International Journal of Medical Microbiology 301 (2011) 34–43 Contents lists available at ScienceDirect International Journal of Medical Microbiology journal homepage: www.elsevier.de/ijmm Stenotrophomonas maltophilia strains from cystic fibrosis patients: Genomic variability and molecular characterization of some virulence determinants Mauro Nicoletti a,1 , Angelo Iacobino b,1 , Gianni Prosseda c , Ersilia Fiscarelli d , Raffaele Zarrilli e , Elena De Carolis f , Andrea Petrucca a , Lucia Nencioni g , Bianca Colonna c , Mariassunta Casalino b,∗ a Dipartimento Scienze Biomediche, Università “G. D’Annunzio”, Via dei Vestini 31 Chieti, Italy b Dipartimento di Biologia, Università “Roma TRE”, Viale Marconi 446 Roma, Italy c Istituto Pasteur-Fondazione Cenci-Bolognetti, Dipartimento di Biologia e Biotecnologie “C. Darwin”, Università Roma “Sapienza”, Via dei Sardi, 70 Rome, Italy d U.O. Microbiologia, Ospedale “Bambino Gesù”, Piazza S. Onofrio 4, Roma, Italy e Dipartimento di Scienze Mediche Preventive, Sezione di Igiene, Università “Federico II”, Via S. Pansini 5, Napoli, Italy f Istituto di Microbiologia, Università Cattolica del “Sacro Cuore”, Largo F. Vito 1, Roma, Italy g Dipartimento di Scienze di Sanità Pubblica, Università Roma “Sapienza”, Piazzale A. Moro 5, Rome, Italy article info Article history: Received 27 April 2010 Received in revised form 30 June 2010 Accepted 5 July 2010 Keywords: Stenotrophomonas maltophilia Cystic fibrosis Molecular typing Virulence factors Extracellular proteases Esterase Galleria mellonella Animal model abstract The genetic relatedness of 52 Stenotrophomonas maltophilia strains, collected from various environmental and clinical sources, including cystic fibrosis (CF) patients, as well as the presence and the expression of some virulence-associated genes were studied. Pulsed-field gel electrophoresis (PFGE) analysis identified 47 profiles and three clusters of isolates with an identical PFGE pattern considered to be indistinguishable strains. Restriction fragment length polymorphism of the gyrB gene grouped the 52 strains into nine different profiles. Most CF clinical isolates (29 out of 41) showed profile 1, while the analysis of the hypervariable regions of the 16S rRNA gene revealed five distinct allelic variations, with the majority of CF isolates (23 out of 41) belonging to sequence group 1. Furthermore, the strains were characterized for motility and expression of virulence-associated genes, including genes encoding type-1 fimbriae, proteases (StmPr1 and StmPr2) and esterase. All S. maltophilia strains exhibited a very broad range of swimming and twitching motility, while none showed swarming motility. A complete smf-1 gene was PCR-amplified only from clinically derived S. maltophilia strains. Finally, the virulence of representative S. maltophilia strains impaired in the expression of proteases and esterase activities was evaluated by infecting larvae of the wax moth Galleria mellonella. The results obtained strongly indicate that the major extracellular protease StmPr1 may be a relevant virulence factor of S. maltophilia. © 2010 Elsevier GmbH. All rights reserved. Introduction Stenotrophomonas maltophilia is a Gram-negative bacterium, found in a great variety of environmental sources and recently recognized as an opportunistic pathogen being isolated from immunocompromised individuals, intensive care and cystic fibro- sis (CF) patients (Crispino et al., 2002; Denton and Kerr, 1998; Lyczak et al., 2002). Inflammation and chronic bacterial infec- tions are responsible for most of the morbidity and mortality of CF patients. Aggressive use of antibiotics has made a major contribution to improve prognosis, however it has been also asso- ciated with a change in the pattern of infecting organisms over decades. When CF was first described, Staphylococcus aureus was the major pathogen and subsequently Pseudomonas aeruginosa ∗ Corresponding author. Tel.: +39 06 57336368; fax: +39 06 57336821. E-mail address: casalino@uniroma3.it (M. Casalino). 1 These authors contributed equally to this work. became increasingly common (Gibson et al., 2003). The use of spe- cific anti-pseudomonal therapies has caused a selective pressure on the CF bacterial polymicrobial community resulting in the emer- gence of new pathogens, including S. maltophilia (Ballestero et al., 1995; Denton and Kerr, 1998; Marchac et al., 2004). Persistent col- onization by P. aeruginosa and damage of the epithelial mucosa has been suggested to enhance the chance of S. maltophilia to colonize the respiratory epithelium of CF patients where a progressive dete- rioration of pulmonary functions has been observed, particularly in patients colonized for longer periods (Denton and Kerr, 1998; Goss et al., 2004; De Vidipò et al., 2001). From the first report, the isolation of S. maltophilia has continued to rise. Currently it is isolated from the lungs of approximately 10% of the CF patients in the USA and up to 25% in Europe (Looney et al., 2009; Steinkamp et al., 2005). Considering its innate resistance to antibiotics, this microorganism has become a cause of great concern within the international CF community (Avison et al., 2001; Canton et al., 2003; Crossman et al., 2008), but despite its high incidence its virulence factors have been poorly characterized. S. maltophilia 1438-4221/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2010.07.003