A Novel Insertion Sequence, ISPa26, in oprD of Pseudomonas aeruginosa Is Associated with Carbapenem Resistance

Carbapenems are frequently used to treat Pseudomonas aeruginosa; however, resistance to the carbapenems is emerging rapidly (9). Mutations or deletions in oprD (3, 2, 8) commonly result in lack of OprD expression, blocking the entry of imipenem into the cell and resulting in resistance to this drug (6). More recently, insertional inactivation of oprD by the insertion sequence (IS) elements ISPa1328 and ISPa1635 was described (7). Previously, we showed that imipenem resistance in P. aeruginosa was due to point mutations and deletions in oprD; IS elements were not detected in the isolates (3). To determine whether IS elements play a role in imipenem resistance in more recent isolates, we screened for their presence in oprD in isolates from two local hospitals.

Fifteen P. aeruginosa isolates were identified as imipenem resistant by disc susceptibility testing (1). Using oprD-specific primers (3), PCR products of the expected size (1,343 bp) were obtained from all but one isolate; strain 8 yielded a product of 2,400 bp. This PCR product was purified and sequenced (3). Analysis of the sequencing data indicated that oprD was disrupted at nucleotide position 35 by the insertion of a 1,193-bp sequence (GenBank accession no. EU000222) containing features of an IS element. The sequence is flanked by 16-bp terminal imperfect inverted repeats and contains an open reading frame (972 bp) with 93.8% identity to TnpA of ISPsy2 from Pseudomonas syringae pv. eriobotryae. No homology with P. aeruginosa PAO1 nor PA14 was detected. This novel IS element, designated ISPa26, resulted in a 4-bp (CTGG) target duplication at the site of insertion in oprD.

To determine whether ISPa26 was present, albeit not in oprD, in the other 14 isolates, PCR assays using primers to specifically amplify tnpA of ISPa26 (ISPa26F, 5′-GATTGCTCTGATCAAACC-3′, and ISPa26R, 5′-ATCCAAATGTTCGACAGG-3′) were carried out. Amplicons corresponding to ISPa26 tnpA were obtained from four additional imipenem-resistant isolates (strains 1, 6, 7, and 286). Interestingly, the element was also detected in two imipenem-susceptible isolates (strains 12 and 529); ISPa26 was not detected in 7 clinical P. aeruginosa isolates from Canada, France, and Japan. Using pulsed-field gel electrophoresis genotyping (www.hpa.org.uk), five of the seven isolates containing ISPa26 (Table 1) were shown to be clonally related.

TABLE 1.

MICs of imipenem and meropenem of P. aeruginosa isolates carrying ISPa26^a

Isolate	Yr of isolation	Hospital	Type of specimen	MIC (μg/ml)		ISPa26		PFGE profileb
				IPM	MPM	Copy no.	Inserted into oprD
1	2000	GSH	Blood	>32	>32	2	−	A
6	2000	GSH	Pus	>32	6	1	−	B
7	2000	GSH	Tracheal aspirate	>32	6	1	−	C
8	2000	GSH	Tracheal aspirate	>32	>32	3	+	C
12	2000	GSH	Tracheal aspirate	2	3	1	−	C
286	2004	GSH	CF sputum	>32	>32	2	−	C
529	2006	RCCH	CF sputum	1	2	1	−	C

^aIPM, imipenem; MPM, meropenem; GSH, Groote Schuur Hospital; RCCH, Red Cross War Memorial Children's Hospital; CF, cystic fibrosis; +, inserted into oprD; −, present in isolate but not inserted into oprD; PFGE, pulsed-field gel electrophoresis.

^bPFGE profiles designated using Tenover criteria (5).

To investigate the ISPa26 copy number in P. aeruginosa, genomic DNA was digested with BamHI, which does not cut within ISPa26, and hybridized (4) to PCR-amplified ISPa26 tnpA (data not shown). One hybridization signal was observed in each of the two imipenem-susceptible isolates, as well as from two of the imipenem-resistant isolates (6 and 7), suggesting that these isolates harbor at least one copy of ISPa26. Two signals were detected in strain 1 and, possibly, in strain 286, suggesting that these isolates harbor at least two copies of ISPa26. Interestingly, the hybridization study indicates that strain 8, which contains an insertionally inactivated oprD, has at least three copies of this element, suggesting mobility of the element. That ISPa26 may be mobile in strain 8 could explain its presence in oprD in this P. aeruginosa isolate.

In conclusion, we have identified a novel IS element, ISPa26, in P. aeruginosa isolates from hospitals in Cape Town. In one isolate, insertional inactivation of oprD by ISPa26 was associated with carbapenem resistance.