Epidemiology and genetics of CTX-M extended-spectrum β-lactamases in Gram-negative bacteria

Abstract

CTX-M enzymes, the plasmid-mediated cefotaximases, constitute a rapidly growing family of extended-spectrum β-lactamases (ESBLs) with significant clinical impact. CTX-Ms are found in at least 26 bacterial species, particularly in Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. At least 109 members in CTX-M family are identified and can be divided into seven clusters based on their phylogeny. CTX-M-15 and CTX-M-14 are the most dominant variants. Chromosome-encoded intrinsic cefotaximases in Kluyvera spp. are proposed to be the progenitors of CTX-Ms, while ISEcp1, ISCR1 and plasmid are closely associated with their mobilization and dissemination.

Introduction

Extended-spectrum β-lactamases (ESBLs) are the most influential mechanism for cephalosporin resistance in Enterobacteriaceae, particularly in Escherichia coli and Klebsiella pneumoniae. ESBLs confer resistance to penicillins, broad-spectrum cephalosporins with an oxyimino side chain (cefotaxime, ceftriaxone and ceftazidime) and the oxyimino-monobactam aztreonam, but can be inhibited by serine-type β-lactamase inhibitors as sulbactam, clavulanate and tazobactam (Philippon et al., 1989; Bradford, 2001). SHV-2 is the first ESBL, identified in a clinical isolate of Klebsiella ozaenae in Germany (Kliebe et al., 1985). To date, over 10 families have been documented to be associated with ESBLs, including CTX-M, SHV, TEM, PER, VEB, BES, GES, TLA, SFO and OXA (Paterson and Bonomo, 2005).

CTX-M enzymes, the plasmid-mediated acquired cefotaximases from a distinct phylogenetic lineage, constitute a rapidly growing family of ESBLs with significant clinical impact (Bonnet, 2004; Cantón and Coque, 2006; Livermore et al., 2007; Naseer and Sundsfjord, 2011). Chromosome-encoded genes of intrinsic cefotaximases in Kluyvera spp. are proposed to be the progenitors of CTX-M family (Humeniuk et al., 2002; Olson et al., 2005; Decousser et al., 2011). Most of CTX-Ms exhibit powerful activity against cefotaxime and ceftriaxone but not ceftazidime. However, some CTX-Ms, such as CTX-M-15 (Poirel et al., 2002a), CTX-M-16 (Bonnet et al., 2001) and CTX-M-19 (Poirel et al., 2001), exhibit enhanced catalytic efficiencies against ceftazidime.

This article summarizes the epidemiology of CTX-M-producing Gram-negative bacteria and the genetics of CTX-M ESBLs, with a focus on the phylogeny, origin and genetic platforms including ISEcp1, ISCR1 and plasmid.

Epidemiology of CTX-M ESBLs

Occurrence and bacterial hosts

A plasmid-mediated cefotaximase was identified from a clinical isolate of E. coli in Munich, Germany, and designated CTX-M in reference to its hydrolytic activity and the region where it was found (Bauernfeind et al., 1990). To date, the numbers of CTX-M variants and the recognized organisms harboring the genes have dramatically increased. At least 109 CTX-M variants, CTX-M-1 to CTX-M-124, have been identified (Table 1) and assigned in the Lahey database (Jacoby and Bush, 2012). The amino-acid sequences of CTX-M-14 and CTX-M-18 and of CTX-M-55 and CTX-M-57 are identical, and CTX-M-118 has been withdrawn. There is no detailed information available for the assigned members CTX-M-70, -73, -100, -103, -115, -119, -120 and -124 so far. In addition, CTX-M-76, -77, -78 and -95 are chromosome-encoded intrinsic cefotaximases in Kluyvera spp., and therefore, they are not counted into the CTX-M family. CTX-M-2, -3 and -37 are plasmid-mediated enzymes but also found on chromosomes in Kluyvera spp. To clarify the differences, the term c-CTX-M is used for such chromosome-encoded CTX-Ms in this article. Of the studied CTX-Ms, at least 19 variants display the enhanced catalytic efficiencies against ceftazidime (Table 1).

Table 1. .

CTX-M ESBLs and their bacterial hosts.

