In vivo synergism of ampicillin, gentamicin, ceftaroline and ceftriaxone against Enterococcus faecalis assessed in the Galleria mellonella infection model

2020 ◽  
Author(s):  
Lara Thieme ◽  
Anita Hartung ◽  
Oliwia Makarewicz ◽  
Mathias W Pletz
Keyword(s):  
Enterococcus Faecalis ◽  
Galleria Mellonella ◽  
Synergistic Effects ◽  
Agar Plate ◽  
Infection Model ◽  
Pharmacokinetic Data ◽  
Multiple Dosing ◽  
Larval Infection

Abstract Background The unfavourable safety profile of aminoglycosides and the synergistic effects observed in vitro have prompted the development of novel dual β-lactam therapies, e.g. ampicillin/ceftriaxone or ampicillin/ceftaroline, for the treatment of Enterococcus faecalis endocarditis. Objectives For comparison with in vitro chequerboard assay results, a partial chequerboard setup of ampicillin/gentamicin, ampicillin/ceftriaxone and ampicillin/ceftaroline against E. faecalis was established in the Galleria mellonella larval infection model. Methods Discrimination of synergistic and additive interactions was based on the evaluation of larval survival, bacterial quantity in the haemolymph and a pathology score index (internal to the workgroup). Single and multiple dosing schemes based on the half-life of ampicillin were applied. Pharmacokinetic data of the antibiotics in the larvae were determined via agar plate diffusion assays. Results Ampicillin and ceftriaxone exhibited strain-specific synergistic interactions in the larvae under both dosing regimens, while the other two combinations showed additive effects. Ampicillin/ceftaroline was inferior to ampicillin/ ceftriaxone. Not all synergistic effects observed in vitro could be replicated in the larvae. Conclusions Our results suggest superior efficacy of ampicillin/ceftriaxone for the treatment of high-inoculum enterococcal infections, for at least some strains, but question the benefit of the current standard of adding the nephrotoxic gentamicin compared with the safer ceftriaxone. This is the first study to develop a scheme for differentiation between additive and synergistic effects in larvae and apply a multiple-antibiotic dosing scheme based on the pharmacokinetics of ampicillin. The model allows the analysis of synergistic effects of antimicrobials in an in vivo setting, but the clinical correlation warrants further study.

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 Combined With Mefloquine, Resurrect Colistin Active in Colistin-Resistant Pseudomonas aeruginosa in vitro and in vivo

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiaodong Zhang ◽  
Yining Zhao ◽  
Luozhu Feng ◽  
Mengxin Xu ◽  
Yiru Ge ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Synergistic Effect ◽  
Biofilm Formation ◽  
Galleria Mellonella ◽  
Synergistic Effects ◽  
Alternative Therapy ◽  
Multidrug Resistant ◽  
Infection Model

Colistin is a polymyxin antibiotic that is widely used for the treatment of multidrug resistant (MDR) Pseudomonas aeruginosa infections, as the last resort. Over the past few years, unreasonable use of antibiotics has resulted in an increase in MDR strains, including colistin-resistant P. aeruginosa. The present study aimed to explore the synergistic effects of mefloquine in combination with colistin for the treatment of colistin-resistant P. aeruginosa in vivo and in vitro. The synergistic effect of the combination of mefloquine and colistin was investigated in vitro using checkerboard method, time-killing assay, biofilm formation inhibition test, and biofilm eradication test. The study also explored the synergistic effects of this combination of drugs in vivo, using a Galleria mellonella infection model. The results for checkerboard method and time killing curve indicated that mefloquine in combination with colistin showed a good antibacterial activity. Furthermore, the combination of these two drugs inhibited biofilm formation and eradicated pre-formed mature biofilms. This synergistic effect was visualized using scanning electron microscopy (SEM), wherein the results showed that the combination of mefloquine and colistin reduced biofilm formation significantly. Further, the application of this combination of drugs to in vivo infection model significantly increased the survival rate of G. mellonella larvae. Altogether, the combination of mefloquine and colistin showed a good synergistic effect in vitro and in vivo, and highlighted its potential to be used as an alternative therapy for the treatment of colistin-resistant P. aeruginosa infection.

