Abstract
The distribution of conjugative-plasmid-mediated 16S rRNA methylase genes among amikacin-resistant Enterobacteriaceae collected between 1995 and 1998 and between 2001 and 2006 at a university hospital in South Korea was examined, and conjugative plasmids carrying the 16S rRNA methylase genes were characterized by PCR-based replicon typing and by determination of their antimicrobial resistance pattern. Among the 7,127 isolates, 463 isolates showed a high level of resistance to amikacin, and 218 of the 463 isolates transferred amikacin resistance by conjugation. Among the 218 isolates, armA was detected in 153 isolates (88 Klebsiella pneumoniae, 28 Escherichia coli, 19 Enterobacter cloacae, and 6 Serratia marcescens isolates and 12 isolates of other organisms), and rmtB was detected in 51 isolates (32 K. pneumoniae isolates, 18 E. coli isolates, and 1 Citrobacter freundii isolate). The first appearance of armA was in 1997. The armA gene was carried by conjugative plasmids of replicon groups IncL/M, IncFIIAs, IncF, IncA/C, IncHI2, and Inc(unidentified) in 38, 20, 7, 9, 4, and 75 strains, respectively. The rmtB gene was carried by conjugative plasmids of groups IncA/C, IncF, and IncI1-Iγ in 43 strains, 7 strains, and 1 strain, respectively. Transconjugants that received the IncL/M plasmid carrying armA or the IncA/C plasmid carrying rmtB showed an additional resistance to cefotaxime. Transconjugants that received the IncFIIA plasmid or Inc(unidentified) plasmid carrying the armA gene showed an additional resistance to cefoxitin and a high MIC50 (0.25 mg/liter) of ciprofloxacin. In conclusion, this study demonstrated that the dissemination of 16S rRNA methylase genes among the Enterobacteriaceae is mediated by conjugative plasmids of various incompatibility groups that confer resistance to multiple drugs, including aminoglycosides, extended-spectrum β-lactams, and/or quinolones.
Aminoglycosides have a high affinity for the 16S rRNA of the bacterial 30S ribosome, and they block protein synthesis (14, 20). Over the past few decades, there have been many studies conducted regarding the mechanisms of resistance to aminoglycosides. One of the most common mechanisms of resistance to aminoglycosides is the production of aminoglycoside-modifying enzymes, such as 1-N-aminoglycoside acetyltransferase (AAC), adenyltransferase, and phosphotransferase (14, 20). Amikacin was developed to suppress a variety of aminoglycoside-modifying enzymes from their accessing target sites (12), and therefore rare, amikacin-resistant bacteria could be expected. Recently, a series of special methylases that protect microbial 16S rRNA, however, were identified in several nosocomial pathogens, and these enzymes are capable of conferring extraordinarily high levels of resistance (MIC > 512 mg/liter) to most clinically important aminoglycosides, including amikacin, isepamicin, arbekacin, kanamycin, tobramycin, and gentamicin (5-8, 21-24). Since the first identification of a gene encoding 16S rRNA methylase, rmtA, from a Pseudomonas aeruginosa isolate in 2003 (20), four major 16S rRNA methylases, rmtA (24), armA (6), rmtB (5), and rmtC (21), have been reported to occur in several nosocomial pathogens, including P. aeruginosa, Serratia marcescens, Klebsiella pneumoniae, Acinetobacter baumannii, and Proteus mirabilis (5, 6, 13, 21, 24). The genes for 16S rRNA methylases are mediated by mobile genetic elements that are carried by transferable large plasmids (5, 7-8); thus, the spread of these genes among gram-negative bacilli has been concerning. Recently, several studies have demonstrated the dissemination of the armA gene to various species of Enterobacteriaceae in European countries (7), the spread of the multidrug-resistant Escherichia coli and K. pneumoniae isolates that produce both extended-spectrum β-lactamases (ESBLs) and 16S rRNA methylases in Taiwan (23), the dissemination of 16S rRNA methylase-mediated amikacin-resistant isolates of K. pneumoniae and A. baumannii in South Korea (13), and the emergence of 16S rRNA methylases in Belgium (2).
In this study, we tried to determine the beginning of the emergence and the current prevalence of plasmid-mediated 16S rRNA methylases among the Enterobacteriaceae. This was done by detecting the 16S rRNA methylase genes in amikacin-resistant Enterobacteriaceae isolates that were collected in 1995 to 1998 and 2001 to 2006 at a university hospital in South Korea. In addition, in order to understand how the genes were disseminated, conjugative plasmids carrying the 16S rRNA methylase genes were identified by PCR-based replicon typing of the major plasmid incompatibility (Inc) groups among the Enterobacteriaceae.
