Staphylococcus aureus small colony variants: A potentially underestimated microbiological challenge in peritoneal dialysis.

INTRODUCTION: Peritonitis remains the major infectious complication in the setting of peritoneal dialysis (PD). Despite known only moderate pathogenicity, the most frequently detected pathogens in PD-related peritonitis are surprisingly coagulase-negative staphylococci. However, this could be explained, at least in part, by Staphylococcus aureus small colony variants (SCVs) induced by PD fluids (PDFs) and misidentified by routinely used microbiological methods. MATERIAL AND METHODS: Bacteria were exposed to commonly used PDFs in various regimens designed to simulate daily use as closely as possible. Wild-type isolates and SCVs were subsequently used to determine minimum inhibitory concentrations (MICs), in vitro biofilm formation capacities, and auxotrophies. Underlying genetic alterations were investigated using whole-genome sequencing, and various microbial identification methods were tested to determine their performance for wild-types and SCVs. RESULTS: Stable SCVs could be isolated most successfully after exposure to glucose-containing PDFs alone. The reading of MICs was significantly affected by the reduced growth of SCVs, resulting in lower MIC values in 44% of all tests. Nonsynonymous mutations were found in all but one SCV, while only two isolates showed typical auxotrophic responses. While MALDI-TOF, PCR and Pastorex Staph-Plus correctly identified all S. aureus SCVs, API-Staph and VITEK-2 yielded identification rates of only 40% and 10%, respectively. CONCLUSIONS: Overall, the present study has shown that commercially available PDFs induce S. aureus SCVs in vitro, which are difficult to identify and test for antimicrobial susceptibility and can potentially lead to recurrent or persistent infections. Thus, they represent a potentially underappreciated challenge not only for microbiologists, but also for clinicians.

Differences in oxazolidinone resistance mechanisms and small colony variants emergence of Staphylococcus aureus induced in an in vitro resistance development model.

Invasive Staphylococcus aureus infections are associated with a high burden of disease, case fatality rate and healthcare costs. Oxazolidinones such as linezolid and tedizolid are considered potential treatment choices for conditions involving methicillin resistance or penicillin allergies. Additionally, they are being investigated as potential inhibitors of toxins in toxin-mediated diseases. In this study, linezolid and tedizolid were evaluated in an in vitro resistance development model for induction of resistance in S. aureus. Whole genome sequencing was conducted to elucidate resistance mechanisms through the identification of causal mutations. After inducing resistance to both linezolid and tedizolid, several partially novel single nucleotide variants (SNVs) were detected in the rplC gene, which encodes the 50S ribosome protein L3 in S. aureus. These SNVs were found to decrease the binding affinity, potentially serving as the underlying cause for oxazolidinone resistance. Furthermore, in opposite to linezolid we were able to induce phenotypically small colony variants of S. aureus after induction of resistance with tedizolid for the first time in literature. In summary, even if different antibiotic concentrations were required and SNVs were detected, the principal capacity of S. aureus to develop resistance to oxazolidinones seems to differ between linezolid and tedizolid in-vivo but not in vitro. Stepwise induction of resistance seems to be a time and cost-effective tool for assessing resistance evolution. Inducted-resistant strains should be examined and documented for epidemiological reasons, if MICs start to rise or oxazolidinone-resistant S. aureus outbreaks become more frequent.

Candida auris in Austria-What Is New and What Is Different.

Candida auris is a novel and emerging pathogenic yeast which represents a serious global health threat. Since its first description in Japan 2009, it has been associated with large hospital outbreaks all over the world and is often resistant to more than one antifungal drug class. To date, five C. auris isolates have been detected in Austria. Morphological characterization and antifungal susceptibility profiles against echinocandins, azoles, polyenes and pyrimidines, as well as the new antifungals ibrexafungerp and manogepix, were determined. In order to assess pathogenicity of these isolates, an infection model in Galleria mellonella was performed and whole genome sequencing (WGS) analysis was conducted to determine the phylogeographic origin. We could characterize four isolates as South Asian clade I and one isolate as African clade III. All of them had elevated minimal inhibitory concentrations to at least two different antifungal classes. The new antifungal manogepix showed high in vitro efficacy against all five C. auris isolates. One isolate, belonging to the African clade III, showed an aggregating phenotype, while the other isolates belonging to South Asian clade I were non-aggregating. In the Galleria mellonella infection model, the isolate belonging to African clade III exhibited the lowest in vivo pathogenicity. As the occurrence of C. auris increases globally, it is important to raise awareness to prevent transmission and hospital outbreaks.

