Ruthenium(II) Polypyridyl Complexes and Metronidazole Derivatives: A Powerful Combination in the Design of Photoresponsive Antibacterial Agents Effective under Hypoxic Conditions.

Ruthenium(II) polypyridyl complexes (RPCs) are gaining momentum in photoactivated chemotherapy (PACT), thanks to the possibility of overcoming the classical reliance on molecular oxygen of photodynamic therapy while preserving the selective drug activation by using light. However, notwithstanding the intriguing perspectives, the translation of such an approach in the development of new antimicrobials has been only barely considered. Herein, MTZH-1 and MTZH-2, two novel analogues of metronidazole (MTZ), a mainstay drug in the treatment of anaerobic bacterial infections, were designed and inserted in the strained ruthenium complexes [Ru(tpy)(dmp)(MTZ-1)]PF6 (Ru2) and [Ru(tpy)(dmp)(MTZ-2)]PF6 (Ru3) (tpy = terpyridine, dmp = 2,9-dimethyl-1,10-phenanthroline) (Chart 1). Analogously to the parental compound [Ru(tpy)(dmp)(5NIM)]PF6 (Ru1) (5-nitroimidazolate), the Ru(II)-imidazolate coordination of MTZ derivatives resulted in promising Ru(II) photocages, capable to easily unleash the bioactive ligands upon light irradiation and increase the antibacterial activity against Bacillus subtilis, which was chosen as a model of Gram-positive bacteria. The photoreleased 5-nitroimidazole-based ligands led to remarkable phototoxicities under hypoxic conditions (<1% O2), with the lead compound Ru3 that exhibited the highest potency across the series, being comparable to the one of the clinical drug MTZ. Besides, the chemical architectures of MTZ derivatives made their interaction with NimAunfavorable, being NimA a model of reductases responsible for bacterial resistance against 5-nitroimidazole-based antibiotics, thus hinting at their possible use to combat antimicrobial resistance. This work may therefore provide fundamental knowledge in the design of novel photoresponsive tools to be used in the fight against infectious diseases. For the first time, the effectiveness of the "photorelease antimicrobial therapy" under therapeutically relevant hypoxic conditions was demonstrated.

Propolis hosts a diversemicrobial community.

Despite the deep knowledge of the honey bee (Apis mellifera) gut microbiome, information on the microbial communities of other hive components is still scarce. Propolis originates from a natural resinous mixture that honeybees collect from different plants sources and modify; it is used mainly to ensure the hygiene of the hive. By virtue of its antimicrobial properties, propolis has been considered relatively aseptic, yet its ability to harbor microorganisms has not been previously investigated. In this study we report the first description of the diversity of the microbial community of propolis by both targeted-metagenomics analysis and cultivation. We demonstrated that propolis hosts a variety of microbial strains belonging to taxa already described in other hive components. Some of them are cultivable in standard laboratory conditions, and show metabolic characteristics compatible with their persistence in different physiological states inside propolis. Isolated bacteria produce antimicrobials against Gram-negative and Gram-positive bacteria, and entomopathogenic fungi, with different spectra of inhibition. Metagenomics analysis shows the presence of bacteria and fungi with great potential to outcompete potentially harmful microorganisms. These findings suggest that the characterized microbiota could contribute to the overall antimicrobial properties of propolis and to its ecological role as "disinfectant" within the hive.

Home economics in an oak gall: behavioural and chemical immune strategies against a fungal pathogen in Temnothorax ant nests.

