The dynamics of colicin E9 release from Escherichia coli in native conditions.

Colicin (Col) plasmid contains colicin encoding genes arranged in an operon controlled by an SOS inducible promoter. Therefore, any external stresses to the host cell can induce the expression of the downstream genes in the Col operon, including a lysis gene. The lysis protein is involved in the extracellular release of colicin through lysis of the producer cells, which causes a decline in culture turbidity. However, it is not yet known that E. coli cells with the native pColE9-J plasmid hold the same level of cell death at the population level following a set of induced conditions. In this study, using a mitomycin C sensitivity assay along with a live dead staining method of detection, we showed that the native pColE9-J plasmid, which unusually carries an extended Col operon (ColE9) containing two lysis genes, did not confer a rapid decline in the culture turbidity following induction with mitomycin C. Interestingly a subset of the cells suffered perturbation of their outer membrane, which was not observed from single lysis mutant (∆celE or ∆celI) cells. This observed heterogeneity in the colicin E9 release leading to differential outer membrane perforation may bring a competitive advantage to these cells in a mixed population.

[Characterization of a Taq DNA polymerase fused with a DNA binding domain of Escherichia coli colicin].

Taq DNA polymerase, which was discovered from a thermophilic aquatic bacterium (Thermus aquaticus), is an enzyme that possesses both reverse transcriptase activity and DNA polymerase activity. Colicin E (CE) protein belongs to a class of Escherichia coli toxins that utilize the vitamin receptor BtuB as a transmembrane receptor. Among these toxins, CE2, CE7, CE8, and CE9 are classified as non-specific DNase-type colicins. Taq DNA polymerase consists of a 5'→3' exonuclease domain, a 3'→5' exonuclease domain, and a polymerase domain. Taq DNA polymerase lacking the 5'→3' exonuclease domain (ΔTaq) exhibits higher yield but lower processivity, making it unable to amplify long fragments. In this study, we aimed to enhance the processivity of ΔTaq. To this end, we fused dCE with ΔTaq and observed a significant improvement in the processivity of the resulting dCE-ΔTaq compared to Taq DNA polymerase and dCE-Taq. Furthermore, its reverse transcriptase activity was also higher than that of ΔTaq. The most notable improvement was observed in dCE8-ΔTaq, which not only successfully amplified 8 kb DNA fragments within 1 minute, but also yielded higher results compared to other mutants. In summary, this study successfully enhanced the PCR efficiency and reverse transcription activity of Taq DNA polymerase by fusing ΔTaq DNA polymerase with dCE. This approach provides a novel approach for modifying Taq DNA polymerase and holds potential for the development of improved variants of Taq DNA polymerase.

Colicins and T6SS-based competition systems enhance enterotoxigenic E. coli (ETEC) competitiveness.

Diarrheal diseases are still a significant problem for humankind, causing approximately half a million deaths annually. To cause diarrhea, enteric bacterial pathogens must first colonize the gut, which is a niche occupied by the normal bacterial microbiota. Therefore, the ability of pathogenic bacteria to inhibit the growth of other bacteria can facilitate the colonization process. Although enterotoxigenic Escherichia coli (ETEC) is one of the major causative agents of diarrheal diseases, little is known about the competition systems found in and used by ETEC and how they contribute to the ability of ETEC to colonize a host. Here, we collected a set of 94 fully assembled ETEC genomes by performing whole-genome sequencing and mining the NCBI RefSeq database. Using this set, we performed a comprehensive search for delivered bacterial toxins and investigated how these toxins contribute to ETEC competitiveness in vitro. We found that type VI secretion systems (T6SS) were widespread among ETEC (n = 47). In addition, several closely related ETEC strains were found to encode Colicin Ia and T6SS (n = 8). These toxins provide ETEC competitive advantages during in vitro competition against other E. coli, suggesting that the role of T6SS as well as colicins in ETEC biology has until now been underappreciated.

Accumulation of colicin M protein and its biological activity in transgenic lettuce and mizuna plants.

