Increasing Prevalence and Diversity of Metallo-β-Lactamases in Pseudomonas spp., Acinetobacter spp., and Enterobacteriaceae from Korea

Abstract

Among imipenem-nonsusceptible isolates, acquired metallo-β-lactamase genes were detected in 36 of 581 (6.2%) Pseudomonas aeruginosa isolates, 42 of 44 (95.4%) other Pseudomonas species, and 136 of 513 (26.5%) Acinetobacter species from 2003 to 2004 at a Korean hospital. Overall, bla_VIM-2-like genes were the most prevalent and were also detected in Enterobacteriaceae, including Klebsiella pneumoniae.

Carbapenems have been the β-lactam antibiotics used most successfully to evade bacterial resistance (8), but acquired carbapenem resistance due to the production of metallo-β-lactamases (MBLs) has been increasingly reported, particularly for Pseudomonas aeruginosa and Acinetobacter spp. (13).

In recent years, carbapenem resistance found in P. aeruginosa and Acinetobacter spp. has gradually increased in Korea, and a significant proportion of these carbapenem-resistant isolates have been shown to produce VIM-2- or IMP-1-type MBL (5). Among the members of the family Enterobacteriaceae, imipenem resistance has been found to be virtually nonexistent.

In 2003 and 2004, an increasing trend in the rates of carbapenem resistance in P. aeruginosa and Acinetobacter spp., along with a sudden increase in imipenem-resistant Klebsiella pneumoniae isolates, was observed at a Korean tertiary-care hospital. In this study, we determined the prevalence of MBL-producing isolates and identified the types of MBL in imipenem-nonsusceptible Pseudomonas spp., Acinetobacter spp., and Enterobacteriaceae isolates from that hospital during that period.

The study included randomly selected imipenem-nonsusceptible Pseudomonas spp., Acinetobacter spp., and Enterobacteriaceae isolates from clinical specimens collected in 2003 and 2004. Isolates were identified by conventional testing (4) or with an ATB 32 GN system (bioMerieux, Marcy-l'Etoile, France). A disk diffusion method (2) was used for routine susceptibility testing. MBL-producing isolates were screened with the modified-imipenem-disk Hodge test using MacConkey agar plates and with the double-disk synergy test using imipenem and EDTA-sodium mercaptoacetic acid disks (6). The bla_IMP-1-, bla_VIM-2-, and bla_SIM-1-like genes were detected by PCR as previously described (1, 7, 11). The bla_VIM-2 gene-carrying integrons from two K. pneumoniae isolates were amplified and sequenced as previously described (12). A template prepared from a transconjugant (the recipient was azide-resistant Escherichia coli J53) and primers SHV-EXT-F (5′-TTCTTTACTCGCCTTTATCG-3′) and SHV-EXT-R (5′-TTTATGGCGTTACCTTTGAC-3′) were used to detect and sequence the bla_SHV allele originally carried in an aztreonam-resistant K. pneumoniae isolate.

In 2004, a notable increase in imipenem resistance rates was observed for P. aeruginosa, other Pseudomonas spp., and Acinetobacter spp. compared to those in 2003 (Table 1). Of the relatively small number of imipenem-resistant isolates of Enterobacteriaceae found from 2003 to 2004, the majority were K. pneumoniae, while the minority consisted of Enterobacter cloacae and Serratia marcescens (Table 1).

TABLE 1.

Results of routine imipenem susceptibility testing and detection of MBL-producing gram-negative bacilli in 2003 and 2004 in Korea

Species	Yr	No. (%) of isolates		No. (%) of nonduplicate imipenem-nonsusceptible isolates
		Total	Imipenem-resistant	Tested	Positive with:
					Hodge test	Double-disk synergy test	blaVIM-2-like genea	blaIMP-1-like genea	blaSIM-1-like genea
P. aeruginosa	2003	2,913	816 (28)	243	27 (11)	16 (7)	16 (100)	0	0
	2004	3,057	1,009 (33)	338	38 (11)	20 (6)	18 (90)	2 (10)	0
Other Pseudomonas spp.	2003	144	79 (55)	14	14 (100)	14 (100)	14 (100)b	0	0
	2004	114	70 (61)	30	28 (93)	28 (93)	28 (100)b	0	0
Acinetobacter spp.	2003	1,765	282 (16)	194	65 (34)	51 (26)	27 (53)	21 (41)	3 (6)
	2004	1,529	459 (30)	319	222 (70)	85 (27)	54 (64)	25 (29)	6 (7)
K. pneumoniae	2003	1,819	36 (2)	22	2 (9)	2 (9)	2 (100)	0	0
	2004	2,006	39 (2)	22	2 (9)	0
E. cloacae	2003	821	2 (<1)	1	1 (100)	1 (100)	1 (100)	0	0
	2004	773	1 (<1)	1	1 (100)	0
S. marcescens	2003	536	1 (<1)	1	1 (100)	1 (100)	1 (100)	0	0
	2004	483	4 (<1)	4	1 (25)	0
Total		15,960	2,797 (18)	1189	402 (34)	217 (18)	161 (74)	48 (22)	9 (4)

^aNumbers in parentheses indicate the proportions of isolates among all MBL-producing isolates.

