Activity of ceftazidime-avibactam alone and in combination with polymyxin B against carbapenem-resistant Klebsiella pneumoniae in a tandem in vitro time-kill/in vivo Galleria mellonella survival model analysis

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00246-20 ◽

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.

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In Vitro and In Vivo Activity of Single and Dual Antimicrobial Agents Against KPC-producing Klebsiella pneumoniae

Open Forum Infectious Diseases ◽

10.1093/ofid/ofx163.936 ◽

2017 ◽

Vol 4 (suppl_1) ◽

pp. S379-S379

Author(s):

Farzad Moussavi ◽

Sarath Nath ◽

Daniel Abraham ◽

David Landman ◽

John Quale

Keyword(s):

Antimicrobial Agents ◽

Galleria Mellonella ◽

Survival Rates ◽

Polymyxin B ◽

The Other ◽

In Vivo Model ◽

Serious Infections ◽

Time Kill

Abstract Background Options for treatment of infections due to KPC-producing K. pneumoniae are limited, and combination therapy is often recommended. In this report, the in vitro and in vivo activity of potential therapeutic agents and combinations was assessed against four KPC-producing K. pneumoniae isolates. Methods Using clinically-relevant concentrations, time-kill experiments and the Galleria mellonella model of infection were used to examine the activity of polymyxin B, ceftazidime-avibactam, meropenem, rifampin, and amikacin alone and in combination. Four isolates of KPC-producing K. pneumoniae were studied, including two isolates that were resistant to polymyxin B and had ceftazidime-avibactam MICs of 8 µg/mL. The other two K. pneumoniae isolates were susceptible to polymyxin B and had lower MICs of ceftazidime-avibactam. Results Two isolates that were resistant to polymyxin B and with ceftazidime-avibactam MICs of 8 µg/mL were also resistant to amikacin and meropenem. When ceftazidime-avibactam was combined with either amikacin or meropenem, synergy was observed in vitro, and these combinations were associated with improved survival with the in vivo model. The other two K. pneumoniae isolates were susceptible to polymyxin B and had lower MICs of ceftazidime-avibactam. At concentrations four times the MIC, ceftazidime-avibactam had bactericidal activity in vitro; at one fourth the MIC, synergy was observed when combined with meropenem. Improved survival rates were observed with therapy with ceftazidime-avibactam, particularly when combined with a second agent for one isolate. In the in vivo model, polymyxin B with or without rifampin or meropenem, was ineffective against polymyxin B resistant strains. Conclusion Pending clinical studies, combining ceftazidime-avibactam with another agent (e.g., a carbapenem) should be encouraged when treating serious infections due to these pathogens, especially for isolates with ceftazidime-avibactam MICs near the susceptibility breakpoint. Disclosures All authors: No reported disclosures.

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In vitro and in vivo synergistic effects of tigecycline combined with aminoglycosides on carbapenem-resistant Klebsiella pneumoniae

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkab122 ◽

2021 ◽

Author(s):

Wentao Ni ◽

Deqing Yang ◽

Jie Guan ◽

Wen Xi ◽

Dexun Zhou ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Synergistic Effects ◽

Molecular Characteristics ◽

Synergistic Activity ◽

Significant Activity ◽

Clinical Pharmacokinetics ◽

Carbapenem Resistant ◽

Time Kill

Abstract Objectives Carbapenem-resistant Klebsiella pneumoniae (CR-KP) infections represent severe threats to public health worldwide. The aim of this study was to assess potential synergistic interaction between tigecycline and aminoglycosides via in vitro and in vivo studies. Methods Antibiotic resistance profiles and molecular characteristics of 168 CR-KP clinical isolates were investigated by susceptibility testing, PCR and MLST. Chequerboard tests and time–kill assays were performed for 20 CR-KP isolates to evaluate in vitro synergistic effects of tigecycline combined with aminoglycosides. A tissue-cage infection model of rats was established to evaluate in vivo synergistic effects. Different doses of tigecycline and aminoglycosides alone or in combination were administered for 7 days via tail vein injection. Antibiotic efficacy was evaluated in tissue-cage fluid and emergence of resistance was screened. Results The chequerboard tests showed that this combination displayed synergistic or partial synergistic activity against CR-KP. The time–kill assays further demonstrated that strong synergistic effects of such a combination existed against isolates that were susceptible to both drugs but for resistant isolates no synergy was observed if clinical pharmacokinetics were taken into consideration. The in vivo study showed that the therapeutic effectiveness of combination therapies was better than that of monotherapy for susceptible isolates, suggesting in vivo synergistic effects. Furthermore, combinations of tigecycline with an aminoglycoside showed significant activity in reducing the occurrence of tigecycline-resistant mutants. Conclusions Compared with single drugs, tigecycline combined with aminoglycosides could exert synergistic effects and reduce the emergence of tigecycline resistance. Such a combination might be an effective alternative when treating CR-KP infections in clinical practice.

