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Library preparation for high-throughput sequencing applications is a critical step in producing representative, unbiased sequencing data. The iGenomX Riptide High Throughput Rapid Library Prep Kit purports to provide high-quality sequencing data with lower costs compared to other Illumina library kits. To test these claims, we compared sequence data quality of Riptide libraries to libraries constructed with KAPA Hyper and NEBNext Ultra. Across several single-source genome samples, mapping performance and de novo assembly of Riptide libraries were similar to conventional libraries prepared with the same DNA. Poor performance of some libraries resulted in low sequencing depth. In particular, degraded DNA samples may be challenging to sequence with Riptide. There was little cross-well plate contamination with the overwhelming majority of reads belong to the proper source genomes. The sequencing of metagenome samples using different Riptide primer sets resulted in variable taxonomic assignment of reads. Increased adoption of the Riptide kit will decrease library preparation costs. However, this method might not be suitable for degraded DNA.

Background In an investigation of hospital-acquired mucormycosis cases among transplant recipients, healthcare linens (HCLs) delivered to our center were found to be contaminated with Mucorales. We describe an investigation and remediation of Mucorales contamination at the laundry supplying our center. Methods We performed monthly RODAC cultures of HCLs upon hospital arrival, and conducted site inspections and surveillance cultures at the laundry facility. Remediation was designed and implemented by infection prevention and facility leadership teams. Results Prior to remediation, 20% of HCLs were culture-positive for Mucorales upon hospital arrival. Laundry facility layout and processes were consistent with industry standards. Significant step-ups in Mucorales and mold culture-positivity of HCLs were detected at the post-dryer step (0% to 12% [P = .04] and 5% to 29% [P = .01], respectively). Further increases to 17% and 40% culture-positivity, respectively, were noted during pre-transport holding. Site inspection revealed heavy Mucorales-positive lint accumulation in rooftop air intake and exhaust vents that cooled driers; intake and exhaust vents that were facing each other; rooftop and plant-wide lint accumulation, including in the pre-transport clean room; uncovered carts with freshly-laundered HCLs. Following environmental remediation, quality assurance measures and education directed toward these sources, Mucorales culture-positivity of newly-delivered HCLs was reduced to 0.3% (P = .0001); area of lint-contaminated rooftop decreased from 918 m2 to 0 m2 on satellite images. Conclusions Targeted laundry facility interventions guided by site inspections and step-wise culturing significantly reduced Mucorales-contaminated HCLs delivered to our hospital. Collaboration between infection prevention and laundry facility teams was crucial to successful remediation.

Background Multidrug-resistant Enterobacterales (MDR-E), including carbapenem-resistant and third-generation cephalosporin-resistant Enterobacterales (CRE, CefR-E), are major pathogens following solid organ transplantation (SOT). Methods We prospectively studied patients who underwent lung, liver, and small bowel transplant from February 2015 through March 2017. Weekly perirectal swabs (up to 100 days post-transplant) were cultured for MDR-E. Whole-genome sequencing (WGS) was performed on gastrointestinal (GI) tract–colonizing and disease-causing isolates. Results Twenty-five percent (40 of 162) of patients were MDR-E GI-colonized. Klebsiella pneumoniae was the most common CRE and CefR-E. Klebsiella pneumoniae carbapenemases and CTX-M were leading causes of CR and CefR, respectively. Thirty-five percent of GI colonizers developed MDR-E infection vs 2% of noncolonizers (P < .0001). The attack rate was higher among CRE colonizers than CefR-E colonizers (53% vs 21%, P = .049). GI colonization and high body mass index were independent risk factors for MDR-E infection (P ≤ .004). Thirty-day mortality among infected patients was 6%. However, 44% of survivors developed recurrent infections; 43% of recurrences were late (285 days to 3.9 years after the initial infection). Long-term survival (median, 4.3 years post-transplant) did not differ significantly between MDR-E–infected and MDR-E–noninfected patients (71% vs 77%, P = .56). WGS phylogenetic analyses revealed that infections were caused by GI-colonizing strains and suggested unrecognized transmission of novel clonal group-258 sublineage CR-K. pneumoniae and horizontal transfer of resistance genes. Conclusions MDR-E GI colonization was common following SOT and predisposed patients to infections by colonizing strains. MDR-E infections were associated with low short- and long-term mortality, but recurrences were frequent and often occurred years after initial infections. Findings provide support for MDR-E surveillance in our SOT program.

We compared the in vitro activities of gentamicin (GEN), tobramycin (TOB), amikacin (AMK), and plazomicin (PLZ) against 13 Enterobacter isolates possessing both Klebsiella pneumoniae carbapenemase and extended-spectrum β-lactamase (KPC+/ESBL+) with activity against 8 KPC+/ESBL−, 6 KPC−/ESBL+, and 38 KPC−/ESBL− isolates. The rates of resistance to GEN and TOB were higher for KPC+/ESBL+ (100% for both) than for KPC+/ESBL− (25% and 38%, respectively), KPC−/ESBL+ (50% and 17%, respectively), and KPC−/ESBL− (0% and 3%, respectively) isolates. KPC+/ESBL+ isolates were more likely than others to possess an aminoglycoside-modifying enzyme (AME) (100% versus 38%, 67%, and 5%; P = 0.007, 0.06, and P < 0.01 for all). KPC+/ESBL+ isolates also had a greater number of AMEs (mean of 4.6 versus 1.5, 0.9, and 0.05, respectively; P < 0.01 for all). GEN and TOB MICs were higher against isolates with >1 AME than with ≤1 AME. The presence of at least 2/3 of KPC, SHV, and TEM predicted the presence of AMEs. PLZ MICs against all isolates were ≤4 μg/ml, regardless of KPC/ESBL pattern or the presence of AMEs. In conclusion, GEN and TOB are limited as treatment options against KPC+ and ESBL+ Enterobacter . PLZ may represent a valuable addition to the antimicrobial armamentarium. A full understanding of AMEs and other aminoglycoside resistance mechanisms will allow clinicians to incorporate PLZ rationally into treatment regimens. The development of molecular assays that accurately and rapidly predict antimicrobial responses among KPC- and ESBL-producing Enterobacter spp. should be a top research priority.

