Colicin Typing Research Articles

Genomic Assessment of Enterobacter mori AYS9: A Potential Plant Growth-Promoting Drought-Resistant Rhizobacteria

Drought stress poses a serious danger to agricultural production. Recent studies have revealed that most of the chemical methods used in the mitigation of its effects on plant production pose a serious threat to humans and the environment. Therefore, the demand for ecologically friendly solutions to ensure the security of the world’s food supply has increased as a result. Plant growth-promoting rhizobacteria (PGPR) treatment may be advantageous in this situation. Enterobacter mori is a promising rhizobacteria in this regard. However, information on the genome analysis of E. mori linked to the rhizosphere soil of the sorghum plant has not been extensively studied. In this study, we present a genomic lens into functional attributes of E. mori AYS9 isolated from sorghum plants, as well as assess its drought tolerance and plant growth-promoting potentials. Our results showed the drought tolerance and plant growth-promoting potentials of the AYS9. Whole genome sequencing (WGS) results revealed that the genome yielded 4,852,175 bp sequence reads, an average read length of 151 bp, 1,845,357 bp genome size, 67 tRNAs, 3 rRNAs, and a G + C content of 55.5%. The functional genes identified in the genome were linked to processes including phosphate solubilization, iron transport, hormone regulation, nitrogen fixation, and resistance to oxidative and osmotic stress. Also, secondary metabolites supporting bacterial biocontrol properties against phytopathogens, and abiotic stress such as aerobactin-type non-ribosomal peptide siderophore, Stewartan-type ladderane, and Colicin type NRPS were discovered in the AYS9 genome. Our findings however establish that the intricate metabolic pathways mediated by the projected new genes in the bacterial genome may offer a genetic foundation for future understanding of rhizosphere biology and the diverse roles that these genes play in plant development and health.

Identification of Three Campylobacter Lysins and Enhancement of Their Anti-Escherichia coli Efficacy Using Colicin-Based Translocation and Receptor-Binding Domain Fusion.

The emergence of multidrug-resistant Escherichia coli, which poses a major threat to public health, has motivated the development of numerous alternative antimicrobials. Lysins are bacteriophage- and bacterium-derived peptidoglycan hydrolases that represent a new antibiotic treatment targeting bacterial cell walls. However, the bactericidal effect of native lysins on Gram-negative bacteria is restricted by the presence of an outer membrane. Here, we first evaluated the antibacterial activity of three Campylobacter-derived lysins (Clysins) against E. coli. To improve their transmembrane ability and antibacterial activities, six engineered Clysins were constructed by fusing with the translocation and receptor-binding (TRB) domains from two types of colicins (colicin A [TRBA] and colicin K [TRBK]), and their biological activities were determined. Notably, engineered lysin TRBK-Cly02 exhibited the highest bactericidal activity against the E. coli BL21 strain, with a reduction of 6.22 ± 0.34 log units of cells at a concentration of 60.1 μg/mL, and formed an observable inhibition zone even at a dose of 6.01 μg. Moreover, TRBK-Cly02 killed E. coli dose dependently and exhibited the strongest bactericidal activity at pH 6. It also exhibited potential bioactivity against multidrug-resistant E. coli clinical isolates. In summary, this study identified three lysins from Campylobacter strains against E. coli, and the enhancement of their antibacterial activities by TRB domains fusion may allow them to be developed as potential alternatives to antibiotics. IMPORTANCE Three lysins from Campylobacter, namely, Clysins, were investigated, and their antibacterial activities against E. coli were determined for the first time. To overcome the restriction of the outer membrane of Gram-negative bacteria, we combined the TRB domains of colicins with these Clysins. Moreover, we discovered that the Clysins fused with TRB domains from colicin K (TRBK) killed E. coli more effectively, and this provides a new foundation for the development of novel bioengineered lysins by employing TRBK constructs that target outer membrane receptor/transport systems. One of the designed lysins, TRBK-Cly02, exhibited potent bactericidal efficacy against E. coli strains and may be used for control of multidrug-resistant clinical isolates. The results suggest that TRBK-Cly02 can be considered a potential antibacterial agent against pathogenic E. coli.

The Biology of Colicin M and Its Orthologs.

