Targeting of Pseudomonas aeruginosa cell surface via GP12, an Escherichia coli specific bacteriophage protein

AbstractBacteriophages are highly abundant molecular machines that have evolved proteins to target the surface of host bacterial cells. Given the ubiquity of lipopolysaccharides (LPS) on the outer membrane of Gram-negative bacteria, we reasoned that targeting proteins from bacteriophages could be leveraged to target the surface of Gram-negative pathogens for biotechnological applications. To this end, a short tail fiber (GP12) from the T4 bacteriophage, which infects Escherichia coli (E. coli), was isolated and tested for the ability to adhere to whole bacterial cells. We found that, surprisingly, GP12 effectively bound the surface of Pseudomonas aeruginosa cells despite the established preferred host of T4 for E. coli. In efforts to elucidate why this binding pattern was observed, it was determined that the absence of the O-antigen region of LPS on E. coli improved cell surface tagging. This indicated that O-antigens play a significant role in controlling cell adhesion by T4. Probing GP12 and LPS interactions further using deletions of the enzymes involved in the biosynthetic pathway of LPS revealed the inner core oligosaccharide as a possible main target of GP12. Finally, we demonstrated the potential utility of GP12 for biomedical applications by showing that GP12-modified agarose beads resulted in the depletion of pathogenic bacteria from solution.

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LpxT-Dependent Phosphorylation of Lipid A in Escherichia coli Increases Resistance to Deoxycholate and Enhances Gut Colonization

Frontiers in Microbiology ◽

10.3389/fmicb.2021.676596 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xudong Tian ◽

Guillaume Manat ◽

Elise Gasiorowski ◽

Rodolphe Auger ◽

Samia Hicham ◽

...

Keyword(s):

Escherichia Coli ◽

Cell Surface ◽

Lipid A ◽

Negative Charge ◽

Polymyxin B ◽

Gram Negative Bacteria ◽

Bacterial Cells ◽

Gram Negative ◽

E Coli ◽

Gut Colonization

The cell surface of Gram-negative bacteria usually exhibits a net negative charge mostly conferred by lipopolysaccharides (LPS). This property sensitizes bacterial cells to cationic antimicrobial peptides, such as polymyxin B, by favoring their binding to the cell surface. Gram-negative bacteria can modify their surface to counteract these compounds such as the decoration of their LPS by positively charged groups. For example, in Escherichia coli and Salmonella, EptA and ArnT add amine-containing groups to the lipid A moiety. In contrast, LpxT enhances the net negative charge by catalyzing the synthesis of tri-phosphorylated lipid A, whose function is yet unknown. Here, we report that E. coli has the intrinsic ability to resist polymyxin B upon the simultaneous activation of the two component regulatory systems PhoPQ and PmrAB by intricate environmental cues. Among many LPS modifications, only EptA- and ArnT-dependent decorations were required for polymyxin B resistance. Conversely, the acquisition of polymyxin B resistance compromised the innate resistance of E. coli to deoxycholate, a major component of bile. The inhibition of LpxT by PmrR, under PmrAB-inducing conditions, specifically accounted for the acquired susceptibility to deoxycholate. We also report that the kinetics of intestinal colonization by the E. coli lpxT mutant was impaired as compared to wild-type in a mouse model of infection and that lpxT was upregulated at the temperature of the host. Together, these findings highlight an important function of LpxT and suggest that a tight equilibrium between EptA- and LpxT-dependent decorations, which occur at the same position of lipid A, is critical for the life style of E. coli.

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High-density transposon libraries utilising outward-oriented promoters identify mechanisms of action and resistance to antimicrobials

FEMS Microbiology Letters ◽

10.1093/femsle/fnaa185 ◽

2020 ◽

Vol 367 (22) ◽

Author(s):

Chris Coward ◽

Gopujara Dharmalingham ◽

Omar Abdulle ◽

Tim Avis ◽

Stephan Beisken ◽

...

