Impact of host factors on susceptibility to antifungal agents

An obstacle to drug development, particularly in this era of multiple drug resistance, is the under-appreciation for the role the host environment plays in microbial response to drugs. With the rise in fungal infection and drug resistance, particularly in individuals with co-morbidities, the influence serum and its components have on antimicrobial susceptibility requires assessment. This study examined the impact of physiologically relevant glucose and insulin levels in the presence and absence of 50 % human plasma on MICs for clinical isolates of Candida lusitaniae, Candida parapsilosis, Candida albicans, Candida tropicalis, Candida glabrata, Candida krusei and Cryptococcus neoformans. The addition of insulin or glucose at physiologic levels in RPMI medium alone altered the MIC in either a positive or negative fashion, depending on the organisms and drug tested, with C. glabrata most significantly altered with a 40, >32- and 46-fold increase in MIC for amphotericin B, itraconazole and miconazole, respectively. The addition of candida-antibody negative plasma also affected MIC, with the addition of glucose and insulin having a tandem effect on MIC. These findings indicate that phenotypic resistance of Candida and Cryptococcus can vary depending on the presence of insulin with glucose and plasma. This modulation of resistance may help explain treatment failures in the diabetic population and facilitate the development of stable drug-resistant strains. Furthermore, these findings indicate the need for a precision approach in the choice of drug treatment and drug development.

Lactobacillus Biofilms Influence Anti-Candida Activity

Lactobacilli are the dominant members of the healthy human vaginal microbiota and represent the first defense line from pathogen infection, including vulvovaginal candidiasis. Biofilm is the predominant microbial growth form in nature, and the formation of biofilms inside the human body has important implications in health and disease. In particular, the formation of biofilm by members of the human resident microbiota is desirable, as it can improve microbial persistence and influence functionality. In the present study, we investigated the capability of 16 vaginal Lactobacillus strains (belonging to Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus vaginalis, and Lactobacillus plantarum species) to form biofilms, and we correlated their mode of growth to anti-Candida activity. L. plantarum strains were the best biofilm producers, and high variability was registered in the level of biofilm formation among L. crispatus and L. gasseri strains. Culture supernatants derived from Lactobacillus biofilm and planktonic growth were tested toward a panel of Candida clinical isolates (Candida albicans, Candida glabrata, Candida lusitaniae, Candida tropicalis, Candida krusei, and Candida parapsilosis) and their metabolome assessed by 1H-NMR. L. crispatus and L. plantarum strains exhibited the best fungistatic profile, and biofilms enhanced their anti-Candida activity; on the contrary, L. gasseri strains were more effective when grown in a planktonic mode. Biofilm/planktonic mode of growth also affects Lactobacillus metabolism, mainly influencing nitrogen and amino acid pathways, and anti-Candida activity is instead strictly related to carbohydrate metabolism. The present study underlined the strict interdependence between microbial mode of growth, metabolism, and functional properties. Biofilm formation by members of the healthy human microbiota represents a crucial issue in the field of microbial physiology and host–microbiota interactions, beyond supporting the development of new antimycotic strategies based on probiotics grown in adherence.

Participation of the ABC Transporter CDR1 in Azole Resistance of Candida lusitaniae

Candida lusitaniae is an opportunistic pathogen in humans that causes infrequent but difficult-to-treat diseases. Antifungal drugs are used in the clinic to treat C. lusitaniae infections, however, this fungus can rapidly acquire antifungal resistance to all known antifungal drugs (multidrug resistance). C. lusitaniae acquires azole resistance by gain-of-function (GOF) mutations in the transcriptional regulator MRR1. MRR1 controls the expression of a major facilitator transporter (MFS7) that is important for fluconazole resistance. Here, we addressed the role of the ATP Binding Cassette (ABC) transporter CDR1 as additional mediator of azole resistance in C. lusitaniae. CDR1 expression in isolates with GOF MRR1 mutations was higher compared to wild types, which suggests that CDR1 is an additional (direct or indirect) target of MRR1. CDR1 deletion in the azole-resistant isolate P3 (V688G GOF) revealed that MICs of long-tailed azoles, itraconazole and posaconazole, were decreased compared to P3, which is consistent with the role of this ABC transporter in the efflux of these azoles. Fluconazole MIC was only decreased when CDR1 was deleted in the background of an mfs7Δ mutant from P3, which underpins the dominant role of MFS7 in the resistance of the short-tailed azole fluconazole. With R6G efflux readout as Cdr1 efflux capacity, our data showed that R6G efflux was increased in P3 compared to an azole-susceptible wild type parent, and diminished to background levels in mutant strains lacking CDR1. Milbemycin oxim A3, a known inhibitor of fungal ABC transporters, mimicked efflux phenotypes of cdr1Δ mutants. We therefore provided evidence that CDR1 is an additional mediator of azole resistance in C. lusitaniae, and that CDR1 regulation is dependent on MRR1 and associated GOF mutations.

