Fatal hemorrhage induced by subtilase cytotoxin from Shiga-toxigenic Escherichia coli

Subtilase cytotoxin (SubAB) is an AB₅ type toxin produced by a subset of Shiga-toxigenic Escherichia coli. The A subunit is a subtilase-like serine protease and cleaves an endoplasmic reticulum chaperone BiP. The B subunit binds to a receptor on the cell surface. Although SubAB is lethal for mice, the cause of death is not clear. In this study, we demonstrate in mice that SubAB induced small bowel hemorrhage and a coagulopathy characterized by thrombocytopenia, prolonged prothrombin time and activated partial thromboplastin time. SubAB also induced inflammatory changes in the small intestine as detected by ¹⁸F-fluoro-2-deoxy-d-glucose positron emission tomography imaging and histochemical analysis. Using RT-PCR and ELISA, SubAB was shown to increase interleukin-6 in a time-dependent manner. Thus, our results indicate that death in SubAB-treated mice may be associated with severe inflammatory response and hemorrhage of the small intestine, accompanied by coagulopathy and IL6 production.     

Low prevalence of STEC autotransporter contributing to biofilm formation (Sab) in verocytotoxin-producing Escherichia coli isolates of humans and raw meats

Escherichia coli autotransporters (AT) are known to confer adherence to eukaryotic extracellular matrix and may, therefore, be virulence-associated. Recently, the plasmid-borne STEC AT contributing to biofilm formation (Sab) was described in verocytotoxin (VT)-producing E. coli (VTEC) strains that do not carry the locus of enterocyte effacement (LEE). Using polymerase chain reaction (PCR), we investigated the prevalence of sab and other virulence genes, VT1 (vtx1), VT2 (vtx2), intimin (eae), enterohemolysin (ehxA), STEC autoagglutinating adhesin (saa), and subtilase cytotoxin (subA), in VTEC isolates from patients (n=263) and raw meats of ruminants and wildlife (n=104) in Belgium from 1990 to 2010. Overall, sab was detected in three (0.82%) of 367 VTEC strains comprising human isolates of serotypes O162:H28 (no clinical data available) and OX183:H18 (patient with abdominal pain), and one ground beef O181:H16 isolate. These three sab-positive isolates were eae-negative, but ehxA-, saa-, and subA-positive. Our data show that sab is uncommon in VTEC isolates. All sab-positive VTEC strains identified to date carried a comparable plasmid-bound virulence profile (ehxA-saa-subA-sab), which may be transmitted to other strains. Sab may mediate intestinal adherence in some LEE-negative VTEC isolates, but more studies on its prevalence and function are needed.     

In Vivo leukocyte changes induced by Escherichia coli subtilase cytotoxin

Subtilase cytotoxin (SubAB) is the prototype of a new family of AB(5) cytotoxins produced by Shiga-toxigenic Escherichia coli. Its cytotoxicity is due to its capacity to enter cells and specifically cleave the essential endoplasmic reticulum chaperone BiP. Previous studies have shown that intraperitoneal injection of mice with purified SubAB causes a pathology that overlaps with that seen in human cases of hemolytic-uremic syndrome, as well as dramatic splenic atrophy, suggesting that leukocytes are targeted. Here we investigated SubAB-induced leukocyte changes in the peritoneal cavity, blood, and spleen. After intraperitoneal injection, SubAB bound peritoneal leukocytes (including T and B lymphocytes, neutrophils, and macrophages). SubAB elicited marked leukocytosis, which peaked at 24 h, and increased neutrophil activation in the blood and peritoneal cavity. It also induced a marked redistribution of leukocytes among the three compartments: increases in leukocyte subpopulations in the blood and peritoneal cavity coincided with a significant decline in splenic cells. SubAB treatment also elicited significant increases in the apoptosis rates of CD4(+) T cells, B lymphocytes, and macrophages. These findings indicate that apart from direct cytotoxic effects, SubAB interacts with cellular components of both the innate and the adaptive arm of the immune system, with potential consequences for disease pathogenesis.     

