Potential novel therapeutic strategies in cystic fibrosis: antimicrobial and anti-biofilm activity of natural and designed α-helical peptides against Staphylococcus aureus, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia.

BACKGROUND: Treatment of cystic fibrosis-associated lung infections is hampered by the presence of multi-drug resistant pathogens, many of which are also strong biofilm producers. Antimicrobial peptides, essential components of innate immunity in humans and animals, exhibit relevant in vitro antimicrobial activity although they tend not to select for resistant strains. RESULTS: Three α-helical antimicrobial peptides, BMAP-27 and BMAP-28 of bovine origin, and the artificial P19(9/B) peptide were tested, comparatively to Tobramycin, for their in vitro antibacterial and anti-biofilm activity against 15 Staphylococcus aureus, 25 Pseudomonas aeruginosa, and 27 Stenotrophomonas maltophilia strains from cystic fibrosis patients. All assays were carried out in physical-chemical experimental conditions simulating a cystic fibrosis lung. All peptides showed a potent and rapid bactericidal activity against most P. aeruginosa, S. maltophilia and S. aureus strains tested, at levels generally higher than those exhibited by Tobramycin and significantly reduced biofilm formation of all the bacterial species tested, although less effectively than Tobramycin did. On the contrary, the viability-reducing activity of antimicrobial peptides against preformed P. aeruginosa biofilms was comparable to and, in some cases, higher than that showed by Tobramycin. CONCLUSIONS: The activity shown by α-helical peptides against planktonic and biofilm cells makes them promising "lead compounds" for future development of novel drugs for therapeutic treatment of cystic fibrosis lung disease.

Phenotypic and genotypic characterization of Stenotrophomonas maltophilia isolates from patients with cystic fibrosis: genome diversity, biofilm formation, and virulence.

BACKGROUND: Stenotrophomonas maltophilia is emerging as one of the most frequently found bacteria in cystic fibrosis (CF) patients. In the present study, phenotypic and genotypic traits of a set of 98 isolates of S. maltophilia obtained from clinical (CF and non-CF patients) and environmental sources were comparatively evaluated. RESULTS: S. maltophilia exhibited a high level of genomic diversity in both CF and non-CF group, thus possibly allowing this bacterium to expand its pathogenic potentials. Strains sharing the same pulsotype infected different patients, thus likely indicating the occurrence of clonal spread or acquisition by a common source. CF isolates differed greatly in some phenotypic traits among each other and also when compared with non-CF isolates, demonstrating increased mean generation time and susceptibility to oxidative stress, but reduced ability in forming biofilm. Furthermore, in CF isolates flagella- and type IV pili-based motilities were critical for biofilm development, although not required for its initiation. Sequential isogenic strains isolated from the same CF patient displayed heterogeneity in biofilm and other phenotypic traits during the course of chronic infection. CF and non-CF isolates showed comparable virulence in a mouse model of lung infection. CONCLUSIONS: Overall, the phenotypic differences observed between CF and non-CF isolates may imply different selective conditions and persistence (adaptation) mechanisms in a hostile and heterogeneous environment such as CF lung. Molecular elucidation of these mechanisms will be essential to better understand the selective adaptation in CF airways in order to design improved strategies useful to counteract and eradicate S. maltophilia infection.

Rapid ESBL NP Test for Rapid Detection of Expanded-Spectrum ß-Lactamase Producers in Enterobacterales.

Objectives: To evaluate the biochemical Rapid ESBL NP® (Liofilchem, Italy) for the rapid detection of extended-spectrum ß-lactamases (ESBLs) in Enterobacterales. Methods: A total of 100 clinical gram-negative strains (40 ESBL producers with or without cephalosporinase, 43 carbapenemase producers with or without additional ESBLs, 8 AmpC-type producers, 6 penicillinase producers, and 3 non-ß-lactamase producers) were tested using this colorimetric technique. Results: The overall sensitivity and specificity of the test were found to be 93.9% and 98.5%, respectively. This test is rapid (turn-around-time of 40-45 minutes), easy to perform, and reliable for identification of ESBL producers. Conclusion: The Rapid ESBL NP test allows differentiation between ESBL producers on one hand, and non-ESBL producers or isolates expressing combined mechanisms of resistance on the other hand.

RapidResa Polymyxin Acinetobacter NP® Test for Rapid Detection of Polymyxin Resistance in Acinetobacter baumannii.

A homemade and culture-based test, relying on the visual detection of the reduction of the resazurin reagent (a cell viability indicator), has been developed for the rapid detection of polymyxin resistance in Acinetobacter baumannii. Here, we evaluated the industrial version of this test, the RapidResa Polymyxin Acinetobacter NP® test (Liofilchem, Italy). A well-characterized panel of 68 clinical A. baumannii strains (36 polymyxin-susceptible, 26 polymyxin-resistant A. baumannii, and 6 colistin-heteroresistant isolates) of worldwide origin was tested. All the colistin-susceptible A. baumannii isolates gave negative results according to the RapidResa Polymyxin Acinetobacter NP® test, except for a single isolate that gave a false-positive result. Out of the 26 colistin-resistant A. baumannii strains, 25 were correctly identified as colistin resistant using the RapidResa Polymyxin Acinetobacter NP® test. Only a single colistin-resistant A. baumannii strain gave a false-negative result. Additionally, the six colistin-heteroresistant isolates tested gave positive results. Altogether, the sensitivity and the specificity of the test were found to be 96% and 97%, respectively. The turn-around-time of this easy-to-perform test was 3-4h, which showed excellent reliability for identification of polymyxin resistance in A. baumannii. The RapidResa Polymyxin Acinetobacter NP® test allows a rapid differentiation between polymyxin-susceptible and -resistant A. baumannii isolates, which may contribute to optimization of the use of polymyxins for treating infections due to multidrug-resistant A. baumannii.

