Search

Enterohaemorrhagic Escherichia coli O157 (O157) is infectious to humans, particularly children, at very low doses and causes not only haemorrhagic colitis but also other serious symptoms. To investigate an association between intestinal bacterial flora and resistance to such infections, we screened faecal samples for the presence of enteric bacteria that are able to suppress the growth of O157. Samples from 303 individuals, 35 children (aged < or =6 years) and 268 adults (aged 20-59 years), were examined. Colonies with different appearances on sorbitol MacConkey agar medium were screened for the production of bacteriocins inhibitory for O157 in an overlay agar plate assay. O157-inhibiting strains were isolated from 52 individuals. The prevalence of these bacteria tended to rise with age, and was significantly higher among 40- to 59-year-old adults (23/101, 22.8%) than among children (3/35, 8.6%; P<0.05). To test the hypothesis that these bacteriocin-producing strains contribute to resistance against O157 in human adults, we examined faecal samples of 25 healthy O157 carriers. Inhibitory bacteria were more prevalent among the latter (9/25, 36.0%) than among age-matched subjects who did not carry O157 (49/268, 18.3%). It appears, therefore, that inhibitory bacteria in the human gut may play a role in inhibiting propagation of O157 and/or suppressing expression of virulence factors by this pathogen.

Holliday junction resolvases (HJRs) are key enzymes of DNA recombination. A detailed computer analysis of the structural and evolutionary relationships of HJRs and related nucleases suggests that the HJR function has evolved independently from at least four distinct structural folds, namely RNase H, endonuclease, endonuclease VII-colicin E and RusA. The endonuclease fold, whose structural prototypes are the phage lambda exonuclease, the very short patch repair nuclease (Vsr) and type II restriction enzymes, is shown to encompass by far a greater diversity of nucleases than previously suspected. This fold unifies archaeal HJRs, repair nucleases such as RecB and Vsr, restriction enzymes and a variety of predicted nucleases whose specific activities remain to be determined. Within the RNase H fold a new family of predicted HJRs, which is nearly ubiquitous in bacteria, was discovered, in addition to the previously characterized RuvC family. The proteins of this family, typified by Escherichia coli YqgF, are likely to function as an alternative to RuvC in most bacteria, but could be the principal HJRs in low-GC Gram-positive bacteria and AQUIFEX: Endonuclease VII of phage T4 is shown to serve as a structural template for many nucleases, including MCR:A and other type II restriction enzymes. Together with colicin E7, endonuclease VII defines a distinct metal-dependent nuclease fold. As a result of this analysis, the principal HJRs are now known or confidently predicted for all bacteria and archaea whose genomes have been completely sequenced, with many species encoding multiple potential HJRs. Horizontal gene transfer, lineage-specific gene loss and gene family expansion, and non-orthologous gene displacement seem to have been major forces in the evolution of HJRs and related nucleases. A remarkable case of displacement is seen in the Lyme disease spirochete Borrelia burgdorferi, which does not possess any of the typical HJRs, but instead encodes, in its chromosome and each of the linear plasmids, members of the lambda exonuclease family predicted to function as HJRs. The diversity of HJRs and related nucleases in bacteria and archaea contrasts with their near absence in eukaryotes. The few detected eukaryotic representatives of the endonuclease fold and the RNase H fold have probably been acquired from bacteria via horizontal gene transfer. The identity of the principal HJR(s) involved in recombination in eukaryotes remains uncertain; this function could be performed by topoisomerase IB or by a novel, so far undetected, class of enzymes. Likely HJRs and related nucleases were identified in the genomes of numerous bacterial and eukaryotic DNA viruses. Gene flow between viral and cellular genomes has probably played a major role in the evolution of this class of enzymes. This analysis resulted in the prediction of numerous previously unnoticed nucleases, some of which are likely to be new restriction enzymes.

Two evolutionary mechanisms have been proposed in the process of protein diversification of the large family of antimicrobial toxins of Escherichia coli, known as the colicins. Data from previous studies suggest that the relatively rare nuclease colicins appear to diversify primarily through the action of positive selection, whilst the more abundant pore-former colicins appear to diversify through the action of recombination. The complete DNA sequence of the newly characterized colicin plasmid, pCol-Let, isolated from a Yanomama Indian of South America, is presented here. This plasmid encodes a newly identified pore-former colicin, colicin Y. DNA and protein sequence comparisons of the colicin Y gene cluster and the encoded proteins with those of published pore-former colicins provide the first evidence that positive selection may also act to increase pore-former colicin diversity.

