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. 2011 Jan 31;55(4):1520–1526. doi: 10.1128/AAC.01595-10

Genomic Analysis of Acinetobacter baumannii A118 by Comparison of Optical Maps: Identification of Structures Related to Its Susceptibility Phenotype▿

Maria Soledad Ramirez 1,2, Mark D Adams 3, Robert A Bonomo 4, Daniela Centrón 2, Marcelo E Tolmasky 1,*
PMCID: PMC3067174  PMID: 21282446

Abstract

Acinetobacter baumannii A118, a naturally competent clinical isolate, is unusually susceptible to several antibiotics. Comparison of the optical map of strain A118 with in silico-generated restriction maps of sequenced genomes and sequence analyses showed that the AbaR region, commonly found inserted within the comM gene in other isolates, is missing in strain A118, which could in part explain the susceptible phenotype exhibited by this isolate. These comparative studies also showed differences in regions where genes coding for functions that may be involved in drug resistance or susceptibility are located. Further sequencing demonstrated that cat and blaADC, named blaADC-55, are present but that a tet(A) gene usually found in other strains is not. In addition, carO and pbp2, which may play a role in susceptibility to carbapenems, are present in strain A118. These findings support the idea that A. baumannii strains possess multiple mechanisms that contribute to antibiotic resistance, and the presence of some of them is not sufficient for a resistant phenotype. The results shown here indicate that optical mapping is a useful tool for preliminary comparative genomic analysis.

Acinetobacter baumannii is an emerging opportunistic human pathogen responsible for a growing number of nosocomial infections mainly affecting patients who are immunosuppressed, who suffer other underlying diseases, or who have been treated using certain invasive procedures (20, 26, 30). The incidence of A. baumannii is steadily growing, and a study indicates that while in 1975 this bacterium was responsible for 1.5% of hospital-acquired pneumonia cases, in 2003 that number had grown to 6.9% (17). The increasing frequency of A. baumannii infections may be due to a combination of factors, such as its ability to survive for a prolonged length of time in different environments and a rise in the number of susceptible individuals as a result of advancements in medical support of critically ill patients. The ability of A. baumannii to form biofilms has also been related to commonly occurring infections associated with medical devices (15, 34). Recent studies identified several other virulence factors and pathogenic islands (6, 38, 41). 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 who were infected with this bacterium (9, 16, 18). Treatment of Acinetobacter infections is becoming increasingly difficult due to the growing number of multidrug-resistant isolates. Compounding the problem, antibiotic drug development to treat infections caused by this bacterium is almost nonexistent (10, 30, 34, 39). Furthermore, the multiresistant nature of most A. baumannii strains makes them difficult to manipulate for genetic studies.

A. baumannii A118, isolated from a culture of blood from a patient admitted to an intensive care unit in a hospital in Buenos Aires, Argentina, is rather exceptional for its susceptibility to antibiotics such as ceftazidime, cefepime, piperacillin, minocycline, amikacin, gentamicin, trimethoprim-sulfamethoxazole, kanamycin, and ciprofloxacin (32). This property, together with its natural competence, led to the suggestion that this strain is a convenient model for genetic studies (33). In this work we analyzed A. baumannii A118 genomic regions known for containing potential resistance or susceptibility determinants in previously studied strains using optical mapping, a powerful tool for comparative genomics (37). Optical maps are full-genome restriction maps obtained after single DNA molecules are immobilized on a charged substrate and digested with the restriction endonuclease of interest, followed by detection and assembly into a high-resolution ordered full-genome restriction map (5). Our results show that the AbaR-type resistance island is missing and suggest that A. baumannii resistance to a variety of antibiotics may be due to a combination of mechanisms, some of which are present in strain A118 but which are not sufficient to confer a resistance phenotype.

MATERIALS AND METHODS

Bacterial strains and genomes.