Two distinct cytotoxic activities of subtilase cytotoxin produced by shiga-toxigenic Escherichia coli

Subtilase cytotoxin (SubAB) is a recently identified AB5 subunit toxin produced by Shiga-toxigenic Escherichia coli. The A subunit is thought to be a subtilase-like, serine protease, whereas the B subunit binds to the toxin receptor on the cell surface. We cloned the genes from a clinical isolate; the toxin was produced as His-tagged proteins. SubAB induced vacuolation at concentrations greater than 1 microg/ml after 8 h, in addition to the reported cytotoxicity induced at a ng/ml level after 48 h. Vacuolation was induced with the B, but not the A, subunit and was dependent on V-type ATPase. The cytotoxicity of SubAB at low concentrations was associated with the inhibition of protein synthesis; the 50% inhibitory dose was approximately 1 ng/ml. The A subunit, containing serine 272, which is thought to be a part of the catalytic triad of a subtilase-like serine protease, plus the B subunit was necessary for this activity, both in vivo and in vitro. SubAB did not cleave azocasein, bovine serum albumin, ovalbumin, or synthetic peptides. These data suggest that SubAB is a unique AB toxin: first, the B subunit alone can induce vacuolation; second, the A subunit containing serine 272 plus the B subunit inhibited protein synthesis, both in vivo and in vitro; and third, the A subunit proteolytic activity may have a strict range of substrate specificity.     

Hemolytic uremic syndrome in Belgium: incidence and association with verocytotoxin-producing Escherichia coli infection

Objective: To evaluate the incidence of hemolytic uremic syndrome (HUS) in Belgium and to determine the role of verocytotoxin-producing Escherichia coli O 157:H7 and other serotypes (non-O 157 VTEC).
Methods: Twenty-two centers, including the seven university hospitals, registered prospectively all cases of HUS; they collected clinical samples for isolation of VTEC strains and serum for detection of specific O-lipopolysaccharide antibodies.
Results: Forty-seven cases of HUS (including five incomplete cases) were recorded. Three cases were seen in nonresidents. The incidence of complete HUS in Belgian residents was 4.3 cases/100 000 in children <5 years old, 1.8 cases/100 000 when all children <15 years were considered, and 0.42/100 000 when patients of all ages were taken into account. By combining bacteriologic and serologic results, evidence of VTEC infection was obtained in 64% of the patients, mainly but not exclusively in children with prodromal diarrhea. The 13 VTEC isolates belonged to serotypes O157:H7 (nine isolates), O26:H11, O121:H-, O145:H- and O172:H- (one each) and all produced VT2 (+VT2vh-a in three O157 strains) and were positive for the eaeA gene.
Conclusions: The incidence rate found in this study and the high mortality and morbidity linked with this syndrome warrant further registration of pediatric and post-diarrheic adult HUS cases and also examination of stools for both O157 and non-O157 VTEC strains. For effective prevention of this disease, further study of the serotypes and accessory virulence factors associated with HUS is needed.     

Multiplex PCR for direct detection of Shiga toxigenic Escherichia coli strains producing the novel subtilase cytotoxin

We have recently described a novel AB₅ subtilase cytotoxin produced by certain Shiga toxigenic Escherichia coli (STEC) strains. This potentially lethal toxin may contribute to severe gastrointestinal and systemic disease in humans. In this study we have developed a trivalent PCR assay for the detection of the novel toxin A subunit gene subA, as well as stx₁ and stx₂. The three primer pairs used in the assay do not interfere with each other and generate amplification products of 556, 180, and 255 bp, respectively. The assay can be used for determining the toxin genotype of STEC isolates, as well as for direct detection of toxin genes in primary fecal culture extracts.     

Protective immunization of mice with an active-site mutant of subtilase cytotoxin of Shiga toxin-producing Escherichia coli

We have recently described a novel AB(5) cytotoxin produced by certain Shiga toxin-producing Escherichia coli strains. The A subunit of this toxin is a subtilase-like serine protease, while the B pentamer mediates binding to host cell glycolipid receptors. The subtilase cytotoxin is lethal for mice, causing extensive microvascular thrombosis as well as necrosis in the brain, kidneys, and liver. In the present study, we have immunized mice with a purified derivative of the toxin with a Ser272 --> Ala mutation in the A subunit which abolishes cytotoxicity. This elicited strong antibody responses, as judged by enzyme-linked immunosorbent assay, which conferred protection against intraperitoneal challenge with purified toxin. Immunized mice were also protected from weight loss resulting from oral challenge with an E. coli K-12 clone expressing the active toxin.     

