In vivo emergence of high-level resistance during treatment reveals the first identified mechanism of amphotericin B resistance in Candida auris

Candida auris has emerged as a healthcare-associated and multidrug-resistant fungal pathogen of great clinical concern. While as much as 50% of C. auris clinical isolates are reported to be resistant to amphotericin B, to date, no mechanisms contributing to this resistance have been identified. We report here mutations in the C. auris sterol-methyltransferase gene, ERG6, as the first identified mechanism of amphotericin B resistance in this emerging pathogen and describe the clinical case in which this high-level amphotericin B resistance was acquired in vivo during therapy. Whole genome sequencing revealed the four C. auris isolates obtained from this single patient case to be genetically related and identified a mutation in ERG6 as being associated with amphotericin B resistance. Cas9-mediated genetic manipulations confirmed this mutation alone to confer a >32-fold increase in amphotericin B resistance, and comprehensive sterol profiling revealed an abrogation of ergosterol biosynthesis and a corresponding accumulation of cholesta-type sterols in isolates and strains harboring the clinically derived ERG6 mutation. Together these findings represent the first significant step forward in the understanding of clinical amphotericin B resistance in C. auris.

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In Vitro and In Vivo Antifungal Activity of AmBisome Compared to Conventional Amphotericin B and Fluconazole against Candida auris

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00306-21 ◽

2021 ◽

Author(s):

Janet Herrada ◽

Ahmed Gamal ◽

Lisa Long ◽

Sonia P. Sanchez ◽

Thomas S. McCormick ◽

...

Keyword(s):

Antifungal Activity ◽

Amphotericin B ◽

Kidney Tissue ◽

Treated Group ◽

Untreated Group ◽

Candida Auris ◽

Fungal Burden ◽

In Vivo Antifungal Activity

Antifungal activity of AmBisome against Candida auris was determined in vitro and in vivo. AmBisome showed MIC50 and MIC90 values of 1 and 2 μg/mL, respectively. Unlike conventional amphotericin B, significant in vivo efficacy was observed in the AmBisome 7.5 mg/kg -treated group in survival and reduction of kidney tissue fungal burden compared to the untreated group. Our data shows that AmBisome shows significant antifungal activity against C. auris in vitro as well as in vivo.

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Rapid Emergence of Daptomycin Resistance in Nonstriatum Corynebacterium Species: A Multicenter Study

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqz112.062 ◽

2019 ◽

Vol 152 (Supplement_1) ◽

pp. S32-S33

Author(s):

Kaitlin Mitchell ◽

Erin McElvania ◽

Meghan Wallace ◽

Amy Robertson ◽

Lars Westblade ◽

...

Keyword(s):

Multicenter Study ◽

Tertiary Care ◽

Total N ◽

Gradient Diffusion ◽

Corynebacterium Species ◽

Short Period ◽

High Level ◽

Level Resistance

Abstract Members of the genus Corynebacterium are increasingly recognized as causes of opportunistic infection; some species can be multidrug resistant, posing a treatment challenge. Daptomycin is frequently used as therapy of last resort in this setting, but previous work from our group demonstrated the ability of C striatum clinical isolates to rapidly develop high-level resistance to daptomycin, both in vivo and in vitro. Here, our objective was to expand this investigation into a multicenter study evaluating multiple Corynebacterium species. Corynebacterium strains from three tertiary-care academic medical centers (total, n = 76; site 1, n = 44; site 2, n = 15; site 3, n = 17) were evaluated, representing 16 species. Isolates were identified during routine clinical testing and reported to species level in accordance with each laboratory’s standard operating procedures. Identification of each species was confirmed using both VITEK MS and Bruker BioTyper MALDI-TOF MS. MICs to daptomycin (Etest), vancomycin (Etest), and telavancin (Liofilchem) at baseline were determined using gradient diffusion methods on Mueller-Hinton agar with blood (Hardy Diagnostics). Each isolate was then inoculated in duplicate to 5 mL Tryptic Soy Broth. A daptomycin Etest was submerged in one tube from each pair, and growth was observed after 24-hour incubation. If turbidity was observed in the tube with daptomycin, MICs for each of the 3 antimicrobials were reassessed. High-level daptomycin resistance emerged in 24 strains: C aurimucosum (1/1 isolate tested), C bovis (1/2), C jeikeium (2/11), C macginleyi (3/3), C resistens (1/1), C simulans (1/1), C striatum (14/14 isolates), and C ulcerans (1/1). The majority of these isolates had MIC values >256 µg/mL following exposure to daptomycin. Forty-eight other isolates remained susceptible to daptomycin: C afermentans (1/1), C amycolatum (19/20), C diphtheriae (1/1), C jeikeium (7/11), C kroppenstedtii (2/2), C propinquum (3/3), C pseudodiphtheriticum (6/6), C tuberculostearicum (0/6), and C urealyticum (0/3). Many of these isolates did not undergo MIC testing postdaptomycin exposure in broth due to complete lack of growth. Among those that did (n = 19), the median daptomycin MIC was 0.38 µg/mL (mean 0.42 µg/mL; range 0.023-1.0 µg/mL). One isolate of C bovis and two isolates of C jeikeium yielded variable susceptibility to daptomycin; a subset of resistant colonies grew adjacent to the gradient diffusion strip. Upon isolation and further MIC testing, these colonies maintained high-level resistance. In addition, one isolate of C amycolatum exhibited high-level daptomycin resistance (MIC >256 µg/mL) prior to in vitro exposure. All isolates in the cohort were susceptible to vancomycin and telavancin, both before and after daptomycin exposure. Our findings suggest that multiple Corynebacterium species can rapidly develop high-level daptomycin resistance after a short period of exposure to this antimicrobial. This finding has important clinical implications, especially in the treatment of invasive infections or infections of indwelling medical devices.