CTX-M (alternate name)	Bacterial host	GenBank accession no.	Reference
CTX-M-1 (MEN-1)	Escherichia coli	X92506	Bauernfeind et al., 1996
	Enterobacter cloacae		al Naiemi et al., 2006
	Klebsiella pneumoniae		Komatsu et al., 2001
	Proteus mirabilis		al Naiemi et al., 2006
	Pseudomonas aeruginosa		al Naiemi et al., 2006
	Salmonella enterica		Rodríguez et al., 2009
	Serratia marcescens		Choi et al., 2007
	Stenotrophomonas maltophilia		al Naiemi et al., 2006
CTX-M-2	Salmonella enterica	X92507	Bauernfeind et al., 1996
	Acinetobacter baumannii		Nagano et al., 2004
	Citrobacter koseri		al Naiemi et al., 2006
	Escherichia coli		Arduino et al., 2003
	Enterobacter cloacae		Arduino et al., 2003
	Klebsiella pneumoniae		Arduino et al., 2003
	Morganella morganii		Power et al., 2005
	Proteus mirabilis		Bonnet et al., 2000
	Providencia stuartii		Minarini et al. 2009
	Pseudomonas aeruginosa		Arduino et al., 2003
	Serratia marcescens		Arduino et al., 2003
	Vibrio cholerae		Soler Bistué et al., 2006
CTX-M-3	Citrobacter freundii	Y10278	Gniadkowski et al., 1998
	Aeromonas caviae		Ye et al., 2010
	Escherichia coli		Yan et al., 2000
	Enterobacter cloacae		De Champs et al., 2000
	Enterobacter aerogenes		Liu et al., 2009
	Klebsiella pneumoniae		Baraniak et al., 2002b
	Klebsiella oxytoca		Baraniak et al., 2002b
	Morganella morganii		Baraniak et al., 2002b
	Proteus mirabilis		Eckert et al., 2006
	Salmonella enterica		Gierczyński et al., 2003
	Sarratia marcescens		Baraniak et al., 2002b
	Shigella flexneri		Galimand et al., 2005
	Shigella sonnei		Acikgoz et al., 2003
CTX-M-4	Salmonella enterica	Y14156	Gazouli et al., 1998b
CTX-M-5	Salmonella enterica	U95364	Bradford et al., 1998
	Acinetobacter baumannii	AF462635
CTX-M-6 (renumbered)	Salmonella enterica	AJ005044	Gazouli et al., 1998a
CTX-M-7 (renumbered)	Salmonella enterica	AJ005045	Gazouli et al., 1998a
CTX-M-8	Citrobacter amalonaticus	AF189721	Bonnet et al., 2000
	Enterobacter cloacae		Bonnet et al., 2000
	Enterobacter aerogenes		Bonnet et al., 2000
	Escherichia coli		Minarini et al. 2009
CTX-M-9	Escherichia coli	AF174129	Sabaté et al., 2000
	Citrobacter freundii		Minarini et al. 2009
	Enterobacter aerogenes	EF441350
	Enterobacter cloacae		Chanawong et al., 2002
	Enterobacter hormaechei		Ho et al., 2005b
	Klebsiella pneumoniae		Chanawong et al., 2002
	Klebsiella oxytoca		Alobwede et al., 2003
	Salmonella enterica		García Fernández et al., 2007
	Serratia marcescens		Choi et al., 2007
CTX-M-10	Escherichia coli	AF255298	Oliver et al., 2001
	Citrobacter freundii		Valverde et al., 2004
	Enterobacter cloacae		Cantón et al., 2002
	Enterobacter gergoviae		Cantón et al., 2002
	Klebsiella pneumoniae		Coque et al., 2002
	Salmonella enterica		Cartelle et al., 2006
CTX-M-11	Klebsiella pneumoniae	AY005110
CTX-M-12	Klebsiella pneumoniae	AF305837	Kariuki et al., 2001
	Escherichia coli		Bae et al., 2006b
	Proteus mirabilis		Song et al., 2011
CTX-M-13	Klebsiella pneumoniae	AF252623	Chanawong et al., 2002
	Escherichia coli	DQ058147
	Enterobacter cloacae	AF462399
	Enterobacter hormaechei		Ho et al., 2005b
	Proteus mirabilis		Ho et al., 2005a
CTX-M-14	Escherichia coli	AF252622	Chanawong et al., 2002
	Citrobacter freundii		Kanamori et al., 2011
	Citrobacter koseri		Kanamori et al., 2011
	Enterobacter cloacae		Chanawong et al., 2002
	Enterobacter hormaechei		Ho et al. 2005b
	Klebsiella pneumoniae		Chanawong et al., 2002
	Proteus mirabilis		Ho et al., 2005a
	Providencia stuartii		Liu et al., 2009
	Salmonella enterica		Romero et al., 2004
	Serratia liquefaciens	AF462398
	Shigella flexneri	DQ350883
	Shigella sonnei		Pai et al., 2001
CTX-M-15 (UOE-1) *	Escherichia coli	AY044436	Karim et al., 2001
	Acinetobacter baumannii		Shakil & Khan, 2010
	Aeromonas hydrophila		Gómez-Garcés et al., 2011
	Citrobacter freundii	HQ214043
	Citrobacter koseri		Kanamori et al., 2011
	Enterobacter aerogenes		Kim et al., 2005
	Enterobacter cloacae		Moubareck et al., 2005
	Enterobacter gergoviae	EU118595
	Klebsiella pneumoniae		Lartigue et al., 2003
	Klebsiella oxytoca		Zhang et al., 2008
	Morganella morganii		al Naiemi et al., 2006
	Pantoea agglomerans		Aibinu et al., 2012
	Proteus mirabilis		Song et al., 2011
	Salmonella enterica		Weill et al., 2004
	Serratia marcescens		Baraniak et al., 2002a
	Shigella flexneri		Zhang et al., 2011
	Shigella sonnei		Hrabák et al., 2008
CTX-M-16 *	Escherichia coli	AY029068	Bonnet et al., 2001
CTX-M-17	Klebsiella pneumoniae	AY033516	Cao et al., 2002
CTX-M-18§	Klebsiella pneumoniae	AF325133	Poirel et al., 2001
CTX-M-19 *	Klebsiella pneumoniae	AF325134	Poirel et al., 2001
CTX-M-20	Proteus mirabilis	AJ416344	Saladin et al., 2002
CTX-M-21	Escherichia coli	AJ416346	Saladin et al., 2002
CTX-M-22	Klebsiella pneumoniae	AY080894	Yu et al., 2007
	Escherichia coli		Yu et al., 2007
	Enterobacter cloacae		Liu et al., 2007
	Serratia liquefaciens	HM470254
	Serratia marcescens	DQ309026
CTX-M-23 *	Escherichia coli	AF488377	Stürenburg et al., 2004
	Klebsiella pneumoniae		Stürenburg et al., 2004
CTX-M-24	Klebsiella pneumoniae	AY143430	Yu et al., 2007
	Escherichia coli		Yu et al., 2007
	Enterobacter aerogenes		Ho et al., 2005b
	Proteus mirabilis		Wu et al., 2008
	Shigella sonnei	FN594520
CTX-M-25 *	Escherichia coli	AF518567	Munday et al., 2004
	Klebsiella pneumoniae		Navon-Venezia et al., 2008
	Proteus mirabilis		Navon-Venezia et al., 2008
CTX-M-26	Klebsiella pneumoniae	AY157676	Brenwald et al., 2003
CTX-M-27 *	Escherichia coli	AY156923	Bonnet et al., 2003
	Salmonella enterica		Bouallègue-Godet et al., 2005
	Shigella sonnei	HM595763
CTX-M-28	Escherichia coli	AJ549244	Galimand et al., 2005
	Enterobacter sp.	EU531513
	Klebsiella pneumoniae		Yu et al., 2007
	Salmonella enterica		Hasman et al., 2005
CTX-M-29	Escherichia coli	AY267213	Yu et al., 2007
CTX-M-30	Citrobacter freundii	AY292654	Abdalhamid et al., 2004
CTX-M-31	Providencia stuartii	AJ567481	Quinteros et al., 2003
	Escherichia coli		Quinteros et al., 2003
CTX-M-32 *	Escherichia coli	AJ557142	Cartelle et al., 2004
	Klebsiella pneumoniae		Mendonça et al., 2009
	Proteus mirabilis		Fernández et al., 2007
CTX-M-33	Escherichia coli	AY238472	Galani et al., 2007
CTX-M-34	Escherichia coli	AY515297	Miró et al., 2005
CTX-M-35 *	Klebsiella pneumoniae	AB176532
	Citrobacter koseri		Tian et al., 2010
	Escherichia coli	AB176533
	Klebsiella oxytoca	AB176534
CTX-M-36	Escherichia coli	AB177384
CTX-M-37 *	Enterobacter cloacae	AY649755
	Salmonella enterica		Govinden et al., 2006
CTX-M-38	Klebsiella pneumoniae	AY822595
CTX-M-39	Escherichia coli	AY954516	Chmelnitsky et al., 2005
	Enterobacter cloacae		Navon-Venezia et al., 2008
	Klebsiella pneumoniae		Navon-Venezia et al., 2008
CTX-M-40 *	Escherichia coli	AY750914	Hopkins et al., 2006
CTX-M-41	Proteus mirabilis	DQ023162	Navon-Venezia et al., 2008
CTX-M-42 *	Escherichia coli	DQ061159	Stepanova et al., 2008
CTX-M-43	Acinetobacter baumannii	DQ102702	Celenza et al., 2006
	Enterobacter aerogenes		Celenza et al., 2006
	Enterobacter cloacae		Celenza et al., 2006
	Morganella morganii		Celenza et al., 2006
	Pseudomonas aeruginosa		Celenza et al., 2006
CTX-M-44 (Toho-1)	Escherichia coli	D37830	Ishii et al., 1995
CTX-M-45 (Toho-2)	Escherichia coli	D89862	Ma et al., 1998
CTX-M-46	Klebsiella pneumoniae	AY847147	Cheng et al., 2008
CTX-M-47	Escherichia coli	AY847143	Cheng et al., 2008
	Klebsiella pneumoniae		Cheng et al., 2008
CTX-M-48	Klebsiella pneumoniae	AY847144	Cheng et al., 2008
	Escherichia coli		Cheng et al., 2008
CTX-M-49	Klebsiella pneumoniae	AY847145	Cheng et al., 2008
CTX-M-50	Klebsiella pneumoniae	AY847146	Cheng et al., 2008
CTX-M-51	Escherichia coli	DQ211987
CTX-M-52	Klebsiella pneumoniae	DQ223685
CTX-M-53 *	Salmonella enterica	DQ268764	Doublet et al., 2009
CTX-M-54 *	Klebsiella pneumoniae	DQ303459	Bae et al., 2006a
CTX-M-55 *	Escherichia coli	DQ885477	Kiratisin et al., 2007
	Klebsiella pneumoniae		Kiratisin et al., 2007
	Shigella sonnei		Zhang et al., 2011
CTX-M-56	Escherichia coli	EF374097	Pallecchi et al., 2007
CTX-M-57§	Salmonella enterica	DQ810789	Hopkins et al., 2008
	Shigella sonnei	EU086736
CTX-M-58 *	Escherichia coli	EF210159
CTX-M-59	Klebsiella pneumoniae	DQ408762	de Oliveira et al., 2008
CTX-M-60	Klebsiella pneumoniae	AM411407
CTX-M-61	Salmonella enterica	EF219142	Brasme et al., 2007
	Klebsiella pneumoniae		Mendonça et al., 2009
CTX-M-62 *	Klebsiella pneumoniae	EF219134	Zong et al., 2008
CTX-M-63	Klebsiella pneumoniae	AB205197
	Morganella morganii	EU660216
	Salmonella enterica		Pornruangwong et al., 2011
CTX-M-64 *	Shigella sonnei	AB284167	Nagano et al., 2009
	Escherichia coli		Sun et al., 2010
	Enterobacter cloacae	GQ300937
CTX-M-65	Escherichia coli	EF418608	Doi et al. 2008
	Citrobacter freundii	EF394372
	Salmonella enterica	FJ907380
CTX-M-66	Proteus mirabilis	EF576988	Wu et al., 2008
CTX-M-67	Escherichia coli	EF581888	Oteo et al., 2008
CTX-M-68	Klebsiella pneumoniae	EU177100	Heffernan et al., 2009
CTX-M-69	Escherichia coli	EU402393
CTX-M-70†		Assigned
CTX-M-71	Klebsiella pneumoniae	FJ815436	Schneider et al., 2009
CTX-M-72	Klebsiella pneumoniae	AY847148	Cheng et al., 2009
CTX-M-73†		Assigned
CTX-M-74	Enterobacter cloacae	GQ149243	Minarini et al., 2009
CTX-M-75	Providencia stuartii	GQ149244	Minarini et al., 2009
c-CTX-M-76‡	Kluyvera ascorbata	AM982520
c-CTX-M-77‡	Kluyvera ascorbata	AM982521
c-CTX-M-78‡	Kluyvera georgiana	AM982522	Rodríguez et al., 2010
CTX-M-79	Escherichia coli	EF426798	Tian et al., 2008
CTX-M-80	Klebsiella pneumoniae	EU202673	Cheng et al., 2010
CTX-M-81	Klebsiella pneumoniae	EU136031	Cheng et al., 2010
CTX-M-82 *	Escherichia coli	DQ256091	Liu et al., 2009
CTX-M-83	Salmonella enterica	FJ214366	Cui et al., 2009
CTX-M-84	Salmonella enterica	FJ214367	Cui et al., 2009
CTX-M-85	Salmonella enterica	FJ214368	Cui et al., 2009
CTX-M-86	Salmonella enterica	FJ214369	Cui et al., 2009
CTX-M-87 (renumbered)	Escherichia coli	EU545409	Yin et al., 2009
CTX-M-88	Salmonella enterica	FJ873739	Ranjbar et al., 2010
CTX-M-89	Proteus mirabilis	FJ971899	McGettigan et al., 2009
	Enterobacter cloacae	FJ966096
CTX-M-90	Salmonella enterica	FJ907381
	Proteus mirabilis		Song et al., 2011
CTX-M-91	Proteus mirabilis	GQ870432
CTX-M-92	Escherichia coli	GU127598	Seputiene et al., 2010
	Klebsiella pneumoniae		Seputiene et al., 2010
CTX-M-93 *	Escherichia coli	HQ166709	Djamdjian et al., 2011
CTX-M-94	Escherichia coli	HM167760
c-CTX-M-95‡	Kluyvera ascorbata	FN813245
CTX-M-96 (CTX-M-12a)	Klebsiella pneumoniae	AJ704396
CTX-M-97	Escherichia coli	HM776707
CTX-M-98	Escherichia coli	HM755448
CTX-M-99	Klebsiella pneumoniae	HM803271
CTX-M-100†		Assigned
CTX-M-101	Escherichia coli	HQ398214
CTX-M-102	Escherichia coli	HQ398215
CTX-M-103†		Assigned
CTX-M-104	Escherichia coli	HQ833652
CTX-M-105	Escherichia coli	HQ833651
CTX-M-106	Escherichia coli	HQ913565
CTX-M-107	Shigella flexneri	JF274244	Zhang et al., 2011
CTX-M-108	Shigella flexneri	JF274245	Zhang et al., 2011
CTX-M-109	Shigella flexneri	JF274248	Zhang et al., 2011
CTX-M-110	Shigella sonnei	JF274242	Zhang et al., 2011
CTX-M-111	Shigella flexneri	JF274243	Zhang et al., 2011
CTX-M-112	Shigella sonnei	JF274246	Zhang et al., 2011
CTX-M-113	Shigella flexneri	JF274247	Zhang et al., 2011
CTX-M-114	Providencia rettgeri	GQ351346
CTX-M-115†		Assigned
CTX-M-116	Proteus mirabilis	JF966749
CTX-M-117	Escherichia coli	JN227085
CTX-M-118		Withdrawn
CTX-M-119†		Assigned
CTX-M-120†		Assigned
CTX-M-121	Escherichia coli	JN790862
CTX-M-122	Escherichia coli	JN790863
CTX-M-123	Escherichia coli	JN790864
CTX-M-124†		Assigned

