Galleria Mellonella Model Research Articles

Evaluating the In Vitro Activity and Safety of Modified LfcinB Peptides as Potential Colon Anticancer Agents: Cell Line Studies and Insect-Based Toxicity Assessments.

Anticancer peptides are increasingly being considered as alternative treatments for cancer due to their potency, selectivity, and low toxicity. Previously, the peptide LfcinB (21-25)Pal showed in vitro anticancer effects against the Caco-2 colon cancer cell line (half-maximal inhibitory concentration (IC50): 86 μM). In this study, we developed modifications to the peptide sequence to increase its anticancer activity. Sequence modifications were made such as the inclusion of amino hexanoic acid (Ahx), N-terminal biotinylation, acetylation, and substitutions of Orn for Arg and/or d-Arg by l-Arg. The molecules were synthesized using manual solid-phase peptide synthesis (SPPS), and their synthetic feasibility (SAScore) ranged from 6.2 to 7.6. The chromatographic purities of the synthesized peptides were greater than 89%. We found that Ahx-RWQWRWQWR and RWQWRWQW-Orn showed activity against both Caco-2 and HT-29 cell lines and decreased IC50 values by approx. 50% in Caco-2 cells (IC50: 40 μM) when compared to the parent peptide RWQWRWQWR. Moreover, the modified peptides demonstrated lower hemolytic effects, with values <10% at 200 μg/mL. Toxicity was assessed using the Galleria mellonella model and the half-maximal lethal dose (LD50) for the best peptides was >100 mg/kg, indicating that their toxicity is classified as moderately toxic or lower. In contrast, cisplatin showed an LD50 of 13 mg/Kg. The designed anticancer peptides presented good in vitro activity and low toxicity, making them promising molecules for future drug development studies.

Polypeptides Targeting Paracoccidioides brasiliensis Drk1.

Considering the toxicity of conventional therapeutic approaches and the importance of precise mechanistic targets, it is important to explore signaling pathways implicated in fungal pathobiology. Moreover, treatment of paracoccidioidomycosis, a systemic mycosis caused by a dimorphic fungus, requires prolonged therapeutic regimens. Among the numerous factors underpinning the establishment of Paracoccidioides spp. infection, the capacity to transition from the mycelial to the yeast form is of pivotal importance. The Drk1 protein of Paracoccidioides brasiliensis likely plays a decisive role in this morphological shift and subsequent virulence. We identified peptides with affinity for the PbDrk1 protein using the phage-display method and assessed the effects of these peptides on P. brasiliensis. The peptides were found to inhibit the phase transition of P. brasiliensis. Furthermore, a substantial proportion of these peptides prevented adhesion to pneumocytes. Although these peptides may not possess inherent antifungal properties, they can augment the effects of certain antifungal agents. Notably, the cell wall architecture of P. brasiliensis appears to be modulated by peptide intervention, resulting in a reduced abundance of glycosylated proteins and lipids. These peptides were also evaluated for their efficacy in a Galleria mellonella model and shown to contribute to enhanced larval survival rates. The role of PbDrk1, which is notably absent in mammals, should be further investigated to improve the understanding of its functional role in P. brasiliensis, which may be helpful for designing novel therapeutic modalities.

Virulence Traits and Azole Resistance in Korean Candida auris Isolates.

We analyzed the virulence traits and azole resistance mechanisms of 104 Candida auris isolates collected from 13 Korean hospitals from 1996 to 2022. Of these 104 isolates, 96 (5 blood and 91 ear isolates) belonged to clade II, and 8 (6 blood and 2 other isolates) belonged to clade I. Fluconazole resistance (minimum inhibitory concentration ≥32 mg/L) was observed in 68.8% of clade II and 25.0% of clade I isolates. All 104 isolates were susceptible to amphotericin B and three echinocandins. In 2022, six clade I isolates indicated the first nosocomial C. auris cluster in Korea. Clade II C. auris isolates exhibited reduced thermotolerance at 42 °C, with diminished in vitro competitive growth and lower virulence in the Galleria mellonella model compared to non-clade II isolates. Of the 66 fluconazole-resistant clade II isolates, several amino acid substitutions were identified: Erg11p in 14 (21.2%), Tac1Ap in 2 (3.0%), Tac1Bp in 62 (93.9%), and Tac1Bp F214S in 33 (50.0%). Although there were a limited number of non-clade II isolates studied, our results suggest that clade II C. auris isolates from Korean hospitals might display lower virulence traits than non-clade II isolates, and their primary fluconazole resistance mechanism is linked to Tac1Bp mutations.