MATERIALS AND METHODS
Bacterial strains.
A total of 7,217 nonduplicate Enterobacteriaceae isolates were collected in 1995 to 1998 and 2001 to 2006 from patients hospitalized at Kyungpook National University (KNU) Hospital in South Korea. Bacterial species were identified by using the Vitek GNI system (bioMerieux, Marcy L'Etoile, France) or the API 20E kit (bioMerieux, Marcy L'Etoile, France). All isolates were tested for resistance to amikacin using the agar dilution method (4).
Transfer of the amikacin resistance determinant by conjugation.
In order to test the transferability of the amikacin resistance determinant to the rifampin-resistant E. coli strain RG 488 and the azide-resistant strain J53, all amikacin-resistant isolates were included as putative donors in a conjugation assay using the broth mating method. The transconjugants were selected on a Muller-Hinton agar plate supplemented with amikacin (64 μg/ml) and rifampin (50 μg/ml) or sodium azide (200 μg/ml).
Detection of 16S rRNA methylase genes and aacA4.
A PCR method was used to detect the genes encoding 16S rRNA methylases (rmtA, rmtB, and armA) and the gene encoding AAC(6′)-Ib (aacA4), which are known to confer resistance to amikacin. The primers used were those described by Yan et al. (23) for rmtA, rmtB, and armA and by Shi et al. (18) for aacA4. PCRs were performed as described previously (18, 23).
PCR-based replicon typing.
All transconjugants obtained from a conjugation assay were subjected to typing by a PCR method, based on replicons of the major plasmid incompatibility groups among Enterobacteriaceae, that was developed by Carattoli et al. (3). The plasmid DNAs from the transconjugants were isolated by an alkaline lysis method (1) and amplified by five multiplex and three simplex PCRs using 18 pairs of primers that recognized Inc replicons FIA, FIB, FIC, HI1, HI2, I1-Iγ, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA. As positive controls in the PCRs, R27 (HI1), R478 (HI2), R483 (I1), R446b (L/M), RN3 (N), Tp181 (FIme), RS-a (W), RP4(P), R40a (A/C), Rts1 (T), R124 (FIV), R387 (K), R16 (B/O), and R6K (X) were used.
Antimicrobial susceptibility testing.
Antimicrobial susceptibility testing and the determination of MICs were performed by the agar dilution method (4). The antimicrobial agents included the following: gentamicin and kanamycin (Duchefa, Haarlem, The Netherlands), amikacin and trimethoprim (ICN Biomedicals, Irvine, CA), streptomycin (Sigma Chemical Co., St. Louis, MO), ampicillin (USB, Cleveland, OH), cefoxitin (Sigma), cefotaxime (Sigma), cefepime (Boryung Inc., Seoul, South Korea), aztreonam (Sigma), ceftazidime (Sigma), ciprofloxacin (Fluka, Buchs, Switzerland), chloramphenicol (Sigma), tetracycline (Sigma), and sulfamethoxazole (Sigma). Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains.
RESULTS
Among the 7,217 clinical isolates of Enterobacteriaceae that were collected from the KNU Hospital in South Korea in 1995 to 1998 and 2001 to 2006, 463 (6.5%) strains showed resistance to amikacin (Table 1). The rate of resistance to amikacin increased from 2.7% in 1995 to 8.4% in 2006. Overall, 269 (13.1%) of the 2,059 Klebsiella pneumoniae, 100 (3.0%) of the 3,326 Escherichia coli, 42 (8.7%) of the 484 Enterobacter cloacae, and 23 (7.5%) of the 307 Serratia marcescens isolates were resistant to amikacin. The rates of resistance to amikacin in Citrobacter freundii, Morganella morganii, and Proteus mirabilis were 5.0% (8/161 isolates), 3.2% (2/76 isolates), and 2.1% (3/140 isolates), respectively.
TABLE 1.