^*with enhanced catalytic efficiencies against ceftazidime;

^†have been assigned in the Lahey database (Jacoby and Bush 2012);

^‡chromosome-encoded intrinsic cefotaximase identified in Kluyvera spp.;

^§CTX-M-18 and CTX-M-14, CTX-M-57 and CTX-M-55 are identical in their amino acid sequences.

CTX-Ms have been detected in at least 26 bacterial species, including Acinetobacter baumannii, Aeromonas caviae, A. hydrophila, Citrobacter amalonaticus, C. freundii, C. koseri, E. coli, Enterobacter cloacae, E. aerogenes, E. gergoviae, E. hormaechei, K. pneumoniae, K. oxytoca, Morganella morganii, Proteus mirabilis, Pantoea agglomerans, Providencia rettgeri, P. stuartii, Pseudomonas aeruginosa, Salmonella enterica, Shigella flexneri, S. sonnei, Serratia marcescens, S. liquefaciens, Stenotrophomonas maltophilia and Vibrio cholera (Table 1).

CTX-M enzymes as the most prevalent ESBLs in E. coli, K. pneumoniae and P. mirabilis

The high prevalence of CTX-M ESBL genes in Enterobacteriaceae, particularly in E. coli, K. pneumoniae and P. mirabilis, has been documented worldwide (Bonnet, 2004; Cantón and Coque, 2006), while the CTX-Ms are not prominent in P. aeruginosa and A. baumannii (Zhao and Hu, 2010, 2012).

A study on the resistance of Enterobacteriaceae to third-generation cephalosporin was undertaken in 16 British hospitals over a 12-week period (Potz et al., 2006). Of 19,252 clinical isolates, CTX-M-producing strains accounted for 1.7%, higher than other ESBLs-producing strains (0.6%) and high-level AmpC-producing strains (0.4%). Particularly, of the resistance isolates of E. coli (n = 574) and Klebsiella spp. (n = 243), the CTX-M-producing strains accounted for 50.9% and 81.9%, respectively, by contrast with other ESBLs-producing strains (15.3% and 11.1%), high-level AmpC-producing strains (7.1% and 0.8%) and non-β-lactamase-producing strains (26.7% and 3.3%).

A rapid occurrence of CTX-M-producing strains in Enterobacteriaceae was documented by several longitudinal surveillances. Of 20,258 E. coli isolates studied in Italy, the prevalence of ESBL-producing strains increased from 0.2% in 1999 to 1.6% in 2003, of which CTX-M-positive strains increased from 12.5% to 38.2% (Brigante et al., 2005). Of 1574 P. mirabilis clinical isolates collected in a Taiwanese hospital during 1999–2005, 44 CTX-M-producing strains were detected at a rate of 0.7% in 1999 and approximately 6% after 2002 (Wu et al., 2008). Of 11,407 E. coli isolates from urine samples of outpatients in the USA, 107 CTX-M-producing strains were detected at a rate of 0.07% in 2003 and 1.66% in 2008 (Qi et al., 2010).

CTX-M-producing strains widespread not only in human but also in animals and in environments. Of 240 E. coli isolates from health and sick pets during 2007–2008 in China, 97 strains (40.4%) harbored ESBL-encoding genes, of which 96 strains were confirmed to be carriers of bla _CTX-M genes (Sun et al., 2010). Of 16 multi-drug resistant E. coli isolates from river water during 2000–2001 in South Korea, 10 strains harbored CTX-M-14 gene (Kim et al., 2008). Of 79 food samples of animal origin in Tunisia, bla _CTX-M-1-positive E. coli strains were isolated from 10 samples (Ben Slama et al., 2010).

A Japanese group surveyed the spread status of CTX-M genes in nosocomial Gram-negative bacteria collected from 132 geographically distant medical facilities during 2001–2003. Of the 1456 isolates resistant to oxyimino-cephalosporins, 21.8% were found to harbor bla _CTX-M genes. The prevalent rates of CTX-Ms in ESBL-producing E. coli, K. pneumoniae and P. mirabilis were 77% (168/218), 56% (50/90) and 99% (71/72), respectively, while the rates of CTX-Ms in ESBL-producing A. baumannii and S. marcescens were 4.5% (4/89) and 7% (10/149), respectively (Shibata et al., 2006).

CTX-M-15 and CTX-M-14 as the most dominant variants in CTX-M family

Although the dominant variants of CTX-Ms are geographically different, CTX-M-15 and CTX-M-14 are the most common variants detected worldwide in clinically important pathogens, followed by CTX-M-2, CTX-M-3 and CTX-M-1 (Table 1). Conjugative plasmid-mediated horizontal transfer and clonal spread contributed to the increased prevalence.

Of 171 CTX-M-producing E. coli isolates from 11 Canadian medical centers in 2007, the positive rates for CTX-M-15, CTX-M-14, CTX-M-3 and CTX-M-27 were 86.5%, 9.9%, 2.9% and 0.6%, respectively (Peirano et al., 2010). Of 202 CTX-M-producing K. pneumoniae isolates from 41 medical centers in Hungary in 2005, 97% were CTX-M-15 producers derived from three genetically distinct clones (Damjanova et al., 2008). Of the CTX-M-producers (288 E. coli and 142 K. pneumoniae isolates) collected from 6 provinces in China during 1998–2002, CTX-M-14 was predominantly detected in 77.4% and 52.8% of the isolates, respectively, followed by CTX-M-3 (18.4% and 29.6%), CTX-M-24 (5.6% and 14.1%) and CTX-M-15 (0.7% and 1.4%) (Yu et al., 2007). An outbreak of CTX-M-producing S. enterica infection occurred in a university hospital in Algeria during 2008–2009, and all of 200 isolates from 138 patients were CTX-M-15 producers, identified to be a single clone (Naas et al., 2011).

Of 44 clinical isolates of CTX-M-producing P. mirabilis from a Taiwanese hospital, CTX-M-14 and CTX-M-3 positive strains accounted for 50% and 40.9%, respectively (Wu et al., 2008). Of 71 CTX-M-producing P. mirabilis isolates collected from 132 geographically distant hospitals in Japan, however, 100% of the strains carried the bla _CTX-M-2-like genes (Shibata et al., 2006). CTX-M-2 was also predominant in C. koseri, accounting for 76.7% of ESBL-producing strains (n = 60) collected from 10 areas throughout Japan in a 5-month period between 2009 and 2010 (Kanamori et al., 2011).