Food-borne illnesses caused by pathogenic Escherichia coli strains, especially enterohaemorrhagic E. coli (EHEC), are a serious public health problem, as debilitating disease and even death from such food poisonings have been repeatedly reported. Colicin M (ColM), a non-antibiotic antimicrobial protein produced by some strains of E. coli, has shown promising activity in controlling multiple enteropathogenic strains of E. coli and related pathogens. As contaminated green leafy vegetables are a frequent source of pathogenic E. coli infections, we genetically modified (GM) two edible crops, lettuce (Lactuca sativa L.) and mizuna (Brassica rapa subsp. nipposinica var. laciniata), to stably express the ColM gene and assessed the antibacterial activity of tissue extracts from these plants against selected E. coli strains in vitro. Transgenic plants of these species were developed using Agrobacterium-mediated transformation with a vector containing the ColM-coding gene (cma) under the control of the 35S promoter. Western blot analysis of recombinant ColM protein was performed in selected transgenic plants to confirm cma gene expression and quantify ColM accumulation. Extracts of transgenic plants expressing ColM showed significant activity against two major strains of EHEC (O157:H7 and O104:H4) as well as E. coli strains resistant to beta-lactam- and carbapenem-class antibiotics. Importantly, the antibacterial activity persisted in several subsequent generations of transgenic lettuce and mizuna plants that stably expressed the ColM gene. In addition, our results also show that the antibacterial activity of dried (up to 40°C) biomass of transgenic plants remained stable without a decrease for at least three months.

Molecular and Genetic Characterization of Colicinogenic Escherichia coli Strains Active against Shiga Toxin-Producing Escherichia coli O157:H7.

The objective of this work was to molecularly and genotypically characterize and test the inhibitory activity of six colicinogenic Escherichia coli strains (ColEc) and their partially purified colicins against STEC O157:H7 isolated from clinical human cases. Inhibition tests demonstrated the activity of these strains and their colicins against STEC O157:H7. By PCR it was possible to detect colicins Ia, E7, and B and microcins M, H47, C7, and J25. By genome sequencing of two selected ColEc strains, it was possible to identify additional colicins such as E1 and Ib. No genes coding for stx1 and stx2 were detected after analyzing the genome sequence. The inhibitory activity of ColEc against STEC O157:H7 used as an indicator showed that colicins are potent growth inhibitors of E. coli O157:H7, being a potential alternative to reduce the presence of pathogens of public health relevance.

Hydrolytic Mechanism of a Metalloenzyme Is Modified by the Nature of the Coordinated Metal Ion.

The nuclease domain of colicin E7 cleaves double-strand DNA non-specifically. Zn2+ ion was shown to be coordinated by the purified NColE7 as its native metal ion. Here, we study the structural and catalytic aspects of the interaction with Ni2+, Cu2+ and Cd2+ non-endogenous metal ions and the consequences of their competition with Zn2+ ions, using circular dichroism spectroscopy and intact protein mass spectrometry. An R447G mutant exerting decreased activity allowed for the detection of nuclease action against pUC119 plasmid DNA via agarose gel electrophoresis in the presence of comparable metal ion concentrations. It was shown that all of the added metal ions could bind to the apoprotein, resulting in a minor secondary structure change, but drastically shifting the charge distribution of the protein. Zn2+ ions could not be replaced by Ni2+, Cu2+ and Cd2+. The nuclease activity of the Ni2+-bound enzyme was extremely high in comparison with the other metal-bound forms, and could not be inhibited by the excess of Ni2+ ions. At the same time, this activity was significantly decreased in the presence of equivalent Zn2+, independent of the order of addition of each component of the mixture. We concluded that the Ni2+ ions promoted the DNA cleavage of the enzyme through a more efficient mechanism than the native Zn2+ ions, as they directly generate the nucleophilic OH- ion.

Comprehensive Analysis of Bacteriocins Produced by the Hypermucoviscous Klebsiella pneumoniae Species Complex.