Numbers of isolates studied and rates of resistance to amikacin among Enterobacteriaceae that were collected from a university hospital in South Korea between 1995 and 2006
| Species | No. of isolates studied (no. of isolates resistant to amikacin) in:
|
Total no. of isolates (total no. resistant to amikacin) | Rate of resistance to amikacin (%) | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1995 | 1996 | 1997 | 1998 | 2001 to ∼2002 | 2003 | 2004 | 2005 | 2006 | |||
| E. coli | 132 (0) | 197 (5) | 157 (1) | 6 (2) | 492 (13) | 156 (9) | 418 (14) | 908 (31) | 860 (25) | 3,326 (100) | 3.0 |
| K. pneumoniae | 60 (2) | 61 (3) | 141 (3) | 42 (3) | 304 (8) | 83 (5) | 226 (26) | 605 (111) | 537 (108) | 2,059 (269) | 13.1 |
| Klebsiella spp. | 2 (0) | 9 (0) | 24 (2) | 4 (0) | 39 (0) | 12 (0) | 18 (0) | 36 (2) | 5 (1) | 149 (5) | 3.4 |
| E. cloacae | 27 (4) | 25 (3) | 29 (9) | 5 (2) | 86 (5) | 13 (2) | 65 (6) | 109 (6) | 125 (5) | 484 (42) | 8.7 |
| Enterobacter spp. | 5 (0) | 7 (1) | 29 (0) | 4 (2) | 45 (0) | 5 (0) | 44 (0) | 107 (2) | 108 (5) | 354 (10) | 2.8 |
| S. marcescens | 16 (0) | 9 (0) | 43 (2) | 7 (0) | 75 (5) | 45 (7) | 23 (2) | 38 (1) | 51 (6) | 307 (23) | 7.5 |
| Serratia spp. | 2 (0) | 2 (0) | 4 (0) | 0 | |||||||
| C. freundii | 4 (0) | 2 (0) | 8 (1) | 1 (1) | 15 (0) | 14 (2) | 17 (0) | 47 (3) | 53 (1) | 161 (8) | 5.0 |
| Citrobacter spp. | 3 (0) | 6 (0) | 4 (1) | 13 (1) | 2.6 | ||||||
| Morganella morganii | 4 (0) | 2 (0) | 4 (0) | 10 (0) | 4 (0) | 25 (1) | 2 (1) | 25 (0) | 76 (2) | 3.2 | |
| P. mirabilis | 6 (1) | 3 (0) | 6 (0) | 3 (0) | 18 (0) | 10 (0) | 15 (0) | 39 (1) | 40 (1) | 140 (3) | 2.1 |
| Proteus spp. | 2 (0) | 1 (0) | 2 (0) | 5 (0) | 2 (0) | 4 (0) | 11 (0) | 16 (0) | 43 (0) | 0 | |
| Providencia spp. | 3 (0) | 3 (0) | 2 (0) | 3 (0) | 11 (0) | 0 | |||||
| Total no. of isolates (total no. resistant to amikacin) | 261 (7) | 316 (12) | 443 (18) | 72 (10) | 1,092 (31) | 344 (25) | 860 (49) | 1,913 (158) | 1,826 (153) | 7,127 (463) | |
| Rate of resistance to amikacin (%) | 2.7 | 3.8 | 4.1 | 13.9 | 2.8 | 7.3 | 5.7 | 8.3 | 8.4 | 6.5 | |
By a conjugation experiment, amikacin resistance could be transferred to 218 (47.1%) of the 463 amikacin-resistant isolates. All 218 strains and their transconjugants were examined for the presence of the genes that encode 16S rRNA methylases (armA, rmtA, and rmtB) and the gene that encodes AAC(6′)-Ib (aacA4). Among the 218 strains, armA, rmtB, and aacA4 were detected in 153 (33.1%), 51 (11.0%), and 69 (31.7%) isolates, respectively, but rmtA was not detected in any of the isolates (Tables 2 and 3). aacA4 was detected with armA (50 strains) or rmtB (6 strains), and it was transferred simultaneously with armA (37 strains) to the recipient E. coli strain, but not with rmtB. The first appearances of armA and rmtB were in 1997 and 2001, respectively (Table 2). Whereas the armA gene was detected among various species of Enterobacteriaceae, rmtB was detected only in E. coli, in K. pneumoniae, and in one Citrobacter freundii isolate (Table 3).
TABLE 2.
Distribution of aacA4 and 16S rRNA methylase genes among the 218 amikacin-resistant isolates of Enterobacteriaceae that were collected from a university hospital in South Korea
| Yr of isolation | No. of strains carrying the following gene(s):
|
Total | |||||
|---|---|---|---|---|---|---|---|
| aacA4 alone | armA alone | armA and aacA4 | rmtB alone | rmtB and aacA4 | Nonea | ||
| 1995 | 2 | 2 | |||||
| 1996 | 9 | 9 | |||||
| 1997 | 1 | 7 | 2 | 10 | |||
| 1998 | 1 | 3 | 1 | 5 | |||
| 2001 | 4 | 2 | 5 | 11 | |||
| 2002 | 1 | 5 | 6 | ||||
| 2003 | 9 | 9 | |||||
| 2004 | 19 | 6 | 1 | 26 | |||
| 2005 | 45 | 8 | 20 | 1 | 1 | 75 | |
| 2006 | 24 | 24 | 17 | 65 | |||
| Total no. of strains | 13 | 103 | 50 | 45 | 6 | 1 | 218 |
No gene was detected.
