CTX-M9 [13,29]. Different class 1 integrons containing ISCR1 and carrying blaCTX-M-2 (In117, InV117) or blaCTX-M-9 (In60 variants) have been fully characterized [13,29,33]. These examples have shown the association of these ISCR1 integrons with different Tn402 derivatives, often linked with mercury-resistance transposons such as Tn21 or Tn1696 (Figure 4). The Tn21 family is disseminated worldwide in both environmental and clinical Gramnegative bacteria, and almost all examples have been found on large conjugative plasmids of Enterobacteriaceae [34]. The mobility of Tn21-derivatives carrying blaCTX-M has been further suggested because of the presence of IS4321, a member of the IS1111 family, linked to a tnp1696 module located upstream the V. cholerae InV117 integron. These ISs target a specific position in the terminal inverted repeats of Tn21 family transposons and could promote transposition of Tn21 along with the IS4321 [33,35,36]. Current Opinion in Microbiology 2006, 9:466–475

It is also worth noting the high diversity found in every module of the genetic platform of blaCTX-M-9 or blaCTX-M0 0 2 genes (5 CS–3 CS1, ISCR1, Tn402–tni module, tnp21/ 1696 and mer21/1696) among isolates from different sources. This heterogeneity might be explained by a continuous recombinatorial exchange between these regions and their corresponding homologous sequences in other elements. In addition, the presence of a multiplicity of hot spots for recombination within those elements (intI1, 30 CS, Tn402–tni module or res site and mer operon in Tn21 derivatives) might yield chimeric structures and localized deletions [13,29,33,37]. It has also been suggested that blaCTX-M genes linked to ISEcp1 insertion sequences might be part of transposon structures, thus hypothesizing their acquisition by transposable events [38]. In the case of blaCTX-M-17 and blaCTXM-19 genes, it was suggested that ISEcp1B could be responsible for the mobilization processes, because of a potential transposon structure comprising ISEcp1B, blaCTX-M, IS903D and part of the gene that encodes an iron transport protein [38]. Nevertheless and as previously stated, in the case of ISEcp1B and blaCTX-M-19 genes, the mobilization process, and thus dissemination, might have been produced by a one-ended transpositional mechanism, able to recognize a variety of DNA sequences rather than the use of a fixed imperfect right inverted repeated (IRR) structure [27

]. In the case of blaCTX-M-15, also linked to ISEcp1 like structures, the study of its genetic environment in Canada revealed the presence of different partial transposon sequences within the MDR region [18

]. However, the role of these structures in the www.sciencedirect.com

The CTX-M b-lactamase pandemic Canto´n and Coque 471

dissemination processes that is putatively associated to transpose-mediated lateral gene transfer remains to be elucidated. blaCTX-M genes and plasmids

Previous studies using mating experiments showed that ESBL blaTEM and blaSHV genes were associated with plasmids belonging to a few specific incompatibility (Inc) groups of variable sizes (80–300 kb) and with variable transfer frequencies (102–109), including IncM, IncC, IncFI and IncHI2. PCR replicon typing assays have recently been used to identify plasmids bearing CTXM b-lactamase genes [13,16,39
]. These PCR assays discriminate plasmids of known incompatibility groups by the presence of specific genes involved in plasmid maintenance. They have also facilitated the recognition of blaCTX-M genes on plasmids of both narrow host-range (IncFI, IncFII, IncHI2 and IncI) and broad host-range (IncN, IncP-1-a, IncL/M and IncA/C) [13,16
] (TM Coque et al., unpublished data). These types of plasmids have been described in early antibiotic resistant isolates, suggesting recent evolution of these elements by acquisition of new resistance genes linked to genetic platforms involving ISs, classic integrons and/or transposons [40]. Dissemination of organisms that produce CTX-M-9, CTX-M-14, CTX-M-15 and CTX-M-32 have been linked with epidemic plasmids. International dissemination of blaCTX-M-15 seems to be associated to those of the incompatibility group FII [13,16
,18

] (TM Coque et al., abstract C2-1666. 46th International Conference on Antimicrobial Agensts and Chemotherapy. ICAAC. San Francisco, CA, September 2006). A 94 kb plasmid designated pC15-1a has been identified among CTX-M-15-producing E. coli isolates from Canada, France and the north of Africa [16
,18

