Alternative Antibacterial Strategies Research Articles

Dual functions of epigallocatechin gallate surface-modified Au nanorods@selenium composites for near-infrared-II light-responsive synergistic antibacterial therapy.

Diseases caused by bacterial infections pose ever-increasing threats to human health, making it important to explore alternative antibacterial strategies. Herein, epigallocatechin gallate (EGCG) surface-modified Au nanorods@selenium composites (ASE NPs) were developed for synergistic NIR-II light-responsive antibacterial therapy. In vitro antibacterial experiments demonstrated the improved antibacterial effect of ASE NPs against Staphylococcus aureus (S. aureus) compared with EGCG alone. In addition, in vivo studies demonstrated that ASE NPs cured skin wound infections and sepsis in mice caused by S. aureus. Au nanorods with excellent photothermal conversion realized synergistic photothermal therapy (PTT) in the NIR-II biowindow with an improved penetration depth at a low power density. More importantly, toxicity analysis showed that the composites had no toxic effects on major organs. Thus, the EGCG surface-modified Au nanorods@selenium composites with an NIR-II light-responsive synergistic activity hold great promise for the effective treatment of drug-resistant bacterial infections.

Essential Oils from Indigenous Iranian Plants: A Natural Weapon vs. Multidrug-Resistant Escherichia coli.

Aim of this study was to investigate the antimicrobial properties of herbal plant essential oils (EOs) from selected Iranian plant species such as Ferulago angulata, Zataria multiflora, Cuminum cyminum, and Mentha longifolia against antibiotic-resistant Escherichia coli (E. coli) strains. For this purpose, the Escherichia coli strains, isolated from raw cow’s milk and local dairy products (yogurt, cream, whey, cheese, and confectionery products) collected from different areas of Hamedan province, Iran, were investigated for their resistance to antibiotics (i.e., streptomycin, tetracycline, gentamicin, chloramphenicol, ciprofloxacin, and cefixime). Thus, the E. coli strains were tested for their susceptibility to the above-mentioned essential oils. Regarding antibiotics, the E. coli strains were highly sensitive to ciprofloxacin. In relation to essential oils, the most effective antibacterial activity was observed with Zataria multiflora; also, the bacteria were semi-sensitive to Cuminum cyminum and Mentha longifolia essential oils. All strains were resistant to Ferulago angulata essential oil. According to the results, the essential oil of Zataria multiflora can be considered as a practical and alternative antibacterial strategy to inhibit the growth of multidrug-resistant E. coli of dairy origin.

Cross-Genus "Boot-Up" of Synthetic Bacteriophage in Staphylococcus aureus by Using a New and Efficient DNA Transformation Method.

Staphylococcus aureus is an opportunistic pathogen that causes a wide range of infections and food poisoning in humans with antibiotic resistance, specifically to methicillin, compounding the problem. Bacteriophages (phages) provide an alternative treatment strategy, but these only infect a limited number of circulating strains and may quickly become ineffective due to bacterial resistance. To overcome these obstacles, engineered phages have been proposed, but new methods are needed for the efficient transformation of large DNA molecules into S. aureus to "boot-up" (i.e., rescue) infectious phages. We presented a new, efficient, and reproducible DNA transformation method, NEST (non-electroporation Staphylococcus transformation), for S. aureus to boot-up purified phage genomic DNA (at least 150 kb in length) and whole yeast-assembled synthetic phage genomes. This method was a powerful new tool for the transformation of DNA in S. aureus and will enable the rapid development of engineered therapeutic phages and phage cocktails against Gram-positive pathogens. IMPORTANCE The continued emergence of antibiotic-resistant bacterial pathogens has heightened the urgency for alternative antibacterial strategies. Phages provide an alternative treatment strategy but are difficult to optimize. Synthetic biology approaches have been successfully used to construct and rescue genomes of model phages but only in a limited number of highly transformable host species. In this study, we used a new, reproducible, and efficient transformation method to reconstitute a functional nonmodel Siphophage from a constructed synthetic genome. This method will facilitate the engineering of Staphylococcus and Enterococcus phages for therapeutic applications and the engineering of Staphylococcus strains by enabling transformation of higher molecular weight DNA to introduce more complex modifications.