Background The majority of bacterial BSIs are believed to stem from a single, clonal organism. We hypothesized that most CRK BSIs are caused by genetically diverse, clonal strains that exhibit different phenotypes. Methods Blood cultures (BCs) that were positive for CRK from each of 10 patients (patients) were streaked onto agar plates, and 100 individual colonies were chosen for Illumina whole-genome sequencing. Strains from 3 patients were tested for in vitro phenotypes and virulence in mice. Results Patients had BSIs due to ST258 K. pneumoniae (Kp; 6), non-ST258 Kp (3), and K. michiganensis (Km; 1). 5 patients were infected with strains that differed by core genome single nucleotide polymorphism phylogeny (2–5 unique genotypes/patient) [figure]. 6 patients were infected with strains that differed by gene or plasmid content, and/or gene deletions [table]. Differences in individual patients encompassed antibiotic resistance and putative virulence genes (including mixtures of blaKPC+ and blaKPC– strains, and various capsular (CPS) and porin mutant strains). In total, BSIs in 8 of 10 patients were caused by a genotypically diverse population. In each of 3 patients, genotypically diverse ST258 Kp strains demonstrated significant differences in antibiotic susceptibility, CPS content, mucoviscosity, adherence, resistance to serum killing, and mortality rates and tissue burdens during BSIs of mice. ST258 strains from a pt with and without a KPC-bearing IncFIA plasmid differed in β-lactam susceptibility, but were equally virulent. Progressive loss of CPS in ST258 strains from another patient enhanced serum killing and adherence, and attenuated virulence. Using PCR markers to test 96 colonies per positive BC bottle, we demonstrated that strains selected by the clinical micro lab accounted for 2% to 98% of a population in different patients. Conclusion CRK causing BSIs in most patients demonstrated remarkable genotypic diversity, which impacted antibiotic susceptibility, virulence and other phenotypes. Differences were not recognized during hospitalization since clinical labs select single, morphologically distinct colonies for evaluation. Studies are needed to understand the clinical implications of our findings, diversity during other BSIs, and whether clinical lab practices need revision. Disclosures All authors: No reported disclosures.

We identified four bla KPC-3 mutations in ceftazidime-avibactam-resistant clinical Klebsiella pneumoniae isolates, corresponding to D179Y, T243M, D179Y/T243M, and EL165-166 KPC-3 variants. Using site-directed mutagenesis and transforming vectors into Escherichia coli , we conclusively demonstrated that mutant bla KPC-3 encoded enzymes that functioned as extended-spectrum β-lactamases; mutations directly conferred higher MICs of ceftazidime-avibactam and decreased the MICs of carbapenems and other β-lactams. Impact was strongest for the D179Y mutant, highlighting the importance of the KPC Ω-loop.

Ceftazidime-avibactam is a novel β-lactam/β-lactamase inhibitor with activity against carbapenem-resistant Enterobacteriaceae (CRE) that produce Klebsiella pneumoniae carbapenemase (KPC). We report the first cases of ceftazidime-avibactam resistance to develop during treatment of CRE infections and identify resistance mechanisms. Ceftazidime-avibactam-resistant K. pneumoniae emerged in three patients after ceftazidime-avibactam treatment for 10 to 19 days. Whole-genome sequencing (WGS) of longitudinal ceftazidime-avibactam-susceptible and -resistant K. pneumoniae isolates was used to identify potential resistance mechanisms. WGS identified mutations in plasmid-borne bla KPC-3 , which were not present in baseline isolates. bla KPC-3 mutations emerged independently in isolates of a novel sequence type 258 sublineage and resulted in variant KPC-3 enzymes. The mutations were validated as resistance determinants by measuring MICs of ceftazidime-avibactam and other agents following targeted gene disruption in K. pneumoniae , plasmid transfer, and bla KPC cloning into competent Escherichia coli . In rank order, the impact of KPC-3 variants on ceftazidime-avibactam MICs was as follows: D179Y/T243M double substitution > D179Y > V240G. Remarkably, mutations reduced meropenem MICs ≥4-fold from baseline, restoring susceptibility in K. pneumoniae from two patients. Cefepime and ceftriaxone MICs were also reduced ≥4-fold against D179Y/T243M and D179Y variant isolates, but susceptibility was not restored. Reverse transcription-PCR revealed that expression of bla KPC-3 encoding D179Y/T243M and D179Y variants was diminished compared to bla KPC-3 expression in baseline isolates. In conclusion, the development of resistance-conferring bla KPC-3 mutations in K. pneumoniae within 10 to 19 days of ceftazidime-avibactam exposure is troubling, but clinical impact may be ameliorated if carbapenem susceptibility is restored in certain isolates.

