Activity of N-Acetylcysteine Alone and in Combination with Colistin against Pseudomonas aeruginosa Biofilms and Transcriptomic Response to N-Acetylcysteine Exposure.

Chronic colonization by Pseudomonas aeruginosa is critical in cystic fibrosis (CF) and other chronic lung diseases, contributing to disease progression. Biofilm growth and a propensity to evolve multidrug resistance phenotypes drastically limit the available therapeutic options. In this perspective, there has been growing interest in evaluating combination therapies, especially for drugs that can be administered by nebulization, which allows high drug concentrations to be reached at the site of infections while limiting systemic toxicity. Here, we investigated the potential antibiofilm activity of N-acetylcysteine (NAC) alone and in combination with colistin against a panel of P. aeruginosa strains (most of which are from CF patients) and the transcriptomic response of a P. aeruginosa CF strain to NAC exposure. NAC alone (8,000 mg/L) showed a limited and strain-dependent antibiofilm activity. Nonetheless, a relevant antibiofilm synergism of NAC-colistin combinations (NAC at 8,000 mg/L plus colistin at 2 to 32 mg/L) was observed with all strains. Synergism was also confirmed with the artificial sputum medium model. RNA sequencing of NAC-exposed planktonic cultures revealed that NAC (8,000 mg/L) mainly induced (i) a Zn2+ starvation response (known to induce attenuation of P. aeruginosa virulence), (ii) downregulation of genes of the denitrification apparatus, and (iii) downregulation of flagellar biosynthesis pathway. NAC-mediated inhibition of P. aeruginosa denitrification pathway and flagellum-mediated motility were confirmed experimentally. These findings suggested that NAC-colistin combinations might contribute to the management of biofilm-associated P. aeruginosa lung infections. NAC might also have a role in reducing P. aeruginosa virulence, which could be relevant in the very early stages of lung colonization. IMPORTANCE Pseudomonas aeruginosa biofilm-related chronic lung colonization contributes to cystic fibrosis (CF) disease progression. Colistin is often a last-resort antibiotic for the treatment of such P. aeruginosa infections, and it has been increasingly used in CF, especially by nebulization. N-acetylcysteine (NAC) is a mucolytic agent with antioxidant activity, commonly administered with antibiotics for the treatment of lower respiratory tract infections. Here, we show that NAC potentiated colistin activity against in vitro biofilms models of P. aeruginosa strains, with both drugs tested at the high concentrations achievable after nebulization. In addition, we report the first transcriptomic data on the P. aeruginosa response to NAC exposure.

Relevant increase of CTX-M-producing Escherichia coli carriage in school-aged children from rural areas of the Bolivian Chaco in a three-year period.

OBJECTIVES: The aim of this study was to perform two cross-sectional surveys on the fecal carriage of CTX-M-producing Enterobacterales in school-aged children from rural areas of the Bolivian Chaco (2016 vs 2019). METHODS: A total of 757 fecal samples were collected from school-aged children living in nine indigenous communities (n=337, 2016; n=420, 2019). After a first passage onto MacConkey agar (MCA), samples were plated onto MCA plus cefotaxime 2 µg/mL (MCA-CTX), and a loopful of the bacterial growth was used as a template for the detection of group 1, 2, 8/25, and 9 blaCTX-M variants by multiplex reverse transcriptase polymerase chain reaction . Positive samples were tested again for detecting, identifying, and characterizing CTX-M-positive isolates. RESULTS: Growth onto MCA-CTX was obtained with 208 samples (27.5%; 62/337, 2016; 146/420, 2019), of which 201 (96.6%) were positive for blaCTX-M genes. Overall, a relevant increase of fecal carriage of CTX-M-producing Enterobacterales was observed in the study period: 17,5% (59/337) in 2016 compared with 33,8% (142/420) in 2019, p<0.01. Nonetheless, the relative group distribution of CTX-M groups remained stable, with group 1 being the prevalent, followed by group 9 and group 8/25. Group 2 was not detected. CONCLUSIONS: The present study demonstrated an alarming spread of CTX-M enzymes in rural areas of the Bolivian Chaco, where antibiotics consumption is limited. Further studies are encouraged to better understand the dissemination dynamics of such relevant resistance determinants.

Activity of fosfomycin/colistin combinations against planktonic and biofilm Gram-negative pathogens.

OBJECTIVES: To investigate the in vitro activity of fosfomycin, colistin and combinations thereof against planktonic and biofilm cultures of Gram-negative pathogens, mostly showing MDR phenotypes, at concentrations achievable via inhalation of aerosolized drugs. METHODS: Activity against planktonic cultures was tested by the chequerboard assay with 130 strains, including 52 Pseudomonas aeruginosa, 47 Klebsiella pneumoniae, 19 Escherichia coli, 7 Stenotrophomonas maltophilia and 5 Acinetobacter baumannii. Activity against biofilm cultures was tested by biofilm chequerboard and quantitative antibiofilm assays with a subset of 20 strains. In addition, 10 of these strains were tested in mutant prevention concentration (MPC) assays. RESULTS: Against planktonic cultures, synergism between fosfomycin and colistin was detected with a minority (10%) of strains (eight K. pneumoniae and five P. aeruginosa), while antagonism was never observed. Synergism between fosfomycin and colistin against biofilms was observed with the majority of tested strains (16/20 in biofilm chequerboard assays, and 18/20 in the quantitative antibiofilm assays), including representatives of each species and regardless of their resistance genotype or phenotype. Furthermore, combination of fosfomycin and colistin was found to significantly reduce the MPC of individual drugs. CONCLUSIONS: Fosfomycin and colistin in combination, at concentrations achievable via inhalation of nebulized drugs, showed notable synergy against MDR Gram-negative pathogens grown in biofilm, and were able to reduce the emergence of fosfomycin- and colistin-resistant subpopulations.

