Colicin Production Research Articles

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., 015:H−, 026:(H11, H−), 0111:(H8, H11, H−), and O157:H7, were evaluated for colicinogeny on Luria agar or Luria agar containing 0.25 μg/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 DH5α. All Col+ DH5α 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.

HIGH LEVELS OF COLICIN RESISTANCE IN ESCHERICHIA COLI.

Colicins are plasmid-encoded antibiotics that are produced by and kill Escherichia coli and other related species. The frequency of colicinogeny is high, on average 30% of E. coli isolates produce colicins. Initial observations from one collection of 72 strains of E. coli (the ECOR collection) suggest that resistance to colicin killing is also ubiquitous, with over 70% of strains resistant to one or more colicins. To determine whether resistance is a common trait in E. coli, three additional strain collections were surveyed. In each of these collections levels of colicin production were high (from 15 to 50% of the strains produce colicins). Levels of colicin resistance were even higher, with most strains resistant to over 10 colicins. A survey of 137 non-E. coli isolates revealed even higher levels of resistance. We discuss a mechanism (pleiotropy) that could result in the co-occurrence of such high levels of colicin production and colicin resistance.

Temporal changes in the frequency of colicinogeny in Escherichia coli from house mice.

Escherichia coli was isolated from feral house mice (Mus domesticus) during the course of a mouse plague in the state of Victoria, Australia. The isolates were characterized for the production of colicins and their resistance to the co-occurring colicins. Of the 447 isolates examined, 59% were found to be colicinogenic. Phenotypic and PCR-based genotypic methods were used to determine the types of colicins being produced. Colicin E2 was the most common, representing 27% of the colicin-producing isolates. Colicin Ia was produced by 3% of the colicinogenic isolates. The remaining colicins could not be identified, but phenotypic and PCR data argue that at least nine different colicin types are present in this collection of E. coli. The frequency of colicinogenic isolates declined from 71% to 43% over the 7 months of the study. All colicin types appeared to decline in frequency. Concurrently, the resistance of isolates to colicin E2 increased from about 50% to 70%. Two hypotheses are proposed to explain the decline in the frequency of colicinogeny in this population of E. coli. The first relates to the within-host interactions occurring among colicinogenic, colicin-susceptible and colicin-resistant populations within a host. The second relates to the among-host interactions between susceptible and colicinogenic populations and the effect of host population densities on these interactions.

Allelopathy in Spatially Distributed Populations

In a homogeneously mixing population ofE. coli, colicin-producing and colicin-sensitive strategies both may be evolutionarily stable for certain parameter ranges, with the outcome of competition determined by initial conditions. In contrast, in a spatially-structured population, there is a unique ESS for any given set of parameters; the outcome is determined by how effective allelopathy is in relation to its costs. Furthermore, in a spatially-structured environment, a dynamic equilibrium may be sustained among a colicin-sensitive type, a high colicin-producing type, and a “cheater” that expends less on colicin production but is resistant.

Potential virulence determinants of Salmonella serovars from poultry and human sources in Spain

A total of 173 Salmonella strains of different serovars isolated during 1992 from poultry and human sources in Zaragoza (NE Spain) were investigated for potential virulence factors. Parameters studied included production of aerobactin, enterobactin, colicin (including colicin V) and hemolysin, serum resistance against serum from man, sheep, cattle and chicken, binding of Congo red and crystal violet, auto-agglutination and calcium dependency at 37°C and double colony morphology. Preliminary studies showed tests with completely negative results (colicins and hemolysin production, double colony morphology, auto-agglutination and calcium dependency at 37°C) and tests with completely positive results (enterobactin production and binding of Congo red). The tests with variability of results were production of aerobactin, serum resistance and crystal violet binding. Aerobactin production was detected in 80% of Salmonella strains of clinical human origin and in 30% of Salmonella strains isolated from healthy slaughtered chickens. Sixty-five per cent of patient isolates were human serum resistant, 73% were ovine serum resistant, 85% were bovine serum resistant and 98% were chicken serum resistant. The percentages of poultry isolates serum resistant were 61, 86, 60 and 89% in human, ovine, bovine and chicken serum, respectively. Crystal violet binding was detected in 22.5% of Salmonella isolates from human origin and in 32.3% of Salmonella strains from poultry origin. This study can form a marker for the prevalence of strains with various characteristics (production of aerobactin, serum resistance and crystal violet binding) for comparison in future epidemiological studies. Furthermore, the data of this work suggested that strains causing enteric salmonellosis in man are partially identical to strains isolated from carrier broilers.