Identification of Filamentous Fungi by MALDI-TOF Mass Spectrometry: Evaluation of Three Different Sample Preparation Methods and Validation of an In-House Species Cutoff.

Invasive infections caused by filamentous fungi constitute a leading cause of morbidity and mortality in immunocompromised patients. Rapid and reliable identification of filamentous fungi is essential for the early initiation of appropriate treatment. In the present study, 230 filamentous fungi isolates identified by conventional methods were investigated using MALDI-TOF MS (Bruker Daltonics, Bremen, Germany) in combination with the Filamentous Fungi Library 3.0 provided by the manufacturer. Three different sample preparation methods were applied as recommended by the manufacturer and identification rates were compared using the criteria provided by the manufacturer. Application of the more time-consuming sample preparation methods clearly improved identification at the species level. Thus, the identification rate increased from 48.9% using the simplest method to 76.1% with the most laborious procedure. Misidentifications did not occur. Furthermore, the reliability of an in-house threshold for species identification was investigated. The reduced threshold increased the rate of isolates correctly identified at the species level by up to 86.4%. As no misidentification was made at the genus level and only one misidentification of minor significance occurred at the species level, this threshold could be validated for routine use in our laboratory. In conclusion, regarding the high identification rates achieved, this commercial platform proved suitable for implementation in routine diagnosis.

Efficacy of octenidine against emerging echinocandin-, azole- and multidrug-resistant Candida albicans and Candida glabrata.

OBJECTIVES: Infections due to Candida species represent a serious threat to healthcare facilities. Only a few classes of antifungal agents are available, and the rapid emergence of (multi)drug resistance eliminates effective treatment options for successful patient outcome. Topically applied antiseptics may represent a suitable tool for infection control and local therapy. This study aimed to investigate the in vitro efficacy of the widely used antiseptic octenidine (OCT) against clinical isolates of emerging azole-, echinocandin- and multi-resistant Candida albicans and Candida glabrata. METHODS: The antifungal activity of different concentrations of OCT ranging from 0.001% to 0.05% and of OCT-containing ready-to-use products was determined against well-characterised (multidrug) resistant C. albicans and C. glabrata isolates, including susceptible wild-type strains. Quantitative suspension tests were performed under "clean conditions" (0.3 g/L bovine serum albumin) and under "dirty conditions" (3 g/L albumin + 3 mL/L defibrinated sheep blood) as well as various contact times (30 s, 1 min, 2 min) according to EN13624:2013. RESULTS: Even in the presence of a high organic load, pure OCT at 0.05% and a contact time of 30 s was fully effective for all Candida strains, with growth kinetics indicating a time- and concentration-dependent activity. Importantly, commercially available OCT-based products achieved the required reduction of ≥4 log10 for all Candida isolates under the most challenging dirty conditions within two minutes, which makes them suitable for routine clinical use. CONCLUSION: These results encourage consideration of the well-tolerated antiseptic molecule OCT in the eradication of emerging (multidrug) resistant C. albicans and C. glabrata.

In vitro evaluation of disease-modifying antirheumatic drugs against rheumatoid arthritis associated pathogens of the oral microflora.