A. baumannii A118 is a bloodstream isolate recovered from a patient in an intensive care unit (28, 33). The available genomes of A. baumannii strains were used for the comparative studies (AYE, GenBank accession no. NC_010410; AB307-0294, GenBank accession no. CP001172; AB0057, GenBank accession no. CP001182; ACICU, GenBank accession no. CP000863; ATCC 17978, GenBank accession no. CP000521; and SDF, GenBank accession no. NC_010400). Escherichia coli TOP10 (Invitrogen, San Diego, CA) was used as host in recombinant cloning.

General procedures.

The A. baumannii A118 NcoI optical map was generated at OpGen Technologies, Inc. (Madison, WI), as described previously (5). Comparative genomic analysis was carried out by comparing the optical map of A. baumannii A118 to NcoI restriction maps of A. baumannii sequenced genomes using the MapSolver software (version 2.1.1; OpGen Technologies, Inc.). PCRs were carried out using the Qiagen Taq master mix, and the products were detected by agarose gel electrophoresis. Cloning into pCR2.1 was performed as recommended by the supplier (Invitrogen). DNA sequencing reactions using amplicons as templates were done at the City of Hope sequencing facility. Genomic DNA was prepared for genome sequencing using a Nextera kit from Epicentre Biotechnologies. Sequencing was performed on an Illumina IIx genome analyzer using paired 76-base reads, resulting in 1,712,408 read pairs. These were assembled using the assembly program Velvet (40), resulting in 186 scaffolds that are at least 500 bases long. The scaffold N50 size is 39.3 kbp, meaning that half of the genome is assembled into scaffolds of at least this length. The total assembled length is 3,824 kbp. Genome annotation was performed using the ISGA web server (21). Amino acid sequence comparisons were performed using the CLUSTAL W program (Pôle Bio-Informatique Lyonnais server [http://npsa-pbil.ibcp.fr/cgi-bin/align_clustalw.pl]) (7).

Nucleotide sequence accession number.

The nucleotide sequence data are available in the GenBank nucleotide database under accession number AEOW00000000.

RESULTS AND DISCUSSION

Regions relevant to the antibiotic susceptibility characteristics of A. baumannii A118 were studied using optical mapping, a technique that is based on immobilization of single DNA molecules on a charged substrate, digestion with a restriction endonuclease, and detection and assembly into a high-resolution ordered restriction map permitting comparison of related genomes (3). The estimated size of the A. baumannii A118 chromosome on the basis of the optical mapping is 3.84 Mb, and the predicted number of NcoI restriction fragments is 465.

AbaR-type resistance island region.

The genomes of five of the six A. baumannii strains for which the complete genome sequence is known include a region that has transposed or inserted into a specific location within comM, a gene that codes for a 495-amino-acid protein that includes an ATPase domain (1, 14). In addition, analysis of other A. baumannii strains showed that most of them also carry a related insertion (31, 36). These inserted regions are usually characterized by the presence of transposase and antibiotic or heavy metal resistance genes and are known to be AbaR-type resistance island regions (1, 31). Comparison of in silico-generated restriction maps of A. baumannii sequenced genomes with the optical map of A. baumannii A118 indicated that there was no AbaR-type resistance island in this strain (Fig. 1a). PCR using the primers, designed before by Shaik et al. (36), located within the comM gene and flanking the location of insertion of AbaR, followed by sequencing of the amplicon, showed that the comM gene was intact and identical to the gene in A. baumannii AB307-0294, a strain known to lack the AbaR-type resistance island (1) (Fig. 1b). This gene has been named comM on the basis of the 49.5% homology found between the proteins from A. baumannii ADP1 and Haemophilus influenzae (2). Mutagenesis of comM in H. influenzae resulted in a reduced ability to take up DNA (19). These results are in agreement with the susceptible phenotype observed for A. baumannii A118 and its natural competency.

FIG. 1.

FIG. 1.

