Dissemination of 16S rRNA methylase-mediated highly amikacin-resistant isolates of Klebsiella pneumoniae and Acinetobacter baumannii in Korea

Diagnostic Microbiology and Infectious Disease 56 (2006) 305 – 312 www.elsevier.com/locate/diagmicrobio

Dissemination of 16S rRNA methylase-mediated highly amikacin-resistant isolates of Klebsiella pneumoniae and Acinetobacter baumannii in Korea Hyukmin Leea, Dongeun Yongb, Jong Hwa Yumb, Kyoung Ho Rohb, Kyungwon Leeb, Kunikazu Yamanec, Yoshichika Arakawac, Yunsop Chongb,4 a Department of Laboratory Medicine, Kwandong University College of Medicine, Kyunggido 412-270, South Korea Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul 120-752, South Korea c Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases, Tokyo 208-0011, Japan Received 2 February 2006; accepted 8 May 2006

b

Abstract Novel 16S rRNA methylase-mediated high-level resistance to amikacin and arbekacin has been reported recently in clinical isolates of Gram-negative bacilli only from several countries. We tested amikacin- or arbekacin-nonsusceptible Gram-negative bacilli isolated in 2003 and 2005 at a tertiary-care hospital in Korea by polymerase chain reaction to detect 16S rRNA methylase genes. armA alleles were detected in 14 isolates of Klebsiella pneumoniae, 10 other species of Enterobacteriaceae, and 16 Acinetobacter baumannii, whereas the rmtB allele was detected in 1 K. pneumoniae isolate. The resistance 1st detected in 2003 persisted in 2005. 16S rRNA methylase-producing isolates were highly resistant to arbekacin and amikacin, and were mostly coresistant to levofloxacin. Most K. pneumoniae isolates also produced extended-spectrum h-lactamases and plasmid-mediated AmpC h-lactamases, and most A. baumannii isolates were nonsusceptible to carbapenems. D 2006 Elsevier Inc. All rights reserved. Keywords: 16S rRNA methylase; armA; rmtB; High-level aminoglycoside resistance

1. Introduction Aminoglycosides are used in the treatment of a broad range of life-threatening infections caused by both Grampositive and aerobic Gram-negative bacteria (MingeotLeclercq et al., 1999). Activity of aminoglycosides depends on binding to a highly conserved motif of 16S rRNA (Yoshizawa et al., 1999). Mechanisms of aminoglycoside resistance include decreased outer membrane permeability (Hancock, 1981), active efflux (Hooper, 2005), amino acid substitutions in ribosomal proteins, and enzymatic modifications of the drug (Shaw et al., 1993). It could be expected that amikacin-resistant clinical isolates are rare, because amikacin was developed to block the access of a variety of aminoglycoside-modifying enzymes to their target sites (Kondo and Hotta, 2005). It was reported that among the 4 Corresponding author. Tel.: +82-2-222-82446; fax: +82-2-313-0908. E-mail address: [email protected] (Y. Chong). 0732-8893/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2006.05.002

strains collected in 1998 to 2003 in North America, 99.4% of Enterobacteriaceae and 85.2% of Acinetobacter isolates were susceptible to amikacin (Sader et al., 2005). The latest semisynthetic aminoglycoside, arbekacin, is active against methicillin-resistant Staphylococcus aureus (You et al., 2000) and has been extensively used in Japan since its approval in 1990 to treat methicillin-resistant S. aureus infections (Kondo and Hotta, 2005). Galimand et al. (2003) reported that a Klebsiella pneumoniae isolate with a putative 16S rRNA methyltransferase was highly resistant to a large number of aminoglycosides. They named the 1st 16S rRNA methylase gene armA. The 16S rRNA methylase confers high-level resistance to 4,6-substituted deoxystreptamines, including arbekacin, amikacin, kanamycin, tobramycin, and gentamicin, by posttranscriptional methylation of rRNA leading to loss of affinity for aminoglycosides. The armA gene is borne by a conjugative plasmid and has the potential to transfer horizontally (Galimand et al., 2003).

