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Dissemination of genetically related IMP-6-producing multidrug-resistant Pseudomonas aeruginosa ST235 in South Korea
International Journal of Antimicrobial Agents 39 (2012) 300–304
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International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Dissemination of genetically related IMP-6-producing multidrug-resistant Pseudomonas aeruginosa ST235 in South Korea夽 Jung Sik Yoo, Ji Woo Yang, Hye Mee Kim, Jeongheum Byeon, Hwa Su Kim, Jae Il Yoo, Gyung Tae Chung, Yeong Seon Lee ∗ Division of Antimicrobial Resistance, Korea National Institute of Health, 187 Osong Saengmyeong 2-ro Chungcheongbuk-do, 363-951, Republic of Korea
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Article history: Received 9 July 2011 Accepted 21 November 2011 Keywords: Carbapenem Metallo--lactamase IMP-6 Non-tertiary care hospital Geriatric hospital
a b s t r a c t The present study aimed to describe the prevalence and molecular epidemiology of metallo-lactamase (MBL)-producing Pseudomonas aeruginosa isolates obtained from non-tertiary care hospitals and geriatric hospitals in South Korea. Of the 644 isolates, 224 were carbapenem-resistant, amongst which 41 (18.3%) were MBL-producers and the major MBL type was IMP-6 (35 isolates). IMP-6producing isolates were multidrug-resistant and showed higher minimum inhibitory concentrations for meropenem than imipenem. All of the IMP-6-producing isolates had class 1 integrons with amplification sizes of 4.5 kb/5.5 kb (34 isolates) or 3.0 kb (1 isolate); 4.5 kb/5.5 kb integrons had blaIMP-6 –qac–aacA4–blaOXA-1 –aadA1 (5.5 kb) and aadB–cmlA–blaOXA-10 –aadA1 (4.5 kb). Pulsed-field gel electrophoresis (PFGE) analysis indicated that all IMP-6-producing P. aeruginosa from various geographic areas had nearly identical patterns with >85% similarity. All IMP-6-producing isolates showed high genetic similarity to those obtained from tertiary care hospitals and had the same integron type, indicating the spread of these strains to the three types of hospitals nationwide. These data show the wide spreading of clonally related IMP-6-producing P. aeruginosa (sequence type235) through tertiary, non-tertiary and geriatric hospitals in South Korea. Continuous monitoring and thorough infection control should be performed in all types of hospitals to prevent further spreading of MBL-producing P. aeruginosa. © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Carbapenems are used as the last choice for treating Gram-negative bacterial infections; however, intensive use of carbapenems in the treatment of these infections has led to the emergence and increase in the occurrence of carbapenem-resistant clinical isolates. The highest level of resistance is exhibited by those isolates producing metallo--lactamases (MBLs). Acquired resistance to carbapenems through the production of MBLs by Pseudomonas and Acinetobacter spp. has been increasingly reported in many countries [1,2]. MBL-associated resistance has caused outbreaks of hospital infections and endemic diseases in several areas worldwide. In 2001, 72% of the carbapenem-resistant Pseudomonas aeruginosa isolates from an Italian hospital were VIM-producers, and 62% of
夽 This study was presented in part at the 20th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), 10–13 April 2010, Vienna, Austria. ∗ Corresponding author. Tel.: +82 43 719 8240; fax: +82 43 719 8269. E-mail address: [email protected] (Y.S. Lee).
the imipenem-resistant isolates from several Greek hospitals had the same type of MBL [3,4]. A previous study conducted between 2004 and 2006 in Hiroshima, Japan, reported that IMP-1-producing multidrug-resistant P. aeruginosa isolates from different hospitals were clonally similar [5]. In 1995, VIM-2 was first detected in P. aeruginosa isolates in South Korea and it has become the most common MBL type since then [6–8]. VIM-2 comprised 71.1% and IMP-1-like type comprised 28.9% in the 2005 survey [7]. However, the IMP type was first found in 2000–2001 isolates [9] and increased to 28.9% of P. aeruginosa isolates in 2005 [7]. There was an outbreak of IMP-6-producing P. aeruginosa in a tertiary care hospital in 2004 [10]. The increasing prevalence of MBL-producing isolates has been attributed to clonal spread and/or horizontal transfer of MBL genes possessing mobile genetic elements [11,12]. However, there have been few studies on the epidemiological situation and characteristics of MBL-producing isolates from all types of hospitals, especially non-tertiary care hospitals and geriatric hospitals in South Korea. The aim of the present study was to assess the prevalence of MBL-producing isolates amongst carbapenem-resistant P. aeruginosa isolates obtained from non-tertiary care and geriatric
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J.S. Yoo et al. / International Journal of Antimicrobial Agents 39 (2012) 300–304
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Table 1 Detection of metallo--lactamase (MBL)-producing Pseudomonas aeruginosa isolates in geriatric hospitals and non-tertiary care hospitals in South Korea. MBL gene
blaIMP-1 blaIMP-6 blaVIM-2 Total a
Geriatric hospitals (n = 271 isolates)
Non-tertiary care hospitals (n = 373 isolates)
No. of isolates
Location(s)a
No. of isolates
Location(s)a
2 13 0 15 (5.5%)
North North, Southeast –
0 22 4 26 (7.0%)
– North, Middle, Southeast Middle, Southeast, Southwest
North, Seoul/Gyeonggi/Incheon/Gangwon; Middle, Chungcheong; Southeast, Busan/Gyungsang; Southwest, Gwangju/Jeolla/Jeju.
