Abstract

A hundred and six Pseudomonas aeruginosa isolates from clinical cases were screened using PCR for the presence of integrons and associated resistance gene cassettes. Forty-four isolates harboured class 1 integrons (41.5%), of which 29 isolates (66%) also carried gene cassettes. The aacA gene was most frequently found within class 1 integrons (69%), followed by blaOXA family genes (52%). From class 1 integron-positive strains, we detected a total of 15 isolates (34%) carrying no gene cassettes. Restriction fragment-length polymorphism analysis of the integrons variable region revealed some identical structures, as well as distinct profiles indicating heterogeneity among these cassette regions. Multiresistance was observed in 71% of isolates, nevertheless no strong correlation was observed between integron presence and multiresistance. This is the first report showing class 1 integron prevalence and gene cassette content in P. aeruginosa isolates from clinical settings in the Brazilian Amazon.

Introduction

The antimicrobial resistance in Pseudomonas aeruginosa is of great interest because, besides its growing involvement with gravis infections, its resistance is a consequence of various mechanisms, from intrinsic resistance to gene acquisition by horizontal transfer mediated by genetic mobile elements [1]. Plasmids, conjugative transposons, and integrons are vehicles and structures for the mobilization, acquisition, and spreading of resistance genes [2].

The integrase (IntI) is the signature of an integron. To date, three classes of integrons (class 1, 2 and 3) have been described to be associated with resistance gene cassettes [3]. Class 1 is recognized as the most widespread among clinical isolates [4,5]. This class is composed of a 5′ conserved segment (CS), which includes the integrase gene (intI), the recombination site (attI) and a promoter region (Pant) [2], and a 3′ CS, which usually includes the qacEΔ1 [2,6] and sulI[2,7] genes. In between these CSs, there is a variable region where gene cassettes are inserted and expressed under Pant control [2]. Class 2 and 3 integrons contain the integrase genes (intI2 and intI3), whose products are 40% and 61% identical to class 1 integrase, respectively [4].

Considering that gene cassettes found in integrons are mobile elements, variability in the number and nature of these structures is to be expected [8]. Various resistance associated genes are harboured in class 1 integrons found in P. aeruginosa, including those encoding extended-spectrum-β-lactamases (ESBLs) and metallo-β-lactamases that hydrolyse third- and fourth-generation cephalosporins and carbapenems, respectively [9].

There are several studies showing class 1 integrons in clinically significant bacterial strains in European and Asian countries [10–13]. To date, the identification of P. aeruginosa carrying class 1 integrons in Brazil, a continental and contrasting country, has been restricted to clinical settings in São Paulo, localized in the Southeast region. There, a high prevalence (63.5%) of class 1 integrons was recorded and the most frequent gene cassette found was aac(3)-Ia [14]. Here, in contrast, clinical samples from settings in the oriental Amazon region were considered. We have determined the incidence of integrons, their class and the presence of cassette arrays.

Materials and methods

Bacterial strains and antimicrobial susceptibility

A hundred and six P. aeruginosa strains were obtained from different specimens of inpatients coming from clinical cases, including hospital infection, colonization and surveillance cultures, which were performed when patients came from other hospitals. These samples were collected in six hospital settings in São Luís of Maranhão city (Brazil) between 2000 and 2003 (Table 2). The isolate identification was determined and confirmed to the species level by phenotypic characteristics and standard biochemical tests using the VITEK automated system (BioMerieux, Hazelhood, Mo). The antimicrobial susceptibility was tested by disk diffusion method on Mueller–Hinton agar plates according to National Committee for Clinical Laboratory Standards NCCLS [15] guidelines. Disks (OXOID, Basingstoke, UK) containing amikacin, aztreonam, cefepime, ceftazidime, ciprofloxacin, imipenem, gentamicin, meropenem, piperacillin/tazobactam, and ticarcillin/clavulanic acid were used.

Table 2

Genotypic and phenotypic profiles of P. aeruginosa strains recovered from clinical specimens from six Brazilian hospitals