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Dissemination of the armA gene to various species of Enterobacteriaceae was reported in European countries (Galimand et al., 2005), and the gene was also detected in an Escherichia coli isolated from an animal (Gonzalez-Zorn et al., 2005). Other 16S rRNA methylase genes, rmtA and rmtB, were detected in Pseudomonas aeruginosa isolates (Yokoyama et al., 2003; Yamane et al., 2004), and in a Serratia marcescens isolate, respectively (Doi et al., 2004). The reported isolates with these genes were small in number, but in a Taiwanese study, armA and rmtB was detected in 12 and 2 isolates of E. coli, respectively, and 16 and 5 isolates of K. pneumoniae, respectively (Yan et al., 2004). The rmtA gene-carrying P. aeruginosa isolate was highly resistant to amikacin and arbekacin (Yokoyama et al., 2003), suggesting usefulness of the resistance for screening the gene-carrying isolates. However, Gram-negative bacilli are usually not tested for arbekacin susceptibility in clinical laboratories, and the usefulness of amikacin resistance for screening armA gene-carrying isolates of Enterobacteriaceae and Acinetobacter spp. is unknown. In a Korean nationwide surveillance, the amikacin resistance rates of K. pneumoniae increased from 8% in 1997 to 13% in 2003 (Lee et al., 2006a). At a Korean tertiary-care hospital in 2004, resistance rates of K. pneumoniae, S. marcescens, P. aeruginosa, and Acinetobacter spp. to amikacin were very high, that is, 46%, 27%, 30%, and 65%, respectively (unpublished data), suggesting the possible presence of 16S rRNA methylase-producing strains. It was reported that the armA gene is flanked by putative transposable elements and is linked to bla CTX-M, suggesting the cotransfer of resistance to broad-spectrum cephalosporins (Galimand et al., 2003). In a Taiwanese study, most, but not all, of the armA- or rmtB-positive E. coli and K. pneumoniae isolates had CTX-M type enzymes (Yan et al., 2004), suggesting that this extended-spectrum h-lactamase (ESBL) gene is not always associated with armA or rmtB genes. The aim of this study was to determine the prevalence of the 16S rRNA methylase gene in amikacin- or arbekacinnonsusceptible clinical isolates of Enterobacteriaceae, P. aeruginosa, and Acinetobacter spp. and to characterize the isolates as to the aminoglycoside resistance level, the coresistance to h-lactams and fluoroquinolone, and the molecular epidemiology. The usefulness of amikacin and arbekacin disk diffusion test in screening for the isolates containing armA gene was also determined.

2. Materials and methods 2.1. Bacterial strains and antimicrobial susceptibility testing Gram-negative bacilli were isolated from clinical specimens at a Korean tertiary-care hospital. The species were identified by conventional methods (Farmer, 2003; Schreckenberger et al., 2003) or by using the Vitek GNI or ATB 32 GN systems (bioMerieux, Marcy L’Etoile, France). In the 1st phase of this study in September 2003, consecutive nonduplicate isolates by each indicated species were used, and in the 2nd phase study in June 2005, 216 consecutive nonduplicate isolates of all indicated species were used. The antimicrobial susceptibility was tested by the disk diffusion test (CLSI, 2006) using commercial disks and Mueller–Hinton II agar (Becton Dickinson, Sparks, MD). Arbekacin susceptibility was tested by using 30-Ag disks (Eiken Chemical, Tokyo, Japan) and interpreted as recommended by the disk manufacturer: V 13 mm, resistant; and z 18 mm, susceptible. MICs of aminoglycosides were determined by the agar dilution method (CLSI, 2006) using arbekacin (Meiji Seika, Tokyo, Japan), amikacin and kanamycin (Dong-A Pharmaceutical, Seoul, Korea), gentamicin and tobramycin (Dong Wha Pharmaceutical, Seoul, Korea), isepamicin and netilmicin (Yuhan, Seoul, Korea), streptomycin (Chong Kun Dang Pharmaceutical, Seoul, Korea), and spectinomycin (Kuk Je Pharmaceutical, Seoul, Korea). E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used for quality control. 2.2. Detection of the 16S rRNA methylase gene by polymerase chain reaction To detect armA, rmtA, and rmtB gene alleles, we tested isolates with arbekacin MIC z 16 Ag/mL in the 1st phase, whereas in the 2nd phase, isolates nonsusceptible to arbekacin or amikacin disk diffusion test were used. Polymerase chain reaction (PCR) primers used for armA and rmtA were as those previously reported (Yokoyama et al., 2003; Yamane et al., 2005), and others are shown in Table 1. The reaction was carried out in a total volume of 20 AL, containing 1 AL of template and PreMix (Bioneer, Cheongwon, Korea). Amplification conditions were the following: for 16S rRNA methylase genes, 94 8C for 5 min followed by 30 cycles of 94 8C for 30 s, 53 8C (armA), 58 8C (rmtA), or 60 8C (rmtB) for 30 s, and 72 8C for 45 s, and a