hospitals in South Korea. The characteristics and genetic similarities between these isolates and the IMP-6-producing P. aeruginosa isolates in tertiary care hospitals were investigated. 2. Materials and methods 2.1. Bacterial strains and susceptibility testing Between 2008 and 2009, 644 consecutive, non-duplicate clinical isolates of P. aeruginosa were collected from 111 non-tertiary care hospitals and 47 geriatric hospitals geographically dispersed across South Korea; 93% of the hospitals sent ≤10 isolates. Nontertiary care hospitals or geriatric hospitals tended to be centralised in some areas, so some hospitals sent more isolates than others. The maximum number of isolates per hospital was 49 and the minimum was 1. However, the mean numbers of isolates per hospital by location were similar (3.1 isolates/hospital in the Southwest, 3.4 in the Southeast, 4.5 in the Middle area and 4.7 in the North). Disk diffusion and agar dilution were performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines [13]. MBL production was detected by the double-disk synergy (DDS) test [14] using a 10 g disk of imipenem and an ethylene diamine tetra-acetic acid (EDTA) disk (1.9 mg) for P. aeruginosa not susceptible to meropenem or imipenem. IMP-6-producing isolates were compared with isolates from tertiary care hospitals (20 IMP-6-possessing P. aeruginosa isolated in 2007–2008 and KM-4 P. aeruginosa isolate, which is one of the first isolated IMP6-producing P. aeruginosa in South Korea [10]) on the basis of characteristics and genetic similarities.
by the unweighted pair-group method using arithmetic averages (UPGMA) with the band position tolerance set at 1.8%. MLST was performed according to the method of Curran et al. [19]. Following PCR and sequence analysis for seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA and trpE) performed using the primers in the MLST database (http://pubmlst.org/paeruginosa), the sequence types (STs) of the isolates were obtained based on the allele sequences in the database. 2.4. S1 nuclease digestion and Southern blot hybridisation PFGE with S1 nuclease (Promega, Madison, WI) digestion of whole genomic DNA was performed as described previously with some modifications [20]. Gel plugs were cut into 5 mm slices, soaked with 1× reaction buffer and then digested with 0.1 U of S1 nuclease for 30 min at 37 ◦ C. The plugs were incubated on ice for 10 min in 200 L of ice-cold 0.5 M EDTA (pH 8.0). Undigested total DNA of the isolates was separated on 0.8% agarose gel by PFGE and transferred onto a Hybond N+ membrane (Amersham Pharmacia Biotech, Little Chalfont, UK) and hybridised separately with a probe for blaIMP-6 (IMP-F, 5 -TGAGCAATGTATCTGTATTC3 ; IMP-R, 5 -TTAGTTGCTTGGTTTTGATG-3 ) and the 16S rRNA gene (16S-F, 5 -CGCGTGTGTGAAGAAGGTCT-3 ; 16S-R, 5 CACCTGTGTCTGAGTTCCCG-3 ). An enhanced chemiluminescence (ECLTM ) Direct Nucleic Acid Labelling and Detection System (GE Healthcare UK Ltd., Little Chalfont, UK) was used. 3. Results
2.2. Molecular detection and analysis of metallo-ˇ-lactamase genes and integrons
3.1. Metallo-ˇ-lactamase-producing Pseudomonas aeruginosa in non-tertiary care and geriatric hospitals
To detect the IMP, VIM, SIM, GIM and SPM types of MBL genes, DDS-positive isolates were tested by multiplex polymerase chain reaction (PCR) as described previously [15]. Then, each MBL type underwent PCR amplification and sequencing of PCR-positive isolates using primers VIM-2A (5 -ATG TTC AAA CTT TTG AGT AGT AAG-3 ), VIM-2B (5 -CTA CTC AAC GAC TGA GCG-3 ), IMP-1F (5 TGA GCA ATG TAT CTG TAT TC-3 ) and IMP1-R (5 -TTA GTT GCT TGG TTT TGA TG-3 ) [16,17]. Detection of class 1 integrons by PCR was also performed as described previously [18]. Amplified integron restriction types were analysed following digestion at 37 ◦ C for 18 h with MboI (New England Biolabs, Beverly, MA). The structure of the variable regions of the MBL-containing integrons was determined by PCR mapping and sequencing.
The resistance rates to meropenem in non-tertiary care and geriatric hospitals were 22.0% and 30.6%, respectively, and those to imipenem were 16.3% and 29.9%, respectively. Of the 644 isolates, 224 (34.8%) were imipenem- or meropenem-resistant and 78 were positive in the EDTA/imipenem synergy test. MBLs were detected in 41 isolates; 35 (85.4%) of the 41 isolates were identified as blaIMP-6 , 2 isolates were identified as blaIMP-1 and 4 isolates were identified as blaVIM-2 (Table 1). IMP-6-possessing isolates were found in 15 of the 111 non-tertiary care hospitals and in 6 geriatric hospitals. Geographically, they were confined to the North (20 isolates in 11 hospitals), Middle (4 isolates in 2 hospitals) and Southeastern (11 isolates in 8 hospitals) areas of South Korea, but were not found in the Southwestern area. Four VIM-2-producing isolates were found in the Middle, Southeast and Southwest.
