Contractile-tailed bacteriophages adsorb to Escherichia coli O128ab lipopolysaccharide that is altered by large plasmids to provide receptors and lipopolysaccharide heterogeneity within the serogroup.

The verotoxigenic Escherichia coli strain H.I.8 (originally O128:B12, now not typeable) contained a ColB+M plasmid and two morphologically identical temperate bacteriophages (H18A and H18B). Both phages were O128ab specific, using the lipopolysaccharide O side chains of susceptible clinical isolates as receptors. SDS polyacrylamide gel electrophoresis with silver staining of O128ab lipopolysaccharide revealed four distinct types of ladder with different interband spacings. No specificity was found between ladder type and sensitivity to either phage. One of the numerous large plasmids present in O128ab isolates was found to modify the structure of the lipopolysaccharide O side chains to provide phage receptors.

A new colicin that adsorbs to outer-membrane protein Tsx but is dependent on the tonB instead of the tolQ membrane transport system.

A new colicin, Col5, was synthesized by an Escherichia coli isolate of human origin from the ECOR Collection. It was unique because it adsorbed to the outer-membrane protein Tsx, but used the tonB rather than the tolQ membrane transport system, which is employed by the only other Tsx-specific colicin, ColK. Col5 was encoded by a 5.2 kb plasmid, p5. It was inducible by mitomycin C, and strains harbouring p5 exhibited quasi-lysis. The bactericidal protein had an M(r) of 56,000.

Synthesis of unusual thick pili by Escherichia coli of EPEC serogroup O119.

Unique very thick pili were found in varying numbers on cells of five out of 11 clinical isolates of enteropathogenic Escherichia coli belonging to the classic EPEC serogroup O119. They were approximately 12.5 nm in diameter, which is thicker than any other known E. coli pilus type. Analysis of the plasmid profiles of the O119 isolates showed that one strain was plasmid-free while the remainder contained numerous plasmids with a wide range of sizes. The thick pilus determinants were located on a 140-kb non-transferrable plasmid. They were not associated with adherence or a putative 90-kb enteroadherence factor plasmid.

Colicinogeny of O55 EPEC diarrhoeagenic Escherichia coli.

Approximately 24% of a sample of pathogenic Escherichia coli strains from different serogroups were found to synthesize colicins. Serogroup O55 had an unusually large proportion of such strains (33%). In a sample of 27 O55 isolates, one synthesized a class A colicin (identified as ColE9), five produced class B colicins (three ColIa, two, unidentifiable), and three a class A and a class B together.

Colicins G and H and their host strains.

Escherichia coli strains CA46(pColG) and CA58(pColH) each apparently synthesized two generally similar bactericidal colicin proteins whose molecular weights were approximately 5,500 and 100,000. These proteins were more resistant to trypsin than representative colicins A, D, E1, and V. The smooth wild-type strains harbouring plasmids pColG and pColH were serotyped O169:NM and O30:NM, respectively, being typically associated with nonpathogenic E. coli of human origin. Rough and semirough variants, which were selected using resistance to novobiocin, were intrinsically insensitive to almost as many colicins (10 tested) as their parents. For this reason the wild-type strains would not be useful for identifying colicins G and H on the basis of immunity. The O antigenic side chains of both wild-type strains shielded three of the six bacteriophage protein receptors tested.

Differences between the LPS cores in adherent and non-adherent strains of enteropathogenic Escherichia coli 0119.

Adherent enteropathogenic Escherichia coli 0119 strains had a larger lipopolysaccharide core than non-adherent strains, although the O-chains were identical. The core from the non-adherent strain 19392 contained five hexose residues in the outer region, with three L-glycero-D-manno-heptose residues and 3-deoxy-D-manno-octulosonic acid (KDO) in the inner region. The core of adherent strain JCP88 had an atypical structure consisting of six hexose residues, KDO, and equimolar amounts of L-glycero-D-manno-heptose and D-glycero-D-manno-heptose. The core of a rough JCP88 mutant resembled an incomplete 19392 core.

Factors related to the presentation of patients with thick primary melanomas.

