Antibacterial and Antibiofilm Activities of Novel Antimicrobial Peptides against Multidrug-Resistant Enterotoxigenic Escherichia Coli

Post-weaning diarrhea due to enterotoxigenic Escherichia coli (ETEC) is a common disease of piglets and causes great economic loss for the swine industry. Over the past few decades, decreasing effectiveness of conventional antibiotics has caused serious problems because of the growing emergence of multidrug-resistant (MDR) pathogens. Various studies have indicated that antimicrobial peptides (AMPs) have potential to serve as an alternative to antibiotics owing to rapid killing action and highly selective toxicity. Our previous studies have shown that AMP GW-Q4 and its derivatives possess effective antibacterial activities against the Gram-negative bacteria. Hence, in the current study, we evaluated the antibacterial efficacy of GW-Q4 and its derivatives against MDR ETEC and their minimal inhibition concentration (MIC) values were determined to be around 2~32 μg/mL. Among them, AMP Q4-15a-1 with the second lowest MIC (4 μg/mL) and the highest minimal hemolysis concentration (MHC, 256 μg/mL), thus showing the greatest selectivity (MHC/MIC = 64) was selected for further investigations. Moreover, Q4-15a-1 showed dose-dependent bactericidal activity against MDR ETEC in time–kill curve assays. According to the cellular localization and membrane integrity analyses using confocal microscopy, Q4-15a-1 can rapidly interact with the bacterial surface, disrupt the membrane and enter cytosol in less than 30 min. Minimum biofilm eradication concentration (MBEC) of Q4-15a-1 is 4× MIC (16 μg/mL), indicating that Q4-15a-1 is effective against MDR ETEC biofilm. Besides, we established an MDR ETEC infection model with intestinal porcine epithelial cell-1 (IPEC-1). In this infection model, 32 μg/mL Q4-15a-1 can completely inhibit ETEC adhesion onto IPEC-1. Overall, these results suggested that Q4-15a-1 may be a promising antibacterial candidate for treatment of weaned piglets infected by MDR ETEC.

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In vitro activity of antimicrobial peptide CDP-B11 alone and in combination with colistin against colistin-resistant and multidrug-resistant Escherichia coli

Scientific Reports ◽

10.1038/s41598-021-81140-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kaitlin S. Witherell ◽

Jason Price ◽

Ashok D. Bandaranayake ◽

James Olson ◽

Douglas R. Call

Keyword(s):

Escherichia Coli ◽

Antimicrobial Peptide ◽

Membrane Integrity ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Multidrug Resistant Bacteria ◽

E Coli ◽

Minimal Media

AbstractMultidrug-resistant bacteria are a growing global concern, and with increasingly prevalent resistance to last line antibiotics such as colistin, it is imperative that alternative treatment options are identified. Herein we investigated the mechanism of action of a novel antimicrobial peptide (CDP-B11) and its effectiveness against multidrug-resistant bacteria including Escherichia coli #0346, which harbors multiple antibiotic-resistance genes, including mobilized colistin resistance gene (mcr-1). Bacterial membrane potential and membrane integrity assays, measured by flow cytometry, were used to test membrane disruption. Bacterial growth inhibition assays and time to kill assays measured the effectiveness of CDP-B11 alone and in combination with colistin against E. coli #0346 and other bacteria. Hemolysis assays were used to quantify the hemolytic effects of CDP-B11 alone and in combination with colistin. Findings show CDP-B11 disrupts the outer membrane of E. coli #0346. CDP-B11 with colistin inhibits the growth of E. coli #0346 at ≥ 10× lower colistin concentrations compared to colistin alone in Mueller–Hinton media and M9 media. Growth is significantly inhibited in other clinically relevant strains, such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In rich media and minimal media, the drug combination kills bacteria at a lower colistin concentration (1.25 μg/mL) compared to colistin alone (2.5 μg/mL). In minimal media, the combination is bactericidal with killing accelerated by up to 2 h compared to colistin alone. Importantly, no significant red blood hemolysis is evident for CDP-B11 alone or in combination with colistin. The characteristics of CDP-B11 presented here indicate that it can be used as a potential monotherapy or as combination therapy with colistin for the treatment of multidrug-resistant infections, including colistin-resistant infections.

