- Volume 3, Issue 12, 2021
Volume 3, Issue 12, 2021
- Research Articles
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Pneumococcal serotypes causing non-invasive pneumonia in adults from a South Indian tertiary care hospital and the impact of the newer conjugate vaccines
Background. Streptococcus pneumoniae is the leading cause of community-acquired pneumonia (CAP) in adults. Ageing, chronic conditions and comorbidities are important risk factors for pneumococcal pneumonia.
Purpose. There is lack of data on the pneumococcal serotypes causing non-invasive pneumonia in India. This study aims to determine the prevalent pneumococcal serotypes causing non-invasive pneumonia, the associated comorbidities, and the coverage of both the available pneumococcal vaccines in India and conjugate vaccines that are currently undergoing clinical trials.
Methods. A total of 280 subjects (aged >16 years) who had clinical symptoms correlating with radiological findings for non-invasive bacteremic pneumonia and microbiological evidence of S. pneumoniae between 2018 and 2020 were included. The clinical, demographic, radiological and microbiological findings were retrieved from the Hospital Information System (HIS).
Results. The common serotypes in order of prevalence were 19F, 9V, 23F, 6B, 11A, 13, 34, 10A, 19A and 6A. The predominant non-vaccine serotypes were 13, 34, 35B, 31 and 16F. The associated radiological findings were pneumonic consolidation and multi-lobar involvement. Other coinfected bacterial pathogens included H. influenzae, S. aureus, K. pneumoniae and P. aeruginosa.
Conclusion. The pneumococcal vaccines: PCV10/GSK, PCV10/SII, PCV13, PCV15, PCV20 and PPSV23 provide an overall serotype coverage of 36, 41, 47, 48, 61 and 69 %, respectively of S. pneumoniae causing non-invasive pneumonia in South India. Increasing catch-up vaccination using PCV10(SII) in pre-school children could have a more significant impact on reducing pneumococcal pneumonia in adults (>50 years) in terms of increased herd immunity at an affordable cost.
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Characteristics of patients with suspected COVID-19 pneumonia and repeatedly negative RT-PCR
Objectives. Challenges remain and there are still a sufficient number of cases with epidemiological, clinical features and radiological data suggestive of COVID-19 pneumonia that persist negative in their RT-PCR results. The aim of the study was to define the distinguishing characteristics between patients developing a serological response to SARS-CoV-2 and those who did not.
Methods. RT-PCR tests used were TaqPath 2019-nCoV Assay Kit v1 (ORF-1ab, N and S genes) from Thermo Fisher Diagnostics and SARS-COV-2 Kit (N and E genes) from Vircell. Serological response was tested using the rapid SARS-CoV2 IgG/IgM Test Cassette from T and D Diagnostics Canada and CMC Medical Devices and Drugs, S.L, CE.
Results. In this cross-sectional study, we included a cohort of 52 patients recruited from 31 March 2020 to 23 April 2020. Patients with positive serology had an older average age (73.29) compared to those who were negative (54.82) (P<0.05). Sat02 in 27 of 34 patients with positive serology were below 94% (P<0.05). There was a frequency of 1.5% negative SARS-CoV-2 RT-PCRs during the study period concurring with 36.7% of positivity.
Conclusions. Clinical features and other biomarkers in a context of a positive serology can be considered crucial for diagnosis.
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Sequence diversity of the Pseudomonas aeruginosa population in loci that undergo microevolution in cystic fibrosis airways
Five hundred and thirty-four unrelated Pseudomonas aeruginosa isolates from inanimate habitats, patients with cystic fibrosis (CF) and other human infections were sequenced in 19 genes that had been identified previously as the hot spots of genomic within-host evolution in serial isolates from 12 CF lungs. Amplicon sequencing confirmed a significantly higher sequence diversity of the 19 loci in P. aeruginosa isolates from CF patients compared to those from other habitats, but this overrepresentation was mainly due to the larger share of synonymous substitutions. Correspondingly, non-synonymous substitutions were either rare (gltT, lepA, ptsP) or benign (nuoL, fleR, pelF) in some loci. Other loci, however, showed an accumulation of non-neutral coding variants. Strains from the CF habitat were often mutated at evolutionarily conserved positions in the elements of stringent response (RelA, SpoT), LPS (PagL), polyamine transport (SpuE, SpuF) and alginate biosynthesis (AlgG, AlgU). The strongest skew towards the CF lung habitat was seen for amino acid sequence variants in AlgG that clustered in the carbohydrate-binding/sugar hydrolysis domain. The master regulators of quorum sensing lasR and rhlR were frequent targets for coding variants in isolates from chronic and acute human infections. Unique variants in lasR showed strong evidence of positive selection indicated by d N/d S values of ~4. The pelA gene that encodes a multidomain enzyme involved in both the formation and dispersion of Pel biofilms carried the highest number of single-nucleotide variants among the 19 genes and was the only gene with a higher frequency of missense mutations in P. aeruginosa strains from non-CF habitats than in isolates from CF airways. PelA protein variants are widely distributed in the P. aeruginosa population. In conclusion, coding variants in a subset of the examined loci are indeed characteristic for the adaptation of P. aeruginosa to the CF airways, but for other loci the elevated mutation rate is more indicative of infections in human habitats (lasR, rhlR) or global diversifying selection (pelA).
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Use of orthogonal serine integrases to multiplex plasmid conjugation and integration from E. coli into Streptomyces
More LessSome major producers of useful bioactive natural products belong to the genus Streptomyces or related actinobacteria. Genetic engineering of these bacteria and the pathways that synthesize their valuable products often relies on serine integrases. To further improve the flexibility and efficiency of genome engineering via serine integrases, we explored whether multiple integrating vectors encoding orthogonally active serine integrases can be introduced simultaneously into Streptomyces recipients via conjugal transfer and integration. Pairwise combinations of Escherichia coli donors containing vectors encoding orthogonal serine integrases were used in each conjugation. Using donors containing plasmids (of various sizes) encoding either the φBT1 or the φC31 integration systems, we observed reproducible simultaneous plasmid integration into Streptomyces coelicolor and Streptomyces lividans at moderate frequencies after conjugation. This work demonstrated how site-specific recombination based on orthogonal serine integrases can save researchers time in genome engineering experiments in Streptomyces .
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Analysis of a Cas12a-based gene-drive system in budding yeast
More LessThe discovery and adaptation of CRISPR/Cas systems within molecular biology has provided advances across biological research, agriculture and human health. Genomic manipulation through use of a CRISPR nuclease and programmed guide RNAs has become a common and widely accessible practice. The identification and introduction of new engineered variants and orthologues of Cas9 as well as alternative CRISPR systems such as the type V group have provided additional molecular options for editing. These include distinct PAM requirements, staggered DNA double-strand break formation, and the ability to multiplex guide RNAs from a single expression construct. Use of CRISPR/Cas has allowed for the construction and testing of a powerful genetic architecture known as a gene drive within eukaryotic model systems. Our previous work developed a drive within budding yeast using Streptococcus pyogenes Cas9. Here, we installed the type V Francisella novicida Cas12a (Cpf1) nuclease gene and its corresponding guide RNA to power a highly efficient artificial gene drive in diploid yeast. We examined the consequence of altering guide length or introduction of individual mutational substitutions to the crRNA sequence. Cas12a-dependent gene-drive function required a guide RNA of at least 18 bp and could not tolerate most changes within the 5′ end of the crRNA.
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- Short Communications
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Phenotypic detection, antimicrobial susceptibility and virulence profile of staphylococci in the pig production setting, Makurdi, Nigeria
More LessLivestock, particularly pigs, have increasingly been recognized as important reservoirs for zoonotic transmission of pathogenic bacteria, including staphylococci. Livestock production systems in developing countries of sub-Saharan Africa, including Nigeria, are characterized by high misuse/abuse of antimicrobials and a close association between humans and these animals, which promotes the emergence and transmission of resistant and potentially virulent bacteria. In the present study, we investigated the occurrence and characteristics (species distribution, virulence and resistance profile) of staphylococci from smallholder backyard pig farms, slaughter slabs and pig handlers in Makurdi, Nigeria. A total of 330 nasal swabs originating from 300 pigs and 30 in-contact humans were collected and processed. One hundred and thirteen samples [34.2 %; 95 % confidence interval (CI): 29.1–39.6] comprising 103 (34.3 %; 95 % CI: 29.0–40.0) and 10 (33.3 %; 95 % CI: 17.3–52.8 %) samples from pigs and humans, respectively, were positive for staphylococci, yielding 120 isolates (pigs n=110, humans n=10). The 120 isolates were distributed into 15 species with Staphylococcus aureus (n=25) followed by Staphylococcus cohnii (n=19) and Staphylococcus sciuri (n=14) occurring more frequently. All isolates were resistant to β-lactam (100 %) antibiotics. Resistance to some critical antimicrobials, including linezolid (22 %), vancomycin (19.2 %), gentamicin (7.5%) and the fluoroquinolones ciprofloxacin (75.8 %) and enrofloxacin (66.7 %), was also observed. Majority (99.2 %) of the isolates displayed a multidrug resistance phenotype with the AMP-C-CIP-E-ENR-FOX-OX-P-S-SXT-TE phenotype being predominant. Overall, 70 % of the isolates expressed the methicillin resistance phenotype, out of which 20 % (n=17) were MRSA. Resistance to serum bactericidal activity and biofilm production were respectively observed in 45 (100 %) and 5 (11.3 %) of the coagulase-positive staphylococci. Our findings demonstrated the occurrence of a high diversity of staphylococci expressing multidrug resistance and potentially virulent phenotypes among healthy swine and pig handlers in small-scale backyard farms in North-Central Nigeria. These findings underscore the potential role of pig production settings in the emergence and dissemination of potentially virulent staphylococci and the importance of the development of antimicrobial resistance monitoring systems/implementation of control measures in developing countries. Proper hygienic practices and control of indiscriminate use and misuse of antibiotics are recommended.
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The ChpR transcriptional regulator of Sinorhizobium meliloti senses 3,5,6-trichloropyridinol, a degradation product of the organophosphate pesticide chlorpyrifos
More LessThe global use of organophosphate insecticides (OPPs) and the growing concern of off-target side effects due to OPP exposure has prompted the need for sensitive and economical detection methods. Here we set out to engineer a previously identified OPP responsive transcription factor, ChpR, from Sinorhizobium melilotii to respond to alternative OPPs and generate a repertoire of whole-cell biosensors for OPPs. The ChpR transcription factor and cognate promoter P chpA, have been shown to activate transcription in the presence of the OPP chlorpyrifos (CPF). Utilizing a GFP reporter regulated by ChpR in a whole-cell biosensor we found that the system responds significantly better to 3,5,6-trichloro-2-pyridinol (TCP), the main degradation product of CPF, compared to CPF itself. This biosensor was able to respond to TCP at 390 nM within 4 h compared to 50 µM of CPF in 7 h. The ChpR-P chpA , and the activating ligand TCP, were able to regulate expression of a kanamycin resistance/sucrose sensitivity (kan/sacB) selection/counterselection module suitable for high throughput mutagenesis screening studies. The ability to control both GFP and the kan/sacB module demonstrates the utility of this reporter for the detection of CPF affected areas. The ChpR-P chpA system serves as an additional positive regulator switch to add to the growing repertoire of controllers available within synthetic biology.
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Immunomodulatory potential of four candidate probiotic Lactobacillus strains from plant and animal origin using comparative genomic analysis
Probiotic strains from different origins have shown promise in recent decades for their health benefits, for example in promoting and regulating the immune system. The immunomodulatory potential of four Lactobacillus strains from animal and plant origins was evaluated in this paper based on their genomic information. Comparative genomic analysis was performed through genome alignment, average nucleotide identity (ANI) analysis and gene mining for putative immunomodulatory genes. The genomes of the four Lactobacillus strains show relative similarities in multiple regions, as observed in the genome alignment. However, ANI analysis showed that L. mucosae LM1 and L. fermentum SK152 are the most similar when considering their nucleotide sequences alone. Gene mining of putative immunomodulatory genes studied from L. plantarum WCFS1 yielded multiple results in the four potential probiotic strains, with L. plantarum SK151 showing the largest number of genes at around 74 hits, followed by L. johnsonii PF01 at 41 genes when adjusted for matches with at least 30 % identity. Looking at the immunomodulatory genes in each strain, L. plantarum SK151 and L. johnsonii PF01 may have wider activity, covering both immune activation and immune suppression, as compared to L. mucosae LM1 and L. fermentum SK152, which could be more effective in activating immune cells and the pro-inflammatory cascade rather than suppressing it. The similarities and differences between the four Lactobacillus species showed that there is no definitive trend based on the origin of isolation alone. Moreover, higher percentage identities between genomes do not directly correlate with higher similarities in potential activity, such as in immunomodulation. The immunomodulatory function of each of the four Lactobacillus strains should be observed and verified experimentally in the future, since some the activity of some genes may be strain-specific, which would not be identified through comparative genomics alone.
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- Abstracts from the Candida and Candidiasis Meeting 2021
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- Oral Abstracts
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Azole uptake in Candida auris is strongly correlated with drug resistance
More LessAnalyses of fluconazole uptake in clinical isolates of C. auris, with wide ranging drug resistance profiles, has revealed interesting differences within the species as well as major distinctions from other yeast species. We previously proposed that prevention of drug uptake is a potential mechanism of drug resistance and our C. auris data provide further support for this.