2.3. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)
3.2. Antimicrobial susceptibilities of IMP-6-producing isolates
Total bacterial DNA was digested using XbaI (New England Biolabs), and DNA fragments were separated on 1.2% agarose gel in a 0.5× TBE (Tris–borate–EDTA) buffer using a CHEF Mapper® apparatus (Bio-Rad Laboratories, Hercules, CA). The conditions were as follows: 14 ◦ C, 6 V/cm, pulses of 10 s to 40 s and a run time of 22 h with a ramping factor of −1.357. Genetic similarities were assessed
Because IMP-6 is the dominant MBL type in tertiary care hospital isolates, IMP-6-producing P. aeruginosa isolates obtained from nontertiary care and geriatric hospitals were analysed. In addition to 35 isolates from non-tertiary and geriatric hospitals, 20 isolates from tertiary care hospitals were included in the study. All of the IMP6-producing P. aeruginosa isolates were resistant to ceftazidime,
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Table 2 Characteristics of 55 IMP-6-producing Pseudomonas aeruginosa isolates from three hospital types in South Korea. Hospital type
No. of isolates Integron structurea
MLST
MIC range (g/mL) IPM
Geriatric 13 Non-tertiary care 22 20 Tertiary care
MEM TZP
Type A (12), type C (1) ST235 16–64 >256 Type A ST235 16–64 >256 Type A (18), type B (2) ST235 8–32 >256
FEP
CAZ
ATM
CIP
TOB
GEN
64 to >256 256 to >256 128 to >256 256 to >256 32–128 64 to >256 256 to >256 32–256 64 to >256 64 to >256 256 to >256 16–128 >256 >256 32–256 64 to >256 64 to >256 32 to >256 16–128 64 to >256 128 to >256
MLST, multilocus sequence type; MIC, minimum inhibitory concentration; IPM, imipenem; MEM, meropenem; TZP, piperacillin/tazobactam; FEP, cefepime; CAZ, ceftazidime; ATM, aztreonam; CIP, ciprofloxacin; TOB, tobramycin; GEN, gentamicin. a Type A, blaIMP-6 –qac–aacA4–blaOXA-1 –aadA1 (5.5 kb)/aadB–cmlA–blaOXA-10 –aadA1 (4.5 kb); type B, blaIMP-6 –qac–aacA4–blaOXA-1 –aadA1 (5.5 kb); and type C, blaIMP-6 –blaOXA-10 –aadA1 (3.0 kb).
cefepime, aztreonam, gentamicin, tobramycin, ciprofloxacin and carbapenems. The minimum inhibitory concentrations (MICs) for meropenem were extremely high (all >256 g/mL). However, the MICs of imipenem ranged from 8 g/mL to 64 g/mL. The MICs for cephalosporins, aminoglycosides and ciprofloxacin were also extremely high (Table 2). Aztreonam also had a high MIC (>256 g/mL) in all but seven IMP-6-producing isolates. 3.3. Structure of the cassette arrays of IMP-6 genes All of the IMP-6-producing isolates had class 1 integrons with amplification sizes of 3.0 kb (1 isolate), 5.5 kb (2 isolates) and 4.5 kb/5.5 kb simultaneously (52 isolates). PCR restriction fragments length polymorphism (PCR-RFLP) analysis of integrons was performed in all IMP-6-producing isolates using MboI. Three types of restriction patterns were observed (types A, B and C). All of the 4.5 kb/5.5 kb integrons belonged to type A, the 5.5 kb integrons belonged to type B and the 3.0 kb integrons belonged to type C. Two representative isolates of type A and one representative isolate of each of types B and C were selected for cassette arrays. Sequencing of several overlapping PCR fragments obtained from each type of integron showed that integron type A had blaIMP-6 –qac–aacA4–blaOXA-1 –aadA1 (5.5 kb) and aadB–cmlA–blaOXA-10 –aadA1 (4.5 kb), type B had blaIMP-6 –qac–aacA4–blaOXA-1 –aadA1 and type C had blaIMP-6 –blaOXA-10 –aadA1. The location of the blaIMP-6 gene was identified by Southern blot hybridisation of S1 nuclease-digested total DNA with a blaIMP-6 probe. All of the isolates showed a high-molecular-weight DNA band, a potential chromosomal DNA that hybridised with the 16S rRNA gene probe as well as the blaIMP-6 gene probes. None of the isolates showed additional DNA bands representing plasmids. 3.4. Molecular epidemiology of IMP-6-producing isolates Fifty-five isolates possessing blaIMP-6 and two isolates possessing blaIMP-1 as well as KM-4 were typed by PFGE through XbaI macrorestriction analysis (Fig. 1). A dendrogram showed that all isolates had a genetic similarity of >85% and were classified as the same pulsotype. All of the IMP-1/IMP-6-producing P. aeruginosa isolates and the KM-4 isolate were classified as the same pulsotype, and some isolates showed an identical pattern, indicating clonality. MLST showed that all of the IMP-1- or IMP-6-producing isolates belonged to the same ST (ST235), which has been reported as a multidrug-resistant strain in many countries. 4. Discussion The rising resistance rate of P. aeruginosa to carbapenem has become a serious problem in treating these pathogens in many countries, including South Korea. The respective resistance rates to imipenem and meropenem increased from 21% and 18% in 2005 to 29% and 27% in 2008 in tertiary care hospitals in South Korea