Covert dissemination of pLVPK-like virulence plasmid in ST29-K54 Klebsiella pneumoniae: emergence of low virulence phenotype strains.

This study aimed to explore the epidemic, clinical characteristics, and molecular and virulence attributes of Klebsiella pneumoniae serotype K54 (K54-Kp). A retrospective study was conducted on 328 strains of Klebsiella pneumoniae screened in a Chinese hospital from January 2016 to December 2019. The virulence genes and antibiotic resistance genes (ARGs) were detected by PCR, and a drug sensitivity test was adopted to detect drug resistance. Multilocus sequence typing (MLST) and PFGE were performed to determine the clonal correlation between isolates. Biofilm formation assay, serum complement-mediated killing, and Galleria mellonella infection were used to characterize the virulence potential. Our results showed that thirty strains of K54-Kp were screened from 328 strains of bacteria, with an annual detection rate of 2.29%. K54-Kp had a high resistance rate to antibiotics commonly used in the clinic, and patients with hepatobiliary diseases were prone to K54-Kp infection. MLST typing showed 10 sequence typing, mainly ST29 (11/30), which concentrated in the B2 cluster. K54-Kp primarily carried virulence genes of aerobactin, silS, allS, wcaG, wabG, and mrkD, among which the terW gene was closely related to ST29 (p<0.05). The strains infected by the bloodstream had strong biofilm formation ability (p<0.05). Most strains were sensitive to serum. Still, the virulence of pLVPK-like virulence plasmid in ST29-K54 Klebsiella pneumoniae was lower than that of ST11 type and NTUH-K2044 in the Galleria mellonella model. Therefore, these findings supply a foundation to roundly comprehend K54-Kp, and clinicians should strengthen supervision and attention.

Impact of diesel exhaust particles on infections with Mycobacterium bovis BCG in in vitro human macrophages and an in vivo Galleria mellonella model

There are strong suggestions for a link between pulmonary tuberculosis (TB) and air quality. Diesel exhaust is one of the main contributors to pollution and it is reported to be able to modify susceptibility to lung infections. In this study we exposed THP-1 human macrophages and Mycobacterium bovis BCG to diesel exhaust particles (DEPs). High cytotoxicity and activation of apoptosis was found in THP-1 cells at 3 and 6 days, but no effect was found on the growth of M. bovis BCG. Infection of THP-1 cells exposed to a non-cytotoxic DEP concentration showed a limited capacity to engulf latex beads. However, M. bovis BCG infection of macrophages did not result in an increase in the bacterial burden, but it did result in an increase in the bacteria recovered from the extracellular media, suggesting a poor contention of M. bovis BCG. We also observed that DEP exposure limited the production of cytokines. Using the Galleria mellonella model of infection, we observed that larvae exposed to low levels of DEPs were less able to survive after infection with M. bovis BCG and had a higher internal bacterial load after 4 days of infection. Unraveling the links between air pollution and impairment of human antimycobacterial immunity is vital, because pollution is rapidly increasing in areas where TB incidence is extremely high.

