Involvement of Acquired Tobramycin Resistance in the Shift to the Viable but Non-Culturable State in Pseudomonas aeruginosa.

Persistent and viable but non-culturable (VBNC) Pseudomonas aeruginosa cells are mainly responsible for the recurrence and non-responsiveness to antibiotics of cystic fibrosis (CF) lung infections. The sub-inhibitory antibiotic concentrations found in the CF lung in between successive therapeutic cycles can trigger the entry into the VBNC state, albeit with a strain-specific pattern. Here, we analyzed the VBNC cell induction in the biofilms of two CF P. aeruginosa isolates, exposed to starvation with/without antibiotics, and investigated the putative genetic determinants involved. Total viable bacterial cells were quantified by the validated ecfX-targeting qPCR protocol and the VBNC cells were estimated as the difference between qPCR and cultural counts. The isolates were both subjected to whole genome sequencing, with attention focused on their carriage of aminoglycoside resistance genes and on identifying mutated toxin-antitoxin and quorum sensing systems. The obtained results suggest the variable contribution of different antibiotic resistance mechanisms to VBNC cell abundance, identifying a major contribution from tobramycin efflux, mediated by MexXY efflux pump overexpression. The genome analysis evidenced putative mutation hotspots, which deserve further investigation. Therefore, drug efflux could represent a crucial mechanism through which the VBNC state is entered and a potential target for anti-persistence strategies.

Efficacy of UV and UV-LEDs Irradiation Models for Microbial Inactivation Applicable to Automated Sterile Drug Compounding.

LEDs development has attracted attention over conventional mercury lamps for the tiny size, high efficiency, long lifetime, low operating temperature. The antimicrobial effectiveness of traditional UV-lamps radiation (wavelength of 254 nm) compared to UV-C LEDs (LED1 wavelength range 275-286 nm and LED2 range 260-270 nm) was carried out, for possible applications to automated sterile drug compounding. The UV lamp and the tested UV-LED devices remarkably reduced microbial load, following a time-dose response, but the best performance was evidenced by LED1, which guaranteed the complete inactivation of high concentrations of bacteria, yeasts, and spores at doses between 200 and 2000 J/m2.

Alkyl-quinolone-dependent quorum sensing controls prophage-mediated autolysis in Pseudomonas aeruginosa colony biofilms.

Pseudomonas aeruginosa is a model quorum sensing (QS) pathogen with three interconnected QS circuits that control the production of virulence factors and antibiotic tolerant biofilms. The pqs QS system of P. aeruginosa is responsible for the biosynthesis of diverse 2-alkyl-4-quinolones (AQs), of which 2-heptyl-4-hydroxyquinoline (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) function as QS signal molecules. Transcriptomic analyses revealed that HHQ and PQS influenced the expression of multiple genes via PqsR-dependent and -independent pathways whereas 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) had no effect on P. aeruginosa transcriptome. HQNO is a cytochrome bc 1 inhibitor that causes P. aeruginosa programmed cell death and autolysis. However, P. aeruginosa pqsL mutants unable to synthesize HQNO undergo autolysis when grown as colony biofilms. The mechanism by which such autolysis occurs is not understood. Through the generation and phenotypic characterization of multiple P. aeruginosa PAO1 mutants producing altered levels of AQs in different combinations, we demonstrate that mutation of pqsL results in the accumulation of HHQ which in turn leads to Pf4 prophage activation and consequently autolysis. Notably, the effect of HHQ on Pf4 activation is not mediated via its cognate receptor PqsR. These data indicate that the synthesis of HQNO in PAO1 limits HHQ-induced autolysis mediated by Pf4 in colony biofilms. A similar phenomenon is shown to occur in P. aeruginosa cystic fibrosis (CF) isolates, in which the autolytic phenotype can be abrogated by ectopic expression of pqsL.

Boosting lung accumulation of gallium with inhalable nano-embedded microparticles for the treatment of bacterial pneumonia.