Background Data on the use of ceftolozane-tazobactam and emergence of ceftolozane-tazobactam resistance during multidrug resistant (MDR)-Pseudomonas aeruginosa infections are limited. Methods We performed a retrospective study of 21 patients treated with ceftolozane-tazobactam for MDR-P. aeruginosa infections. Whole genome sequencing and quantitative real-time polymerase chain reaction were performed on longitudinal isolates. Results Median age was 58 years; 9 patients (43%) were transplant recipients. Median simplified acute physiology score-II (SAPS-II) was 26. Eighteen (86%) patients were treated for respiratory tract infections; others were treated for bloodstream, complicated intraabdominal infections, or complicated urinary tract infections. Ceftolozane-tazobactam was discontinued in 1 patient (rash). Thirty-day all-cause and attributable mortality rates were 10% (2/21) and 5% (1/21), respectively; corresponding 90-day mortality rates were 48% (10/21) and 19% (4/21). The ceftolozane-tazobactam failure rate was 29% (6/21). SAPS-II score was the sole predictor of failure. Ceftolozane-tazobactam resistance emerged in 3 (14%) patients. Resistance was associated with de novo mutations, rather than acquisition of resistant nosocomial isolates. ampC overexpression and mutations were identified as potential resistance determinants. Conclusions In this small study, ceftolozane-tazobactam was successful in treating 71% of patients with MDR-P. aeruginosa infections, most of whom had pneumonia. The emergence of ceftolozane-tazobactam resistance in 3 patients is worrisome and may be mediated in part by AmpC-related mechanisms. More research on treatment responses and resistance during various types of MDR-P. aeruginosa infections is needed to define ceftolozane-tazobactam's place in the armamentarium.

The pathogenesis of Candida glabrata infections is poorly understood. We studied the pathogenesis of intra-abdominal candidiasis (IAC) in mice that were infected intraperitoneally with C. glabrata and sterile feces. C. glabrata BG2 (5 × 10 8 CFU) caused a 100% mortality rate. Sublethal inocula of BG2 (1 × 10 8 or 1 × 10 7 CFU) caused peritonitis that progressed to abscesses. Three clinical C. glabrata strains (5 × 10 8 CFU) caused 80 to 100% mortality rates, while a fourth (strain 346) caused a 29% mortality rate. Following sublethal inocula (1 × 10 7 CFU), the intra-abscess burdens of virulent strain 356 were ∼1 log greater than those of strain 346. A C. glabrata Δ plb1-2 mutant (phospholipase B genes disrupted) killed mice as well as BG2 did. When sublethal inocula were used, however, the Δ plb1-2 mutant was associated with more rapid abscess resolution and lower intra-abscess burdens; these findings were reversed by PLB1 and PLB2 reinsertion. The Δ plb1-2 mutant was also more susceptible than BG2 to killing by human neutrophils in vitro . BG2 and the Δ plb1-2 mutant were indistinguishable during hematogenously disseminated candidiasis. C. albicans SC5314 was more virulent than C. glabrata BG2 during IAC, causing a 100% mortality rate following a challenge with 5 × 10 7 CFU. In contrast, a sublethal inoculum (1 × 10 7 CFU) of BG2 caused less neutrophil infiltration and greater burdens in peritoneal fluid than SC5314 did and abscesses that persisted longer and contained greater burdens. In conclusion, a mouse model of C. glabrata IAC mimics disease in humans and distinguishes the relative virulence of clinical and gene disruption strains. C. glabrata differed from C. albicans during IAC by being less lethal and eliciting dampened neutrophil responses but resulting in more persistent peritonitis and abscesses.

Background IAC is a common manifestation of invasive candidiasis, but its pathogenesis is poorly understood. We developed a mouse model of C. albicans IAC, in which disease progresses from peritonitis to abscesses (IAA) in a manner that recapitulates human infection. Our goal was to use the model to identify C. albicans TFs that regulate virulence during IAC. Methods We screened a signature-tagged library (48 unique oligonucleotide markers) of homozygous deletion mutants for 165 C. albicans TF genes, created in duplicate in strain SC5314 (S. Noble). Mice were infected intra-peritoneally in triplicate with pools of 24 mutants and wild-type, and strains harvested at 72 hours in IAA. Results Twenty-one TF mutants were significantly attenuated for virulence in both libraries, and 2 TF mutants were significantly more virulent in both libraries, as measured by tissue burdens (figure). Biologic processes over-represented among attenuated mutants were regulation of pH responses, biofilm, hyphal formation, echinocandin responses, and copper metabolism. pH responses are likely to be crucial to pathogenesis of IAC, as C. albicans transitions from pH 8 during peritonitis to pH 6.8 within IAA. 9 pH response regulators contributing to virulence included RIM101, STP2 (alkaline), ASH1, SFL1, SFL2 (neutral), MNL1, SKO1, PHO4 (weak acid), and CSR1 (acid). We created rim101 null mutant and reconstitution strains, and demonstrated that the gene was essential for complete virulence during peritonitis and IAA. Transcriptional profiling of strains by RT-PCR during peritonitis and in vitro showed both conserved and rewired Rim101 targets. SAP5, which encodes an aspartyl protease, is a major Rim101 target in vivo and in vitro; over-expression of SAP5 in rim101 restored virulence during IAA at 3, 7, and 10 days, but not during peritonitis. Other pH regulatory TF genes are currently being validated as virulence determinants, and pathway relationships between MNL1, SKO1, and PHO4 during IAA formation are being explored through epistasis approaches. Conclusion Screening of a C. albicans TF mutant library identified pH responses and other biologic processes as important during pathogenesis of IAC. Rim101, an alkaline pH response regulator, contributes to both peritonitis and IAA, the latter at least in part through its effects on Sap5. Disclosures All Authors: No reported Disclosures.