[21,22]. In the present study, the resistance rates to imipenem and meropenem in non-tertiary care hospitals were 16.3% and 22.0%, respectively; these values are much higher than the 8.1% and 9.3% reported in 2006 (P = 0.0028, 2 test) [23]. Thus, the mechanisms responsible for the resistance to carbapenem of P. aeruginosa, especially of MBL-producing isolates, are of special interest in South Korea. However, most studies have focused on the trends for MBL-producing isolates in tertiary care hospitals [7,24,25] and few studies have focused on the prevalence of MBL-producers in geriatric and non-tertiary care hospitals. The objectives of the present study were to determine the prevalence of MBL-producers in geriatric and non-tertiary care hospitals and to compare the characteristics and genetic similarities of these isolates with those from tertiary care hospital isolates. In South Korea, IMP-6 was first reported in a 2004 outbreak that occurred in a tertiary care hospital in the Southeast. In the 2005 Korean Nationwide Surveillance of Antimicrobial Resistance (KONSAR) tertiary care hospital survey, three (6.7%) of the 45 MBLproducing P. aeruginosa isolates were IMP-6-producers [7]. In the present study, 41 (18.3%) of the 224 imipenem- or meropenemresistant isolates were MBL-producers. IMP-6 was the dominant type (85.4%) both in geriatric and non-tertiary care hospitals, although recent studies have shown that IMP-1 is an isolated MBL type in South Korea [7,26,27]. IMP-6, which has high activity against carbapenems, especially meropenem, was first detected in Japan in 1996 in Serratia marcescens KU3838, located on a plasmid [28]. In the present study, 97.1% of the IMP-6-producing P. aeruginosa isolated from the three types of hospitals had the same class 1 integrons (blaIMP-6 –qac–aacA4–blaOXA-1 –aadA1 and aadB–cmlA–blaOXA-10 –aadA1) in their chromosomes. All of the IMP-6-possessing isolates detected in all three types of hospitals were classified as a single pulsotype, with a similarity of >85% and of the same ST (ST235), which has recently been found internationally in a multidrug-resistant clone [29,30]. It is noteworthy that the isolates also had the same integron structure and were classified as the same pulsotype as KM-4, which was the first reported IMP-6-producing P. aeruginosa in South Korea [10]. Both the PFGE type and analysis of the blaIMP-6 -possessing integrons confirmed that IMP-6-producing P. aeruginosa, which is multidrug-resistant, was spread by clonal dissemination to the three types of hospitals tested. This suggests that the disseminated IMP-6-producing P. aeruginosa originated from the outbreak strain, which included KM-4. Although the cause of dissemination has not been identified, advances in the population’s public transportation system may be a contributing factor. Since 2004, improved transportation, including bullet trains, has enabled the centralised treatment of patients and the movement of patients into metropolitan areas. Although we do not have hard evidence, there have been many news reports about increasing numbers of patients from the Southeast area receiving medical examinations and treatments in the North area since the introduction of the bullet trains. In addition, overuse of carbapenems in general hospitals may act as a selection pressure for these strains.
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Fig. 1. Dendrogram analysis of IMP-6/IMP-1-producing Pseudomonas aeruginosa isolated from three hospital classes using pulsed-field gel electrophoresis with XbaI digestion. All of the isolates clustered to one group (A) with 87.1% similarity. KM4 is one of the first outbreak isolates carrying IMP-6 in South Korea, and M0901 and M0903 are IMP-1-producing isolates. T, tertiary care hospital; NT, non-tertiary care hospital; G, geriatric hospital.
This is the first study on the nationwide dissemination of clonally related IMP-6-producing P. aeruginosa in the three types of hospitals in South Korea. The results of the present study indicate that infection control is needed to hinder the circulation of MBLproducing organisms in non-tertiary care and geriatric hospitals as well as in tertiary care hospitals. In conclusion, genetically related blaIMP-6 -possessing isolates were found in the three types of hospitals nationwide. The results of the present study suggest that blaIMP-6 -possessing P. aeruginosa may lead to high-level endemicity. Therefore, continuous
monitoring, suitable intervention and prudent use of carbapenems in the three types of hospitals are needed to prevent further spread of MBL-producing P. aeruginosa.
Acknowledgments The authors thank Seok Hoon Jeong for providing the IMP-6 producing Pseudomonas aeruginosa isolates from tertiary care hospitals in 2008 as well as KM-4 strain.
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Funding: This work was supported by a research grant from the Korea Centers for Disease Control and Prevention (2009-N4400300). Competing interests: None declared. Ethical approval: Not required. References [1] Walsh TR, Toleman MA, Poirel L, Nordmann P. Metallo--lactamases: the quiet before the storm? Clin Microbiol Rev 2005;18:306–25. [2] Maltezou HC. Metallo--lactamases in Gram-negative bacteria: introducing the era of pan-resistance? Int J Antimicrob Agents 2009;33:405.e1–7. [3] Giakkoupi P, Petrikkos G, Tzouvelekis LS, Tsonas S, Legakis NJ, Vatopoulos AC, et al. Spread of integron-associated VIM-type metallo--lactamase genes among imipenem-nonsusceptible Pseudomonas aeruginosa strains in Greek hospitals. J Clin Microbiol 2003;41:822–5. [4] Ragatolla C, Tonin EA, Monti-Bragadin C, Dolzani L, Gombac F, Bearzi C, et al. Endemic carbapenem-resistant Pseudomonas aeruginosa with acquired metallo--lactamase determinants in a European hospital. Emerg Infect Dis 2004;10:535–8. [5] Kouda S, Ohara M, Onodera M, Fujiue Y, Sasaki M, Kohara T, et al. Increased prevalence and clonal dissemination of multidrug-resistant Pseudomonas