Diversity, Epidemiology, and Genetics of Class D β-Lactamases

Abstract

Class D β-lactamase-mediated resistance to β-lactams has been increasingly reported during the last decade. Those enzymes also known as oxacillinases or OXAs are widely distributed among Gram negatives. Genes encoding class D β-lactamases are known to be intrinsic in many Gram-negative rods, including Acinetobacter baumannii and Pseudomonas aeruginosa, but play a minor role in natural resistance phenotypes. The OXAs (ca. 150 variants reported so far) are characterized by an important genetic diversity and a great heterogeneity in terms of β-lactam hydrolysis spectrum. The acquired OXAs possess either a narrow spectrum or an expanded spectrum of hydrolysis, including carbapenems in several instances. Acquired class D β-lactamase genes are mostly associated to class 1 integron or to insertion sequences.

Class D β-lactamases, also known as oxacillinases or OXA-type β-lactamases (OXAs), are active-serine-site enzymes like Ambler class A and class C β-lactamases, differing from class A and C enzymes in amino acid structure, whereas class B β-lactamases are metalloenzymes with a Zn²⁺ ion(s) in the active site (4, 71, 78). Even though class D includes mostly enzymes with higher hydrolysis rates for cloxacillin and oxacillin than for benzylpenicillin (hence the name oxacillinases), not all class D β-lactamases have this characteristic. Most of the class D enzymes belong to group 2d of the Bush functional classification scheme for β-lactamases (23). Among the four β-lactamase molecular classes, class D β-lactamases are the most diverse enzymes (107). This diversity is observed at both the genetic and biochemical levels, with enzymes possessing either a narrow or expanded spectrum of hydrolysis. In addition, several class D β-lactamases have an expanded spectrum of activity resulting from point mutations.

Although many class D β-lactamase genes are embedded into class 1 integrons, recent reports indicated that other specific genetic structures, including insertion sequences and transposons, may be associated with class D β-lactamase genes. Numerous class D β-lactamase genes have been identified as a source of acquired resistance in gram-negative bacteria, but recent studies have shown that class D β-lactamases are also naturally produced in clinically significant pathogens and environmental species (107).

This review focuses on the diversity and substrate profiles of class D β-lactamases, their sources, and the genetics of acquisition of the corresponding genes. All the class D β-lactamases for which a sequence is available in the GenBank databases are listed in Table 1.

TABLE 1.