Table 1 Primers used for PCR detection of 16S rRNA methylase genes and h-lactamase genes Primer

Target

Oligonucleotides (5V to 3V)

Expected size (bp)

Reference

RMTB-F RMTB-R CTX-MUNI-F CTX-MUNI-R CTX-M14-F CTX-M14-R

rmtB

CCC AAA CAG ACC GTA GAG GC CTC AAA CTC GGC GGG CAA GC CVA TGT GCA GYA CCA GTA A ARG TSA CCA GAA YMA GCG G ACA ATG ACG CTG GCA GAA CTG TTA CAG CCC TTC GGC GAT GA

584

This study

585

A. Baurnfeinda

512

This study

a

bla CTX-M bla CTX-M-14

Personal communication.

H. Lee et al. / Diagnostic Microbiology and Infectious Disease 56 (2006) 305 – 312

307

Table 2 armA and rmtB alleles detected in clinical isolates of Gram-negative bacilli isolated in 2003 and in 2005 Species

No. of isolatesa September 2003

E. coli K. pneumoniae C. freundii E. cloacae S. marcescens Acinetobacter spp. P. aeruginosa Total

June 2005

Total

ARB or AMK I/R

armA allele positive

rmtB allele positive

Total

ARB or AMK I/R

armA allele positive

rmtB allele positive

30 30 15 15 14 15 30 148

2 14 3 4 6 4 9 42

0 9 1 2 4 2c 0 18

0 1 0 0 0 0 0 1

54 40 7 17 7 33 58 216

9 12 1 0 2 26 18 68

3 5b 0 NT 0 14c 0 22

0 0 0 NT 0 0 0 0

ARB = arbekacin; AMK = amikacin; I/R = intermediate or resistant; NT = not tested. a Consecutive isolates by each indicated species in 2003 and consecutive isolates by all indicated species in 2005. b One K. pneumoniae isolate in 2005 had bla CTX-M-14 allele. c All armA allele-positive isolates were identified to be A. baumannii.

final extension at 72 8C for 6 min; for bla CTX-M, 94 8C for 5 min followed by 30 cycles of 94 8C for 30 s, 55 8C for 30 s, and 72 8C for 1 min, and a final extension at 72 8C for 7 min. Plasmid-mediated AmpC h-lactamase genes were amplified as previously reported (Lee et al., 2005). All armA and rmtB allele-positive isolates were tested for ESBL production by a double-disk synergy test using ceftazidime, cefotaxime, aztreonam, cefepime, and amoxicillin–clavulanate disks (Pitout et al., 2003). Polymerase chain reaction (PCR) was performed to detect the bla CTX-M gene in the ESBL-producing isolates and to detect the plasmid-mediated AmpC h-lactamase gene in cefoxitin-

nonsusceptible E. coli and K. pneumoniae isolates (PerezPerez and Hanson, 2002). Imipenem-nonsusceptible isolates were tested for metallo-h-lactamase production by the imipenem disk Hodge (cloverleaf) test and the imipenem and ethylenediaminetetraacetic acid (EDTA) plus sodium mercaptoacetic acid (SMA) double-disk synergy test (Lee et al., 2003). 2.3. Conjugation and pulsed-field gel electrophoresis An agar mating method was used to transfer high-level aminoglycoside resistance from randomly selected clinical isolates to an azide-resistant E. coli J53 recipient. Azide