Twenty-seven diarrheagenic Escherichia coli (DEC) strains from five closely related, genetically distinct clones (DEC 3, 4, 8, 9, and 10), representing serotypes commonly associated with Shiga-like toxin production, i.e., O15:H-, O26:(H11, H-), O111:(H8, H11, H-), and O157:H7, were evaluated for colicinogeny on Luria agar or Luria agar containing 0.25 microgram/ml mitomycin C to induce colicin production. Ten (37%) of the DEC strains tested were colicinogenic. One of 11 serotype O157:H7 strains, DEC strain 4E, produced a colicin identified as Col D. DEC strains 8B, 9D, and 10B produced Col E1, whereas DEC strain 10A produced Col E2. DEC strains 8A, 8E, 10C, 10E, and 10F produced "untypable" colicins that killed almost all Pugsley Colicin Reference Set strains and the other DEC strains tested. To aid with further characterization of the colicins, plasmids extracted from each colicin-producing (Col+) DEC strain were used to transform E. coli strain DH5 alpha. All Col+ DH5 alpha transformants contained one plasmid ranging in size from 1.3 to 10 kb. Some transformants were stable colicin producers whereas others were unstable. The inhibitory activity and colicin sensitivity and insensitivity profiles of the Col+ transformants were similar to those of the corresponding Col+ donor DEC strains. It appears that the untypable colicins are novel and, thus, warrant further study. Colicin production by some of the DEC strains evaluated partly explains why they were insensitive to standard colicins in a previous study.

We noted that eight outbreaks of Shigella sonnei from an unknown source occurred sequentially in Aichi Prefecture, Japan, between October 1992-June 1993. For comparative purposes we analyzed 53 outbreak-related isolates of Shigella sonnei using different subtyping methods and studied the epidemiology of the outbreaks. It appeared from our study that DNA-based techniques such as plasmid typing and pulsed-field gel electrophoresis (PFGE) were more useful tools for subtyping Shigella sonnei than colicin typing and the antimicrobial susceptibility test. Moreover, according to PFGE analysis, four genetically related isolates of Shigella sonnei were responsible for the eight sequential outbreaks. To further investigate the epidemiology of outbreaks, 58 sporadic isolates of Shigella sonnei from overseas travelers with shigellosis during the same period were also examined. We found that some sporadic isolates from travelers in Asia were genetically related to those of the outbreak-related isolates, indicating that genetically related isolates prevailed in Asia during this period, probably because of the extensive movement of people or food.

Colicins are toxic exoproteins produced by bacteria of colicinogenic strains of Escherichia coli and some related species of Enterobacteriaceae, during the growth of their cultures. They inhibit sensitive bacteria of the same family. About 35% E. coli strains appearing in human intestinal tract are colicinogenic. Synthesis of colicins is coded by genes located on Col plasmids. Until now more than 34 types of colicins have been described, 21 of them in greater detail, viz. colicins A, B, D, E1-E9, Ia, Ib, JS, K, M, N, U, 5, 10. In general, their interaction with sensitive bacteria includes three steps: (1) binding of the colicin molecule to a specific receptor in the bacterial outer membrane; (2) its translocation through the cell envelope; and (3) its lethal interaction with the specific molecular target in the cell. The classification of colicins is based on differences in the molecular events of these three steps.

An extended scheme of colicin typing of Shigella sonnei strains includes 70 colicin types. Colicin type No. 56 is the producer of a new colicin, for which T marking was proposed. A total number 105 of indicator strains was prepared for typing.

Colicins are unusual bacterial toxins because they are directed against close relatives of the producing strain. They kill their targets in one of three distinct ways; via a ribonuclease or deoxyribonuclease activity or by forming pores in the target cell's membrane. This review deals with the steps involved in pore-forming colicin activity including, initial synthesis of the toxin, toxin release, receptor binding, translocation across the periplasm and pore formation in the cytoplasmic membrane. Special reference is made to the role of colicin in vivo, the structural changes occurring during pore formation and the role of the immunity protein.