Hemagglutination and adhesiveness of toxigenic Escherichia coli isolated from humans.

Toxigenic strains of Escherichia coli isolated from humans were studied for adherence to human buccal mucosal epithelial cells. The E. coli strains were labeled with 3H-amino acids or fluorescein isothiocyanate. Toxigenic E. coli strains varied in their ability to adhere in the presence of mannose. Of 32 toxigenic strains examined, 52% bound to the buccal cells, whereas none of 8 control strains did so (Mann-Whitney U test, P =0.007). The control strains were nontoxigenic E. coli isolates from humans, enterotoxigenic E. coli isolates from animals, and E. coli K-12 containing the K88 or K99 plasmid; these strains exhibited only background-level adherence in this assay. Among the toxigenic E. coli strains that bound to human buccal mucosal cells, there was no correlation with mannose-resistant hemagglutination (MR-HA) of guinea pig and human erythrocytes. Screening 32 strains, we found the following phenotypes: (i) MR-HA+, buccal adherent; (ii) MR-HA+, buccal nonadherent; (iii) MR-HA-, buccal adherent. Presumably the third group represents strains with another type(s) of surface attachment components not involved in the MR-HA reaction. Our findings indicate that a number of bacterial surface structures can function in MR-HA and buccal adherence.     

Virulence genes in verocytotoxigenic Escherichia coli strains isolated from humans and cattle

Verocytotoxigenic Escherichia coli (VTEC) causing diarrhoea, haemorrhagic colitis and haemolytic-uremic syndrome usually have additional traits such as the adhesin intimin and a large plasmid that seems to increase virulence. There are, however, isolates of VTEC causing serious symptoms that do not harbour these traits. In the present study we have used PCR with primers detecting adhesin genes other than eaeA, namely fimA, papC, sfaD/sfaE and daaE. We have also used PCR to detect the genes hlyA and iutA that besides the plasmid-borne gene E-hly possibly support the bacterial access to iron. The aim of the study was to identify and compare the presence of virulence genes in VTEC isolates of human and cattle origin. The main finding was that the absence of E-hly might be compensated for by the gene iutA coding for aerobactin or hlyA coding for alpha-haemolysin as 94% of the human VTEC isolates had at least one of these genes. Interestingly, only 45% of VTEC isolated from cattle had any of these genes. We propose that this might be the reason for the relatively low incidence of symptomatic VTEC infections among humans in relation to the high number of VTEC among cattle.     

Type II heat-labile enterotoxin-producing Escherichia coli isolated from animals and humans.

Heat-labile enterotoxin (LT)-producing Escherichia coli strains, as identified by the Y1 adrenal cell assay, were examined with a DNA probe coding for type I and type II LTs. Of 236 LT-producing E. coli isolates, 60% hybridized with LT-I, 17% hybridized with LT-II, and 23% did not hybridize with either probe and no longer produced LT as determined by the Y1 adrenal cell assay. These isolates presumably lost plasmids coding for LT-I during storage. A total of 75% of LT-producing E. coli isolates (27 of 36) from cows, 64% of LT-producing E. coli isolates (7 of 11) from buffalo, 31% of LT-producing E. coli isolates (4 of 13) from beef obtained in markets, and 2% of LT-producing E. coli isolates (3 of 168) from humans contained genes coding for LT-II. Genes coding for LT-II were not found in 50 LT-I-producing and heat-stable enterotoxin-producing E. coli isolates from 11 children with diarrhea and 44 LT-nonproducing and heat-stable enterotoxin-producing E. coli isolates from 12 other children with diarrhea. A total of 9% of LT-II-producing E. coli isolates (3 of 34) from cows and buffalo hybridized with DNA probes for genes coding for verocytotoxin 2 (VT2), and 18% (6 of 34) hybridized with a DNA probe coding for enterohemorrhagic E. coli (EHEC) adhesin fimbriae. E. coli SA-53, the original isolate in which LT-II was found, contained genes coding for VT2 and EHEC adhesin fimbriae. Five VT-producing, LT-II-producing E. coli isolates that hybridized with the EHEC probe did not contain DNA sequences coding for VT1 or VT2. LT-II-producing E. coli strains were frequently isolated from cattle and buffalo but were rarely isolated from humans.     

