Metallo-beta-lactamase-producing Pseudomonas spp. in Korea: high prevalence of isolates with VIM-2 type and emergence of isolates with IMP-1 type.

PURPOSE: Two Korean nationwide studies showed that metallo-beta-lactamases (MBLs)-producing-Pseudomonas spp. are not rare. The aim of this study was to assess the trends of MBL-producing isolates among imipenem-resistant isolates of Pseudomonas spp.
MATERIALS AND METHODS: Imipenem-resistant clinical isolates were collected from 23 hospitals and one commercial laboratory participating in the KONSAR program in 2005. Polymerase chain reaction (PCR) was used to detect MBL genes.
RESULTS: Alleles of MBL genes were detected in 10.8% of 415 Pseudomonas aeruginosa and 66.7% of 12 P. putida isolates from 18 of 24 hospitals/laboratory. Among the 14 IMP-1-like and 39 VIM-2-like MBLs, emergence of IMP-6 was detected for the first time.
CONCLUSION: Prevalence of MBL-producing P. aeruginosa has not significantly increased, but IMP-6 emerged in P. aeruginosa.

INTRODUCTION

Pseudomonas aeruginosa is one of the most important nosocomial pathogens, while other Pseudomonas spp. are occasional causes of infections. Current isolates of P. aeruginosa are often multiresistant to many classes of potent antimicrobial agents, including β-lactams, aminoglycosides, and fluoroquinolones. Carbapenems are the most potent β-lactams, which are active even against extended-spectrum β-lactamase- and AmpC β-lactamase-producing gram-negative bacilli. Due to acquired metallo-β-lactamase (MBL) production, however, carbapenem resistance in P. aeruginosa and Pseudomonas spp., has increasingly been reported in some countries.1,2 Richet et al.3 initiated an international network and included carbapenemase-producing gram-negative bacilli in the early warning system list for emerging antimicrobial drug-resistant pathogens.

The Korean Nationwide Surveillance of Antimicrobial Resistance (KONSAR) program conducted in the years between 1998 and 2003 showed that P. aeruginosa ranked the 3rd by the number of isolates.4,5 The imipenem resistance rate of P. aeruginosa in 1999 was already relatively high (19%), but the rate rose further to reach 24% in 2004. Two previous studies with clinical isolates of P. aeruginosa, which were collected in 2000-2001 and in 2003 from hospitals participating in the program of KONSAR, showed that over 10% of all imipenem-resistant isolates were VIM-2 type MBL producers.6,7 Other studies also documented the presence of MBL-producing P. aeruginosa in Korean hospitals8,9 as well as the occasional presence of VIM-2-type MBL-producing isolates of Enterobacteriaceae.10,11 Moreover, the emergence of IMP-1-like-producing P. aeruginosa and finding of the fifth MBL, SIM-1-producing Acinetobacter baumannii isolates, were reported from a hospital in Seoul.12

The aim of this study was to determine any change in the prevalence of VIM-2- and IMP-1-like-producing clinical isolates of Pseudomonas spp. and possible emergence of new MBL variants two years after the second nationwide study. The possible presence of MBL-producing Enterobacteriaceae and spreading of blaSIM-1 type MBL gene to Pseudomonas spp. were also investigated.

MATERIALS AND METHODS

Bacterial strains and susceptibility testing

Non-duplicate, imipenem-resistant strains of P. aeruginosa, Pseudomonas spp., and Enterobacteriaceae, isolated during January to August 2005, were collected from 23 hospitals and one commercial laboratory participating in the KONSAR program. The number of isolates collected from any one hospital was limited to 25 for an individual species. Identification of the species was reconfirmed at the coordinating laboratory through the use of a conventional method13,14 or the ATB 32GN system (bioMerieux, Marcy l'Etoile, France). The MICs of antimicrobial agents for MBL-producing isolates were determined by the NCCLS agar dilution method.15

Screening MBL-producing isolates

Carbapenemase production was screened by the imipenem-disk Hodge (cloverleaf) test, using MacConkey agar instead of previously used Mueller-Hinton agar.16 MBL production was screened by the double-disk synergy test using an imipenem disk and an EDTA (750 µg) plus sodium mercaptoacetic acid (SMA, 2 mg) disk on Mueller-Hinton agar with 10 mm distance from the edge to the edge of the disk.16 Commercial imipenem disks and media (Becton-Dickinson, Sparks, MD, USA) were used for these tests, while the EDTA-SMA disks were prepared from commercially available chemicals (Sigma Chemical, St. Louis, MO, USA).

