The fungal pathogen Candida glabrata has emerged as a major health threat since it readily acquires resistance to multiple drug classes, including triazoles and/or echinocandins. Thus far, cellular mechanisms promoting the emergence of resistance to multiple drug classes have not been described in this organism. Here we demonstrate that a mutator phenotype caused by a mismatch repair defect is prevalent in C. glabrata clinical isolates. Strains carrying alterations in mismatch repair gene MSH2 exhibit a higher propensity to breakthrough antifungal treatment in vitro and in mouse models of colonization, and are recovered at a high rate (55% of all C. glabrata recovered) from patients. This genetic mechanism promotes the acquisition of resistance to multiple antifungals, at least partially explaining the elevated rates of triazole and multi-drug resistance associated with C. glabrata. We anticipate that identifying MSH2 defects in infecting strains may influence the management of patients on antifungal drug therapy.

has simultaneously emerged on five continents as a fungal pathogen causing nosocomial outbreaks. The challenges in the treatment of infections are the variable antifungal susceptibility profiles among clinical isolates and the development of resistance to single or multiple classes of available antifungal drugs. Here, the susceptibility to echinocandin antifungal drugs was determined and sequencing was performed on 106 clinical isolates. Four isolates were identified to be resistant to all tested echinocandins (MIC ≥ 4 mg/liter) and harbored an S639F mutation in hot spot region 1. All remaining isolates were wild type (WT) and echinocandin susceptible, with micafungin being the most potent echinocandin (MIC = 0.125 mg/liter). Antifungal susceptibility testing with caspofungin was challenging due to the fact that all WT isolates exhibited an Eagle effect (also known as the paradoxical growth effect), which occurred at various intensities. To assess whether the Eagle effect resulted in pharmacodynamic resistance, 8 representative isolates were evaluated for their drug response in a murine model of invasive candidiasis. All isolates were susceptible to caspofungin at a human therapeutic dose, except for those harboring the S639F mutation. The data suggest that only isolates carrying mutations in are echinocandin resistant and that routine testing of isolates for susceptibility to caspofungin by the broth microdilution method should be viewed cautiously or avoided.

Candida auris is an emerging multidrug-resistant fungal pathogen causing nosocomial and invasive infections associated with high mortality. C. auris is commonly misidentified as several different yeast species by commercially available phenotypic identification platforms. Thus, there is an urgent need for a reliable diagnostic method. In this paper, we present fast, robust, easy-to-perform and interpret PCR and real-time PCR assays to identify C. auris and related species: Candida duobushaemulonii, Candida haemulonii, and Candida lusitaniae. Targeting rDNA region nucleotide sequences, primers specific for C. auris only or C. auris and related species were designed. A panel of 140 clinical fungal isolates was used in both PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The identification results from the assays were 100% concordant with DNA sequencing results. These molecular assays overcome the deficiencies of existing phenotypic tests to identify C. auris and related species.

Our study demonstrated that the circulating miRNAs miR-199a, miR-122, miR-145*, and miR-542-3p could potentially serve as noninvasive biomarkers for endometriosis. miR-199a may also play an important role in the progression of the disease. This is the first report that circulating miRNAs serve as biomarkers of endometriosis.

Invasive fungal infections remain a major source of global morbidity and mortality, especially among patients with underlying immune suppression. Successful patient management requires antifungal therapy. Yet, treatment choices are restricted due to limited classes of antifungal agents and the emergence of antifungal drug resistance. In some settings, the evolution of multidrug-resistant strains insensitive to several classes of antifungal agents is a major concern. The resistance mechanisms responsible for acquired resistance are well characterized and include changes in drug target affinity and abundance, and reduction in the intracellular level of drug by biofilms and efflux pumps. The development of high-level and multidrug resistance occurs through a stepwise evolution of diverse mechanisms. The genetic factors that influence these mechanisms are emerging and they form a complex symphony of cellular interactions that enable the cell to adapt and/or overcome drug-induced stress. Drivers of resistance involve a complex blend of host and microbial factors. Understanding these mechanisms will facilitate development of better diagnostics and therapeutic strategies to overcome and prevent antifungal resistance.

KPC variants. The assay consists of six molecular beacons and two oligonucleotide primer pairs, allowing for detection and classification of all currently described bla KPC variants (bla KPC-2 to bla KPC-11 ). The MB-PCR detection limit was 5 to 40 DNA copies per reaction and 4 CFU per reaction using laboratory-prepared samples. The MB-PCR probes were highly specific for each bla KPC variant, and cross-reactivity was not observed using DNA isolated from several bacterial species. A total of 457 clinical Gram-negative isolates were successfully characterized by our MB-PCR assay, with bla KPC-3 and bla KPC-2 identified as the most common types in the New York/New Jersey metropolitan region. The MB-PCR assay described herein is rapid, sensitive, and specific and should be useful for understanding the ongoing evolution of carbapenem resistance in Gram-negative bacteria. As novel bla KPC variants continue to emerge, the MB-PCR assay can be modified in response to epidemiologic developments.

We conducted a retrospective study of 17 transplant recipients with carbapenem-resistant Klebsiella pneumoniae bacteremia, and described epidemiology, clinical characteristics and strain genotypes. Eighty-eight percent (15/17) of patients were liver or intestinal transplant recipients. Outcomes were death due to septic shock (18%), cure (24%) and persistent (>7 days) or recurrent bacteremia (29% each). Thirty- and 90-day mortality was 18% and 47%, respectively. Patients who were cured received at least one active antimicrobial agent and underwent source control interventions. Forty-one percent (7/17) of patients had intra-abdominal infections; all except one developed persistent/recurrent bacteremia despite drainage. Two patients tolerated persistent bacteremia for >300 days. All patients except one were infected with sequence type 258 (ST258), K. pneumoniae carbapenemase (KPC)-2-producing strains harboring a mutant ompK35 porin gene; the exception was infected with an ST37, KPC-3-producing strain. Seventy-one percent (12/17) of patients were infected with ST258 ompK36 mutant strains. In two patients, persistent bacteremia was caused by two strains with different ompK36 genotypes. Three ompK36 mutations were associated with significantly higher carbapenem minimum inhibitory concentrations than wild-type ompK36. Pulse-field gel electrophoresis identified a single ST258 lineage; serial strains from individual patients were indistinguishable. In conclusion, KPC-K. pneumoniae bacteremia exhibited highly diverse clinical courses following transplantation, and was caused by clonal ST258 strains with different ompK36 genotypes.

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