]. This plasmid is a derivative of R100, an IncFII self-transmissible multiresistance plasmid isolated from a Shigella flexneri strain in Japan in the late 1950s. Recent findings have also identified blaCTX-M-15 on mosaic plasmids carrying replicons from pC15-1a and pBRS107 (an IncFII plasmid isolated from activated sludge bacteria of a wastewater treatment plant) [41
], suggesting interaction among specific plasmids of IncFI and IncFII groups. Spread of blaCTX-M-9 in a Spanish hospital from 1996 through 2003 was as a result of dissemination of narrow host-range plasmids of the HI2 incompatibility group. IncHI2 plasmids were first reported in Serratia marcescens in the United States in 1969 and were recovered from environmental and human Salmonella enterica serovar Panama in Chile in the 1980s [42
]. Dissemination of blaCTX-M-9, as well as blaCTX-M-32, also occur linked to epidemic spread of broad host-range incompatibility plasmids such as IncP-1a and IncN, respectively [13]. The former were first detected in 1969 from both Pseudomonas aeruginosa and Enterobacteriaceae isolates in the UK and www.sciencedirect.com

have recently been detected in a waste-water treatment plant in Germany [43
]. IncN plasmids have been recently found in environmental samples. These facts are an alert of the possibility of the spread of the production of CTX-M enzymes to species other than Enterobacteriaceae and might be responsible for the presence of blaCTX-M genes in P. aeruginosa and Acinetobacter species, which has been observed in recent surveillance studies [44]. The possibility of multiple recombinatorial events within the same plasmid or among different plasmids has been suggested recently with the study of different isolates harbouring the blaCTX-M-15 or blaCTX-M-9 [13,16
]. In the first case, different CTX-M-15-producing E. coli isolates in the Paris area, Tunisia and Banui were studied and compared with those from Canada. In this study, and despite a clonal dissemination of CTX-M-15-producing isolates (at least in the Paris area), a mosaic plasmid was recognized, suggesting genetic interactions among different plasmids. In the case of CTX-M-9-producing isolates recovered in our institution, all representing different clones, plasmids of the same incompatibility group carrying different blaCTX-M-9 integron variants (defined in relation to the In60 backbone, and the flanking Tn402 and Tn21 sequences) were observed. This also highlights the role of recombinatorial shuffling of sequences in the evolution of antibiotic resistance plasmids despite similar origin [13].

CTX-M-producing isolates, phylogenetic groups and virulence factors As previously stated most of the CTX-M enzymes have been found in E. coli isolates and, to a lesser extent, in K. pneumoniae and other Enterobacteriaceae isolates [4
,6
]. The potential relationship of a specific phylogenetic group with a specific ESBL-type producing isolate has been investigated in different collections of ESBL-producing E. coli isolates [45–47]. E. coli are grouped in four main phylogenetic groups: A, B1, B2 and D. Groups B2 and D are mainly associated with extraintestinal pathogenic strains recovered from patients with urinary tract infections, bacteremia, meningitis and other infections, whereas groups A and B1 are more associated with animal and human commensal strains. The B2 phylogenetic group often carries virulence determinants that are not present in other phylogroups. On the contrary, antimicrobial resistance was shown to be greater in isolates of non-B2 phylogenetic group. Branger et al. [45] found that group B2 represented about 40% of the ESBL-producing strains, but this percentage was lower when only CTX-Mproducing E. coli isolates were considered. In our experience, TEM-, SHV- and CTX-M-ESBLproducing isolates were over-represented in group D (50% overall of ESBL-producing isolates) [46]. However, this result could be biased because of the origin of the Current Opinion in Microbiology 2006, 9:466–475