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In Vitro Activities of Various Antimicrobials Alone and in Combination with Tigecycline against Carbapenem-Intermediate or -Resistant Acinetobacter baumannii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01099-06 ◽

2007 ◽

Vol 51 (5) ◽

pp. 1621-1626 ◽

Cited By ~ 67

Author(s):

Marc H. Scheetz ◽

Chao Qi ◽

John R. Warren ◽

Michael J. Postelnick ◽

Teresa Zembower ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Antimicrobial Susceptibility ◽

Bloodstream Infections ◽

Polymyxin B ◽

In Vitro Susceptibility ◽

Carbapenem Resistant ◽

Susceptibility Profiles ◽

Time Kill

ABSTRACT The activities of tigecycline alone and in combination with other antimicrobials are not well defined for carbapenem-intermediate or -resistant Acinetobacter baumannii (CIRA). Pharmacodynamic activity is even less well defined when clinically achievable serum concentrations are considered. Antimicrobial susceptibility testing of clinical CIRA isolates from 2001 to 2005 was performed by broth or agar dilution, as appropriate. Tigecycline concentrations were serially increased in time-kill studies with a representative of the most prevalent carbapenem-resistant clone (strain AA557; imipenem MIC, 64 mg/liter). The in vitro susceptibility of the strain was tested by time-kill studies in duplicate against the average free serum steady-state concentrations of tigecycline alone and in combination with various antimicrobials. Ninety-three CIRA isolates were tested and were found to have the following antimicrobial susceptibility profiles: tigecycline, MIC50 of 1 mg/liter and MIC90 of 2 mg/liter; minocycline, MIC50 of 0.5 mg/liter and MIC90 of 8 mg/liter; doxycycline, MIC50 of 2 mg/liter and MIC90 of ≥32 mg/liter; ampicillin-sulbactam, MIC50 of 48 mg/liter and MIC90 of 96 mg/liter; ciprofloxacin, MIC50 of ≥16 mg/liter and MIC90 of ≥16 mg/liter; rifampin, MIC50 of 4 mg/liter and MIC90 of 8 mg/liter; polymyxin B, MIC50 of 1 mg/liter and MIC90 of 1 mg/liter; amikacin, MIC50 of 32 mg/liter and MIC90 of ≥32 mg/liter; meropenem, MIC50 of 16 mg/liter and MIC90 of ≥128 mg/liter; and imipenem, MIC50 of 4 mg/liter and MIC90 of 64 mg/liter. Among the tetracyclines, the isolates were more susceptible to tigecycline than minocycline and doxycycline, according to FDA breakpoints (95%, 88%, and 71% of the isolates were susceptible to tigecycline, minocycline, and doxycycline, respectively). Concentration escalation studies with tigecycline revealed a maximal killing effect near the MIC, with no additional extent or rate of killing at concentrations 2× to 4× the MIC for tigecycline. Time-kill studies demonstrated indifference for tigecycline in combination with the antimicrobials tested. Polymyxin B, minocycline, and tigecycline are the most active antimicrobials in vitro against CIRA. Concentration escalation studies demonstrate that tigecycline may need to approach concentrations higher than those currently achieved in the bloodstream to adequately treat CIRA bloodstream infections. Future studies should evaluate these findings in vivo.

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Activity of Antimicrobial Combinations against KPC-2-Producing Klebsiella pneumoniae in a Rat Model and Time-Kill Assay

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00323-15 ◽

2015 ◽

Vol 59 (7) ◽

pp. 4301-4304 ◽

Cited By ~ 14

Author(s):

Paula Virginia Michelon Toledo ◽

Ayrton Alves Aranha Junior ◽

Lavinia Nery Arend ◽

Vanessa Ribeiro ◽

Alexandre Prehn Zavascki ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Triple Therapy ◽

Rat Model ◽

Polymyxin B ◽

Content Type ◽

Culture Positivity ◽

Time Kill ◽

Antimicrobial Combinations

ABSTRACTThis study evaluated the efficacy of tigecycline (TIG), polymyxin B (PMB), and meropenem (MER) in 80 rats challenged withKlebsiella pneumoniaecarbapenemase (KPC)-producingK. pneumoniaeinfection. A time-kill assay was performed with the same strain. Triple therapy and PMB+TIG were synergistic, promoted 100% survival, and produced negative peritoneal cultures, while MER+TIG showed lower survival and higher culture positivity than other regimens (P= 0.018) and was antagonistic.In vivoandin vitrostudies showed that combined regimens, except MER+TIG, were more effective than monotherapies for this KPC-producing strain.

