All Title Author
Keywords Abstract

PLOS ONE  2013 

Whole Transcriptome Analysis of Acinetobacter baumannii Assessed by RNA-Sequencing Reveals Different mRNA Expression Profiles in Biofilm Compared to Planktonic Cells

DOI: 10.1371/journal.pone.0072968

Full-Text   Cite this paper   Add to My Lib


Acinetobacter baumannii has emerged as a dangerous opportunistic pathogen, with many strains able to form biofilms and thus cause persistent infections. The aim of the present study was to use high-throughput sequencing techniques to establish complete transcriptome profiles of planktonic (free-living) and sessile (biofilm) forms of A. baumannii ATCC 17978 and thereby identify differences in their gene expression patterns. Collections of mRNA from planktonic (both exponential and stationary phase cultures) and sessile (biofilm) cells were sequenced. Six mRNA libraries were prepared following the mRNA-Seq protocols from Illumina. Reads were obtained in a HiScanSQ platform and mapped against the complete genome to describe the complete mRNA transcriptomes of planktonic and sessile cells. The results showed that the gene expression pattern of A. baumannii biofilm cells was distinct from that of planktonic cells, including 1621 genes over-expressed in biofilms relative to stationary phase cells and 55 genes expressed only in biofilms. These differences suggested important changes in amino acid and fatty acid metabolism, motility, active transport, DNA-methylation, iron acquisition, transcriptional regulation, and quorum sensing, among other processes. Disruption or deletion of five of these genes caused a significant decrease in biofilm formation ability in the corresponding mutant strains. Among the genes over-expressed in biofilm cells were those in an operon involved in quorum sensing. One of them, encoding an acyl carrier protein, was shown to be involved in biofilm formation as demonstrated by the significant decrease in biofilm formation by the corresponding knockout strain. The present work serves as a basis for future studies examining the complex network systems that regulate bacterial biofilm formation and maintenance.


[1]  Corbella X, Montero A, Pujol M, Domínguez MA, Ayats J et al. (2000) Emergence and rapid spread of carbapenem resistance during a large and sustained hospital outbreak of multiresistant Acinetobacter baumannii. J Clin Microbiol 38: 4086-4095. PubMed: 11060073.
[2]  del Mar Tomas M, Cartelle M, Pertega S, Beceiro A, Llinares P et al. (2005) Hospital outbreak caused by a carbapenem-resistant strain of Acinetobacter baumannii: patient prognosis and risk-factors for colonisation and infection. Clin Microbiol Infect 11: 540-546. doi:10.1111/j.1469-0691.2005.01184.x. PubMed: 15966971.
[3]  Domenech de Cellès M, Salomon J, Marinier A, Lawrence C, Gaillard JL et al. (2012) Identifying more epidemic clones during a hospital outbreak of multidrug-resistant Acinetobacter baumannii. PLOS ONE 7: e45758. doi:10.1371/journal.pone.0045758. PubMed: 23029226.
[4]  Montero A, Ariza J, Corbella X, Doménech A, Cabellos C et al. (2002) Efficacy of colistin versus beta-lactams, aminoglycosides, and rifampin as monotherapy in a mouse model of pneumonia caused by multiresistant Acinetobacter baumannii. Antimicrob Agents Chemother 46: 1946-1952. doi:10.1128/AAC.46.6.1946-1952.2002. PubMed: 12019113.
[5]  Raad II, Mohamed JA, Reitzel RA, Jiang Y, Dvorak TL et al. (2011) The prevention of biofilm colonization by multidrug-resistant pathogens that cause ventilator-associated pneumonia with antimicrobial-coated endotracheal tubes. Biomaterials 32: 2689-2694. doi:10.1016/j.biomaterials.2010.12.015. PubMed: 21295343.
