Emergence of ceftazidime/avibactam resistance in KPC-3-producing Klebsiella pneumoniae in vivo

2019 ◽  
Vol 74 (11) ◽  
pp. 3211-3216 ◽  
Author(s):  
Stephan Göttig ◽  
Denia Frank ◽  
Eleonora Mungo ◽  
Anika Nolte ◽  
Michael Hogardt ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Klebsiella Pneumoniae ◽  
Selection Pressure ◽  
Galleria Mellonella ◽  
Phenotypic Characterization ◽  
Infection Model ◽  
Development Of Resistance ◽  
Time Kill

Abstract Objectives The β-lactam/β-lactamase inhibitor combination ceftazidime/avibactam is active against KPC-producing Enterobacterales. Herein, we present molecular and phenotypic characterization of ceftazidime/avibactam resistance in KPC-3-producing Klebsiella pneumoniae that emerged in vivo and in vitro. Methods Sequence analysis of blaKPC-3 was performed from clinical and in vitro-generated ceftazidime/avibactam-resistant K. pneumoniae isolates. Time–kill kinetics and the Galleria mellonella infection model were applied to evaluate the activity of ceftazidime/avibactam and imipenem alone and in combination. Results The ceftazidime/avibactam-resistant clinical K. pneumoniae isolate revealed the amino acid change D179Y in KPC-3. Sixteen novel mutational changes in KPC-3 among in vitro-selected ceftazidime/avibactam-resistant isolates were described. Time–kill kinetics showed the emergence of a resistant subpopulation under selection pressure with either imipenem or ceftazidime/avibactam. However, combined selection pressure with imipenem plus ceftazidime/avibactam prevented the development of resistance and resulted in bactericidal activity. Concordantly, the G. mellonella infection model revealed that monotherapy with ceftazidime/avibactam is prone to select for resistance in vivo and that combination therapy with imipenem results in significantly better survival. Conclusions Ceftazidime/avibactam is a valuable antibiotic against MDR and carbapenem-resistant Enterobacterales. Based on time–kill kinetics as well as an in vivo infection model we postulate a combination therapy of ceftazidime/avibactam and imipenem as a strategy to prevent the development of ceftazidime/avibactam resistance in KPC-producing Enterobacterales in vivo.

scholarly journals Polymyxin Triple Combinations against Polymyxin-Resistant, Multidrug-Resistant, KPC-Producing Klebsiella pneumoniae

2020 ◽  
Vol 64 (8) ◽  
Author(s):  
Su Mon Aye ◽  
Irene Galani ◽  
Heidi Yu ◽  
Jiping Wang ◽  
Ke Chen ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Triple Therapy ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Infection Model ◽  
Bacterial Killing ◽  
Standard Dosage ◽  
Time Kill

ABSTRACT Resistance to polymyxin antibiotics is increasing. Without new antibiotic classes, combination therapy is often required. We systematically investigated bacterial killing with polymyxin-based combinations against multidrug-resistant (including polymyxin-resistant), carbapenemase-producing Klebsiella pneumoniae. Monotherapies and double- and triple-combination therapies were compared to identify the most efficacious treatment using static time-kill studies (24 h, six isolates), an in vitro pharmacokinetic/pharmacodynamic model (IVM; 48 h, two isolates), and the mouse thigh infection model (24 h, six isolates). In static time-kill studies, all monotherapies (polymyxin B, rifampin, amikacin, meropenem, or minocycline) were ineffective. Initial bacterial killing was enhanced with various polymyxin B-containing double combinations; however, substantial regrowth occurred in most cases by 24 h. Most polymyxin B-containing triple combinations provided greater and more sustained killing than double combinations. Standard dosage regimens of polymyxin B (2.5 mg/kg of body weight/day), rifampin (600 mg every 12 h), and amikacin (7.5 mg/kg every 12 h) were simulated in the IVM. Against isolate ATH 16, no viable bacteria were detected across 5 to 25 h with triple therapy, with regrowth to ∼2-log10 CFU/ml occurring at 48 h. Against isolate BD 32, rapid initial killing of ∼3.5-log10 CFU/ml at 5 h was followed by a slow decline to ∼2-log10 CFU/ml at 48 h. In infected mice, polymyxin B monotherapy (60 mg/kg/day) generally was ineffective. With triple therapy (polymyxin B at 60 mg/kg/day, rifampin at 120 mg/kg/day, and amikacin at 300 mg/kg/day), at 24 h there was an ∼1.7-log10 CFU/thigh reduction compared to the starting inoculum for all six isolates. Our results demonstrate that the polymyxin B-rifampin-amikacin combination significantly enhanced in vitro and in vivo bacterial killing, providing important information for the optimization of polymyxin-based combinations in patients.

