Impact ofIn VivoTriazole and Echinocandin Combination Therapy for Invasive Pulmonary Aspergillosis: Enhanced Efficacy againstCyp51Mutant Isolates

ABSTRACTPrevious studies examining combination therapy for invasive pulmonary aspergillosis (IPA) have revealed conflicting results, including antagonism, indifference, and enhanced effects. The most commonly employed combination for this infection includes a mold-active triazole and echinocandin. Few studies have evaluated combination therapy from a pharmacodynamic (PD) perspective, and even fewer have examined combination therapy against both wild-type and azole-resistantCyp51mutant isolates. The current studies aim to fill this gap in knowledge. FourAspergillus fumigatusisolates were utilized, including a wild-type strain, anFks1mutant (posaconazole susceptible and caspofungin resistant), and twoCyp51mutants (posaconazole resistant). A neutropenic murine model of IPA was used for the treatment studies. The dosing design included monotherapy with posaconazole, monotherapy with caspofungin, and combination therapy with both. Efficacy was determined using quantitative PCR, and results were normalized to known quantities of conidia (conidial equivalents [CE]). The static dose, 1-log kill dose, and associated PD target area under the curve (AUC)/MIC ratio were determined for monotherapy and combination therapy. Monotherapy experiments revealed potent activity for posaconazole, with reductions of 3 to 4 log10AspergillusCE/ml with the two “low”-MIC isolates. Posaconazole alone was less effective for the two isolates with higher MICs. Caspofungin monotherapy did not produce a significant decrease in fungal burden for any strain. Combination therapy with the two antifungals did not enhance efficacy for the two posaconazole-susceptible isolates. However, the drug combination produced synergistic activity against both posaconazole-resistant isolates. Specifically, the combination resulted in a 1- to 2-log10decline in burden that would not have been predicted based on the monotherapy results for each drug. This corresponded to a reduction in the free-drug posaconazole AUC/MIC ratio needed for stasis of up to 17-fold. The data suggest that combination therapy using a triazole and an echinocandin may be a beneficial treatment strategy for triazole-resistant isolates.

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Posaconazole Pharmacodynamic Target Determination against Wild-Type andCyp51Mutant Isolates of Aspergillus fumigatus in anIn VivoModel of Invasive Pulmonary Aspergillosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01279-12 ◽

2012 ◽

Vol 57 (1) ◽

pp. 579-585 ◽

Cited By ~ 42

Author(s):

Alexander J. Lepak ◽

Karen Marchillo ◽

Jaimie VanHecker ◽

David R. Andes

Keyword(s):

Dose Range ◽

Invasive Pulmonary Aspergillosis ◽

Free Drug ◽

Lung Homogenate ◽

Study Endpoint ◽

Pharmacokinetic Data ◽

Wild Type ◽

Pulmonary Aspergillosis ◽

Time Curve ◽

Content Type

ABSTRACTInvasive pulmonary aspergillosis (IPA) is a devastating disease of immunocompromised patients. Pharmacodynamic (PD) examination of antifungal drug therapy in IPA is one strategy that may improve outcomes. The current study explored the PD target of posaconazole in an immunocompromised murine model of IPA against 10A. fumigatusisolates, including 4Cyp51wild-type isolates and 6 isolates carryingCyp51mutations conferring azole resistance. The posaconazole MIC range was 0.25 to 8 mg/liter. Following infection, mice were given 0.156 to 160 mg/kg of body weight of oral posaconazole daily for 7 days. Efficacy was assessed by quantitative PCR (qPCR) of lung homogenate and survival. At the start of therapy, mice had 5.59 ± 0.19 log10Aspergillusconidial equivalents (CE)/ml of lung homogenate, which increased to 7.11 ± 0.29 log10CE/ml of lung homogenate in untreated animals. The infection was uniformly lethal prior to the study endpoint in control mice. A Hill-type dose response function was used to model the relationship between posaconazole free drug area under the concentration-time curve (AUC)/MIC and qPCR lung burden. The static dose range was 1.09 to 51.9 mg/kg/24 h. The free drug AUC/MIC PD target was 1.09 ± 0.63 for the group of strains. The 1-log kill free drug AUC/MIC was 2.07 ± 1.02. The PD target was not significantly different for the wild-type and mutant organism groups. Mortality mirrored qPCR results, with the greatest improvement in survival noted at the same dosing regimens that produced static or cidal activity. Consideration of human pharmacokinetic data and the current static dose PD target would predict a clinical MIC threshold of 0.25 to 0.5 mg/liter.