TABLE 3.
Distribution of aacA4 and 16S rRNA methylase genes among the 218 amikacin-resistant isolates of Enterobacteriaceae collected from a university hospital in South Korea between 1995 and 2006
| Species | No. of strains carrying the following amikacin resistance gene(s):
|
Total no. of strains | |||||
|---|---|---|---|---|---|---|---|
| aacA4 alone | armA alone | armA and aacA4 | rmtB alone | rmtB and aacA4 | Nonea | ||
| E. coli | 5 | 14 | 14 | 17 | 1 | 1 | 52 |
| K. pneumoniae | 3 | 65 | 23 | 27 | 5 | 123 | |
| Klebsiella spp. | 1 | 1 | 2 | ||||
| E. cloacae | 4 | 17 | 2 | 23 | |||
| Enterobacter spp. | 1 | 5 | 1 | 7 | |||
| S. marcescens | 6 | 6 | |||||
| C. freundii | 3 | 1 | 4 | ||||
| M. morganii | 1 | 1 | |||||
| Total no. of strains | 13 | 103 | 50b | 45 | 6c | 1 | 218 |
No gene was detected.
Thirteen of 50 strains transferred the armA gene alone to the recipient E. coli strain, as determined by the conjugation assay.
All six strains transferred the rmtB gene alone to the recipient E. coli strain, as determined by the conjugation assay.
In order to identify the conjugative plasmids carrying armA or rmtB, PCR-based replicon typing was performed using 18 pairs of primers that recognized the Inc replicons FIA, FIB, FIC, HI1, HI2, I1-Iγ, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA (Table 4). IncL/M, IncFIIAs, and IncHI2 were detected in 37 (24.2%), 20 (13.1%), and 4 (2.6%) of the 153 transconjugants that carry armA, respectively, but not in the transconjugants that carry rmtB. IncA/C was detected in 41 (80.3%) of the 51 transconjugants that carry rmtB and in 7 (4.6%) of the 153 transconjugants that carry armA. IncF was detected in 6 (11.7%) of the 51 transconjugants that carry rmtB and in 7 (4.6%) of the 153 transconjugants that carry armA. IncFIB and IncB/O were detected only in the transconjugants that carry aacA4 alone. In 75 (49.0%) of the 153 transconjugants that carry armA, none of the 18 replicons were detected. Replicons such as N, P, W, T, K, and X were not detected in any of the strains that carry armA, rmtB, or aacA4.
TABLE 4.
Incompatibility groups of conjugative plasmids which confer a high level of resistance to amikacin due to the presence of the 16S rRNA methylase gene and/or aacA4 gene
| Incompatibility group(s) | No. of transconjugants carrying the following gene(s):
|
Total | No. (%) of transconjugants with:
|
P valuec | |||||
|---|---|---|---|---|---|---|---|---|---|
| aacA4 alone | armA alone | armA and aacA4 | rmtB alone | Nonea | armA | rmtB | |||
| FIIAs | 20 (13.1) | 0.007 | |||||||
| HI2 | 4 | 4 | 4 (2.6) | ||||||
| L/M | 8 | 29 | 37 | 37 (24.2) | <0.001 | ||||
| L/M, F | 1 | 1 | 1 (0.7) | ||||||
| A/C | 2 | 4 | 3 | 41 | 1 | 51 | 7 (4.6) | 41 (80.3) | <0.001 |
| A/C, I1-Iγ | 1 | 1 | 2 | 2 (1.3) | |||||
| A/C, F | 1 | 1 | 1 (2.0) | ||||||
| A/C, F, FIA | 1 | 1 | 1 (2.0) | ||||||
| F | 7 | 6 | 1 | 14 | 7 (4.6) | 6 (11.7) | 0.069 | ||
| F, Y | 1 | 1 | 1 (2.0) | ||||||
| I1-Iγ | 1 | 1 | 1 (2.0) | ||||||
| F, FIB | 1 | 1 | |||||||
| B/O | 4 | 4 | |||||||
| NIb | 4 | 72 | 3 | 1 | 80 | 75 (49.0) | <0.001 | ||
| Total no. of transconjugants | 11 | 116 | 37 | 51 | 3 | 218 | 153 | 51 | |
No gene was detected.
