Geraldine A. Willshaw

A simple method for the preparation of large quantities of pure plasmid DNA.

Polyethylene glycol quantitatively precipitates plasmid DNA of molecular weight 6-123-10-6, from cleared lysates of plasmid-carrying bacterial strains, After resuspension and density-gradient centrifugation of the precipitated DNA, it is unchanged in length and in transformation efficiency for Escherichia coli K12. Plasmid DNA can be easily prepared in large quantities by including a polyethylene glycol precipitation step in standard plasmid isolation procedures.

The emergence and spread of antibiotic resistance in food-borne bacteria.

Since the early 1990s there has been a dramatic increase in resistance to antimicrobial drugs in Salmonella enterica and Campylobacter spp., and to a lesser extent in Vero cytotoxin-producing Escherichia coli O157 from cases of human infection in developed countries. For S. Typhimurium a particularly important aspect of this increase has been the widespread dissemination of a multiply drug-resistant (MR) strain of definitive phage type (DT) 104 in food animals since the early 1990s. The use of antimicrobials for prophylaxis in food producing animals has been an important factor in the emergence of strains with resistance to certain antimicrobials. It is hoped that recently introduced Codes of Practice for the prophylactic use of antimicrobials in food animals will result in a decline in the occurrence of drug resistant strains in the food chain.

Vero cytotoxin-producing strains of Escherichia coli from children with haemolytic uraemic syndrome and their detection by specific DNA probes

Faecal specimens from 66 children with haemolytic uraemic syndrome in the United Kingdom were examined for strains of Escherichia coli producing Vero cytotoxin (VT). Initially, conventional bacteriological methods were used to identify colonies of E. coli which were then tested for VT production. Subsequently, specific DNA probes for VT1 and VT2 were used in hybridisation tests to detect VT-producing E. coli (VTEC). VTEC strains were isolated from 19 cases and in 15 they belonged to serogroup O157. Fourteen of these O157 strains possessed the flagellar antigen H7 and one was non-motile. The VTEC strains from the remaining four cases belonged to serotypes O26:H11, O104:H2, O153:H25, and O163:H19 together with a rough VT+ strain with flagellar antigen H51. The O157 strains hybridised with either the VT2 probe or both VT1 and VT2 probes. The other VTEC strains hybridised with either the VT1 or VT2 probe. Confirmation of the production of VT1 and VT2 in vivo was obtained by the neutralisation of faecal VT with specific antisera raised against these two cytotoxins.

Heterogeneity of Escherichia coli phages encoding Vero cytotoxins: comparison of cloned sequences determining VT1 and VT2 and development of specific gene probes.

Phages coding for production of Vero cytotoxins VT1 or VT2 in strains of Escherichia coli serotype O157.H7 or O157.H- were morphologically indistinguishable. Their genome size and restriction enzyme digests of the phage DNA were similar. These phages were clearly different in these respects from a VT1-encoding phage isolated from a strain of E. coli O26.H11 (H19). However the VT1 region cloned from the phage originating in the E. coli O157.H7 strain was identical to the VT1 region previously cloned from the phage carried by H19. Sequences encoding VT2 that were cloned from the phage in E. coli O157.H- have been mapped and the VT2 region identified by transposon insertion. The cloned regions coding for VT1 or VT2 production had no similarities in the presence of restriction enzyme sites over a distance of about 2 kb, and two VT1-specific probes spanning a region of about 1.4 kb did not hybridize under stringent conditions with cloned VT2 DNA. A 2 kb HincII fragment contained the VT2 genes but hybridized to VT1-encoding phages and recombinant plasmids via flanking phage DNA. A 0.85 kb AvaI-PstI fragment was a specific probe for VT2 sequences and did not hybridize under stringent conditions to phages or plasmid recombinants encoding VT1.

Properties of strains of Escherichia coli belonging to serogroup O157 with special reference to production of Vero cytotoxins VT1 and VT2

Fifty-four strains of Escherichia coli belonging to serogroup O157 were examined for the production of Vero cytotoxins VT1 and VT2, and for other properties such as plasmid content, resistance to antimicrobial agents and colicin production. Twenty-six strains from cases of diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome in humans produced VT. By serum neutralization tests and hybridization with DNA probes for VT1 or VT2, three classes were recognized which produced either VT1 alone or VT2 alone or both VT1 and VT2. These strains were of H type 7 or non-motile. The strains producing VT were sensitive to all the antimicrobial agents tested, and all carried at least one plasmid which had a molecular weight of c. 60 X 10(6). Seven strains of porcine origin and 21 strains of human origin did not produce VT or hybridize with either DNA probe. None of these strains was of H type 7. Of the 21 human VT- strains, 17 were of extra-intestinal origin and 18 were of H type 45. Twenty-three of the 28 VT-strains were resistant to at least one antimicrobial agent.

Mutagenesis of plasmid DNA with hydroxylamine: Isolation of mutants of multi-copy plasmids

SummaryAn investigation of in vitro mutagenesis of plasmid DNA with hydroxylamine is described. The treated plasmid DNA was used to transform Escherichia coli K12. Mutants of the plasmid NTP3, which codes for resistance to ampicillin and sulphonamides, were isolated and characterised. They were classified according to the reduction in level of their β-lactamase activity. Hydroxylamine-induced mutants of NTP14 were also isolated. This plasmid codes for ampicillin resistance, synthesis of colicin E1, and the EcoRI restriction and modification enzymes. One class of mutants is lethal to the host strain at temperatures above 33° C, but carrier strains grow well at 28° C. There is evidence that these mutants code for a temperature-sensitive EcoRI modification activity: the lethal effect probably results from the cleavage of the host-cell DNA by the restriction enzyme at non-permissive temperatures. The possible genetic uses of the mutant plasmids for the production of hybrid plasmids in the bacterial cell are discussed.