Features of oxacillinases

Namea	Alternate name	OXA group	Type	Original host	A or Nb	Associated mobile element		Gene GC content (%)	Isoelectric pointd		GenBank accession no.e	Referencef
						Transposon or insertion sequence	Integronc		Exptl	Theoretical
OXA-1	OXA-30		Narrow spectrum	E. coli	A	Tn2603	+	34.4	7.4	7.7	J02967	113
OXA-2		OXA-2	Narrow spectrum	S. Typhimurium	A		+	50	7.7	9.1	X07260	97
OXA-3		OXA-2	Narrow spectrum	K. pneumoniae	A	Tn1411	+	50	7.1	8.1	L07945	142
OXA-4	OXA-35		Narrow spectrum	E. coli	A	Tn1409	+		7.5		AY162283	142
OXA-5			Narrow spectrum	P. aeruginosa	A	Tn1406	+	40.2	7.6	8.4	X58272	32
OXA-6			Narrow spectrum	P. aeruginosa	A				7.7			UP
OXA-7		OXA-10	Narrow spectrum	E. coli	A		+	40.6	7.7	9.3	X75562	145
OXA-8
OXA-9			Narrow spectrum	K. pneumoniae	A	Tn1331	+	49.5	6.9	7.1	M55547	155
LCR-1			Narrow spectrum	P. aeruginosa	A	Tn1412	−	52	6.5	7.1	X56809	32
OXA-10			Narrow spectrum	P. aeruginosa	A	Tn1404	+	42.1	6.1	7.0	U37105	70
OXA-11		OXA-10	ES-OXA	P. aeruginosa	A		+	42	6.4	6.3	Z22590	60
OXA-12			Narrow spectrum	A. jandaei	N		−	62.3	8.6	8.4	U10251	139
AmpS			Narrow spectrum	A. hydrophila	N		−	63	7.9	7.1	X80276	165
OXA-13		OXA-10	Narrow spectrum	P. aeruginosa	A		+	41.2	8.0	8.7	U59183	99
OXA-14		OXA-10	ES-OXA	P. aeruginosa	A		+	42.1	6.2	6.3	L38523	38
OXA-15		OXA-2	ES-OXA	P. aeruginosa	A		+	50	8.7	9.3	U63835	36
OXA-16		OXA-10	ES-OXA	P. aeruginosa	A		+	42.1	6.2	6.3	AF043100	39
OXA-17		OXA-10	ES-OXA	P. aeruginosa	A		+	42.1	6.1	7.0	AF060206	37
OXA-18			ES-OXA	P. aeruginosa	A	ISCR19	−	61.2	5.5	5.9	U85514	118
OXA-19		OXA-13	ES-OXA	P. aeruginosa	A		+	41.2	7.6	8.4	AF043381	98
OXA-20			Narrow spectrum	P. aeruginosa	A		+	45.1	7.4	9.0	AF024602	108
OXA-21		OXA-3	Narrow spectrum	A. baumannii	A		+	50.1	7.0	8.1	Y10693	162
OXA-22			Narrow spectrum	R. pickettii	N		−	65.5	7.0	6.4	AF064820	112
OXA-23			CHDL	A. baumannii	A	Tn2006/Tn2007	−	38	6.7	7.0	AJ132105	42
OXA-24	OXA-40		CHDL	A. baumannii	A		−	34.4	8.6	9.0	AJ239129	19
OXA-25		OXA-40	CHDL	A. baumannii	A		−	34.4	8.0	8.5	AF201826	1
OXA-26		OXA-40	CHDL	A. baumannii	A		−	34.4	7.9	9.0	AF201827	1
OXA-27		OXA-23	CHDL	A. baumannii	A		−	38	6.8	8.0	AF201828	1
OXA-28		OXA-13	ES-OXA	P. aeruginosa	A		+	41.2	8.1	8.7	AF231133	125
OXA-29			Narrow spectrum	L. gormanii	N		−	36.6	9.0	9.4	AJ400619	49
OXA-30	OXA-1		Narrow spectrum	E. coli	A		+	34.4	7.3	6.8	AF255921	148
OXA-31		OXA-1	ES-OXA	P. aeruginosa	A		+	34.4	7.5	6.8	AF294653	9
OXA-32		OXA-2	ES-OXA	P. aeruginosa	A		+	50	7.7	9.0	AF315351	124
OXA-33	OXA-40		CHDL	A. baumannii	A		−	34.4	8.6	9.0	AY008291
OXA-34		OXA-2	ES-OXA	P. aeruginosa	A		+	50		8.9	AF350424	UP
OXA-35		OXA-10	ES-OXA	P. aeruginosa	A		+	41.2	8.0	8.7	AF315786	8
OXA-36		OXA-2	ES-OXA	P. aeruginosa	A		+	49.4		9.2	AF300985	UP
OXA-37		OXA-20	Narrow spectrum	A. baumannii	A		+	44.8	7.4	8.9	AY007784	109
OXA-38
OXA-39
OXA-40			CHDL	A. baumannii	A		−	34.4	8.6	9.0	AF509241	65
OXA-41
OXA-42			Narrow spectrum	B. pseudomallei	N		−	66.3	9.2	9.3	AJ488302	111
OXA-43			Narrow spectrum	B. pseudomallei	N		−	65.9	9.2	9.3	AJ488303	111
OXA-44
OXA-45			ES-OXA	P. aeruginosa	A	ISCR5	−	61.8	8.8	9.4	AJ519683	153
OXA-46			Narrow spectrum	P. aeruginosa	A		+	47.1	7.8	8.7	AF317511	57
OXA-47		OXA-1	Narrow spectrum	K. pneumoniae	A		+	34.1	7.4	6.8	AY237830	127
OXA-48			CHDL	K. pneumoniae	A	Tn1999	−	44.5	7.2	8.0	AY236073	127
OXA-49		OXA-23	CHDL	A. baumannii	A		−	38		6.0	AY288523	UP
OXA-50			Narrow spectrum	P. aeruginosa	N		−	64.8	8.6	9.0	AY306130	54
OXA-51	OXA-Ab1		CHDL	A. baumannii	A		−	39.3	7.0	8.0	AJ309734	22
OXA-52
OXA-53		OXA-2	ES-OXA	S. Agona	A		+	50.2	6.9	7.2	AY289608	103
OXA-54			CHDL	S. oneidensis	N		−	46.6	6.8	6.7	AY500137	126
OXA-55			CHDL	S. algae	N		−	53.8	8.6	8.6	AY343493	68
OXA-56			Narrow spectrum	P. aeruginosa	A		+	40.7	6.5	8.7	AY445080	25
OXA-57			Narrow spectrum	B. pseudomallei	N		−	66		9.3	AJ631966	73
OXA-58			CHDL	A. baumannii	A		−	37.4	7.2	7.2	AY665723	130
OXA-59			Narrow spectrum	B. pseudomallei	N		−	65.9		9.3	AJ632249	73
OXA-60			Narrow spectrum	R. pickettii	N		−	64.9	5.1	5.4	AF525303	55
OXA-61			Narrow spectrum	C. jejuni	N		−	27.4		9.1	AY587956	2
OXA-62			CHDL	P. pnomenusa	N		−	65.3	>9.0	9.5	AY423074	144
OXA-63			Narrow spectrum	B. pilosicoli	N		−	24.9		6.0	AY619003	94
OXA-64	OXA-Ab2	OXA-51	CHDL	A. baumannii	N		−	39.6		8.0	AY750907	21
OXA-65	OXA-Ab3	OXA-51	CHDL	A. baumannii	N		−	39.2		8.8	AY750908	21
OXA-66	OXA-Ab4	OXA-51	CHDL	A. baumannii	N		−	39.4		9.0	AY750909	21
OXA-67	OXA-Ab5	OXA-51	CHDL	A. baumannii	N		−	39		8.0	DQ491200	UP
OXA-68	OXA-Ab6	OXA-51	CHDL	A. baumannii	N		−	39		7.1	AY750910	21
OXA-69	OXA-Ab7	OXA-51	CHDL	A. baumannii	N		−	39.3	8.4	8.6	AY750911	66
OXA-70	OXA-Ab8	OXA-51	CHDL	A. baumannii	N		−	39.3		9.0	AY750912	21
OXA-71	OXA-Ab9	OXA-51	CHDL	A. baumannii	N		−	39.7		8.0	AY750913	21
OXA-72		OXA-40	CHDL	A. baumannii	A		−	36.4		8.8	EF534256	166
OXA-73		OXA-23	CHDL	K. pneumoniae	A		−	37.6		8.0	AY762325	UP
OXA-74		OXA-10	Unknown	P. aeruginosa	A		−	41.9	6.5	7.0	AJ854182	46
OXA-75	OXA-Ab10	OXA-51	CHDL	A. baumannii	N		−	38.7		8.6	AY859529	66
OXA-76	OXA-Ab11	OXA-51	CHDL	A. baumannii	N		−	39.3		9.2	AY949203	66
OXA-77	OXA-Ab12	OXA-51	CHDL	A. baumannii	N		−	39.2		8.6	AY949202	66
OXA-78	OXA-Ab13	OXA-51	CHDL	A. baumannii	N		−	39.2		8.9	AY862132	UP
OXA-79	OXA-Ab14	OXA-51	CHDL	A. baumannii	N		−	39.5		9.0	EU019534	47
OXA-80	OXA-Ab15	OXA-51	CHDL	A. baumannii	N		−	39.3		9.0	EU019535	47
OXA-81
OXA-82	OXA-Ab16	OXA-51	CHDL	A. baumannii	N		−	39.4		9.0	EU019536	158
OXA-83	OXA-Ab17	OXA-51	CHDL	A. baumannii	N		−	39.5		9.0	DQ309277	158
OXA-84	OXA-Ab18	OXA-51	CHDL	A. baumannii	N		−	39.4		9.0	DQ309276	158
OXA-85			Narrow spectrum	F. nucleatum	N		−	24.6	5.3	6.1	AY227054	164
OXA-86	OXA-Ab19	OXA-51	CHDL	A. baumannii	N		−	38.8		8.0	DQ149247	159
OXA-87	OXA-Ab20	OXA-51	CHDL	A. baumannii	N		−	38.9		8.0	DQ348075	159
OXA-88	OXA-Ab21	OXA-51	CHDL	A. baumannii	N		−	39.2		9.2	DQ392963	75
OXA-89	OXA-Ab22	OXA-51	CHDL	A. baumannii	N		−	38.4	7.0	8.6	DQ445683	94
OXA-90	OXA-Ab23	OXA-51	CHDL	A. baumannii	N		−	39.2		8.6	EU433382	UP
OXA-91	OXA-Ab24	OXA-51	CHDL	A. baumannii	N		−	39		8.0	DQ519083	75
OXA-92	OXA-Ab25	OXA-51	CHDL	A. baumannii	N		−	39.3		8.6	DQ335566	156
OXA-93	OXA-Ab26	OXA-51	CHDL	A. baumannii	N		−	39.3		8.0	DQ519087	75
OXA-94	OXA-Ab27	OXA-51	CHDL	A. baumannii	N		−	39.3		8.9	DQ519088	75
OXA-95	OXA-Ab28	OXA-51	CHDL	A. baumannii	N		−	39.5		8.6	DQ519089	75
OXA-96		OXA-58	CHDL	A. baumannii	A		−	37.5		7.2	DQ519090	75
OXA-97		OXA-58	CHDL	A. baumannii	A		−	37.8		7.2	EF102240	129
OXA-98	OXA-Ab29	OXA-51	CHDL	A. baumannii	N		−	39.2		8.6	AM279652	UP
OXA-99	OXA-Ab30	OXA-51	CHDL	A. baumannii	N		−	39.4		8.0	DQ888718	UP
OXA-100
OXA-101		OXA-10	Unknown	C. freundii	A		+	40.7		8.8	AM412777	UP
OXA-102		OXA-23	CHDL	A. radioresistens	N		−	38		5.8	Unknown	123
OXA-103		OXA-23	CHDL	A. radioresistens	N		−	38		5.8	Unknown	123
OXA-104	OXA-Ab31	OXA-51	CHDL	A. baumannii	N		−	39.3		8.6	EF581285	47
OXA-105		OXA-23	CHDL	A. radioresistens	N		−	38		7.0	Unknown	UP
OXA-106	OXA-Ab32	OXA-51	CHDL	A. baumannii	N		−	39.3		8.9	EF650032	47
OXA-107	OXA-Ab33	OXA-51	CHDL	A. baumannii	N		−	39.3		8.6	EF650033	47
OXA-108	OXA-Ab34	OXA-51	CHDL	A. baumannii	N		−	39		8.5	EF650034	47
OXA-109	OXA-Ab35	OXA-51	CHDL	A. baumannii	N		−	39.3		9.0	EF650035	47
OXA-110	OXA-Ab36	OXA-51	CHDL	A. baumannii	N		−	39.3		8.6	EF650036	47
OXA-111	OXA-Ab37	OXA-51	CHDL	A. baumannii	N		−	39.4		7.1	EF650037	47
OXA-112	OXA-Ab38	OXA-51	CHDL	A. baumannii	N		−	39.4		8.6	EF650038	47
OXA-113	OXA-Ab39	OXA-51	CHDL	A. baumannii	N		−	39.3		8.0	EF653400	106
OXA-114			Narrow spectrum	A. xylosoxidans	N		−	70.4	8.6	9.0	EU188842	41
OXA-115	OXA-Ab40	OXA-51	CHDL	A. baumannii	N		−	39.3		9.0	EU029998	UP
OXA-116	OXA-Ab41	OXA-51		A. baumannii	N		−	39.3		8.6	EU220744	UP
OXA-117	OXA-Ab42	OXA-51		A. baumannii	N		−	39.2		8.6	EU220745	UP
OXA-118			Narrow spectrum	B. cepacia	A		+	49.3		7.3	AF371964	33
OXA-119			Narrow spectrum	Uncultured bacterium	A		+	49.4		6.7	AY139598	150
OXA-120
OXA-121
OXA-122
OXA-123
OXA-124
OXA-125
OXA-126
OXA-127
OXA-128		OXA-10	CHDL	A. baumannii	N		+	39.1		8.0	EU375515	52a
OXA-129	OXA-Ab43	OXA-5	Unknown	S. Bredeney	A		+	39.9		9.1	AM932669	95
OXA-130	OXA-Ab44	OXA-51		A. baumannii	N		−	39.1		8.5	EU547445	UP
OXA-131	OXA-Ab45	OXA-51		A. baumannii	N		−	39.4		9.0	EU547446	UP
OXA-132	OXA-Ab46	OXA-51		A. baumannii	N		−	39.3		8.0	EU547447	UP
OXA-133		OXA-23	CHDL	A. radioresistens	N		−	39.3		6.1	EU571228	123
OXA-134			CHDL	A. lwoffii	N		−	46.2		5.3		UP
OXA-135
OXA-136		OXA-63	Narrow spectrum	B. pilosicoli	N		−	25.1		5.3	EU086830	96
OXA-137		OXA-63	Narrow spectrum	B. pilosicoli	N		−	24.9		5.7	EU086834	96
OXA-138
OXA-139
OXA-140
OXA-141		OXA-2	ES-OXA	P. aeruginosa	A		+	49.9		9.1	EF552405	UP
OXA-142		OXA-10	ES-OXA	P. aeruginosa	A		+	42		6.3	EU358785	UP
OXA-143			CHDL	A. baumannii	A		−	34.4		8.7		UP
OXA-144
OXA-145		OXA-10	ES-OXA	P. aeruginosa	A		+	41.1		8.7	FJ790516	UP
OXA-146
OXA-147		OXA-10	ES-OXA	P. aeruginosa	A			41		8.1	FJ848783	UP

