Skip Navigation


JAC Advance Access originally published online on May 2, 2006
Journal of Antimicrobial Chemotherapy 2006 58(1):178-182; doi:10.1093/jac/dkl178
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
58/1/178    most recent
dkl178v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (13)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Naas, T.
Right arrow Articles by Nordmann, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Naas, T.
Right arrow Articles by Nordmann, P.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Emergence of PER and VEB extended-spectrum ß-lactamases in Acinetobacter baumannii in Belgium

Thierry Naas1,*, Pierre Bogaerts2, Caroline Bauraing2, Yves Degheldre2, Youri Glupczynski2 and Patrice Nordmann1

1 Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris XI 78 rue du Général Leclerc, 94275 K.-Bicêtre, France 2 Laboratoire de Bactériologie, Cliniques Universitaires UCL de Mont-Godinne Belgium

*Corresponding author. Tel: +33-1-45-21-29-86; Fax: +33-1-45-21-63-40; E-mail: thierry.naas{at}bct.ap-hop-paris.fr

Received 27 February 2006; returned 3 April 2006; revised 7 April 2006; accepted 10 April 2006


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 Transparency declarations
 References
 
Objectives: Strains of Acinetobacter baumannii producing the extended-spectrum ß-lactamase (ESBL) PER-1 are widespread in Turkey and have also been reported from Korea and France. In contrast, A. baumannii producing the ESBL VEB-1 have only been reported from France, where one strain was responsible for a nationwide outbreak in 2003–2004. Here we describe the emergence of strains of A. baumannii producing VEB-1 and PER- 1 in Belgium.

Methods: Belgian hospitals were alerted in December 2003 to the emergence in France of VEB-1-producing A. baumannii susceptible only to meropenem and colistin. Isolates with a compatible susceptibility profile were sent to a single central laboratory for VEB-1 confirmation, molecular characterization and typing.

Results: From December 2003 to March 2005, three hospitals located close to the French border and one in the Brussels area reported isolation of eight A. baumannii isolates compatible with the French epidemic clone. Using PCR, six were identified as VEB-1-positive and two as PER-1-positive. All the VEB-1-positive isolates were clonally related by PFGE and by integron analysis to the French epidemic strain. The PER-1-positive strains were indistinguishable by PFGE but not related to the known French isolate or to several Turkish isolates. Both genes were chromosomally encoded.

Conclusions: This work illustrates the inter-country spread of VEB-1-producing A. baumannii isolates as well as the emergence of PER-1-producing A. baumannii strains in Belgium.

Keywords: ESBLs , inter-country spread , outbreak , multidrug resistance


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 Transparency declarations
 References
 
Over the past decade, nosocomial outbreaks of Acinetobacter baumannii have been described with increasing frequency, mostly in intensive care units (ICUs), burn units or surgical wards.1 A. baumannii is not the most common antimicrobial resistant pathogen in hospitalized patients; however, increasing therapeutic difficulties due to the emergence of highly multiresistant strains have become a serious clinical concern.1,2

The main mechanisms of resistance to broad-spectrum ß-lactams in A. baumannii are overexpression of chromosomal cephalosporinases3 and production of plasmid-encoded Ambler class A, B and D ß-lactamases.2,4–6 Strains of A. baumannii producing the extended-spectrum ß-lactamase (ESBL) PER-1 have been reported from Turkey, Korea and France,5–7 while strains producing VEB-1 and CTX-M-2 have been reported from France8,9 and Korea,4 respectively.

The blaVEB-1 is an integron-located ESBL gene initially detected in Enterobacteriaceae and in Pseudomonas aeruginosa strains from south-east Asia.10,11 Subsequently, it was described in clonally related A. baumannii isolates recovered during an outbreak that lasted over a 9 month period (August 2001–April 2002) in the ICU of the Valenciennes Hospital in the northern part of France.8,9 In these strains, the blaVEB-1 gene was chromosomal and integron-located.8,9 The ESBL PER-1 was initially detected in 1993 in a P. aeruginosa isolate from a Turkish patient in France,12 and it has also been detected in P. aeruginosa isolates from ICU patients in Belgium13 and in Italy.14 The blaPER-1 gene is widespread in Acinetobacter spp., P. aeruginosa and Salmonella enterica serovar Typhimurium in Turkey.5,6,15

In this article we report the first characterization of these ESBL-producing A. baumannii isolates in Belgian hospitals, further supporting the global spread of such strains.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 Transparency declarations
 References
 
Bacterial strains, antimicrobial agents and MIC determinations

All A. baumannii isolates were recovered from routine clinical cultures or from colonization samples, identified by hospital laboratories and then sent for confirmatory testing to a central laboratory (Cliniques Universitaires UCL de Mont-Godinne, University of Louvain, Belgium).

