Enrique A. González

Serotypes, Virulence Genes, and Intimin Types of Shiga Toxin (Verotoxin)-Producing Escherichia coli Isolates from Cattle in Spain and Identification of a New Intimin Variant Gene (eae-ξ)

ABSTRACT A total of 514 Shiga toxin-producing Escherichia coli (STEC) isolates from diarrheic and healthy cattle in Spain were characterized in this study. PCR showed that 101 (20%) isolates carried stx1 genes, 278 (54%) possessed stx2 genes, and 135 (26%) possessed both stx1 and stx2. Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 326 (63%) and in 151 (29%) of the isolates, respectively. STEC isolates belonged to 66 O serogroups and 113 O:H serotypes (including 23 new serotypes). However, 67% were of one of these 15 serogroups (O2, O4, O8, O20, O22, O26, O77, O91, O105, O113, O116, O157, O171, O174, and OX177) and 52% of the isolates belonged to only 10 serotypes (O4:H4, O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and ONT:H19). Although the 514 STEC isolates belonged to 164 different seropathotypes (associations between serotypes and virulence genes), only 12 accounted for 43% of isolates. Seropathotype O157:H7 stx2eae-γ1 ehxA (46 isolates) was the most common, followed by O157:H7 stx1stx2eae-γ1 ehxA (34 isolates), O113:H21 stx2 (25 isolates), O22:H8 stx1stx2ehxA (15 isolates), O26:H11 stx1eae-β1 ehxA (14 isolates), and O77:H41 stx2ehxA (14 isolates). Forty-one (22 of serotype O26:H11) isolates had intiminβ 1, 82 O157:H7 isolates possessed intimin γ1, three O111:H- isolates had intimin type γ2, one O49:H- strain showed intimin type δ, 13 (six of serotype O103:H2) isolates had intimin type ε and eight (four of serotype O156:H-) isolates had intimin ζ. We have identified a new variant of the eae intimin gene designated ξ (xi) in two isolates of serotype O80:H-. The majority (85%) of bovine STEC isolates belonged to serotypes previously found for human STEC organisms and 54% to serotypes associated with STEC organisms isolated from patients with hemolytic uremic syndrome. Thus, this study confirms that cattle are a major reservoir of STEC strains pathogenic for humans.

Serotypes, Virulence Genes, and Intimin Types of Shiga Toxin (Verotoxin)-Producing Escherichia coli Isolates from Healthy Sheep in Spain

ABSTRACT Fecal swabs obtained from 1,300 healthy lambs in 93 flocks in Spain in 1997 were examined for Shiga toxin-producing Escherichia coli (STEC). STEC O157:H7 strains were isolated from 5 (0.4%) animals in 4 flocks, and non-O157 STEC strains were isolated from 462 (36%) lambs in 63 flocks. A total of 384 ovine STEC strains were characterized in this study. PCR showed that 213 (55%) strains carried the stx1 gene, 10 (3%) possessed the stx2 gene, and 161 (42%) carried both the stx1 and the stx2 genes. Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 106 (28%) and 23 (6%) of the STEC strains, respectively. The STEC strains belonged to 35 O serogroups and 64 O:H serotypes (including 18 new serotypes). However, 72% were of 1 of the following 12 serotypes: O5:H−, O6:H10, O91:H−, O117:H−, O128:H−, O128:H2, O136:H20, O146:H8, O146:H21, O156:H−, O166:H28, and ONT:H21 (where NT is nontypeable). Although the 384 STEC strains belonged to 95 different seropathotypes (associations between serotypes and virulence genes), 49% of strains belonged to only 11. O91:H− stx1stx2 (54 strains) was the most common seropathotype, followed by O128:H− stx1stx2 (33 strains) and O6:H10 stx1 (25 strains). Three strains of serotypes O26:H11, O156:H11, and OX177:H11 had intimin type β1; 5 strains of serotype O157:H7 possessed intimin type γ1; and 15 strains of serotypes O49:H−, O52:H12, O156:H− (12 strains), and O156:H25 had the new intimin, intimin type ζ. The majority (82%) of ovine STEC strains belonged to serotypes previously found to be associated with human STEC strains, and 51% belonged to serotypes associated with STEC strains isolated from patients with hemolytic-uremic syndrome. Thus, this study confirms that healthy sheep are a major reservoir of STEC strains pathogenic for humans.

