European Journal of Epidemiology 10:707-711, 1994. © 1994 Kluwer Academic Publishers. Printed in the Netherlands. Serotypes of CNFl-producing Escherichia coli strains that cause extraintestinal infections in humans J. E. Blanco 1, J. Blanco 1, M. Blanco 1, M. P. Alonso 2 & W. H. Jansen 3 t Departamento de Microbioloxia e Parasitoloxia, Facultade de Veterinaria, Universidade de Santiago, Lugo, Spain; 2 Unidade de Microbioloxia, Complexo Hospitalario Xeral-Calde, Lugo, Spain; 3Laboratory of Bacteriology and Antimicrobial Agents, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands Accepted in revised form 15 July 1994 Abstract. The O:K:H serotypes of 137 neerotoxi- genic Escherichia coli (NTEC) producing the cyto- toxic necrotizing factor type 1 (CNF1) isolated from human extraintestinal infection were determined. Although NTEC producing CNF1 belonged to 58 different serotypes, only 10 of them accounted for 54% of strains. The most common serotypes, in order of frequency, were: O4:K?:H5, O6:KI3:H1, O83:Kl:H31, O75:K95:H5, O2:Kl:H6, O2:K7:H-, O75:Kl:H7, O2:K?:H1, O4:K12:H1 and O22:K13:H1. CNF1 strains of serotypes O2:K7:H- and O4:K12:H1 express P-fimbriae, whereas CNF1 strains of serotypes O2:K?:H1, O2:Kl:H6 and O75:K95:H5 possess the adhesin responsible for MRHA type III. Among CNF1 strains of serotype O4:K?:H5 there exist some that express P-fimbriae and others that possess MRHA type III. Lastly, the majority of CNF1 strains of serotypes O6:K13:H1, O22:KI3:H1, O75:Kl:H7 and O83:Kl:H31 do not express P-fimbriae nor the adhesin responsible to MRHA type III. Our results show that extraintestinal infections are caused by a limited number of virulent clones, as suggested by the theory of special patho- genicity. Key words: Necrotoxigenic Escherichia coli, Pathogenicity, Toxins, Virulence Introduction Necrotoxigenic Escherichia coli (NTEC) strains are able to elaborate two types of cytotoxic necrotizing factors (CNF1 and CNF2) that induce cell multinu- cleation in Vero and HeLa tissue cultures, cause necrosis in rabbit skin and are lethal to mice [1 ]. Both necrotic toxins are cell-associated products easy to detect when bacteria are sonicated or when grown in the presence of mitomycin C [2]. CNF1 and CNF2 are heat-labile proteins of 115 and 110 kilodaltons, respectively [3-6]. Whereas CNF1 is encoded by chromosomic genes [7], CNF2 is encoded by trans- missible plasmids [5]. NTEC were associated with human extraintestinal infections [8-13] and isolated from calves with diarrhoea or septicaemia [14-16]. NTEC of human origin usually produce CNF1, whereas bovine NTEC generally synthesize CNF2 [17]. We have recently established that the majority of human CNF1 strains belong to serogroups 02, 04, 06, O14, 022, 075 or 083, and that the bovine NTEC producing CNF2 usually belong to serogroups O1, 03, O15, 055, 088 or O123 [17]. The present report is the first one to describe the O:K:H serotypes of CNF1 E.coli strains that cause human extrain- testinal infections in Spain. Materials and methods Bacterial strains. A total of 137 CNF1 NTEC strains isolated from patients with extraintestinal infections between 1986 and 1992 were investigated, Strains from asymptomatic bacteriuria, cystitis, pyelo- nephritis, bacteremia, peritonitis, appendicitis, chole- cystitis, wound and respiratory infections and other miscellaneous sources were isolated from both out- patients and inpatients attending three hospitals in Galicia (northwestern Spain): Residencia Sanitaria Juan Canalejo of La Corufia, Hospital Xeral-Calde of Lugo and Hospital Comarcal da Costa of Burela. A single E. coli strain was obtained from each patient. Isolation and identification of E. coli was performed by standard bacteriological methods. Reference E. coli strains used as positive and negative controls were: MR48 (O75:K95, MRHA III ÷, CNF1 ÷, Hly+), MR199 (O6:K13, MRHA HI+, CNF1 ÷, Hly+), C1212- 77 (O6:K2:H1, P-fimbriated) and C1976-79 (OI:KI:H7, P-fimbriated). Strains were stored at room temperature in nutrient broth (Difco, USA) with 0.75% of agar. Production and detection of CNF1. For the detec- tion of CNF1 the filtrates of cultures treated with mitomycin C were assayed on Vero and HeLa cells as previously described [2]. The Vero and HeLa cell