JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2010, p. 1488–1490 0095-1137/10/$12.00 doi:10.1128/JCM.01264-09 Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 48, No. 4
Naturally Competent Acinetobacter baumannii Clinical Isolate as a Convenient Model for Genetic Studies䌤 Maria Soledad Ramirez,1,2 Michelle Don,1 Andrea K. Merkier,2 Alfonso J. Soler Bistue´,3 Angeles Zorreguieta,3 Daniela Centro ´n,2 and Marcelo E. Tolmasky1* Center for Applied Biotechnology Studies, Department of Biological Science, California State University, Fullerton, Fullerton, California1; Departamento de Microbiología, Facultad de Medicina, UBA, Buenos Aires, Argentina2; and Fundacio ´n Instituto Leloir, Instituto de Investigaciones Bioquímicas de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Te´cnicas and Departamento de Química Biolo ´gica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina3 Received 28 June 2009/Returned for modification 1 September 2009/Accepted 2 February 2010
Acinetobacter baumannii A118 was isolated from a patient’s blood culture. It is susceptible to several antibiotics, is naturally competent, and supports replication and stable maintenance of four plasmid replicons. A. baumannii A118 took up a fluorophore-labeled oligonucleotide analog. These characteristics make this isolate a convenient model for genetic studies. replicons, some of them widely used as genetic tools. The plasmids pJHCMW1 (16), pMET1 (17), pAADA1KN, pAADB, and pVK102 (9) (Table 1) were used in transformation and stability assays. Plasmid DNA was isolated using the QIAfilter midi kit (Qiagen). Plasmid stability assays were carried out for 40 generations, as described previously (20). Briefly, plasmid-containing cells in late log phase were diluted 10⫺6-fold in fresh nonselective medium (LB broth) and incubated at 37°C until the culture reached the same optical density (20 generations). This culture was again diluted, and the procedure was repeated to reach 40 generations. These cultures were diluted and spread on selective and nonselective plates to determine the percentage of plasmid-containing cells. The case of pAADA1KN is discussed below. Experiments were done twice; plasmid DNA was prepared from several resistant colonies and analyzed in agarose gel electrophoresis and by using it to transform competent Escherichia coli cells. Transformation of untreated cells with plasmid DNA. Natural competency assays were carried out by adding 100 ng of plasmid DNA to a mix containing 50 l of fresh LB broth and 50 l of culture in stationary phase, followed by incubation at 25 or 37°C for 1 h and plating on L agar plates containing 12.5 or 20 g/ml Kan. All five plasmids transformed A. baumannii A118 at frequencies ranging from 7.4 ⫻ 102 to 29.6 ⫻ 102 transformants/g DNA (Table 1). Colonies were subcultured in liquid medium, and plasmid DNA was extracted. With the exception of pAADA1KN, the plasmids were detected in agarose gel electrophoresis (Fig. 1B). Although pAADA1KN could not be detected in agarose gels, the presence of a low concentration of this plasmid in the extracts was evident because it could be used to transform E. coli and recover pAADA1KN from the transformant cells. As will be discussed in the following section, the resistance to KAN but low concentration of plasmid recovered could be due to integration of the plasmid in the chromosome in a significant percentage of the cells. Stability of plasmid DNA. Selection after transformation does not give an accurate indication of plasmid stability. A plasmid may be replicated, but it needs other stabilizing factors