Incidence and virulence determinants of verocytotoxin-producing Escherichia coli infections in the Brussels-Capital Region, Belgium, in 2008-2010

The incidence of verocytotoxin-producing Escherichia coli (VTEC) was investigated by PCR in all human stools from Universitair Ziekenhuis Brussel (UZB) and in selected stools from six other hospital laboratories in the Brussels-Capital Region, Belgium, collected between April 2008 and October 2010. The stools selected to be included in this study were those from patients with hemolytic-uremic syndrome (HUS), patients with a history of bloody diarrhea, patients linked to clusters of diarrhea, children up to the age of 6 years, and stools containing macroscopic blood. Verocytotoxin genes (vtx) were detected significantly more frequently in stools from patients with the selected conditions (2.04%) than in unselected stools from UZB (1.20%) (P = 0.001). VTEC was detected most frequently in patients with HUS (35.3%), a history of bloody diarrhea (5.15%), or stools containing macroscopic blood (1.85%). Stools from patients up to the age of 17 years were significantly more frequently vtx positive than those from adult patients between the ages of 18 and 65 years (P = 0.022). Although stools from patients older than 65 years were also more frequently positive for vtx than those from patients between 18 and 65 years, this trend was not significant. VTEC was isolated from 140 (67.9%) vtx-positive stools. One sample yielded two different serotypes; thus, 141 isolates could be characterized. Sixty different O:H serotypes harboring 85 different virulence profiles were identified. Serotypes O157:H7/H- (n = 34), O26:H11/H- (n = 21), O63:H6 (n = 8), O111:H8/H- (n = 7), and O146:H21/H- (n = 6) accounted for 53.9% of isolates. All O157 isolates carried vtx2, eae, and a complete O island 122 (COI-122); 15 also carried vtx1. Non-O157 isolates (n = 107), however, accounted for the bulk (75.9%) of isolates. Fifty-nine (55.1%) isolates were positive for vtx1, 36 (33.6%) were positive for vtx2, and 12 (11.2%) carried both vtx1 and vtx2. Pulsed-field gel electrophoresis revealed wide genetic diversity; however, small clusters of O157, O26, and O63:H6 VTEC that could have been part of unidentified outbreaks were identified. Antimicrobial resistance was observed in 63 (44.7%) isolates, and 34 (24.1%) showed multidrug resistance. Our data show that VTEC infections were not limited to patients with HUS or bloody diarrhea. Clinical laboratories should, therefore, screen all stools for O157 and non-O157 VTEC using selective media and a method for detecting verocytotoxins or vtx genes.     

Genotypic characterization of enteropathogenic Escherichia coli (EPEC) isolated in Belgium from dogs and cats

Enteropathogenic Escherichia coli (EPEC) are isolated from man and farm animals but also from dogs and cats. They produce typical histological lesions called 'attaching and effacing' lesions. Both plasmid and chromosomal elements are involved in the pathogenesis of EPEC infection. The presence of these genetic elements was investigated in 14 dog and three cat EPEC isolates. A bfpA-related gene was detected in five of the 17 isolates in association with high molecular weight plasmids, and a locus of enterocyte effacement (LEE) was present in all isolates. The LEE was inserted in the selC region in only 12% of the isolates. The eae, tir, espA and espB genes were analyzed by multiplex PCR. The results indicated the presence of those genes in the tested isolates with heterogeneity in the gene subtypes present: eae gamma-tir alpha-espA alpha-espB alpha (65%), eae beta-tir beta-espA beta-espB beta (29%), eae alpha-tir alpha-espA alpha-espB alpha (6%). Moreover, the espD gene was also present in dog and cat EPEC. The DEPEC and CEPEC form a heterogeneous group and five of them are closely related to human EPEC.     

Serotypes of attachment pili of enterotoxigenic Escherichia coli isolated from humans.

