Identification of an atypical integron carrying an IS26-disrupted aadA1 gene cassette in Acinetobacter baumannii

International Journal of Antimicrobial Agents 32 (2008) 165–169

Short communication

Identification of an atypical integron carrying an IS26-disrupted aadA1 gene cassette in Acinetobacter baumannii Hu Lin Han a,1 , Sook Jin Jang a,b,∗ , Geon Park b , Joong Ki Kook c , Jong Hee Shin d , Sung Heui Shin a , Dong Min Kim e , Ji Seon Cheon f , Dae Soo Moon b , Young Jin Park b a

Research Center for Resistant Cells, Chosun University Medical School, Gwang-Ju, South Korea Department of Laboratory Medicine, Chosun University Medical School, Gwang-Ju, South Korea Department of Oral Biochemistry, College of Dentistry, Chosun University, Gwang-Ju, South Korea Department of Laboratory Medicine, Chonnam University Medical School, Gwang-Ju, South Korea e Department of Internal Medicine, Chosun University Medical School, Gwang-Ju, South Korea f Department of Plastic and Reconstructive Surgery, Chosun University Medical School, Gwang-Ju, South Korea

b c d

Received 1 February 2008; accepted 14 March 2008

Abstract An unusual class 1 integron was identified that carries an IS26-disrupted aadA1 gene cassette (designated as ‘integron-IS26’) in an imipenemresistant Acinetobacter baumannii (IRAB) outbreak strain. DNA sequencing revealed that integron-IS26 contained two gene cassettes, the aac(6’)-Im cassette and a peculiar aadA1 cassette that was disrupted by IS26 (disrupted aadA1 cassette). Southern blotting localised integronIS26 to the chromosome. Nested polymerase chain reaction (PCR) was performed to define the frequency of integron-IS26 in five groups of bacteria. Nested PCR identified integron-IS26 in 19 (73.1%) of 26 clinical outbreak strains of IRAB, 10 (100%) of 10 IRAB isolated from environmental cultures, 3 (13.0%) of 23 imipenem-susceptible A. baumannii (ISAB) non-outbreak strains, 1 (3.6%) of 28 netilmicin- and tobramycin-resistant A. baumannii and none of the netilmicin- and tobramycin-resistant Pseudomonas aeruginosa. In conclusion, we have identified a novel class I integron that carries the aac(6’)-Im cassette and an IS26-disrupted copy of aadA1 (integron-IS26) in most IRAB outbreak strains and in a few ISAB non-outbreak control strains. Integron-IS26 is located chromosomally. © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: IS26; Aac(6’)-Im; Chromosome; Molecular characterisation; Mobile element; Resistance determinant

1. Introduction Acinetobacter baumannii is an opportunistic human pathogen that causes nosocomial infections and frequently exhibits multidrug resistance. The integron is considered to play a major role in multidrug resistance dissemination among Gram-negative bacteria [1]. We identified an unusual class 1 integron that carries an IS26-disrupted aadA1 ∗ Corresponding author. Present address: Department of Laboratory Medicine, Chosun University Medical School, 375 Seoseok-dong, Donggu, Gwang-Ju 501-759, South Korea. Tel.: +82 62 220 3259/82 10 9614 8853; fax: +82 62 232 2063. E-mail address: [email protected] (S.J. Jang). 1 Present address: Department of Clinical Pathology, China Yanji City Hospital, Yanji, China.

gene cassette (termed ‘integron-IS26’) in an imipenemresistant A. baumannii (IRAB) outbreak strain. The outbreak of IRAB involved 77 patients in the Intensive Care Unit of Chosun University Hospital, South Korea, during 2004. While investigating this outbreak, we discovered integron-IS26 in IRAB outbreak strain CM-128. Class 1 integrons are the most commonly found integrons and they contain single or multiple antimicrobial resistance gene cassettes in a variable region located between two conserved segments, 5’CS and 3’CS [1]. The association of multidrug resistance with this type of the integron is of great concern given the ability of the integron to capture DNA. In the present study, we describe the composition of the gene cassette of integron-IS26 in a representative IRAB

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outbreak strain, the location of the integron and the frequency of the integron in five groups of bacteria.

v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and the 3730XL capillary DNA sequencer (Applied Biosystems).

