Mary Ann Jabra-Rizk

Polymicrobial Interactions: Impact on Pathogenesis and Human Disease

SUMMARY Microorganisms coexist in a complex milieu of bacteria, fungi, archaea, and viruses on or within the human body, often as multifaceted polymicrobial biofilm communities at mucosal sites and on abiotic surfaces. Only recently have we begun to appreciate the complicated biofilm phenotype during infection; moreover, even less is known about the interactions that occur between microorganisms during polymicrobial growth and their implications in human disease. Therefore, this review focuses on polymicrobial biofilm-mediated infections and examines the contribution of bacterial-bacterial, bacterial-fungal, and bacterial-viral interactions during human infection and potential strategies for protection against such diseases.

Antimicrobial Peptides: Primeval Molecules or Future Drugs?

The authors have declared that no competing interests exist. This work was supported by NIH grants DE14424 and DE016257. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Cross-kingdom interactions: Candida albicans and bacteria

Bacteria and fungi are found together in a myriad of environments and particularly in a biofilm, where adherent species interact through diverse signaling mechanisms. Yet, despite billions of years of coexistence, the area of research exploring fungal-bacterial interactions, particularly within the context of polymicrobial infections, is still in its infancy. However, reports describing a multitude of wide-ranging interactions between the fungal pathogen Candida albicans and various bacterial pathogens are on the rise. An example of a mutually beneficial interaction is coaggregation, a phenomenon that takes place in oral biofilms where the adhesion of C. albicans to oral bacteria is considered crucial for its colonization of the oral cavity. In contrast, the interaction between C. albicans and Pseudomonas aeruginosa is described as being competitive and antagonistic in nature. Another intriguing interaction is that occurring between Staphylococcus aureus and C. albicans, which although not yet fully characterized, appears to be initially synergistic. These complex interactions between such diverse and important pathogens would have significant clinical implications if they occurred in an immunocompromised host. Therefore, understanding the mechanisms of adhesion and signaling involved in fungal-bacterial interactions may lead to the development of novel therapeutic strategies for impeding microbial colonization and development of polymicrobial disease.

Effect of Farnesol on Staphylococcus aureus Biofilm Formation and Antimicrobial Susceptibility

ABSTRACT Staphylococcus aureus is among the leading pathogens causing bloodstream infections able to form biofilms on host tissue and indwelling medical devices and to persist and cause disease. Infections caused by S. aureus are becoming more difficult to treat because of increasing resistance to antibiotics. In a biofilm environment particularly, microbes exhibit enhanced resistance to antimicrobial agents. Recently, farnesol was described as a quorum-sensing molecule with possible antimicrobial properties. In this study, the effect of farnesol on methicillin-resistant and -susceptible strains of S. aureus was investigated. With viability assays, biofilm formation assessment, and ethidium bromide uptake testing, farnesol was shown to inhibit biofilm formation and compromise cell membrane integrity. The ability of farnesol to sensitize S. aureus to antimicrobials was assessed by agar disk diffusion and broth microdilution methods. For both strains of staphylococci, farnesol was only able to reverse resistance at a high concentration (150 μM). However, it was very successful at enhancing the antimicrobial efficacy of all of the antibiotics to which the strains were somewhat susceptible. Therefore, synergy testing of farnesol and gentamicin was performed with static biofilms exposed to various concentrations of both agents. Plate counts of harvested biofilm cells at 0, 4, and 24 h posttreatment indicated that the combined effect of gentamicin at 2.5 times the MIC and farnesol at 100 μM (22 μg/ml) was able to reduce bacterial populations by more than 2 log units, demonstrating synergy between the two antimicrobial agents. This observed sensitization of resistant strains to antimicrobials and the observed synergistic effect with gentamicin indicate a potential application for farnesol as an adjuvant therapeutic agent for the prevention of biofilm-related infections and promotion of drug resistance reversal.

Fungal biofilms and drug resistance.

Candida species, including the novel opportunistic pathogen Candida dubliniensis, are now emerging as major agents of nosocomial infections. Many such manifestations of infections associated with the formation of Candida biofilms include those occurring on devices such as indwelling intravascular catheters. Fungal biofilm-associated infections are frequently refractory to conventional therapy because of resistance to antimicrobial agents. This resistance could be in part due to the surface-induced upregulation of drug efflux pumps. Biofilm-associated Candida show uniform resistance to a wide spectrum of the currently available conventional antifungal agents, which implies that antimicrobial drugs that specifically target biofilm-associated infections are needed. The novel classes of antifungal agents, the lipid formulation of amphotericins, and the echinocandins have demonstrated unique antifungal activity against the resistant Candida biofilms, providing a breakthrough in the treatment of life-threatening invasive systemic mycoses. The use of drugs effective in combating biofilm-associated infections could lead to major developments in the treatment of fungal implant infections.

Peptide Nucleic Acid Fluorescence In Situ Hybridization-Based Identification of Candida albicans and Its Impact on Mortality and Antifungal Therapy Costs

ABSTRACT The impact of rapid identification of Candida albicans blood isolates by peptide nucleic acid fluorescence in situ hybridization (PNA FISH) on the selection and expenditure of antifungal therapy was evaluated. PNA FISH was 100% sensitive and specific in the rapid identification of 31 out of 72 candidemias as C. albicans and resulted in a significant reduction of caspofungin usage, with an overall cost savings of

Microbial interactions and differential protein expression in Staphylococcus aureus–Candida albicans dual-species biofilms

1,729 per patient.