Phylogeny, origin and evolution of CTX-M enzymes

Amino-acid identity and phylogeny

The deduced amino-acid sequences of CTX-Ms comprise 291 residues, with the exceptions of CTX-M-11 (282), CTX-M-107 and -108 (288), CTX-M-45 and -109 (289), CTX-M-40, -63 and -106 (290) and CTX-M-110 (292). Based on the phylogenetic tree of amino-acid sequences, CTX-M enzymes may be divided into seven clusters (Figure 1).

Figure 1. .

Phylogenetic tree of CTX-M family based on amino-acid sequences. DNASIS Pro v2.10 (Hitachi Software Engineering Co., Tokyo, Japan) was used to align the amino-acid sequences and construct the phylogenetic tree. The amino-acid sequences were downloaded from GenBank under the accession numbers cited in Table 1. The branch lengths are drawn to scale and are proportional to the number of different amino-acid residues. The scale bars of 0.05 and 0.005 represent 5% and 0.5% amino-acid difference, respectively.

CTX-M-3 cluster includes 42 members, sharing 97.6–99.7% identity in amino-acid sequences. The other clusters are as follows: CTX-M-14 cluster, 38 members, 97.3–99.7% identity; CTX-M-2 cluster, 16 members, 95.2–99.7% identity; CTX-M-25 cluster, 7 members, 98.6–99.7% identity; CTX-M-8 cluster, 3 members, 97.9–99.7% identity; CTX-M-64 cluster, 2 members, 95.9% identity. There is only one member in CTX-M-45 cluster. Among CTX-M variants, CTX-M-4 and CTX-M-45 are most divergent with 91 amino-acid substitutions.

Variations of amino-acid sequences

Based on the central positions in phylogenetic tree (Figure 1), CTX-M-2, -3, -8, -14, -25, -45 and -64 are chosen as the representative enzymes in each cluster. The amino-acid sequences of the seven enzymes are aligned, and numbered according to the standard numbering scheme for the class A serine β-lactamases, giving the active site serine residue the Ambler number 70 (Ambler et al., 1991) (Figure 2). The sequences of CTX-M variants are then compared with their representative in each cluster (Table 2). In the CTX-M-3 cluster, for example, a single amino-acid is substituted between CTX-M-3 and CTX-M-15, -22, -42, -54, -62, -66, -72 or -80, while 5 amino-acids are substituted between CTX-M-3 and CTX-M-58.

Figure 2. .

Comparison of amino-acid sequences of seven representative enzymes in the CTX-M family. Amino-acids are numbered according to the standard numbering scheme for the class A serine β-lactamases, giving the active site serine residue the Ambler number 70. Dots indicate identical amino-acids compared to CTX-M-2. Deletion mutations are expressed with short lines. The underlined amino-acids, ⁷⁰SXXK⁷³, ¹⁰⁷P, 130SDN¹³², ¹⁴³GG¹⁴⁴, ¹⁶⁶E and ²³⁴KXG²³⁶, represent the conserved residues in typical class A serine β-lactamases.

Table 2. .

Amino acid substitutions of CTX-M variants compared to their representative enzymes.

CTX-M	Amino acid substitution	CTX-M	Amino acid substitution
Cluster 2	vs. CTX-M-2	Cluster 8	vs. CTX-M-8
CTX-M-4	L48Q, R61V, K98R, K99A, A125G, T171S, L225M, V230G	CTX-M-40	K89N, T109A, N158D, N192H
CTX-M-5	A26T, V230G, E253A, I278V	CTX-M-63	K89N, T109A, N158D, N192H, S274N
CTX-M-6	R61L, K99A, A125G, T171S, S228C, I278V	Cluster 14	vs. CTX-M-14
CTX-M-7	R61V, K98R, K99A, E121Q, A125G, T171S, V230G, I278V	CTX-M-9	V231A
CTX-M-20	I278F	CTX-M-13	V2M, A52K, A154E
CTX-M-31	T159S	CTX-M-16	V231A, D240G
CTX-M-35	P167S	CTX-M-17	E288K
CTX-M-43	D240G, S274R	CTX-M-19	P167S
CTX-M-44	S274R	CTX-M-21	A9G, A10G, C12G, L22F, V29G
CTX-M-56	S274N	CTX-M-24	S274R
CTX-M-59	H89L	CTX-M-27	D240G
CTX-M-74	P167T	CTX-M-38	S220R
Cluster 2	vs. CTX-M-2	Cluster 14	vs. CTX-M-14
CTX-M-75	P14S	CTX-M-46	S27N, A47P
CTX-M-92	A205T	CTX-M-47	G42R
CTX-M-97	R3G	CTX-M-48	S27N
Cluster 3	vs. CTX-M-3	CTX-M-49	G42R, A47P
CTX-M-1	A77V, N114D, A140S, D288N	CTX-M-50	A47P
CTX-M-10	A27V, R38Q	CTX-M-51	A77V, V231A
CTX-M-11	E35G, L119P, D277H, deletion of 282AAKIVTDGL290	CTX-M-65	A77V, S274R
CTX-M-12	T12A, N89S, V278I	CTX-M-67	N106S
CTX-M-15	D240G	CTX-M-81	K82E, K98Q, N132H
CTX-M-22	D288N	CTX-M-83	Q56H
CTX-M-23	A77V, P167T, D288N	CTX-M-84	T209A
CTX-M-28	D240G, D288N	CTX-M-85	L119P
CTX-M-29	T12A, N114D, D240G, D288N	CTX-M-86	I108F
CTX-M-30	T12A, N114D	CTX-M-87	A77V, P167L
CTX-M-32	A77V, N114D, A140S, D240G, D288N	CTX-M-90	A77V
CTX-M-33	N106S, D240G	CTX-M-93	L169Q, D240G
CTX-M-34	A27V, R38Q, G238C	CTX-M-98	A77V, D240G
CTX-M-36	N114D, A140S, D288N	CTX-M-99	P167S, S274R
CTX-M-37	Y23H, R38Q, N114D	CTX-M-102	A205E, D240G
CTX-M-42	P167T	CTX-M-104	S274N
CTX-M-52	A77V, P167S	CTX-M-105	A77V, A205E, D240G
CTX-M-53	A27V, R38Q, A77V, D240G, T263I	CTX-M-106	K234R, R276H, deletion of 290L
CTX-M-54	P167Q	CTX-M-110	K111E, insertion of N before 290L
CTX-M-55	A77V, D240G	CTX-M-111	P145Q
CTX-M-58	A77V, N114D, A140S, P167T, D288N	CTX-M-112	S123G
CTX-M-60	T12A, N89S, V278I, A77V	CTX-M-113	Q83R
CTX-M-61	A77V, N114D, A140S	CTX-M-121	A109T, D240G
CTX-M-62	P167S	CTX-M-122	A154S, S274R
CTX-M-66	S19N	Cluster 25	vs. CTX-M-25
CTX-M-68	Y23H, A27V, E158D	CTX-M-26	V77A, Q222R, G240D
CTX-M-69	A77V, D240G, K271N, D288N	CTX-M-39	V77A, G240D
CTX-M-71	G238C, D240G	CTX-M-41	V77A, I103V, S123I
CTX-M-72	R164G	CTX-M-89	G240D
CTX-M-79	A77V, D240G, D288N	CTX-M-91	A189S, G240D
CTX-M-80	A27V	CTX-M-94	V77A, F119L
CTX-M-82	A67P, D240G	Cluster 64	vs. CTX-M-64
CTX-M-88CTX-M-96	D240G, R276HT12A, N89S, D240G, V278I	CTX-M-123	P67A, Q83K, T86S, Q87E, K88P, Q89N, P94R, P99K, A100S, T118S, A227T, V230T
CTX-M-101	S123I, D240G		T118S, A227T, V230T
CTX-M-107	K234R, D240G, deletion of 288DGL290
CTX-M-108	V95A, D240G, deletion of 288DGL290
CTX-M-109	Q56R, D240G, D288K, deletion of 289GL290
CTX-M-114	V74A, A77V, D240G
CTX-M-116	A77V, D288N
CTX-M-117	P174Q, D240G

Origin of CTX-M family

In the family Enterobacteriaceae, the genus Kluyvera is a relatively new member, which has been isolated from various clinical specimens and regarded as a potentially virulent pathogen (Sarria et al., 2001). Some Kluyvera spp. harbor chromosome-encoded intrinsic genes of cefotaximases which are closely associated with CTX-Ms (Decousser, et al., 2001; Humeniuk et al., 2002; Rodríguez et al., 2004). Generally, Kluyvera spp. are susceptible to cefotaxime in despite of the presence of naturally occurring cefotaximases. However, the recombinant clones of E. coli with Kluyvera-derived cefotaximase genes exhibited a significant increase in resistance to cefotaxime (Decousser et al., 2001; Humeniuk et al., 2002; Rodríguez et al., 2004), suggesting that a proper genetic platform is necessary for the gene expression. The chromosome-encoded cefotaximases identified in Kluyvera spp. include KLUA, KLUG, KLUY, KLUC, c-CTX-M-2, c-CTX-M-3, c-CTX-M-37, c-CTX-M-76, c-CTX-M-77, c-CTX-M-78 and c-CTX-M-95. All of them comprise 291 amino-acid residues. An aspartate aminotransferase-encoding gene is found commonly upstream of these chromosomal bla genes, which is replaced by ISEcp1 or ISCR1 in the plasmid-harbored bla _CTX-M genes (see the details under next section).

