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Mycetoma, a neglected infection of subcutaneous tissues, poses a significant health burden, especially in tropical regions. It is caused by fungal (eumycetoma) and bacterial (actinomycetoma) pathogens, with current treatments often providing unsatisfactory outcomes. This study aims to discover novel broad-spectrum antimicrobial agents to circumvent the lengthy and costly diagnostic procedures. Eight essential oils (EOs) from the roots and aerial parts of Geigeria alata, Lavandula angustifolia, Melaleuca alternifolia, Myristica fragrans, Pimpinella anisum, Syzigum aromaticum, and Thymus vulgaris were prepared using steam distillation. The in vitro antimycetomal activity against Madurella mycetomatis and Actinomadura madurae strains was assessed using resazurin assays. The chemical compositions of the EOs were analyzed using gas chromatography and mass spectrometry (GC–MS). Promising EOs underwent further in vivo toxicity and efficacy testing in Galleria mellonella larvae models. EOs of G. alata roots, M. fragrans, P. anisum, S. aromaticum, and T. vulgaris showed wide-spectrum dual in vitro antimycetomal activity against all tested strains, with minimum inhibitory concentrations (MICs) ranging from 0.004 to 0.125% v/v. G. alata aerial parts and L. angustifolia EOs demonstrated activity predominantly against A. madurae, while M. alternifolia EO did not inhibit any tested strains. M. fragrans and P. anisum EOs significantly enhanced the survival of M. mycetomatis-infected larvae without inducing toxicity in uninfected larvae. Notably, P. anisum EO tended to enhance the survival of A. madurae-infected larvae, ranking it as the most promising EO among those tested. The investigated EOs, particularly P. anisum, exhibited promising broad-spectrum antimycetomal activity against fungal and bacterial pathogens responsible for mycetoma. These findings highlight the potential of essential oils as a basis for developing novel antimycetomal agents, offering hope for improved treatment strategies for this neglected disease.

Objective: To investigate the source of infection in a patient with recurrent severe neck infections caused by Klebsiella pneumoniae and to analyze the virulence of isolates obtained from different sites of the patient. Methods: We collected preoperative neck abscess puncture fluid, intraoperative neck drainage fluid, sputum, intestinal fecal specimens, and blood samples from a patient who visited Wuxi Second People's Hospital twice between 2017 and 2018. We conducted isolation, identification, drug sensitivity tests, and string tests on the isolates. Capsule serotyping and virulence gene analysis were performed using PCR. The genetic relationship of different isolates was assessed by Multilocus Sequence Typing and virulence was evaluated using the Galleria mellonella infection model. Additionally, whole-genome sequencing was used to analyze the chromosomal and plasmid genes of one isolate. Results: Klebsiella pneumoniae was detected in the sputum and fecal specimens from both hospitalizations, as well as the preoperative ultrasound-guided puncture fluid and intraoperative drainage fluid from the first hospitalization, resulting in six isolates. These isolates were all K16 serotype, positive in the string test, and identified as ST660 by Multilocus Sequence Typing, indicating they belonged to the same clone. Virulence gene analysis showed that wcaG, iucB, iroNB, rmpA, rmpA2, Aer, kfuBC, ureA, fimH, mrkD, uge, and peg344 were positive, while allS, cf29a, and Wzy_K1 were negative. In the Galleria mellonella virulence assay, the lethality of different isolates was dose-dependent. The K16 group showed significantly higher larval mortality compared to other control groups (including K1, K2, K5, K20, and K57 groups). Genome sequencing revealed that plasmid p17388 carried numerous virulence genes and insertion sequences, particularly ISKPN74, and showed high homology with other Klebsiella plasmids. Conclusion: This study is the first to report severe cervical necrotizing fasciitis caused by the K16-ST660 Klebsiella pneumoniae Isolate. The high virulence of these isolates was confirmed by the Galleria mellonella virulence assay and the detection of numerous virulence genes. In-depth analysis of plasmid p17388 suggests that ISKPN74 may enhance stable integration of the plasmid into the bacterial chromosome through recombinases and transposases, thereby reducing the likelihood of plasmid loss and increasing bacterial virulence. Additionally, IS5 family insertion sequences may carry extra promoters or enhancers that, when inserted upstream of mucoviscosity-associated genes such as rmpA, may increase the transcription levels of downstream genes. This ISKPN74-mediated integration or insertion reveals a complex genetic mechanism that may contribute to the severity of infections caused by ST660 isolates. Our findings offer new insights into the virulence and structure of ST660-K16 Klebsiella pneumoniae, suggesting that further investigation into the specific mechanisms by which these insertion sequences enhance virulence could aid in developing novel infection management strategies.

ABSTRACT Histones possess significant antimicrobial potential, yet their activity against biofilms remains underexplored. Moreover, concerns regarding adverse effects limit their clinical implementation. We investigated the antibacterial efficacy of human recombinant histone H1 subtypes against Pseudomonas aeruginosa PAO1, both planktonic and in biofilms. After the in vitro tests, toxicity and efficacy were assessed in a P. aeruginosa PAO1 infection model using Galleria mellonella larvae. Histones were also evaluated in combination with ciprofloxacin (Cpx) and gentamicin (Gm). Our results demonstrate antimicrobial activity of all three histones against P. aeruginosa PAO1, with H1.0 and H1.4 showing efficacy at lower concentrations. The bactericidal effect was associated with a mechanism of membrane disruption. In vitro studies using static and dynamic models showed that H1.4 had antibiofilm potential by reducing cell biomass. Neither H1.0 nor H1.4 showed toxicity in G. mellonella larvae, and both increased larvae survival when infected with P. aeruginosa PAO1. Although in vitro synergism was observed between ciprofloxacin and H1.0, no improvement over the antibiotic alone was noted in vivo. Differences in antibacterial and antibiofilm activity were attributed to sequence and structural variations among histone subtypes. Moreover, the efficacy of H1.0 and H1.4 was influenced by the presence and strength of the extracellular matrix. These findings suggest histones hold promise for combating acute and chronic infections caused by pathogens such as P. aeruginosa.IMPORTANCEThe constant increase of multidrug-resistant bacteria is a critical global concern. The inefficacy of current therapies to treat bacterial infections is attributed to multiple mechanisms of resistance, including the capacity to form biofilms. Therefore, the identification of novel and safe therapeutic strategies is imperative. This study confirms the antimicrobial potential of three histone H1 subtypes against both Gram-negative and Gram-positive bacteria. Furthermore, histones H1.0 and H1.4 demonstrated in vivo efficacy without associated toxicity in an acute infection model of Pseudomonas aeruginosa PAO1 in Galleria mellonella larvae. The bactericidal effect of these proteins also resulted in biomass reduction of P. aeruginosa PAO1 biofilms. Given the clinical significance of this opportunistic pathogen, our research provides a comprehensive initial evaluation of the efficacy, toxicity, and mechanism of action of a potential new therapeutic approach against acute and chronic bacterial infections.

Mucormycosis is an invasive fungal infection caused by certain members of the fungal order of Mucorales. The species most frequently identified as the etiological agents of mucormycosis belong to the genera Rhizopus, Lichtheimia, and Mucor. The frequency of systemic mucormycosis has been increasing, mainly because of increasing numbers of susceptible patients. Furthermore, Mucorales display intrinsic resistance to the majority of routinely used antifungal agents (e.g., echinocandins and short-tailed azoles), which limits the number of possible therapeutic options. All the above-mentioned issues urge the improvement of molecular identification methods and the discovery of new antifungal targets and strategies. Spore coat proteins (CotH) constitute a kinase family present in many pathogenic bacteria and fungi and participate in the spore formation in these organisms. Moreover, some of them can act as virulence factors being receptors of the human GRP78 protein during Rhizopus delemar-induced mucormycosis. We identified 17 cotH-like genes in the Mucor lusitanicus genome database. Successful disruption of five cotH genes in Mucor was performed using the CRISPR-Cas9 system. The CotH3 and CotH4 proteins play a role in adaptation to different temperatures as well as in developing the cell wall structure. We also show CotH4 protein is involved in spore wall formation by affecting the total chitin content and, thus, the composition of the spore wall. The role of CotH3 and CotH4 proteins in virulence was confirmed in two invertebrate models and a diabetic ketoacidosis (DKA) mouse model. IMPORTANCE Current treatment options for mucormycosis are inadequate, resulting in high mortality rates, especially among immunosuppressed patients. The development of novel therapies for mucormycosis has been hampered by lack of understanding of the pathogenetic mechanisms. The importance of the cell surface CotH proteins in the pathogenesis of Rhizopus-mediated mucormycosis has been recently described. However, the contribution of this family of proteins to the virulence of other mucoralean fungi and their functionality in vital processes remain undefined. Through the use of the CRISPR-Case9 gene disruption system, we demonstrate the importance of several of the CotH proteins to the virulence of Mucor lusitanicus by using three infection models. We also report on the importance of one of these proteins, CotH4, to spore wall formation by affecting chitin content. Therefore, our studies extend the importance of CotH proteins to Mucor and identify the mechanism by which one of the CotH proteins contributes to the development of a normal fungal cell wall, thereby indicating that this family of proteins can be targeted for future development of novel therapeutic strategies of mucormycosis.