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Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates: In Vivo Virulence Assessment in Galleria mellonella and Potential Therapeutics by Polycationic Oligoethyleneimine

Antibiotics ◽

10.3390/antibiotics10010056 ◽

2021 ◽

Vol 10 (1) ◽

pp. 56

Author(s):

Dalila Mil-Homens ◽

Maria Martins ◽

José Barbosa ◽

Gabriel Serafim ◽

Maria J. Sarmento ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Galleria Mellonella ◽

Multidrug Resistant ◽

In Vitro Models ◽

Clinical Isolates ◽

In Vivo Model ◽

Virulence Potential ◽

Hospital Acquired

Klebsiella pneumoniae, one of the most common pathogens found in hospital-acquired infections, is often resistant to multiple antibiotics. In fact, multidrug-resistant (MDR) K. pneumoniae producing KPC or OXA-48-like carbapenemases are recognized as a serious global health threat. In this sense, we evaluated the virulence of K. pneumoniae KPC(+) or OXA-48(+) aiming at potential antimicrobial therapeutics. K. pneumoniae carbapenemase (KPC) and the expanded-spectrum oxacillinase OXA-48 isolates were obtained from patients treated in medical care units in Lisbon, Portugal. The virulence potential of the K. pneumonia clinical isolates was tested using the Galleria mellonella model. For that, G. mellonella larvae were inoculated using patients KPC(+) and OXA-48(+) isolates. Using this in vivo model, the KPC(+) K. pneumoniae isolates showed to be, on average, more virulent than OXA-48(+). Virulence was found attenuated when a low bacterial inoculum (one magnitude lower) was tested. In addition, we also report the use of a synthetic polycationic oligomer (L-OEI-h) as a potential antimicrobial agent to fight infectious diseases caused by MDR bacteria. L-OEI-h has a broad-spectrum antibacterial activity and exerts a significantly bactericidal activity within the first 5-30 min treatment, causing lysis of the cytoplasmic membrane. Importantly, the polycationic oligomer showed low toxicity against in vitro models and no visible cytotoxicity (measured by survival and health index) was noted on the in vivo model (G. mellonella), thus L-OEI-h is foreseen as a promising polymer therapeutic for the treatment of MDR K. pneumoniae infections.

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Macrocolony of NDM-1 Producing Enterobacter hormaechei subsp. oharae Generates Subpopulations with Different Features Regarding the Response of Antimicrobial Agents and Biofilm Formation

Pathogens ◽

10.3390/pathogens8020049 ◽

2019 ◽

Vol 8 (2) ◽

pp. 49 ◽

Cited By ~ 2

Author(s):

Flávia Roberta Brust ◽

Luana Boff ◽

Danielle da Silva Trentin ◽

Franciele Pedrotti Rozales ◽

Afonso Luís Barth ◽

...

Keyword(s):

Antimicrobial Agents ◽

Galleria Mellonella ◽

Polymyxin B ◽

Multidrug Resistant ◽

Biofilm Production ◽

Depth Study ◽

Enterobacter Hormaechei ◽

Type 3

Enterobacter cloacae complex has been increasingly recognized as a nosocomial pathogen representing the third major Enterobacteriaceae species involved with infections. This study aims to evaluate virulence and antimicrobial susceptibility of subpopulations generated from macrocolonies of NDM-1 producing Enterobacter hormaechei clinical isolates. Biofilm was quantified using crystal violet method and fimbrial genes were investigated by PCR. Susceptibility of antimicrobials, alone and combined, was determined by minimum inhibitory concentration and checkerboard assays, respectively. Virulence and efficacy of antimicrobials were evaluated in Galleria mellonella larvae. Importantly, we verified that some subpopulations that originate from the same macrocolony present different biofilm production ability and distinct susceptibility to meropenem due to the loss of blaNDM-1 encoding plasmid. A more in-depth study was performed with the 798 macrocolony subpopulations. Type 3 fimbriae were straightly related with biofilm production; however, virulence in larvae was not statistically different among subpopulations. Triple combination with meropenem–rifampicin–polymyxin B showed in vitro synergistic effect against all subpopulations; while in vivo this treatment showed different efficacy rates for 798-1S and 798-4S subpopulations. The ability of multidrug resistant E. hormaechei isolates in generating bacterial subpopulations presenting different susceptible and virulence mechanisms are worrisome and may explain why these infections are hardly overcome.