Table 3 Comparison of MICs of aminoglycosides for armA or rmtB allele-positive and allele-negative Gram-negative bacilli Species (no. of isolates tested)

Antimicrobial agent

MIC (Ag/mL) Range

Arbekacin Amikacin Kanamycin Tobramycin Gentamicin Netilmicin Isepamicin Spectinomycin Streptomycin Acinetobacter spp. Arbekacin Amikacin Kanamycin Tobramycin Gentamicin Netilmicin Isepamicin Spectinomycin Streptomycin b c d

MIC (Ag/mL) 50%

90%

b

Enterobacteriaceae

a

armA or rmtB allele-negative isolatesa

armA or rmtB allele-positive isolates

(n = 25) 512 to N 1024 N 1024 N 1024 512 to N 1024 256 to N 1024 N 1024 512 to N 1024 V 8 to N 1024 V 8 to 512 (n = 16)d 1024 to N 1024 N 1024 N 1024 N 1024 N 1024 N 1024 512 to N 1024 N 1024 512 to N 1024

Range

50%

90%

c

N 1024 N 1024 N 1024 N 1024 1024 N 1024 N 1024 512 256

N 1024 N 1024 N 1024 N 1024 N 1024 N 1024 N 1024 N 1024 512

N 1024 N 1024 N 1024 N 1024 N 1024 N 1024 N 1024 N 1024 N 1024

N 1024 N 1024 N 1024 N 1024 N 1024 N 1024 N 1024 N 1024 N 1024

(n = 10) 1 to 64 1 to 64 2 to 1024 1 to 128 1 to 512 1 to 256 1 to 8 8 to N 1024 4 to 1024 (n = 9) 8 to 32 32 to 256 16 to N 1024 8 to 128 64 to 256 128 to N 1024 32 to 512 256 to N 1024 512 to N 1024

Arbekacin- or amikacin-nonsusceptible isolates by disk diffusion test. Number of isolates were K. pneumoniae 15, S. marcescens 4, E. coli 3, E. cloacae 2, and C. freundii 1. All isolates were K. pneumoniae. All armA or rmtB allele-positive isolates were identified to be A. baumannii.

8 32 256 32 1 128 2 256 128

64 32 1024 64 256 256 4 N 1024 512

16 64 N 1024 128 256 N 1024 128 N 1024 1024

32 256 N 1024 128 256 N 1024 512 N 1024 N 1024

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100 Ag/mL and amikacin 30 Ag/mL were used to select for transconjugants. Plugs of genomic DNA were prepared according to the protocols from the manufacturer of the CHEF-DR II system (Bio-Rad, Hercules, CA) and digested using SmaI for isolates of Acinetobacter baumannii and XbaI for isolates of Enterobacteriaceae. Pulsed-field gel electrophoresis (PFGE) was performed for 20 h at 6 V with a 3-s initial pulse and a 10-s final pulse for A. baumannii; pulses of 0.5 and 60 s were used for other species. After ethidium bromide staining, the DNA band patterns were analyzed using the UVIband/Map software (UVItech, Cambridge, United Kingdom) to generate a dendrogram that was based on the unweighted pair group method using an arithmetic average from the Dice coefficient. 3. Results and discussion In the 1st phase study in 2003, to determine presence of Gram-negative bacilli with 16S rRNA methylase-mediated high level aminoglycosides resistance, we detected armA and rmtB alleles by PCR in 18 isolates and 1 isolate,