Pili from enterotoxigenic Escherichia coli isolated from humans have been partially purified, and antisera have been prepared. These pili were initially attached to erythrocytes and then removed by thermal elution for purification. Three distinct antigenic types of pili have been identified. Antisera against these three pili types reacted with 60 of 106 (56%) enterotoxigenic E. coli isolated from humans but not with nontoxigenic, normal human fecal isolates of E. coli nor with enterotoxigenic E. coli strains isolated from animals. There was no correlation between pili serogroup and any of the following toxin production (heat labile, heat stable, or both), O antigenic type, geographical source of isolation, or mannose-resistant hemagglutination patterns of various erythrocyte types.     

Prevalence of quinolone resistance mechanisms and associations to minimum inhibitory concentrations in quinolone-resistant Escherichia coli isolated from humans and swine in Denmark

Prevalence of quinolone resistance mechanisms and associations to minimum inhibitory concentrations (MICs) of nalidixic acid (NAL) and ciprofloxacin (CIP) were investigated in 124 Escherichia coli isolated from humans (n=85) and swine (n=39) in Denmark. The collection included 59 high-level CIP-resistant isolates (MIC >or= 4) from human (n=51) and pig origin (n=8) and 65 low-level CIP-resistant isolates (MIC >or= 0.125) from human (n=34) and pig origin (n=31). Resistance by target modification was screened by PCR amplification and sequencing of the quinolone resistance determining regions (QRDRs) of gyrA, gyrB, parC, and parE. QRDR mutations occurred in all except two isolates (98%). All high-level CIP-resistant E. coli had one or two mutations in gyrA in combination with mutations in parC or parE. Mutations in parC and parE were only found in combination with gyrA mutations, and no mutations were observed in gyrB. Efflux pump mechanisms were detected in 10 human (11.8%) and 29 porcine (74.4%) isolates by an efflux pump inhibitor (EPI) agar dilution assay. The aac(6')-Ib-cr gene mediating resistance by enzymatic modification was found in 12 high-level CIP-resistant human isolates. The qnrA and qnrS genes conferring quinolone resistance by target protection were detected in two human low-level CIP-resistant isolates that did not display NAL resistance. As expected, target mutation in QRDRs was the most prevalent mechanism of quinolone resistance. This mechanism was complemented by efflux mechanisms in most porcine isolates. Transferable resistance by target protection or enzymatic modification was less common (10%) and restricted to human isolates.     

HeLa cell adherence and cytotoxin production by enteropathogenic Escherichia coli isolated from infants with diarrhea in Thailand.

Enteropathogenic Escherichia coli (EPEC) strains isolated from hospitalized infants with diarrhea in Thailand were examined for HeLa cell adherence and cytotoxin production. Of 101 strains examined, 56 adhered to HeLa cells in a localized pattern (LA), 27 adhered in a diffuse pattern (DA), and 18 did not adhere. All 56 LA EPEC strains were O:K serotype O119:K69. A total of 20 (83%) of 24 EPEC O86:K61 strains and 7 (38%) of 19 EPEC strains belonging to six other O:K serotypes exhibited DA. All LA EPEC strains hybridized with a DNA probe for genes encoding EPEC adherence factor, whereas none of the 27 DA or 18 nonadherent EPEC strains hybridized with EPEC adherence factor probe. Sonic extracts of 57 (58%) of 98 EPEC strains tested at a dilution of 1:100 caused greater than 25% mortality of HeLa cell monolayers. A total of 50 (88%) of 57 cytotoxic sonic extracts were inhibited to various degrees by a 1:500 dilution of polyclonal rabbit antisera to purified Shiga toxin. The mean percent inhibition of cytotoxic sonic extracts by anti-Shiga toxin was 67% (range, 29 to 89%). Fifty percent (38 of 56) of LA EPEC strains, fifty-two percent (14 of 27) of DA EPEC strains, and fifty-three percent (8 of 15) of nonadherent EPEC strains produced Shiga-like toxins. Both adherence and low levels of cell-associated cytotoxins were identified in EPEC strains from Thailand, but there did not appear to be an association between these two factors.     

Properties of an Escherichia coli cytotoxin.