472 Antimicrobials

isolates (87% were recovered from extra-intestinal sources) or because of ESBL distribution (48% TEMESBL or SHV-ESBL and 52% CTX-M-ESBL type). Nevertheless, this figure changes when specific ESBLs are focused on. The CTX-M-9-producing isolates were mainly associated with phylogenetic group A (38%) and to a lesser extent with group D (28%), which are mainly associated with nosocomial isolates [13]. Also in our institution, CTX-M-15-producing E. coli isolates were mainly associated with group D (A Novais et al., unpublished data). This association was also the case of faecal CTX-M-15-producing E. coli isolates from healthy children in Bolivia and Peru [48], but not in clinical isolates from Canada, France and Portugal, which mainly belonged to group B2 [47,49,50]. These results might reflect different availability of phylogenetic groups in different geographic areas or the influence of different origin (age or gender of patients) in the studied collections as previously observed in E. coli population studies [51]. In one study specific virulence traits, including virulence markers of extraintestinal pathogenic E. coli, were investigated in CTX-M-producing E. coli isolates compared with non-ESBL-producing isolates [49]. There were significant prevalence differences from individual virulence factors among CTX-M producers and non-producers; however, aggregate virulence factor scores were similar. This result was also the case in another study comparing colonizing and infective strains, with the exception of aerobactin production, which was significantly higher in the strains responsible for infection than in the strains responsible for colonization [45]. In CTX-M-15-producing epidemic clones in long-term care facilities in France and in Spain, specific virulence factor genotypes, including aer, fyuA and irp2, were associated with these clones [45,47,52].

CTX-M-producing isolates, coresistance and therapeutic options The frequent phenotype of resistance to multiple antibiotics among ESBL-producing isolates is as a result of the presence of other genes, normally present in the same plasmid carrying blaCTX–M genes. This contributes to maintaining ESBL-producing populations under different antibiotic selective pressures; some of these associations are of growing interest. Traditionally, ESBL-producing isolates, mostly TEMand SHV-producers, display co-resistance to aminoglycosides, tetracyclines and sulfonamides. In addition to these compounds, most of the CTX-M producers are also resistant to fluoroquinolones [53]. This resistance is not only associated with topoisomerase mutations but also with the presence of different qnr genes and/or the production of the new variant of the aminoglycosidemodifying enzyme AAC(60 )-Ib that also modifies certain Current Opinion in Microbiology 2006, 9:466–475

fluoroquinolones [54
,55

]. The qnrA gene has been associated with blaCTX-M-9, blaCTX-M-14 and other nonblaCTX-M genes such as blaVEB, whereas qnrB was associated with blaCTX-M-15 or blaSHV-12 genes [54
,56]. Interestingly, qnrA-carrying isolates might also carry the aac(60 )-Ib-cr gene [55

]. The product of this gene facilitates the acetylation of the amino nitrogen of fluoroquinolones with a piperazinyl substituent (ciprofloxacin and norfloxacin but not levofloxacin). Using multivariant analysis, these findings partially explain the association of previous fluoroquinolone use with the presence of an infection by CTX-M-producing isolates [12
,57]. It is of note that the CTX-M-15, one of the most spread CTX-M enzymes in the community and in long-term care facilities [16
,18

,47,52,58], is also associated with aac(60 )-Ib-cr [50]. In a recent study of ESBL-producing E. coli as a cause of nosocomial infection or colonization, the use of oxyiminob-lactams was found to be an independent risk factor associated with the isolation of CTX-M-producing isolates [57]. This use was previously associated with TEMor SHV-ESBL-producing isolates. Therapeutic options in hospitalized patients infected with CTX-M-producing isolates are limited to only carbapenems (imipenem, meropenem and ertapenem) and b-lactam/b-lactamase inhibitor combinations (piperacillin and tazobactam, amoxicillin and clavulante, ticarcillin and clavulanate, and ampicillin and sulbactam) within the b-lactams. The use of the latter combinations is controversial and depends on the site of infection and the concomitant presence of other b-lactamase or resistance mechanisms (i.e. porin deficiency) affecting b-lactam activity in the infecting isolate. Non b-lactam antibiotics might include tigecycline, a new glycylcycline derivative with good activity against ESBL producers [53] and, under in vitro susceptibility testing guidance, quinolones or aminoglycosides. Alternatives in the community are more limited, particularly if oral antibiotics are used [7
], and includes b-lactams/b-lactamase inhibitor combinations with oral formulations and quinolones and trimethoprim-sulfamethoxazole if isolates are not resistant. Fosfomycin and nitrofurantoin are also proposed in urinary tract infections due to CTX-M producing isolates.

Conclusions CTX-M-producing isolates have risen to prominence during the past few years, particularly in the community setting. This cannot be explained simply as a result of selective pressure exerted by the use of expanded spectrum cephalosporins. Other factors influencing this evolution at molecular level include recombinatorial events of blaCTX-M genes with ISs or putative transposases, and inclusion on complex structures harboured by transposable elements might have established modular plastic platforms able to be efficiently maintained in bacterial populations. The enclosure of these complex genetic www.sciencedirect.com

The CTX-M b-lactamase pandemic Canto´n and Coque 473

structures into well-established plasmids in different environments might have facilitated the spread of blaCTX-M. Future efforts should be directed towards understanding the rules governing the interplay among all discrete sequences involved in recombinatorial exchanges, and the relative weight of drift and selection in the spread of particular combinations, which will help us understand the CTX-M pandemics.