Disrupting iron homeostasis can potentiate colistin activity and overcome colistin resistance mechanisms in Gram-Negative Bacteria

Acinetobacter baumannii is a Gram-negative priority pathogen that can readily overcome antibiotic treatment through a range of intrinsic and acquired resistance mechanisms. Treatment of carbapenem-resistant A. baumannii largely relies on the use of colistin in cases where other treatment options have been exhausted. However, the emergence of resistance against this last-line drug has significantly increased amongst clinical strains. In this study, we identify the phytochemical kaempferol as a potentiator of colistin activity. When administered singularly, kaempferol has no effect on growth but does impact biofilm formation. Nonetheless, co-administration of kaempferol with sub-inhibitory concentrations of colistin exposes bacteria to a metabolic Achilles heel, whereby kaempferol-induced dysregulation of iron homeostasis leads to bacterial killing. We demonstrate that this effect is due to the disruption of Fenton’s reaction, and therefore to a lethal build-up of toxic reactive oxygen species in the cell. Furthermore, we show that this vulnerability can be exploited to overcome both intrinsic and acquired colistin resistance in clinical strains of A. baumannii and E. coli in vitro and in the Galleria mellonella model of infection. Overall, our findings provide a proof-of-principle demonstration that targeting iron homeostasis is a promising strategy for enhancing the efficacy of colistin and overcoming colistin-resistant infections.

Coexistence of c-rmpA with p-rmpA and p-rmpA2 rather than excessive siderophores confers higher virulence in K1 Klebsiella pneumoniae

Pyogenic liver abscess (PLA), which is particularly endemic in East Asia, is a relatively common and fatal infectious disease. Over the last 30-40 years, Klebsiella pneumoniae has replaced Escherichia coli as the dominant and overwhelming pathogen. To investigate the survival advantage of serotype K1 K.pneumoniae, we determined sequence types (STs), serotypes, and 11 virulence genes (allS, entB, irp2, iroN, iucA, fimH, mrkD, p-rmpA2, c-rmpA, p-rmpA, and peg-344). Virulence genes c-rmpA, p-rmpA, and p-rmpA2 in K.pneumoniae NTUH-K2044, which all confer hypercapsule and consequent hypervirulence, were deleted individually, and the consequent effects were evaluated. The lethality of various K1 K.pneumoniae strains was compared by using the Galleria mellonella model. In total, 31 K1 K.pneumoniae strains causing PLA and 30 causing non-PLA were identified. A significantly higher rate of c-rmpA was presented in PLA-derived K.pneumoniae strains than in non-PLA-derived strains. Similar ST23 (which dominates K1 strains) and string test-positive rates were observed in the two groups. Deletion of c-rmpA, p-rmpA, and p-rmpA2 individually did not confer significant effects on morphologies, such as positive string test, hypercapsule, and growth speed. Δc-rmpA presented weaker expressions of p-rmpA/p-rmpA2 than NTUH-K2044 and showed a higher expression of manC than Δp-rmpA and Δp-rmpA2. Three rmpAs conferred more virulence than one or two rmpAs, which presented an equally lethal effect in K1 K.pneumoniae. Klebsiella pneumoniae strains (H19 and H34) with the same genetic backgrounds except for siderophores showed equal virulence, but were less virulent than strain NTUH-K2044. Thus, the coexistence of c-rmpA with p-rmpA and p-rmpA2 enhances the lethality of K1 K.pneumoniae strains and the development of PLA. Excessive siderophores are not vital for the hypervirulence of K1 K.pneumoniae strains, although K1 strains usually harbour them on a molecular basis.

Antibacterial Activity of ZnO Nanoparticles in a Staphylococcus-aureus-Infected Galleria mellonella Model Is Tuned by Different Apple-Derived Phytocargos.

This research investigates pH changes during the green synthesis of ZnO nanoparticles (NPs) and emphasises its importance in their physicochemical, antibacterial, and biological properties. Varying the synthesis pH from 8 to 12 using "Bravo de Esmolfe" apple extracts neither affected the morphology nor crystallinity of ZnO but impacted NP phytochemical loads. This difference is because alkaline hydrolysis of phytochemicals occurred with increasing pH, resulting in BE-ZnO with distinct phytocargos. To determine the toxicity of BE-ZnO NPs, Galleria mellonella was used as an alternative to non-rodent models. These assays showed no adverse effects on larvae up to a concentration of 200 mg/kg and that NPs excess was relieved by faeces and silk fibres. This was evaluated by utilising fluorescence-lifetime imaging microscopy (FLIM) to track NPs' intrinsic fluorescence. The antibacterial efficacy against Staphylococcus aureus was higher for BE-ZnO12 than for BE-ZnO8; however, a different trend was attained in an in vivo infection model. This result may be related to NPs' residence in larvae haemocytes, modulated by their phytocargos. This research demonstrates, for the first time, the potential of green synthesis to modulate the biosafety and antibacterial activity of NPs in an advanced G. mellonella infection model. These findings support future strategies to overcome antimicrobial resistance by utilizing distinct phytocargos to modulate NPs' action over time.