The potential of intra-venous gallium nitrate (GaN) administration against Pseudomonas aeruginosa pneumonia was recently demonstrated in mice and in cystic fibrosis (CF) patients. Likewise, the added value of direct lung delivery of Ga(III) has been shown in rats. Therefore, the design of a drug delivery system specifically engineered for Ga(III) inhalation is imperative to improve its accumulation in lungs. To this purpose, Ga(III) was efficiently encapsulated into hyaluronic acid/chitosan nanoparticles (Ga_HA/CS NPs), whose features were tuned to facilitate access to the target by overcoming mucus and biofilm surrounding bacteria. Then, to improve in vivo lung deposition, Ga_HA/CS NPs were engineered into mannitol-based NEM (Ga_Man NEM). The powders showed optimal in vitro aerosol performance, and sustained release kinetics in lung lining fluids. Moreover, good tolerability and antimicrobial properties were shown in vitro. Intratracheal insufflation of Ga_Man NEM in rats resulted in a significant improvement of Ga(III) persistence in the lungs coupled to a lower Ga(III) concentration in plasma and urine, compared to GaN solution. Noteworthy, the developed formulation significantly modifies the unfavorable Ga(III) kinetic increasing the Ga(III) to the lung and preventing Ga(III) accumulation in the kidney, key responsible for adverse effects, conclusively demonstrating the benefit of Ga_Man NEM to exploit the therapeutic effect of Ga(III) via inhalation route.

Class 1 integron and Enterococcus spp. abundances in swine farms from the " Suckling piglets" to the "Fatteners" production category.

Swine farms are considered a hotspot of antimicrobial resistance and may contribute to the spread of antibiotic-resistant and/or pathogenic bacteria into the environment as well as to farm workers. In this study, swine fecal samples have been collected over the primary production, selecting three categories, i.e., "Suckling piglets", "Weaning pigs" and "Fatteners", in six intensive swine farms, for two years. Feces were analysed for the detection and abundance of class 1 integrons (used as proxy of antibiotic resistance and of anthropogenic pollution), and of enterococci [fecal indicator bacteria (FIB) and potentially pathogenic for humans] by quantitative Real Time PCR. Furthermore, Enterococcus faecalis and Enterococcus faecium were isolated, analysed for the presence of the intI1 gene by Real Time PCR and genetically typed by Pulsed-Field Gel Electrophoresis. Both enterococci and class 1 integrons were significantly more abundant in the Suckling piglets (p = 0.0316 and 0.0242, respectively). About 8% of the isolated enterococci were positive for the intI1 gene by Real Time PCR. E. faecalis and E. faecium were found genetically heterogeneous and no specific pattern could be identified as the driver for their presence along the pig primary production. These findings suggest that the "Suckling piglets" category of production represents the key point where to mitigate the risk of transmission of enterococci and class 1 integrons with associated antibiotic resistance genes to humans and spread into the environment.

Comparative Analysis of the Antimicrobial Activity of Essential Oils and Their Formulated Microemulsions against Foodborne Pathogens and Spoilage Bacteria.

The antimicrobial activity of several essential oils (EOs) and their related microemulsions (MEs) was investigated. EOs were obtained from Cannabis sativa L. cv CS (C. sativa), Carum carvi L. (C. carvi), Crithmum maritimum L. (C. maritimum), Cuminum cyminum L. (C. cyminum), x Cupressocyparis leylandii A.B. Jacks & Dallim. (C. leylandii), Cupressus arizonica Greene (C. arizonica), Ferula assa-foetida L. (F. assa-foetida)., Ferula gummosa Boiss. (F. gummosa), Juniperus communis L. (J. communis), Juniperus x pfitzeriana (Spath) P.A. Schmidt (J. pfitzeriana), Pimpinella anisum L (P. anisum). Preliminary screening revealed that Cuminum cyminum, Crithmum maritimum, and Pimpinella anisum (10% v/v) were effective against all tested microorganisms (Escherichia coli ATCC 35218, Listeria monocytogenes ATCC 7644, Staphylococcus aureus ATCC 29213, Pseudomonas fluorescens DSM 4358, and Candida albicans ATCC 10231), with growth inhibition diameter from 10 to 25 mm. These EOs were used to formulate the MEs with an average size < 50 nm and a good stability over 30 days. EOs' antimicrobial activity was further enhanced in the MEs, with a generalized lowering of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values. C. cyminum-ME reached, in most cases, MIC two times lower (0.312%) than the corresponding EO (0.625%) and even eight times lower against S. aureus (0.156 vs. 1.25%). A more remarkable microbicide effect was noted for C. cyminum-ME, with MBC values eight times lower (from 0.312 to 0.625%) than the corresponding EO (from 2.5 to 5%). Overall, MEs resulted in an efficient system for EOs encapsulation, enhancing solubility and lowering concentration to exert antimicrobial efficacy.