^aThe nomenclature is in accordance with that provided by G. Jacoby on the Lahey website (http://www.lahey.org/Studies/other.asp#table1). Lacking variants (in boldface) are those for which a number has been assigned on this website but for which no information is yet available.

^bA, acquired; N, natural.

^c+, the oxacillinase gene was found to be associated with an integron-borne gene cassette; −, the gene is not associated with an integron-borne gene cassette.

^dExperimentally obtained pI values (when available) versus calculated values. Theoretical values were calculated using software found at the ExPASy proteomics tools website (http://www.expasy.ch/tools/) and the amino acid sequences of the mature proteins only. Peptide cleavage site identification was performed with SignalP (http://www.cbs.dtu.dk/services/SignalP/), and pI computing was performed with the Compute pI/M_w tool (http://www.expasy.ch/tools/pi_tool.html).

^eUP, unpublished.

GENERAL PROPERTIES

OXAs are β-lactamases belonging to class D according to the molecular nomenclature based on amino acid identity comparison. Similar to class A and C β-lactamases, they possess an active-site serine which is located at position 70 in the class D β-lactamase numbering system (the DBL numbering system) (72, 97) (Fig. 1). The DBL numbering system has been established to analyze the molecular structures of the class D β-lactamases, in analogy to the numbering system adopted for class A enzymes (5, 32). The two groups of enzymes share the serine and lysine residues which are part of the highly conserved motif S-T-F-K found at positions 70 to 73 (79). The Y-G-N motif (positions 144 to 146) and the K-T-G motif (positions 216 to 218) are highly conserved in class D β-lactamases, but the F-G-N motif may replace Y-G-N in several OXAs (Fig. 1).

FIG. 1.

Amino acid alignment of 13 representative class D β-lactamases from the different groups. Stars indicate residues identical among all the amino acid sequences. Amino acid motifs which are well conserved (even if possibly variable) among class D β-lactamases are indicated by gray shading. Numbering is according to DBL numbering (32).

Class D β-lactamases are usually not inhibited by clavulanic acid, tazobactam, and sulbactam, whereas their activities may be inhibited in vitro by sodium chloride (NaCl). This property is not shared by β-lactamases of other classes, thus defining it as a useful characteristic for in vitro identification. NaCl at a concentration of 100 mM inhibits totally the activities of most class D β-lactamases (9, 54, 65, 127, 130). This property is not clearly explained, but it has been related at least to the presence of a Tyr residue at position 144. In vitro mutagenesis showed that the replacement of a Tyr residue by a Phe at that position makes a mutant resistant to NaCl inhibition (65).

Class D β-lactamases were first defined as β-lactamases hydrolyzing cloxacillin and oxacillin faster than benzylpenicillin (23). This definition does not seem valuable anymore since recently described enzymes actually inactivate cloxacillin and oxacillin poorly, even sometimes sparing those substrates. However, all class D β-lactamases significantly hydrolyze amino- and carboxypenicillins.

Although all the OXAs first characterized corresponded to acquired genes, it has been demonstrated lately that many gram-negative species naturally possess genes in their genomes coding for OXAs, which are therefore considered to be resident enzymes.

ACQUIRED NARROW-SPECTRUM CLASS D β-LACTAMASES

The OXA-1 subgroup.

As mentionned by Boyd and Mulvey (20), β-lactamases OXA-1 and OXA-30 are the same enzyme, since an original sequencing error in the OXA-1 sequence had introduced a mistake (113). The bla_OXA-1 gene has been found in plasmid and integron locations in a large variety of gram-negative organisms (see below). OXA-1 β-lactamase, like most OXAs, hydrolyzes amino- and ureidopenicillins significantly and hydrolyzes narrow-spectrum cephalosporins weakly (9). In addition, OXA-1 slightly hydrolyzes broad-spectrum cephalosporins, conferring reduced susceptibility to cefepime and cefpirome (9). It may therefore be classified as a β-lactamase possessing either a narrow or an expanded spectrum of hydrolysis (see below).

The bla_OXA-1 gene has often been identified in ampicillin-resistant enterobacterial strains such as isolates of Escherichia coli, Shigella flexneri, and Salmonella spp., with spread occuring among community-acquired enterobacterial species (44, 62, 148). It has been identified in human and animal isolates. It was, for instance, identified in Shigella dysenteriae strains from diverse geographical origins (45) and in Salmonella enterica serovar Typhimurium isolates from humans and food products of animal origin in Portugal (6). Noteworthily, it seems to be a frequent source of resistance to ampicillin in Shigella spp. and Salmonella spp. isolated from humans in the United States (63).

The bla_OXA-1 gene has frequently been found to be associated with genes encoding extended-spectrum β-lactamases (ESBLs). Plasmids coharboring the bla_OXA-1 and bla_CTX-M-1 genes have been identified in animal E. coli isolates from Spain (31), and recent studies have reported very frequent association of bla_OXA-1 with the worldwide-spread CTX-M-15 ESBL determinant in human E. coli isolates from diverse geographical origins (29). This association of bla_OXA-1 with bla_CTX-M genes makes isolates resistant to β-lactam-β-lactamase inhibitor combinations. Some OXA-1 variants such as OXA-47, carrying up to seven substitutions compared to OXA-1 (none located inside the conserved motifs of class D β-lactamases), possess a narrow spectrum of hydrolysis (127). The bla_OXA-47 gene has been identified as plasmid borne and is carried on a gene cassette inserted into an integron in a Klebsiella pneumoniae isolate from Turkey coproducing OXA-48 (123) (see below).