Ultrasmall Fe-doped carbon dots nanozymes for photoenhanced antibacterial therapy and wound healing

Pathogenic bacteria pose a devastating threat to public health. However, because of the growing bacterial antibiotic resistance, there is an urgent need to develop alternative antibacterial strategies to the established antibiotics. Herein, iron-doped carbon dots (Fe-CDs, ∼3 nm) nanozymes with excellent photothermal conversion and photoenhanced enzyme-like properties are developed through a facile one-pot pyrolysis approach for synergistic efficient antibacterial therapy and wound healing. In particular, Fe doping endows CDs with photoenhanced peroxidase (POD)-like activity, which lead to the generation of heat and reactive oxygen species (ROS) for Gram-positive and Gram-negative bacteria killing. This study demonstrates Fe-CDs have significant wound healing efficiency of Fe-CDs by preventing infection, promoting fibroblast proliferation, angiogenesis, and collagen deposition. Furthermore, the ultrasmall size of Fe-CDs possesses good biocompatibility favoring clinical translation. We believe that the nanozyme-mediated therapeutic platform presented here is expected to show promising applications in antibacterial.

Selective photodynamic inactivation of Helicobacter pylori by a cationic benzylidene cyclopentanone photosensitizer - an in vitro and ex vivo study.

The rise in the antibiotic resistance rate of Helicobacter pylori has led to an increasing eradication failure of this carcinogenic bacterial pathogen worldwide. This underlines the need for alternative antibacterial strategies against H. pylori infection. Antimicrobial photodynamic therapy (aPDT) is a promising non-pharmacological antibacterial technology. In this study, the selective killing activities of three benzylidene cyclopentanone (BCP) photosensitizers (Y1, P1 and P3) towards H. pylori over normal human gastric epithelial GES-1 cells were evaluated and the ex vivo photodynamic inactivation effect was preliminarily assessed on twelve H. Pylor-infected mice. Results showed that under the irradiation of 24J/cm2 532nm laser, Y1, P1 and P3 at 2.5μM induced a 3-log10 reduction of H. pylori CFU (99.9% killing). Confocal images showed that P3, unlike Y1 and P1, could not be uptaken by GES-1 cells. P3 at 2.5 to 20μM showed not significant (p>0.05) phototoxicity to GES-1 cells, nevertheless, Y1 and P1 under the same concentrations exhibited remarkable phototoxicity to GES-1 cells. In the co-culture of H. pylori and GES-1 cells, P3 at 2.5μM led to a complete eradication of H. pylori under the irradiation of 24J/cm2 532nm laser. While for the GES-1 cells, no significant (p>0.05) phototoxicity was observed under the same aPDT dosage. The ex vivo experiments showed that P3 mediated aPDT resulted in 82.4% to 100% reduction of H. pylori CFU without damaging the gastric mucosa. To sum up, P3 is a promising anti-H. pylori photosensitizer with the ability to selectively photo-inactivate H. pylori while sparing normal gastric tissues.

Engineering a customizable antibacterial T6SS-based platform in Vibrio natriegens.

Bacterial pathogens are a major risk to human, animal, and plant health. To counteract the spread of antibiotic resistance, alternative antibacterial strategies are urgently needed. Here, we construct a proof‐of‐concept customizable, modular, and inducible antibacterial toxin delivery platform. By engineering a type VI secretion system (T6SS) that is controlled by an externally induced on/off switch, we transform the safe bacterium, Vibrio natriegens, into an effective antibacterial weapon. Furthermore, we demonstrate that the delivered effector repertoire, and thus the toxicity range of this platform, can be easily manipulated and tested. We believe that this platform can serve as a foundation for novel antibacterial bio‐treatments, as well as a unique tool to study antibacterial toxins.

Evolution of Bacterial Cross-Resistance to Lytic Phages and Albicidin Antibiotic.