Characterization of Salmonella dublin and Salmonella typhimurium (Copenhagen) isolates from cattle.

Eight Salmonella typhimurium (Copenhagen) and eight Salmonella dublin isolates from cattle were compared by their antibiotic resistance patterns, by their production of colicin, aerobactin, haemolysin and capsule, by their possession of transmissible R plasmids and the spvC gene, and by their ability to invade and replicate within cultured epithelial cells. The two groups differed in their antibiotic resistance profiles, with more of the host-adapted S. dublin isolates resistant to tetracycline than were the non-host-adapted S. typhimurium (Copenhagen) group, but more of the S. typhimurium (Copenhagen) isolates resistant to the other antibiotics tested. None of the isolates produced colicin, but all produced aerobactin. One isolate in each group was encapsulated. All of the S. typhimurium (Copenhagen) and S. dublin isolates contained plasmids, and all of them contained the spvC-homologous sequences. Four of the S. typhimurium (Copenhagen) isolates were able to transfer an R plasmid to a recipient organism by conjugation. One of the five S. dublin isolates, which showed resistance to some of the antibiotics tested, was able to transfer an R plasmid by conjugation. Both groups of isolates invaded cultured epithelial cells to a similar degree after 1 h, but the S. dublin isolates reached significantly higher levels within the cells than did S. typhimurium (Copenhagen) after 9 h. This ability may, in part, explain the association of S. dublin with more severe forms of salmonellosis and prolonged carrier states. Further study of the intracellular growth of these isolates seems warranted.

Plasmid distribution in Escherichia coli urinary isolates with special reference to aerobactin and colicin production

Plasmids were detected in 31 out of 35 strains of Escherichia coli isolated from unclassified cases of urinary tract infection at a median value of 1.88 plasmid bands per isolate. The isolates showed an association of aerobactin and colicin production with the distribution of plasmid bands having a median value of 2.33 and 1.72 (plasmid bands per isolate) in aerobactin-positive and aerobactin-negative strains respectively. For colicin producers, the median plasmid bands per isolate was 3.66 compared to 1.80 for colicin-negative strains.

Evaluation of colicins for inhibitory activity against diarrheagenic Escherichia coli strains, including serotype O157:H7.

Twenty-four Escherichia coli strains producing standard colicins were evaluated for inhibitory activity against 27 diarrheagenic E. coli strains of serotypes O15:H-, O26:(H11, H-), and O111:(H8, H11, H-), including O157:H7, representing diarrheagenic E. coli clones, 3, 4, 8, 9, and 10. Overlay techniques were used to assess inhibition on Luria agar and Luria agar supplemented with 0.25 micrograms of mitomycin C per ml to induce colicin production. As a group, the A colicins (Col) E1 to E8, K, and N inhibited 23 to 25 (85.2 to 92.6%) of the 27 diarrheagenic strains on mitomycin C-containing agar, whereas the most active group B colicins, Col D and Ia, inhibited 9 and 12 (33.3 and 44.4%), of the diarrheagenic strains, respectively. Col G and H and Mcc B17 inhibited 22 to 27 (81.5 to 100%) of the diarrheagenic strains on Luria agar but were suppressed on mitomycin C-containing agar medium. All O157:H7 strains evaluated were sensitive to Col E1 to E8, K, and N on mitomycin C-containing agar and to Col G and H and Mcc B17 on Luria agar. Sensitivity to colicins of the selected set of diarrheagenic strains was in the order diarrheagenic E. coli clone 9 > 4 > 3 > 10 > 8 and was not restricted to strains of a single clone or serotype. Strain 8C from clone 8 was resistant to most test colicins. There is potential for using colicins in foods and agriculture to inhibit sensitive diarrheagenic E. coli strains, including serotype O157:H7.

Escherichia coli cellulitis in broiler chickens: clonal relationships among strains and analysis of virulence-associated factors of isolates from diseased birds

