Identification of Acinetobacter baumannii by Detection of the blaOXA-5
http://www.100md.com
《微生物临床杂志》
Laboratory of HealthCare Associated Infection
Antibiotic Resistance Monitoring and Reference Laboratory, Centre for Infections, Health Protection Agency, London NW9 5EQ, United Kingdom
ABSTRACT
blaOXA-51-like was sought in clinical isolates of Acinetobacter species in a multiplex PCR, which also detects blaOXA-23-like and class 1 integrase genes. All isolates that gave a band for blaOXA-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.
TEXT
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 blaOXA-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 "blaOXA-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 blaOXA-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 blaOXA-51-like genes by PCR with group-specific primers (13) and compare the results with those obtained by ARDRA. Detection of blaOXA-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 blaOXA-51-like, blaOXA-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 blaOXA-51-like, blaOXA-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 blaOXA-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.
All 106 isolates of the main outbreak strains of A. baumannii were PCR positive for blaOXA-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 blaOXA-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 blaOXA-51-like (18 sporadic isolates and a representative of strain F) identified as other Acinetobacter species.
Although integrons are a good marker for outbreak strains of A. baumannii, increasingly we have found multiple representatives of blaOXA-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 blaOXA-23-like, which confer high level carbapenem resistance, and we will continue to monitor this.
blaOXA-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.
Many variants of blaOXA-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 blaOXA-75 (there is a single base mismatch with the forward primer in blaOXA-75). In the present study, all 141 isolates of A. baumannii found, representing 23 genotypes, gave a band in the blaOXA-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 blaOXA-51-like can be used as a simple and reliable way of identifying A. baumannii. We have found blaOXA-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 blaOXA-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 blaOXA-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 blaOXA-23-like (Fig. 1). Results from the multiplex can be obtained rapidly and should prove highly useful to clinicians and infection control staff.
ACKNOWLEDGMENTS
We are grateful to Juliana Coelho and Lenie Dijkshoorn for providing the Acinetobacter species isolates in Table 4.
FOOTNOTES
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@hpa.org.uk.
REFERENCES
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.
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.
Brown, S., and S. G. B. Amyes. 2006. OXA (beta)-lactamases in Acinetobacter: the story so far. J. Antimicrob. Chemother. 57:1-3.
Da Silva, G. J., S. Quinteira, E. Bertolo, 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.
Heritier, 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.
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.
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.
Poirel, L., O. Menuteau, N. Agoli, C. Cattoen, and P. Nordmann. 2003. Outbreak of extended-spectrum -lactamase VEB-1-producing isolates of Acinetobacter baumannii in a French hospital. J. Clin. Microbiol. 41:3542-3547.
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.
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.
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.
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.
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.(Jane F. Turton, Neil Woodford, Judith Gl)
Antibiotic Resistance Monitoring and Reference Laboratory, Centre for Infections, Health Protection Agency, London NW9 5EQ, United Kingdom
ABSTRACT
blaOXA-51-like was sought in clinical isolates of Acinetobacter species in a multiplex PCR, which also detects blaOXA-23-like and class 1 integrase genes. All isolates that gave a band for blaOXA-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.
TEXT
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 blaOXA-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 "blaOXA-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 blaOXA-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 blaOXA-51-like genes by PCR with group-specific primers (13) and compare the results with those obtained by ARDRA. Detection of blaOXA-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 blaOXA-51-like, blaOXA-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 blaOXA-51-like, blaOXA-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 blaOXA-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.
All 106 isolates of the main outbreak strains of A. baumannii were PCR positive for blaOXA-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 blaOXA-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 blaOXA-51-like (18 sporadic isolates and a representative of strain F) identified as other Acinetobacter species.
Although integrons are a good marker for outbreak strains of A. baumannii, increasingly we have found multiple representatives of blaOXA-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 blaOXA-23-like, which confer high level carbapenem resistance, and we will continue to monitor this.
blaOXA-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.
Many variants of blaOXA-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 blaOXA-75 (there is a single base mismatch with the forward primer in blaOXA-75). In the present study, all 141 isolates of A. baumannii found, representing 23 genotypes, gave a band in the blaOXA-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 blaOXA-51-like can be used as a simple and reliable way of identifying A. baumannii. We have found blaOXA-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 blaOXA-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 blaOXA-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 blaOXA-23-like (Fig. 1). Results from the multiplex can be obtained rapidly and should prove highly useful to clinicians and infection control staff.
ACKNOWLEDGMENTS
We are grateful to Juliana Coelho and Lenie Dijkshoorn for providing the Acinetobacter species isolates in Table 4.
FOOTNOTES
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@hpa.org.uk.
REFERENCES
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.
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.
Brown, S., and S. G. B. Amyes. 2006. OXA (beta)-lactamases in Acinetobacter: the story so far. J. Antimicrob. Chemother. 57:1-3.
Da Silva, G. J., S. Quinteira, E. Bertolo, 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.
Heritier, 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.
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.
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.
Poirel, L., O. Menuteau, N. Agoli, C. Cattoen, and P. Nordmann. 2003. Outbreak of extended-spectrum -lactamase VEB-1-producing isolates of Acinetobacter baumannii in a French hospital. J. Clin. Microbiol. 41:3542-3547.
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.
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.
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.
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.
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.(Jane F. Turton, Neil Woodford, Judith Gl)