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CRISPR-Mediated Knockout of the ABCC2 Gene in Ostrinia furnacalis Confers High-Level Resistance to the Bacillus thuringiensis Cry1Fa Toxin

Toxins ◽

10.3390/toxins12040246 ◽

2020 ◽

Vol 12 (4) ◽

pp. 246 ◽

Cited By ~ 6

Author(s):

Xingliang Wang ◽

Yanjun Xu ◽

Jianlei Huang ◽

Wenzhong Jin ◽

Yihua Yang ◽

...

Keyword(s):

Bacillus Thuringiensis ◽

Deletion Mutation ◽

Ostrinia Furnacalis ◽

Corn Borer ◽

Resistance Evolution ◽

Loop Region ◽

Truncating Mutation ◽

High Level ◽

Level Resistance

The adoption of transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal crystalline (Cry) proteins has reduced insecticide application, increased yields, and contributed to food safety worldwide. However, the efficacy of transgenic Bt crops is put at risk by the adaptive resistance evolution of target pests. Previous studies indicate that resistance to Bacillus thuringiensis Cry1A and Cry1F toxins was genetically linked with mutations of ATP-binding cassette (ABC) transporter subfamily C gene ABCC2 in at least seven lepidopteran insects. Several strains selected in the laboratory of the Asian corn borer, Ostrinia furnacalis, a destructive pest of corn in Asian Western Pacific countries, developed high levels of resistance to Cry1A and Cry1F toxins. The causality between the O. furnacalis ABCC2 (OfABCC2) gene and resistance to Cry1A and Cry1F toxins remains unknown. Here, we successfully generated a homozygous strain (OfC2-KO) of O. furnacalis with an 8-bp deletion mutation of ABCC2 by the CRISPR/Cas9 approach. The 8-bp deletion mutation results in a frame shift in the open reading frame of transcripts, which produced a predicted protein truncated in the TM4-TM5 loop region. The knockout strain OfC2-KO showed much more than a 300-fold resistance to Cry1Fa, and low levels of resistance to Cry1Ab and Cry1Ac (<10-fold), but no significant effects on the toxicities of Cry1Aa and two chemical insecticides (abamectin and chlorantraniliprole), compared to the background NJ-S strain. Furthermore, we found that the Cry1Fa resistance was autosomal, recessive, and significantly linked with the 8-bp deletion mutation of OfABCC2 in the OfC2-KO strain. In conclusion, in vivo functional investigation demonstrates the causality of the OfABCC2 truncating mutation with high-level resistance to the Cry1Fa toxin in O. furnacalis. Our results suggest that the OfABCC2 protein might be a functional receptor for Cry1Fa and reinforces the association of this gene to the mode of action of the Cry1Fa toxin.