Identification was confirmed using the API 32GN system (BioMérieux, Marcy-l'Étoile, France) and by the ability to grow at 44°C in Trypticase soy broth.9 Electro-competent E. coli DH10B (Invitrogen, Eragny, France) was used as a recipient in electroporation experiments and E. coli strain J53AzR, which is resistant to sodium azide, and rifampicin-resistant A. baumannii CIP7010 (Pasteur Institut, Paris, France) were used for the conjugation experiments. A. baumannii AMA-1 from France5 and A. baumannii IST-1 and A. baumannii IST-2 from Turkey6 were used as PER-1-producing A. baumannii isolates and A. baumannii VAL-1 from France was used as a VEB-1-producing A. baumannii isolate.9

Routine antibiograms were determined by the disc diffusion method on Mueller–Hinton (MH) agar (Bio-Rad, Marnes-La-Coquette, France) and the susceptibility breakpoints were as recommended by the French Society for Microbiology guidelines (SFM; http://www.sfm.asso.fr/nouv/general.php?pa=2). The presence of an ESBL was suggested by a synergy image using the double-disc synergy test performed with cefepime, ceftazidime and ticarcillin/clavulanic acid discs on cloxacillin (200 mg/L)-containing MH agar plates, which partially inhibits the cephalosporinase activity.8,9

IEF and PFGE analyses

Analytical isoelectric focusing (IEF) was performed with an ampholine polyacrylamide gel, as described previously.5 PFGE was performed as previously described9 and ApaI-macrorestriction patterns were interpreted according to Tenover et al.16

PCR analyses and sequencing

Genomic DNA extraction and PCR experiments were performed as described previously.8,9 The PCR amplification and the primers used to search for known ß-lactamase genes (blaTEM, blaSHV, blaPER-1/2, blaVEB-1, blaGES-1 and blaCTX-M) in A. baumannii isolates have been described previously.8,9

For PCR-mapping experiments of the genetic environment of blaVEB-1 and blaPER-1, 500 ng of total DNA of A. baumannii isolates was used in standard PCR mixtures.9 The PCR primers are listed in Table 1 and Figure 1 (a and b). The following amplification program was used: 10 min at 94°C; 35 cycles of 1 min at 94°C, 1 min at 55°C and 3–5 min at 72°C; followed by a final extension of 10 min at 72°C.


View this table:
[in this window]
[in a new window]
  		Table 1. Primers used for PCR mapping of the genetic environment of blaVEB-1 and blaPER-1 genes

 

Figure 1
View larger version (22K):
[in this window]
[in a new window]
  		Figure 1. Molecular characterization of ESBL-producing A. baumannii strains. (a) Schematic representation of the genetic environment of (a) blaVEB-1 and (b) blaPER-1 genes found in the Belgian A. baumannii isolates.5,8 The coding regions are represented as boxes, the arrows indicate their translational orientation and the gene names are indicated above the boxes. The thick bold horizontal lines represent the amplified fragments, and the estimated sizes of the different PCR products are indicated between divergent arrows. Triangles at the end of coding regions indicate the inverted repeats of insertion sequences. Small arrows with a number represent the primers used and are listed in Table 1. (c) PFGE patterns of VEB-1- and PER-1-expressing A. baumannii isolates. The assigned numbers of A. baumannii isolates are shown over the lanes of the gel and correspond to those of Table 1. The positions of molecular size markers in kb (M) are shown on the right-hand side of the gel. Lanes 1–3 and lanes 4–6 correspond to VEB-expressing A. baumannii isolates from Tournai and Chimay, respectively, and lanes 8 and 9 correspond to PER-1-expressing A. baumannii isolates from Brussels. Lane 7, corresponds to A. baumannii VAL-1 epidemic clone from Northern France,9 and lanes 10, 11 and 12 correspond to A. baumannii AMA-1, A. baumannii IST-1 and A. baumannii IST-2, respectively.5,6

 
The blaVEB-1 and blaPER-1 amplicons were purified using a Qiaquick PCR purification kit (Qiagen) and subsequently sequenced on both strands on an Applied Biosystem sequencer (ABI 3100). Nucleotide sequence analysis was performed at the National Center of Biotechnology Information website (http://www.ncbi.nlm.nih.gov).