The misuse of antibiotics during the last decades led to the emergence of multidrug resistant pathogenic bacteria. This phenomenon constitutes a major public health issue. Consequently, the discovery of new antibacterials in the short term is crucial. Colicins, due to their antibacterial properties, thus constitute good candidates. These toxin proteins, produced by E. coli to kill enteric relative competitors, exhibit cytotoxicity through ionophoric activity or essential macromolecule degradation. Among the 25 colicin types known to date, colicin M (ColM) is the only one colicin interfering with peptidoglycan biosynthesis. Accordingly, ColM develops its lethal activity in E. coli periplasm by hydrolyzing the last peptidoglycan precursor, lipid II, into two dead-end products, thereby leading to cell lysis. Since the discovery of its unusual mode of action, several ColM orthologs have also been identified based on sequence alignments; all of the characterized ColM-like proteins display the same enzymatic activity of lipid II degradation and narrow antibacterial spectra. This publication aims at being an exhaustive review of the current knowledge on this new family of antibacterial enzymes as well as on their potential use as food preservatives or therapeutic agents.

Colicin Z, a structurally and functionally novel colicin type that selectively kills enteroinvasive Escherichia coli and Shigella strains

Colicin production in Escherichia coli (E. coli) strains represents an important trait with regard to microbial survival and competition in the complex intestinal environment. A novel colicin type, colicin Z (26.3 kDa), was described as a product of an original producer, extraintestinal E. coli B1356 strain, isolated from the anorectal abscess of a 17 years-old man. The 4,007 bp plasmid (pColZ) was completely sequenced and colicin Z activity (cza) and colicin Z immunity (czi) genes were identified. The cza and czi genes are transcribed in opposite directions and encode for 237 and 151 amino acid-long proteins, respectively. Colicin Z shows a narrow inhibitory spectrum, being active only against enteroinvasive E. coli (EIEC) and Shigella strains via CjrC receptor recognition and CjrB- and ExbB-, ExbD-mediated colicin translocation. All tested EIEC and Shigella strains isolated between the years 1958–2010 were sensitive to colicin Z. The lethal effect of colicin Z was found to be directed against cell wall peptidoglycan (PG) resulting in PG degradation, as revealed by experiments with Remazol Brilliant Blue-stained purified peptidoglycans and with MALDI-TOF MS analyses of treated PG. Colicin Z represents a new class of colicins that is structurally and functionally distinct from previously studied colicin types.

Transfer-messenger RNA and SmpB mediate bacteriostasis in Escherichia coli cells against tRNA cleavage.

RNAs, such as mRNA, rRNA and tRNA, are essential macromolecules for cell survival and maintenance. Any perturbation of these molecules, such as by degradation or mutation, can be toxic to cells and may occasionally induce cell death. Therefore, cells have mechanisms known as quality control systems to eliminate abnormal RNAs. Although tRNA is a stable molecule, the anticodon loop is quite susceptible to tRNA-targeting RNases such as colicin E5 and colicin D. However, the mechanism underlying cellular reaction to tRNA cleavage remains unclear. It had long been believed that tRNA cleavage by colicins E5 and D promptly induces cell death because colony formation of the sensitive cells is severely reduced; this indicates that cells do not resist the tRNA cleavage. Here, we show that Escherichia coli cells enter a bacteriostatic state against the tRNA cleavage of colicins D and E5. The bacteriostasis requires small protein B (SmpB) and transfer-messenger RNA (tmRNA), which are known to mediate trans-translation. Furthermore, another type of colicin, colicin E3 cleaving rRNA, immediately reduces the viability of sensitive cells. Moreover, nascent peptide degradation has an additive effect on bacteriostasis. Considering the recent observation that tRNA cleavage may be used as a means of cell-to-cell communication, tRNA cleavage could be used by bacteria not only to dominate other bacteria living in the same niche, but also to regulate growth of their own or other cells.

Activity spectrum of colicins produced by Shigella sonnei and genetic mechanism of colicin resistance in conspecific S. sonnei strains and Escherichia coli.