aeruginosa with the blaIMP-1 gene cassette in Hiroshima. J Antimicrob Chemother 2009;64:46–51. [6] Lee K, Lim JB, Yum JH, Yong D, Chong Y, Kim JM, et al. blaVIM-2 cassettecontaining novel integrons in metallo--lactamase-producing Pseudomonas aeruginosa and Pseudomonas putida isolates disseminated in a Korean hospital. Antimicrob Agents Chemother 2002;46:1053–8. [7] Lee K, Park AJ, Kim MY, Lee HJ, Cho JH, Kang JO, et al. Metallo--lactamaseproducing Pseudomonas spp. in Korea: high prevalence of isolates with VIM-2 type and emergence of isolates with IMP-1 type. Yonsei Med J 2009;50: 335–9. [8] Lee K, Ha GY, Shin BM, Kim JJ, Kang JO, Jang SJ, et al. Metallo-lactamase-producing Gram-negative bacilli in Korean Nationwide Surveillance of Antimicrobial Resistance group hospitals in 2003: continued prevalence of VIM-producing Pseudomonas spp. and increase of IMP-producing Acinetobacter spp. Diagn Microbiol Infect Dis 2004;50:51–8. [9] Oh EJ, Lee S, Park YJ, Park JJ, Park K, Kim SI, et al. Prevalence of metallo-lactamase among Pseudomonas aeruginosa and Acinetobacter baumannii in a Korean university hospital and comparison of screening methods for detecting metallo--lactamase. J Microbiol Methods 2003;54:411–18. [10] Ryoo NH, Lee K, Lim JB, Lee YH, Bae IK, Jeong SH. Outbreak by meropenemresistant Pseudomonas aeruginosa producing IMP-6 metallo--lactamase in a Korean hospital. Diagn Microbiol Infect Dis 2009;63:115–17. [11] Riccio ML, Pallecchi P, Docquier J-D, Cresti S, Catania MR, Pagani L, et al. Clonal relatedness and conserved integron sequences in epidemiologically unrelated Pseudomonas aeruginosa strains producing the VIM-1 metallo-lactamase from different Italian hospitals. Antimicrob Agents Chemother 2005;49:104–10. [12] Toleman MA, Biedenbach D, Bennett DM, Jones RN, Walsh TR. Italian metallo-lactamases: a national problem? Report from the SENTRY Antimicrobial Surveillance Programme. J Antimicrob Chemother 2005;55:61–70. [13] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; nineteenth informational supplement. Document M100-S19. Wayne, PA: CLSI; 2009.
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International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Dissemination of genetically related IMP-6-producing multidrug-resistant Pseudomonas aeruginosa ST235 in South Korea夽 Jung Sik Yoo, Ji Woo Yang, Hye Mee Kim, Jeongheum Byeon, Hwa Su Kim, Jae Il Yoo, Gyung Tae Chung, Yeong Seon Lee ∗ Division of Antimicrobial Resistance, Korea National Institute of Health, 187 Osong Saengmyeong 2-ro Chungcheongbuk-do, 363-951, Republic of Korea
a r t i c l e
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Article history: Received 9 July 2011 Accepted 21 November 2011 Keywords: Carbapenem Metallo--lactamase IMP-6 Non-tertiary care hospital Geriatric hospital
a b s t r a c t The present study aimed to describe the prevalence and molecular epidemiology of metallo-lactamase (MBL)-producing Pseudomonas aeruginosa isolates obtained from non-tertiary care hospitals and geriatric hospitals in South Korea. Of the 644 isolates, 224 were carbapenem-resistant, amongst which 41 (18.3%) were MBL-producers and the major MBL type was IMP-6 (35 isolates). IMP-6producing isolates were multidrug-resistant and showed higher minimum inhibitory concentrations for meropenem than imipenem. All of the IMP-6-producing isolates had class 1 integrons with amplification sizes of 4.5 kb/5.5 kb (34 isolates) or 3.0 kb (1 isolate); 4.5 kb/5.5 kb integrons had blaIMP-6 –qac–aacA4–blaOXA-1 –aadA1 (5.5 kb) and aadB–cmlA–blaOXA-10 –aadA1 (4.5 kb). Pulsed-field gel electrophoresis (PFGE) analysis indicated that all IMP-6-producing P. aeruginosa from various geographic areas had nearly identical patterns with >85% similarity. All IMP-6-producing isolates showed high genetic similarity to those obtained from tertiary care hospitals and had the same integron type, indicating the spread of these strains to the three types of hospitals nationwide. These data show the wide spreading of clonally related IMP-6-producing P. aeruginosa (sequence type235) through tertiary, non-tertiary and geriatric hospitals in South Korea. Continuous monitoring and thorough infection control should be performed in all types of hospitals to prevent further spreading of MBL-producing P. aeruginosa. © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Carbapenems are used as the last choice for treating Gram-negative bacterial infections; however, intensive use of carbapenems in the treatment of these infections has led to the emergence and increase in the occurrence of carbapenem-resistant clinical isolates. The highest level of resistance is exhibited by those isolates producing metallo--lactamases (MBLs). Acquired resistance to carbapenems through the production of MBLs by Pseudomonas and Acinetobacter spp. has been increasingly reported in many countries [1,2]. MBL-associated resistance has caused outbreaks of hospital infections and endemic diseases in several areas worldwide. In 2001, 72% of the carbapenem-resistant Pseudomonas aeruginosa isolates from an Italian hospital were VIM-producers, and 62% of
夽 This study was presented in part at the 20th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), 10–13 April 2010, Vienna, Austria. ∗ Corresponding author. Tel.: +82 43 719 8240; fax: +82 43 719 8269. E-mail address: [email protected] (Y.S. Lee).