Acknowledgements We are grateful for support from the Fondo de Investigaciones Sanitarias (FIS) from the Ministry of Health (grants C03/103 and PI020943), from the Ministry of Science and Technology of Spain (grant SAF 2003-09285), and from the European Commission (grant LSHM-CT-2003-503335). The authors would like to thank Juan Carlos Gala´n for critical reading of the manuscript.

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resolution structures of CTX-M b-lactamases.,;1; extended spectrum activities from increased mobility and decreased stability. J Mol Biol 2005, 348:349-362. www.sciencedirect.com

The recognition of specific amino acid residues in the interaction with oxyimino side chains of third generation cephalosporins explains the extraordinary ESBL activity of CTX-Ms. The enhancement of the spectrum towards ceftazidime is also explained. 11. Canto´n R, Coque TM, Baquero F: Multi-resistant Gram-negative bacilli: from epidemics to endemics. Curr Opin Infect Dis 2003, 16:315-325. 12. Ben-Ami R, Schwaber MJ, Navon-Venezia S, Schwartz D,
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Willey BM, Bryce E, Gardam M, Nordmann P, Mulvey MR: Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extended-spectrum b-lactamase involved in an outbreak in long-term-care facilities in Toronto, Canada. Antimicrob Agents Chemother 2004, 48:3758-3764. This study provides one of the first complete sequence of a plasmid carrying blaCTX-M. 19. Olson AB, Silverman M, Boyd DA, McGeer A, Willey BM, Pong-Porter V, Daneman N, Mulvey MR: Identification of a progenitor of the CTX-M-9 group of extended-spectrum b-lactamases from Kluyvera georgiana isolated in Guyana. Antimicrob Agents Chemother 2005, 49:2112-2115. 20. Rodrı´guez MM, Power P, Radice M, Vay C, Famiglietti A, Galleni M, Ayala JA, Gutkind G: Chromosome-encoded CTX-M-3 from Kluyvera ascorbata: a possible origin of plasmid-borne CTXM-1-derived cefotaximases. Antimicrob Agents Chemother 2004, 48:4895-4897. 21. Power P, Galleni M, Di Conza J, Ayala JA, Gutkind G: Description of In116, the first blaCTX-M-2-containing complex class 1 integron found in Morganella morganii isolates from Buenos Aires, Argentina. J Antimicrob Chemother 2005, 55:461-465. 22. Oliver A, Pere´z-Dia´z JC, Coque TM, Baquero F, Canto´n R: Nucleotide sequence and characterization of a novel cefotaxime-hydrolyzing b-lactamase (CTX-M-10) isolated in Spain. Antimicrob Agents Chemother 2001, 45:616-620. 23. Garcı´a A, Navarro F, Miro E, Mirelis B, Campoy S, Coll P: Characterization of the highly variable region surrounding the bla(CTX-M-9)gene in non-related Escherichia coli from Barcelona. J Antimicrob Chemother 2005, 56:819-826. Current Opinion in Microbiology 2006, 9:466–475

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24. Toleman MA, Bennett PM, Walsh TR: ISCR elements: novel

gene-capturing systems of the 21st century? Microbiol Mol Biol Rev 2006, 70:296-316. An excellent review summarizing recent knowledge in modular structures carrying CR elements, proposing a new nomenclature for these elements. 25. Oliver A, Coque TM, Alonso D, Valverde A, Baquero F, Canto´n R: CTX-M-10 linked to a phage-related element is widely disseminated among Enterobacteriaceae in a Spanish hospital. Antimicrob Agents Chemother 2005, 49:1567-1571. 26. Lartigue MF, Poirel L, Aubert D, Nordmann P: In vitro analysis of ISEcp1B-mediated mobilization of naturally occurring b-lactamase gene blaCTX-M of Kluyvera ascorbata. Antimicrob Agents Chemother 2006, 50:1282-1286. 27. Poirel L, Lartigue MF, Decousser JW, Nordmann P: ISEcp1B