Evaluation of Virulence of Salmonella Infantis and Salmonella Enteritidis with In Vitro and In Vivo Models.

Salmonella Infantis and Enteritidis serovars have been reported as important causes of salmonellosis in humans worldwide. However, the virulence of these two serovars has yet to be compared. To evaluate the virulence of Salmonella Infantis (n = 23) and Salmonella Enteritidis (n = 7), we used two models: the Caco2 cells model (in vitro) and the Galleria mellonella model (in vivo). Additionally, the virulence genes of all tested strains were contrasted with phenotypic outcomes. Results showed that adhesion means were 18.2% for Salmonella Enteritidis and 38.2% for Salmonella Infantis strains. Invasion means were 77.1% for Salmonella Enteritidis and 56.2% for Salmonella Infantis strains. Significant differences were found between serovars in adherence and invasion assays. Mortality rates (58% for Salmonella Enteritidis and 62.6% for Salmonella Infantis) were not significantly different between serotypes. The distribution of virulence genes showed that genes fae (fimbrial adherence determinants) and shdA (nonfimbrial adherence determinants) were only found in Salmonella Infantis strains. On the other hand, the rck gene (invasion) and Plasmid-encoded fimbriae genes (pef A, B, C, D) were present in Salmonella Enteritidis exclusively. In conclusion, this study shows that Salmonella Enteritidis has a higher virulence potential under experimental conditions than Salmonella Infantis. However, more studies are needed to determine the risk that Salmonella Infantis could represent compared with Salmonella Enteritidis. Moreover, other in vivo models should be considered to assess the virulence of these serovars.

Doxycycline PEP can induce doxycycline resistance in Klebsiella pneumoniae in a Galleria mellonella model of PEP.

Four randomized controlled trials have now established that doxycycline post exposure (sex) prophylaxis (PEP) can reduce the incidence of chlamydia and syphilis in men who have sex with men. These studies have concluded that the risk of selecting for antimicrobial resistance is low. We evaluated this risk in vitro and in vivo using a Galleria mellonella infection model. We evaluated how long it took for doxycycline resistance to emerge during passage on doxycycline containing agar plates in 4 species - Escherichia coli, Klebsiella pneumoniae, Neisseria gonorrhoeae and Neisseria subflava. We then assessed if K. pneumoniae could acquire resistance to doxycycline (and cross resistance to other antimicrobials) during intermittent exposure to doxycycline in a Galleria mellonella model of doxycycline PEP. In our passage experiments, we found that resistance first emerged in K. pneumoniae. By day 7 the K. pneumoniae MIC had increased from 2 mg/L to a median of 96 mg/L (IQR 64-96). Under various simulations of doxycycline PEP in the G. mellonella model, the doxycycline MIC of K. pneumoniae increased from 2 mg/L to 48 mg/L (IQR 48-84). Ceftriaxone and ciprofloxacin MICs increased over ten-fold. Whole genome sequencing revealed acquired mutations in ramR which regulates the expression of the AcrAB-TolC efflux pump. Doxycycline PEP can select for doxycycline, ceftriaxone and ciprofloxacin resistance in K. pneumoniae in a G. mellonella model. The emergent ramR mutations were similar to those seen in circulating strains of K. pneumoniae. These findings suggest that we need to assess the effect of doxycycline PEP on resistance induction on a broader range of bacterial species than has hitherto been the case.

