Ann Microbiol (2012) 62:493–500 DOI 10.1007/s13213-011-0281-y ORIGINAL ARTICLE Analysis of the antimicrobial susceptibility of the ionizing radiation-resistant bacterium Deinococcus radiodurans: implications for bioremediation of radioactive waste Haïtham Sghaier & Ons Bouchami & Claus Desler & Hadeer Lazim & Mouldi Saidi & Lene Juel Rasmussen & Assia Ben Hassen Received: 8 March 2011 /Accepted: 6 May 2011 /Published online: 24 May 2011 # Springer-Verlag and the University of Milan 2011 Abstract Deinococcus radiodurans R1 is one of the most tions using Subsystems Technology (RAST), and the resistant bacteria to chronic ionizing radiation ever identi- automatic hierarchical classification of proteins (ProtoNet) fied. Therefore, Deinococcus radiodurans shows promise servers. Among 19 retrieved sequences of D. radiodurans, in the bioremediation of mixed radioactive wastes. Nuclear six genes were functionally re-annotated as antimicrobial wastes have been demonstrated to host bacteria containing resistance genes, and only two genes were found to possess genes for antimicrobial production (Bagwell et al. PloS the radiation/desiccation response motif (RDRM). In vitro, ONE 3:e3878, 2008; Bai et al. Chem Biol 13:387–397, we found that D. radiodurans is sensitive to 40 tested 2006; Phillips et al. Int J Syst Evol Microbiol 52:933–938, antimicrobials, and it was resistant to one macrolide 2002), which may represent a constraint on the bioremedi- (spiramycin) and to three quinolones (ofloxacin, ciproflox- ation potential of Deinococcus radiodurans. In this context, acin, and nalidixic acid). The resistance of D. radiodurans the aim of this work is to investigate the antimicrobial to these quinolones was discussed based on different susceptibility of D. radiodurans through in silico and in possibilities. Moreover, in silico analyses indicated that the vitro approaches. In silico, we initiated our analyses by investigated 19 genes of D. radiodurans from ARDB, RAST, retrieving genes that are predicted to confer to D. radio- and ProtoNet encode translatable messenger ribonucleic durans resistance to antimicrobials based on the Antibiotic acids (mRNAs) related to resistance to antimicrobials. These Resistance Genes Database (ARDB), the Rapid Annota- data together with the antibiograms of D. radiodurans represent a suggestive evidence that the majority of these genes encode non-functional proteins or belong to inefficient H. Sghaier (*) : H. Lazim : M. Saidi biochemical pathways. The susceptibility of D. radiodurans Research Unit UR04CNSTN01 “Medical and Agricultural to these antimicrobials limits its potential for bioremediation Applications of Nuclear Techniques”, of nuclear waste and further genetic engineering is needed National Center for Nuclear Sciences and Technology (CNSTN), depending on the site to be depolluted. Sidi Thabet Technopark, 2020 Sidi Thabet, Tunisia e-mail: [email protected] Keywords Antimicrobial susceptibility. Bioremediation . Deinococcus radiodurans . Radioactive waste H. Sghaier e-mail: [email protected] : O. Bouchami A. Ben Hassen Introduction Laboratory of the National Bone Marrow Transplantation Centre, Street Djebel-Lakdhar, Bab-Saadoun, 1006 Tunis, Tunisia Presently, there is significant interest in microbiologically : mediated remediation because it promises to be simpler, C. Desler L. J. Rasmussen cheaper, and more environmentally friendly than the Center for Healthy Aging, Faculty of Health Sciences, Copenhagen University, more commonly used non-biological options (Lovley DK-2200 Copenhagen N, Denmark 2003). In 2002, Kineococcus radiotolerans SRS30216 494 Ann Microbiol (2012) 62:493–500 was discovered in a high-level radioactive environment at With regard to other classes of bactericidal antimicrobials, the Savannah River Site in the United States of America quinolones target DNA replication and repair by binding (USA) (Phillips et al. 2002). In 2004, Gram-positive DNA gyrase complexed with DNA, which drives double- bacteria high in G + C content, including members of strand DNA break formation and cell death as demonstrated in Arthrobacter, Bacillus, Streptomyces,andNocardioides, previous experimental work (Kohanski et al. 2007, and were among the most common genera represented among references therein). the cloned sequences from Hanford radioactive waste sites To develop bio-cleanup technologies, numerous bacteria, in the USA (Fredrickson et al. 2004). For instance, including members of the extremely radiation-resistant members of the genus Streptomyces account for 70–80% family Deinococcaceae, have been studied for their ability of secondary metabolites within the actinomycetes which to transform, detoxify, or immobilize a variety of metallic are responsible for more than two-thirds