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Improvement of the Enterotoxigenic Escherichia coli Infection Model for Post-Weaning Diarrhea by Controlling for Bacterial Adhesion, Pig Breed and MUC4 Genotype

Veterinary Sciences ◽

10.3390/vetsci7030106 ◽

2020 ◽

Vol 7 (3) ◽

pp. 106

Author(s):

Hiroki Matsumoto ◽

Masashi Miyagawa ◽

Sayaka Takahashi ◽

Ryouichi Shima ◽

Takayuki Oosumi

Keyword(s):

Escherichia Coli ◽

Experimental Infection ◽

Antibacterial Agents ◽

Experimental Models ◽

Enterotoxigenic Escherichia Coli ◽

Infection Model ◽

Swine Industry ◽

Pig Breeds ◽

Adhesion Ability ◽

Escherichia Coli Infection

Enterotoxigenic Escherichia coli (ETEC) is a major cause of post-weaning diarrhea (PWD) in pigs and causes significant damage to the swine industry worldwide. In recent years, there has been increased regulation against the use of antibacterial agents in swine due to their health risks. Utilizing experimental models that consistently recapitulate PWD is important for the development of non-antibacterial agents against PWD in pigs. In this study, we established a highly reproducible PWD infection model by examining differences in adhesion of ETEC to the intestinal tissue as well as the association between MUC4 polymorphisms and sensitivity to PWD. Post-weaning diarrhea differences between pig breeds were also examined. The adhesion to enterocytes varied from 104.0 to 106.4 CFU/mL even among the F4 ETEC strains. Experimental infection revealed that PWD can be induced in all MUC4 genotypes after infection with 1010 CFU/pig of highly adherent ETEC, although there were variable sensitivities between the genotypes. Lowly adherent ETEC did not cause PWD as efficiently as did highly adherent ETEC. The incidence of PWD was confirmed for all pigs with the ETEC-susceptible MUC4 genotypes in all of the breeds. These results indicate that high-precision and reproducible experimental infection is possible regardless of pig breeds by controlling factors on the pig-end (MUC4 genotype) and the bacterial-end (adhesion ability).

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Efficacy of mecillinam against clinical multidrug-resistant Escherichia coli in a murine urinary tract infection model

International Journal of Antimicrobial Agents ◽

10.1016/j.ijantimicag.2019.11.008 ◽

2020 ◽

Vol 55 (2) ◽

pp. 105851

Author(s):

Ilya Nikolaevich Zykov ◽

Niels Frimodt-Møller ◽

Lars Småbrekke ◽

Arnfinn Sundsfjord ◽

Ørjan Samuelsen

Keyword(s):

Escherichia Coli ◽

Urinary Tract Infection ◽

Urinary Tract ◽

Tract Infection ◽

Multidrug Resistant ◽

Infection Model

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Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity

Molecules ◽

10.3390/molecules26237142 ◽

2021 ◽

Vol 26 (23) ◽

pp. 7142

Author(s):

Junhao Cheng ◽

Marhaba Ahmat ◽

Henan Guo ◽

Xubiao Wei ◽

Lulu Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Antibacterial Activity ◽