   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
Transparency declarations
 References
 
Belgian hospitals were alerted in December 2003 of the emergence of VEB-1-producing A. baumannii, mostly in the northern part of France. Recommendations for surveillance and control were sent to all hospitals through the National Scientific Institute of Public Health. All hospital-based laboratories were requested to send clinical A. baumannii isolates with a compatible susceptibility profile, being susceptible to meropenem and to colistin only, to a reference laboratory (Cliniques Universitaires UCL de Mont-Godinne, Belgium) for confirmatory testing. From December 2003 to March 2005, three hospitals located close to the French border, two in Tournai and one in the Chimay area, and one in the Brussels area reported eight ESBL-producing A. baumannii strains with a compatible phenotype.

The antibiogram of the eight ESBL-producing A. baumannii isolates was very similar and a synergy image, signature of the presence of an ESBL, could not be observed with most isolates between clavulanic acid and cefepime or ceftazidime discs on a routine antibiogram. In contrast, the use of cloxacillin-containing plates that inhibit cephalosporinase activity allowed detection of this synergy image in all the eight strains.9

blaVEB-1-specific primers gave PCR products of expected size for A. baumannii isolates 1–3 from Tournai and isolates 4–6 from Chimay (Figure 1c) while A. baumannii isolates 7 and 8 from Brussels were positive for the blaPER-1 gene (Figure 1c). Direct sequencing of the amplicons revealed 100% identity with the blaVEB-1 gene of A. baumannii AYE8 and with the blaPER-1 gene of P. aeruginosa PER0.12 Negative PCR results were obtained using blaTEM, blaSHV, blaGES-1 and blaCTX-M. IEF results showed the presence of three distinct pI values (6.3 for OXA-10, 7.4 for VEB-1 and >8.5 for AmpC)8 in A. baumannii isolates 1–6 and two distinct pI values (5.3 for PER-1 and >8.5 for AmpC)5 in A. baumannii isolates 7 and 8.

Since the blaVEB-1 gene is integron-borne in A. baumannii AYE, class 1 integron specific-, blaVEB-1 intragenic- and cassette specific-primers were used to amplify the blaVEB-1-containing integron of Belgian A. baumannii VEB-1 isolates and to compare their size with that of A. baumannii AYE (Figure 1a).8 The amplimers were similar in size suggesting that the genetic environment of blaVEB-1 genes was identical to that previously published for A. baumannii AYE.8 Similarly, the genetic environment of the blaPER-1 gene was compared with that recently characterized by Poirel et al.15 The blaPER-1 gene in A. baumannii isolates 7 and 8 was also located on a composite transposon, Tn1213, bracketed by two insertion sequences, ISPa12 and ISPa13 (Figure 1b), that is itself inserted into another insertion sequence, ISPa14.15 Attempts to demonstrate plasmids or transfer genes encoding PER-1 or VEB-1 were unsuccessful (data not shown), suggesting that the genes were chromosomally encoded.

The VEB-1-positive isolates were clonally related according to PFGE results and to integron analysis to the French epidemic strain (Figure 1a and c). Investigations demonstrated that frequent patient transfers between French hospitals and long-term care facilities located in Belgium close to the French border might be responsible for the inter-country spread of the strains. As observed during the French national outbreak several subclones of A. baumannii were circulating (T. Naas, personal communication). Finding of small differences (one band) between the A. baumannii isolates from Chimay and Tournai (Belgium) suggested that at least two different events led to A. baumannii VEB-1 introduction into the Belgian hospitals (Figure 1c).

The two PER-1-positive strains were indistinguishable by PFGE but not related to the French or to the Turkish isolates (Figure 1c). In contrast to A. baumannii VEB-1 isolates that are clonally related, a genetic heterogeneity was identified among the A. baumannii PER-1 isolates, even though the immediate genetic environment was similar. Interestingly, patient 9, a 59-year-old Belgian patient, suffered a traffic injury while on holiday in Turkey and had to be repatriated to a hospital in Brussels. It is thus very likely that this isolate was imported from Turkey and that it was eventually transmitted to patient 8 who stayed in the same ICU.