Variable Susceptibility to Gallium Compounds of Major Cystic Fibrosis Pathogens.

The decreasing efficacy of existing antibiotics against pulmonary pathogens that affect cystic fibrosis (CF) patients calls for the development of novel antimicrobials. Iron uptake and metabolism are vital processes for bacteria, hence potential therapeutic targets. Gallium [Ga(III)] is a ferric iron-mimetic that inhibits bacterial growth by disrupting iron uptake and metabolism. In this work we evaluate the efficacy of three Ga(III) compounds, namely, Ga(NO3)3, (GaN), Ga(III)-maltolate (GaM), and Ga(III)-protoporphyrin IX (GaPPIX), against a collection of CF pathogens using both reference media and media mimicking biological fluids. All CF pathogens, except Streptococcus pneumoniae, were susceptible to at least one Ga(III) compound. Notably, Mycobacterium abscessus and Stenotrophomonas maltophilia were susceptible to all Ga(III) compounds. Achromobacter xylosoxidans, Burkholderia cepacia complex, and Pseudomonas aeruginosa were more susceptible to GaN and GaM, whereas Staphylococcus aureus and Haemophilus influenzae were more sensitive to GaPPIX. The results of this study support the development of Ga(III)-based therapy as a broad-spectrum strategy to treat CF lung infections.

Berberine Derivatives as Pseudomonas aeruginosa MexXY-OprM Inhibitors: Activity and In Silico Insights.

The natural alkaloid berberine has been demonstrated to inhibit the Pseudomonas aeruginosa multidrug efflux system MexXY-OprM, which is responsible for tobramycin extrusion by binding the inner membrane transporter MexY. To find a structure with improved inhibitory activity, we compared by molecular dynamics investigations the binding affinity of berberine and three aromatic substituents towards the three polymorphic sequences of MexY found in P. aeruginosa (PAO1, PA7, and PA14). The synergy of the combinations of berberine or berberine derivatives/tobramycin against the same strains was then evaluated by checkerboard and time-kill assays. The in silico analysis evidenced different binding modes depending on both the structure of the berberine derivative and the specific MexY polymorphism. In vitro assays showed an evident MIC reduction (32-fold and 16-fold, respectively) and a 2-3 log greater killing effect after 2 h of exposure to the combinations of 13-(2-methylbenzyl)- and 13-(4-methylbenzyl)-berberine with tobramycin against the tobramycin-resistant strain PA7, a milder synergy (a 4-fold MIC reduction) against PAO1 and PA14, and no synergy against the ΔmexXY strain K1525, confirming the MexY-specific binding and the computational results. These berberine derivatives could thus be considered new hit compounds to select more effective berberine substitutions and their common path of interaction with MexY as the starting point for the rational design of novel MexXY-OprM inhibitors.

A Highly Sensitive Luminescent Biosensor for the Microvolumetric Detection of the Pseudomonas aeruginosa Siderophore Pyochelin.

The pyochelin (PCH) siderophore produced by the pathogenic bacterium Pseudomonas aeruginosa is an important virulence factor, acting as a growth promoter during infection. While strong evidence exists for PCH production in vivo, PCH quantification in biological samples is problematic due to analytical complexity, requiring extraction from large volumes and time-consuming purification steps. Here, the construction of a bioluminescent whole cell-based biosensor, which allows rapid, sensitive, and single-step PCH quantification in biological samples, is reported. The biosensor was engineered by fusing the promoter of the PCH biosynthetic gene pchE to the luxCDABE operon, and the resulting construct was inserted into the chromosome of the ΔpvdAΔpchDΔfpvA siderophore-null P. aeruginosa mutant. A bioassay was setup in a 96-well microplate format, enabling the contemporary screening of several samples in a few hours. A linear response was observed for up to 40 nM PCH, with a lower detection limit of 1.64 ± 0.26 nM PCH. Different parameters were considered to calibrate the biosensor, and a detailed step-by-step operation protocol, including troubleshooting specific problems that can arise during sample preparation, was established to achieve rapid, sensitive, and specific PCH quantification in both P. aeruginosa culture supernatants and biological samples. The biosensor was implemented as a screening tool to detect PCH-producing P. aeruginosa strains on a solid medium.

Intra-tracheal administration increases gallium availability in lung: implications for antibacterial chemotherapy.