We developed an assay using radio-labeled fluconazole to measure intracellular azole accumulation in fungal cells. The assay is performed under glucose-replete conditions to inhibit ATP-dependent efflux. A comparative study measuring fluconazole uptake in 63 C. auris isolates as well as a panel of other species such as C. albicans, S. cerevisiae, C. glabrata, C. krusie, C. lusitanea, C. tropicalis, and C. dublinienses revealed striking C. auris phenotypes that we have not seen in other fungal species.
There is a strong correlation between fluconazole resistance and reduced drug uptake in C. auris. Fluconazole-resistant C. auris isolates had reduced levels of intracellular fluconazole accumulation compared to susceptible isolates. Drug-resistant C. auris isolates had the lowest drug accumulation of any of the yeast species tested. Fluconazole-susceptible C. auris isolates had dramatically increased fluconazole accumulation compared to the resistant isolates as well as when compared to other yeast species.
Of the 63 C. auris isolates, 28 of 32 (∼88%) resistant isolates had extremely low fluconazole uptake, whereas 15 of 18 (∼83%) susceptible isolates had high fluconazole uptake. This association between reduced drug uptake and resistance could be a C. auris-distinctive mechanism of drug resistance.
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Candida albicans phosphate transport, facilitating nucleotide sugar biosynthesis, contributes to cell wall stability.
The Candida albicans high-affinity phosphate transporter Pho84 is required for normal Target of Rapamycin signaling, oxidative stress resistance and virulence of this fungal pathogen. It also contributes to C. albicans’ tolerance of two antifungal drug classes, polyenes and echinocandins. Echinocandins inhibit biosynthesis of a major cell wall component, beta-1,3-glucan. Cells lacking Pho84 were hypersensitive to other forms of cell wall stress beyond echinocandin exposure, while their cell wall integrity signaling response was weak. Metabolomics experiments showed that levels of phosphoric intermediates, including nucleotides like ATP and nucleotide sugars, were low in pho84 mutant compared to wild type cells recovering from phosphate starvation. Non-phosphoric precursors like nucleobases and nucleosides were elevated. Outer cell wall phosphomannan biosynthesis requires a nucleotide sugar,GDP-mannose. The nucleotide sugar UDP-glucose is the substrate of enzymes that synthesize two major structural cell wall polysaccharides, beta-1,3- and beta-1,6-glucan. Another nucleotide sugar, UDP-N-acetylglucosamine, is the substrate of chitin synthases which produce a stabilizing component of the intercellular septum and of lateral cell walls. Lack of Pho84 activity, and phosphate starvation, potentiated pharmacological or genetic perturbation of these enzymes. Our model is that low substrate concentrations of beta-D-glucan- and chitin synthases diminish enzymatic reaction rates and potentiate pharmacologic inhibitors to decrease the yield of their cell wall-stabilizing products. Phosphate import is not conserved between fungal and human cells, and humans do not synthesize beta-D-glucans or chitin. Hence inhibiting these processes simultaneously could yield potent antifungal effects with low toxicity to humans.
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The colon microcosm: a novel in vitro model to study Candida albicans colonisation of the human colon
More LessCandida albicans colonises the gastro-intestinal (GI) tract of over 60% of the population. In severely ill or immune compromised patients, this fungus can escape the gut, disseminate through the body and cause systemic disease. Most research in the field has focused on defining traits that contribute directly to virulence; there are comparatively few studies which have addressed how C. albicans colonises and persists in the gut. Furthermore, such studies have typically been performed mouse models devoid of resident GI bacteria, completely neglecting the major impact of the local microbiota on GI colonisation. How, then, does C. albicans persist in the GI tract in the presence of the normal gut microbiota?
To address this question, a novel in vitro two-phase anaerobic fermentation system that simulates the human colon microenvironment has been developed. This “colon microcosm” supports the growth of human faecal microbiota in liquid anaerobic colon medium (phase 1) and C. albicans growth on agar plugs which are added to the medium to mimic the epithelial surface (phase 2). The impact of C. albicans upon the faecal microbiota is monitored by examining the planktonic phase (phase 1), whilst the effect of the microbiota on the growth of C. albicans is monitored after extracting C. albicans cells from the agar plugs (phase 2).
The results of assays carried out to validate the model will be presented, as will data from pilot studies which illustrate the potentially exploitable impact of the human GI microbiota from healthy individuals on C. albicans growth.
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Unveiling azole resistance mechanisms in Candida glabrata clinical isolates encoding wild-type or gain-of-function CgPdr1 alleles
The relevance of C. glabrata as a human pathogen is linked with its poor susceptibility to azoles as well as its extreme genomic plasticity that allows the rapid acquisition of resistance. Extensive characterization of azole-resistant C. glabrata strains unveiled the central role of the transcriptional regulator CgPdr1 in the resistance phenotype, with many strains encoding hyperactive (or gain-of-function; GOF) CgPdr1 alleles. Large scale profiling of a collection of clinical C. glabrata isolates recovered in hospitals of the Lisbon area, in Portugal, led to the identification of 11 strains exhibiting resistance to fluconazole and voriconazole, while 2 were only resistant to fluconazole. Among these strains, 10 were found to encode alleles of the CgPDR1 gene harbouring multiple non-synonymous SNPs that were not found in the alleles encoded by susceptible strains, including K274Q, I392M and I803T not previously described as GOF mutations. The isolates encoding these alleles were found to over-express several CgPdr1 target genes including the azole efflux pump CgCDR1 sustaining the idea that these represent new gain-of-function CgPdr1 alleles. Only one of the identified azole-resistant strains was found to encode a CgPDR1 allele fully identical to the one encoded by susceptible strains. To better understand the resistance phenotype of this strain, its transcriptome was compared with the one of a susceptible strain and of strains encoding CgPdr1 GOF alleles. The results of this comparative transcriptomic analysis will be discussed shedding light into the different azole-resistance mechanisms evolved by C. glabrata, including those independent of CgPdr1 GOF strains.
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Exploiting diverse chemical collections to uncover novel antifungals
Kali Iyer, Kaddy Camara, Martin Daniel-Ivad, Nicole Revie, Jennifer Lou, Sheena Li, Richard Trilles, Sheila Elardo, Yoko Yashiroda, Jen Fossen, Karen Marchillo, Zhongle Liu, Shakti Singh, Jose Muñoz, Sang Hu Kim, Hiroyuki Hirano, Minoru Yoshida, Hiroyuki Osada, Christina Cuomo, Noelle Williams, Ashraf Ibrahim, Jack Edwards, David Andes, Justin Nodwell, John Porco, Charlie Boone, Mohammad Mazhab-Jafari, Lauren Brown, Luke Whitesell, Nicole Robbins and Leah CowenThe rise in drug resistance amongst pathogenic fungi, paired with the limited arsenal of antifungals available is an imminent threat to our medical system. To address this, we screened two distinct compound libraries to identify novel strategies to expand the antifungal armamentarium. The first collection wasthe RIKEN Natural Product Depository (NPDepo), which was screened for antifungal activity against four major human fungal pathogens: Candida albicans, Candida glabrata, Candida auris, and Cryptococcus neoformans. Through a prioritization pipeline, one compound, NPD6433, emerged as having broad-spectrum antifungal activity and minimal mammalian cytotoxicity. Chemical-genetic and biochemical assays demonstrated that NPD6433 inhibits the essential fungal enzyme fatty acid synthase 1 (Fas1). Treatment with NPD6433 inhibited various virulence traits in C. neoformans and C. auris, and rescued mammalian cell growth in a co-culture model with C. auris. The second compound library screened was adiversity-oriented collectionfrom Boston University. This chemical screen was focused on identifying novel molecules that enhance the activity of the widely deployed antifungal, fluconazole, against C. auris. Through this endeavour, we discovered a potent compound that enhanced fluconazole efficacy against C. auris through increasing azole intracellular accumulation. This activity was dependent on expression of the multidrug transporter geneCDR1, suggesting that this compound targets efflux mechanisms. Furthermore, this molecule significantly reduced fungal burden alone and in combination with fluconazole in a murine model of C. auris disseminated infection. Overall, this work identifies novel compounds with bioactivity against fungal pathogens, revealing important biology, and paving the way for the critical development of therapeutic strategies.
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Identification of genome-wide alternative splicing events in sequential, isogenic clinical isolates of Candida albicans reveals a mechanism important for drug resistance and tolerance to cellular stress
More LessAlternative gene splicing (AS) is a process by which a single gene can give rise to different protein isoforms, generating proteome diversity. Despite recent advances in our understanding of AS in basic cellular processes, the role of AS in drug resistance and fungal pathogenesis is poorly understood. In Candida albicans, approximately 6% of the genes contain introns. Considering this low and random distribution of introns, we focused our study on alternative splicing (AS) and its impact on the development of drug resistance, an area largely unexplored in this yeast. We performed comparative RNA sequencing of sequential isogenic azole sensitive and resistant isolates of C. albicans. The analysis revealed differential expression of splice junctions/isoforms in 14 genes, between the drug sensitive and resistant isolates. Furthermore, C. albicans WT cells exposed to antifungal drugs, heat stress or metal deficiency also showed differential expression of isoforms for the genes undergoing AS. In this study we present data on the effect of AS on the function of SOD3. The C. albicans SOD3 has a single intron and is important for the removal of superoxide radicals. The overexpression of the two isoforms of SOD3 in its null background highlighted importance of spliced isoform in complementing the susceptibility to menadione. However, the two isoforms did not differ in rescuing the susceptibility of sod3Δ/Δto Amphotericin B. Collectively, these data suggest that AS may be a novel mechanism in C. albicans for stress adaptation and overcoming drug resistance.
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Quorum sensing plays a key role in controlling beta-glucan exposure in response to environmental pH
Candida albicans is a commensal yeast of the human gut, which is tolerated by the immune system, but has the potential to become an opportunistic pathogen. One way in which C. albicans achieves this duality is through concealing, or exposing cell wall pathogen-associated molecular patterns (PAMPs) in response to host derived environment cues (pH, hypoxia, lactate). This cell wall remodelling allows C. albicans to evade or hyperactivate the host’s innate immune responses leading to disease. Previously, we identified that adaptation of C. albicans to acidic environments, conditions encountered during colonisation of the female reproductive tract, induce significant cell wall remodelling resulting in the exposure of two key fungal PAMPs (glucan and chitin). Here we report that this pH-dependent cell wall remodelling is time dependent with the initial change in pH driving cell wall unmasking, which is then remasked at later time points. Remasking ofglucan was mediated via the cell density dependent fungal quorum sensing molecule farnesol, while chitin remasking was mediated via a small, heat-stable, non-proteinaceous secreted molecule(s). Transcript profiling identified a core set of 42 genes significantly regulated by pH over time, and identified the transcription factor Efg1 as a regulator of chitin exposure through regulation of CHT2. This dynamic cell wall remodelling influenced innate immune recognition of C. albicans, suggesting that during infection C. albicans can manipulate the host innate immune responses.
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The first multi-omic interaction network in the human pathogen Candida glabrata provides novel insights into the virulence regulator, Hsp90
More LessCandida glabrata-caused candidiasis is growing but treatments remain limited by paucity of drug targets, intrinsic azole resistance and increasing resistance to other drug classes. Drug resistance is one of numerous virulence traits regulated by the chaperone, heat shock protein 90 (Hsp90) in Candida albicans via its interactions with 5% of the genome. Hsp90 also regulates key drug resistance mechanisms in C. glabrata, but little else was known about Hsp90 in this organism. Therefore, CgHsp90 interactions were elucidated by genetic and proteomic methods.
A genetic network was produced by a chemical-genetic, synthetic-sick screen on a gene-deletion library covering 16% of the genome; whilst quantitative proteomics was undertaken by tandem mass tagging on wild-type cells. Both experiments were undertaken at 30°C, 37°C and 39°C and Hsp90 was perturbed using sub-lethal concentrations of Hsp90 inhibitor. Efforts to identify Hsp90 interactors at these host-infection associated temperatures produced a genetic network of 68 genes and a protein network of 2298 proteins. Of these, 4 genes and 261 proteins interacted with Hsp90 at all three temperatures, indicating a core Hsp90 interaction network. Intriguingly, both networks had enrichment for the “antibiotic biosynthesis” GO term. Two genes, BCY1 and MCM16, were found to interact with Hsp90 at multiple temperatures in both networks. These data indicate the divergence of Hsp90 networks between C. glabrata and its close relatives and offer important targets for further research. Presented here is the first multi-omic interaction network in C. glabrata, focused on the virulence and drug resistance regulator, Hsp90.
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Spatial mapping of immunological epitopes in the Candida cell wall using C-type lectin probes
The primary recognition event between a fungal pathogen and the immune system normally involves the engagement of a pattern recognition receptor with specific components of the cell wall. However, the cell wall is a complex three dimensional structure whose composition changes rapidly in accordance with environmental stimuli. Therefore it is important to know what is the precise nature of the primary recognition event, how many events occur to activate the immune response and how these recognition events are affected by changes in cell wall architecture, cellular morphogenesis and physiological adaptation of the pathogen to specific niches in the human body. We address this fundamental question using four soluble immune C-Type lectin receptor-probes which recognize specific mannans and β-1,3 glucan in the cell wall. We use these C-type lectin probes to demonstrate that mannan epitopes are differentially distributed in the inner and outer layers of fungal cell wall in a clustered or diffuse manner. Immune reactivity of fungal cell surfaces did not correlate with relatedness of different fungal species, and mannan-detecting receptor-probes discriminated between cell surface mannans generated by the same fungus growing under different conditions. These studies demonstrate that mannan-epitopes within fungal cell walls are differentially distributed and dynamically expressed as the fungus adapted to microenvironments that would be encountered in vivo.