Nest architecture is a fundamental character shaping immune strategies of social insects. The arboreal ant Temnothorax unifasciatus nests in cavities such as oak galls where the entire colony lives in a unique small chamber. In these conditions, physiological and behavioural strategies likely prevail over compartmentalisation and are presumably tuned with colony size. We designed two experiments to study chemical and behavioural immune strategies against the entomopathogenic fungus Metarhizium anisopliae in colonies of different sizes. First, we compared spore germination and length of germinal tubes inside artificial nests, designed to impede the contact between the ants and the fungus, in colonies of different size. In the absence of direct contact, Temnothorax unifasciatus colonies inhibit fungal growth inside their nests, presumably through volatile compounds. The analysis revealed a positive correlation between fungistatic activity and colony size, indicating that workers of smaller colonies do not invest a higher per capita effort in producing such substances compared to larger colonies. Second, we performed a removal experiment of contaminated and non-contaminated items introduced inside the nests of colonies of different size. Small colonies challenged with contaminated fibres showed an increased removal of all the items (both contaminated and non-contaminated) compared to small colonies challenged with non-contaminated fibres only. Conversely, larger colonies moved items regardless of the presence of the spores inside the nest. Colony size qualitatively affected removal of waste items showing a pathogen elicited reaction in small colonies to optimise the reduced workforce, while the removal behaviour in larger colonies revealed to be expressed constitutively.

Trans-generational immunization in the acrobat ant Crematogaster scutellaris.

Trans-generational immunization is defined as the transmission of an enhanced resistance to a pathogen from parents to offspring. By using the host-parasite system of the ant Crematogaster scutellaris and the entomopathogenic fungus Metarhizium anisopliae, we describe this phenomenon for the first time in ants. We exposed four groups of hibernating queens to different treatments (i) a non-lethal dose of live conidiospores in Triton, (ii) a dose of heat-killed conidiospores in Triton, (iii) a control Triton solution, and (iv) a naive control. We exposed their first workers to a high dose of conidiospores and measured mortality rates. Workers produced by queens exposed to live conidiospores survived longer than those belonging to the other groups, while exposure to Triton and dead spores had no effect. Starved workers showed a significantly higher mortality. The treatments did not influence queen mortality, nor the number of offspring they produced at the emergence of the first worker, showing no evidence of immunization costs-at least for these parameters in the first year of colony development. We propose that trans-generational immunization represents an important component of social immunity that could affect colony success, particularly during the critical phase of claustral foundation.

Characterization of the Community of Black Meristematic Fungi Inhabiting the External White Marble of the Florence Cathedral.

Meristematic black fungi are a highly damaging group of microorganisms responsible for the deterioration of outdoor exposed monuments. Their resilience to various stresses poses significant challenges for removal efforts. This study focuses on the community of meristematic fungi inhabiting the external white marble of the Cathedral of Santa Maria del Fiore, where they contribute to its darkening. Twenty-four strains were isolated from two differently exposed sites of the Cathedral, and their characterization was conducted. Phylogenetic analysis using ITS and LSU rDNA regions revealed a wide diversity of rock-inhabiting fungal strains within the sampled areas. Eight strains, belonging to different genera, were also tested for thermal preferences, salt tolerance, and acid production to investigate their tolerance to environmental stressors and their interaction with stone. All tested strains were able to grow in the range of 5-30 °C, in the presence 5% NaCl, and seven out of eight strains were positive for the production of acid. Their sensitivities to essential oils of thyme and oregano and to the commercial biocide Biotin T were also tested. The essential oils were found to be the most effective against black fungi growth, indicating the possibility of developing a treatment with a low environmental impact.

Essential oils to contrast biodeterioration of the external marble of Florence Cathedral.

The search for more sustainable strategies to contrast biodeterioration of stone cultural heritage has been developing in recent years to find alternatives to synthetic biocides, since their toxicity and potential impact on the environment and health. In this study, the application of the oregano and thyme essential oils (EOs) was tested to control microbial growth on the external marble of Florence Cathedral affected by extended darkening. Before in situ application, preliminary tests were carried out to evaluate the interference of the EOs with marble (colorimetric and water absorption assays on marble specimens) and their efficacy in inhibiting marble microbiota (sensitivity test on nutrient media). EOs inhibited the whole cultivable microbiota sampled from the Cathedral marble at a very low concentration, while they did not interfere with colour and water absorption capability of uncolonised marble samples when applied as a 2 % solution. Then the two EOs and the commercial biocide Biotin T were used in in situ trials on marble in two outdoor study sites of Florence Cathedral. The effectiveness of the treatments was assessed through short- and mid-term evaluation by multidisciplinary in situ non-invasive (colorimetric and ATP assays, microscopy) and ex situ (microbial viable titer) tests. Concerning results, we found a good correspondence between parameters for evaluation of viability (bacterial and fungi viable titer) and activity (ATP determination) and some correspondence among these and microscopy and colorimetry. Considering the whole data, treatments with oregano and thyme EOs were effective against microbial community, in more cases comparably to the commercial biocide. Some differences found, particularly by viable titer, in the two study sites or in bacterial and fungal components of the microbiota can be partly attributed to differences in structure and colonization pattern of the microbial community due to the peculiar climatic conditions of the differently exposed study areas.

