In117, an Unusual In0-Like Class 1 Integron Containing CR1 and bla_CTX-M-2 and Associated with a Tn21-Like Element

Abstract

An unusual In0-like class 1 integron containing a common region that includes the putative recombinase gene named orf513 (CR1) and bla_CTX-M-2 was characterized from Escherichia coli. The integron contained an unusual gene cassette array, estX-aadA1, embedded between the 5′-conserved segment (5′-CS) and 3′-CS1 regions and was flanked by mer-Tn21 sequences downstream of the tni truncated module. This element constitutes one of the few examples of CR1-bearing class 1 integrons that has been fully characterized.

The CTX-M enzymes are among the most widespread extended-spectrum β-lactamases of Ambler class A (5). Five clusters of CTX-M β-lactamases (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25) have been described on the basis of their amino acid sequences (http://www.lahey.org/studies/webt.htm). Chromosomal genes from Kluyvera species have been identified as progenitors of each CTX-M group, and mobilization seems to have occurred by the association of these genes to CR1, ISECp1, or phage-related elements (3, 5, 16, 25). The bla_CTX-M-2 and bla_CTX-M-9 genes have been mainly associated with class 1 integrons containing CR1. Their backbone structure consists on the 5′-conserved segments (5′-CS) and 3′-CS flanking variable gene cassette arrays, CR1, several antibiotic resistance genes that do not resemble gene cassettes, and a second partial copy of the 3′-CS designated 3′-CS2 (2, 5, 24). The sequences upstream of the 5′-CS and beyond the second copy of qacEΔ1 at the 3′-CS2 have been described only for In6 and In34 (19). The aim of this work was to characterize the genetic environment of bla_CTX-M-2 in Escherichia coli strain VS27, one of the few CTX-M-2-producing isolates described in Spain.

E. coli VS27 (resistant to β-lactams and streptomycin, sulfonamide, tetracycline, nalidixic acid, and ciprofloxacin) was recovered from the feces of a healthy volunteer without recent hospitalization or antibiotic exposure in 2003 (29). Transfer of bla_CTX-M-2 by broth and filter-mating methods using E. coli BM21R (nalidixic acid and rifampin resistant, lactose fermentation positive, and plasmid free) or E. coli HB101 (kanamycin and azide resistant, lactose fermentation negative, and plasmid free) as the recipient strain was unsuccessful. The whole-plasmid profile determined by the Kado and Liu method using E. coli V517 and E. coli NCTC 50192 as control strains for the estimation of plasmid sizes consisted of three plasmids of 70, 40, and 5 kb and three plasmids of less than 3 kb (27). Hybridization of plasmid DNA (26) with an intragenic bla_CTX-M-2 probe labeled and detected by ECL kits according to the manufacturer's instructions (Amersham Life Sciences, Uppsala, Sweden) was negative.

Although bla_CTX-M-2 has been previously associated with class 1 integrons bearing CR1, these elements have been only partially characterized (2, 3, 23). An overlapping PCR assay based on the sequence of In35 containing bla_CTX-M-2 and Tn21, often associated with class 1 integrons, was designed (GenBank accession numbers AY079169 and AF071413) (3, 12, 13) (Fig. 1). PCR assays were performed in volumes of 50 μl with a mixture containing 1.5 mM MgCl₂, 0.2 mM of each deoxynucleoside triphosphate, 0.1 μM of each primer, and 1.5 units of Taq DNA polymerase (AmpliTaq Gold; PE Applied Biosystems, Norwalk, Conn.) for 12 min at 94°C and for 35 cycles at 94°C (1 min), 56 to 65°C (1 to 2 min), and 72°C (1 to 3 min) followed by a final step for 10 min at 72°C for standard PCR assays and with a mixture containing 2.5 mM MgCl₂, 0.1 μM of each primer, and 2.5 units of Takara LA Taq polymerase (Takara Bio Inc, Shiga, Japan) for 1 min at 94°C and for 35 cycles of 96°C (20 s), 60°C (1 min), and 72°C (3 to 5 min) followed a final step for 10 min at 72°C for long PCRs (>3 kb). Amplified products were purified using the QIAquick PCR purification kit (QIAGEN) and sequenced on an ABI Prism 377 automated sequencer (PE Applied Biosystems). The oligonucleotide sequences used in PCR assays are listed in Table 1.

FIG. 1.