Disseminated Candida lusitaniae: Nosocomial Acquisition Secondary to an Indwelling Urinary Catheter

Candida lusitaniae is a rare opportunistic pathogen, and its most common risk factors include immunocompromised patients often with an underlying malignancy. It commonly displays resistance to amphotericin B, and historically, echinocandins have been considered first-line treatment. We present a 77-year-old male with a history of diabetes mellitus. He was treated for cellulitis and discharged to a skilled nursing facility with an indwelling urinary catheter. Despite recommendations from the medicine team to remove the catheter, the patient refused even after discussing the risks and benefits. He returned to the hospital 3 weeks later with symptoms of dysarthria, right-sided facial droop, and right-sided weakness. Ultimately, he was determined to have fungemia and native valve endocarditis due to Candida lusitaniae stemming from his indwelling urinary catheter. He was treated with micafungin, but repeated blood cultures continued to grow C. lusitaniae, and he eventually expired following withdrawal of care. We present this case report to illustrate a rare occurence of Candida lusitaniae in a patient without typical risk factors. C. lusitaniae fungemia is an extremely uncommon disease in patients without underlying malignancy. Despite this lack of apparent, classic risk factors, our patient developed endocarditis of his native valve due to C. lusitaniae fungemia from an indwelling urinary catheter. The ability of this organism to form biofilms, and its rapid mutation rate, makes treating C. lusitaniae very difficult. The treatment of choice for C. lusitaniae endocarditis is surgical intervention due to biofilm formation on the cardiac valves. Medical treatment recommendations are currently fluconazole, which is in contrast to the historical use of echinocandins. Infection due to Candida lusitaniae, though rare, should be remembered by clinicians. This particular fungal agent is especially difficult to treat due to its multiple virulence factors. Additionally, the use of indwelling urinary catheters should only occur when proper indications are present and should be promptly discontinued when their placement is no longer necessary.

A rare cause of osteomyelitis of distal phalanx: Candida lusitaniae

Osteomyelitis of the phalanx caused by Candida species are rare. A 49-year-old female patient was admitted to an external center with a splinter injury of the third phalanx of the middle finger of her left hand about 45 days ago. She was referred to our clinic with persistent pain and discharge, despite four-week antibiotherapy. Debridement and curettage were performed and partial excision of the distal phalanx at an appropriate level was done. Her complaints gradually resolved postoperatively with prescribed antibiotics for the pathogen identified as Candida lusitaniae based on the intraoperative cultures. At her three-month follow-up visit, treatment yielded near-excellent results. To the best of our knowledge, this is the first case of osteomyelitis of the distal phalanx caused by Candida lusitaniae in the literature, highlighting the importance of definitive diagnosis and pathogen-specific treatment, rather than empirical treatment, to achieve favorable results with cure.

CONTRIBUTION OF THE MYCOLOGY LABORATORY IN THE MANAGEMENT OF ORAL CANDIDOSIS IN IMMUNODEPRESSED PATIENTS

The authors report a series of 49 cases of oral candidiasis including 20 symptomatic, listed on 100 immunocompromised patients collected at the Mohammed V Military Hospital in Rabat over a 12-month period. The objective of this work was to define the risk factors that lay the foundation for fungal proliferation in the mouth, through early detection in asymptomatic or non-asymptomatic patients. Mycological analysis in the laboratory was based on direct examination and culture on Sabouraud chloramphenicol medium with and without cycloheximide, then identification of the fungal species by API 20 C AUX galleries and the VITEK 2 compact® . The prevalence of oral candidiasis was 49%. The mean age of the patients was 54 years with a sex ratio M / F of 1.04. The contributing factors identified were hyposialia (p = 0.0337), corticosteroid therapy (p = 0.025 and dental removable prostheses (p = 0.000791). The fungal species identified were Candida albicans (79%), Candida dubliniensis (7%), Candida ciferrii (4%), Candida famata (4%), Candida glabrata (4%) and Candida lusitaniae (2%). Conclusion: The oral localization of candidiasis remains very frequent in immunocompromised subjects. Their treatment involves first of all the search for contributing factors and early detection in the presence of asymptomatic forms that only mycological analysis can identify the variety.

Hygiene quality of traditional and industrial table olives from markets in Rabat-Salé and Temara cities in Morocco