Colicin-mediated killing is an example of allelopathy, which has been found among several bacteria. Screening of 42 strains of Shigella sonnei isolated from diarrheal patients revealed that 39 (93%) S. sonnei strains were positive for colicin production against Escherichia coli DH5α. In the PCR-based detection of the colicin types, 36 (92.3%) were identified as E3, 2 (5.1%) as E3 and E8, and 1 (2.6%) as E3 and E2. Representative S. sonnei strains producing heterologous colicins exhibited antagonism against diarrheagenic Escherichia coli (DEC) groups. Although it is known that mutation in the colicin receptor renders the host resistant to colicin, there is a dearth of information on the genetic characterization of such mutants. In the fluctuation test, colicin-resistant E. coli mutants were found to occur spontaneously at the rates of 2.51 × 10(-8) and 5.52 × 10(-8) per generation when exposed to colicins E3 and E8 and colicins E3 and E2, respectively. Genotypic characterization of colicin-resistant E. coli (EC(Cr)) and S. sonnei (SS(Cr)) strains displayed mutations in the btuB gene, which encodes the receptor for vitamin B12 uptake. This gene was interrupted by various insertion sequences, such as IS1, IS2, and IS911. Complementation of EC(Cr) and SS(Cr) with plasmid-borne btuB (pbtuB) accomplished restoration of the colicin-susceptible phenotype. The vitamin B12 uptake assay gave an insight into the physiological relevance of the btuB mutation. Our studies provide insights into the latent influence of S. sonnei colicins in governing the existence of some of the shigellae and all of the DEC and the genetic mechanism underlying the emergence of resistance.

Colicin type 7 produced by majority of Shigella sonnei isolated from Thai patients with diarrhoea

Thirty one out of 153 strains of Shigella sonnei isolated from Thai patients with diarrhoea showed antibacterial activity against S. sonnei by agar well diffusion method. All of them harbor plasmids with the genetic determination of colicin type 7 (Js) gene but without colicin E and colicin U gene. The PCR product obtained from strain 35/44 was shown to be the gene for colicin type 7 lytic protein (cja). The partially purified bacteriocin (PPB) containing colicin type 7 of strain 35/44 was prepared and used for characterization. The antibacterial activity of PPB against a total of 17 selected Gram-positive and Gram-negative bacteria was tested. It was found that PPB of strain 35/44 was active against E. coli O157, S. sonnei and S. boydii. The sensitivity of PPB from this strain to proteinase K, trypsin and α-chymotrypsin suggests the proteinaceous nature of these antimicrobial substances. Therefore, this isolated bacterium can be regarded as bacteriocin producing bacteria. The bacteriocin produced by this isolated S. sonnei was heat stable as evidenced by its ability to maintain the activity at 80 °C for 60 min. In addition, it was stable within a wide range of pH (3-9). The molecular weight of colicin type 7 from isolated S. sonnei strain 35/44 analyzed by SDS-PAGE was 54.4 kDa composing of at least five subunits. It is to our knowledge; the first report of Thai patients with diarrhoea that S. sonnei isolated from them contained colicin type 7.

Novel Colicin F Y of Yersinia frederiksenii Inhibits Pathogenic Yersinia Strains via YiuR-Mediated Reception, TonB Import, and Cell Membrane Pore Formation

A novel colicin type, designated colicin Fy, was found to be encoded and produced by the strain Yersinia frederiksenii Y27601. Colicin Fy was active against both pathogenic and nonpathogenic strains of the genus Yersinia. Plasmid YF27601 (5,574 bp) of Y. frederiksenii Y27601 was completely sequenced. The colicin Fy activity gene (cfyA) and the colicin Fy immunity gene (cfyI) were identified. The deduced amino acid sequence of colicin Fy was very similar in its C-terminal pore-forming domain to colicin Ib (69% identity in the last 178 amino acid residues), indicating pore forming as its lethal mode of action. Transposon mutagenesis of the colicin Fy-susceptible strain Yersinia kristensenii Y276 revealed the yiuR gene (ykris001_4440), which encodes the YiuR outer membrane protein with unknown function, as the colicin Fy receptor molecule. Introduction of the yiuR gene into the colicin Fy-resistant strain Y. kristensenii Y104 restored its susceptibility to colicin Fy. In contrast, the colicin Fy-resistant strain Escherichia coli TOP10F' acquired susceptibility to colicin Fy only when both the yiuR and tonB genes from Y. kristensenii Y276 were introduced. Similarities between colicins Fy and Ib, similarities between the Cir and YiuR receptors, and the detected partial cross-immunity of colicin Fy and colicin Ib producers suggest a common evolutionary origin of the colicin Fy-YiuR and colicin Ib-Cir systems.

