- Email: [email protected]
Dissemination and genetic context analysis of blaVIM-6 among Pseudomonas aeruginosa isolates in Asian-Pacific Nations
186
Clinical Microbiology and Infection, Volume 16 Number 2, February 2010
Transparency Declaration The authors declare that there was no source of funding and that they have no conflicts of interest.
CMI
Dissemination and genetic context analysis of blaVIM-6 among Pseudomonas aeruginosa isolates in Asian-Pacific Nations M. Castanheira1, J. M. Bell2, J. D. Turnidge2, R. E. Mendes1 and R. N. Jones1
References
1) JMI Laboratories, North Liberty, IA, USA and 2) Women’s and Children’s Hospital, Adelaide, SA, Australia
1. Weber DJ, Raasch R, Rutala WA. Nosocomial infections in the ICU: the growing importance of antibiotic-resistant pathogens. Chest 1999; 115: 34S–41S. 2. Zhanel GG, DeCorby M, Laing N et al. Antimicrobial-resistant pathogens in intensive care units in Canada: results of the Canadian National Intensive Care Unit (CAN-ICU) study, 2005–2006. Antimicrob Agents Chemother 2008; 52: 1430–1437. 3. Al Naiemi N, Heddema ER, Bart A et al. Emergence of multidrugresistant Gram-negative bacteria during selective decontamination of the digestive tract on an intensive care unit. J Antimicrob Chemother 2006; 58: 853–856. 4. Antoniadou A, Kontopidou F, Poulakou G et al. Colistin-resistant isolates of Klebsiella pneumoniae emerging in intensive care unit patients: first report of a multiclonal cluster. J Antimicrob Chemother 2007; 59: 786–790. 5. Martins IS, Pessoa-Silva CL, Nouer SA et al. Endemic extended-spectrum beta-lactamase-producing Klebsiella pneumoniae at an intensive care unit: risk factors for colonization and infection. Microb Drug Resist 2006; 12: 50–58. 6. Falagas M, Bliziotis I, Kasiakou S et al. Outcome of infections due to pandrug-resistant (PDR) Gram-negative bacteria. BMC Infect Dis 2005; 5: 24. 7. Michalopoulos A, Fotakis D, Virtzili S et al. Aerosolized colistin as adjunctive treatment of ventilator-associated pneumonia due to multidrug-resistant Gram-negative bacteria: a prospective study. Respir Med 2008; 102: 407–412. 8. Matthaiou DK, Michalopoulos A, Rafailidis PI et al. Risk factors associated with the isolation of colistin-resistant gram-negative bacteria: a matched case–control study. Crit Care Med 2008; 36: 807– 811. 9. Markogiannakis H, Pachylaki N, Samara E et al.. Infections in a surgical intensive care unit of an university hospital in Greece. Int J Infect Dis 2009; 13: 145–153. 10. Tharavichitkul P, Khantawa B, Bousoung V, Boonchoo M. Activity of fosfomycin against extended-spectrum-b-lactamase-producing Klebsiella pneumoniae and Escherichia coli in Maharaj Nakorn Chiang Mai Hospital. J Infect Dis Antimicrob Agents 2005; 22: 121– 126. 11. Falagas ME, Kanellopoulou MD, Karageorgopoulos DE et al. Antimicrobial susceptibility of multi-drug resistant Gram-negative bacteria to fosfomycin. Eur J Clin Microbiol Infect Dis 2008; 27: 439–443. 12. Hardisson C, Villar CJ, Llaneza J, Mendoza MC. Prevalence and dispersion of plasmids conferring fosfomycin resistance in enterobacteria. Pathol Biol (Paris) 1984; 32: 755–758. 13. Bedirdjian JP, Morin JP, Fouchet B, Fillastre JP. Effect of fosfomycin on respiration by rat kidney mitochondria. Drugs Exp Clin Res 1978; 4: 57–62. 14. de Cueto M, Lo´pez L, Herna´ndez JR, Morillo C, Pascual A. In vitro activity of fosfomycin against extended-spectrum-beta-lactamaseproducing Escherichia coli and Klebsiella pneumoniae: comparison of susceptibility testing procedures. Antimicrob Agents Chemother 2006; 50: 368–370.
Abstract VIM-6, previously reported in two strains from Singapore recovered in 2000, was detected in 16 isolates collected in 2006 in India (12 isolates), Indonesia (two), Korea and the Philippines (one each). High genetic variability was observed among VIM-6producing isolates (12 ribotypes and 11 pulsed-field gel electrophoresis types), but clones were observed in India and Indonesia; blaVIM-6-carrying integrons of 3.9 kb and 5 kb were detected, and two of five Indian hospitals yielded isolates with both integrons. These two integrons, blaVIM-6 was located in the first position, followed by blaOXA-10 and aacA4. The 5-kb integrons also harboured aadA1 and a 331-bp open reading frame encoding a putative efflux pump.
