Prevalence of enteric bacteria that inhibit growth of enterohaemorrhagic Escherichia coli O157 in humans.

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.

SURVEY AND SUMMARY: holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories.

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.

The newly characterized colicin Y provides evidence of positive selection in pore-former colicin diversification.

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.

Colicinogeny among Escherichia coli serotypes, including O157:H7, representing four closely related diarrheagenic clones.

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.

Epidemiologic study of Shigella sonnei from sequential outbreaks and sporadic cases using different typing techniques.

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--exocellular lethal proteins of Escherichia coli.

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.

Conocimiento y posibles aplicaciones del estudio de las colicinas / Knowledge and possible aplications of the study of colicins

Las colicinas son sustancias proteicas con actividad bactericida, producidas por miembros de la familia Enterobacteriaceae y que presentan un espectro de acción limitado únicamente a especies de la misma familia. El estudio de las colicinas data desde 1925, aunque su primera clasificación, basada en sus propiedades químicas, físicas y biológicas, fue la realizada en 1948 por Frédériq. Posteriormente, Davies y Reeves en 1975 las dividieron en dos grandes grupos: A y B, de acuerdo con los diferentes patrones de resistencia obtenidos para una serie de mutantes resistentes a cada una de las colicinas conocidas. Se demostró que las mutatantes de resistencia a colicinas del grupo A nunca son resistentes a colicinas del grupo B, y viceversa. Actualmente se han hecho avances notables en el conocimiento del modo de acción de las colicinas. En efecto, se ha determinado que las colicinas pueden actuar en tres formas diferentes: 1) aquellas que actúan específicamente en la membrana celular, induciendo canales dependientes de diferencias de voltaje; 2) las que presentan actividad endonucleolítica, y 3) el caso especial de la colicina M, que es capaz de inhibir específicamente la síntesis de mureína. En general, se ha determinado que el efecto bactericida de las colicinas en las células sensibles precisa de tres partes de la molécula: 1) la unión específica de la colicina con su receptor en la membrana celular externa, que requiere de la parte central de la proteína; 2) la parte -NH2 terminal de la colicina, la cual es necesaria para que ésta atraviese la membrana celular, y 3) la parte -COOH terminal, que es indispensable para el reconocimiento de la colicina con su blanco específico. Las colicinas están codificadas en plásmidos; se han localizado varios de los genes involucrados tanto en la producción de la colicina, como de la proteína de liberación, así como de la proteína que le confiere inmunidad a la célula portadora de dicho plásmido. Los genes estructurales que codifican para dichas proteínas están bajo el control del regulón SOS, que se encuentra reprimido por la proteína LexA. El conocimiento de los plásmido colicinogénicos (factores col+) ha facilitado el desarrollo de la biotecnología, debido a que son multicopia y autorreplicables, por lo que son empleados como vectores de clonación en diferentes estudios del DNA. No obstante el uso más frecuente del conocimiento del fenómeno de colicinogenia en diferentes partes del mundo, ha sido en estudios epidemiológicos Así, se ha establecido que la frecuencia de cepas colicinogénicas es mayor entre los aislados de individuos enfermos, en comparación con los obtenidos de individuos sanos de la misma especie y de la misma ubicación geográfica. Por su parte, la colicina V ha sido ampliamente reconocida como un marcador de patogenicidad entre cepas de Escherichia coli, mientras que la colicina E1 se ha identificado frecuentemente entre sujetos sanos. Por otro lado, a pesar de que las colicinas se han detectado desde hace más de setenta y cinco años, aún no se conocen los efectos biológicos que puedan tener sobre el hospedero humano portador de cepas colinogénicas. Consecuentemente, para establecer si las colicinas pueden conferir alguna ventaja no sólo a las cepas productoras, sino también al hospedero, se requieren aún numerosos trabajos de investigación biomédica

Seventy colicin types of Shigella sonnei and an indicator system for their determination.

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.