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Pathogenic Bacteria ◽

Selective Advantage ◽

Mechanisms Of Action ◽

Over Expression ◽

E Coli ◽

Synthase Inhibitor ◽

Established Technique ◽

Bacterial Transposon

ABSTRACT The use of bacterial transposon mutant libraries in phenotypic screens is a well-established technique for determining which genes are essential or advantageous for growth in conditions of interest. Standard, inactivating, transposon libraries cannot give direct information about genes whose over-expression gives a selective advantage. We report the development of a system wherein outward-oriented promoters are included in mini-transposons, generation of transposon mutant libraries in Escherichia coli and Pseudomonas aeruginosa and their use to probe genes important for growth under selection with the antimicrobial fosfomycin, and a recently-developed leucyl-tRNA synthase inhibitor. In addition to the identification of known mechanisms of action and resistance, we identify the carbon–phosphorous lyase complex as a potential resistance liability for fosfomycin in E. coli and P. aeruginosa. The use of this technology can facilitate the development of novel mechanism-of-action antimicrobials that are urgently required to combat the increasing threat worldwide from antimicrobial-resistant pathogenic bacteria.

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Changes in Cell Surface Properties of Shiga Toxigenic Escherichia coli by Quercus infectoria G. Olivier

Journal of Food Protection ◽

10.4315/0362-028x-72.8.1699 ◽

2009 ◽

Vol 72 (8) ◽

pp. 1699-1704 ◽

Cited By ~ 7

Author(s):

SUPAYANG PIYAWAN VORAVUTHIKUNCHAI ◽

SAKOL SUWALAK

Keyword(s):

Escherichia Coli ◽

Cell Surface ◽

Surface Properties ◽

Cell Surface Hydrophobicity ◽

Ethanolic Extract ◽

Bacterial Cells ◽

Electron Microscopic ◽

Cell Surface Properties ◽

E Coli ◽

Quercus Infectoria

The effects of Quercus infectoria (family Fagaceae) nutgalls on cell surface properties of Shiga toxigenic Escherichia coli (STEC) were investigated with an assay of microbial adhesion to hydrocarbon. The surface of bacterial cells treated with Q. infectoria exhibited a higher level of cell surface hydrophobicity (CSH) toward toluene than did the surface of untreated cells. With 50% ethanolic extract, the CSH of the three strains of STEC O157:H7 treated with 4× MIC of the extract resulted in moderate or strong hydrophobicity, whereas at 2× MIC and MIC, the CSH of only one strain of E. coli O157:H7 was significantly affected. The 95% ethanolic extract had a significant effect on CSH of all three strains at both 4× MIC and 2× MIC but not at the MIC. The effect on bacterial CSH was less pronounced with the other STEC strains. At 4× MIC, the 50% ethanolic extract increased the CSH of all non-O157 STEC strains significantly. At 2× MIC and 4× MIC, the 95% ethanolic extract affected the CSH of E. coli O26:H11 significantly but did not affect E. coli O111:NM or E. coli O22. Electron microscopic examination revealed the loss of pili in the treated cells. The ability of Q. infectoria extract to modify hydrophobic domains enables this extract to partition the lipids of the bacterial cell membrane, rendering the membrane more permeable and allowing leakage of ions and other cell contents, which leads to cell death. Further studies are required to evaluate the effects of Q. infectoria extract in food systems or in vivo and provide support for the use of this extract as a food additive for control of these STEC pathogens.

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In Vitro Activity of Newer and Conventional Antimicrobial Agents, Including Fosfomycin and Colistin, against Selected Gram-Negative Bacilli in Kuwait

Pathogens ◽

10.3390/pathogens7030075 ◽

2018 ◽

Vol 7 (3) ◽

pp. 75 ◽

Cited By ~ 5

Author(s):