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- Poster Presentations
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Suppressor genetics reveals novel inter-domain crosstalk within the multidrug transporter Mdr1 protein
More LessThe Multidrug resistance-1 protein (Mdr1p) of Candida albicans is a crucial drug/H+ antiporter within the Major Facilitator Superfamily of proteins involved in the efflux of a broad spectrum of structurally diverse xenobiotic compounds. As a member of the DHA1 subfamily, Mdr1p consists of 12 transmembrane helices (TMHs), divided equally into two Transmembrane Domains (TMDs). How the pseudo-symmetrically positioned TMHs, and the TMDs they compose, communicate with each other remains poorly characterized. In that direction, the recovery of spontaneous chromosomal mutants that negatively affect the primary mutant phenotype can provide essential inter-domain communication insights. For this purpose, in the current study we have performed a suppressor screen for a critically transport deficient mutant G230A, located within TMH-4 of Mdr1p, predicted towards the intracellular space. The recovered suppressor (P528H), that restores the transport capacity of this initially drug susceptible mutant, map to TMH-12, very close to the extracellular space. Since the mutant and suppressor sites occupy the N-domain and C-domain, respectively, and locate at a pseudo-symmetrical position, these results hint to a novel pattern of crosstalk. Additionally, the recovered suppressor mutation restores wild type phenotypes for all tested xenobiotic substrates except cycloheximide, thus implying substrate selectivity. Furthermore, the molecular modeling and docking results suggest a novel compensatory mechanism which is independent of drug binding. Altogether, the present study is a first attempt to gain insights into the transport mechanism of drug/H+ antiporter using the suppressor genetics approach.
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Exploration of anti EFG1 locked nucleic acid gapmers to control Candida albicans filamentation
Introduction:Antisense oligonucleotides (ASOs) have been successfully utilized to silence gene expression for the treatment of many genetic human diseases, and particularly the locked nucleic acid (LNA) chemical modification is extensively used with this propose. However, LNA-modified ASOs have never been exploited for controlling virulence genes of Candida.
EFG1is an important determinant of virulence that is involved in the switch from yeast to filamentous forms in C. albicans. Thus, our main goal was to explore LNA antisense gapmers for controlling EFG1gene expression and to block C. albicans filamentation.
Methods:A set of five LNA-modified gapmers were designed with different chemical modifications (phosphorothioate backbone (PS) and/or palmitoyl-2’-amino-LNA) and ASO length. The in vitro performance of the different ASOs was evaluatedon their ability to control EFG1 gene expression, by qRT-PCR, and to reduce C. albicans’ filamentation, through filaments’ enumeration by microscopy. The in vivo therapeutic potential of ASOs was assessed using a G. mellonella model of infection, through a survival assay.
Results:In vitro results showed that all ASOs were able to reduce the levels of EFG1gene expression, consequently reducing the levels of C. albicans filamentation around 50%. Interestingly, in vivo tests showed that the LNA-modified gapmer with PS backbone and palmitoyl-2’-amino-LNA was more effective at preventing G. mellonella infections.
Conclusions:Undeniably, this work promotes the development of a novel approach for the treatment of Candida infections based on the delivery of ASOs coupled with LNA chemical modification.
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Genome-wide analysis of experimentally evolved Candida auris reveals multiple novel mechanisms of multidrug-resistance
Candida auris is globally recognized as an opportunistic fungal pathogen of high concern, due to its extensive multidrug-resistance (MDR). Still, molecular mechanisms of MDR are largely unexplored. This is the first account of genome wide evolution of MDR in C. auris obtained through serial in vitro exposure to azoles, polyenes and echinocandins. We show the stepwise accumulation of multiple novel mutations in genes known and unknown in antifungal drug resistance, albeit almost all new for C. auris. Echinocandin resistance was accompanied by a codon deletion in FKS1hot spot 1 and a substitution in FKS1 ‘novel’ hot spot 3. Mutations in ERG3 and CIS2 further increased the echinocandin MIC. Decreased azole susceptibility was linked to a mutation in transcription factor TAC1b and overexpression of the drug efflux pump Cdr1; a segmental duplication of chromosome 1 containing ERG11; and a whole chromosome 5 duplication, which contains TAC1b. The latter was associated with increased expression of ERG11, TAC1band CDR2, but not CDR1. The simultaneous emergence of nonsense mutations in ERG3 and ERG11 was shown to decrease amphotericin B susceptibility, accompanied with fluconazole cross resistance. A mutation in MEC3, a gene mainly known for its role in DNA damage homeostasis, further increased the polyene MIC. Overall, this study shows the alarming potential and diversity for MDR development in C. auris, even in a clade until now not associated with MDR (clade II),hereby stressing its clinical importance and the urge for future research.
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FunHoMic: A Marie Skłodowska-Curie Innovative Training Network for the study of the Fungus-Host-Microbiota interplay
Nathaniel Cole, Benoît Marsaux, Diletta Rosati, Margot Delavy, Daria Kosmala, Zixuan Xie, Ann-Kristin Kaune, Marisa Valentine, Sayoni Chakraborty, Manjyot Kaur, Leovigildo Rey Alaban, Moran Morelli, Ricardo Fróis-Martins, Alan Walker, Pieter Van den Abbeele, Mihai Netea, Marie-Elisabeth Bougnoux, Chaysavanh Manichanh, Carol Munro, Bernhard Hube, Ilse Jacobsen, Karine Roget, Vincent Thomas, Karla Queiroz, Salomé Leibundgut-Landmann, Alistair Brown and Christophe d'EnfertIntroductionThe FunHoMic project is a Marie Skłodowska-Curie Innovative Training Network comprising 13 PhD students, 8 academic partners and 3 industry partnersaimingto understand the interplay between fungi, hostsand microbiota to improve prevention and treatment of fungal infections.
ImportanceAbout 2 billion people suffer fungal infections, which have a mortality rate close to that of malaria or breast cancer. Candida albicans has a high clinical and economic burden, making it of particular interest to the FunHoMic project. 70% of women experience at least one episode of vulvovaginal candidiasis (“thrush”) during their lifetime; 8% suffer recurring infections. C. albicans may live as a commensal but can cause symptoms when the fungus-host-microbiota equilibrium is disrupted. Infections by C. albicans have a significant clinical impact, with fatalities in severe cases. Many factors are associated with C. albicans infections; intensive care, neutropenic and diabetic patients are most at risk of systemic infection. Rising antifungal drug resistance has led to certain C. albicans infections having no treatment option.
AimThe FunHoMic consortium combines projectson fungal pathogenesis, immunology, microbial ecology and’omics technologies to understand and exploit interactions between fungus, host and microbiota. Identification of novel bio markers on the fungal side such as genetic polymorphisms or on the host side such asmicrobiota profiles, metabolites and/or immune markers can lead to patient classification based on relative risk of infection. This could be the beginning of personalised management for fungal infections using preventive or therapeutic interventions like new antifungals, immune modulators or Live Biotherapeutic Products (LBPs).
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the MarieSklodowska-Curie grant agreement No 812969.
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A novel CRISPRi platform to study the role of essential genes in antifungal drug-resistant Candida albicans isolates
With the emergence of antifungal resistant Candida albicans strains, the need for new antifungal drugs is critical in combating this fungal pathogen. Investigating essential genes in C. albicans is a vital step in characterizing putative antifungal drug targets. As some of these essential genes are conserved between fungal organisms, developed therapies targeting these genes have the potential to be broad range antifungals. In order to study these essential genes, classical genetic knockout or CRISPR-based approaches cannot be used as disrupting essential genes leads to lethality in the organism. Fortunately, a variation of the CRISPR system (CRISPR interference or CRISPRi) exists that enables precise transcriptional repression of the gene of interest without introducing genetic mutations. CRISPRi utilizes an endonuclease dead Cas9 protein that can be targeted to a precise location but lacks the ability to create a double-stranded break. The binding of the dCas9 protein to DNA prevents the binding of RNA polymerase to the promoter through steric hindrance thereby reducing expression. We recently published the novel use of this technology in C. albicans and are currently working on expanding this technology to large scale repression of essential genes. Through the construction of an essential gene CRISPRi-sgRNA library, we can begin to study the function of essential genes under different conditions and identify genes that are involved in critical processes such as drug tolerance in antifungal resistant background strains. These genes can ultimately be characterized as putative targets for novel antifungal drug development, or targeted as a means to sensitize drug-resistant strains to antifungal treatment.
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Extracellular RNA as a potential activator of Neutrophil Extracellular Traps by Candida albicans biofilm
More LessNeutrophils represent the first line of innate host defense. The ability to inhibit the development of infections is associated with the involvement of several fighting strategies. The still poorly understood mechanism is netosis, involving the release of Extracellular Neutrophil Traps (NETs). NETs are complexes of chromosomal DNA and granule content. Such a web-like structure inhibits the spread of invaders.
Netosis plays a significant role in combating Candida albicans infections. It has been shown that several factors, composing C. albicans cell surface mediate NETs production. However, the development of difficult to eradicate fungal infection is associated with the formation of the biofilm structure, which partially protects the pathogen cells from contact with the host’s immune system. One of the reasons for the creation of a such protective environment is the production of the extracellular matrix (ECM). The major components of the C. albicans ECM layer are lipids, proteins, carbohydrates but also extracellular nucleic acids, among which we observed a significant RNA content.
Considering that the ECM consisting of RNA molecules is one of the first lines of contact between biofilms and neutrophils, our current studies aimed to assess the potential role of extracellular RNA in the triggering of the netosis process by human neutrophils in vitro. We showed that RNA purified from C. albicans biofilm structure and the whole cells have the capability to induction of ROS-dependent netosis pathway. Additionally, cell migration analysis indicate that RNA molecules may also be an effective chemotactic agent. This work was supported by NCN (2019/33/B/NZ6/02284).
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The impact of lemongrass, oregano, and thyme essential oils on Candida albicans’ virulence factors
More LessIncreased systemic infections and growing resistance of Candida species in immunosuppressed people have prompted research for additional treatment options. The purpose of this study was to investigate the potential of lemongrass, oregano, and thyme essential oils tested individually, combined, and combined with the antifungal agents fluconazole and caspofungin to kill Candida albicans isolates in a controlled laboratory setting. A quantitative design was used to analyze the experimental data collected and investigate risk factors related to age, gender, race, and co-morbidities. Kill rates of lemongrass, oregano, and thyme essential oils individually and combined, kill rates of fluconazole, caspofungin, and the kill rates when the antifungals were each combined with the three essential oils were compared using 117 isolates recovered from bloodstream infections between January 2009 through August 1, 2017. There were statistically significant increases in kill rates when the isolates were exposed to any of the combinations of essential oils tested. Data on the covariates age, gender, race, and co-morbidities were assessed for risk factors related to Candida albicans bloodstream infections. The age group 25-34, kidney failure, and solid organ tumor cancer, were all statistically significantly associated with an increased risk for Candida albicans bloodstream infections and multiple organ failure was negatively associated with the risk. Through the use of essential oils, the ability to reduce the number of patients becoming infected with life-threatening yeast infections could not only reduce mortality but also reduce the costs associated with serious infections.
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Recovery of Candida spp. from a denture rat model
Denture stomatitis is a common infection in denture wearers. This study evaluated the recovery of Candida spp. from the palate of Wistar rats after using an acrylic device with single and mixed-species of Candida spp. After approval of the Ethics Committee, 84 male and female Wistar rats were used. Custom-made acrylic devices were fabricated for each animal and sterilized by microwave irradiation. Single and mixed species biofilms of C. albicans (Ca), C. glabrata (Cg), and C. tropicalis (Ct) were grown on the devices for 48 h at 37°C. Rats were anesthetized and the devices were cemented on the molar teeth (n=5 for each sex and Candida spp.). Rats received a carbohydrate-rich diet. Single and mixed species were inoculated in the oral cavity thrice after three-day intervals. Controls received only dentures without Candida spp. After 4 weeks, the devices were removed, the palates were swabbed, and diluted samples were plated on Agar Sabouraud Dextrose and CHROMAgar Candida for colony counting and presumptive identification, respectively, after 48 h. Data were analyzed by 3way ANOVA (α=5%). There was a significant interaction (p=0.003) between sex and species. For females, all groups recovered significant values (p≤0.027) compared with controls. For males, groups with Ct as single and dual-species showed the lowest values without difference (p≥0.183) with the control. The groups with triple-species showed the highest values but without difference (p≥0.071) with the groups with single and dual-species, except males with Ct. Ct alone showed reduced recovery from palate of male rats.
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Molecular epidemiology of Candida auris in Qatar using whole genome sequence data
Candida auris is an emerging, multidrug resistant fungal pathogen that has become a public health threat worldwide. Candida auris spreads easily among patients within and between hospitals, and the incidence of infections has increased substantially in the last decade. Multiple C. auris outbreaks have been reported worldwide including India, USA and United Kingdom. Infections and outbreaks caused by C. auris have also been reported in the Middle East region including Kuwait, Oman, Saudi Arabia, and Qatar; however, the origin of these isolates is largely unknown. This study uses whole genome sequencing (WGS) data to determine the epidemiology and the drug resistance mutations from C. auris in Qatar. Forty samples isolated from the patients and the hospital environment were sequenced by Illumina Nextseq. Core genome SNPs revealed that all isolates belonged to the Indian lineage, which could be originated from the expatriate labour from South Asia. The genetic variability among the isolates was low but comprised of more than one genetic cluster. The environmental isolates were identical to the clinical isolates, and the isolates from patients of different hospitals/outbreaks clustered together, suggesting the transmission of C. auris could be linked to infected/colonized patients and the hospital environment. Mutations associated with azole and echinocandin resistance were discussed.