KLUA-1 to -5 and -8 to -12 (GenBank accession no. AJ272538, AJ251722, AJ427461, AJ427462, AJ427463, AJ427465, AJ427466, AJ427467, AJ427468, AJ427469) are a group of chromosomal cefotaximases identified in K. ascorbata, with minor variations (<5%) in their amino-acid sequences (Humeniuk et al., 2002). KLUA-2 shares 100% identity with plasmid-mediated CTX-M-5. CTX-M-2 and CTX-M-3 originally identified on plasmids were also found on the chromosomes of K. ascorbata (Rodríguez et al., 2004; Lartigue et al., 2006). The immediate upstream- and downstream-sequences of bla _KLUA-1 and plasmid-mediated bla genes in CTX-M-2 cluster (bla _{CTX-M-2, -4, -5, -6, -7,-44}) share 85 to 100% identities (Di Conza et al., 2002; Humeniuk et al., 2002). The architectures of the flanking regions corresponding to c-CTX-M-3 and plasmid-mediated CTX-M-3 are identical, including a 128 bp immediate upstream region and the first 373 bp of the downstream region of the bla gene (Rodríguez et al., 2004). The c-CTX-M-76, -77 and -95 (AM982520, AM982521, FN813245) identified in K. ascorbata also share high identities with the enzymes in CTX-M-2 cluster.

KLUY-1 to -4 (AY623932, AY623935, AY623934, AY623933) are a group of chromosomal cefotaximases identified in K. Georgiana (Olson et al., 2005). They share high homology with the enzymes in CTX-M-14 cluster. Typically, KLUY-1 exhibits 100% amino-acid identity with CTX-M-14. The upstream- and downstream-sequences of bla _KLUY and bla _{CTX-M-9, -13, -14} also share consistent identity. A 42 bp upstream region of bla _CTX-M-14 is identical to the corresponding region of bla _KLUY genes. A 347 bp downstream region of bla _CTX-M-9 and bla _CTX-M-13 shares 95.7–98.6% identities with the corresponding region of bla _KLUY genes (Olson et al., 2005).

KLUG-1 (AF501233) and c-CTX-M-78 (AM982522) are the chromosomal cefotaximases identified in K. Georgiana. KLUG-1 shares 99% amino-acid identity with the plasmid-mediated CTX-M-8 (Poirel et al., 2002b). The c-CTX-M-78 possesses high homology with the known members of CTX-M-25 cluster, sharing 95.2–96.2% identities (Rodríguez et al., 2010).

CTX-M-37 was also found on the chromosome of K. cryocrescens (FN813246), suggesting the c-CTX-M-37 as an origin of CTX-M-3 cluster. KLUC-1 (AY026417) and KLUC-2 (EF057432), with a single amino-acid substitution, are two chromosome-encoded cefotaximases identified in K. cryocrescens (Decousser et al., 2001). KLUC-1 and -2 are diverse from the known CTX-Ms, sharing only 87.6% identity with CTX-M-3. Notably, KLUC-2 was also identified on a plasmid carried by a clinical isolate of E. cloacae, indicating the transfer of bla _KLUC from chromosome to the plasmid (Petrella et al., 2008). We would like to suggest the plasmid-mediated KLUC-2 as a novel cluster or member of CTX-M family.

CTX-M-64 shows a chimeric sequence of both CTX-M-14 (central portion) and CTX-M-15 (N- and C-terminal moieties), suggesting an origination owing to homologous recombination between the bla _{CTX-M-14 and -15} genes (Nagano et al., 2009).

Taken together, the origins of the acquired CTX-Ms in various clusters can be traced back to the intrinsic cefotaximase genes harbored by Kluyvera spp., of which the CTX-M-2 cluster appears to be derived from K. ascorbata, the CTX-M-14, CTX-M-8 and CTX-M-25 clusters from K. georgiana, while the CTX-M-3 cluster from both K. ascorbata and K. cryocrescens (Figure 3).

Figure 3. .

Identification of intrinsic cefotaximase genes in Kluyvera spp. as the original sources of acquired CTX-Ms based on their amino-acid identities and the homologies of neighboring sequences of the associated genes. c-CTX-M, CTX-M identified on chromosome of Kluyvera spp.; p-KLUC-2, KLUC-2 identified on plasmid in a clinical isolate of Enterobacter cloacae.

Genetic platforms of CTX-M enzymes

ISEcp1

Insertion sequences (ISs) are the smallest transposable elements (<2.5 kb) capable of independent transposition in an organism, thereby causing insertion mutations and genome rearrangements (Mahillon and Chandler, 1998). ISs play three basic roles in bacteria: encoding a transposase which makes a genetic element mobile; providing promoters to activate silent genes or enhance expression of downstream determinants; moving IS-mobilized genes among integrons, transposons, plasmids and chromosomes, thereby greatly increasing the opportunity a resistance determinant becomes transferable.

Of the genetic platforms associated with CTX-Ms, ISEcp1 is one of the most important elements (Table 3). ISEcp1 was first identified on the plasmid pST01 in E. coli strain 79 (AJ242809), hence its name (Stapleton, 1999). ISEcp1 is composed of an orf encoding a transposase with 420 amino-acids and two imperfect and inverted repeats. ISEcp1 can mobilize the downstream-located bla _CTX-M gene and provide a promoter for its expression (Karim et al., 2001; Cao et al., 2002; Poirel et al., 2003, 2005; Dhanji et al., 2011b).

Table 3. .

Genetic platforms of CTX-M enzymes.