2. Materials and methods

2.3. PFGE and Southern hybridisation

2.1. Bacterial isolates

Molecular typing by PFGE was performed using the CHEF DR III system (Bio-Rad, Hercules, CA). After digestion with I-CeuI, which has a recognition site in the 23S rDNA, PFGE of whole-cell DNA was carried out to produce chromosomal banding patterns [7]. These bands were blotted and then hybridised using gene-specific probes for 23S rRNA, integrase I1 and the disrupted aadA1 cassette of the integron-IS26, respectively. Preparation of the genespecific probes and Southern hybridisation were performed using the DIG High Prime DNA Labeling and Detection Kits (Roche Diagnostics, Mannheim, Germany) [8]. Amplicons of the 23S rRNA, integrase I1 and the second-round PCR product of nested PCR for integron-IS26 (517 bp product, which encompasses a portion of the front part of the disrupted aadA1 and IS26 transposase) were used as template DNA for the preparation of gene-specific probes.

Five groups of bacteria were used in this study. Initially, three groups of isolates were collected during the IRAB outbreak in 2004: clinical outbreak strains of IRAB (Group 1); IRAB isolated from environmental culture (Group 2); and imipenem-susceptible A. baumannii (ISAB) non-outbreak strains (Group 3). The remaining two groups were netilmicin- and tobramycin-resistant A. baumannii (Group 4) and netilmicin- and tobramycinresistant Pseudomonas aeruginosa (Group 5) isolated in 2005. The aminoglycosides were used for strain selection because aac(6’)-Im, which is one of the gene cassettes in integron-IS26, confers resistance to these antibiotics [2]. Most of the IRAB outbreak strains in Groups 1 and 2, including CM-128, were susceptible to colistin and resistant to almost all the antibiotics in the Vitek AST-N017 or AST-N022 kits (bioM´erieux, Durham, NC), including all the ␤-lactams tested (e.g. imipenem), gentamicin, netilmicin, tobramycin, isepamicin, ciprofloxacin, pefloxacin and trimethoprim/sulfamethoxazole. ISAB non-outbreak strains (Group 3) were susceptible to colistin and imipenem, and most of these strains showed multiresistance patterns, i.e. resistant to representative antibiotics of at least three different classes of antimicrobial agent [3]. Most of the netilmicin- and tobramycin-resistant A. baumannii (Group 4) and P. aeruginosa (Group 5) strains also showed multiresistance patterns. The CM-128 IRAB strain was selected for the sequencing study because it was isolated from the early stage of the outbreak and presented a pulsed-field gel electrophoresis (PFGE) pattern typical of the major clone of the outbreak. CM-128 was isolated from a wound culture specimen of a 63year-old female patient with panperitonitis due to a perforated gastric ulcer. For the Southern blot hybridisation, five Group 1, four Group 2 and three Group 3 strains were examined. 2.2. Integron polymerase chain reaction (PCR) and sequencing

2.4. Nested PCR Based on the gene cassette sequence of integronIS26 in CM-128, a nested PCR for integron-IS26 was developed to investigate the frequency of this integron among five selected groups of bacteria. PCR was carried out using the following specific primers for the integron: for the first-round primers, aac(6 )Im F 5’-TGCTTGGGAATATGTCTGG-3’ and aadA1 R2 5’-AGCGCCTCAAATAGATCCTGTTC-3’ (1837 bp amplicon); and for the second-round primers, aadA1 F 5’-CTCCGCGCTGTAGAAGTCACC-3’ and IS26 R 5’-TTCTGGGTAAAATCCTCAACAAC-3’ (517 bp amplicon). 2.5. Nucleotide sequence The nucleotide sequence reported in this paper has been deposited in EMBL/GenBank with accession no. DQ861641.

3. Results and discussion 3.1. Gene cassette composition of integron-IS26

The variable region of integron-IS26 from CM-128 was amplified by integron PCR using primers specific for the 5’CS and 3’CS [4,5]. To extend the sequence outward from the amplicon of the integron, thermal asymmetric interlaced PCR (TAIL-PCR) [6] was performed by DNA walking using the SpeedUpTM Premix Kit (Seegene, Rockville, MD). The resulting PCR products were cloned into pGEM-T Easy (Promega, Madison, WI) and sequenced at the Solgent Laboratory (Daejeon, South Korea) using the BigDye Terminator

DNA sequencing of the integron PCR product (2567 bp amplicon) of CM-128 showed that integron-IS26 in CM-128 contained two gene cassettes (Fig. 1): (i) the aac(6 )-Im cassette, which exhibited 99% identity with the sequence of a Citrobacter freundii strain isolated in Belgium [2]; and (ii) a peculiar aadA1 cassette that was disrupted by the insertion of IS26 (i.e. a disrupted aadA1 cassette), which was located 356 bp downstream of the start codon of aadA1. The integron-

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Fig. 1. Schematic representation of the class 1 integron identified in Acinetobacter baumannii CM-128. The gene cassettes are shown as boxes, with arrows indicating the direction of transcription. White ovals indicate the attI and attC (59-bp element) recombination sites. The 5 -conserved segment contains the intI1 integrase gene and the attI1 recombination site. The 3 -conserved segment, downstream of the gene cassettes, includes the qacEΔ1 and sul1 genes. aac(6 )-Im and aadA1 indicate aminoglycoside resistance genes. Asterisks show the 8-bp target duplication that occurs in aadA1 during IS26 integration.