Background We developed and validated a mouse model of C. albicans IAC that mimics peritonitis and abscesses (IAA) in humans, and that is amenable to temporal–spatial transcriptional profiling and virulence studies. Methods We measured C. albicans SC5314 gene expression by RNA-Seq (RiboPure extraction; Illumina MiSeq) in triplicate during early peritonitis (within 30 minutes of infection), late peritonitis (24 hours) and IAA (48 hours). Differential expression was defined by ≥2-fold differences at false discovery rate ≤0.01. Results ≥7 million C. albicans reads were detected in each experiment. 67% of C. albicans reads mapped to coding sequences, covering 93% of open reading frames. The 50 C. albicans genes most highly expressed during early peritonitis were associated with pH (including RIM101 and PHR1) and oxidative stress responses, and adhesion/hyphal growth (e.g., ALS3, HWP1, ECM331, SAP6). The corresponding 50 C. albicans late peritonitis genes were associated with neutrophil/macrophage responses and nutrient acquisition (glyoxylate cycle, fatty acid β-oxidation, iron homeostasis). Responses within IAA included DNA damage and iron metabolism, reflecting stress response and nutrient/metal limitation. The top 50 core gene responses for all stages were associated with adhesion, stress response, and glucose transport. Among the most up-regulated genes in late peritonitis and IAA compared with early peritonitis were those involved antifungal drug resistance (CDR family, MDR1, FLU1, and ERG family), although mice were not exposed to antifungals. Null and reconstitution mutants for genes involved in adhesion (ALS3), copper transport (CCC2), DNA (DDI1) and cell wall damage responses (DAP1 homologs), and glycerol biosynthesis (RHR2) were attenuated for virulence in temporal-spatial fashion during peritonitis and IAA, and/or hyper-susceptible to phagocytosis and echinocandins (table). Conclusion C. albicans relies upon diverse biologic processes to cause peritonitis and IAA. Multiple genes induced in response to stress during IAC mediate virulence, phagocyte, and echinocandin resistance. Therefore, pathogenic strategies used by C. albicans during IAC may lessen responses to echinocandin treatment, even in the absence of drug exposure or FKS mutations. Disclosures All authors: No reported disclosures.

Doripenem-colistin exerts synergy against some, but not all, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains in vitro . We determined if doripenem MICs and/or ompK36 porin gene mutations impacted the responses of 23 sequence type 258 (ST258), KPC-2-producing strains to the combination of doripenem (8 μg/ml) and colistin (2 μg/ml) during time-kill assays. The median doripenem and colistin MICs were 32 and 4 μg/ml. Doripenem MICs did not correlate with KPC-2 expression levels. Five and 18 strains had wild-type and mutant ompK36 , respectively. The most common mutations were IS 5 promoter insertions ( n = 7) and insertions encoding glycine and aspartic acid at amino acid (aa) positions 134 and 135 (ins aa134-135 GD; n = 8), which were associated with higher doripenem MICs than other mutations or wild-type ompK36 (all P values ≤ 0.04). Bactericidal activity (24 h) was achieved by doripenem-colistin against 12%, 43%, and 75% of ins aa134-135 GD, IS 5 , and wild-type/other mutants, respectively ( P = 0.04). Doripenem-colistin was more active in time-kill studies than colistin at 12 and 24 h if the doripenem MIC was ≤8 μg/ml ( P = 0.0007 and 0.09, respectively), but not if the MIC was >8 μg/ml ( P = 0.10 and 0.16). Likewise, doripenem-colistin was more active at 12 and 24 h against the wild type/other mutants than ins aa134-135 GD or IS 5 mutants ( P = 0.007 and 0.0007). By multivariate analysis, the absence of ins aa134-135 GD or IS 5 mutations was the only independent predictor of doripenem-colistin responses at 24 h ( P = 0.002). In conclusion, ompK36 genotypes identified ST258 KPC- K. pneumoniae strains that were most likely to respond to doripenem-colistin.

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.

Background. The pathogenesis of intra-abdominal candidiasis is poorly understood. Methods. Mice were intraperitoneally infected with Candida albicans (1 × 106 colony-forming units) and sterile stool. nanoString assays were used to quantitate messenger RNA for 145 C. albicans genes within the peritoneal cavity at 48 hours. Results. Within 6 hours after infection, mice developed peritonitis, characterized by high yeast burdens, neutrophil influx, and a pH of 7.9 within peritoneal fluid. Organ invasion by hyphae and early abscess formation were evident 6 and 24 hours after infection, respectively; abscesses resolved by day 14. nanoString assays revealed adhesion and responses to alkaline pH, osmolarity, and stress as biologic processes activated in the peritoneal cavity. Disruption of the highly-expressed gene RIM101, which encodes an alkaline-regulated transcription factor, did not impact cellular morphology but reduced both C. albicans burden during early peritonitis and C. albicans persistence within abscesses. RIM101 influenced expression of 49 genes during intra-abdominal candidiasis, including previously unidentified Rim101 targets. Overexpression of the RIM101-dependent gene SAP5, which encodes a secreted protease, restored the ability of a rim101 mutant to persist within abscesses. Conclusions. A mouse model of intra-abdominal candidiasis is valuable for studying pathogenesis and C. albicans gene expression. RIM101 contributes to persistence within intra-abdominal abscesses, at least in part through activation of SAP5.