CTX-M	Genetic platform	Bacterial host	Reference/GenBank accession no.
CTX-M-1	ISEcp1–bla CTX-M-1 –orf477	E. coli	Eckert et al., 2006
	ISEcp1Δ----IS26–ISEcp1Δ–bla CTX-M-1	K. pneumoniae	Diestra et al., 2009
	IS26–ISEcp1Δ–bla CTX-M-1 – orf477Δ	E. coli	Cullik et al., 2010
	intI1–dfrA17–aadA5–qacEΔ1–sul1–ISCR1–bla CTX-M-1 –orf3–IS3000–qacEΔ1–sul1-like–orf5	E. coli	Su et al., 2008
CTX-M-2	intI1–aacA4–bla OXA-2 –orfD–qacEΔ1–sul1–ISCR1–bla CTX-M-2 –orf3Δ–qacEΔ1–sul1	P. mirabilis	Arduino et al., 2002
	intI1–aacA4–bla OXA-2 –orfD–qacEΔ1–sul1–ISCR1–bla CTX-M-2 –orf3Δ–qacEΔ1–sul1–orf5	V. cholera	Soler Bistué et al., 2006
	intI1–aacA4–bla OXA-2 –orfD–qacEΔ1–sul1–ISCR1–dfrA10–bla CTX-M-2 –orf3Δ–qacEΔ1–sul1–orf5–tniBΔ–IS1326	S. enterica	AJ311891
	intI1–dfrA12–orfF–aadA2–qacEΔ1–sul1–ISCR1–bla CTX-M-2 –orf3Δ–qacEΔ1–sul1–orf5–IS1326	K. pneumoniae	EU780013
	intI1–estX–aadA1–qacEΔ1–sul1–ISCR1–bla CTX-M-2 –orf3Δ–qacEΔ1–sul1–orf5–IS1326	E. coli	Valverde et al., 2006
	intI1–aac(6′)-Iq–aadA1–qacEΔ1–sul1–ISCR1–bla CTX-M-2 –orf3Δ–qacEΔ1	K. pneumoniae	EU622037
	intI1–aadA1–qacEΔ1–sul1–ISCR1–bla CTX-M-2 –orf3Δ–qacEΔ1	K. pneumoniae	EU622040
	intI1–aadA2–qacEΔ1–sul1–ISCR1–bla CTX-M-2 –orf3Δ–qacEΔ1	K. pneumoniae	EU622038
	intI1–dhfrh1–aadA2–qacEΔ1–sul1–ISCR1–bla CTX-M-2 –orf3Δ–qacEΔ1–sul1	E. coli	Eckert et al., 2006
	intI1–dfrA1–aadA1–qacEΔ1–sul1–ISCR1–bla CTX-M-2 –orf3Δ–qacEΔ1–sul1	S. enterica	EF592570
	intI1–dfrA12–orfF–aadA2–qacEΔ1–sul1–ISCR1–bla CTX-M-2 –orf3Δ–qacEΔ1–sul1	S. enterica	EF592571
	intI1–dfrA21–qacEΔ1–sul1–ISCR1–bla CTX-M-2 –orf3Δ–qacEΔ1	K. pneumoniae	EU622039
	intI1–dfr22–qacEΔ1–sul1–ISCR1–bla CTX-M-2 –orf3Δ–qacEΔ1	K. pneumoniae	EU622041
	intI1–orf1–cat–orf2–aadA1–qacEΔ1–sul1–ISCR1–bla CTX-M-2 –orf3Δ–qacEΔ1–sul1	P. mirabilis	Song et al., 2011
	ISEcp1–bla CTX-M-2	P. mirabilis	Harada et al., 2012
CTX-M-3	ISEcp1–bla CTX-M-3 –orf477	K. pneumoniae	Eckert et al., 2006
	ISEcp1–bla CTX-M-3 –orf477–mucA	K. pneumoniae	Eckert et al., 2006
	ISEcp1-like–bla CTX-M-3 –orf477-like	P. mirabilis	Wu et al., 2008
	ISEcp1–bla CTX-M-3	E. coli	Diestra et al., 2009
	ISEcp1–IS1–bla CTX-M-3 –orf477–mucA	K. pneumoniae	Eckert et al., 2006
	ISEcp1–bla CTX-M-3 –orf–mucA	C. freundii	Lartigue et al., 2004
	IS26–ISEcp1Δ–bla CTX-M-3	E. coli	Diestra et al., 2009
	IS26–ISEcp1–bla CTX-M-3 –orf477–mucA	P. mirabilis	Eckert et al., 2006
CTX-M-9	intI1–aadB–qacEΔ1–sul1–ISCR1–bla CTX-M-9 –orf3-like–IS3000	E. cloacae	DQ108615
	intI1–dhfr12–orfX–aadA8–qacEΔ1–sul1–ISCR1–bla CTX-M-9 –orf3–orf339Δ	E. coli	Eckert et al., 2006
	intI1–dfrA16–aadA2–qacEΔ1–sul1–ISCR1–bla CTX-M-9 –orf3-like–IS3000–qacEΔ1–sul1	E. coli	Sabaté et al., 2002
	ISCR1–bla CTX-M-9	E. coli	Diestra et al., 2009
	ISEcp1–bla CTX-M-9	C. freundii	Minarini et al. 2009
CTX-M-10	Tn1000-like–orf2–orf3–orf4–DNA-invertase-gene–bla CTX-M-10 –orf7–orf8–IS4321–orf10–orf11–IS5	K. pneumoniae	Oliver et al., 2005
	ISEcp1–bla CTX-M-10 –orf–Tn5396	E. coli	Lartigue et al., 2004
CTX-M-12	ISEcp1–bla CTX-M-12	P. mirabilis	Song et al., 2011
CTX-M-13	ISEcp1B–bla CTX-M-13	E. coli	DQ058147
CTX-M-14	ISEcp1–bla CTX-M-14 –IS903	E. coli	Lartigue et al., 2004
	ISEcp1-like–bla CTX-M-14 –IS903-like	P. mirabilis	Wu et al., 2008
	ISEcp1–IS10–bla CTX-M-14 –IS903	E. coli	Eckert et al., 2006
	ISEcp1–IS10–bla CTX-M-14 –IS903D	E. coli	EU136400
	IS26–ISEcp1–bla CTX-M-14	K. pneumoniae	Eckert et al., 2006
	IS26–ISEcp1–bla CTX-M-14 –IS903	K. pneumoniae	GQ385317
	IS26–bla CTX-M-14 –IS903D	S. enterica	Izumiya et al., 2005
	ISEcp1B–bla CTX-M-14	E. coli	Billard-Pomares et al., 2011
	intI1–dfrA12–orfF–aadA2–qacEΔ1–sul1–ISCR1–bla CTX-M-14 –IS903-like	E. coli	Bae et al., 2007
	intI1–dfrA12–orfF–aadA2–qacEΔ1–sul1–orf5–IS6100–ISCR1–ISEcp1Δ–bla CTX-M-14 –IS903D	E. coli	Bae et al., 2008
CTX-M-15	ISEcp1–bla CTX-M-15	A. hydrophila	Gómez-Garcés et al., 2011
	ISEcp1–bla CTX-M-15 –orf477	E. coli	Eckert et al., 2006
	ISEcp1–bla CTX-M-15 –orf477Δ–Tn3	A. baumannii	JN788267
	Tn3Δ–ISEcp1–bla CTX-M-15 –orf–Tn3Δ	E. coli	Lartigue et al., 2004
	IS26–ISEcp1–bla CTX-M-15 –orf477	E. coli	Eckert et al., 2006
	IS26–ISEcp1–bla CTX-M-15 –orf477Δ	S. enterica	Fabre et al., 2009
	bla TEM-1 –tnpR–tnpA–ISEcp1–bla CTX-M-15 –orf477	E. coli	Eckert et al., 2006
CTX-M-16	ISEcp1–bla CTX-M-16 –IS903	E. coli	Brasme et al., 2007
	ISEcp1–bla CTX-M-16 –orf3–orf339–orf477	E. coli	AM910790
CTX-M-17	ISEcp1-like–bla CTX-M-17 –IS903C	K. pneumoniae	Cao et al., 2002
CTX-M-19	intI1-like–aacA4–cmlA1–qacEΔ1–sul1–Tn1721–ISEcp1B–bla CTX-M-19 –IS903D	K. pneumoniae	Poirel et al., 2003
CTX-M-20	ISEcp1–bla CTX-M-20	P. mirabilis	AJ416344
CTX-M-21	ISEcp1–bla CTX-M-21	E. coli	AJ416346
CTX-M-22	ISEcp1Δ–IS26–bla CTX-M-22 –orf477–ISEcp1Δ	S. liquefaciens	HM470254
CTX-M-24	ISEcp1–bla CTX-M-24 –IS903	E. coli	Eckert et al., 2006
	ISEcp1-like–bla CTX-M-24 –IS903-like	P. mirabilis	Wu et al., 2008
CTX-M-25	intI1–aacA4–bla OXA-2 –ISEcp1–bla CTX-M-25 –qacEΔ1–sul1	P. mirabilis	Navon-Venezia et al. 2008
	ISEcp1Δ–IS50-A–ISEcp1Δ–bla CTX-M-25 –orfX	E. coli	Munday et al. 2004
CTX-M-26	intI1–dhfr7–ISEcp1–bla CTX-M-26 –qacEΔ1–sul1	K. pneumoniae	Navon-Venezia et al. 2008
	ISEcp1–bla CTX-M-26 –orfX	K. pneumoniae	Munday et al. 2004
CTX-M-27	ISEcp1–bla CTX-M-27	S. enterica	Bouallègue-Godet et al., 2005
	ISEcp1–bla CTX-M-27 –IS903	E. coli	Sun et al., 2010
CTX-M-32	ISEcp1Δ–IS5–IS1A–ISEcp1Δ–bla CTX-M-32 –orf477	E. coli	Fernández et al., 2007
	ISEcp1Δ–IS5–ISEcp1Δ–bla CTX-M-32	E. coli	Diestra et al., 2009
CTX-M-39	intI1–dhfr7–ISEcp1–bla CTX-M-39 –qacEΔ1–sul1	E. coli	Navon-Venezia et al. 2008
	intI1–aadA1–ISEcp1–bla CTX-M-39 –qacEΔ1–sul1	E. coli	Navon-Venezia et al. 2008
CTX-M-40	ISEcp1-like–bla CTX-M-40	E. coli	Hopkins et al., 2006
CTX-M-42	ISEcp1–bla CTX-M-42	E. coli	DQ061159
CTX-M-53	ISSen2---bla CTX-M-53 –orf477Δ–IS26	S. enterica	Doublet et al., 2009
CTX-M-54	ISEcp1–bla CTX-M-54 –IS903-like	K. pneumoniae	Bae et al., 2006a
CTX-M-55	ISEcp1–bla CTX-M-55 –orf477	E. coli	Sun et al., 2010
	ISEcp1Δ–IS1294–bla CTX-M-55 –orf477	E. coli	JN977127
CTX-M-59	intI1–dfr15b–cmlA4-like–aadA2–qacEΔ1–sul1–ISCR1–bla CTX-M-59 –orf3Δ–qacEΔ1	K. pneumoniae	EU622856
CTX-M-62	ISEcp1–bla CTX-M-62 –ISEcp1Δ1/Δ2	K. pneumoniae	Zong et al., 2010
CTX-M-64	ISEcp1–bla CTX-M-64 –orf477	S. sonnei	Nagano et al., 2009
CTX-M-65	ISEcp1–bla CTX-M-65 –IS903	E. coli	Sun et al., 2010
CTX-M-66	ISEcp1-like–bla CTX-M-66 –orf477-like	P. mirabilis	Wu et al., 2008
CTX-M-74	ISCR1–bla CTX-M-74 –orf3Δ–qacEΔ1–sul1	E. cloacae	Minarini et al. 2009
CTX-M-75	ISCR1–bla CTX-M-75 –orf3Δ–qacEΔ1–sul1	P. stuartii	Minarini et al. 2009
CTX-M-79	ISEcp1–bla CTX-M-79	E. coli	FJ169498
CTX-M-82	ISEcp1–bla CTX-M-82	E. coli	GU477621
CTX-M-89	ISEcp1-like–bla CTX-M-89 –orf477-like	E. cloacae	FJ966096
CTX-M-90	ISEcp1–bla CTX-M-90 –IS903-like	P. mirabilis	Song et al., 2011
	ISEcp1–bla CTX-M-90	P. mirabilis	Song et al., 2011
CTX-M-93	ISEcp1–bla CTX-M-93 –IS903	E. coli	Djamdjian et al., 2011
CTX-M-98	ISEcp1–bla CTX-M-98 –IS903	E. coli	HM755448
CTX-M-101	ISEcp1–bla CTX-M-101	E. coli	HQ398214
CTX-M-102	ISEcp1–bla CTX-M-102 –IS903	E. coli	HQ398215
CTX-M-104	ISEcp1–bla CTX-M-104 –IS903	E. coli	HQ833652
CTX-M-105	ISEcp1–bla CTX-M-105 –IS903	E. coli	HQ833651
CTX-M-116	ISEcp1–bla CTX-M-116	P. mirabilis	JF966749
CTX-M-121	ISEcp1–bla CTX-M-121 –IS903	E. coli	JN790862
CTX-M-122	ISEcp1–bla CTX-M-122 –IS903	E. coli	JN790863
CTX-M-123	ISEcp1–bla CTX-M-123	E. coli	JN790864

