Alternative models to study bacteria-fungi interaction

Juliana Campos Junqueira

Microbiology and Immunology Department of Biosciences and Oral Diagnosis

Institute of Science and Technology Unesp-São José dos Campos

Models hosts

Study of infectious diseases: In vitro In vivo

Colonize and invade host

tissues

In vivo

Escape host immune system

Virulence mechanisms Junqueira et al. 2012

Rossoni et al. 2013

Experimental candidiasis

The importance of understanding the nature of host-pathogen interactions in animal models

Alternative Models hosts

(Rats and Mice) Vertebrate models Ethical restrictions

Logistical difficulties Long time of experiment

Large number of: -Mutants strains (Genetic information) -Antimicrobial compounds

Alternative Models hosts

Large-scale studies Easier and faster experiments

Invertebrate models

Acanthamoeba castellanii

Invertebrates:

Ideal candidates for large-scale studies

Low cost Simplicity of use Short life span

No ethical restrictions

Caenorhabditis elegans

Drosophila melanogaster

Galleria mellonella

Insect (Larvae of the moth) 5 cm

Nematode 1 mm

Insect (Fruit fly) 3 mm

Amoebae unicellular organisms

Innate immune mechanisms are evolutionarily conserved between invertebrates and

mammalian

Available model hosts offer

advantages and disadvantages

G. mellonella

C. elegans

Desalermos et al. 2012

G. mellonella

Life cycle

Eggs Larvae

Pupa Moth

Natural habitat: beehives (pollen, honey and beeswax)

Lepidoptera

Injection of specific amount of a pathogen

Survive at the mammalian temperature (37°C)

Immune response (phagocytosis)

MEV: G. mellonella Vilcinskas 2011

G. mellonella

Positive correlation between mammalian and G. mellonella

Acinobacter baumanii

Pseudomonas aeruginosa

Staphylococcus aureus

Streptococcus pyogenes

Streptococcus mutans

Enterococcus faecalis

Candida albicans

Cryptococcus neoformans

C. elegans

Brenner 1960: Neurobiology and genetics

Hermaphrodite lifestyle

Life cycle:

Embryonic stage

Larval stages (L1, L2, L3, L4)

Adulthood

Culture: solid NGM (Nematode Growth Media)

Fed with E. coli OP50

C. elegans

-Rapid life cycle

-Physiological simplicity

-Production of genetically identical progeny

-Transparency of its cuticle

-Sequenced genoma

-Several mutant strains of C. elegans

Virulence genes

Antimicrobial compounds SCREENING

Studies using invertebrate as model host

Galleria mellonella (2010)

Pathogenicity of bacteria and fungi

Antimicrobial agents

Bacteria-fungi interactions

RESEARCH ARTICLE Open Access

Selective photoinactivation of Candida albicans inthe non-vertebrate host infection model GalleriamellonellaJosé Chibebe Junior1,2,3†, Caetano P Sabino4,5†, Xiaojiang Tan6,2†, Juliana C Junqueira1*, Yan Wang2,7,Beth B Fuchs2, Antonio OC Jorge1, George P Tegos4,8,9, Michael R Hamblin4,9,10 and Eleftherios Mylonakis2,11

Abstract

Background: Candida spp. are recognized as a primary agent of severe fungal infection in immunocompromisedpatients, and are the fourth most common cause of bloodstream infections. Our study explores treatment withphotodynamic therapy (PDT) as an innovative antimicrobial technology that employs a nontoxic dye, termed aphotosensitizer (PS), followed by irradiation with harmless visible light. After photoactivation, the PS produces eithersinglet oxygen or other reactive oxygen species (ROS) that primarily react with the pathogen cell wall, promotingpermeabilization of the membrane and cell death. The emergence of antifungal-resistant Candida strains hasmotivated the study of antimicrobial PDT (aPDT) as an alternative treatment of these infections. We employed theinvertebrate wax moth Galleria mellonella as an in vivo model to study the effects of aPDT against C. albicansinfection. The effects of aPDT combined with conventional antifungal drugs were also evaluated in G. mellonella.