The emergence of pan-resistant strains in nosocomial settings underscores the urgent need of novel therapies targeting vital bacterial functions. Bacterial iron metabolism is a fascinating target for new antimicrobials. Iron mimetic metal Ga(III) has been repurposed as an antimicrobial drug, in pre-clinical studies and recent clinical studies have raised the possibility of using Ga(III) for the treatment of P. aeruginosa pulmonary infection. Ga(III) has been approved by FDA for the treatment of cancer, autoimmune and bone resorption disorders. However, some critical issues affect the therapeutic schedule of Ga(III), principally the intra-venous (i.v.) administration, and the nephrotoxicity caused by prolonged administration. Ga(III) aerosolization could represent a viable alternative for treatment of lung infections, since delivery of antimicrobial agents to the airways maximizes drug concentration at the site of infection, improves the therapeutic efficacy, and alleviates systemic toxic effects. We demonstrate the advantage of inhaled vs i.v. administered Ga(III), in terms of bio-distribution and lung acute toxicity, by using a rat model. In vivo results support the use of Ga(III) for inhalation since intra-tracheal Ga(III) delivery improved its persistence in the lung, while the i.v. administration caused rapid clearance and did not allow to attain a significant Ga(III) concentration in this organ. Moreover, local and systemic acute toxicity following intra-tracheal administration was not observed, since no significant signs of inflammation were found. At this stage of evidence, the direct administration of Ga(III) to the lung appears feasible and safe, boosting the development of Ga(III)-based drugs for inhalation therapy.

Draft Genome Sequence and Polyhydroxyalkanoate Biosynthetic Potential of Jeongeupia naejangsanensis Type Strain DSM 24253.

Jeongeupia naejangsanensis is a Gram-negative, cellulose-degrading betaproteobacterium. Here, we report the draft genome sequence of the type strain J. naejangsanensis DSM 24253 and identify the genes implicated in the biosynthesis of polyhydroxyalkanoate bioplastic polymers.

3D printed clotrimazole intravaginal ring for the treatment of recurrent vaginal candidiasis.

Vulvovaginal candidiasis is a vaginal infection caused by the fungal pathogen Candida albicans that, most commonly, affects women of reproductive age. Its first-line treatment consists in topical applications of conventional drug formulations (e.g., creams, gels, tablets) containing imidazole drugs. The treatment involves single or multiple daily applications and, in the case of recurrences, daily administration of oral antifungal drugs for up to one month. Intravaginal rings are flexible, biocompatible medical devices that, compared to conventional drug formulations, offer the possibility of a controlled vaginal drug delivery over a determined period with a single application, thus increasing patient compliance. Among innovative manufacturing techniques, in recent years, fused deposition modeling 3D printing has emerged in the pharmaceutical field to produce different therapeutics combining drugs and polymers. This technique allows to print objects layer by layer with many different thermoplastic materials after a computer-assisted design. Thermoplastic polyurethanes are flexible and biocompatible materials that can be efficiently employed for the manufacturing of drug release systems, already utilized to prepare vaginal devices. In this work, we produced a clotrimazole-loaded intravaginal ring by fused deposition modeling 3D printing combining the drug with thermoplastic polyurethane using hot melt extrusion. The rings were computer-designed and then printed with two different drug concentrations (i.e., 2% and 10% w/w). The intravaginal rings were first tested in an agar-diffusion test to evaluate their effectiveness against C. albicans; and the 10% loaded ring was selected for further studies. Drug release was evaluated in two different media (i.e., 50% ethanol and vaginal fluid simulant) showing a sustained release over a period of seven days. Next, in vitro time-kill analysis against C. albicans in simulated vaginal fluid was performed and displayed a complete growth inhibition after 5 days, compared to the control. These results suggest a potential application of these 3D printed intravaginal rings for the treatment of vulvovaginal candidiasis and for the long-time treatment of recurrences.

Draft Genome Sequence and Secondary Metabolite Biosynthetic Potential of the Lysobacter niastensis Type Strain DSM 18481.

Lysobacter niastensis belongs to a group of bacterial predators that produce a number of bioactive small molecules endowed with lytic properties toward other microorganisms. Here, we report the draft genome sequence of the type strain DSM 18481 and the identification of gene clusters implicated in the biosynthesis of secondary metabolites.

Draft Genome Sequence of the Carboxydotrophic Alphaproteobacterium Aminobacter carboxidus Type Strain DSM 1086.