Characterization of the Bacterial Communities Inhabiting Tropical Propolis of Puerto Rico.

Propolis is a resinous material produced by honeybees from different plant sources and used in the hive as a building material and to protect the colony from parasites and pathogens. Despite its antimicrobial properties, recent studies showed that propolis hosts diverse microbial strains, some with great antimicrobial potential. In this study, the first description of the bacterial community of propolis produced by the gentle Africanized honeybee was reported. Propolis was sampled from hives of two different geographic areas of Puerto Rico (PR, USA), and the associated microbiota investigated by both cultivation and metataxonomic approaches. Metabarcoding analysis showed appreciable bacterial diversity in both areas and statistically significant dissimilarity in the taxa composition of the two areas, probably due to the different climatic conditions. Both metabarcoding and cultivation data revealed the presence of taxa already detected in other hive components and compatible with the bee's foraging environment. Isolated bacteria and propolis extracts showed antimicrobial activity against Gram-positive and Gram-negative bacterial tester strains. These results support the hypothesis that the propolis microbiota could contribute to propolis' antimicrobial properties.

Molecular basis of bacterial calcium carbonate precipitation.

Calcium carbonate precipitation is a widespread process, occurring in different bacterial taxonomic groups and in different environments, at a scale ranging from the microscopic one of cells to that of geological formations. It has relevant implications in natural processes and has great potentiality in numerous applications. For these reasons, bacterial precipitation has been investigated extensively both in natural environments and under laboratory conditions. Different mechanisms of bacterial involvement in precipitation have been proposed. There is an agreement that the phenomenon can be influenced by the environmental physicochemical conditions and it is correlated both to the metabolic activity and the cell surface structures of microorganisms. Nevertheless, the role played by bacteria in calcium mineralization remains a matter of debate. This chapter reviews the main mechanisms of the process with particular focus on what is known on molecular aspects, and discusses the significance of the precipitation event also from an evolutionary point of view.

MinION technology for microbiome sequencing applications for the conservation of cultural heritage.

The MinION single-molecule sequencing system has been attracting the attention of the community of microbiologists involved in the conservation of cultural heritage. The use of MinION for the conservation of cultural heritage is extremely recent, but surprisingly the only few applications available have been exploring many different substrates: stone, textiles, paintings and wax. The use of MinION sequencing is mainly used to address the metataxonomy (with special emphasis on non-cultivable microorganisms) with the effort to identify species involved in the degradation of the substrates. In this review, we show the current applications available on different artworks, showing how this technology can be a useful tool for microbiologists and conservators also in light of its low cost and the easy chemistry.

Bacterial Calcium Carbonate Mineralization in situ Strategies for Conservation of Stone Artworks: From Cell Components to Microbial Community.

Calcareous stones have been widely used in artworks and buildings by almost all human cultures. Now, more than ever, the increased environmental pollution and global warming are threatening the stone cultural heritage. Weathering due to physical, chemical and biological factors results in monumental calcareous stone deterioration. These agents induce a progressive dissolution of the mineral matrix, increase porosity, and lead to structural weakening. Bacterial Calcium Carbonate Mineralization is a widespread naturally occurring process which in the last decades was proposed as an environmentally friendly tool to protect monumental and ornamental calcareous stones. The advantage of this treatment is that it mimics the natural process responsible for stone formation, producing a mineral product similar to the stone substrate. This mini review highlights the milestones of the biomineralization approaches with focus on in situ stone artworks protection. The strategies explored to date are based on three main approaches: (i) the use of allochthonous and (ii) autochthonous alive cells that, due to the bacterial metabolism, foster biomineralization; (iii) the cell-free approach which uses fractionated cellular components inducing biomineralization. We discuss the challenging aspects of all these techniques, focusing on in situ applications and suggesting perspectives based on recent advances.