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Farnesol secretion as a possible driving force for maintaining Candida albicans as a diploid
More LessCandida albicans is a pathogenic dimorphic fungus which is invariably found as a diploid in patients. C. albicans secretes the sesquiterpene farnesol both as a quorum sensing molecule which blocks the yeast to hypha conversion and as a virulence factor for pathogenicity. 20-25 μM farnesol kills other competing yeasts and fungi, often by triggering apoptosis, and yet wild type diploid C. albicans tolerates 300-500 μM farnesol. The recent availability of 10 haploid strains of C. albicans (5 mating type aand 5 mating type α) allowed us to compare their production of and sensitivity to farnesol. On average, the heterozygous diploid strains of C. albicans were 2.4 times more resistant to 20-40 μM farnesol than MTLa haploid cells and 4.6 times more resistant than MTLα haploid cells. Furthermore, the MTLa haploids produce approximately 10 times more farnesol than do the MTLα haploids. Prior work concluded that haploid strains exhibited such low fitness that C. albicans was thought to be an obligate diploid. We now suggest that increased farnesol secretion by the MTLa haploids and increased farnesol sensitivity of the MTLα haploids is a mechanism for maintaining the dominant heterozygous diploid status of C. albicans. This idea is based on the observation that the a-factor peptide pheromone is farnesylated but the α-factor pheromone is not farnesylated. Our working hypothesis is that farnesol is secreted in part via Ste6 and imported in part via Ste3, the proteins which export and import the farnesylated a-pheromone. We also examined whether farnesol was excreted in extracellular vesicles.
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Characterization of the mechanism of action of the Enterococcus faecalis bacteriocin EntV on Candida albicans
Candida albicans shares communal niches with multiple bacterial species. Previous work from our group demonstrated that the Gram-positive bacterium Enterococcus faecalis, a normal constituent of the oral and gut microbiome that is often co-isolated with C. albicans, antagonizes hyphal morphogenesis, biofilm formation, and virulence in C. albicans. These effects are mediated by EntV, a bacteriocin and antimicrobial peptide produced by E. faecalis. The main aim of this work is to unveil the molecular mechanism behind the activity of EntV on C. albicans. Using fluorescence microscopy, we determined that EntV binds to the cell walls of several Candida species, including both yeast and hyphae of C. albicans. Contrary to other antimicrobial peptides, it does not cause cell lysis and does not synergize with cell wall damaging agents. Moreover, we screened a library of C. albicans mutants for strains with altered susceptibility to the peptide; most of the positive hits had functions related to cell wall maintenance and were further screened to ascertain changes in the staining patterns. Furthermore, to identify the target layer on the cell wall, pull-down assays were performed. Mannan was identified as the major wall component able to bind the peptide. Finally, live imaging of macrophages incubated with Candida was carried out in order to assess any change in the phagocytic behaviour in presence of the peptide. Identifying the molecular target of EntV in regard to the anti-virulence mechanisms of C. albicans is an important step in its further development as a therapeutic addition to the classical antifungal agents.
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Candida albicans cell wall mannosidases, Dfg5 and Dcw1, regulate chitin synthesis
In Candida albicans chitin synthesis is important for cell wall integrity and may also have a role in emergence of drug-resistance. Our past studies showed that cell wall mannosidases, Dfg5 and Dcw1, regulate HOG MAPK signaling. In this study, we investigated how Dfg5 and Dcw1 regulate chitin synthesis by affecting HOG, PKC and Calcium-Calcineurin signaling pathways. DFG5 and DCW1 heterologous mutants (ES1 & ES195) and a conditional mutant (ES195+methionine/cysteine) were utilized. WT SC5314 served as negative control and Hog1 knock-out mutant as positive control. Fluorescence microscopy of calcofluor white (CFW) stained mutant and control strains was performed to observe chitin accumulation. Quantitative PCR analysis was performed to measure the relative expression of chitin synthases CHS1, CHS2, CHS3 and CHS8. Incubation with chitinase was done to determine cell separation using light microscopy and scanning electron microscopy (SEM) analysis. Fluorescence microscopy showed significantly increased chitin accumulation in the mutants as compared to wild type. Chitin accumulation was observed mainly at the budding sites indicating a cause for defective cell separation phenotype. Incubation with chitinase led to cell separation in the mutants. CHS2, CHS3 and CHS8 expression was observed to be significantly upregulated in the conditional mutant and HOG1 mutant as compared to the wild type. This upregulation was also observed when the cell wall integrity PKC pathway was activated. However, activation of the Calcium-calcineurin pathway downregulated chitin synthase expression in the mutants. Our data indicates that Dfg5 and Dcw1 regulate expression of chitin synthases via HOG MAPK, PKC and Calcium-calcineurin signaling pathways.
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Liposomes functionalized with fungi-targeting peptide demonstrate increased interaction with Candida albicans
More LessCandida albicans infections can be challenging to treat, as current antifungal drugs exhibit poor water solubility and host toxicity. To overcome these issues, new methods of drug delivery are needed. Liposomes have been shown to be an effective method for administrating antifungals and can increase bioavailability and solubility while decreasing toxicity. However, existing antifungal liposomal formulations lack infection specificity. For example, AmBisome, a liposomal formulation of amphotericin B, relies on passive accumulation to infection sites. We have developed antifungal liposomes that display fungi-targeting moieties to promote interaction with Candida;we predict that these formulations can increase fungal eradication and decrease off-site toxicity. Here, the C. albicans-targeting peptide P-113Q2.10 (AQRHHGYKRQFH), a derivative of the antifungal peptide histatin 5, was incorporated into liposomes via conjugation to palmitic acid (PA). PA-P-113Q2.10 conjugates were synthesized using solid phase peptide synthesis, confirmed by liquid-chromatography-mass spectrometry. Liposomes composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine and cholesterol with 1% w/w PA-P-113Q2.10 were formed via thin film-hydration and extrusion, yielding ∼100 nm liposomes with a polydispersity index of ∼0.1. Flow cytometry demonstrated that interaction with C. albicans SC5314 was enhanced for P-113Q2.10 liposomes, increasing from ∼60% in cells incubated with liposomes lacking peptide to ∼79%. These liposomes preferentially interact with C. albicans compared to NIH 3T3 murine fibroblasts; on average, only ∼15% of fibroblasts incubated with liposomes (with and without peptide) showed positive liposome interaction. This liposome formulation has the potential to serve as an antifungal delivery platform that selectively targets C. albicans cells for increased efficacy in treatment of fungal infections.
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Candida auris gene expression: modulation upon caspofungin treatment
More LessCandida auris has emerged as a serious worldwide threat by causing invasive infections in humans that are frequently resistant to one or more conventional antifungal medications, resulting in high mortality rates. Against this backdrop, health warnings around the world have focused efforts on understanding C. auris fungal biology and effective treatment approaches to combat this fungus. To date, there is little information about C. auris gene expression regulation in response to antifungal treatment. Our integrated analyses focused on the comparative transcriptomics of C. auris in the presence and absence of caspofungin as well as a detailed analysis of the yeast's extracellular vesicle (EV)-RNA composition. The results showed that genes coding oxidative stress response, ribosomal proteins, cell wall, and cell cycle were significantly up-regulated in the presence of caspofungin, whereas transcriptional regulators and proteins related to nucleus were down-regulated. The mRNAs in the EVswere associated with the stress responses induced by caspofungin and the ncRNA content of the EVs shifted during caspofungin treatment. Altogether, the results provide further insights into the fungal response to caspofungin and demonstrate that analyses of C. auris growth under antifungal stress can elucidate resistance and survival mechanisms of this fungus in response to medical therapy.
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CO2 accelerates Candida albicans biofilm formation and reveals novel approaches to their inhibition on airway management devices
More LessC. albicans is the predominant human fungal pathogen worldwide and frequently colonises medical devices, such as voice prosthesis, as a biofilm. It is a dimorphic yeast that can switch between yeast and hyphal forms in response to environmental cues, a property that is essential during biofilm establishment and maturation. One such cue is elevation of CO2 levels, as observed in exhaled breath for example. However, despite the clear medical relevance the effects of high CO2 levels on C. albicans biofilm growth has not been investigated to date. Here, we show that 5% CO2 significantly enhances each stage of the C. albicans biofilm forming process; from attachment through maturation to dispersion, via stimulation of the Ras/cAMP/PKA signalling pathway. Transcriptome analysis of biofilm formation under elevated CO2 conditions revealed the activation of key biofilm formation pathways governed by the central biofilm regulators Efg1, Brg1, Bcr1 and Ndt80. Biofilms grown in under elevated CO2 conditions also exhibit increases in azole resistance, tolerance to nutritional immunity and enhanced glucose uptake capabilities. We thus characterise the mechanisms by which elevated CO2 promote C. albicans biofilm formation. We also investigate the possibility of re-purposing drugs that can target the CO2 activated metabolic enhancements observed in C. albicans biofilms. Using this approach we can significantly reduce multi-species biofilm formation in a high CO2 environment and demonstrate a significant extension of the lifespan of voice prostheses in a patient trial. Our research demonstrates a bench to bedside approach to tackle Candida albicans biofilm formation.
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Investigation of the zinc uptake system of the human fungal pathogen Candida parapsilosis
More LessCandida parapsilosis is the second or third most commonly isolated Candida species from blood cultures and is frequently associated with infections in neonatal intensive care units. Candida species have several virulence factors enabling them to adapt to host environmental conditions and cause infections. These factors include adhesion, biofilm formation, and secretion of hydrolytic enzymes, such as acidic proteinases and lipases. Candida species also obtain heavy metal ions from their environment, such as zinc. Zinc is a cofactor of several proteins and a vital element in cellular mechanisms of the fungi. On the one hand, the host niche represents a zinc-limited environment, that indirectly inhibits microbial growth. In order to survive in such an environment, these pathogens have evolved a zinc transport system that allows them to access bound zinc ions during infection. On the other hand, high zinc ion concentration within the host can also be toxic to microbes e.g. in the phagosomes of Mycobacterium tuberculosis infected macrophages. In case of C. albicans, zinc acquisition processes are intensively studied, but we lack information of the zinc uptake, transfer and homeostasis mechanisms in C. parapsilosis. Here, predicted potential zinc transporters in C. parapsilosis using in silico analyses, generated homozygous knock out mutants and performed their phenotypical characterization by exposing them to various types of stressors and zinc limiting conditions. Furthermore, we analyzed their virulence traits by examining kinetics of fungal cell uptake by macrophages, their killing efficiency and also investigated zinc ion levels in the phagolysosome during in vitro infections
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Genetic analysis of sirtuin deacetylases in hyphal growth of Candida albicans
More LessCandida albicans is a major human fungal pathogen that encounters varied host environments during infection. In response to environmental cues, C. albicans switches between ovoid yeast and elongated hyphal growth forms, and this morphological plasticity contributes to virulence. Environmental changes that alter the cell’s metabolic state could be sensed by sirtuins, which are NAD+-dependent deacetylases. Here we studied the roles of three sirtuin deacetylases, Sir2, Hst1, and Hst2, in hyphal growth of C. albicans. We made single, double, and triple sirtuin knockout strains and tested their ability to switch from yeast to hyphae. We found that true hyphae formation was significantly reduced by the deletion of SIR2 but not HST1 or HST2. Moreover, the expression of hyphal-specific genes HWP1, ALS3, and ECE1 decreased in the sir2Δ/Δ mutant compared to wild-type. This regulation of hyphae formation was dependent on the deacetylase activity of Sir2, as a point mutant lacking deacetylase activity had a similar defect in hyphae formation as the sir2Δ/Δ mutant. Finally, we found that Sir2 and Hst1 were localized to the nucleus, with Sir2 specifically focused in the nucleolus. This nuclear localization suggests a role for Sir2 and Hst1 in regulating gene expression. In contrast, Hst2 was localized to the cytoplasm. In conclusion, our results suggest that Sir2 plays a critical and non-redundant role in hyphal growth of C. albicans.
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Genotypic and phenotypic portrait of Candida albicans clinical isolates colonizing the airways of patients with cystic fibrosis
Candida albicans colonizes the respiratory tract of patients with Cystic Fibrosis (CF). It competes with CF-associated pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, and contributes to disease severity. We serially recovered 160 C. albicans clinical isolates over a period of 30 months from the sputum of 23 pediatric and 2 adult antifungal-naive CF patients at Children’s Hospital Tunis and characterized the genotype and phenotype of a subset of strains using multilocus sequence typing (MLST) and growth assays on multiple stress-, filamentous growth- and biofilm-inducing media. Out of 16 patients regularly sampled for at least 9 months, 8 and 4 were chronically and transiently colonized with C. albicans, respectively. MLST analyses of 56 strains originating from 15 patients indicated that each patient was colonized with a single strain, while 8 patients (53%) carried isolates from clade 4 known to be enriched with strains from Middle East-Africa. A subset of these isolates with the same sequence type and colonizing 3 unrelated patients displayed altered susceptibility to cell wall-perturbing agents, suggesting changes in cell wall structure/function during growth in the CF lung. We also observed differential ability to filament and/or form biofilms in a set of identical isolates from clade 10 sampled over a period of 9 months in a pediatric CF patient, suggesting alterations in phenotypes associated with virulence. Our findings will rely on future whole-genome sequencing analyses to identify polymorphisms that could explain the emergence of new traits in C. albicans strains thriving in the CF host environment.