scholarly journals Designing P. aeruginosa synthetic phages with reduced genomes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Diana P. Pires ◽  
Rodrigo Monteiro ◽  
Dalila Mil-Homens ◽  
Arsénio Fialho ◽  
Timothy K. Lu ◽  
...  
Keyword(s):  
Galleria Mellonella ◽  
Antibacterial Properties ◽  
Genetically Engineered ◽  
In Vivo Studies ◽  
Infection Model ◽  
Promising Therapeutic Approach ◽  
Genes Encoding ◽  
First Time

AbstractIn the era where antibiotic resistance is considered one of the major worldwide concerns, bacteriophages have emerged as a promising therapeutic approach to deal with this problem. Genetically engineered bacteriophages can enable enhanced anti-bacterial functionalities, but require cloning additional genes into the phage genomes, which might be challenging due to the DNA encapsulation capacity of a phage. To tackle this issue, we designed and assembled for the first time synthetic phages with smaller genomes by knocking out up to 48% of the genes encoding hypothetical proteins from the genome of the newly isolated Pseudomonas aeruginosa phage vB_PaeP_PE3. The antibacterial efficacy of the wild-type and the synthetic phages was assessed in vitro as well as in vivo using a Galleria mellonella infection model. Overall, both in vitro and in vivo studies revealed that the knock-outs made in phage genome do not impair the antibacterial properties of the synthetic phages, indicating that this could be a good strategy to clear space from phage genomes in order to enable the introduction of other genes of interest that can potentiate the future treatment of P. aeruginosa infections.

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 ace, Which Encodes an Adhesin in Enterococcus faecalis, Is Regulated by Ers and Is Involved in Virulence

2009 ◽  
Vol 77 (7) ◽  
pp. 2832-2839 ◽  
Author(s):  
Francois Lebreton ◽  
Eliette Riboulet-Bisson ◽  
Pascale Serror ◽  
Maurizio Sanguinetti ◽  
Brunella Posteraro ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Galleria Mellonella ◽  
Animal Experiments ◽  
Opportunistic Pathogen ◽  
Transcriptional Analysis ◽  
Binding Assays ◽  
Mobility Shift ◽  
Reverse Transcription Quantitative Pcr

ABSTRACT Enterococcus faecalis is an opportunistic pathogen that causes numerous infectious diseases in humans and is a major agent of nosocomial infections. In this work, we showed that the recently identified transcriptional regulator Ers (PrfA like), known to be involved in the cellular metabolism and the virulence of E. faecalis, acts as a repressor of ace, which encodes a collagen-binding protein. We characterized the promoter region of ace, and transcriptional analysis by reverse transcription-quantitative PCR and mobility shift protein-DNA binding assays revealed that Ers directly regulates the expression of ace. Transcription of ace appeared to be induced by the presence of bile salts, probably via the deregulation of ers. Moreover, with an ace deletion mutant and the complemented strain and by using an insect (Galleria mellonella) virulence model, as well as in vivo-in vitro murine macrophage models, we demonstrated for the first time that Ace can be considered a virulence factor for E. faecalis. Furthermore, animal experiments revealed that Ace is also involved in urinary tract infection by E. faecalis.

scholarly journals An Optimized Bacteriophage Cocktail Can Effectively Control Salmonella in vitro and in Galleria mellonella

2021 ◽  
Vol 11 ◽  
Author(s):  
Janet Y. Nale ◽  
Gurinder K. Vinner ◽  
Viviana C. Lopez ◽  
Anisha M. Thanki ◽  
Preeda Phothaworn ◽  
...  
Keyword(s):  
Galleria Mellonella ◽  
Future Application ◽  
Infection Model ◽  
Prophylactic Regimen ◽  
Phage Cocktail ◽  
Resistant Strains ◽  
Future Work ◽  
Further Development