Bacteriologic and serologic studies were carried out on the etiology of coli infections in newborn and young lambs. Ascertained was the participation of some serogroups of Escherichia coli in cases of lamb septicaemia, such as 019, 020, 025, 078, and 0101. Colicin was found to be produced by a total of 64 strains as well as colicin V by 14 strains, all belonging to serogroup 025.

In a series of published articles, colicin A has been mistakenly labeled as colicin K.

When 1420 isolates of Shigella sonnei recovered in Dundee, Scotland between 1971 and 1976 were examined with indicator strains of known immune status, four new colicin types, not previously described among the 26 types of Horák (8), were observed. The types were: 11 variant (colicins B, M), 4 variant (Ib, B, M), SH1 (E3) and SH3 (E5). Only type 4 variant strains were of epidemiological significance in the Dundee area.

The main forms of the epidemic manifestation of dysentery induced by different causative agents in 1975-1980 were revealed. During these years the cases of dysentery induced by Shigella sonnei, biovar II, were found to prevail (82.0-90.1%) both at the periods between epidemics and at the periods of the seasonal rise of morbidity. The experimental infection of white mice by intraperitoneal inoculation revealed no relationship between the seasonal rise of morbidity in dysentery and the virulence of its causative agents.

Fifty-four S. sonnei colicin types and 92 indicator strains used for their determination are listed. Most of the indicators were prepared by conjugational transfer of Col factors from shigellae to the recipient strains of E. coli K13 HfrR nalr and E. coli K12 ROW or by selection of resistant mutants of these strains or by combination of both methods. Ten indicators are S. sonnei strains. The indicator strains are suitable for E. coli colicin typing.

Colicins A, E1, E2, E3, E4, E5, E6, and E7 exhibited reduced activity against BtuB mutants of Escherichia coli K-12 and also against wild-type cells in the presence of vitamin B12. Plasmids encoding representatives of these colicins were specifically immune to high levels of the homologous colicin. Col(+) cells grown in media containing mitomycin C accumulated large amounts of colicin polypeptide. ColE2(+), ColE3(+), ColE4(+), ColE5(+), and ColE6(+) cultures also synthesized large amounts of second, lower-molecular-weight protein under these conditions. Colicins E2 through E7, but not A or E1, reacted with antiserum raised against purified colicin E3. Colicins E2 and E7 induced synthesis of beta-galactosidase encoded by lacZ under the control of the colicin Ib gene promotor on a derivative of Col plasmid ColIb.P9. This promotor is usually active only when the cells are treated with agents which damage DNA or block replication. Plasmids encoding various mutant forms of colicin E3 (M. Mock and M. Schwartz, J. Bacteriol. 142:384-390, 1980) recombined with ColE2, ColE4, ColE5, or ColE6 plasmids at a frequency of 10(-4) per cell to produce a colicin active against ColE2(+), E4(+), E5(+), or E6(+) cells. ColE5 and ColE6 plasmids recombined with ColE3 plasmids bearing mutations affecting colicin E3 receptor recognition, envelope penetration, and catalytic activities. ColE2 and ColE4 plasmids recombined only with ColE3 plasmids bearing mutations affecting receptor recognition and envelope penetration. Recombinants between mutant ColE3 plasmids and ColA, ColE1, or ColE7 plasmids were not detected. We propose the designation BtuB group for the colicins described here, and we divide the group into two classes comprising colicins A and E1, which act on the cytoplasmic membrane, and the related colicins E2 through E7, which have known or putative nuclease activities.

A set of 2227 strains of Shigella sonnei isolated from dysentery patients in East-Slovakian region from 1975 through 1977 was analyzed by age and sex of patients, place and time of isolation, and by phage type, colicin type and antibiogram patterns of strains. The study showed that some phage types tended to occur in association with certain colicin types, the most common combination being that of phage type 75 and col factór Ei (86% of strains). In 1976 and 1977 this phage type gradually replaced col factor Ia that in 1975 was predominant. The rise in the incidence of these strains was striking and pointed to their intensive circulation among the population of East-Slovakian districts, particularly among children of preschool age. A hypothetic assumption is that such changes in the phage type and colicin type patterns might precede the new epidemic wave of dysentery outbreaks in the population. That would also explain e.g. the irregularity of dysentery epidemic cycles encountered in Czechoslovakia during the decade from 1972 through 1982. The analysis of strains by pattern of antibiogram showed that the percentage of strains resistant to all antimicrobials and sulphonamides tested remained virtually constant over the three years under study and did not exceed 6% of strains. Only the strains monoresistant to tetracycline were found to show a striking rise in their incidence from 5% in 1975 to 23% in 1977. In the majority of cases they were S. sonnei strains with col factor Ei.

