The fluorescent probe-based recombinase-aided amplification for rapid detection of Escherichia coli O157:H7

Escherichia coli O157:H7 (E. coli O157:H7) is a common foodborne morbigenous microorganism, which can spread through fecal-oral transmission. Humans can be infected by ingesting foods and water contaminated with E. coli O157:H7, which can cause various symptoms. In present study, we have successfully developed a quick and hypersensitive fluorescent probe-based Recombinase-aided amplification (RAA) method and applied in E. coli O157:H7 detection at 39 °C in 20 min. The sensitivity of the assay in pure E. coli O157:H7 suspension was 5.6 × 10⁰ CFU/mL. The fluorescent probe-based RAA assay was further applied in three samples, and the limit of detection (LOD) in skimmed milk, lettuces and lake water was 5.4 × 10¹ CFU/mL, 7.9 × 10¹ CFU/mL and 5.2 × 10¹ CFU/mL, separately. This method showed a high sensitivity and short detection time, which has the feasible application in on-site test in real samples.     

Development of an oligonucleotide-based microarray to detect multiple foodborne pathogens

Escherichia coli O157:H7, Salmonella enterica, Listeria monocytogenes and Campylobacter jejuni are considered important pathogens causing the most food-related human illnesses worldwide. Current methods for pathogen detection have limitations in the effectiveness of identifying multiple foodborne pathogens. In this study, a pathogen detection microarray was developed using various 70-mer oligonucleotides specifically targeting the above pathogens. To reduce the cost of detection, each microarray chip was designed and fabricated to accommodate 12 identical arrays which could be used for screening up to 12 different samples. To achieve high detection sensitivity and specificity, target-specific DNA amplification instead of whole genome random amplification was used prior to microarray analysis. Combined with 14-plex PCR amplification of target sequences, the microarray unambiguously distinguished all 4 pathogens with a detection sensitivity of 1 × 10^?4 ng (approximately 20 copies) of each genomic DNA. Applied the assay to 39 fresh meat samples, 16 samples were found to be contaminated by either 1 or 2 of these pathogens. The co-occurrences of Salmonella and E. coli O157:H7, Salmonella and L. monocytogenes in the same meat samples were also observed. Overall, the microarray combined with multiplex PCR method was able to effectively screen single or multiple pathogens in food samples and to provide important genotypic information related to pathogen virulence.     

Detection of verotoxin-producing Escherichia coli in stool specimens by the polymerase chain reaction

In this retrospective study a total of 404 stools kept at −70°C were tested for the presence of verotoxin-producing Escherichia coli (VTEC) by the polymerase chain reaction (PCR). Thirteen positive samples from 11 patients were identified by PCR which correlated with previous isolation of E. coli O157:H7. There was no failure to detect VTEC by PCR but PCR did not identify further VTEC isolates. We concluded that (a) the occurrence of VTEC other than serotype O157:H7 is rare in our demographic area, (b) PCR is effective in the identification of E. coli O157:H7, and (c) PCR has the additional advantage over conventional culture methods of identifying VTEC, including sorbitol-fermenting serotypes, which might have been detected if we had extended our sample size.     

Neutralizing activity of bovine colostral antibody against verotoxin derived from enterohemorrhagic Escherichia coli O157:H7 in mice

The neutralization efficacy of bovine colostral antibody against verotoxin (VT) 1 and 2 was investigated. Cows were immunized with VT1 or VT2 fourteen times at 7-day intervals. A colostral antibody exhibiting high titers was obtained from immunized cows. Survival rates were evaluated in mice administered VT1 or VT2, and those infected with Escherichia coli (E. coli) O157:H7 producing VT1 or VT2. Survival rates after VT1 administration were 100% in the single-administration group, 90% in the repeat-administration group, and 78.6% in the control group. Survival rates after VT2 were 75.0% in the single-administration group, and 100% in the repeat-administration group. All mice in the control group died. Colostral antibody and fosfomycin (FOM) in the colostral antibody group and FOM and skim milk in the control group were administered three times per day for 5 days to mice infected with E. coli O157:H7 producing VT1 or VT2. Survival rates after inoculation with E. coli O157:H7 producing VT1 were 80.0% in the colostral antibody group, and 63.6% in the control group. Survival rates after inoculation with E. coli O157:H7 producing VT2 were 83.3% in the colostral antibody group, and 20.0% in the control group. The survival rate in mice without treatment following inoculation with E. coli O157:H7 producing VT2 was 88.2%.The survival rates in mice infected with E. coli O157:H7 strains producing VT1 or VT2 improved after administration of this colostral antibody, which exhibited neutralization efficacy against VT.     