[6]  Jones ME, Draghi DC, Thornsberry C, Karlowsky JA, Sahm DF et al. (2004) Emerging resistance among bacterial pathogens in the intensive care unit--a European and North American Surveillance study (2000-2002). Ann Clin Microbiol Antimicrob 3: 14. doi:10.1186/1476-0711-3-14. PubMed: 15283864.
[7]  Ong CW, Lye DC, Khoo KL, Chua GS, Yeoh SF et al. (2009) Severe community-acquired Acinetobacter baumannii pneumonia: an emerging highly lethal infectious disease in the Asia-Pacific. Respirology 14: 1200-1205. doi:10.1111/j.1440-1843.2009.01630.x. PubMed: 19909464.
[8]  Bou G, Cerveró G, Domínguez MA, Quereda C, Martínez-Beltrán J (2000) PCR-based DNA fingerprinting (REP-PCR, AP-PCR) and pulsed-field gel electrophoresis characterization of a nosocomial outbreak caused by imipenem- and meropenem-resistant Acinetobacter baumannii. Clin Microbiol Infect 6: 635-643. doi:10.1046/j.1469-0691.2000.00181.x. PubMed: 11284921.
[9]  Dijkshoorn L, Nemec A, Seifert H (2007) An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat Rev Microbiol 5: 939-951. doi:10.1038/nrmicro1789. PubMed: 18007677.
[10]  Santillana E, Beceiro A, Bou G, Romero A (2007) Crystal structure of the carbapenemase OXA-24 reveals insights into the mechanism of carbapenem hydrolysis. Proc Natl Acad Sci U S A 104: 5354-5359. doi:10.1073/pnas.0607557104. PubMed: 17374723.
[11]  Higgins PG, Dammhayn C, Hackel M, Seifert H (2010) Global spread of carbapenem-resistant Acinetobacter baumannii. J Antimicrob Chemother 65: 233-238. doi:10.1093/jac/dkp428. PubMed: 19996144.
[12]  De la Cruz MA, Calva E (2010) The complexities of porin genetic regulation. J Mol Microbiol Biotechnol 18: 24-36. doi:10.1159/000274309. PubMed: 20068355.
[13]  Fernández-Cuenca F, Martínez-Martínez L, Conejo MC, Ayala JA, Perea EJ et al. (2003) Relationship between β-lactamase production, outer membrane protein and penicillin-binding protein profiles on the activity of carbapenems against clinical isolates of Acinetobacter baumannii. J Antimicrob Chemother 51: 565-574. doi:10.1093/jac/dkg097. PubMed: 12615856.
[14]  Srinivasan VB, Rajamohan G, Gebreyes WA (2009) Role of AbeS, a novel efflux pump of the SMR family of transporters, in resistance to antimicrobial agents in Acinetobacter baumannii. Antimicrob Agents Chemother 53: 5312-5316. doi:10.1128/AAC.00748-09. PubMed: 19770280.
[15]  Roca I, Marti S, Espinal P, Martínez P, Gibert I et al. (2009) CraA, a major facilitator superfamily efflux pump associated with chloramphenicol resistance in Acinetobacter baumannii. Antimicrob Agents Chemother 53: 4013-4014. doi:10.1128/AAC.00584-09. PubMed: 19581458.
[16]  Coyne S, Guigon G, Courvalin P, Périchon B (2010) Screening and quantification of the expression of antibiotic resistance genes in Acinetobacter baumannii with a microarray. Antimicrob Agents Chemother 54: 333-340. doi:10.1128/AAC.01037-09. PubMed: 19884373.
[17]  Rajamohan G, Srinivasan VB, Gebreyes WA (2010) Novel role of Acinetobacter baumannii RND efflux transporters in mediating decreased susceptibility to biocides. J Antimicrob Chemother 65: 228-232. doi:10.1093/jac/dkp427. PubMed: 20008046.
[18]  Costerton JW, Stewart PS, Greenberg EP (1999) Bacterial biofilms: a common cause of persistent infections. Science 284: 1318-1322. doi:10.1126/science.284.5418.1318. PubMed: 10334980.