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Genomic comparison. (a) The A. baumannii A118 optical map was compared to the A. baumannii strain AB0057 and AYE NcoI restriction maps obtained in silico around the location of the AbaR-type resistance island region using the MapSolver software. The white regions represent DNA fragments missing in strain A118. The AbaR3 and AbaR1 regions are the genomic islands present in strains AB0057 and AYE (1, 14). Vertical lines represent NcoI restriction sites. (b) Diagram showing the point of insertion of AbaR genomic islands within the comM gene, which was shown to be intact in A. baumannii A118 by sequencing an amplicon generated using total DNA as the template and the primers 5′-TCCATTTTACCGCCACTTTC and 5′-AATCGATGCGGTCGAGTAAC (36). Nucleotides shown in red are directly repeated in those strains where AbaR has been inserted.

Other loci related to antibiotic resistance.

The genomes of all sequenced A. baumannii isolates of human origin include a cat gene. In addition, strains AB0057 and AYE include a second cat gene within the AbaR-type resistance island (1, 14). A. baumannii A118 lacks this island, and therefore, this strain must lack at least one of the cat genes. Comparative analysis of the A. baumannii A118 optical map at the region where the cat gene present outside the AbaR-type resistance island is located in the sequenced strains showed some heterogeneity, with apparent insertions and deletions. This is best illustrated by the comparison of the optical map of strain A118 with the in silico-generated NcoI restriction maps of strains AB0057 and AYE. While comparison of strains A118 and AB0057 suggests that the fragment, including cat in A118, although it is not identical, is present, comparison of strains A118 and AYE suggests that there is a deletion in strain A118 that includes the fragment where cat should be located. To confirm the presence of cat in strain A118, an amplicon of 1,261 bp obtained using a pair of primers located within the flanking greA and uspA genes was sequenced. The results indicated that the genome of A. baumannii A118 includes the cat gene with nearly perfect identity to those present in other A. baumannii strains (strain AYE, locus tag ABAYE0798; strain AB0057, locus tag AB57_3104; strain ATCC 17978, locus tag A1S_2691) (Fig. 2b). These results indicate that there must be some variability at the nucleotide region that resulted in modifications in the NcoI restriction site patterns that led to the apparent deletion of a DNA fragment in strain A118. We hypothesize that the lower MIC of chloramphenicol exhibited by strain A118 is due to the absence of the cat gene located within the AbaR-type resistance island. However, comparison of the MICs of chloramphenicol for A. baumannii A118 and ATCC 17978, which also lacks the cat present within the AbaR-type resistance island, showed that they were 12 and 48 μg/ml, respectively. Although it is possible that this cat gene contributes to resistance to chloramphenicol, it is most likely that other factors may also contribute to the overall resistance to chloramphenicol, of which some must be absent in strain A118.

FIG. 2.

FIG. 2.

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Genomic comparison. (a) Comparison of the A. baumannii A118 optical map with the A. baumannii strain AB0057 and AYE NcoI restriction maps obtained in silico around the location of the cat gene using the MapSolver software. White fragments represent putative missing/inserted fragments. The locations of the greA, cat, and uspA genes and relevant NcoI sites are shown. The numbers indicate the coordinates of NcoI sites or the locations of the specified genes in the GenBank entries for A. baumannii AB0057 and AYE genome annotations (accession numbers CP001182 and NC_010410, respectively). (b) CLUSTAL W comparison of chloramphenicol acetyltransferase amino acid sequences. Strain AYE, locus tag ABAYE0798; strain AB0057, locus tag AB57_3104; strain ATCC 17978, locus tag A1S_2691.