IntroductionManaging burn injuries is a challenge in healthcare. Due to the alarming increase in antibiotic resistance, new prophylactic and therapeutic strategies are being sought. This study aimed to evaluate the potential of live Lactic Acid Bacteria for managing burn infections, using Galleria mellonella larvae as an alternative preclinical animal model and comparing the outcomes with a common antibiotic.MethodsThe antimicrobial activity of LAB isolated from human breast milk was assessed in vitro against Pseudomonas aeruginosa ATCC 27853. Additionally, the immunomodulatory effects of LAB were evaluated in vivo using the G. mellonella burn wound infection model.Results and discussionIn vitro results demonstrated the antimicrobial activity of Lactic Acid Bacteria against P. aeruginosa. In vivo results show that their prophylactic treatment improves, statistically significant, larval survival and modulates the expression of immunity-related genes, Gallerimycin and Relish/NF-κB, strain-dependently. These findings lay the foundation and suggest a promising alternative for burn wound prevention and management, reducing the risk of antibiotic resistance, enhancing immune modulation, and validating the potential G. mellonella as a skin burn wound model.

ABSTRACTClostridium perfringens is a prevalent bacterial pathogen in poultry, and due to the spread of antimicrobial resistance, alternative treatments are needed to prevent and treat infection. Bacteriophages (phages), viruses that kill bacteria, offer a viable option and can be used therapeutically to treat C. perfringens infections. The aim of this study was to isolate phages against C. perfringens strains currently circulating on farms across the world and establish their virulence and development potential using host range screening, virulence assays, and larva infection studies. We isolated 32 phages of which 19 lysed 80%–92% of our global C. perfringens poultry strain collection (n = 97). The virulence of these individual phages and 32 different phage combinations was quantified in liquid culture at multiple doses. We then developed a multi-strain C. perfringens larva infection model, to mimic an effective poultry model used by the industry. We tested the efficacy of 16/32 phage cocktails in the larva model. From this, we identified that our phage cocktail consisting of phages CPLM2, CPLM15, and CPLS41 was the most effective at reducing C. perfringens colonization in infected larvae when administered before bacterial challenge. These data suggest that phages do have significant potential to prevent and treat C. perfringens infection in poultry.IMPORTANCEClostridium perfringens causes foodborne illness worldwide, and 95% of human infections are linked to the consumption of contaminated meat, including chicken products. In poultry, C. perfringens infection causes necrotic enteritis, and associated mortality rates can be up to 50%. However, treating infections is difficult as the bacterium is becoming antibiotic-resistant. Furthermore, the poultry industry is striving toward reduced antibiotic usage. Bacteriophages (phages) offer a promising alternative, and to progress this approach, robust suitable phages and laboratory models that mimic C. perfringens infections in poultry are required. In our study, we isolated phages targeting C. perfringens and found that many lyse C. perfringens strains isolated from chickens worldwide. Consistent with other published studies, in the model systems we assayed here, when some phages were combined as cocktails, the infection was cleared most effectively compared to individual phage use.

Carbapenem-resistant Acinetobacter baumannii (CRAB) has become a new threat in recent years, owing to its rapidly increasing resistance to antibiotics and new effective therapies are needed to combat this pathogen. Phage therapy is considered to be the most promising alternative for treating CRAB infections. In this study, a novel phage, Ab_WF01, which can lyse clinical CRAB, was isolated and characterized from hospital sewage. The multiplicity of infection, morphology, one-step growth curve, stability, sensitivity, and lytic activity of the phage were also investigated. The genome of phage Ab_WF01 was 41, 317 bp in size with a GC content of 39.12% and encoded 51 open reading frames (ORFs). tRNA, virulence, and antibiotic resistance genes were not detected in the phage genome. Comparative genomic and phylogenetic analyses suggest that phage Ab_WF01 is a novel species of the genus Friunavirus, subfamily Beijerinckvirinae, and family Autographiviridae. The in vivo results showed that phage Ab_WF01 significantly increased the survival rate of CRAB-infected Galleria mellonella (from 0% to 70% at 48 h) and mice (from 0% to 60% for 7 days). Moreover, after day 3 post-infection, phage Ab_WF01 reduced inflammatory response, with strongly ameliorated histological damage and bacterial clearance in infected tissue organs (lungs, liver, and spleen) in mouse CRAB infection model. Taken together, these results show that phage Ab_WF01 holds great promise as a potential alternative agent with excellent stability for against CRAB infections.

The larvae of the insect Galleria mellonella have gained wide acceptance as an in vivo model for the study of virulence in Gram-negative and Gram-positive bacteria and fungi. Importantly, G. mellonella offers an alternative, low-cost, and ethically acceptable method of assessing pathogens. These insects have a number of advantages, including ease of breeding, a rapid developmental cycle, low maintenance costs, and ease of experimental manipulation. Most importantly, G. mellonella larvae have a complete immune system and a nonspecific immune response similar to that of humans, making them a valuable model for studying pathogen-host interactions. Furthermore, using this model, it is possible to test the efficacy of various chemical compounds or natural substances that show antimicrobial activity. In this literature review, we provide a comprehensive overview of recent studies of the use of G. mellonella in virulence analyses and as a platform for testing the efficacy of new drugs.

ABSTRACTThe ability of many human pathogens to infect requires their ability to adhere to the host surfaces as a first step in the process. Porphyromonas gingivalis, a keystone oral pathogen, uses adhesins to adhere to the surface of the gingival epithelium and other members of the oral microbiome. In a previous study, we identified several proteins potentially linked to virulence whose mRNA levels are regulated by CRISPR-Cas type I-C. Among those, PGN_1547 was highly upregulated in the CRISPR-Cas 3 mutant. PGN_1547 is annotated as a hypothetical protein. Employing homology searching, our data support that PGN_1547 resembles an auto-transporter adhesin of P. gingivalis based on containing the DUF2807 domain. To begin to characterize the function of PGN_1547, we found that a deletion mutant displayed a significant decrease in virulence using a Galleria mellonela model. Furthermore, this mutant was significantly impaired in forming biofilms and attaching to the macrophage-like cell THP-1. Luminex revealed that the PGN_1547 mutant elicited a less robust cytokine and chemokine response from THP-1 cells, and TLR2 predominantly sensed that recombinant PGN_1547. Taken together, these findings broaden our understanding of the toolbox of virulence factors possessed by P. gingivalis. Importantly, PGN_1547, a hypothetical protein, has homologs in another member of the order Bacteroidales whose function is unknown, and our results could shed light on the role of this family of proteins as auto-transport adhesins in this phylogenetic group.IMPORTANCEPeriodontal diseases are among humans’ most common infections, and besides their effect on the oral cavity, they have been associated with systemic inflammatory conditions. Among members of the oral microbiome implicated in the development of periodontitis, Porphyromonas gingivalis is considered a keystone pathogen. We have identified a new adhesin that acts as a virulence factor, PGN_1547, which contains the DUF2807 domain, which belongs to the putative auto-transporter adhesin, head GIN domain family. Deletion of this gene lowers the virulence of P. gingivalis and impacts the ability of P. gingivalis to form biofilm and attach to host cells. Furthermore, the broad distribution of these receptors in the order Bacteroidales suggests their importance in colonization by this important group of organisms.

ABSTRACTExtensively drug-resistant (XDR) Klebsiella pneumoniae inflict a notable burden on healthcare worldwide. Of specific concern are strains producing carbapenem-hydrolyzing enzymes, as the therapeutic options for these strains are still very limited. Specific sequence types of K. pneumoniae have been noted for their epidemic occurrence globally, but the mechanisms behind the success of specific clones remain unclear. Herein, we have characterized 20 high-risk clones (HiRCs) and 10 non-HiRCs of XDR K. pneumoniae, exploring factors connected to the epidemiological success of some clones. Isolates were subjected to core genome multilocus sequence typing analysis to determine the clonal relationships of the isolates and subsequently characterized with regard to features known to be linked to overall bacterial fitness and virulence. The genomes were analyzed in silico for capsule types, O antigens, virulence factors, antimicrobial resistance genes, prophages, and CRISPR-Cas loci. In vitro growth experiments were conducted to retrieve proxies for absolute and relative fitness for 11 HiRC and 9 non-HiRC isolates selected based on the clonal groups they belonged to, and infections in a Galleria mellonella insect model were used to evaluate the virulence of the isolates in vivo. This study did not find evidence that virulence factors, prophages, CRISPR-Cas loci, or fitness measured in vitro alone would contribute to the global epidemiological success of specific clones of carbapenemase-producing XDR K. pneumoniae. However, this study did find the HiRC group to be more virulent than the non-HiRC group when measured in vivo in a model with G. mellonella. This suggests that the virulence and epidemiological success of certain clones of K. pneumoniae cannot be explained by individual traits investigated in this study and thus warrant further experiments in the future.IMPORTANCEHerein, we explored potential explanations for the successfulness of some epidemic or high-risk clones of carbapenemase-producing Klebsiella pneumoniae. We found differences in mortality in a larva model but found no clear genomic differences in known virulence markers. Most of the research on virulence in K. pneumoniae has been focused on hypervirulent strains, but here, we try to understand differences within the group of highly resistant strains. The results from the larva virulence model could be used to design experiments in higher animals. Moreover, the data could provide further support to a differentiated infection control approach against extensively drug-resistant strains, based on their classification as high-risk clones.

Mucormycosis is still regarded a rare fungal infection, but the high incidences of COVID-associated cases in India and other countries have shown its potential threat to large patient cohorts. In addition, infections by these fast-growing fungi are often fatal and cause disfigurement, badly affecting patients’ lives. In advancing our understanding of pathogenicity factors involved in this disease, to enhance the diagnostic toolset and to evaluate novel treatment regimes, animal models are indispensable. As ethical and practical considerations typically favor the use of alternative model systems, this review provides an overview of alternative animal models employed for mucormycosis and discusses advantages and limitations of the respective model.