Keywords: Asia-Pacific, metallo-b-lactamases, Pseudomonas aeruginosa, SENTRY, VIM Original Submission: 23 October 2008; Revised Submission: 4 February 2009; Accepted: 11 February 2009 Editor: D. Mack Article published online: 10 August 2009
Clin Microbiol Infect 2010; 16: 186–189
10.1111/j.1469-0691.2009.02903.x
Corresponding author and reprint requests: M. Castanheira, JMI Laboratories, 345 Beaver Kreek Center, Suite A, North Liberty Iowa 52317, IA, USA E-mail: [email protected]
Acquired metallo-b-lactamase (MBL)-producing isolates have been increasingly reported in the Asia-Pacific (APAC) region.
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 179–199
Research Notes
CMI
Several MBL types were found in Korea (IMP-1, IMP-8, VIM2, and SIM-1), China (IMP-4, IMP-9, and VIM-2) and Taiwan (IMP-8, VIM-2, and VIM-3), among other countries from that region [1,2]. A new VIM-type enzyme was identified in two Pseudomonas putida isolates recovered in Singapore in 2000 [3]. This enzyme, named VIM-6, showed only two amino acid differences from VIM-2, which is probably its genetic ancestor. VIM-6 has not been reported in any other geographical location, and its description was succinct, focusing on the clinical findings. In this study, we report 16 blaVIM-6-carrying Pseudomonas aeruginosa isolates from four countries in the APAC region, and their genetic context. A total of 719 P. aeruginosa isolates were consecutively collected from bloodstream, respiratory tracts and skin and skin structure infections in 57 hospitals located in the APAC region during the SENTRY Antimicrobial Surveillance Program (2006). Isolates were susceptibility-tested by CLSI broth microdilution [4,5]. Among those strains, 196 (27.3%) were non-susceptible (MIC of ‡8 mg/L) to imipenem and/or meropenem and were tested using generic primers designed to align multiple variants of IMP, VIM and other MBL genes in a multiplex PCR approach [6]. All amplicons were sequenced on both strands and analysed. The blaVIM-6 gene was detected in 16 (8.1%) isolates recovered in India (12 isolates), Indonesia (two), Korea (one), and the Philippines (one; Table 1). All isolates were resistant to three or more classes of antipseudomonal agents. Two isolates from India were susceptible to fluoroquinolones (ciprofloxacin MIC of 0.12 and 0.5 mg/L), whereas polymyxin B and colistin remained active against all strains (MIC of 0.5–1 mg/L).
187
Primers annealing in the two conserved regions (CS) of class 1 integrons [7] were used in combination with MBL primers to determine the size of the integron structure. The blaVIM-6 gene was carried as a gene cassette in the first position of 3.9-kb and 5-kb class 1 integrons (Table 1). Amplicons generated with blaVIM primers anchored in the 3¢CS were digested with DraI and XbaI in separate reactions. Integrons of the same size generated identical restriction fragment length polymorphism patterns, indicating a similar content and arrangement. Sequencing showed that the 3.9kb integrons carried blaVIM-6, blaOXA-10 and aacA4 followed by qacED1/sul1 in the 3¢CS (Fig. 1). The 5-kb integrons possessed the same gene content but two additional gene cassettes: a 331-bp open reading frame (ORF) and aadA1 upstream of the 3¢CS (Fig. 1). The 5-kb integrons found in this study appear to be identical to the blaVIM-6 integron carried by the isolate found in Singapore, which has been deposited in GenBank (no. EF522838) but not yet described. In two isolates, the 3¢CS could not be amplified with several different primers annealing in different structures found in the 3¢-end of class 1 integrons. However, primers annealing in aacA4 and aadA1 showed that these genetic structures were similar to the other blaVIM-6-carrying integrons described here (Table 1). The putative protein encoded by the 331-bp ORF carried in the 5-kb integrons showed 78% similarity with the efflux pump QACED1, which provides resistance to quaternary ammonium compounds. The orf was cloned into pPCRScriptCam SK+ (Stratagene Cloning Systems, La Jolla, CA, USA), and recombinant strains were tested by MIC methods [4] for several clinically available antimicrobial agents and other
TABLE 1. Characteristics of 16 blaVIM-6-carrying Pseudomonas aeruginosa isolates obtained in 2006 in the Asia-Pacific (APAC) region Isolate
Country
Hospital
Source
Ribotypea
PFGE pattern
Integron size
225-22-D 228-23-D 241-09-D 241-73.2-D 244-07-D 244-10-D 244-18-C 244-24-D 244-29-D 247-03-D 247-06-D 248-11-A 248-26-C 248-77-C 252-05-C 252-09-C
Korea Philippines Indonesia Indonesia India India India India India India India India India India India India
A B C C D D D D D E E F F F G G
Abscess Skin and soft tissue Wound Abscess Skin and soft tissue Wound Tracheal aspirate Tissue Unknown Wound Wound Blood culture Unknown Unknown Sputum Endotracheal tube
1034-2 35-7 566-6 566-6 566-2 347-6 347-8 352-2 352-2 348-6 348-6 45-6 45-6 114-4 147-6 134-4
A B C C1 D E E1 E E F F G H I J K
5 kb-likeb 3.9 kb-likeb 5 kb 5 kb 3.9 kb 5 kb 5 kb 5 kb 5 kb 5 kb 5 kb 3.9 kb 3.9 kb 5 kb 3.9 kb 3.9 kb
PFGE, pulsed-field gel electrophoresis. a Isolates were considered to be grouped in the same ribotype if the similarity coefficient was ‡0.93. Ribotypes were named according to the batch (first three digits) and sample position (last single digit) of the first isolate identified with that pattern. b The same gene cassette arrangement was observed, but the 3¢CS could not be amplified.