The genomes of A. baumannii AYE, AB0057, AB307-0294, and ATCC 17978 include a tet(A) gene outside the AbaR-type resistance island that may be involved in tetracycline resistance (strain AYE, locus tag ABAYE0369; strain AB0057, locus tag AB57_3570; strain AB307-0294, locus tag ABBFA_00039; strain ATCC 17978, locus tag A1S_3117). In addition, strains AB0057 and AYE include one gene [tet(A)] and two genes [tet(A) and tet(G)] within the AbaR-type resistance island, respectively (1, 14, 24, 31; http://faculty.washington.edu/marilynr/tetweb1.pdf). A comparison of the optical map of strain A118 and the in silico-generated NcoI restriction maps of A. baumannii genomes at the location of tet(A) showed that strains ACICU and A118 have a different pattern than the rest of the strains (Fig. 3). To investigate if the differences observed correlated with the presence or absence of the tet(A) gene, the sequences of the ACICU, AYE, AB0057, AB307-0294, and ATCC 17978 strains were compared among themselves and to the drafts of strain A118. The results indicated that while the genes glyS and glyQ were present and highly homologous in all strains, ACICU and A118 do not include the tet(A) gene; instead, there is a short open reading frame with no homology to tet(A) upstream of glyQ (Fig. 3). Furthermore, BLAST analyses comparing the tet(A) nucleotide sequence or the coded amino acid sequence against the available sequences of strain A118 showed no homology, confirming that this strain lacks the tet(A) gene.

FIG. 3.

FIG. 3.

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Genomic comparison. Comparison of the A. baumannii A118 optical map with the A. baumannii strain ATCC 17978, ACICU, AB307-0294, AB0057, and AYE NcoI restriction maps obtained in silico at the location of the tet(A) gene using the MapSolver software. The locations of glyS, glyQ, and tet(A) in the A. baumannii ACICU and AYE strains and the positions of the relevant NcoI sites are shown. The numbers indicate the coordinates of NcoI sites or the locations of the specified genes in the GenBank entries for each strain. Strain AYE, locus tag ABAYE0369; strain AB0057, locus tag AB57_3570; strain AB307-0294, locus tag ABBFA_00039; strain ATCC 17978, locus tag A1S_3117.

Other genes of interest with respect to drug resistance were also found in the genome of A. baumannii A118, such as blaOXA-51-like, which codes for a β-lactamase that has weak catalytic activity against penicillins and carbapenems but not expanded-spectrum cephalosporins (22); blaADC, a gene coding for the noninducible ADC cephalosporinase that has been named blaADC-55 according to the nomenclature proposed elsewhere (8, 23); carO, a gene coding for CarO, an outer membrane protein that participates in the influx of carbapenems (29); and pbp2, a gene coding for the key protein, PBP 2, which leads to carbapenem resistance when it is expressed at low levels (12).

Comparison of the optical map of strain A118 and the in silico-generated NcoI restriction maps of A. baumannii genomes at the region where carO is located showed that all six genomes were similar (Fig. 4a). Therefore, it was expected that the gene was present in strain A118. The nucleotide sequence confirmed this expectation, and the amino acid sequences of all CarO proteins were highly related (Fig. 4b). The results of optical map comparison in the case of the pbp2 gene were not as straightforward. A first look at the comparison showed an apparent deletion (Fig. 5). However, nucleotide sequencing showed that there is a complete copy of the gene in A. baumannii A118 but that it includes a number of point mutations that are silent and do not result in amino acid changes (data not shown). Two NcoI restriction sites are not present in the strain A118 version of the gene due to two of these point mutations, and a third one was not detected by the optical mapping, which resulted in an apparent missing fragment inside the gene sequence. Interestingly, these results are in agreement with those of a recent analysis of penicillin-binding proteins (PBPs) in all the A. baumannii genomes deposited in GenBank that showed that several point mutations were present but that >90% of them were silent (4). Carbapenem resistance in A. baumannii has been reported to be due to one or a combination of the following factors: enzymatic modification by β-lactamases of different classes, a decrease in permeability as a consequence of alterations in the structure and number of porins, the presence of efflux pumps, and changes in the structure or expression of PBPs (30). In particular, a recent study of strains isolated from blood samples in a hospital in Spain found that PBP 2 was expressed at very low levels in a group of A. baumannii strains highly resistant to carbapenems (imipenem and meropenem) compared to the expression level in another group that showed significantly higher susceptibility to these antibiotics (12). Our results suggest that PBP 2 is present in A. baumannii A118, but since the levels of expression are not known, the role of this protein in the susceptible phenotype remains undetermined.

FIG. 4.

FIG. 4.