Due to concerns over the global increase of antibiotic-resistant bacteria, alternative antibacterial strategies, such as phage therapy, are increasingly being considered. However, evolution of bacterial resistance to new therapeutics is almost a certainty; indeed, it is possible that resistance to alternative treatments might result in an evolved trade-up such as enhanced antibiotic resistance. Here, we hypothesize that selection for Escherichia coli bacteria to resist phage T6, phage U115, or albicidin, a DNA gyrase inhibitor, should often result in a pleiotropic trade-up in the form of cross-resistance, because all three antibacterial agents interact with the Tsx porin. Selection imposed by any one of the antibacterials resulted in cross-resistance to all three of them, in each of the 29 spontaneous bacterial mutants examined in this study. Furthermore, cross-resistance did not cause measurable fitness (growth) deficiencies for any of the bacterial mutants, when competed against wild-type E. coli in both low-resource and high-resource environments. A combination of whole-genome and targeted sequencing confirmed that mutants differed from wild-type E. coli via change(s) in the tsx gene. Our results indicate that evolution of cross-resistance occurs frequently in E. coli subjected to independent selection by phage T6, phage U115 or albicidin. This study cautions that deployment of new antibacterial therapies such as phage therapy, should be preceded by a thorough investigation of evolutionary consequences of the treatment, to avoid the potential for evolved trade-ups.

Secretory System Components as Potential Prophylactic Targets for Bacterial Pathogens.

Bacterial secretory systems are essential for virulence in human pathogens. The systems have become a target of alternative antibacterial strategies based on small molecules and antibodies. Strategies to use components of the systems to design prophylactics have been less publicized despite vaccines being the preferred solution to dealing with bacterial infections. In the current review, strategies to design vaccines against selected pathogens are presented and connected to the biology of the system. The examples are given for Y. pestis, S. enterica, B. anthracis, S. flexneri, and other human pathogens, and discussed in terms of effectiveness and long-term protection.

An Investigation into the Potential of Targeting Escherichia coli rne mRNA with Locked Nucleic Acid (LNA) Gapmers as an Antibacterial Strategy.

The increase in antibacterial resistance is a serious challenge for both the health and defence sectors and there is a need for both novel antibacterial targets and antibacterial strategies. RNA degradation and ribonucleases, such as the essential endoribonuclease RNase E, encoded by the rne gene, are emerging as potential antibacterial targets while antisense oligonucleotides may provide alternative antibacterial strategies. As rne mRNA has not been previously targeted using an antisense approach, we decided to explore using antisense oligonucleotides to target the translation initiation region of the Escherichia coli rne mRNA. Antisense oligonucleotides were rationally designed and were synthesised as locked nucleic acid (LNA) gapmers to enable inhibition of rne mRNA translation through two mechanisms. Either LNA gapmer binding could sterically block translation and/or LNA gapmer binding could facilitate RNase H-mediated cleavage of the rne mRNA. This may prove to be an advantage over the majority of previous antibacterial antisense oligonucleotide approaches which used oligonucleotide chemistries that restrict the mode-of-action of the antisense oligonucleotide to steric blocking of translation. Using an electrophoretic mobility shift assay, we demonstrate that the LNA gapmers bind to the translation initiation region of E. coli rne mRNA. We then use a cell-free transcription translation reporter assay to show that this binding is capable of inhibiting translation. Finally, in an in vitro RNase H cleavage assay, the LNA gapmers facilitate RNase H-mediated mRNA cleavage. Although the challenges of antisense oligonucleotide delivery remain to be addressed, overall, this work lays the foundations for the development of a novel antibacterial strategy targeting rne mRNA with antisense oligonucleotides.

Rough Carbon-Iron Oxide Nanohybrids for Near-Infrared-II Light-Responsive Synergistic Antibacterial Therapy.

Infections caused by multidrug resistant bacteria are still a serious threat to human health. It is of great significance to explore effective alternative antibacterial strategies. Herein, carbon-iron oxide nanohybrids with rough surfaces (RCF) are developed for NIR-II light-responsive synergistic antibacterial therapy. RCF with excellent photothermal property and peroxidase-like activity could realize synergistic photothermal therapy (PTT)/chemodynamic therapy (CDT) in the NIR-II biowindow with improved penetration depth and low power density. More importantly, RCF with rough surfaces shows increased bacterial adhesion, thereby benefiting both CDT and PTT through effective interaction between RCF and bacteria. In vitro antibacterial experiments demonstrate a broad-spectrum synergistic antibacterial effect of RCF against Gram-negative Escherichia coli (E. coli), Gram-positive Staphylococcus aureus (S. aureus), and methicillin-resistant Staphylococcus aureus (MRSA). In addition, satisfactory biocompatibility makes RCF a promising antibacterial agent. Notably, the synergistic antibacterial performances in vivo could be achieved employing the rat wound model with MRSA infection. The current study proposes a facile strategy to construct antibacterial agents for practical antibacterial applications by the rational design of both composition and morphology. RCF with low power density NIR-II light responsive synergistic activity holds great potential in the effective treatment of drug-resistant bacterial infections.