Evaluation of SuperCAZ/AVI® Medium for Screening Ceftazidime-avibactam Resistant Gram-negative Isolates.

The industrial version of SuperCAZ/AVI® medium developed for screening CAZ/AVI resistant Gram-negative isolates has been evaluated here using a collection of 87 well-characterized clinical isolates of worldwide origin. In addition, testing was performed by spiking stools with a series of resistant and susceptible isolates. In those conditions, the SuperCAZ/AVI® medium exhibited a sensitivity and specificity of 100 %, down to the lower limit of detection of 101 to 102 CFU/ml. The SuperCAZ/AVI® medium is a sensitive and specific screening medium for detection of CZA-resistant bacteria regardless of their resistance mechanisms.

Gold standard susceptibility testing of fosfomycin in Staphylococcus aureus and Enterobacterales using a new agar dilution panel®.

OBJECTIVES: Many clinical laboratories have difficulty in routinely performing in vitro fosfomycin susceptibility testing using the agar dilution (AD) method, considered to be the gold standard method. The objective of our work was to evaluate a rapid commercial fosfomycin agar dilution panel against clinical Staphylococcus aureus and Enterobacterales strains, in two different centres located in Italy and in the UK. METHODS: A total of 99 Enterobacterales (mostly Escherichia coli and Klebsiella pneumoniae) and 80 S. aureus clinical isolates was used to evaluate the commercial device, a 12-well panel containing fosfomycin incorporated into CA-MH agar supplemented with 25mg/L of glucose-6-phosphate (Liofilchem S.r.l., Roseto degli Abruzzi, Italy). Testing was performed in two centres (Italy and UK) and kit results were compared against the gold standard in-house AD MIC method. RESULTS: According to the EUCAST breakpoints, fosfomycin inhibited 61% of the S. aureus strains, and 76% of the Enterobacterales isolates tested by the AD reference method. There was a Categorical Agreement (CA) of 100% and an Essential Agreement (EA) of 91.25% for S. aureus; while the Enterobacterales strains showed a CA of 94% and an EA of 97%. No evaluation errors were observed among S. aureus, while 5% Major Error and 1% Very Major Error were observed for the Enterobacterales. CONCLUSIONS: Our results confirmed the feasibility of determining fosfomycin susceptibility using a commercial AD panel as a routine substitution for the AD test. The few differences observed were only in strains with MICs around the breakpoint used.

Evaluation of antibacterial and antibiofilm mechanisms by usnic acid against methicillin-resistant Staphylococcus aureus.

AIM: To evaluate the antibacterial and antibiofilm mechanisms of usnic acid (USN) against methicillin-resistant Staphylococcus aureus from cystic fibrosis patients. MATERIALS & METHODS: The effects exerted by USN at subinhibitory concentrations on S. aureus Sa3 strain was evaluated by proteomic, real-time PCR and electron microscopy analyses. RESULTS & CONCLUSION: Proteomic analysis showed that USN caused damage in peptidoglycan synthesis, as confirmed by microscopy. Real-time PCR analysis showed that antibiofilm activity of USN is mainly due to impaired adhesion to the host matrix binding proteins, and decreasing lipase and thermonuclease expression. Our data show that USN exerts anti-staphylococcal effects through multitarget inhibitory effects, thus confirming the rationale for considering it 'lead compound' for the treatment of cystic fibrosis infections.

In vitro activity of colistin against biofilm by Pseudomonas aeruginosa is significantly improved under "cystic fibrosis-like" physicochemical conditions.

The impact of physicochemical conditions observed in cystic fibrosis (CF) lung on colistin activity against both planktonic and biofilm P. aeruginosa cells was evaluated. MIC, minimum bactericidal concentration (MBC), and minimum biofilm eradication concentration (MBEC) values were assessed against 12 CF strains both under "CF-like" (anaerobiosis, pH6.4) and "standard" (aerobiosis, pH7.4) conditions. The activity of colistin was significantly higher under "CF-like" conditions compared to "standard" ones, both against planktonic (MIC90: 1 and 4 µg/mL, respectively) and biofilm (MBEC90: 512 and 1.024 µg/mL, respectively) cells, as confirmed by scanning electron microscopy. Improved activity was not related to biofilm matrix amount. It may be necessary to adequately "rethink" the protocols used for in vitro assessment of colistin activity, by considering physicochemical and microbiological features in the CF lung at the site of infection. This could provide a more favorable therapeutic index, rationale for administration of lower doses, probably resulting in reduced toxicity and emergence of resistant clones.

Antimicrobial and antibiofilm activity of secondary metabolites of lichens against methicillin-resistant Staphylococcus aureus strains from cystic fibrosis patients.

AIM: Three secondary metabolites of lichens - usnic acid, atranorin and fumarprotocetraric acid - were evaluated for their in vitro antibacterial and antibiofilm activities against three strains each of methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MRSA) from cystic fibrosis patients. MATERIALS & METHODS: Antibacterial activity was assessed by broth microdilution, while antibiofilm activity was evaluated by spectrophotometry or viable count. RESULTS: Usnic acid was significantly more active than atranorin against planktonic cells, while fumarprotocetraric acid exhibited no activity. Atranorin was the most effective in counteracting adhesion to polystyrene, although usnic acid was more active against MRSA. Usnic acid and atranorin showed comparable activity against biofilm formation, although atranorin was more active against MRSA. Usnic acid was significantly more active than atranorin against preformed biofilms. CONCLUSION: Secondary metabolites of lichens may be considered to be 'lead compounds' for the development of novel molecules for the treatment of S. aureus infections in cystic fibrosis patients.