Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Turton, J. F. Articles by Pitt, T. L. Search for Related Content PubMed PubMed Citation Articles by Turton, J. F. Articles by Pitt, T. L.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, August 2006, p. 2974-2976, Vol. 44, No. 8
0095-1137/06/$08.00+0     doi:10.1128/JCM.01021-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification of Acinetobacter baumannii by Detection of the bla_OXA-51-like Carbapenemase Gene Intrinsic to This Species

Jane F. Turton,^1* Neil Woodford,² Judith Glover,¹ Susannah Yarde,¹ Mary E. Kaufmann,¹ and Tyrone L. Pitt¹

Laboratory of HealthCare Associated Infection,¹ Antibiotic Resistance Monitoring and Reference Laboratory, Centre for Infections, Health Protection Agency, London NW9 5EQ, United Kingdom²

Received 16 May 2006/ Returned for modification 6 June 2006/ Accepted 8 June 2006

Top Abstract Text References

 
bla_OXA-51-like was sought in clinical isolates of Acinetobacter species in a multiplex PCR, which also detects bla_OXA-23-like and class 1 integrase genes. All isolates that gave a band for bla_OXA-51-like identified as A. baumannii. This gene was detected in each of 141 isolates of A. baumannii but not in those of 22 other Acinetobacter species.

Top Abstract Text References

 
Acinetobacter baumannii is an increasingly important nosocomial pathogen that particularly affects critically ill patients. It is associated with multiple antibiotic resistance, and many widespread strains are resistant to almost all antibiotics currently in use (4, 7, 8, 9, 10). There is mounting evidence that A. baumannii has a naturally occurring carbapenemase gene intrinsic to this species (5, 11, 13). The first report of this gene described bla_OXA-51 (2), but since then a large number of closely related variants have been found (with OXA numbers 64, 65, 66, 67, 68, 69, 70, 71, 75, 76, 77, 83, 84, 86, 87, 88, 89, 91, 92, 94, and 95) (1, 5, 11), and we have referred to them collectively as "bla_OXA-51-like" genes. Carbapenem resistance has only been associated with these genes when the insertion sequence ISAba1 is upstream (11) and is not an indicator of whether an isolate has such a gene.

Although it is clear that bla_OXA-51-like genes are present in at least the vast majority of isolates of A. baumannii, there has been some debate as to whether they are present in all isolates of this species (3). If they are consistently found and are also unique to this species, then their detection could provide a simple and convenient method of identifying A. baumannii which could more easily be carried out than current definitive methods, such as amplified rRNA gene restriction analysis (12) (ARDRA), and which would be more reliable than biochemical identification (e.g., by API), which is most commonly used. Since A. baumannii is clinically by far the most significant of the Acinetobacter species, the ability to distinguish it rapidly from other members of the genus would be highly valuable.

Here we describe the results of testing large numbers of well-characterized clinical Acinetobacter isolates for bla_OXA-51-like genes by PCR with group-specific primers (13) and compare the results with those obtained by ARDRA. Detection of bla_OXA-51-like can be carried out as part of a multiplex, and two such multiplexes (one detects all the known groups of OXA carbapenemase genes in Acinetobacter [13], and the second detects bla_OXA-51-like, bla_OXA-23-like, and the class 1 integrase gene) are in current use in our laboratories; the class 1 integrase gene is a useful marker for outbreak strains of A. baumannii (6, 10).

All isolates of Acinetobacter received by the United Kingdom reference laboratories between November 2005 and March 2006 were included (170 isolates). Every isolate was compared with previous isolates by pulsed-field gel electrophoresis (PFGE) of ApaI-digested DNA, using BioNumerics software, as described previously (9, 10). The set included 64 isolates of OXA-23 clone 1 (the most prevalent genotype in the United Kingdom), 22 isolates of the South East clone, 18 isolates of the T strain, and 1 isolate each of the W strain (known to belong to European clone 1) and the "uncertain" strain. These outbreak strains have all been described previously and iden-tified as A. baumannii (9, 10). The remaining isolates received consisted of sporadic strains (40 isolates) (defined by unique PFGE profiles and the absence of the class 1 integrase gene) and minor strains (24 isolates). Thirty-eight of these were further characterized by ARDRA (12).