Strains (n= 106) Patient (n= 90) C S Isolation date Hosp Resistance profile IntI1 Variable region aadA aacA blaOXA RFLP type  
PS1 Mar/02/00 H.U. Atm, Fep, Caz, Cip, Gen, Imp, Mrp, Tzp, Tcc 800 bp − − − II  
PS2 May/03/00 H.A. Ami, Atm, Fep, Caz, Cip, Gen, Imp, Mrp, Tzp, Tcc −      
PS8 Jun/02/00 H.U. Ami, Fep, Gen − −      
PS11 Jun/12/00 H.S.D. Total susceptibility − −      
PS20 Jun/30/00 H.S.D. Ami, Fep, Caz, Cip, Gen, Imp, Tcc −      
PS21 Jun/20/00 H.A. Ami, Caz, Gen, Imp −      
PS23 May/23/00 H.U. Caz, Gen, Imp 850 bp − − −  
PS25 Nov/03/00 H.S.D. Ami, Caz, Cip, Gen, Imp − −      
PS26 Nov/11/00 H.S.D. Ami, Fep, Caz, Cip, Gen, Imp, Mrp, Tzp, Tcc 800 bp − − − II  
PS27 10 Nov/03/00 Atm, Caz, Cip, Tzp, Tcc − −      
PS29 Dec/05/00 H.S.D. Atm, Fep, Caz, Gen, Imp, Tzp, Tcc 1.7 kb − − − VI  
PS34 Oct/24/00 H.S.D. Ami, Fep, Caz, Cip, Gen, Imp, Tzp, Tcc −      
PS35 11 Oct/13/00 Atm, Caz, Cip, Imp, Tzp, Tcc − −      
PS36 12 Oct/18/00 H.S.D. Ami, Atm, Fep, Caz, Cip, Gen, Imp, Mrp, Tzp, Tcc 900 bp − − − VII  
PS37 13 Nov/03/00 H.S.D. Ami, Atm, Fep, Caz, Cip, Gen, Imp, Mrp, Tzp, Tcc 1.6 kb − − − VIII  
PS38 14 Dec/11/00 U.D.I. Cip, Mrp − −      
PS39 15 Dec/11/00 H.A. Ami, Atm, Fep, Cip, Gen, Tcc 800 bp − − − II  
PS42 16 Jan/24/01 U.D.I. Ami, Fep, Caz, Cip, Gen, Imp, Mrp, Tzp, Tcc −      
PS44 17 Jan/24/01 U.D.I. Ami, Atm, Fep, Caz, Gen, Imp, Mrp − −      
PS52 18 Dec/20/00 H.U. Ami, Atm, Fep, Caz, Cip, Gen, Imp −      
PS65 19 Mar/14/01 H.S.D. Ami, Cip, Gen, Imp, Tcc 1.8 kb −  
PS66 20 Mar/12/01 Total susceptibility − −      
PS67 21 Mar/12/01 Gen − −      
PS68 21 Mar/05/01 Total susceptibility − −      
PS69 22 Feb/23/01 H.U. Total susceptibility − −      
PS71 23 Feb/23/01 H.U. Total susceptibility − −      
PS72 24 Feb/09/01 H.U. Atm, Caz, Tcc − −      
PS73 25 Feb/16/01 H.U. Total susceptibility − −      
PS76 26 Feb/23/01 H.U. Ami, Fep, Gen, Imp, Tcc − −      
PS79 27 Feb/23/01 H.U. Atm, Fep, Caz, Cip, Imp, Tcc 1 kb − − IX  
PS80 28 Feb/15/01 H.U. Ami, Atm, Fep, Caz, Cip, Gen −      
PS83 29 Feb/05/01 H.U. Ami, Atm, Fep, Caz, Cip, Gen 3 kb − − III  
PS90 30 Mar/06/01 C.M. Ami, Atm, Fep, Caz, Cip, Gen, Imp −      
PS91 31 Feb/03/01 H.A. Atm, Fep, Cip, Gen, Tzp, Tcc −      
PS94 32 Mar/06/01 H.A. Gen − −      
PS95 33 Mar/17/01 U.D.I. Gen − −      
PS96 34 Mar/05/01 U.D.I Total susceptibility − −      
PS99 35 Apr/08/01 U.D.I Ami, Cip, Gen, Tzp, Tcc 2 kb IV  
PS103 36 Apr/12/01 Ami, Atm, Cip, Gen, Tzp, Tcc − −      
PS105 37 Apr/16/01 U.D.I. Atm, Fep, Caz, Cip, Imp, Tcc − −      
PS108 31 Apr/18/01 H.A. Atm, Fep, Caz, Imp − −      
PS109 38 Apr/25/01 U.D.I. Ami, Atm, Fep, Caz, Cip, Gen, Tcc 1.8 kb −  
PS110 31 Apr/25/01 H.A. Atm, Fep, Caz, Cip, Imp −      
PS113 39 Apr/25/01 U.D.I. Ami, Atm, Fep, Caz, Cip, Gen, Imp, Mrp, Tzp, Tcc −      
PS115 40 Jul/18/00 H.S.D. Cip, Gen, Tcc − −      
PS117 41 Apr/19/01 H.S.D. Atm, Gen, Tcc − −      
PS118 42 Apr/11/01 H.U. Atm, Fep, Caz, Gen, Tcc − −      
PS120 43 Mar/21/01 C.M. Ami, Atm, Fep, Caz, Cip, Gen, Imp 2.5 kb − − −  
PS121 44 Apr/02/01 C.M. Ami, Atm, Fep, Caz, Cip, Gen, Imp, Tcc − −      
PS122 45 Apr/06/01 C.M. Ami, Atm, Fep, Caz, Cip, Gen, Tzp, Tcc − −      