Results: We verified that methylene blue-mediated aPDT prolonged the survival of C. albicans infected G. mellonellalarvae. The fungal burden of G. mellonella hemolymph was reduced after aPDT in infected larvae. A fluconazole-resistant C. albicans strain was used to test the combination of aPDT and fluconazole. Administration of fluconazoleeither before or after exposing the larvae to aPDT significantly prolonged the survival of the larvae compared toeither treatment alone.

Conclusions: G. mellonella is a useful in vivo model to evaluate aPDT as a treatment regimen for Candida infections.The data suggests that combined aPDT and antifungal therapy could be an alternative approach to antifungal-resistant Candida strains.

Keywords: Candida albicans, Photodynamic therapy, Galleria mellonella

BackgroundCandida albicans and other Candida species commonlycolonize the epithelial surfaces of the human body [1].One-half of humans have oral cavities colonized by Can-dida species in a commensal relationship with the host[2]. Although few healthy carriers develop clinical can-didiasis, when the host becomes immunocompromiseddue to cancer, HIV/AIDS, diabetes, major surgery, trans-plantation of solid organs or hematopoietic stem cells,these opportunistic pathogens can cause superficial

infections that may be cutaneous, subcutaneous or mu-cosal. In progressive cases, the fungus can penetrate theepithelial surface and be disseminated by the blood-stream with serious consequences [1,3-7].C. albicans is the most common species isolated from

superficial and systemic candidiasis and it is consideredthe most pathogenic species of the Candida genus[5,8-11]. In vitro investigations indicate that C. albicansexpresses higher levels of putative virulence factors com-pared to other Candida species. It has been proposedthat several virulence factors are involved in the patho-genicity of C. albicans, such as adhesion to host sur-faces, hyphal formation and secretion of proteinases[11]. In addition, C. albicans cells employ mechanisms

* Correspondence: [email protected]†Equal contributors1Department of Biosciences and Oral Diagnosis, Univ Estadual Paulista/UNESP, São José dos Campos, SP 12245000, BrazilFull list of author information is available at the end of the article

© 2013 Chibebe Junior et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of theCreative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly cited.

Chibebe Junior et al. BMC Microbiology 2013, 13:217http://www.biomedcentral.com/1471-2180/13/217

Photodynamic and Antibiotic Therapy Impair thePathogenesis of Enterococcus faecium in a Whole AnimalInsect ModelJose Chibebe Junior1,2,3*, Beth B. Fuchs2, Caetano P. Sabino4,5, Juliana C. Junqueira1, Antonio O. C. Jorge1,

Martha S. Ribeiro5, Michael S. Gilmore6, Louis B. Rice7, George P. Tegos4,8,9, Michael R. Hamblin4,8,10,

Eleftherios Mylonakis7*

1 Department of Biosciences and Oral Diagnosis, Univ Estadual Paulista/UNESP, Sao Jose dos Campos, Sao Paulo, Brazil, 2 Division of Infectious Diseases, Massachusetts

General Hospital, Boston, Massachusetts, United States of America, 3 Department of Restorative Dentistry, Faculty of Pindamonhangaba, Pindamonhangaba, Sao Paulo,

Brazil, 4 Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 5 Center for Lasers and Applications,

Nuclear and Energy Research Institute, Sao Paulo, Sao Paulo, Brazil, 6 Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, United States

of America, 7 Warren Alpert Medical School, Brown University/Rhode Island and Miriam Hospitals, Providence, Rhode Island, United States of America, 8 Department of

Dermatology, Harvard Medical School, Boston, Massachusetts, United States of America, 9 Department of Pathology and Center for Molecular Discovery, University of New

Mexico, Albuquerque, New Mexico, United States of America, 10 Division of Health Sciences and Technology, Harvard-Massachusetts Institute of Technology, Cambridge,

