Virulence traits in Cronobacter species isolated from different sources Ariadnna Cruz, Juan Xicohtencatl-Cortes, Bertha González-Pedrajo, Miriam Bobadilla, Carlos Eslava, and Irma Rosas Abstract: Cronobacter spp. (Enterobacter sakazakii) includes gram-negative opportunistic foodborne pathogens known as rare but important causes of life-threatening neonatal infections. However, the pathogenic mechanism is not yet clear. In this study, 43 isolates of Cronobacter, from human and nonhuman sources, were analyzed. A total of four clusters were identi- fied and 32 DNA pulsotypes were observed by pulsed-field gel electrophoresis. In addition, 86% of the Cronobacter isolates were able to adhere to HEp-2 cells and 35% were invasive, Cronobacter sakazakii isolates being the most efficient. Twenty- six percent of Cronobacter isolates were able to form biofilms, mainly those from nonhuman sources, such as Cronobacter dublinensis and Cronobacter malonaticus. Three putative virulence genes (siderophore-interacting protein (sip), type III he- molysin (hly), and plasminogen activator (cpa)) were identified by bioinformatic analysis and then detected by PCR. The sip gene was the most frequently detected (60%; 26/43), followed by the hly gene (37%; 16/43) and the cpa gene (28%; 12/ 43). The three genes were identified primarily in C. sakazakii. Our data show that Cronobacter species harbor different viru- lence traits. Key words: Cronobacter spp., adherence, invasion, biofilm, putative virulence genes. Résumé : Cronobacter spp. (Enterobacter sakazakii) comprend des pathogènes alimentaires opportunistes négatifs à Gram, connus pour être des causes rares mais importantes d’infections néonatales potentiellement mortelles. Cependant, les méca- nismes pathogènes ne sont pas encore clairs. Dans cette étude, 43 isolats de Cronobacter provenant de sources humaines et non humaines ont été analysés. Un total de quatre grappes a été identifié et 32 pulsotypes d’ ADN ont été observés en élec- trophorèse sur gel en champ pulsé (PFGE). De plus, 86 % des isolats de Cronobacter étaient capables d’adhérer aux cellules HEp-2, et 35 % étaient invasives, les isolats de Cronobacter sakazakii étant les plus efficaces. Vingt-six pourcent des isolats de Cronobacter pouvaient former des biofilms, principalement ceux d’ origine non humaine, comme Cronobacter dublinensis et Cronobacter malonaticus Trois gènes de virulence présumés (sip, codant la « siderophore-interacting protein », hly codant l’hémolysine de type III, et cpa codant l’activateur du plasminogène) ont été identifiés par analyse bioinformatique, puis dé- tectés par PCR. Le gène sip était le plus fréquemment détecté, à 60 % (26/43), suivi par le gène hly, à 37 % (16/43) et le gène cpa, à 28 % (12/43). Ces trois gènes ont été d’abord identifiés chez C. sakazakii. Nos données montrent que les espèces de Cronobacter possèdent différents traits de virulence. Mots‐clés : Cronobacter spp., adhérence, invasion, biofilm, gènes de virulence présumés. [Traduit par la Rédaction] Introduction Cronobacter spp. has been proposed as a new genus with five species (Cronobacter sakazakii, Cronobacter malonati- cus, Cronobacter dublinensis, Cronobacter muytjensii, and Cronobacter turicensis) and one genomospecies (Crono- bacter genomospecies 1). It encompasses the organisms pre- viously identified as Enterobacter sakazakii (Iversen et al. 2008). Cronobacter spp. are opportunistic foodborne patho- gens that have been associated with sporadic cases of menin- gitis, septicemia, and necrotizing enterocolitis in neonates and immunocompromised infants (Bowen and Braden 2006). There are few reports of Cronobacter spp. associated with these infections in adults, but in contrast to neonatal infec- tion, it is not usually life threatening (Hunter et al. 2008). The mechanisms of transmission of these bacteria have been Received 13 January 2011. Revision received 6 June 2011. Accepted 6 June 2011. Published at www.nrcresearchpress.com/cjm on 22 August 2011. A. Cruz. Facultad de Medicina-Centro de Ciencias de la Atmósfera, Universidad Nacional Autónoma de México, 04510 México D.F. J. Xicohtencatl-Cortes. Laboratorio de Bacteriología Intestinal, Hospital Infantil de México Federico Gómez, 01900 México D.F. B. González-Pedrajo. Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 70-243, 04510 México, D.F. M. Bobadilla. Microbiología Clínica, Instituto Nacional de Ciencias Médicas y Nutrición “Salvador Zubirán”, 14000 México, D.F. C. Eslava. Facultad de Medicina, Salud Pública, Universidad Nacional Autónoma de México, 04510 México, D.F. I. Rosas. Laboratorio de Aerobiología, Centro de Ciencias de la Atmósfera, Universidad Nacional Autónoma de México, 04510 México, D.F. Corresponding author: Irma Rosas (e-mail: iarp@atmosfera.unam.mx). 735 Can. J. Microbiol. 57: 735–744 (2011) doi:10.1139/W11-063 Published by NRC Research Press