CASE REPORT Acinetobacter baumannii A118 was isolated from a blood culture of a patient admitted to an intensive care unit in a hospital in Buenos Aires, Argentina. The isolate was identified at the species level using several criteria: (i) the biochemical scheme described by Bouvet and Grimont (1); (ii) amplified ribosomal DNA restriction analysis (ARDRA) (12, 22); the restriction pattern obtained with CfoI, AluI, and MboI, which is 111, characteristic for A. baumannii (http://users.ugent.be /⬃mvaneech/ARDRA/Acinetobacter.html) (Fig. 1A); (iii) amplification and sequencing of the 16S rRNA; (iv) amplification of the rpoB gene; and (v) identification and sequencing of blaOXA-51-like, a carbapenemase gene intrinsic to A. baumannii (12, 21). A. baumannii A118 lacks the integrase genes intI1, intI2, and intI3 and includes the competence genes comA, comM, and pilC, as determined by PCR amplification and DNA sequencing. Determination of antibiotic susceptibilities using the agar dilution method according to the CLSI guidelines (2) indicated that A. baumannii A118 is susceptible to ceftazidime, cefepime, piperacillin, minocycline, amikacin (Amk), gentamicin, trimethoprim-sulfamethoxazole, and ciprofloxacin. Although there is no CLSI guideline for using kanamycin (Kan) on A. baumannii, we determined the MIC, 1.5 g/ml, because this antibiotic is widely used as a selective drug in laboratory experiments. Bacteria of the genus Acinetobacter have been shown to be naturally competent (4). However, while data on natural competence of A. baylyi abound, studies of natural competence, as well as stability of plasmid replicons that could be used as potential cloning vehicles for researching A. baumannii, are scarce (13). Here we determined the competency of the A. baumannii A118 isolate and the stability of several plasmid * Corresponding author. Mailing address: Center for Applied Biotechnology Studies, Department of Biological Science, California State University, Fullerton, 800 N. State College Boulevard, Fullerton, CA 92831-3599. Phone: (657) 278-5263. Fax: (657) 278-3426. E-mail:
[email protected]. 䌤 Published ahead of print on 24 February 2010. 1488
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FIG. 1. (A) Amplified ribosomal DNA restriction analysis (ARDRA). Ethidium bromide-stained 1.5% agarose gel electrophoresis of products obtained after treatment of the amplified 16S rRNA gene with CfoI, AluI, or MboI is shown. Molecular size DNA standard values are shown on the right side of the gel. Numbers at the bottom of each lane correspond to the ARDRA pattern (http://users.ugent.be/⬃mvaneech/ARDRA /Acinetobacter.html). Ab, A. baumannii. (B) Plasmid DNA extracted from A. baumannii A118. Ethidium bromide-stained 0.7% agarose gel electrophoresis of linearized plasmid DNA isolated from transformed A. baumannii A118 is shown. Molecular size DNA standard values are shown on the right side of the gel. The enzymes used to digest the plasmids were as follows: XhoI (pVK102 and pHCMW1), EcoRV (pAADB and pAADA1KN), and SpeI (pMET1). (C) Internalization of fluorescent PS. Bright field (upper) and fluorescent (lower) microscopy of A. baumannii A118 cells incubated in the presence of Oregon green-labeled PS (green). The membranes were labeled using FM5-95 (red) as described before (14). Cells were visualized with a ⫻100 objective on a Nikon Eclipse TE2000-U microscope equipped with a Photometrics Cool-SNAP HQ charge-coupled-device camera. The images were analyzed and processed using the Adobe Photoshop software program. Arrows show cells that took up labeled PS. (D) Internalization of fluorescent LNA/DNA. Cells in stationary phase were incubated in saline buffer with Alexa Fluor 488-conjugated LNA/DNA for 30 min at 37°C in the absence of light. Then, the cells were washed twice by centrifugation, resuspended in saline buffer, and labeled with FM5-95. Images were taken by laser scanning confocal microscopy (LSCM) in a Zeiss Pascal confocal microscope. Deconvolution of the overlay picture focused at 1.6 m was performed at the Oregon Health and Science University Molecular Microbiology and Immunology Imaging Core Facility Microscopy.