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 179–199
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Clinical Microbiology and Infection, Volume 16 Number 2, February 2010
attI1
CMI
59’-be 3´CS
5´CS
intI1
bla VIM-6
bla OXA-10
aacA4
qacE 1/sul1
FIG. 1. Schematic representation of blaVIM-6carrying integrons detected in carbapenemresistant Pseudomonas aeruginosa isolates from the Asia-Pacific region. The 5-kb integrons possessed a 331-bp open reading frame (ORF) and aadA1 that were not present in 3.9-kb
331 bp ORF
antibacterial compounds, such as surfactants and detergents (Brij58, deoxicolate Tween-80, chlorhexidine, benzalconium, sodium dodecylsulphate and sarkosyl), dyes (ethidium bromide and crystal violet), and heavy metals (cadmium, lithium, nickel and chromium). The results showed no consistent differences in the MIC values for the Escherichia coli strains harbouring the plasmid containing the 331-bp ORF or the plasmid without the insert. The expression of orf was confirmed in the clinical isolate and in the recombinant colonies by RT-PCR (OneStep RT-PCR kit; Qiagen, Hilden, Germany) in RNA preparations (RNeasy Mini kit; Qiagen) previously treated with RNase-free DNase (Promega, Madison, WI, USA). Although this gene did not confer a phenotype of resistance to the compounds tested, this putative efflux pump could still play a role in the selection of strains harbouring these mobile elements. MBL-producing isolates were ribotyped using PvuII in the Riboprinter Microbial Characterization System (Qualicon, Wilmington, DE, USA). Isolates were also evaluated using pulsed-field gel electrophoresis (PFGE), as previously described [8]. Isolates carrying blaVIM-6 were clustered in 12 ribotypes and 11 PFGE patterns (Table 1). Three clones of two isolates each were identified in India by ribotyping, but PFGE showed that isolates from one suspected clone were not related. Whereas ribotyping identified a clone with two isolates in one Indian hospital, PFGE demonstrated that four isolates from the same hospital were genetically related (clone E/E1). The two other clonal strains from India and the two isolates from Indonesia were found to be genetically related by both epidemiological methods. The other five strains, including the strains found in Korea and the Philippines, showed unique ribotypes and PFGE patterns. Six isolates harboured the 3.9-kb blaVIM-6-carrying integrons, and all but one isolate were found in three Indian medical sites (Table 1). The remaining organism carrying a 3.9-kb integron was recovered in the Philippines, and this integron lacked the conventional 3¢CS of class 1 integrons. All isolates were geneti-
aadA1
structures.