Verocytotoxin-producing Escherichia coli (VTEC) O157 and other VTEC from human infections in England and Wales: 1995-1998.

A total of 3429 isolations of verocytotoxin-producing Escherichia coli O157 (VTEC O157) was confirmed from human sources in England and Wales during the period 1995-1998. The largest annual total was 1087 in 1997. Most infections occurred in the third quarter of each year. The overall rate of infection ranged from 1.28 to 2.10/100,000 population and showed regional variation. The highest incidence was in children aged 1-4 years. Annually, between 5% and 11% of strains were from patients who had travelled abroad. There were 67 general outbreaks of infection represented by 407 (11.9%) VTEC O157 isolates. Outbreaks involved transmission by contaminated food or water, person-to-person spread and direct or indirect animal contact, and five were associated with foreign travel. The majority (76%) of strains carried verocytotoxin (VT) 2 genes and 23.3% were VT1+VT2. Most strains had the flagellar antigen H7, but c. 14% were non-motile. Approximately 20% of isolates were resistant to antimicrobial agents, predominantly streptomycin, sulphonamides and tetracycline. In addition to VTEC O157, strains of serogroup O157 that did not possess VT genes were identified. These were either derivatives of VTEC O157 that had lost VT genes or were strains with H antigens other than H7 that have never been associated with VT production. Strains of VTEC other than O157 were characterised. Most were associated with diarrhoea, bloody diarrhoea or haemolytic uraemic syndrome and had virulence markers in addition to VT.

Cloning of genes determining the production of vero cytotoxin by Escherichia coli

Sequences encoding the production of a cytotoxin (VT) active on Vero cells were cloned in Escherichia coli K12 from a VT-determining phage that originated in E. coli strain H19 of serotype O26.H11. Subcloning resulted in the identification of a 2.5 kb fragment that still coded for VT production. Mutagenesis with transposon Tn1000 was used to map VT sequences and a 0.75 kb probe was developed. In colony hybridization tests with strains isolated from patients with haemolytic uraemic syndrome or diarrhoea, this probe derived from the H19 VT genes detected only some of the VT+ strains belonging to serogroup 0157. A VT+ strain, E32511, serotype 0157.H-, which was negative in colony hybridization was the source of another VT-determining phage from which VT sequences were cloned. Southern hybridization of the VT genes from E32511 with the H19 probe was negative under stringent conditions but there was weak homology under conditions of low stringency. These results indicate that there are differences in the VT genes of pathogenic E. coli.

Non-radioactively labelled polynucleotide and oligonucleotide DNA probes, for selectively detecting Escherichia coli strains producing Vero cytotoxins VT1, VT2 and VT2 variant

Vero cytotoxin producing Escherichia coli (VTEC) were detected in faecal specimens and bacterial isolates, using non-radioactively labelled polynucleotide and oligonucleotide DNA probes specific for Vero cytotoxin (VT) genes. VT1 and VT2 structural gene sequences, previously cloned and used for radioactive probes, were labelled with digoxigenin or biotin. Oligonucleotide gene sequences coding for the A subunit of VT1, VT2 and VT2 variant were labelled with digoxigenin. The VT1 and VT2 probes were specific for detecting VT1 and VT2 gene sequences and gave very similar results to those obtained using the radioactive label 35S as a standard. The VT2 variant probe hybridized only with the strains of porcine origin. For the range of isolates tested, there was little significant difference in specificity and sensitivity between the digoxigenin-labelled polynucleotide and oligonucleotide probes. The biotin system gave rise to more non-specific effects, particularly with some non-E. coli strains, and was therefore less reliable. All of the digoxigenin-labelled probes gave satisfactory results after several times re-use, which is of importance when considering cost.

Identification of enteropathogenic Escherichia coli isolated in Britain as enteroaggregative or as members of a subclass of attaching-and-effacing E. coli not hybridising with the EPEC adherence-factor probe

Strains of Escherichia coli from sporadic cases of diarrhoea and belonging to serotypes O44:H18, O55:H7, O111ab:H21, O111ab:H25 or O126:H27 were examined for virulence properties. With the exception of O111ab:H25 these are considered to be classical enteropathogenic E. coli (EPEC) serotypes. The strains had been isolated in Britain from the faeces of children less than 3 years old. Of the serotypes examined, 7 of 13 O44:H18 strains, all of 10 O111ab:H21 strains and 13 of 21 O126:H27 strains belonged to the enteroaggregative class of E. coli (EAggEC) that attached to HEp-2 cells in the characteristic aggregative pattern and hybridised with the EAggEC probe. They also caused mannose-resistant haemagglutination of rat erythrocytes, a property which may be a useful marker for their identification. Strains of O44:H18 with similar properties were also isolated from three small outbreaks in Britain, one of which involved elderly patients. EAggEC have not been considered previously as aetiological agents of diarrhoea in developed countries and have rarely been reported as belonging to EPEC serotypes. All 15 O55:H7 strains and seven of eight O111ab:H25 strains were also considered to be potentially diarrhoeagenic as they gave localised attachment (LA) to HEp-2 cells that resulted in a positive fluorescence actin-staining test. This test is considered to correlate with the attaching-and-effacing virulence mechanisms of EPEC in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)