[19]  Moreno-Paz M, Gómez MJ, Arcas A, Parro V (2010) Environmental transcriptome analysis reveals physiological differences between biofilm and planktonic modes of life of the iron oxidizing bacteria Leptospirillum spp. in their natural microbial community. BMC Genomics 11: 404. doi:10.1186/1471-2164-11-404. PubMed: 20576116.
[20]  Vidal R, Dominguez M, Urrutia H, Bello H, Gonzalez G et al. (1996) Biofilm formation by Acinetobacter baumannii. Microbios 86: 49-58. PubMed: 8771775.
[21]  Lee HW, Koh YM, Kim J, Lee JC, Lee YC et al. (2008) Capacity of multidrug-resistant clinical isolates of Acinetobacter baumannii to form biofilm and adhere to epithelial cell surfaces. Clin Microbiol Infect 14: 49-54. doi:10.1111/j.1469-0691.2007.01842.x. PubMed: 18005176.
[22]  Rodríguez-Ba?o J, Martí S, Soto S, Fernández-Cuenca F, Cisneros JM et al. (2008) Biofilm formation in Acinetobacter baumannii: associated features and clinical implications. Clin Microbiol Infect 14: 276-278. doi:10.1111/j.1469-0691.2007.01916.x. PubMed: 18190568.
[23]  Martí S, Rodríguez-Ba?o J, Catel-Ferreira M, Jouenne T, Vila J et al. (2011) Biofilm formation at the solid-liquid and air-liquid interfaces by Acinetobacter species. BMC Res Notes 4: 5. doi:10.1186/1756-0500-4-5. PubMed: 21223561.
[24]  Espinal P, Martí S, Vila J (2012) Effect of biofilm formation on the survival of Acinetobacter baumannii on dry surfaces. J Hosp Infect 80: 56-60. doi:10.1016/j.jhin.2011.08.013. PubMed: 21975219.
[25]  Tomaras AP, Dorsey CW, Edelmann RE, Actis LA (2003) Attachment to and biofilm formation on abiotic surfaces by Acinetobacter baumannii: involvement of a novel chaperone-usher pili assembly system. Microbiology 149: 3473-3484. doi:10.1099/mic.0.26541-0. PubMed: 14663080.
[26]  Gaddy JA, Actis LA (2009) Regulation of Acinetobacter baumannii biofilm formation. Future Microbiol 4: 273-278. doi:10.2217/fmb.09.5. PubMed: 19327114.
[27]  Tomaras AP, Flagler MJ, Dorsey CW, Gaddy JA, Actis LA (2008) Characterization of a two-component regulatory system from Acinetobacter baumannii that controls biofilm formation and cellular morphology. Microbiology 154: 3398-3409. doi:10.1099/mic.0.2008/019471-0. PubMed: 18957593.
[28]  Petrova OE, Sauer K (2009) A novel signaling network essential for regulating Pseudomonas aeruginosa biofilm development. PLOS Pathog 5: e1000668.
[29]  Loehfelm TW, Luke NR, Campagnari AA (2008) Identification and characterization of an Acinetobacter baumannii biofilm-associated protein. J Bacteriol 190: 1036-1044. doi:10.1128/JB.01416-07. PubMed: 18024522.
[30]  Gaddy JA, Tomaras AP, Actis LA (2009) The Acinetobacter baumannii 19606 OmpA protein plays a role in biofilm formation on abiotic surfaces and in the interaction of this pathogen with eukaryotic cells. Infect Immun 77: 3150-3160. doi:10.1128/IAI.00096-09. PubMed: 19470746.
[31]  McConnell MJ, Actis L, Pachón J (2013) Acinetobacter baumannii: human infections, factors contributing to pathogenesis and animal models. FEMS Microbiol Rev 37: 130-155. PubMed: 22568581.
