REVIEW. Genetic support of extended-spectrum b-lactamases L. Poirel, T. Naas and P. Nordmann

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1 REVIEW Genetic support of extended-spectrum b-lactamases L. Poirel, T. Naas and P. Nordmann Service de Bactériologie-Virologie, Hôpital de Bicêtre, South-Paris Medical School, University Paris XI, Le Kremlin-Bicêtre, France ABSTRACT Genes encoding extended-spectrum b-lactamases (ESBLs) have been reported in a variety of Gramnegative species, mostly in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. They are mostly either TEM or SHV derivatives, CTX-M-like enzymes now emerging worldwide or, less frequently, VEB, GES, and PER ESBLs. The mechanisms responsible for their acquisition are very diverse, and mostly are related to insertion sequences (ISs), transposons, class 1 integrons, and also sul1- type integrons containing the ISCR1 element. This diversity of genetic vehicles at the origin of these mobilisation acquisition processes enhances the spread of ESBLs. Keywords b-lactamases, insertion sequence, integron, review, transposon Clin Microbiol Infect 2008; 14 (Suppl. 1): INTRODUCTION Expanded-spectrum cephalosporins are b-lactams with a broad spectrum of activity against most Gram-negative bacteria [1]. However, they are sensitive to hydrolysis by several b-lactamases, including extended-spectrum b-lactamases (ES- BLs). Most ESBLs belong to class A of the Ambler nomenclature [2], possessing an active site serine and being mostly susceptible to inhibition by clavulanic acid. The genes encoding these acquired b-lactamases are mostly plasmid-mediated and are increasingly reported. The most common ESBLs identified in the past were those of the TEM and SHV types, which evolved via mutation of earlier penicillinases. The SHV-type ESBLs were considered, until recently, to be the most frequent ESBLs, although TEM ESBLs are also extremely common [1]. Other ESBLs did not evolve by point mutations of a narrow-spectrum b-lactamase, the most important group being the CTX-M enzymes, which comprise at least 50 variants distributed in five subgroups represented by CTX-M-1, CTX-M-3, CTX-M-8, CTX-M-9, and CTX-M-25. The other ESBLs identified so far in Gram-negative bacteria Corresponding author and reprint requests: L. Poirel, Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, Le Kremlin-Bicêtre, France laurent.poirel@bct.aphp.fr are the VEB, PER and GES b-lactamases and, in addition, a series of rarely reported enzymes, e.g., BES-1, BEL-1, TLA-1 and TLA-2, and SFO-1. Several GES variants have weak but significant carbapenemase activity. This review considers the genetic bases of ESBL gene acquisition. GENETIC SUPPORT OF bla TEM GENES Over 150 TEM b-lactamase variants have been identified so far, many of them displaying an ESBL phenotype ( All are TEM-1 or TEM-2 derivatives, and are carried by three of the earliest bacterial transposons to be identified, namely Tn1, Tn2 and Tn3 [3,4]. Tn1 and Tn3 contain transposase and resolvase genes, namely tnpa and tnpr, and a res resolution site (Fig. 1). The Tn3 class II transposon possesses 38-bp inverted repeats (IRs) and is able to efficiently transpose the bla TEM ampicillin resistance gene marker, together with resistance to expanded-spectrum b-lactams when the TEM determinant encodes an ESBL variant. Most of the structures surrounding the bla TEM ESBL genes derive from a common Tn3-like structure rather than from DNA segments assembled during several distinct mobilisation events [5]. These structures may have evolved significantly, as observed in a Pseudomonas aeruginosa isolate from France with the bla TEM-21 ESBL gene located in a Tn801 transposon (Tn3 derivative) disrupted by

2 76 Clinical Microbiology and Infection, Volume 14, Supplement 1, January 2008 (a) (b) (c) tnpa tnpr bla TEM-1 P inti1 bla VEB-1 qaceδ1 sul1 orf5 5 -CS atti bla inti1 qaceδ1 sul1 orf513 CTX-M-9 qaceδ1 sul1 ISCR1 3 -CS Fig. 1. Schematic representation of three main genetic structures of extended-spectrum b-lactamase genes acquisition. (a) A Tn3 transposon harbouring the bla TEM-1 gene (its inverted repeats are represented by black rectangles). (b) A class 1 integron (with its 5 -CS and 3 -CS conserved regions) harbouring the bla VEB-1 gene cassette (inti1, integrase gene; black circle, atti recombination site; white arrows, gene cassettes; white circles, respective 59-be of gene cassettes; thin arrow, the promoter sequences provided by the 