Biosensing systems for the detection and quantification of methane gas.

Climate change due to the continuous increase in the release of green-house gasses associated with anthropogenic activity has made a significant impact on the sustainability of life on our planet. Methane (CH4) is a green-house gas whose concentrations in the atmosphere are on the rise. CH4 measurement is important for both the environment and the safety at the industrial and household level. Methanotrophs are distinguished for their unique characteristic of using CH4 as the sole source of carbon and energy, due to the presence of the methane monooxygenases that oxidize CH4 under ambient temperature conditions. This has attracted interest in the use of methanotrophs in biotechnological applications as well as in the development of biosensing systems for CH4 quantification and monitoring. Biosensing systems using methanotrophs rely on the use of whole microbial cells that oxidize CH4 in presence of O2, so that the CH4 concentration is determined in an indirect manner by measuring the decrease of O2 level in the system. Although several biological properties of methanotrophic microorganisms still need to be characterized, different studies have demonstrated the feasibility of the use of methanotrophs in CH4 measurement. This review summarizes the contributions in methane biosensing systems and presents a prospective of the valid use of methanotrophs in this field. KEY POINTS: • Methanotroph environmental relevance in methane oxidation • Methanotroph biotechnological application in the field of biosensing • Methane monooxygenase as a feasible biorecognition element in biosensors.

Application of Phage Therapy in a Case of a Chronic Hip-Prosthetic Joint Infection due to Pseudomonas aeruginosa: An Italian Real-Life Experience and In Vitro Analysis.

Background: Prosthetic joint infection (PJI) caused by Pseudomonas aeruginosa represents a severe complication in orthopedic surgery. We report the case of a patient with chronic PJI from P. aeruginosa successfully treated with personalized phage therapy (PT) in combination with meropenem. Methods: A 62-year-old woman was affected by a chronic right hip prosthesis infection caused by P. aeruginosa since 2016 . The patient was treated with phage Pa53 (I day 10 mL q8h, then 5 mL q8h via joint drainage for 2 weeks) in association with meropenem (2gr q12h iv) after a surgical procedure. A 2-year clinical follow up was performed. An in vitro bactericidal assay of the phage alone and in combination with meropenem against a 24-hour-old biofilm of bacterial isolate was also carried out. Results: No severe adverse events were observed during PT. Two years after suspension, there were no clinical signs of infection relapse, and a marked leukocyte scan showed no pathological uptake areas. In vitro studies showed that the minimum biofilm eradicating concentration of meropenem was 8 µg/mL. No biofilm eradication was observed at 24 hours incubation with phages alone (108 plaque-forming units [PFU]/mL). However, the addition of meropenem at suberadicating concentration (1 µg/mL) to phages at lower titer (103 PFU/mL) resulted in a synergistic eradication after 24 hours of incubation. Conclusions: Personalized PT, in combination with meropenem, was found to be safe and effective in eradicating P. aeruginosa infection. These data encourage the development of personalized clinical studies aimed at evaluating the efficacy of PT as an adjunct to antibiotic therapy for chronic persistent infections.

Antibacterial Efficacy of Two Commercially Available Bacteriophage Formulations, Staphylococcal Bacteriophage and PYO Bacteriophage, Against Methicillin-Resistant Staphylococcus aureus: Prevention and Eradication of Biofilm Formation and Control of a Systemic Infection of Galleria mellonella Larvae.

