The Novel CTX-M-116 β-Lactamase Gene Discovered in Proteus mirabilis Is Composed of Parts of the CTX-M-22 and CTX-M-23 Genes

Abstract

The novel β-lactamase gene bla_CTX-M-116 was identified in a Proteus mirabilis nosocomial isolate recovered from the urine of a patient in Moscow in 2005. DNA sequence analysis showed bla_CTX-M-116 to be a hybrid gene consisting of 5′ bla_CTX-M-23 (nucleotides 1 to 278) and 3′ bla_CTX-M-22 (nucleotides 286 to 876) moieties separated by an intervening putative site of recombination (GTTAAAT). A retrospective analysis of available bla_CTX-M genes in the GenBank database revealed 19 bla_CTX-M genes that display the same hybrid structure.

TEXT

CTX-M extended-spectrum β-lactamases (ESBLs) are a major cause of β-lactam resistance among Gram-negative bacteria worldwide (1–3). A total of 130 CTX-M sequence types have been identified (www.lahey.org/studies) grouped into five major clusters (CTX-M-1, -2, -8, -9, and -25) based on their amino acid sequences (4). The CTX-M-1 cluster is the most diversified group and also includes the most prevalent CTX-M variants described to date (5, 6). The family of bla_CTX-M genes is an excellent model for studying evolution in real time (7–9). It has been shown in natural CTX-M variants and in phylogenetic reconstruction experiments that new CTX-M enzymes arise by point mutation of their genes (10). One clear example of a recombination event that generated a novel bla_CTX-M variant found in Shigella sonnei is bla_CTX-M-64, which evolved as a result of homologous recombination between the bla_CTX-M-15-like gene (N- and C-terminal moieties) and the bla_CTX-M-14-like gene (central moieties) (11).

In this report, we describe a novel bla_CTX-M-116 gene that was identified in Proteus mirabilis strain K-27 (State Collection of Pathogenic Microbes—Obolensk, accession number B-6773) isolated from the urine of a patient in the Urology Unit of the Sechenov Moscow Medical Academy in March 2005 (12). The bacterial strain was resistant to ampicillin (MIC > 256 μg/ml), ampicillin/sulbactam (MIC = 32 μg/ml), cefuroxime (MIC > 256 μg/ml), cefoxitin (MIC = 32 μg/ml), cefotaxime (MIC = 32 μg/ml), cefepime (MIC = 32 μg/ml), gentamicin (MIC = 32 μg/ml), ciprofloxacin (MIC = 256 μg/ml), doxycycline (MIC > 256 μg/ml), chloramphenicol (MIC = 128 μg/ml), and trimethoprim-sulfamethoxazole (MIC = 16/304 μg/ml); showed intermediate resistance to aztreonam (MIC = 2 μg/ml); and was sensitive to ceftazidime (MIC = 0.25 μg/ml), meropenem (MIC = <0.12 μg/ml), and amikacin (MIC = 0.5 μg/ml) as determined by broth microdilution in Mueller-Hinton broth (Difco) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (13). Genetic determinants carried by Proteus mirabilis K-27 revealed by PCR using specific primers (12, 14–19) included bla_TEM, ISEcp1, bla_CTX-M, class 1 integrase, and an integron insertion carrying the aadA1 gene. A specific 45-bp sequence was located between the ISEcp1 and bla_CTX-M-116 sequences (20). The PCR was carried out in a 25-μl reaction mixture using GradientPalmCycler (Corbert Research, Australia).

