ABSTRACT
Eleven clinical class 1 integron-containing Pseudomonas aeruginosa isolates from Australia and Uruguay were investigated for the genomic locations of these elements. Several novel class 1 integrons/transposons were found in at least four distinct locations in the chromosome, including genomic islands. These elements seem to be undergoing successful dispersal by lateral gene transfer since integrons were identified across several lineages and more than one clonal line.
TEXT
Clinically, Pseudomonas aeruginosa is one of the most important nosocomial and opportunistic pathogens (4, 13). The acquisition of virulence factors and antibiotic resistance genes in recent years by this species has seen the evolution of pathogenic strains that are difficult to treat with antibiotics (2, 17). This acquired accessory genome includes integrons carrying a variety of gene cassettes, transposons, and genomic islands (GIs). Class 1 integrons are commonly highly mobilized, being found in plasmids, and conjugation is a major mechanism by which resistance genes are spread between cells and across genera. However, class 1 resistance integrons can also be found in chromosomes, frequently in GIs of pathogenic bacteria. Probably the best example of this is Salmonella genomic island 1 (SGI1), which was found in multiple serovars of Salmonella enterica (11). Although the association between class 1 integrons and GIs has been reported in other bacteria (5), including P. aeruginosa (8), the extent of the association in P. aeruginosa is less clear. Recently, we identified a transposon containing two class 1 integrons, Tn6060 (Fig. 1), in a genomic island (here referred to as GI1) of the P. aeruginosa clinical isolate 37308 (21). The cassette arrays in Tn6060 have been commonly reported elsewhere, although the genetic context is, in most cases, not known (19, 20, 24). To investigate whether Tn6060 or relatives are dispersed in P. aeruginosa, intI1-positive clinical isolates derived from Australia and Uruguay were examined. We identified novel class 1 integrons/transposons in multiple chromosomal locations in several distinct clonal lines, suggesting that nonplasmid lateral exchange of resistance regions may be common in P. aeruginosa.
Structure of Tn6162 in comparison to that of Tn6060. Numbered horizontal lines indicate regions common to Tn6060 and Tn6162. (A) Tn6060. This is a modified version of Fig. 1 from Roy Chowdhury et al. (21). See also accession number GQ161847. Top line depicts the transposon backbone in which the integron (bottom line) is inserted. Vertical arrow indicates the point of insertion. The filled vertical rectangles indicate inverted repeats (IRs) as shown. Filled horizontal arrows represent genes or operons and the direction of transcription. The filled diamond is the attI1 site, and the filled ovals are the attC sites. Gene designations are as described in the text. (B) Tn6162. The general organization is as for Tn6060.
P. aeruginosa isolates were recovered from three hospitals, two in Sydney, Australia (2010), and one in Montevideo, Uruguay (2008). Eleven intI1-positive isolates (nine from Sydney and two from Montevideo) known to carry class 1 integrons were screened for the presence of the transposition module of Tn6060 and for the insertion of it in GI1, as reported previously (21). Two isolates positive from this screen, C79 from Sydney and U09 from Montevideo, were selected to investigate the context of class 1 integrons via the construction of fosmid genomic libraries (21). These two isolates were epidemiologically unrelated and comprised two different genetic clones based on pulsed-field gel electrophoresis (PFGE) analysis (Table 1). Both clonal types were different from the P. aeruginosa strain 37308, in which Tn6060 was identified. Sequencing revealed that both C79 and U09 possessed two class 1 integrons and that all were contained within mercury resistance transposons. A class 1 integron was present in both strains that, like Tn6060, is linked to a Tn1403 transposition module (Fig. 1). The sequences of the class 1 integrons/transposons in each of C79 and U09 were identical despite their geographical and clonal origins, thus implying lateral transfer. This transposon was designated Tn6162 (Fig. 1). Also, Tn6162 transposons in both C79 and U09 were inserted into GI1 at the same location as Tn6060 in strain 37308 from Sydney (21). In both C79 and U09, PCR with appropriate primers (see Table S1 in the supplemental material) and sequencing confirmed that the GI in these strains is located in the same position in the chromosome as that reported in the cystic fibrosis strain PACS171b in which this GI (without an integron) was first found (7) and in the Tn6060-containing strain 37308. The cassette array common to Tn6162 in C79 and U09, aadA6-gcuD (formerly orfD), is different from both of the cassette arrays in Tn6060 (21) (Fig. 1). This array was recovered in a P. aeruginosa isolate from France in 1998 (17), although the sequence context in which the array was found was not reported.
Features of class 1 integron-containing P. aeruginosa isolates
Based on the information obtained from C79 and U09, the remaining nine isolates were examined for the presence of Tn6162 by sequencing or PCR analysis (Table 1). All nine possessed Tn6162 in GI1. The second Uruguay isolate was the same PFGE clonal type as U09. Seven of the eight remaining Sydney isolates were the same PFGE clonal type as C79, with one comprising another distinct grouping based on PFGE, again implying lateral movement of this GI and associated resistance region between strains.
The second class 1 integron and surrounding sequence in strains C79 and U09 were recovered from appropriate fosmid clones and sequenced. In C79, this second integron was not in GI1 but, rather, a second GI, here designated GI2. A GI closely related to GI2, LESGI-3, has previously been reported in LESB58, an epidemic strain in the United Kingdom, and is located in the chromosome (26). The second integron in C79 (Fig. 2 and Table 1) contained a four-cassette array that included blaGES-5. The integron had acquired a number of insertion sequence (IS) elements, including a variant of the attC targeting ISPa21 (23), here named ISPa21e, as well as IS6100 and an IS4-like element (18). This integron was embedded in a mercury resistance transposon, Tn4380. This transposon, without an integron, has a small number of precedents in the databases, including in the plasmid pMOL30, present in the environmental soil bacterium Cupriavidus metallidurans strain CH34 (accession number CP000354.2). This implied mobility is consistent with a report (9) that showed that GIs in P. aeruginosa can move between unrelated genera. In C79, the transposon, here designated Tn6163 (Fig. 2), was flanked by direct repeats, implying insertion into GI2 by transposition. Tn6163 was not present in the two Uruguay isolates but was present in six of the remaining eight Sydney isolates. All isolates with Tn6163 had indistinguishable PFGE profiles and may thus represent clonal spread within the Sydney region. Of the two Sydney isolates lacking Tn6163, one possessed GI2 based on PCR analysis (Table 1, PFGE profile D) and the second lacked GI2 (PFGE profile B).