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Isavuconazole (BAL4815) Pharmacodynamic Target Determination in anIn VivoMurine Model of Invasive Pulmonary Aspergillosis against Wild-Type andcyp51Mutant Isolates of Aspergillus fumigatus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01355-13 ◽

2013 ◽

Vol 57 (12) ◽

pp. 6284-6289 ◽

Cited By ~ 54

Author(s):

Alexander J. Lepak ◽

Karen Marchillo ◽

Jamie VanHecker ◽

David R. Andes

Keyword(s):

Dose Range ◽

Invasive Pulmonary Aspergillosis ◽

Mortality Rates ◽

Free Drug ◽

Lung Homogenate ◽

Infection Model ◽

Wild Type ◽

Pulmonary Aspergillosis ◽

Content Type ◽

Target Determination

ABSTRACTInvasive pulmonary aspergillosis (IPA) continues to rise in concert with increasing numbers of immune suppression techniques to treat other medical conditions and transplantation. Despite these advances, morbidity and mortality rates remain unacceptably high. One strategy used to optimize outcomes is antifungal pharmacodynamic (PD) examination. We explored the pharmacodynamics of a new triazole in development, isavuconazole, in a murine neutropenic IPA model. TenA. fumigatusisolates were used, including four wild-type isolates and sixcyp51mutants. The MIC range was 0.125 to 8 mg/liter. Following infection, groups of mice were treated orally with the prodrug (BAL8557) at 40 to 640 mg/kg/12 h for 7 days. Efficacy was determined by quantitative PCR of lung homogenates. At the start of therapy, mice had 4.97 log10conidial equivalents (CE)/ml of lung homogenate, and this increased to 6.82 log10CE/ml of lung homogenate in untreated animals. The infection model was uniformly lethal in untreated control mice. The PD target endpoints examined included the static-dose AUC/MIC ratio and the 1-log10killing AUC/MIC ratio. A stasis endpoint was achieved for all isolates with an MIC of ≤1 mg/liter and 1-log10killing in all isolates with an MIC of ≤0.5 mg/liter, regardless of the presence or absence of thecyp51mutation. The static-dose range was 65 to 617 mg/kg/12 h. The corresponding median free-drug AUC/MIC ratio was near 5. The 1-log10killing dose range was 147 to 455 mg/kg/12 h, and the corresponding median free-drug AUC/MIC ratio was 11.1. These values are similar to those previously reported for other triazoles.

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Posaconazole Prophylaxis in Experimental Azole-Resistant Invasive Pulmonary Aspergillosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03850-14 ◽

2014 ◽

Vol 59 (3) ◽

pp. 1487-1494 ◽

Cited By ~ 11

Author(s):

Seyedmojtaba Seyedmousavi ◽

Johan W. Mouton ◽

Willem J. G. Melchers ◽

Paul E. Verweij

Keyword(s):

Invasive Aspergillosis ◽

Aspergillus Fumigatus ◽

Murine Model ◽

Pulmonary Infection ◽

Invasive Pulmonary Aspergillosis ◽

Clinical Isolates ◽

Wild Type ◽

Breakthrough Infection ◽

Pulmonary Aspergillosis ◽

Content Type

ABSTRACTWe investigated the efficacy of posaconazole prophylaxis in preventing invasive aspergillosis due to azole-resistantAspergillus fumigatusisolates. Using a neutropenic murine model of pulmonary infection, posaconazole prophylaxis was evaluated using three isogenic clinical isolates, with posaconazole MICs of 0.063 mg/liter (wild type), 0.5 mg/liter (F219I mutation), and 16 mg/liter. A fourth isolate harboring TR34/L98H (MIC of 0.5 mg/liter) was also tested. Posaconazole prophylaxis was effective inA. fumigatuswith posaconazole MICs of ≤0.5 mg/liter, where 100% survival was reached. However, breakthrough infection was observed in mice infected with the isolate for which the posaconazole MIC was >16 mg/liter.

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A Novel CO-Responsive Transcriptional Regulator and Enhanced H2Production by an Engineered Thermococcus onnurineus NA1 Strain

Applied and Environmental Microbiology ◽

10.1128/aem.03019-14 ◽

2014 ◽

Vol 81 (5) ◽

pp. 1708-1714 ◽

Cited By ~ 25

Author(s):

Min-Sik Kim ◽

Ae Ran Choi ◽

Seong Hyuk Lee ◽

Hae-Chang Jung ◽

Seung Seob Bae ◽

...