Massachusetts, United States of America

Abstract

Enterococcus faecium has emerged as one of the most important pathogens in healthcare-associated infections worldwidedue to its intrinsic and acquired resistance to many antibiotics, including vancomycin. Antimicrobial photodynamic therapy(aPDT) is an alternative therapeutic platform that is currently under investigation for the control and treatment of infections.PDT is based on the use of photoactive dye molecules, widely known as photosensitizer (PS). PS, upon irradiation withvisible light, produces reactive oxygen species that can destroy lipids and proteins causing cell death. We employed Galleriamellonella (the greater wax moth) caterpillar fatally infected with E. faecium to develop an invertebrate host model systemthat can be used to study the antimicrobial PDT (alone or combined with antibiotics). In the establishment of infection by E.faecium in G. mellonella, we found that the G. mellonella death rate was dependent on the number of bacterial cells injectedinto the insect hemocoel and all E. faecium strains tested were capable of infecting and killing G. mellonella. Antibiotictreatment with ampicillin, gentamicin or the combination of ampicillin and gentamicin prolonged caterpillar survivalinfected by E. faecium (P = 0.0003, P = 0.0001 and P = 0.0001, respectively). In the study of antimicrobial PDT, we verified thatmethylene blue (MB) injected into the insect followed by whole body illumination prolonged the caterpillar survival(P = 0.0192). Interestingly, combination therapy of larvae infected with vancomycin-resistant E. faecium, with antimicrobialPDT followed by vancomycin, significantly prolonged the survival of the caterpillars when compared to either antimicrobialPDT (P = 0.0095) or vancomycin treatment alone (P = 0.0025), suggesting that the aPDT made the vancomycin resistant E.faecium strain more susceptible to vancomycin action. In summary, G. mellonella provides an invertebrate model host tostudy the antimicrobial PDT and to explore combinatorial aPDT-based treatments.

Citation: Chibebe Junior J, Fuchs BB, Sabino CP, Junqueira JC, Jorge AOC, et al. (2013) Photodynamic and Antibiotic Therapy Impair the Pathogenesis ofEnterococcus faecium in a Whole Animal Insect Model. PLoS ONE 8(2): e55926. doi:10.1371/journal.pone.0055926

Editor: Tarek Msadek, Institut Pasteur, France

Received October 18, 2012; Accepted January 3, 2013; Published February 14, 2013

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone forany lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Funding: The first author thanks CAPES (PDEE 2507-11-0) for the scholarship during the ‘‘Sandwich’’ PhD Program at Harvard Medical School. Researchconducted in the Mylonakis Laboratory was supported by NIH (RO1 AI050875 to EM, and the Harvard-wide Program on Antibiotic Resistance [P01 AI083214] toEM and MSG). Research conducted in the Hamblin Laboratory was supported by NIH (RO1 AI050875 to MRH) and US Air Force MFEL Program (FA9550-04-1-0079).George P. Tegos is supported by the NIH (grant 5U54MH084690-02) and DTRA (contract HDTRA1-13-C-0005). The funders had no role in study design, datacollection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected] (JCJ); [email protected] (EM)

Introduction

Enterococci are part of the gastrointestinal tract of humans [1–3], but due to intrinsic and acquired resistance to many antibiotics,they have become leading causes of nosocomial infectionsworldwide [4–7]. Enterococcus faecalis and Enterococcus faecium accountfor 95% of clinical isolates from the genus Enterococcus, and areisolated from patients with endocarditis, bloodstream infection,wound and surgical-site infection, and intra-abdominal andurinary tract infection [3,8,9]. In dentistry, they are frequently

associated with chronic periodontitis and persistent endodonticinfections [10–12]. In the 1980s and early 1990s, more than 90%of all enterococcal infections were caused by E. faecalis and only 5–10% by E. faecium. Due to the acquisition of the virulencedeterminants as well as acquired antibiotic resistance, this ratio haschanged, and currently, E. faecium is associated with between 38–75% of all enterococcal infections [4,13].