Escherichia coli Bacteriocins: Antimicrobial Efficacy and Prevalence among Isolates from Patients with Bacteraemia

Bacteriocins are antimicrobial peptides generally active against bacteria closely related to the producer. Escherichia coli produces two types of bacteriocins, colicins and microcins. The in vitro efficacy of isolated colicins E1, E6, E7, K and M, was assessed against Escherichia coli strains from patients with bacteraemia of urinary tract origin. Colicin E7 was most effective, as only 13% of the tested strains were resistant. On the other hand, 32%, 33%, 43% and 53% of the tested strains exhibited resistance to colicins E6, K, M and E1. Moreover, the inhibitory activity of individual colicins E1, E6, E7, K and M and combinations of colicins K, M, E7 and E1, E6, E7, K, M were followed in liquid broth for 24 hours. Resistance against individual colicins developed after 9 hours of treatment. On the contrary, resistance development against the combined action of 5 colicins was not observed. One hundred and five E. coli strains from patients with bacteraemia were screened by PCR for the presence of 5 colicins and 7 microcins. Sixty-six percent of the strains encoded at least one bacteriocin, 43% one or more colicins, and 54% one or more microcins. Microcins were found to co-occur with toxins, siderophores, adhesins and with the Toll/Interleukin-1 receptor domain-containing protein involved in suppression of innate immunity, and were significantly more prevalent among strains from non-immunocompromised patients. In addition, microcins were highly prevalent among non-multidrug-resistant strains compared to multidrug-resistant strains. Our results indicate that microcins contribute to virulence of E. coli instigating bacteraemia of urinary tract origin.

Study the toxic effect of non-bound colicins extracted from Escherichia coil on transplanted Murine adenocarcinoma (AM3)

Four types of non-bound colicins (H5, H9, H13, H19) extracted from intestinal normal flora E.coli isolated from apparently healthy people’s stool. Then cytotoxic effect of colicins on transplanted murine adenocarcinoma (AM3) was studied. The results showed,that the four colicins had inhibitory effects on growth of AM3 cells when injected intratumorally at (200) mg/kg. Percentages of tumor inhibition were (98.79, 90.45, 90.08 and 88.26) % for colicins (H19, H13, H9 and H5) respectively. Intraperitoneal injection caused less inhibition, (57.47, 36.25, 60.26 and 53.25) % respectively using a daily dose of (200) mg/kg for period of 24 days.

Isolation and Characterization of Potential Probiotic Escherichia coli Strains from Rat Faecal Samples

Problem statement: Gastrointestinal disorders mainly diarrhea is the most common cause of morbidity and mortality among infants and children in the developing countries occurs due to infection by enteropathogens. Ccontrol of these pathogens could be achieved by nonpathogenic Escherichia coli producing bacteriocins. Approach: Primary aim of this study was to isolate E. coli strains which inhibit enteropathogens. For this purpose, E. coli strains were isolated and tested for probiotic properties such as antimicrobial activity against enteropathogens, antibiotic susceptibility and resistance to low pH, absence of virulence traits, susceptibility to proteolytic activity and detection of colicins type. Results: Approximately 280 E. coli strains were collected from rat feacal samples at different ages and were screened for their antimicrobial activity. Out of these, 47 isolates showed antimicrobial effect against E. coli DH5α and BL21 strains and 16 were effective against different pathogens viz. Salmonella sp., Klebisella pneumoniae, Staphylococcus aureus and Enterobacter sp. These 16 isolates were susceptible to the common Antibiotics and most of them better survived at pH 3 and a few survived even at pH 2. Colony PCR of these isolates with colicinogenic primer demonstrated the presence of multiple colicin types. Out of 16 isolates, 12 had multiple pore forming colicins of E1 and 1a/1b type. In addition to the pore forming E1 and 1a/1b colicins, four E. coli strains 16, 2P and 14, 20 had Emix (nuclease type)/BD type (translation blocker) colicins, respectively. Conclusion: This study showed that E. coli isolates 10, 14 and 14 P possess good probiotic properties and could be effective against enteropathogens.