Prevalence and characteristics of Escherichia coli serotype O157:H7 and other verotoxin-producing E. coli in healthy cattle

From February to July of 1994, 328 faecal samples from 32 herds were collected and verotoxin-producing Escherichia coli (VTEC) found on 84% of the farms. The proportion of animals infected varied from 0-63%. VTEC were recovered from 52 (20%) of 257 cows and from 16 (23%) of 71 calves. Although the VTEC belonged to 25 different serogroups, 7 (O8, O20, O22, O77, O113, O126 and O162) accounted for 46% of strains. Nearly 45% of the strains. Nearly 45% of the 83 bovine VTEC strains belonged to serogroups associated with haemorrhagic colitis and haemolytic uraemic syndrome in humans. However, only 2 (2%) of 83 VTEC strains isolated from cattle belonged to enterohaemorrhagic E. coli (EHEC) serotypes (O26:H11 and O157:H7), and only 8 (10%) were positive for the attaching and effacing E. coli (eae) gene sequence. Polymerase chain reaction (PCR) showed that 17 (20%) of VTEC strains carried VT1 genes, 43 (52%) possessed VT2 genes, and 23 (28%) carried both VT1 and VT2 genes. Characterization of VTEC isolates revelated a heterogeneous population in terms of serogroup and toxin type in the positive herds. This study confirms that healthy cattle are a reservoir of VTEC, but, the absence of eae genes in most bovine VTEC strains suggests that they may be less virulent for humans than eae-positive EHEC.

Serotypes, phage types and virulence genes of Shiga-producing Escherichia coli isolated from sheep in Spain

PROBLEM ADDRESSED Shiga toxin-producing Escherichia coli (STEC), have emerged as food poisoning pathogens which can cause severe diseases in humans. OBJECTIVE The aim of this study was to determinate the serotypes and virulence genes of STEC strains isolated from sheep in Spain, with the purpose of determining whether sheep represent a potential source of STEC pathogenic for humans. METHODS AND APPROACH Faecal swabs obtained from 697 healthy lambs on 35 flocks in Spain during the years 2000 and 2001 were examined for STEC using phenotypic (Vero cells) and genotypic (PCR) methods. RESULTS STEC O157:H7 strains were isolated from seven (1%) animals in six flocks, whereas non-O157 STEC strains were isolated from 246 (35%) lambs in 33 flocks. A total of 253 ovine STEC strains were identified in this study. PCR showed that 110 (43%) strains carried stx(1) genes, 10 (4%) possessed stx(2) genes and 133 (53%) both stx(1) and stx(2). Enterohaemolysin (ehxA) and intimin (eae) virulence genes were detected in 120 (47%) and in 9 (4%) of the STEC strains. STEC strains belonged to 22 O serogroups and 44 O:H serotypes. However, 70% were of one of these six serogroups (O6, O91, O117, O128, O146, O166) and 71% belonged to only nine serotypes (O6:H10, O76:H19, O91:H-, O117:H-, O128:H-, O128:H2, O146:H21, O157:H7, O166:H28). A total of 10 new O:H serotypes not previously reported in STEC strains were found in this study. Seven strains of serotype O157:H7 possessed intimin type gamma1, and two strains of serotype O156:H- had the new intimin zeta. STEC O157:H7 strains were phage types 54 (four strains), 34 (two strains) and 14 (one strain). CONCLUSIONS This study confirms that healthy sheep are a major reservoir of STEC pathogenic for humans. However, because the eae gene is present only in a very small proportion of ovine non-O157 STEC, most ovine strains may be less pathogenic.

Verotoxin-producing Escherichia coli in Spain: prevalence, serotypes, and virulence genes of O157:H7 and non-O157 VTEC in ruminants, raw beef products, and humans.

In Spain, as in many other countries, verotoxin-producing Escherichia coli (VTEC) strains have been frequently isolated from cattle, sheep, and foods. VTEC strains have caused seven outbreaks in Spain (six caused by E. coli O157:H7 and one by E. coli O111:H– [nonmotile]) in recent years. An analysis of the serotypes indicated serological diversity. Among the strains isolated from humans, serotypes O26:H11, O111:H–, and O157:H7 were found to be more prevalent. The most frequently detected serotypes in cattle were O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and OUT (O untypeable):H19. Different VTEC serotypes (e.g., O5:H–, O6:H10, O91:H–, O117:H–, O128:H–, O128:H2, O146:H8, O146:H21, O156:H–, and OUT:H21) were found more frequently in sheep. These observations suggest a host serotype specificity for some VTEC. Numerous bovine and ovine VTEC serotypes detected in Spain were associated with human illnesses, confirming that ruminants are important reservoirs of pathogenic VTEC. VTEC can produce one or two toxins (VT1 and VT2) that cause human illnesses. These toxins are different proteins encoded by different genes. Another virulence factor expressed by VTEC is the protein intimin that is responsible for intimate attachment of VTEC and effacing lesions in the intestinal mucosa. This virulence factor is encoded by the chromosomal gene eae. The eae gene was found at a much less frequency in bovine (17%) and ovine (5%) than in human (45%) non-O157 VTEC strains. This may support the evidence that the eae gene contributes significantly to the virulence of human VTEC strains and that many animal non-O157 VTEC strains are less pathogenic to humans.