Salmonella spp. is a leading cause of gastrointestinal enteritis in humans where it is largely contracted via contaminated poultry and pork. Phages can be used to control Salmonella infection in the animals, which could break the cycle of infection before the products are accessible for consumption. Here, the potential of 21 myoviruses and a siphovirus to eliminate Salmonella in vitro and in vivo was examined with the aim of developing a biocontrol strategy to curtail the infection in poultry and swine. Together, the phages targeted the twenty-three poultry and ten swine prevalent Salmonella serotype isolates tested. Although individual phages significantly reduced bacterial growth of representative isolates within 6 h post-infection, bacterial regrowth occurred 1 h later, indicating proliferation of resistant strains. To curtail bacteriophage resistance, a novel three-phage cocktail was developed in vitro, and further investigated in an optimized Galleria mellonella larva Salmonella infection model colonized with representative swine, chicken and laboratory strains. For all the strains examined, G. mellonella larvae given phages 2 h prior to bacterial exposure (prophylactic regimen) survived and Salmonella was undetectable 24 h post-phage treatment and throughout the experimental time (72 h). Administering phages with bacteria (co-infection), or 2 h post-bacterial exposure (remedial regimen) also improved survival (73–100% and 15–88%, respectively), but was less effective than prophylaxis application. These pre-livestock data support the future application of this cocktail for further development to effectively treat Salmonella infection in poultry and pigs. Future work will focus on cocktail formulation to ensure stability and incorporation into feeds and used to treat the infection in target animals.

scholarly journals Enterococcus faecalis persists and replicates within epithelial cells in vitro and in vivo during wound infection

2021 ◽  
Author(s):  
Wei Hong Tay ◽  
Ronni A.G. da Silva ◽  
Foo Kiong Ho ◽  
Kelvin K.L. Chong ◽  
Alexander Ludwig ◽  
...  
Keyword(s):  
Wound Infection ◽  
Enterococcus Faecalis ◽  
Opportunistic Pathogen ◽  
Small Gtpase ◽  
Infection Model ◽  
Robust Immune Response ◽  
Lysosomal Protease ◽  
The Face

Enterococcus faecalis is a frequent opportunistic pathogen of wounds, whose infections are associated with biofilm formation, persistence, and recalcitrance toward treatment. We have previously shown that E. faecalis wound infection persists for at least 7 days. Here we report that viable E. faecalis are present within both immune and non-immune cells at the wound site up to 5 days after infection, raising the prospect that intracellular persistence contributes to chronic E. faecalis infection. Using an in vitro keratinocyte infection model, we show that a subpopulation of E. faecalis becomes internalized via macropinocytosis into single membrane-bound compartments, where they can survive and replicate. These intracellular E. faecalis can persist in late endosomes up to 72 hours after infection in the absence of colocalization with the lysosomal protease cathepsin D or apparent fusion with the lysosome, suggesting that E. faecalis blocks endosomal maturation. Indeed, intracellular E. faecalis infection results in a marked reduction in Rab7 expression, a small GTPase required for endosome-lysosome fusion. Finally, we demonstrate that intracellular E. faecalis derived from infected keratinocytes are significantly more efficient in reinfecting new keratinocytes. Together, these data suggest that intracellular proliferation of E. faecalis may contribute to its persistence in the face of a robust immune response, providing a primed reservoir of bacteria for subsequent reinfection.