Among the OXA-1 derivatives that have been identified, no known variant possesses the ability to hydrolyze ceftazidime. OXA-1 possesses the ability to hydrolyze cefepime and cefpirome slightly, and OXA-31, which differs from OXA-1 by two amino acid substitutions and has been identified in a Pseudomonas aeruginosa isolate from France (9), also possesses that property. We have demonstrated previously that a transfer of the natural plasmid harboring the bla_OXA-31 gene into a P. aeruginosa recipient strain confers resistance to cefepime and cefpirome but that susceptibility to ceftazidime is not modified. By comparing the MICs of β-lactams for E. coli recombinant clones producing OXA-1 or OXA-31, as well the kinetic parameters, we showed that OXA-1 and OXA-31 possess similar hydrolysis profiles, including the ability to hydrolyze cefepime and cefpirome (9). Therefore, OXA-1 and OXA-31 may be considered to be class D β-lactamases characterized by expanded-spectrum profiles with significant impacts on MICs for bacterial species that possess high-level intrinsic impermeability (such as P. aeruginosa) and not on those for bacterial species with low-level intrinsic impermeability (such as E. coli).

All the genes for the OXA-1-like β-lactamases described above have been identified in the form of gene cassettes inserted into class 1 integrons (9, 44, 107, 127).

The OXA-2 subgroup.

OXA-2 β-lactamase is another narrow-spectrum β-lactamase that shares ca. 30% amino acid identity with OXA-1 (77). It constitutes, together with its derivatives OXA-3, OXA-15, OXA-21, OXA-32, OXA-34, OXA-36, and OXA-53, a distinct genetic cluster (Fig. 2). The bla_OXA-2 gene has been identified often in P. aeruginosa and S. Typhimurium isolates producing the ESBL PER-1 (46), as has the variant OXA-3 (142). Recently, bla_OXA-2 has been identified in P. aeruginosa isolates from Serbia and Hungary that also produced the ESBL PER-1 (81). In addition, it has been identified in other species such as Morganella morganii in Argentina (135), K. pneumoniae in Uruguay (161), and E. coli in France (GenBank accession no. CAJ13583). Surprisingly, it has also been identified in distantly related species such as Bordetella bronchiseptica (GenBank accession no. ABD63309) and Aeromonas hydrophila (GenBank accession no. ABF69297) and even the gram-positive species Corynebacterium amycolatum (GenBank accession no. CAI40608) (unpublished data). The OXA-21 variant, a point mutation derivative of the OXA-3 enzyme, has been identified once in Acinetobacter baumannii (163). The genes for these β-lactamases have been also identified in the form of gene cassettes inserted into class 1 integrons.

FIG. 2.

Dendrogram obtained for 73 class D β-lactamases by using Phylip (ClustalW). Branch lengths are to scale and proportional to the number of amino acid changes. The distance along the vertical axis has no significance. The different clusters identified allowed the identification of nine main groups, considering that proteins from the same group have more than 80% amino acid identity.

The OXA-10 subgroup.

OXA-10 β-lactamase (formerly known as PSE-2) also possesses the ability to hydrolyze cephalosporins, hydrolyzing cefotaxime, ceftriaxone, and aztreonam at low levels but sparing ceftazidime, cephamycins, and carbapenems (70). The bla_OXA-10 gene is encountered in a large variety of gram-negative species, being the bla_OXA gene identified most frequently in P. aeruginosa (8, 25, 37-39, 46, 60, 99, 125).

There are some point mutation derivatives of OXA-10, such as OXA-11, OXA-13, OXA-16, OXA-28, OXA-35, and OXA-74, possessing increased activities toward expanded-spectrum cephalosporins (see below) (Fig. 2). Very few OXA-10-like enzymes possessing a narrow spectrum of hydrolysis have been described, one of which is OXA-7, encoded by a gene reported to be plasmid borne in E. coli. OXA-7 has 95% amino acid identity to OXA-10 (91, 145).

Other subgroups.

Among the narrow-spectrum OXAs, some variants are not, or are weakly, related to the OXA-1, OXA-2, and OXA-10 subgroups (Fig. 2). This is the case for OXA-9, which shares 45 and 54% amino acid identity with the most closely related enzymes, OXA-12 and OXA-18 (155). OXA-9 has the property, uncommon for an OXA enzyme, of being inhibited by clavulanic acid and cloxacillin but not by NaCl (16). Its hydrolytic profile remains to be precisely determined, even if OXA-9 would likely correspond to a narrow-spectrum β-lactamase (our unpublished data). The bla_OXA-9 gene was first identified on a plasmid in a K. pneumoniae isolate (155). The bla_OXA-9 gene has been identified in a Pseudomonas putida isolate from France coexpressing the metallo-β-lactamase VIM-2 (122), in an Enterobacter cloacae isolate from Canada (119), and in an Enterobacter aerogenes isolate from France (120). Recently, it has also been identified in a carbapenem-resistant E. cloacae strain originating in the Untied States and coproducing the class A carbapenemase KPC-3 (43) and in a carbapenem-resistant K. pneumoniae isolate in Turkey coproducing the class D carbapenemase OXA-48 (127).

Another narrow-spectrum class D β-lactamase is LCR-1, which was one of the very first class D β-lactamases to be identified on the basis of biochemical properties only (146), was recovered from P. aeruginosa, and hydrolyzes only penicillins, nitrocefin, and oxacillin (168). Its recently determined amino acid sequence (32) shows that it belongs to a separate cluster among class D enzymes (Fig. 2), sharing 40% or less amino acid identity with other OXAs (40% with OXA-55 and 37% with OXA-48). NPS-1 is another narrow-spectrum enzyme which is related to LCR-1 (carrying eight amino acid differences) (114). The bla_NPS-1 gene was identified on a conjugative plasmid from P. aeruginosa isolates in the United Kingdom (83).

The product of the bla_OXA-5 gene represents another subgroup of narrow-spectrum class D β-lactamases (Fig. 2). After the gene was phenotypically described as plasmid borne in a P. aeruginosa isolate recovered in the United Kingdom (91), its sequence was determined (32). The OXA-5 determinant shares less than 81% identity with OXA-10 derivatives. The bla_OXA-5 gene was found as a gene cassette inserted into a class 1 integron and was later found in association with the bla_GES-2 gene, encoding a peculiar ESBL with carbapenemase properties, in carbapenem-resistant P. aeruginosa isolates from South Africa during an outbreak period (134). The hydrolysis spectrum of OXA-5 is limited to narrow-spectrum penicillins, oxacillin, and cephalothin (cefalotin). Its activity is inhibited by NaCl but not by clavulanate (91). Recently, OXA-129 was identified in S. enterica subsp. enterica serovar Bredeney porcine isolates from Brazil (95). It differs from OXA-5 by 12 amino acids.

OXA-20 is another narrow-spectrum class D β-lactamase that has weak amino acid identity to other class D β-lactamases (108). It shares less than 75% identity with the most closely related enzyme, OXA-2. OXA-20 hydrolyzes penicillins, cephalothin, and cephaloridine but spares expanded-spectrum cephalosporins. Interestingly, OXA-20 is inhibited by clavulanic acid (108). The bla_OXA-20 gene was identified in a P. aeruginosa isolate from Sicily, Italy, that coexpressed OXA-18, the latter OXA possessing an expanded-spectrum profile (118) (see below). Noteworthily, the bla_OXA-20 gene was also identified in A. baumannii isolates from France and Italy and was also located inside integron structures (53, 58, 121, 169). A point mutation derivative of OXA-20, OXA-37, has been identified in an A. baumannii isolate from Spain (109).

OXA-46 was identified in a P. aeruginosa isolate from Italy. In fact, this determinant was identified in a multidrug-resistant isolate that coexpressed the metallo-β-lactamase VIM-1 (57). OXA-46 shares 78% amino acid identity with OXA-2 and OXA-53, the most closely related enzymes. It hydrolyzes mostly penicillins, oxacillin, and narrow-spectrum cephalosporins. Interestingly, it is inactivated by tazobactam and carbapenems but exhibits low susceptibility to NaCl (57).

ACQUIRED ES-OXAs

Whereas most of the previously mentioned class D β-lactamases exhibit a substrate profile limited mostly to penicillins, nitrocefin, oxacillin, and cloxacillin, class D β-lactamases able to hydrolyze expanded-spectrum cephalosporins have also been identified (though their distribution remains quite limited). There are two main types of expanded-spectrum class D β-lactamases (ES-OXAs) (Table 1). Some ES-OXAs are just point mutation derivatives of narrow-spectrum class D β-lactamases. Others may be completely different enzymes with weak amino acid sequence identity. Interestingly, most of the ES-OXAs identified so far are from P. aeruginosa.