This work illustrates the European inter-country spread of VEB-1- and possibly also of PER-1-producing A. baumannii strains in Belgium. It further underlines the usefulness of cloxacillin-containing plates for detection of ESBL-producing A. baumannii strains and of detailed molecular analysis of antibiotic resistance mechanisms to avoid confusion between strains displaying similar resistance phenotypes. The emergence and spread of these ESBL-producing A. baumannii strains is worrisome, since it will enhance extensive use of carbapenems, thus increasing the risk of emergence of carbapenem-resistant isolates. Thus, this work emphasizes the need for hospitals to strictly adhere to existing recommendations for the prevention and control of multidrug-resistant bacteria.

Although most of the hospitals of Northern France were large reservoirs of VEB-1-positive A. baumannii strains, this was not the case for Belgian hospitals that were in the near vicinity. Belgian public health authorities were informed about the French national outbreak through the French national health authorities ‘Institut de Veille Sanitaire’, thus allowing early detection and control of a cluster in Belgian hospitals close to the French border.


   Transparency declarations
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Transparency declarations
References
 
None to declare.


   Acknowledgements
 
We are very grateful to B. Coignard from the French National Health Institute and to B. Jans and C. Suetens from the Belgian Scientific Institute of Public Health for their assistance. We are also indebted to J.-F. Marchal, I. Mansoor and M.-F. Parmentier for sending the strains and for reporting the cases included in the study. This work was funded by a grant from the Ministère de l'Education Nationale et de la Recherche (UPRES-EA3539), Université Paris XI, by the Assistance Publique-Hôpitaux de Paris, France, and by the European Community (6th PCRD, LSHMCT- 2003-503-335).


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Transparency declarations
 References
 
1 Bergogne-Bérézin E and Towner KJ. (1996) Acinetobacter spp. as nosocomial pathogens: microbiological, clinical and epidemiological features. Clin Microbiol Rev 9:148–65.[Web of Science][Medline]

2 Nordmann P and Poirel L. (2002) Emerging carbapenemases in Gram-negative aerobes. Clin Microbiol Infect 8:321–31.[CrossRef][Web of Science][Medline]

3 Corvec S, Caroff N, Espaze E, et al. (2003) AmpC cephalosporinase hyperproduction in Acinetobacter baumannii clinical strains. J Antimicrob Chemother 52:629–35.[Abstract/Free Full Text]

4 Nagano N, Nagano Y, Cordevant C, et al. (2004) Nosocomial transmission of CTX-M-2 ß-lactamase-producing Acinetobacter baumannii in a neurosurgery ward. J Clin Microbiol 42:3978–84.[Abstract/Free Full Text]

5 Poirel L, Karim A, Mercat A, et al. (1999) Extended-spectrum ß-lactamase-producing strain of Acinetobacter baumannii isolated from a patient in France. J Antimicrob Chemother 43:157–65.[Free Full Text]

6 Vahaboglu H, Öztürk R, Aygün G, et al. (1997) Widespread detection of PER-1-type extended-spectrum ß-lactamases among nosocomial Acinetobacter and Pseudomonas aeruginosa isolates in Turkey: a nationwide multicenter study. Antimicrob Agents Chemother 41:2265–9.[Abstract]

7 Yong D, Shin JH, Kim S, et al. (2003) High prevalence of PER-1 extended-spectrum ß-lactamase-producing Acinetobacter spp. in Korea. Antimicrob Agents Chemother 47:1749–51.[Abstract/Free Full Text]

8 Poirel L, Menuteau O, Agoli N, et al. (2003) Outbreak of extended-spectrum ß-lactamase VEB-1-producing isolates of Acinetobacter baumannii in a French hospital. J Clin Microbiol 41:3542–7.[Abstract/Free Full Text]

9 Carbonne A, Naas T, Blanckaert K, et al. (2005) Investigation of a nosocomial outbreak of extended-spectrum ß-lactamase VEB-1-producing isolates of Acinetobacter baumannii in a hospital setting. J Hosp Infect 60:14–8.[CrossRef][Medline]

10 Naas T, Poirel L, Karim A, et al. (1999) Molecular characterization of In50, a class 1 integron encoding the gene for the extended-spectrum ß-lactamase VEB-1 in Pseudomonas aeruginosa. FEMS Microbiol Lett 176:411–9.[Web of Science][Medline]

11 Naas T, Mikami Y, Imai T, et al. (2001) Characterization of In53, a class 1 plasmid- and composite transposon-located integron of Escherichia coli, which carries an unusual array of gene cassettes. J Bacteriol 183:235–49.[Abstract/Free Full Text]

