Ramirez 2011 Optical Mapping of Ab

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2011, p. 15201526 Vol. 55, No. 40066-4804/11/$12.00 doi:10.1128/AAC.01595-10Copyright 2011, American Society for Microbiology. All Rights Reserved.

Genomic Analysis of Acinetobacter baumannii A118 by Comparisonof Optical Maps: Identification of Structures Related to

Its Susceptibility Phenotype

Maria Soledad Ramirez,1,2 Mark D. Adams,3 Robert A. Bonomo,4Daniela Centron,2 and Marcelo E. Tolmasky1*

Center for Applied Biotechnology Studies, Department of Biological Science, California State University Fullerton, Fullerton,California1; Departamento de Microbiologa, Facultad de Medicina, UBA, Buenos Aires, Argentina2; Department of

Genetics, Case Western Reserve University School of Medicine, Cleveland, Ohio3; and Departments of Pharmacology andMolecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio, and

Louis Stokes Cleveland Department of Veteran Affairs Medical Center, Cleveland, Ohio4

Received 17 November 2010/Returned for modification 2 January 2011/Accepted 23 January 2011

Acinetobacter baumannii A118, a naturally competent clinical isolate, is unusually susceptible to severalantibiotics. Comparison of the optical map of strain A118 with in silico-generated restriction maps of sequencedgenomes and sequence analyses showed that the AbaR region, commonly found inserted within the comM genein other isolates, is missing in strain A118, which could in part explain the susceptible phenotype exhibited bythis isolate. These comparative studies also showed differences in regions where genes coding for functions thatmay be involved in drug resistance or susceptibility are located. Further sequencing demonstrated that cat andblaADC, 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. Thesefindings support the idea that A. baumannii strains possess multiple mechanisms that contribute to antibioticresistance, and the presence of some of them is not sufficient for a resistant phenotype. The results shown hereindicate that optical mapping is a useful tool for preliminary comparative genomic analysis.

Acinetobacter baumannii is an emerging opportunistic hu-man pathogen responsible for a growing number of nosocomialinfections mainly affecting patients who are immunosup-pressed, who suffer other underlying diseases, or who havebeen treated using certain invasive procedures (20, 26, 30). Theincidence of A. baumannii is steadily growing, and a studyindicates that while in 1975 this bacterium was responsible for1.5% of hospital-acquired pneumonia cases, in 2003 that num-ber 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 indifferent environments and a rise in the number of susceptibleindividuals as a result of advancements in medical support ofcritically ill patients. The ability of A. baumannii to form bio-films has also been related to commonly occurring infectionsassociated with medical devices (15, 34). Recent studies iden-tified several other virulence factors and pathogenic islands (6,38, 41). A. baumannii infections have also gained attention dueto the high number of soldiers serving in Iraq and Afghanistanand victims of the 2004 Asian tsunami who were infected withthis bacterium (9, 16, 18). Treatment of Acinetobacter infec-tions is becoming increasingly difficult due to the growing num-ber 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 makesthem difficult to manipulate for genetic studies.

A. baumannii A118, isolated from a culture of blood from apatient admitted to an intensive care unit in a hospital inBuenos Aires, Argentina, is rather exceptional for its suscep-tibility to antibiotics such as ceftazidime, cefepime, piperacil-lin, minocycline, amikacin, gentamicin, trimethoprim-sulfame-thoxazole, kanamycin, and ciprofloxacin (32). This property,together with its natural competence, led to the suggestion thatthis strain is a convenient model for genetic studies (33). In thiswork we analyzed A. baumannii A118 genomic regions knownfor containing potential resistance or susceptibility determi-nants in previously studied strains using optical mapping, apowerful tool for comparative genomics (37). Optical maps arefull-genome restriction maps obtained after single DNA mol-ecules are immobilized on a charged substrate and digestedwith the restriction endonuclease of interest, followed by de-tection and assembly into a high-resolution ordered full-ge-nome restriction map (5). Our results show that the AbaR-typeresistance island is missing and suggest that A. baumannii re-sistance to a variety of antibiotics may be due to a combinationof mechanisms, some of which are present in strain A118 butwhich are not sufficient to confer a resistance phenotype.

MATERIALS AND METHODS

Bacterial strains and genomes. A. baumannii A118 is a bloodstream isolaterecovered from a patient in an intensive care unit (28, 33). The available ge-nomes 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 ac-

* Corresponding author. Mailing address: Center for Applied Bio-technology Studies, Department of Biological Science, California StateUniversity Fullerton, 800 N. State College Boulevard, Fullerton, CA92831-3599. Phone: (657) 278-5263. Fax: (657) 278-3426. E-mail:[email protected].

Published ahead of print on 31 January 2011.