The novel bla_CTX-M-116 gene was first identified as bla_CTX-M-23 using a PCR-restriction fragment length polymorphism (PCR-RFLP) algorithm (21). However, DNA sequence analysis using Vector NTI 9 software (Invitrogen) and nucleotide alignments using the BLAST Web resource (http://blast.ncbi.nlm.nih.gov/Blast.cgi) showed that only the 5′ moiety (nucleotides 1 to 278, starting from the start site of translation) of bla_CTX-M-116 was identical to the corresponding segment of bla_CTX-M-23 (including specific single nucleotide polymorphisms [SNP] in positions 21, 138, 139, and 239), while the 3′ moiety (nucleotides 286 to 876) matched bla_CTX-M-22 (including specific SNP in positions 313, 315, 508, and 609), suggesting that bla_CTX-M-116 originated through a recombination event. The putative 7-bp site of recombination (GTTAAAT) is located between the bla_CTX-M-23-associated 5′ moiety and the bla_CTX-M-22-associated 3′ moiety from nucleotide 279 to nucleotide 285 (Fig. 1A). So we propose that the bla_CTX-M-116 gene originated as a result of recombination between bla_CTX-M-23 and bla_CTX-M-22.

Fig 1.

Alignments of the CTX-M-116 nucleotide sequence (A) and amino acid sequence (B) with those of CTX-M-23 and CTX-M-22. Nucleotides in positions bp 50 to 100, bp 550 to 600, and bp 651 to 876 are not shown because of 100% identity. Bold type highlights specific nucleotides and amino acids; the proposed core site at nucleotides 279 to 285 is indicated by gray shading. The amino acids are numbered according to the standard numbering scheme for the class A β-lactamases of Ambler et al. (26). The underlined area represents the active-site omega loop. Bold type in gray boxes shows specific amino acids.

The hybrid bla_CTX-M-116 gene confers a phenotype of resistance that differs from the phenotypes of both bla_CTX-M-23 and bla_CTX-M-22 genes. The CTX-M-23 β-lactamase contains the amino acid substitution at position 167 (Pro to Thr) associated with ceftazidime-hydrolyzing activity resulting in an elevated MIC of ceftazidime (MIC = 64 to 128 μg/ml), but a lower MIC of cefotaxime (MIC = 2 to 16 μg/ml) (22). However, the CTX-M-22 β-lactamase, a variant of CTX-M-1, typically hydrolyzes cefotaxime (MIC = 64 μg/ml) more efficiently than ceftazidime (MIC = 2 μg/ml) (23). Consequently, the cephalosporin resistance profile of P. mirabilis K-27 (cefotaxime MIC = 32 μg/ml and ceftazidime MIC = 0.25 μg/ml) conferred by the hybrid bla_CTX-M-116 gene may be influenced by the amino acid at position 167 (Pro) of the CTX-M-22 enzyme because of just this SNP located at the active-site omega loop (Fig. 1B).

Comparison of available bla_CTX-M sequences (http://www.lahey.org/studies/other.asp#table1 and GenBank) showed that three other bla_CTX-M genes are organized in a similar manner with 5′ and 3′ moieties matching two different bla_CTX-M genes: bla_CTX-M-57 includes 5′-bla_CTX-M-52 and 3′-bla_CTX-M-15; bla_CTX-M-79 includes 5′-bla_CTX-M-52 and 3′-bla_CTX-M-28; and bla_CTX-M-89 includes 5′-bla_CTX-M-25 and 3′-bla_CTX-M-39 (Table 1). Moreover, 13 bla_CTX-M genes belonging to cluster 1 and 3 genes from cluster 8/25 display similar basic hybrid structures with some differences. In these instances, while the 5′ moiety originated from other bla_CTX-M genes, the 3′ moiety is unique for the particular variant (Table 1). Finally, there are bla_CTX-M genes from cluster 1 and cluster 8/25 with an entirely unique sequence for the particular variant (Table 1). The contribution of 5′ and 3′ moieties may be more disseminated for some bla_CTX-M genes than others. For example, the 5′ and 3′ moieties associated with the gene encoding the epidemic CTX-M-15 enzyme that is globally disseminated among bacterial pathogens (6) were identified in 8 and 4 genes, respectively (Table 2). The basic hybrid structure of bla_CTX-M genes was found only in genes belonging to clusters 1 and 8/25, but not in genes in clusters 2 and 9. Proposed sites of recombination are specific for bla_CTX-M cluster 1 (GTTAAAT) and cluster 8/25 (GTTGAGT) (Table 1).

Table 1.