IS26 sequence of the isolate shared 99% identity with the IS26 sequences of a wide variety of Gram-negative bacteria and, following removal of the element, aadA1 showed 100% identity with the aadA1 sequences of other Gram-negative bacteria. IS26, which is a non-autonomous transposable element originally found in transposon Tn2680 [9], is associated with several antibiotic resistance genes and is implicated in the dissemination of resistance genes via several routes [10]. In addition, IS26 can mobilise chromosomally located genes [11]. IS26 generates an 8-bp target duplication upon inte-

gration [12], which was also observed in our CM-128 strain (Fig. 1). Previously, insertion of IS26 was observed 39 bases downstream of aadA4 [13], the 5 -end of intI1 [14] and the 5 -end of frdB [15]. In strain CM-128, IS26 was inserted near the middle of the aadA1 gene cassette. Taken together, the results of the present study and other studies [12–15] confirm that the IS26 insertion sites are variable. Given the apparently random distribution of IS26 integration sites [12], with insertion even into the gene cassette, as well as its ability to mobilise chromosomally located resistance genes, this element may change the features of

Fig. 2. Pulsed-field gel electrophoresis (PFGE) and Southern hybridisation of 12 Acinetobacter baumannii strains. (A) PFGE profiles of whole-cell DNA digested with I-CeuI. (B–D) Southern hybridisation using gene-specific probes for 23S rRNA (B), integrase I1 (C) and the disrupted aadA1 cassette (D). Lanes 1–5, clinical outbreak strains of imipenem-resistant A. baumannii (IRAB) (Group 1); lanes 6–9, IRAB isolated from an environmental culture (Group 2); lanes 10–12, imipenem-susceptible A. baumannii non-outbreak strains (Group 3); lane M, lambda DNA size marker. The following strains are shown: lane 1, CM-128; lane 2, CM-106; lane 3, CM-111; lane 4, CM-130; lane 5, CM-132; lane 6, CM-302; lane 7, CM-304; lane 8, CM-309; lane 9, CM-310; lane 10, CM-202; lane 11, CM-204; lane 12, CM-225.

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Table 1 Nested polymerase chain reaction (PCR) for the detection of IS26 in different groups of bacteria Group

Species and features of bacteria

Year

No. tested

No. (%) PCR-positive for integron-IS26

Group 1 Group 2 Group 3 Group 4 Group 5

Clinical outbreak strains of IRAB IRAB isolated from environmental culture ISAB non-outbreak strains NET- and TOB-resistant A. baumannii NET- and TOB-resistant Pseudomonas aeruginosa

2004 2004 2004 2005 2005

26 10 23 28 28

19 (73.1) 10 (100) 3 (13.0) 1 (3.6) 0

PCR, polymerase chain reaction; IRAB, imipenem-resistant Acinetobacter baumannii; ISAB, imipenem-susceptible A. baumannii; NET, netilmicin; TOB, tobramycin.

aadA1 or of other IS26 located in sites other than the integron.

resistance genes thereby contributing to the evolution and dissemination of resistance genes. The presence of IS26 in outbreak strains, as in the present study, may facilitate the spread of resistance.

3.3. Prevalence of integron-IS26 in five groups of bacteria

3.2. Localisation of integron-IS26 The result of nested PCR for integron-IS26 showed that most of the IRAB outbreak strains (Groups 1 and 2 collected in 2004) contained integron-IS26 (Table 1). Since integronIS26 was also found in 3 (13.0%) of 23 ISAB non-outbreak strains (Group 3 collected in 2004), it is possible that this integron was transferred among different clones of A. baumannii. For the strains of Group 4 and Group 5 collected in 2005, 1 (3.6%) of 28 Group 4 strains and none of the Group 5 strains contained integron-IS26. The characteristics of 12 A. baumannii strains tested in PFGE–Southern blotting are shown in Table 2. The result of nested PCR for integron-IS26 was concordant with the Southern hybridisation reaction with the disrupted aadA1 probe (Table 2; Fig. 2D) in all 12 A. baumannii strains. Among the ISAB strains (Group 3), CM-202 containing integron-IS26 was more resistant to antibiotics than CM-204 and CM-225. The same findings were also observed in Group 3 and Group 4. Three strains of Group 3 and one strain of Group 4 A. baumannii carrying integron-IS26 were more resistant to