PS123 45 Apr/06/01 C.M. Ami, Atm, Fep, Caz, Cip, Gen, Tcc − −      
PS133 38 May/29/01 U.D.I. Ami, Atm, Fep, Caz, Cip, Gen, Imp, Tcc 1.8 kb −  
PS142 46 May/25/01 Ami, Cip, Gen, Tcc 1.8 kb −  
PS147 47 May/17/01 Ami, Cip, Gen, Tzp, Tcc − −      
PS149 48 May/19/01 H.S.D. Ami, Atm, Cip, Gen, Tzp, Tcc −      
PS150 49 May/17/01 H.S.D. Ami, Atm, Cip, Gen, Tzp, Tcc − −      
PS151 50 May/10/01 Ami, Atm, Caz, Cip, Gen, Tzp, Tcc − −      
PS152 51 Jun/01/01 Ami, Cip, Gen, Mrp, Tcc, Caz 1.8 kb −  
PS155 52 Sept/09/01 H.S.D. Ami, Gen − −      
PS158 53 Jun/24/01 H.S.D Ami, Atm, Cip, Gen, Tcc 2.5 kb − − XI  
PS160 51 May/31/01 Ami, Cip, Gen, Mrp, Tcc 1.8 kb −  
PS163 54 Jul/11/01 H.A. Atm, Fep, Caz, Cip, Gen, Tzp, Tcc − −      
PS164 55 Jul/18/01 U.D.I. Fep, Caz − −      
PS167 56 Jul/20/01 U.D.I. Ami, Atm, Fep, Caz, Cip, Gen, Tzp − −      
PS168 57 Jul/20/01 H.S.D. Atm, Caz, Gen, Tzp, Tcc − −      
PS169 58 Jul/22/01 H.S.D. Gen − −      
PS172 59 Jul/25/01 C.M. Atm, Cip, Gen, Tzp, Tcc − −      
PS173 60 Jul/25/01 H.A. Imp − −      
PS180 61 Aug/12/01 C.M. Fep, Mrp − −      
PS182 62 Aug/04/01 H.U. Ami, Cip, Gen, Tcc 3 kb − − III  
PS184 63 Aug/20/01 H.U. Fep, Caz, Tzp, Tcc − −      
PS185 64 Jul/15/01 H.U. Imp − −      
PS186 65 Aug/20/01 U.D.I. Atm, Gen, Tcc − −      
PS189 66 Aug/14/01 U.D.I. Atm, Gen, Tcc − −      
PS191 67 Aug/17/01 H.S.D. Ami, Atm, Fep, Gen, Mrp − −      
PS192 68 Feb/08/01 H.S.D. Total susceptibility − −      
PS193 69 Jul/27/01 Gen − −      
PS196 70 Aug/27/01 U.D.I. Total susceptibility − −      
PS197 71 Aug/24/01 H.A. Ami, Atm, Fep, Cip, Gen, Tcc 1.8 kb −  
PS200 72 Aug/30/01 C.M. Total susceptibility − −      
PS201 73 Sep/09/01 H.U. Atm, Tcc − −      
PS204 74 Aug/15/01 Ami, Atm, Cip, Gen, Mrp, Tcc 1.8 kb −  
PS210 74 Sep/12/01 Ami, Cip, Gen, Mrp, Tcc 1.8 kb −  
PS212 74 Sep/11/01 Ami, Cip, Gen, Mrp, Tcc 1.8 kb −  
PS219 75 Sep/15/01 H.U. Ami, Atm, Cip, Gen, Tcc −      
PS262 76 Oct/03/01 H.U. Ami, Atm, Fep, Cip, Gen, Imp, Tzp, Tcc 3 kb − − III  
PS278 77 Dec/06/01 H.U. Ami, Atm, Fep, Cip, Gen, Imp − −      
PS281 78 Dec/08/01 H.U. Total susceptibility − −      
PS296 79 Jan/25/02 H.A. Atm − −      
PS297 79 Jan/23/02 H.A. Atm − −      
PS298 80 Jan/18/02 H.A. Ami, Cip, Gen, Tcc 2 kb IV  
PS299 81 Jan/19/02 H.A. Atm, Cip, Gen, Tzp, Tcc − −      
PS312 76 Jan/08/02 H.U. Imp − −      
PS314 82 Jan/18/02 H.U. Total susceptibility −      
PS316 83 Dec/26/01 H.U. Ami, Atm, Fep, Cip, Gen, Tzp, Tcc 2 kb IV  
PS333 84 Mar/14/02 U.D.I. Ami, Atm, Fep, Caz, Cip, Gen, Imp, Tcc −      
PS375 85 Mar/15/02 H.U. Ami, Atm, Cip, Gen, Imp, Tcc − −      
PS408 85 Jun/04/02 H.U. Ami, Atm, Cip, Gen, Tcc 1 kb − − XII  
PS438 85 Dec/13/02 H.U. Ami, Atm, Fep, Cip, Gen, Imp, Tcc − −      
PS446 85 Nov/27/02 H.U. Ami, Atm, Cip, Gen, Imp, Tcc − −      
PS472 86 Jan/07/03 H.U. Ami, Fep, Caz, Cip, Gen, Tzp, Tcc − −      
PS493 87 Feb/13/03 H.A. Atm, Fep, Caz, Cip, Imp, Tcc − −      
PS503 88 Feb/22/03 U.D.I. Atm, Fep, Caz, Imp, Tzp, Tcc − −      
PS509 85 Mar/14/03 H.U. Cip, Gen, Tcc 1.8 kb −  
PS516 89 Feb/28/03 H.U. Ami, Atm, Fep, Caz, Cip, Gen, Imp, Tzp, Tcc 1.8 kb −  
PS546 90 Mar/19/03 H.A. Atm, Tzp − −      