^bAll bla_VIM-2-like-gene-positive isolates were P. putida.

Among the imipenem-nonsusceptible isolates tested, 36 of 581 (6%) P. aeruginosa isolates and 42 of 44 (95%) other Pseudomonas spp. produced an acquired MBL (Table 1). In Korea, VIM-2 has been the only MBL type detected in Pseudomonas spp. since 1995 (5; K. Lee, unpublished results). In the present study, most MBL genes detected in P. aeruginosa were bla_VIM-2-like, but we also detected for the first time in Korea the presence of two isolates carrying bla_IMP-1-like genes. This may suggest dissemination from Japan, where IMP-type enzymes are the most prevalent MBLs in Pseudomonas spp. (10). Interestingly, all 42 MBL-producing isolates of other Pseudomonas spp. were found to be Pseudomonas putida (Table 1). In this study, the number of MBL-producing isolates of P. aeruginosa was surpassed by the number of P. putida isolates, which suggests that P. putida plays a significant role as a reservoir of MBL genes, even though it is a species that is rarely involved in infection (4). The MBL genes detected in all 42 P. putida isolates were bla_VIM-2-like.

Overall, 136 of the 513 (26.5%) imipenem-nonsusceptible Acinetobacter sp. isolates tested carried an acquired MBL determinant (Table 1). In 1998, only bla_IMP-1-like genes were present in Acinetobacter spp. (15), but since 2001, the proportion of isolates with bla_VIM-2-like genes has gradually increased (J. H. Yum, unpublished results). In the present study, 64% of the MBL-producing Acinetobacter isolates in 2004 had bla_VIM-2-like genes, while only 29% of them had bla_IMP-1-like genes (Table 1). Two isolates of Acinetobacter baumannii with a novel MBL, SIM-1, in addition to seven previously reported isolates, were also detected (7). It is important to note that many imipenem-nonsusceptible but MBL-negative isolates of P. aeruginosa and Acinetobacter spp. were detected in this study. These findings suggest a wide dissemination of isolates with other resistance mechanisms. Carbapenem-hydrolyzing OXA-23 β-lactamase was reported for Acinetobacter strains from another Korean hospital (3).

Only bla_VIM-2-like genes were detected in enterobacterial isolates (Table 1), and to the best of our knowledge, this is the first detection of similar determinants in K. pneumoniae. The structures of class 1 integrons carrying bla_VIM-2 from these two isolates (GenBank accession no. DQ153217 and DQ153218) were identical to those of bla_VIM-2-containing integrons from a Providencia rettgeri isolate (GenBank accession no. AY887109), suggesting the dissemination of these integrons among Enterobacteriaceae by horizontal transfer.

The MICs of imipenem for VIM-4-producing K. pneumoniae and for VIM-2-producing Citrobacter freundii were reported to be low, 2 μg/ml and 1 μg/ml, respectively (9, 14). However, the imipenem MICs for the two VIM-2-producing K. pneumoniae isolates in our study, determined by an agar dilution method (2), were much higher, namely, 32 μg/ml and >128 μg/ml, suggesting the contribution of additional resistance mechanisms. Although MBLs do not hydrolyze aztreonam, the MIC of aztreonam for one of the bla_VIM-2-positive K. pneumoniae isolates (YMC 03/10/U1702) was significantly higher (128 μg/ml) than that for the other isolate (0.5 μg/ml). The double-disk synergy test was positive for the aztreonam-resistant isolate, and the bla_SHV-12 gene was detected in a transconjugant, which explains the high-level resistance to the monobactam.

In this study, 90% of the MBL-producing isolates were from patients in a general ward or an intensive care unit, while 10% were from patients in an emergency room or another outpatient department (data not shown). The majority (82%) of MBL-producing isolates were from sputum and urine specimens, as reported in a previous study (5).

In conclusion, imipenem-nonsusceptible P. aeruginosa isolates carrying bla_VIM-2-like genes remain highly prevalent, while the incidence of Acinetobacter spp. positive for bla_VIM-2-like genes has increased significantly. The spread of bla_IMP-1-like genes to P. aeruginosa and bla_VIM-2-like genes to Acinetobacter spp. or even to K. pneumoniae is a cause for concern and warrants continuous surveillance to control the further spread of these resistance mechanisms.