OBJECTIVE: To identify any characteristics of patients that are associated with presentation with thick primary melanoma. DESIGN: This was a retrospective survey of the clinical records of 1300 patients attending the Newcastle Melanoma Unit. Characteristics of 131 patients with thick melanomas (defined as 3 mm or greater in thickness) were compared with those of 543 patients with thin melanomas (defined as 0.75 mm or less in thickness). Comparisons were made using contingency table analysis, Wilcoxon rank sum tests, log rank analysis and logistic regression. SETTING: The Newcastle Melanoma Unit is a tertiary referral centre for the treatment of primary melanoma. PATIENTS: We surveyed all 1300 patients attending the Newcastle Melanoma Unit over the years 1981-1990. They represented approximately 90% of the patients in the Hunter region of New South Wales who developed melanoma during this period. Excluded from analysis were 39 patients with occult primary melanomas, 79 with multiple primary melanomas, 51 with primary melanomas of unknown thickness and seven with incomplete records, leaving 1124 patients in the study. MAIN OUTCOME MEASURES: These were selected before the results were known. The hypothesis was generated following analysis of the data. RESULTS: Patients with thick primary melanoma were more likely to be men (68% men and 32% women in the thick melanoma group, compared with 45% and 55% respectively in the thin melanoma group, P less than 0.005) over 60 (75% were over 50 years of age in the thick group versus 33% in the thin melanoma group, P less than 0.001) with nodular melanoma (62%, versus 2% in the thin melanoma group, P less than 0.001) and with melanoma on the head and neck (27%, versus 12% in patients with thin melanoma, P less than 0.005). The time from detection of a change in skin to diagnosis was not longer for those with thick compared to those with thin melanomas. CONCLUSION: The greatest problem of those with thin melanomas. CONCLUSION: The greatest problem of detecting melanoma at an early (surgically curable) stage appears to be in patients over the age of 50 who have nodular melanoma, particularly in the head and neck.

Colicinogeny of O157:H7 enterohemorrhagic Escherichia coli and the shielding of colicin and phage receptors by their O-antigenic side chains.

Twenty O157:H7 enterohemorrhagic Escherichia coli strains from patients with different clinical conditions were tested for colicinogeny and the presence of Verotoxin (VT) genes. From bloody diarrhea cases, 7/8 isolates and from hemolytic uremic syndrome cases 3/5 isolates all synthesized what appeared to be colicin D. The remaining strains, which included 7 from asymptomatic sources, were noncolicinogenic. The plasmid determining the colicin was found to be 1.4 kb larger than the 5.2-kb pColD. The colicin D protein had a molecular weight of about 90,000, whereas the O157 colicin was 87,000. The plasmid was designated pColD157 to reflect these differences. Of O157:H7 isolates 17/20 had genes for both of the Verotoxins VT1 and VT2, and the remaining 3/20 for VT1 only. There was no correlation between the presence of VT determinants and colicinogeny or symptoms. The O157:H7 strains exhibited significant resistance to other colicins and bacteriophages.

Characterization of the F-plasmid conjugative transfer gene traU.

We characterized the traU gene of the Escherichia coli K-12 conjugative plasmid F. Plasmids carrying segments of the F transfer operon were tested for their capacity to complement F lac traU526. The protein products of TraU+ clones were identified, and the nucleotide sequence of traU was determined. traU mapped between traW and trbC. It encodes a 330-amino-acid, Mr36,786 polypeptide that is processed. Ethanol caused accumulation of a precursor polypeptide; removal of ethanol permitted processing of the protein to occur. Because F lac traU526 strains appear to be resistant to F-pilus-specific phages, traU has been considered an F-pilus assembly gene. However, electron microscopic analysis indicated that the traU526 amber mutation caused only a 50% reduction in F-piliation. Since F lac traU526 strains also retain considerable transfer proficiency, new traU mutations were constructed by replacing a segment of traU with a kanamycin resistance gene. Introduction of these mutations into a transfer-proficient plasmid caused a drastic reduction in transfer proficiency, but pilus filaments remained visible at approximately 20% of the wild-type frequency. Like traU526 strains, such mutants were unable to plaque F-pilus-specific phages but exhibited a slight sensitivity on spot tests. Complementation with a TraU+ plasmid restored the wild-type transfer and phage sensitivity phenotypes. Thus, an intact traU product appears to be more essential to conjugal DNA transfer than to assembly of pilus filaments.

Conjugation system of IncC plasmid RA1, and the interaction of RA1 pili with specific RNA phage C-1.

RA1 was the only IncC plasmid that was slightly temperature-sensitive for replication and transfer. At 30 degrees C, RA1 determined constitutive synthesis of conjugative pili and yet was transfer-repressed. Attachment of shaft-adsorbing RNA phage C-1 virions prevented the probable retraction of pili under heat stimulus (55 degrees C). Electron microscopy showed single adsorbed virions at pilus bases where they were thought to have stopped retraction.

Interaction of drug resistance plasmids and bacteriophages with diarrheagenic strains of Escherichia coli.