We characterized carbapenem resistance mechanisms among 12 Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (referred to here as KPC K. pneumoniae ) clinical isolates and evaluated their effects on the activity of 2- and 3-drug combinations of colistin, doripenem, and ertapenem. All isolates were resistant to ertapenem and doripenem; 75% (9/12) were resistant to colistin. Isolates belonged to the ST258 clonal group and harbored bla KPC-2 , bla SHV-12 , and bla TEM-1 . As determined by time-kill assays, doripenem (8 μg/ml) and ertapenem (2 μg/ml) were inactive against 92% (11/12) and 100% (12/12) of isolates, respectively. Colistin (2.5 μg/ml) exerted some activity (range, 0.39 to 2.5 log 10 ) against 78% (7/9) of colistin-resistant isolates. Colistin-ertapenem, colistin-doripenem, and colistin-doripenem-ertapenem exhibited synergy against 42% (5/12), 50% (6/12), and 67% (8/12) of isolates, respectively. Expression of ompK35 and ompK36 porins correlated with each other ( R 2 = 0.80). Levels of porin expression did not correlate with colistin-doripenem or colistin-ertapenem synergy. However, synergy with colistin-doripenem-ertapenem was more likely against isolates with high porin expression than those with low expression (100% [8/8] versus 0% [0/4]; P = 0.002). Moreover, bactericidal activity (area under the bacterial killing curve) against isolates with high porin expression was greater for colistin-doripenem-ertapenem than colistin-doripenem or colistin-ertapenem ( P ≤ 0.049). In conclusion, colistin-carbapenem combinations may provide optimal activity against KPC K. pneumoniae , including colistin-resistant isolates. Screening for porin expression may identify isolates that are most likely to respond to a triple combination of colistin-doripenem-ertapenem. In the future, molecular characterization of KPC K. pneumoniae isolates may be a practical tool for identifying effective combination regimens.

Caspofungin exerts candidacidal activity by inhibiting cell wall (1,3)-β- d -glucan synthesis. We investigated the physiologic mechanisms of caspofungin-induced Candida albicans cell death. Apoptosis (programmed cell death) and necrosis were studied after C. albicans SC5314 cells were exposed to caspofungin at 0.06, 0.125, and 0.5 μg/ml (0.5×, 1×, and 4× the MIC, respectively) for 3 h. Caspofungin at 0.125 and 0.5 μg/ml reduced cellular viability by >50%, as measured by colony counts and methylene blue exclusion. Apoptosis and necrosis were demonstrated by annexin V and propidium iodide staining for phosphatidylserine externalization and loss of membrane integrity, respectively. At all concentrations of caspofungin, 20 to 25% and 5 to 7% of C. albicans cells exhibited early apoptosis and late apoptosis/necrosis, respectively ( P value was not significant [NS]). Necrosis, on the other hand, was significantly greater at 0.125 (43%) and 0.5 (48%) μg/ml than at 0.06 μg/ml (26%) ( P values of 0.003 and 0.003, respectively). The induction of apoptosis at concentrations less than or equal to the MIC was corroborated by dihydrorhodamine 123 (DHR-123) and dihydroethidium (DHE) staining (reactive oxygen species production), JC-1 staining (mitochondrial membrane potential dissipation), and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining (DNA damage and nuclear fragmentation). Moreover, electron microscopy of cells exposed to 0.125 μg/ml of caspofungin showed hallmark apoptotic features like chromatin margination and condensation and nuclear blebs. Apoptosis was associated with metacaspase 1 activation, as demonstrated by D2R staining. Caspofungin exerts activity against C. albicans by directly killing cells (resulting in necrosis) and causing others to undergo programmed cell death (apoptosis). Apoptosis is initiated at subinhibitory concentrations, suggesting that strategies to target this process may augment the benefits of antifungal agents.

We used ribonucleic acid sequencing to profile Candida albicans transcription within biliary fluid from a patient with cholangitis; samples were collected before and after treatment with fluconazole and drainage. Candida albicans transcriptomes at the infection site distinguished treated from untreated cholangitis. After treatment, 1131 C. albicans genes were differentially expressed in biliary fluid. Up-regulated genes were enriched in hyphal growth, cell wall organization, adhesion, oxidation reduction, biofilm, and fatty acid and ergosterol biosynthesis. This is the first study to define Candida global gene expression during deep-seated human infection. Successful treatment of cholangitis induced C. albicans genes involved in fluconazole responses and pathogenesis.