All isolates were subjected to the multiplex PCR to detect bla_OXA-51-like, bla_OXA-23-like, and class 1 integrase genes (Table 1). PCRs were carried out as previously described (10) in 25-µl reaction volumes with 3 µl of extracted DNA, 12.5 pmol of each primer, and 1.5 U of Taq DNA polymerase in 1x PCR buffer containing 1.5 mM MgCl₂ (QIAGEN) and 200 µM of each deoxynucleoside triphosphate. Conditions for the multiplex were the following: 94°C for 3 min, and then 35 cycles at 94°C for 45 s, at 57°C for 45 s, and at 72°C for 1 min, followed by a final extension at 72°C for 5 min. A single PCR (using only the OXA-51-like primers) was also used to seek bla_OXA-51-like in representatives of Acinetobacter genomic species 1 to 17 (inclusive) as well as other Acinetobacter species. Conditions were the same, except that an annealing temperature of 60°C was used.

View this table: [in this window] [in a new window]

TABLE 1. Primers used in the multiplex PCR

All 106 isolates of the main outbreak strains of A. baumannii were PCR positive for bla_OXA-51-like genes (Fig. 1). However, it was the minor strains, most of which were negative for the class 1 integrase gene, and the sporadic strains, detailed in Table 2, which provided a greater challenge for this method, since these were the most diverse. Thirty-eight of these isolates (which included representatives of minor strains A to F) were investigated further (Table 3). All those that were positive for bla_OXA-51-like (13 sporadic isolates and representatives of strains A to E) identified as A. baumannii by ARDRA. All those that were PCR negative for bla_OXA-51-like (18 sporadic isolates and a representative of strain F) identified as other Acinetobacter species.

View larger version (35K): [in this window] [in a new window]

FIG. 1. Multiplex PCR results to amplify fragments of blaOXA-23-like, blaOXA-51-like, and the class 1 integrase gene from Acinetobacter spp. Representatives of OXA-23 clone 1 (lanes 1, 4, 5, 7, 8, 9, 11, and 12), the SE clone (lanes 16 and 17), and strains A (lane 6), C (lanes 2 and 3), and D (lane 10) are included. Isolates in lanes 13, 14, and 15 were sporadic. All isolates except that in lane 13 identified as A. baumannii and had blaOXA-51-like; the isolate in lane 13 was identified as A. lwoffii and lacked this gene. M, 123-bp ladder.

View this table: [in this window] [in a new window]

TABLE 2. PFGE and PCR results obtained on sporadic and minor outbreak strains

View this table: [in this window] [in a new window]

TABLE 3. Results of identification tests on isolates of sporadic and minor strains

Although integrons are a good marker for outbreak strains of A. baumannii, increasingly we have found multiple representatives of bla_OXA-23-like-positive genotypes which are PCR negative for the class 1 integrase gene, such as strains B and C (Table 2). In today's climate of carbapenem therapy, the association between outbreak strains and integrons may break down among isolates with carbapenemase genes such as bla_OXA-23-like, which confer high level carbapenem resistance, and we will continue to monitor this.

bla_OXA-51-like was also sought in reference isolates of Acinetobacter genomic species 1 to 17 and other Acinetobacter species (Table 4). These included all those likely to be encountered clinically. Only the isolate of genomic species 2 (A. baumannii) (type strain ATCC 19606) gave a band. Clinical isolates of other gram-negative organisms tested (Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Escherichia coli, and Moraxella nonliquefaciens) failed to give a band.

View this table: [in this window] [in a new window]

TABLE 4. Results of the blaOXA-51-like PCR carried out on reference isolates of Acinetobacter species

Many variants of bla_OXA-51 have been described, and a possible limitation of this PCR is whether it can detect every variant. By design, it should detect all the variants in GenBank to date, with the possible exception of bla_OXA-75 (there is a single base mismatch with the forward primer in bla_OXA-75). In the present study, all 141 isolates of A. baumannii found, representing 23 genotypes, gave a band in the bla_OXA-51-like PCR, clearly suggesting that this PCR does detect these genes in all the isolates of A. baumannii we currently encounter, but we remain alert to the possibility of nondetection of some variants. A further potential problem is that these genes are sometimes associated with ISAba1 (11), which may render them mobile.

These results provide evidence that detection of bla_OXA-51-like can be used as a simple and reliable way of identifying A. baumannii. We have found bla_OXA-51-like in every isolate of A. baumannii we have investigated in both this and a previous study (11). Furthermore, it is present in the type strain, isolated decades ago. GenBank submissions describing variants are from isolates of A. baumannii from many different countries (France, Greece, Turkey, Spain, United Kingdom, South Africa, Hong Kong, Singapore, and Argentina) (e.g., DQ335566 and DQ445683) distributed over four continents (1), clearly suggesting bla_OXA-51-like is ubiquitous in A. baumannii.