cally distinct according to PFGE analysis; nonetheless, two isolates from one hospital were clustered in the same ribotype, showing genetic relatedness. Less genetic variability was noted in the isolates carrying 5-kb integrons (three clones). Plasmid DNA from the VIM-6-producing strains was extracted using the Plasmid DNA Midi Kit (Qiagen) and resolved on agarose gels. E. coli NTCC 50192 was used as a molecular weight marker. Transfer of plasmid DNA to a nylon membrane was performed by Southern blot as described previously [9]. The blaVIM-6 probe generated by PCR was labelled and membranes were hybridized using the non-radioactive DIG high Prime DNA labelling and detection kit (Roche Diagnostics GmbH, Mannheim, Germany). All isolates showed at least one plasmid band, and the blaVIM-6 probe hybridization demonstrated that this MBL gene was carried by plasmids of approximately 35 kb in all isolates, including isolates carrying different blaVIM-6 integrons. This suggests that these plasmids continually undergo genetic events, such as rearrangements, incisions, and excisions. Our results show that a genetic structure carrying blaVIM-6 is probably responsible for the dissemination of this MBL gene in the four APAC countries; however, we also observed clonal spread of VIM-6-producing strains in specific hospitals. The blaVIM-6 gene was initially reported from two P. putida clinical isolates recovered in Singapore, but the context of this gene has not been described [3]. In this study, this MBL gene was found in four more countries of the APAC region, including Indonesia and the Philippines, where MBLs have not previously been reported. The spread and high genetic diversity observed among blaVIM-6-harbouring isolates can be attributed to a promiscuous mobile structure that carries this MBL gene.
Nucleotide Sequence Accession Number The nucleotide sequence of the 3.9-kb integron carrying blaVIM-6 has been submitted to GenBank and assigned accession number FM994936.
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 179–199
Research Notes
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189
Tunisia, 2) Laboratoire de Bacte´riologie, UPRES EA 2392, Faculte´ de Me´d-
Acknowledgements
ecine Pierre et Marie Curie, Universite´ Paris VI and 3) Service de Bacte´riologie-Hygie`ne, Hoˆpital Tenon, Assistance Publique-Hoˆpitaux de Paris,
This study was partially presented at the 47th Interscience Conference of Antimicrobial Agents and Chemotherapy (ICAAC), 17–20 September 2007, Chicago, IL, USA. The authors thank the following individuals for assistance in testing and/or manuscript preparation: L. M. Deshpande, H. S. Sader, and T. R. Fritsche.
Paris, France
Abstract From 2002 to 2006, 35 of 73 multidrug-resistant Pseudomonas aeruginosa isolates from different wards at Charles Nicolle hospital of Tunis were positive for class B carbapenemase (using the
Transparency Declaration
imipenem–EDTA test), owing to a blaVIM-2 gene cassette in a class 1 integron. Twenty-three isolates additionally produced the
The authors have no conflict of interest to declare.
extended-spectrum b-lactamase SHV2a. DNA sequences imme-
References
Klebsiella pneumoniae plasmid sequence. Despite belonging to the
diately surrounding blaSHV2a shared extensive identity with a same chromosomal type, as shown by pulsed-field gel electrophoresis (PFGE), the VIM-2 producing P. aeruginosa isolates 1. Walsh TR, Toleman MA, Poirel L et al. Metallo-b-lactamases: the quiet before the storm? Clin Microbiol Rev 2005; 18: 306–325. 2. Queenan AM, Bush K. Carbapenemases: the versatile b-lactamases. Clin Microbiol Rev 2007; 20: 440–458. 3. Koh TH, Wang GC, Sng LH. IMP-1 and a novel metallo-b-lactamase, VIM-6, in fluorescent pseudomonads isolated in Singapore. Antimicrob Agents Chemother 2004; 48: 2334–2336. 4. Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, seventh edition. M7-A7. Wayne, PA: CLSI, 2006. 5. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing, 18th informational supplement. M100S18. Wayne, PA: CLSI, 2008. 6. Mendes RE, Kiyota KA, Monteiro J et al. Rapid detection and identification of metallo-b-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis. J Clin Microbiol 2007; 45: 544–547. 7. Castanheira M, Toleman MA, Jones RN et al. Molecular characterization of a b-lactamase gene, blaGIM-1, encoding a new subclass of metallo-b-lactamase. Antimicrob Agents Chemother 2004; 48: 4654–4661. 8. Tenover FC, Arbeit RD, Goering RV et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995; 33: 2233– 2239. 9. Sambrook J, MacCallum P, Russell D. Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2001.
Diversity in VIM-2-encoding class 1 integrons and occasional blaSHV2a carriage in isolates of a persistent, multidrugresistant Pseudomonas aeruginosa clone from Tunis S. Hammami1, V. Gautier2, R. Ghozzi1, A. Da Costa2, S. Ben-Redjeb1 and G. Arlet2,3 1) Laboratoire de Recherche ‘Re´sistance aux Antimicrobiens’, LR99ES09, De´partement de Microbiologie, Faculte´ de Me´decine de Tunis, Tunis,
prevalent at Charles Nicolle hospital displayed a diversity of VIM-2-carrying integrons.