the imipenem-resistant isolates from several Greek hospitals had the same type of MBL [3,4]. A previous study conducted between 2004 and 2006 in Hiroshima, Japan, reported that IMP-1-producing multidrug-resistant P. aeruginosa isolates from different hospitals were clonally similar [5]. In 1995, VIM-2 was first detected in P. aeruginosa isolates in South Korea and it has become the most common MBL type since then [6–8]. VIM-2 comprised 71.1% and IMP-1-like type comprised 28.9% in the 2005 survey [7]. However, the IMP type was first found in 2000–2001 isolates [9] and increased to 28.9% of P. aeruginosa isolates in 2005 [7]. There was an outbreak of IMP-6-producing P. aeruginosa in a tertiary care hospital in 2004 [10]. The increasing prevalence of MBL-producing isolates has been attributed to clonal spread and/or horizontal transfer of MBL genes possessing mobile genetic elements [11,12]. However, there have been few studies on the epidemiological situation and characteristics of MBL-producing isolates from all types of hospitals, especially non-tertiary care hospitals and geriatric hospitals in South Korea. The aim of the present study was to assess the prevalence of MBL-producing isolates amongst carbapenem-resistant P. aeruginosa isolates obtained from non-tertiary care and geriatric
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Table 1 Detection of metallo--lactamase (MBL)-producing Pseudomonas aeruginosa isolates in geriatric hospitals and non-tertiary care hospitals in South Korea. MBL gene
blaIMP-1 blaIMP-6 blaVIM-2 Total a
Geriatric hospitals (n = 271 isolates)
Non-tertiary care hospitals (n = 373 isolates)
No. of isolates
Location(s)a
No. of isolates
Location(s)a
2 13 0 15 (5.5%)
North North, Southeast –
0 22 4 26 (7.0%)
– North, Middle, Southeast Middle, Southeast, Southwest
North, Seoul/Gyeonggi/Incheon/Gangwon; Middle, Chungcheong; Southeast, Busan/Gyungsang; Southwest, Gwangju/Jeolla/Jeju.
hospitals in South Korea. The characteristics and genetic similarities between these isolates and the IMP-6-producing P. aeruginosa isolates in tertiary care hospitals were investigated. 2. Materials and methods 2.1. Bacterial strains and susceptibility testing Between 2008 and 2009, 644 consecutive, non-duplicate clinical isolates of P. aeruginosa were collected from 111 non-tertiary care hospitals and 47 geriatric hospitals geographically dispersed across South Korea; 93% of the hospitals sent ≤10 isolates. Nontertiary care hospitals or geriatric hospitals tended to be centralised in some areas, so some hospitals sent more isolates than others. The maximum number of isolates per hospital was 49 and the minimum was 1. However, the mean numbers of isolates per hospital by location were similar (3.1 isolates/hospital in the Southwest, 3.4 in the Southeast, 4.5 in the Middle area and 4.7 in the North). Disk diffusion and agar dilution were performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines [13]. MBL production was detected by the double-disk synergy (DDS) test [14] using a 10 g disk of imipenem and an ethylene diamine tetra-acetic acid (EDTA) disk (1.9 mg) for P. aeruginosa not susceptible to meropenem or imipenem. IMP-6-producing isolates were compared with isolates from tertiary care hospitals (20 IMP-6-possessing P. aeruginosa isolated in 2007–2008 and KM-4 P. aeruginosa isolate, which is one of the first isolated IMP6-producing P. aeruginosa in South Korea [10]) on the basis of characteristics and genetic similarities.
by the unweighted pair-group method using arithmetic averages (UPGMA) with the band position tolerance set at 1.8%. MLST was performed according to the method of Curran et al. [19]. Following PCR and sequence analysis for seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA and trpE) performed using the primers in the MLST database (http://pubmlst.org/paeruginosa), the sequence types (STs) of the isolates were obtained based on the allele sequences in the database. 2.4. S1 nuclease digestion and Southern blot hybridisation PFGE with S1 nuclease (Promega, Madison, WI) digestion of whole genomic DNA was performed as described previously with some modifications [20]. Gel plugs were cut into 5 mm slices, soaked with 1× reaction buffer and then digested with 0.1 U of S1 nuclease for 30 min at 37 ◦ C. The plugs were incubated on ice for 10 min in 200 L of ice-cold 0.5 M EDTA (pH 8.0). Undigested total DNA of the isolates was separated on 0.8% agarose gel by PFGE and transferred onto a Hybond N+ membrane (Amersham Pharmacia Biotech, Little Chalfont, UK) and hybridised separately with a probe for blaIMP-6 (IMP-F, 5 -TGAGCAATGTATCTGTATTC3 ; IMP-R, 5 -TTAGTTGCTTGGTTTTGATG-3 ) and the 16S rRNA gene (16S-F, 5 -CGCGTGTGTGAAGAAGGTCT-3 ; 16S-R, 5 CACCTGTGTCTGAGTTCCCG-3 ). An enhanced chemiluminescence (ECLTM ) Direct Nucleic Acid Labelling and Detection System (GE Healthcare UK Ltd., Little Chalfont, UK) was used. 3. Results
2.2. Molecular detection and analysis of metallo-ˇ-lactamase genes and integrons
3.1. Metallo-ˇ-lactamase-producing Pseudomonas aeruginosa in non-tertiary care and geriatric hospitals
To detect the IMP, VIM, SIM, GIM and SPM types of MBL genes, DDS-positive isolates were tested by multiplex polymerase chain reaction (PCR) as described previously [15]. Then, each MBL type underwent PCR amplification and sequencing of PCR-positive isolates using primers VIM-2A (5 -ATG TTC AAA CTT TTG AGT AGT AAG-3 ), VIM-2B (5 -CTA CTC AAC GAC TGA GCG-3 ), IMP-1F (5 TGA GCA ATG TAT CTG TAT TC-3 ) and IMP1-R (5 -TTA GTT GCT TGG TTT TGA TG-3 ) [16,17]. Detection of class 1 integrons by PCR was also performed as described previously [18]. Amplified integron restriction types were analysed following digestion at 37 ◦ C for 18 h with MboI (New England Biolabs, Beverly, MA). The structure of the variable regions of the MBL-containing integrons was determined by PCR mapping and sequencing.