Sessile bacteria growing on surfaces are more resistant to standard antibiotics than their planktonic counterpart. Due to their antimicrobial properties, bacteriophages have re-emerged as a promising approach to treat bacterial biofilm-associated infections. Here, we evaluated the ability of two commercially available phage formulations, Staphylococcal bacteriophage (containing the monophage Sb-1) and PYO bacteriophage (a polyphage), in preventing and eradicating an in vitro biofilm of methicillin-resistant Staphylococcus aureus (MRSA) by isothermal microcalorimetry and high-resolution confocal laser scanning microscopy (CLSM). Moreover, to assess the potential in vivo efficacy of both phage preparations, a Galleria mellonella model of MRSA systemic infection was used. Microcalorimetry measurement showed that 107 PFU/ml (the highest tested titer) of both phage formulations were able to inhibit planktonic growth in a concentration-dependent manner. However, MRSA biofilm was eradicated only by co-incubation of 5-7 days with the highest phage titers, respectively. In the experiments of biofilm prevention, isothermal microcalorimetry revealed that the heat production was completely abolished in the presence of sub-inhibitory titers (104 PFU/ml) of phages. These data were also confirmed by confocal laser scanning microscopy. Both phage formulations increased the survival of G. mellonella larvae preventing or treating MRSA infection compared to untreated control. In conclusion, tested phage formulations are promising for preventing device colonization and killing biofilm bacteria attached on a surface. Novel strategies for direct coating and release of phages from material should be investigated.

TbD1 deletion as a driver of the evolutionary success of modern epidemic Mycobacterium tuberculosis lineages.

Mycobacterium tuberculosis (Mtb) strains are classified into different phylogenetic lineages (L), three of which (L2/L3/L4) emerged from a common progenitor after the loss of the MmpS6/MmpL6-encoding Mtb-specific deletion 1 region (TbD1). These TbD1-deleted "modern" lineages are responsible for globally-spread tuberculosis epidemics, whereas TbD1-intact "ancestral" lineages tend to be restricted to specific geographical areas, such as South India and South East Asia (L1) or East Africa (L7). By constructing and characterizing a panel of recombinant TbD1-knock-in and knock-out strains and comparison with clinical isolates, here we show that deletion of TbD1 confers to Mtb a significant increase in resistance to oxidative stress and hypoxia, which correlates with enhanced virulence in selected cellular, guinea pig and C3HeB/FeJ mouse infection models, the latter two mirroring in part the development of hypoxic granulomas in human disease progression. Our results suggest that loss of TbD1 at the origin of the L2/L3/L4 Mtb lineages was a key driver for their global epidemic spread and outstanding evolutionary success.

A CRISPR/Cas9-based strategy to simultaneously inactivate the entire ALS gene family in Candida orthopsilosis.

Aim: In this study, the CRISPR gene-editing approach was used to simultaneously inactivate all three members of the ALS gene family in the opportunistic pathogen Candida orthopsilosis. Materials & methods: Using a single gRNA and repair template, CRISPR-edited clones were successfully generated in a one-step process in both C. orthopsilosis reference and clinical strains. Results: The phenotypic characterization of the ALS triple-edited strains revealed no impact on growth in liquid or solid media. However, pseudohyphal formation and the ability to adhere to human buccal epithelial cells were significantly decreased in triple-edited clones. Conclusion: Our CRISPR/Cas9 system is a powerful tool for simultaneous editing of fungal gene families, which greatly accelerates the generation of multiple gene-edited Candida strains. Data deposition: Nucleotide sequence data are available in the GenBank databases under the accession numbers MK875971, MK875972, MK875973, MK875974, MK875975, MK875976, MK875977.

The synthetic killer peptide KP impairs Candida albicans biofilm in vitro.