respectively, of 42 strains, for which arbekacin MICs were 16 to N 128 Ag/mL. Most armA allele-positive isolates were K. pneumoniae (Table 2). In the 2nd phase study in 2005, to determine persistence of the resistance, we detected armA alleles in 3, 5, and 14 isolates of E. coli, K. pneumoniae, and A. baumannii, respectively, among 68 amikacin- or arbekacin-nonsusceptible isolates by the disk test. It is interesting that armA allele-positive A. baumannii became predominant and that rmtA allele was not detected, although it was present in P. aeruginosa isolates from Japan (Yamane et al., 2005; Yokoyama et al., 2003). High-level aminoglycoside resistance was transferred from 1 strain each of E. coli, K. pneumoniae, Enterobacter cloacae, and Citrobacter freundii, but not from S. marcescens and A. baumannii isolates (data not shown). Partial sequencing of nucleotides confirmed the amplicons from 1 isolate each of all 4 species, and 1 transconjugant had armA alleles (data not shown). The MIC50s of all aminoglycosides, including arbekacin and amikacin, but not streptomycin and spectinomycin, were z 1024 Ag/mL for armA and rmtB allele-positive isolates of Enterobacteriaceae (Table 3). 16S rRNA

Table 4 Pulsed-field gel electrophoresis types of endonuclease-restricted genomic DNA and antimicrobial susceptibility of armA or rmtB allele-positive Gram-negative bacilli Isolates with alleles of

K. pneumoniae

E. coli

E. cloacae C. freundii S. marcescens A. baumannii

PFGE type

DDS test

Antimicrobial susceptibility

YMC strain no.

FOX (P-M AmpC)

IPM

MEM

LVX

armA armA

A1 A1

Pos Pos

R (CMY-2) R (CMY-2)

I S

I S

R R

armA armA armA armA armA armA armA rmtB armA armA armA armA armA armA armA armA armA armA

A1 A2 A3 B C1 C2 D E F A B C A B NT NT NT A1, 3–6

Neg Pos Pos Pos Posb Pos Pos Pos Neg Pos Neg Pos Pos Neg Neg Pos Pos Neg

R (CMY-2) R (CMY-2) R (DHA-1) R (DHA-1) I (Neg) R (Neg) R (Neg) S (NA) R (DHA-1) R (DHA-1) R (Neg) R (DHA-1) NA NA NA NA NA NA

S R S S S S S S S S S S S S S S S R

S R S S S S S S S S S S S S S R I R

R R R R R R S S R R R R R S I R R R

armA

A2

Neg

NA

I

R

R

armA armA armA armA

A7 B C1 C2

Pos Neg Neg Neg

NA NA NA NA

S S I S

R S R S

R R R I

03/9/U1015 03/9/R533, 03/9/U818, 03/9/U840, 03/9/U1041 03/9/T86, 03/9/U959 03/9/R615 03/9/R547 05/6/U1062Ka 05/6/U1162 05/6/P359 05/6/P300 03/9/U756 05/6/U1190 05/6/U1062Ea 05/6/U1079 05/6/U1173 03/9/P218 03/9/P254 03/9/T109 03/9/U630, 03/9/U1649 03/9/U837, 03/9/U881 03/9/R496, 05/6/R672, 05/6/T127, 05/6/R667, 05/6/P365, 05/6/R912 05/6/R660, 05/6/R749, 05/6/R752, 05/6/R873, 05/6/R891, 05/6/U990 05/6/R783 03/9/P225 05/6/R933 05/6/P364

DDS = double-disk synergy test for ESBL; FOX = cefoxitin; P-M = plasmid-mediated; IPM = imipenem; MEM = meropenem; LVX = levofloxacin; Pos = positive; Neg = negative; NA = not applicable; S = susceptible; I = intermediate; R = resistant; YMC = Yonsei Medical Center. a An isolate each of E. coli and K. pneumoniae were isolated from a patient. b A bla CTX-M-14 allele was detected.

