Production Of Cytotoxic Necrotizing Factor Research Articles

Fator necrosante citotóxico em Escherichia coli isolada de mastite clínica bovina

This report describes the production of cytotoxic necrotizing factor (CNF) by an Escherichia coli strain isolated from clinical bovine mastitis with clinical signs of toxemia The animal had hemorrhages and necrosis of the mammary glands, and died within 24 hours after the onset of clinical signs. In addition to CNF identification, alpha-haemolysin and siderophores production were also characterized in this strain. This report reinforce the association of CNF and alpha-haemolysin production in E. coli virulence associated with clinical cases of severe bovine mastitis.

Gene block encoding production of cytotoxic necrotizing factor 1 and hemolysin in Escherichia coli isolates from extraintestinal infections

Cytotoxic necrotizing factors (CNFs) are Escherichia coli protein toxins causing cell multinucleation and enlargement in tissue cultures and necrosis in rabbit skin. In E. coli isolates causing urinary tract infections in humans, the production of CNF1 is closely associated with hemolysin production. In this study, we obtained data suggesting that this phenotypic association is due to the genetic linkage of the determinants of the two toxins on the chromosome of uropathogenic E. coli. The genes encoding hemolysin and CNF1 were shown to be closely linked in a 37-kb cloned DNA fragment from an E. coli urinary tract isolate of serotype O4:K12:H5 (E-B35). A DNA region encoding CNF1 production but not hemolysin production was further subcloned as a 12-kb SalI-EcoRI fragment and used as a CNF1-specific gene probe. DNA hybridization experiments indicated that the CNF1 and hemolysin determinants were closely linked on the chromosomes of isolate E-B35 and six additional extraintestinal isolates belonging to serogroups O2, O4, O6, O22, O75, and O85.

Relative Importance of Eight Virulence Characteristics of Pyelonephritogenic Escherichia Coli Strains Assessed by Multivariate Statistical Analysis

We have previously reported univariate statistical analysis of the prevalences of putative virulence determinants in Escherichia coli isolated from children and adults with acute pyelonephritis. The expression of P-fimbriae, cell surface hydrophobicity, mannose resistant haemagglutination, haemolysin synthesis, cytotoxic necrotizing factor production and aerobactin mediated iron uptake occurred more often in a collection of 115 Escherichia coli strains isolated from children and women with acute non-obstructive pyelonephritis compared to 96 strains isolated from the commensal fecal flora. With the aim to study which of these virulence markers were independently associated with strains causing infection we performed a multivariate statistical analysis with the data from these strains. The previously proposed virulence factors, expression of type 1 fimbriae and adhesion to HeLa cells were also included in the analysis. P-fimbriae, mannose resistant haemagglutination and the production of haemolysin were, in the multivariate analysis, associated with strains isolated from patients with acute pyelonephritis.

Characteristics of α-hemolytic strains of Escherichia coli isolated from dogs with gastroenteritis

Twenty-four hemolysin producing (Hly +) strains of Escherichia coli isolated from dogs with gastroenteritis were investigated for their virulence markers and their phenotypic properties. The strains were distributed over eleven known E. coli O-serogroups and most of them were heterogeneous for their phenotypes. All strains were found to produce α-hemolysin which was detected by Southern hybridization and colony immunoblotting using a specific gene probe and a monoclonal antibody. Eight strains were carrying plasmids encoding α-hemolysin sequences ( hly-plasmids) and 16 strains carried chromosomal hly-determinants. Twelve of the strains showed enterotoxic activities which were tested for in different assays. Among these, three O42:H37 and two O7O:H- strains carrying hyl-plasmids were found to harbour other plasmids encoding the heat-stable enterotoxin ST A1. The other seven strains showing enterotoxicity in the ileal loop or the suckling mouse assay were negative for ST A1, ST A2, or LT. None of the 24 strains were positive for invasiveness or for production of Vero (Shiga-like) toxins. The production of α-hemolysin was closely associated with the production of cytotoxic necrotizing factor (CNF), which was detected in 17 of 24 strains. Of these, 16 elaborated CNF1 and one strain produced an unknown CNF type. Surprisingly, all strains carrying ST-plasmids and six of eight strains carrying hly-plasmids were negative for CNF. Thus, in canine E. coli strains CNF production seems to be closely associated with production of chromosomally encoded α-hemolysin whereas hly-plasmids are more often associated with ST-producing, CNF negative isolates.

Production of cytotoxic necrotizing factor, verocytotoxin and haemolysin by pyelonephritogenicEscherichia coli

Two hundred and thirty-two strains of Escherichia coli isolated from children with non-obstructive acute pyelonephritis (n = 65), women with non-obstructive acute pyelonephritis (n = 63) and the faecal flora of healthy children (n = 33) and adults (n = 71) were examined for cytotoxic necrotizing factor production, haemolysin synthesis, verocytotoxin production and expression of mannose-resistant haemaglutination of human erythrocytes. Forty-eight per cent of the pyelonephritogenic Escherichia coli strains produced cytotoxic necrotizing factor and 61% produced haemolysin compared to 25% and 27% of faecal control strains (p less than 0.001 and p less than 0.001 respectively). Cytotoxic necrotizing factor production did not occur among the non-haemolytic Escherichia coli strains which confirms the close association between these two toxic factors. The bacterial phenotypes producing both haemolysin and cytotoxic necrotizing factor, and the phenotype expressing both these toxic factors and mannose-resistant haemagglutination occurred significantly more often in pyelonephritogenic strains than in faecal isolates (p less than 0.001). Haemolytic strains without the ability to produce cytotoxic necrotizing factor were more common in faecal isolates than in uropathogenic strains (p = 0.05). Strains lacking the ability to synthesize both these toxins were also over-represented in faecal isolates (p less than 0.01).