[32]  Hamilton S, Bongaerts RJ, Mulholland F, Cochrane B, Porter J et al. (2009) The transcriptional programme of Salmonella enterica serovar Typhimurium reveals a key role for tryptophan metabolism in biofilms. BMC Genomics 10: 599. doi:10.1186/1471-2164-10-599. PubMed: 20003355.
[33]  Valle J, Da Re S, Schmid S, Skurnik D, D’Ari R et al. (2008) The amino acid valine is secreted in continuous-flow bacterial biofilms. J Bacteriol 190: 264-274. doi:10.1128/JB.01405-07. PubMed: 17981982.
[34]  Cabral MP, Soares NC, Aranda J, Parreira JR, Rumbo C et al. (2011) Proteomic and functional analyses reveal a unique lifestyle for Acinetobacter baumannii biofilms and a key role for histidine metabolism. J Proteome Res 10: 3399-3417. doi:10.1021/pr101299j. PubMed: 21612302.
[35]  Kolodkin-Gal I, Romero D, Cao S, Clardy J, Kolter R et al. (2010) D-amino acids trigger biofilm disassembly. Science 328: 627-629. doi:10.1126/science.1188628. PubMed: 20431016.
[36]  Veb? HC, Snipen L, Nes IF, Brede DA (2009) The transcriptome of the nosocomial pathogen Enterococcus faecalis V583 reveals adaptive responses to growth in blood. PLOS ONE 4: e7660. doi:10.1371/journal.pone.0007660. PubMed: 19888459.
[37]  Toledo-Arana A, Dussurget O, Nikitas G, Sesto N, Guet-Revillet H et al. (2009) The Listeria transcriptional landscape from saprophytism to virulence. Nature 459: 950-956. doi:10.1038/nature08080. PubMed: 19448609.
[38]  Whiteley M, Bangera MG, Bumgarner RE, Parsek MR, Teitzel GM et al. (2001) Gene expression in Pseudomonas aeruginosa biofilms. Nature 413: 860-864. doi:10.1038/35101627. PubMed: 11677611.
[39]  Hood MI, Jacobs AC, Sayood K, Dunman PM, Skaar EP (2010) Acinetobacter baumannii increases tolerance to antibiotics in response to monovalent cations. Antimicrob Agents Chemother 54: 1029-1041. doi:10.1128/AAC.00963-09. PubMed: 20028819.
[40]  Eijkelkamp BA, Hassan KA, Paulsen IT, Brown MH (2011) Investigation of the human pathogen Acinetobacter baumannii under iron limiting conditions. BMC Genomics 12: 126. doi:10.1186/1471-2164-12-126. PubMed: 21342532.
[41]  Mao C, Evans C, Jensen RV, Sobral BW (2008) Identification of new genes in Sinorhizobium meliloti using the Genome Sequencer FLX system. BMC Microbiol 8: 72. doi:10.1186/1471-2180-8-72. PubMed: 18454850.
[42]  Droege M, Hill B (2008) The Genome Sequencer FLX System--longer reads, more applications, straight forward bioinformatics and more complete data sets. J Biotechnol 136: 3-10. doi:10.1016/j.jbiotec.2008.07.1845. PubMed: 18616967.
[43]  Sittka A, Lucchini S, Papenfort K, Sharma CM, Rolle K et al. (2008) Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq. PLOS Genet 4: e1000163.
[44]  Liu JM, Livny J, Lawrence MS, Kimball MD, Waldor MK et al. (2009) Experimental discovery of sRNAs in Vibrio cholerae by direct cloning, 5S/tRNA depletion and parallel sequencing. Nucleic Acids Res 37: e46. doi:10.1093/nar/gkp080. PubMed: 19223322.
[45]  Camarena L, Bruno V, Euskirchen G, Poggio S, Snyder M (2010) Molecular mechanisms of ethanol-induced pathogenesis revealed by RNA-sequencing. PLOS Pathog 6: e1000834. PubMed: 20368969.