CuO(1−x)ZnO x nanocomposite with broad spectrum antibacterial activity: application in medical devices and acrylic paints

Inappropriate and disproportionate use of antibiotics have led to a rapid increase in antibacterial resistance. Therefore, alternative antibacterial strategies and solutions are sought to overcome any form of resistance to effectively treat and/or prevent the spread of infections. In this study, we report an eco-friendly and scalable approach to produce highly antibacterial CuO(1−x)ZnO x nanocomposite and its inclusion in medical devices and acrylic paint. Nanocomposite has nanoporous structure composed of primary nanocrystallites of Zn+2 ion doped CuO (∼15 nm) phase and pure ZnO (∼10 nm) phase. Nanocomposite exhibit strong antibacterial activity against broad spectrum of bacteria relevant to the biomedical and food industries. At 100 ppm concentration and 2 h contact period, over 5 log reduction was observed against Escherichia coli, Listeria monocytogenes, Methicillin-resistant Staphylococcus aureus and Salmonella enterica Serovar Typhimurium. Nanocomposite incorporated in medical gauze, topical formulation, and acrylic paint exhibit over 4 log reduction against S. aureus. Bactericidal activity is governed by synergetic combination of electrostatic interaction of nanocomposite with bacterial cell envelope and simultaneous generation of reactive oxygen species. Results described here would be of great benefit in developing medical devices, coatings, and paints to eradicate the growth of a wide range of bacterial pathogens.

Synergistic antibacterial effects of colistin in combination with aminoglycoside, carbapenems, cephalosporins, fluoroquinolones, tetracyclines, fosfomycin, and piperacillin on multidrug resistant Klebsiella pneumoniae isolates.

Multidrug resistant Enterobacterales have become a serious global health problem, with extended hospital stay and increased mortality. Antibiotic monotherapy has been reported ineffective against most drug resistant bacteria including Klebsiella pneumoniae, thus encouraging the use of multidrug therapies as an alternative antibacterial strategy. The present works assessed the antibacterial activity of colistin against K. pneumoniae isolates. Resistant isolates were tested against 16 conventional antibiotics alone and in combination with colistin. The results revealed that all colistin resistant isolates demonstrated multidrug resistance against the tested antibiotics except amikacin. At sub-inhibitory concentrations, combinations of colistin with amikacin, or fosfomycin showed synergism against 72.72% (8 of 11 isolates). Colistin with either of gentamicin, meropenem, cefoperazone, cefotaxime, ceftazidime, moxifloxacin, minocycline, or piperacillin exhibited synergism against 81.82% (9 of 11 isolates). Combinations of colistin with either of tobramycin or ciprofloxacin showed synergism against 45.45% (5 in 11 isolates), while combinations of colistin with imipenem or ceftolozane and tazobactam displayed 36.36% (4 of 11 isolates) and 63.64% (7 of 11 isolates) synergism. In addition, combinations of colistin with levofloxacin was synergistic against 90.91% (10 of 11 isolates). The results revealed that combinations of colistin with other antibiotics could effectively inhibit colistin resistant isolates of K. pneumoniae, and thus could be further explore for the treatment of multidrug resistant pathogens.

EFFECT OF EFFLUX PUMP INHIBITORS ON ANTIMICROBIAL ACTIVITY OF FLORFENICOL AGAINST RESISTANT STRAINS OF PORCINE ACTINOBACILLUS PLEUROPNEUMONIAE AND PASTEURELLA MULTOCIDA

