Colicin Production Research Articles

Assessment of virulence potential of <i>Escherichia coli</i> isolated from clinical and non-clinical sources in port Harcourt, Nigeria.

Escherichia coli commonly occurs as a normal flora found in a variety of gastrointestinal tracts. However, certain strains acquire virulence characteristics. Potentially pathogenic species have also been isolated in non-clinical environments.This often raises concerns on the role these environments play in transmission. This study aimed to assess the occurrence of virulence traits in Escherichia coli isolated from non-clinical and clinical sources in Port Harcourt, Nigeria. Two hundred samples from clinical (urine and stool) and non-clinical (soil and poultry) sources were analyzed. 8 isolation, purification and identification of E. coli isolates were performed using standard bacteriological and biochemical tests. Out of the 200 samples collected, 98 isolates (49%) were confirmed as E. coli. Confirmed E. coli isolates were then tested for virulence using standard phenotypic tests (protease, colicin, congo red, hemolysin, haemagglutination, adhesin and mannose resistant/sensitive haemagglutination). E. coli strains from clinical samples displayed a number of virulence traits ranging frombiofilm producing capabilities (53%), congo red binding propensity (24%), colicin (34%), protease (15%) and hemolysin production (4%) compared to their non-clinical counterpart. All samples were negative for haemagglutination. The total virulence prevalence of E. coli was 81%. Clinical samples possessed more virulence traits (71%) than samples from non-clinical sources (29%), with urine having a higher occurrence among the clinical samples (36%). There is need for an in-depth study on virulence attributes of E. coli and ways to control these factors to minimize the rate of spread and the degree of infections.

Analysis of In-Patient Evolution of Escherichia coli Reveals Potential Links to Relapse of Bone and Joint Infections.

Bone and joint infections (BJIs) are difficult to treat and affect a growing number of patients, in which relapses are observed in 10-20% of case. These relapses, which call for prolonged antibiotic treatment and increase resistance emergence risk, may originate from ill-understood adaptation of the pathogen to the host. Here, we investigated 3 pairs of Escherichia coli strains from BJI cases and their relapses to unravel adaptations within patients. Whole-genome comparison presented evidence for positive selection and phenotypic characterization showed that biofilm formation remained unchanged, contrary to what is usually described in such cases. Although virulence was not modified, we identified the loss of 2 virulence factors contributing to immune system evasion in one of the studied strains. Other strategies, including global growth optimization and colicin production, likely allowed the strains to outcompete competitors. This work highlights the variety of strategies allowing in-patient adaptation in BJIs.

Colicin production, antibacterial effect and molecular sequencing of Escherichia coli isolated form different sources

Bacteria especially Escherichia coli (E. coli) possess several mechanisms enabling them to respond to changing environment and to out compete other bacteria. E. coli strains are known to produce bacteriocins, one of which is colicin. Colicins are effective against closely related enterobacteriaceae and are considered viable alternatives to antibiotics. In this study Twelve (12) E. coli strains were isolated from food, water soil and anal swab. They were characterized, confirmed and identified as E. coli. The isolates were subjected to stress condition to stimulate colicin production. The colicin produced was tested against three indicator organisms namely Salmonella Sp, Klebsiella Sp and Staphylococcus aureus. Comparative antibiotic susceptibility test against standard antibiotics and the colicin produced was carried out to ascertain the efficacy of the colicin. E. coli isolates were also subjected to antibiotic sensitivity test to determine the resistance profile of the colicin producing strains of E. coli. Plasmid amplification to identify colicin plasmid was also carried out to determine the relationship between colicin production and resistance to antibiotics of colicinogenic E. coli. Result obtained showed that six out of the twelve strains of E. coli isolated from water produced colicin that showed high antibacterial activity against the indicator organisms. The comparative antibiotic resistance profile revealed that the colicin had better antibacterial activities than some of the antibiotics used. Furthermore the colicinogenic E. coli also showed resistance to some of antibiotics used. The plasmid amplification showed the colicinogenic E. coli possess high molecular weight (4kb) colicin plasmid while the gene sequencing revealed the presence of ColF and ColR plasmids. The result of this work underscores the fact that the use of colicin as an alternative to standard antibiotics can be further explored, especially in the search for alternatives to antibiotics considering the high level of bacteria resistance to standard antibiotics.