Antimicrobial Peptides ◽

Bacterial Infections ◽

Expression System ◽

Enterotoxigenic Escherichia Coli ◽

Proteinase K ◽

Hybrid Peptide ◽

Tev Protease ◽

High Level

CLP is a novel hybrid peptide derived from CM4, LL37 and TP5, with significantly reduced hemolytic activity and increased antibacterial activity than parental antimicrobial peptides. To avoid host toxicity and obtain high-level bio-production of CLP, we established a His-tagged SUMO fusion expression system in Escherichia coli. The fusion protein can be purified using a Nickel column, cleaved by TEV protease, and further purified in flow-through of the Nickel column. As a result, the recombinant CLP with a yield of 27.56 mg/L and a purity of 93.6% was obtained. The purified CLP exhibits potent antimicrobial activity against gram+ and gram- bacteria. Furthermore, the result of propidium iodide staining and scanning electron microscopy (SEM) showed that CLP can induce the membrane permeabilization and cell death of Enterotoxigenic Escherichia coli (ETEC) K88. The analysis of thermal stability results showed that the antibacterial activity of CLP decreases slightly below 70 °C for 30 min. However, when the temperature was above 70 °C, the antibacterial activity was significantly decreased. In addition, the antibacterial activity of CLP was stable in the pH range from 4.0 to 9.0; however, when pH was below 4.0 and over 9.0, the activity of CLP decreased significantly. In the presence of various proteases, such as pepsin, papain, trypsin and proteinase K, the antibacterial activity of CLP remained above 46.2%. In summary, this study not only provides an effective strategy for high-level production of antimicrobial peptides and evaluates the interference factors that affect the biological activity of hybrid peptide CLP, but also paves the way for further exploration of the treatment of multidrug-resistant bacterial infections.

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Activity of Tigecycline or Colistin in Combination with Zidovudine against Escherichia coli Harboring tet(X) and mcr-1

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01172-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01172-20 ◽

Cited By ~ 1

Author(s):

Yu-Feng Zhou ◽

Ping Liu ◽

Shu-He Dai ◽

Jian Sun ◽

Ya-Hong Liu ◽

...

Keyword(s):

Escherichia Coli ◽

Multidrug Resistant ◽

Therapeutic Options ◽

Infection Model ◽

Synergistic Activity ◽

Colistin Resistance ◽

Content Type ◽

E Coli ◽

Antiretroviral Drug

ABSTRACTAlternative therapeutic options are urgently needed against multidrug-resistant Escherichia coli infections, especially in situations of preexisting tigecycline and colistin resistance. Here, we investigated synergistic activity of the antiretroviral drug zidovudine in combination with tigecycline or colistin against E. coli harboring tet(X) and mcr-1 in vitro and in a murine thigh infection model. Zidovudine and tigecycline/colistin combinations achieved synergistic killing and significantly decreased bacterial burdens by >2.5-log10 CFU/g in thigh tissues compared to each monotherapy.

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Experimental Infection of Human Volunteers with the Heat-Stable Enterotoxin-Producing Enterotoxigenic Escherichia coli Strain TW11681

Pathogens ◽

10.3390/pathogens8020084 ◽

2019 ◽

Vol 8 (2) ◽

pp. 84 ◽

Cited By ~ 2

Author(s):

Sunniva Todnem Sakkestad ◽

Hans Steinsland ◽

Steinar Skrede ◽

Elisabeth Kleppa ◽

Kristine Lillebø ◽

...

Keyword(s):

Escherichia Coli ◽

T Cell ◽

Protective Efficacy ◽

T Cell Responses ◽

Enterotoxigenic Escherichia Coli ◽

Cd4 T Cell ◽

Infection Model ◽

Serological Response ◽

Cell Responses ◽

Heat Stable

Infection with enterotoxigenic Escherichia coli (ETEC) producing the heat-stable enterotoxin (ST) is one of the most important causes of childhood diarrhoea in low- and middle-income countries. Here, we undertook a controlled human infection model (CHIM) study to investigate whether ST-producing ETEC strain TW11681 would be suitable for testing the protective efficacy of new ST-based vaccine candidates in vaccine challenge models. In groups of three, nine volunteers ingested 1 × 106, 1 × 107, or 1 × 108 colony-forming units (CFU) of TW11681. Flow cytometry-based assays were used to measure CD4+ T cell responses and antibody levels targeting virulence factors expressed by the strain. We found that infection with TW11681 elicited few and mild symptoms, including mild diarrhoea in two volunteers, both of whom ingested 1 × 106 CFU. Averaged across all volunteers, the CD4+ T cell responses specific for E. coli YghJ mucinase peaked 10 days after infection (3.2-fold (p = 0.016)), while the CD4+ T cell responses specific for Colonization Factor Antigen I (CFA/I) major fimbrial subunit (CfaB) peaked after 28 days (3.6-fold (p = 0.063)). The serum CfaB-specific anti-IgA and anti-IgG/IgM levels were significantly increased and peaked 3 months after infection. Both remained elevated for the duration of the 12-month follow-up. The corresponding anti-YghJ serological response was strongest after 10 days, although a significant increase was seen only for IgA levels (3.2-fold (p = 0.008)). In conclusion, due to its low diarrhoea attack risk, TW11681 is probably not suitable for testing the efficacy of new vaccines in human challenge studies at doses 1 × 106 to 1 × 108. However, the strain may still be useful in CHIMs for studying ETEC host-pathogen interactions.