The aim of this study is to investigate whether combinational use of efflux pump inhibitors (EPIs) could improve florfenicol (FF) antimicrobial activity. Five EPIs including Carbonyl Cyanide Chlorophenylhydrazone (CCCP), omeprazole, Phenylalanine-arginine [Formula: see text]-naphthylamide (PA[Formula: see text]N), reserpine and verapamil were individually combined with FF and the minimum inhibitory concentration (MIC) against porcine Actinobacillus pleuropneumoniae and Pasteurella multocida were evaluated by the broth microdilution assay. The results indicated that CCCP demonstrated substantial improvement on the antimicrobial activity (FF MIC reduction [Formula: see text] folds) while PAßN showed minimal effect at high concentrations (80–120[Formula: see text][Formula: see text]g/mL). The MICs of FF were further examined with CCCP at 5[Formula: see text][Formula: see text]g/mL against resistant A. pleuropneumoniae and at 2[Formula: see text][Formula: see text]g/mL against resistant P. multocida. With this combination, a total of 75% (6/8) of A. pleuropneumoniae and 100% (8/8) of P. multocida resistant isolates showed significantly reduced ([Formula: see text] folds) FF MICs. In addition, 100% of the 16 resistant bacterial strains carried the floR gene that regulates the phenicol-specific efflux pump (FloR pump). The time kill experiments were used to verify the effect of CCCP demonstrating a bactericidal effect in resistant A. pleuropneumoniae and synergistic effect in resistant P. multocida. In contrast, the FF MICs were not significantly affected in the FF susceptible strains of these two bacteria. These findings suggested that despite small sample sizes, consistent beneficial effects of CCCP were evident such that the combinational use of selective EPI with FF might be an alternative antibacterial strategy against FF resistant A. pleuropneumoniae and P. multocida. The anti-efflux mechanism was indicated, but more researches to further decipher the mechanisms of CCCP’s effects are warranted.

Copper-Silver Alloy Coated Door Handles as a Potential Antibacterial Strategy in Clinical Settings

Coating surfaces with a copper-silver alloy in clinical settings can be an alternative or complementary antibacterial strategy to other existing technologies and disinfection interventions. A newly developed copper-silver alloy coating has a high antibacterial efficacy against common pathogenic bacteria in laboratory setups, and the purpose of this study is to determine the antibacterial efficacy of this copper-silvery alloy in real-world clinical settings. Two field trials were carried out at a private clinic and a wound care center. Door handles coated with the copper-silver alloy had a lower total aerobic plate count (1.3 ± 0.4 Log CFU/cm2 and 0.8 ± 0.3 Log CFU/cm2, CFU stands for Colony Forming Units) than the reference uncoated material on-site (2.4 ± 0.4 Log CFU/cm2 for the stainless steel and 1.7 ± 0.4 Log CFU/cm2 for the satin brass). The copper-silver alloy did not selectively reduce specific bacterial species. This study points to the possibility of a successful long-term implementation of the copper-silver alloy coating as an antibacterial strategy.

Lysin LysMK34 of Acinetobacter baumannii Bacteriophage PMK34 Has a Turgor Pressure-Dependent Intrinsic Antibacterial Activity and Reverts Colistin Resistance.

The prevalence of extensively and pandrug-resistant strains of Acinetobacter baumannii leaves little or no therapeutic options for treatment for this bacterial pathogen. Bacteriophages and their lysins represent attractive alternative antibacterial strategies in this regard. We used the extensively drug-resistant A. baumannii strain MK34 to isolate the bacteriophage PMK34 (vB_AbaP_PMK34). This phage shows fast adsorption and lacks virulence genes; nonetheless, its narrow host spectrum based on capsule recognition limits broad application. PMK34 is a Fri1virus member of the Autographiviridae and has a 41.8-kb genome (50 open reading frames), encoding an endolysin (LysMK34) with potent muralytic activity (1,499.9 ± 131 U/μM), a typical mesophilic thermal stability up to 55°C, and a broad pH activity range (4 to 10). LysMK34 has an intrinsic antibacterial activity up to 4.8 and 2.4 log units for A. baumannii and Pseudomonas aeruginosa strains, respectively, but only when a high turgor pressure is present. The addition of 0.5 mM EDTA or application of an osmotic shock after treatment can compensate for the lack of a high turgor pressure. The combination of LysMK34 and colistin results in up to 32-fold reduction of the MIC of colistin, and colistin-resistant strains are resensitized in both Mueller-Hinton broth and 50% human serum. As such, LysMK34 may be used to safeguard the applicability of colistin as a last-resort antibiotic.IMPORTANCEA. baumannii is one of the most challenging pathogens for which development of new and effective antimicrobials is urgently needed. Colistin is a last-resort antibiotic, and even colistin-resistant A. baumannii strains exist. Here, we present a lysin that sensitizes A. baumannii for colistin and can revert colistin resistance to colistin susceptibility. The lysin also shows a strong, turgor pressure-dependent intrinsic antibacterial activity, providing new insights in the mode of action of lysins with intrinsic activity against Gram-negative bacteria.