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cession no. CP000863; ATCC 17978, GenBank accession no. CP000521; andSDF, 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). Com-parative genomic analysis was carried out by comparing the optical map of A.baumannii A118 to NcoI restriction maps of A. baumannii sequenced genomesusing the MapSolver software (version 2.1.1; OpGen Technologies, Inc.). PCRswere carried out using the Qiagen Taq master mix, and the products weredetected by agarose gel electrophoresis. Cloning into pCR2.1 was performed asrecommended by the supplier (Invitrogen). DNA sequencing reactions usingamplicons as templates were done at the City of Hope sequencing facility.Genomic DNA was prepared for genome sequencing using a Nextera kit fromEpicentre Biotechnologies. Sequencing was performed on an Illumina IIx genomeanalyzer using paired 76-base reads, resulting in 1,712,408 read pairs. These wereassembled using the assembly program Velvet (40), resulting in 186 scaffolds that areat least 500 bases long. The scaffold N50 size is 39.3 kbp, meaning that half of thegenome is assembled into scaffolds of at least this length. The total assembled lengthis 3,824 kbp. Genome annotation was performed using the ISGA web server (21).Amino acid sequence comparisons were performed using the CLUSTAL W pro-gram (Pole 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 numberAEOW00000000.

RESULTS AND DISCUSSION

Regions relevant to the antibiotic susceptibility characteris-tics of A. baumannii A118 were studied using optical mapping,a technique that is based on immobilization of single DNAmolecules on a charged substrate, digestion with a restrictionendonuclease, and detection and assembly into a high-resolu-tion ordered restriction map permitting comparison of relatedgenomes (3). The estimated size of the A. baumannii A118chromosome 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 genomesequence is known include a region that has transposed or in-serted into a specific location within comM, a gene that codes fora 495-amino-acid protein that includes an ATPase domain (1, 14).In addition, analysis of other A. baumannii strains showed thatmost of them also carry a related insertion (31, 36). These in-serted regions are usually characterized by the presence of trans-posase and antibiotic or heavy metal resistance genes and areknown to be AbaR-type resistance island regions (1, 31). Com-parison of in silico-generated restriction maps of A. baumanniisequenced genomes with the optical map of A. baumannii A118indicated that there was no AbaR-type resistance island in thisstrain (Fig. 1a). PCR using the primers, designed before by Shaiket al. (36), located within the comM gene and flanking the loca-tion of insertion of AbaR, followed by sequencing of the ampli-con, showed that the comM gene was intact and identical to thegene in A. baumannii AB307-0294, a strain known to lack theAbaR-type resistance island (1) (Fig. 1b). This gene has beennamed comM on the basis of the 49.5% homology found betweenthe proteins from A. baumannii ADP1 and Haemophilus influen-zae (2). Mutagenesis of comM in H. influenzae resulted in a re-duced ability to take up DNA (19). These results are in agree-ment with the susceptible phenotype observed for A. baumanniiA118 and its natural competency.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 asecond cat gene within the AbaR-type resistance island (1, 14).A. baumannii A118 lacks this island, and therefore, this strainmust lack at least one of the cat genes. Comparative analysis ofthe A. baumannii A118 optical map at the region where the catgene present outside the AbaR-type resistance island is locatedin the sequenced strains showed some heterogeneity, with ap-parent insertions and deletions. This is best illustrated by thecomparison of the optical map of strain A118 with the insilico-generated NcoI restriction maps of strains AB0057 andAYE. While comparison of strains A118 and AB0057 suggeststhat the fragment, including cat in A118, although it is notidentical, is present, comparison of strains A118 and AYEsuggests that there is a deletion in strain A118 that includes thefragment where cat should be located. To confirm the presenceof cat in strain A118, an amplicon of 1,261 bp obtained usinga pair of primers located within the flanking greA and uspAgenes was sequenced. The results indicated that the genome ofA. baumannii A118 includes the cat gene with nearly perfectidentity to those present in other A. baumannii strains (strainAYE, locus tag ABAYE0798; strain AB0057, locus tagAB57_3104; strain ATCC 17978, locus tag A1S_2691) (Fig.2b). These results indicate that there must be some variabilityat the nucleotide region that resulted in modifications in theNcoI restriction site patterns that led to the apparent deletionof a DNA fragment in strain A118. We hypothesize that thelower MIC of chloramphenicol exhibited by strain A118 is dueto the absence of the cat gene located within the AbaR-typeresistance island. However, comparison of the MICs of chlor-amphenicol for A. baumannii A118 and ATCC 17978, whichalso lacks the cat present within the AbaR-type resistance