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Assessment of Candida auris Response to Antifungal Drugs Using Time–Kill Assays and an Animal Model

Open Forum Infectious Diseases ◽

10.1093/ofid/ofx163.003 ◽

2017 ◽

Vol 4 (suppl_1) ◽

pp. S73-S73 ◽

Cited By ~ 1

Author(s):

Ronen Ben-Ami ◽

Liat Ashkenazi ◽

Judith Berman ◽

Nuphar Korolker ◽

Anna Novikov

Keyword(s):

Amphotericin B ◽

Fungicidal Activity ◽

Antifungal Drugs ◽

Rank Test ◽

Infection Model ◽

Candida Auris ◽

Fungistatic Activity ◽

Kill Curve ◽

Time Kill

Abstract Background Candida auris is an emerging nosocomial pathogen that is resistant to Fluconazole and variably susceptible to other systemic drug classes. Treatment with echinocandins has been recommended based on MICs in the susceptible range, but supporting in vivo data is lacking. Methods We tested the MIC of C. auris strains (n = 12) to fluconazole, voriconazole, posaconazole. anidulafungin, amphotericin B and flucytosine. Representative C. auris strains from Israel and South Africa, and a reference C. albicans strain were analysed using time–kill curve assays. Fungicidal activity was defined as reduction of ≥3 log from baseline CFU/ml. Response to caspofungin treatment was assessed in BALB/c mice immunosuppressed with cyclophosphamide and inoculated with 7 × 107C. auris cells by tail vein injection. Mice were treated from day +1 to day +7 with caspofungin (IP) at doses of 1 or 5 mg/kg and compared with sham-treated controls. Survival was assessed daily. Kaplan-Meier survival analyses were performed and treatment arms were compared using the log-rank test. Results Drug susceptibility results (MIC50 and MIC90) were: fluconazole, 64 and 128 mg/l; voriconazole, 0.5 and 24 mg/l; posaconazole, 0.5 and 27 mg/l; anidulafungin, 0.03 and 0.06 mg/l; amphotericin B, 2 and 8 mg/l; flucytosine, 0.3 and 1 mg/l. Time–kill curve analyses showed log reduction from baseline CFU concentration of −3.0 to −2.8 for fluconazole (MIC ×1), 5.6–6.1 for amphotericin B (MIC ×4) and −0.4 to −0.9 for caspofungin (MIC ×16), consistent with fungicidal activity of amphotericin B and weak fungistatic activity of caspofungin. In the mouse model, survival rate was similar with sham treatment (33%) and treatment with caspofungin 1 mg/kg/day (44%) and 5 mg/kg/day (22%), P = 0.7. Conclusion Despite generally low MIC, caspofungin has only mild fungistatic activity on C. auris and no effect on survival in a mouse infection model. Amphotericin B has fungicidal activity against C. auris. Disclosures All authors: No reported disclosures.

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Efficacy of Ampicillin plus Ceftriaxone in Treatment of Experimental Endocarditis Due to Enterococcus faecalis Strains Highly Resistant to Aminoglycosides

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.3.639 ◽

1999 ◽

Vol 43 (3) ◽

pp. 639-646 ◽

Cited By ~ 82

Author(s):

Joan Gavaldà ◽

Carmen Torres ◽

Carmen Tenorio ◽

Pedro López ◽

Myriam Zaragoza ◽

...

Keyword(s):

Aortic Valve ◽

Enterococcus Faecalis ◽

Pharmacokinetic Parameters ◽

Initial Inoculum ◽

High Level ◽

Time Kill ◽

Disk Method ◽

Level Resistance

The purpose of this work was to evaluate the in vitro possibilities of ampicillin-ceftriaxone combinations for 10 Enterococcus faecalis strains with high-level resistance to aminoglycosides (HLRAg) and to assess the efficacy of ampicillin plus ceftriaxone, both administered with humanlike pharmacokinetics, for the treatment of experimental endocarditis due to HLRAg E. faecalis. A reduction of 1 to 4 dilutions in MICs of ampicillin was obtained when ampicillin was combined with a fixed subinhibitory ceftriaxone concentration of 4 μg/ml. This potentiating effect was also observed by the double disk method with all 10 strains. Time-kill studies performed with 1 and 2 μg of ampicillin alone per ml or in combination with 5, 10, 20, 40, and 60 μg of ceftriaxone per ml showed a ≥2 log10 reduction in CFU per milliliter with respect to ampicillin alone and to the initial inoculum for all 10E. faecalis strains studied. This effect was obtained for seven strains with the combination of 2 μg of ampicillin per ml plus 10 μg of ceftriaxone per ml and for six strains with 5 μg of ceftriaxone per ml. Animals with catheter-induced endocarditis were infected intravenously with 108 CFU of E. faecalis V48 or 105 CFU of E. faecalisV45 and were treated for 3 days with humanlike pharmacokinetics of 2 g of ampicillin every 4 h, alone or combined with 2 g of ceftriaxone every 12 h. The levels in serum and the pharmacokinetic parameters of the humanlike pharmacokinetics of ampicillin or ceftriaxone in rabbits were similar to those found in humans treated with 2 g of ampicillin or ceftriaxone intravenously. Results of the therapy for experimental endocarditis caused by E. faecalis V48 or V45 showed that the residual bacterial titers in aortic valve vegetations were significantly lower in the animals treated with the combinations of ampicillin plus ceftriaxone than in those treated with ampicillin alone (P < 0.001). The combination of ampicillin and ceftriaxone showed in vitro and in vivo synergism against HLRAgE. faecalis.