Strains (n= 106) Patient (n= 90) C S Isolation date Hosp Resistance profile IntI1 Variable region aadA aacA blaOXA RFLP type  
PS1 Mar/02/00 H.U. Atm, Fep, Caz, Cip, Gen, Imp, Mrp, Tzp, Tcc 800 bp − − − II  
PS2 May/03/00 H.A. Ami, Atm, Fep, Caz, Cip, Gen, Imp, Mrp, Tzp, Tcc −      
PS8 Jun/02/00 H.U. Ami, Fep, Gen − −      
PS11 Jun/12/00 H.S.D. Total susceptibility − −      
PS20 Jun/30/00 H.S.D. Ami, Fep, Caz, Cip, Gen, Imp, Tcc −      
PS21 Jun/20/00 H.A. Ami, Caz, Gen, Imp −      
PS23 May/23/00 H.U. Caz, Gen, Imp 850 bp − − −  
PS25 Nov/03/00 H.S.D. Ami, Caz, Cip, Gen, Imp − −      
PS26 Nov/11/00 H.S.D. Ami, Fep, Caz, Cip, Gen, Imp, Mrp, Tzp, Tcc 800 bp − − − II  
PS27 10 Nov/03/00 Atm, Caz, Cip, Tzp, Tcc − −      
PS29 Dec/05/00 H.S.D. Atm, Fep, Caz, Gen, Imp, Tzp, Tcc 1.7 kb − − − VI  
PS34 Oct/24/00 H.S.D. Ami, Fep, Caz, Cip, Gen, Imp, Tzp, Tcc −      
PS35 11 Oct/13/00 Atm, Caz, Cip, Imp, Tzp, Tcc − −      
PS36 12 Oct/18/00 H.S.D. Ami, Atm, Fep, Caz, Cip, Gen, Imp, Mrp, Tzp, Tcc 900 bp − − − VII  
PS37 13 Nov/03/00 H.S.D. Ami, Atm, Fep, Caz, Cip, Gen, Imp, Mrp, Tzp, Tcc 1.6 kb − − − VIII  
PS38 14 Dec/11/00 U.D.I. Cip, Mrp − −      
PS39 15 Dec/11/00 H.A. Ami, Atm, Fep, Cip, Gen, Tcc 800 bp − − − II  
PS42 16 Jan/24/01 U.D.I. Ami, Fep, Caz, Cip, Gen, Imp, Mrp, Tzp, Tcc −      
PS44 17 Jan/24/01 U.D.I. Ami, Atm, Fep, Caz, Gen, Imp, Mrp − −      
PS52 18 Dec/20/00 H.U. Ami, Atm, Fep, Caz, Cip, Gen, Imp −      
PS65 19 Mar/14/01 H.S.D. Ami, Cip, Gen, Imp, Tcc 1.8 kb −  
PS66 20 Mar/12/01 Total susceptibility − −      
PS67 21 Mar/12/01 Gen − −      
PS68 21 Mar/05/01 Total susceptibility − −      
PS69 22 Feb/23/01 H.U. Total susceptibility − −      
PS71 23 Feb/23/01 H.U. Total susceptibility − −      
PS72 24 Feb/09/01 H.U. Atm, Caz, Tcc − −      
PS73 25 Feb/16/01 H.U. Total susceptibility − −      
PS76 26 Feb/23/01 H.U. Ami, Fep, Gen, Imp, Tcc − −      
PS79 27 Feb/23/01 H.U. Atm, Fep, Caz, Cip, Imp, Tcc 1 kb − − IX  
PS80 28 Feb/15/01 H.U. Ami, Atm, Fep, Caz, Cip, Gen −      
PS83 29 Feb/05/01 H.U. Ami, Atm, Fep, Caz, Cip, Gen 3 kb − − III  
PS90 30 Mar/06/01 C.M. Ami, Atm, Fep, Caz, Cip, Gen, Imp −      
PS91 31 Feb/03/01 H.A. Atm, Fep, Cip, Gen, Tzp, Tcc −      
PS94 32 Mar/06/01 H.A. Gen − −      
PS95 33 Mar/17/01 U.D.I. Gen − −      
PS96 34 Mar/05/01 U.D.I Total susceptibility − −      
PS99 35 Apr/08/01 U.D.I Ami, Cip, Gen, Tzp, Tcc 2 kb IV  
PS103 36 Apr/12/01 Ami, Atm, Cip, Gen, Tzp, Tcc − −      
PS105 37 Apr/16/01 U.D.I. Atm, Fep, Caz, Cip, Imp, Tcc − −      
PS108 31 Apr/18/01 H.A. Atm, Fep, Caz, Imp − −      
PS109 38 Apr/25/01 U.D.I. Ami, Atm, Fep, Caz, Cip, Gen, Tcc 1.8 kb −  
PS110 31 Apr/25/01 H.A. Atm, Fep, Caz, Cip, Imp −      
PS113 39 Apr/25/01 U.D.I. Ami, Atm, Fep, Caz, Cip, Gen, Imp, Mrp, Tzp, Tcc −      
PS115 40 Jul/18/00 H.S.D. Cip, Gen, Tcc − −      
PS117 41 Apr/19/01 H.S.D. Atm, Gen, Tcc − −      
PS118 42 Apr/11/01 H.U. Atm, Fep, Caz, Gen, Tcc − −      
PS120 43 Mar/21/01 C.M. Ami, Atm, Fep, Caz, Cip, Gen, Imp 2.5 kb − − −  
PS121 44 Apr/02/01 C.M. Ami, Atm, Fep, Caz, Cip, Gen, Imp, Tcc − −      
PS122 45 Apr/06/01 C.M. Ami, Atm, Fep, Caz, Cip, Gen, Tzp, Tcc − −      