MBL gene detection by PCR

To detect alleles of blaIMP-1, blaVIM-2, and blaSIM-1, DDS-positive isolates were tested by PCR as described previously.12 Briefly, primers used were: IMP1-F 5'-CAT GGT TTG GTG GTT CTT GT-3' and IMP1-R 5'-ATA ATT TGG CGG ACT TTG GC-3'; VIM2-F 5'-ATG TTC AAA CTT TTG AGT AAG-3' and VIM2-R 5'-CTA CTC AAC GAC TGA GCG-3'; SIM1-F 5'-TAC AAG GGA TTC GGC ATC G-3' and SIM1-R 5'-TAA TGG CCT GTT CCC ATG TG-3'. PCR was performed with 1 µL of heat-extracted DNA template, 20 pmol of each primer, and PreMix (Bioneer, Cheongwon, Korea) containing 1 U of Taq DNA polymerase in a total volume of 20 µL. A Mastercycler instrument (Eppendorf, Hamburg, Germany) was used with the following reaction conditions: 94℃ for 5 min, 25 cycles of 94℃ for 30 s, 56℃ for 30 s, and 72℃ for 45 s, and finally, 72℃ for 7 min. Amplicons were visualized by electrophoresis and ethidium bromide staining.

Pulsed field gel electrophoresis and MBL gene sequencing

For PFGE, χbaI-digested genomic DNA was prepared according to the instruction of Bio-Rad (Hercules, Calif., USA), and fragments were separated using a CHEF-DR II system (Bio-Rad). We selected 17 isolates which represented PFGE types and performed the sequencing of PCR amplicons using IMP1-F or VIM2-F and Int2 (5'-AAG CAG ACT TGA CCT GA-3'), and IMP1-R or VIM2-R and Int1 (5'-GGC ATC CAA GCA GCA AG-3').

RESULTS

Among the 443 imipenem-resistant isolates collected, the majority were P. aeruginosa (93.7%), whereas 16 isolates were species of Enterobacteriaceae (9 K. pneumoniae, 2 each of E. aerogenes, E. coli, and P. mirabilis, and 1 C. freundii). Of these isolates, 127 were positive for the imipenem-disk Hodge test and 53 were positive for the DDS test (Table 1). Alleles of blaVIM-2 or blaIMP-1 were detected in all 53 DDS-positive isolates. Among all the isolates tested, the MBL-positive isolates comprised 10.8% of P. aeruginosa and 66.7% (8 of 12) of P. putida. MBL was not detected in any of the 16 isolates of Enterobacteriaceae.

Table 1

Metallo-β-Lactamase Genes Detected in Imipenem-Resistant Isolates of Pseudomonas spp. and Enterobacteriaceae by PCR

PCR, polymerase chain reaction; DDS, double disk synergy.

Overall, the proportion of blaVIM-2 allele-positive isolates was greater (73.6%) than blaIMP-1 allele-positive isolates (Table 1). blaSIM-1-allele-positive isolate was not detected. All MBL-producing isolates of Pseudomonas spp. other than P. aeruginosa were identified as P. fluorescens/P. putida by the ATB 32 GN system. However, they were confirmed to have biochemical characteristics of P. putida by conventional tests. Nucleotide sequencing revealed that all the blaVIM-2-like and blaIMP-1-like in P. aeruginosa were VIM-2 and IMP-6. MBLs in P. putida were identified as VIM-2 and IMP-1. We found 13 different PFGE patterns in 23 blaVIM-2-like Pseudomonas spp. and 4 patterns in 7 blaIMP-1-like Pseudomonas spp., indicating that multiple MBL-producing Pseudomonas clones exist in Korea. The MIC50s of various β-lactams for MBL-producing P. aeruginosa were equal to over 64 µg/mL except that of aztreonam (Table 2). The % resistances for aztreonam, piperacillin, amikacin, ciprofloxacin and other β-lactams were 67%, 83%, 87%, 96%, and 100%, respectively.

Table 2

Antimicrobial Activities Against MBL-Producing P. aeruginosa and P. putida Strains

MBL, metallo-β-lactamase.

MBL-producing isolates were detected in 18 of 24 (75%) hospitals/laboratory, as well as in all four regions (Table 3). MBL-producing isolates were mostly derived from specimens of urine, sputum, and wounds (Table 4). All 8 MBL-producing P. putida isolates came either from urine or sputum. The MBL-producers were isolated mainly from inpatients (80: 63% isolates from general ward and 17% isolates from intensive-care unit); while 20% of isolates were from outpatient department.

Table 3

Metallo-β-Lactamase Genes Detected in Isolates of Pseudomonas spp. by the Location of Hospitals

*City or province: North, Seoul, Gyunggi and Gangwon; Southeast, Busan, Daegu, Wulsan and Gyungsang; Southwest, Jeolla; Southwestern island, Jeju; Commercial Laboratory collected specimens from clinics located all over the country.

†Some hospitals had isolates with blaVIM-2 and with blaIMP-1.

Table 4

Source of Isolation of Metallo-β-Lactamase Gene-Positive Pseudomonas spp.