Enhancement of Shiga Toxin Production in Enterohemorrhagic Escherichia coli Serotype O157:H7 by DNase Colicins

Colicins are proteins produced by and active against several strains of Escherichia coli. Previously we reported that colicinogenic bacteria seemed beneficial in preventing the clinical manifestations of infectious disease caused by enterohemorrhagic E. coli O157 in humans. The inhibitory effects could be due to a decrease in O157 levels and/or pathogenicity. This study investigated the effects of colicinogenic E. coli on the production of Shiga toxin (Stx) by O157. Standard strains of colicinogenic bacteria carrying plasmids for each type of colicin (E3/5/8/9) were used for the study. The O157 strains were cultured in the presence of colicinogenic bacteria or extracted colicins. Compared with results for controls, DNase colicins (E8/9) facilitated an 8- to 64-fold increase in production of Stx2, while RNase colicins (E3/5) suppressed Stx production in only two strains. Stx prophages were induced in synchrony with Stx production. Semiquantitative real-time reverse transcription-PCR (RT-PCR) was then performed to examine SOS gene expression. The RT-PCR results clearly indicated a marked increase in mRNA levels of SOS reaction-associated genes after the addition of DNase colicins. We believe that Stx prophages are induced by the SOS response to DNA damage caused by DNase colicins, thus leading to higher Stx production. These findings suggest that while colicinogenic bacteria can be antagonistic to O157 infection, DNase colicins may enhance Stx production. Thus, colicinogenic flora is likely to be involved in the complex pathogenic pathways of O157 infection, and further investigation should be performed before the use of colicinogenic bacteria as an intervention method.

赤痢菌型別検査方法としてのAmplified Fragment Length Polymorphism (AFLP) 法の有用性の検討

Shigella is an etiological agent of communicable and food-borne disease worldwide, so it is important to develop typing for Shigella in epidemiological studies. We compared amplified fragment length polymorphism (AFLP), molecular epidemiological typing, to pulsed-field gel electrophoresis (PFGE) and colicin typing in typeability, reproducibility, discriminatory power, ease of interpretation, and ease of use for 51 Shigella isolates to determine AFLP applicability to Shigella. AFLP showed less reproducibility and ease of interpretation although it was superior to PFGE and colicin typing in typeability and discriminatory power. Specifying the reproducibility of these typing methods, the intrastrain similarity of AFLP was 81.9%-90.5% in each of three strains tested in triplicate trials, while PFGE showed higher similarity, ranging from 92.3%-100%. AFLP created a phylogenetic tree and classified four Shigella species taxonomically, despite its lower reproducibility. These results suggest that AFLP is inferior to PFGE as molecular typing for Shigella epidemiologically in outbreaks or sporadic cases, although AFLP can create a phylogenetic tree for taxonomical purposes.

Assessment of resistance to colicinogenic Escherichia coli by E. coli O157:H7 strains

To assess a collection of 96 Escherichia coli O157:H7 strains for their resistance potential against a set of colicinogenic E. coli developed as a probiotic for use in cattle. Escherichia coli O157:H7 strains were screened for colicin production, types of colicins produced, presence of colicin resistance and potential for resistance development. Thirteen of 14 previously characterized colicinogenic E. coli strains were able to inhibit 74 serotype O157:H7 strains. Thirteen E. coli O157:H7 strains were found to be colicinogenic and 11 had colicin D genes. PCR products for colicins B, E-type, Ia/Ib and M were also detected. During in vitro experiments, the ability to develop colicin resistance against single-colicin producing E. coli strains was observed, but rarely against multiple-colicinogenic strains. The ability of serotype O157:H7 strains to acquire colicin plasmids or resistance was not observed during a cattle experiment. Escherichia coli O157:H7 has the potential to develop single-colicin resistance, but simultaneous resistance against multiple colicins appears to be unlikely. Colicin D is the predominant colicin produced by colicinogenic E. coli O157:H7 strains. The potential for resistance development against colicin-based strategies for E. coli O157:H7 control may be very limited if more than one colicin type is used.