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Investigation of the MFS drug transporter family in Candida parapsilosis using CRISPR-Cas9
More LessThe function of specific transporters is a key feature underlying drug resistance in Candida species. Drug transporters fall into two main classes – ATP-binding cassette (ABC) transporters, and the major facilitator superfamily (MFS) transporters. Some members of the drug/H (+) antiporter (DHA1) of the MFS superfamily have been shown to function as multidrug transporters. We targeted 16 genes belonging to five families that compose one branch of the DHA1 transporter group. These include MDR1/FLR1, associated with multidrug resistance in C. albicans (3 members); TPO4, associated with polyamine transport (1 member); NAG3/4, associated with transport of N-acetyl glucosamine (2 members); TPO2/3, associated with polyamine transport (1 member); and TPO1/FLU1, possibly associated with fluconazole resistance (9 members). We used CRISPR-Ca9 based gene editing to explore the function of of the five families in C. parapsilosis.
All 16 members were individually disrupted by introducing stop codons in the first third of the open reading frames (editing), or by deleting the whole gene. In addition, members of each family were disrupted together, including all 9 members of the TPO1/FLU1 family. CPAR2_603010, CPAR2_207540, and CPAR2_301760 all belonged to the MDR1 family. Editing CPAR2_603010 conferred sensitivity to fluconazole and voriconazole, though disrupting the other two genes had no effect. The azole sensitivity of the CPAR2_603010 edited strain was reverted by introducing the wild type sequence. Disrupting CPAR2_603010 or CPAR2_301760 individually did not affect sensitivity to 4-nitroquinoline 1-oxide. However, the double disruptant was sensitive. Disrupting CPAR2_300760, a member of the TPO1/FLU1 family, resulted in sensitivity to mycophenolic acid. Whole genome sequencing analysis of a strain in which all nine TPO1 genes were disrupted revealed that few off-target effects introduced by the CRISPR-Cas9 system, as few unexpected changes were found after eight rounds of transformation.
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Short peptides derived from EntV inhibit virulence and biofilm formation in Candida albicans
More LessCandida albicans exists as a member of the commensal flora of the skin and gut where many complex polymicrobial interactions occur with genera such as Pseudomonas, Staphylococcus, and Streptococcus. Some of these interactions potentiate or inhibit virulence. The bacterial gastrointestinal commensal speciesEnterococcus faecalisproduces a small peptide, EntV, that modulates C. albicans virulence. The active 68 amino acid EntV peptide inhibits biofilm formationin vitro; biofilm-related infections are difficult to treat with current therapeutics. EntV also attenuates fungal virulence in a Caenorhabditis elegansinfection model and a murine oral candidiasis model. We sought to identify the regions of EntV responsible for the anti-fungal activity, and based on structural information, we hypothesized that it could be localized to a single helix of the mature peptide. In this study, we report that smaller peptides derived from this helix ranging from 12 to 16 amino acids have equal to improved efficacy in inhibiting C. albicans virulence andbiofilm formation. These smaller peptides attenuate virulence in the C. elegans infection model, inhibit initial adhesion to abiotic surfaces, and reduce the size of mature biofilms measured by confocal microscopy. Further trimming of these peptides to fewer than 11 amino acids reduces and eventually eliminates activity. These data indicate that EntV-derived peptides warrant further investigation as potential non-fungicidal additives to medical devices and antifungal therapeutics.
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Complex sphingolipids: Vital determinants of drug susceptibility, membrane integrity and pathogenesis of Candida glabrata
Complex Sphingolipids (SLs) are unique to fungi, which apart from being novel drug targets, also appear to act as molecular signals, in diverse biological processes. In this study, we have specifically blocked the key synthesis step of SLs metabolism by disruption of the uncharacterized CgIPT1 gene, which based on homology with other Candida spp., predicted to mediate the conversion of MIPC to M(IP)2C. We followed fusion based PCR homologous recombination method for IPT1 deletion by using dominant markerNAT1. The knockout was selected on a nourseothricin drug plate and confirmed by gene specific PCR and by checking M(IP)2C levels. We observed that the specific accumulation of MIPC or lack of M(IP)2C in C. glabrata displayed increased susceptibility to both imidazole’s (ketoconazole, miconazole and clotrimazole) and triazoles (fluconazole, itraconazole and posaconazole). RNA Sequencing of Cgipt1Δcells revealed no major impact on of expression levels of common MDR determinants albeit a distinct imbalances in expression of lipid homeostasis genes was evident. The Fluorescence Recovery after Photobleaching (FRAP) experiments confirmed that plasma membrane in Cgipt1Δ cells display a reduction in micro-viscosity leading to increase in drug diffusion and susceptibility of Cgipt1Δcells. Interestingly, the Cgipt1Δ also exhibit attenuated virulence in a murine model. Together, our data confirms the relevance of M(IP)2C in governing drug susceptibility and virulence in C. glabrata.
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Precise editing using CRISPR-Cas9 to explore the contribution of clinically-derived mutations to antifungal resistance in the pathogenic yeast Candida parapsilosis
More LessIntroductionCandida parapsilosis is both a commensal/saprophytic yeast of the human skin and an opportunistic pathogen which can be responsible for life-threatening infections. The increasing reports of clonal outbreaks involving azole-resistant C. parapsilosis in the clinical setting is worrisome and urges for a better understanding of antifungal resistance in this species. Previous studies have identified mutations in key genes which can explain acquired fluconazole resistance. Reverse genetics approaches are now warranted to confirm their involvement and to determine whether they can affect other clinically-licensed antifungals. Here, we used a CRISPR-Cas9 technique to study the relative contributions of clinically-derived mutations to antifungal resistance and provide answers to these questions.
Materials and MethodsSix clinically-derived mutations were selected (ERG11Y132F, ERG11K143R,ERG11R398I, TAC1G650E, MRR1G583R, ERG3G111R) to be engineered in two C. parapsilosis fluconazole-susceptible backgrounds (ATCC22019, STZ5) using a previously described CRISPR-Cas9 method. In vitro susceptibility of the transformants to fluconazole, voriconazole, posaconazole, isavuconazole and micafungin was determined by Etest®.
Results/DiscussionThe impact on fluconazole susceptibility was highly variable depending on the residue/gene involved, but roughly similar between the two genetic backgrounds. All but two(ERG11R398I, ERG3G111R) conferred fluconazole resistance, though the highest MIC increase was observed for MRR1G583R (≥650 fold). As expected in a diploid species, we noted an impact of allelic dosage. Some kind of cross-resistance to the other azoles was noted from some mutations, although the impact was lower for posaconazole and isavuconazole, except for MRR1G583R which led to multi-azole resistance. Finally, ERG3G111R increased tolerance to both azoles and echinocandins.
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Conducting genetic interaction analysis with CRISPR-Cas9-based strategies in Candida albicans
More LessCandida albicans is an opportunistic fungal pathogen found in the oral mucosa, the gut, the vaginal mucosa, and humans' skin. While C. albicans can cause superficial infections, severe invasive infections can occur in immunocompromised individuals. Understanding the survival mechanisms and pathogenesis of C. albicans is critical for novel antifungal drug discovery. Determining the relationships between different genes can create a genetic interaction map, which can identify complementary gene sets, central to C. albicans survival, as potential drug targets in combination therapy. A genetic approach using the CRISPR-Cas9-based genome editing platform will focus on genetic interaction analysis of C. albicans stress response genes. The ultimate goal is to create a stress response gene deletion library to study its pathogen survival role. This library of single and double stress response gene mutants will be screened under diverse growth conditions to assess their relative fitness. Genetic interaction analysis will help map out epistatic interactions between fungal genes involved in growth, survival, and pathogenesis and uncover putative targets for combination antifungal therapy based on negative or synthetic lethal genetic interactions.
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Discovering the chlamydospore regulatory network in Candida albicans
More LessA normal resident of healthy humans and warm-blooded animals, C. albicans is a commensal fungus that is also among the most common opportunistic pathogens of humans. C. albicans forms unique morphological structures called chlamydospores, which are large, spherical, thick-walled structures formed at the ends of hyphae that have unknown biological function. My goal is to discover the regulatory network controlling chlamydospore formation in C. albicans. By determining this network, we can gain insight into the biological roles of chlamydospores in the C. albicans lifestyle, better understand C. albicans morphological transitions, and determine the selective advantage (if any) provided by chlamydospores to this pathogenic fungus. To determine this regulatory network, I have screened a library of 211 C. albicans transcription factor (TF) homozygous deletion mutants to assay for their abilities to form chlamydospores under standard chlamydospore-inducing growth conditions. I have identified seven TF mutants that fail to produce any chlamydospores andthree TF mutants that produce high levels of chlamydospores relative to WT. To characterize the transcriptional changes occurring during chlamydospore formation, I have performed RNA sequencing (RNA-seq) on these identified regulator mutants to uncover the differentially regulated target genes of each chlamydospore regulator. I will use genome-wide chromatin immunoprecipitation followed by sequencing (ChIP-seq) on epitope-tagged versions of these regulators to determine which genes are directly under the control of each TF. RNA-seq coupled with ChIP-seq will allow me to determine the regulatory network controlling chlamydospore formation in C. albicans.
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Evaluation of antifungal activity of Terminalia subspathulata bark and fruit extracts against azole-resistant Candida isolates
More LessBackground:Emerging resistant Candida species become a severe problem for public health. The study aims to examine the efficacy of Terminalia subspathulata bark and fruit extracts as an alternative source of antifungals for resistant strains of Candida species.
Methodology:Candida albicans was isolated between 4–5th interdigital foot space and C. parapsilosis were isolated from the left-hand third finger of male patients. Morphological and molecular methods identified the isolates. Antifungal susceptibility testing (AFST) was determined using azole derivatives, amphotericin B (AmpB) and echinocandins based on E-test and broth microdilution (BMD) methods. The AFST activity of the 10 μL methanolic extracts with concentrations between 1.25–10 mg/mL was assessed by disk diffusion method according to CLSI guidelines.
Results:The AFST results showed total resistance to all azoles (FLU, MIC ≥ 256 μg/ml and VOR, ITR and POS each with MIC ≥ 32 μg/ml). The BMD method agreed with E-test MICs. The T. subspathulata bark extract tested indicated activity on the C. albicans lawn with the zone of inhibition between 9–13 mm. The fruit extract indicated zone between 8–10 mm. Candida parapsilosis indicated susceptibility to bark extract with the zone of inhibition between 10–15 mm, while the fruit extract showed zone between 8–13 mm.
Conclusion:Despite azole resistance, the extracts showed good activity against the isolates. Findings showed alternatives to AmpB and echinocandins because of toxicity and parenteral administration, respectively. Based on our findings, our study first reports the medicinal value of T. subspathulata.
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Drug tolerance facilitates the evolution of drug resistance in Candida albicans
BackgroundFor Candida albicans and Candidiasis, drug resistance is sometimes due to the pre-existence of genetic polymorphisms that bypass the mode of action of the drug, thus conferring a long-term survival benefit. In other cases, resistance is acquired via the evolution of de novo genetic polymorphisms. There is evidence that C. albicans possess a drug tolerance response which “buys time” for individuals to evolve beneficial mutations. Our goal here is to characterize this poorly understood epigenetic cytoprotective program at the single cell molecular level.
MethodsWe developed a nano-litre droplet based Candida single cell sequencing platform capable of transcriptionally profiling several thousand individual cells in an efficient manner. We exploit this platform to profile both untreated and drug exposed (incl. fluconazole, caspofungin and nystatin) populations at early time points post-treatment (tolerance) and late time points (resistance) in order to understand survival trajectories. The profile are compared with the matched sequenced genomes.
ResultsWe show that untreated Candida populations exhibit “bet hedging”, stochastically expressing cytoprotective transcriptional programs, and drug tolerant individuals partition into distinct subpopulations, each with a unique survival strategy involving different transcriptional programs. We observe a burst of chromosomal aberrations at two days post-treatment that differ between survivor subpopulation.
DiscussionOur single cell approach highlights that survivor subpopulations pass through a tolerance phase that involves a multivariate transcriptional response including upregulation of efflux pumps, chaperones and transport mechanisms, and cell wall maintenance. Together this suggests that targeting the tolerance response concomitantly with standard therapies could represent an efficient approach to ablating clinical persistence.
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In silico screening leads to novel scaffolds with both antifungal and anti-NLRP3 inflammasome activity
More LessDue to structural similarities that exist between established inhibitors of the NLRP3-inflammasome, sulfonylureas Glyburide and MCC-950, and herbicidal-sulfonylureas, that specifically target fungal acetohydroxyacid synthase (AHAS), we sought to determine the potential for compounds to block both inflammation and inhibit fungal growth.
In silico screening of ∼250,000 compounds was used to identify a prioritized list of chemical structures capable of inhibiting both targets. Prioritization of the top 1% of scores identified ∼70 compounds with a diverse set of scaffolds for testing in vitro. Selected hits were used to assess anti-inflammatory function in a THP-1 challenge model with LPS+ATP and resulting IC50 values were obtained. MIC and hyphal-growth assays were conducted to determine potential antifungal activity using media depleted of branched chain amino acids isoleucine and valine, to confirm on target AHAS inhibition.
Identification of hits that exhibited low micromolar activity for NLRP3 and AHAS inhibition were selected for SAR study. In vitro testing of the analogs along with molecular docking led to increased knowledge for lead optimization of the potential hits.