Klebsiella pneumoniae produces several kinds of bacteriocins that have antimicrobial effects against closely related species, but few studies have comprehensively reported bacteriocin distribution among the Klebsiella population. In this study, we identified bacteriocin genes in 180 K. pneumoniae species complex genomes, including 170 hypermucoviscous isolates, and investigated the antibacterial activity against 50 strains, including antimicrobial-resistant organisms, belonging to multiple species, namely, Klebsiella spp., Escherichia coli, Pseudomonas spp., Acinetobacter spp., Enterobacter cloacae, Stenotrophomonas maltophilia, Chryseobacterium indologenes, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus mutans. Our study determined that 32.8% (59/180) of isolates carried at least one bacteriocin type. Different types of bacteriocin were usually present in different specific sequence types (STs); meanwhile, bacteriocins were not detected in certain STs. Microcin E492 was the most prevalent bacteriocin (14.4%), mostly in ST23 isolates, and displayed a wide spectrum of activity, including against Klebsiella spp., E. coli, Pseudomonas spp., and Acinetobacter spp. Cloacin-like bacteriocin was detected in 7.2% of strains, all of which were non-ST23 isolates, and exhibited inhibitory activity against closely related species, mainly Klebsiella spp. Klebicin B-like bacteriocin was detected at a rate of 9.4%, although 82.4% of these strains carried a disrupted bacteriocin gene, and an inhibitory effect could not be observed from the intact-gene-carrying isolates. Other bacteriocins, such as microcin S-like, microcin B17, and klebicin C-like, were detected at lower rates and had limited inhibitory activity. Our findings suggested that Klebsiella strains that carry different bacteriocin types may affect the composition of the surrounding bacterial community. IMPORTANCE Klebsiella pneumoniae is a Gram-negative commensal bacterium that asymptomatically colonizes human mucosal membranes, such as the intestinal tract, but it is also a leading cause of health care- and community-associated infections. Additionally, multidrug-resistant K. pneumoniae has been continuously evolving, which significantly challenges the available chemotherapeutic treatment for its infections. K. pneumoniae produces several kinds of antimicrobial peptides known as bacteriocins, which have antibacterial activity against closely related species. This work was the first comprehensive report of bacteriocin distribution among the hypermucoviscous K. pneumoniae species complex population and the inhibitory activity of each bacteriocin type against various species, including multidrug-resistant strains. Our findings provide a foundation for future studies on the K. pneumoniae species complex, including studies on the competition within the microflora and the potential applications of bacteriocins in treating multidrug-resistant bacteria.

Pairing Colicins B and E5 with Bdellovibrio bacteriovorus To Eradicate Carbapenem- and Colistin-Resistant Strains of Escherichia coli.

While diverse antibacterials are available in nature, each possesses their own strengths and limitations. One such antibacterial is colicins, proteinaceous toxins that are produced by strains of E. coli to subvert the growth or viability of other E. coli strains. Similarly, predatory bacteria, of which Bdellovibrio bacteriovorus is well-known, are microbes that actively predate on and consume other Gram-negative bacterial strains. While they are all quite active as antibacterials, they also present some limitations: rapid resistance development to colicins while predation does not completely kill their prey. Within this study, therefore, we evaluated the impact of two different colicins (colicin B [ColB] and colicin E5 [ColE5]) and B. bacteriovorus HD100 either individually or together against four clinical isolates of E. coli that are resistant to either colistin or carbapenem. While the ColB and ColE5 were quickly active when used alone, causing a significant loss in viability (>3-log) in susceptible populations after only 3 h, the pathogens always grew afterwards and had final cell densities that were similar with their respective controls. Predation with B. bacteriovorus HD100, in contrast, was most pronounced after 24 h (>3-log reduction in each pathogen viability but never complete). When combined, better killing efficiencies were observed with several of the pathogens, with complete eradication realized for two (<100 viable pathogens per mL). Given the diversity of colicins in nature and the broad-spectrum activities of B. bacteriovorus strains, the results presented here suggest there is a massive potential to control pathogens when they are used together. IMPORTANCE The coupled impact of drug resistance with reduced antibiotic development has placed humankind at a postantibiotic crossroads where antibiotic alternatives are desperately needed. Consequently, we discuss here the combined effectiveness of two vastly different classes of antibacterials, namely, colicins and a predatory bacterium (i.e.,Bdellovibrio bacteriovorus HD100), against two priority pathogenic groups, colistin- and carbapenem-resistant strains of E. coli. While each is effective in its own manner, these antibacterials also display limitations, i.e., the rapid appearance of mutations that confer resistance to the colicins while predatory bacteria do not completely kill their prey. Here, we show these limitations can be overcome using combined treatments of these antibacterials, with two pathogenic E. coli populations completely eradicated within 24 h. Given the diversity of colicins and the broad-spectrum activities of B. bacteriovorus strains, the results presented here suggests there is a massive potential to control pathogens when they are used together.