Keywords: Carbapenem resistance, Pseudomonas aeruginosa, SHV-2a extended-spectrum b-lactamase, VIM-2 metallo-blactamase Original Submission: 17 March 2009; Revised Submission: 29 July 2009; Accepted: 30 July 2009 Editor: P. Tassios Article published online: 17 August 2009 Clin Microbiol Infect 2010; 16: 189–193
10.1111/j.1469-0691.2009.03023.x
Corresponding author and reprint requests: G. Arlet, Service de Bacte´riologie, Hoˆpital Tenon, 4 rue de la Chine, 75970 Paris Cedex 20, France E-mail: [email protected]
Carbapenems are among the drugs of choice for the treatment of infections due to multidrug-resistant (MDR) Pseudomonas aeruginosa. However, their efficacy is being increasingly compromised by the emergence of P. aeruginosa strains producing metallo-b-lactamases (MBLs) [1], which are implicated in large outbreaks, as described in Greece, Italy, Canada, Korea and Kenya [2–6]. We report the first molecular characterization of Tunisian MBL-producing P. aeruginosa isolates, some of which were also extended-spectrum b-lactamase producers.
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 179–199
Clinical Microbiology and Infection, Volume 16 Number 2, February 2010
Transparency Declaration The authors declare that there was no source of funding and that they have no conflicts of interest.
CMI
Dissemination and genetic context analysis of blaVIM-6 among Pseudomonas aeruginosa isolates in Asian-Pacific Nations M. Castanheira1, J. M. Bell2, J. D. Turnidge2, R. E. Mendes1 and R. N. Jones1
References
1) JMI Laboratories, North Liberty, IA, USA and 2) Women’s and Children’s Hospital, Adelaide, SA, Australia
1. Weber DJ, Raasch R, Rutala WA. Nosocomial infections in the ICU: the growing importance of antibiotic-resistant pathogens. Chest 1999; 115: 34S–41S. 2. Zhanel GG, DeCorby M, Laing N et al. Antimicrobial-resistant pathogens in intensive care units in Canada: results of the Canadian National Intensive Care Unit (CAN-ICU) study, 2005–2006. Antimicrob Agents Chemother 2008; 52: 1430–1437. 3. Al Naiemi N, Heddema ER, Bart A et al. Emergence of multidrugresistant Gram-negative bacteria during selective decontamination of the digestive tract on an intensive care unit. J Antimicrob Chemother 2006; 58: 853–856. 4. Antoniadou A, Kontopidou F, Poulakou G et al. Colistin-resistant isolates of Klebsiella pneumoniae emerging in intensive care unit patients: first report of a multiclonal cluster. J Antimicrob Chemother 2007; 59: 786–790. 5. Martins IS, Pessoa-Silva CL, Nouer SA et al. Endemic extended-spectrum beta-lactamase-producing Klebsiella pneumoniae at an intensive care unit: risk factors for colonization and infection. Microb Drug Resist 2006; 12: 50–58. 6. Falagas M, Bliziotis I, Kasiakou S et al. Outcome of infections due to pandrug-resistant (PDR) Gram-negative bacteria. BMC Infect Dis 2005; 5: 24. 7. Michalopoulos A, Fotakis D, Virtzili S et al. Aerosolized colistin as adjunctive treatment of ventilator-associated pneumonia due to multidrug-resistant Gram-negative bacteria: a prospective study. Respir Med 2008; 102: 407–412. 8. Matthaiou DK, Michalopoulos A, Rafailidis PI et al. Risk factors associated with the isolation of colistin-resistant gram-negative bacteria: a matched case–control study. Crit Care Med 2008; 36: 807– 811. 9. Markogiannakis H, Pachylaki N, Samara E et al.. Infections in a surgical intensive care unit of an university hospital in Greece. Int J Infect Dis 2009; 13: 145–153. 10. Tharavichitkul P, Khantawa B, Bousoung V, Boonchoo M. Activity of fosfomycin against extended-spectrum-b-lactamase-producing Klebsiella pneumoniae and Escherichia coli in Maharaj Nakorn Chiang Mai Hospital. J Infect Dis Antimicrob Agents 2005; 22: 121– 126. 11. Falagas ME, Kanellopoulou MD, Karageorgopoulos DE et al. Antimicrobial susceptibility of multi-drug resistant Gram-negative bacteria to fosfomycin. Eur J Clin Microbiol Infect Dis 2008; 27: 439–443. 12. Hardisson C, Villar CJ, Llaneza J, Mendoza MC. Prevalence and dispersion of plasmids conferring fosfomycin resistance in enterobacteria. Pathol Biol (Paris) 1984; 32: 755–758. 13. Bedirdjian JP, Morin JP, Fouchet B, Fillastre JP. Effect of fosfomycin on respiration by rat kidney mitochondria. Drugs Exp Clin Res 1978; 4: 57–62. 14. de Cueto M, Lo´pez L, Herna´ndez JR, Morillo C, Pascual A. In vitro activity of fosfomycin against extended-spectrum-beta-lactamaseproducing Escherichia coli and Klebsiella pneumoniae: comparison of susceptibility testing procedures. Antimicrob Agents Chemother 2006; 50: 368–370.