Virulence factors of bacteraemic Escherichia coli with particular reference to production of cytotoxic necrotising factor (CNF) by P-fimbriate strains

Thirty-seven strains of Escherichia coli isolated from bacteraemia and 40 faecal strains isolated from healthy individuals were O serogrouped and investigated for the production of colicins, haemolysin (Hly), cytotoxic necrotising factor (CNF), lethal activity for mice, the expression of P fimbriae, mannose-resistant (MRHA) and mannose-sensitive (MSHA) haemagglutination, and relative cell surface hydrophobicity. Virulence factors significantly associated with bacteraemic strains were: serogroups O2, O4, O6, O7, O8 and O75 (54% versus 10%, p less than 0.001), production of Hly (32% versus 8%, p less than 0.02) and CNF (38% versus 10%, p less than 0.01), expression of P fimbriae (27% versus 5%, p less than 0.02), MRHA types III, IVa and IVb (51% versus 8%, p less than 0.001), and possession of a moderate cell surface hydrophobic charge (35% versus 13%, p less than 0.05). Virulence factors were strongly associated with strains expressing defined MRHA types. Thus, all strains belonging to MRHA types III and IVa were toxigenic, whereas only 11% of strains belonging to MRHA types IVb, V or VI were toxigenic (p less than 0.001). Virulence factors were concentrated in strains belonging to O serogroups usually found in E. coli that cause extra-intestinal infections, especially in strains of O4 and O6 groups. The most interesting result of this study was that all 12 P-fimbriate strains expressed the MRHA type IVa and 11 of them synthesised CNF.

Production of toxins by Escherichia coli strains isolated from calves with diarrhoea in Galicia (North-western Spain)

A total of 289 Escherichia coli colonies isolated from 78 diarrhoeic calves were studied for production of heat-labile (LT) and heat-stable (STa) enterotoxins, verotoxin (VT), cytotoxic necrotizing factor (CNF) and K99 antigen, and they were serotyped. Production of STa was detected in a single strain possessing both K99 and F41 antigens; the serotype was 09:K (A) 35. LT-producing strains were not detected. From 16 (20.5%) calves, 51 VT-producing colonies of E. coli were isolated. Production of the necrotic factor was detected in 33 E. coli colonies isolated from 14 (17.9%) calves. Serotype was a useful marker for production of VT and CNF. Among the 51 VT-producing colonies, 24 were untypable and the remainder belonged to serotypes O2:K?, O103:K--, O104:K?, O128:K?, O153:K-- and O157:K--:H7. Four of the 33 CNF-producing colonies were untypable and the majority of the remaining colonies belonged to serotypes O15:K14, O78:(K80), O123:K-- and O139:K--. Both VT and CNF were lethal for mice, but only CNF showed necrotizing reaction in rabbit skin. Our results indicate that VT-producing and CNF-producing E. coli strains are frequently isolated from diarrhoeic calves and that according to the serotypes exhibited, some of them might be considered potential pathogens for humans. The role of VT-producing and CNF-producing strains in calf diarhoea remains to be established.

Serotypes, virulence genes and intimin types of Shiga toxin (verocytotoxin)-producing Escherichia coli isolates from minced beef in Lugo (Spain) from 1995 through 2003

BackgroundShiga toxin-producing Escherichia coli (STEC) have emerged as pathogens that can cause food-borne infections and severe and potentially fatal illnesses in humans, such as haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). In Spain, like in many other countries, STEC strains have been frequently isolated from ruminants, and represent a significant cause of sporadic cases of human infection. In view of the lack of data on STEC isolated from food in Spain, the objectives of this study were to determine the level of microbiological contamination and the prevalence of STEC O157:H7 and non-O157 in a large sampling of minced beef collected from 30 local stores in Lugo city between 1995 and 2003. Also to establish if those STEC isolated from food possessed the same virulence profiles as STEC strains causing human infections.ResultsSTEC were detected in 95 (12%) of the 785 minced beef samples tested. STEC O157:H7 was isolated from eight (1.0%) samples and non-O157 STEC from 90 (11%) samples. Ninety-six STEC isolates were further characterized by PCR and serotyping. PCR showed that 28 (29%) isolates carried stx1 genes, 49 (51%) possessed stx2 genes, and 19 (20%) both stx1 and stx2. Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 43 (45%) and in 25 (26%) of the isolates, respectively. Typing of the eae variants detected four types: γ1 (nine isolates), β1 (eight isolates), ε1 (three isolates), and θ (two isolates). The majority (68%) of STEC isolates belonged to serotypes previously detected in human STEC and 38% to serotypes associated with STEC isolated from patients with HUS. Ten new serotypes not previously described in raw beef products were also detected. The highly virulent seropathotypes O26:H11 stx1eae-β1, O157:H7 stx1stx2eae-γ1 and O157:H7 stx2eae-γ1, which are the most frequently observed among STEC causing human infections in Spain, were detected in 10 of the 96 STEC isolates. Furthermore, phage typing of STEC O157:H7 isolates showed that the majority (seven of eight isolates) belonged to the main phage types previously detected in STEC O157:H7 strains associated with severe human illnesses.ConclusionThe results of this study do not differ greatly from those reported in other countries with regard to prevalence of O157 and non-O157 STEC in minced beef. As we suspected, serotypes different from O157:H7 also play an important role in food contamination in Spain, including the highly virulent seropathotype O26:H11 stx1eae-β1. Thus, our data confirm minced beef in the city of Lugo as vehicles of highly pathogenic STEC. This requires that control measures to be introduced and implemented to increase the safety of minced beef.