5 -CS). (c) A sul1-type integron carrying the bla CTX-M-9 gene, comprising the class 1 integron and its gene cassettes associated with the ISCR1 element (containing the orf513 transposase gene) and a duplication of the qaced1 sul1 tandem. an insertion sequence IS6100 element [6]. However, it is hypothesised that the b-lactamase gene has mostly evolved into a defined genetic structure, with which it is quite systematically associated. The bla TEM genes have never been identified inside integron structures, probably because they are not associated with features compatible with the formation of a gene cassette. GENETIC SUPPORT OF bla SHV GENES Few studies have investigated the genetics of acquisition of bla SHV -like genes, despite the fact that these genes clearly originate from the chromosome of Klebsiella pneumoniae, are common in Gram-negative bacteria and have spread worldwide. As SHV ESBLs are just point mutants of either narrow-spectrum chromosomal SHV-1 or SHV-2 b-lactamases, it is not surprising that some DNA fragments originate from the K. pneumoniae chromosome in the immediate vicinity of the bla SHV -like genes. Thus, the plasmid-borne bla SHV-5 gene identified in a Salmonella Typhimurium isolate from Greece had been acquired, together with seven other co-linear genes originating from K. pneumoniae, this locus being bracketed by two IS26 elements inserted in the same orientation [7]. The IS26 element located at the 5 end of bla SHV-5 was associated with the 5 -end of a class 1 integron structure harbouring five gene cassettes, including one encoding the metallo-blactamase VIM-1. It is likely that the IS26 element identified downstream of bla SHV-5 was responsible for its acquisition through a homologous recombination, rather than a transposition event, as suggested by the absence of target site duplication. A similar structure was also identified on plasmid pacm1 but, this time, the two direct repeated IS26 elements were both truncated, forming a defective compound transposon [8]. In several enterobacterial and P. aeruginosa isolates, only a single IS26 element was identified upstream of the bla SHV -like gene. Thus, as suggested by Ford et al. [9], it is very likely that IS26 is a key feature in acquisition of bla SHV genes, with mobilisation having occurred through at least two separate events. Like bla TEM genes, the bla SHV genes have never been identified inside integron structures. GENETIC SUPPORT OF bla CTX-M GENES The dissemination of genes encoding CTX-M-like ESBLs is extremely worrisome, and the problem has appeared recently, as compared with the dissemination of bla TEM and bla SHV ESBL genes. The bla CTX-M genes originate from the chromosomes of Kluyvera species, Kluyvera georgiana probably being the progenitor of the bla CTX-M-8 - like and bla CTX-M-9 -like genes, and Kluyvera ascorbata and Kluyvera cryocrescens the progenitors of the bla CTX-M-1 -like and bla CTX-M-2 -like genes [10]. Different genetic elements have been found to be involved in the acquisition of bla CTX-M genes, including insertion sequences ISEcp1 and ISCR1 (formerly CR1 element), and also phage-related elements. ISEcp1-like insertion sequence elements have been identified in association with genes encoding enzymes of the CTX-M-1, CTX-M-2 and CTX- M-9 clusters. ISEcp1 belongs to the IS1380 family and is able to mobilise bla CTX-M genes by a peculiar transposition process [11]. It mobilises adjacent sequences by transposition, after recognition of a variety of DNA sequences as right inverted repeats (IRRs). Thus, a single copy of ISEcp1 located upstream of a bla CTX-M gene is sufficient to mobilise the gene from the chromo-

3 Poirel et al. Genetics of ESBLs 77 some of a Kluyvera strain [12]. In addition, by providing promoter sequences, ISEcp1 enhances the expression of the bla CTX-M gene, which is low in its natural source species but high once mobilised onto a plasmid [11]. The duplication of a 5-bp sequence is the signature of the ISEcp1- mediated transposition, and defines the boundaries of the bla CTX-M -borne transposon. Several genetic elements, e.g., IS91 and Tn2, are able to mobilise adjacent sequences by a one-ended transposition process, recognising different variable sequences used as IRRs that define the right extremity of the transposed fragment. The IRRlike sequences used by ISEcp1 during transposition are not absolutely random but share a significant degree of identity with the original RIR