All in the family: the toxic activity of pore-forming colicins.

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.

Bacteriocinogenia, un sistema de antagonismo bacteriano / Bacteriocinogenia, a system for bacterial antagonism

Bacterial antagonism depends on many factors. Among others, for example we have competition for an essential nutrient, the production of toxic metabolites or the release of antibiotic substances. The production of bacteriocines by "killer" strains correspons to the latrer, being determined by plasmidial or chromosomal genes. On the other hand, the susceptibility to a given bacteriocine depends on the presence of surface recptors that are codified by the cellular DNA. The bacteriocine particles correspond to protein conglomerates which are sometimes associated with glucolipids. With the death of the productive cell, these particles are livated motivated by synthesis. The similarities which they share with the bacteriophage are many. However, their fundamental differences are that they lack nuclic acid and their incapcity to transmit themselves. Cellular death caused by the action of the bacteriocine is extremely quck, acting on the permeability of the plasmatic membrane, on the macromolecular syntesis (DNA, RNA an proteins) and on cellular oxidation. The wide range of bacteriocine and their respective spectre of action over the more or less related strains are excellent parameters to detect, identify and classify the productive bacteria and the susceptible ones. With only a few exceptions, the bacteriocines not lethal to their own productive cell nor to taxonomically distant species. This has led to intersting ecological and epidemiological outlooks. The amplitude of this phenomenon in Gram+ and Gram- bacteria has led to the study of fine mechanisms which have triggered the genetic expression to elaborate bacteriocines, determining the one responsible for this are the genes and localized signals in transmissible episomes or in chromosomal operons

Bacteriocinogenia, un sistema de antagonismo bacteriano / Bacteriocinogenia, a system for bacterial antagonism

Bacterial antagonism depends on many factors. Among others, for example we have competition for an essential nutrient, the production of toxic metabolites or the release of antibiotic substances. The production of bacteriocines by "killer" strains correspons to the latrer, being determined by plasmidial or chromosomal genes. On the other hand, the susceptibility to a given bacteriocine depends on the presence of surface recptors that are codified by the cellular DNA. The bacteriocine particles correspond to protein conglomerates which are sometimes associated with glucolipids. With the death of the productive cell, these particles are livated motivated by synthesis. The similarities which they share with the bacteriophage are many. However, their fundamental differences are that they lack nuclic acid and their incapcity to transmit themselves. Cellular death caused by the action of the bacteriocine is extremely quck, acting on the permeability of the plasmatic membrane, on the macromolecular syntesis (DNA, RNA an proteins) and on cellular oxidation. The wide range of bacteriocine and their respective spectre of action over the more or less related strains are excellent parameters to detect, identify and classify the productive bacteria and the susceptible ones. With only a few exceptions, the bacteriocines not lethal to their own productive cell nor to taxonomically distant species. This has led to intersting ecological and epidemiological outlooks. The amplitude of this phenomenon in Gram+ and Gram- bacteria has led to the study of fine mechanisms which have triggered the genetic expression to elaborate bacteriocines, determining the one responsible for this are the genes and localized signals in transmissible episomes or in chromosomal operons (AU)

Colicin Js of Shigella sonnei: classification of type colicin "7".

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".

Antibiotic resistance and plasmid profiles of Escherichia coli and Klebsiella isolated from in-patients receiving prolonged antibiotic therapy.

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.

Escherichia coli K12 strains for use in the identification and characterization of colicins.

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.

[Separation of colicins from E. coli strains isolated from lambs]. / Otdeliane na kolitsin ot shtamove E. coli, izolirani ot agneta.

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.

To shigellosis epidemicity in East-Slovakian region.

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.

Fifty-four Shigella sonnei colicin types and their typing by specific indicator strains.

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.

[Relation of the epidemiological characteristics of dysentery to the biological properties of the causative agents]. / Sviaz' nekotorykh osobennostei épidemiologii dizenterii s biologicheskimi svoistvami vozbuditelei.

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.