<em>Pseudomonas aeruginosa</em> Biofilm Lung Infection in Cystic Fibrosis: The Challenge of Persisters

Pseudomonas aeruginosa lung infection is difficult to eradicate due to the multiple (intrinsic and acquired) antibiotic resistance of bacteria and to their ability to produce a thick biofilm. Antibiotic treatment is hampered by poor antibiotic diffusion, efflux pump overexpression and the development of a persistent subpopulation with low metabolic activity. This is a cause for special concern in Cystic Fibrosis (CF) patients, where P. aeruginosa lung infection is the chief cause of morbidity and mortality. Combined tobramycin-ciprofloxacin treatment is routinely adopted due to the low frequency of resistant strains and its ostensible ability to control the infection. Nevertheless, symptoms usually recur, mainly due to the antibiotic persisters, which are difficult to detect in routine cultural microbiological assays. This chapter describes the issues involved in the microbiological diagnosis of P. aeruginosa lung infection in CF patients and the possible role of subinhibitory antibiotic concentrations in persister development and infection recurrence

Defence strategies and antibiotic resistance gene abundance in enterococci under stress by exposure to low doses of peracetic acid

partially_open12noPeracetic acid (PAA) is an organic compound used efficiently as disinfectant in wastewater treatments. Yet, at low doses it may cause selection; thus, the effect of low doses of PAA on Enterococcus faecium as a proxy of human-related microbial waste was evaluated. Bacteria were treated with increasing doses of PAA (from 0 to 25 mg L1 min) and incubated in regrowth experiments under non-growing, limiting conditions and under growing, favorable conditions. The changes in bacterial abundance, in bacterial phenotype (number and composition of small cell clusters), and in the abundance of an antibiotic resistance gene (ARG) was evaluated. The experiment demonstrated that the selected doses of PAA efficiently removed enterococci, and induced a long-lasting effect after PAA inactivation. The relative abundance of small clusters increased during the experiment when compared with that of the inoculum. Moreover, under growing favorable conditions the relative abundance of small clusters decreased and the number of cells per cluster increased with increasing PAA doses. A strong stability of the measured ARG was found, not showing any effect during the whole experiment. The results demonstrated the feasibility of low doses of PAA to inactivate bacteria. However, the stress induced by PAA disinfection promoted a bacterial adaptation, even if potentially without affecting the abundance of the ARG.openTurolla, Andrea; Sabatino, Raffaella; Fontaneto, Diego; Eckert, Ester M.; Colinas, Noemi; Corno, Gianluca; Citterio, Barbara; Biavasco, Francesca; Antonelli, Manuela; Mauro, Alessandro; Mangiaterra, Gianmarco; Di Cesare, AndreaTurolla, Andrea; Sabatino, Raffaella; Fontaneto, Diego; Eckert, Ester M.; Colinas, Noemi; Corno, Gianluca; Citterio, Barbara; Biavasco, Francesca; Antonelli, Manuela; Mauro, Alessandro; Mangiaterra, Gianmarco; Di Cesare, Andre

Structural Modifications of the Quinolin-4-yloxy Core to Obtain New Staphylococcus aureus NorA Inhibitors

: Tackling antimicrobial resistance (AMR) represents a social responsibility aimed at renewing the antimicrobial armamentarium and identifying novel therapeutical approaches. Among the possible strategies, efflux pumps inhibition offers the advantage to contrast the resistance against all drugs which can be extruded. Efflux pump inhibitors (EPIs) are molecules devoid of any antimicrobial activity, but synergizing with pumps-substrate antibiotics. Herein, we performed an in silico scaffold hopping approach starting from quinolin-4-yloxy-based Staphylococcus aureus NorA EPIs by using previously built pharmacophore models for NorA inhibition activity. Four scaffolds were identified, synthesized, and modified with appropriate substituents to obtain new compounds, that were evaluated for their ability to inhibit NorA and synergize with the fluoroquinolone ciprofloxacin against resistant S. aureus strains. The two quinoline-4-carboxamide derivatives 3a and 3b showed the best results being synergic (4-fold MIC reduction) with ciprofloxacin at concentrations as low as 3.13 and 1.56 µg/mL, respectively, which were nontoxic for human THP-1 and A549 cells. The NorA inhibition was confirmed by SA-1199B ethidium bromide efflux and checkerboard assays against the isogenic pair SA-K2378 (norA++)/SA-K1902 (norA-). These in vitro results indicate the two compounds as valuable structures for designing novel S. aureus NorA inhibitors to be used in association with fluoroquinolones

Unravelling antibiotic resistance and persistence of Pseudomonas aeruginosa cystic fibrosis pulmonary infections