Abstract VIM-6, previously reported in two strains from Singapore recovered in 2000, was detected in 16 isolates collected in 2006 in India (12 isolates), Indonesia (two), Korea and the Philippines (one each). High genetic variability was observed among VIM-6producing isolates (12 ribotypes and 11 pulsed-field gel electrophoresis types), but clones were observed in India and Indonesia; blaVIM-6-carrying integrons of 3.9 kb and 5 kb were detected, and two of five Indian hospitals yielded isolates with both integrons. These two integrons, blaVIM-6 was located in the first position, followed by blaOXA-10 and aacA4. The 5-kb integrons also harboured aadA1 and a 331-bp open reading frame encoding a putative efflux pump.
Keywords: Asia-Pacific, metallo-b-lactamases, Pseudomonas aeruginosa, SENTRY, VIM Original Submission: 23 October 2008; Revised Submission: 4 February 2009; Accepted: 11 February 2009 Editor: D. Mack Article published online: 10 August 2009
Clin Microbiol Infect 2010; 16: 186–189
10.1111/j.1469-0691.2009.02903.x
Corresponding author and reprint requests: M. Castanheira, JMI Laboratories, 345 Beaver Kreek Center, Suite A, North Liberty Iowa 52317, IA, USA E-mail: [email protected]
Acquired metallo-b-lactamase (MBL)-producing isolates have been increasingly reported in the Asia-Pacific (APAC) region.
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 179–199
Research Notes
CMI
Several MBL types were found in Korea (IMP-1, IMP-8, VIM2, and SIM-1), China (IMP-4, IMP-9, and VIM-2) and Taiwan (IMP-8, VIM-2, and VIM-3), among other countries from that region [1,2]. A new VIM-type enzyme was identified in two Pseudomonas putida isolates recovered in Singapore in 2000 [3]. This enzyme, named VIM-6, showed only two amino acid differences from VIM-2, which is probably its genetic ancestor. VIM-6 has not been reported in any other geographical location, and its description was succinct, focusing on the clinical findings. In this study, we report 16 blaVIM-6-carrying Pseudomonas aeruginosa isolates from four countries in the APAC region, and their genetic context. A total of 719 P. aeruginosa isolates were consecutively collected from bloodstream, respiratory tracts and skin and skin structure infections in 57 hospitals located in the APAC region during the SENTRY Antimicrobial Surveillance Program (2006). Isolates were susceptibility-tested by CLSI broth microdilution [4,5]. Among those strains, 196 (27.3%) were non-susceptible (MIC of ‡8 mg/L) to imipenem and/or meropenem and were tested using generic primers designed to align multiple variants of IMP, VIM and other MBL genes in a multiplex PCR approach [6]. All amplicons were sequenced on both strands and analysed. The blaVIM-6 gene was detected in 16 (8.1%) isolates recovered in India (12 isolates), Indonesia (two), Korea (one), and the Philippines (one; Table 1). All isolates were resistant to three or more classes of antipseudomonal agents. Two isolates from India were susceptible to fluoroquinolones (ciprofloxacin MIC of 0.12 and 0.5 mg/L), whereas polymyxin B and colistin remained active against all strains (MIC of 0.5–1 mg/L).