Detection and characterization of thefimAgene ofEscherichia coliO157:H7

An expected 850-bp DNA fragment containingfimA, the structural gene for type 1 fimbriae, and flanking sequences was amplified from 39 (of 46) pathogenic and commensal strains ofEscherichia coliusing the polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) analysis of the amplified products showed 13HinPI and fourSau96I restriction profiles among these 39E. colistrains, revealing the polymorphic nature of this allele. A unique RFLP pattern was shared byE. coliO157:H7, O157:H^- and a few O55 serotype strains. DNA sequence analysis of thefimAregion demonstrated thatE. coliO157:H7 strain 933 and O157:H⁻strain E32511 contained identical DNA sequences that were distinct from otherE. colistrains, especially a 16-bp sequence 5′ tofimAthat was conspicuously absent only inE. coliO157 strains. Exploiting these differences, a PCR assay was developed that amplifies a 936-bp fragment from allE. coliO157:H7 strains examined to date. This PCR assay offers a simple, rapid, and reliable means to detectE. colistrains of the O157:H7 serotype.     

Restrictive Streptomycin Resistance Mutations Decrease the Formation of Attaching and Effacing Lesions in Escherichia coli O157:H7 Strains

Streptomycin binds to the bacterial ribosome and disrupts protein synthesis by promoting misreading of mRNA. Restrictive mutations on the ribosomal subunit protein S12 confer a streptomycin resistance (Str^r) phenotype and concomitantly increase the accuracy of the decoding process and decrease the rate of translation. Spontaneous Str^r mutants of Escherichia coli O157:H7 have been generated for in vivo studies to promote colonization and to provide a selective marker for this pathogen. The locus of enterocyte effacement (LEE) of E. coli O157:H7 encodes a type III secretion system (T3SS), which is required for attaching and effacing to the intestinal epithelium. In this study, we observed decreases in both the expression and secretion levels of the T3SS translocated proteins EspA and EspB in E. coli O157:H7 Str^r restrictive mutants, which have K42T or K42I mutations in S12. However, mildly restrictive (K87R) and nonrestrictive (K42R) mutants showed slight or indistinguishable changes in EspA and EspB secretion. Adherence and actin staining assays indicated that restrictive mutations compromised the formation of attaching and effacing lesions in E. coli O157:H7. Therefore, we suggest that E. coli O157:H7 strains selected for Str^r should be thoroughly characterized before in vivo and in vitro experiments that assay for LEE-directed phenotypes and that strains carrying nonrestrictive mutations such as K42R make better surrogates of wild-type strains than those carrying restrictive mutations.     

Antigenic cross-reactions between Escherichia coli O157, Vibrio cholerae O1 (Inaba) and group N salmonella

Lipopolysaccharide antigen-antibody cross-reactions between Escherichia coli O157 and other members of the Enterobacteriaceae were examined. Using serotyping schemes established in the Laboratory of Enteric Pathogens (LEP), cross-reactions were demonstrated between E. coli O157, Vibrio cholerae O1-Inaba and group N (O = 30) salmonella. SDS-PAGE and immunoblotting showed that the lipopolysaccharide (LPS) of E. coli O157 contained epitopes shared with LPS expressed by group N salmonella. These cross-reactions were also detected using sera from patients with haemolytic uraemic syndrome (HUS) caused by E. coli O157:H7. Rabbit antibodies, prepared to the LPS of V. cholerae O1-Inaba reacted with the LPSs of both E. coli O157 and V. cholerae O1-Inaba, whilst rabbit antisera prepared to E. coli O157 did not react with the LPS of V. cholerae O1-Inaba. Antibodies in sera from patients with HUS, which reacted with the LPS of E. coli O157, did not react with the LPS from V. cholerae O1-Inaba. We concluded from our study that cross-reactions between E. coli O157 and other bacterial species are rare; and that patients infected with bacteria known to share epitopes with E. coli O157 are unlikely to give false positive results when their sera were tested for E. coli O157 serology.     