Enduring pathogenicity of African strains of Salmonella on plastics and glass in simulated peri-urban environmental waste piles

In low- and middle-income countries, plastic has become a major constituent of landfills and urban dump sites. Environmental plastic pollution can also provide a novel surface for the formation of microbial biofilm, which often includes pathogenic bacteria and viruses. Here, under conditions simulating a peri-urban waste pile typical of an African informal settlement, we aimed to determine if pathogenic Salmonella spp. can retain their virulence following a prolonged period of desiccation on the surfaces of environmental plastic and glass. We show that clinically (and environmentally) relevant strains of Salmonella including S. Enteritidis, S. Typhimurium and S. Typhi can persist on plastic and glass for at least 28-days and that temperature (which increases with the depth of an urban waste pile) is a key determinant of this survival. All three strains of Salmonella retained their pathogenicity (determined by using a Galleria mellonella model of infection) following their recovery from the plastisphere indicating that plastics in the environment can act as reservoirs for human pathogens and could facilitate their persistence for extended periods of time. Pathogens colonising environmental plastic waste therefore pose a heightened public health risk, particularly in areas where people are frequently exposed to plastic pollution.

In vitro and in vivo comparative analysis of antibacterial activity of green-synthesized silver nanoparticles.

This study aims to compare antibacterial effects of green-synthesized silver nanoparticles (AgNPs) with silver nitrate (AgNO3 ). AgNPs were successfully synthesized using Eucalyptus camaldulensis leaf extract as a reducing and stabilizing agent. Minimum inhibitory concentrations (MIC) of AgNPs and AgNO3 against Staphylococcus aureus and Pseudomonas aeruginosa ranged between 4.8-6.75μg/mL. Growth curves demonstrated that inhibition of P. aeruginosa occurred right after AgNPs were added and throughout the period of the study (72h). Antibacterial effects of both AgNPs and AgNO3 could be abrogated by cysteine and 2-mercaptoethanol, thiol-containing compounds. Galleria mellonella model revealed relatively low toxic effects of both AgNPs and AgNO3 . At 20MIC of AgNPs (≈137.8mg/kg), more than 80% survival of G. mellonella was observed. Unexpectedly, silver-containing agents could not rescue larvae after S. aureus infection. Further ex vivo experiments in the presence of coelomic larval fluid demonstrated the reduction of antibacterial activity of both AgNPs and AgNO3 . We speculated that anionic molecules present in the coelomic fluid might neutralize the action of Ag ions. Binding of AgNPs or AgNO3 to albumin, a major protein in human blood which transport several endogenous compounds was not detected, indicating that the silver-containing agents could be applied as an antimicrobial agent. This article is protected by copyright. All rights reserved.

Powerful and Real-Time Quantification of Antifungal Efficacy against Triazole-Resistant and -Susceptible Aspergillus fumigatus Infections in Galleria mellonella by Longitudinal Bioluminescence Imaging.

Aspergillus fumigatus is an environmental mold that causes life-threatening respiratory infections in immunocompromised patients. The plateaued effectiveness of antifungal therapy and the increasing prevalence of triazole-resistant isolates have led to an urgent need to optimize and expand the current treatment options. For the transition of in vitro research to in vivo models in the time- and resource-consuming preclinical drug development pipeline, Galleria mellonella larvae have been introduced as a valuable in vivo screening intermediate. Despite the high potential of this model, the current readouts of fungal infections in G. mellonella are insensitive, irreproducible, or invasive. To optimize this model, we aimed for the longitudinal quantification of the A. fumigatus burden in G. mellonella using noninvasive bioluminescence imaging (BLI). Larvae were infected with A. fumigatus strains expressing a red-shifted firefly luciferase, and the substrate dosage was optimized for the longitudinal visualization of the fungal burden without affecting larval health. The resulting photon flux was successfully validated for fungal quantification against colony forming units (CFU) analyses, which revealed an increased dynamic range from BLI detection. Comparison of BLI to survival rates and health index scores additionally revealed improved sensitivity for the early discrimination of differences in fungal burdens as early as 1 day after infection. This was confirmed by the improved detection of treatment efficacy against triazole-susceptible and -resistant strains. In conclusion, we established a refined G. mellonella aspergillosis model that enables the noninvasive real-time quantification of A. fumigatus by BLI. This model provides a quick and reproducible in vivo system for the evaluation of treatment options and is in line with 3Rs recommendations. IMPORTANCE Triazole-resistant Aspergillus fumigatus strains are rapidly emerging, and resistant infections are difficult to treat, causing mortality rates of up to 88%. The recent WHO priority list underscores A. fumigatus as one of the most critical fungal pathogens for which innovative antifungal treatment should be (urgently) prioritized. Here, we deliver a Galleria mellonella model for triazole-susceptible and -resistant A. fumigatus infections combined with a statistically powerful quantitative, longitudinal readout of the A. fumigatus burden for optimized preclinical antifungal screening. G. mellonella larvae are a convenient invertebrate model for in vivo antifungal screenings, but so far, the model has been limited by variable and insensitive observational readouts. We show that bioluminescence imaging-based fungal burden quantification outperforms these readouts in reliability, sensitivity, and time to the detection of treatment effects in both triazole-susceptible and -resistant infections and can thus lead to better translatability from in vitro antifungal screening results to in vivo confirmation in mouse and human studies.