Colicins of Escherichia coli Lead to Resistance against the Diarrhea-Causing Pathogen Enterotoxigenic E. coli in Pigs.

ABSTRACTGut microbes can affect host adaptation to various environment conditions. Escherichia coli is a common gut species, including pathogenic strains and nonpathogenic strains. This study was conducted to investigate the effects of different E. coli strains in the gut on the health of pigs. In this study, the complete genomes of two E. coli strains isolated from pigs were sequenced. The whole genomes of Y18J and the enterotoxigenic E. coli strain W25K were compared to determine their roles in pig adaptation to disease. Y18J was isolated from feces of healthy piglets and showed strong antimicrobial activity against W25K in vitro. Gene knockout experiments and complementation analysis followed by modeling the microbe-microbe interactions demonstrated that the antagonistic mechanism of Y18J against W25K relied on the bacteriocins colicin B and colicin M. Compared to W25K, Y18J is devoid of exotoxin-coding genes and has more secondary-metabolite-biosynthetic gene clusters. W25K carries more genes involved in genome replication, in accordance with a shorter cell cycle observed during a growth experiment. The analysis of gut metagenomes in different pig breeds showed that colicins B and M were enriched in Laiwu pigs, a Chinese local breed, but were scarce in boars and Duroc pigs.IMPORTANCE This study revealed the heterogeneity of E. coli strains from pigs, including two strains studied by both in silico and wet experiments in detail and 14 strains studied by bioinformatics analysis. E. coli Y18J may improve the adaptability of pigs toward disease resistance through the production of colicins B and M. Our findings could shed light on the pathogenic and harmless roles of E. coli in modern animal husbandry, leading to a better understanding of intestinal-microbe–pathogen interactions in the course of evolution.

Bacteriocin Production by Escherichia coli during Biofilm Development.

Escherichia coli is a highly versatile bacterium ranging from commensal to intestinal pathogen, and is an important foodborne pathogen. E. coli species are able to prosper in multispecies biofilms and secrete bacteriocins that are only toxic to species/strains closely related to the producer strain. In this study, 20 distinct E. coli strains were characterized for several properties that confer competitive advantages against closer microorganisms by assessing the biofilm-forming capacity, the production of antimicrobial molecules, and the production of siderophores. Furthermore, primer sets for E. coli bacteriocins–colicins were designed and genes were amplified, allowing us to observe that colicins were widely distributed among the pathogenic E. coli strains. Their production in the planktonic phase or single-species biofilms was uncommon. Only two E. coli strains out of nine biofilm-forming were able to inhibit the growth of other E. coli strains. There is evidence of larger amounts of colicin being produced in the late stages of E. coli biofilm growth. The decrease in bacterial biomass after 12 h of incubation indicates active type I colicin production, whose release normally requires E. coli cell lysis. Almost all E. coli strains were siderophore-producing, which may be related to the resistance to colicin as these two molecules may use the same transporter system. Moreover, E. coli CECT 504 was able to coexist with Salmonella enterica in dual-species biofilms, but Shigella dysenteriae was selectively excluded, correlating with high expression levels of colicin (E, B, and M) genes observed by real-time PCR.

Population structure and uropathogenic potential of extended-spectrum cephalosporin-resistant Escherichia coli from retail chicken meat