Aminobacter carboxidus is a soil Gram-negative alphaproteobacterium belonging to the physiological group of carboxydobacteria which aerobically oxidize CO to CO2 Here, we report the draft genome sequence of the A. carboxidus DSM 1086 type strain and the identification of both form I and form II CO dehydrogenase systems in this strain.

The microbial community of a biofilm lining the wall of a pristine cave in Western New Guinea.

Caves are extreme environments inhabited by microbial communities adapted to thrive oligotrophic conditions. Cave microbes are organised in complex ecological networks and have developed survival strategies involving the production and release of a large variety of secondary metabolites, including antibiotic-like compounds. In this study, the structure and the metabolic features of a biofilm-like microbial community lining the walls of a pristine karst cavity (the Yumugi river cave) located in a remote region of the Western New Guinea were investigated. 16S rRNA and shotgun sequence analyses highlighted the prevalence of chemoorganotrophic phyla (Proteobacteria, Actinobacteria, Firmicutes and Acidobacteria), consistent with metabolic predictions inferred from the cave metagenome analysis. Few clinically relevant antimicrobial resistance genes were detected. A culture-based approach allowed the isolation of some heterotrophic members of the bacterial community, and antimicrobial susceptibility testing revealed an overall high level of resistance to different antimicrobials classes. Isolates presumptively representing new uncharacterized members of genus Pseudomonas displayed interesting antibiotic properties against Gram-positive indicator strains. Our work supports the hypothesis that caves represent a reservoir for new bacterial species and drug discovery research.

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.

Moulds on cementitious building materials-problems, prevention and future perspectives.

Materials rich in organic and inorganic compounds, such as building materials or paints, represent an excellent substrate for the development of moulds. Several conditions affect mould's growth on cementitious materials, such as nutrient and water availability, temperature, pH and moisture. Microorganisms, and especially moulds, attack these surfaces and contribute to their erosion, thereby reducing the life of the structure itself and negatively affecting human health through inhalation, ingestion and dermal contact with spores. Interventions are based on The European Communities Council Directive 89/106/EEC, that obliges the use of materials, products and building elements that are resistant to fungi and other forms of degradation, and that do not constitute a health risk for users and the environment. This mini-review summarises the current state of problems related to mould growth on cementitious building materials, emphasising new innovative approaches for limiting or contrasting their growth. In particular, the use of nanoparticles and the related nanomaterials as well as the potential use of new "biocides" from natural sources is discussed.

Geometrical-optics approach to measure the optical density of bacterial cultures using a LED-based photometer.

We develop a suitable geometrical-optics approach and demonstrate that it is possible to measure the optical density (OD) of bacterial cultures using a light emitting diode (LED)-based photometer. We measure both attenuation and spot-size variation, and we compensate for diffraction and stray-light impairment related to the incoherent source and large detection area. The approach is validated for different concentrations of two bacterial species, Escherichia coli and Staphylococcus aureus, that present different shapes and clustering organization.

Geometrical-optics approach to increase the accuracy in LED-based photometers for point-of-care testing.

A geometrical-optics approach is proposed to increase the accuracy in photometric measurements, using a point-of-care testing (POCT) LED-based sensor. Due to stray-light effects, the measurement accuracy depends on the dimension of the CMOS area, where the radiation is detected. We propose two image processing approaches and evaluate the influence of the sensor area. In addition, we demonstrate that with the same measurement, both absorption coefficient and refractive index can be determined, measuring the beam attenuation and the spot-size enlargement due to ray refraction.

Bacterial Cheaters Evade Punishment by Cyanide.

In all domains of life, mechanisms exist that protect cooperating groups from exploitation by cheaters. Recent observations with the bacterium Pseudomonas aeruginosa have suggested a paradigmatic cheater control mechanism in which cooperator cells punish or "police" cheater cells by cyanide poisoning. These cheater cells are deficient in a pleiotropic quorum-sensing regulator that controls the production of cooperative secretions including cyanide, and presumably also cyanide resistance. In this study, we directly tested and refuted the cyanide policing model. Contrary to the hypothesis, cheater fitness was unaffected by the presence of cyanide. Cheater mutants grew equally well in co-cultures with either cyanide-proficient or cyanide-deficient cooperators, and they were as resistant to exogenous cyanide as wild-type cells. We show that these behaviors are the result of quorum-sensing-independent and cyanide-responsive resistance gene regulation. Our results highlight the role of genetic architecture in the evolution of cooperative behavior.