Multiplex PCR for detection of trait and virulence factors in enterohemorrhagic Escherichia coli serotypes

A multiplex PCR assay was developed which allowed the simultaneous detection of five trait genes or virulence markers in enterohemorrhagic Escherichia coli (EHEC) serotypes. A primer pair, designed to detect a single base-pair mutation in the uidA gene, is specific only for the prototypic EHEC of O157:H7 serotype and its toxigenic, non-motile variants. In a similar way, primers to the eaeA gene of the γ-intimin derivative specifically detects strains in the EHEC 1 clonal group, which consists mostly of O157:H7 and some O55:H7 serotypes. The other three primer pairs, specific for stx1, stx2 and both variants of ehxA genes, will detect the presence of these virulence genes in all EHEC serotypes. Analysis of 34 strains, including various serotypes of EHEC, Shiga toxin-producing E. coli and enteropathogenic E. coli, confirmed that the multiplex PCR assay detected the presence of these genes in a manner consistent with the known genotype of each respective strains.     

Synergistic Effect between Colistin and Bacteriocins in Controlling Gram-Negative Pathogens and Their Potential To Reduce Antibiotic Toxicity in Mammalian Epithelial Cells

Pathogens resistant to most conventional antibiotics are a harbinger of the need to discover novel antimicrobials and anti-infective agents and develop innovative strategies to combat them. The aim of this study was to assess the in vitro activity of colistin alone or in combination with two bacteriocins, nisin A and pediocin PA-1/AcH, against Salmonella choleraesuis ATCC 14028, Pseudomonas aeruginosa ATCC 27853, Yersinia enterocolitica ATCC 9610, and Escherichia coli ATCC 35150 (O157:H7). The strain most sensitive to colistin was enterohemorrhagic E. coli O157:H7, which was inhibited at a concentration of about 0.12 μg/ml. When nisin A (1.70 μg/ml) or pediocin PA-1/AcH (1.56 μg/ml) was combined with colistin, the concentrations required to inhibit E. coli O157:H7 were 0.01 and 0.03 μg/ml, respectively. The in vitro antigenotoxic effect of colistin was determined by using the comet assay method to measure the level of DNA damage in freshly isolated human peripheral blood leukocytes (PBLs) incubated with colistin for 1 h at 37°C. Changes in the tail extents of PBLs of about 69.29 ± 0.08 μm were observed at a final colistin concentration of about 550 ng/ml. Besides the synergistic effect, the combination of colistin (1 mg/ml) and nisin (2 mg/ml) permitted us to re-evaluate the toxic effect of colistin on Vero (monkey kidney epithelial) cells.     

昆明市首次检出肠出血性大肠杆菌O157∶H7

为了解昆明市是否存在肠出血性大肠杆菌O15 7∶H7及流行情况 ,了解蔬菜、食物、水等的污染情况 ,2 0 0 1年 10月采集昆明市鸡、鸭、牛 3个养殖场、西式快餐店 2个和市区 9个农贸市场的鸡、鸭、牛粪 ,生牛奶 ,生、熟肉馅 ,生蔬菜 ,生、熟猪、牛肉 ,海产品 ,以及猪、鱼、鸡内脏等 14种共 15 5份样品。按照大肠杆菌O15 7∶H7的常规分离鉴定方法和分子生物学和血清学试验鉴定 ,结果共发现 19株大肠杆菌O15 7∶H7,并且 19株菌株均含有志贺样毒素VT2的致病基因。其中从蔬菜 (33份 )分离到 9株、鸡内脏 (9份 )分离到 3株、生猪肠 (17份 )和卤猪肉 (12份 )各分离到 2株 ,发干菜 (6份 )、鱼内脏 (6份 )、海产品 (6份 )各分离到 1株大肠杆菌O15 7∶H7。食品中检出阳性菌株数之多在全国尚属少见。特别是蔬菜 ,其检出O15 7∶H7大肠杆菌阳性数 ,占总检出数的 4 3 37% ,与国内其它地区有所不同。昆明市存在发生肠出血性大肠杆菌O15 7∶H7感染暴发或流行的潜在危险.     