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309

Table 5 Comparison of efficiency of arbekacin and amikacin disk diffusion test to screen armA and rmtB allele-positive Gram-negative bacilli armA or rmtB

Organism (no. of isolates)

Aminoglycosides

Positive

Enterobacteriaceae (15)

Arbekacin Amikacin Arbekacin Amikacin Arbekacin Amikacin Arbekacin Amikacin Arbekacin Amikacin

6

A. baumannii (14) Negative

Enterobacteriaceae (15) Acinetobacter spp. (12) P. aeruginosa (17)

a

No. of isolates with inhibition zone diameter (mm)a

15 14 12 14

7

8

9

10

12

13

14

15

16

17

18

19

z 20

4 4

2 4 1

1 2

1 2

2

1

2

1 1

1

1 2 4 1

1

1 1

1 2

2

1

3

1 2

2 11

2

2 1

1 7

11

1

1 1

1

8 1

Data in boldface indicate number of isolates resistant to arbekacin or amikacin.

methylases do not confer high-level resistance to streptomycin. The MICs of the aminoglycosides were much lower for arbekacin- or amikacin-nonsusceptible isolates that were 16S rRNA methylase gene negative. MICs for 4 transconjugants from 1 isolate each of K. pneumoniae, E. coli, C. freundii, and E. cloacae were all z 1024 Ag/mL, except for those of streptomycin and spectinomycin, which were 256 to 512 Ag/mL and 32 to N1024 Ag/mL, respectively (data not shown). Among the 41 armA or rmtB allele-positive isolates, 12 (80.0%) of 15 K. pneumoniae, 2 of 3 E. coli, 1 of 2 E. cloacae, and all 4 S. marcescens isolates were doubledisk synergy test positive, indicating production of ESBLs (Table 4). The armA gene was reported to be linked to bla CTX-M gene in a K. pneumoniae isolate (Galimand et al., 2003), and in a Taiwanese study, 94.3% of armA- or rmtBpositive E. coli and K. pneumoniae isolates had CTX-M type ESBLs (Yan et al., 2004). However, in our study, the bla CTX-M-14 allele was detected in only 1 K. pneumoniae isolate (case 12), suggesting that 16S rRNA methylase gene

is not always linked to bla CTX-M. In a Taiwanese study, 2 armA-positive E. coli isolates had the plasmid-mediated AmpC h-lactamase CMY-2 (Yan et al., 2004). Of the 18 isolates of armA or rmtB allele-positive K. pneumoniae and E. coli in our study, 16 were resistant to cefoxitin, and among them, 8 and 5 were positive for bla CMY-2 and bla DHA-1 alleles, respectively (Table 4). This finding may not be unusual because our previous study in 2003 showed that among the cefoxitin-resistant isolates, 32.7% of E. coli and 76.2% of K. pneumoniae had either bla CMY-2 or bla DHA-1 alleles (Lee et al., 2006b). Two of 15 K. pneumoniae isolates and 13 (81.3%) of 16 A. baumannii isolates were nonsusceptible to imipenem by the disk diffusion test, but they were negative to the imipenem disk Hodge test, and the imipenem and EDTA–SMA double-disk synergy test (Lee et al., 2003), indicating nonproduction of a metalloh-lactamase (data not shown). The levofloxacin resistance rates of armA or rmtB allele-positive isolates of K. pneumoniae and A. baumannii were much higher, that is, 86.7% and 93.8%, respectively (Table 4), compared with

Table 6 Clinical features of the patients from whom armA or rmtB allele-positive isolates were detected Age (year) (no. of patients)

Male/female

b 15 (4)

1/3

2

2

1

15–29 30–39 40–49 50–59

1/1 4/0 3/0 6/3

1 3 2 6

1 1 1 3

60–69 (10)

5/5

5

N 70 (8)

5/3

Total (40)

25/15

(2) (4) (3) (9)