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Genomic comparison. (a) Comparison of the A. baumannii A118 optical map with the A. baumannii strain ATCC 17978, ACICU, AB307-0294, AB0057, and AYE NcoI restriction maps obtained in silico at the location of the carO gene using the MapSolver software. The locations of carO and relevant NcoI sites are shown. The numbers indicate the coordinates of NcoI sites or the locations of the specified genes in the GenBank entries for each strain. (b) CLUSTAL W comparison of CarO amino acid sequences.

FIG. 5.

FIG. 5.

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Genomic comparison. Comparison of the A. baumannii A118 optical map with the A. baumannii strain ATCC 17978, ACICU, AB307-0294, AB0057, and AYE NcoI restriction maps obtained in silico at a fragment of the pbp2 gene using the MapSolver software. White fragments represent putative missing/inserted fragments. The locations of relevant NcoI sites are shown. The numbers indicate the coordinates of NcoI sites or the locations of the specified genes in the GenBank entries for each strain.

The comparative analysis at the region where blaADC is located exhibited similar patterns with minor differences (Fig. 6a). The presence of this gene in strain A118 was confirmed by sequencing. Figure 6b shows the CLUSTAL W comparison of ADC proteins from complete A. baumannii genomes, which are highly homologous. A detailed analysis and discussion of A. baumannii ADC proteins has recently been published (35). A factor contributing to the high susceptibility of strain A118 to expanded-spectrum cephalosporins and to carbapenems, in spite of harboring blaADC-55 and the blaOXA-51-like gene blaOXA-89 (28, 33), may be the lack of copies of ISAba1 or ISAba9 (27) upstream of the structural genes to provide a strong promoter necessary for high levels of expression (8, 13).

FIG. 6.

FIG. 6.

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Genomic comparison. (a) Comparison of the A. baumannii A118 optical map with the A. baumannii strain ATCC 17978, ACICU, AB307-0294, AB0057, and AYE NcoI restriction maps obtained in silico at the location of the blaADC gene using the MapSolver software. The locations of blaADC and relevant NcoI sites are shown. The numbers indicate the coordinates of NcoI sites or the locations of the specified genes in the GenBank entries for each strain. (b) CLUSTAL W comparison of amino acid sequences.

Concluding remarks.

Our work shows that the comparative analysis of optical maps is of help for an initial comparative analysis of a genome but that the results must be further confirmed by other means, such as amplification and sequencing of the regions in question. A. baumannii A118 is susceptible to several antibiotics. A distinguishing characteristic that we found in this preliminary study is the lack of the AbaR-type resistance island region and the tet(A) gene. In addition, two of the genes that may be responsible for resistance to carbapenems and to expanded-spectrum cephalosporins in other strains, a blaOXA51-like gene and the blaADC gene, lack the insertion sequences described to provide a promoter for significant expression. Other genes present in multidrug-resistant strains, such as carO, pbp2, and cat, are present in strain A118. These results partially explain the susceptible nature of strain A118 but also indicate that drug resistance in A. baumannii is a complex process where many factors influence the phenotype, including the presence of genes coding for different functions that contribute to resistance or susceptibility to a given antibiotic as well as their level of expression. Further studies, including analysis of the complete A. baumannii A118 genome sequence, when it is available, will permit us to better understand the factors responsible for its susceptibility phenotype. Furthermore, a long-term project consisting of systematic gene deletion in multiresistant strains, an approach successfully used in the past with other bacteria (11, 25), could reveal genes involved in resistance that had not been considered as such in the past.

Acknowledgments

This study was supported by Public Health Service grant 2R15AI047115 (to M.E.T.) from the National Institutes of Health and grant PICT 0354 (to M.S.R.). R.A.B. was supported by a Merit Review Award from the U.S. Department of Veterans Affairs and grants from the National Institutes of Health (NIH/NIAID AI072219 and AI063517). M.S.R. and D.C. are career investigators of CONICET.

Footnotes

▿

Published ahead of print on 31 January 2011.

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