ES-OXAs as point mutation derivatives of narrow-spectrum OXAs.

The first variant described to possess an extended-spectrum profile was OXA-15, a point mutation derivative of OXA-2 (with a change from Asp to Gly at position 150 in the DBL numbering system). The bla_OXA-15 gene was identified inside a class 1 integron structure in a P. aeruginosa isolate from Turkey (36). OXA-15 has increased ability to hydrolyze ceftazidime compared to that of OXA-2, but it also hydrolyzes cefepime and aztreonam at a lower level than OXA-2. OXA-32 is another OXA-2 derivative possessing an expanded-spectrum hydrolysis profile, identified in a ceftazidime-resistant P. aeruginosa isolate recovered from a patient originating from Guadeloupe in the French West Indies (124). The bla_OXA-32 gene was integron borne and located on a 250-kb conjugative plasmid. OXA-32 differs from OXA-2 by a Leu169Ile substitution (DBL numbering) that extends its hydrolysis spectrum toward ceftazidime but not cefotaxime (124). Interestingly, this substitution also modifies the behavior of the class D β-lactamase OXA-32 toward inhibitors, since imipenem and cefoxitin were found to significantly inhibit the activity of OXA-32 compared to that of OXA-2.

A large number of class D β-lactamases with expanded-spectrum activity have been identified among OXA-10 variants. OXA-11, identified in a P. aeruginosa isolate from a Turkish patient, was found to be encoded by a plasmid (60). Compared to OXA-10, it possesses two substitutions (Asn146Ser and Gly167Asp [DBL numbering]) that extend its ability to hydrolyze ceftazidime. Other OXA-10-like ES-OXAs have been identified in P. aeruginosa strains from Turkey, including OXA-14 (carrying a Gly167Asp change) (38), OXA-16 (with Ala114Thr and Gly167Asp changes) (39), and OXA-17, the latter having an Asn76Ser substitution conferring an increased hydrolysis rate for cefotaxime and a decreased rate for ceftazidime (37). OXA-35, which has been identified in a P. aeruginosa isolate from France (8), is a point mutation derivative of both OXA-19 (95) and OXA-28 (125). OXA-35 possesses hydrolytic activity toward penicillins and cefsulodin but spares narrow- and expanded-spectrum cephalosporins. In contrast, OXA-19 and OXA-28, differing from OXA-10 by 9 and 10 amino acid substitutions, respectively, confer resistance to ceftazidime (98, 125).

Other ES-OXAs.

Several ES-OXAs are not structurally related to narrow-spectrum OXAs. OXA-18 was the first enzyme of this type to be identified, in a P. aeruginosa isolate obtained in 1995 in Paris from a patient previously hospitalized in Sicily (118). OXA-18 shares less than 50% amino acid identity with the other OXAs. It is peculiar since it confers high-level resistance to expanded-spectrum cephalosporins and is surprisingly well inhibited (for a class D β-lactamase) by clavulanic acid (118). Its substrate profile does not include cephamycins and carbapenems. OXA-18 can therefore be considered a typical ESBL. The bla_OXA-18 gene is very likely chromosomally located. Other OXA-18-producing P. aeruginosa isolates have been recovered recently in Belgium, as sources of outbreaks (105). The structures surrounding the bla_OXA-18 gene in those strains were identical to those discovered in the OXA-18-producing Italian isolate (105).

OXA-45 is another ES-OXA, identified in a multidrug-resistant Texan P. aeruginosa isolate coexpressing the class B β-lactamase VIM-7 (153). OXA-45 shows the highest degrees of identity to OXA-18 (66%), OXA-9 (43%), and OXA-22 (40%). The bla_OXA-45 gene, located on a 24-kb plasmid, was not in the form of a gene cassette but was associated with two copies of an ISCR5-like element. OXA-45 includes in its substrate profile expanded-spectrum cephalosporins, such as ceftazidime, cefotaxime, and aztreonam, but spares carbapenems and cephamycins. The activity of OXA-45, like that of OXA-18, is inhibited by clavulanic acid (153).

Finally, OXA-53 is another ES-OXA that has been identified recently in an S. enterica serovar Agona isolate from Brazil (103). It shares 90% amino acid identity with the narrow-spectrum OXA-2 but exhibits unusual properties since it is well inhibited by clavulanic acid and confers reduced susceptibility to ceftazidime. The bla_OXA-53 gene was plasmid and integron borne (103).

ACQUIRED CHDLs

Some acquired class D β-lactamases may also hydrolyze carbapenems. None of these carbapenem-hydrolyzing class D β-lactamases (CHDLs) (131, 133) significantly hydrolyze expanded-spectrum cephalosporins, therefore indicating that currently known class D β-lactamases are unable to combine extended-spectrum and carbapenem-hydrolyzing properties. Most of these enzymes are from Acinetobacter spp.

As noticed by Queenan and Bush (137), the level of hydrolysis of carbapenems by CHDLs remains low, due to poor turnover of these β-lactams. In general, the hydrolysis of imipenem, even if slow, is faster than that of meropenem. Usually, the K_m values for imipenem are low, indicating that CHDLs have very high apparent affinities for that substrate (65, 130). Due to these peculiar properties, the exact contribution of these enzymes to phenotypic resistance has been debated. However, it has been demonstrated recently using knockout or complementation experiments that acquired CHDLs such as OXA-23, OXA-40, and OXA-58 identified in A. baumannii significantly contribute to carbapenem resistance (67).

The first reported acquired class D β-lactamase with carbapenemase activity was OXA-23 (also known as ARI-1 [43, 115]), detected in an A. baumannii isolate from Scotland and found to be plasmid mediated after its transfer to Acinetobacter junii (143). The ability of OXA-23 to hydrolyze carbapenems was weak, since hydrolytic measurements showed only low-level activity against imipenem. However, compared to the activities of other class D β-lactamases, this hydrolysis was significant and was allowed to define the novel subgroup of OXAs currently named CHDLs (133). OXA-23, weakly related to other class D β-lactamases with only 36% amino acid identity to OXA-5 and OXA-10, belongs to the first group of CHDLs. Although kinetic parameters of hydrolysis have been determined with crude extracts of OXA-23 (115), precise parameters are still not available. The other member of the first group is OXA-27, which possesses two substitutions (Thr86Ala and Asn250Lys [DBL numbering]) compared to OXA-23 (1). The corresponding bla_OXA-27 gene, identified in a carbapenem-resistant A. baumannii isolate from Singapore, was likely chromosomally located (1). Whereas bla_OXA-27 has been identified in only a single isolate, bla_OXA-23 is widespread and identified only in Acinetobacter spp., with a single exception, since this gene was also identified as chromosomally carried in a carbapenem-resistant Proteus mirabilis isolate from France (17). Recent data indicate that OXA-23-positive A. baumannii strains have spread throughout the world, to locations including France (30), Bulgaria (149), Iran (48), the United Arab Emirates (101), Tunisia (86), Brazil (35), and Australia (160). In addition, OXA-23 producers have been at the origin of hospital outbreaks in French Polynesia (104), Colombia (163), the United Kingdom (28), Turkey (92), China (166, 170), and Korea (74). OXA-23-positive A. baumannii isolates and one A. junii strain have been also identified in Romania (87).

A second group of CHDLs is made up of OXA-25, OXA-26, OXA-40, and OXA-72 (an original sequencing error in OXA-24 makes it now OXA-40 [see the Lahey website at http://www.lahey.org/Studies/]). These enzymes differ by a few amino acid substitutions only. OXA-24/OXA-40 was originally identified as chromosomally encoded in a carbapenem-resistant A. baumannii isolate recovered from Spain (18, 19). The bla_OXA-40 gene has since been identified in different areas, especially in Portugal and Spain (40, 116, 138, 141), but also in the Untied States (84, 136). Interestingly, the bla_OXA-40 gene has been identified as either chromosomally carried or plasmid borne (138). Surprisingly, it has been very recenty identified as plasmid located in two carbapenem-resistant P. aeruginosa isolates in Spain (146). OXA-40 is not inhibited by NaCl (65). OXA-25 and OXA-26 are point mutation derivatives of OXA-40 that have been identified in carbapenem-resistant A. baumannii isolates recovered from Spain and Belgium, respectively (1). OXA-72 has been identified in A. baumannii isolates from China, South Korea, Taiwan, and Bahrain (80, 85, 102, 166).