Thirty-nine Escherichia coli isolates from broiler chickens with cellulitis were serotyped and analyzed for clonal relationships by multilocus enzyme electrophoresis. The isolates were further characterized with respect to hemagglutination (HA); serum resistance; antibiotic susceptibility; production of aerobactin, colicin V, and hemolysin; expression of K1 or K5 capsule; sensitivity to cloacin DF13 after treatment with diphenylamine; expression of iron-regulated outer membrane proteins; and virulence in 1-day-old chickens. In addition, the isolates were examined for the presence of DNA sequences related to F1A (fim) and P (pap) fimbriae, aerobactin synthesis (iuc) and transport (iut), hemolysin operon hly, and TraT lipoprotein-induced serum resistance (traT). Only 38.4% of the isolates were typeable with standard O antisera, and of these, serogroups O25 and O78 were the most frequently observed. Multilocus enzyme electrophoresis, based on 20 enzymes, resolved 17 electrophoretic types, forming seven clusters. Isolates from four of these clusters fell into E. coli clone complexes that have been previously reported to be commonly associated with avian colibacillosis. All isolates expressed two to five iron-regulated outer membrane proteins, were resistant to serum and cloacin DF13, and possessed DNA sequences homologous to fim and iuc/iut. Most isolates (72%) were positive for traT, and a majority produced colicin V and aerobactin (92 and 82%, respectively). Assays for the presence of fim and pap DNA sequences, for HA, and for virulence gave variable results but suggest that cellulitis isolates may express F1A and/or other mannose-resistant HA fimbriae different from P and may be virulent in 1-day-old chickens. Our results support the hypothesis that cellulitis in broilers in many cases is caused by E. coli clones identical to other pathogenic avian E. coli strains. Certain clones may be specific to cellulitis, because 25% of the isolates tested belong to clusters not related to known clone complexes.

Rapid invasion by colicinogenic Escherichia coli with novel immunity functions.

Bacteriocins have been suggested to play an important role in the invasion dynamics of bacteria. Recently, the 'diversifying selection' hypothesis has been proposed, which addresses the origin and diversification of one group of bacteriocins, the colicins of Escherichia coli. According to this hypothesis, novel colicin gene clusters arise from mutations generating expanded immunity functions. Positive selection, favouring these novel immunities, then rapidly drives strains carrying the evolved colicin gene clusters to fixation in the local population. To test this fixation step driven by selection, invasion experiments were carried out by introducing novel colicinogenic strains into established colicinogenic populations. In all cases, invasion by strains expressing novel immunity functions occurred rapidly, even when initial frequencies of the invader were quite low. These invasions were attributed primarily to colicin killing effect. Other factors, such as growth rate, level of colicin production and stationary-phase survival rate, were shown to play very minor roles in the invasion process. These results provide direct evidence for the hypothesis of diversifying selection acting on colicin gene clusters and shed light on the ecological role of colicins.

Analysis of Plasmids Cloned from a Virulent Avian Escherichia coli and Transformed into Escherichia coli DH5a

Three of four plasmids from a virulent wild-type avian Escherichia coli were cloned or transformed into an avirulent laboratory recipient E. coli DH5 alpha and tested for the ability to confer a virulence phenotype. The three plasmids transformed into E. coli DH5 alpha were 5, 6, and 56 kb. A fourth plasmid of 64 kb was not successfully transformed. Parameters used to measure virulence included presence of type 1 pili and a smooth lipopolysaccharide (LPS) layer, motility, production of Colicin V, resistance to host complement, and embryo lethality. The 5-kb plasmid encoded for ampicillin resistance, whereas the 6-kb plasmid encoded for tetracycline resistance. The 56-kb plasmid encoded for streptomycin, sulfisoxazole, and tetracycline resistance. Twelve-day embryos inoculated with 467 colony-forming units of E. coli DH5 alpha containing the 56-kb plasmid had increased death rates (45%) in the embryo lethality assay and a decreased weight of surviving embryos with cranial hemorrhages as compared with embryos inoculated with similar amounts of E. coli DH5 alpha (0%) and phosphate-buffered saline (0%). Embryos inoculated with the wild-type virulent E. coli had 90% deaths. The 56-kb plasmid also had homology with a probe for Colicin V production (cvaC). No differences in LPS layer, complement resistance, motility, Colicin V activity, type 1 pili, cell-free supernatant proteins, or outer membrane proteins were observed in the transformants when compared with nontransformed E. coli DH5 alpha.

Characteristics of Escherichia coli Isolates from Avian Cellulitis

The purpose of this study was to characterize Escherichia coli isolates from avian cellulitis, particularly with respect to the occurrence of potential virulence factors. At slaughter, five broilers with lesions of cellulitis were selected from each of 20 farms from among the broilers processed. One hundred E. coli isolates from the lesions were characterized with respect to serotype; biotype; drug susceptibility; plasmid profile; ability to produce aerobactin, colicin, colicin V, and hemolysin; and cytotoxicity for Vero cells and chicken fibroblasts. The same properties were determined from a collection of 25 E. coli from the feces of chickens. Serotyping showed that, among the cellulitis isolates, 23 belonged to O group 78, 14 belonged to O2, eight belonged to O115, and seven belonged to O(21.83); 25 were untypable. Isolates from a single farm typically belonged to three to five O groups. More than half of the fecal isolates were untypable, and the rest were distributed among seven O groups. Biotype, drug-resistance pattern, and plasmid profile could not be used as markers of avian cellulitis E. coli. No plasmid was detected in 12% of cellulitis isolates and 48% of fecal isolates. No isolate was hemolytic or showed cytotoxic effects. Aerobactin was produced by 90% of cellulitis isolates, and colicin was produced by 85% of these isolates; the corresponding percentages for the fecal isolates were 16% and 40%. Production of colicin V was detected in 21% of cellulitis isolates and 24% of fecal isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