scholarly journals In VitroandIn VivoActivities of OP0595, a New Diazabicyclooctane, against CTX-M-15-Positive Escherichia coli and KPC-Positive Klebsiella pneumoniae

2016 ◽  
Vol 60 (5) ◽  
pp. 3001-3006 ◽  
Author(s):  
Akihiro Morinaka ◽  
Yuko Tsutsumi ◽  
Keiko Yamada ◽  
Yoshihiro Takayama ◽  
Shiro Sakakibara ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Infection Model ◽  
Gram Negative Bacteria ◽  
Content Type ◽  
Effective Combination ◽  
Time Kill ◽  
Lactamase Inhibitor

ABSTRACTGram-negative bacteria are evolving to produce β-lactamases of increasing diversity that challenge antimicrobial chemotherapy. OP0595 is a new diazabicyclooctane serine β-lactamase inhibitor which acts also as an antibiotic and as a β-lactamase-independent β-lactam “enhancer” againstEnterobacteriaceae. Here we determined the optimal concentration of OP0595 in combination with piperacillin, cefepime, and meropenem, in addition to the antibacterial activity of OP0595 alone and in combination with cefepime, inin vitrotime-kill studies and anin vivoinfection model against five strains of CTX-M-15-positiveEscherichia coliand five strains of KPC-positiveKlebsiella pneumoniae. An OP0595 concentration of 4 μg/ml was found to be sufficient for an effective combination with all three β-lactam agents. In bothin vitrotime-kill studies and anin vivomodel of infection, cefepime-OP0595 showed stronger efficacy than cefepime alone against all β-lactamase-positive strains tested, whereas OP0595 alone showed weaker or no efficacy. Taken together, these data indicate that combinational use of OP0595 and a β-lactam agent is important to exert the antimicrobial functions of OP0595.

scholarly journals Pharmacodynamics of linezolid-plus-fosfomycin against vancomycin-susceptible and -resistant enterococci in vitro and in vivo of a Galleria mellonella larval infection model

2019 ◽  
Author(s):  
Caifen Qi ◽  
Shuangli Xu ◽  
Maomao Wu ◽  
Shuo Zhu ◽  
Yanyan Liu ◽  
...  
Keyword(s):  
Combination Treatment ◽  
Cell Morphology ◽  
Bacterial Cell ◽  
Galleria Mellonella ◽  
Infection Model ◽  
Bacterial Killing ◽  
Bacterial Cell Morphology ◽  
Time Kill

AbstractObjectiveTo explore the in vitro and in vivo antibacterial activity of linezolid/fosfomycin combination against vancomycin-susceptible and -resistant enterococci (VSE and VRE), providing theoretical basis for the treatment of VRE.MethodsThe checkerboard method and time-kill curve study were used to evaluate the synergistic effect of linezolid combined with fosfomycin against VSE and VRE. The transmission electron microscopy (TEM) was employed to observe the bacterial cell morphology followed by each drug alone and in combination, elucidating the possible result of antibiotic combination therapy. The Galleria mellonella infection model was constructed to demonstrate the in vivo efficacy of linezolid plus fosfomycin for VSE and VRE infection.ResultsThe fractional inhibitory concentration index (FICI) values of all strains suggested that linezolid showed synergy or additivity in combination with fosfomycin against five of the six strains. Time-kill experiments demonstrated that the combination of linezolid-fosfomycin at 1×MIC or 2×MIC led to higher degree of bacterial killing without regrowth for all isolates tested than each monotherapy. TEM imaging showed that the combination treatment damaged the bacterial cell morphology more obviously than each drug alone. In the Galleria mellonella infection model, the enhanced survival rate of the combination treatment was revealed compared to linezolid monotherapy (P<0.05).ConclusionsOur data manifest that the combination of linezolid and fosfomycin may be a possible therapeutic regimen for VRE infection. The combination displays excellent bacterial killing and inhibits amplification of fosfomycin-resistant subpopulations.