[46]  Smith MG, Gianoulis TA, Pukatzki S, Mekalanos JJ, Ornston LN et al. (2007) New insights into Acinetobacter baumannii pathogenesis revealed by high-density pyrosequencing and transposon mutagenesis. Genes Dev 21: 601-614. doi:10.1101/gad.1510307. PubMed: 17344419.
[47]  J?ger D, Sharma CM, Thomsen J, Ehlers C, Vogel J et al. (2009) Deep sequencing analysis of the Methanosarcina mazei G?1 transcriptome in response to nitrogen availability. Proc Natl Acad Sci U S A 106: 21878-21882. doi:10.1073/pnas.0909051106. PubMed: 19996181.
[48]  Sharma CM, Hoffmann S, Darfeuille F, Reignier J, Findeiss S et al. (2010) The primary transcriptome of the major human pathogen Helicobacter pylori. Nature 464: 250-255. doi:10.1038/nature08756. PubMed: 20164839.
[49]  Filiatrault MJ, Stodghill PV, Bronstein PA, Moll S, Lindeberg M et al. (2010) Transcriptome analysis of Pseudomonas syringae identifies new genes, noncoding RNAs, and antisense activity. J Bacteriol 192: 2359-2372. doi:10.1128/JB.01445-09. PubMed: 20190049.
[50]  D?tsch A, Eckweiler D, Schniederjans M, Zimmermann A, Jensen V et al. (2012) The Pseudomonas aeruginosa transcriptome in planktonic cultures and static biofilms using RNA sequencing. PLOS ONE 7: e31092. doi:10.1371/journal.pone.0031092. PubMed: 22319605.
[51]  Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10: R25. doi:10.1186/gb-2009-10-3-r25. PubMed: 19261174.
[52]  Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11: R106. doi:10.1186/gb-2010-11-10-r106. PubMed: 20979621.
[53]  G?tz S, García-Gómez JM, Terol J, Williams TD, Nagaraj SH et al. (2008) High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res 36: 3420-3435. doi:10.1093/nar/gkn176. PubMed: 18445632.
[54]  Tendolkar PM, Baghdayan AS, Gilmore MS, Shankar N (2004) Enterococcal surface protein, Esp, enhances biofilm formation by Enterococcus faecalis. Infect Immun 72: 6032-6039. doi:10.1128/IAI.72.10.6032-6039.2004. PubMed: 15385507.
[55]  Héritier C, Poirel L, Lambert T, Nordmann P (2005) Contribution of acquired carbapenem-hydrolyzing oxacillinases to carbapenem resistance in Acinetobacter baumannii. Antimicrob Agents Chemother 49: 3198-3202. doi:10.1128/AAC.49.8.3198-3202.2005. PubMed: 16048925.
[56]  Hamad MA, Zajdowicz SL, Holmes RK, Voskuil MI (2009) An allelic exchange system for compliant genetic manipulation of the select agents Burkholderia pseudomallei and Burkholderia mallei. Gene 430: 123-131. doi:10.1016/j.gene.2008.10.011. PubMed: 19010402.
[57]  Aranda J, Poza M, Pardo BG, Rumbo S, Rumbo C et al. (2010) A rapid and simple method for constructing stable mutants of Acinetobacter baumannii. BMC Microbiol 10: 279. doi:10.1186/1471-2180-10-279. PubMed: 21062436.
[58]  Soares NC, Cabral MP, Gayoso C, Mallo S, Rodriguez-Velo P et al. (2010) Associating growth-phase-related changes in the proteome of Acinetobacter baumannii with increased resistance to oxidative stress. J Proteome Res 9: 1951-1964. doi:10.1021/pr901116r. PubMed: 20108952.