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Effect of drug dose and timing of treatment on the emergence of drug resistance in vivo in a malaria model

Evolution Medicine and Public Health ◽

10.1093/emph/eoaa016 ◽

2020 ◽

Vol 2020 (1) ◽

pp. 196-210 ◽

Cited By ~ 1

Author(s):

Mónica M Acosta ◽

Joshua T Bram ◽

Derek Sim ◽

Andrew F Read

Keyword(s):

Drug Resistance ◽

Drug Treatment ◽

Drug Dose ◽

Treatment Timing ◽

Treatment Regimens ◽

Resistance Evolution ◽

Timing Of Treatment ◽

High Level ◽

Level Resistance

Abstract Background and objectives There is a significant interest in identifying clinically effective drug treatment regimens that minimize the de novo evolution of antimicrobial resistance in pathogen populations. However, in vivo studies that vary treatment regimens and directly measure drug resistance evolution are rare. Here, we experimentally investigate the role of drug dose and treatment timing on resistance evolution in an animal model. Methodology In a series of experiments, we measured the emergence of atovaquone-resistant mutants of Plasmodium chabaudi in laboratory mice, as a function of dose or timing of treatment (day post-infection) with the antimalarial drug atovaquone. Results The likelihood of high-level resistance emergence increased with atovaquone dose. When varying the timing of treatment, treating either very early or late in infection reduced the risk of resistance. When we varied starting inoculum, resistance was more likely at intermediate inoculum sizes, which correlated with the largest population sizes at time of treatment. Conclusions and implications (i) Higher doses do not always minimize resistance emergence and can promote the emergence of high-level resistance. (ii) Altering treatment timing affects the risk of resistance emergence, likely due to the size of the population at the time of treatment, although we did not test the effect of immunity whose influence may have been important in the case of late treatment. (iii) Finding the ‘right’ dose and ‘right’ time to maximize clinical gains and limit resistance emergence can vary depending on biological context and was non-trivial even in our simplified experiments. Lay summary In a mouse model of malaria, higher drug doses led to increases in drug resistance. The timing of drug treatment also impacted resistance emergence, likely due to the size of the population at the time of treatment.

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High-Level Pacidamycin Resistance in Pseudomonas aeruginosa Is Mediated by an Opp Oligopeptide Permease Encoded by theopp-fabIOperon

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01198-13 ◽

2013 ◽

Vol 57 (11) ◽

pp. 5565-5571 ◽

Cited By ~ 19

Author(s):