Carbapenem-Resistant and ESBL-Producing Enterobacterales Emerging in Central Texas.

Purpose: Carbapenem-resistant Enterobacterales (CRE) are subject to intense global monitoring in an attempt to maintain awareness of prevalent and emerging resistance mechanisms and to inform treatment and infection prevention strategies. CRE and extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales are not usually examined collectively in regards to their shared pool of resistance determinants. Here, we genetically and phenotypically assess clinical isolates of CRE and extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales in the growing region of Central Texas, where CRE are emergent and occurrence of non-carbapenemase-producing-CRE (non-CP-CRE) infections is increasing. Methods: CRE (n=16) and ESBL-producing Enterobacterales (n=116) isolates were acquired from a regional hospital in Central Texas between December 2018 and January 2020. Isolates were assessed genetically and phenotypically using antibiotic susceptibility testing, targeted PCR, and whole genome sequencing. Results: CRE infections are increasing in incidence in Central Texas, and Klebsiella pneumoniae is causing the majority of these infections. Moreover, K. pneumoniae sequence type (ST) 307 is commonly found among both non-CP-CRE and EBSL-producing strains. Isolates carry similar plasmids harboring the gene for the ESBL CTX-M-15 and belong to the global lineage, rather than the Texas lineage, of ST307. Antibiotic resistance profiles, sequence data, and clinical records suggest that porin mutations may promote the transition of ST307 isolates from ESBL-producing to non-CP-CRE. In addition to antibiotic resistance mechanisms, several CRE isolates harbor active colicinogenic plasmids, which might influence the competitiveness of these bacteria during patient colonization. Conclusion: K. pneumoniae of the global ST307 lineage is circulating in Central Texas and is responsible for both non-CP CRE and ESBL-producing Enterobacterales infections. Enhanced surveillance is needed to understand the possible routes for the emergence of non-CP-CRE from EBSL-producing strains.

Genomic and phenotypic characterization of multidrug-resistant Salmonella enterica serovar Reading isolates involved in a turkey-associated foodborne outbreak.