Admission wards

Underlying diseases or clinical conditions

ICUs

Malignancy

Others

IC bleeding

Sources of isolation

UGI bleeding

Paraplegia

Other conditions

0

0

0

0 1 0 1

0 1 2 3

0 1 0 1

1 0 0 0

One each of spleen rupture, dextrocardia, and foot injury SLE Septic shock

4

4

3

0

1

5

4

0

2

1

2

24

16

7

11

3

4

One each of DM, liver cirrhosis, epilepsy, and herniated cervical disk One each of dermatomyositis and pulmonary edema One each of septic shock, cholecystitis, and pneumonia

Sputum

Urine

Wound

1

1

2

0 2 0 4

0 2 2 3

2 0 1 2

5

5

0

5

3

0

17

16

7

ICU = intensive care unit; IC = intracranial; UGI = upper gastrointestinal; SLE = systemic lupus erythematosus; DM = diabetes mellitus.

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Fig. 1. Pulsed-field gel electrophoresis patterns of endonuclease-restricted genomic DNAs from 16S rRNA methylase-positive isolates of K. pneumoniae (A) and A. baumannii (B). Seven of 10 armA gene-positive K. pneumoniae isolates from 2003 showed identical PFGE patterns (A1). Among the 14 A. baumannii isolates with armA genes from 2005, 6 showed identical patterns (A2).

H. Lee et al. / Diagnostic Microbiology and Infectious Disease 56 (2006) 305 – 312

those of 31% and 60%, respectively, in 2003, at the hospital (unpublished data). The resistance rates of armA or rmtB allele-positive isolates to trimethoprim–sulfamethoxazole and tetracycline were 100% for A. baumannii, and 76% and 72%, respectively, for Enterobacteriaceae (data not shown). Correlation of inhibition zone diameter of b9 mm and presence of armA or rmtB allele (Table 5) showed that arbekacin was more useful than amikacin for screening isolates with these resistance determinants; positive rates were 29 of 32 isolates (90.6%) with arbekacin compared with 29 of 48 isolates (60.4%) with amikacin. Retrospective analysis of clinical features showed that the armA and rmtB allele-positive isolates were from 25 (62.5%) male and 15 (37.5%) female patients, and 27 (67.5%) were older than 49 years (Table 6). All were inpatients with various morbidities, and 24 (60.0%) were admitted to intensive care units. The impact of antimicrobial resistance on the clinical outcomes of study patients was not evaluated because many patients received various antimicrobial agents, and the etiologic role of the sputum isolates is difficult to determine (Thomson and Miller, 2003). The sources of the isolates were sputum (42.5%), urine (40.0%), and wound (17.5%). Seven of 10 armA gene-positive K. pneumoniae isolates from 2003 showed identical PFGE patterns (A1), suggesting that a small outbreak occurred from a clone (Fig. 1A). However, all 5 isolates in 2005 had different patterns, suggesting that the horizontal spread of the resistance determinant had occurred. The presence of armA allele-positive E. coli and K. pneumoniae in a patient (case No. 15) suggested that in vivo horizontal transfer of the resistance determinant had possibly occurred. Among the 14 A. baumannii isolates in 2005, 6 showed identical patterns (Fig. 1B and A2), again suggesting a clonal outbreak. Isolates in 2003 and 2005 showed distantly related patterns suggesting that horizontal spread of the resistance had occurred. In conclusion, 16S rRNA methylase-producing isolates of K. pneumoniae and A. baumannii were relatively more prevalent in a hospital in Korea than in other countries. 16S rRNA methylase-producing isolates were highly resistant to arbekacin and amikacin, and are mostly coresistant to levofloxacin. Most K. pneumoniae isolates also produced ESBLs and plasmid-mediated AmpC h-lactamases, but bla CTX-M was detected in only 1 isolate. Most A. baumannii isolates were also nonsusceptible to imipenem or meropenem. Further studies are urgently required to determine the resistance prevalence in other Korean hospitals to establish measures to control further spread of the resistance. Acknowledgments This work was supported in part by the Brain Korea 21 Project for Medical Sciences, Yonsei University, Seoul, South Korea, in 2004. The authors thank Myungsook Kim for collecting the isolates and Younghee Seon and Chasoon Lee for technical assistance.

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