187
Primers annealing in the two conserved regions (CS) of class 1 integrons [7] were used in combination with MBL primers to determine the size of the integron structure. The blaVIM-6 gene was carried as a gene cassette in the first position of 3.9-kb and 5-kb class 1 integrons (Table 1). Amplicons generated with blaVIM primers anchored in the 3¢CS were digested with DraI and XbaI in separate reactions. Integrons of the same size generated identical restriction fragment length polymorphism patterns, indicating a similar content and arrangement. Sequencing showed that the 3.9kb integrons carried blaVIM-6, blaOXA-10 and aacA4 followed by qacED1/sul1 in the 3¢CS (Fig. 1). The 5-kb integrons possessed the same gene content but two additional gene cassettes: a 331-bp open reading frame (ORF) and aadA1 upstream of the 3¢CS (Fig. 1). The 5-kb integrons found in this study appear to be identical to the blaVIM-6 integron carried by the isolate found in Singapore, which has been deposited in GenBank (no. EF522838) but not yet described. In two isolates, the 3¢CS could not be amplified with several different primers annealing in different structures found in the 3¢-end of class 1 integrons. However, primers annealing in aacA4 and aadA1 showed that these genetic structures were similar to the other blaVIM-6-carrying integrons described here (Table 1). The putative protein encoded by the 331-bp ORF carried in the 5-kb integrons showed 78% similarity with the efflux pump QACED1, which provides resistance to quaternary ammonium compounds. The orf was cloned into pPCRScriptCam SK+ (Stratagene Cloning Systems, La Jolla, CA, USA), and recombinant strains were tested by MIC methods [4] for several clinically available antimicrobial agents and other
TABLE 1. Characteristics of 16 blaVIM-6-carrying Pseudomonas aeruginosa isolates obtained in 2006 in the Asia-Pacific (APAC) region Isolate
Country
Hospital
Source
Ribotypea
PFGE pattern
Integron size
225-22-D 228-23-D 241-09-D 241-73.2-D 244-07-D 244-10-D 244-18-C 244-24-D 244-29-D 247-03-D 247-06-D 248-11-A 248-26-C 248-77-C 252-05-C 252-09-C
Korea Philippines Indonesia Indonesia India India India India India India India India India India India India
A B C C D D D D D E E F F F G G
Abscess Skin and soft tissue Wound Abscess Skin and soft tissue Wound Tracheal aspirate Tissue Unknown Wound Wound Blood culture Unknown Unknown Sputum Endotracheal tube
1034-2 35-7 566-6 566-6 566-2 347-6 347-8 352-2 352-2 348-6 348-6 45-6 45-6 114-4 147-6 134-4
A B C C1 D E E1 E E F F G H I J K
5 kb-likeb 3.9 kb-likeb 5 kb 5 kb 3.9 kb 5 kb 5 kb 5 kb 5 kb 5 kb 5 kb 3.9 kb 3.9 kb 5 kb 3.9 kb 3.9 kb
PFGE, pulsed-field gel electrophoresis. a Isolates were considered to be grouped in the same ribotype if the similarity coefficient was ‡0.93. Ribotypes were named according to the batch (first three digits) and sample position (last single digit) of the first isolate identified with that pattern. b The same gene cassette arrangement was observed, but the 3¢CS could not be amplified.
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 179–199
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Clinical Microbiology and Infection, Volume 16 Number 2, February 2010
attI1
CMI
59’-be 3´CS
5´CS
intI1
bla VIM-6
bla OXA-10
aacA4
qacE 1/sul1
FIG. 1. Schematic representation of blaVIM-6carrying integrons detected in carbapenemresistant Pseudomonas aeruginosa isolates from the Asia-Pacific region. The 5-kb integrons possessed a 331-bp open reading frame (ORF) and aadA1 that were not present in 3.9-kb
331 bp ORF
antibacterial compounds, such as surfactants and detergents (Brij58, deoxicolate Tween-80, chlorhexidine, benzalconium, sodium dodecylsulphate and sarkosyl), dyes (ethidium bromide and crystal violet), and heavy metals (cadmium, lithium, nickel and chromium). The results showed no consistent differences in the MIC values for the Escherichia coli strains harbouring the plasmid containing the 331-bp ORF or the plasmid without the insert. The expression of orf was confirmed in the clinical isolate and in the recombinant colonies by RT-PCR (OneStep RT-PCR kit; Qiagen, Hilden, Germany) in RNA preparations (RNeasy Mini kit; Qiagen) previously treated with RNase-free DNase (Promega, Madison, WI, USA). Although this gene did not confer a phenotype of resistance to the compounds tested, this putative efflux pump could still play a role in the selection of strains harbouring these mobile elements. MBL-producing isolates were ribotyped using PvuII in the Riboprinter Microbial Characterization System (Qualicon, Wilmington, DE, USA). Isolates were also evaluated using pulsed-field gel electrophoresis (PFGE), as previously described [8]. Isolates carrying blaVIM-6 were clustered in 12 ribotypes and 11 PFGE patterns (Table 1). Three clones of two isolates each were identified in India by ribotyping, but PFGE showed that isolates from one suspected clone were not related. Whereas ribotyping identified a clone with two isolates in one Indian hospital, PFGE demonstrated that four isolates from the same hospital were genetically related (clone E/E1). The two other clonal strains from India and the two isolates from Indonesia were found to be genetically related by both epidemiological methods. The other five strains, including the strains found in Korea and the Philippines, showed unique ribotypes and PFGE patterns. Six isolates harboured the 3.9-kb blaVIM-6-carrying integrons, and all but one isolate were found in three Indian medical sites (Table 1). The remaining organism carrying a 3.9-kb integron was recovered in the Philippines, and this integron lacked the conventional 3¢CS of class 1 integrons. All isolates were geneti-
aadA1
structures.