In silico screening has resulted in IC50 (IL-1β release) and MIC50 (fungal growth) values with low μM potency against several Candida species. In vivo validation will further confirm the potential of the scaffolds for further synthetic-modification for the rationale design of novel dual-purpose drugs
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Investigation of the interaction of keratinocytes and Candida species
More LessOur skin provides immunological protection against several pathogens. Skin epithelial cells respond to microbial stimuli in various ways, such as through the production of antimicrobial peptides or secretion of cytokines, although phagocytosis of potentially evading microbes was also reported.
Relatively little is known about how skin keratinocytes differentiate between the presence of pathogenic and commensal fungi. In this project, we aimed to investigate how human keratinocytes interact with different Candida species, as common colonizers of the skin. While C. albicans is a common cause of cutaneous candidiasis, C. parapsilosisis rarely associated with this disease.For the experimentshuman skin keratinocyte cell lines (HaCaT, HPV-KER)were applied andchallengedwith C. albicans (SC5314 and WO1 strains) and C. parapsilosis (GA1 and CLIB214 strains)strains.We aimedto determine the extent to which C. albicans and C. parapsilosis damage human keratinocytes, their attachment to host cells, the keratinocytes’ ability to internalize these fungi and to examinecytokine production in response to stimuli.
Our results suggest that C. albicans causes significantly more damage to human keratinocytes than C. parapsilosis and the HPV-KER cell line was more susceptibleto the infection. In both HaCaT and HPV-KER cells, the production of IL-6, IL-8, and CCL5 increased primarilyafter C. albicans infection. Based on the adhesion studies, there was a low degree of association in case of C. parapsilosis GA1 and CLIB214 compared to C. albicans SC5314 and WO1.
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Identification and characterization of a fungal-selective glutaminyl tRNA synthetase inhibitor with potent activity against Candida albicans
Candida albicans is the leading cause of systemic candidiasis. Effective treatment is threatened by a dearth of antifungal options and the emergence of resistance. Thus, there is an urgent need to identify novel therapeutic targets to expand our antifungal armamentarium. A promising approach is the discovery of essential genes, as most antimicrobials target essential bioprocesses. Despite detailed characterization of gene essentiality in Saccharomyces cerevisiae,defining essential targets in the pathogen of interest is necessary due to the high level of divergence between these organisms. Thus, using a machine learning algorithm we generated a comprehensive prediction of all genes essential in C. albicans. We leveraged our essentiality predictions with high-throughput screening and chemogenomic datasets to assign the mechanism of action of a previously uncharacterized compound. We identified T-035897 as a molecule with potent bioactivity against C. albicans. Prior chemogenomic profiling in S. cerevisiae suggested that T-035897 targets the glutaminyl tRNA synthetase Gln4, whose homolog in C. albicans was predicted and verified to be required for viability. To confirm the mechanism of T-035897 in C. albicans, we performed haploinsufficiency profiling,which supported Gln4as the target. In parallel, selection of resistant mutants and targeted sequencing uncovered substitutions in the Gln4 catalytic domain. Moreover, T-035897 inhibited translation in afluorescence-based reporter assay. Finally, T-035897 selectively abrogated fungal cell growth in a co-culture model with mammalian cells. Thus, we highlight the power of leveraging essentiality datasets in order to characterize compounds with potent antifungal activity in an effort to unveil novel therapeutic strategies.
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Role of SAGA complex subunits in gene regulation of Candida albicans
More LessThe SAGA (Spt-Ada-Gcn5-acetyltransferase) is an evolutionary conserved multidomain co-activator complex involved in gene regulation through its histone acetyltransferase (HAT) and deubiquitinase (DUB) functions. It is well studied in Saccharomyces cerevisiae, and recent reports from humans and Drosophila expand its importance from gene transcription regulation to transcription elongation, protein stability and telomere maintenance. In Candida albicans, little is known about the components of the SAGA complex and their influence in morphogenesis and stress response. In this work, we analysed individual components of the SAGA complex, their role in morphogenesis and responses to different signalling cues. We initially analysed conditionally repressed strains of SAGA complex subunits involved in the HAT function of the complex: Tra1, Ngg1, Spt7, Spt8, Taf5, Taf6, Taf9, and Taf10. It appears that the Tra1 might be essential for the viability of C. albicans, as we failed to obtain homozygous deletions although it showed detectible growth in the conditionally repressed strain. Also, we observed that TBP- associated factors are essential in C. albicans, possibly due to their role in the transcription initiation factor TFIID instead of SAGA. We also detected that the Spt8 repressed mutant was extensively invasive in YPD at 300C while a repressed Ngg1 was considerably less invasive compared to its wild type. Also, we have seen that the mutations affecting TBP-binding ability confer susceptibility to drugs, temperature, osmotic, oxidative and DNA damage stress. Further, it seems that the modules of SAGA complex might have antagonistic roles in expression regulation but this needs more in-depth study.
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A variant ECE1 allele contributes to reduced pathogenicity of Candida albicans during vulvovaginal candidiasis via altered secretion of candidalysin
More LessBackground:Candida albicans is the primary etiological agent of vulvovaginal candidiasis (VVC) and exerts its pathogenicity through secretion of the peptide toxin candidalysin encoded by the ECE1 gene. A highly conserved variant ECE1 sequence exists across a diverse set of clinical isolates. Thus, we sought to determine the relative pathogenicity and mechanism(s) associated with this alternative ECE1 allele.
Methods:Isogenic strains harboring WT or variant ECE1 sequences were engineered in an Δ/Δece1 background. After confirmation of equivalent expression by qPCR, pathogenicity of strains were tested using in vitro epithelial cell and in vivo VVC models of infection and LDH, IL-1β, neutrophil levels monitored. Follow up studies using synthetic candidalysin peptide were also performed. Lastly, a panel of ECE1 chimeras were constructed to assess potential processing defects and detected by a novel HiBiT-tagging approach.
Results:Strains transformed with either the variant full length ECE1 or candidalysin allele, as compared to the WT sequence, demonstrated significantly reduced immunopathogenicity during in vitro or in vivo infection despite equivalent fungal burden. Interestingly, epithelial challenge with WT or variant synthetic peptide revealed similar capacity to elicit damage and IL-1β. Allele profiling and ECE1 chimera experiments demonstrated that defects in pathogenicity are at least partly due to inefficient ECE1 processing at the peptide 2-peptide 3 junction.
Discussion:The ECE1 gene displays conserved polymorphisms that alter candidalysin secretion and strain pathogenicity. Future work is focused on determining specific amino acid sequences that contribute to these affects across clinical isolates and disease states.
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ID-CARD: A clade-specific molecular assay for the detection of 25 Candida spp. causing candidemia based on antifungal susceptibility patterns and in vitro testing of the antifungal activity of a synthetic antimicrobial peptide derived from human lactoferrin (hLF1-11).
More LessFungal infections are a serious health concern affecting over 1.5 million individuals annually. ID-CARD aims to improve diagnostics taking into account phylogeny and antifungal susceptibility patterns of Candida spp. involved in candidemia.Twenty-five Candida spp. were chosen. Based on ribosomal DNA sequences, clade-specific primers/Taqman probes were designed. Different multiplex panels consisting of four clades that exhibited similar antifungal susceptibility profiles were created. To create the groups, we tested fluconazole and anidulafungin with broth microdilution according to EUCAST against 3-5 isolates/species (n=121), which were also used for specificity testing of the molecular assay. Furthermore, we tested the in vitro activity of hLF(1-11) peptide against isolates that exhibited elevated minimum inhibitory concentrations (MICs) for one or both of the drugs. The groups created are : i. Lodderomyces, Kluyveromyces, Metschnikowiaceae Sensitive, Internal control, (all with low MICs) ii. Pichiaceae, Nakaseomyces, Wickerhamomycetaceae, Debaryomyces & Diutina, (all with high MICs to azoles) and iii. Yarrowia, Wickerhamiella & Meyerozyma, Candida auris, Candida haemulonii complex (all with high MICs to both azoles & echinocandins). The primers/probes showed 100% specificity and capacity for multiplexing. In vitro experiments indicated that hLF(1-11) is fungicidal against various Candidaspp. A synergistic effect of antifungal and hLF(1-11) against various Candida species was shown as combinations of the peptide with antifungals were more effective than these alone ID-CARD will contribute to a fast and reliable molecular detection of yeasts involved in candidiasis. AMPs is a novel way to treat Candida spp. exhibiting high MICs to commonly used antifungal drugs.
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A systemic analysis of amino acid transporters identifies Gnp2 as the main proline permease in Candida albicans
More LessThe tight association of Candida albicans with the human host has driven the evolution of mechanisms that permit metabolic flexibility. Amino acids, present in free form or peptide bound, are an abundant carbon and nitrogen source in many host niches. Further,the capacity to sense and utilize certain amino acids, like proline, is directly linked to virulence. The C. albicans genome encodes for at least 24 amino acid permeases (AAPs), highlighting the importance of flexible amino acid uptake for fungal growth and virulence. Although the substrate specificity and role of certain AAPs has been investigated, a comprehensive characterization was missing. Therefore, we assembled a library of AAP deletion strains, which was tested for resistance to toxic amino acid analogs. Most striking was the specific resistance of gnp2Δ to the proline analog 3,4-dehydroproline. Subsequent tests validated that Gnp2 is a specific proline permease in C. albicans, which is contrary to the model yeast Saccharomyces cerevisiae where proline transport is mediated by four permeases. Furthermore, the induction of GNP2 appears to be independent of the SPS (Ssy1-Ptr3-Ssy5) regulatory pathway that controls proline utilization in the model yeast, pointing towards rewired proline uptake in C. albicans. Additionally, strains lacking GNP2were unable to respond to proline-induced filamentation, displayed decreased cytotoxicity to macrophages and showed increased sensitivity to oxidative stress, underlining the importance of proline uptake for fungal virulence. Taken together, the role of Gnp2-mediated proline uptake illustrates the importance of metabolism-driven virulence in C. albicans.
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The microbial ecology of Candida albicans strains CHN1 and SC5314 in mice
More LessStrain SC5314 is the most widely studied strain of Candida albicans. Despite C. albicans being the most commonly isolated yeast from the human gastrointestinal (GI) microbiome, strain SC5314 does not stably colonize the mouse GI tract long term, even after antibiotic disruption. In contrast, strain CHN1 will stably colonize the mouse GI tract long term. Comparative genomic analysis of strain CHN1 indicates that it belongs to a different evolutionary clade of C. albicans than strain SC5314. Previous studies from our laboratory have shown that colonization by strain CHN1 causes a change in the GI bacterial microbiome of mice and predisposes them to more robust Th2 immune responses. Despite this, little is known about the GI microbial ecology of SC5314 vs. CHN1 and subsequent host responses. Using a short-term antibiotic disruption model in C57BL/6 mice, we have been able to observe significantly different colonization kinetics between these two C. albicans strains, with CHN1 establishing stable long-term colonization. In contrast, colonization by SC5314 was lower, highly variable and cage-dependent. C. albicans colonization kinetics impacted the composition of the bacterial microbiome with a marked effect on the levels of Lactobacillus and Enterococcus. qPCR analysis of 46 host immune response genes did not detect significant differences in host gene expression between SC5134 and CHN1 colonized mice, except for chitinase expression. Thus, these studies suggest that yeast-bacteria interactions in the microbiome may be far more important in determining long-term colonization potential of C. albicans and secondary immunomodulatory effects.
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Tell me what type of extracellular vesicles you secrete, and I will tell you who you are: yeast or hypha
The transition between yeast and hyphal morphologies plays a crucial role in the pathogenicity of Candida albicans. Recent studies have pointed out the great relevance of extracellular vesicles (EVs) secreted by microorganisms in a wide variety of biological processes including interaction with the host. Therefore, the main objective of this work was to compare the EVs secreted by yeast and hyphal forms to shed light on C. albicans-host interaction.
EVs were obtained by ultracentrifugation of the culture medium supernatant and analysed by mass spectrometry. They were characterized by transmission electronic microscopy (TEM) and dynamic light scattering (DLS).
DLS and TEM analysis showed that yeast EVs were significantly bigger than hyphal EVs, being most of them in the range between 400 to 500nm while hyphal EVs were ranged mostly around 100-200nm.
Proteomic analysis showed greater protein diversity in hyphal EVs when compared to yeast EVs (up to 1700 different proteins identified versus 300), although less amount of total protein was obtained. Gene Ontology (GO) analysis showed that yeast EVs were enriched in surface proteins while hyphal EVs, although containing also most of these surface proteins, were also significantly and exclusively enriched in proteins involved in protein metabolism (ribosomal proteins, many aminoacid-pathway enzymes and proteasome) and cellular transport. The differences between YEVs and HEVs also prompted a different immune host response, as tested with macrophage cell cultures and human sera from patients with invasive candidiasis.
All these differences point out a possible different biogenesis and roles of EVs secreted by both morphologies.
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Candida glabrata MSH2 deletion increases antifungal tolerance in vitro and during intra-abdominal candidiasis (IAC), it does not impact pathogenesis of peritonitis or intra-abdominal abscesses
More LessBackground:IAC is the second most common type of invasive Candidiasis, but its pathogenesis is poorly understood. We have shown that Candida albicans DNA damage response genes are strongly induced within intra-abdominal abscesses. Deletion of C. glabrata MSH2, A DNA mismatch repair (MMR) gene, results in a mutator phenotype that facilitates multidrug resistance in vitro and in mouse gastrointestinal tracts. Our goal was to determine if CGMSH2 Contributed to pathogenesis or resistance to the new antifungal rezafungin during IAC.
Methods:We createdΔMSH2 in BG2 using SAT-Flipper, and tested virulence and rezafungin responses in a mouse model of IAC.