OBJECTIVES: In the past, the human microbiome has consistently been associated with rheumatoid arthritis (RA) and disease activity. Here, we investigate the antimicrobial activity of disease-modifying antirheumatic drugs (DMARDs) against typical representatives of the oral microflora that have been associated with RA. METHODS: DMARDs were screened for antimicrobial activity against bacteria that are associated with the pathogenesis of the disease and/or frequently isolated from the oral microflora of patients with RA. Screening was done by an agar diffusion assay and minimum inhibitory concentrations (MICs) of antimicrobial active substances were then determined by broth dilution. RESULTS: Aurothiomalate and sulfasalazine demonstrated broad-spectrum antimicrobial activity, but with MICs ranging from 18 to >280 µg/mL and 150 to >600 µg/mL, respectively, only at supratherapeutic concentrations. Methotrexate showed antimicrobial activity only against Fusobacterium nucleatum and Viridans streptococci. The corresponding MICs were 3.75 to >30 µg/mL and 0.5-15 µg/mL, respectively, thus at least for streptococci, within the therapeutically achievable range. No other DMARD tested showed antimicrobial activity in the agar diffusion screening assay. CONCLUSION: Methotrexate, sulfasalazine and aurothiomalate showed antimicrobial activity against a broad spectrum of RA associated pathogens of the oral microflora. While methotrexate showed relevant antimicrobial activity, and to a more limited extent aurothiomalate, sulfasalazine was active only at far supratherapeutic systemic concentrations. Nevertheless, given the highly species-dependent antimicrobial activity and the multiple ways it can affect the human microbiome, our results suggest a link between antimicrobially active antirheumatic drugs and their potential effect in the treatment of RA.

Do Candida albicans Isolates with Borderline Resistant Micafungin MICs Always Harbor FKS1 Hot Spot Mutations?

Antifungal susceptibility testing is important in guiding patient therapy due to an increasing number of resistant Candida isolates. In the clinical strain collection of the Austrian resistance report (AURES), a high number of micafungin-resistant C. albicans isolates (18.2% 49/269) was detected in seven different centres in Austria from 2011-2016. Most of these isolates showed a micafungin MIC value that was just above the clinical breakpoint (CB) established by EUCAST (0.016 mg/L). The aim of this study was to analyse whether C. albicans strains showing a micafungin MIC value of 1-2 dilutions above the CB (0.032 mg/L and 0.064 mg/L) are associated with mutations in FKS1 hotspot (HS) regions. 115 C. albicans candidemia strains showing a micafungin MIC one or two dilutions above the EUCAST CB (0.032 mg/L and 0.064 mg/L) were categorized as borderline resistant and screened for mutations in FKS1 HS1, HS2, and HS3 regions, which are known locations for the development of echinocandin resistance. For this purpose, we implemented targeted resequencing utilizing a next generation sequencing technology. No missense mutations could be detected in FKS1 HS1, HS2, and HS3 in any of the 115 isolates, which indicated that resistance conferred by alteration of FKS1 seems unlikely.

Molecular Methods for the Diagnosis of Invasive Candidiasis.

Invasive infections caused by members of the genus Candida are on the rise. Especially patients in intensive care units, immunocompromised patients, and those recovering from abdominal surgery are at risk for the development of candidemia or deep-seated candidiasis. Rapid initiation of appropriate antifungal therapy can increase survival rates significantly. In the past, most of these infections were caused by C. albicans, a species that typically is very susceptible to antifungals. However, in recent years a shift towards infections caused by non-albicans species displaying various susceptibly patterns has been observed and the prompt diagnosis of the underlying species has become an essential factor determining the therapeutic outcome. The gold standard for diagnosing invasive candidiasis is blood culture, even though its sensitivity is low and the time required for species identification usually exceeds 48 h. To overcome these issues, blood culture can be combined with other methods, and a large number of tests have been developed for this purpose. The aim of this review was to give an overview on strengths and limitations of currently available molecular methods for the diagnosis of invasive candidiasis.

Clinical evaluation of an in-house panfungal real-time PCR assay for the detection of fungal pathogens.

PURPOSE: Due to an increasing incidence of invasive fungal infections, the availability of reliable diagnostic tools for the fast detection of a wide spectrum of fungal pathogens is of vital importance. In this study, we aimed to conduct an extensive clinical evaluation of a recently published in-house panfungal PCR assay on samples from suspected invasive fungal infections. METHODS: Overall 265 clinical samples from 232 patients with suspected invasive fungal disease (96 deep airway samples, 60 sterile fluids, 50 tissue biopsies, and 59 blood samples) were included. All samples underwent standard culture-based diagnostics and were additionally analyzed with our panfungal PCR assay. RESULTS: Overall, 55.1% of agreement between culture and the panfungal PCR was observed; in 17% of all samples partial concordance was noted, while results between culture and our PCR assay were not in agreement in 27.9%. Our panfungal assay performed better in samples from normally sterile sites, while samples from the deep airways yielded the highest rate of discordant (39.6%) results. In two tissue and three blood samples an invasive pathogen was only detected by PCR while cultures remained negative. CONCLUSION: In combination with routine methods, our panfungal PCR assay is a valuable diagnostic tool. Patients at risk for invasive fungal infections might profit from the reduced time to pathogen identification.