By using each of the available colicins, we have isolated a large number of colicin-resistant mutants. They included both receptor and tolerant mutants and each was screened for cross-resistance to all other colicins. On the basis of the cross-resistance of these mutants it was possible to place known colicins into two mutually exclusive groups, group A and group B. Mutants selected as resistant to colicins of group A may or may not be cross-resistant to other colicins of group A, BUT Are never resistant to colicins of group B. The reverse also applies. The mutants isolated as resistant to colicins of group A (A, E1, E2, E3, K, L, N, S4, and X) have been divided into 21 phenotypic classes on the basis of their colicin resistance patterns. These include most of the tolerant and receptor mutants previously isolated, some of which were previously shown to also have an increased sensitivity to certain antibiotics and detergents. Type strains from each of the phenotypic classes were therefore tested for sensitivity to a range of antibiotics, detergents, and surfactants that included all those previously used. With these new data, it has been possible to speculate informatively on the mode of action of the different colicins. We have confirmed the position of previously isolated mutations on the Escherichia coli K-12 genetic map, and located approximately the loci conferring colicin resistance in some of the newly isolated mutants.

Distribution by serogroup, phage type, colicin production, colicin type, sensitivity to antibiotics and plasmid characteristics of 74 Escherichia coli and 11 Klebsiella strains isolated from hospitalized patients receiving prolonged antibiotic therapy indicated that the infections were not associated with the hospital environment. Resistance was tested to 26 antibiotics, some of them being not generally used in therapy; 30 strains were resistant to 4 to 17 antibiotics. There was a significant difference in the antibiotic resistance of strains derived from patients with urinary-tract infections (UTI) and with leukaemia (LP). As compared to the UTI group, among E. coli strains in the LP group the frequency of multiple resistance was significantly higher, the MIC values were higher and R-plasmids were more frequent. Out of 30 multiple resistant E. coli strains 27 were R-plasmid carriers. Three different kinds of plasmid profile were shown in more than one strain (2 out of 10 UTI strains and 3 and 2 out of 10 LP strains). The rest of the isolates differed in plasmid profile from these and from one another; the presence of "epidemic plasmid" was not demonstrated. Plasmid epidemiological examinations may forecast the efficacy of an antibiotic or of a group of antibiotics.

A collection of strains derived from Escherichia coli K12 W3110 and harbouring various colicin or microcin plasmids (18 and 2 representatives, respectively), or carrying well-characterized mutations conferring reduced colicin/microcin sensitivity is described. The strains can be used in typing schemes based on the identification of colicins, in the detection of new types of colicins/microcins, and in the further characterization of previously identified colicins/microcins and their plasmids.

Escherichia coli strain CL137, a K-12 derivative made E colicinogenic by contact with Fredericq's strain K317, was unaffected by colicin E2-P9, but K-12 carrying ColE2-P9 was sensitive to the E colicin made by strains CL137 and K317. This colicin we named E7-K317 because by the test of colicinogenic immunity it differed from colicins E1-K30, E2-P9, and E3-CA38 and from recently recognized colicins termed E4Horak, E5, and E6. Strain K317 as conjugational donor transmitted E7 colicinogeny; about half the E7-colicinogenic transconjugants were immune to colicin E2-P9. A spontaneous variant of CL137 retained E7 colicinogeny but was sensitive to E2 colicins. We attribute the E2 immunity of strain CL137 and some E7-coliconogeic transconjugants to a "colicin-immunity plasmid," ColE2imm-K317, from strain K317. Tra+ E7-colicinogenic transconjugants restricted phage BF23 in the same way as strains carrying ColIb-P9. We attribute Tra+ and restricting ability to a plasmid, pRES-K317, acquired from strain K317, and related to the ColI plasmids.