The combination of markers detected in the multiplex described provides powerful information not only on identification as A. baumannii but also on probable outbreak potential and allows, to some extent, prediction of likely genotype. For example, the vast majority of isolates of OXA-23 clone 1, the most prevalent genotype in the United Kingdom, give all three bands in this multiplex, while sporadic and more minor strains with bla_OXA-23-like lack the class 1 integrase gene. Other common outbreak genotypes of A. baumannii are positive for the class 1 integrase gene but lack bla_OXA-23-like (Fig. 1). Results from the multiplex can be obtained rapidly and should prove highly useful to clinicians and infection control staff.

 
We are grateful to Juliana Coelho and Lenie Dijkshoorn for providing the Acinetobacter species isolates in Table 4.

 
* Corresponding author. Mailing address: Laboratory of HealthCare Associated Infection, Centre for Infections, Health Protection Agency, 61 Colindale Ave., London NW9 5EQ, United Kingdom. Phone: 44 (0)208 327 7276. Fax: 44 (0)208 200 7449. E-mail: jane.turton{at}hpa.org.uk.

Top Abstract Text References

 

1
1. Brown, S., and S. G. B. Amyes. 2005. The sequences of seven class D beta-lactamases isolated from carbapenem-resistant Acinetobacter baumannii from four continents. Clin. Microbiol. Infect. 11:326-329.[CrossRef][Medline]
2
2. Brown, S., H. K. Young, and S. G. B. Amyes. 2005. Characterisation of OXA-51, a novel class D carbapenemase found in genetically unrelated clinical strains of Acinetobacter baumannii from Argentina. Clin. Microbiol. Infect. 11:15-23.[Medline]
3
3. Brown, S., and S. G. B. Amyes. 2006. OXA (beta)-lactamases in Acinetobacter: the story so far. J. Antimicrob. Chemother. 57:1-3.[Abstract/Free Full Text]
4
4. Da Silva, G. J., S. Quinteira, E. Bértolo, J. C. Sousa, L. Gallego, A. Duarte, and L. Peixe on behalf of the Portuguese Resistance Study Group. 2004. Long-term dissemination of an OXA-40 carbapenemase-producing Acinetobacter baumannii clone in the Iberian Peninsula. J. Antimicrob. Chemother. 54:255-258.[Abstract/Free Full Text]
5
5. Héritier, C., L. Poirel, P.-E. Fournier, J.-M. Claverie, D. Raoult, and P. Nordmann. 2005. Characterization of the naturally occurring oxacillinase of Acinetobacter baumannii. Antimicrob. Agents Chemother. 49:4174-4179.[Abstract/Free Full Text]
6
6. Koeleman, J. G. M., J. Stoof, M. W. van der Bijl, C. M. J. E. Vandenbroucke-Grauls, and P. H. M. Savelkoul. 2001. Identification of epidemic strains of Acinetobacter baumannii by integrase gene PCR. J. Clin. Microbiol. 39:8-13.[Abstract/Free Full Text]
7
7. Landman, D., J. M. Quale, D. Mayorga, A. Adedeji, K. Vangala, J. Ravishankar, C. Flores, and S. Brooks. 2002. Citywide clonal outbreak of multiresistant Acinetobacter baumannii and Pseudomonas aeruginosa in Brooklyn, N.Y.: the preantibiotic era has returned. Arch. Intern. Med. 162:1515-1520.[Abstract/Free Full Text]
8
8. Poirel, L., O. Menuteau, N. Agoli, C. Cattoen, and P. Nordmann. 2003. Outbreak of extended-spectrum ß-lactamase VEB-1-producing isolates of Acinetobacter baumannii in a French hospital. J. Clin. Microbiol. 41:3542-3547.[Abstract/Free Full Text]
9
9. Turton, J. F., M. E. Kaufmann, M. Warner, J. M. Coelho, L. Dijkshoorn, T. van der Reijden, and T. L. Pitt. 2004. A prevalent, multiresistant, clone of Acinetobacter baumannii in South East England. J. Hosp. Infect. 58:170-179.[CrossRef][Medline]
10
10. Turton, J. F., M. E. Kaufmann, J. Glover, J. M. Coelho, M. Warner, R. Pike, and T. L. Pitt. 2005. Detection and typing of integrons in epidemic strains of Acinetobacter baumannii found in the United Kingdom. J. Clin. Microbiol. 43:3074-3082.[Abstract/Free Full Text]
11
11. Turton, J. F., M. E. Ward, N. Woodford, M. E. Kaufmann, R. Pike, D. M. Livermore, and T. L. Pitt. 2006. The role of ISAba1 in expression of OXA carbapenemase genes in Acinetobacter baumannii. FEMS Microbiol. Lett. 258:72-77.[CrossRef][Medline]
12
12. Vaneechoutte, M., L. Dijkshoorn, I. Tjernberg, A. Elaichouni, P. de Vos, G. Claeys, and G. Verschraegen. 1995. Identification of Acinetobacter genomic species by amplified ribosomal DNA restriction analysis. J. Clin. Microbiol. 33:11-15.[Abstract]
13
13. Woodford, N., M. J. Ellington, J. M. Coelho, J. F. Turton, M. E. Ward, S. Brown, S. G. B. Amyes, and D. M. Livermore. 2006. Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp. Int. J. Antimicrob. Agents 27:351-353.[CrossRef][Medline]