PS123 45 Apr/06/01 C.M. Ami, Atm, Fep, Caz, Cip, Gen, Tcc − −      
PS133 38 May/29/01 U.D.I. Ami, Atm, Fep, Caz, Cip, Gen, Imp, Tcc 1.8 kb −  
PS142 46 May/25/01 Ami, Cip, Gen, Tcc 1.8 kb −  
PS147 47 May/17/01 Ami, Cip, Gen, Tzp, Tcc − −      
PS149 48 May/19/01 H.S.D. Ami, Atm, Cip, Gen, Tzp, Tcc −      
PS150 49 May/17/01 H.S.D. Ami, Atm, Cip, Gen, Tzp, Tcc − −      
PS151 50 May/10/01 Ami, Atm, Caz, Cip, Gen, Tzp, Tcc − −      
PS152 51 Jun/01/01 Ami, Cip, Gen, Mrp, Tcc, Caz 1.8 kb −  
PS155 52 Sept/09/01 H.S.D. Ami, Gen − −      
PS158 53 Jun/24/01 H.S.D Ami, Atm, Cip, Gen, Tcc 2.5 kb − − XI  
PS160 51 May/31/01 Ami, Cip, Gen, Mrp, Tcc 1.8 kb −  
PS163 54 Jul/11/01 H.A. Atm, Fep, Caz, Cip, Gen, Tzp, Tcc − −      
PS164 55 Jul/18/01 U.D.I. Fep, Caz − −      
PS167 56 Jul/20/01 U.D.I. Ami, Atm, Fep, Caz, Cip, Gen, Tzp − −      
PS168 57 Jul/20/01 H.S.D. Atm, Caz, Gen, Tzp, Tcc − −      
PS169 58 Jul/22/01 H.S.D. Gen − −      
PS172 59 Jul/25/01 C.M. Atm, Cip, Gen, Tzp, Tcc − −      
PS173 60 Jul/25/01 H.A. Imp − −      
PS180 61 Aug/12/01 C.M. Fep, Mrp − −      
PS182 62 Aug/04/01 H.U. Ami, Cip, Gen, Tcc 3 kb − − III  
PS184 63 Aug/20/01 H.U. Fep, Caz, Tzp, Tcc − −      
PS185 64 Jul/15/01 H.U. Imp − −      
PS186 65 Aug/20/01 U.D.I. Atm, Gen, Tcc − −      
PS189 66 Aug/14/01 U.D.I. Atm, Gen, Tcc − −      
PS191 67 Aug/17/01 H.S.D. Ami, Atm, Fep, Gen, Mrp − −      
PS192 68 Feb/08/01 H.S.D. Total susceptibility − −      
PS193 69 Jul/27/01 Gen − −      
PS196 70 Aug/27/01 U.D.I. Total susceptibility − −      
PS197 71 Aug/24/01 H.A. Ami, Atm, Fep, Cip, Gen, Tcc 1.8 kb −  
PS200 72 Aug/30/01 C.M. Total susceptibility − −      
PS201 73 Sep/09/01 H.U. Atm, Tcc − −      
PS204 74 Aug/15/01 Ami, Atm, Cip, Gen, Mrp, Tcc 1.8 kb −  
PS210 74 Sep/12/01 Ami, Cip, Gen, Mrp, Tcc 1.8 kb −  
PS212 74 Sep/11/01 Ami, Cip, Gen, Mrp, Tcc 1.8 kb −  
PS219 75 Sep/15/01 H.U. Ami, Atm, Cip, Gen, Tcc −      
PS262 76 Oct/03/01 H.U. Ami, Atm, Fep, Cip, Gen, Imp, Tzp, Tcc 3 kb − − III  
PS278 77 Dec/06/01 H.U. Ami, Atm, Fep, Cip, Gen, Imp − −      
PS281 78 Dec/08/01 H.U. Total susceptibility − −      
PS296 79 Jan/25/02 H.A. Atm − −      
PS297 79 Jan/23/02 H.A. Atm − −      
PS298 80 Jan/18/02 H.A. Ami, Cip, Gen, Tcc 2 kb IV  
PS299 81 Jan/19/02 H.A. Atm, Cip, Gen, Tzp, Tcc − −      
PS312 76 Jan/08/02 H.U. Imp − −      
PS314 82 Jan/18/02 H.U. Total susceptibility −      
PS316 83 Dec/26/01 H.U. Ami, Atm, Fep, Cip, Gen, Tzp, Tcc 2 kb IV  
PS333 84 Mar/14/02 U.D.I. Ami, Atm, Fep, Caz, Cip, Gen, Imp, Tcc −      
PS375 85 Mar/15/02 H.U. Ami, Atm, Cip, Gen, Imp, Tcc − −      
PS408 85 Jun/04/02 H.U. Ami, Atm, Cip, Gen, Tcc 1 kb − − XII  
PS438 85 Dec/13/02 H.U. Ami, Atm, Fep, Cip, Gen, Imp, Tcc − −      
PS446 85 Nov/27/02 H.U. Ami, Atm, Cip, Gen, Imp, Tcc − −      
PS472 86 Jan/07/03 H.U. Ami, Fep, Caz, Cip, Gen, Tzp, Tcc − −      
PS493 87 Feb/13/03 H.A. Atm, Fep, Caz, Cip, Imp, Tcc − −      
PS503 88 Feb/22/03 U.D.I. Atm, Fep, Caz, Imp, Tzp, Tcc − −      
PS509 85 Mar/14/03 H.U. Cip, Gen, Tcc 1.8 kb −  
PS516 89 Feb/28/03 H.U. Ami, Atm, Fep, Caz, Cip, Gen, Imp, Tzp, Tcc 1.8 kb −  
PS546 90 Mar/19/03 H.A. Atm, Tzp − −      