Wadha Alfouzan ◽

Rita Dhar ◽

David Nicolau

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Antimicrobial Agents ◽

The Other ◽

In Vitro Activity ◽

Limited Data ◽

Gram Negative ◽

E Coli ◽

Microbroth Dilution

Limited data are available on susceptibilities of these organisms to some of the recently made accessible antimicrobial agents. The in vitro activities of newer antibiotics, such as, ceftolozane/tazobactam (C/T) and ceftazidime/avibactam (CZA) along with some “older” antibiotics, for example fosfomycin (FOS) and colistin (CL) were determined against selected strains (resistant to ≥ 3 antimicrobial agents) of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Minimum inhibitory concentrations (MIC) were determined by Clinical and Laboratory Standards Institute microbroth dilution. 133 isolates: 46 E. coli, 39 K. pneumoniae, and 48 P. aeruginosa were tested. Results showed that E. coli isolates with MIC50/90, 0.5/1 μ g / mL for CL; 4/32 μ g / mL for FOS; 0.25/32 μ g / mL for C/T; 0.25/8 μ g / mL for CZA, exhibited susceptibility rates of 95.7%, 97.8%, 76.1%, and 89.1%, respectively. On the other hand, K. pneumoniae strains with MIC50/90, 0.5/1 μ g / mL for CL; 256/512 μ g / mL for FOS; 2/128 μ g / mL for C/T; 0.5/128 μ g / mL for CZA showed susceptibility rates of 92.3%, 7.7%, 51.3%, and 64.1%, respectively. P. aeruginosa isolates with MIC50/90, 1/1 μ g / mL for CL; 128/128 μ g / mL for C/T; 32/64 μ g / mL for CZA presented susceptibility rates of 97.9%, 33.3%, and 39.6%, respectively. Higher MICs were demonstrated against most of the antibiotics. However, CL retained efficacy at low MICs against most of the isolates tested.

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National Surveillance of Antimicrobial Susceptibility of Bacteremic Gram-Negative Bacteria with Emphasis on Community-Acquired Resistant Isolates: Report from the 2019 Surveillance of Multicenter Antimicrobial Resistance in Taiwan (SMART)

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01089-20 ◽

2020 ◽

Vol 64 (10) ◽

Author(s):

Po-Yu Liu ◽

Yu-Lin Lee ◽

Min-Chi Lu ◽

Pei-Lan Shao ◽

Po-Liang Lu ◽

...

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Antimicrobial Resistance ◽

Klebsiella Pneumoniae ◽

Gram Negative Bacteria ◽

National Surveillance ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Carbapenem Resistant

ABSTRACT A multicenter collection of bacteremic isolates of Escherichia coli (n = 423), Klebsiella pneumoniae (n = 372), Pseudomonas aeruginosa (n = 300), and Acinetobacter baumannii complex (n = 199) was analyzed for susceptibility. Xpert Carba-R assay and sequencing for mcr genes were performed for carbapenem- or colistin-resistant isolates. Nineteen (67.8%) carbapenem-resistant K. pneumoniae (n = 28) and one (20%) carbapenem-resistant E. coli (n = 5) isolate harbored blaKPC (n = 17), blaOXA-48 (n = 2), and blaVIM (n = 1) genes.

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Antibiotic susceptibility of intra-abdominal infection isolates from Indian hospitals during 2008

Journal of Medical Microbiology ◽

10.1099/jmm.0.020784-0 ◽

2010 ◽

Vol 59 (9) ◽

pp. 1050-1054 ◽

Cited By ~ 14

Author(s):

Stephen P. Hawser ◽

Robert E. Badal ◽

Samuel K. Bouchillon ◽

Daryl J. Hoban ◽

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Antimicrobial Resistance ◽

Gram Negative ◽

E Coli ◽

Abdominal Infection ◽

Intra Abdominal Infection ◽

Abdominal Infections ◽

Hospital Acquired ◽

Very High

A total of 542 clinical isolates of aerobic Gram-negative bacilli from intra-abdominal infections were collected during 2008 from seven hospitals in India participating in the Study for Monitoring Antimicrobial Resistance Trends (SMART). Isolates were from various infection sources, the most common being gall bladder (30.1 %) and peritoneal fluid (31.5 %), and were mostly hospital-associated isolates (70.8 %) as compared to community-acquired (26.9 %). The most frequently isolated pathogens were Escherichia coli (62.7 %), Klebsiella pneumoniae (16.7 %) and Pseudomonas aeruginosa (5.3 %). Extended-spectrum β-lactamase (ESBL) rates in E. coli and K. pneumoniae were very high, at 67 % and 55 %, respectively. Most isolates exhibited resistance to one or more antibiotics. The most active drugs were generally ertapenem, imipenem and amikacin. However, hospital-acquired isolates in general, as well as ESBL-positive isolates, exhibited lower susceptibilities than community-acquired isolates. Further surveillance monitoring of intra-abdominal isolates from India is recommended.