Relationship between cytotoxic necrotizing factor production and serotype in hemolytic Escherichia coli.

We examined the relationship between serotype and cytotoxic necrotizing factor (CNF) production in 123 hemolytic strains of Escherichia coli isolated from both stools and extraintestinal infections. Of 76 strains producing both hemolysin (Hly) and CNF, 66 (87%) belonged to one of six serogroups (O2, O4, O6, O22, O75, and O83). In contrast, 47 E. coli strains producing Hly only belonged to 21 different O serogroups, and only 2 of these (O6 and O18ac) were widely represented. Generally, CNF-positive and CNF-negative hemolytic isolates were assigned to different O serogroups, with the exception of O6, often present in both categories of isolates. Serogroups O4 and O18ac were significantly more prevalent among strains from extraintestinal infections than among those from stools. In contrast, the Hly-positive, CNF-negative isolates, belonging to numerous less common serogroups, were hardly ever isolated from extraintestinal infections. Serological typing further confirmed that hemolytic isolates of E. coli may grossly be divided into two main populations on the basis of the ability to produce CNF. Examination of hemolytic E. coli for this property may also be useful in achieving a more detailed characterization of pathogenic clones.

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 prossessing 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 untype 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 wre 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 diarrhoea remains to be established.

Toxins and serotypes of faecal non-enterotoxigenic and non-enteropathogenic Escherichia coli strains causing mannose-resistant haemagglutination: Relation with haemagglutination patterns

Forty-three faecal non-enterotoxigenic and non-enteropathogenic human Escherichia coli strains causing mannose-resistant haemagglutination (MRHA) were tested for production of cytotoxic necrotizing factor (CNF), haemolysis (Hly), Verotoxin (VT) and lethal activity for mice. The serotypes of the strains were also determined. Of the total strains investigated, 49% synthesized CNF, 53% were haemolytic and 40% were lethal for mice. No strain producing VT was detected. Striking differences in the production of Hly and CNF were observed when MRHA strains were grouped according to their lethal or non-lethal activity. Thus, 82% of lethal strains produced Hly and/or CNF whereas only 35% (p < 0.01) and 27% (p < 0.01) of non-lethal strains produced Hly and CNF, respectively. The production of toxins was specially associated with strains possessing defined MRHA types. Thus, 100%, 82% and 50% of strains belonging to MRHA types III, IVa and V, respectively, were toxigenic, whereas no toxigenic strains from MRHA types IVb and VI were detected. The majority (77%) of MRHA strains possessed typical O groups usually reported to be present in pathogenic extraintestinal E. coli or in facultatively enteropathogenic E. coli. Furthermore, these O groups were more frequently detected in toxigenic (93%) than in non-toxigenic (47%) strains (p < 0.01). Our results suggest that faecal non-enterotoxigenic E. coli strains belonging to MRHA types III, IVa and V may be responsible for extraintestinal infections as well as for sporadic intestinal infections, and that certain O groups are specially associated with E. coli strains belonging to particular MRHA types.

Frequent production of toxins byEscherichia colistrains isolated from human urinary tract infections: relation with haemagglutination

We have investigated the production of heat-labile enterotoxin (LT), Verotoxin (VT), cytotoxic necrotizing factor (CNF), haemolysis (Hly) and lethal activity for mice in 48 Escherichia coli strains isolated from patients with urinary tract infections. Mannose-resistant and mannose-sensitive haemagglutination in these strains were also studied. Among the total strains investigated, 50% were haemolytic, 50% synthesized CNF and 58% were lethal for mice. A total of 33 (69%) strains were toxigenic, showing positive results at least in one of the tests employed for toxin detection. No strain was positive for LT and VT production. We conclude that, in addition to haemolytic and haemagglutinating activities, the production of CNF was closely associated to virulence in E. coli strains isolated from urinary tract infections.

Cytotoxic necrotizing factor production by hemolytic strains of Escherichia coli causing extraintestinal infections.

Two hundred and nineteen strains of Escherichia coli from extraintestinal infections and feces of healthy subjects were examined for hemolysin (Hly) and cytotoxic necrotizing factor (CNF) production and for mannose-resistant hemagglutination. Of 105 strains from extraintestinal infections, 42 (40.0%) were positive for production of both Hly and CNF, and 21 (20.0%) were positive for Hly alone; on the contrary, only 1 Hly- and CNF-positive strain and 2 Hly-positive strains were found among 114 strains from normal stools. CNF production was not found to occur among the nonhemolytic strains, confirming the close association existing between these toxic factors. Hemolytic strains positive for CNF showed mannose-resistant hemagglutination less frequently than did Hly-positive, CNF-negative strains (25.6 versus 82.6%), suggesting the existence of two distinct classes among hemolytic strains of E. coli.