Resistance profile of multiresistant strains is indicated in boldface.

Blank spaces indicate not tested strains. Samples without class 1 integrons or without inserted gene cassettes were not included in RFLP analysis and in gene cassette screening.

+ (plus), positive result; − (minus), negative result.

Clinical specimens, A — central venous catheter; B — bronchoalveolar lavage; C — wound biopsy; D — urine; E — wound swab; F — blood; G — nasal swab; H — other specimens.

Hospitals of isolation.

Abbreviations: Ami, amikacin; Atm, aztreonam; Fep, cefepime; Caz, ceftazidime; Cip, ciprofloxacin; Gen, gentamicin; Imp, imipenem; Mrp, meropenem; Tzp, piperacillin/tazobactam; Tcc, ticarcillin/clavulanic acid.

DNA extraction and PCR assay

Bacterial cultures were centrifuged and the pellet was washed twice with 500 µl of a saline solution (0.85%). Cells were suspended in 500 µl of Milli-Q water and subsequently boiled in a water bath for 15 min. After, the cell extracts were immediately stored at −20 °C for their use as DNA template in PCR reactions.

We have designed primers to detect the class 1, 2 and 3 integrase, aadA, blaOXA, blaIMP and aacA genes (Table 1). The amplification of gene cassette regions (5′CS/3′CS), as well as the 3′ conserved segment, corresponding to qacEΔ1 and sulI genes (qacEΔ1-F/Sul1-B), was performed by using the primers described in Table 1.

Table 1

Oligonucleotides used in PCR analysis of integrons and gene cassettes

Primers Nucleotide sequence (5′–3′) PCR targets Reference 
IntI f AAA ACC GCC ACT GCG CCG TTA Class 1 integrase gene This work 
IntI r GAA GAC GGC TGC ACT GAA CG 
    
qacEΔ1-F ATC GCA ATA GTT GGC GAA GT 3′CS of class 1 integron [16] 
SulI-B GCA AGG CGG AAA CCC GCG CC 
    
Int2 f GCG TTT TAT GTC TAA CAG TCC Class 2 integrase gene This work 
Int2 r AAG TAG CAT CAG TCC ATC C 
    
Int3 f ACT TTC AGC ACA TGC G Class 3 integrase gene This work 
Int3 r TCT GTG GAC CCA CAA AC 
    
5′CS GGC ATC CAA GCA GCA AG Gene cassettes associated with integron [17] 
3′CS AAG CAG ACT TGA CCT 
    
AadA f CTT GAT GAA ACA AGG CGG aadA resistance genes This work 
AadA r TAC CAA ATG CGG GAC AAC 
    
Oxa f AAG AAA CGC TAC TCG CCT GC blaOXA resistance genes [18] 
Oxa r CCA CTC AAC CCA TCC TAC CC 
    
AacA f CGT ACA GGA ACA GTA CTT GC aacA resistance genes This work 
AacA r TTT GAA CCA TGT ACA CGG C 
    
IMP f CTA CCG CAG CAG AGT CTT GG blaIMP resistance genes This work 
IMP r AAC CAG TTC TGC CTT ACC AT 
Primers Nucleotide sequence (5′–3′) PCR targets Reference 
IntI f AAA ACC GCC ACT GCG CCG TTA Class 1 integrase gene This work 
IntI r GAA GAC GGC TGC ACT GAA CG 
    
qacEΔ1-F ATC GCA ATA GTT GGC GAA GT 3′CS of class 1 integron [16] 
SulI-B GCA AGG CGG AAA CCC GCG CC 
    
Int2 f GCG TTT TAT GTC TAA CAG TCC Class 2 integrase gene This work 
Int2 r AAG TAG CAT CAG TCC ATC C 
    