NI, not identified.
The chi-square test was performed.
MICs of various kinds of antimicrobial agents for the transconjugants that received plasmids carrying armA or rmtB were determined. All transconjugants showed high levels of resistance (MICs, >256 μg/ml) to amikacin, kanamycin, and gentamicin. MIC50s of 12 kinds of antimicrobial agents other than aminoglycosides were calculated among the strains that were grouped with regard to the replicon types of plasmid incompatibility groups and the presence of armA or rmtB (Table 5). Groups with fewer than five strains were excluded from the calculation of MIC50s. Surprisingly, a very distinctive antimicrobial resistance pattern was revealed in each group. Compared to other groups, transconjugants that received the IncL/M plasmid carrying armA and the IncA/C plasmid carrying rmtB showed higher MIC50 values for cefotaxime (64 μg/ml) and cefepime (8 μg/ml). In the transconjugants that received the Inc(unidentified) plasmid carrying armA, higher MIC50 values of aztreonam (32 μg/ml), ceftazidime (32 μg/ml), cefoxitin (32 μg/ml), and ciprofloxacin (0.25 μg/ml) were revealed. Transconjugants that received the IncFIIAs plasmid carrying armA showed higher MIC50 values of cefoxitin (32 μg/ml) and ciprofloxacin (0.25 μg/ml). Transconjugants that received the IncA/C plasmid carrying rmtB showed additional resistance to multiple antimicrobial agents, including ampicillin, cefotaxime, chloramphenicol, streptomycin, sulfamethoxazole, trimethoprim, and tetracycline.
TABLE 5.
Antimicrobial susceptibilities of transconjugants grouped by presence of the armA or rmtB gene and by plasmid incompatibility groupa
| Antimicrobial agent | MIC50 for isolates with the indicated gene and Inc plasmidb:
|
||||||
|---|---|---|---|---|---|---|---|
|
armA (n = 143)
|
rmtB (n = 47)
|
||||||
| FIIAs (n = 20) | L/M (n = 37) | A/C (n = 7) | F (n = 7) | NI (n = 75) | A/C (n = 41) | F (n = 6) | |
| Ampicillin | 512 | >512 | >512 | >512 | >512 | >512 | >512 |
| Aztreonam | <1 | 8 | <1 | <1 | 32 | 8 | <1 |
| Ceftazidime | <1 | 2 | <1 | <1 | 32 | 2 | <1 |
| Cefotaxime | <1 | 64 | <1 | <1 | 4 | 64 | <1 |
| Cefepime | <1 | 8 | <1 | <1 | <1 | 8 | <1 |
| Cefoxitin | 32 | 4 | 1 | 2 | 32 | 4 | 4 |
| Ciprofloxacin | 0.25 | 0.03 | 0.015 | 0.03 | 0.25 | 0.015 | 0.25 |
| Chloramphenicol | 4 | 4 | 64 | 4 | 4 | 128 | 4 |
| Streptomycin | 256 | 4 | 512 | 256 | 256 | 256 | 4 |
| Sulfamethoxazole | 16 | 16 | >512 | 16 | 16 | >512 | 16 |
| Trimethoprim | <1 | <1 | <1 | 8 | <1 | >512 | <1 |
| Tetracycline | <1 | <1 | 1 | <1 | <1 | 32 | <1 |
Groups with fewer than five strains were excluded from calculations of MIC50s.
Numbers of strains included in each group are shown in parentheses. NI, not identified.
