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β-Lactamase-mediated resistance to ceftazidime-avibactam (CZA) is a serious limitation in the treatment of Gram-negative bacteria harboring Klebsiella pneumoniae carbapenemase (KPC). Herein, the basis of susceptibility to carbapenems and resistance to ceftazidime (CAZ) and CZA of the D179Y variant of KPC-2 and -3 was explored. First, we determined that resistance to CZA in a laboratory strain of Escherichia coli DH10B was not due to increased expression levels of the variant enzymes, as demonstrated by reverse transcription PCR (RT-PCR). Using timed mass spectrometry, the D179Y variant formed prolonged acyl-enzyme complexes with imipenem (IMI) and meropenem (MEM) in KPC-2 and KPC-3, which could be detected up to 24 h, suggesting that IMI and MEM act as covalent β-lactamase inhibitors more than as substrates for D179Y KPC-2 and -3. This prolonged acyl-enzyme complex of IMI and MEM by D179Y variants was not observed with wild-type (WT) KPCs. CAZ was studied and the D179Y variants also formed acyl-enzyme complexes (1 to 2 h). Thermal denaturation and differential scanning fluorimetry showed that the tyrosine substitution at position 179 destabilized the KPC β-lactamases (KPC-2/3 melting temperature [T(m)] of 54 to 55°C versus D179Y T(m) of 47.5 to 51°C), and the D179Y protein was 3% disordered compared to KPC-2 at 318 K. Heteronuclear (1)H/(15)N-heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy also revealed that the D179Y variant, compared to KPC-2, is partially disordered. Based upon these observations, we discuss the impact of disordering of the Ω loop as a consequence of the D179Y substitution. These conformational changes and disorder in the overall structure as a result of D179Y contribute to this unanticipated phenotype.

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.

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.

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.

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 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.

Candida albicans IRS4 encodes a protein that regulates phosphatidylinositol-(4,5)-bisphosphate, which was shown to contribute to hematogenously disseminated candidiasis (DC) after several days in the standard mouse model. Our objective was to more accurately define the temporal contributions of IRS4 to pathogenesis. During competition assays in vitro , an irs4 -null (Δ irs4 ) mutant exhibited wild-type fitness. In DC experiments, mice were infected intravenously with the Δ irs4 mutant, strain CAI-12 (1 × 10 5 CFU), or a mixture of the strains (0.5 × 10 5 CFU each). In single-strain infections, quantitative PCR revealed reduced Δ irs4 mutant burdens within kidneys at days 1, 4, and 7 but not 6 h. In competitive infections, the Δ irs4 mutant was outcompeted by CAI-12 in each mouse at ≥6 h (competitive indices, P ≤ 0.0001). At 4 and 7 days, the Δ irs4 mutant burdens during competitive infections were significantly lower than those during single-strain infections ( P = 0.01 and P < 0.001, respectively), suggesting increased susceptibility to inflammatory responses. Phagocytic infiltration of kidneys in response to CAI-12 or competitive infections was significantly greater than that in response to Δ irs4 mutant infection at days 1 and 4 ( P < 0.001), and the Δ irs4 mutant was more susceptible to phagocytosis and killing by human polymorphonuclear cells ( P = 0.01 and P = 0.006, respectively) and mouse macrophages in vitro ( P = 0.04 and P = 0.01, respectively). Therefore, IRS4 contributes to tissue invasion at early stages of DC and mediates resistance to phagocytosis as DC progresses. Microarray analysis revealed remarkably similar gene expression by the Δ irs4 mutant and reference strain CAI-12 within blood, suggesting that IRS4 is not significantly involved in the hematogenous stage of disease. A competitive DC model detects attenuated virulence that is not evident with the standard model.

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 previously showed that phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and septin regulation play major roles in maintaining Candida albicans cell wall integrity in response to caspofungin and other stressors. Here, we establish a link between PI(4,5)P2 signaling and septin localization and demonstrate that rapid redistribution of PI(4,5)P2 and septins is part of the natural response of C. albicans to caspofungin. First, we studied caspofungin-hypersusceptible C. albicans irs4 and inp51 mutants, which have elevated PI(4,5)P2 levels due to loss of PI(4,5)P2-specific 5′-phosphatase activity. PI(4,5)P2 accumulated in discrete patches, rather than uniformly, along surfaces of mutants in yeast and filamentous morphologies, as visualized with a green fluorescent protein (GFP)-pleckstrin homology domain. The patches also contained chitin (calcofluor white staining) and cell wall protein Rbt5 (Rbt5-GFP). By transmission electron microscopy, patches corresponded to plasma membrane invaginations that incorporated cell wall material. Fluorescently tagged septins Cdc10 and Sep7 colocalized to these sites, consistent with well-described PI(4,5)P2-septin physical interactions. Based on expression patterns of cell wall damage response genes, irs4 and inp51 mutants were firmly positioned within a group of caspofungin-hypersusceptible, septin-regulatory protein kinase mutants. irs4 and inp51 were linked most closely to the gin4 mutant by expression profiling, PI(4,5)P2-septin-chitin redistribution and other phenotypes. Finally, sublethal 5-min exposure of wild-type C. albicans to caspofungin resulted in redistribution of PI(4,5)P2 and septins in a manner similar to those of irs4 , inp51 , and gin4 mutants. Taken together, our data suggest that the C. albicans Irs4-Inp51 5′-phosphatase complex and Gin4 function upstream of PI(4,5)P2 and septins in a pathway that helps govern responses to caspofungin.

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.

We measured time-kills and postantifungal effects (PAFEs) of caspofungin against Candida albicans , C. parapsilosis , and C. glabrata isolates. One-hour exposure to caspofungin during PAFE experiments accounted for the majority of killing during time-kill experiments. Regrowth of all isolates was inhibited for at least 24 h following drug washout.

We previously identified Candida albicans Not5p as an immunogenic protein expressed during oropharyngeal candidiasis (OPC). In this study, we demonstrate that C. albicans NOT5 reverses the growth defects of a Saccharomyces cerevisiae not5 mutant strain at 37°C, suggesting that the genes share at least some functional equivalence. We implicate C. albicans NOT5 in the pathogenesis of disseminated candidiasis (DC) induced by intravenous infection among neutropenic and nonimmunosuppressed mice, as well as in that of OPC in mice immunosuppressed with corticosteroids. We find no role in virulence, however, among neutropenic and corticosteroid-suppressed mice with DC resulting from gastrointestinal translocation, nor do we implicate the gene in vulvovaginal candidiasis among mice in pseudoestrus. These findings suggest that the role of NOT5 in virulence depends on the specific in vivo environment and is influenced by diverse factors such as tissue site, portal of entry, and the status of host defenses. NOT5 is necessary for normal adherence to colonic and cervical epithelial cells in vitro, demonstrating that such assays cannot fully replicate disease processes in vivo. Lastly, antibody responses against Not5p do not differ in the sera of patients with OPC, patients with DC, and healthy controls, suggesting that the protein is associated with both commensalism and the pathogenesis of disease.

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.