From November 1972 to the end of 1975, Shigella sonnei types implicated in dysentery morbidity in a number of districts of East Bohemia region were studied. 301 strains subjected to phage typing and colicine typing were classified into 45 "combined" types. During the study five types prevailed in succession, each for a protracted period of time. Four of these types retained, in the great majority of cases, their original high drug sensitivity throughout the period of their prevalence. Other types were only encountered transiently and sporadically. As a result of a phagerestrictive effect of newly acquired plasmids there occurred, in rare instances in some strains, a change in phage type; or the colicine type changed while the phage type was retained as a result of another col factor having been acquired by the strain. The changes occurred in the course of epidemics.

Inthe years 1976-1978 3,552 strains of Shigella sonnei obtained from the whole Czechoslovak territory by the method of colicin typing and phage typing was examined. Each strain represented always one focus. 81.3% of the strains were colicinogenic. Twenty-one colicin types, 41 characteristic and 105 non-characteristic phage types participated in bacillary dysentery. Of the total number, 77.4% belonged to 9 phage types (2, 3, 6, 65, 67, 75, NC IV-VIII- and NC III-IV-VIII-), which are endemic in Czechoslovakia. Of the 2,889 colicinogenic strains, 85% belonged to 3 colicin types: 2 = Ia (31%), 6/11 = E1 (27%), 12 = E6 (27%). Some phage types produced predominantly colicin of a certain type.

A total of 550 E. coli isolates--250 from apparently healthy, and 300 from diarrhoeic West African pigmy goats were tested for colicinogenicity. 33.2% of strains from apparently healthy animals were colicinogenic as against 56% recorded for strains from animals with diarrhoea. Of the 251 colicinogenic E. coli strains from both groups of animals, 76.5% were Type I while 23.5% belonged to Type II. Identified colicins from the healthy animals consisted of types G, K, E2, A and V in decreasing frequency of occurrence, whereas those from goats with diarrhoea were made up of types V, B, E1, G, E2, E3, and Ia also in decreasing frequency of occurrence. In contrast to isolates from healthy animals, there was a marked variation in the colicin spectra of Types I and II E. coli from the diarrhoeic animals--that of Type I being much broader. The Public Health significance of possible transfer of multiple drug resistance from colicinogenic E. coli strains to other enterobacteria is also discussed.

Colicinotype 7 of Shigella sonnei, introduced by Abbott and Graham, is one of those most frequently found in epidemiological screenings. It is represented by the production of a single colicin (acidic protein of m. w. 46 kD) endowed with some striking features. It is unstable at pH 8.0 and at the temperature of 70 degrees C. Its inhibitive activity is specific for the species Shigella sonnei, most strains of which seem to be sensitive. It is the only colicin known so far inactive towards Escherichia coli, including the indicator strains of broad-spectrum sensitivity, such as K12 Row. It shows no cross-resistance with any other colicin type. Its adsorption on sensitive bacteria and inhibitive effect on them proceed extremely quickly. As indicated by indirect fluorimetry, its mode of action is extremely quickly. As indicated by indirect fluorimetry, its mode of action is probably different from those known for other colicins: it does not concern the plasma membrane of sensitive bacteria under aerobiosis, but it interferes with it under anaerobic conditions. These data show clearly that it is a unique colicin type. It is proposed to include it into the current system of classified colicins as "colicin Js".

The paper presents data obtained during the year-long medico-engineering experiment (in which three test subjects participated) with respect to changes in the Escherichia composition as judged by colicinogenic and hemolytic activity. At the beginning of the experiment colicinogenic E. coli (col/D and col/B) was found in one of the three test subjects (U-85%); he also showed the highest stability of intestinal microflora throughout the experiment. The two other test subjects (B and M) displayed colicinogenic micro-organisms of the same type plus an unidentified type only during and immediately after emergency situations. Hemolytically active E. coli was isolated from the subjects with unstable microflora under emergency situations and at the end of the experiment. The results obtained allowed to note the periodicity in predominance of Escherichia with antagonistic and hemolytic activity as related to the changes in the physiological state of the macro-organism and the rate of mechanical and possibly genetic exchange within the microbial associations in the human intestine.

The effect of colicins on deoxyribonucleic acid and protein synthesis, and also their effect on the ability of T4 phage to replicate in Escherichia coli K-12, were studied. Colicins of type K inhibited deoxyribonucleic acid synthesis, protein synthesis, and phage growth. Among colicins of type E, there was an absolute correlation between mode of action and subdivision into types E(1), E(2), and E(3).