The year of isolation and the species of the Enterobacteriaceae isolates that were able to transfer armA or rmtB to the recipient E. coli isolate are shown in Table 6. Whereas 9 of the 13 strains that were isolated from 1997 and 1998 were Enterobacteriaceae, including 5 Enterobacter cloacae strains, 2 Citrobacter freundii strains, 1 Klebsiella oxytoca strain, and 1 Enterobacter agglomerans strain, 162 of the 191 strains that were isolated after 2001 were E. coli and K. pneumoniae. The major conjugative plasmids carrying armA were IncA/C and IncHI2 plasmids until 1998, but after 2001, they were replaced by the conjugative plasmids of incompatibility groups IncFIIAs, IncL/M, and Inc(unidentified). Between 2003 and 2004, a rapid increase in the number of IncL/M and Inc(unidentified) conjugative plasmids carrying armA among the Enterobacteriaceae, especially in K. pneumoniae, was revealed. Among the K. pneumoniae isolates, the armA-carrying conjugative plasmids of groups Inc(unidentified), IncL/M, IncFIIAs, IncF, and IncA/C were detected in 55, 17, 11, 3, and 2 isolates, respectively. Among the E. coli isolates, the armA-carrying conjugative plasmids of groups IncL/M, Inc(unidentified), IncFIIAs, IncF, and IncA/C were detected in 10 isolates, 9 isolates, 3 isolates, 2 isolates, and 1 isolate, respectively. Among the E. cloacae isolates, the armA-carrying conjugative plasmids of groups IncFIIAs, Inc(unidentified), IncHI2, IncL/M, and IncF were detected in six, six, three, two, and two isolates, respectively. The IncL/M conjugative plasmid carrying armA was detected in six of six S. marcescens isolates, and the IncA/C conjugative plasmid carrying armA was detected in three of the four C. freundii isolates. The IncA/C conjugative plasmid carrying rmtB was detected in 31 K. pneumoniae isolates, 11 E. coli isolates, and 1 C. freundii isolate. The IncF conjugative plasmid carrying rmtB was detected in six E. coli isolates and one K. pneumoniae isolate. The IncI1-Iγ conjugative plasmid carrying rmtB was detected in one E. coli isolate.
TABLE 6.
Years of isolation and species of Enterobacteriaceae isolates which were able to transfer the plasmids of various incompatibility groups carrying armA or rmtB
| Yr of isolation and speciesa | No. of strains that transfer a conjugative plasmid of the indicated Inc group carrying gene:
|
||||||||
|---|---|---|---|---|---|---|---|---|---|
|
armA
|
rmtB
|
||||||||
| FIIAs | HI2 | L/M | A/C | F | NIb | A/C | F | I1-Iγ | |
| 1997-1998 | |||||||||
| E. coli | 3d | 1 | |||||||
| K. pneumoniae | |||||||||
| Others | 4 | 3 | 2 | ||||||
| 2001 | |||||||||
| E. coli | 1 | ||||||||
| K. pneumoniae | 1 | 6 | |||||||
| Others | 3 | ||||||||
| 2002 | |||||||||
| E. coli | 4e | 1g | |||||||
| K. pneumoniae | |||||||||
| Others | 1 | ||||||||
| 2003 | |||||||||
| E. coli | 2 | ||||||||
| K. pneumoniae | 1 | ||||||||
| Others | 6 | ||||||||
| 2004 | |||||||||
| E. coli | 1 | 1 | 1 | 2 | 1 | ||||
| K. pneumoniae | 1 | 1 | 2 | 10 | |||||
| Others | 1 | 5 | |||||||
| 2005 | |||||||||
| E. coli | 1 | 1 | 1 | 5 | 4f | 4 | 1 | ||
| K. pneumoniae | 6 | 2 | 1 | 1 | 30 | 10 | 1 | ||
| Others | 2 | 1 | 1 | 1 | 1 | ||||
| 2006 | |||||||||
| E. coli | 1 | 6 | 2 | 2 | |||||
| K. pneumoniae | 4 | 13c | 1 | 14 | 15 | ||||
| Others | 4 | 3 | |||||||
| Total no. of strains | 20 | 4 | 38c | 9d | 7 | 75 | 43e,f | 7g | 1 |
Others, Enterobactericeae isolates other than E. coli and K. pneumoniae, including 19 E. cloacae isolates, 6 Enterobacter species isolates, 6 S. marcescens isolates, 4 C. freundii isolates, 2 K. oxytoca isolates, and 1 M. morganii isolate.
NI, not identified.
One isolate was positive for L/M and F.
Two isolates were positive for A/C and I1-Iγ.
One isolate was positive for A/C and F.
One isolate was positive for A/C, F, and FIA.
One isolate was positive for F and Y.
DISCUSSION
We have studied the distribution of conjugative-plasmid-mediated 16S rRNA methylase genes, such as armA, rmtA, and rmtB, among the amikacin-resistant Enterobacteriaceae that were collected in 1995 to 1998 and 2001 to 2006 at KNU Hospital in South Korea. In addition, the conjugative plasmids that are responsible for disseminating the 16S rRNA methylase genes were identified by replicon typing of plasmid incompatibility groups. The results indicate that the widespread presence of armA and rmtB among amikacin-resistant Enterobacteriaceae has been mediated by conjugative plasmids of various incompatibility groups conferring additional resistance to other kinds of antimicrobial agents, including ESBLs and quinolones.