Results:ΔMSH2 displayed no growth defects at 30°C in liquid (YPD, Ypglycerol) or solid media (YPD+0.02% MMS, 1MM H2O2, 1M NACL, 20 UG/ML CW, 250 UG/ML OR 0.02% SDS). ΔMSH2 longevity in YPD was comparable to BG2. Caspofungin-, Rezafungin- and Fluconazole-resistant mutants arose 24-, 16- and 3-fold more often, respectively, for ΔMSH2 than BG2 (108-106 CFU overnight in YPD, selected on 8XMIC-Containing plates). However, respective minimum inhibitory concentrations (MICS) were not different, nor were rezafungin time-kills.ΔMSH2 was comparable to BG2 in peritonitis and abscess burdens in mouse IAC.ΔMSH2 demonstrated significantly greater caspofungin- and fluconazole-tolerance than BG2 in abscesses. Rezafungin reduced peritonitis and abscess burdens ofΔMSH2,BG2 ANDFKS mutant strains to similar extents.
Conclusions:CgMSH2 deletionincreased the frequency of spontaneously-arising echinocandin- and fluconazole-resistant colonies in vitro and tolerance in intra-abdominal abscesses, but it did not attenuate virulence or rezafungin responses during IAC.
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Ambisome shows potent anti-Candida auris in vitro and in vivo activity
More LessCandida auris is an emerging multidrug resistant Candida species that has been reported in many parts of the world for causing severe illness in hospitalized patients especially bloodstream infections. The challenge with this type of yeast is its resistance to commonly used antifungal drugs, thus identifying antifungals that are effective against this species is critical.
In this study, we determined the in vitro activity of Ambisome against 35 clinical isolates of C. auris using minimum inhibitory concentration (MIC) assay as well as the efficacy of Ambisome compared to Amphotericin B and fluconazole using a C. auris murine disseminated model.
Antifungal activity of C. auris against Ambisome and comparators was assessed using amicrodilution method performed according to the Clinical and Laboratory Standard Institute (CLSI) M27-A4 methodology. Mice were immunocompromised and challenged with 3 x 107 C. auris blastopores in 0.1 ml of normal saline (via the tail vein). Treatment efficacy was assessed by determining reductions in mortality as well as decrease in tissue fungal burden (CFUs).
Ambisome showed lower MIC50 and MIC90 values (1 and 2 μg/mL, respectively) than the comparators tested. Significant efficacy was observed in the Ambisome 7.5 mg/kg -treated group (100% and 90% survival by day 7- and 14-days post inoculation, respectively). Additionally, Ambisome and fluconazole treated groups showed significant reduction in CFUs in the kidneys (P- values of 0.028 and 0.022, respectively) compared to the untreated group.
Our data shows that Ambisome shows significant antifungal activity against C. auris in vitro as well as in vivo.
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Farnesol abrogates biofilm- and efflux pump- associated genes in drug resistant Candida auris strains
More LessBackground:Candida auris, a decade old Candida species, has been identified globally as a significant nosocomial multidrug resistant (MDR) pathogen responsible for causing invasive outbreaks. Biofilms and over expression of efflux pumps such as Major Facilitator Superfamily and ATP Binding Cassette are known to cause multidrug resistance in Candida species, including C. auris. Therefore, targeting these factors may prove an effective approach to combat MDR in C. auris.
Methods:In this study, 25 clinical isolates of C. auris from different hospitals of South Africa were used. Antifungal susceptibility profile of all the isolates against commonly used drugs was determined following CLSI recommended guidelines. Rhodamine-6-G extracellular efflux and intracellular accumulation assays were used to study active drug efflux mechanism. We further studied the role of farnesol in modulating development of biofilms and drug efflux in C. auris. Down-regulation of biofilm- and efflux pump- associated genes by farnesol was also investigated. CLSM analysis for examining C. auris biofilm architecture among treated and untreated isolates.
Results:Most of the isolates (twenty-two) were found resistant to FLZ whereas five were resistant to AmB. All the isolates were found capable of biofilm formation and ornamented with active drug efflux mechanism. The MIC for planktonic cells ranged from 62.5-125 mM and for sessile cells was 125 mM (0 h and 4 h biofilm) and 500 mM (12 h and 24 h biofilm), CLSM studies also confirmed these findings. Farnesol also blocked efflux pumps and down-regulated biofilm- and efflux pump- associated genes.
Conclusion:Modulation of biofilm- and efflux pump- associated genes by farnesol represent a promising approach in combating C. auris infection.
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Role for the phosphatidylinositol 3-phosphate 5-kinase in antifungal tolerance in Candida glabrata
More LessCandida glabrata is an opportunistic fungal pathogen of humans, which is intrinsically less susceptible to widely used azole antifungals, that block ergosterol biosynthesis. The major azole resistance mechanisms include mitochondrial dysfunction and multidrug efflux pump overexpression. In the current study, we have uncovered an essential role for the actin cytoskeletal network reorganization in survival of the azole stress. We demonstrate for the first time that the azole antifungal fluconazole induces remodelling of the actin cytoskeleton in C. glabrata, and genetic or chemical perturbation of actin structures results in intracellular sterol accumulation and azole susceptibility. Further, we showed that the vacuolar membrane-resident phosphatidylinositol 3-phosphate 5-kinase (CgFab1) is pivotal to this process, as CgFAB1 disruption impaired vacuole homeostasis and actin organization. We also showed that the actin depolymerization factor CgCof1 binds to phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), and CgCof1 distribution along with the actin filament-capping protein CgCap2 is altered upon both CgFAB1disruption and fluconazole exposure. Additionally, while the F-actin-stabilizing compound jasplakinolide rescued azole toxicity in cytoskeleton defective-mutants, the actin polymerization inhibitor latrunculin B rendered fluconazole fully and partially fungicidal in azole-susceptible and azole-resistant C. glabrata clinical isolates, respectively. These data underscore the essentiality of actin cytoskeleton reorganization for azole stress survival. Lastly, we have also shown a pivotal role of CgFab1 kinase activity regulators, CgFig4, CgVac7 and CgVac14, through genetic analysis, in azole and echinocandin antifungal tolerance. Altogether, I shall present our findings on functions and metabolism of the PI(3,5)P2 lipid in antifungal tolerance and virulence of C. glabrata.
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Novel antifungal activity of Q-Griffithsin, a broad-spectrum antiviral lectin
More LessBackgroundThere is a rising global trend in candida strains with high resistance to fluconazole and other antifungal drugs, hence the need for novel agents. Here, we investigated the anti-Candida activity of Q-Griffithsin (Q-GRFT), a lectin naturally produced by the red-sea algae, Griffithsia spp.
MethodsTo assess in vitro growth inhibitory activity, C. albicans was incubated with Q-GRFT on agar plates and in broth media. We investigated GFP-bound Q-GRFT’s ability to adhere to C. albicans using fluorescence microscopy and fluorescence intensity assessments. To demonstrate in vivogrowth inhibitory activity, CBA/J mice were treated per vaginam with Q-GRFT followed by challenge with C. albicans, and fungal burden determined following vaginal lavage.
ResultsWild type fluorescently labeled Q-GRFT displayed higher fluorescence than the lectin-binding site deficient variant following incubation with C. albicans. Q-GRFT localized around the fungal cells and bound to α-mannan in the cell wall. Q-GRFT significantly inhibited C. albicans growth in broth and on agar plates, disrupted the integrity of the cell wall, and induced ROS formation. The lectin significantly inhibited the growth of C. glabrata, C. parapsilosis and C. krusei, with modest activity against C. auris CDC388 and C. auris CDC389 strains in vitro. Topical treatment resulted in a lower fungal burden compared to the vehicle control group in vaginal candidiasis.
ConclusionQ-GRFT binds to and inhibits C. albicans growth both in vitro and in vivo. Further studies are needed to establish the mechanism of growth inhibition.
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CRISPR-Cas9 mutagenesis and single gene reintegration suggests functional diversity within the Candida albicans TLO gene family
More LessCandida albicans has between 10-15 Telomere-associated ORF family(TLO)genes, whereas its closest relative, Candida dubliniensis, has two. The Tlo proteins are components of the Mediator complex which plays an important role in transcriptional regulation. CRISPR-Cas9 mutagenesis was used to generate a TLOnull mutant of C. albicans. Phenotypic analysis of the mutant showed significantly reduced fitness, with major defects in growth rate, morphogenesis, stress resistance and virulence in a Galleria mellonellamodel. Clade representative TLOα1, TLOβ2 and TLOγ11constructs were reintroduced into the null mutant background to determine if members of the TLO gene family exhibit functional differences. The genes were reintroduced under the control of the TET1 and ENO1promoters. TLOα1and TLOβ2expression restored stress tolerance and growth rate, in some cases to the level of the WT. TLOβ2expression also showed a dramatic effect on morphology resulting in constitutive true hyphal growth. Moderate expression of TLOγ11 had no detectable effect on many of the phenotypes tested, however overexpression increased biofilm formation in Spider medium, and also conferred increased resistance to cell wall stressors. These data suggest that individual TLO genes have distinct functions and that the diversity within the TLO family may contribute to the relative success of C. albicans as a coloniser and pathogen of humans.
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Tolerance to fluconazole in Candida albicans is regulated by temperature and aneuploidy
More LessCandida albicans is a prevalent human fungal pathogen. Azoles are the most widely used antifungal drugs. Drug tolerance in bacteria is well defined and thoroughly studied, but in fungi, the definition of drug tolerance and the mechanism that drive it are not well understood. Here, we found that a large proportion of clinical isolates were intrinsically tolerant to fluconazole, and/or could be induced by high temperature (37°C) to become tolerant (conditionally tolerant). When treated with inhibitory doses of fluconazole, non-tolerant strains became tolerant by forming aneuploids involving different chromosomes, with chromosome R duplication as the most recurrent mechanism. Tolerance determines the ability to grow in the presence of fluconazole and other azoles, in a manner independent of the MIC. Both temperature conditional tolerance and the associated aneuploidy were sensitive to FK506, an inhibitor of calcineurin. Intrinsic and conditional tolerance were also abolished by deletions of genes encoding the calcineurin (CMP1 and CNB1). However, the dependence of tolerance on calcineurin could be bypassed by a different aneuploid chromosome. Thus, fluconazole tolerance in C. albicans is regulated by temperature and by aneuploidy and is dependent upon aneuploidy, but this dependence can be bypassed by an additional aneuploidy.
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Preliminary study into the effects of tobacco smoke on Candida albicans
More LessBackground:Denture-stomatitis (DS) is the most common form of oral candidosis with increased prevalence in cigarette smokers (Akram et al. 2018). Interestingly, tobacco condensate (TC) increases Candida albicans adhesion, growth, biofilm-formation, virulence gene expression (Semlali et al. 2014)and hyphal production (Awad and Karuppayil 2018). We hypothesised that TC-treated denture acrylic would therefore affect C. albicans within acrylic biofilms.
Methods:Acrylic discs (pre-conditioned with TC, artificial saliva (AS) or water) were incubated at 37°C with C. albicans (n=6) for 90 min or 24 h. Adherent Candida were stained with calcofluor white and confocal laser scanning microscopy (CLSM) used to assess levels of adherence, biofilm and hyphal numbers. Expressed virulence genes (n=7) were measured by qPCR.
Results:CLSM showed that effects of TC-treatment were strain dependent. Adherence of C. albicans PTR/94 to TC-treated surfaces was significantly (P<0.002) lower than on the untreated control. Biofilm levels of PTR/94 after 24 h were found to be significantly higher on AS-treated acrylic than the TC-treated and untreated control. Five strains had significantly fewer filamentous forms after 90 min on TC-treated surfaces. TC-treatment promoted hyphal levels for strain 705/93 after 24h.
Conclusion:TC pre-conditioning altered adherence and biofilm coverage of C. albicans to acrylic surfaces and influenced hyphal development. Work is ongoing to ascertain the significance of these effects on C. albicans pathogenicity.
Akram et al. (2018). Journal of Oral Science 60(1):115–120.
Awad and Karuppayil (2018). American Journal of Clinical Microbiology and Antimicrobials1(3):1–6.
Semlali et al. (2014). BMC Microbiology. 14:61
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Proteomic study of the effect of metformin on C. albicans
More LessFungal infections are a global health problem. Of them, those produced by Candida albicans are the most important, with a reduced arsenal of antifungals and an increasing problem of antifungal resistance. Thus, the discovery of new antifungal targets and drugs remains interesting. Metformin is a biguanide administered as a first-line treatment for Type II Diabetes Mellitus and it has recently been published its anti-Candida action, especially against C. glabrata, and its synergistic effect with other antifungals. Our studies of the effect of metformin on C. albicans have revealed an inhibition of growth, filamentation and other phenotypes important for virulence. Although metformin has been described as an AMPK agonist, its mechanism of action is partly unknown. To deepen into the anti-Candida mechanism of action, we have addressed the differential proteomic study. A set-up of the conditions for the proteomic study has been carried out, fixing a concentration of 50mM of metformin, 6 h of treatment at 37°C in RPMI medium and with 60 rpm of agitation. The proteomic study using the Labelfree technique and 4 biological replicas, allowed the identification and quantification of a total of 1899 proteins, 206 of them presenting differences in abundance due to metformin exposure. Of these, 127 increased and 79 decreased due to the action of the drug. The most relevant functions of these proteins are related to antifungal response, filamentation, biofilm formation and metabolism, being 9 essential proteins for the microorganism that could be new antifungal targets.
