Characterization of the Genetic Environment of the bla VEB-4 Gene,Associated with a Transposable Region in a Proteus mirabilis Clinical Isolate

Introduction

Proteus mirabilis is the second most common cause of urinary tract infections, such as symptomatic cystitis and pyelonephritis, and is present in cases of asymptomatic bacteriuria, particularly in the elderly and patients with type 2 diabetes. P. mirabilis is also an important cause of nosocomial infections, infection in the respiratory tract, eye, ear, nose, skin, throat, burns, and wounds, and has been implicated in neonatal meningoencephalitis, empyema, and osteomyelitis. ¹

Resistance to extended-spectrum cephalosporins in P. mirabilis is mainly due to the production of extended-spectrum β-lactamases (ESBLs) that belong to Ambler class A. TEM-type and CTX-M-type ESBLs are the most widely distributed among this species, but minor ESBLs, such as the VEB type, have also been identified. ²

VEB-type β-lactamases, which represent one of the smaller subgroups of class A β-lactamases,^3,4 classified as group 2be enzymes ⁵ are a rare type of enzyme responsible for conferring high-level resistance to ceftazidime, cefotaxime, and aztreonam, although not carbapenems. There are 16 variants of VEB enzymes reported to date, derived from VEB-1 by one or more amino acid substitutions (www.lahey.org/studies/other.asp#table1). The bla_VEB genes have been isolated from various species of Enterobacteriaceae and nonfermenter gram-negative bacilli (Pseudomonas aeruginosa, Acinetobacter baumannii, Achromobacter xylosoxidans)^4,6–8 from Southeast Asia, Europe, the Middle East, Africa, and North and South America. ⁹ In P. mirabilis three variants have been reported: VEB-1, ² VEB-4, ¹⁰ and VEB-6. ¹¹ They are usually inserted within the variable regions of class 1 integrons and horizontally transmitted at the intra- and interspecific levels. ¹² Genetic analysis of bla_VEB-1 has revealed both chromosome and plasmid locations. ¹³ Interestingly, bla_VEB-1 has also been reported on a class 1 integron located in an 86 kb resistance island. ¹⁴ The bla_VEB-1 gene was first reported in an Escherichia coli isolate from a Vietnamese patient, where it was plasmid- and integron-located, ¹⁵ and subsequently in two P. aeruginosa isolates from Thailand, where it was chromosomally and integron-located. ¹¹

We report in this study the genetic environment of the bla_VEB-4 gene in a P. mirabilis clinical isolate N2231 recovered from a patient treated in a Spanish hospital.

Materials and Methods

Results

Genetic location of the bla_VEB-4 gene

Genomic DNA digestion with S1 nuclease and subsequent PFGE analysis and hybridization with the bla_VEB probe were negative, pointing to a chromosomal localization for this gene. The bla_VEB-4 chromosomal location was screened by Southern hybridization using the I-CeuI-PFGE profile with bla_VEB and 16S rDNA probes. bla_VEB and 16S rDNA PCR amplicons hybridized in the same ca. 291 kb I-CeuI fragment, suggesting that bla_VEB-4 was located on the chromosome of P. mirabilis N2231 (Fig. 1).

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FIG. 1.

Southern hybridization of I-CeuI-PFGE. a, PFGE-I-CeuI; b, hybridization with 16S rDNA probe; c, hybridization with bla_VEB probe, M, lambda ladder PFE marker. PFGE, pulsed-field gel electrophoresis.

Genetic context of the bla_VEB-4 gene

Sequence analysis derived from the inverse PCR and PCR mapping in P. mirabilis N2231 revealed that the obtained 15 kb segment corresponded to the multidrug resistance (MDR) region of a Salmonella genomic island 1 (SGI1), and included a previously described complex class 1 integron. ¹⁹ The bla_VEB-4 described herein was located in a similar integron as part of a truncated gene cassette in opposite orientation (Fig. 2). Interestingly, this 15 kb segment was 98% identical to those described by Siebor and Neuwirth ¹⁹ in a bla_VEB-6-producing P. mirabilis VB1248 (HQ888851) and 100% identical to the deposited sequence described by Zong et al., ² who also found bla_VEB-6 in the P. mirabilis JIE273 chromosome. The bla_VEB-4 gene was bracketed by two 135-bp repeated elements named Re1 and Re2, found in opposite orientation in comparison with the P. mirabilis VB1248. A 28-bp segment found between Re1 and the bla_VEB-4 was absent in the same region in the P. mirabilis VB1248. The structure found downstream of Re1 until the insertion sequence IS6100 (transposase) was identical to P. mirabilis VB1248 (Fig. 2).

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FIG. 2.

Schematic view of the genetic context of the bla_VEB-4 gene in Proteus mirabilis N2231 and comparative structures with the MDR region within the SGI1 in P. mirabilis SGI1-PmSCO (GenBank accession number JX121639), P. mirabilis VB1248 (SGI1-V) (GenBank accession number HQ888851) and P. stuartii B1. Conserved segments (5′-CS and 3′-CS). The repeat elements (Re1, Re2, and Re3). Regions of homology are shaded in gray scale.^19,28 MDR, multidrug resistance; SGI1, Salmonella genomic island 1.