Antifungal susceptibility of yeast bloodstream isolates collected during a 10-year period in Austria.

BACKGROUND: Candida-associated infections put a significant burden on western healthcare systems. Development of (multi-)resistant fungi can become untreatable and threaten especially vulnerable target groups, such as the immunocompromised. OBJECTIVES: We assessed antifungal susceptibility and explored possible influence factors of clinical Candida isolates collected from Austrian hospitals between 2007 and 2016. METHODS: Thousand three hundred and sixty clinical Candida spp. isolated from blood cultures were subjected to antifungal susceptibility testing (AFST) in a liquid-handling aided continuous microdilution assay. We tested against fluconazole, voriconazole, posaconazole, itraconazole, isavuconazole, anidulafungin, caspofungin and micafungin according to EUCAST with additional recording of growth curves. We performed rigid quality control on each assay via growth curve assessment and included two standard reference strains. Minimal inhibitory concentrations (MIC) were quantified according to EUCAST guideline E.DEF 7.3.1, and susceptibility was evaluated using EUCAST clinical breakpoints. RESULTS: The isolate collection consisted of Candida albicans (59%), C. glabrata (19%), C. parapsilosis (9%), C. tropicalis (5%) and C. krusei (3%) and few other Candida species and fungi (5%). During the observed time period, species abundance and antifungal resistance rates remained constant. Multi-resistance was rare and we found no single isolate which was resistant to both azoles and echinocandins. Within the antifungal resistance profile of our strain collection, we observed clusters along species boundaries. CONCLUSIONS: Over the last decade, the distribution of Candida species and its level of antifungal resistance remained constant in Austria. Our data compare well with other European countries. Principal component analysis of the susceptibility profile of this collection revealed species-specific clusters and substantial intra-species variation, especially for C. glabrata.

Analysis of antifungal resistance genes in Candida albicans and Candida glabrata using next generation sequencing.

INTRODUCTION/OBJECTIVES: An increase in antifungal resistant Candida strains has been reported in recent years. The aim of this study was to detect mutations in resistance genes of azole-resistant, echinocandin-resistant or multi-resistant strains using next generation sequencing technology, which allows the analysis of multiple resistance mechanisms in a high throughput setting. METHODS: Forty clinical Candida isolates (16 C. albicans and 24 C. glabrata strains) with MICs for azoles and echinocandins above the clinical EUCAST breakpoint were examined. The genes ERG11, ERG3, TAC1 and GSC1 (FKS1) in C. albicans, as well as ERG11, CgPDR1, FKS1 and FKS2 in C. glabrata were sequenced. RESULTS: Fifty-four different missense mutations were identified, 13 of which have not been reported before. All nine echinocandin-resistant Candida isolates showed mutations in the hot spot (HS) regions of FKS1, FKS2 or GSC1. In ERG3 two homozygous premature stop codons were identified in two highly azole-resistant and moderately echinocandin-resistant C. albicans strains. Seven point mutations in ERG11 were determined in azole-resistant C. albicans whereas in azole-resistant C. glabrata, no ERG11 mutations were detected. In 10 out of 13 azole-resistant C. glabrata, 12 different potential gain-of-function mutations in the transcription factor CgPDR1 were verified, which are associated with an overexpression of the efflux pumps CDR1/2. CONCLUSION: This study showed that next generation sequencing allows the thorough investigation of a large number of isolates more cost efficient and faster than conventional Sanger sequencing. Targeting different resistance genes and a large sample size of highly resistant strains allows a better determination of the relevance of the different mutations, and to differentiate between causal mutations and polymorphisms.