Abstract Background Acinetobacter baumannii is one of the main causes of healthcare-associated infections that threaten public health, and carbapenems, such as meropenem, have been a therapeutic option for these infections. Therapeutic failure is mainly due to the antimicrobial resistance of A. baumannii, as well as the presence of persister cells. Persisters constitute a fraction of the bacterial population that present a transient phenotype capable of tolerating supra-lethal concentrations of antibiotics. Some proteins have been suggested to be involved in the onset and/or maintenance of this phenotype. Thus, we investigated the mRNA levels of the adeB (AdeABC efflux pump component), ompA, and ompW (outer membrane proteins) in A. baumannii cells before and after exposure to meropenem. Results We found a significant increase (p-value 5.5-fold) and ompW (> 10.5-fold) in persisters. However, adeB did not show significantly different expression levels when comparing treated and untreated cells. Therefore, we suggest that these outer membrane proteins, especially OmpW, could be part of the mechanism of A. baumannii persisters to deal with the presence of high doses of meropenem. We also observed in the Galleria mellonella larvae model that persister cells are more virulent than regular ones, as evidenced by their LD50 values. Conclusions Taken together, these data contribute to the understanding of the phenotypic features of A. baumannii persisters and their relation to virulence, as well as highlight OmpW and OmpA as potential targets for drug development against A. baumannii persisters.

IntroductionCandida albicans is the primary cause of systemic candidiasis, which is involved in high morbidity and mortality. Drug resistance exacerbates these problems. In addition, there are limited antifungal drugs available. In order to solve these problems, combination therapy has aroused great interest. Teriflunomide is an immunosuppressant. In the present work, we aimed to identify whether teriflunomide can reverse the resistance of Candida albicans in the presence of sub-inhibitory concentrations of fluconazole in vitro and in vivo.MethodsSeven Candida albicans isolates were used in this study. Susceptibility of Candida albicans in vitro to the drugs was determined using a checkerboard microdilution assay in accordance with the recommendations of the Clinical and Laboratory Standards Institute. The effects of drugs on biofilm biomass of Candida albicans were determined by crystal violet staining. The development ability of Candida albicans hyphae was performed using a modified broth microdilution method. Galleria mellonella was used for testing the in vivo efficacy of the combination therapies. ResultsWe found that the combination of teriflunomide (64 µg/mL) and fluconazole (0.5-1 µg/mL) has a significant synergistic effect in all resistant Candida albicans isolates (n=4). Also, this drug combination could inhibit the immature biofilm biomass and hyphae formation of resistant Candida albicans. Galleria mellonella was used for testing the in vivo efficacy of this combination therapies. As for the Galleria mellonella larvae infected by resistant Candida albicans, teriflunomide (1.6 µg/larvae) combined with fluconazole (1.6 µg/larvae) significantly increased their survival rates, and reduced the fungal burden, as well as damage of tissue in comparison to that in the control group or drug monotherapy group. ConclusionThese results expand our knowledge about the antifungal potential of teriflunomide as an adjuvant of existing antifungal drugs, and also open new perspectives in the treatment of resistant Candida albicans based on repurposing clinically available nonantifungal drugs.

ABSTRACT Due to the increasing incidence of antibiotic-resistant bacterial pathogens, the need for the development of alternative compounds is of utmost importance. Bacteriocins are considered a promising alternative to antibiotics. In this study, we determined the in vitro activities of 65 bacteriocins from the PARAGEN collection along with microbisporicin (NAI-107) against 14 WHO reference isolates of Neisseria gonorrhoeae, one isolate of Neisseria meningitidis, and four commensal Neisseria spp. The initial screening of the bacteriocins was carried out using a spot-on-lawn assay wherein 12 bacteriocins (12/66) showed a zone of inhibition against the WHO-P N. gonorrhoeae isolate. Four bacteriocins (4/12), Garvicin KS (GarKS) (ABC), lacticin (lcn) Q, lcn Z, and NAI-107, demonstrated higher activity, and minimum inhibitory concentrations (MICs) using agar dilutions were determined for 19 Neisseria spp. GarKS (ABC), lcn Q, and lcn Z had an MIC range of 4–16 µg/mL, whereas NAI-107 had an MIC range of

IntroductionCandida parapsilosis, a pathogenic yeast associated with systemic infections, exhibits metabolic adaptability in response to nutrient availability.MethodsWe investigated the impact of RPMI glucose supplemented (RPMId), TSB, BHI and YPD media on C. parapsilosis growth, morphology, susceptibility (caspofungin and amphotericin B), and in vivo virulence (Galleria mellonella) in planktonic and biofilm states.ResultsHigh-glucose media favors growth but hinders metabolic activity and filamentation. Media promoting carbohydrate production reduces biofilm susceptibility. Virulence differences between planktonic cells and biofilm suspensions from the same media shows that biofilm-related factors influence infection outcome depending on nutrient availability. Pseudohyphal growth occurred in biofilms under low oxygen and shear stress, but its presence is not exclusively correlated with virulence.DiscussionThis study provides valuable insights into the intricate interplay between nutrient availability and C. parapsilosis pathogenicity. It emphasizes the importance of considering pathogen behavior in diverse conditions when designing research protocols and therapeutic strategies.

ABSTRACT Carbapenem resistance among Enterobacterales has become a global health concern. Clinical Escherichia coli isolates producing the metallo β-lactamase NDM have been isolated from two hospitals in Faisalabad, Pakistan. These E. coli strains were characterized by MALDI-TOF, PCR, antimicrobial susceptibility testing, XbaI and S1 nuclease pulsed-field gel electrophoresis (PFGE), conjugation assay, DNA hybridization, whole genome sequencing, bioinformatic analysis, and Galleria mellonella experiments. Thirty-four bla NDM producing E. coli strains were identified among 52 nonduplicate carbapenem-resistant strains. More than 90% of the isolates were found to be multidrug resistant by antimicrobial susceptibility testing. S1 PFGE confirmed the presence of bla NDM gene on plasmids ranging from 40 kbps to 250 kbps, and conjugation assays demonstrated transfer frequencies of bla NDM harboring plasmids ranging from 1.59 × 10−1 to 6.46 × 10−8 per donor. Whole genome sequencing analysis revealed bla NDM-5 as the prominent NDM subtype with the highest prevalence of bla OXA-1, bla CTX-M-15, aadA2, aac(6')-Ib-cr, and tet(A) associated resistant determinants. E. coli sequence types: ST405, ST361, and ST167 were prominent, and plasmid Inc types: FII, FIA, FIB, FIC, X3, R, and Y, were observed among all isolates. The genetic environment of bla NDM region on IncF plasmids included partial ISAba125, the bleomycin ble gene, and a class I integron. The virulence genes terC, traT, gad, fyuA, irp2, capU, and sitA were frequently observed, and G. mellonella experiments showed that virulence correlated with the number of virulence determinants. A strong infection control management in the hospital is necessary to check the emergence of carbapenem resistance in Gram-negative bacteria. IMPORTANCE We describe a detailed analysis of highly resistant clinical E. coli isolates from two tertiary care centers in Pakistan including carbapenem resistance as well as common co-resistance mechanisms. South Asia has a huge problem with highly resistant E. coli. However, we find that though these isolates are very difficult to treat they are of low virulence. Thus the Western world has an increasing problem with virulent E. coli that are mostly of low antibiotic resistance, whereas, South Asia has an increasing problem with highly resistant E. coli that are of low virulence potential. These observations allow us to start to devise methodologies to limit both virulence and resistance and combat problems in developing nations as well as the Western world.

Candida auris has globally emerged as a multidrug-resistant fungus linked to healthcare-associated outbreaks. There is still limited evidence on its virulence, pathogenicity determinants, and complex host–pathogen interactions. This study analyzes the in vivo fungal behaviour, immune response, and host–pathogen interactions upon C. auris infection compared to C. albicans and C. parapsilosis in G. mellonella. This was performed by immunolabelling fungal structures and larval plasmatocytes and using a quantitative approach incorporating bioinformatic morphometric techniques into the study of microbial pathogenesis. C. auris presents a remarkably higher immunogenic activity than expected at its moderate degree of tissue invasion. It induces a greater inflammatory response than C. albicans and C. parapsilosis at the expense of plasmatocyte nodule formation, especially in non-aggregative strains. It specifically invades the larval respiratory system, in a pattern not previously observed in other Candida species, and presents inter-phenotypic tissue tropism differences. C. auris filaments in vivo less frequently than C. albicans or C. parapsilosis mostly through pseudohyphal growth. Filamentation might not be a major pathogenic determinant in C. auris, as less virulent aggregative phenotypes form pseudohyphae to a greater extent. C. auris has important both interspecific and intraspecific virulence and phenotype heterogeneity, with aggregative phenotypes of C. auris sharing characteristics with low pathogenic species such as C. parapsilosis. Our work suggests that C. auris owns an important morphogenetic plasticity that distinguishes it from other yeasts of the genus. Routine phenotypic identification of aggregative or non-aggregative phenotypes should be performed in the clinical setting as it may impact patient management.

ABSTRACT 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 past decade witnessed the emergence in Shiga toxin-producing Escherichia coli (STEC) infections linked to the consumption of unpasteurized milk and raw milk cheese. The virulence of STEC is primarily attributed to the presence of Shiga toxin genes (stx1 and stx2) carried by Stx-converting bacteriophages, along with the intimin gene eae. Most of the available information pertains to the “Top 7” serotypes associated with STEC infections. The objectives of this study were to characterize and investigate the pathogenicity potential of E. coli UC4224, a STEC O174:H2 strain isolated from semi-hard raw milk cheese and to develop surrogate strains with reduced virulence for use in food-related studies. Complete genome sequence analysis of E. coli UC4224 unveiled the presence of a Stx1a bacteriophage, a Stx2a bacteriophage, the Locus of Adhesion and Autoaggregation (LAA) pathogenicity island, plasmid-encoded virulence genes, and other colonization facilitators. In the Galleria mellonella animal model, E. coli UC4224 demonstrated high pathogenicity potential with an LD50 of 6 CFU/10 μL. Upon engineering E. coli UC4224 to generate single and double mutant derivatives by inactivating stx1a and/or stx2a genes, the LD50 increased by approximately 1 Log-dose in the single mutants and 2 Log-doses in the double mutants. However, infectivity was not completely abolished, suggesting the involvement of other virulence factors contributing to the pathogenicity of STEC O174:H2. Considering the possibility of raw milk cheese serving as a reservoir for STEC, cheesemaking model was developed to evaluate the survival of UC4224 and the adequacy of the respective mutants as reduced-virulence surrogates. All tested strains exhibited the ability to survive the curd cooking step at 48°C and multiplied (3.4 Log CFU) in cheese within the subsequent 24 h. These findings indicate that genomic engineering did not exert any unintended effect on the double stx1-stx2 mutant behaviour, making it as a suitable less-virulent surrogate for conducting studies during food processing.