scholarly journals «Enhanced antibacterial activity of repurposed mitomycin C and imipenem in combination with the lytic phage vB_KpnM-VAC13 against clinical isolates of Klebsiella pneumoniae »

2021 ◽  
Author(s):  
Olga Pacios ◽  
Laura Fernández-García ◽  
Ines Bleriot ◽  
Lucia Blasco ◽  
Mónica González-Bardanca ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Mitomycin C ◽  
Galleria Mellonella ◽  
Synergistic Effects ◽  
Resistant Isolate ◽  
Infection Model ◽  
Lytic Phage ◽  
Broth Microdilution Method

Klebsiella pneumoniae is an opportunistic Gram-negative pathogen that employs different strategies (resistance and persistence) to counteract antibiotic treatments. This study aimed to search for new means of combatting imipenem-resistant and persister strains of K. pneumoniae by repurposing the anticancer drug mitomycin C as an antimicrobial agent and by combining the drug and the conventional antibiotic imipenem with the lytic phage vB_KpnM-VAC13. Several clinical K. pneumoniae isolates were characterized, and an imipenem-resistant isolate (harbouring OXA245 ß-lactamase) and a persister isolate were selected for study. The mitomycin C and imipenem MICs for both isolates were determined by the broth microdilution method. Time-kill curve data were obtained by OD 600 measurement and CFU enumeration in the presence of each drug alone and with the phage. The frequency of occurrence of mutants resistant to each drug and the combinations was also calculated, and the efficacy of the combination treatments was evaluated using an in vivo infection model ( Galleria mellonella ). The lytic phage vB_KpnM-VAC13 and mitomycin C had synergistic effects on imipenem-resistant and persister isolates, both in vitro and in vivo. The phage-imipenem combination successfully killed the persisters but not the imipenem-resistant isolate harbouring OXA245 ß-lactamase. Interestingly, the combinations decreased the emergence of in vitro resistant mutants of both isolates. Combinations of the lytic phage vB_KpnM-VAC13 with mitomycin C and imipenem were effective against the persister K. pneumoniae isolate. The lytic phage/mitomycin C combination was also effective against imipenem-resistant K. pneumoniae strains harbouring OXA245 ß-lactamase.

scholarly journals Phages from Ganges River curtail in vitro biofilms and planktonic growth of drug resistant Klebsiella pneumoniae in a zebrafish infection model

2020 ◽  
Author(s):  
Niranjana Sri Sundaramoor ◽  
Subramanian Thothathri ◽  
Muthu Meenakshi Bhaskaran ◽  
ArunKumar GaneshPrasad ◽  
Saisubramanian Nagarajan
Keyword(s):  
Klebsiella Pneumoniae ◽  
Infection Model ◽  
Histopathological Analysis ◽  
Drug Resistant ◽  
Ganges River ◽  
Promising Alternative ◽  
Planktonic Growth ◽  
Time Kill

Abstract Bacteriophages are a promising alternative for curtailing infections caused by multi drug resistant (MDR) bacteria. The objective of the present study is to evaluate phage populations from water bodies to inhibit planktonic and biofilm mode of growth of drug resistant Klebsiella pneumoniae in vitro and curtail planktonic growth in vivo in a zebrafish model. Phage specific to K. pneumoniae (MTCC 432) was isolated from Ganges river (designated as KpG). One-step growth curve, in vitro time kill curve study and in vivo infection model were performed to evaluate the efficacy of phage to curtail planktonic growth. Crystal violet assay and colony biofilm assay was done to determine the action of phages on biofilms. KpG phages had a greater burst size, better bactericidal potential and enhanced inhibitory effect against biofilms formed at liquid air and solid air interfaces. In vivo injection of KpG phages revealed that it did not pose any toxicity to zebrafish as evidenced by liver/brain enzyme profiles and by histopathological analysis. In vitro time kill assay showed a 3 log decline and a 6 log decline in K. pneumoniae colony counts, when phages were administered individually and in combination with streptomycin, respectively. The muscle tissue of zebrafish, infected with K. pneumoniae and treated with KpG phages showed a significant 2 log decline in bacterial counts relative to untreated control. Our study reveals that KpG phages has the potential to curtail plantonic and biofilm mode of growth in vivo in higher animal models.