Rapid PCR detection of enterohemorrhagic Escherichia coli (EHEC) in bovine food products and feces

Although Escherichia coli (E. coli) O157:H7 is a major cause of foodborne illness, other types of E. coli can also cause illness. E. coli that possess the eae gene for attachment and effacing have the potential to cause disease. Many real-time, molecular-based assays have been developed to detect Enterohemorrhagic E. coli (EHEC) including E. coli O157:H7. However, no assay currently exists to detect the eae gene present in E. coli O157:H7 and other EHEC strains with a confirmed positive or negative result in less than 12 h. Raw beef food products (raw ground beef and raw boneless beef) at 25 and 375 g samples and bovine fecal samples at 2 g were inoculated with 10(1), 10(3), 10(4), and 10(5) organisms of E. coli O157:H7 to test the sensitivity of this assay. Fourteen different foodborne bacteria (including E. coli O157:H7) and 19 various E. coli strains, obtained from the United States Department of Agriculture-Agricultural Research Service (USDA-ARS) were tested for specificity. E. coli O157:H7 was detected at the level of 10(1) organisms in both 25 and 375 g samples of raw ground and raw boneless beef products as well as 2 g samples of bovine feces after pre-enrichment and concentration. None of the 14 foodborne bacteria screened for cross-reactivity was detected. All USDA E. coli strains confirmed to contain the eae gene were detected.     

Prevalence, Serogroups, Shiga-toxin Genes and Pulsed Field Gel Electrophoresis Analyses of Escherichia coli Isolated from Bovine Milk

Shiga toxin-producing Escherichia coli (STEC) including non-O157 strains have been linked to outbreaks and sporadic cases of illness worldwide. A total of 647 milk samples were collected at different levels of collection and processing (udder, milking utensils, milk collection centres and receiving dock) within West Coast region of India. The milk samples were screened for the presence of E. coli and further tested for the Shiga-toxin (stx) genes by PCR. The isolates were characterized for their serogroups and XbaI digestion patterns of total DNA separated by pulsed-field gel electrophoresis (PFGE). A total of 77 (11.90 %) isolates were confirmed as having E. coli. The serogroups reported were O4, O60, O112, O56, O159, O120, O2, O83, O88, O95, O141, O21, O25, O80, O140, O97, O24, O166, O146, O51, O169, O147, O103, O18, O100, O15, O69, O43, O7, O3, O45, O124, O110, O84, and O114. Out of the 77 E. coli isolates, 25 (32.46 %) could be classified as Shiga-toxigenic based on PCR results. Of these 11, 3 and 11 isolates were positive for stx1, stx2, and both stx1 and stx2, respectively. PFGE profiles indicated genetic diversity of E. coli strains. Much variation was observed among isolates recovered at different levels of collection. Further research is needed to uncover unique characteristics and resistance of non-O157 STEC strains.     

Comparative study of five different techniques for epidemiological typing of Escherichia coli O157

A set of 47 Austrian human, food, and veterinary Escherichia coli O157:H7 isolates was used to evaluate five different epidemiological typing methods. Ribotyping using an automated microbial characterization system (RiboPrinter^™) was not suitable for detection of epidemiological relatedness. All but one E. coli strain were typeable by phage typing. Random amplified polymorphic DNA-PCR fingerprinting was performed using primer M13 containing the sequence 5′-GAG GGT GGC GGT TCT-3′ and primer 1247 (5′-AAGAGCCCGT-3′). Although both methods recognized only two clusters, both dendrograms grouped most of the EHEC O157 isolates into epidemiologically related subgroups. Pulsed-field gel electrophoresis of XbaI digested total DNA was a valuable subtyping system. We found that major differences can exist between results of multiple subtyping methods. E. coli O157 isolates should not be classified as epidemiologically related or nonrelated on the basis of a single typing method alone.     