The resistance rates to meropenem in non-tertiary care and geriatric hospitals were 22.0% and 30.6%, respectively, and those to imipenem were 16.3% and 29.9%, respectively. Of the 644 isolates, 224 (34.8%) were imipenem- or meropenem-resistant and 78 were positive in the EDTA/imipenem synergy test. MBLs were detected in 41 isolates; 35 (85.4%) of the 41 isolates were identified as blaIMP-6 , 2 isolates were identified as blaIMP-1 and 4 isolates were identified as blaVIM-2 (Table 1). IMP-6-possessing isolates were found in 15 of the 111 non-tertiary care hospitals and in 6 geriatric hospitals. Geographically, they were confined to the North (20 isolates in 11 hospitals), Middle (4 isolates in 2 hospitals) and Southeastern (11 isolates in 8 hospitals) areas of South Korea, but were not found in the Southwestern area. Four VIM-2-producing isolates were found in the Middle, Southeast and Southwest.
2.3. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)
3.2. Antimicrobial susceptibilities of IMP-6-producing isolates
Total bacterial DNA was digested using XbaI (New England Biolabs), and DNA fragments were separated on 1.2% agarose gel in a 0.5× TBE (Tris–borate–EDTA) buffer using a CHEF Mapper® apparatus (Bio-Rad Laboratories, Hercules, CA). The conditions were as follows: 14 ◦ C, 6 V/cm, pulses of 10 s to 40 s and a run time of 22 h with a ramping factor of −1.357. Genetic similarities were assessed
Because IMP-6 is the dominant MBL type in tertiary care hospital isolates, IMP-6-producing P. aeruginosa isolates obtained from nontertiary care and geriatric hospitals were analysed. In addition to 35 isolates from non-tertiary and geriatric hospitals, 20 isolates from tertiary care hospitals were included in the study. All of the IMP6-producing P. aeruginosa isolates were resistant to ceftazidime,
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Table 2 Characteristics of 55 IMP-6-producing Pseudomonas aeruginosa isolates from three hospital types in South Korea. Hospital type
No. of isolates Integron structurea
MLST
MIC range (g/mL) IPM
Geriatric 13 Non-tertiary care 22 20 Tertiary care
MEM TZP
Type A (12), type C (1) ST235 16–64 >256 Type A ST235 16–64 >256 Type A (18), type B (2) ST235 8–32 >256
FEP
CAZ
ATM
CIP
TOB
GEN
64 to >256 256 to >256 128 to >256 256 to >256 32–128 64 to >256 256 to >256 32–256 64 to >256 64 to >256 256 to >256 16–128 >256 >256 32–256 64 to >256 64 to >256 32 to >256 16–128 64 to >256 128 to >256
MLST, multilocus sequence type; MIC, minimum inhibitory concentration; IPM, imipenem; MEM, meropenem; TZP, piperacillin/tazobactam; FEP, cefepime; CAZ, ceftazidime; ATM, aztreonam; CIP, ciprofloxacin; TOB, tobramycin; GEN, gentamicin. a Type A, blaIMP-6 –qac–aacA4–blaOXA-1 –aadA1 (5.5 kb)/aadB–cmlA–blaOXA-10 –aadA1 (4.5 kb); type B, blaIMP-6 –qac–aacA4–blaOXA-1 –aadA1 (5.5 kb); and type C, blaIMP-6 –blaOXA-10 –aadA1 (3.0 kb).
cefepime, aztreonam, gentamicin, tobramycin, ciprofloxacin and carbapenems. The minimum inhibitory concentrations (MICs) for meropenem were extremely high (all >256 g/mL). However, the MICs of imipenem ranged from 8 g/mL to 64 g/mL. The MICs for cephalosporins, aminoglycosides and ciprofloxacin were also extremely high (Table 2). Aztreonam also had a high MIC (>256 g/mL) in all but seven IMP-6-producing isolates. 3.3. Structure of the cassette arrays of IMP-6 genes All of the IMP-6-producing isolates had class 1 integrons with amplification sizes of 3.0 kb (1 isolate), 5.5 kb (2 isolates) and 4.5 kb/5.5 kb simultaneously (52 isolates). PCR restriction fragments length polymorphism (PCR-RFLP) analysis of integrons was performed in all IMP-6-producing isolates using MboI. Three types of restriction patterns were observed (types A, B and C). All of the 4.5 kb/5.5 kb integrons belonged to type A, the 5.5 kb integrons belonged to type B and the 3.0 kb integrons belonged to type C. Two representative isolates of type A and one representative isolate of each of types B and C were selected for cassette arrays. Sequencing of several overlapping PCR fragments obtained from each type of integron showed that integron type A had blaIMP-6 –qac–aacA4–blaOXA-1 –aadA1 (5.5 kb) and aadB–cmlA–blaOXA-10 –aadA1 (4.5 kb), type B had blaIMP-6 –qac–aacA4–blaOXA-1 –aadA1 and type C had blaIMP-6 –blaOXA-10 –aadA1. The location of the blaIMP-6 gene was identified by Southern blot hybridisation of S1 nuclease-digested total DNA with a blaIMP-6 probe. All of the isolates showed a high-molecular-weight DNA band, a potential chromosomal DNA that hybridised with the 16S rRNA gene probe as well as the blaIMP-6 gene probes. None of the isolates showed additional DNA bands representing plasmids. 3.4. Molecular epidemiology of IMP-6-producing isolates Fifty-five isolates possessing blaIMP-6 and two isolates possessing blaIMP-1 as well as KM-4 were typed by PFGE through XbaI macrorestriction analysis (Fig. 1). A dendrogram showed that all isolates had a genetic similarity of >85% and were classified as the same pulsotype. All of the IMP-1/IMP-6-producing P. aeruginosa isolates and the KM-4 isolate were classified as the same pulsotype, and some isolates showed an identical pattern, indicating clonality. MLST showed that all of the IMP-1- or IMP-6-producing isolates belonged to the same ST (ST235), which has been reported as a multidrug-resistant strain in many countries. 4. Discussion The rising resistance rate of P. aeruginosa to carbapenem has become a serious problem in treating these pathogens in many countries, including South Korea. The respective resistance rates to imipenem and meropenem increased from 21% and 18% in 2005 to 29% and 27% in 2008 in tertiary care hospitals in South Korea