Characteristics of haemolytic Escherichia coli with particular reference to production of cytotoxic necrotizing factor type 1 (CNF1)

A total of 1,106 Escherichia coli strains isolated in Spain between 1986 and 1991 from extraintestinal infections and faeces of healthy controls were examined for production of alpha-haemolysin (Hly). Among strains causing urinary tract infections, sepsis and other extraintestinal infections, Hly production was detected in 51% (P < 0.001), 32% (P < 0.001) and 18% (P < 0.02), respectively. In contrast, only 9% of faecal isolates from healthy individuals synthesized Hly. The 356 haemolytic E. coli strains characterized in this study belonged to 28 different serogroups. However, 284 (80%) were of one of eight serogroups (02, 04, 06, 08, 018, 022, 075 and 083); 40% and 31% of haemolytic strains expressed P fimbriae and mannose-resistant haemagglutination (MRHA) type III, respectively. We have found that haemolytic isolates of E. coli may clearly be divided into two categories on the basis of the ability to produce cytotoxic necrotizing factor type 1 (CNF1). The serogroups and adhesins determined in Hly+CNF1+ strains were generally different from those found in Hly+CNF1- strains. Thus, serogroups 02, 06 and 075 were associated with haemolytic E. coli producing CNF1+, whereas serogroups 01, 08, 018, 028 and 086 were established more frequently among Hly+CNF1- strains. While expression of P fimbriae was more frequently detected in Hly+CNF1- strains (70 versus 29%, P < 0.001), MRHA type III was usually identified in Hly+CNF1+ E. coli (42 versus 1%, P < 0.001). Furthermore, the sonic extracts of Hly+CNF1+ strains caused necrosis in rabbit skin (96 versus 25%, P < 0.001) and death in intraperitoneally injected mice (73 versus 11%, P < 0.001) more frequently than sonic extracts of Hly+CNF1- strains.

Serogroups of Escherichia coli isolated from piglets in Spain.

Serogroups of 1334 E. coli colonies isolated in Spain between 1986 and 1991 from piglets with diarrhoea, oedema disease and from healthy piglets, were determined. The serogroups determined in E. coli from diarrhoea were: O1, O2, O4, O5, O7, O8, O9, O12, O20, O21, O23, O25, O26, O32, O39, O45, O54, O75, O78, O80, O83, O91, O92, O101, O103, O113, O115, O116, O118, O125, O127, O138, O139, O141, O149, O153 and O157; from pigs with oedema disease: O8, O101, O138, O149 and O157; and from healthy piglets: O1, O2, O5, O7, O8, O9, O20, O21, O26, O29, O45, O64, O71, O80, O81, O91, O101, O105, O113, O115, O116, O126, O128, O132, O138, O139, O142, O146, O149, O152, O153, and O168. Serogroups O138, O141 and O149 were found to be statistically associated with enteric porcine colibacillosis (diarrhoea and oedema disease) (0.025 > P < 0.01). In addition, enterotoxigenic (ETEC) strains belonged to serogroups: O8, O9, O20, O101, O138, O141 and O149; verotoxigenic (VTEC) strains (VTEC) strains to serogroups 091 and 0138; and necrotizing (NTEC) strains to serogroups O2, O4, O8, O54, O78 and O83. Furthermore, 91.2% (249 out 273) of the ETEC (LT and/or STa) and/or VTEC strains belonged to only seven different serogroups. These major serogroups to which the ETEC and VTEC strains belonged, were determined in a lower percentage (21.2%) among non-toxigenic E. coli colonies isolated from sick and healthy piglets.