The armA gene was initially sequenced from a C. freundii strain that was isolated in Poland (GenBank accession no. AF550415) and later characterized from a K. pneumoniae strain that was isolated in 2000 from a patient in France (6). The rmtB gene was first identified in S. marcescens S-95 isolated in 2002 from a patient in Japan (5). In the current study, the armA gene was first detected among three E. coli strains, three E. cloacae strains, one C. freundii strain, and one K. oxytoca strain that were isolated in 1997. The year of the first appearance of rmtB could not be correctly determined due to the exclusion of isolates distributed in 1999 and 2000; however, the emergence of rmtB as early as 2001 was assumed because rmtB was detected in seven strains that were isolated in 2001. Thus, this study has demonstrated that armA and rmtB emerged earlier than what has been reported so far.
The overall prevalence rates of armA and rmtB among the Enterobacteriaceae at KNU Hospital were 2.1% (153/7,127 isolates) and 0.7% (51/7,127 isolates), respectively. The overall prevalence rates of armA were 4.3% (88/2,059 isolates) for K. pneumoniae, 3.9% (19/484) for E. cloacae, 2.0% (6/307) for S. marcescens, 2.0% (3/161) for C. freundii, and 0.8% (28/3,326) for E. coli, thus indicating that armA is widespread among the various species of Enterobacteriaceae. The overall prevalence rates of rmtB were 1.6% (32/2,059 isolates) for K. pneumoniae and 0.5% (18/3,326) for E. coli, indicating the limited spread of rmtB among the Enterobacteriaceae compared to the spread of armA. The prevalence rates of armA and rmtB among K. pneumoniae and E. coli were much higher than those reported in a previous study performed in Taiwan (23), in which the prevalence rates were 0.9% (15/1,624 isolates) and 0.3% (5/1,624) for K. pneumoniae and 0.4% (10/2,559) and 0.04% (1/2,559) for E. coli, respectively.
The dissemination mechanisms of the genes that encode 16S rRNA methylases are of clinical significance since the genes confer a high level of resistance to all clinically available aminoglycosides, except streptomycin, and they were often linked to other resistance determinants, such as blaTEM-1, blaCTX-M-3, blaCTX-M-14, sul1, and dfrXII (2, 7). From studies of the genetic environments of armA and rmtB, the armA gene was part of functional composite transposon Tn1548 in plasmid pIP1204 (7) and the rmtB gene was found in the flanking region of the Tn3-like structure (5), suggesting that the spread of armA and rmtB was by transposition. Another mechanism, the dissemination of armA and rmtB by conjugative plasmids, has been demonstrated in a few studies: armA by a broad-host-range IncL/M conjugative plasmid (2, 5, 7, 23) and by a self-transferable IncN plasmid in an E. coli pig isolate from Spain (9) and rmtB by an IncF1 plasmid (2). The present study, however, demonstrated that conjugative plasmids that belonged to a variety of incompatibility groups were involved in the dissemination of armA and rmtB among Enterobacteriaceae. armA was carried by IncA/C and IncHI2 conjugative plasmids until 1998 but, after 2001, carried by plasmids of groups Inc(unidentified), IncL/M, IncFIIAs, and IncF. rmtB was carried by IncA/C, IncF, and IncI1-Iγ plasmids. In addition, the major plasmids that were responsible for the dissemination of armA and rmtB were completely different in terms of the incompatibility groups of the plasmids. The incompatibility groups of major conjugative plasmids that were involved in the dissemination of armA were Inc(unidentified) (75/153 isolates [49.0%]), IncL/M (37/153 isolates [24.2%]), and IncFIIAs (20/153 isolates [13.1%]), whereas the major conjugative plasmids that were involved in the dissemination of rmtB were IncA/C (41/51 isolates [80.3%]) and IncF (6/51 isolates [11.7%]). This finding suggests that the dissemination of armA and rmtB among Enterobacteriaceae isolates has been independently carried out by conjugative plasmids of different incompatibility groups. This suggestion could be supported with another finding, namely, that armA was detected among various species of Enterobacteriaceae but that rmtB was detected only in E. coli and K. pneumoniae, as well as in one C. freundii isolate. Interestingly, the aacA4 gene, which was the main amikacin resistance determinant before the emergence of armA and rmtB, was identified along with armA or rmtB and transferred simultaneously with armA but not rmtB. An IncL/M plasmid was detected in most of the transconjugants that carried both aacA4 and armA, suggesting the colocalization of aacA4 and armA on a broad-host-range IncL/M conjugative plasmid. Localization of aacA4 on an IncL/M plasmid that was carried by gentamicin- and amikacin-resistant Salmonella enterica serotype Typhimurium isolates has been reported in a previous study (19), and the localization of armA on an IncL/M plasmid has been reported repeatedly (2, 5, 7, 23). However, the colocalization of aacA4 and armA on an IncL/M plasmid has never before been demonstrated.