of ISEcp1, which should lead to a transposition frequency lower than that of a true oneended transposition process [13]. The second main mechanism of acquisition of bla CTX-M genes is linked to a peculiar insertion sequence-mediated transposition process called rolling-circle (RC) transposition [14,15]. Some bla CTX-M genes occur in sul1-type integron structures that correspond to class 1 integrons bracketed at their 3 -extremity by the orf513 gene and a duplication of the qaced1 sul1 tandem. The bla CTX-M genes are not present as gene cassettes but are located downstream of the orf513 gene, between the two qaced1 sul1 repeats (Fig. 1). The orf513 gene was originally defined, together with a 33-bp recombination crossover site located at its right-hand boundary, as part of the so-called common region CR1 [16]. The putative Orf513 recombinase was proposed to mediate acquisition of adjacent genes by homologous recombination. Recently, a novel definition of the CR1 element has been proposed and the element itself is now recognised as an insertion sequence element. ISCR1 belongs to the IS91 family and Orf513 is a transposase that shares the structural and functional features of the IS91-like transposases [15]. ISCR1 lacks the terminal IRs typical of most insertion sequence elements and probably transposes differently. Its mechanism of transposition, called RC transposition, implicates the transposase responsible for initiating, and probably terminating, replication of the element before the final recombination step of the transposition process. One end of the ISCR1 element, named oriis, serves as an origin of replication, whereas the other, teris, is a replication terminator. ISCR1, like other IS91-like elements, may mobilise adjacent sequences when the RC replication mechanism misidentifies teris and proceeds to replicate the DNA adjacent to it. Thus, ISCR1 may mobilise chromosomal genes (e.g., the bla CTX-M gene from Kluyvera spp.) by first transposing into a position adjacent to them and then transposing again but aberrantly, thus mobilising the adjacent sequence onto a conjugative plasmid. The formation of the sul1-type integron could then be consequent to a homologous recombination process between the ISCR1-associated DNA sequence and another ISCR1 element originally associated with the 3 -extremity of a class 1 integron [14,15]. Notably, ISCR1 was experimentally shown to additionally enhance expression of the bla CTX-M-9 gene [17]. Besides these ISEcp1- or ISCR1-related elements, phage-related elements have been identified upstream of the bla CTX-M-10 gene in several enterobacterial isolates recovered from Spain [18]. Several open reading frames (ORFs) exhibiting structural similarities with conserved phage tail proteins, including a DNA invertase, have been identified upstream of bla CTX-M-10. These ORFs are preceded by a Tn1000-like transposase. Downstream of bla CTX-M-10, sequences have been identified that share significant identity with DNA sequences of K. cryocrescens followed by an IS5 element. Thus, transfer of the bla CTX-M-10 gene from the chromosome of Kluyvera spp. to a transferable plasmid may have been mediated by a bacteriophage-mediated transduction process. Notably, none of the bla CTX-M genes has been reported as a form of gene cassette in a class 1 integron. Recent identification of the bla CTX-M-2 gene in an Acinetobacter baumannii isolate may indicate spread of its related genetic support to an unrelated Gram-negative bacterium [19]. GENETIC SUPPORT OF bla VEB GENES The bla VEB-1 was identified first in an Escherichia coli isolate recovered in France from a Vietnamese child [20]. This was the first identification of an ESBL-encoding gene in the form of a gene cassette in a class 1 integron structure. An integron is a genetic unit capable of capturing, mobilising and expressing genes that are contained in mobile elements called gene cassettes [21]. Its essential components are an int1 gene, encoding a