L’infezione polmonare da Pseudomonas aeruginosa, caratterizzata da cronicità e ricorrenti infiammazioni polmonari, costituisce la principale causa di mortalità nella fibrosi cistica. Nonostante ne siano note le principali dinamiche, alcuni aspetti microbiologici devono essere ancora descritti nel dettaglio, in particolare: la presenza di eventuali forme batteriche persistenti e dormienti, probabile causa di infezioni ricorrenti, e l’identificazione di fattori e pattern genetici coinvolti nella loro induzione e mantenimento. Altre problematiche non ancora chiarite risultano la caratterizzazione dei principali meccanismi di antibioticoresistenza e l’identificazione di possibili inibitori, al fine di sviluppare trattamenti antibiotici in grado di contrastare l’insorgenza di ceppi multiresistenti. Il presente lavoro di tesi ha permesso lo studio e l’approfondimento di tali tematiche, fornendo i seguenti risultati: I) lo sviluppo di un efficiente ed affidabile protocollo di diagnosi molecolare, in grado di rilevare P. aeruginosa e le possibili forme vitali ma non coltivabili, in campioni di espettorato di pazienti affetti da fibrosi cistica; II) l’induzione di tali forme in modelli in vitro di biofilm, soggetti a concentrazioni subinibenti di farmaci comunemente utilizzati nel trattamento delle infezioni da P. aeruginosa; III) lo studio della regolazione genica nell’insorgenza di popolazioni batteriche persistenti; IV) la caratterizzazione delle pompe di efflusso come determinanti di antibioticoresistenza in P. aeruginosa e l’identificazione di tre possibili inibitori dei principali sistemi di efflusso del patogeno, da usare in combinazione con antibiotici. I dati ottenuti forniscono nuove informazioni sulle dinamiche dell’infezione da P. aeruginosa e permettono lo sviluppo di protocolli terapeutici adeguati a contrastare lo sviluppo e la persistenza del patogeno, assicurando una migliore aspettativa di vita ai pazienti affetti da fibrosi cistica.Pseudomonas aeruginosa pulmonary infection, often chronic and characterized by recurrent exacerbations, is the main cause of mortality in cystic fibrosis. Although its main dynamics have been defined, many features still represent a riddle for the microbiological research, in particular: the insurgence of possible persister or dormant bacterial forms, possibly involved in the infection recurrence, and the identification of their inducing factors and regulatory genetic pathways; the characterization of the main antibiotic resistance mechanisms and of their putative inhibitors, to develop novel antibiotic treatments against multidrug resistant P. aeruginosa strains. This thesis project has allowed the investigation of these topics, providing the following results: I) the development of an efficient and reliable molecular diagnostic protocol, able to detect P. aeruginosa presence, even the viable but non-culturable forms, in cystic fibrosis sputum samples; II) these forms have been subsequently induced in in vitro P. aeruginosa biofilms, exposed to subinhibitory concentrations of antibiotics usually adopted in P. aeruginosa infections treatment; III) the study of the role of specific regulatory genetic pathways in the formation of bacterial persister cell forms, responsible for the infection recurrence; IV) the definition of the contribution of efflux pumps to P. aeruginosa antibiotic resistance and the identification of 3 putative efflux pumps inhibitors, to be used in combination with antibiotics against the main efflux systems of this pathogen in multidrug resistant strains. The obtained data have provided new insights on the dynamics of P. aeruginosa pulmonary infections in cystic fibrosis and allow the development of new therapeutic protocols, to limit the pathogen infectiveness and mortality and to assure a better life expectansy to cystic fibrosis patients

A Fluorinated Analogue of Marine Bisindole Alkaloid 2,2-Bis(6-bromo-1H-indol-3-yl)ethanamine as Potential Anti-Biofilm Agent and Antibiotic Adjuvant Against Staphylococcus aureus

Methicillin resistant Staphylococcus aureus (MRSA) infections represent a major global healthcare problem. Therapeutic options are often limited by the ability of MRSA strains to grow as biofilms on medical devices, where antibiotic persistence and resistance is positively selected, leading to recurrent and chronic implant-associated infections. One strategy to circumvent these problems is the co-administration of adjuvants, which may prolong the efficacy of antibiotic treatments, by broadening their spectrum and lowering the required dosage. The marine bisindole alkaloid 2,2-bis(6-bromo-1H-indol-3-yl)ethanamine (1) and its fluorinated analogue (2) were tested for their potential use as antibiotic adjuvants and antibiofilm agents against S. aureus CH 10850 (MRSA) and S. aureus ATCC 29213 (MSSA). Both compounds showed antimicrobial activity and bisindole 2 enabled 256-fold reduction (ΣFICs = 0.5) in the minimum inhibitory concentration (MIC) of oxacillin for the clinical MRSA strain. In addition, these molecules inhibited biofilm formation of S. aureus strains, and compound 2 showed greater eradicating activity on preformed biofilm compared to 1. None of the tested molecules exerted a viable but non-culturable cells (VBNC) inducing effect at their MIC values. Moreover, both compounds exhibited no hemolytic activity and a good stability in plasma, indicating a non-toxic profile, hence, in particular compound 2, a potential for in vivo applications to restore antibiotic treatment against MRSA infections