Keyword(s):

Production Rate ◽

Gene Cluster ◽

Type Strain ◽

Wild Type Strain ◽

Regulatory System ◽

Transcriptional Regulator ◽

Bioreactor Culture ◽

Wild Type ◽

Content Type ◽

Transcriptional Regulatory

ABSTRACTGenome analysis revealed the existence of a putative transcriptional regulatory system governing CO metabolism inThermococcus onnurineusNA1, a carboxydotrophic hydrogenogenic archaeon. The regulatory system is composed of CorQ with a 4-vinyl reductase domain and CorR with a DNA-binding domain of the LysR-type transcriptional regulator family in close proximity to the CO dehydrogenase (CODH) gene cluster. Homologous genes of the CorQR pair were also found in the genomes ofThermococcusspecies and “CandidatusKorarchaeum cryptofilum” OPF8. In-frame deletion of eithercorQorcorRcaused a severe impairment in CO-dependent growth and H2production. WhencorQandcorRdeletion mutants were complemented by introducing thecorQRgenes under the control of a strong promoter, the mRNA and protein levels of the CODH gene were significantly increased in a ΔCorR strain complemented with integratedcorQR(ΔCorR/corQR↑) compared with those in the wild-type strain. In addition, the ΔCorR/corQR↑strain exhibited a much higher H2production rate (5.8-fold) than the wild-type strain in a bioreactor culture. The H2production rate (191.9 mmol liter−1h−1) and the specific H2production rate (249.6 mmol g−1h−1) of this strain were extremely high compared with those of CO-dependent H2-producing prokaryotes reported so far. These results suggest that thecorQRgenes encode a positive regulatory protein pair for the expression of a CODH gene cluster. The study also illustrates that manipulation of the transcriptional regulatory system can improve biological H2production.

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Activation of 2,4-Diaminoquinazoline in Mycobacterium tuberculosis by Rv3161c, a Putative Dioxygenase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01505-18 ◽

2018 ◽

Vol 63 (1) ◽

Author(s):

Eduard Melief ◽

Shilah A. Bonnett ◽

Edison S. Zuniga ◽

Tanya Parish

Keyword(s):

Mycobacterium Tuberculosis ◽

Mutant Strain ◽

Type Strain ◽

Wild Type Strain ◽

Type A ◽

Wild Type ◽

Metabolite Analysis ◽

Content Type ◽

Data Support ◽

In The Wild

ABSTRACT The diaminoquinazoline series has good potency against Mycobacterium tuberculosis. Resistant isolates have mutations in Rv3161c, a putative dioxygenase. We carried out metabolite analysis on a wild-type strain and an Rv3161c mutant strain after exposure to a diaminoquinazoline. The parental compound was found in intracellular extracts from the mutant but not the wild type. A metabolite consistent with a monohydroxylated form was identified in the wild type. These data support the hypothesis that Rv3161c metabolizes diaminoquinazolines in M. tuberculosis.

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The Riemerella anatipestiferAS87_01735Gene Encodes Nicotinamidase PncA, an Important Virulence Factor

Applied and Environmental Microbiology ◽

10.1128/aem.01829-16 ◽

2016 ◽

Vol 82 (19) ◽

pp. 5815-5823 ◽

Cited By ~ 7

Author(s):

Xiaolan Wang ◽

Beibei Liu ◽

Yafeng Dou ◽

Hongjie Fan ◽

Shaohui Wang ◽

...

Keyword(s):

Type Strain ◽

Wild Type Strain ◽

Vaccine Candidate ◽

Wild Type ◽

Salvage Pathway ◽

Effective Protection ◽

Domestic Ducks ◽

Content Type ◽

Key Enzyme ◽

Important Virulence Factor

ABSTRACTRiemerella anatipestiferis a major bacterial pathogen that causes septicemic and exudative diseases in domestic ducks. In our previous study, we found that deletion of theAS87_01735gene significantly decreased the bacterial virulence ofR. anatipestiferstrain Yb2 (mutant RA625). TheAS87_01735gene was predicted to encode a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD+salvage pathway. In this study, theAS87_01735gene was expressed and identified as the PncA-encoding gene, using an enzymatic assay. Western blot analysis demonstrated thatR. anatipestiferPncA was localized to the cytoplasm. The mutant strain RA625 (named Yb2ΔpncAin this study) showed a similar growth rate but decreased NAD+quantities in both the exponential and stationary phases in tryptic soy broth culture, compared with the wild-type strain Yb2. In addition, Yb2ΔpncA-infected ducks showed much lower bacterial loads in their blood, and no visible histological changes were observed in the heart, liver, and spleen. Furthermore, Yb2ΔpncAimmunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Our results suggest that theR. anatipestiferAS87_01735gene encodes PncA, which is an important virulence factor, and that the Yb2ΔpncAmutant can be used as a novel live vaccine candidate.IMPORTANCERiemerella anatipestiferis reported worldwide as a cause of septicemic and exudative diseases of domestic ducks. ThepncAgene encodes a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD+salvage pathway. In this study, we identified and characterized thepncA-homologous geneAS87_01735inR. anatipestiferstrain Yb2.R. anatipestiferPncA is a cytoplasmic protein that possesses similar PncA activity, compared with other organisms. Generation of thepncAmutant Yb2ΔpncAled to a decrease in the NAD+content, which was associated with decreased capacity for invasion and attenuated virulence in ducks. Furthermore, Yb2ΔpncAimmunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Altogether, these results suggest that PncA contributes to the virulence ofR. anatipestiferand that the Yb2ΔpncAmutant can be used as a novel live vaccine candidate.