Salmonella is a global bacterial foodborne pathogen associated with a variety of contaminated food products. Poultry products are a common source of Salmonella-associated foodborne illness, and an estimated 7% of human illnesses in the United States are attributed to turkey products. From November 2017 to March 2019, the Centers for Disease Control and Prevention reported a turkey-associated outbreak of multidrug-resistant (MDR; resistant to ≥3 antimicrobial classes) Salmonella enterica serovar Reading (S. Reading) linked to 358 human infections in 42 US states and Canada. Since S. Reading was seldom linked to human illness prior to this outbreak, the current study compared genomic sequences of S. Reading isolates prior to the outbreak (pre-outbreak) to isolates identified during the outbreak period, focusing on genes that were different between the two groups but common within a group. Following whole-genome sequence analysis of five pre-outbreak and five outbreak-associated turkey/turkey product isolates of S. Reading, 37 genes located within two distinct chromosomal regions were identified only in the pre-outbreak isolates: (1) an ~5 kb region containing four protein-coding genes including uidA which encodes beta-glucuronidase, pgdA encoding peptidoglycan deacetylase, and two hypothetical proteins and (2) an ~28 kb region comprised of 32 phage-like genes and the xerC gene, which encodes tyrosine recombinase (frequently associated with phage genes). The five outbreak isolates also had a deletional event within the cirA gene, introducing a translational frame shift and premature stop codon. The cirA gene encodes a protein with dual receptor functions: a siderophore receptor for transport of dihydroxybenzoylserine as well as a colicin Ia/b receptor. Significant differences for the identified genetic variations were also detected in 75 S. Reading human isolates. Of the 41 S. Reading isolates collected before or in 2017, 81 and 90% of the isolates contained the uidA and pgdA genes, respectively, but only 24% of the isolates collected after 2017 harbored the uidA and pgdA genes. The truncation event within the cirA gene was also significantly higher in isolates collected after 2017 (74%) compared to before or in 2017 (5%). Phenotypic analysis of the S. Reading isolates for colicin and cefiderocol sensitivities (CirA) and ß-methyl-D-glucuronic acid utilization (UidA and accessory proteins) supported the genomic data. Overall, a similar genome reduction pattern was generally observed in both the turkey and human isolates of S. Reading during the outbreak period, and the genetic differences were present in genes that could potentially promote pathogen dissemination due to variation in Salmonella colonization, fitness, and/or virulence.

Colicin E2 expression in Escherichia coli biofilms: Induction and regulation revisited.

Colicins, bacteriocins produced by the gram-negative bacterium Escherichia coli, are tightly regulated by the DNA damage response regulatory system (SOS), and are thus triggered at times of stress. Colicins' regulation and expression profiles were primarily studied in suspended (planktonic) cultures yet, in their natural environments E. coli cells are sessile, assembled in biofilms. We hypothesized that colicin expression would differ between planktonic and biofilm E. coli cultures, even when induced by the same triggers. To test our hypothesis, we compared colicin E2 expression and SOS regulated genes in planktonic and biofilm cultures of E. coli, in response to DNA damaging agents and oxygen depletion. The results indicate that uninduced biofilms express more transcripts of the colicin operon than uninduced planktonic cells. Whole genome expression profiles confirmed that in uninduced biofilms, SOS genes are upregulated compared to planktonic cultures. However, DNA damaging agents and oxygen depletion augmented colicin expression in planktonic cells, while only marginal increase was recorded in biofilms. Our results suggest that the regulation of colicin E2 expression in E. coli biofilms considerably differ from planktonic cells, thus the induction of colicins in their host natural environment, i.e., the gastrointestinal tract, needs to be re-evaluated.

The effect of Escherichia coli ZP strain with a conjugation-based colicin E7 delivery on growth performance, hematological, biochemical, and histological parameters, gut microbiota, and nonspecific immunity of broilers.

The Escherichia coli ZP strain (ZP) was constructed based on the known probiotic E. coli strain Nissle 1917. It was genetically modified to carry the colicin E7 synthesis gene encoding DNase on a conjugative plasmid and the colicin E7 immunity gene in the chromosome. The aim of this study was to evaluate the effects of the daily ZP per oral administration (5 × 108 or 5 × 1010 CFU per bird) on the growth performance, hematological, biochemical, histological parameters, gut microbiota, and nonspecific immunity of the 4-24 days old broilers. The ZP administration increased the abundance of genera Bacillus, Butyrivibrio, and Clostridium and did not influence the weight gain of 4-16 days old broilers. The biochemical parameters were within normal ranges for poultry in experimental and control groups. The ZP administration had no effect on the erythrocyte numbers, hemoglobin and immunoglobulin Y concentrations, but significantly increased the serum lysozyme concentration, leukocyte numbers, and reactive oxygen species production by phagocytes compared with the control group. It did not cause inflammatory changes in intestinal mucosa, Peyer's patches, and spleen. Thus, the ZP had no detrimental effects on broiler health and could be an efficient probiotic for the broiler colibacillosis prophylaxis.