scholarly journals Phages from Ganges River curtail in vitro biofilms and planktonic growth of drug resistant Klebsiella pneumoniae in a zebrafish infection model

2020 ◽  
Author(s):  
Niranjana Sri Sundaramoor ◽  
Subramanian Thothathri ◽  
Muthu Meenakshi Bhaskaran ◽  
ArunKumar GaneshPrasad ◽  
Saisubramanian Nagarajan
Keyword(s):  
Klebsiella Pneumoniae ◽  
Infection Model ◽  
Histopathological Analysis ◽  
Drug Resistant ◽  
Ganges River ◽  
Promising Alternative ◽  
Planktonic Growth ◽  
Time Kill

Abstract Bacteriophages are a promising alternative for curtailing infections caused by multi drug resistant (MDR) bacteria. The objective of the present study is to evaluate phage populations from water bodies to inhibit planktonic and biofilm mode of growth of drug resistant Klebsiella pneumoniae in vitro and curtail planktonic growth in vivo in a zebrafish model. Phage specific to K. pneumoniae (MTCC 432) was isolated from Ganges river (designated as KpG). One-step growth curve, in vitro time kill curve study and in vivo infection model were performed to evaluate the efficacy of phage against planktonic growth. Crystal violet assay and colony biofilm assay was done to determine the action of phages on biofilms. KpG phages had a greater burst size, better bactericidal potential and enhanced inhibitory effect against biofilms formed at liquid air and solid air interfaces. In vivo injection of KpG phages revealed that it did not pose any toxicity to zebrafish as evidenced by liver/brain enzyme profiles and by histopathological analysis. In vitro time kill assay showed a 3 log decline and a 6 log decline in K. pneumoniae colony counts, when phages were administered individually and in combination with streptomycin, respectively. The muscle tissue of zebrafish, infected with K. pneumoniae and treated with KpG phages showed a significant 2 log decline in bacterial counts relative to untreated control. Our study reveals that KpG phages has the potential to curtail plantonic and biofilm mode of growth in vivo in higher animal models.

scholarly journals Phages from Ganges River curtail in vitro biofilms and planktonic growth of drug resistant Klebsiella pneumoniae in a zebrafish infection model

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Niranjana Sri Sundaramoorthy ◽  
Subramaniam Thothathri ◽  
Muthumeenakshi Bhaskaran ◽  
ArunKumar GaneshPrasad ◽  
Saisubramanian Nagarajan
Keyword(s):  
Klebsiella Pneumoniae ◽  
Infection Model ◽  
Histopathological Analysis ◽  
Drug Resistant ◽  
Ganges River ◽  
Promising Alternative ◽  
Planktonic Growth ◽  
Time Kill

AbstractBacteriophages are a promising alternative for curtailing infections caused by multi drug resistant (MDR) bacteria. The objective of the present study is to evaluate phage populations from water bodies to inhibit planktonic and biofilm mode of growth of drug resistant Klebsiella pneumoniae in vitro and curtail planktonic growth in vivo in a zebrafish model. Phage specific to K. pneumoniae (MTCC 432) was isolated from Ganges River (designated as KpG). One-step growth curve, in vitro time kill curve study and in vivo infection model were performed to evaluate the ability of phage to curtail planktonic growth. Crystal violet assay and colony biofilm assay were performed to determine the action of phages on biofilms. KpG phages had a greater burst size, better bactericidal potential and enhanced inhibitory effect against biofilms formed at liquid air and solid air interfaces. In vitro time kill assay showed a 3 log decline and a 6 log decline in K. pneumoniae colony counts, when phages were administered individually and in combination with streptomycin, respectively. In vivo injection of KpG phages revealed that it did not pose any toxicity to zebrafish as evidenced by liver/brain enzyme profiles and by histopathological analysis. The muscle tissue of zebrafish, infected with K. pneumoniae and treated with KpG phages alone and in combination with streptomycin showed a significant 77.7% and 97.2% decline in CFU/ml, respectively, relative to untreated control. Our study reveals that KpG phages has the potential to curtail plantonic and biofilm mode of growth in higher animal models.