Background Candida parapsilosis fungemia typically occurs in patients with intravascular catheters or prosthetic devices. In 2017, we noted an increase in C. parapsilosis infective endocarditis (IE). Methods We retrospectively reviewed C. parapsilosis fungemia and IE from January 2015 to February 2018. Species were identified using MALDI-TOF, and confirmed by ITS sequencing. Results Between 2010 and 2017, there was no increase in cases of C. parapsilosis fungemia (mean: 13/year), but there was a significant increase in C. parapsilosis IE (P = 0.048) (Figure 1). From January 2015 to February 2018, 22% (12/54) of C. parapsilosis fungemia was complicated by IE. Demographics of C. parapsilosis fungemia included: community-acquired infection (87%), presence of vascular catheters (80%), opiate noninjection drug use (non-IDU, 44%), IDU (20%), and presence of cardiac devices (18%). Ninety-one percent (49/54) of C. parapsilosis fungemia was caused by C. parapsilosis sensu strictu (Cpss); C. orthopsilosis and C. metapsilosis accounted for 4% (2/54) each (1 isolate could not be subtyped). Cpss, C. orthopsilosis, and C. metapsilosis accounted for 83% (10/12), 8% (1/12), and 8% (1/12) of IE, respectively. Ninety-two% (11/12) of C. parapsilosis IE was left-sided, and 33% (4/12) involved multiple valves. Risk factors for C. parapsilosis IE were past or active IDU (P < 0.001), community-acquired fungemia (P = 0.02), prosthetic heart valve (P = 0.01) or implanted cardiac device (P = 0.03). Receipt of an antibiotic within 30 days was a risk for C. parapsilosis fungemia without IE (P = 0.001). Median age for IE vs. fungemia was 38 vs. 57 years (P = 0.09). By multivariate logistic regression, IDU (P < 0.0001), prosthetic valve (P = 0.006) or implanted cardiac device (P = 0.04) were independent risks for C. parapsilosis IE. 70% (7/10), 20% (2/10), and 10% (1/10) of patients with IDU and C. parapsilosis IE primarily used heroin, buprenorphine/naltrexone, and cocaine, respectively. 50% (6/12) of patients with C. parapsilosis IE underwent surgery; most common initial AF regimens were caspofungin and amphotericin B. Nonsurgical patients were suppressed with long-term azole; one relapsed requiring surgery. Thirty-day and in-hospital mortality for patients with fungemia vs. IE were 32% vs. 17% and 26% vs. 17%, respectively. Conclusion C. parapsilosis IE has emerged at our center. Unique aspects of C. parapsilosis pathogenesis that may account for emergence are a propensity to colonize skin, adhere to prosthetic material and form biofilm. C. parapsilosis IE may be an under-appreciated consequence of IDU and opioid abuse. Disclosures All authors: No reported disclosures.

In this study, we measured time-kills and post-antifungal effects (PAFEs) for micafungin against Candida albicans (n=4), Candida glabrata (n=3), Candida parapsilosis (n=3) and Candida krusei (n=2) isolates and further characterised the PAFEs. Minimum inhibitory concentrations (MICs) were 0.5-1.0 mg/L against C. parapsilosis and 0.008-0.125 mg/L against the other species. Micafungin caused kills >1 log at 1 x MIC, 4 x MIC (range 1.19-3.10 log) and 16 x MIC (2.27-3.68 log), achieving fungicidal levels (> or = 3 log) against nine isolates. One-hour drug exposure during PAFE experiments resulted in kills of 0.73-2.88 log and 1.72-3.55 log at 4 x and 16 x MIC, respectively, achieving fungicidal levels against five isolates. Isolates of each species collected 8 h after a 1-h exposure to micafungin (4 x and 16 x MIC) were hypersusceptible to sodium dodecyl sulphate (SDS) and Calcofluor White. Cells tested during the PAFE period demonstrated cell wall disturbances as evident on electron micrographs as well as significant reductions in adherence to epithelial cells. Phagocytosis by J774 macrophages was significantly enhanced for three PAFE isolates tested. Micafungin is fungicidal and exerts PAFEs that kill diverse Candida spp., disturb cell walls of viable organisms, reduce adherence and enhance susceptibility to phagocytosis.

Anidulafungin targets the cell walls of Candida species by inhibiting β-1,3-glucan synthase, thereby killing isolates and exerting prolonged postantifungal effects (PAFEs). We performed time-kill and PAFE experiments on Candida albicans ( n = 4), C. glabrata ( n = 3), C. parapsilosis ( n = 3), and C. krusei ( n = 2) isolates and characterized the PAFEs in greater detail. MICs were 0.008 to 0.125 μg/ml against C. albicans , C. glabrata , and C. krusei and 1.0 to 2.0 μg/ml against C. parapsilosis . During time-kill experiments, anidulafungin caused significant kills at 16× MIC (range, log 2.68 to 3.89) and 4× MIC (log 1.87 to 3.19), achieving fungicidal levels (≥log 3) against nine isolates. A 1-hour drug exposure during PAFE experiments resulted in kills ranging from log 1.55 to 3.47 and log 1.18 to 2.89 (16× and 4× MIC, respectively), achieving fungicidal levels against four isolates. Regrowth of all 12 isolates was inhibited for ≥12 h after drug washout. Isolates of each species collected 8 h after a 1-hour exposure to anidulafungin (16× and 4× MIC) were hypersusceptible to sodium dodecyl sulfate (0.01 to 0.04%) and calcofluor white (40 μg/ml). Moreover, PAFEs were associated with major cell wall disturbances, as evident in electron micrographs of viable cells, and significant reductions in adherence to buccal epithelial cells ( P ≤ 0.01). Finally, three of four PAFE isolates tested were hypersusceptible to killing by J774 macrophages ( P ≤ 0.007). Our data suggest that the efficacy of anidulafungin in the treatment of candidiasis might stem from both direct fungicidal activity and indirect PAFEs that lessen the ability of Candida cells to establish invasive disease and to persist within infected hosts.