Integrons are located either episomally or chromosomally. Southern blotting was performed to locate the integron-IS26. PFGE of whole-cell DNA digested with I-CeuI revealed five chromosomal bands in all 12 A. baumannii strains tested (Fig. 2A). All five chromosomal bands hybridised with the 23S rRNA probe (Fig. 2B). Of these, the uppermost chromosomal band of 1087.5 kb in all 12 A. baumannii strains hybridised with the integrase I1 probes (Fig. 2C). These integron-containing chromosomal bands of all nine outbreak A. baumannii strains (Groups 1 and 2) and one of three non-outbreak A. baumannii strains (Group 3) hybridised with the disrupted aadA1 probes (Fig. 2D). These results indicate that the integron-IS26 is located chromosomally and that all nine outbreak strains and one non-outbreak strain of A. baumannii carry integron-IS26. Below them, another faint band of 533.5 kb hybridised with disrupted aadA1 probes in most A. baumannii strains tested (Fig. 2D). This is probably the result of the existence of an intact

Table 2 Characteristics of 12 Acinetobacter baumannii strains tested in pulsed-field gel electrophoresis (PFGE)–Southern blotting Group

Strain no.

Genotypea

Integron-IS26 PCR

Specimen

CAZ

CIP

GEN

IPM

NET

TZP

TOB

TIC

TIM

SXT

Group 1

CM-128 CM-106 CM-111 CM-130 CM-132

A A B A A

Pos. Pos. Pos. Pos. Pos.

Sputum Sputum Stool Sputum Sputum

R R R R R

R R R R R

R R R R R

R R R R R

R R R R R

R R R R R

R R R R R

R R R R R

R R R R R

R R R R R

Group 2

CM-302 CM-304 CM-309 CM-310

A A A A

Pos. Pos. Pos. Pos.

Env. C. Env. C. Env. C. Env. C.

N.D. N.D. N.D. N.D.

R R R R

R R R R

R R R R

R R R R

R R R R

R R R R

N.D. N.D. N.D. N.D.

R R R R

N.D. N.D. N.D. N.D.

Group 3

CM-202 CM-204 CM-225

A A C

Pos. Neg. Neg.

Sputum Sputum Sputum

R R S

R R S

R S S

S S S

R I S

I S S

R I S

R R S

R R S

R R S

Antibiogram

PCR, polymerase chain reaction; CAZ, ceftazidime; CIP, ciprofloxacin; GEN, gentamicin; IPM, imipenem; NET, netilmicin; TZP, piperacillin/tazobactam; TOB, tobramycin; TIC, ticarcillin; TIM, ticarcillin/clavulanic acid; SXT, trimethoprim/sulfamethoxazole; Pos., positive; Neg., negative; Env. C., environmental culture; R, resistant; S, susceptible; N.D., not done. a PFGE pattern after complete digestion of the A. baumannii strains using I-CeuI.

H.L. Han et al. / International Journal of Antimicrobial Agents 32 (2008) 165–169

antibiotics than other members of each group that did not carry the integron (data not shown). Although insertion of IS26 would have been expected to inactivate aadA1, which confers resistance both to streptomycin and spectinomycin, strain CM-128 showed resistance to both of these antibiotics. This may be explained by the presence of an intact copy of aadA1 in most of the outbreak strains, including CM-128, as verified by PCR. If the gene cassette composition of certain integrons is unique like integron-IS26, it may be useful as a marker in epidemiological studies of outbreak strains that contain such integrons. In summary, we have identified a novel class 1 integron that carries aac(6’)-Im and an IS26-disrupted copy of aadA1 (integron-IS26) in most of the IRAB outbreak strains tested, as well as in a few ISAB non-outbreak control strains. Integron-IS26 is located chromosomally.

Acknowledgments The authors thank Prof. Kyungwon Lee and Jong Hwa Yum, Yonsei University College of Medicine, Seoul, South Korea, and Dr Kyung Won Cho for their helpful discussion. This study was presented in part at the 47th Congress of the Korean Society for Laboratory Medicine, 21–23 September 2006, Seoul, South Korea. Funding: This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean government (MOST) through the Research Center for Resistant Cells (R13-2003-009). Competing interests: None declared. Ethical approval: Not required.

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