mediated transposition of blaCTX-M in Escherichia coli. Antimicrob Agents Chemother 2005, 49:447-450. A demonstration of the participation of the ISEcp1-like structure in the mobilization of blaCTX-M genes by a one-end transpositional mechanism. 28. Wachino J, Yamane K, Shibayama K, Kurokawa H, Shibata N, Suzuki S, Doi Y, Kimura K, Ike Y, Arakawa Y: Novel plasmidmediated 16S rRNA methylase, RmtC, found in a Proteus mirabilis isolate demonstrating extraordinary high-level resistance against various aminoglycosides. Antimicrob Agents Chemother 2006, 50:178-184. 29. Valverde A, Canto´n R, Galan JC, Nordmann P, Baquero F, Coque TM: In117, an unusual In0-like class 1 integron containing CR1 and bla(CTX-M-2) and associated with a Tn21like element. Antimicrob Agents Chemother 2006, 50:799-802. 30. Partridge SR, Hall RM: In34, a complex In5 family class 1 integron containing orf513 and dfrA10. Antimicrob Agents Chemother 2003, 47:342-349. 31. D’Andrea MM, Nucleo E, Luzzaro F, Giani T, Migliavacca R, Vailati F, Kroumova V, Pagani L, Rossolini GM: CMY-16, a novel acquired AmpC-type b-lactamase of the CMY/LAT lineage in multifocal monophyletic isolates of Proteus mirabilis from Northern Italy. Antimicrob Agents Chemother 2006, 50:618-624. 32. Rodrı´guez-Martı´nez JM, Poirel L, Canto´n R, Nordmann P: Common region CR1 for expression of antibiotic resistance genes. Antimicrob Agents Chemother 2006, 50:2544-2546. 33. Soler Bistue AJ, Martin FA, Petroni A, Faccone D, Galas M, Tolmasky ME, Zorreguieta A: Vibrio cholerae InV117, a class 1 integron harboring aac(60 )-Ib and blaCTX-M-2, is linked to transposition genes. Antimicrob Agents Chemother 2006, 50:1903-1907.

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50. Machado E, Coque TM, Canto´n R, Baquero F, Sousa JC, Peixe L, The Portuguese Resistance Study Group: Dissemination in Portugal of CTX-M-15-, OXA-1-, and TEM-1-producing enterobacteriaceae strains containing the aac(60 )-Ib-cr gene, which encodes an aminoglycoside- and fluoroquinolonemodifying enzyme. Antimicrobial Agents Chemother 2006, 50:3220-3221.

37. Partridge SR, Hall RM: Complex multiple antibiotic and mercury resistance region derived from the r-det of NR1 (R100). Antimicrob Agents Chemother 2004, 8:4250-4255. 38. Poirel L, Decousser JW, Nordmann P: Insertion sequence ISEcp1B is involved in expression and mobilization of a bla(CTX-M) b-lactamase gene. Antimicrob Agents Chemother 2003, 47:2938-2945. 39. Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ:
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51. Gordon DM, Stern SE, Collignon PJ: Influence of the age and sex of human hosts on the distribution of Escherichia coli ECOR groups and virulence traits. Microbiol 2005, 151:15-23. 52. Oteo J, Navarro C, Cercenado E, Delgado-Iribarren A, Wilhelmi I, Orden B, Garcı´a C, Miguelanez S, Pe´rez-Vazquez M, Garcı´a-Cobos S et al.: Spread of Escherichia coli strains with high-level cefotaxime and ceftazidime resistance between the community, long-term care facilities, and hospital institutions. J Clin Microbiol 2006, 44:2359-2366. 53. Morosini MI, Garcı´a-Castillo M, Coque TM, Valverde A, Novais A, Loza E, Baquero F, Canto´n R: Antibiotic coresistance in extended-spectrum b-lactamase-producing Enterobacteriaceae and the in vitro activity of tigecycline. Antimicrob Agents Chemother 2006, 50:2695-2699. www.sciencedirect.com

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Park CH, Bush K, Hooper DC: Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat Med 2006, 12:83-88. This is the first recognition of a modifying enzyme involved in fluoroquinolone resistance. 56. Jacoby GA, Walsh KE, Mills DM, Walker VJ, Oh H, Robicsek A, Hooper DC: qnrB, another plasmid-mediated gene for

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Current Opinion in Microbiology 2006, 9:466–475