Immunomagnetic separation and size-based detection of Escherichia coli O157 at the meniscus of a membrane strip

We developed a facile method for the detection of pathogenic bacteria using gold-coated magnetic nanoparticle clusters (Au@MNCs) and porous nitrocellulose strips. Au@MNCs were synthesized and functionalized with half-fragments of Escherichia coli O157 antibodies. After the nanoparticles were used to capture E. coli O157 in milk and dispersed in a buffer solution, one end of a test strip was dipped into the solution. Due to the size difference between the E. coli–Au@MNC complexes (approximately 1 μm) and free Au@MNCs (approximately 180 nm), only E. coli–Au@MNC complexes accumulated at the meniscus of the test strip and induced a color change. The color intensity of the meniscus was proportional to the E. coli concentration, and the detection limit for E. coli in milk was 10³ CFU mL⁻¹ by the naked eye. The presence of E. coli–Au@MNC complexes at the meniscus was confirmed using a real-time PCR assay. The developed method was highly selective for E. coli when compared with Salmonella typhimurium, Listeria monocytogenes, and Staphylococcus aureus.

E. coli–Au/MNC complexes accumulate at the meniscus of the test strip where the flow velocity reaches a maximum.     

Antibiotic Susceptibility of Enterohemorrhagic Escherichia coli O157:H7 Isolated from an Outbreak in Japan in 1996

The antibiotic susceptibilities of 43 strains of Escherichia coli O157:H7 identified in the summer of 1996 in Japan were investigated. Growth of 90% of O157 strains was inhibited at a concentration of ≤0.5 μg/ml by several agents including fosfomycin with glucose-6-phosphate.     

Consanguineous hemolytic uremic syndrome secondary to Escherichia coli O157:H7 infection treated with aggressive therapeutic plasma exchange

We describe the successful management of an elderly husband and wife with Escherichia coli O157:H7 associated hemolytic uremic syndrome (HUS) treated with aggressive therapeutic plasma exchange (TPE) with replacement with fresh frozen plasma. Following twelve TPEs (three 1.5 volume; nine 1 volume), the husband's platelet count increased from 45 × 10⁹/L to 183 × 10⁹/L. Following ten 1.5 volume TPEs, the wife's platelet count increased from 30 × 10⁹/L to 193 × 10⁹/L. This is the first known occurrence of E. coli O157:H7 associated HUS in an elderly husband and wife successfully treated with aggressive TPE. We conclude that early, aggressive TPE should be considered and may be life‐saving for E coli O157:H7 associated HUS in the elderly. J. Clin. Aperesis 16:155–156, 2001 © 2001 Wiley‐Liss, Inc.     

Comparison between ImmunoCard STAT! and real-time PCR as screening tools for both O157:H7 and non-O157 Shiga toxin-producing Escherichia coli in Southern Alberta,Canada

An increasing number of non-O157 Shiga toxin-producing Escherichia coli (STEC) infections and outbreaks have been reported. In this study, we evaluated the performance of ImmunoCard STAT!^® (Meridian Bioscience, Inc., Cincinnati, OH, USA) as a method to screen stool specimens for STEC (O157 and non-O157). An in-house real-time PCR method was used as the “gold standard”. We also evaluated the prevalence and clinical characteristics of STEC infections in the Alberta South West Zone. From July to November 2011, 819 stool specimens submitted for routine stool culture were tested. With our in-house real-time PCR, 7 O157:H7 and 10 non-O157 STEC isolates were identified for a total of 17 STECs. In comparison, ImmunoCard STAT!^® identified a total of 6, resulting in a sensitivity and specificity of 35% and 99%, respectively (P < 0.05). Because of the low sensitivity, ImmunoCard STAT!^® cannot be recommended as a routine screening test for STEC from enriched stool specimens. The rate of STEC positivity as detected by PCR was 2.08%, of which 0.86% was O157:H7 and 1.22% non-O157 STEC. Five of the 7 cases of STEC O157 infection experienced bloody diarrhea, and 1 developed hemolytic uremic syndrome.     