BackgroundFood-producing animals and their products are considered a source for human acquisition of antimicrobial resistant (AMR) bacteria, and poultry are suggested to be a reservoir for Escherichia coli resistant to extended-spectrum cephalosporins (ESC), a group of antimicrobials used to treat community-onset urinary tract infections in humans. However, the zoonotic potential of ESC-resistant E. coli from poultry and their role as extraintestinal pathogens, including uropathogens, have been debated. The aim of this study was to characterize ESC-resistant E. coli isolated from domestically produced retail chicken meat regarding their population genetic structure, the presence of virulence-associated geno- and phenotypes as well as their carriage of antimicrobial resistance genes, in order to evaluate their uropathogenic potential.ResultsA collection of 141 ESC-resistant E. coli isolates from retail chicken in the Norwegian monitoring program for antimicrobial resistance in bacteria from food, feed and animals (NORM-VET) in 2012, 2014 and 2016 (n = 141) were whole genome sequenced and analyzed. The 141 isolates, all containing the beta-lactamase encoding gene blaCMY-2, were genetically diverse, grouping into 19 different sequence types (STs), and temporal variations in the distribution of STs were observed. Generally, a limited number of virulence-associated genes were identified in the isolates. Eighteen isolates were selected for further analysis of uropathogen-associated virulence traits including expression of type 1 fimbriae, motility, ability to form biofilm, serum resistance, adhesion- and invasion of eukaryotic cells and colicin production. These isolates demonstrated a high diversity in virulence-associated phenotypes suggesting that the uropathogenicity of ESC-resistant E. coli from chicken meat is correspondingly highly variable. For some isolates, there was a discrepancy between the presence of virulence-associated genes and corresponding expected phenotype, suggesting that mutations or regulatory mechanisms could influence their pathogenic potential.ConclusionOur results indicate that the ESC-resistant E. coli from chicken meat have a low uropathogenic potential to humans, which is important knowledge for improvement of future risk assessments of AMR in the food chains.

Non-lactose-fermenting uropathogenic Escherichia coli from dogs: virulence profile characterization.

Non-lactose-fermenting Escherichia coli (NLFEC) has a few descriptive studies restricted to human infections. In the present study, isolates of NLFEC obtained from urine samples of dogs with hyperadrenocorticism were characterized regarding their virulence ability, biofilm formation capacity and antimicrobial susceptibility profile. Escherichia coli lactose-fermenting strains from urinary infection in dogs with the same conditions were analysed to provide comparisons. The non-lactose-fermenting E. coli strains were classified as belonging to clade I E. coli, whereas the lactose-fermenting strains were classified in phylogroup B2. All strains presented virulence markers to adhesion, iron acquisition, toxins, colicin and cytotoxin production, and biofilm regulation. Components of the extracellular matrix in addition to the in vitro biofilm formation ability were observed in the strains. Multidrug resistance (MDR) profiles were observed by in vitro susceptibility tests to all NLFEC strains. In summary, non-lactose-fermenting uropathogenic E. coli from dogs behaves similar to lactose-fermenting E. coli, exhibiting MDR profile, and pathogenic potential of promote animal infections.

Antimicrobial Activity of Non-bond Colicin on Candida albicans Biofilm

Two hundred fifty mid-stream urine specimens were collected from Baqubah Teaching Hospital and Al-Batool Teaching Hospital from patients with urinary tract infections (UTI). Of these investigated urine specimens, 66 (26.4%) specimens showed positive growth culture of Gram-negative bacteria. From these, Escherichia coli was the most prevalent bacteria of the examined culture (41, 62.12%). Additionally, the cup assay was used to determine colicin producers while the most efficient colicin producers were estimated by the formation of larger inhibition zone. Approximately half of the investigated E. coli isolates (20, 49 %) was colicin producers. Colicins was extracted after induction by mitomycin-C showed a concentration of 3020 μg/ml, as estimated utilizing the Lowry method, while its activity was 80 U/ml. Our study results showed that colicin had significant antibiofilm activity (P≤ 0.05) against Candida albicans and the effect seemed to be concentration dependent. . However, the values of biofilm inhibition varied depending on the different tested isolates. The biofilm of isolate 5 showed the most significant inhibition (P≤ 0.05) by colicin with a value of 46%, while isolate 3 was less affected with an inhibition rate of 19% at the concentration of 2500 μl/ml.

Deciphering the Role of Colicins during Colonization of the Mammalian Gut by Commensal E. coli.