Cryptococcus neoformansis a common cause of meningoencephalitis among AIDS patients. SeveralC. neoformansvirulence factors have been identified, but the relative importance of particular factors is unknown. This study examined the corrrelation of the virulence of 18C. neoformansvar.grubiiisolates from AIDS patients with the expression of several well-described virulence factors. The LD50at 15 days after intracranial inoculation of ICR mice was 20 000 for 39 %. Higher cryptococcal concentrations in brains were noted for isolates with lower LD50(P=0.002). In survival studies, no immunocompetent BALB/c mice (nu/−) infected with 3×LD50of three virulent isolates (LD50=62, 99, 1280) survived beyond 23 days, whereas 100 %, 90 % and 90 % of mice infected with 20 000 c.f.u. of three hypovirulent isolates (LD50>20 000) survived for 60 days (Pnu/nu) mice, survival rates over 60 days were 100 %, 70 % and 50 %, respectively, for the hypovirulent isolates. Growth rate at 37 °C and capsule size within brains correlated with LD50by univariate (P=0.0001 and 0.028, respectively) and multivariate (P=0.017 and 0.016, respectively) analyses. There was no correlation between LD50and capsule sizein vitro, phospholipase activity, melanin formation, proteinase activity and fluconazole MIC. In conclusion, AIDS patients are susceptible to infection byC. neoformansisolates of wide-ranging virulence, including isolates that are markedly hypovirulent. The virulence of a given isolate reflects a composite of factors rather than the contribution of a dominant factor. Growth at 37 °C and capsule sizein vivomake particularly important contributions.

Candida albicansis a common cause of mucosal and bloodstream infections. As a screening strategy to identify novel candidal virulence factors, sera recovered from HIV-infected patients with active oropharyngeal candidiasis (OPC) were previously used to probe aC. albicansgenomic expression library.IRS4was identified as a gene that encodes an immunogenic protein. In the present study, the presence ofIRS4transcripts was verified within OPC pseudomembranes recovered from patients. Having confirmed that the gene is expressed during human candidiasis, gene disruption strains were created and this implicatedIRS4in diverse processes, including hyphal formation on solid media and under embedded conditions, cell wall integrity and structure, and adherence to human epithelial cellsin vitro.IRS4disruption, however, did not influence hyphal formation or virulence in a murine model of OPC. Rather, the gene was found to be necessary for normal morphogenesis and full virulence during murine intravenously disseminated candidiasis (DC).IRS4's effects on hyphal formation and virulence during DC were not evident on the first day after intravenous inoculation, even though transcripts were detected within murine kidneys. After 4 days, however, anirs4null mutant strain was associated with attenuated mortality, diminished tissue burdens, less extensive infections, impairedC. albicanshyphal formation and decreased kidney damage. Taken together, these findings suggest thatIRS4makes distinct temporal-spatial contributions to the pathogenesis of candidiasis, which appear to vary between different tissue sites as well as within a given tissue over time.

We developed a high-performance liquid chromatography (HPLC) assay to validate time-kill and postantifungal-effect (PAFE) experiments for voriconazole against Candida albicans , Candida glabrata , and Candida parapsilosis isolates. Voriconazole exerted prolonged fungistatic activity but no PAFE at concentrations achievable in human sera. HPLC confirmed that experiments were conducted at the desired steady-state voriconazole concentrations.

Echinocandins are frontline agents against invasive candidiasis (IC), but predictors for echinocandin therapeutic failure have not been well defined. Mutations in Candida FKS genes, which encode the enzyme targeted by echinocandins, result in elevated MICs and have been linked to therapeutic failures. In this study, echinocandin MICs by broth microdilution and FKS1 and FKS2 mutations among C. glabrata isolates recovered from patients with IC at our center were correlated retrospectively with echinocandin therapeutic responses. Thirty-five patients with candidemia and 4 with intra-abdominal abscesses were included, 92% (36/39) of whom received caspofungin. Twenty-six percent (10) and 74% (29) failed and responded to echinocandin therapy, respectively. Caspofungin, anidulafungin, and micafungin MICs ranged from 0.5 to 8, 0.03 to 1, and 0.015 to 0.5 μg/ml, respectively. FKS mutations were detected in 18% (7/39) of C. glabrata isolates ( FKS1 , n = 2; FKS2 , n = 5). Median caspofungin and anidulafungin MICs were higher for patients who failed therapy ( P = 0.04 and 0.006, respectively). By receiver operating characteristic (ROC) analyses, MIC cutoffs that best predicted failure were >0.5 (caspofungin), >0.06 (anidulafungin), and >0.03 μg/ml (micafungin), for which sensitivity/specificity were 60%/86%, 50%/97%, and 40%/90%, respectively. Sensitivity/specificity of an FKS mutation in predicting failure were 60%/97%. By univariate analysis, recent gastrointestinal surgery, prior echinocandin exposure, anidulafungin MIC of >0.06 μg/ml, caspofungin MIC of >0.5 μg/ml, and an FKS mutation were significantly associated with failure. The presence of an FKS mutation was the only independent risk factor by multivariate analysis ( P = 0.002). In conclusion, detection of C. glabrata FKS mutations was superior to MICs in predicting echinocandin therapeutic responses among patients with IC.

The ability to identify patients at particularly low risk for invasive aspergillosis (IA) would facilitate more efficient targeting of antifungal prophylaxis. We measured baseline serum immunoglobulin responses against 6 purified recombinant Aspergillus fumigatus proteins before hematopoietic stem cell transplantation (HSCT) or chemotherapy in 73 subjects, including 19 patients who subsequently developed proven or probable IA and 54 uninfected controls. We also assessed responses at the time of IA diagnosis and 4 weeks later (acute and convalescent sera, respectively). Baseline IgG responses against enolase, Ahp1, Hsp90, Crf1, and Cdc37 were significantly higher in the patients with IA compared with controls (P < .05). Cutoff concentrations identified by reveiver-operating characteristic curve analysis were 67%-84% sensitive and 52%-67% specific. In a population with a 15% likelihood of developing IA, positive and negative predictive values would be 22%-26% and 92%-95%, respectively. Positive IgG responses against Hsp90, Pep2, Crf1, and Cdc37 were specifically associated with early-onset IA (