cally distinct according to PFGE analysis; nonetheless, two isolates from one hospital were clustered in the same ribotype, showing genetic relatedness. Less genetic variability was noted in the isolates carrying 5-kb integrons (three clones). Plasmid DNA from the VIM-6-producing strains was extracted using the Plasmid DNA Midi Kit (Qiagen) and resolved on agarose gels. E. coli NTCC 50192 was used as a molecular weight marker. Transfer of plasmid DNA to a nylon membrane was performed by Southern blot as described previously [9]. The blaVIM-6 probe generated by PCR was labelled and membranes were hybridized using the non-radioactive DIG high Prime DNA labelling and detection kit (Roche Diagnostics GmbH, Mannheim, Germany). All isolates showed at least one plasmid band, and the blaVIM-6 probe hybridization demonstrated that this MBL gene was carried by plasmids of approximately 35 kb in all isolates, including isolates carrying different blaVIM-6 integrons. This suggests that these plasmids continually undergo genetic events, such as rearrangements, incisions, and excisions. Our results show that a genetic structure carrying blaVIM-6 is probably responsible for the dissemination of this MBL gene in the four APAC countries; however, we also observed clonal spread of VIM-6-producing strains in specific hospitals. The blaVIM-6 gene was initially reported from two P. putida clinical isolates recovered in Singapore, but the context of this gene has not been described [3]. In this study, this MBL gene was found in four more countries of the APAC region, including Indonesia and the Philippines, where MBLs have not previously been reported. The spread and high genetic diversity observed among blaVIM-6-harbouring isolates can be attributed to a promiscuous mobile structure that carries this MBL gene.
Nucleotide Sequence Accession Number The nucleotide sequence of the 3.9-kb integron carrying blaVIM-6 has been submitted to GenBank and assigned accession number FM994936.
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 179–199
Research Notes
CMI
189
Tunisia, 2) Laboratoire de Bacte´riologie, UPRES EA 2392, Faculte´ de Me´d-
Acknowledgements
ecine Pierre et Marie Curie, Universite´ Paris VI and 3) Service de Bacte´riologie-Hygie`ne, Hoˆpital Tenon, Assistance Publique-Hoˆpitaux de Paris,
This study was partially presented at the 47th Interscience Conference of Antimicrobial Agents and Chemotherapy (ICAAC), 17–20 September 2007, Chicago, IL, USA. The authors thank the following individuals for assistance in testing and/or manuscript preparation: L. M. Deshpande, H. S. Sader, and T. R. Fritsche.
Paris, France
Abstract From 2002 to 2006, 35 of 73 multidrug-resistant Pseudomonas aeruginosa isolates from different wards at Charles Nicolle hospital of Tunis were positive for class B carbapenemase (using the
Transparency Declaration
imipenem–EDTA test), owing to a blaVIM-2 gene cassette in a class 1 integron. Twenty-three isolates additionally produced the
The authors have no conflict of interest to declare.
extended-spectrum b-lactamase SHV2a. DNA sequences imme-
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Diversity in VIM-2-encoding class 1 integrons and occasional blaSHV2a carriage in isolates of a persistent, multidrugresistant Pseudomonas aeruginosa clone from Tunis S. Hammami1, V. Gautier2, R. Ghozzi1, A. Da Costa2, S. Ben-Redjeb1 and G. Arlet2,3 1) Laboratoire de Recherche ‘Re´sistance aux Antimicrobiens’, LR99ES09, De´partement de Microbiologie, Faculte´ de Me´decine de Tunis, Tunis,
prevalent at Charles Nicolle hospital displayed a diversity of VIM-2-carrying integrons.
Keywords: Carbapenem resistance, Pseudomonas aeruginosa, SHV-2a extended-spectrum b-lactamase, VIM-2 metallo-blactamase Original Submission: 17 March 2009; Revised Submission: 29 July 2009; Accepted: 30 July 2009 Editor: P. Tassios Article published online: 17 August 2009 Clin Microbiol Infect 2010; 16: 189–193
10.1111/j.1469-0691.2009.03023.x
Corresponding author and reprint requests: G. Arlet, Service de Bacte´riologie, Hoˆpital Tenon, 4 rue de la Chine, 75970 Paris Cedex 20, France E-mail: [email protected]
Carbapenems are among the drugs of choice for the treatment of infections due to multidrug-resistant (MDR) Pseudomonas aeruginosa. However, their efficacy is being increasingly compromised by the emergence of P. aeruginosa strains producing metallo-b-lactamases (MBLs) [1], which are implicated in large outbreaks, as described in Greece, Italy, Canada, Korea and Kenya [2–6]. We report the first molecular characterization of Tunisian MBL-producing P. aeruginosa isolates, some of which were also extended-spectrum b-lactamase producers.
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 179–199