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Elimination of Extracellular Adenosine Triphosphate for the Rapid Prediction of Quantitative Plate Counts in 24 h Time-Kill Studies against Carbapenem-Resistant Gram-Negative Bacteria

Microorganisms ◽

10.3390/microorganisms8101489 ◽

2020 ◽

Vol 8 (10) ◽

pp. 1489

Author(s):

Yiying Cai ◽

Jonathan J. Ng ◽

Hui Leck ◽

Jocelyn Q. Teo ◽

Jia-Xuan Goh ◽

...

Keyword(s):

Gram Negative Bacteria ◽

Gram Negative ◽

Carbapenem Resistant ◽

Infection Models ◽

Rapid Prediction ◽

Atp Bioluminescence ◽

Plate Counts ◽

Time Kill

Traditional in vitro time-kill studies (TKSs) require viable plating, which is tedious and time-consuming. We used ATP bioluminescence, with the removal of extracellular ATP (EC-ATP), as a surrogate for viable plating in TKSs against carbapenem-resistant Gram-negative bacteria (CR-GNB). Twenty-four-hour TKSs were conducted using eight clinical CR-GNB (two Escherichia coli, two Klebsiella spp., two Acinetobacter baumannii, two Pseudomonas aeruginosa) with multiple single and two-antibiotic combinations. ATP bioluminescence and viable counts were determined at each timepoint (0, 2, 4, 8, 24 h), with and without apyrase treatment. Correlation between ATP bioluminescence and viable counts was determined for apyrase-treated and non-apyrase-treated samples. Receiver operator characteristic curves were plotted to determine the optimal luminescence threshold to discriminate between inhibitory/non-inhibitory and bactericidal/non-bactericidal combinations, compared to viable counts. After treatment of bacteria with 2 U/mL apyrase for 15 min at 37 °C, correlation to viable counts was significantly higher compared to untreated samples (p < 0.01). Predictive accuracies of ATP bioluminescence were also significantly higher for apyrase-treated samples in distinguishing inhibitory (p < 0.01) and bactericidal (p = 0.03) combinations against CR-GNB compared to untreated samples, when all species were collectively analyzed. We found that ATP bioluminescence can potentially replace viable plating in TKS. Our assay also has applications in in vitro and in vivo infection models.

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Comparative Effectiveness of Aminoglycosides, Polymyxin B, and Tigecycline for Clearance of Carbapenem-Resistant Klebsiella pneumoniae from Urine

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00387-11 ◽

2011 ◽

Vol 55 (12) ◽

pp. 5893-5899 ◽

Cited By ~ 79

Author(s):

Michael J. Satlin ◽

Christine J. Kubin ◽

Jill S. Blumenthal ◽

Andrew B. Cohen ◽

E. Yoko Furuya ◽

...

Keyword(s):

New York ◽

Klebsiella Pneumoniae ◽

Urine Culture ◽

Active Agent ◽

Polymyxin B ◽

Content Type ◽

Carbapenem Resistant ◽

Tract Infections

ABSTRACTCarbapenem-resistantKlebsiella pneumoniae(CRKP) is an increasingly common cause of health care-associated urinary tract infections. Antimicrobials within vitroactivity against CRKP are typically limited to polymyxins, tigecycline, and often, aminoglycosides. We conducted a retrospective cohort study of cases of CRKP bacteriuria at New York-Presbyterian Hospital from January 2005 through June 2010 to compare microbiologic clearance rates based on the use of polymyxin B, tigecycline, or an aminoglycoside. We constructed three active antimicrobial cohorts based on the active agent used and an untreated cohort of cases that did not receive antimicrobial therapy with Gram-negative activity. Microbiologic clearance was defined as having a follow-up urine culture that did not yield CRKP. Cases without an appropriate follow-up culture or that received multiple active agents or less than 3 days of the active agent were excluded. Eighty-seven cases were included in the active antimicrobial cohorts, and 69 were included in the untreated cohort. The microbiologic clearance rate was 88% in the aminoglycoside cohort (n= 41), compared to 64% in the polymyxin B (P= 0.02;n= 25), 43% in the tigecycline (P< 0.001;n= 21), and 36% in the untreated (P< 0.001;n= 69) cohorts. Using multivariate analysis, the odds of clearance were lower for the polymyxin B (odds ratio [OR], 0.10;P= 0.003), tigecycline (OR, 0.08;P= 0.001), and untreated (OR, 0.14;P= 0.003) cohorts than for the aminoglycoside cohort. Treatment with an aminoglycoside, when activein vitro, was associated with a significantly higher rate of microbiologic clearance of CRKP bacteriuria than treatment with either polymyxin B or tigecycline.

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