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The gut commensal Bacteroides vulgatus mpk reduces Candida albicans pathogenicity towards epithelial cells
The human body is colonized by various microbes, among them the yeast Candida albicans. Mostly harmless, this opportunist causes also disease, ranging from superficial infections to sepsis. Risk factors are disturbed host defenses, mucosal barrier breakdown, and antibiotic-induced dysbiosis. Hence, residing bacteria are important to protect from Candida-mediated damage or inflammation. Bacteroides vulgatus mpk, e.g., is described as positively immunomodulatory in mouse models of inflammatory bowel disease, but its effect on the mycobiota is unknown.
In this study we aimed to determine if B. vulgatus mpk affects C. albicans pathogenicity.
Therefore, intestinal and oral epithelial cellswere pre-infectedin vitrowith B. vulgatus mpk and then challenged with C. albicans SC5314. The role of soluble factors was investigated by spatial separation or use of Bacteroides-conditioned medium (BCM).
Preincubation of host cells with B. vulgatus mpk strongly reduced C. albicans-mediated damage while fungal burden and hyphal length were unaffected by the bacterium. The protective effect did not depend on direct contact of Bacteroidesto host cellsor Candida and could be mimicked using BCM. Contact independency suggests that diffusible factors modulate host cell susceptibility.
Ongoing experiments aim to identifykey soluble Bacteroides mediators as well as subsequent host cell signaling. Additionally, co-colonization experiments of germ-free mice are planned to investigate B. vulgatus mpk’s potential to mediate colonization resistance towards C. albicans. This will contribute to our understanding of how commensal bacteria affect C. albicans and host protection.
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The effect of acquired triazole resistance on abiotic stress tolerance and virulence in Candida auris micro evolved strains
More LessRecently, C. auris become one of the most prominent members of the genus Candida. Since its occurrence, several C. auris outbreaks have been reported worldwide. These outbreaks were associated with isolates displaying decreased susceptibility towards fluconazole, the first-line agent for prophylaxis. Fluconazole is the most frequently used antifungal drug to treat bloodstream Candida infections.
The physiological effects of acquired antifungal resistance was investigated in this species using fluconazole, posaconazole and voriconazole resistant mutant strains generated by the in vitro microevolution method. Alterations in antifungal susceptibility and cross resistance were determined by the microdilution method, utilizing azoles (fluconazole, voriconazole, posaconazole), echinocandins (caspofungin, micafungin, anidulafungin) and a polyene (amphotericin B). Changes in the abiotic stress tolerance was examined by spotting assay, using osmotic stressors, cell wall perturbants and a membrane detergent. To evaluate the impact of the acquired resistance on sterol biosynthesis, ergosterol composition of all generated mutant strains were examined. A potential relationship between virulence and acquired antifungal resistance was also studied both in vitro and in vivo. Phagocytosis of the generated strains by J774.2 mouse macrophage-like cells was measured and analyzed by flow cytometry. In the murine infection model fungal burden of the triazole evolved strains was determined in spleen, kidney, liver and brain and compared to the fungal burden associated with the initial azole susceptible strain. Significant differences in virulence of the initial and the generated strains was observed suggesting a potential connection between the virulence and antifungal susceptibility of the emerging fungal pathogen, C. auris.
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Extending the proteomic characterization of Candida albicans exposed to stress and apoptotic inducers through data-independent acquisition mass espectrometry
More LessCandida albicans is a commensal fungus that causes systemic infections in immunosuppressed patients. In order to deal with the changing environment during commensalism or infection, C. albicans must reprogram its proteome. Characterizing the stress-induced changes in the proteome that C. albicans uses to survive should be very useful in the development of new antifungal drugs. We studied the C. albicans global proteome after exposure to hydrogen peroxide (H2O2) and acetic acid (AA), using a DIA-MS strategy. More than 2000 C. albicans proteins were quantified using an ion library previously constructed using DDA-MS. C. albicans responded to treatment with H2O2 with an increase in the abundance of many proteins involved in the oxidative stress response, protein folding and proteasome-dependent catabolism, which led to an increased proteasome activity. The data revealed a previously unknown key role for Prn1, a protein similar to pirins, in the oxidative stress response. Treatment with AA resulted in a general decrease in the abundance of proteins involved in amino acid biosynthesis, protein folding, and rRNA processing. Almost all proteasome proteins declined, as did proteasome activity. Apoptosis was observed after treatment with H2O2, but not AA. A targeted proteomic study of 32 proteins related to apoptosis in yeast supported the results found by DIA-MS and allowed the creation of an efficient method to quantify relevant proteins after treatment with stressors (H2O2, AA, and amphotericin B). This approach also uncovered a main role for Oye32, an oxidoreductase, suggesting this protein as a possible apoptotic marker common to many stressors.
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- Personal Views
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- Case Reports
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Detection of invasive Trichosporon asahii in patient blood by a fungal PCR array
Rare invasive fungal infections are increasingly emerging in hosts with predisposing factors such as immunodeficiency. Their timely diagnosis remains difficult, as their clinical picture may initially mimic infections with more common fungal species and species identification may be difficult with routine methods or may require time-consuming subcultures. This often results in ineffective drug administration and fatal outcomes. We report on a patient in their early twenties with mixed cellularity classical Hodgkin lymphoma with a disseminated Trichosporon asahii (T. asahii) infection. Even though pathogen detection and identification was possible via the standard procedure consisting of culture followed by matrix-assisted laser desorption ionisation–time of flight (MALDI-TOF) mass spectrometry, the patient passed away in the course of multi organ failure. Herein, we report on a retrospectively applied experimental diagnostic fungal PCR-analysis used on an EDTA blood sample and consisting of two pan-fungal reactions and seven branch-specific reactions. Regarding invasive T. asahii infection, this PCR array could considerably shorten time to diagnosis and switch to a targeted therapy with triazoles.
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Sneathia amnii bacteraemia and chorioamnionitis leading to second trimester abortion: a case report
More LessBackground. Sneathia amnii (formerly designated as Leptotrichia amnionii ) was first described in 2002 in the USA. Members of the genus Sneathia can be part of the normal flora of the genitourinary tract, but have been implicated in invasive (mostly gynaecological) infections.
Case presentation. To the best of our knowledge, here we present the first case of S. amnii infection in Belgium, in a young woman presenting with fever leading to second trimester septic abortion.
Conclusions. Despite its pathogenicity, S. amnii remains an underrated cause of infections due to inherent difficulties with conventional laboratory methods. By extracting the bacterial DNA directly from the blood culture broth and performing a 16S ribosomal RNA gene sequence analysis we succeeded in identifying S. amnii as the most probable cause of the septic abortion in our patient.
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Penile implant infection resulting in Staphylococcus aureus bacteraemia and infective endocarditis
More LessIntroduction. Penile implant infections are a possible surgical complication that has historically been most commonly associated with Gram-positive bacteria. Staphylococcus aureus is a Gram-positive bacteria and is the most common cause of endocarditis.
Case Presentation. A male patient in his 50s with a past medical history of hypertension, diabetes, end-stage renal disease (ESRD) on peritoneal dialysis (PD) and erectile dysfunction with a penile implant placed 6 years prior to the admission date presented with complaints of scrotal pain. The pump for his implant had eroded through his scrotum and was draining pus. Blood cultures returned positive for Gram-positive cocci in clusters in 4/4 bottles, which was eventually identified as methicillin-sensitive Staphylococcus aureus (MSSA). A transthoracic echocardiogram (TTE) was performed due to concern for infective endocarditis (IE) but did not show any valvular abnormalities. Due to high clinical suspicion, a transesophageal echocardiogram (TEE) was performed and revealed a vegetation on the native mitral valve. His penile implant was removed by urology and intraoperative cultures grew MSSA. Surgical valve replacement was not recommended, and the patient was sent home with IV antibiotics for 6 weeks.
Discussion. Post-operative site infections are a quite uncommon point of entry for infective endocarditis, with penile implant infections being an even rarer cause. While a TTE is often used initially to attempt to diagnose infective endocarditis, it has lower sensitivity than a TEE. If clinical suspicion for infective endocarditis remains high after negative imaging with TTE, then TEE should be performed for better visualization of the heart valves.
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Reactivation of Q fever: case report of osteoarticular infection developing at the site of a soft tissue injury
More LessCoxiella burnetii , the causative agent of Q fever, is known to cause acute and persistent infection, but reactivation of infection is rarely reported. This case demonstrates reactivation of a distant, untreated Q fever infection after a relatively innocuous soft tissue injury in an adjacent joint without pre-existing pathology. A 52-year-old male abbatoir worker sustained an adductor muscle tear in a workplace injury. He was unable to walk thereafter, and developed a chronic, progressive, destructive septic arthritis of the adjacent hip with surrounding osteomyelitis of the femur and acetabulum. He had evidence of prior Q fever infection, with a positive skin test and serology 15 years beforehand. He was diagnosed with chronic osteoarticular Q fever on the basis of markedly elevated phase I antibodies, and symptomatic and serological response to prolonged antibiotic treatment with doxycycline and hydroxychloroquine. He required a two-stage hip arthroplasty. This case illustrates reactivation of latent C. burnetii infection at the site of a soft tissue injury. Clinicians need to be aware of this possibility in patients with previous Q fever infection, and in the setting of undiagnosed osteoarticular pathology following soft tissue injury.
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Dengue 2 serotype and yellow fever coinfection
Case Presentation. Arboviruses primarily consist of RNA, which favours greater genetic plasticity, with a higher frequency of mutations that allow the virus to adapt to different hosts. The initial symptomatology is nonspecific, in that the patient can present fever, myalgia, arthralgia, rash and headache. This makes a clinical diagnosis using laboratory tests difficult and time-consuming. In Brazil, the main arboviruses involved in epidemics belong to the family Flaviviridae. The patient in this case is from the municipality of São Bernardo do Campo, an area endemic for arboviruses. He presented symptoms of fever, myalgia and headache.
Results. The multiplex assay for arboviruses detected genetic material from the dengue 2 and yellow fever viruses.
Conclusion. This result confirms the importance of molecular tests showing high sensitivity and specificity that can assist clinical diagnosis, particularly in endemic areas during periods of outbreak for other arboviruses, like the epidemiological picture in Brazil in 2018, when significant co-circulation of dengue virus and yellow fever virus occurred. The presence of co-circulating arboviruses increases the chance of coinfection and demonstrates the importance of differential diagnosis.
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Corynebacterium striatum thrombophlebitis: a nosocomial multidrug-resistant disease?
Introduction. Corynebacterium striatum is a non-Diphteriae commensal bacterium with a wide range of pathogenicity. The identification of multidrug-resistant (MDR) C. striatum is concerning because drug susceptibility testing is not usually performed in microbiology laboratories. There is no consensus yet on the treatment of septic thrombophlebitis in this situation.
Case report. We report here the first case of a quinquagenarian patient with a history of AIDS and fungic endocarditis, who was diagnosed with a nosocomial thrombophlebitis in the right jugular vein caused by C. striatum . Bitherapy with daptomycin for 12 days and linezolid for 23 days was combined with a therapeutic anticoagulant. The follow-up included weekly cervical ultrasound controls. The efficiency of the treatment and the stability of the lesions allowed us to alleviate the medication with a prophylactic dose of anticoagulant. The patient was discharged from hospital and showed no signs of recurrence after 12 months.
Conclusion. The lack of consensus relative to the management of septic thrombophlebitis precludes the validation of a specific treatment for the condition. Our results suggest that a combination that includes removal of the medical device is needed. A total of 6 weeks of antibiotherapy should be applied, starting with 2 weeks of vancomycin or a combination of antibiotitherapy with daptomycin in order to reduce the bacterial load and avoid resistance. Six weeks of anticoagulation therapy is effective.
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Rapid identification of mcr-1-positive Escherichia coli from patient urine using a novel lipid-based MALDI-TOF-MS assay
More LessMobilized colistin resistance (mcr) genes confer resistance to colistin, a last-resort antibiotic for multidrug-resistant Gram-negative infections. In this case report, we describe a novel lipid-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) diagnostic used to rapidly identify an mcr-1-positive Escherichia coli directly from a patient with a urinary tract infection without the need for ex vivo growth.
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- Case Series
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Spectrum of mycobacterial pathogens responsible for head and neck tuberculosis-like presentation
More LessTuberculosis (TB) of the head and neck can be contained in the lymph nodes, larynx, oropharynx, salivary glands, nose and paranasal sinuses, ear, skin and skull. Head and neck TB presentations are varied in nature and thus difficult to diagnose. The clinical features, radiological findings, microbiological diagnostic modalities, surgical and medical management and outcomes of nine cases of head and neck TB are discussed in detail here, together with a thorough review of the literature. Patients presented with atypical symptoms such as discharging sinus, ear lobule swelling, otitis media, vision loss and facial weakness, long refractory otorrhoea and granulation tissue in the ear canal. We diagnosed tubercular skull base osteomyelitis (one case) and laryngeal tuberculosis (two cases), mastoid tuberculosis (one case) and non-tubercular mycobacterial infection involving the temporal bone (two cases), sino-nasal region (one case), maxilla (one cases) and ear lobule (one case) over a period of 8 months. All patients were managed successfully with a combination of surgery and a well-planned treatment regimen for non-tuberculous mycobacteria (NTM) or anti-tubercular drugs for TB. All had successful outcomes except one patient with tubercular skull base osteomyelitis who expired before the initiation of anti-tubercular therapy (ATT). High clinical suspicion followed by thorough diagnostic work-up for both TB and NTM would enable early diagnosis and complete treatment.
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