FIG. 1. Genomic comparison. (a) The A. baumannii A118 opticalmap was compared to the A. baumannii strain AB0057 and AYE NcoIrestriction maps obtained in silico around the location of the AbaR-type resistance island region using the MapSolver software. The whiteregions represent DNA fragments missing in strain A118. The AbaR3and AbaR1 regions are the genomic islands present in strains AB0057and AYE (1, 14). Vertical lines represent NcoI restriction sites. (b)Diagram showing the point of insertion of AbaR genomic islandswithin the comM gene, which was shown to be intact in A. baumanniiA118 by sequencing an amplicon generated using total DNA as thetemplate and the primers 5-TCCATTTTACCGCCACTTTC and 5-AATCGATGCGGTCGAGTAAC (36). Nucleotides shown in red aredirectly repeated in those strains where AbaR has been inserted.

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island, showed that they were 12 and 48 g/ml, respectively.Although it is possible that this cat gene contributes to resis-tance to chloramphenicol, it is most likely that other factorsmay also contribute to the overall resistance to chloramphen-icol, of which some must be absent in strain A118.

The genomes of A. baumannii AYE, AB0057, AB307-0294,and ATCC 17978 include a tet(A) gene outside the AbaR-typeresistance island that may be involved in tetracycline resistance(strain AYE, locus tag ABAYE0369; strain AB0057, locus tagAB57_3570; strain AB307-0294, locus tag ABBFA_00039;strain ATCC 17978, locus tag A1S_3117). In addition, strainsAB0057 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 ofstrain A118 and the in silico-generated NcoI restriction mapsof A. baumannii genomes at the location of tet(A) showed that

strains ACICU and A118 have a different pattern than the restof the strains (Fig. 3). To investigate if the differences observedcorrelated with the presence or absence of the tet(A) gene, thesequences of the ACICU, AYE, AB0057, AB307-0294, andATCC 17978 strains were compared among themselves and tothe drafts of strain A118. The results indicated that while thegenes glyS and glyQ were present and highly homologous in allstrains, ACICU and A118 do not include the tet(A) gene;instead, there is a short open reading frame with no homologyto tet(A) upstream of glyQ (Fig. 3). Furthermore, BLAST anal-yses comparing the tet(A) nucleotide sequence or the codedamino acid sequence against the available sequences of strainA118 showed no homology, confirming that this strain lacksthe tet(A) gene.

Other genes of interest with respect to drug resistance werealso found in the genome of A. baumannii A118, such asblaOXA-51-like, which codes for a -lactamase that has weak

FIG. 2. Genomic comparison. (a) Comparison of the A. baumannii A118 optical map with the A. baumannii strain AB0057 and AYE NcoIrestriction maps obtained in silico around the location of the cat gene using the MapSolver software. White fragments represent putativemissing/inserted fragments. The locations of the greA, cat, and uspA genes and relevant NcoI sites are shown. The numbers indicate the coordinatesof NcoI sites or the locations of the specified genes in the GenBank entries for A. baumannii AB0057 and AYE genome annotations (accessionnumbers 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.

1522 RAMIREZ ET AL. ANTIMICROB. AGENTS CHEMOTHER.

catalytic activity against penicillins and carbapenems but not ex-panded-spectrum cephalosporins (22); blaADC, a gene coding forthe noninducible ADC cephalosporinase that has been namedblaADC-55 according to the nomenclature proposed elsewhere (8,23); carO, a gene coding for CarO, an outer membrane proteinthat participates in the influx of carbapenems (29); and pbp2, agene coding for the key protein, PBP 2, which leads to carbap-enem resistance when it is expressed at low levels (12).

Comparison of the optical map of strain A118 and the insilico-generated NcoI restriction maps of A. baumannii ge-nomes at the region where carO is located showed that all sixgenomes were similar (Fig. 4a). Therefore, it was expected thatthe gene was present in strain A118. The nucleotide sequenceconfirmed this expectation, and the amino acid sequences of allCarO proteins were highly related (Fig. 4b). The results ofoptical map comparison in the case of the pbp2 gene were notas straightforward. A first look at the comparison showed anapparent deletion (Fig. 5). However, nucleotide sequencingshowed that there is a complete copy of the gene in A. bau-mannii A118 but that it includes a number of point mutationsthat are silent and do not result in amino acid changes (datanot shown). Two NcoI restriction sites are not present in thestrain A118 version of the gene due to two of these pointmutations, and a third one was not detected by the opticalmapping, which resulted in an apparent missing fragment in-side the gene sequence. Interestingly, these results are inagreement with those of a recent analysis of penicillin-bindingproteins (PBPs) in all the A. baumannii genomes deposited inGenBank that showed that several point mutations were pres-ent but that 90% of them were silent (4). Carbapenem re-

FIG. 3. Genomic comparison. Comparison of the A. baumanniiA118 optical map with the A. baumannii strain ATCC 17978, ACICU,AB307-0294, AB0057, and AYE NcoI restriction maps obtained insilico 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 andAYE strains and the positions of the relevant NcoI sites are shown.The numbers indicate the coordinates of NcoI sites or the locations ofthe specified genes in the GenBank entries for each strain. StrainAYE, locus tag ABAYE0369; strain AB0057, locus tag AB57_3570;strain AB307-0294, locus tag ABBFA_00039; strain ATCC 17978,locus tag A1S_3117.