Sensitive and specific discrimination of pathogenic and nonpathogenic Escherichia coli using Raman spectroscopy—a comparison of two multivariate analysis techniques

The determination of bacterial identity at the strain level is still a complex and time-consuming endeavor. In this study, visible wavelength spontaneous Raman spectroscopy has been used for the discrimination of four closely related Escherichia coli strains: pathogenic enterohemorrhagic E. coli O157:H7 and non-pathogenic E. coli C, E. coli Hfr K-12, and E. coli HF4714. Raman spectra from 600 to 2000 cm⁻¹ were analyzed with two multivariate chemometric techniques, principal component-discriminant function analysis and partial least squares-discriminant analysis, to determine optimal parameters for the discrimination of pathogenic E. coli from the non-pathogenic strains. Spectral preprocessing techniques such as smoothing with windows of various sizes and differentiation were investigated. The sensitivity and specificity of both techniques was in excess of 95%, determined by external testing of the chemometric models. This study suggests that spontaneous Raman spectroscopy with visible wavelength excitation is potentially useful for the rapid identification and classification of clinically-relevant bacteria at the strain level.OCIS codes: (300.6450) Spectroscopy, Raman; (170.0170) Medical optics and biotechnology; (170.1420) Biology; (170.1580) Chemometrics; (170.5660) Raman spectroscopy; (170.1530) Cell analysis     

An Engineered R-Type Pyocin Is a Highly Specific and Sensitive Bactericidal Agent for the Food-Borne Pathogen Escherichia coli O157:H7

