Contents lists available at ScienceDirect Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath Occurrence of virulence factors in Cronobacter sakazakii and Cronobacter malonaticus originated from clinical samples Ondřej Holý a,* , Ariadnna Cruz-Córdova b , Juan Xicohtencatl-Cortes b , Igor Hochel c , Julio Parra-Flores d , Jana Petrželová e , Kamila Fačevicová f , Stephen Forsythe g , Abdlrhman Alsonosi h a Department of Public Health, Palacký University Olomouc, Olomouc, Czech Republic b Laboratorio de Investigación en Bacteriología Intestinal, Hospital Infantil de México Federico Gómez, Ciudad de México, Mexico c Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Czech Republic d Departament of Nutrition and Public Health, Universidad del Bío-Bío, Chillán, Ñuble, 3780000, Chillán, Chile e Department of Microbiology, Palacký University Olomouc, Olomouc, Czech Republic f Department of Mathematical Analysis and Applications of Mathematics, Faculty of Science, Palacký University Olomouc, Olomouc, Czech Republic g Adams Hill, Keyworth, Nottinghamshire, NG12 5GY, United Kingdom h Microbiology Department, Faculty of Biomedical Sciences, Sabha University, Sabha, Libya ARTICLE INFO Keywords: Cronobacter sakazakii Cronobacter malonaticus Virulence factors Adherence Invasion Clinical strains ABSTRACT Background: Cronobacter spp. are Gram-negative, facultative-anaerobic, non-spore forming, enteric coliform bacteria, which belongs to the Enterobacteriaceae family. Cronobacter spp. are opportunistic pathogens that have brought rare but life-threatening infections such as meningitis, necrotizing enterocolitis and bloodstream in- fections in neonates and infants. Information on the diversity, pathogenicity and virulence of Cronobacter species obtained from various sources is still relatively scarce and fragmentary. The aim of this study was to examine and analyse different pathogenicity and virulence factors among C. sakazakii and C. malonaticus strains isolated from clinical samples. Methods: The thirty-six clinical Cronobacter strains have been used in this study. This bacterial collection consists of 25 strains of C. sakazakii and 11 strains of C. malonaticus, isolated from different clinical materials. Seven genes (ompA, inv, sip, aut, hly, fliC, cpa) were amplified by PCR. Moreover, the motility and the ability of these strains to adhere and invade human colorectal adenocarcinoma (HT-29) and mouse neuroblastoma (N1E-115) cell lines were investigated. Results: Our results showed that all tested strains were able to adhere to both used cell lines, HT-29 and N1E- 115 cells. The invasion assay showed that 66.7% (24/36) of isolates were able to invade N1-E115 cells while 83% (30/36) of isolates were able to invade HT-29 cells. On the average, 68% of the C. sakazakii strains ex- hibited seven virulence factors and only 18% in C. malonaticus. All strains amplified ompA and fliC genes. The other genes were detected as follow: sip 97% (35/36), hlyA 92% (33/36), aut 94% (34/36), cpa 67% (24/36), and inv 69% (25/36). Conclusions: C. sakazakii and C malonaticus strains demonstrate the diversity of the virulence factors present among these pathogens. It is necessary to permanently monitor the hospital environment to appropriately treat and resolve cases associated with disease. Furthermore, in-depth knowledge is needed about the source and transmission vehicles of pathogens in hospitals to adopt pertinent prevention measures. 1. Introduction Cronobacter spp., formerly known as Enterobacter sakazakii [16] are Gram-negative, facultative-anaerobic, non-spore forming, enteric coli- form bacteria, which belongs to the Enterobacteriaceae family. At pre- sent, seven species are known: C. sakazakii, C. malonaticus, C. https://doi.org/10.1016/j.micpath.2018.12.011 Received 18 October 2018; Received in revised form 6 December 2018; Accepted 6 December 2018 * Corresponding author. Department of Public Health, Faculty of Medicine and Dentistry, Palacký University Olomouc, Hněvotínská 3, 77515, Olomouc, Czech Republic. E-mail addresses: holy.ondrej@seznam.cz (O. Holý), steve.forsythe@foodmicrobe.com (S. Forsythe). URL: http://foodmicrobe.com (S. Forsythe). Microbial Pathogenesis 127 (2019) 250–256 Available online 11 December 2018 0882-4010/ © 2018 Elsevier Ltd. All rights reserved. T