The Presence of Virulent and Multidrug-Resistant Clones of Carbapenem-Resistant Klebsiella pneumoniae in Southeastern Brazil.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) represents an urgent threat to global public health due to the limited therapeutic options available to control this pathogen. This study aims to analyze the molecular epidemiology, antimicrobial resistance and virulence profile of CRKP isolated from patients at hospitals in Southeastern Brazil. KPC and other beta-lactamase genes were detected in all strains, which were also multidrug-resistant (MDR). In addition, 11 strains showed resistance to last-resort antimicrobials, such as colistin and tigecycline. MLST analysis revealed eight different sequence types (ST11, ST37, ST147, ST340, ST384, ST394, ST437, and ST628), being two (ST628 and ST394) reported for the first time in Brazil. Strains belonging to the clonal complex 258 (CC258) "high-risk clones" were prevalent in this study. The Galleria mellonella model showed the emergence of virulent CRKP strains in the healthcare environment and, suggests that colistin-resistant strains were associated with higher virulence. This study shows the presence of virulent CRKP-MDR strains in hospitals across Southeastern Brazil, and draws attention to the presence of highly virulent emerging CRKP-MDR ST628 strains, showing that virulent and resistant clones can emerge quickly, requiring constant monitoring.

Probiotic Properties and Safety Evaluation in the Invertebrate Model Host Galleria mellonella of the Pichia kudriavzevii YGM091 Strain Isolated from Fermented Goat Milk.

Potential probiotic yeast strains isolated from fermented food need to meet safe and beneficial conditions for the host's health. The Pichia kudriavzevii YGM091 strain isolated from fermented goat milk has outstanding probiotic characteristics, including: the high survival percentage in digestive system conditions (reaching up 247.13 ± 0.12 and 145.03 ± 0.06% at pH 3.0 and bile salt 0.5%, respectively); good tolerance to temperature, salt, phenol, ethanol; good surface properties such as high hydrophobicity percentage (> 60%), the high auto-aggregation percentage rate (66.56 ± 1.45% after 45min of incubation) and the high co-aggregation percentage rate with pathogenic bacteria in a short time (> 40% after 2h of incubation); biofilm forming after 24h of incubation on abiotic surfaces; antioxidant activity reached excellent level after only 24h of incubation (The percentage free radical scavenging and the Trolox equivalent reaching up 79.86 ± 0.70% and 92.09 ± 0.75µg/mL after 72h of incubation); extracellular enzymes production protease and cellulase with high activity, amylase and pectinase with moderate activity and non-lipase activity. Simultaneously, the YGM091 strain is the in vitro safety yeast: insensitive to antibiotics and fluconazole, negative for gelatinase, phospholipase, coagulase, and non-hemolysis activities. Furthermore, this strain is in vivo safety yeast with the dosages below 106CFU/larva in the Galleria mellonella model with over 90% survival larvae and the yeast density reduced to just 102-103CFU/larva after 72h post-injection. Research results have demonstrated that the Pichia kudriavzevii YGM091 strain is a safe potential probiotic yeast and could become a candidate probiotic food to be used in the future.