to be stably maintained in the absence of selection. Furthermore, since it has been described that integration of foreign DNA into the chromosome of Acinetobacter species by homologous as well as illegitimate mechanisms is a very common event (4, 8), it is possible that in a significant number of cells, the plasmids may be integrated into the chromosome. To achieve a more thorough characterization of the fate of the
plasmids introduced by transformation in A. baumannii A118, we determined the stability of the transforming plasmids and confirmed their extrachromosomal nature in colonies that conserved the resistance. Plasmids pJHCMW1, pMET1, pAADB, and pVK102 were stably maintained after 40 generations (Table 1). Instead, plasmid pAADA1KN was lost in a significant percentage of A. baumannii A118 cells (Table 1). This was an
TABLE 1. Plasmid characteristics and stability Plasmid
Relevant characteristics (reference)
Transformation frequencya (CFU/g DNA)
Stability (%)b
pJHCMW1 pMET1 pAADA1KN pAADB pKV102
pJHCMW1 replicon, 11,354 bp, Kanr Amkr (aac(6⬘)-Ib) (16) pMET1 replicon, 41,723 bp, Kanr Amkr (aac(6⬘)-Ib) (17) p15A replicon, ca. 11,000 bp, Kanr (aph) derivative of pACYC184 (this work) pUC replicon, 5,672 bp, Kanr (aph) (this work) RK2 replicon (IncP), ca. 23,000 bp, Kanr (aph) (9)
(11.3 ⫾ 2.4) ⫻ 102 (7.4 ⫾ 0.3) ⫻ 102 (7.9 ⫾ 0.8) ⫻ 102 (13.7 ⫾ 2.9) ⫻ 102 (26.9 ⫾ 7.4) ⫻ 102
99.5 ⫾ 1 94.5 ⫾ 2 72.5 ⫾ 3 99.0 ⫾ 1 100
Transformation frequencies were determined as described by Zinniel et al. (23). Values are the means of results from three assays ⫾ standard deviations. Percentage of A. baumannii A118 cells harboring plasmid after 40 generations without selective pressure. Values are the means of results from two assays ⫾ standard deviations. The experimental procedure is described in the text. a b
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expected result, since it is well known that pACYC184 is unstable (10). However, our results show that pAADA1KN is lost at a higher rate in E. coli (36.5% of the cells carry the plasmid after 40 generations) than in A. baumannii A118. This could be due to a higher stability of the plasmid in A. baumannii A118 or to integration of the plasmid into the chromosome. However, since no pAADA1KN was detected after plasmid extraction from A. baumannii A118 resistant to Kan (Fig. 1B), we think that the apparent increase in stability is due to integration into the chromosome. Uptake of oligodeoxynucleotide analogs. A. baumannii A118 was also able to take up a fluorophore-labeled 10-mer phosphorothioate oligodeoxynucleotide analog (PS). Figure 1C shows a typical field where some cells had taken up the fluorescent compound. Quantification indicated that the PS accumulates within 1.8% ⫾ 0.9% of the cells (the total number of cells analyzed is 2,929). To confirm that the oligomer was taken into the cytoplasm, we examined cells after exposure to an Alexa Fluor 488-conjugated locked nucleic acid (LNA)/DNA oligomer using laser scanning confocal microscopy, capturing a series of images focused at 0.2-m intervals through the depth of the cell, and deconvolution of the 1.6-m overlay picture (Fig. 1D). These results indicate that after appropriate conditions are found, this strain could potentially be used as a model for technologies, such as antisense gene silencing, which require internalization of oligonucleotides or oligonucleotide analogs. A recent antisense study has been published using a hyperpermeable E. coli strain that was able to take up LNA/DNA oligomers (18).
Conclusions. Acinetobacter baumannii is an emerging opportunistic human pathogen responsible for a growing number of community and nosocomial infections (7, 11, 13). The incidence of A. baumannii has steadily grown, and there is a frightening rise in the number of multidrug-resistant isolates (13). As a consequence, treatment of these infections is becoming increasingly difficult. Compounding the problem, drug development to treat this bacterium is almost nonexistent (5, 13, 15, 19). A. baumannii infections have also gained attention due to the high number of soldiers serving in Iraq and Afghanistan and victims of the 2004 Asian tsunami that were infected with this bacterium (3, 6). Molecular genetic studies of A. baumannii clinical strains have often been limited because they are usually resistant to most antibiotics. The ability of A. baumannii A118 to be transformed and stably support the replication of several plasmids together with its susceptibility profile make this strain a useful model for genetic analyses and studies on virulence and antibiotic resistance. This study was supported by Public Health Service grant 2R15AI047115 (to M.E.T.) from the National Institutes of Health, California State University Program for Education and Research on Biotechnology (CSUPERB), BID 1728 OC/AR PICT 0690 (to D.C.), and PICT 0354 (to M.S.R.). D.C. and A.Z. are career members of CONICET. M.S.R. was supported in part by fellowships from the International Union of Microbiological Societies and CONICET (postdoctoral). A.J.S.B. was supported by fellowships from the American Society for Microbiology (International Fellowship for Latin America) and CONICET. M.D. was supported in part by grant MHIRT T37MD001368 from the National Center on Minority Health and Health Disparities, National Institutes of Health.
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