Characterization of the novel OXA-213-like β-lactamase OXA-822 from Acinetobacter calcoaceticus

2020 ◽  
Author(s):  
Manuela Tietgen ◽  
Laura Leukert ◽  
Julian Sommer ◽  
Jan S Kramer ◽  
Steffen Brunst ◽  
...  
Keyword(s):  
Galleria Mellonella ◽  
Sequence Similarity ◽  
Acinetobacter Calcoaceticus ◽  
Amino Acid Sequence Similarity ◽  
Control Group ◽  
Infection Model ◽  
The Novel ◽  
High Turnover

Abstract Objectives This study analysed the novel carbapenem-hydrolysing class D β-lactamase OXA-822 identified in the clinical Acinetobacter calcoaceticus isolate AC_2117. Methods WGS was employed for identification of β-lactamases. Micro-broth dilution was used for evaluation of antibiotic susceptibility of AC_2117 and transformants containing blaOXA-822. After heterologous purification of OXA-822, OXA-359 and OXA-213, enzyme kinetics were determined using spectrometry. The effect of OXA-822 upon meropenem treatment was analysed in the Galleria mellonella in vivo infection model. Results OXA-822 is a member of the intrinsic OXA-213-like family found in A. calcoaceticus and Acinetobacter pittii. Amino acid sequence similarity to the nearest related OXA-359 was 97%. Production of OXA-822, OXA-359 and OXA-213 in Acinetobacter baumannii ATCC® 19606T resulted in elevated MICs for carbapenems (up to 16-fold). Penicillinase activity of the purified OXA-822 revealed high KM values, in the millimolar range, combined with high turnover numbers. OXA-822 showed the highest affinity to carbapenems, but affinity to imipenem was ∼10-fold lower compared with other carbapenems. Molecular modelling revealed that imipenem does not interact with a negatively charged side chain of OXA-822, as doripenem does, leading to the lower affinity. Presence of OXA-822 decreased survival of infected Galleria mellonella larvae after treatment with meropenem. Only 52.7% ± 7.7% of the larvae survived after 24 h compared with 90.9% ± 3.7% survival in the control group. Conclusions The novel OXA-822 from a clinical A. calcoaceticus isolate displayed penicillinase and carbapenemase activity in vitro, elevated MICs in different species and decreased carbapenem susceptibility in A. baumannii in vivo.

scholarly journals A Galleria mellonella infection model reveals double and triple antibiotic combination therapies with enhanced efficacy versus a multidrug-resistant strain of Pseudomonas aeruginosa

2014 ◽  
Vol 63 (7) ◽  
pp. 945-955 ◽  
Author(s):  
Jessica Krezdorn ◽  
Sophie Adams ◽  
Peter J. Coote
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Resistant Strain ◽  
Galleria Mellonella ◽  
Multidrug Resistant ◽  
Therapeutic Benefit ◽  
Infection Model ◽  
Combination Therapies ◽  
Multidrug Resistant Strain

The aim of this study was to compare the inhibitory effect of antibiotic combinations in vitro with efficacy in Galleria mellonella larvae in vivo to identify efficacious combinations that target Pseudomonas aeruginosa. P. aeruginosa NCTC 13437, a multidrug-resistant strain resistant to β-lactams and aminoglycosides, was used. Susceptibility to cefotaxime, piperacillin, meropenem, amikacin, levofloxacin and colistin alone, or in dual or triple combinations, was measured in vitro via a 24 h time-kill assay. In vitro results were then compared with the efficacy of the same dual or triple antibiotic combinations versus G. mellonella larvae infected with P. aeruginosa. G. mellonella haemolymph burden of P. aeruginosa was determined over 96 h post-infection and treatment with the most potent combination therapies. Many dual and triple combinations of antibiotics displayed synergistic inhibition of multidrug-resistant P. aeruginosa in vitro. There was little correlation between combinations that were synergistic in vitro and those that showed enhanced efficacy in vivo versus infected G. mellonella larvae. The most potent dual and triple combinations in vivo were cefotaxime plus piperacillin, and meropenem plus piperacillin and amikacin, respectively. Fewer combinations were found to offer enhanced therapeutic benefit in vivo compared with in vitro. The therapeutic benefit arising from treatment with antibiotic combinations in vivo correlated with reduced larval burden of P. aeruginosa. This study has identified antibiotic combinations that merit further investigation for their clinical potential and has demonstrated the utility of using G. mellonella to screen for novel antibiotic treatments that demonstrate efficacy in vivo.

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