Colicins are specific and potent toxins produced by Enterobacteriaceae that result in the rapid elimination of sensitive cells. Colicin production is commonly found throughout microbial populations, suggesting its potential importance for bacterial survival in complex microbial environments. Nonetheless, as colicin biology has been predominately studied using synthetic models, it remains unclear how colicin production contributes to survival and fitness of a colicin-producing commensal strain in a natural environment. To address this gap, we took advantage of MP1, an E. coli strain that harbors a colicinogenic plasmid and is a natural colonizer of the murine gut. Using this model, we validated that MP1 is competent for colicin production and then directly interrogated the importance of colicin production and immunity for MP1 survival in the murine gut. We showed that colicin production is dispensable for sustained colonization in the unperturbed gut. A strain lacking colicin production or immunity shows minimal fitness defects and can resist displacement by colicin producers. This report extends our understanding of the role that colicin production may play for E. coli during gut colonization and suggests that colicin production is not essential for a commensal to persist in its physiologic niche in the absence of exogenous challenges.

Bacteriocin production by mucosal bacteria in current and previous colorectal neoplasia

BackgroundOptimal therapy for colorectal carcinoma (CRC), a frequently diagnosed malignancy, does not exist. Some of colicins and microcins, ribosomally synthesized peptides by gramnegative bacteria, have shown significant biological activity specifically against different cancer cells in vitro and in vivo conditions. The aim of this prospective study was to evaluate natural colicin and microcin production by large intestinal mucosal bacteria in each stage of colorectal neoplasia and in those with a history of colorectal neoplasia.MethodsA total of 21 patients with non-advanced adenoma (non-a-A; 16/21 with current and 5/21 with history of non-a-A), 20 patients with advanced colorectal adenoma (a-A; 11/20 with current and 9/20 with history of a-A), 22 individuals with CRC (9/22 with current and 13/22 with history of CRC) and 20 controls were enrolled. Mucosal biopsies from the caecum, transverse colon and the rectum were taken during colonoscopy in each individual. Microbiological culture followed. Production of colicins and microcins was evaluated by PCR methods.ResultsA total of 239 mucosal biopsies were taken. Production of colicins and microcins was significantly more frequent in individuals with non-a-A, a-A and CRC compared to controls. No significant difference in colicin and microcin production was found between patients with current and previous non-a-A, a-A and CRC. Significantly more frequent production of colicins was observed in men compared to women at the stage of colorectal carcinoma. A later onset of increased production of microcins during the adenoma-carcinoma sequence has been observed in males compared to females.ConclusionsStrains isolated from large intestinal mucosa in patients with colorectal neoplasia produce colicins and microcins more frequently compared to controls. Bacteriocin production does not differ between patients with current and previous colorectal neoplasia. Fundamental differences in bacteriocin production have been confirmed between males and females.

The Effects of Colicin Production Rates on Allelopathic Interactions in Escherichia coli Populations.

Allelopathic interactions mediated by bacteriocins production serve microorganisms in the never-ending battle for resources and living space. Competition between the bacteriocin producer and sensitive populations results in the exclusion of one or the other depending on their initial frequencies, the structure of their habitat, their community density and their nutrient availability. These interactions were extensively studied in bacteriocins produced by Escherichia coli, the colicins. In spatially structured environments where interactions are local, colicin production has been shown to be advantageous to the producer population, allowing them to compete even when initially rare. Yet, in a well-mixed, unstructured environment where interactions are global, rare producer populations cannot invade a common sensitive population. Here we are showing, through an experimental model, that colicin-producers can outcompete sensitive and producer populations when the colicin production rates are enhanced. In fact, colicin production rates were proportional to the producer competitive fitness and their overall success in out-competing opponents when invading at very low initial frequencies. This ability of rare populations to invade established communities maintains diversity and allows the dispersal of beneficial traits.