4 78 Clinical Microbiology and Infection, Volume 14, Supplement 1, January 2008 site-specific recombinase belonging to the integrase family, an atti adjacent site recognised by the integrase and acting as a receptor site for gene cassettes, and a promoter for gene cassette expression (Fig. 1). The cassettes are mobile elements that include a gene (often encoding antibiotic resistance), and an integrase-specific recombination site known as the 59-be element [21]. The bla VEB-1 ESBL gene was identified inside a gene cassette structure possessing perfect core and inverse core sites, together with a 133-bp 59-be recombination site [20]. The bla VEB-1 gene cassette was located inside the In53 class 1 integron, which carries an unusual array of eight functional gene cassettes encoding other antibiotic resistance determinants [22]. In53 was identified as plasmid-borne and as part of a composite transposon structure, named Tn2000. It was flanked on both of its extremities by an IS26 element. On both extremities of Tn2000, an 8-bp duplication of the target site was the signature of the transposition process. Thus, In53, instead of being located in a defective Tn402-based transposon, as is mostly the case for class 1 integrons, may have acquired mobility by the insertion of two IS26 elements. By analysing the genetic environments of bla VEB-1 genes identified in enterobacterial and P. aeruginosa isolates recovered worldwide and in an A. baumannii epidemic clone from France [23], it was shown that this ESBL-encoding gene was present in a variety of class 1 integron structures. However, the features underlying the genetic mobility of those integrons have not been studied, except in the A. baumannii strain. In that latter organism, the bla VEB-1 -containing integron was associated, at its 3 -extremity, with a sul1-type integron structure containing an ISCR1 element at the origin of acquisition of a dfrx trimethoprim resistance gene [24]. Notably, that overall structure was not part of a Tn402-like or Tn2000-like transposon, but had integrated into the chromosome of the A. baumannii strain, together with other transposons or operons encoding antiseptic resistance as parts of a resistance island, named AbaR1 [24]. This island was 86 kb long and had been integrated through a transposition process, as revealed by the 5-bp target site duplication identified on its extremities [24]. Besides these class 1 integron-borne bla VEB-1 genes, other interesting structures have been found at the origin of the acquisition of this gene. Specifically, the bla VEB-1a gene was inserted between two truncated 3 -CS regions in a direct repeat and bracketed by four 135-bp repeated DNA sequences (named repeated elements (Res), two of them being in direct orientation upstream of bla VEB-1 )inap. aeruginosa isolate from India [25]. A very similar structure was found in a bla VEB-1 -positive Providencia stuartii isolate from Algeria, although with only a single Re element (containing the promoter sequences) present upstream of the b-lactamase gene [26]. Another similar structure was identified in a P. aeruginosa isolate from Bangladesh, but in that case associated with an ISCR1 element, itself associated with an integron, thus forming an In121 sul1-type integron at the origin of bla VEB-1 acquisition [27]. The origin of these Res and their function in the mobilisation process of bla VEB-1 remains unknown; nonetheless, they have been shown to be involved in its expression by providing promoter sequences. To summarise, the bla VEB-1 gene may be associated with either class 1 integrons or other genetic structures. GENETIC SUPPORT OF bla GES GENES The bla GES-1 gene was initially identified as plasmid-borne in a K. pneumoniae clinical isolate recovered in France from a patient originating from French Guyana [28]. Interestingly, this gene was found to be part of a gene cassette, but with a truncated recombination site. The bla GES-1 gene cassette was identified at the first position in a class 1 integron structure named In52. Subsequently, this gene cassette was identified in a P. aeruginosa isolate from France, at the second position of a class 1 integron [29]. Eight other GES (or IBC)-like encoding genes have been subsequently identified in Enterobacteriaceae and P. aeruginosa, always located inside class 1 integrons [30], with the exception of a bla GES-1 gene from a K. pneumoniae strain from Portugal, which was embedded in a class 3 integron [31]. GENETIC SUPPORT OF bla PER GENES Analysis of the sequences bracketing the chromosomally located bla PER-1 gene in several Gramnegative isolates revealed that this b-lactamase gene was part of a composite transposon named Tn1213 [32]. This composite transposon