Seven transfer-derepressed plasmids from different incompatibility groups in Escherichia coli K-12 were tested for their ability to enter 43 strains of diarrheagenic E. coli (mostly enteropathogenic E. coli clinical isolates) representing 12 serogroups and including rough and semirough mutants (characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Strains in some serogroups were more competent as recipients of plasmids than were those in others. Five test plasmids in an E. coli K-12 (rough) donor transferred significantly less efficiently to two smooth strains than to their rough or semirough isogenic derivatives. When the same smooth and rough strains were used as donors, the plasmids transferred to E. coli K-12 equally well. These results suggested that the O-antigenic lipopolysaccharide side chains of diarrheagenic E. coli isolates shielded the outer membrane receptors for conjugative pili, thus preventing plasmid entry. The different receptors for eight bacteriophages were also covered by O side chains. In addition, a limited survey of clinical isolates for drug resistance markers and resident plasmids was carried out.

Escherichia coli tolQ mutants are resistant to filamentous bacteriophages that adsorb to the tips, not the shafts, of conjugative pili.

The tolQ (previously fii) mutation in Escherichia coli K12 inhibits infection by filamentous bacteriophages f1 and IKe but not by RNA-containing phage f2. This work extends these observations to other plasmid-specific bacteriophages including various filamentous. RNA-containing, and lipid-containing isolates. Only tip-adsorbing filamentous phages were affected by tolQ and not shaft-adsorbing ones. Electron microscopy showed that RP4-specific filamentous phage Pf3 was one of the latter kind. Several tip-adsorbing filamentous phages inhibited conjugation between tolQ strains carrying their specific plasmids, implicating the phage receptors (conjugative pili) as mating organelles. tolQ mutant strains were as proficient as their parents in conjugation mediated by a wide range of plasmids.

Bacteriophage X-2: a filamentous phage lysing IncX-plasmid-harbouring bacterial strains.

Phage X-2, a filamentous rod about 950 nm in length, was isolated from sewage as plating on strains of Escherichia coli, Salmonella typhimurium or Serratia marcescens carrying either the IncX plasmid R6K, or the unique plasmid R775. Phage X-2 differs morphologically from a previously described very broad host range filamentous phage X which also lyses plasmid R6K-carrying strains and the phages differ in their resistance to inactivation by diethyl ether. Phage X-2 is serologically unrelated to phage X and the X-like phages IKe and I2-2. The adsorption site of the phage on the plasmid-bearing strains could not be determined but evidence implicating conjugative pili is presented.

Characteristics of RP4 tellurite-resistance transposon Tn521.

A restriction map of the tellurite-resistance (Ter) transposon Tn521 (parent plasmid RP4Ter) was prepared. Five sites from RP4Ter, including the EcoRI origin, were found in pIN25::Tn521. Tn521 was inserted into a transferable 27.5 kb vector (pCU109) to make three different insertion mutants, in which the size of Tn521 was measured accurately at 4.5 kb. Unlike the Ter of IncHI2 plasmids, that of Tn521 in RP4Ter was non-inducible. Ter was expressed in five widely differing bacterial species to which RP4Ter was transferred from Escherichia coli. Electron micrographs of bacteria expressing the Ter of RP4Ter, H complex plasmids, and chromosomal mutants, all revealed similar tellurium metal crystallites when the bacteria were grown in potassium tellurite medium. No other Ter determinants were found amongst 54 plasmids representing most incompatibility groups (excluding the H complex).

Organization and regulation of the conjugation genes of IncI1 plasmid colIb-P9.

The IncI1 plasmid ColIb-P9 is among a group of related plasmids that encode the I1 type of conjugation system. The I1 system is known to include two morphologically distinct types of pilus, a DNA primase gene (sog) and an exclusion determinant (exc). Transposon mutagenesis and analysis of cloned fragments of ColIb were used to identify the location of these determinants with respect to an EcoRI restriction map. Also identified were the location of the origin of transfer (oriT) and a gene determining an EDTA-resistant nuclease, which is coordinately regulated with the transfer genes. The results indicate that the ColIb tra genes are separated into at least three Tra regions. The pleiotropic nature of transposon insertion mutations in two of these regions suggests that two positive regulators are required for expression of the transfer genes and evidence is also found for a trans-acting repressor. It is suggested that the I1 conjugation system may have evolved following fusion of two distinct types of conjugative plasmid.

Location on RP4 of a tellurite resistance determinant not normally expressed in IncP alpha plasmids.

The tellurite resistance (Ter) determinant of RP4 is not normally expressed unless variants are selected on medium containing tellurite. The determinant was mapped in the variant plasmid RP4Ter by Tn7 insertion mutagenesis. Based on a 56.4-kilobase (kb) replicon, it covered the region from 56 kb, across the EcoRI site at 0 kb, to 1.5 kb.

Phage tf-1: a filamentous bacteriophage specific for bacteria harbouring the IncT plasmid pIN25.