of the total and organic pollutants (Bonaventura and Johnson 1997; number of antimicrobials produced by bacteria and fungi Lovley 2003;LovleyandCoates1997;Daly2000). (more than 5,000) (Challis and Hopwood 2003). These Particularly, research aimed at developing Deinococcus discoveries, together with genomic evidence that Kine- species for bioremediation of nuclear waste began in 1997 ococcus radiotolerans, for example, has open reading with the demonstration that, unlike most organisms, frames (ORFs) with significant homology to genes implicated their representative, D. radiodurans, can grow in the in validamycin biosynthesis (e.g., Krad_0836) (Bai et al. presence of chronic ionizing radiation at 60 grays (Gy) 2006), antimicrobial biosynthesis (e.g., Krad_2675), and per hour, comparable to the most radioactive waste sites streptomycin biosynthesis (e.g., Krad_3239, Krad_1553, of the Department of Energy (DOE) in the USA Krad_3509, Krad_4156) (Bagwell et al. 2008), strengthens (Fredrickson et al. 2000;Daly2000; Lange et al. 1998; our proposal in this work that the resistance to antimicrobials Brim et al. 2000, 2003, 2006). must be evoked when discussing bioremediation issues of The bioremediation potential of radioactive waste sites radioactive sites. Ideally, bioremediation strategies would be by Deinococcus radiodurans due to its extreme resistance designed based on knowledge of the contaminated environ- to chronic ionizing radiation (Brim et al. 2000, 2006; Daly ments (Lovley 2003). For example, knowledge of predom- 2000; Lange et al. 1998; Fredrickson et al. 2000) and the inant antimicrobial-producing organisms is important to presence in nuclear wastes of bacteria (Phillips et al. 2002) define the biological constraints for bioremediation of a with antimicrobial biosynthesis pathways (Bagwell et al. contaminated radioactive site. Consequently, an efficient 2008; Bai et al. 2006) led to this study. Here, our strategy bioremediation strategy will require the construction of for elucidating the antimicrobial susceptibility profile of D. genetic constructs related to antimicrobial resistance to be radiodurans was to retrieve and analyze, in silico, genes introduced to bioremediation bacteria. that could confer resistance to antimicrobials and to collect Previously, it was proposed that there is a common information about the functional status of these genes mechanism of cellular death underlying all classes of from the antibiograms of D. radiodurans. Based on this bactericidal antimicrobials (Kohanski et al. 2007; Liochev work, it should be possible in future to engineer D. and Fridovich 1999; Touati 2000). Briefly, highly reactive radiodurans using appropriate antimicrobial resistance hydroxyl radicals (HO•) are formed as a function of gene determinants for bioremediation within contaminated metabolism related to three reactions (1) transfer of radioactive environments. electrons from reducing substrates such as nicotinamide- adenine dinucleotide (NADH) to oxygen (O2), (2) leaching of ferrous iron(II) (Fe2+) from iron-sulfur (Fe-S) clusters, Materials and methods and (3) stimulation of the Fenton reaction—the reaction of 2+ Fe with hydrogen peroxide (H2O2) to yield ferric iron(III) Bioinformatics (Fe3+), hydroxyl anion (OH−), and HO•—which will readily damage membrane lipids, proteins, and DNA Genes of D. radiodurans related to resistance to anti- (Kohanskietal.2007; Liochev and Fridovich 1999; microbials were retrieved from the ARDB database Touati 2000;Daly2009; Chen and Schopfer 1999). Some accessible at http://ardb.cbcb.umd.edu/ (Liu and Pop antimicrobials inhibit ribosome function, targeting both 2009). Re-annotation work of the genome of D. radiodurans the 30S (tetracycline family) and 50S (macrolide family was done using the RAST server available at http://rast. and chloramphenicol) ribosome subunits. Cell-wall syn- nmpdr.org/ (Aziz et al. 2008). Proteins were retrieved from thesis inhibitors (such as beta-lactams), which interact the curated protein sequence database SWISS-PROT with penicillin-binding proteins and glycopeptides that (Bairoch and Apweiler 2000). Proteic clusters were deter- interact with peptidoglycan building blocks, interfere with mined using ProtoNet accessible at http://www.protonet.cs. normal cell-wall synthesis and induce lysis and cell death. huji.ac.il (Kaplan et al. 2005). Also, when indicated, Ann Microbiol (2012) 62:493–500 495 homology search was done using default settings and (Battista 1997). Antibiotic susceptibility testing was per- published recommendations of BLAST at NCBI (Altschul formed by using the Kirby-Bauer disc diffusion method et al. 1990; Pertsemlidis et al. 2001; Sayers et al. 2009). (Sanofi Dignostics Pasteur, France) after inoculation into Protein