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Influence of Chemotaxis and Swimming Patterns on the Virulence of the Coral PathogenVibrio coralliilyticus

Journal of Bacteriology ◽

10.1128/jb.00791-17 ◽

2018 ◽

Vol 200 (15) ◽

Cited By ~ 6

Author(s):

Blake Ushijima ◽

Claudia C. Häse

Keyword(s):

Type Strain ◽

Wild Type Strain ◽

Bacterial Colonization ◽

Valuable Insight ◽

Wild Type ◽

Coral Mucus ◽

Content Type ◽

Swimming Pattern ◽

Vibrio Coralliilyticus ◽

Insight Into

ABSTRACTChemotaxis, the directed movement toward or away from a chemical signal, can be essential to bacterial pathogens for locating hosts or avoiding hostile environments. The coral pathogenVibrio coralliilyticuschemotaxes toward coral mucus; however, chemotaxis has not been experimentally demonstrated to be important for virulence. To further examine this, in-frame mutations were constructed in genes predicted to be important forV. coralliilyticuschemotaxis. MostVibriogenomes contain multiple homologs of various chemotaxis-related genes, and two paralogs of each forcheB,cheR, andcheAwere identified. Based on single mutant analyses, the paralogscheB2,cheR2, andcheA1were essential for chemotaxis in laboratory assays. As predicted, the ΔcheA1and ΔcheR2strains had a smooth-swimming pattern, while the ΔcheB2strain displayed a zigzag pattern when observed under light microscopy. However, these mutants, unlike the parent strain, were unable to chemotax toward the known attractants coral mucus, dimethylsulfoniopropionate, andN-acetyl-d-glucosamine. The ΔcheB2strain and an aflagellate ΔfliG1strain were avirulent to coral, while the ΔcheA1and ΔcheR2strains were hypervirulent (90 to 100% infection within 14 h on average) compared to the wild-type strain (66% infection within 36 h on average). Additionally, the ΔcheA1and ΔcheR2strains appeared to better colonize coral fragments than the wild-type strain. These results suggest that although chemotaxis may be involved with infection (the ΔcheB2strain was avirulent), a smooth-swimming phenotype is important for bacterial colonization and infection. This study provides valuable insight into understandingV. coralliilyticuspathogenesis and how this pathogen may be transmitted between hosts.IMPORTANCECorals are responsible for creating the immense structures that are essential to reef ecosystems; unfortunately, pathogens like the bacteriumVibrio coralliilyticuscan cause fatal infections of reef-building coral species. However, compared to related human pathogens, the mechanisms by whichV. coralliilyticusinitiates infections and locates new coral hosts are poorly understood. This study investigated the effects of chemotaxis, the directional swimming in response to chemical signals, and bacterial swimming patterns on infection of the coralMontipora capitata. Infection experiments with different mutant strains suggested that a smooth-swimming pattern resulted in hypervirulence. These results demonstrate that the role of chemotaxis in coral infection may not be as straightforward as previously hypothesized and provide valuable insight intoV. coralliilyticuspathogenesis.

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Loss of Ethanolamine Utilization in Enterococcus faecalis Increases Gastrointestinal Tract Colonization

mBio ◽

10.1128/mbio.00790-18 ◽

2018 ◽

Vol 9 (3) ◽

Cited By ~ 8

Author(s):

Karan Gautam Kaval ◽

Kavindra V. Singh ◽

Melissa R. Cruz ◽

Sruti DebRoy ◽

Wade C. Winkler ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Opposite Effect ◽