It is very interesting to note that a distinctive antimicrobial resistance pattern is revealed in each group whose transconjugants were grouped by the presence of armA or rmtB and by a plasmid incompatibility group, suggesting the linkage of armA and rmtB to other plasmid-located antimicrobial resistance genes and the association of these genes with specific plasmid backbones. The armA-carrying IncL/M plasmid and the rmtB-carrying IncA/C plasmid were associated with resistance to cefotaxime but not to ceftazidime, strongly suggesting the presence of blaCTX-M. These results were in agreement with those of a previous study in which the association of armA with blaCTX-M-3 on the IncL/M plasmid was demonstrated (7). The association of rmtB with blaCTX-M-14 was also demonstrated in a previous study (2), but the conjugative plasmid that carried these genes was the IncF plasmid. Besides being associated with resistance to cefotaxime, the rmtB-carrying IncA/C plasmids were associated with additional resistance to multiple drugs, including chloramphenicol, streptomycin, sulfamethoxazole, trimethoprim, and tetracycline, suggesting the presence of an integron. The armA-carrying Inc(unidentified) and the IncFIIAs plasmids were associated with resistance to cefoxitin, suggesting the presence of plasmid-mediated AmpC-type β-lactamase (pACBL). A similar association between armA and pACBL, such as with CMY-2 or DHA-1, was reported in a previous study (13). The armA-carrying IncFIIAs and Inc(unidentified) plasmids and rmtB-carrying IncF plasmid were associated with a relatively high MIC50 of ciprofloxacin (0.25 mg/liter), suggesting the presence of the plasmid-mediated quinolone resistance determinant qnr. Although the association between qnr determinants and ESBL or pACBL has been demonstrated in previous reports (10, 15-17), the connection of qnr determinants with armA or rmtB has not yet been determined. In fact, transconjugants that received an Inc(unidentified) plasmid carrying armA showed additional resistance to oxyimino-cephalosporins, such as aztreonam and ceftazidime, suggesting the production of ESBL. Therefore, the association of armA, pACBL, qnr, and ESBL on the conjugative Inc(unidentified) plasmid is suggested, and a high rate of such a conjugative plasmid among Enterobacteriaceae isolated after 2004 threatened the spread of multidrug-resistant Enterobacteriaceae, which are resistant to almost all clinically important antimicrobial agents, such as aminoglycosides, ESBLs, and quinolones. In order to confirm the hypotheses put forth in this paper, the identification of antimicrobial resistance determinants and integrons that are associated with armA or rmtB on specific plasmid backbones should be conducted.
Notably, there was an increase in the proportion of amikacin-resistant isolates to total isolates in 2005 and 2006. It appears that most of the increase was due to an increase in the proportion of amikacin-resistant K. pneumoniae isolates to total K. pneumoniae isolates in 2005 and 2006 (around 18 to 20% of total isolates in 2005 and 2006 versus about 11% of total isolates in 2004 and about 6% of total isolates in 2003). Indeed, among the 139 Enterobacteriaceae isolates carrying armA or rmtB in 2005 and 2006, 98 (70.5%) isolates were K. pneumoniae and, surprisingly, 94 (95.9%) of the 98 K. pneumoniae isolates carried one of the following plasmids: armA-carrying Inc(unidentified), IncL/M, and IncFIIAs plasmids and an rmtB-carrying IncA/C plasmid, all of which were assumed to carry additional ESBLs and/or pACBLs. Therefore, an increase in the rate of resistance to amikacin in K. pneumoniae isolates in 2005 and 2006 seemed to result from the dissemination of armA and rmtB, which was mediated by the four kinds of conjugative plasmids that were assumed to carry additional ESBLs and/or pACBLs. Because ESBLs and pACBLs are prevalent among K. pneumoniae isolates in South Korea (11, 15), the association of armA and rmtB with ESBLs and/or pACBLs on conjugative plasmids might have contributed to the increase in the presence of armA and rmtB among K. pneumoniae strains in recent years.
Acknowledgments
This work was supported by a grant from the South Korean Health 21 R&D Project, Ministry of Health and Welfare, South Korea (03-PJ1-PG1-CH03-0002), and in part by the Brain Korea 21 Project (2006).
Footnotes
Published ahead of print on 19 December 2007.
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