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Syndemics of Fluoroquinolone Resistance in N. gonorrhoeae, E. coli and K. pneumoniae: A Global Ecological Analysis

10.20944/preprints202108.0361.v1 ◽

2021 ◽

Author(s):

Chris Kenyon

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

General Population ◽

Fluoroquinolone Resistance ◽

Selection Methods ◽

Ecological Analysis ◽

Gram Negative ◽

E Coli ◽

Country Level ◽

National Prevalence

It is unclear how important it is to reduce fluoroquinolone consumption in the general population to prevent the spread of fluoroquinolone resistance in Neisseria gonorrhoeae (bystander selection). Methods We assessed bystander selection by using Spearman’s correlation to assess if the country-level prevalence of fluoroquinolone resistance in N. gonorrhoeae was correlated with the prevalence of fluoroquinolone resistance in four other gram-negative species - Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Results Fluoroquinolone resistance in N. gonorrhoeae was positively associated with homologous resistance in all 4 species - A. baumanii. (ρ=0.61, P=0.0003, E. coli (ρ=0.67, P<0.0001), K. pneumoniae (ρ=0.52, P=0.0004) and P. aeruginosa (ρ=0.40, P=0.0206). Positive associations were also found between the national prevalence of fluoroquinolone resistance and fluoroquinolone consumption in the general population in the preceding year for 4 of the 5 species. Conclusions Gonococcal fluoroquinolone resistance can be productively viewed as being part of a syndemic of fluoroquinolone resistance. Strengthening antimicrobial stewardship programs may help retard the spread of fluoroquinolone resistance in N. gonorrhoeae.

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Colonization resistance against potentially pathogenic bacteria in hexaflora-associated gnotobiotic mice

Canadian Journal of Microbiology ◽

10.1139/m78-016 ◽

1978 ◽

Vol 24 (2) ◽

pp. 79-83 ◽

Cited By ~ 3

Author(s):

Kunwar K. Srivastava

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Pathogenic Bacteria ◽

Enterobacter Aerogenes ◽

Lactobacillus Brevis ◽

Colonization Resistance ◽

Post Challenge ◽

E Coli ◽

Gnotobiotic Mice ◽

Akr Mice

A study of colonization resistance against potentially pathogenic bacteria (Escherichia coli and Pseudomonas aeruginosa) was conducted in hexaflora-associated gnotobiotic mice. Groups of germfree AKR mice were swabbed with five bacterial and a single gastrointestinal yeast species: Streptococcus faecalis, Lactobacillus brevis, Staphylococcus epidermidis, Enterobacter aerogenes, Bacteroides fragilis var. vulgatus, and Torulopsis sp. All species became established in the gut in 8 weeks. Later these associated mice were divided and challenged by four graded doses of E. coli or P. aeruginosa. The presence of challenge organism was monitored specifically in the freshly voided fecal specimens of the challenged mice. Escherichia coli colonized the gut of each mouse at each level up to 60 days post challenge. Pseudomonas aeruginosa was completely eliminated from each mouse at each dose level after 30 days post challenge. Evidence suggests that all six species were sufficient to prevent the colonization of P. aeruginosa and not of E. coli in the gut of the gnotobiotic mice.

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The lipopolysaccharide of the mastitis isolate Escherichia coli strain 1303 comprises a novel O-antigen and the rare K-12 core type

Microbiology ◽

10.1099/mic.0.046912-0 ◽

2011 ◽

Vol 157 (6) ◽

pp. 1750-1760 ◽

Cited By ~ 13

Author(s):

Katarzyna A. Duda ◽

Buko Lindner ◽

Helmut Brade ◽

Andreas Leimbach ◽

Elżbieta Brzuszkiewicz ◽

...