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Effects of curcumin on growth performance, jejunal mucosal membrane integrity, morphology and immune status in weaned piglets challenged with enterotoxigenic Escherichia coli

International Immunopharmacology ◽

10.1016/j.intimp.2015.04.038 ◽

2015 ◽

Vol 27 (1) ◽

pp. 46-52 ◽

Cited By ~ 44

Author(s):

Wenjuan Xun ◽

Liguang Shi ◽

Hanlin Zhou ◽

Guanyu Hou ◽

Ting Cao ◽

...

Keyword(s):

Escherichia Coli ◽

Growth Performance ◽

Immune Status ◽

Membrane Integrity ◽

Enterotoxigenic Escherichia Coli ◽

Weaned Piglets ◽

Mucosal Membrane

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Antimicrobial peptides in the duodenum at the acute and convalescent stages in patients with diarrhea due to Vibrio cholerae O1 or enterotoxigenic Escherichia coli infection

Microbes and Infection ◽

10.1016/j.micinf.2011.06.014 ◽

2011 ◽

Vol 13 (12-13) ◽

pp. 1111-1120 ◽

Cited By ~ 17

Author(s):

Tahmina Shirin ◽

Arman Rahman ◽

Åke Danielsson ◽

Taher Uddin ◽

Taufiqur Rahman Bhuyian ◽

...

Keyword(s):

Escherichia Coli ◽

Antimicrobial Peptides ◽

Vibrio Cholerae ◽

Enterotoxigenic Escherichia Coli ◽

Escherichia Coli Infection ◽

Vibrio Cholerae O1

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Emergence of a Multidrug-Resistant Shiga Toxin-Producing Enterotoxigenic Escherichia coli Lineage in Diseased Swine in Japan

Journal of Clinical Microbiology ◽

10.1128/jcm.03141-15 ◽

2016 ◽

Vol 54 (4) ◽

pp. 1074-1081 ◽

Cited By ~ 23

Author(s):

Masahiro Kusumoto ◽

Yuna Hikoda ◽

Yuki Fujii ◽

Misato Murata ◽

Hirotsugu Miyoshi ◽

...

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Significant Risk ◽

Multidrug Resistant ◽

Limited Range ◽

Enterotoxigenic Escherichia Coli ◽

Content Type ◽

E Coli ◽

Shigella Boydii ◽

High Level

EnterotoxigenicEscherichia coli(ETEC) and Shiga toxin-producingE. coli(STEC) are important causes of diarrhea and edema disease in swine. The majority of swine-pathogenicE. colistrains belong to a limited range of O serogroups, including O8, O138, O139, O141, O147, O149, and O157, which are the most frequently reported strains worldwide. However, the circumstances of ETEC and STEC infections in Japan remain unknown; there have been few reports on the prevalence or characterization of swine-pathogenicE. coli. In the present study, we determined the O serogroups of 967E. coliisolates collected between 1991 and 2014 from diseased swine in Japan, and we found that O139, O149, O116, and OSB9 (O serogroup ofShigella boydiitype 9) were the predominant serogroups. We further analyzed these four O serogroups using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, and virulence factor profiling. Most of the O139 and O149 strains formed serogroup-specific PFGE clusters (clusters I and II, respectively), whereas the O116 and OSB9 strains were grouped together in the same cluster (cluster III). All of the cluster III strains belonged to a single sequence type (ST88) and carried genes encoding both enterotoxin and Shiga toxin. This PFGE cluster III/ST88 lineage exhibited a high level of multidrug resistance (to a median of 10 antimicrobials). Notably, these bacteria were resistant to fluoroquinolones. Thus, this lineage should be considered a significant risk to animal production due to the toxigenicity and antimicrobial resistance of these bacteria.

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