Characterization of Colicinogenic Escherichia coli Strains Inhibitory to Enterohemorrhagic Escherichia coli

A previously identified set of anti– Escherichia coli O157:H7 colicinogenic E. coli were characterized to assess the suitability of these isolates as a preharvest food safety intervention in cattle. This collection of 23 E. coli strains were screened for virulence factors, antibiotic resistance, type of colicin(s) present, and their ability to inhibit other pathogenic E. coli. With the use of PCR, pathogen genes were detected in six of the 23 colicinogenic E. coli. When the nonpathogenic strains were assessed for antibiotic resistance, four strains showed resistance to at least one antibiotic. The remaining set of 14 strains were evaluated for the presence of previously identified colicins. Seven colicins (B, E1, E2/E7, E7, Ia/Ib, K, and M) were detected. One half of the strains possessed multiple types of colicins. The most commonly detected colicins were B, E2/E7, and M, which were found in six strains each. DNA sequencing was also performed in order to classify the E2/E7 colicins separately from E7 colicins. The 14 colicinogenic E. coli also were evaluated for their ability to inhibit 10 different non-O157 pathogenic E. coli. Six of the colicinogenic E. coli were capable of inhibiting all 10 pathogens, and the remaining eight strains could each inhibit between six to eight of the pathogenic E. coli. This strain collection has great potential for inhibiting E. coli O157:H7 in cattle.

Is there any function for colicinogeny in the post-weaning diarrhoea of piglets?

Using seven experimental approaches, we attempted to solve the question of possible participation of colicinogeny and colicin-sensitivity in the pathology of the post-weaning diarrhoeic enteritis of piglets. In our research, both enterotoxic E. coli strains (ETEC) and normal, commensal E. coli strains in the intestinal microflora of 803 weaned piglets were followed. In diarrhoeic piglets, colicinogeny was more frequent in the ETEC strains than in the simultaneously isolated commensal ones. ETEC strains were largely insensitive to the most frequently appearing colicin types and the inhibitive effect of their colicins on commensal E. coli was less likely than the opposite (inhibition of ETEC strains by colicins of the commensal ones). Bolstering the diet of healthy piglets with a mixture of symbiotic colicinogenic strains and colicin-sensitive ETEC strains could not prevent diarrhoeic enteritis due to established dominance of ETEC. In all parts of the intestinal tract during the post-weaning diarrhoea (PWD), mostly non-colicinogenic strains of the commensal flora survived. In six serotypes of ETEC strains, the frequency of colicinogenic strains ranged from 7% (in the serotype O139) to 66% (in serotype O141). From 9 frequent colicin types, colicin V (mainly against the serotypes O8 and O147), E2 (against O139 and O8), D (mainly against O8) and E3 (mainly against O138) met most sensitive strains among ETEC. Hence, colicinogeny was no pathogenetic factor of PWD. Nevertheless, colicinogenic commensal strains gradually regained dominance during the decline phase of the disease in surviving piglets.

Human tumor cells are selectively inhibited by colicins.

The activity in vitro of four types of colicins (A, E1, E3, U) against one human standard fibroblast line and against 11 human tumor-cell lines carrying defined mutations of the p53 gene was quantified by MTT (tetrazolium bromide) assay. Flow cytometry showed that the pore-forming colicins A, E1 and U affected the cell cycle of 5 of these cell lines. Colicins E3 and U did not show any distinct inhibitory effects on the cell lines, while colicins E1 and especially A inhibited the growth of all of them (with one exception concerning colicin E1). Colicin E1 inhibited the growth of the tumor lines by 17-40% and standard fibroblasts MRC5 by 11%. Colicin A exhibited a differentiated 16-56% inhibition, the growth of standard fibroblasts being inhibited by 36%. In three of the lines, colicins A and E1 increased the number of cells in the G1 phase (by 12-58%) and in apoptosis (by 7-58%). These results correlated with the data from sensitivity assays. Hence, the inhibitory effect of colicins on eukaryotic cells in cell-selective, colicin-specific and can be considered to be cytotoxic.

Incidence of colicinogenic strains among human Escherichia coli.