A review of the antimicrobial potential of herbal drugs used in popular Italian medicine (1850s-1950s) to treat bacterial skin diseases.

Before the advent of modern antibiotics, microbial infections were treated with herbal medicine or cauterization. Literature from the latter half of the nineteenth to the early mid-twentieth century, when antibiotics became widely available, arguably holds the most progressive information about herbal remedies to treat bacterial skin diseases. The corpus of literature produced in Italy during that period is not easily accessible and mostly out of print. Plant-based remedies utilized in popular Italian medicine to treat anthrax, boils, erysipelas, impetigo, pustules, and whitlow were sourced from literature indexed in and available through the National Library Service website of the Italian Libraries Network. The remedies are assessed for their antimicrobial potential based on a detailed search of the herbal drug species in scientific databases. A considerable part of the reviewed recipes included specific excipients (41 out of 139) and others were produced with fresh plant material (48 out of 139). Out of the 52 identified herbal drug species used in popular Italian medicine against dermatologic infections, extracts of 43 were shown to have moderate in vitro activity against Gram-positive and Gram-negative bacteria. The antibacterial activity of the extracts and pure compounds as reported in the reviewed literature is mostly based on in vitro assays and generally does not encourage follow up studies. The effectiveness of the reported recipes, which include fresh plant material and excipients can only be assessed through in vivo studies. Those remedies including herbal drugs with reported antimicrobial activity might have the potential as complementary therapies. The reviewed plant based antimicrobial recipes might serve as inspirations in the search for alternative topical antibacterial strategies and the search for their synergistic and potentiating ingredients.

Quorum quenching intervened in vivo attenuation and immunological clearance enhancement by Solanum torvum root extract against Pseudomonas aeruginosa instigated pneumonia in Sprague Dawley rats

BackgroundPseudomonas aeruginosa have been delineated for its disreputable charisma in nosocomial infections leading to pulmonary pneumonia. The recent developments in multi-drug resistance accelerate the need of alternative antibacterial strategies. In this study we analysed the efficacy of quorum quenching mediated bacterial silencing in effective immunological clearance upon pulmonary infected rat models.ResultsQuorum sensing inhibitor was extracted from Solanum torvum which is a native plant in Kerala, India. The in vitro pilot studies upon Pseudomonas aeruginosa strain jb07 revealed the ability of Solanum torvum root extract (STRE) to successfully inhibit quorum sensing controlled virulent factors such as chromogenesis and biofilm formation; though the bacterial growth was unaffected. In vivo analysis upon Sprague Dawley rats shown the potential of STRE in aiding successful bacterial clearance where in cent percentage of survival was evident that compared with untreated group. Subjects treated with STRE possessed natural immunological response with inflammation, MNC infiltration and BALT (bronchus associated lymphoid tissue) hyperplasia unlike the Ciprofloxacin treated comparative group.ConclusionsThe observations of this study suggests that STRE is not having any bactericidal activity rather its silencing the infectant in a system which in turn disables the virulence of bacteria and remain attenuated; therefore immune system get adequate time for successful countermeasures.

Carbon@polypyrrole nanotubes as a photosensitizer in laser phototherapy of Pseudomonas aeruginosa

Phototherapy has been offered as an alternative and promising antibacterial strategy to overcome the antibiotic resistance problem. This study evaluated the antibacterial and phototherapy effects of carbon nanotubes with a polypyrrole coating in a core@shell structure (CNTs@PPy) on Pseudomonas aeruginosa (P. aeruginosa). P. aeruginosa was treated with CNTs@PPy at different concentrations (50–500 μg mL−1) in dark or laser light irradiation with a wavelength of 808 nm, a power density of 1000 mW cm−2 for 20 min. Temperature increment, cell viability, formation of reactive oxygen species (ROS) and protein/nucleic acid leakage subsequent the P. aeruginosa treatment were evaluated. The results showed that near-infrared laser irradiation of CNTs@PPy caused to a temperature increment confirming the ability of powerful photokilling of P. aeruginosa in a photothermal route. On the other hand, while CNTs@PPy represented just a 30–50% P. aeruginosa killing rate in dark, laser irradiation of 250 and 500 μg mL−1 concentrations of CNTs@PPy resulted in a ˜70% P. aeruginosa killing rate, along with significant ROS production into the medium and protein and nucleic acid leakage from P. aeruginosa. These later effects were assigned to a photodynamic route activity of CNTs@PPy upon laser irradiation. Therefore, CNTs@PPy acted as a photosensitizer in both photothermal and photodynamic therapies to present an enhanced bactericidal activity to annihilate and destroyed the gram-negative bacteria P. aeruginosa, a cause of many infectious diseases.