Candida albicans is a commensal organism, commonly inhabiting mucosal surfaces of healthy individuals, as a part of the resident microbiota. However, in susceptible hosts, especially hospitalized and/or immunocompromised patients, it may cause a wide range of infections. The presence of abiotic substrates, such as central venous or urinary catheters, provides an additional niche for Candida attachment and persistence, particularly via biofilm development. Furthermore, Candida biofilm is poorly susceptible to most antifungals, including azoles. Here we investigated the effects of a synthetic killer peptide (KP), known to be active in vitro, ex vivo and/or in vivo against different pathogens, on C. albicans biofilm. Together with a scrambled peptide used as a negative control, KP was tested against Candida biofilm at different stages of development. A reference strain, two fluconazole-resistant and two fluconazole-susceptible C. albicans clinical isolates were used. KP-induced C. albicans oxidative stress response and membrane permeability were also analysed. Moreover, the effect of KP on transcriptional profiles of C. albicans genes involved in different stages of biofilm development, such as cell adhesion, hyphal development and extracellular matrix production, was evaluated. Our results clearly show that the treatment with KP strongly affected the capacity of C. albicans to form biofilm and significantly impairs preformed mature biofilm. KP treatment resulted in an increase in C. albicans oxidative stress response and membrane permeability; also, biofilm-related genes expression was significantly reduced. Comparable inhibitory effects were observed in all the strains employed, irrespective of their resistance or susceptibility to fluconazole. Finally, KP-mediated inhibitory effects were observed also against a catheter-associated C. albicans biofilm. This study provides the first evidence on the KP effectiveness against C. albicans biofilm, suggesting that KP may be considered as a potential novel tool for treatment and prevention of biofilm-related C. albicans infections.

Inhibition of Candida albicans Biofilm Formation by the Synthetic Lactoferricin Derived Peptide hLF1-11.

The aim of this study was to evaluate the in vitro activity of the synthetic peptide hLF1-11 against biofilm produced by clinical isolates of Candida albicans with different fluconazole susceptibility. The antibiofilm activity of the peptide hLF1-11 was assessed in terms of reduction of biofilm cellular density, metabolic activity and sessile cell viability. The extent of morphogenesis in hLF1-11 treated and untreated biofilms was also investigated microscopically. Transcription levels of genes related to cell adhesion, hyphal development and extracellular matrix production were analysed by qRT-PCR in hLF1-11 treated and untreated biofilms. Exogenous dibutyryl-cAMP (db-cAMP) was used to rescue morphogenesis in cells exposed to the peptide. The results revealed that hLF1-11 exhibited an inhibitory effect on biofilm formation by all C. albicans isolates tested in a dose-dependent manner, regardless of their fluconazole susceptibility. Visual inspection of treated or untreated biofilm cells with an inverted microscope revealed a significant reduction in hyphal formation by hLF1-11 treated cells, as early as 3 hours of incubation. Moreover, hLF1-11 showed a reduced activity on preadherent cells. hLF1-11 induced the down-regulation of biofilm and hyphal-associated genes, which were predominantly regulated via the Ras1-cAMP-Efg1 pathway. Indeed, exogenous db-cAMP restored morphogenesis in hLF1-11 treated cells. The hLF1-11 peptide significantly inhibited biofilm formation by C. albicans mainly at early stages, interfering with biofilm cellular density and metabolic activity, and affected morphogenesis through the Ras1-cAMP-Efg1 pathway. Our findings provide the first evidence that hLF1-11 could represent a potential candidate for the prevention of biofilm formation by C. albicans.

Targeted gene disruption in Candida parapsilosis demonstrates a role for CPAR2_404800 in adhesion to a biotic surface and in a murine model of ascending urinary tract infection.

Candida parapsilosis is an emerging opportunistic pathogen, second in frequency only to C. albicans and commonly associated with both mucosal and systemic infections. Adhesion to biotic surfaces is a key step for the development of mycoses. The C. parapsilosis genome encodes 5 predicted agglutinin-like sequence proteins and their precise role in the adhesion process still remains to be elucidated. In this study, we focused on the putative adhesin Cpar2_404800, in view of its high homology to the most important adhesion molecule in C. albicans. Two independent lineages of C. parapsilosis CPAR2_404800 heterozygous and null mutants were obtained by site-specific deletion. CPAR2_404800 mutants did not differ from wild-type strain in terms of in vitro growth or in their ability to undergo morphogenesis. However, when compared for adhesion to a biotic surface, CPAR2_404800 null mutants exhibited a marked reduction in their adhesion to buccal epithelial cells (>60% reduction of adhesion index). Reintroduction of one copy of CPAR2_404800 gene in the null background restored wild type phenotype. A murine model of urinary tract infection was used to elucidate the in vivo contribution of CPAR2_404800. A 0.5 and 1 log10 reduction in colony forming unit numbers (per gram) was observed respectively in bladder and kidneys obtained from mice infected with null mutant compared to wild-type infected ones. Taken together, these findings provide the first evidence for a direct role of CPAR2_404800 in C. parapsilosis adhesion to host surfaces and demonstrate its contribution to the pathogenesis of murine urinary candidiasis.