Twenty-two atypical enteroaggregative Escherichia coli isolates from a previous epidemiological study harboring EAEC virulence genes were examined for their adhesion properties. Nine strains showed a typical aggregative adherence (AA) pattern, while 13 strains showed variant AA, such as AA with lined up cells characteristic of the chain-like adhesion (CLA) and AA mainly to HeLa cells characteristic of the diffuse adherence (DA). The aggregative forming pilus (AFP) genes afpA2 and afpR were detected only in strain Q015B, which exhibited an AA/DA pattern. Using Tn5-based transposon mutagenesis on Q015B strain, we identified a 5517-bp open reading frame (ORF) encoding a predicted 1838-amino-acid polypeptide that is genetically related to a putative filamentous hemagglutinin identified in E. coli strain 7-233-03_S3_C2. Therefore, the ORF was named orfHA. The regions flanking orfHA were sequenced and two ORFs were found; upstream, an ORF that encodes a 603-amino-acid polypeptide with 99% identity to hemolysin secretion/activation proteins of the ShlB/FhaC/HecB family, and downstream, another ORF, which encodes a 632-amino-acid polypeptide with 72% identity to the glycosyltransferase EtpC. An orfHA mutant (Q015BΔorfHA) was constructed from strain Q015B. Q015BΔorfHA strain did not adhere to HeLa cells, whereas Q015BΔ orfHA transformed with a pACYC184 plasmid carrying orfHA restored the AA/DA phenotype of strain Q015B. Furthermore, the Q015ΔorfHA mutant had a marked effect on the ability of strain Q015B to kill the larvae of Galleria mellonella. Our results suggest that the AA/DA pattern of strain Q015B is mediated by a hemagglutinin-associated protein which also contributes to its virulence in the G. mellonella model.

ABSTRACT Acinetobacter baumannii is an antibiotic-resistant, Gram-negative pathogen that causes a multitude of nosocomial infections. However, pathogenicity mechanisms and the host cell response during infection remain unclear. In this study, we determined virulence traits of A. baumannii clinical isolates belonging to the most widely disseminated international clonal lineage, international cluster 2 (IC2), in vitro and in vivo. Complexome profiling of primary human endothelial cells with A. baumannii revealed that mitochondria, and in particular complexes of the electron transport chain, are important host cell targets. Infection with highly virulent A. baumannii remodelled assembly of mitochondrial protein complexes and led to metabolic adaptation. These were characterized by reduced mitochondrial respiration and glycolysis in contrast to those observed in infection with low-pathogenicity A. baumannii. Perturbation of oxidative phosphorylation, destabilization of mitochondrial ribosomes, and interference with mitochondrial metabolic pathways were identified as important pathogenicity mechanisms. Understanding the interaction of human host cells with the current global A. baumannii clone is the basis to identify novel therapeutic targets. IMPORTANCE Virulence traits of Acinetobacter baumannii isolates of the worldwide most prevalent international clonal lineage, IC2, remain largely unknown. In our study, multidrug-resistant IC2 clinical isolates differed substantially in their virulence potential despite their close genetic relatedness. Our data suggest that, at least for some isolates, mitochondria are important target organelles during infection of primary human endothelial cells. Complexes of the respiratory chain were extensively remodelled after infection with a highly virulent A. baumannii strain, leading to metabolic adaptation characterized by severely reduced respiration and glycolysis. Perturbations of both mitochondrial morphology and mitoribosomes were identified as important pathogenicity mechanisms. Our data might help to further decipher the molecular mechanisms of A. baumannii and host mitochondrial interaction during infection.

Enterobacter cloacae produces insecticidal proteins capable of causing toxicity in pests, but the insecticidal mechanisms of these proteins for insect control remain unclear. To elucidate the mechanisms, the purified insecticidal protein from E. cloacae NK was administered to Galleria mellonella larvae either by intraperitoneal injection or by feeding. The number of hemocytes, apoptosis in immune cells, and polyphenol oxidase (PO) activity of G. mellonella larvae were detected by hemocytometer, Annexin V-FITC/PI, and UV–vis spectrophotometer, respectively. With the extension of the invasion time of NK insecticidal protein, the number of hemocytes in G. mellonella larvae decreased significantly (p

ABSTRACT Ribonucleotide reductases (RNRs) are key enzymes which catalyze the synthesis of deoxyribonucleotides, the monomers needed for DNA replication and repair. RNRs are classified into three classes (I, II, and III) depending on their overall structure and metal cofactors. Pseudomonas aeruginosa is an opportunistic pathogen which harbors all three RNR classes, increasing its metabolic versatility. During an infection, P. aeruginosa can form a biofilm to be protected from host immune defenses, such as the production of reactive oxygen species by macrophages. One of the essential transcription factors needed to regulate biofilm growth and other important metabolic pathways is AlgR. AlgR is part of a two-component system with FimS, a kinase that catalyzes its phosphorylation in response to external signals. Additionally, AlgR is part of the regulatory network of cell RNR regulation. In this study, we investigated the regulation of RNRs through AlgR under oxidative stress conditions. We determined that the nonphosphorylated form of AlgR is responsible for class I and II RNR induction after an H2O2 addition in planktonic culture and during flow biofilm growth. We observed similar RNR induction patterns upon comparing the P. aeruginosa laboratory strain PAO1 with different P. aeruginosa clinical isolates. Finally, we showed that during Galleria mellonella infection, when oxidative stress is high, AlgR is crucial for transcriptional induction of a class II RNR gene (nrdJ). Therefore, we show that the nonphosphorylated form of AlgR, in addition to being crucial for infection chronicity, regulates the RNR network in response to oxidative stress during infection and biofilm formation. IMPORTANCE The emergence of multidrug-resistant bacteria is a serious problem worldwide. Pseudomonas aeruginosa is a pathogen that causes severe infections because it can form a biofilm that protects it from immune system mechanisms such as the production of oxidative stress. Ribonucleotide reductases are essential enzymes which synthesize deoxyribonucleotides used in the replication of DNA. RNRs are classified into three classes (I, II, and III), and P. aeruginosa harbors all three of these classes, increasing its metabolic versatility. Transcription factors, such as AlgR, regulate the expression of RNRs. AlgR is involved in the RNR regulation network and regulates biofilm growth and other metabolic pathways. We determined that AlgR induces class I and II RNRs after an H2O2 addition in planktonic culture and biofilm growth. Additionally, we showed that a class II RNR is essential during Galleria mellonella infection and that AlgR regulates its induction. Class II RNRs could be considered excellent antibacterial targets to be explored to combat P. aeruginosa infections.

Objective. Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) affect 150 million people annually.Purpose: Characteristics of non-hospital strains of UPEC isolated from patients with UTI in Yaroslavl in 2016– 2017.Materials and methods. Susceptibility of UPEC strains (n = 20) to antibacterials was measured by the serial dilution method; the antibiotic resistance and virulence genes, phylogroups, O-serogroups and sequence types were identified by PCR and whole genome sequencing. The virulence of the strains was studied using the model of Galleria mellonella larvae.Results. UPEC strains were classified as resistant (n = 11) and multi-drug resistant (n = 9) pathogens. Betalactamase genes blaTEM (n = 10), blaCTX-M (n = 6), class 1 integrons (n = 8), and gene cassettes dfrA17-aadA5 (n = 2), dfrA1 (n = 1) and aacA4-cmlA1 (n = 1) were identified. UPEC-virulence genetic determinants coding adhesins fimH, papG, sfaS, focG, afa/draBC, csgA, siderophores iroN, fyuA, iutA, counteracting factors of host immunity ompT, traT, toxins hlyA, cnf1, usp, capsule transporter kpsMTII, colicin cvaC, and pathogenicity islands I536, II536, III536, IV536, IIJ96 и IICFT073 were detected. Highly virulent and slightly virulent for G. mellonella larvae UPEC strains were obtained with LD50 104–105 and 106–107 CFU, respectively. The phylogroups A, B1, B2, E and F, serogroups О2, О4, О6, O9, O11, О15, О18, О25, О75 and O89, known sequence types ST14, ST58, ST69, ST73, ST93, ST127, ST131, ST-141, ST165, ST297, ST457, ST537, ST744, ST1434 and novel ST9239 and ST10102 were revealed.Conclusions. The identified genetic diversity of non-hospital UPEC strains is consistent with the observed global trend in the spread of human pathogens, which are characterized with both high virulence and multiple drug resistance. This makes possible to assess prospectively the current epidemiological situation, give a forecast for its development in the future, as well as determine the optimal therapeutic options.