5 Poirel et al. Genetics of ESBLs 79 was formed by two structurally related but significantly different insertion sequences, ISPa12 and ISPa13. An 8-bp duplication of the insertion site was noticed at the left-hand extremity of ISPa12 and at the right-hand extremity of ISPa13, clearly showing that this structure had transposed. Analysis of Tn1213-surrounding sequences in P. aeruginosa, A. baumannii and P. stuartii isolates revealed that the transposition had occurred inside an IS element named ISPa14, by interrupting its transposase gene. The Tn4176 transposon, very similar to Tn1213 but inserted into a Tn5393 derivative, was also identified in an Alcaligenes faecalis isolate from Italy [33]. In two S. Typhimurium isolates and a single A. baumannii isolate, where it was plasmid-located, the bla PER-1 gene was not part of Tn1213. Rather, only ISPa12 was identified upstream of the bla PER-1 gene, providing efficient promoter sequences for b-lactamase gene expression, thus suggesting that another mechanism was responsible for bla PER-1 gene acquisition in those isolates. Analysis of the limited flanking DNA sequences associated with the bla PER-2 gene that are available in the GenBank databases revealed that this gene, encoding b-lactamase PER-2 (86% amino acid identity with PER-1) in an S. Typhimurium isolate from Argentina, was also associated with an ISPa12 element, thus suggesting a mobilisation mechanism similar to those deduced for bla PER-1 in distant continents (South America vs. Europe and Asia). Interestingly, the bla PER-3 gene encoding PER- 3 (with single amino-acid change as compared to PER-1) identified in an Aeromonas punctata isolate from France was identified in association with the ISCR1 element inside the In39 sul1-type integron (C. Neuwirth, unpublished data, Gen- Bank no. AY740681). This finding may indicate another means of spread of bla PER genes, thus enhancing their potential for dissemination and underscoring the great ability of ISCR1 elements to spread antibiotic resistance genes. GENETIC SUPPORT OF OTHER ESBL- ENCODING GENES The bla BEL-1 gene was identified only in a P. aeruginosa isolate from Belgium in 2004 [34]. This gene was part of a gene cassette structure defined by perfect core and inverse core sites and a 63-bp 59-be element. The bla BEL-1 gene cassette, together with three other resistance gene cassettes, was part of the In120 class 1 integron, which was itself part of a Tn402 derivative with an ISPa7 element at its 5 -extremity. In120 was found inserted into the resolution site of Tn1404, identified at its 3 -end [34]. The linkage of the ISPa7 element with P. aeruginosa-specific genes confirmed its chromosomal location in that isolate. There is no information regarding the genetic environment of the bla BES-1 gene identified in a Serratia marcescens isolate from Brazil, or the bla TLA-1 gene from an E. coli isolate from Mexico (analysis of DNA database sequences showed part of an IS10-like element upstream of the b-lactamase gene), or that of the plasmid-borne bla SFO-1 gene identified in an Enterobacter cloacae isolate from Japan, but originating from the chromosome of Serratia fonticola. The bla TLA-2 gene, encoding TLA-2 (only 51% identity with TLA-1), isolated from an unidentified bacterium from sludge was not embedded in a class 1 integron but was bracketed by 145-bp direct repeats of unknown function [35]. CONCLUSION Analysis of the genetic supports at the origin of acquisition and diffusion of ESBL genes reveals a variety of genetic elements, including most of the tools known to be implicated in bacterial genetic plasticity as a whole. These elements have the potential to disseminate rapidly in a wide range of bacterial species. Some of them (e.g., ISEcp1) may mobilise genes very easily and efficiently, while others are mosaic structures, probably as a result of more complex events at the origin of their mobility. Several of these structures may lead to multidrug resistance to antibiotics, whereas others are associated with just a single resistance marker. All these features make the analysis of the genetic processes complex. The variety of genetic vehicles associated with ESBL genes make them perfect tools for analysing the spread of antibiotic resistance genes, and the factors that may trigger either their emergence or their expression. Experimental studies such as the in-vitro mobilisation of a bla CTX-M gene from the chromosome of K. ascorbata to an E. coli recipient strain [12] may significantly contribute to our understanding of the genetics that drive in-vivo mobilisation of antibiotic resistance genes.

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