---

Journal of Clinical Microbiology, August 2006, p. 2974-2976, Vol. 44, No. 8
0095-1137/06/$08.00+0     doi:10.1128/JCM.01021-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Chen, Y., Pi, B., Zhou, H., Yu, Y., Li, L. (2009). Triclosan resistance in clinical isolates of Acinetobacter baumannii. J Med Microbiol 58: 1086-1091 [Abstract] [Full Text]  
• Akers, K. S., Mende, K., Yun, H. C., Hospenthal, D. R., Beckius, M. L., Yu, X., Murray, C. K. (2009). Tetracycline Susceptibility Testing and Resistance Genes in Isolates of Acinetobacter baumannii-Acinetobacter calcoaceticus Complex from a U.S. Military Hospital. Antimicrob. Agents Chemother. 53: 2693-2695 [Abstract] [Full Text]  
• Bogaerts, P., Cuzon, G., Naas, T., Bauraing, C., Deplano, A., Lissoir, B., Nordmann, P., Glupczynski, Y. (2008). Carbapenem-Resistant Acinetobacter baumannii Isolates Expressing the blaOXA-23 Gene Associated with ISAba4 in Belgium. Antimicrob. Agents Chemother. 52: 4205-4206 [Full Text]  
• Bratu, S., Landman, D., Martin, D. A., Georgescu, C., Quale, J. (2008). Correlation of Antimicrobial Resistance with {beta}-Lactamases, the OmpA-Like Porin, and Efflux Pumps in Clinical Isolates of Acinetobacter baumannii Endemic to New York City. Antimicrob. Agents Chemother. 52: 2999-3005 [Abstract] [Full Text]  
• Peleg, A. Y., Seifert, H., Paterson, D. L. (2008). Acinetobacter baumannii: Emergence of a Successful Pathogen. Clin. Microbiol. Rev. 21: 538-582 [Abstract] [Full Text]  
• Ikonomidis, A., Ntokou, E., Maniatis, A. N., Tsakris, A., Pournaras, S. (2008). Hidden VIM-1 Metallo- -Lactamase Phenotypes among Acinetobacter baumannii Clinical Isolates. J. Clin. Microbiol. 46: 346-349 [Abstract] [Full Text]  
• Koh, T. H., Sng, L.-H., Wang, G. C. Y., Hsu, L.-Y., Zhao, Y. (2007). Carbapenemase and efflux pump genes in Acinetobacter calcoaceticus Acinetobacter baumannii complex strains from Singapore. J Antimicrob Chemother 60: 1173-1174 [Full Text]  
• Hu, W. S., Yao, S.-M., Fung, C.-P., Hsieh, Y.-P., Liu, C.-P., Lin, J.-F. (2007). An OXA-66/OXA-51-Like Carbapenemase and Possibly an Efflux Pump Are Associated with Resistance to Imipenem in Acinetobacter baumannii. Antimicrob. Agents Chemother. 51: 3844-3852 [Abstract] [Full Text]  
• Wang, H., Guo, P., Sun, H., Wang, H., Yang, Q., Chen, M., Xu, Y., Zhu, Y. (2007). Molecular Epidemiology of Clinical Isolates of Carbapenem-Resistant Acinetobacter spp. from Chinese Hospitals. Antimicrob. Agents Chemother. 51: 4022-4028 [Abstract] [Full Text]  
• Queenan, A. M., Bush, K. (2007). Carbapenemases: the Versatile {beta}-Lactamases. Clin. Microbiol. Rev. 20: 440-458 [Abstract] [Full Text]  
• Corvec, S., Poirel, L., Naas, T., Drugeon, H., Nordmann, P. (2007). Genetics and Expression of the Carbapenem-Hydrolyzing Oxacillinase Gene blaOXA-23 in Acinetobacter baumannii. Antimicrob. Agents Chemother. 51: 1530-1533 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Turton, J. F. Articles by Pitt, T. L. Search for Related Content PubMed PubMed Citation Articles by Turton, J. F. Articles by Pitt, T. L.