In the quest for mitigators of bacterial virulence, cell-free supernatants (CFS) from 25 human commensal and associated bacteria were tested for activity against Pseudomonas aeruginosa. Among these, Escherichia coli Nissle 1917 CFS significantly inhibited biofilm formation and dispersed extant pseudomonas biofilms without inhibiting planktonic bacterial growth. eDNA was reduced in biofilms following exposure to E. coli Nissle CFS, as visualized by confocal microscopy. E. coli Nissle CFS also showed a significant protective effect in a Galleria mellonella-based larval virulence assay when administrated 24 h before challenge with the P. aeruginosa. No inhibitory effects against P. aeruginosa were observed for other tested E. coli strains. According to proteomic analysis, E. coli Nissle CFS downregulated the expression of several P. aeruginosa proteins involved in motility (Flagellar secretion chaperone FliSB, B-type flagellin fliC, Type IV pilus assembly ATPase PilB), and quorum sensing (acyl-homoserine lactone synthase lasI and HTH-type quorum-sensing regulator rhlR), which are associated with biofilm formation. Physicochemical characterization of the putative antibiofilm compound(s) indicates the involvement of heat-labile proteinaceous factors of greater than 30 kDa molecular size.

ABSTRACT 12-Bis-THA Cl2 [12,12′-(dodecane-1,12-diyl)-bis-(9-amino-1,2,3,4-tetrahydroacridinium) chloride] is a cationic bolalipid adapted from dequalinium chloride (DQC), a bactericidal anti-infective indicated for bacterial vaginosis (BV). Here, we used a structure-activity-relationship study to show that the factors that determine effective killing of bacterial, fungal, and mycobacterial pathogens differ, to generate new analogues with a broader spectrum of activity, and to identify synergistic relationships, most notably with aminoglycosides against Acinetobacter baumannii and Pseudomonas aeruginosa, where the bactericidal killing rate was substantially increased. Like DQC, 12-bis-THA Cl2 and its analogues accumulate within bacteria and fungi. More hydrophobic analogues with larger headgroups show reduced potential for DNA binding but increased and broader spectrum antibacterial activity. In contrast, analogues with less bulky headgroups and stronger DNA binding affinity were more active against Candida spp. Shortening the interconnecting chain, from the most lipophilic twelve-carbon chain to six, improved the selectivity index against Mycobacterium tuberculosis in vitro, but only the longer chain analogue was therapeutic in a Galleria mellonella infection model, with the shorter chain analogue exacerbating the infection. In vivo therapy of Escherichia coli ATCC 25922 and epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA-15) infections in Galleria mellonella was also achieved with longer-chain analogues, as was therapy for an A. baumannii 17978 burn wound infection with a synergistic combination of bolaamphiphile and gentamicin. The present study shows how this class of bolalipids may be adapted further to enable a wider range of potential applications. IMPORTANCE While we face an acute threat from antibiotic resistant bacteria and a lack of new classes of antibiotic, there are many effective antimicrobials which have limited application due to concerns regarding their toxicity and which could be more useful if such risks are reduced or eliminated. We modified a bolalipid antiseptic used in throat lozenges to see if it could be made more effective against some of the highest-priority bacteria and less toxic. We found that structural modifications that rendered the lipid more toxic against human cells made it less toxic in infection models and we could effectively treat caterpillars infected with either Mycobacterium tuberculosis, methicillin resistant Staphylococcus aureus, or Acinetobacter baumannii. The study provides a rationale for further adaptation toward diversifying the range of indications in which this class of antimicrobial may be used.

ABSTRACT In recent years, Stenotrophomonas maltophilia (S. maltophilia) has become an important pathogen of clinically acquired infections accompanied by high pathogenicity and high mortality. Moreover, infections caused by multidrug-resistant S. maltophilia have emerged as a serious challenge in clinical practice. Bacteriophages are considered a promising alternative for the treatment of S. maltophilia infections due to their unique antibacterial mechanism and superior bactericidal ability compared with traditional antibiotic agents. Here, we reported a new phage BUCT700 that has a double-stranded DNA genome of 43,214 bp with 70% GC content. A total of 55 ORFs and no virulence or antimicrobial resistance genes were annotated in the genome of phage BUCT700. Phage BUCT700 has a broad host range (28/43) and can lyse multiple ST types of clinical S. maltophilia (21/33). Furthermore, bacteriophage BUCT700 used the Type IV fimbrial biogenesis protein PilX as an adsorption receptor. In the stability test, phage BUCT700 showed excellent thermal stability (4 to 60°C) and pH tolerance (pH = 4 to 12). Moreover, phage BUCT700 was able to maintain a high titer during long-term storage. The adsorption curve and one-step growth curve showed that phage BUCT700 could rapidly adsorb to the surface of S. maltophilia and produce a significant number of phage virions. In vivo, BUCT700 significantly increased the survival rate of S. maltophilia-infected Galleria mellonella (G. mellonella) larvae from 0% to 100% within 72 h, especially in the prophylactic model. In conclusion, these findings indicate that phage BUCT700 has promising potential for clinical application either as a prophylactic or therapeutic agent. IMPORTANCE The risk of Stenotrophomonas maltophilia infections mediated by the medical devices is exacerbated with an increase in the number of ICU patients during the Corona Virus Disease 2019 (COVID-19) epidemic. Complications caused by S. maltophilia infections could complicate the state of an illness, greatly extending the length of hospitalization and increasing the financial burden. Phage therapy might be a potential and promising alternative for clinical treatment of multidrug-resistant bacterial infections. Here, we investigated the protective effects of phage BUCT700 as prophylactic and therapeutic agents in Galleria mellonella models of infection, respectively. This study demonstrates that phage therapy can provide protection in targeting S. maltophilia-related infection, especially as prophylaxis.

Carbapenem-resistant Klebsiella pneumoniae is one of the primary bacterial pathogens that pose a significant threat to global public health because of the lack of available therapeutic options. Phage therapy shows promise as a potential alternative to current antimicrobial chemotherapies. In this study, we isolated a new Siphoviridae phage vB_KpnS_SXFY507 against KPC-producing K. pneumoniae from hospital sewage. It had a short latent period of 20 min and a large burst size of 246 phages/cell. The host range of phage vB_KpnS_SXFY507 was relatively broad. It has a wide range of pH tolerance and high thermal stability. The genome of phage vB_KpnS_SXFY507 was 53,122 bp in length with a G + C content of 49.1%. A total of 81 open-reading frames (ORFs) and no virulence or antibiotic resistance related genes were involved in the phage vB_KpnS_SXFY507 genome. Phage vB_KpnS_SXFY507 showed significant antibacterial activity in vitro. The survival rate of Galleria mellonella larvae inoculated with K. pneumoniae SXFY507 was 20%. The survival rate of K. pneumonia-infected G. mellonella larvae was increased from 20 to 60% within 72 h upon treatment with phage vB_KpnS_SXFY507. In conclusion, these findings indicate that phage vB_KpnS_SXFY507 has the potential to be used as an antimicrobial agent for the control of K. pneumoniae.

Azole resistance in Aspergillus fumigatus (Af) has become a widespread threat and a major concern for optimal management of patients with invasive aspergillosis (IA). Combination of echinocandins with azoles is an attractive alternative option for the treatment of IA due to azole-resistant Af strains. The aim of this study was to evaluate the in vitro and in vivo combination of caspofungin (CAS) with either voriconazole (VRZ) or posaconazole (PSZ). In vitro interactions were assessed by two methods, and an animal model of IA in Galleria mellonella was used for in vivo evaluation. Assessment of efficacy was based on larvae mortality. Groups of 10 larvae were infected by 3 clinical strains of Af (azole susceptible, AfS; PSZ resistant, AfR1; VRZ and PSZ resistant strain, AfR2). In vitro, combination of CAS and azoles was indifferent against AfS, and AfR2, and a synergy was found for AfR1. When compared to VRZ monotherapy, the combination of VRZ at 4 µg/larva with CAS at 4 µg/larva improved survival of AfR2-infected larvae (p=0.0066). Combination of PSZ at 4µg/larva with CAS at 4 µg/larva improved survival of AfR1-infected larvae compared to CAS (p=0.0002) and PSZ (0.0024) monotherapy. Antagonism was never observed. In conclusion, the combination of caspofungin with azoles is a promising alternative for the treatment of azole resistant strains of Af.

ABSTRACT Gram-negative bacteria have a robust cell envelope that excludes or expels many antimicrobial agents. However, during infection, host soluble innate immune factors permeabilize the bacterial outer membrane. We identified two small molecules that exploit outer membrane damage to access the bacterial cell. In standard microbiological media, neither compound inhibited bacterial growth nor permeabilized bacterial outer membranes. In contrast, at micromolar concentrations, JAV1 and JAV2 enabled the killing of an intracellular human pathogen, Salmonella enterica serovar Typhimurium. S. Typhimurium is a Gram-negative bacterium that resides within phagosomes of cells from the monocyte lineage. Under broth conditions that destabilized the lipopolysaccharide layer, JAV2 permeabilized the bacterial inner membrane and was rapidly bactericidal. In contrast, JAV1 activity was more subtle: JAV1 increased membrane fluidity, altered reduction potential, and required more time than JAV2 to disrupt the inner membrane barrier and kill bacteria. Both compounds interacted with glycerophospholipids from Escherichia coli total lipid extract-based liposomes. JAV1 preferentially interacted with cardiolipin and partially relied on cardiolipin production for activity, whereas JAV2 generally interacted with lipids and had modest affinity for phosphatidylglycerol. In mammalian cells, neither compound significantly altered mitochondrial membrane potential at concentrations that killed S. Typhimurium. Instead, JAV1 and JAV2 became trapped within acidic compartments, including macrophage phagosomes. Both compounds improved survival of S. Typhimurium-infected Galleria mellonella larvae. Together, these data demonstrate that JAV1 and JAV2 disrupt bacterial inner membranes by distinct mechanisms and highlight how small, lipophilic, amine-substituted molecules can exploit host soluble innate immunity to facilitate the killing of intravesicular pathogens. IMPORTANCE Innovative strategies for developing new antimicrobials are needed. Combining our knowledge of host-pathogen interactions and relevant drug characteristics has the potential to reveal new approaches to treating infection. We identified two compounds with antibacterial activity specific to infection and with limited host cell toxicity. These compounds appeared to exploit host innate immunity to access the bacterium and differentially damage the bacterial inner membrane. Further, both compounds accumulated within Salmonella-containing and other acidic vesicles, a process known as lysosomal trapping, which protects the host and harms the pathogen. The compounds also increased host survival in an insect infection model. This work highlights the ability of host innate immunity to enable small molecules to act as antibiotics and demonstrates the feasibility of antimicrobial targeting of the inner membrane. Additionally, this study features the potential use of lysosomal trapping to enhance the activities of compounds against intravesicular pathogens.