Phage tf-1 is a filamentous phage which is about 800 nm in length, 10 nm in width and has slightly tapered ends. The phage was isolated from sewage and formed plaques or propagated only on Escherichia coli, Salmonella typhimurium and Klebsiella oxytoca strains harbouring the IncT plasmid pIN25 at 30 degrees C. It adsorbed in large numbers to pIN25-encoded long thick flexible conjugative pili formed at 30 degrees C and also to the short form of these pili synthesized at 37 degrees C. The reason for the failure to form plaques at 37 degrees C is not known. The adsorption site is a short length of the pilus shaft extending 100-200 nm back from the distal tip. Efficient phage tf-1 adsorption to the same site was found for pili determined by other IncT plasmids in spite of the fact that phage tf-1 did not plate or propagate on strains harbouring them. However, areas of specific partial clearing on lawns of these plasmid-containing bacteria were produced by phage in high concentrations. Lack of plaque-formation could be due to inefficient intracellular assembly coupled to avid adsorption of any liberated phage to pili. The phage differs from all but one other filamentous phage by being sensitive to diethyl ether.

Apramycin and gentamicin resistance in Escherichia coli and salmonellas isolated from farm animals.

Since the aminoglycoside antibiotic apramycin was licensed for veterinary use in 1980, all isolates of Escherichia coli and salmonellas received at the Central Veterinary Laboratory have been monitored for resistance to apramycin and the related antibiotic gentamicin. During the period 1982-4, the incidence of resistance in E. coli to apramycin increased from 0.6% in 1982 to 2.6% in 1984. In salmonellas the incidence of resistance to apramycin increased from 0.1% in 1982 to 1.4% in 1984. Resistance to both apramycin and gentamicin was detected in six different salmonella serotypes, although an isolate of Salmonella thompson from poultry was resistant to gentamicin but not apramycin. Most of the cultures were isolated from pigs, although the incidence of apramycin resistance in S. typhimurium (DT 204C) from calves has shown a recent dramatic increase. All the isolates with one exception produced the enzyme aminoglycoside 3-N-acetyltransferase IV (ACC(3)IV). The resistance was transferable by conjugation in most of the strains examined, and the plasmids specifying the resistance have been found to belong to a number of different incompatibility groups. Plasmids from three E. coli strains were compatible with all the reference plasmids and belonged to a previously undescribed group which was investigated further. It is suggested that bacteria from humans should be examined for resistance to apramycin and gentamicin to determine the possibility of the antibiotic-resistance bacteria, and their genes, spreading from animals to humans.

Bacteriophages F0lac h, SR, SF: phages which adsorb to pili encoded by plasmids of the S-complex.

Phage F0lac is an RNA-containing phage which plates only on strains carrying the plasmid EDP208, a pilus derepressed derivative of the unique incompatibility plasmid F0lac. A host range mutant, phage F0lac h, was selected which plated on strains carrying the ungrouped plasmid pPLS::Tn5 and lysed strains carrying another ungrouped plasmid TP224::Tn10 or the Com9 plasmid R71. An RNA-containing phage, SR, was isolated from sewage on bacteria harbouring plasmid pPLS::Tn5. It was antigenically distinct from the above two phages but had the same host range as phage F0lac h. Phages F0lac h and SR adsorbed unevenly to the shafts of the conjugative pili. Another phage, SF, was filamentous and plated or propagated on strains carrying any of the above plasmids as well as on strains harbouring IncD or F-complex plasmids. Plasmids TP224::Tn10 and pPLS::Tn5 were compatible with representative plasmids of all Inc groups also encoding thick flexible pili. The four plasmids EDP208, R71, TP224::Tn10 and pPLS::Tn5 were compatible with one another except for the reaction of TP224::Tn10 in the presence of pPLS::Tn5 which was slightly ambiguous. The host ranges of the bacteriophages, together with the serological relatedness of the thick flexible pili determined by these four compatible plasmids, suggested that they constitute a new complex, here designated S.

The unique conjugation system of IncHI3 plasmid MIP233.

The conjugation system of the IncHI3 plasmid MIP233 was studied using a transfer-derepressed Tn5-insertion mutant. The conjugative pili of this plasmid were short pointed rods resembling rigid pili, with a well-defined modal length. Unlike plasmids with rigid pili, the MIP233 mutant mediated a surface + liquid conjugation system. The pili were serologically different from all known pilus types including H pili, and did not act as receptors for any known pilus-specific bacteriophage. They converted the surface conjugation system of RP4 to a surface + liquid one. Antiserum to pili of the mutant plasmid inhibited transfer of the wild-type plasmid MIP233, demonstrating that it contained only one transfer system.