Structure of Tn6163 and its genetic context. The general organization and symbols are as for Fig. 1. Tn6163, IS4-like element with 62% protein identity to accession number NC_007336.1. aacA4-like gene cassette with 25 nucleotides replacing nucleotides 1 to 24 of the standard cassette. GI2, genomic island 2. PAO2583 is a gene located in the core P. aeruginosa genome (accession number AE004091) and is the inferred position of GI2 based on the known location of LESGI-3 (26), which is highly similar to GI2.
In U09, the insertion point of the second class 1 integron/transposon was within the chromosomal gene oprD (Fig. 3A). Insertion into oprD is noteworthy since loss of this gene is associated with increased levels of resistance to carbapenems (6, 12, 25). The sequence beyond the inverted repeat transposon end revealed a Tn21-like mer operon beyond which was a sequence of a GI similar to PAGI-2C (10) (Fig. 3). The integron in U09 possessed a five-cassette array that included the blaOXA-129 gene cassette. This cassette has been seen only once previously, in a Salmonella enterica serovar Bredeney isolate from a pig in Brazil (14). PCR analysis revealed that U61, even though it has a PFGE profile indistinguishable from that of U09, has a complete and uninterrupted oprD gene. Extensive sequencing revealed that the second integron in U61 (Fig. 3B) was identical to that of U09 with respect to IS26 and IS26-linked sequences (that is, the sequence to the left of IS26, as shown in Fig. 3). In U61, the sequence immediately beyond IS26 consisted of about 4 kb and was identical to a sequence from another GI that is located in the chromosome of the betaproteobacterium Herminiimonas arsenicoxydans (16). H. arsenicoxydans is a recently characterized bacterium associated with arsenic-contaminated water and sediments. In U61, this 4-kb region is followed by a tRNAgly gene located in the P. aeruginosa chromosome (Fig. 3B). The integrons associated with IS26 in strains U09 and U61 are likely to be moving in a way that is mediated by this IS since the insertion point is different in each of the two strains. This IS26-associated mobilized region includes the integron, associated mer region, and adjacent PAGI-2c-like GI. It has been hypothesized that IS26 can initiate nonstandard transposition that results in only a single copy appearing in the transposed product (3), and it is possible that such an event is responsible for the mobilization seen here.
Structure and location of IS26-associated integrons. The general organization and symbols are as for Fig. 1. (A) Strain U09. oprDΔ is an insertionally inactivated oprD gene. PAO958 is a gene located in the core P. aeruginosa genome (accession number AE004091). (B) HEAR2053 refers to a conserved hypothetical protein (accession number CAL62196.1), and HEAR2054 refers to a DNA integrase (nonintegron) protein (accession number CAL62197.1) located in the genome of a Herminiimonas arsenicoxydans strain. The corresponding encoded proteins found here match most closely to these. PAO2820 is a gene located in the core P. aeruginosa genome (accession number AE004091).
We have identified several multidrug-resistant class 1 integrons/transposons within the chromosomes of P. aeruginosa pathogenic isolates. Class 1 integrons/transposons were found in at least four chromosomal locations and in different clonal lines based on PFGE analysis (Table 1). This locus variability is partly region specific. It is also noteworthy that lateral gene transfer is probably occurring between clinical isolates and bacteria from food production animals and the general environment, given that a blaOXA-129 gene containing an IS26-linked element is present in animals (14) and that the U61 strain has a sequence identical to that found in the environment (16).
The chromosome may be an important platform in the dispersal of complex resistance regions in P. aeruginosa. All 20 integrons across the 11 isolates examined were linked, based on sequencing or PCR analysis, either to a core region of the P. aeruginosa chromosome or to a GI that is only known to be present in the chromosome. The U09 isolate is also noteworthy in that the insertion of the integron into the oprD gene is in itself extending the antibiotic resistance profile of this strain despite the fact that inactivation of this gene is likely to reduce fitness outside an infection context (1). In contrast, in a clinical context, insertion into oprD may be selected for, and we note that recently, a P. aeruginosa isolate from Japan was found to have a class 1 integron linked to IS26 inserted at oprD (15). In this strain, the insertion point was different from that seen in the U09 isolate, as was the cassette array. We predict that the chromosomal spread of diverse, complex, multidrug-resistant regions is likely to be a common theme globally in P. aeruginosa pathogenic isolates.
ACKNOWLEDGMENTS
This work was supported by the National Health and Medical Research Council of Australia and a University of Technology, Sydney, postgraduate scholarship to E.M.
We thank Nola Hitchick from the Sydney Adventist Hospital for the supply of some strains used in this study and Natasha Vanegas for assistance with PFGE analysis. The RAC database (http://www2.chi.unsw.edu.au/rac) assisted in the annotation and identification of gene cassettes and arrays.
FOOTNOTES
- Received 23 April 2011.
- Returned for modification 26 June 2011.
- Accepted 6 January 2012.
- Accepted manuscript posted online 23 January 2012.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.06048-11.
- Copyright © 2012, American Society for Microbiology. All Rights Reserved.