ABSTRACT In the current scenario of growing antibiotic resistance, understanding the interplay between resistance mechanisms and biological costs is crucial for designing therapeutic strategies. In this regard, intrinsic AmpC β-lactamase hyperproduction is probably the most important resistance mechanism of Pseudomonas aeruginosa, proven to entail important biological burdens that attenuate virulence mostly under peptidoglycan recycling alterations. P. aeruginosa can acquire resistance to new β-lactam–β-lactamase inhibitor combinations (ceftazidime-avibactam and ceftolozane-tazobactam) through mutations affecting ampC and its regulatory genes, but the impact of these mutations on the associated biological cost and the role that β-lactamase activity plays per se in contributing to the above-mentioned virulence attenuation are unknown. The same questions remain unsolved for plasmid-encoded AmpC-type β-lactamases such as FOX enzymes, some of which also provide resistance to new β-lactam–β-lactamase inhibitor combinations. Here, we assessed from different perspectives the effects of changes in the active center and, thus, in the hydrolytic spectrum resistance to inhibitors of AmpC-type β-lactamases on the fitness and virulence of P. aeruginosa, using site-directed mutagenesis; the previously described AmpC variants T96I, G183D, and ΔG229-E247; and, finally, blaFOX-4 versus blaFOX-8. Our results indicate the essential role of AmpC activity per se in causing the reported full virulence attenuation (in terms of growth, motility, cytotoxicity, and Galleria mellonella larvae killing), although the biological cost of the above-mentioned AmpC-type variants was similar to that of the wild-type enzymes. This suggests that there is not an important biological burden that may limit the selection/spread of these variants, which could progressively compromise the future effectiveness of the above-mentioned drug combinations. IMPORTANCE The growing antibiotic resistance of the top nosocomial pathogen Pseudomonas aeruginosa pushes research to explore new therapeutic strategies, for which the resistance-versus-virulence balance is a promising source of targets. While resistance often entails significant biological costs, little is known about the bases of the virulence attenuations associated with a resistance mechanism as extraordinarily relevant as β-lactamase production. We demonstrate that besides potential energy and cell wall alterations, the enzymatic activity of the P. aeruginosa cephalosporinase AmpC is essential for causing the full attenuation associated with its hyperproduction by affecting different features related to pathogenesis, a fact exploitable from the antivirulence perspective. Less encouraging, we also show that the production of different chromosomal/plasmid-encoded AmpC derivatives conferring resistance to some of the newest antibiotic combinations causes no significantly increased biological burdens, which suggests a free way for the selection/spread of these types of variants, potentially compromising the future effectiveness of these antipseudomonal therapies.

The RNA chaperone Hfq promotes the association of small RNAs (sRNAs) with cognate mRNAs, controlling the expression of bacterial phenotype. Actinobacillus pleuropneumoniae hfq mutants strains are attenuated for virulence in pigs, impaired in the ability to form biofilms, and more susceptible to stress, but knowledge of the extent of sRNA involvement is limited. Here, using A. pleuropneumoniae strain MIDG2331 (serovar 8), 14 sRNAs were identified by co-immunoprecipitation with Hfq and the expression of eight, identified as trans-acting sRNAs, were confirmed by Northern blotting. We focused on one of these sRNAs, named Rna01, containing a putative promoter for RpoE (stress regulon) recognition. Knockout mutants of rna01 and a double knockout mutant of rna01 and hfq, both had decreased biofilm formation and hemolytic activity, attenuation for virulence in Galleria mellonella, altered stress susceptibility, and an altered outer membrane protein profile. Rna01 affected extracellular vesicle production, size and toxicity in G. mellonella. qRT-PCR analysis of rna01 and putative cognate mRNA targets indicated that Rna01 is associated with the extracytoplasmic stress response. This work increases our understanding of the multilayered and complex nature of the influence of Hfq-dependent sRNAs on the physiology and virulence of A. pleuropneumoniae.

Azole resistance is becoming increasingly serious due to the frequent recurrence of fungal infections and the need for long-term clinical prevention. In our previous study, we discovered ORF19.5274 with an unknown function by TMT™ quantitative proteomics technology after fluconazole (FLC) treatment of Candida albicans. In this study, we created the target gene deletion strain using CRISPR-Cas9 editing technology to see if ORF19.5274 regulates azole sensitivity. The data showed that ORF19.5274 was involved in hyphal development and susceptibility to antifungal azoles. Deleting this gene resulted in defective hyphal growth in solid medium, while only a weak lag in the initiation of hyphal development and restoring hyphal growth during the hyphal maintenance phase under liquid conditions. Moreover, intracellular reactive oxygen species (ROS) assay and propidium iodide staining assays showed increased endogenous ROS levels and membrane permeability, but decreased metabolic activity of biofilm in orf19.5274Δ/Δ after treatment with FLC in comparison with either SC5314 or orf19.5274Δ/Δ::ORF19.5274 strains. More importantly, orf19.5274Δ/Δ significantly enhanced the FLC efficacy against C. albicans in infected Galleria mellonella larvae. The above characteristics were fully or partially restored in the complemented strain indicating that the changes caused by ORF19.5274 deletion were specific. In summary, the ORF19.5274 gene is required for hyphal development of C. albicans, and is correlated with the response to antifungal azoles in vitro and in vivo. The identification of ORF19.5275 is promising to expand the potential candidate targets for azoles.

Over the past decades, the spread of multi-drug-resistant Klebsiella pneumoniae (MDR-KP) is becoming a new threat and new effective therapies against this pathogen are needed. Bacteriophage (phage) therapy is considered to be a promising alternative treatment for MDR-KP infections compared with antibacterial drug usage. Here, we reported a new phage BUCT541 which can lyse MDR-KP ST23. The genome of BUCT541 is a double-stranded linear 46,100-bp long DNA molecule with 48% GC content through the Next generation sequencing (NGS) data. A total of 81 open reading frames and no virulence or antimicrobial resistance genes are annotated in the BUCT541 genome. BUCT541 was able to lyse 7 of the 30 tested MDR-KP according to the host range analysis. And the seven sensitive strains belonged to the K. pneumoniae K1-ST23. BUCT541 exhibited high thermal stability (4–70°C) and broad pH tolerance (pH 3-11) in the stability test. The in vivo results showed that BUCT541 (4 × 105 plaque-forming units (PFU)/each) significantly increased the survival rate of K. pneumoniae infected Galleria mellonella from 5.3% to 83.3% within 48 h. Moreover, in the mouse lung infection model, high doses of BUCT541 (2 × 107 PFU/each) cured 100% of BALB/c mice that were infected with K. pneumoniae. After 30 h of treatment with phage BUCT541 of the multiplicity of infection (MOI) = 10, the K. pneumoniae in the lungs of mice was lower than 104 CFU/mL, compared to the control group 109 CFU/mL. Together, these findings indicate that phage BUCT541 holds great promise as an alternative therapy with excellent stability and a wide lysis range for the treatment of MDR-KP ST23 infection.

Hypervirulent Klebsiella pneumoniae (hvKp) pathotype is emerging worldwide in pyogenic liver abscesses (PLAs). However, the role of virulence factors in pathogenicity remains unclear. On the other hand, the epidemiology of PLAs in Iran is unknown. From July 2020 to April 2022, bacterial species were isolated and identified from the drainage samples of 54 patients with PLAs. K. pneumoniae as the most common pathogen of pyogenic liver abscesses was identified in 20 (37%) of the 54 patients. We analyzed the clinical and microbiological characteristics of K. pneumoniae-related pyogenic liver abscesses. Antibiotic susceptibility testes and string test were performed. 16S rRNA, antibiotic resistance, and virulence genes were determined by polymerase chain reaction amplification. Clonal relatedness of isolates was identified by multilocus sequence typing. Virulence levels were assessed in the Galleria mellonella larval infection model. Four hvKp isolates (K1/K2) were found to be responsible for cryptogenic PLAs, and 16 classical K. pneumoniae isolates (non-K1/K2) were associated with non-cryptogenic PLAs. Three capsular serotype K1 strains belonged to sequence type 23 (ST23) and one K2 strain to ST65. Meanwhile, the non-K1/K2 strains belonged to other STs. ST231 was the most common strain among the classical K. pneumoniae strains. Compared with the non-K1/K2 strains, capsular serotypes K1/K2 strains were less resistant to antibiotics, had positive string test results, and had more virulence genes. In Galleria mellonella, a concentration of 106 colony-forming units of the K1 hvKp strain resulted in 100% death at 24 hours, confirming the higher virulence of the hvKp strain compared with cKp. K. pneumoniae isolates represented that the acquisition of any plasmid or chromosomal virulence genes contributes to pathogenicity and high prevalence in PLAs. Meanwhile, hvKp isolates with a specific genetic background were detected in cryptogenic PLAs.