Background. The sensitivity of blood cultures for diagnosing invasive candidiasis (IC) is poor. Methods. We performed a validated Candida real-time polymerase chain reaction (PCR) and the Fungitell 1,3-b-D-glucan (BDG) assay on blood samples collected from prospectively identified patients with IC (n 5 55) and hospitalized controls (n 5 73). Patients with IC had candidemia (n 5 17), deep-seated candidiasis (n 5 33), or both (n 55). Controls had mucosal candidiasis (n 55), Candida colonization (n 5 48), or no known Candida colonization (n 5 20). Results. PCR using plasma or sera was more sensitive than whole blood for diagnosing IC (P 5 .008). Plasma or sera PCR was more sensitive than BDG in diagnosing IC (80% vs 56%; P 5 .03), with comparable specificity (70% vs 73%; P 5 .31). The tests were similar in diagnosing candidemia (59% vs 68%; P 5 .77), but PCR was more sensitive for deep-seated candidiasis (89% vs 53%; P 5 .004). PCR and BDG were more sensitive than blood cultures among patients with deep-seated candidiasis (88% and 62% vs 17%; P 5 .0005 and .003, respectively). PCR and culture identified the same Candida species in 82% of patients. The sensitivity of blood cultures combined with PCR or BDG among patients with IC was 98% and 79%, respectively. Conclusions. Candida PCR and, to a lesser extent, BDG testing significantly enhanced the ability of blood cultures to diagnose IC.

Animal models are powerful tools to study the pathogenesis of diverse types of candidiasis. Murine models are particularly attractive because of cost, ease of handling, technical feasibility, and experience with their use. In this chapter, we describe methods for two of the most popular murine models of disease caused by Candida albicans. In an intravenously disseminated candidiasis (DC) model, immunocompetent mice are infected by lateral tail vein injections of a C. albicans suspension. Endpoints include mortality, tissue burdens of infection (most importantly in the kidneys, although spleens and livers are sometimes also assessed), and histopathology of infected organs. In a model of oral/esophageal candidiasis, mice are immunosuppressed with cortisone acetate and inoculated in the oral cavities using swabs saturated with a C. albicans suspension. Since mice do not die from oral candidiasis in this model, endpoints are tissue burden of infection and histopathology. The DC and oral/esophageal models are most commonly used for studies of C. albicans virulence, in which the disease-causing ability of a mutant strain is compared with an isogenic parent strain. Nevertheless, the basic techniques we describe are also applicable to models adapted to investigate other aspects of pathogenesis, such as spatiotemporal patterns of gene expression, specific aspects of host immune response and assessment of antifungal agents, immunomodulatory strategies, and vaccines.

Despite shortcomings, cultures of blood and sterile sites remain the “gold standard” for diagnosing systemic candidiasis. Alternative diagnostic markers, including antibody detection, have been developed, but none are widely accepted. In this study, we used an enzyme-linked immunosorbent assay to measure serum antibody responses against 15 recombinant Candida albicans antigens among 60 patients with systemic candidiasis due to various Candida spp. and 24 uninfected controls. Mean immunoglobulin G (IgG) responses against all 15 antigens were significantly higher among patients with systemic candidiasis than among controls, whereas IgM responses were higher against only seven antigens. Using discriminant analysis that included IgG responses against the 15 antigens, we derived a mathematical prediction model that identified patients with systemic candidiasis with an error rate of 3.7%, a sensitivity of 96.6%, and a specificity of 95.6%. Furthermore, a prediction model using a subset of four antigens (SET1, ENO1, PGK1-2, and MUC1-2) identified through backward elimination and canonical correlation analyses performed as accurately as the full panel. Using the simplified model, we predicted systemic candidiasis in a separate test sample of 32 patients and controls with 100% sensitivity and 87.5% specificity. We also demonstrated that IgG titers against each of the four antigens included in the prediction model were significantly higher in convalescent-phase sera than in paired acute-phase sera. Taken together, our findings suggest that IgG responses against a panel of candidal antigens might represent an accurate and early marker of systemic candidiasis, a hypothesis that should be tested in future trials.

We developed a pharmacokinetic/pharmacodynamic (PK/PD) mathematical model that fits voriconazole time–kill data against Candida isolates in vitro and used the model to simulate the expected kill curves for typical intravenous and oral dosing regimens. A series of Emax mathematical models were used to fit time–kill data for two isolates each of Candida albicans, Candida glabrata and Candida parapsilosis. PK parameters extracted from human data sets were used in the model to simulate kill curves for each isolate. Time–kill data were best fit by using an adapted sigmoidal Emax model that corrected for delays in candidal growth and the onset of voriconazole activity, saturation of the number of Candida and the steepness of the concentration–response curve. The rates of maximal killing by voriconazole (kmax) were highly correlated with the growth rates (ks) of the isolates (Pearson’s correlation coefficient = 0.9861). Simulations using PK parameters derived from the human data sets showed fungistatic effects against each of the isolates. In conclusion, we demonstrated that the activity of voriconazole against Candida isolates can be accurately described using a mathematical model. In the future, it might be possible to devise optimal dosing regimens of voriconazole using the model and PK data collected in vivo.

We showed that a Candida albicans petite mutant in which oxidative phosphorylation is uncoupled was eightfold more resistant to fluconazole and voriconazole than SC5314 but equally susceptible to ketoconazole, itraconazole, and amphotericin B. Strain P5 significantly overexpressed MDR1 , which likely accounts for the decreased drug susceptibility.