Some strains of Pseudomonas aeruginosa produce R-type pyocins, which are high-molecular-weight phage tail-like protein complexes that have bactericidal activity against other Pseudomonas strains. These particles recognize and bind to bacterial surface structures via tail fibers, their primary spectrum determinant. R-type pyocins kill the cell by contracting a sheath-like structure and inserting their hollow core through the cell envelope, resulting in dissipation of the cellular membrane potential. We have retargeted an R-type pyocin to Escherichia coli O157:H7 by fusing a tail spike protein from an O157-specific phage, φV10, to the pyocin tail fiber. The φV10 tail spike protein recognizes and degrades the O157 lipopolysaccharide. This engineered pyocin, termed AVR2-V10, is sensitive and specific, killing 100% of diverse E. coli O157:H7 isolates but no other serotypes tested. AVR2-V10 can kill E. coli O157:H7 on beef surfaces, making it a candidate agent for the elimination of this pathogen from food products. All rare AVR2-V10-resistant mutants isolated and examined have lost the ability to produce the O157 antigen and are expected to have compromised virulence. In addition, E. coli O157:H7 exposed to and killed by AVR2-V10 do not release Shiga toxin, as is often the case with many antibiotics, suggesting potential therapeutic applications. The demonstration that a novel R-type pyocin can be created in the laboratory by fusing a catalytic tail spike from the family Podoviridae to a tail fiber of a member of the family Myoviridae is evidence that the plasticity observed among bacteriophage tail genes can, with modern molecular techniques, be exploited to produce nonnatural, targeted antimicrobial agents.Shiga toxin (Stx)-producing Escherichia coli (STEC) strain O157:H7 is a major cause of food-borne outbreaks of colitis, which can sometimes lead to hemolytic-uremic syndrome (19). Cattle are a major, unaffected reservoir for the pathogen, and outbreaks are often associated with contaminated (adulterated) beef. However, outbreaks are also associated with dairy and other food products, including fresh leafy vegetables and fruit juices, possibly as a result of contamination with water from manure runoff or wildlife intrusions (26). The application of a method that specifically and rapidly kills this food-borne pathogen without compromising food quality might decrease the occurrence of these outbreaks.R-type pyocins are high-molecular-weight protein complexes, produced by some Pseudomonas aeruginosa strains, which specifically kill other strains of the same species (for a review, see reference 17). These complexes resemble the tail structures of bacteriophages of members of the family Myoviridae, particularly the P2-like phages, and consist of a hollow core surrounded by a sheath, at one end of which is a base plate with six tail fibers (Fig. ​(Fig.1).1). R-type pyocins are encoded in the bacterial genome on a single locus of 16 open reading frames, of which less than 10 probably encode structural proteins of the pyocin (18, 25). Pyocin production is induced by the SOS response, which yields about 100 to 200 particles per cell (15). The cells then lyse by a process similar to phage-mediated lysis, releasing the pyocin particles into the surroundings. The particles kill the target bacteria by first binding to a cell surface structure via their tail fibers, followed by contraction of the sheath and insertion of their hollow core through the cell envelope. The resulting channel leads to the depolarization of the cytoplasmic membrane and cell death (29). A single pyocin particle can kill a cell (6, 28, 29). In many respects, R-type pyocins can be considered defective prophages, and it is highly likely that they share a common ancestry with related phages. However, pyocins do not contain a genome and they do not infect target cells; replication occurs only as a genetic component of the producer strain chromosome (9, 10, 24). Cells carrying an R-type pyocin gene cluster are genetically resistant to their own pyocin. However, cells that express pyocin must lyse to release the particles. Thus, it seems that the selective advantage is to protect kin strains from nonkin strains, a form of altruism or kin selection.Open in a separate windowFIG. 1.Cartoon representation of the R-type pyocin structure. R2 is the wild type and kills certain P. aeruginosa strains. AVR2-P2 has been retargeted to kill some rough strains of E. coli by using the tail fiber of phage P2. AVR2-V10 is the engineered pyocin characterized in this study, in which the phage φV10 tail spike was fused to the pyocin tail fiber and is specific for E. coli strains that produce the O157 antigen.Five naturally occurring R-type pyocins, termed R1 to R5, each with a unique bactericidal spectrum, have been described (7). Each pyocin spectrum is related to that of the others, such that R4 encompasses the spectrum of R3, R2 encompasses the spectra of R3 and R4, and R5 has the broadest spectrum and includes the spectra of all the other pyocins, plus those of some additional strains. The spectrum of R1 is unrelated to that of R2, R3, or R4; but it is still a subset of that of R5. This places the R5 receptor at the root of a spectrum tree with two branches, one consisting of the R1 receptor and the other consisting of the receptors for R2, R4, and R3. The well-studied strain P. aeruginosa PAO1 produces R2 pyocin and is the platform R-type pyocin used in this study.Because of their specific and potent bactericidal activities, R-type pyocins are candidate antibacterial agents. Although a few studies have shown that parenterally administered R-type pyocins can rescue mice from lethal infections (4, 16, 22), interest has been limited. One of the concerns about the use of R-type pyocins as antibacterial agents is their limited spectrum, which is determined primarily by the specificity of the tail fiber binding to bacterial surface structures or receptors. While it is unlikely that an R-type pyocin could be engineered to have a very broad spectrum, we have shown that it is possible to retarget their spectra to other bacterial species and genera by making fusions between the pyocin tail fibers and the tail fibers of related bacteriophages (32). One such pyocin, AVR2-P2 was made by fusing the C-terminal portion of the phage P2 tail fiber to the N-terminal portion of the R2 tail fiber, thereby retargeting the bactericidal activity toward rough E. coli strains that serve as the host for P2, including E. coli strain C. Unfortunately, AVR2-P2 does not kill any E. coli O157:H7 isolates. Here we describe some of the properties and applications of a new O157-specific R-type pyocin created by fusing to the R2 tail fiber an O157-specific tail spike protein from an unrelated phage from the family Podoviridae.