Production of recombinant colicin M in Nicotiana tabacum plants and its antimicrobial activity

Antibiotic-resistant microorganisms causing a life-threatening infection pose a serious challenge for modern science. The rapidly growing number of incidents for which the use of standard antibiotics is ineffective forces us to develop new alternative methods of killing microorganisms. Antimicrobial proteins and peptides (AMPs) can be promising candidates to solve this problem. Colicin-M is one of the representatives of this group and is naturally produced by Escherichia coli acting on other closely related bacterial strains by disrupting their outer cell membrane. This bacteriocin has huge potential as a potent antimicrobial agent, especially, since it was recognized by the FDA as safe. In this work, we present the expression of colicin M in model transgenic Nicotiana tabacum L. plants. We demonstrate that purified colicin retains its antibacterial activity against the control Escherichia coli strains and clinical isolates of Escherichia coli and Klebsiella pneumoniae. Our results also show that plant-derived ColM is not toxic for L929 and HeLa cell line, which allows us to suppose that plant-based expression could be an alternative production method of such important proteins.

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.

Optimizing Cell-Free Protein Synthesis for Increased Yield and Activity of Colicins.

Colicins are antimicrobial proteins produced by Escherichia coli that hold great promise as viable complements or alternatives to antibiotics. Cell-free protein synthesis (CFPS) is a useful production platform for toxic proteins because it eliminates the need to maintain cell viability, a common problem in cell-based production. Previously, we demonstrated that colicins produced by CFPS based on crude Escherichia coli lysates are effective in eradicating antibiotic-tolerant bacteria known as persisters. However, we also found that some colicins have poor solubility or low cell-killing activity. In this study, we improved the solubility of colicin M from 16% to nearly 100% by producing it in chaperone-enriched E. coli extracts, resulting in enhanced cell-killing activity. We also improved the cytotoxicity of colicin E3 by adding or co-expressing the E3 immunity protein during the CFPS reaction, suggesting that the E3 immunity protein enhances colicin E3 activity in addition to protecting the host strain. Finally, we confirmed our previous finding that active colicins can be rapidly synthesized by observing colicin E1 production over time in CFPS. Within three hours of CFPS incubation, colicin E1 reached its maximum production yield and maintained high cytotoxicity during longer incubations up to 20 h. Taken together, our findings indicate that colicin production can be easily optimized for improved solubility and activity using the CFPS platform.

Porcine pathogenic Escherichia coli strains differ from human fecal strains in occurrence of bacteriocin types

Enterotoxigenic and Shiga-toxigenic Escherichia coli (i.e., ETEC and STEC) are important causative agents of human and animal diseases. In humans, infections range from mild diarrhea to severe life-threating conditions, while infections of piglets result in lower weight gain and higher pig mortality with the accompanying significant economic losses.In this study, frequencies of four phylogenetic groups, fourteen virulence- and thirty bacteriocin determinants were analyzed in a set of 443 fecal E. coli isolates from diseased pigs and compared to a previously characterized set of 1283 human fecal E. coli isolates collected in the same geographical region. In addition, these characteristics were compared among ETEC, STEC, and non-toxigenic porcine E. coli isolates.Phylogenetic group A was prevalent among porcine pathogenic E. coli isolates, whereas the frequency of phylogroup B2, adhesion/invasion (fimA, pap, sfa, afaI, ial, ipaH, and pCVD432) and iron acquisition (aer and iucC) determinants were less frequent compared to human fecal isolates.Additionally, porcine isolates differed from human isolates relative to the spectrum of produced bacteriocins. While human fecal isolates encoded colicins and microcins with a similar prevalence, porcine pathogenic E. coli isolates produced predominantly colicins (94% of isolates); especially colicins B (42.6%), M (40.1%), and Ib (34.0%), which are encoded on large conjugative plasmids. The observed high prevalence of these colicin determinants suggests the importance of large colicinogenic plasmids and/or the importance of colicin production in intestinal inflammatory conditions.