Metarhizium anisopliae E6 secretome reveals molecular players in host specificity and toxicity linked to cattle tick infection

Although Metarhizium anisopliae is one of the most studied fungal biocontrol agents, its infection mechanism is far from being completely understood. Using multidimensional protein identification technology (MudPIT), we evaluated the differential secretome of M. anisopliae E6 induced by the host Rhipicephalus microplus cuticle. The proteomic result showed changes in the expression of 194 proteins after exposure to host cuticle, such as proteins involved in adhesion, penetration, stress and fungal defense. Further, we performed a comparative genomic distribution of differentially expressed proteins of the M. anisopliae secretome against another arthropod pathogen, using the Beauveria bassiana ARSEF2860 protein repertory. Among 47 analyzed protein families, thirty were overexpressed in the M. anisopliae E6 predicted genome compared to B. bassiana. An in vivo toxicity assay using a Galleria mellonella model confirmed that the M. anisopliae E6 secretome was more toxic in cattle tick infections compared to other secretomes, including B. bassiana with cattle ticks and M. anisopliae E6 with the insect Dysdereus peruvianus, which our proteomic results had also suggested. These results help explain molecular aspects associated with host infection specificity due to genetic differences and gene expression control at the protein level in arthropod-pathogenic fungi.

Nanoemulsion Increases the Antifungal Activity of Amphotericin B against Four Candida auris Clades: In Vitro and In Vivo Assays.

Candida auris is an emerging yeast of worldwide interest due to its antifungal resistance and mortality rates. The aim of this study was to analyse the in vitro and in vivo antifungal activity of a nanoemulsion loaded with amphotericin B (NEA) against planktonic cells and biofilm of C. auris clinical isolates belonging to four different clades. In vivo assays were performed using the Galleria mellonella model to analyse antifungal activity and histopathological changes. The in vitro results showed that NEA exhibited better antifungal activity than free amphotericin B (AmB) in both planktonic and sessile cells, with >31% inhibition of mature biofilm. In the in vivo assays, NEA demonstrated superior antifungal activity in both haemolymph and tissue. NEA reduced the fungal load in the haemolymph more rapidly and with more activity in the first 24 h after infection. The histological analysis of infected larvae revealed clusters of yeast, immune cells, melanisation, and granulomas. In conclusion, NEA significantly improved the in vitro and in vivo antifungal activity of AmB and could be considered a promising therapy for C. auris infections.

Small molecules restore azole activity against drug-tolerant and drug-resistant Candida isolates.

Each year, fungi cause more than 1.5 billion infections worldwide and have a devastating impact on human health, particularly in immunocompromised individuals or patients in intensive care units. The limited antifungal arsenal and emerging multidrug-resistant species necessitate the development of new therapies. One strategy for combating drug-resistant pathogens is the administration of molecules that restore fungal susceptibility to approved drugs. Accordingly, we carried out a screen to identify small molecules that could restore the susceptibility of pathogenic Candida species to azole antifungals. This screening effort led to the discovery of novel 1,4-benzodiazepines that restore fluconazole susceptibility in resistant isolates of Candida albicans, as evidenced by 100-1,000-fold potentiation of fluconazole activity. This potentiation effect was also observed in azole-tolerant strains of C. albicans and in other pathogenic Candida species. The 1,4-benzodiazepines selectively potentiated different azoles, but not other approved antifungals. A remarkable feature of the potentiation was that the combination of the compounds with fluconazole was fungicidal, whereas fluconazole alone is fungistatic. Interestingly, the potentiators were not toxic to C. albicans in the absence of fluconazole, but inhibited virulence-associated filamentation of the fungus. We found that the combination of the potentiators and fluconazole significantly enhanced host survival in a Galleria mellonella model of systemic fungal infection. Taken together, these observations validate a strategy wherein small molecules can restore the activity of highly used anti-infectives that have lost potency. IMPORTANCE In the last decade, we have been witnessing a higher incidence of fungal infections, due to an expansion of the fungal species capable of causing disease (e.g., Candida auris), as well as increased antifungal drug resistance. Among human fungal pathogens, Candida species are a leading cause of invasive infections and are associated with high mortality rates. Infections by these pathogens are commonly treated with azole antifungals, yet the expansion of drug-resistant isolates has reduced their clinical utility. In this work, we describe the discovery and characterization of small molecules that potentiate fluconazole and restore the susceptibility of azole-resistant and azole-tolerant Candida isolates. Interestingly, the potentiating 1,4-benzodiazepines were not toxic to fungal cells but inhibited their virulence-associated filamentous growth. Furthermore, combinations of the potentiators and fluconazole decreased fungal burdens and enhanced host survival in a Galleria mellonella model of systemic fungal infections. Accordingly, we propose the use of novel antifungal potentiators as a powerful strategy for addressing the growing resistance of fungi to clinically approved drugs.