Isoelectric focusing of a heat-labile cytotoxin of Escherichia coli H30 revealed the presence of two molecular variants, pI 7.2 and a comparatively small quantity of pI 6.8. Predominant component pI 7.2 had a molecular weight of 28,000, induced some fluid accumulation in rabbit ileal loops, and showed no morphological response in Y-1 cells but a strong cytotoxic effect on Vero cells.     

Distinct pathotypes of O113 Escherichia coli strains isolated from humans and animals in Brazil

Two distinct diarrheagenic Escherichia coli pathotypes, enteroaggregative E. coli (EAEC) and Shiga toxin-producing E. coli, were observed in association with O113 strains isolated from human and nonhuman sources in Brazil, respectively. The O113 strains from human diarrhea belonged to a diversity of serotypes, and nine (53%) of them harbored virulence traits of typical EAEC.     

Characteristics of Shiga toxin producing- and enteropathogenic Escherichia coli of the emerging serotype O80:H2 isolated from humans and diarrhoeic calves in Belgium

Objectives

Recently a highly virulent Escherichia coli O80:H2 pathotype carrying Shiga toxin genes, the intimin subtype eaeξ, and genes associated with the extraintestinal pathogenic E. coli (ExPEC) pS88 plasmid was described in France. In this study we examine the relatedness of Belgian E. coli O80:H2 isolated from humans and diarrhoeic calves as well their similarities with the French pathotype.

Prevalence of beta-lactamases among ampicillin-resistant Escherichia coli and Salmonella isolated from food animals in Denmark

The genetic background for beta-lactamase-mediated resistance to beta-lactam antibiotics was examined by PCR and sequencing in 160 ampicillin-resistant isolates (109 Escherichia coli and 51 Salmonella) obtained from healthy and diseased food animals in Denmark. Sequencing revealed three different variants of bla (TEM-1), of which bla (TEM-1b) was the most frequently detected (80 E. coli and 47 Salmonella), followed by bla (TEM-1a) (eight E. coli, one Salmonella) and bla (TEM-1c) (seven E. coli). A few isolates were found to express OXA, TEM-30, or PSE beta-lactamases. Mutations in the ampC promoter leading to increased production of the AmpC beta-lactamase were demonstrated in 11 cefoxitin-resistant or intermediate E. coli isolates. Nine of these isolates did not contain any bla (TEM) genes, whereas the remaining two did. No genes encoding SHV or extended-spectrum beta-lactamases were detected. Two new variants of bla (TEM) were detected, which have been designated bla (TEM-127) and bla (TEM-128). In TEM-127, amino acid 158 is substituted from His to Asn, whereas a substitution from Asp to Glu is seen at amino acid 157 in TEM-128. According to MIC determinations, these novel enzymes do not possess activity against extended-spectrum beta-lactams.     

Signal transduction responses following adhesion of verocytotoxin-producing Escherichia coli.

Attaching and effacing adhesion to epithelial cells is a pathognomonic feature of infection by both enteropathogenic Escherichia coli (EPEC) and certain strains of verocytotoxin-producing E. coli (VTEC). EPEC adhesion to tissue culture epithelial cells results in activation of the phosphatidylinositol pathway, with elevated levels of inositol 1,4,5-triphosphate and cytosolic free calcium. In this report, we show that VTEC also activate this signal transduction pathway in infected epithelial cells. Specifically, increased levels of inositol 1,4,5-triphosphate and intracellular free calcium were observed in HEp-2 cells infected with VTEC of serotype O157:H7. VTEC of serotypes O157:H7 and O113:H21 also induced increases in intracellular calcium levels in the human intestinal crypt-like cell line T84, even with minimal or no attaching and effacing activity as monitored by transmission electron microscopy. In contrast to EPEC, VTEC failed to induce tyrosine phosphorylation of epithelial cell proteins in HEp-2 and T84 cells, as determined by indirect immunofluorescence microscopy. These findings suggest that signal transduction responses to VTEC, including elevated levels of inositol triphosphates and intracellular free calcium, are independent of formation of the attaching and effacing lesion. Our findings also show that VTEC pathogenesis may involve signal transduction pathways that are distinct from those induced by EPEC infection.