Schematic representation of the bla_CTX-M-2 genetic loci. A comparison with other gene array cassettes located within the 5′-CS-3′-CS1 region described to date is represented at the bottom of the left side (2). The location of the primers used for the identification of the element by PCR-overlapping assay are represented with black arrows. Vertical bars symbolize inverted repeats of the integron (gray) or Tn21 (black). Gray shaded open reading frames represent the 20,032-bp region fully sequenced, with 15,882 bp corresponding to In117. Circles represent 59-bp elements of the corresponding gene cassettes. GenBank accession numbers are in parentheses.

TABLE 1.

Oligonucleotides used in this study

Primer no.	Primer	Sequence	Positions	GenBank accession no.	Reference or source
1	5′-CS	5′-GGCATCCAAGCAGCAAG-3′	5298-5314	AF071413	11
2	3′-CS	5′-AAGCAGACTTGACCTGAT-3′	6306-6289	AF071413	11
3	aadA1F	5′-GCTGGCCGTGCATTTGTACG-3′	5487-5506	AF071413	This study
4	ORF513rF1R	5′-GAGCTCTGCACCATCCCAC-3′	527-507	AY079169.1	This study
5	qacEΔ2	5′-ATCGCAATAGTTGGCGAAGT-3′	6383-6392	AF071413	This study
6	ORF513-4F	5′-CTCGCTTGAGGCGTTGCAT-3′	2106-2088	AY079169.1	This study
7	ORF513-4R	5′-ATGCAACGCCTCAAGCGAG-3′	2088-2106	AY079169.1	This study
8	CTX-M-2R/P2b	5′-TCCCGACGGCTTTCCGCCTT-3′	3655-3637	AY079169.1	31
9	CTX-M-2F/P3	5′-ATGATGACTCAGAGCATTCG-3′	2823-2842	AY079169.1	31
10	qacEΔ1B	5′-CAAGCTTTTGCCCATGAAGC-3′	5050-5031	AY079169.1	This study
11	orf5-R	5′-AGTTCTAGGCGTTCTGCG-3′	8157-8140	AF071413	This study
12	orf5-F	5′-CGATATCGACGAGGTTGTGC-3′	7712-7730	AF071413	This study
13	IS1326-F	5′-TACCGGGTCTTATGACCGAGT-3′	10357-10337	AF071413	This study
14	IS1326-R	5′-ACTGTCATAGCGGTTCACGTT-3′	9141-9161	AF071413	This study
15	tniBΔ1F	5′-ATCATCGACCTGTCCCACCT-3′	13201-13182	AF071413	This study
16	tniBΔ1R	5′-AGGTGGGACAGGTCGATGAT-3′	13182-13201	AF071413	This study
17	tniAF	5′-TCGTGCGGAGATCATCAGTCC-3′	14821-14801	AF071413	This study
18	merA1	5′-ACCATCGGCGGCACCTGCGT-3′	17597-17578	AF071413	13
19	merA5	5′-ACCATCGTCAGGTAGGGGAACAA-3′	16360-16382	AF071413	13
20	merR1	5′-GCGGATTTGCCTCCACGTTGA-3′	19278-19260	AF071413	13
21	merT1	5′-CCAGGCAGCAGGTCGATGCAAG-3′	19055-19076	AF071413	13
22	Tn21IR/38	5′-GGGCACCTCAGAAAACGGAAA-3′	19669-19649	AF071413	14
23	TnpR-Fa	5′-ATGCTATGCACCACCACGG-3′	3376-3394	AF071413	14
24	intF1	5′-GGGTCAAGGATCTGGATTTCG-3′	4774-4754	AF071413	This study
25	merA6	5′-GCCGACCAGTTGTTCCCCTACCTGACG-3′	16391-16365	AF071413	13
26	merD1	5′-CGCACGATATGCACGCTCACCC-3′	16211-16233	AF071413	13
27	merA0	5′-GTCGCAGGTCATGCCGGTGATTTT-3′	178950-17974	AF071413	13
28	merP1	5′-GGCTATCCGTCCAGCGTCAA-3′	18520-18501	AF071413	13
29	merC1	5′-CATCGGGCTGGGCTTCTTGAG-3′	18361-18351	AF071413	13
30	merC2	5′-CATCGTTCCTTATTCGTGTGG-3′	17987-18007	AF071413	13
31	IRIn2R	5′-TGGTGCAGTCGTCTTCTGAAAA-3′	15012-15033	AF071413	14
32	tniAR	5′-GGACTGATGATCTCCGCACGA-3′	14801-14821	AF071413	This study
33	IRTn21Fa	5′-GGGTCGTCTCAGAAAACGG-3′	1-38	AF071413	This study
34	TnpR-Ra	5′-CCGTGGTGGTGCATAGCAT-3′	3394-3376	AF071413	This study
35	IRIn2F	5′-TTTCAGAAGACGGCTGCACTG-3′	4046-4066	AF071413	14

^aPrimers 23, 33, and 34 were used in combination with primers 24 and 35 in order to characterize the 5′ end of In117 using appropriate controls.