Background: Table olives are one of the most important vegetable canning products in Morocco, which is considered one of the world's largest producing countries. Currently, many outlets prepare table olives by different methods that do not comply with standard hygiene practices. Hence, this research was conducted to assess the quality standard of these olives by evaluating their physico-chemical and microbiological properties.Methodology: A total of 108 samples of table olives (pitted green olives and blacks) obtained from Rabat-Salé and Rabat-Temara markets in Morocco were evaluated. Physico-chemical properties of the olives including pH, oxido-reduction potential (ORP) and titrable acidity were determined using the analytical methods of the Association of Official Analytical Chemists (AOAC). Microbiological analyses including standard plate count (SPC) for total aerobic mesophilic flora (TAMB), total coliforms (TC), faecal coliforms (FC), yeasts, clostridia, Staphylococcus aureus, faecal streptococci and salmonella counts, were performed using standard microbiological methods. The identification of yeast isolates was carried out with the commercial API 20C biochemical identification kit.Results: The average microbial loads for traditional olive samples were 3.2x106 CFU/ml for SPC, 1.7x104 CFU/ml for TC, 8.7x103 CFU/ml for FC, and 2.5x106 CFU/ml for yeast, which were higher compared to the average microbial loads of industrial olives with values of 5.9x105 CFU/ml, 5x101 CFU/ml, 0 CFU/ml and 0 CFU/ml respectively. One hundred percent (56 of 56) of the traditional olives (pitted green and black) from Temara-Rabatmarkets were contaminated with coliforms while 50% of green and 65% of black olives in Salé-Rabat were contaminated with coliforms. Five percent (5%) each of the traditional green and black olives in Salé-Rabat markets were contaminated with clostridia (spore forming bacteria). No FC or other bacteria and yeasts were present in the industrial olives, and none of the olives was contaminated with S. aureus, faecal streptococci and salmonella. Of the total of 8 yeast strains isolated from the traditional olives, 4 (50%) were Candida guilliermondii, 2 (25%) Candida lusitaniae and 2 (25%) Candida famata.Conclusion: The contamination of olive oil products may be due to different sources such as water, processing materials, storage condition, cleaning, labour and others. There is need for increase awareness and control of these at the points of sale of these traditional olives. Keywords: hygiene; physico-chemical properties; microbiology; traditional olives; quality French Title: Qualité Hygiène des olives de table traditionnelles et industrielles des marchés des villes de Rabat-Salé et Témara au Maroc Contexte: Les olives de table sont l'un des produits de mise en conserve de légumes les plus importants au Maroc, qui est considéré comme l'un des plus grands pays producteurs du monde. Actuellement, de nombreux points de vente préparent les olives de table par différentes méthodes non conformes aux pratiques d'hygiène standard. Ainsi, cette recherche a été menée pour évaluer le standard de qualité de ces olives en évaluant leurs propriétés physico-chimiques et microbiologiques.Méthodologie: Un total de 108 échantillons d'olives de table (olives vertes dénoyautées et noires) obtenus sur les marchés de Rabat-Salé et Rabat-Témara au Maroc ont été évalués. Les propriétés physico-chimiques des olives, y compris le pH, le potentiel d'oxydoréduction (ORP) et l'acidité titrable ont été déterminées en utilisant les méthodes analytiques de l'Association of Official Analytical Chemists (AOAC). Les analyses microbiologiques, y compris la numération sur plaque standard (SPC) pour la flore mésophile aérobie totale (FMAT), les coliformes totaux (CT), les coliformes fécaux (CF), les levures, les clostridies, Staphylococcus aureus, les streptocoques fécaux et les numérations de salmonelles, ont été effectuées à l'aide de méthodes microbiologiques standard. L'identification des isolats de levure a été réalisée avec le kit d'identification biochimique API 20E du commerce.Résultats: Les charges microbiennes moyennes pour les échantillons d'olives traditionnelles étaient de 3,2x106 UFC/ml pour le SPC, 1,7x104 UFC/ml pour le TC, 8,7x103 UFC/ml pour le FC et 2,5x106 UFC/ml pour la levure, qui étaient plus élevées par rapport aux charges microbiennes moyennes des olives industrielles avec des valeurs respectives de 5,9x105 UFC/ml, 5x101 UFC/ml, 0 UFC/ml et 0 UFC/ml. Cent pour cent (56 sur 56) des olivestraditionnelles (dénoyautées vertes et noires) des marchés de Témara-Rabat étaient contaminées par des coliformes tandis que 50% des olives vertes et 65% des olives noires de Salé-Rabat étaient contaminées par des coliformes. Cinq pour cent (5%) de chacune des olives vertes et noires traditionnelles des marchés de Salé-Rabat étaient contaminées par des clostridia (bactéries sporulantes). Aucune FC ni aucune autre bactérie et levure n'étaient présentes dans les olives industrielles, et aucune des olives n'était contaminée par S. aureus, des streptocoques fécaux et des salmonelles. Sur un total de 8 souches de levure isolées des olives traditionnelles, 4 (50%) étaient Candida guilliermondii, 2 (25%) Candida lusitaniae et 2 (25%) Candida famata.Conclusion: La contamination des produits à base d'huile d'olive peut être due à différentes sources telles que l'eau, les matériaux de traitement, les conditions de stockage, le nettoyage, la main-d'œuvre et autres. Il est nécessaire d'accroître la sensibilisation et le contrôle de ceux-ci dans les points de vente de ces olives traditionnelles. Mots-clés: hygiène; propriétés physico-chimiques; microbiologie; olives traditionnelles; qualité