Int3 f ACT TTC AGC ACA TGC G Class 3 integrase gene This work 
Int3 r TCT GTG GAC CCA CAA AC 
    
5′CS GGC ATC CAA GCA GCA AG Gene cassettes associated with integron [17] 
3′CS AAG CAG ACT TGA CCT 
    
AadA f CTT GAT GAA ACA AGG CGG aadA resistance genes This work 
AadA r TAC CAA ATG CGG GAC AAC 
    
Oxa f AAG AAA CGC TAC TCG CCT GC blaOXA resistance genes [18] 
Oxa r CCA CTC AAC CCA TCC TAC CC 
    
AacA f CGT ACA GGA ACA GTA CTT GC aacA resistance genes This work 
AacA r TTT GAA CCA TGT ACA CGG C 
    
IMP f CTA CCG CAG CAG AGT CTT GG blaIMP resistance genes This work 
IMP r AAC CAG TTC TGC CTT ACC AT 

F, forward primer; r, reverse primer; CS, conserved segment.

PCRs were performed in a final volume of 50 µl containing 3 µl of total DNA as template, 3 mM of MgCl2 (Promega), 50 pmol of each primer (Invitrogen) and 1.6 U of Taq polymerase (Promega). Amplification reactions consisted of denaturation at 94 °C for 5 min followed by 35 cycles of denaturation at 94 °C/30 s, annealing at 55 °C/30 s and elongation at 72 °C/1 min, for class 1 and 2 integrons; 30 s for 3′CS, resistance genes and class 3 integrons, and 3 min for 5′CS/3′CS regions. Amplification products were analyzed using 1.5% agarose gel electrophoresis (1 ×TAE buffer at 60 mA), stained with ethidium bromide solution and visualized using UV transilluminator.

Restriction fragment-length polymorphism

Amplicons corresponding to gene cassette regions were cleaved with ClaI and HaeIII (Invitrogen) restriction enzymes. The products were separated in a 1.5% agarose gel.

DNA sequencing

Both PCR product strands were directly sequenced. Amplicons were purified using Wizard SV Gel and PCR Clean-Up system kit (Promega). Sequencing reactions were performed with Big Dye Terminator RR Mix (PE Applied Biosystems) in an ABI Prism DNA Sequencer 377 (Applied Biosystems). Nucleotide sequences were compared to those available in the GenBank database accessible on the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov).

Results

Antibiotic resistance profiles

Clinical P. aeruginosa isolates (n= 106) collected from 90 patients were obtained from six different hospitals in São Luís, MA between 2000 and 2003. All isolates were tested for susceptibility to 10 antimicrobial agents indicated for treating P. aeruginosa infections. No selection criteria were applied for the screening of the isolates. Seventy-six isolates (approximately 71%) were considered multiresistant (MR). Eleven strains (14.5%) were resistant to all antibiotic groups tested. The most common resistance was observed towards aminoglycosides (70%), while resistance to carbapenems was less observed among isolates (43%) as shown in Table 2. MR was established as resistance to at least three different antimicrobial groups.

Integron detection and characterization

All 106 P. aeruginosa isolates tested for susceptibility to antibiotics were screened for the presence of class 1, 2 and 3 integrases. Forty-four isolates (41.5%) were identified as being positive for class 1 integrase (Table 2). Class 2 and 3 integrases were not detected. The integrase type was confirmed by amplicon sequencing (data not shown).

In order to verify the presence of gene cassettes within class 1 integrons, PCR reactions were performed using primers targeting the conserved sequences flanking the variable region. Ten distinct variable regions were detected among 29 isolates (66%), with sizes ranging from 800 bp to 3 kb (Table 2). The rest 34% of isolates harboured class 1 integron elements without any gene cassette attached (Table 2). The 3′CS of class 1 integrons, normally characterized by qacEΔ1 and sulI, was present in all but two isolates, one of which carried no gene cassette (data not shown).

Identification of resistance genes associated with class 1 integron

The unique report about prevalence of gene cassettes associated to class 1 integrons in Brazil screened aadA, blaOXA alleles, aac (3)-Ia and blaIMP alleles [14]. In order to establish the prevalence of these genes in association with class 1 integrons in samples from another geographic region, PCR was performed targeting the aacA, blaOXA, blaIMP and aadA genes. Reactions combining the primer for 5′CS with one specific to each of these genes were also carried out in order to establish the link between these genes and integrons. Of 29 strains, 12 were positive for aacA and blaOXA genes; three were positive for three genes tested (aacA, blaOXA, aadA); five harbour only the aacA gene and one isolate carried the aadA gene (Table 2). PCR reactions with IMP primers failed to give any positive results (data not shown). Therefore, the most prevalent gene cassette was the aac, followed by blaOXA.