Co-existence of ISEcp1 and bla _CTX-M at a high rate in CTX-M-producing E. coli isolates is well documented. ISEcp1 was identified upstream of bla _CTX-M genes in 86.9% of the isolates (93/107) recovered from health and sick pets in China, and no major clonal relatedness was observed (Sun et al., 2010). Similarly, ISEcp1 was identified upstream of bla _CTX-M-14 in 91.4% of the clinical isolates (32/35) in Korea (Kim et al., 2011), and upstream of bla _CTX-M-1 in 69.2% of the isolates (9/13) from food samples in Tunisia (Ben Slama et al., 2010). In addition, variations of ISEcp1 were also observed. ISEcp1B, originally identified upstream of a bla _CTX-M-19 gene cassette (AF458080), differs from ISEcp1 by three nucleotide substitutions (Poirel, et al., 2003). Of the 174 ISEcp1-like and bla _CTX-M-15 complex from E. coli isolates, the intact ISEcp1, truncated ISEcp1 with various lengths and a 24 bp remnant of ISEcp1 accounted for 62%, 33.3% and 4.6%, respectively (Dhanji et al., 2011b). Notably, ISEcp1 was also detected upstream of chromosomal bla _CTX-M-2 genes in 4 P. mirabilis isolates in Japan (Harada et al., 2012), highlighting the ISEcp1-mediated movement of bla _CTX-M genes between plasmids and chromosomes.

ISEcp1-bla _CTX-M-IS903 (Figure 4A) and ISEcp1-bla _CTX-M-orf477 (Figure 4B) are two major genetic platforms. In some cases, ISEcp1-mobilized bla _CTX-M is inserted in a class 1 integron (Figure 4C). IS903 (V00359) encodes a transposase with 307 amino-acids and was originally found on a kanamycin resistance transposon Tn903 (Oka et al., 1981). IS903 and IS903-like elements, such as IS903C and IS903D, are located downstream of bla _CTX-M genes (Table 3), including bla _CTX-M-14-like genes (blaCTX-M-14, -16_{, -17, -19, -24, -27, -65, -90, -93, -98, -102, -104, -105, -121, -122}) and bla _CTX-M-3-like gene (bla _CTX-M-54). orf477 encodes a protein of 158 amino-acids with unknown function and the orf477 and orf477-like elements were found downstream of plasmid-harbored bla _CTX-M-3-like genes (bla _{CTX-M-1, -3, -15, -22, -32, -53, -55, -66}), bla _CTX-M-89 and bla _CTX-M-64 (Table 3). The orf477 was also identified downstream of the chromosomal bla _CTX-M-3 in K. ascorbata, of the chromosomal bla _{KLUY-1, -2, -3, -4} in K. georgiana, and of the chromosomal bla _CTX-M-37 (FN813246) in K. cryocrescens (Rodriguez et al., 2004; Olson et al., 2005), footnoting the ISEcp1-mediated transfer of bla _CTX-M genes together with the orf477 from the chromosomes of Kluyvera spp. to plasmids.

Figure 4. .

Typical genetic platforms of CTX-M enzymes. A & B: the bla _CTX-M gene cassettes bracketed upstream by ISEcp1/ISEcp1-like and downstream by IS903/IS903-like (A) or orf477/orf477-like (B); C: bla _CTX-M genes associated with class 1 integron-ISEcp1; D & E: bla _CTX-M genes associated with class 1 integron-ISCR1 complex. CS, conserved segment; intI, integrase gene; qacE▵1, quaternary ammonium resistance gene; sul1, sulphonamide resistance gene; 3′-CS2, the second copy of 3′-conserved segment.

Class 1 integron-ISCR1 complex

Integrons are defined as mobile DNA elements that can capture genes by site-specific recombination (Stokes and Hall, 1989). A typical class 1 integron consists of a 5′ conserved segment (5′-CS), a variable region and a 3′ conserved segment (3′-CS). The 5′-CS consists of the gene encoding integrase (intI1), the site adjacent to intI1 for the insertion of captured genes (attI), and a promoter region (Pc). The 3′-CS often consists of a partially deleted qac gene (qacEΔ1) fused to a sul1 gene, and confers resistance to antiseptics and sulfonamide, respectively. Class 1 integrons play a critical role in acquiring and spreading metallo-β-lactamases (Mazel, 2006; Zhao and Hu, 2011a,b). The role of integrons in CTX-M gene acquisition and dissemination, however, is still unclear. The physical link of some bla _CTX-M genes with class 1 integron-ISEcp1 complex (Figure 4C) and class 1 integron-ISCR1 complex (Figure 4D, 4E) indicates a possible association among the three genetic elements.

ISCR1 is another important element in the genetic platforms associated with the mobilization and dissemination of CTX-M genes (Rodriguez-Martinez et al. 2006; Toleman et al., 2006). Common region 1 (CR1) was first found as element associated with but distinct from class 1 integrons (Stokes et al., 1993). The CR1 element was renamed ISCR1 because it possesses the key motifs of IS91-like element and accommodates orf513 gene which codes a putative transposase of 513 amino-acids (Toleman et al., 2006). ISCR1 is particularly important for CTX-M-2 and CTX-M-9 genes (Table 3). In most instance, the ISCR1-bla _CTX-M-2 is located between a typical class 1 integron and a fuse type of orf3Δ and qacEΔ1/sul1 (Table 3, Figure 4D). Notably, the genes harbored by class 1 integrons in their variable regions, such as bla _OXA-2, aacA4, cmlA and dfr, are also associated with bacterial resistance to β-lactam, aminoglycoside, chloramphenicol and trimethoprim, respectively.

Molecular epidemiological study performed in Argentine during 1993–2000 showed that class 1 integron-ISCR1 complex was adjacent to bla _CTX-M-2 in all the CTX-M-2 producers (n = 35), including Acinetobacter spp., E. cloacae, E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa, S. enterica and S. marcescens, while only 1.5% of the bla _CTX-M-2-negative isolates (n = 65) harbored ISCR1 (Arduino et al., 2003). These data strongly implicate the association of ISCR1 with the emergence and dissemination of bla _CTX-M-2 gene. In addition, ISCR1 is also related to bla _{CTX-M-59, -74, -75} (members of CTX-M-2 cluster) and bla _{CTX-M-1, -9, -14} (Table 3).

Other IS and phage-related sequences

Besides ISEcp1, IS903 and ISCR1 described above, IS1, IS5, IS10, IS26, IS50A, IS1294, IS1326, IS3000, IS4321 and IS6100 were also found to be adjacent to bla _CTX-M genes (Table 3). In some cases, several IS elements co-existed in a gene complex, for example, intI1-dfrA12-orfF-aadA2-qacEΔ1-sul1-ISCR1-IS6100-ISCR1-ISEcp1Δ-bla _CTX-M-14-IS903D (Bae et al., 2008). Such heterogeneity may be explained by a continuously recombinatorial exchange of gene cassettes, denoting the sophisticated genetic rearrangement strategies that organisms acquire and dispense resistance genes.