Characterization of Two Pseudomonas aeruginosa Viruses vB_PaeM_SCUT-S1 and vB_PaeM_SCUT-S2.

The sophisticated antibiotic resistance mechanism of Pseudomonas aeruginosa has urged the development of alternative antibacterial strategies. Phage therapy has been proven successful for the treatment of multidrug-resistant infections. In this study, we reported two virulent P. aeruginosa phages, vB_PaeM_SCUT-S1 (S1) and vB_PaeM_SCUT-S2 (S2), which were characterized at morphological, genomic, and proteomic levels. Phages S1 and S2 were assigned to the Myoviridae family. The genome sequencing showed that the genome size of Phage S1 was 66,046 bp and that of Phage S2 was 94,434 bp. The phylogenetic tree indicated that the two phages were distantly related to each other and were classified in the genera Pbunavirus and Pakpunavirus respectively. Thirty-one proteins were identified for each phage by mass spectrometry and were used to substantiate the function of the predicted coding genes. The two phages inhibited the growth of P. aeruginosa strain PAO1 at low multiplicity of infection levels and had good performance both on preventing biofilm formation and eradicating preformed biofilms. They were also stable over a wide range of temperature and pH values, supporting their potential use in the treatment of P. aeruginosa infections.

Activity and Impact on Resistance Development of Two Antivirulence Fluoropyrimidine Drugs in Pseudomonas aeruginosa.

The rise in antibiotic resistance among bacterial pathogens has prompted the exploitation of alternative antibacterial strategies, such as antivirulence therapy. By inhibiting virulence traits, antivirulence drugs are expected to lessen pathogenicity without affecting bacterial growth, therefore avoiding the spread of resistance. However, some studies argued against this assumption, and the lack of antivirulence drugs in clinical use hampers the empirical assessment of this concept. Here we compared the mode of action and range of activity of two drugs which have been proposed for repurposing as quorum sensing and pyoverdine inhibitors in the human pathogen Pseudomonas aeruginosa: the anticancer drug 5-fluorouracil (5-FU) and the antimycotic drug 5-fluorocytosine (5-FC), respectively. The effect on bacterial growth, emergence and spread of resistance, and activity against clinical isolates were assessed. Our results confirm that 5-FU has growth inhibitory activity on reference strains and can rapidly select for spontaneous resistant mutants with loss-of-function mutations in the upp gene, responsible for uracil conversion into UMP. These mutants were also insensitive to the anti-pyoverdine effect of 5-FC. Conversely, 5-FC did not cause relevant growth inhibition, likely because of poor enzymatic conversion into 5-FU by cytosine deaminase. However, coculturing experiments showed that 5-FU resistant mutants can outcompete sensitive cells in mixed populations, in the presence of not only 5-FU but also 5-FC. Moreover, we observed that serial passages of wild-type cells in 5-FC-containing medium leads to the appearance and spread of 5-FC insensitive sub-populations of 5-FU resistant cells. The different effect on growth of 5-FU and 5-FC was overall conserved in a large collection of cystic fibrosis (CF) isolates, corresponding to different infection stages and antibiotic resistance profiles, although high variability was observed among strains. Notably, this analysis also revealed a significant number of pyoverdine-deficient isolates, whose proportion apparently increases over the course of the CF infection. This study demonstrates that the efficacy of an antivirulence drug with no apparent effect on growth can be significantly influenced by the emergence of insensitive mutants, and highlights the importance of the assessment of resistance-associated fitness cost and activity on clinical isolates for the development of “resistance-proof” antivirulence drugs.