Keyword(s):

Escherichia Coli ◽

Inner Core ◽

Bovine Mastitis ◽

Outer Core ◽

Coli Strain ◽

Core Oligosaccharide ◽

2D Nmr Spectroscopy ◽

E Coli ◽

O Antigen ◽

K 12

Mastitis represents one of the most significant health problems of dairy herds. The two major causative agents of this disease are Escherichia coli and Staphylococcus aureus. Of the first, its lipopolysaccharide (LPS) is thought to play a prominent role during infection. Here, we report the O-antigen (OPS, O-specific polysaccharide) structure of the LPS from bovine mastitis isolate E. coli 1303. The structure was determined utilizing chemical analyses, mass spectrometry, and 1D and 2D NMR spectroscopy methods. The O-repeating unit was characterized as -[→4)-β-d-Quip3NAc-(1→3)-α-l-Fucp2OAc-(1→4)-β-d-Galp-(1→3)-α-d-GalpNAc-(1→]- in which the O-acetyl substitution was non-stoichiometric. The nucleotide sequence of the O-antigen gene cluster of E. coli 1303 was also determined. This cluster, located between the gnd and galF genes, contains 13 putative open reading frames, most of which represent unknown nucleotide sequences that have not been described before. The O-antigen of E. coli 1303 was shown to substitute O-7 of the terminal ld-heptose of the K-12 core oligosaccharide. Interestingly, the non-OPS-substituted core oligosaccharide represented a truncated version of the K-12 outer core – namely terminal ld-heptose and glucose were missing; however, it possessed a third Kdo residue in the inner core. On the basis of structural and genetic data we show that the mastitis isolate E. coli 1303 represents a new serotype and possesses the K-12 core type, which is rather uncommon among human and bovine isolates.

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Electroceutical disinfection strategies impair the motility of pathogenic Pseudomonas aeruginosa and Escherichia coli

10.1101/088120 ◽

2016 ◽

Cited By ~ 1

Author(s):

Kristina Doxsee ◽

Ryan Berthelot ◽

Suresh Neethirajan

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Acetic Acid ◽

Mammalian Cells ◽

Pathogenic Bacteria ◽

Electrical Potential ◽

Microfluidic Platform ◽

E Coli ◽

Therapeutic Benefits ◽

The Impact

Electrotaxis or galvanotaxis refers to the migration pattern of cells induced in response to electrical potential. Although it has been extensively studied in mammalian cells, electrotaxis has not been explored in detail in bacterial cells; information regarding the impact of current on pathogenic bacteria is severely lacking. Therefore, we designed a series of single and multi-cue experiments to assess the impact of varying currents on bacterial motility dynamics in pathogenic multi-drug resistant (MDR) strains of Pseudomonas aeruginosa and Escherichia coli using a microfluidic platform. Motility plays key roles in bacterial migration and the colonization of surfaces during the formation of biofilms, which are inherently recalcitrant to removal and resistant to traditional disinfection strategies (e.g. antibiotics). Use of the microfluidic platform allows for exposure to current, which can be supplied at a range that is biocidal to bacteria, yet physiologically safe in humans (single cue). This system also allows for multi-cue experiments where acetic acid, a relatively safe compound with anti-fouling/antimicrobial properties, can be combined with current to enhance disinfection. These strategies may offer substantial therapeutic benefits, specifically for the treatment of biofilm infections, such as those found in the wound environment. Furthermore, microfluidic systems have been successfully used to model the unique microfluidic dynamics present in the wound environment, suggesting that these investigations could be extended to more complex biological systems. Our results showed that the application of current in combination with acetic acid has profound inhibitory effects on MDR strains of P. aeruginosa and E. coli, even with brief applications. Specifically, E. coli motility dynamics and cell survival were significantly impaired starting at a concentration of 125 μA DC and 0.31% acetic acid, while P. aeruginosa was impaired at 70 μA and 0.31% acetic acid. As these strains are relevant wound pathogens, it is likely that this strategy would be effective against similar strains in vivo and could represent a new approach to hasten wound healing.

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