12 Nordmann P, Ronco E, Naas T, et al. (1993) Characterization of a novel extended-spectrum ß-lactamase from Pseudomonas aeruginosa. Antimicrob Agents Chemother 37:962–9.[Abstract/Free Full Text]

13 Claeys G, Verschraegen G, de Baere T, et al. (2000) PER-1 ß-lactamase-producing Pseudomonas aeruginosa in an intensive care unit. J Antimicrob Chemother 45:924–5.[Free Full Text]

14 Pagani L, Mantengoli E, Migliavacca R, et al. (2004) Multifocal detection of multidrug-resistant Pseudomonas aeruginosa producing the PER-1 extended-spectrum ß-lactamase in Northern Italy. J Clin Microbiol 42:2523–9.[Abstract/Free Full Text]

15 Poirel L, Cabanne L, Vahaboglu H, et al. (2005) Genetic environment and expression of the extended-spectrum ß-lactamase blaPER-1 gene in gram-negative bacteria. Antimicrob Agents Chemother 49:1708–13.[Abstract/Free Full Text]

16 Tenover FC, Arbeit RD, Goering RV, et al. (1995) Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 33:2233–9.[Web of Science][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Antimicrob. Agents Chemother.Home page
Z. Zong, S. R. Partridge, and J. R. Iredell
A blaVEB-1 Variant, blaVEB-6, Associated with Repeated Elements in a Complex Genetic Structure
Antimicrob. Agents Chemother., April 1, 2009; 53(4): 1693 - 1697.
[Abstract] [Full Text] [PDF]

Home page
 J Med MicrobiolHome page
T. W. Boo, F. Walsh, and B. Crowley
Molecular characterization of carbapenem-resistant Acinetobacter species in an Irish university hospital: predominance of Acinetobacter genomic species 3
J. Med. Microbiol., February 1, 2009; 58(2): 209 - 216.
[Abstract] [Full Text] [PDF]

Home page
 J Antimicrob ChemotherHome page
D. M. Livermore, S. Mushtaq, M. Warner, C. Miossec, and N. Woodford
NXL104 combinations versus Enterobacteriaceae with CTX-M extended-spectrum {beta}-lactamases and carbapenemases
J. Antimicrob. Chemother., November 1, 2008; 62(5): 1053 - 1056.
[Abstract] [Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
Z. Zong, S. R. Partridge, L. Thomas, and J. R. Iredell
Dominance of blaCTX-M within an Australian Extended-Spectrum {beta}-Lactamase Gene Pool
Antimicrob. Agents Chemother., November 1, 2008; 52(11): 4198 - 4202.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Microbiol. Rev.Home page
A. Y. Peleg, H. Seifert, and D. L. Paterson
Acinetobacter baumannii: Emergence of a Successful Pathogen
Clin. Microbiol. Rev., July 1, 2008; 21(3): 538 - 582.
[Abstract] [Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
Z. Zong, S. R. Partridge, and J. R. Iredell
RmtC 16S rRNA Methyltransferase in Australia
Antimicrob. Agents Chemother., February 1, 2008; 52(2): 794 - 795.
[Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
F. Perez, A. M. Hujer, K. M. Hujer, B. K. Decker, P. N. Rather, and R. A. Bonomo
Global Challenge of Multidrug-Resistant Acinetobacter baumannii
Antimicrob. Agents Chemother., October 1, 2007; 51(10): 3471 - 3484.
[Full Text] [PDF]

Home page
 J. Clin. Microbiol.Home page
J. Empel, K. Filczak, A. Mrowka, W. Hryniewicz, D. M. Livermore, and M. Gniadkowski
Outbreak of Pseudomonas aeruginosa Infections with PER-1 Extended-Spectrum {beta}-Lactamase in Warsaw, Poland: Further Evidence for an International Clonal Complex
J. Clin. Microbiol., September 1, 2007; 45(9): 2829 - 2834.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Microbiol.Home page
J. M. Coelho, J. F. Turton, M. E. Kaufmann, J. Glover, N. Woodford, M. Warner, M.-F. Palepou, R. Pike, T. L. Pitt, B. C. Patel, et al.
Occurrence of Carbapenem-Resistant Acinetobacter baumannii Clones at Multiple Hospitals in London and Southeast England.
J. Clin. Microbiol., October 1, 2006; 44(10): 3623 - 3627.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
58/1/178    most recent
dkl178v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (13)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Naas, T.
Right arrow Articles by Nordmann, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Naas, T.
Right arrow Articles by Nordmann, P.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?