The formation of persister cells is associated with recalcitrance and infections. In this study, we examined the antimicrobial property of alpha mangostin, a natural xanthone molecule, against methicillin-resistant Staphylococcus aureus (MRSA) persisters and biofilm. The MIC of alpha mangostin against MRSA persisters was 2 µg/ml, and activity was mediated by causing membrane permeabilization within 30 min of exposure. The membrane activity of alpha mangostin was further studied by fast-killing kinetics of MRSA persiste r cells and found that the compound exhibited 99.99% bactericidal activity within 30 min. Furthermore, alpha mangostin disrupted established MRSA biofilms and inhibited bacterial attachment as biofilm formation. Alpha mangostin down-regulated genes associated with the formation of persister cells and biofilms, such as norA, norB, dnaK, groE, and mepR, ranging from 2 to 4-folds. Alpha mangostin at 16 μg/ml was non-toxic (> 95% cell survival) to liver-derived HepG2 and lung-derived A549 cells, similarly. Still, alpha mangostin exhibited 50% cell lysis of human RBC at 16 μg/ml. Interestingly, alpha mangostin was effective in vivo at increasing the survival up to 75% (p

Enterotoxigenic Escherichia coli (ETEC) causing post-weaning diarrhea (PWD) in piglets have a detrimental impact on animal health and economy in pig production. ETEC strains can adhere to the host’s small intestinal epithelial cells using fimbriae such as F4 and F18. Phage therapy could represent an interesting alternative to antimicrobial resistance against ETEC infections. In this study, four bacteriophages, named vB_EcoS_ULIM2, vB_EcoM_ULIM3, vB_EcoM_ULIM8 and vB_EcoM_ULIM9, were isolated against an O8:F18 E. coli strain (A-I-210) and selected based on their host range. These phages were characterized in vitro, showing a lytic activity over a pH (4–10) and temperature (25–45 °C) range. According to genomic analysis, these bacteriophages belong to the Caudoviricetes class. No gene related to lysogeny was identified. The in vivo Galleria mellonella larvae model suggested the therapeutic potential of one selected phage, vB_EcoS_ULIM2, with a statistically significant increase in survival compared to non-treated larvae. To assess the effect of this phage on the piglet gut microbiota, vB_EcoS_ULIM2 was inoculated in a static model simulating the piglet intestinal microbial ecosystem for 72 h. This study shows that this phage replicates efficiently both in vitro and in vivo in a Galleria mellonella model and reveals the safety of the phage-based treatment on the piglet microbiota.

Abstract Background New therapeutics are urgently needed for infectious diseases, especially for the fungal infection like Fonsecaea monophora. Photodynamic therapy has been showing antimicrobial activity on some pathogens. The combination of antimicrobial medicines and photodynamic therapy (PDT) might be a practical approach. However, whether the treatment of PDT could do benefits to the host immunity remains poorly documented. Results In this study, Galleria mellonella larvae were employed as a model organism to evaluate the activity of PDT, and also to investigate the regulation of humoral immunity by PDT. Photosensitizer 5-aminolevulinic acid (ALA) was applied to the G. mellonella infection model. It was found that ALA-mediated PDT was non-toxic to G. mellonella, and could extend the median survival of infected larvae from 3 days to 5.5 days. We observed that larval hemocytes inhibited the growth of Candida albicans and Staphylococcus aureus, without any contribution by ALA-PDT. Furthermore, the application of ALA-PDT demonstrated the immunomodulation of larval innate immunity as increased hemocyte density counting, cell morphological transformation, and sensitivity to pathogens. Conclusions G. mellonella could be considered as a useful model to study the immunoregulation of PDT. This model revealed that ALA-PDT positively defense against infections through inducing humoral immune responses of larvae.

Entomopathogenic nematodes (EPNs) were collected from soil using Galleria mellonella as bait and propagated on Wouts medium as well as new semi-solid feed developed from dog feed. The EPN was isolated from the cucumber soil from Kadayar, Thadiyoor, Kerala for the Mass production. The strains collected were identified as Steinernema siamkayai KUT1 and the symbiont bacteria identified as Xenorhabdus stockiae using advanced DNA sequencing method. Nematodes were grown on two different sets of medium viz., Wouts medium on sponge and Semi-solid dog feed in T-flasks. The Steinernema siamkayai KUT1 grown increasingly in semi-solid dog feed medium than other medium. This research suggests that EPN can be used to for the inhibition of malarial parasites in open stagnant water bodies. Breeding grounds for this species could be controlled efficiently using EPNs. Our research concludes the use of newly modified semi solid Dog feed medium containing essential nutrients can be safe and cheap enough for the mass production of EPN.

The rise of antimicrobial resistant (AMR) bacteria is a major health concern, especially with regard to members of the ESKAPE group, to which vancomycin-resistant (VRE) Enterococcus faecium belongs. Phage therapy has emerged as a novel alternative for the treatment of AMR infections. This, however, relies on the isolation and characterisation of a large collection of phages. This work describes the exploration of human faeces as a source of new E. faecium-infecting phages. Phage vB_EfaH_163 was isolated and characterised at the microbiological, genomic, and functional levels. vB_EfaH_163 phage, a new member of Herelleviridae, subfamily Brockvirinae, has a dsDNA genome of 150,836 bp that does not harbour any virulence factors or antibiotic resistance genes. It infects a wide range of E. faecium strains of different origins, including VRE strains. Interestingly, it can also infect Enterococcus faecalis strains, even some that are linezolid-resistant. Its capacity to control the growth of a clinical VRE isolate was shown in broth culture and in a Galleria mellonella animal model. The discovery and characterisation of vB_EfaH_163 increases the number of phages that might be used therapeutically against AMR bacteria.

Purpose: Zinc oxide nanoparticles (ZnO-NPs) have exerted antimicrobial properties. However, there is insufficient evaluation regarding the in vivo antifungal activity of ZnO-NPs. This study aimed to investigate the efficacy and mechanism of ZnO-NPs in controlling Candida albicans in the invertebrate Galleria mellonella.Methods:Galleria mellonella larvae were injected with different doses of ZnO-NPs to determine their in vivo toxicity. Non-toxic doses of ZnO-NPs were chosen for prophylactic injection in G. mellonella followed by C. albicans infection. Then the direct in vitro antifungal effect of ZnO-NPs against C. albicans was evaluated. In addition, the mode of action of ZnO-NPs was assessed in larvae through different assays: quantification of hemocyte density, morphology observation of hemocytes, characterization of hemocyte aggregation and phagocytosis, and measurement of hemolymph phenoloxidase (PO) activity.Results: Zinc oxide nanoparticles were non-toxic to the larvae at relatively low concentrations (≤20 mg/kg). ZnO-NP pretreatment significantly prolonged the survival of C. albicans-infected larvae and decreased the fungal dissemination and burden in the C. albicans-infected larvae. This observation was more related to the activation of host defense rather than their fungicidal capacities. Specifically, ZnO-NP treatment increased hemocyte density, promoted hemocyte aggregation, enhanced hemocyte phagocytosis, and activated PO activity in larvae.Conclusion: Prophylactic treatment with lower concentrations of ZnO-NPs protects G. mellonella from C. albicans infection. The innate immune response primed by ZnO-NPs may be part of the reason for the protective effects. This study provides new evidence of the capacity of ZnO-NPs in enhancing host immunity and predicts that ZnO-NPs will be attractive for further anti-infection applications.

The increasing interest for Galleria mellonella larvae as an infection model is evidenced by the number of papers reporting its use, which increases exponentially since the early 2010s. This popularity was initially linked to limitation of conventional animal models due to financial, technical and ethical aspects. In comparison, alternative models (e.g. models using Caenorhabditis elegans, Drosophila melanogaster or G. mellonella) were cheap, simple to use and not limited by ethical regulation. Since then, similar results have been established with G. mellonella model comparatively to vertebrates, and it is more and more often used as a robust model per se, not only as an alternative to the murine model. This review attempts to summarize the current knowledge supporting the development of this model, both on immunological and microbiological aspects. For that, we focus on investigation of virulence and new therapies for the most important pathogenic bacteria. We also discuss points out directions for standardization, as well as recent advances and new perspectives for monitoring host-pathogen interactions.

Antimicrobial compounds can combat microbes through modulating host immune defense, inhibiting bacteria survival and growth, or through impeding or inhibiting virulence factors. In the present study, a panel of substituted diphenyl amide compounds previously found to disrupt bacterial quorum sensing were investigated and several were found to promote survival in the Galleria mellonella model when provided therapeutically to treat a Gram-positive bacterial infection from methicillin-resistant Staphylococcus aureus strain MW2. Out of 21 tested compounds, N-4-Methoxyphenyl-3-(4-methoxyphenyl)-propanamide (AMI 82B) was the most potent at disrupting S. aureus virulence and promoted 50% larvae survival at 120 and 96 h when delivered at 0.5 and 5 mg/Kg, respectively, compared to untreated controls (p < 0.0001). AMI 82B did not exhibit G. mellonella toxicity (LC50 > 144 h) at a delivery concentration up to 5 mg/Kg. Further assessment with mammalian cells suggest AMI 82B hemolytic effects against erythrocytes has an HL50 greater than the highest tested concentration of 64 μg/mL. Against HepG2 hepatic cells, AMI 82B demonstrated an LD50 greater than 64 μg/mL. AMI 82B lacked direct bacteria inhibition with a minimal inhibitory concentration that exceeds 64 μg/mL and no significant reduction in S. aureus growth curve at the same concentration. Assessment via qPCR revealed that AMI 82B significantly depressed quorum sensing genes agr, spa, and icaA (p < 0.05). Thus, AMI 82B therapeutic effect against S. aureus in the G. mellonella infection model is likely an influence on bacterial quorum sensing driven virulence factors and provides an interesting hit compound for this medically important pathogen.