Genomic Epidemiology of Carbapenem-Resistant Klebsiella in Qatar: Emergence and Dissemination of Hypervirulent Klebsiella pneumoniae Sequence Type 383 Strains.

The emergence of carbapenem-resistant, hypervirulent Klebsiella pneumoniae is a new threat to health care. We studied the molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae isolates in Qatar using whole-genome sequence data. We also characterized the prevalence and genetic basis of hypervirulent phenotypes and established the virulence potential using a Galleria mellonella model. Of 100 Klebsiella isolates studied, NDM and OXA-48 were the most common carbapenemases. Core genome single-nucleotide polymorphism (SNP) analysis indicated the presence of diverse sequence types and clonal lineages; isolates belonging to Klebsiella quasipneumoniae subsp. quasipneumoniae sequence type 196 (ST196) and ST1416 may be disseminated among several health care centers. Ten K. pneumoniae isolates carried rmpA and/or truncated rmpA2, and 2 isolates belonged to KL2, indicating low prevalence of classical hypervirulent isolates. Isolates carrying both carbapenem resistance and hypervirulence genes were confined mainly to ST231 and ST383 isolates. One ST383 isolate was further investigated by MinION sequencing, and the assembled genome indicated that blaNDM was located on an IncHI1B-type plasmid (pFQ61_ST383_NDM-5) which coharbored several virulence factors, including the regulator of the mucoid phenotype (rmpA), the regulator of mucoid phenotype 2 (rmpA2), and aerobactin (iucABCD and iutA), likely resulting from recombination events. Comparative genomics indicated that this hybrid plasmid may be present in two additional Qatari ST383 isolates. Carbapenem-resistant, hypervirulent K. pneumoniae ST383 isolates pose an emerging threat to global health due to their simultaneous hypervirulence and multidrug resistance.

Identification by Reverse Vaccinology of Three Virulence Factors in Burkholderia cenocepacia That May Represent Ideal Vaccine Antigens.

The Burkholderia cepacia complex comprises environmental and clinical Gram-negative bacteria that infect particularly debilitated people, such as those with cystic fibrosis. Their high level of antibiotic resistance makes empirical treatments often ineffective, increasing the risk of worst outcomes and the diffusion of multi-drug resistance. However, the discovery of new antibiotics is not trivial, so an alternative can be the use of vaccination. Here, the reverse vaccinology approach has been used to identify antigen candidates, obtaining a short-list of 24 proteins. The localization and different aspects of virulence were investigated for three of them-BCAL1524, BCAM0949, and BCAS0335. The three antigens were localized in the outer membrane vesicles confirming that they are surface exposed. We showed that BCAL1524, a collagen-like protein, promotes bacteria auto-aggregation and plays an important role in virulence, in the Galleria mellonella model. BCAM0949, an extracellular lipase, mediates piperacillin resistance, biofilm formation in Luria Bertani and artificial sputum medium, rhamnolipid production, and swimming motility; its predicted lipolytic activity was also experimentally confirmed. BCAS0335, a trimeric adhesin, promotes minocycline resistance, biofilm organization in LB, and virulence in G. mellonella. Their important role in virulence necessitates further investigations to shed light on the usefulness of these proteins as antigen candidates.