The increased resistance of bacteria to antibiotics has emergedas one of the most important clinical challenges of this century,highlighting the need for new and effective antimicrobial

PLOS ONE | www.plosone.org 1 February 2013 | Volume 8 | Issue 2 | e55926

Lactobacillus acidophilus ATCC 4356inhibits biofilm formation by C. albicans andattenuates the experimental candidiasis in

Galleria mellonellaSimone FG Vilela1, J!unia O Barbosa1, Rodnei D Rossoni1, J!essica D Santos1, Marcia CA Prata2, Ana Lia Anbinder1,

Antonio OC Jorge1, and Juliana C Junqueira1,*

1 Department of Biosciences and Oral Diagnosis; Institute of Science and Technology; UNESP - Univ Estadual Paulista; S~ao Jos!e dos Campos, Brazil;2Empresa Brasileira de Agropecu!aria; EMBRAPA; Juiz de Fora; Brazil

Keywords: biofilm, candidiasis, Candida albicans, filamentation, probiotic, Galleria mellonella, Lactobacillus acidophilus

Abbreviations: ATCC, American type culture collection; YNB, Yeast nitrogen base; MRS, Man, Rogosa and Sharpe; PBS, phosphatebuffered saline; BHI, Brain heart infusion; CFU, colony-forming unit; SEM, Scanning electron microscopy; PAS, periodic acid-

Schiff; HE, hematoxylin-eosin; pH, potential hydrogen ion; NIH, National Institutes of Health.

Probiotic strains of Lactobacillus have been studied for their inhibitory effects on Candida albicans. However, fewstudies have investigated the effect of these strains on biofilm formation, filamentation and C. albicans infection. Theobjective of this study was to evaluate the influence of Lactobacillus acidophilus ATCC 4356 on C. albicans ATCC 18804using in vitro and in vivo models. In vitro analysis evaluated the effects of L. acidophilus on the biofilm formation and onthe capacity of C. albicans filamentation. For in vivo study, Galleria mellonella was used as an infection model to evaluatethe effects of L. acidophilus on candidiasis by survival analysis, quantification of C. albicans CFU/mL, and histologicalanalysis. The direct effects of L. acidophilus cells on C. albicans, as well as the indirect effects using only a Lactobacillusculture filtrate, were evaluated in both tests. The in vitro results showed that both L. acidophilus cells and filtrate wereable to inhibit C. albicans biofilm formation and filamentation. In the in vivo study, injection of L. acidophilus into G.mellonella larvae infected with C. albicans increased the survival of these animals. Furthermore, the number of C.albicans CFU/mL recovered from the larval hemolymph was lower in the group inoculated with L. acidophilus comparedto the control group. In conclusion, L. acidophilus ATCC 4356 inhibited in vitro biofilm formation by C. albicans andprotected G. mellonella against experimental candidiasis in vivo.

Introduction

Fungal infections are one of the most common diseases causedby microorganisms, especially in hospitalized and immunocom-promised patients. Among species of the genus Candida, C. albi-cans is the pathogen most frequently isolated from the humanbody, including the oral cavity and gastrointestinal and genitouri-nary tract. This species can cause infections that range from super-ficial lesions of the mucosa or skin to severe systemic infections.1,2

C. albicans shows a great capacity of biofilm formation on oralstructures and its presence in the oral cavity may serve as a reservoirof this fungus for infections in other parts of the body.3

Current treatment for candidiasis consists of the administra-tion of topical antifungal agents such as nystatin, amphotericin Band clotrimazole, or systemic antifungal agents such as flucona-zole, ketoconazole and itraconazole. However, the use of thesedrugs can cause side effects and lead to microbial development of

resistance.4,5 The increase in the resistance of microorganisms toconventional antifungal drugs has encouraged studies designed todiscover new treatments for infections caused by Candida spp.6,7

The use of probiotics to prevent or treat Candida infections maybe an interesting strategy. In this respect, certain Lactobacillusstrains have been shown to exert anti-Candida activity by produc-ing antimicrobial molecules, such as organic acids, hydrogen per-oxide and bacteriocins.8

Probiotics are defined by the World Health Organization aslive microorganisms that confer health benefits on the host whenadministered in adequate amounts. They are included in a varietyof products such as foods, dietary supplements and medications.In addition to Lactobacillus, other microorganisms have beenused as probiotics, including Bifidobacterium, Saccharomyces cere-visiae, Escherichia coli and Bacillus.9

The anti-Candida effects of Lactobacillus have been investi-gated in several in vitro studies using different strains of L.