In Vitro Synergism of Colistin and N-acetylcysteine against Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is an emerging global opportunistic pathogen, responsible for a wide range of human infections, including respiratory tract infections. Intrinsic multidrug resistance and propensity to form biofilms make S. maltophilia infections recalcitrant to treatment. Colistin is among the second-line options in case of difficult-to-treat S. maltophilia infections, with the advantage of being also administrable by nebulization. We investigated the potential synergism of colistin in combination with N-acetylcysteine (NAC) (a mucolytic agent with antioxidant and anti-inflammatory properties) against S. maltophilia grown in planktonic phase and biofilm. Eighteen S. maltophilia clinical isolates (comprising three isolates from cystic fibrosis (CF) and two trimethoprim-sulfamethoxazole (SXT)-resistant strains) were included. Checkerboard assays showed a synergism of colistin/NAC combinations against the strains with colistin Minimum Inhibitory Concentration (MIC) >2 µg/mL (n = 13), suggesting that NAC could antagonize the mechanisms involved in colistin resistance. Nonetheless, time-kill assays revealed that NAC might potentiate colistin activity also in case of lower colistin MICs. A dose-dependent potentiation of colistin activity by NAC was also clearly observed against S. maltophilia biofilms, also at sub-MIC concentrations. Colistin/NAC combinations, at concentrations likely achievable by topical administration, might represent a valid option for the treatment of S. maltophilia respiratory infections and should be examined further.

Tailored Ag-Cu-Mg multielemental nanoparticles for wide-spectrum antibacterial coating.

Bactericidal nanoparticle coatings are very promising for hindering the indirect transmission of pathogens through cross-contaminated surfaces. The challenge, limiting their employment in nosocomial environments, is the ability of tailoring the coating's physicochemical properties, namely, composition, cytotoxicity, bactericidal spectrum, adhesion to the substrate, and consequent nanoparticles release into the environment. We have engineered a new family of nanoparticle-based bactericidal coatings comprising Ag, Cu, and Mg and synthesized by a green gas-phase technique. These coatings present wide-spectrum bactericidal activity on both Gram-positive and Gram-negative reference strains and tunable physicochemical properties of relevance in view of their "on-field" deployment. The link between material and functional properties is rationalized based on a multidisciplinary and multitechnique approach. Our results pave the way for engineering biofunctional, fully tunable nanoparticle coatings, exploiting an arbitrarily wide number of elements in a straightforward, eco-friendly, high-throughput, one-step process.

In vitro synergism of colistin in combination with N-acetylcysteine against Acinetobacter baumannii grown in planktonic phase and in biofilms.

Objectives: To investigate the potential synergism of colistin in combination with N-acetylcysteine against Acinetobacter baumannii strains grown in planktonic phase or as biofilms. Methods: Sixteen strains were investigated, including nine colistin-susceptible (MIC range 0.5-1 mg/L) and seven colistin-resistant (MIC range 16-256 mg/L) strains. Synergism of colistin in combination with N-acetylcysteine was investigated by chequerboard assays. The activity of colistin/N-acetylcysteine combinations was further evaluated by time-kill assays with planktonic cultures (three colistin-resistant strains and one colistin-susceptible strain) and by in vitro biofilm models (three colistin-resistant and three colistin-susceptible strains). Results: Chequerboard assays revealed a relevant synergism of colistin/N-acetylcysteine combinations with all colistin-resistant strains, whereas no synergism was observed with colistin-susceptible strains. Time-kill assays showed a concentration-dependent potentiation of colistin activity by N-acetylcysteine against colistin-resistant strains, with eradication of the culture by combinations of N-acetylcysteine at 8000 mg/L plus colistin at 2 or 8 mg/L. A static effect during the first 8 h of incubation was demonstrated with the colistin-susceptible strain exposed to 0.25 × MIC colistin plus 8000 mg/L N-acetylcysteine. A remarkable antibiofilm synergistic activity of 8 mg/L colistin plus 8000 mg/L N-acetylcysteine was demonstrated with all colistin-resistant and colistin-susceptible strains. The effects were greater with colistin-resistant strains (marked reduction of viable biofilm cells was observed at sub-MIC colistin concentrations). Conclusions: N-acetylcysteine, at concentrations achievable by topical administration, was shown to revert the colistin-resistant phenotype in A. baumannii, and to exert a relevant activity against biofilms of colistin-susceptible and colistin-resistant A. baumannii strains.