A third identified group of CHDLs is represented by OXA-58, which has also been detected only in Acinetobacter spp. so far. OXA-58 was first identified in a multidrug-resistant A. baumannii isolate from France (130), that strain being at the origin of an outbreak in the local hospital (64). OXA-58 shares 35, 33, and 18% amino acid identity with OXA-5, OXA-10, and OXA-1, respectively. OXA-58 hydrolyzes penicillins and carbapenems at low levels. Whereas weak hydrolysis of cefpirome was detected, hydrolysis of cefepime, ceftazidime, and cefotaxime was not. The rate of hydrolysis of imipenem was 10-fold lower, and that of meropenem was 100-fold lower, than that of benzylpenicillin (130). The bla_OXA-58 gene was found to be plasmid borne in A. baumannii MAD (130), as well as in most of the OXA-58-producing A. baumannii isolates reported worldwide, including those in Europe, Argentina, Australia, and the United States (26-28, 69, 87, 117); OXA-58 is often associated with hospital outbreaks and has been involved in such outbreaks in France, Belgium, Italy, Turkey, Greece, and the United States (12, 15, 53, 64, 69, 128, 159). Noteworthily, OXA-58 has also been identified in other Acinetobacter species, such as A. junii in Romania (87) and Australia (117), Acinetobacter genomospecies 3 in Spain (88), and Acinetobacter phenon 6/ct13TU in Spain (89). OXA-97 is a point mutation variant of OXA-58 that shares the same hydrolytic properties and has been recently identified in A. baumannii isolates from Tunisia (129). The bla_OXA-97 gene was plasmid carried and identified in a clonal strain at the origin of an outbreak at the Salhoul Hospital in Sousse, Tunisia. Another point mutation derivative is OXA-96, identified in A. baumannii in Singapore (75).

Very recently, the novel CHDL OXA-143 was identified in a clinical A. baumannii isolate that had been recovered in Brazil (68a). It shares 88% amino acid identity with OXA-40, 63% with OXA-23, and 52% with OXA-58. It may therefore correspond to a new subgroup of CHDLs encountered in A. baumannii and involved in carbapenem resistance in that species. Its substrate profile was similar to those of other CHDLs, and the corresponding gene was not integron or transposon carried but was likely acquired by a homologous recombination process.

Another unrelated CHDL is OXA-48, which has been identified in a carbapenem-resistant K. pneumoniae isolate from Istanbul, Turkey (127), thus, in a member of the Enterobacteriaceae. That isolate coexpressed several β-lactamases, including the class A ESBL SHV-2a and the narrow-spectrum β-lactamases TEM-1 and OXA-47. OXA-48 β-lactamase hydrolyzes penicillins and, at a lower level, imipenem but is not active against expanded-spectrum cephalosporins (127). This enzyme is the class D β-lactamase with the highest known catalytic efficiency for imipenem (having a k_cat value of 2 s⁻¹) (127). It shares less than 46% amino acid identity with other class D β-lactamases and has weak identity to OXA-23 and OXA-40 (36 and 32%, respectively). The bla_OXA-48 gene was found to be plasmid located in K. pneumoniae 11978 (127). Very recently, we reported an important outbreak of infection with OXA-48-producing K. pneumoniae that occurred in Istanbul from May 2006 to January 2007 (24). Two distinct clones differing from the first OXA-48 producer and producing different ESBL determinants (SHV-12 and CTX-M-15) were identified in the same hospital. In addition, the bla_OXA-48 gene has been identified in E. coli and Citrobacter freundii strains in Turkey (59, 110). The spread of the bla_OXA-48 gene may be much more important than expected. Indeed, the gene has been found in K. pneumoniae isolates from Lebanon and from Belgium (34, 90). Its detection may be difficult since the level of acquired resistance to carbapenems may remain quite low.

Overall, the hydrolysis spectra of these class D β-lactamases are quite consistent, all enzymes hydrolyzing imipenem at low levels but not broad-spectrum cephalosporins and aztreonam.

NATURALLY OCCURRING CLASS D β-LACTAMASES

Recent studies have shown that many bacterial species possess chromosomally located and naturally occurring class D β-lactamase genes. The first such OXA gene to be identified was that from Aeromonas jandaei (formerly A. sobria). This species naturally produces the class D β-lactamase OXA-12, which has strong activity against cloxacillin and oxacillin and whose expression is inducible (3, 139). Another class D β-lactamase, AmpS, lacking significant activity against cloxacillin and oxacillin but hydrolyzing penicillins, was also detected in A. jandaei (A. sobria) (165). AmpS shares 96% amino acid identity with OXA-12. However, analysis using current GenBank databases indicates that AmpS likely corresponds to the intrinsic class D β-lactamase of another Aeromonas species whose genome sequences have been released, namely, A. hydrophila (GenBank accession no. YP_858675). β-Lactamases OXA-12 and AmpS both possess the property, peculiar for a class D enzyme, of being inhibited by clavulanic acid (139, 165).

Ralstonia pickettii is naturally resistant to penicillins, narrow-spectrum cephalosporins, ceftazidime, and aztreonam or shows intermediate susceptibilities to these agents. Two types of class D β-lactamases have been identified in that species. OXA-22 exhibits narrow-spectrum β-lactam hydrolysis activity that does not explain resistance to monobactams in its natural host (112). The gene encoding OXA-22 is chromosomally located, and induction experiments demonstrated that its expression is inducible (linked to a peculiar open reading frame [ORF] located upstream of bla_OXA-22) but also revealed the overexpression of another β-lactamase gene. Further cloning experiments identified a second class D β-lactamase gene, bla_OXA-60, which is also located on the chromosome of R. pickettii (55). PCR screening of multiple R. pickettii isolates identified variants of both bla_OXA-22 and bla_OXA-60 genes. OXA-60 possesses only 19% amino acid identity to OXA-22 and is weakly related to the other OXAs. It exhibits a narrow-spectrum hydrolysis profile, including activity against penicillins and nitrocefin and weak activity against carbapenems (55). The molecular basis of the induction process requires the presence of ORF RP3, located 190 bp upstream of bla_OXA-60 and divergently transcribed. Disruption of ORF RP3 abolishes induction of both β-lactamases, suggesting that ORF RP3 may be a global regulator, although the 532-amino-acid-long product displays no obvious sequence homology to known regulatory proteins (56).

The bla_OXA-61 gene was identified on the chromosome of Campylobacter jejuni and encodes a narrow-spectrum β-lactamase not inhibited by clavulanic acid (2). Considering the MICs of β-lactams for the original C. jejuni isolates, it seems likely that OXA-61-like enzymes are poorly expressed in their natural host and consequently play a minor role in susceptibility to β-lactams (2).

The bla_OXA-62 gene was identified on the chromosome of a carbapenem-resistant Pandoarea pnomenusa isolate recovered from a cystic fibrosis patient (145). Pandoraea spp. are gram-negative, non-glucose-fermenting rods. The bla_OXA-62 gene, which possesses an unusually high GC content of 65.3%, encodes a CHDL that is well inhibited by NaCl and hydrolyzes penicillins and oxacillin, as well as imipenem and meropenem at low levels, but does not hydrolyze expanded-spectrum cephalosporins. OXA-62 shares weak amino acid identity with other class D β-lactamases, the highest degree of similarity being that to OXA-50 (43% amino acid identity). Related bla_OXA-62 genes have been identified in other P. pnomenusa isolates but not in other Pandoraea species (144).

The causative agent of melioidosis, Burkholderia pseudomallei, naturally produces OXA-42 (111) and derivatives, such as OXA-43 (111) and OXA-57 and OXA-59 (73). OXA-57, which has also been identified in Bulkholderia thailandensis, is the only enzyme among these variants that has been studied biochemically, and it hydrolyzes penicillins, oxacillin, cephalothin, and nitrocefin but spares ceftazidime. It is uninhibited by clavulanate and uninhibited by NaCl, the latter property being quite uncommon (73).