Effects of TolC on the pathogenicity of porcine extraintestinal pathogenic Escherichia coli.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a well-known critical pathogenic zoonosis that causes extraintestinal infections in humans and animals by affecting their immune organs. Recently, research on the outer membrane protein of E. coli, tolerant colicin (TolC), a virulent protein in the formation of the ExPEC efflux pump, has been an attractive subject. However, the pathogenic mechanisms remain unclear. This study aimed to explore the role of TolC in the pathogenesis of the ExPEC strain PPECC42; a complementation strain (Cm-TolC) and an isogenic mutant (ΔTolC) were constructed. Loss of TolC drastically impaired the virulence of ExPEC in an experimental mouse model. ΔTolC showed a substantial decrease in the porcine aortic vascular endothelial cell (PAVEC) adherence, invasion, and pro-inflammatory response, in contrast to that of the wild type, with a reduced survival ratio in both the bacterial load and whole blood in mice. ΔTolC also showed decreased expression of necroptosis signals such as receptor-interacting protein kinase 1, phosphorylated mixed-lineage kinase domain-like protein, and mitochondrial proteins such as phosphoglycerate mutase family member 5. Our data suggest that TolC is closely associated with ExPEC pathogenesis. These results provide scientific grounds for exploring the potential of TolC as an effective drug target for controlling ExPEC infection, screening new inhibitors, and developing new drugs. This will allow for further prevention and control of ExPEC infection.

Heterogeneity in the spontaneous induction of the promoter of the ColE9 operon in Escherichia coli.

Spontaneous production of E colicins is known to occur in only a small fraction of colicinogenic population. The current study aimed to determine if the same holds true for the production of colicin E9 in real time, by investigating the induction dynamics of the promoter of the ColE9 operon which results in the expression of the ColE9 activity and functional genes. A novel fluorescent reporter was constructed which carries the fusion of the ColE9 promoter and the gfpmut2 gene in a low copy number plasmid that was compatible with the native ColE9-J plasmid. Using the fluorescent reporter construct in the non colicinogenic E. coli cells, the induction of the ColE9 promoter was investigated. The current study demonstrates that the spontaneous induction of the ColE9 promoter occurs in a heterogenous manner and this heterogeneity is maintained in a bacterial population for several generations suggesting that it is unlikely due to any irreversible mutation in the bacterial culture. Furthermore, the same investigations were repeated using the colicin E9 producing E. coli cells. Flow cytometry analysis revealed that 7.1 ± 0.68% of the colicin E9 producing E. coli cells expressed GFP albeit only 2.45 ± 0.30% was observed from non colicinogenic E. coli cells. The considerable increase in the number of the fluorescent cells was likely due to the DNase activity of colicin E9 produced by their clonemates, resulting the auto-induction, which can be abolished with the inactivation of the DNase activity of the colicin E9.

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.

Shooting hoops: globetrotting plasmids spreading more than just antimicrobial resistance genes across One Health.

Our study provides novel insights into the global nature of antimicrobial resistance (AMR) plasmids across the food chain. We provide compelling evidence of the globetrotting nature of AMR plasmids and the need for surveillance to sequence plasmids with a template of analyses for others to expand these data. The AMR plasmids analysed were detected in 63 countries and in samples from humans, animals and the environment. They contained a combination of known and novel AMR genes, metal resistance genes, virulence factors, phage and replicon types.

In Vitro Screening for Cytotoxic Effect of Pore Forming Colicin N and Its Domains on Human Cancer Cells.