Comparison of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis adhesive properties and pathogenicity.

Retrospective studies indicate that Candida metapsilosis and Candida orthopsilosis each represents 1-10% of the infections/colonisations attributed to C. parapsilosis by conventional biochemical tests. Little is known on the virulence properties of these fungi and on their role in the establishment/progression of the infection. In this study, the adhesive properties of clinical isolates belonging to the 'psilosis' species were assessed in an in vitro model of co-incubation with human buccal epithelial cells (HBECs). Ectophosphatase activity was also measured for all isolates, since the activity of this enzyme has previously been linked to adhesion properties in C. parapsilosis. The results indicate that whilst C. parapsilosis and C. orthopsilosis strains showed similar adhesion abilities, C. metapsilosis isolates displayed a significantly lower ability to adhere to HBECs (P<0.05). No evidence of a correlation between ectophosphatase activity and adhesion was observed, and this finding was also confirmed by phosphatase inhibition experiments. Experimental vaginal candidiasis induced in oestrogen-treated mice with representative isolates of the 3 species indicated that mice infected with C. metapsilosis displayed a reduced vaginal fungal burden, especially in the early stages of the infection. The overall findings confirm that C. orthopsilosis has a comparable behaviour to C. parapsilosis, whilst C. metapsilosis seems to possess a reduced virulence potential.

Adaptive response of microglial cells to in vitro infection by Candida albicans isolates with different genomic backgrounds.

It has been recently demonstrated that Candida albicans isolates with distinct genomic backgrounds (namely, b and c genotypes) express different susceptibility to antifungal activity by human monocytes in vitro. We show here that, although comparable in their ability to undergo dimorphic transition and in susceptibility to phagocytosis by microglial cells, the b and c isolates show striking differences in terms of intracellular survival. Only the c genotype resists indeed to intracellular killing and eventually replicates inside microglial cells, that in turn respond to fungal infection, preferentially towards the c genotype, with nuclear factor-kappaB (NF-kappaB) activation and increased Mip1alpha production. These data indicate that C. albicans-microglial cell interaction is strictly dependent upon fungal genotype, strengthening the potential significance of genotyping as prognostic parameter in clinical infections by C. albicans.

Candida albicans isolates with different genomic backgrounds display a differential response to macrophage infection.

Few human pathogens possess the ability exhibited by Candida albicans to colonize and cause symptomatic infections at different body sites. The host immune system is the major factor determining whether this opportunistic yeast behaves as a commensal or as a pathogen, since C. albicans strains appear capable of expressing similar virulence factors in response to specific body-district cues. This report provides evidence showing that C. albicans isolates with diverse genomic backgrounds (b and c karyotypes) differently modulate their pathogenic potential when assayed in cocultures with human monocytic derived macrophages (THP-1 cells). Striking differences were observed in the ability to undergo bud-hypha transition, a relevant C. albicans virulence factor, between b and c karyotypes (P<0.0001) upon their internalization by macrophages. All c types were able to develop hyphal forms, resist intracellular killing, replicate, and escape from macrophages. The b type isolates, which were shown to be more efficiently ingested by THP-1 cells than the c type strains (P=0.013), were susceptible to intracellular killing and predominantly found as blastoconidia inside macrophages. Despite their different intracellular disposition, both b and c type isolates were equally able to undergo morphogenesis and to express NRG1 and HWP1 genes, markers of the bud-hypha transition program, during in vitro propagation. Since macrophages play a critical role in the host resistance to C. albicans, the different response of b and c isolates to macrophage infection suggests that the c type strains are better suited to behave as a more virulent strain cluster.