The bla_OXA-63 gene was identified in a clinical isolate of the anaerobic spirochete species Brachyspira pilosicoli (94). It encodes a narrow-spectrum β-lactamase that possesses the unusual property of being well inhibited by clavulanic acid and resistant to NaCl inhibition. OXA-63 shows the closest identity (53%) to FUS-1 (OXA-85) (see below). Very recently, Mortimer-Jones and colleagues (96) analyzed B. pilosicoli srains recovered from humans and pigs in Australia and Papua New Guinea. They identified genes encoding OXA-136 and OXA-137, sharing 94 to 95% amino acid identity with OXA-63 and conferring similar patterns of resistance once the genes were cloned and expressed in E. coli. Interestingly, in that study bla_OXA-63-like genes were identified in penicillin-resistant strains only and not in penicillin-susceptible isolates (96).

FUS-1 (also named OXA-85) corresponds to the naturally occurring class D β-lactamase of Fusobacterium nucleatum subsp. polymorphum, a gram-negative anaerobic rod (164). OXA-85 shares only 25 to 44% identity with other class D β-lactamases. It also possesses a narrow spectrum of β-lactam hydrolysis and is not inhibited by NaCl or by clavulanate.

The bla_OXA-29 gene was identified in a Legionella (Fluoribacter) gormanii strain recovered from soil (52). OXA-29 shares less than 50% amino acid identity with other OXA enzymes (44% with OXA-18) and hydrolyzes oxacillin, penicillins, cefazolin, and at lower levels, cefuroxime, cefotaxime, ceftazidime, and aztreonam, sparing carbapenems. OXA-29 activity is inhibited by tazobactam but not by clavulanic acid and is also surprisingly resistant to NaCl (52).

The bla_OXA-114 gene was cloned from the genome of Achromobacter xylosoxidans (formerly Alcaligenes denitrificans subsp. xylosoxydans) (41). It encodes OXA-114, which shares 56% amino acid identity with the naturally occurring class D β-lactamase of Burkholderia cepacia (as determined by GenBank in silico analysis) and 42% with β-lactamases OXA-9 and OXA-18. OXA-114 has a narrow-spectrum hydrolysis profile, although it shows a very low level of activity against imipenem. Several bla_OXA-114-like genes have been identified in all five isolates of A. xylosoxidans tested, making such genes a feature of that species (41). In contrast to the expression of some other natural bla_OXA genes, the expression of bla_OXA-114 is not inducible (41).

Interestingly, we recently identified Acinetobacter radioresistens, a nonpathogenic and environmental Acinetobacter species, as the progenitor of the acquired CHDL-encoding gene bla_OXA-23 (122). Similar bla_OXA genes identified in other A. radioresistens isolates encode OXA-23 variants (namely, OXA-102, OXA-103, and OXA-105) exhibiting no more than six amino acid substitutions compared to OXA-23 and conferring similar levels of resistance to carbapenems once expressed in E. coli (123). Since both A. baumannii and A. radioresistens naturally possess class D β-lactamase genes, we believe that other Acinetobacter species may act as reservoirs for other clinically relevant class D β-lactamase genes. Preliminary investigations identified the bla_OXA-134 gene in the chromosome of Acinetobacter lwoffii (51). bla_OXA-134 encodes a CHDL that shares 63, 58, 57, and 53% amino acid identity with OXA-23, OXA-51, OXA-24, and OXA-58, respectively. The substrate profile of OXA-134, including penicillins and carbapenems, is similar to those of other CHDLs.

Expansion of the knowledge of entire genome sequences leads to the identification of putative ORFs that may encode class D β-lactamases. Some of these ORFs have subsequently been shown to encode functional β-lactamases, while the products of others are not β-lactamases even if such designations are falsely used in the databases, constituting a source of confusion. Some other products are good candidates for β-lactamases on the basis of in silico analysis and require further investigations, in particular accurate enzymatic analyses.

Sequencing analysis of the entire genome of A. baumannii isolate AYE revealed a chromosomal gene encoding OXA-69 that was further characterized (66). OXA-69 shares 62 and 56% amino acid identity with OXA-40 and OXA-23, respectively, and 97% identity with OXA-51, identified in several unrelated A. baumannii isolates from Argentina (22). OXA-69 hydrolyzes penicillins and, at low levels, carbapenems. OXA-51/OXA-69 enzymes do not contribute significantly to the natural resistance pattern observed in A. baumannii. The bla_OXA genes for these enzymes are neither inducible nor regulated. Genes encoding OXA-51/OXA-69 derivatives were identified in different collections of A. baumannii isolates recovered from widespread geographical areas (66, 93). Some of these variants had also been identified in carbapenem-resistant A. baumannii isolates disseminated worldwide (21, 47, 158). Up to 45 OXA-51/OXA-69 variants are now known (Table 1), and most if not all probably have identical biochemical properties. Due to the very large and increasing number of OXA-51/OXA-69 variants identified, we propose here to define an alternative nomenclature for these class D β-lactamases specific for A. baumannii, which may include OXA-AB (for OXA-A. baumannii) followed by a number and which would start with OXA-51's being consequently renamed OXA-Ab1, with the next enzyme being called OXA-Ab2, etc. (this nomenclature is presented in Table 1). Interestingly, even though the role of OXA-51-like β-lactamases in conferring the natural resistance pattern is small, they may be at least partially the source of acquired resistance. Indeed, it has been reported previously that insertion sequences ISAba1 and ISAba9 may sometimes be located upstream of the bla_OXA-51-like gene in carbapenem-resistant A. baumannii isolates, providing promoter sequences for high-level expression of OXA-51-like enzymes (49, 50, 157).

In silico analysis has identified a putative class D β-lactamase gene in the chromosome of P. aeruginosa. The gene, bla_OXA-50, was cloned and expressed in E. coli (54). OXA-50 (also referred to as PoxB [76]) hydrolyzes β-lactams at a very low level (54) and plays only a minor role in the phenotype of natural resistance to β-lactams in P. aeruginosa. OXA-50 variants have been identified in diverse P. aeruginosa clinical isolates recovered from different geographical areas (54). However, the expression of bla_OXA-50 in P. aeruginosa is not inducible, as opposed to that of the bla_OXA-22 gene in R. pickettii (56).

In silico analysis revealed that the chromosome of Shewanella oneidensis strain MR-1, an environmental gram-negative isolate from lake sediment, harbored a class D β-lactamase gene (126). This bla_OXA-54 gene was cloned and expressed in E. coli. OXA-54 shares 92% amino acid identity with OXA-48, the gene for which was identified in K. pneumoniae (see above). OXA-54 possesses enzymatic properties similar to those of OXA-48, significantly hydrolyzing imipenem (with even a higher k_cat/K_m ratio due to better affinity for this substrate) and not being inhibited by clavulanic acid. Thus, S. oneidensis may be considered a reservoir of CHDLs that are able to spread among Enterobacteriaceae (126).

More generally, the Shewanella genus may be considered to be a progenitor of other related enzymes, since OXA-55 (with 55% amino acid identity to OXA-54) was further identified in Shewanella algae and possesses biochemical properties similar to those of OXA-54, in particular the ability to hydrolyze carbapenems (68). In addition, with the recent release of many genome sequences, it appears after in silico analysis that Shewanella-related species are often (if not always) potential sources of class D β-lactamase genes, as observed for Shewanella pealeana, Shewanella loihica, Shewanella baltica, and Shewanella putrefaciens (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

In addition, in silico analyses of several other genomes of very unrelated species, such as Limnobacter sp. (Burkholderiaceae family), Idiomarina loihiensis (Idiomarinaceae family), Bradyrhizobium japonicum and Rhodopseudomonas palustris (Bradyrhizobiaceae family), Delftia acidovorans (Comamonadaceae family), Geobacter uraniireducens (Geobacteraceae family), Agrobacterium tumefaciens (Rhizobiaceae family), and Cytophaga hutchinsonii (“Flexibacteraceae” family), reveal some ORFs that may correspond to class D β-lactamases. However, due to the lack of adequate cloning experiments, the ability of all these putative proteins to act as β-lactamases has not yet been demonstrated. These observations, however, suggest that such ORFs may play a physiological but still unidentified role in many gram-negative organisms.