Antibacterial Activity of Synergistic Effect of colicin and Gold Nanoparticles against Klebsiella pneumonia

Fifty of urine samples were collected from patients with urinary tract infection (UTI). The samples were collected from AL-Yarmuk hospital in Baghdad. All of the isolates were diagnosed using biochemical test and vitek. The result showed that 30 (60%) isolates identified as E.coli from 50 urine samples. The colicinogenic isolates were determined using cup assay methods. The results showed that 10 out of 30 isolates (33.3%) were detect as colicin producers from 30 isolate identified as E.coli depending on the clear zone that observed against the sensitive isolate. Colicin was extracted from the efficient isolate. Colicin activity (320 U/ml) was determined by well assay method. The protein concentration (520 μg/ml) estimated by using Bradford assay. The watery extract of Chilli papers (Capsicum baccatum) was extracted and used it as reducing and capping agent for gold nanoparticles synthesis. The characterization of the gold nanoparticles was done by UV-Visible Spectrophotometer, Transmission Electron Microscope (TEM), and resulting spherical nanoparticles with diameter ranging between (35–70 nm). The antibacterial activity of colicin alone and gold nanoparticles alone and combination of colicin with gold nanoparticles against ten isolates Klebsiella pneumonia isolated from urine samples, using tube method.

Rapid production and characterization of antimicrobial colicins using Escherichia coli-based cell-free protein synthesis.

Colicins are antimicrobial proteins produced by Escherichia coli, which, upon secretion from the host, kill non-host E. coli strains by forming pores in the inner membrane and degrading internal cellular components such as DNA and RNA. Due to their unique cell-killing activities, colicins are considered viable alternatives to conventional antibiotics. Recombinant production of colicins requires co-production of immunity proteins to protect host cells; otherwise, the colicins are lethal to the host. In this study, we used cell-free protein synthesis (CFPS) to produce active colicins without the need for protein purification and co-production of immunity proteins. Cell-free synthesized colicins were active in killing model E. coli cells with different modes of cytotoxicity. Pore-forming colicins E1 and nuclease colicin E2 killed actively growing cells in a nutrient-rich medium, but the cytotoxicity of colicin Ia was low compared to E1 and E2. Moreover, colicin E1 effectively killed cells in a nutrient-free solution, while the activity of E2 was decreased compared to nutrient-rich conditions. Both colicins E1 and E2 decreased the level of persister cells (metabolically dormant cell populations that are insensitive to antibiotics) by up to six orders of magnitude compared to that of the rifampin pretreated persister cells. This study finds that colicins can eradicate non-growing cells including persisters, and that CFPS is a promising platform for rapid production and characterization of toxic proteins.

What Does Not Kill You Makes You Stronger

Colicin production is an extreme form of labor division; cells lyse after making the toxin! Stochastic phenotype switching allows producers to outcompete sensitive strains since colicin release frees up vacancy. If patch dynamics does not kill you, it stimulates adaptation to a dynamic habitat landscape which selects for rapid dispersal.

Bet-hedging in bacteriocin producing Escherichia coli populations: the single cell perspective

Production of public goods in biological systems is often a collaborative effort that may be detrimental to the producers. It is therefore sustainable only if a small fraction of the population shoulders the cost while the majority reap the benefits. We modelled this scenario using Escherichia coli populations producing colicins, an antibiotic that kills producer cells’ close relatives. Colicin expression is a costly trait, and it has been proposed that only a small fraction of the population actively expresses the antibiotic. Colicinogenic populations were followed at the single-cell level using time-lapse microscopy, and showed two distinct, albeit dynamic, subpopulations: the majority silenced colicin expression, while a small fraction of elongated, slow-growing cells formed colicin-expressing hotspots, placing a significant burden on expressers. Moreover, monitoring lineages of individual colicinogenic cells showed stochastic switching between expressers and non-expressers. Hence, colicin expressers may be engaged in risk-reducing strategies—or bet-hedging—as they balance the cost of colicin production with the need to repel competitors. To test the bet-hedging strategy in colicin-mediated interactions, competitions between colicin-sensitive and producer cells were simulated using a numerical model, demonstrating a finely balanced expression range that is essential to sustaining the colicinogenic population.

A broad-host range coliphage against a clinically isolated E. coli O157: isolation and characterization

Isolation of colicinogenic E. coli, optimization of colicin production and transformation of Col plasmidArumugam Suresh, Manikkam Radhakrishnan1, Gopal Suresh, Nainangu Prasannabalaji, Sivaraj Anbarasu, Jerrine Joseph