In opposite orientation to bla_VEB-4, a 432 bp segment corresponding to a hypothetical protein was found, followed by the Tn1721Δ-like truncated tetracycline resistance transposon, including the tet(A) gene (encoding a class A tetracycline efflux protein), ²⁰ which was bracketed by the repeated elements Re2 and Re3. Upstream to Re3 was detected a third copy of a 3′-conserved segment (3′-CS1) and the conservative structure of a class 1 integron containing four gene cassettes: aacA4 and aadB genes (conferring resistance to aminoglycosides), dfrA1 (conferring resistance to trimethoprim), and orfC with unknown function. At the terminal left-hand end, a fragment of a 5′-conserved segment (5′-CS) containing intI1 (integrase) was found.

Discussion

The aim of this study was to characterize the genetic surroundings of the bla_VEB-4 gene found in the P. mirabilis N2231 clinical isolate. ¹⁰ The well-known VEB β-lactamase has a wide dissemination and an increasing number of novel variants recovered from humans or food indicate a rapid evolution and spread.^19,21 P. mirabilis isolates naturally lack bla genes on their chromosome and are therefore generally susceptible to all β-lactam antibiotic agents. However, a progressive increase of multiresistant strains has been occurring in this species, ²² which has demonstrated a great ability to acquire resistance genes, such as inhibitor-resistant TEM β-lactamases, ESBLs, and ampC β-lactamases. ²³

Sequence analysis of the genetic context of the bla_VEB-4 described herein revealed a 15 kb segment corresponding to an MDR region of a SGI1, including a previously described class 1 integron belonging to the In104 family. ¹⁹ SGI1-like variants have been detected among bacteria other than Salmonella, including P. mirabilis.^24–26 This MDR region included frequently described resistance genes, such as aacA4, aadB, sul1, dfrA1, and tet(A) in addition to the bla_VEB-4 and qnrA genes. Our results were very similar (98% identity) to those reported by Siebor and Neuwirth, ¹⁹ who found bla_VEB-6 in the MDR region of a SGI1-V from a P. mirabilis clinical isolate, and 100% identical to the reported sequence described by Zong et al. ² The latter found bla_VEB-6 in the P. mirabilis JIE273 chromosome in a truncated gene cassette, similarly flanked by three repeated elements Re1, Re2, and Re3, including the Tn1721Δ-like transposon and duplications of the 3′-CSs of a class 1 integron.

Siebor and Neuwirth ²⁶ have hypothesized that P. mirabilis is involved in the dissemination of SGI1, since they found that the same modifications of the SGI1 backbones occur in Salmonella. A schematic comparison of the MDR regions among P. mirabilis strains, N2231 from this study, SGI1-PmSCO, ²⁶ and VB1248¹⁹ (Fig. 2), might suggest the presence of an SGI1 element in our strain, considering the similarity of the structures, particularly with P. mirabilis VB1248 (98% identity). Interestingly, the bla_VEB-6 variant is essentially identical to bla_VEB-4, varying in only one nucleotide (A52G), which predicts only a conservative amino acid substitution (Ile18Val) in the leader peptide.

The bla_VEB-1 gene is often part of a gene cassette located in class 1 integrons. ²⁷ Worthy of mention, the immediate genetic environment (3′-CS-Re3-Tn1721Δ-Re2-bla_VEB-Re1-3′-CS) of the bla_VEB-4 in P. mirabilis N2231 included the 135-bp repeat elements (Re1, Re2, and Re3) bracketing the bla_VEB-4 gene and duplications of the 3′-CS of class 1 integrons. Notably, this structure is similar to that observed for bla_VEB-1a in P. aeruginosa, ⁶ bla_VEB-1b in P. stuartii B1, ²⁸ and bla_VEB-6 in P. mirabilis JIE273² and P. mirabilis VB1248¹⁹ (Fig. 2), suggesting that bla_VEB is associated with highly conserved genetic structures. In addition, the presence of bla_VEB in different bacterial species illustrates how resistance genes may spread via conjugative plasmids and/or integrons, ¹¹ as well as transposable elements. This is of concern in the context of dissemination of resistance genes via horizontal gene transfer.

The high similarity between these genetic contexts, particularly in three P. mirabilis strains with a chromosomally located bla_VEB gene, JIE273, ² VB1248, ¹⁹ and N2231 of our study, indicate a common ancestry, possibly mediated by SGI1 elements, taking into account that P. mirabilis is an important host of SGI1. ²⁶ The association of bla_VEB variants with MDR regions of SGI1, cassette arrays, repeat elements bracketing the bla_VEB genes, duplications of the 3′-CS of a class 1 integron, and components of transposons further support this hypothesis. The presence of these components could also play a role in the movement of the bla_VEB gene and suggest possible recombinations and rearrangements of a limited set of different elements, as described by Zong et al. ²

In addition, differences in genes at both ends of the bla_VEB structures could be explained by these recombination events, which can mediate large evolutionary jumps in bacterial genomes by rapidly spreading variants associated with increased virulence, fitness, and antibiotic resistance. Interestingly, a recent study reported transmission of bla_VEB-6 through the food chain to humans: Seiffert et al. ²¹ identified bla_VEB-6 in a P. mirabilis isolate from poultry meat in Switzerland. The bla_VEB-6 from that study had a nucleotide sequence identical to that found in the human VEB-6-positive P. mirabilis VB1248 reported in France.

This is the first report describing the genetic environment of the bla_VEB-4 gene, detected in a P. mirabilis N2231 clinical isolate from Spain. The chromosomally located bla_VEB-4 gene was inserted in a complex structure of a class 1 integron, which is part of an MDR region of an SGI1, possibly involved in the mobilization of the gene and homologous recombination.

This structure carried several genes whose products confer resistance to aminoglycosides, tetracycline, sulfonamides, and all tested β-lactams, except carbapenems. Therefore, multiresistant strains harboring several resistant genes and complex structures require special attention.