Analysis of the 15,882-bp sequence between inverted repeats of Tn402 located upstream of intI1 and downstream of tniA revealed the presence of an integron belonging to the In0 group that we called In117. The 5′-CS region includes a copy of the integrase intI1 with a P_c promoter identical to that of In1 in R46 (GenBank accession number AY046276), consisting of TGGACA(−35) and TAAACT(−10) hexamers separated by 17 bp (22). This P_c promoter is of intermediate strength in In1, and although it has been described less frequently than P_c promoters of weak or strong strength, it is increasingly being found in specific class 1 integrons carrying bla_IMP, bla_VIM, or bla_GES (10, 28, 30). A gene cassette array, estX-aadA1, within the 5′-CS-3′-CS1 region was identified. The deduced amino acid sequence of the estX gene displayed 90% amino acid identity with sat-1 of Tn1825 conferring resistance to streptothricin, 40% amino acid identity with proteins annotated as putative esterases or hydrolases of the α/β fold superfamily, and 42% amino acid identity with a protein encoded in E. coli multiresistance plasmids. The estX gene was initially considered to be a sat cassette because of its similarity with sat-1; however, Partridge and Hall have demonstrated that sat-1 resulted from the fusion of estX and sat-2 genes, suggesting a change in the nomenclature of these genes (18). The estX-aadA1 gene cassette combination has not been previously linked to CTX-M-2-producing isolates, although it has been associated with class 1 integrons from Shigella sonnei clinical strains, and estX has been found in different class 1 and class 2 integrons from community isolates at different locations (1, 4, 8).

The 5,585-bp region from 3′-CS1 to the second copy of qacEΔ1 in 3′-CS2 showed 100% homology with that of In35 (GenBank accession number AY079169). The extent of 3′-CS2 was identified as a 6,900-bp sequence with 100% homology with class 1 integron In0 (GenBank accession number U49101). This sequence includes the typical 3′-CS (qacEΔ1, sul1, and orf5) followed by the insertion sequence IS1326, a member of the IS21 family, and a truncated tni module of Tn402 (7). Although several members of the IS21 family are widely distributed, IS1326 remains associated with the class 1 integron lineage In0-In2-In5 (7, 19, 22), which differ one from another in the promoter of intI1 and in the truncated tni module sequences originating from the insertion of IS1326 and further deletion events. Tn21 sequences (left inverted repeats and tnpR) upstream of intI1 were not detected. Interestingly, amplification with primers specific for the mer locus and inverted repeats of Tn21 and the integron showed the presence of mer-Tn21 sequences downstream of tniA (Fig. 1) (12, 32).

Our results revealed the presence of bla_CTX-M-2 in a defective transposon derivative of the Tn402 family and constitute, besides In34 and In6, one of the few examples of class 1 integrons containing CR1 in which the structure beyond the 3′-CS2 has been established (19). In0, In2, and In5 are Tn402 derivatives located in plasmids and/or transposons, often in mercury resistance transposons such as Tn21. These transposons are considered to be a worldwide disseminated population composed of a few variants shared by gram-negative environmental and clinical bacteria (32). The absence of a Tn21-like transposition module upstream of intI1 was not surprising, since Tn21 subgroup transposons are frequently inactivated or yield mosaic structures by exchanging transposition modules by recombination at the res site (15, 20, 32). The great polymorphism within the 5′-CS-3′-CS1 region of integrons carrying bla_CTX-M-2 (2) suggests recombinatorial exchange either among cassettes of different class 1 integrons or among CR1 and class 1 integrons containing the 3′-CS1 (6, 17, 19, 21).

The presence of bla_CTX-M-2 in Spain increases the diversity of bla_CTX-M genes described in our area, already epidemic for those of CTX-M-9 (bla_CTX-M-9 and bla_CTX-M-14) and CTX-M-1 (bla_CTX-M-1, bla_CTX-M-3, bla_CTX-M-10, bla_CTX-M-15, and bla_CTX-M-32) clusters (9; unpublished results). The genetic elements containing bla_CTX-M-2 may fuel the dissemination of this gene, as has recently occurred for carbapenemase genes in Europe (28) or bla_CTX-M-9 in Spain (unpublished results), both of which are associated with composite transposon platforms.

Nucleotide sequence accession number.

The sequence for In117 was deposited in the GenBank database under accession number DQ125241.