Universal barrier to lateral spread of specific genes among microorganisms.

A genetic circuit to suppress the lateral spread of cloned genes from recombinant to indigenous microorganisms in the environment has been developed. It is based on the endonucleolytic activity of the bacterial toxin colicin E3, which has a distinct target at the 3' end of the 16S ribosomal RNA; this sequence is conserved in virtually all prokaryotic and many eukaryotic genera. Cleavage at this sequence separates the mRNA binding sites from the remainder of the 16S rRNA, thereby inhibiting protein synthesis. While host bacteria carrying the genes for both colicin production and colicin immunity are perfectly viable, lateral transfer of the E3 gene to non-immune recipients results in killing of such recipients. This genetic circuit decreases operational transfer frequencies of cloned genes linked to the E3 gene among a variety of bacterial genera by four to five orders of magnitude. In combination with transposon cloning vectors, the circuit is predicted to reduce the rate of lateral spread of specific genes to ecologically insignificant levels. This system therefore represents a useful tool both to explore the evolutionary and ecological consequences of experimentally reducing lateral gene spread among microorganisms, and to increase the ecological predictability of novel recombinant microorganisms.

Virulence-associated factors in Escherichia coli strains isolated from children with urinary tract infections.

One hundred and sixty-eight strains of Escherichia coli were isolated from cases of pyelonephritis (24) and lower urinary tract infections (UTI) (144) from hospitalised and outpatient children up to 2 years old. These strains were investigated for the expression of P fimbriae (PF), mannose-resistant and mannose-sensitive haemagglutination, cell-surface hydrophobicity, serum resistance and the production of alpha-haemolysin (AH), colicins and aerobactin. PF, AH, aerobactin production and serum resistance were significantly more frequent amongst strains expressing mannose-resistant haemagglutination. PF and AH production was significantly more frequent in pyelonephritogenic strains than in lower UTI strains. Serotypes O6 and O112 were isolated most frequently and plasmids were found in the majority of strains tested.

Characteristics associated with pathogenicity of avian septicaemic Escherichia coli strains

Seventeen strains of E. coli, isolated from chickens with colisepticaemia, were studied with respect to their pathogenic characteristics including: serum resistance, toxin production, pathogenicity for one-day-old chicks, colicin production, adherence to and invasiveness of HeLa cells, plasmid DNA profile and SDS-PAGE electrophoresis of membrane proteins, as well as electron microscope studies and hemagglutination tests for fimbriae. We concluded that the adherence to and the invasiness of HeLa cells were not related to the pathogenicity of these strains for chickens. Plasmid profiles were not related to the bacterial activity of the serum. Toxin production was correlated to the highest levels of pathogenicity. Some of the strains had mannose-resistant fimbriae. SDS-PAGE of membrane proteins of all the strains which were either not pathogenic or which had a very high LD 50 lacked two major protein subunits of 40.7 kDa and 28.8 kDa found only in pathogenic strains.

In vitro comparison of bovine mastitis and fecal Escherichia coli isolates

In vitro methods were used to test the hypothesis that Escherichia coli from bovine mastitis are essentially no different from isolates from bovine feces. Fifty E. coli isolates from bovine mastitic milk, 50 from feces of mastitic cows and 50 from feces of healthy cows were compared with respect to biochemical properties and certain potential virulence factors. There were no significant differences among the groups in tests for biotype; production of colicins, colicin V, or Vero cell cytotocity; and growth in 90% gnotobiotic calf serum or 90% normal milk whey. Resistance to killing in 90% gnotobiotic calf serum varied from 66 to 84%. Most isolates grew in normal whey: the percentage in a group varied from 86 to 96. Mastitic milk isolates were significantly different from the fecal isolates in adonitol fermentation ( P⩽0.006), production of aerobactin ( P⩽0.026), and ability to grow in 90% mastitic whey ( P⩽0.00004). However, only 40% of mastitis E. coli fermented adonitol and only 20% produced aerobactin. Ninety-six percent of mastitic milk E. coli grew in mastitic whey, whereas 64% and 60%, respectively, of mastitic fecal and normal fecal isolates grew in this medium. It is concluded that none of the properties that were investigated constitute potential virulence factors or markers for ability to induce mastitis; the data are consistent with the hypothesis that mastitic E. coli are simply opportunistic pathogens.