A 12.2-kb DNA fragment containing bla _CTX-M-10 gene in plasmid pRYCE21 was cloned from K. pneumoniae, and further detected in other bacterial species including E. coli, E. cloacae and E. gergoviae. Analysis of the sequence showed a phage-related 3.5-kb element immediately upstream of the bla _CTX-M-10 gene cassettes. This phage-related fragment corresponds to four orfs, of which orf2, orf3 and orf4 display homology to the genes of conserved phage tail proteins (Oliver et al., 2005). Although there is a limited report on phage-related CTX-M genes, this finding indicates that phages may also function as a tool for bla _CTX-M-associated genetic elements to become transferable.

Plasmids

The movement of IS-mobilized genes between chromosomes and plasmids greatly increase the opportunity a resistance determinant becomes transferable. Particularly, conjugative plasmid is one of the most important mechanisms for intra-species, inter-species and inter-genus gene transfers.

Plasmids are usually classified on their incompatibility (Inc), defined as the inability of two plasmids to be propagated stably in the same bacterial strain; thus, only compatible plasmids can be rescued in transconjugants (Novick et al., 1976). At least 29 Inc groups have been recognized among plasmids of enteric bacteria, including IncFI, IncFII, IncFIII, IncFIV, IncFV, IncFVI, IncI1, IncI2, IncIy, IncHI1, IncHI2, IncHI3, IncA/C, IncB, IncD, IncJ, IncK, IncL/M, IncN, IncO, IncP, IncS, IncT, IncU, IncV, IncW, IncX, IncY and com9 (Novick et al., 1976; Couturier et al., 1988). The IncFII, IncA/C, IncL/M, and IncI1 plasmids show the highest occurrence among the typed resistance plasmids (Carattoli, 2009).

Molecular epidemiological studies have revealed a close and significant linkage of bla _CTX-M genes to plasmids, mainly belonged to IncF, IncI, IncN, IncHI2, IncL/M and IncK groups (Table 4). The IncF group (FIA, FIB and FII) is the most prevalent in transmitting bla _CTX-M-15 genes_, while IncF, IncK and IncI1 are closely related to the widespread of bla _CTX-M-14 genes. In addition, the bla _CTX-M-1 gene is dominantly harbored by IncN and IncI1, bla _CTX-M-3 gene by IncL/M and IncI1, and bla _CTX-M-9 gene by IncHI2.

Table 4. .

Plasmids associated with the spread of CTX-M genes.

CTX-M gene (No. of isolates)	Inc group (No. of isolates)	Rate*	Resource	Reference
bla CTX-M-1 (119)	N (119)	100%	E. coli from bovine on a dairy farm with high consumption of cephalosporins in Czech Republic, 2008	Dolejska et al., 2011
bla CTX-M-1 (10)	I1 (10)	100%	S. enterica from poultry and humans in France, 2003–08	Cloeckaert et al., 2010
bla CTX-M-3 (14)	L/M (13)	92.9%	Enterobacteriaceae from Bulgaria, Poland and France	Galimand et al., 2005
bla CTX-M-9 (41)	HI2 (24) P1-α (10) FIB (4) HI2, F1 (2) I1 (1)	58.5% 24.4% 9.8% 4.9% 2.4%	Enterobacteriaceae from a university hospital in Spain, 1996–03	Novais et al., 2006
bla CTX-M-14 (40)	K (27) I1 (11) HI2 (2)	67.5% 27.5% 5%	E. coli from patients and healthy volunteers in Spain, 2000–05	Valverde et al., 2009
bla CTX-M-14 (25)	F (8) I1 (5) F, I1 (3) N (1) Q (1)	32% 20% 12% 4% 4%	E. coli from 20 hospitals in 15 provinces in China, 2007–08	Cao et al., 2011
bla CTX-M-14 (23)	FII (13) I1-Iγ (4) FIB (2) FII, I1-Iγ (1) K (1)	56.5% 17.4% 8.7% 4.3% 4.3%	E. coli from outpatients in Hong Kong, 2002–04	Ho et al., 2011
bla CTX-M-15 (18)	FII (17) FI (1)	94.4% 5.6%	E. coli from a hospital in Turkey, 2002–04	Gonullu et al., 2008
bla CTX-M-15 (36)	FI (36)	100%	E. coli from a university hospital in Germany, 2006–07	Mshana et al., 2009
bla CTX-M-15 (55)	FIIA (41) A/C (3) FIIA, A/C (4)	74.5% 5.5% 7.3%	K. pneumoniae from patients in 9 Asian countries, 2008–09	Lee et al., 2011
bla CTX-M-1 (11) bla CTX-M-14 (15) bla CTX-M-15 (19)	N (8) I1 (3) F (9) K (2) F (12) I1 (1) L/M (1) N (1)	72.7% 27.3% 60% 13.3% 63.2% 5.3% 5.3% 5.3%	E. coli from different areas in France, 1997–02	Marcadé et al., 2009
bla CTX-M-1 (7) bla CTX-M-9 (14) bla CTX-M-14 (13) bla CTX-M-15 (4) bla CTX-M-32 (3)	N (5) FII (2) I1 (4) I1, P (3) HI2 (4) FIB (2) K (12) F (4) N (3)	71.4% 28.6% 28.6% 21.4% 28.6% 14.3% 92.3% 100% 100%	E. coli and K. pneumoniae from 11 hospitals in Spain, 2004	Diestra et al., 2009
bla CTX-M-2 (16) bla CTX-M-14 (8)	A/C (11) FVII (1) I1 (1) I1 (6)	68.8% 6.3% 6.3% 75%	E. coli from a survey among 3193 healthy children in Peru & Bolivia, 2005	Pallecchi et al., 2007
bla CTX-M-3 (49) bla CTX-M-15 (11)	I1 (36) FI (8) Y (3) N (2) FI (11)	73.5% 16.3% 6.1% 4.1% 100%	E. coli from faeces of residents in16 nursing homes in the UK, 2004–06	Dhanji et al., 2011a

^*Rate = (No. in the 2nd column/No. in the 1st column) × 100%.

Unlike the plasmids with broad host range, such as IncP, IncA/C and IncQ, IncF plasmids are limited by host range to the genera of Enterobacteriaceae (Toukdarian, 2004), footnoting the high prevalence and widespread of CTX-M genes in Enterobacteriaceae, but not in Acinetobacter and Pseudomonas.

Various resistance genes frequently co-exist on a plasmid, facilitating the dissemination of resistance determinants and the survival of bacteria under the pressure of various antibiotics. For example, plasmid pEK499 (a fusion of type FII and FIA replicons) identified in a UK variant of the internationally prevalent E. coli O25:H4-ST131 lineage is confirmed to harbor 10 resistance genes, conferring resistance to seven antibiotic classes, β-lactams (bla _CTX-M-15, bla _OXA-1, bla _TEM-1), aminoglycoside (aac6′-Ib-cr, aadA5), macrolides (mph(A)), chloramphenicol (catB4), tetracycline (tet(A)), trimethoprim (dfrA7) and sulfonamide (sul1) (Woodford et al., 2009).

Secondary chromosomal integration

Most of the bla _CTX-M genes are harbored by plasmids and the secondary chromosomal insertions of bla _CTX-M genes are also confirmed, particularly in P. mirabilis. Of 25 clinical isolates of CTX-M-producing P. mirabilis collected in Korea, 21 strains harbored bla _CTX-Ms on their chromosomes (Song et al., 2011). The genes of bla _{CTX-M-25 and-41} were also found on the chromosomes of P. mirabilis in Israel (Navon-Venezia et al., 2008).

In addition, chromosomal integration of bla _CTX-M-15 gene was reported in E. coli, K. pneumoniae and S. enterica (Coque et al., 2008; Coelho et al., 2010; Fabre et al., 2009). Chromosomal bla _CTX-M-9 was observed in one strain of 30 E. coli isolates collected in Barcelona during 1996–1999 (García et al., 2005).

Conclusion

Plasmid-mediated CTX-M enzymes are the most prevalent ESBLs, particularly in E. coli, K. pneumoniae and P. mirobilis. At least 109 members in CTX-M family are identified and can be divided into seven clusters based on their phylogeny. CTX-M-15 and CTX-M-14 are the most dominant variants in the family, followed by CTX-M-2, CTX-M-3 and CTX-M-1.

The CTX-M genes can be traced back to the chromosome-encoded cefotaximas genes in Kluyvera spp., strongly indicating that the plasmid-mediated CTX-M enzymes are originally from Kluyvera. Multiple genetic elements, especially ISEcp1 and ISCR1, are involved in the mobilization of bla _CTX-M genes from the chromosomes to plasmids. Conjugative plasmids are responsible for the transfer of the bla _CTX-M genes to new hosts, while the properties of plasmid incompatibility and host range are closely associated with the high prevalence and widespread of the CTX-M genes in Enterobacteriaceae, but not in Acinetobacter and Pseudomonas.