Protein-based drugs have increasingly become an important segment of cancer treatment. In comparison with chemotherapy, they offer high efficacy and fewer side effects due to specifically targeting only cancer cells. Monoclonal antibodies are currently the main protein-based drugs in the market but their complexity and limitations in tumour penetration led to the development of alternative protein therapeutics such as pore-forming toxins. Colicin N (ColN), a pore-forming protein produced by E. coli, was previously found to exhibit cytotoxicity and selectivity in human lung cancer cells with promising potential for further development. Here we aimed to screen for the cytotoxicity of ColN in breast (MCF-7 and MDA-MB-231), lung (A549) and colon cancer cells (HT-29 and HCT-116) by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay with various concentrations for 72 h and to investigate the cytotoxic effect of ColN domains on cancer cells. It showed that ColN mildly mediated the decrease in cell viability except for MCF-7. The highest effect was seen in A549 and HCT-116 cells which showed 31.9% and 31.5% decrease in cell viability, respectively. The mild inhibition or promotion of cancer cell proliferation by ColN tend to be based on the cell types. Furthermore, to search for the functional domain of ColN used for cytotoxicity, full-length ColN and truncated ColN with deletion of translocating, receptor binding and pore-forming domains were also tested on HCT-116 colon cancer cells. The findings indicated that HCT-116 cells were not significantly sensitive to ColN but full length ColN caused slight decrease in cancer cell viability. The data in this study will benefit the further development of ColN for alternative protein-based cancer therapy.

Acacia Fiber Protects the Gut from Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli Colonization Enabled by Antibiotics.

Novel approaches to combating antibiotic resistance are needed given the ever-continuing rise of antibiotic resistance and the scarce discovery of new antibiotics. Little is known about the colonization dynamics and the role of intrinsic plant-food characteristics in this process. We sought to determine whether plant fiber could alter colonization dynamics by antibiotic-resistant bacteria in the gut. We determined that ingestion of antibiotics in mice markedly enhanced gut colonization by a pathogenic extended-spectrum beta-lactamase-producing Escherichia coli strain of human origin, E. coli JJ1886 (ST131-H30Rx). Furthermore, ingestion of soluble acacia fiber before and after antibiotic exposure significantly reduced pathogenic E. coli colonization. 16S rRNA analysis and ex vivo cocultures demonstrated that fiber protected the microbiome by serving as a prebiotic, which induced native gut E. coli to inhibit pathogenic E. coli via colicin M. Fiber may be a useful prebiotic with which to administer antibiotics to protect human and livestock gut microbiomes against colonization from antibiotic-resistant, pathogenic bacteria. IMPORTANCE A One Health-based strategy-the concept that human health and animal health are interconnected with the environment-is necessary to determine the drivers of antibiotic resistance from food to the clinic. Moreover, humans can ingest antibiotic-resistant bacteria on food and asymptomatically, or "silently," carry such bacteria in the gut long before they develop an opportunistic extraintestinal infection. Here, we determined that fiber-rich foods, in particular acacia fiber, may be a new, promising, and inexpensive prebiotic to administer with antibiotics to protect the mammalian (i.e., human and livestock) gut against such colonization by antibiotic-resistant, pathogenic bacteria.

Stenocins: Novel modular bacteriocins from opportunistic pathogen Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is a global emerging pathogenic bacillus that is highly drug resistant and known to cause nosocomial infections in immunocompromised hosts. Because of their novel modes of action, bacteriocins are being proposed as alternatives to antibiotics for the treatment of infections caused by multidrug resistant bacteria. This study is the first report of modular bacteriocins called stenocins, which were discovered in the genomes of S. maltophilia. These two novel peptidoglycan-degrading bacteriocins were identified, cloned, and expressed in plants. We demonstrate that plant-expressed stenocins are functional and inhibit the growth of Stenotrophomonas strains in vitro.

Encapsulating IM7-Displaying Yeast Cells in Calcium Alginate Beads for One-Step Protein Purification and Multienzyme Biocatalysis.

There are several commercial chromatographic systems for protein purification; however, development of cost-effective 3H-grade (high yield, high purity, and high activity) purification approaches is highly demanded. Here, we establish a methodology for encapsulating the IM7-displaying yeast cells in calcium alginate beads. Taking advantage of this biomaterial-based affinity chromatography, rapid and cost-effective purification of proteins with over 90% purity in a single step is achieved. Moreover, our system enables coating the multienzyme complex to produce reusable immobilized cells for efficient cascade biotransformation. Together, the present method has great application potentials not only in the laboratory but also in the industry for production of protein products as well as biocatalysis.