FIG. 4. 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. Thelocations of carO and relevant NcoI sites are shown. The numbers indicate the coordinates of NcoI sites or the locations of the specified genesin the GenBank entries for each strain. (b) CLUSTAL W comparison of CarO amino acid sequences.

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sistance in A. baumannii has been reported to be due to one ora combination of the following factors: enzymatic modificationby -lactamases of different classes, a decrease in permeabilityas a consequence of alterations in the structure and number ofporins, the presence of efflux pumps, and changes in the struc-ture or expression of PBPs (30). In particular, a recent study ofstrains isolated from blood samples in a hospital in Spain foundthat PBP 2 was expressed at very low levels in a group of A.

baumannii strains highly resistant to carbapenems (imipenemand meropenem) compared to the expression level in anothergroup that showed significantly higher susceptibility to theseantibiotics (12). Our results suggest that PBP 2 is present in A.baumannii A118, but since the levels of expression are notknown, the role of this protein in the susceptible phenotyperemains undetermined.

The comparative analysis at the region where blaADC is located

FIG. 6. 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. Thelocations of blaADC and relevant NcoI sites are shown. The numbers indicate the coordinates of NcoI sites or the locations of the specified genesin the GenBank entries for each strain. (b) CLUSTAL W comparison of amino acid sequences.

FIG. 5. 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. Whitefragments represent putative missing/inserted fragments. The locations of relevant NcoI sites are shown. The numbers indicate the coordinates ofNcoI sites or the locations of the specified genes in the GenBank entries for each strain.

1524 RAMIREZ ET AL. ANTIMICROB. AGENTS CHEMOTHER.

exhibited similar patterns with minor differences (Fig. 6a). Thepresence of this gene in strain A118 was confirmed by sequencing.Figure 6b shows the CLUSTAL W comparison of ADC proteinsfrom complete A. baumannii genomes, which are highly homol-ogous. A detailed analysis and discussion of A. baumannii ADCproteins has recently been published (35). A factor contributingto the high susceptibility of strain A118 to expanded-spectrumcephalosporins and to carbapenems, in spite of harboringblaADC-55 and the blaOXA-51-like gene blaOXA-89 (28, 33), may bethe lack of copies of ISAba1 or ISAba9 (27) upstream of thestructural genes to provide a strong promoter necessary for highlevels of expression (8, 13).Concluding remarks. Our work shows that the comparative

analysis of optical maps is of help for an initial comparativeanalysis of a genome but that the results must be furtherconfirmed by other means, such as amplification and sequenc-ing of the regions in question. A. baumannii A118 is susceptibleto several antibiotics. A distinguishing characteristic that wefound in this preliminary study is the lack of the AbaR-typeresistance island region and the tet(A) gene. In addition, two ofthe genes that may be responsible for resistance to carbapen-ems and to expanded-spectrum cephalosporins in other strains,a blaOXA51-like gene and the blaADC gene, lack the insertionsequences described to provide a promoter for significant ex-pression. Other genes present in multidrug-resistant strains,such as carO, pbp2, and cat, are present in strain A118. Theseresults partially explain the susceptible nature of strain A118but also indicate that drug resistance in A. baumannii is acomplex process where many factors influence the phenotype,including the presence of genes coding for different functionsthat contribute to resistance or susceptibility to a given antibi-otic as well as their level of expression. Further studies, includ-ing analysis of the complete A. baumannii A118 genome se-quence, when it is available, will permit us to better understandthe factors responsible for its susceptibility phenotype. Fur-thermore, a long-term project consisting of systematic genedeletion in multiresistant strains, an approach successfullyused in the past with other bacteria (11, 25), could reveal genesinvolved in resistance that had not been considered as such inthe past.

ACKNOWLEDGMENTS

This study was supported by Public Health Service grant2R15AI047115 (to M.E.T.) from the National Institutes of Health andgrant PICT 0354 (to M.S.R.). R.A.B. was supported by a Merit ReviewAward from the U.S. Department of Veterans Affairs and grants fromthe National Institutes of Health (NIH/NIAID AI072219 andAI063517). M.S.R. and D.C. are career investigators of CONICET.

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