No evidence of queen immunisation despite transgenerational immunisation in Crematogaster scutellaris ants.

Like vertebrates, invertebrates evolved acquired immunity based on memory-like mechanisms, known as immunisation. Immunisation and its transmission among individuals are phylogenetically ancestral and conserved characters that have been reported in different insect orders. Physiological mechanisms are still largely unknown, and the high variability in responses in different host-parasite systems led to different conclusions. In social insect species, the complex organisation of colonies further complicates the interpretation of the immune responses. In ants, it has been shown that the expression of immunisation depends on species, caste and physiological status of individuals. In this study, we investigate the occurrence of immunisation in queens of Crematogaster scutellaris ants using the fungus Metarhizium anisopliae as elicitor. Foundation in C. scutellaris is claustral and monogynic, allowing us to test the existence of the phenomenon in two distinct physiological conditions, corresponding to the claustral and colonial phases of queens. Queens and foundresses challenged with heavy doses of the pathogen showed higher mortality if previously exposed to light doses, indicating the absence of immunisation in our experimental settings. On the other hand, evidence of the trans-generational immunisation in the same host-parasite system has been recently found, where workers produced by queens exposed to light doses of M. anisopliae survived longer than those belonging to the control group. These results indicate that foundresses exposed to M. anisopliae can elicit an increased resistance in the offspring without providing themselves with a similar increased immune response and that immunisation and trans-generational immunisation are uncoupled phenomena in this host-parasite system.

A method to assess bioavailability of antibiotics in anthropogenic polluted ecosystems by using a bacterial fitness test.

Antibiotics released in the environment exert a selective pressure on the resident microbiota. It is well accepted that the mere measurement of antibiotics does not reflect the actual bioavailability. In fact, antibiotics can be adsorbed or complexed to particles and/or chemicals in water and soil. Bioavailable concentrations of antibiotics in soil and water are subjected to great uncertainty, therefore biological assays are increasingly recognized as that allow an indirect determination of the residual antibiotic activity. Here we propose how a fitness test for bacteria can be used to qualitatively assess the bioavailability of a specific antibiotic in the environment. The findings show that by using a pair of resistant and sensitive bacterial strains, the resulting fitness can indirectly reflect antibiotic bioavailability. Hence, this test can be used as a complementary assay to other biological and chemical tests to assess bioavailability of antibiotics.

Dominulin A and B: two new antibacterial peptides identified on the cuticle and in the venom of the social paper wasp Polistes dominulus using MALDI-TOF, MALDI-TOF/TOF, and ESI-ion trap.

Two new antibacterial peptides, denominated as Dominulin A and B, have been found on the cuticle and in the venom of females of the social paper wasp Polistes dominulus. The amino acidic sequence of the two peptides, determined by mass spectrometry, is INWKKIAE VGGKIL SSL for Dominulin A (MW = 1854 Da) and INWKKIAEIGKQVL SAL (MW = 1909 Da) for Dominulin B. Their presence on the cuticle was confirmed using MALDI-TOF by means of micro-extractions and direct analyses on body parts. The presence in the venom and the primary structure of the dominulins suggest their classification in the mastoparans, a class of peptides found in the venom of other Aculeate hymenoptera. Their antimicrobial action against Gram+ and Gram- bacteria fits in the range of the best natural antimicrobial peptides. Dominulins can represent an important defense of the colony of Polistes dominulus against microbial pathogens.

Antibacterial activity of silver nanoparticles obtained by pulsed laser ablation in pure water and in chloride solution.