[21,22]. In the present study, the resistance rates to imipenem and meropenem in non-tertiary care hospitals were 16.3% and 22.0%, respectively; these values are much higher than the 8.1% and 9.3% reported in 2006 (P = 0.0028, 2 test) [23]. Thus, the mechanisms responsible for the resistance to carbapenem of P. aeruginosa, especially of MBL-producing isolates, are of special interest in South Korea. However, most studies have focused on the trends for MBL-producing isolates in tertiary care hospitals [7,24,25] and few studies have focused on the prevalence of MBL-producers in geriatric and non-tertiary care hospitals. The objectives of the present study were to determine the prevalence of MBL-producers in geriatric and non-tertiary care hospitals and to compare the characteristics and genetic similarities of these isolates with those from tertiary care hospital isolates. In South Korea, IMP-6 was first reported in a 2004 outbreak that occurred in a tertiary care hospital in the Southeast. In the 2005 Korean Nationwide Surveillance of Antimicrobial Resistance (KONSAR) tertiary care hospital survey, three (6.7%) of the 45 MBLproducing P. aeruginosa isolates were IMP-6-producers [7]. In the present study, 41 (18.3%) of the 224 imipenem- or meropenemresistant isolates were MBL-producers. IMP-6 was the dominant type (85.4%) both in geriatric and non-tertiary care hospitals, although recent studies have shown that IMP-1 is an isolated MBL type in South Korea [7,26,27]. IMP-6, which has high activity against carbapenems, especially meropenem, was first detected in Japan in 1996 in Serratia marcescens KU3838, located on a plasmid [28]. In the present study, 97.1% of the IMP-6-producing P. aeruginosa isolated from the three types of hospitals had the same class 1 integrons (blaIMP-6 –qac–aacA4–blaOXA-1 –aadA1 and aadB–cmlA–blaOXA-10 –aadA1) in their chromosomes. All of the IMP-6-possessing isolates detected in all three types of hospitals were classified as a single pulsotype, with a similarity of >85% and of the same ST (ST235), which has recently been found internationally in a multidrug-resistant clone [29,30]. It is noteworthy that the isolates also had the same integron structure and were classified as the same pulsotype as KM-4, which was the first reported IMP-6-producing P. aeruginosa in South Korea [10]. Both the PFGE type and analysis of the blaIMP-6 -possessing integrons confirmed that IMP-6-producing P. aeruginosa, which is multidrug-resistant, was spread by clonal dissemination to the three types of hospitals tested. This suggests that the disseminated IMP-6-producing P. aeruginosa originated from the outbreak strain, which included KM-4. Although the cause of dissemination has not been identified, advances in the population’s public transportation system may be a contributing factor. Since 2004, improved transportation, including bullet trains, has enabled the centralised treatment of patients and the movement of patients into metropolitan areas. Although we do not have hard evidence, there have been many news reports about increasing numbers of patients from the Southeast area receiving medical examinations and treatments in the North area since the introduction of the bullet trains. In addition, overuse of carbapenems in general hospitals may act as a selection pressure for these strains.
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Fig. 1. Dendrogram analysis of IMP-6/IMP-1-producing Pseudomonas aeruginosa isolated from three hospital classes using pulsed-field gel electrophoresis with XbaI digestion. All of the isolates clustered to one group (A) with 87.1% similarity. KM4 is one of the first outbreak isolates carrying IMP-6 in South Korea, and M0901 and M0903 are IMP-1-producing isolates. T, tertiary care hospital; NT, non-tertiary care hospital; G, geriatric hospital.
This is the first study on the nationwide dissemination of clonally related IMP-6-producing P. aeruginosa in the three types of hospitals in South Korea. The results of the present study indicate that infection control is needed to hinder the circulation of MBLproducing organisms in non-tertiary care and geriatric hospitals as well as in tertiary care hospitals. In conclusion, genetically related blaIMP-6 -possessing isolates were found in the three types of hospitals nationwide. The results of the present study suggest that blaIMP-6 -possessing P. aeruginosa may lead to high-level endemicity. Therefore, continuous
monitoring, suitable intervention and prudent use of carbapenems in the three types of hospitals are needed to prevent further spread of MBL-producing P. aeruginosa.
Acknowledgments The authors thank Seok Hoon Jeong for providing the IMP-6 producing Pseudomonas aeruginosa isolates from tertiary care hospitals in 2008 as well as KM-4 strain.
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