GENETIC PLATFORMS FOR ACQUIRED CLASS D β-LACTAMASE GENES

Most of the class D β-lactamase genes identified in Enterobacteriaceae and Pseudomonas species are located on large, transferable plasmids. Some class D β-lactamase genes found in P. aeruginosa, such as those encoding OXA-13, OXA-17, OXA-18, and OXA-20, have been found to be chromosomally located (37, 99, 108, 118). However, negative results from mating-out assays and the lack of plasmid detection in P. aeruginosa do not rule out plasmid origins for a class D β-lactamase gene.

Most plasmid-located class D β-lactamase genes have been found in association with genetic vehicles responsible for their mobilization. These vehicles are mobile elements, often transposons belonging to the Tn3 family (10, 82). These transposons enable integrons (which are not self-mobile elements) and, consequently, their associated resistance gene cassettes to diffuse and spread. Integrons are genetic structures capable of integrating individual genes in the form of gene cassettes (61).

Among class D β-lactamase genes which have been identified in the form of gene cassettes (140), the most common are the bla_OXA-1-like, bla_OXA-2-like, and bla_OXA-10-like genes (107). There are peculiar features associated with the bla_OXA-9 gene identified in a plasmid from a K. pneumoniae isolate (155), since the bla_OXA-9 gene was in the form of a gene cassette but, instead of being associated with an integron, was part of the Tn3-type transposon Tn1331 also carrying the bla_TEM-1 gene and lacking an integrase gene.

Besides all these bla_OXA gene cassettes, there are other bla_OXA genes which have been identified in other genetic environments. The bla_OXA-23 gene originating from the chromosome of A. radioresistens may be mobilized onto plasmids diffusing into A. baumannii through different genetic structures, such as the composite transposon Tn2006, formed by two ISAba1 elements (30, 100). Alternatively, ISAba4 was found upstream of the bla_OXA-23 gene in a single copy (14, 30).

The bla_OXA-48 gene has been identified in association with insertion sequence IS1999 in K. pneumoniae (127). The bla_OXA-48 gene was part of composite transposon Tn1999, made of two copies of IS1999, with the copy located upstream of bla_OXA-48 providing promoter sequences for bla_OXA-48 expression (7).

The bla_OXA-58 gene, which has been identified only in Acinetobacter sp. isolates, has been associated with a variety of different genetic structures. By analyzing a series of OXA-58-positive A. baumannii isolates recovered in different countries (France, Spain, Romania, Greece, and Turkey), 11 distinct structures were identified (128, 130, 132). Nevertheless, there are always common features, since bla_OXA-58 is always bracketed by two ISAba3 elements. The ISAba3 copy upstream of bla_OXA-58, as well as other insertion sequences, such as IS18, ISAba1, and ISAba2, may provide promoter sequences playing a role in bla_OXA-58 expression (132). For the A. baumannii isolate MAD, which has been at the origin of a clonal outbreak in a French hospital (64, 130), we demonstrated that the entire structure encompassing the ISAba2-bla_OXA-58-ISAba3 fragment had very likely integrated into the plasmid backbone by a recombination process (132). In addition to being dependent on IS elements, the level of expression of the bla_OXA-58 gene may be related to the gene copy number, as exemplified in one Italian clone exhibiting various levels of resistance to imipenem as a consequence of the bla_OXA-58 copy number (13).

Finally, another mechanism of acquisition of class D β-lactamase genes has been identified recently. It is mobilization by peculiar IS elements named ISCRs, which mobilize adjacent sequences by rolling-circle (RC) transposition (151, 152). bla_OXA-18 gene acquisition is likely the result of RC transposition mediated by the ISCR19 element (105), and the aquisition of the bla_OXA-45 gene is likely mediated by ISCR5 (154).

DETECTION OF CLASS D β-LACTAMASE GENES

One of the major concerns for controlling the spread of class D β-lactamase producers is the absence of phenotypic tests that could contribute to their easy recognition. Although tools are available for the detection of class A ESBLs (clavulanic acid-based synergy tests), metallo-β-lactamases (EDTA-based approaches), and AmpC enzymes (cloxacillin-based tests), there is no phenotypic tool allowing rapid and easy detection of class D β-lactamase producers. However, some class D β-lactamases possess peculiar properties that may facilitate their detection. For example, OXA-13 and its extended-spectrum variant OXA-19 both possess the ability to be inhibited by imipenem (98). Once the enzyme is produced in P. aeruginosa, it is possible to demonstrate this characteristic by placing a disk of imipenem next to one of cefsulodin, a substrate well hydrolyzed by OXA-13 (99). We have observed that this specific property is shared by other class D β-lactamases, such as those belonging to the OXA-10 subgroup (unpublished data). Also, the production of some OXAs whose activity is inhibited by clavulanate or tazobactam, such as OXA-12 from A. jandaei (139) and OXA-18, OXA-45, and OXA-46 from P. aeruginosa (57, 118, 153), may be identified by synergy tests using clavulanic acid-containing disks. However, the result of these synergy tests may be misinterpretated and the corresponding isolates may be considered to be class A ESBL producers.

One alternative approach that may be performed in specialized laboratories is spectrophometric analysis. By using crude extracts and UV spectrophotometry, it is possible to evaluate the abilities of β-lactamase extracts to hydrolyze oxacillin. It is also possible to use the NaCl inhibition property with a referenced substrate such as benzylpenicillin. However, as indicated above, there are some limits in this approach since (i) some class D β-lactamases do not hydrolyze oxacillin, (ii) the in vitro inhibition of their activities by NaCl is not always observed, and (iii) OXA-positive clinical isolates often express additional non-class D β-lactamases. In addition, many CHDLs hydrolyze carbapenems at a low level, interfering negatively with the sensitivity of the technique. PCR-based methods remain, therefore, the “gold standard” for the identification of class D β-lactamases (133, 167).

CONCLUSIONS

OXA β-lactamases are distributed in a large variety of gram-negative species. Whereas their acquisition is detected mostly in Enterobacteriaceae, Pseudomonas spp., and Acinetobacter spp., naturally occurring chromosomal genes are increasingly discovered in gram-negative organisms, such as Aeromonas, Legionella, Shewanella, and Campylobacter species. The identification of the progenitor of a clinically significant OXA is an exciting perspective, since it may help to better control the sources of emerging class D β-lactamases, to better evaluate their dissemination, and eventually to better control their spread.

According to Barlow and Hall (11), this diversity of class D β-lactamases, at least those whose genes have been incorporated onto plasmids, is the result of ancient events that occurred millions of years ago. That hypothesis is debatable, since for example, the widely distributed OXA-23 determinant has been found with almost perfect identity in the chromosome of an A. radioresistens strain which corresponds to its natural reservoir. It is very likely that the diversity of plasmid-borne bla_OXA genes simply reflects the diversity of their natural progenitors.

It is likely that antibiotic selective pressure is a driving force for the spread of several bla_OXA genes. The bla_OXA genes identified as having been aquired are often expressed at high levels, and in contrast, the genes are very often silent in their natural progenitors. Mobilization elements (e.g., integrons and IS elements) contribute to the spread and expression of these genes by providing strong promoters. Future work may be directed toward the role of antibiotic concentrations in given environments as a source of enhancement of the spread of bla_OXA genes from their reservoirs to secondary hosts.

From a clinical point of view, these OXA β-lactamases should be considered a threat similar to β-lactamases of the other Ambler classes, since many of them possess an expanded spectrum of activity. Currently, CHDLs in A. baumannii, ES-OXAs in P. aeruginosa, and the OXA-48 CHDL in Enterobacteriaceae represent potential sources of clinical failure for many β-lactams (70a). Lack of detection may enhance their hidden and rapid spread among clinical isolates.