Anita Mistry ◽

Mark S. Warren ◽

John K. Cusick ◽

RoxAnn R. Karkhoff-Schweizer ◽

Olga Lomovskaya ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

High Frequency ◽

Cross Resistance ◽

Peptide Antibiotics ◽

Content Type ◽

Resistant Mutants ◽

High Level ◽

Level Resistance ◽

Resistance Mutants

ABSTRACTPacidamycins (or uridyl peptide antibiotics) possess selectivein vivoactivity againstPseudomonas aeruginosa. An important limitation for the therapeutic use of pacidamycins withP. aeruginosais the high frequency (10−6to 10−7) at which resistant mutants emerge. To elucidate the mechanism(s) of this resistance, pacidamycin-resistantP. aeruginosamutants were isolated. Two types of mutants were obtained. Type 1, or high-level resistance mutants with a pacidamycin MIC of 512 μg/ml, were more abundant, with a frequency of ∼2 × 10−6, and did not show cross-resistance with other antibiotics. Type 2, low-level resistance mutants, were isolated with a frequency of ∼10−8and had a pacidamycin MIC of 64 μg/ml (the MIC for the wild-type strain was 4 to 16 μg/ml). These mutants were cross-resistant to levofloxacin, tetracycline, and erythromycin and were shown to overexpress either the MexAB-OprM or MexCD-OprJ multidrug resistance efflux pumps. High-level resistant mutants were isolated by transposon mutagenesis and one insertion was localized tooppB, one of two periplasmic binding protein components of an oligopeptide transport system which is encoded by theopp-fabIoperon. The Opp system is required for uptake of pacidamycin across the inner membrane, since variousopp, but notfabI, mutants were resistant to high levels of pacidamycin. Both of the two putative Opp periplasmic binding proteins, OppA and OppB, were required for pacidamycin uptake. Although both impaired uptake into and efflux from the cell can cause pacidamycin resistance inP. aeruginosa, our data suggest that impaired uptake is the primary reason for the high-frequency and high-level pacidamycin resistance.

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Candida auris Bloodstream Infections in Russia

Antibiotics ◽

10.3390/antibiotics9090557 ◽

2020 ◽

Vol 9 (9) ◽

pp. 557 ◽

Cited By ~ 1

Author(s):

Natalia E. Barantsevich ◽

Antonina V. Vetokhina ◽

Natalia I. Ayushinova ◽

Olga E. Orlova ◽

Elena P. Barantsevich

Keyword(s):

Mortality Rate ◽

Antifungal Susceptibility ◽

Bloodstream Infections ◽

Attributable Mortality ◽

Candida Auris ◽

D2 Domain ◽

All Cause Mortality ◽

Report Data ◽

High Level ◽

Level Resistance

Candida auris—a fungus (yeast) that can cause hospital outbreaks was first recognized in 2009. The authors report data on 38 cases of C. auris bloodstream infections in multidisciplinary hospitals situated in two distantly located regions of Russia, considering predisposing factors, antifungal susceptibility of isolates, treatment, and outcomes. Interhospital transfers of patients and labor migration contributed to the spread of C. auris. The South Asian lineage of the studied strains was indicated by K143R substitution in ERG11 gene and phylogenetic analysis of internal transcribed spacer and D1-D2 domain. All isolates from C. auris candidemia cases were susceptible to echinocandins. High-level resistance to fluconazole and resistance to amphotericin B were present in the majority of strains. The overall all-cause mortality rate in C. auris bloodstream infections was 55.3% and the 30-day all-cause mortality rate 39.5%. The attributable mortality was 0%. Eradication of C. auris from blood was associated with the favourable outcomes in patients. It was achieved irrespective of whether antifungal preparations within or outside the susceptibility range were administered. Further international surveillance and studies providing consensus guidelines for the management of C. auris infections are needed.

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Efficacy of vancomycin and teicoplanin alone and in combination with streptomycin in experimental, low-level vancomycin-resistant, VanB-type Enterococcus faecalis endocarditis.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.1.55 ◽

1996 ◽

Vol 40 (1) ◽

pp. 55-60 ◽

Cited By ~ 9

Author(s):

D P Nicolau ◽

M N Marangos ◽

C H Nightingale ◽

K B Patel ◽

B W Cooper ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Resistant Strain ◽

Low Level ◽

Vancomycin Resistant ◽

Low Levels ◽

High Level ◽

Isogenic Strains ◽

Level Resistance

The efficacy of vancomycin (VM) and teicoplanin (TE), alone and in combination with streptomycin (SM), against enterococci that express low-level VanB-type VM resistance was investigated in experimental endocarditis using isogenic strains of Enterococcus faecalis susceptible to glycopeptides and aminoglycosides or inducibly resistant to low levels of VM (MIC = 16 micrograms/ml). VM was significantly less active against the resistant strain than against the susceptible strain, establishing that low-level VanB-type VM resistance can influence therapeutic efficacy. By contrast, TE had equally good activity against both strains. VM or TE combined with SM was synergistic and bactericidal against the resistant strain in vitro. While both combinations were efficient in reducing bacterial density in vivo, TE plus SM was significantly superior to VM plus SM if valve sterilization was considered. These data suggest that despite the presence of low-level VanB-type resistance, combination therapy with a glycopeptide and SM (and presumably other aminoglycosides to which there is not high-level resistance) will nevertheless provide effective bactericidal activity.

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