Facteurs et marqueurs de virulence de souche <em>Escherichia coli</em> isolées de diarrhées chez des veaux âgés de 4 à 45 jours en Algérie

The study involved 492 strains of Escherichia coli isolated from the feces of 44 diarrhoeal calves and 4 healthy calves in 7 wilayates in Algeria (Tipaza, Ain Defla, Bejaïa, Borj Bou Arreridj, Bouira, Medea and Algiers). The authors looked for the surface proteins K99, CS31A, Vir, F17 (FY), 20K and certain factors or markers of virulence such as the production of colicins, particularly colicin V, the aerobactin siderophore, alpha hemolysin and enterohemolysin. They also studied the frequency of certain 0 serogroups and evaluated the resistance of the E. coli strains to 10 antibiotics. The results showed that the majority of diarrhoeal calves were colonized by Escherichia coli expressing one or more factors of virulence and that the stocks which produce the antigen CS31A are usually resistant to 4 or 6 antibiotics.

Non-Association of Macrophage Phagocytosis and Oxidant Stimulation with Complement Resistance and Colicin V Production by Avian Escherichia coli

Avian isolates of Escherichia coli were classified as virulent based on their isolation from chickens with natural cases of colisepticemia, production of colicin V, and complement resistance. A second group of isolates was designated as avirulent based on their isolation from healthy chickens, their inability to produce colicin, and their classification as sensitive or intermediate to the action of complement. In vitro assays of phagocytosis and oxidant production were performed in an attempt to correlate these activities with the ability of each group of bacteria to escape the specific host defense mechanisms of phagocytosis and killing. Although oxidant production regressed with significant linearity on percent phagocytosis, neither group (virulent or avirulent) differed in ability to stimulate peritoneal macrophage phagocytic and oxidant activity when opsonized with normal chicken serum. These results differ from those in mammalian species.

Effect of Normal Intestinal Flora of Chickens on Colonization by Virulent Colicin V-Producing, Avirulent, and Mutant Colicin V-Producing Avian Escherichia coli

Colonization of the intestinal tracts of newly hatched chicks with Escherichia coli was attempted by swabbing test organisms onto the air-shell of 19-day-old embryos. Test organisms consisted of two virulent E. coli isolates, one avirulent isolate, and one laboratory-derived mutant of the avirulent isolate carrying a recombinant plasmid coding for Colicin V production. Chicks were cultured weekly for 3 weeks for total E. coli and for the test organisms using selective media. Control chicks were sampled on weeks 1 and 5, and the normal E. coli intestinal microflora were examined for the production of colicins. The two virulent E. coli isolates maintained colonization of the chicks for the 3-week test period, with titers decreasing from 10' to 10'- colony-forming units (CFU)/g of intestine. The avirulent isolate and laboratory mutant did not consistently colonize the intestinal tracts. The majority of intestinal samples taken from the control chicks at 1 and 5 weeks had colicin-producing E. coli that were inhibitory to the test organisms.

Virulence factors associated with F165-positive Escherichia coli strains isolated from piglets and calves

In this study, 91 F165-positive Escherichia coli isolated from calves and piglets with diarrhea or septicemia were characterized with respect to receptor binding specificity, presence of the aerobactin system, production of colicin V, resistance to the bactericidal effects of serum. Although most F165-positive isolates shared similar DNA sequences with pap operon sequences, less than half of these isolates demonstrated MRHA to P antigen of human red blood cells and Forssman antigen of sheep red blood cells recognized by P and F (or Prs) adhesins respectively. Certain F165-positive isolates sharing similar DNA sequences with both pap and sfa operon sequences demonstrated mannose-resistant hemagglutination of sheep erythrocytes, as observed in human uropathogenic E. coli possessing the prs operon. Most isolates caused mannose-resistant, neuraminidase-resistant hemagglutination of human, equine, feline, and bovine erythrocytes. Thus, F165-positive isolates express one or more adhesins with different receptor binding specificities. An association was observed between the various receptor binding specificities and serogroup. Most F165-positive isolates possessed the aerobactin system and were resistant to the bactericidal effects of serum, but only 3.8.5% isolates produced colicin V.