The dissemination of antibiotic-resistant priority pathogens beyond hospital settings is both a public health and an environmental problem. In this regard, high-risk clones exhibiting a multidrug-resistant (MDR) or extensively drug-resistant (XDR) phenotype have shown rapid adaptation at the human-animal-environment interface. In this study, we report genomic data and the virulence potential of the carbapenemase, São Paulo metallo-β-lactamase (SPM-1)-producing Pseudomonas aeruginosa strains (Pa19 and Pa151) isolated from polluted urban rivers, in Brazil. Bioinformatic analysis revealed a wide resistome to clinically relevant antibiotics (carbapenems, aminoglycosides, fosfomycin, sulfonamides, phenicols, and fluoroquinolones), biocides (quaternary ammonium compounds) and heavy metals (copper), whereas the presence of exotoxin A, alginate, quorum sensing, types II, III, and IV secretion systems, colicin, and pyocin encoding virulence genes was associated with a highly virulent behavior in the Galleria mellonella infection model. These results confirm the spread of healthcare-associated critical-priority P. aeruginosa belonging to the MDR sequence type 277 (ST277) clone beyond the hospital, highlighting that the presence of these pathogens in environmental water samples can have clinical implications for humans and other animals.

Candida albicans is the main fungal species associated with the development of oral candidiasis. Currently, therapeutic options for these infections are limited by the adverse effects of antifungal drugs and by the emergence of drug resistant strains. Thus, the development of new antifungal agents is needed for the prevention and treatment of oral Candida infections. Caffeic acid phenethyl ester (CAPE) is a natural compound from propolis polyphenolic groups that exhibits many pharmacological properties. In this study, we investigated whether CAPE can have antifungal and immunomodulatory effects on oral candidiasis. Preliminary tests to assess the antifungal activity of CAPE were performed using the Minimum Inhibitory Concentration (MIC) assay that demonstrated inhibition in a range from 16 to 32 μg/mL, confirming its antifungal activity on several C. albicans strains isolated from the oral cavity. Subsequently, we analyzed Candida spp biofilms formed in vitro, in which CAPE treatment at 5 x MIC caused a reduction of 68.5% in the total biomass and ~2.60 Log in the viable cell count (CFU/mL) in relation to the untreated biofilm (p

Acinetobacter baumannii is an important pathogen of nosocomial infection. Recently, a group of genes, named “gig” (for Growth in Galleria), have been identified in a contemporary multi-drug resistant clinical isolate of A. baumannii—strain AB5075. Among these so-called gig genes, gigA and gigB were found to promote antibiotic resistance, stress survival, and virulence of AB5075 by interacting with the nitrogen phosphotransferase system (PTSNtr). This study aimed to investigate the roles of gigA/gigB, which appear to comprise a stress-signaling pathway (encoding for an atypical two-component system response regulator and a predicted anti-anti-sigma factor, respectively), and the involvement of ptsP (encoding the Enzyme I component of the PTSNtr) in the growth, stress resistance, and virulence of the widely studied A. baumannii strain ATCC 17978. Genetic analyses of strains harboring mutations of gigA and gigB were performed to investigate the roles of these genes in bacterial growth, stress resistance, evading macrophage defense, and killing of Galleria mellonella larva. In contrast with findings from strain AB5075 where gigA and gigB contribute to aminoglycoside resistance, the data presented herein indicate that the loss of gigA/gigB does not impact antibiotic resistance of strain ATCC 17978. Interestingly, however, we found that deletion of gigA/gigB in the ATCC 17978 background imparts a general growth in laboratory medium and also conferred growth and replication defects within murine macrophages and an inability to kill G. mellonella larvae. Importantly, studies as well as the loss of ptsP restored the phenotypes of the gigA/gigB mutant to that of the wild-type. The data presented herein indicate that in A. baumannii ATCC 17978, the gigA/gigB genes play a key role in both growth and virulence traits, but are dispensable for other stress-resistance survival phenotypes, including aminoglycoside resistance. Our findings thus highlight several similarities and also important differences between the gigA/gigB stress-signaling pathway in two commonly studied isolates of this troublesome pathogen.

Antibiotic resistance is a major public health concern in many countries worldwide. The rapid spread of multidrug-resistant (MDR) bacteria is the main driving force for the development of novel non-antibiotic antimicrobials as a therapeutic alternative. Here, we isolated and characterized three virulent bacteriophages that specifically infect and lyse MDR Klebsiella pneumoniae with K23 capsule type. The phages belonged to the Autographiviridae (vB_KpnP_Dlv622) and Myoviridae (vB_KpnM_Seu621, KpS8) families and contained highly similar receptor-binding proteins (RBPs) with polysaccharide depolymerase enzymatic activity. Based on phylogenetic analysis, a similar pattern was also noted for five other groups of depolymerases, specific against capsule types K1, K30/K69, K57, K63, and KN2. The resulting recombinant depolymerases Dep622 (phage vB_KpnP_Dlv622) and DepS8 (phage KpS8) demonstrated narrow specificity against K. pneumoniae with capsule type K23 and were able to protect Galleria mellonella larvae in a model infection with a K. pneumoniae multidrug-resistant strain. These findings expand our knowledge of the diversity of phage depolymerases and provide further evidence that bacteriophages and phage polysaccharide depolymerases represent a promising tool for antimicrobial therapy.

Abstract Background The popularity of Galleria mellonella as invertebrate model is increasing rapidly, because it forms an attractive alternative to study bacterial, fungal and viral infections, toxin biology, and to screen antimicrobial drugs. For a number of vertebrate and invertebrate animal and plant models, it has been established that the commensals present within the microbial communities on various host surfaces will influence the host’s immune and growth development state and the colonization capacity of newly introduced micro-organisms. The microbial communities of Galleria mellonella larvae have, however, not yet been well characterized. Results In this study, we present the bacterial communities that were found by 16S rRNA amplicon sequencing on different body sites of G. mellonella larvae. These communities showed very little diversity and were mostly dominated by one Enterococcus taxon. In addition, we found that the production conditions (as ‘bait’ for fishing or under more controlled ‘research grade’ conditions - with or without hormones and antibiotics) appear to have little impact on the microbiota of the larvae. Conclusions Establishment of the simplicity of the microbiota of G. mellonella larvae underlines the potential of the larvae as a model host system for microbiome-host interactions.

Abstract Background Acinetobacter baumannii is an opportunistic pathogen that causes serious nosocomial infection in intensive care units. In particular, carbapenem-resistant A. baumannii (CRAB) strains have been increasing in the past decade, and they have caused major medical problems worldwide. In this study, a novel A. baumannii lytic phage, the YMC 13/03/R2096 ABA BP (phage Βϕ-R2096), which specifically causes the lysis of CRAB strains, was characterized in detail in vitro and in silico, and the in vivo effectiveness of phage therapy was evaluated using Galleria mellonella and a mouse model of acute pneumonia. Results The A. baumannii phage Βϕ-R2096 was isolated from sewage water using CRAB clinical strains selected from patients at a university hospital in South Korea. The complete genome of the phage Βϕ-R2096, which belongs to the Myoviridae family, was analyzed. Phage Βϕ-R2096 inhibited bacterial growth in a dose-dependent manner and exhibited high bacteriolytic activity at MOI = 10. In the evaluation of its therapeutic potential against CRAB clinical isolates using two in vivo models, phage Βϕ-R2096 increased the survival rates of both G. mellonella larvae (from 0 to 50% at 24 h) and mice (from 30% with MOI = 0.1 to 100% with MOI = 10 for 12 days) in post-infection of CRAB. In particular, phage Βϕ-R2096 strongly ameliorated histologic damage to infected lungs, with bacterial clearance in the lungs observed on day 3 postinfection in the mouse acute pneumonia model. Moreover, in vivo studies revealed no mortality or serious side effects in phage-treated groups. Conclusion The results of this study strongly suggest that phage Βϕ-R2096, a novel A. baumannii lytic phage, could be an alternative antibacterial agent to control CRAB infections. This study is the first report to compare in vivo evaluations (G. mellonella larvae and a mouse acute pneumonia model) of the therapeutic efficacy of a phage against CRAB infections.

Cryptococcus neoformans is the leading cause of cryptococcosis, an invasive and potentially fatal infectious disease. Therapeutic failures are due to the increase in antifungal resistance, the adverse effects of drugs, and the unavailability of therapeutic regimens in low-income countries, which limit the treatment of cryptococcosis, increasing the morbidity and mortality associated with these infections. Thus, new antifungal drugs and innovative strategies for the cryptococcosis treatment are urgently needed. The aim of the present study was to evaluate the effect of ethyl acetate fraction (EAF) of Poincianella pluviosa stem bark on planktonic and biofilm mode of growth of C. neoformans. Furthermore, the interaction between the EAF and amphotericin B (AmB) was evaluated in vitro and in Galleria mellonella infection model. Minimal inhibitory concentrations (MICs) of EAF ranged from 125.0 to >1,000.0 μg/ml and >1,000.0 μg/ml for planktonic and sessile cells, respectively. The combination between EAF and AmB exhibited a synergistic fungicidal activity toward C. neoformans, with a fractional inhibitory concentration index (FICI) ranging from 0.03 to 0.06 and 0.08 to 0.28 for planktonic and sessile cells, respectively. Microscopy analyses of planktonic C. neoformans cells treated with EAF, alone or combined with AmB, revealed morphological and ultrastructural alterations, including loss of integrity of the cell wall and cell membrane detachment, suggesting leakage of intracellular content, reduction of capsule size, and presence of vacuoles. Moreover, EAF alone or combined with AmB prolonged the survival rate of C. neoformans-infected G. mellonella larvae. These findings indicate that P. pluviosa may be an important source of new compounds that can be used as a fungus-specific adjuvant for the treatment of cryptococcosis.