5′ moiety-proposed site of recombination-3′ moiety structure of bla_CTX-M genes^a

blaCTX-M gene (gene no.)	GenBank accession no.	5′ moiety (gene no.)	RS (oligonucleotides)	3′ moiety (gene no.)
Cluster 1
1	X92506	1	GTTAAAT	1
10	AF255298	10	GTTAAAT	10
11	AY005110	11	GTTAAAT	11
12	AF305837	12	GTTAAAT	12
15	AY044436	15	GTTAAAT	15
23	AF488377	23	GTTAAAT	23
28	AJ549244	28	GTTAAAT	28
29	AY267213	29	GTTAAAT	29
30	AY292654	30	GTTAAAT	30
33	AY238472	33	GTTAAAT	33
36	AB177384	36	GTTAAAT	36
37	AY649755	37	GTTAAAT	37
52	DQ223685	52	GTTAAAT	52
53	DQ268764	53	GTTAAAT	53
60	AM411407	60	GTTAAAT	60
62	EF219134	62	GTTAAAT	62
66	EF576988	66	GTTAAAT	66
68	EU177100	68	GTTAAAT	68
80	EU202673	80	GTTAAAT	80
82	DQ256091	82	GTTAAAT	82
101	HQ398214	101	GTTAAAT	101
114	GQ351346	114	GTTAAAT	114
3*	Y10278	15	GTTAAAT	3
22*	AY080894	15	GTTAAAT	22
32*	AJ557142	1	GTTAAAT	32
34*	AY515297	10	GTTAAAT	34
42*	DQ061159	15	GTTAAAT	42
54*	DQ303459	15	GTTAAAT	54
58*	EF210159	1	GTTAAAT	58
61*	EF219142	1	GTTAAAT	61
69*	EU402393	52	GTTAAAT	69
71*	FJ815436	15	GTTAAAT	71
72*	AY847148	15	GTTAAAT	72
88*	FJ873739	15	GTTAAAT	88
96*	AJ704396	12	GTTAAAT	96
57**	DQ810789	52	GTTAAAT	15
79**	EF426798	52	GTTAAAT	28
116**	JF966749	23	GTTAAAT	22
Cluster 8/25
8	AF189721	8	GTTGAGT	8
25	AF518567	25	GTTGAGT	25
26	AY157676	26	GTTGAGT	26
40	AY750914	40	GTTGAGT	40
63	AB205197	63	GTTGAGT	63
91	GQ870432	91	GTTGAGT	91
39*	AY954516	26	GTTGAGT	39
41*	DQ023162	26	GTTGAGT	41
94*	HM167760	26	GTTGAGT	94
89**	FJ971899	25	GTTGAGT	39

^a*, the sequence of the 5′ moiety is contributed by another bla_CTX-M gene, but the sequence of the 3′ moiety is unique.

^**, both the 5′ and 3′ moieties are contributed by other bla_CTX-M genes. RS, proposed site of recombination.

Table 2.

Prevalence of 5′ and 3′ moieties among bla_CTX-M genes

5′ moiety of blaCTX-M (gene no.)	No. of genes	3′ moiety of blaCTX-M (gene no.)	No. of genes
Cluster 1
15	8	15	4
1	4	3	3
52	4	28	2
12	2	12	2
23	2	22	2
10	2
Cluster 8/25
26	4	39	2
25	2

Since the presence of two different genes is needed for the hybrid recombination event, it is interesting that two and three different CTX-M genes in the same strain were described in some studies, namely, in 5.3% (24), 10.4% (25), and 1.0% (12) of CTX-M-positive Enterobacteriaceae isolates.

In conclusion, we describe the novel hybrid bla_CTX-M-116 gene and propose that it was derived through the recombination of bla_CTX-M-23 and bla_CTX-M-22. Thus, recombination is a powerful mechanism driving the evolution of resistance genes encoding CTX-M β-lactamases along with the accumulation of point mutations.

Nucleotide sequence accession number.

The nucleotide sequence of the bla_CTX-M-116 gene is available under GenBank accession number JF966749.