Silver nanoparticles (AgNPs) have increasingly gained importance as antibacterial agents with applications in several fields due to their strong, broad-range antimicrobial properties. AgNP synthesis by pulsed laser ablation in liquid (PLAL) permits the preparation of stable Ag colloids in pure solvents without capping or stabilizing agents, producing AgNPs more suitable for biomedical applications than those prepared with common, wet chemical preparation techniques. To date, only a few investigations into the antimicrobial effect of AgNPs produced by PLAL have been performed. These have mainly been performed by ablation in water with nanosecond pulse widths. We previously observed a strong surface-enhanced Raman scattering (SERS) signal from such AgNPs by "activating" the NP surface by the addition of a small quantity of LiCl to the colloid. Such surface effects could also influence the antimicrobial activity of the NPs. Their activity, on the other hand, could also be affected by other parameters linked to the ablation conditions, such as the pulse width. The antibacterial activity of AgNPs was evaluated for NPs obtained either by nanosecond (ns) or picosecond (ps) PLAL using a 1064 nm ablation wavelength, in pure water or in LiCl aqueous solution, with Escherichia coli and Bacillus subtilis as references for Gram-negative and Gram-positive bacteria, respectively. In all cases, AgNPs with an average diameter less than 10 nm were obtained, which has been shown in previous works to be the most effective size for bactericidal activity. The measured zeta-potential values were very negative, indicating excellent long-term colloidal stability. Antibacterial activity was observed against both microorganisms for the four AgNP formulations, but the ps-ablated nanoparticles were shown to more effectively inhibit the growth of both microorganisms. Moreover, LiCl modified AgNPs were the most effective, showing minimum inhibitory concentration (MIC) values in a restricted range of 1.0-3.7 µg/mL. An explanation is proposed for this result based on the increased surface reactivity of the metal surface due to the presence of positively charged active sites.

Antibiotic delivery by liposomes from prokaryotic microorganisms: Similia cum similis works better.

To date the effectiveness of antibiotics is undermined by microbial resistance, threatening public health worldwide. Enhancing the efficacy of the current antibiotic arsenal is an alternative strategy. The administration of antimicrobials encapsulated in nanocarriers, such as liposomes, is considered a viable option, though with some drawbacks related to limited affinity between conventional liposomes and bacterial membranes. Here we propose a novel "top-down" procedure to prepare unconventional liposomes from the membranes of prokaryotes (PD-liposomes). These vectors, being obtained from bacteria with limited growth requirements, also represent low-cost systems for scalable biotechnology production. In depth physico-chemical characterization, carried out with dynamic light scattering (DLS) and Small Angle X-ray Scattering (SAXS), indicated that PD-liposomes can be suitable for the employment as antibiotic vectors. Specifically, DLS showed that the mean diameter of loaded liposomes was ∼200-300nm, while SAXS showed that the structure was similar to conventional liposomes, thus allowing a direct comparison with more standard liposomal formulations. Compared to free penicillin G, PD-liposomes loaded with penicillin G showed minimal inhibitory concentrations against E. coli that were up to 16-times lower. Noteworthy, the extent of the bacterial growth inhibition was found to depend on the microorganisms from which liposomes were derived.

Growth phase-dependent expression of an endoglucanase encoding gene (eglS) in Streptomyces rochei A2.

Different growth phases of Streptomyces rochei A2, a cellulolytic strain isolated from termite gut, were determined in liquid culture. The strain exhibited a multiphasic growth curve typical of streptomycetes. In S. rochei A2, the secreted endoglucanase EglS is coded by a TTA-containing gene, conserved in other Streptomyces. The onset of S. rochei A2 eglS gene expression was determined by RT-PCR using total RNA extracted at different growth phases. The eglS gene was expressed only during transition, second rapid growth and stationary phases, the timing of expression being transcriptionally regulated.

[Compulsory and additional parameters in water control]. / Applicazione dei parametri obbligatori ed accessori nel controllo di acque di diversa origine.

The authors have analyzed 22 water samples from the Arno River (both river and drinking samples), as well as 11 effluent samples from an urban plant. Chemical and microbiological features of river samples were in line with the A3 class, needing treatment for drinking use (Executive Order n. 152/99); drinkable water met requirements (Presidents Decree n degrees 236/1988 and Executive Order n degrees 31/2001). As regards chemical parameters, effluents complied with the law (Executive Order n. 152/1999) but their bacteriological figures exceeded the standards set by the Authorities due to a lack in chlorination. Enteric viruses reacted to the RT-PCR test in 5 of the samples, including a Coxsackievirus Type B2 and a Poliovirus. No association was shown between bacteriophage parameters and virus.