scholarly journals Repurposing the anti-viral drug zidovudine (AZT) in combination with meropenem as an effective treatment for infections with multi-drug resistant, carbapenemase-producing strains of Klebsiella pneumoniae

2020 ◽  
Vol 78 (9) ◽  
Author(s):  
Alexandra E DeSarno ◽  
Benjamin J Parcell ◽  
Peter J Coote
Keyword(s):  
Combination Therapy ◽  
Klebsiella Pneumoniae ◽  
Galleria Mellonella ◽  
Larval Mortality ◽  
Therapeutic Benefit ◽  
Drug Resistant ◽  
Bacterial Proliferation ◽  
Global Threat ◽  
Time Kill

Abstract Multi-drug resistant (MDR) Klebsiella pneumoniae represent a global threat to healthcare due to lack of effective treatments and high mortality rates. The aim of this research was to explore the potential of administering zidovudine (AZT) in combination with an existing antibiotic to treat resistant K. pneumoniae infections. Two MDR K. pneumoniae strains were employed, producing either the NDM-1 or KPC-3 carbapenemase. Efficacy of combinations of AZT with meropenem were compared with monotherapies against infections in Galleria mellonella larvae by measuring larval mortality and bacterial burden. The effect of the same combinations in vitro was determined via checkerboard and time-kill assays. In vitro, both K. pneumoniae strains were resistant to meropenem but were susceptible to AZT. In G. mellonella, treatment with either AZT or meropenem alone offered minimal therapeutic benefit against infections with either strain. In contrast, combination therapy of AZT with meropenem presented significantly enhanced efficacy compared to monotherapies. This was correlated with prevention of bacterial proliferation within the larvae but not elimination. Checkerboard assays showed that the interaction between AZT and meropenem was not synergistic but indifferent. In summary, combination therapy of AZT with meropenem represents a potential treatment for carbapenemase-producing MDR K. pneumoniae and merits further investigation.

Antisense inhibition of lpxB gene expression in Acinetobacter baumannii by peptide–PNA conjugates and synergy with colistin

2019 ◽  
Vol 75 (1) ◽  
pp. 51-59 ◽  
Author(s):  
Marta Martínez-Guitián ◽  
Juan Carlos Vázquez-Ucha ◽  
Laura Álvarez-Fraga ◽  
Kelly Conde-Pérez ◽  
Germán Bou ◽  
...  
Keyword(s):  
Survival Rate ◽  
Acinetobacter Baumannii ◽  
Lipid A ◽  
Galleria Mellonella ◽  
A549 Cells ◽  
Infection Model ◽  
Kill Curve ◽  
Time Kill

Abstract Background LpxB is an enzyme involved in the biosynthesis pathway of lipid A, a component of LPS. Objectives To evaluate the lpxB gene in Acinetobacter baumannii as a potential therapeutic target and to propose antisense agents such as peptide nucleic acids (PNAs) as a tool to combat bacterial infection, either alone or in combination with known antimicrobial therapies. Methods RNA-seq analysis of the A. baumannii ATCC 17978 strain in a murine pneumonia model was performed to study the in vivo expression of lpxB. Protein expression was studied in the presence or absence of anti-lpxB (KFF)3K-PNA (pPNA). Time–kill curve analyses and protection assays of infected A549 cells were performed. The chequerboard technique was used to test for synergy between pPNA and colistin. A Galleria mellonella infection model was used to test the in vivo efficacy of pPNA. Results The lpxB gene was overexpressed during pneumonia. Treatment with a specific pPNA inhibited LpxB expression in vitro, decreased survival of the ATCC 17978 strain and increased the survival rate of infected A549 cells. Synergy was observed between pPNA and colistin in colistin-susceptible strains. In vivo assays confirmed that a combination treatment of anti-lpxB pPNA and colistin was more effective than colistin in monotherapy. Conclusions The lpxB gene is essential for A. baumannii survival. Anti-lpxB pPNA inhibits LpxB expression, causing bacterial death. This pPNA showed synergy with colistin and increased the survival rate in G. mellonella. The data suggest that antisense pPNA molecules blocking the lpxB gene could be used as antibacterial agents.

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