Wild Type Strain ◽

Food Poisoning ◽

Wild Type ◽

Gi Tract ◽

Human Gut ◽

Content Type ◽

Serovar Typhimurium ◽

High Concentrations

ABSTRACT Enterococcus faecalis is paradoxically a dangerous nosocomial pathogen and a normal constituent of the human gut microbiome, an environment rich in ethanolamine. E. faecalis carries the eut (ethanolamine utilization) genes, which enable the catabolism of ethanolamine (EA) as a valuable source of carbon and/or nitrogen. EA catabolism was previously shown to contribute to the colonization and growth of enteric pathogens, such as Salmonella enterica serovar Typhimurium and enterohemorrhagic Escherichia coli (EHEC), in the gut environment. We tested the ability of eut mutants of E. faecalis to colonize the gut using a murine model of gastrointestinal (GI) tract competition and report the surprising observation that these mutants outcompete the wild-type strain. IMPORTANCE Some bacteria that are normal, harmless colonizers of the human body can cause disease in immunocompromised patients, particularly those that have been heavily treated with antibiotics. Therefore, it is important to understand the factors that promote or negate these organisms’ ability to colonize. Previously, ethanolamine, found in high concentrations in the GI tract, was shown to promote the colonization and growth of bacteria associated with food poisoning. Here, we report the surprising, opposite effect of ethanolamine utilization on the commensal colonizer E. faecalis , namely, that loss of this metabolic capacity made it a better colonizer.

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Identification of Crp as a novel regulator of the Std fimbrial expression in Salmonella

Microbiology ◽

10.1099/mic.0.001022 ◽

2021 ◽

Author(s):

Karine Dufresne ◽

France Daigle

Keyword(s):

Wild Type Strain ◽

Distal Region ◽

Transcriptional Factor ◽

Major Influence ◽

Wild Type ◽

Content Type ◽

Link Type ◽

Serovar Typhimurium ◽

Metabolic Regulators

The Salmonella enterica serovar Typhi genome contains 14 putative fimbrial systems. The Std fimbriae belong to the chaperone-usher family and its regulation has not been investigated in S. Typhi. Several regulators of Std were previously identified in the closely related serovar Typhimurium. We hypothesize that regulators of S. Typhimurium may be shared with S. Typhi, but that several other regulators remain to be discovered. Here, we describe the role of more than 50 different candidate regulators on std expression. Three types of regulators were investigated: known regulators in S. Typhimurium, in silico predicted regulators and virulence/metabolic regulators. Expression of std was determined in the regulator mutants and compared with the wild-type strain. Overall, 21 regulator mutations affect std promoter expression. The role of Crp, a newly identified factor for std expression, was further investigated. Crp acted as an activator of std expression on a distal region of the std promoter region. Together, our results demonstrate the major influence of Crp as a novel transcriptional factor on std promoter expression and later production of Std fimbriae in Salmonella .

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The Ser/Thr Kinase PrkC Participates in Cell Wall Homeostasis and Antimicrobial Resistance inClostridium difficile

Infection and Immunity ◽

10.1128/iai.00005-19 ◽

2019 ◽

Vol 87 (8) ◽

Cited By ~ 6

Author(s):

Elodie Cuenot ◽

Transito Garcia-Garcia ◽

Thibaut Douche ◽

Olivier Gorgette ◽

Pascal Courtin ◽

...

Keyword(s):

Cell Wall ◽

Type Strain ◽

Wild Type Strain ◽

Cell Envelope ◽

Wild Type ◽

Hamster Model ◽

Content Type ◽

Physiological Processes ◽

Signal Transduction Networks ◽

Mean Size

ABSTRACTClostridium difficileis the leading cause of antibiotic-associated diarrhea in adults. During infection,C. difficilemust detect the host environment and induce an appropriate survival strategy. Signal transduction networks involving serine/threonine kinases (STKs) play key roles in adaptation, as they regulate numerous physiological processes. PrkC ofC. difficileis an STK with two PASTA domains. We showed that PrkC is membrane associated and is found at the septum. We observed that deletion ofprkCaffects cell morphology with an increase in mean size, cell length heterogeneity, and presence of abnormal septa. A ΔprkCmutant was able to sporulate and germinate but was less motile and formed more biofilm than the wild-type strain. Moreover, a ΔprkCmutant was more sensitive to antimicrobial compounds that target the cell envelope, such as the secondary bile salt deoxycholate, cephalosporins, cationic antimicrobial peptides, and lysozyme. This increased susceptibility was not associated with differences in peptidoglycan or polysaccharide II composition. However, the ΔprkCmutant had less peptidoglycan and released more polysaccharide II into the supernatant. A proteomic analysis showed that the majority ofC. difficileproteins associated with the cell wall were less abundant in the ΔprkCmutant than the wild-type strain. Finally, in a hamster model of infection, the ΔprkCmutant had a colonization delay that did not significantly affect overall virulence.

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