[59]  Petrova OE, Schurr JR, Schurr MJ, Sauer K (2011) The novel Pseudomonas aeruginosa two-component regulator BfmR controls bacteriophage-mediated lysis and DNA release during biofilm development through PhdA. Mol Microbiol 81: 767-783. doi:10.1111/j.1365-2958.2011.07733.x. PubMed: 21696457.
[60]  Liou ML, Soo PC, Ling SR, Kuo HY, Tang CY et al. (2013) The sensor kinase BfmS mediates virulence in Acinetobacter baumannii. J Microbiol Immunol Infect. PubMed: 23453128. In press.
[61]  McQueary CN, Actis LA (2011) Acinetobacter baumannii biofilms: variations among strains and correlations with other cell properties. J Microbiol 49: 243-250. doi:10.1007/s12275-011-0343-7. PubMed: 21538245.
[62]  Gordon NC, Wareham DW (2010) Multidrug-resistant Acinetobacter baumannii: mechanisms of virulence and resistance. Int J Antimicrob Agents 35: 219-226. doi:10.1016/j.ijantimicag.2009.10.024. PubMed: 20047818.
[63]  Marti S, Nait Chabane Y, Alexandre S, Coquet L, Vila J et al. (2011) Growth of Acinetobacter baumannii in pellicle enhanced the expression of potential virulence factors. PLOS ONE 6: e26030. doi:10.1371/journal.pone.0026030. PubMed: 22046254.
[64]  de Breij A, Gaddy J, van der Meer J, Koning R, Koster A et al. (2009) CsuA/BABCDE-dependent pili are not involved in the adherence of Acinetobacter baumannii ATCC19606(T) to human airway epithelial cells and their inflammatory response. Res Microbiol 160: 213-218. doi:10.1016/j.resmic.2009.01.002. PubMed: 19530313.
[65]  Domka J, Lee J, Bansal T, Wood TK (2007) Temporal gene-expression in Escherichia coli K-12 biofilms. Environ Microbiol 9: 332-346. doi:10.1111/j.1462-2920.2006.01143.x. PubMed: 17222132.
[66]  Camilli A, Bassler BL (2006) Bacterial small-molecule signaling pathways. Science 311: 1113-1116. doi:10.1126/science.1121357. PubMed: 16497924.
[67]  Anbazhagan D, Mansor M, Yan GO, Yusof MY, Hassan H, et al. (2012) Detection of quorum sensing signal molecules and identification of an autoinducer synthase gene among biofilm forming clinical isolates of Acinetobacter spp. PLOS ONE 7: e36696.
[68]  Clemmer KM, Bonomo RA, Rather PN (2011) Genetic analysis of surface motility in Acinetobacter baumannii. Microbiology 157: 2534-2544. doi:10.1099/mic.0.049791-0. PubMed: 21700662.
[69]  Saroj SD, Rather PN (2013) Streptomycin inhibits quorum sensing in Acinetobacter baumannii. Antimicrob Agents Chemother 57: 1926-1929. doi:10.1128/AAC.02161-12. PubMed: 23318804.
[70]  Niu C, Clemmer KM, Bonomo RA, Rather PN (2008) Isolation and characterization of an autoinducer synthase from Acinetobacter baumannii. J Bacteriol 190: 3386-3392. doi:10.1128/JB.01929-07. PubMed: 18281398.
[71]  McAllister KA, Peery RB, Zhao G (2006) Acyl carrier protein synthases from gram-negative, gram-positive, and atypical bacterial species: Biochemical and structural properties and physiological implications. J Bacteriol 188: 4737-4748. doi:10.1128/JB.01917-05. PubMed: 16788183.
[72]  Moré MI, Finger LD, Stryker JL, Fuqua C, Eberhard A et al. (1996) Enzymatic synthesis of a quorum-sensing autoinducer through use of defined substrates. Science 272: 1655-1658. doi:10.1126/science.272.5268.1655. PubMed: 8658141.


comments powered by Disqus