Physiological requirements for carbonate precipitation during biofilm development of Bacillus subtilis etfA mutant.

Although the implications of calcium carbonate (CaCO(3)) precipitation by microorganisms in natural environments are quite relevant, the physiology and genetics of this phenomenon are poorly understood. We have chosen Bacillus subtilis 168 as our model to study which physiological aspects are associated with CaCO(3) (calcite) formation during biofilm development when grown on precipitation medium. A B. subtilis eftA mutant named FBC5 impaired in calcite precipitation was used for comparative studies. Our results demonstrate that inactivation of etfA causes a decrease in the pH of the precipitation medium during biofilm development. Further analysis demonstrated that eftA extrudes an excess of 0.7 mol H(+) L(-1) with respect to B. subtilis 168 strain. Using media buffered at different pH values, we were able to control calcite formation. Because etfA encodes the alpha-subunit of a putative flavoprotein involved in fatty acid metabolism, we compared the intracellular levels of NADH among strains. Our physiological assay showed that FBC5 accumulated up to 32 times more NADH than the wild-type strain. We propose that the accumulation of NADH causes a deregulation in the generation of the proton motive force (DeltamicroH(+)) in FBC5 producing the acidification.

Bacillus subtilis gene cluster involved in calcium carbonate biomineralization.

Calcium carbonate precipitation, a widespread phenomenon among bacteria, has been investigated due to its wide range of scientific and technological implications. Nevertheless, little is known of the molecular mechanisms by which bacteria foster calcium carbonate mineralization. In our laboratory, we are studying calcite formation by Bacillus subtilis, in order to identify genes involved in the biomineralization process. A previous screening of UV mutants and of more than one thousand mutants obtained from the European B. subtilis Functional Analysis project allowed us to isolate strains altered in the precipitation phenotype. Starting from these results, we focused our attention on a cluster of five genes (lcfA, ysiA, ysiB, etfB, and etfA) called the lcfA operon. By insertional mutagenesis, mutant strains carrying each of the five genes were produced. All of them, with the exception of the strain carrying the mutated lcfA operon, were unable to form calcite crystals. By placing transcription under IPTG (isopropyl-beta-d-thiogalactopyranoside) control, the last gene, etfA, was identified as essential for the precipitation process. To verify cotranscription in the lcfA operon, reverse transcription-PCR experiments were performed and overlapping retrocotranscripts were found comprising three adjacent genes. The genes have putative functions linked to fatty acid metabolism. A link between calcium precipitation and fatty acid metabolism is suggested.

Characterization of bacterial pectinolytic strains involved in the water retting process.

Pectinolytic microorganisms involved in the water retting process were characterized. Cultivable mesophilic anaerobic and aerobic bacteria were isolated from unretted and water-retted material. A total of 104 anaerobic and 23 aerobic pectinolytic strains were identified. Polygalacturonase activity was measured in the supernatant of cell cultures; 24 anaerobic and nine aerobic isolates showed an enzymatic activity higher than the reference strains Clostridium felsineum and Bacillus subtilis respectively. We performed the first genotypic characterization of the retting microflora by a 16S amplified ribosomal DNA restriction analysis (ARDRA). Anaerobic isolates were divided into five different groups, and the aerobic isolates were clustered into three groups. 84.6% of the anaerobic and 82.6% of the aerobic isolates consisted of two main haplotypes. Partial 16S rRNA gene sequences were determined for 12 strains, representative of each haplotype. All anaerobic strains were assigned to the Clostridium genus, whereas the aerobic isolates were assigned to either the Bacillus or the Paenibacillus genus. Anaerobic isolates with high polygalacturonase (PG) activity belong to two clearly distinct phylogenetic clusters related to C. acetobutylicum-C. felsineum and C. saccharobutylicum species. Aerobic isolates with high PG activity belong to two clearly distinct phylogenetic clusters related to B. subtilisT and B. pumilusT.