DISCUSSION

Antimicrobial resistance surveillance has become increasingly important with the recent increase in the resistant organisms.17 Analysis of the data generated by the participating hospitals in 1998 and 2004 showed an alarming rise in the imipenem resistance rates of P. aeruginosa; from 19% to 24%.4,5 Decrease of carbapenem susceptible P. aeruginosa was also reported in Japan; from 80.7% to 62.0% over the period of 1998-2002.18

Among the β-lactamases, the genetically mobile MBL are among the most feared, because of their ability to hydrolyze virtually all β-lactams, except monobactams,19 and to spread horizontally. As a result of their geographic spread, these enzymes are among the greatest concerns to the medical communities.1,20 The rates of occurrence and number of enzyme types of MBL have escalated since 2000 in Asia, Europe, and Latin America.1,2,21 In the present study, 10.8% of imipenem-resistant-isolates of P. aeruginosa were MBL producers. These proportions were similar to those in the previous study in 2000-2001 and in 2003.6,7 The rate was also similar to that in a Japanese study in 1998-2002.18

In this study, only 12 imipenem-resistant isolates of Pseudomonas spp. other than P. aeruginosa were collected. It is of an interest to note that all 8 MBL-producing isolates were identified as P. putida. P. putida is a species difficult to differentiate from P. fluorescens by commonly used biochemical tests. Although P. putida is an only organism occasionally isolated from clinical specimens,13 the detection of MBL gene in 8 of 12 imipenem-resistant isolates suggests that this organism may play a significant role as an important reservoir of MBL genes.

Only 16 imipenem-resistant Enterobacteriaceae isolates could be collected in this study, and MBL was not detected in any of these isolates. This indicates rarity of Enterobacteriaceae isolates not only producing MBL, but also resistant to imipenem. MBL has been detected only in a few isolates of Enterobacteriaceae in Korea.11,13,22

It is interesting that the first acquired MBL discovered in the early 1990s was IMP-1 in nearby Japan,23 whereas in Korea, VIM-2 was the first MBL type detected and IMP-1 was detected in Acinetobacter spp. in the early 2000s.6 In a study at a hospital in Seoul, IMP-1-like-producing P. aeruginosa was detected in strains isolated in 2003-2004, but the number was only two of 76 isolates of Pseudomonas spp.12 In the present study, 14 of 53 MBL-producing isolates of P. aeruginosa and P. putida had blaIMP-1, indicating spread of this MBL gene to these species (Table 1). However, blaSIM-1 which was discovered in A. baumannii isolates in Seoul has not yet spread to Pseudomonas spp.

Detection of MBL-producing Pseudomonas spp. in isolates collected from 18 of 24 (75.0%) hospitals/laboratory in the present study (Table 3) is a great concern. As in previous studies,6,7 MBL-producing isolates were isolated mostly from urine and sputum specimens (Table 4), indicating that these specimens can serve as the important reservoirs for dissemination of the resistant organisms.

In conclusion, clinical isolates of P. aeruginosa in Korea are increasingly resistant to imipenem, and a significant proportion of the resistance is due to MBL production. blaVIM-2 remains the dominant type, however, IMP-1 variant, IMP-6-producing P. aeruginosa emerged. Extensive amount of effort is required to control further spread of MBL-producing Pseudomonas spp. in hospitals.

OTHER KONSAR PARTICIPANTS

Namhee Ryoo, Keimyong University Dong San Medical Center, Daegu; Seok Hoon Jeong, Kosin University Gospel Hospital, Busan; Gyoung-Yim Ha, Dongguk University Kyongju Hospital, Gyeongju; Mi-Na Kim, University of Ulsan College of Medicine, Seoul; Wee-Gyo Lee, Ajou University Hospital, Suwon; Young-Ae Hong, Ulsan Dong-Kang General Hospital, Ulsan; Soung-Eun Cho, Ewha Womans University College of Medicine, Seoul; Young Uh, Yonsei University Wonju Christian Hospital, Wonju; Sook Jin Jang, Chosun University Hospital, Gwangju; Myung Hee Lee, Korea Veterans Hospital, Seoul; Wonkeun Song, Hallym University College of Medicine, Seoul; Tae Yeal Choi, Hanyang University Medical College, Seoul; Jong Hee Shin, Chonnam National University Hospital, Gwangju; Seong Geun Hong, Pochon CHA University Hopsital, Seongnam; Young Ah Kim, National Health Insurance Corporation Ilsan Hospital, Goyang; Dong Hee Cho, Samsung Cheil Hospital, Seoul; Dong Hee Whang, Inje University College of Medicine, Seoul; Seungok Lee, Seoul Clinical Laboratory, Seoul; Seonghee Lee, Hanmaeum Hospital, Jeju.