During the years 1993-1999, altogether 1,043 Escherichia coli strains from colons of different persons were screened for colicinogeny using a most susceptible procedure and indicator system. In control persons (with healthy colons), 41.37% producers of colicins were found. In patients suffering from salmonelloses, the proportion of colicinogenic Escherichia coli was the same. In patients with non-specific inflammatory colon diseases, the proportion of colicinogenic Escherichia coli strains appeared slightly though weakly, significantly or unsignificantly increased: to 47.50% in morbus Crohn and to 56.10% in colitis ulcerosa. These results suggest some sort of engagement of colicinogeny in the pathogenesis thereof. In malignant tumours of the colon, the incidence of colicinogenic Escherichia coli was not altered (40.58%). This does not indicate any colicin participation in the pathology of malignant tumours. In colitis ulcerosa, the incidence of colicinogenic Escherichia coli strains inhibiting Shigella sonei 17 (the indicator for colicin Js which generally inhibits interoinvasive strains of both species) increased from 21.94% (control samples) to 41.46%. Uropathogenic Escherichia coli strains shared the same incidence of colicinogeny as controls (42.08%), if they were not haemolytic; haemolytic ones were colicinogenic with only 22.37%. This difference was highly significant. The patterns of some colicin activities in the set of five indicator strains used suggested that several wild strains produced new, so far unknown types of colicins and/or combinations thereof.

Genetic organization of plasmid ColJs, encoding colicin Js activity, immunity, and release genes.

The 5.2-kb ColJs plasmid of a colicinogenic strain of Shigella sonnei (colicin type 7) was isolated and sequenced. pColJs was partly homologous to pColE1 and to pesticin-encoding plasmid pPCP1, mainly in the rep, mob, and cer regions. A 1.2-kb unique region of pColJs showed significantly different G+C content (34%) compared to the rest of pColJs (53%). Within the unique region, seven open reading frames (ORFs) were identified. ORF94 was shown to code for colicin Js activity (cja), a 94-amino-acid polypeptide (molecular mass, 10.4 kDa); ORF129 (cji) was shown to code for the 129-amino-acid colicin Js immunity protein (molecular mass, 14.3 kDa); and ORF65 was shown to be involved in colicin Js release by producer bacteria (cjl) coding for a 65-amino-acid polypeptide (molecular mass, 7.5 kDa). In contrast to the gene order in other colicin operons, the cjl gene was found upstream from cja. Moreover, the promoter upstream from cjl was similar to promoters described upstream from several colicin activity genes. The cji gene was found to be located downstream from cja with a transcription polarity opposite to that of the cjl and cja genes. The cja, cji, and cjl genes were not similar to other known colicin genes. Colicin Js was purified as an inactive fusion protein with an N-terminal histidine tag. Activity of the purified fusion form of colicin Js was restored after cleavage of the amino acids fused to the colicin Js N terminus.

Epidemiological analysis of Shiga toxin-producing Escherichia coli O157 isolates from familial infection

A total of 201 Shiga toxin-producing Escherichia coli (STEC) O157:H7 isolates from 22 epidemiologically unrelated familial infections in Osaka were analyzed by various epidemiological markers, such as Shiga toxin (STx) typing, antimicrobial resistant patterns, colicine typing, plasmid profiles and pulsed-field gel electrophoresis (PFGE) typing. There were two cases where different type strains were detected in a family (family No. 21 and 22). In the family No. 21, three different strains were isolated from a 5-year-old male infant; one identical with that from his mother, and the others different in 4 markers except STx type. In the family No. 22, two kinds of strain were detected in a 48-year-old father; one identical with those from other members of the family, and the other different in STx, plasmid profile and PFGE types. These facts showed the possibility of a simultaneous double infection from the common sources of infectious factors or a successive reinfection with different types of the agents. Identical marker strains were detected from 8 out of 12 familial infection cases from July to September. Although infectious sources of these cases are not yet clearly identified, these results of epidemiological markers analysis indicate a probable circulation of the common contaminated foodstuffs. A combined use of phenotypic and genotypic tests were shown to be useful for the epidemiological analysis. Further, it seemed necessary to examine epidemiological markers of more than one strain in familial infection or identical facilities generation cases. And also a collective analysis of the relating factors such as biological markers of the causative agents, the list of eaten foodstuffs, and successive outbreaks of the patients was thought most important.