Characterization of cassette arrays by RFLP

Restriction fragment-length polymorphism (RFLP) analysis of the variable segments of 29 integrons revealed several structures. Amplicons of the same size, yielding the same RFLP pattern, were considered to harbour the same cassette array. Twelve amplicons of 1.8 kb, three of 800 bp, three of 3 kb and three of 2 kb showed an equal and discriminatory RFLP pattern with the two restriction enzymes used (data not shown). The cassette arrays with identical RFLP pattern were clustered into groups named I, II, III and IV, respectively (Table 2). The four groups were found to be present in strains circulating in all hospitals while group III was found in a unique hospital in an eight-month period. Group I, II and IV were isolated from different hospitals from March/2001 to March/2003, March to December/2000 and April/2001 to January/2002, respectively. The remaining eight amplicons presented unique RFLP patterns when compared with the four groups and were spread throughout various hospitals (Table 2). The most prevalent gene, the aacA, was identified in strains carrying groups I, III and IV variable segments (Table 2).

Discussion

Currently, in the investigation of genetic bases of P. aeruginosa multiresistance, an important aspect that has been considered is the integron and the associated gene cassettes. The role of these elements in the horizontal acquisition and expression of genes, and as a gene reservoir, has been associated with the emergence of antibiotic resistance among clinical isolates of bacteria.

The screening for integrons in clinical P. aeruginosa isolates (76 MR and 30 non-MR) from the Amazon region revealed that 44/106 isolates carried class 1 integrons. From these 76 MR isolates, 43 were positive for the presence of class 1 integrons. However, the analysis of the integron variable region revealed that 14/43 (32.5%) were devoid of gene cassettes, indicating a high occurrence of empty class 1 integrons among MR strains (Table 2). These data are in contrast with Severino and Magalhães [14] findings in P. aeruginosa from clinical settings in São Paulo, where only one (1.8%) of 54 class 1 integron-positive isolates was empty.

Several reports have shown the presence of aminoglycoside resistance genes associated with integrons found in gram-negative bacteria [4,14,19], but in our study was observed that aminoglycoside resistance was spread in the majority of isolates, including those integron-negative (Table 2). The absence of class 2 and 3 integrons among our samples confirmed the restricted distribution of these two genetic elements among bacterial populations.

In the present study, a clear difference was shown in the distribution and frequency of class 1 integrons among MR and non-MR clinical strains of P. aeruginosa from hospitals in the Amazon region, since only MR isolates (38%) harboured class 1 integrons carrying gene cassettes (Table 2). Heir et al. [20] showed a higher prevalence of class 1 integrons among the MR strains within three distinct sets of samples including MR and non-MR from clinical settings in Norway. However, in our study, a surprisingly and unpredicted high occurrence of empty integrons (18%) was, for the first time, observed among MR isolates. Furthermore, the multiresistance itself did not have a strong correlation with the integron presence, once 62% of MR isolates did not present class 1 integrons or any cassettes (Table 2). van Loon et al. [21] analysed 69 P. aeruginosa clinical isolates and found only one strain carrying a class 1 integron, indicating that integrons played only a minor role in drug resistance in this species. Actually, the association of multidrug resistance with integrons has been specially shown in Enterobacteriaceae[21,22], despite the lack, in some studies, of experimental evidences showing the presence or content of the variable region [22]. In fact, it is well-known that multiple mechanisms are related to antibiotic resistance in P. aeruginosa[1], and the integrons role in this species is an additional element in the dynamics of the resistance acquisition.

In our work, a large proportion of class 1 integrons carrying resistance gene cassettes was detected with sizes ranging from 800 bp to 3 kb. They were clustered in different groups according to RFLP profiles. The results indicate the presence of equal structures among strains and, at the same time, heterogeneity in organization and/or composition of these elements, considering the number and nature of gene cassettes within integrons circulating in one specific bacterial group.

Genes conferring resistance to aminoglycosides and β-lactams are frequently found in integrons from Pseudomonas and Enterobacteriaceae, and the most common aminoglycoside resistance gene cassettes belong to aad and aac families [4,19]. The most frequent gene cassette found in our isolates was from the aacA family (69%), followed by blaOXA (52%). Severino and Magalhães [14] showed a predominance of the aac(3)-Ia (32%) gene cassette followed by aadA (23%) in strains isolated in São Paulo, the most industrialized city in Brazil. The high percentage of blaOXA gene associated to class 1 integrons in the Amazon region (52%) contrasts with the low presence of this gene in other Brazilian settings (6%) [14]. In fact, many of the members of the OXA β-lactamase family have been found in bacterial isolates originated in some European countries and Turkey [9,23]. It is not certain whether these countries represent foci of strains harbouring these enzymes, but our data contributes to show the extension of the OXA gene family in world. Another discrepancy is the difference in the occurrence of the aadA gene between our samples (13%) and those from the São Paulo clinical setting (23%) [14]. As observed by Severino and Magalhães [14], blaIMP alleles were also absent from Amazonian P. aeruginosa strains, suggesting that these genes are restricted to clinical settings in Asia and Europe [24]. These data indicate that integron gene content varies as consequence of distinct selective pressures occurring in different geographic areas.

This work is the first report on the prevalence and characterization of class 1 integrons found in P. aeruginosa isolates from clinical settings in the Amazon region, where this issue had not yet been addressed.

Acknowledgements

We thank Ana Beatriz Robotton for reviewing the manuscript. This work was supported by CAPES fellowship and Oswaldo Cruz Institute grants.

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