Antibiotic and heavy metal resistance in enterococci from coastal marine sediment

Sediment samples from three coastal sites - two beach resorts (Beach 1 and Beach 2 sites) and an area lying between an oil refinery and a river estuary (Estuarine site) - were analyzed for antibiotic- and heavy metal (HM)-resistant enterococci. A total of 123 enterococci, 36 E. faecium, 34 E. casseliflavus, 33 E. hirae, 5 E. faecalis, 3 E. durans, 3 E. gallinarum, and 9 Enterococcus spp, were recovered. Strains resistant to erythromycin, tetracycline and quinupristin/dalfopristin (Q/D) were recovered from all sites, whereas multidrug-resistant isolates were recovered only from “Beach 2” (14%) and “Estuarine” (3.7%). As regards HM resistance, the strains showed a high frequency (68%) of cadmium and/or copper resistance and uniform susceptibility to mercury. The prevalence of cadmium-resistant strains was significantly higher among erythromycin-resistant than among erythromycin-susceptible strains. A significant association between cadmium or copper resistance and Q/D resistance was also observed at “Estuarine” site. The levels of the two HMs in sediment from all sites were fairly low, ranging from 0.070 to 0.126 μg/g, for cadmium and from 1.00 to 7.64 μg/g for copper. Mercury was always undetectable. These findings are consistent with reports that low HM concentrations may contribute to co-selection of antibiotic-resistant bacterial strains, including enterococci

Contribution of Drugs Interfering with Protein and Cell Wall Synthesis to the Persistence of <em>Pseudomonas aeruginosa</em> Biofilms: An In Vitro Model

The occurrence of Pseudomonas aeruginosa (PA) persisters, including viable but non-culturable (VBNC) forms, subpopulations of tolerant cells that can survive high antibiotic doses, is the main reason for PA lung infections failed eradication and recurrence in Cystic Fibrosis (CF) patients, subjected to life-long, cyclic antibiotic treatments. In this paper, we investigated the role of subinhibitory concentrations of different anti-pseudomonas antibiotics in the maintenance of persistent (including VBNC) PA cells in in vitro biofilms. Persisters were firstly selected by exposure to high doses of antibiotics and their abundance over time evaluated, using a combination of cultural, qPCR and flow cytometry assays. Two engineered GFP-producing PA strains were used. The obtained results demonstrated a major involvement of tobramycin and bacterial cell wall-targeting antibiotics in the resilience to starvation of VBNC forms, while the presence of ciprofloxacin and ceftazidime/avibactam lead to their complete loss. Moreover, a positive correlation between tobramycin exposure, biofilm production and c-di-GMP levels was observed. The presented data could allow a deeper understanding of bacterial population dynamics during the treatment of recurrent PA infections and provide a reliable evaluation of the real efficacy of the antibiotic treatments against the bacterial population within the CF lung

Properties of an innovative multi-functional finish for the improvement of indoor air quality

Due to lifestyle changes, people spend most of their time indoors at present; thus, Indoor Air Quality (IAQ) is a matter of utmost importance. Multi-functional and innovative finishes can help to passively improve the IAQ, benefitting the health and comfort of occupants. For this study, reference and pre-mixed commercial mortars are compared to a new multi-functional hydraulic lime mortar for indoor finishes, in which conventional aggregates are substituted by a highly porous adsorbent material and biomass waste ashes. The up to 20% higher accessible porosity of the multi-functional finish led to lower density (30%), higher thermal insulation properties (30%), higher water vapor permeability (more than 40%), and improved moisture buffering capacity (three times higher), when compared to the reference mortar. Different types of photocatalytic agents (TiO2) were also added into the new multi-functional hydraulic lime mortar, in order to investigate their effect on the de-polluting properties of the finish. Even if the photocatalytic efficiency remained unexpressed under indoor conditions, due to the predominance of the adsorption process, the de-polluting properties of the new mix were more than 30% higher than that of the reference mortar. The obtained results confirm that the developed innovative multifunctional finish—besides fulfilling the ordinary requirements—is better than commercial mortars, as it can improve the IAQ passively, thus benefitting the health and comfort of occupants