Streptococcus mutans, Candida albicans, and the human mouth: a sticky situation

The fungal species Candida albicans and the bacterial species Staphylococcus aureus are responsible for a majority of hospital-acquired infections and often coinfect critically ill patients as complicating polymicrobial biofilms. To investigate biofilm structure during polymicrobial growth, dual-species biofilms were imaged with confocal scanning laser microscopy. Analyses revealed a unique biofilm architecture where S. aureus commonly associated with the hyphal elements of C. albicans. This physical interaction may provide staphylococci with an invasion strategy because candidal hyphae can penetrate through epithelial layers. To further understand the molecular mechanisms possibly responsible for previously demonstrated amplified virulence during coinfection, protein expression studies were undertaken. Differential in-gel electrophoresis identified a total of 27 proteins to be significantly differentially produced by these organisms during coculture biofilm growth. Among the upregulated staphylococcal proteins was l-lactate dehydrogenase 1, which confers resistance to host-derived oxidative stressors. Among the downregulated proteins was the global transcriptional repressor of virulence factors, CodY. These findings demonstrate that the hyphae-mediated enhanced pathogenesis of S. aureus may not only be due to physical interactions but can also be attributed to the differential regulation of specific virulence factors induced during polymicrobial growth. Further characterization of the intricate interaction between these pathogens at the molecular level is warranted, as it may aid in the design of novel therapeutic strategies aimed at combating fungal–bacterial polymicrobial infection.

A Novel Immune Evasion Strategy of Candida albicans : Proteolytic Cleavage of a Salivary Antimicrobial Peptide

The human mouth with its diverse niches and ample supply of nutrients is undoubtedly conducive for the unrestricted formation of natural microbial biofilms. The oral microbial communities are some of the most complex microbial floras in the human body, consisting of more than 700 different bacterial species [1], [2]. Occurrence of disease results from disturbance of the equilibrium of this complex ecosystem, where population shifts lead to overrepresentation of pathogenic species which contribute to the onset and progression of the most common oral diseases, caries and periodontal disease [3]. Culture-independent molecular methods such as proteomics and 16S rRNA sequencing aiming to determine the bacterial diversity in the human oral cavity have demonstrated that in the supragingival plaque, S. mutans was the dominant species, with elevated levels of other streptococci including S. sanguinis, S. mitis, and S. salivarius in addition to lactobacilli and Veillonella. In contrast, the subgingival plaque was made up primarily of Gram-negative anaerobic bacteria such as Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia which are known to be periodontal pathogens [3]–[5]. The dental tissues—enamel, dentin, and cementum—constitute the oral solid surfaces coated by a pellicle to which the microbial cells attach. The primary colonizers and secondary organisms stick to each other on the surface of teeth and generate a matrix of exopolysaccharide within which cells grow, forming a community with a collective physiology [6]. The resulting biofilm formed, known as dental plaque, subjects the teeth and gingival tissues to high concentrations of microbial metabolites which result in dental disease [2], [7]. The interactions between the various species in these mixed biofilms can be synergistic in that the presence of one microorganism generates a niche for other pathogenic microorganisms which can serve to facilitate the retention of organisms, an oral phenomenon known as coaggregation [3], [8]. The bacteria in the biofilm are always metabolically active which causes fluctuations in pH and loss of minerals from the tooth, ultimately resulting in dissolution of the dental hard tissues and formation of lesions known as dental caries [6], [9]. Interestingly, metabolic communications among oral bacteria may occur where the excretion of a metabolite by one organism is used as a nutrient by other organisms and breakdown of a substrate by enzymatic activity of one organism creates available substrates for different organisms [10], [11].

Staphylococcus aureus adherence to Candida albicans hyphae is mediated by the hyphal adhesin Als3p

Oropharyngeal candidiasis is an opportunistic infection considered to be a harbinger of AIDS. The etiologic agent Candida albicans is a fungal species commonly colonizing human mucosal surfaces. However, under conditions of immune dysfunction, colonizing C. albicans can become an opportunistic pathogen causing superficial or even life-threatening infections. The reasons behind this transition, however, are not clear. In the oral cavity, salivary antimicrobial peptides are considered to be an important part of the host innate defense system in the prevention of microbial colonization. Histatin-5 specifically has exhibited potent activity against C. albicans. Our previous studies have shown histatin-5 levels to be significantly reduced in the saliva of HIV+ individuals, indicating an important role for histatin-5 in keeping C. albicans in its commensal stage. The versatility in the pathogenic potential of C. albicans is the result of its ability to adapt through the regulation of virulence determinants, most notably of which are proteolytic enzymes (Saps), involved in tissue degradation. In this study, we show that C. albicans cells efficiently and rapidly degrade histatin-5, resulting in loss of its anti-candidal potency. In addition, we demonstrate that this cellular activity is due to proteolysis by a member of the secreted aspartic proteases (Sap) family involved in C. albicans pathogenesis. Specifically, the proteolysis was attributed to Sap9, in turn identifying histatin-5 as the first host-specific substrate for that isoenzyme. These findings demonstrate for the first time the ability of a specific C. albicans enzyme to degrade and deactivate a host antimicrobial peptide involved in the protection of the oral mucosa against C. albicans, thereby providing new insights into the factors directing the transition of C. albicans from commensal to pathogen, with important clinical implications for alternative therapy. This report characterizes the first defined mechanism behind the enhanced susceptibility of HIV+ individuals to oral candidiasis since the emergence of HIV.