*Correspondence to: Juliana Junqueira; Email: [email protected]: 07/08/2014; Revised: 10/02/2014; Accepted: 10/24/2014http://dx.doi.org/10.4161/21505594.2014.981486

www.landesbioscience.com 29Virulence

Oral Candida albicans isolates from HIV-positiveindividuals have similar in vitro biofilm-formingability and pathogenicity as invasive CandidaisolatesJuliana C Junqueira1*, Beth B Fuchs2, Maged Muhammed2, Jeffrey J Coleman2, Jamal MAH Suleiman3,Simone FG Vilela1, Anna CBP Costa1, Vanessa MC Rasteiro1, Antonio OC Jorge1 and Eleftherios Mylonakis2

Abstract

Background: Candida can cause mucocutaneous and/or systemic infections in hospitalized andimmunosuppressed patients. Most individuals are colonized by Candida spp. as part of the oral flora and theintestinal tract. We compared oral and systemic isolates for the capacity to form biofilm in an in vitro biofilmmodel and pathogenicity in the Galleria mellonella infection model. The oral Candida strains were isolated from theHIV patients and included species of C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. krusei, C. norvegensis, andC. dubliniensis. The systemic strains were isolated from patients with invasive candidiasis and included species of C.albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. lusitaniae, and C. kefyr. For each of the acquired strains, biofilmformation was evaluated on standardized samples of silicone pads and acrylic resin. We assessed the pathogenicityof the strains by infecting G. mellonella animals with Candida strains and observing survival.

Results: The biofilm formation and pathogenicity in Galleria was similar between oral and systemic isolates. Thequantity of biofilm formed and the virulence in G. mellonella were different for each of the species studied. Onsilicone pads, C. albicans and C. dubliniensis produced more biofilm (1.12 to 6.61 mg) than the other species (0.25to 3.66 mg). However, all Candida species produced a similar biofilm on acrylic resin, material used in dentalprostheses. C. albicans, C. dubliniensis, C. tropicalis, and C. parapsilosis were the most virulent species in G. mellonellawith 100% of mortality, followed by C. lusitaniae (87%), C. novergensis (37%), C. krusei (25%), C. glabrata (20%), andC. kefyr (12%).

Conclusions: We found that on silicone pads as well as in the Galleria model, biofilm formation and virulencedepends on the Candida species. Importantly, for C. albicans the pathogenicity of oral Candida isolates was similarto systemic Candida isolates, suggesting that Candida isolates have similar biofilm-forming ability and virulenceregardless of the infection site from which it was isolated.

BackgroundFungi are increasingly recognized as major pathogens incritically ill patients. Candida spp. are the fourth leadingcause of bloodstream infections in the U.S. and dissemi-nated candidiasis is associated with a mortality in excessof 25% [1-3]. Oropharyngeal candidiasis (OPC) is the

most frequent opportunistic infection encountered inhuman immunodeficiency virus (HIV) infected indivi-duals with 90% at some point experiencing OPC duringthe course of HIV disease [4]. Among Candida species,C. albicans is the most commonly isolated and responsi-ble for the majority of superficial and systemic infec-tions. However, many non-albicans species, such as C.glabrata, C. parapsilosis and C. tropicalis have recentlyemerged as important pathogens in suitably debilitatedindividuals [5].

* Correspondence: [email protected] of Biosciences and Oral Diagnosis, Univ Estadual Paulista/UNESP, 777 Av. Eng. Francisco José Longo, São José dos Campos, SP12245000, BrazilFull list of author information is available at the end of the article

Junqueira et al. BMC Microbiology 2011, 11:247http://www.biomedcentral.com/1471-2180/11/247

© 2011 Junqueira et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.