scholarly journals Phage Resistance Is Associated with Decreased Virulence in KPC-Producing Klebsiella pneumoniae of the Clonal Group 258 Clade II Lineage

2021 ◽  
Vol 9 (4) ◽  
pp. 762
Author(s):  
Lucia Henrici De Angelis ◽  
Noemi Poerio ◽  
Vincenzo Di Pilato ◽  
Federica De Santis ◽  
Alberto Antonelli ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Parental Strain ◽  
Bacterial Infections ◽  
Capsular Polysaccharide ◽  
Galleria Mellonella ◽  
Bacterial Pathogen ◽  
Phage Resistance ◽  
Infection Model ◽  
Lytic Phage ◽  
Susceptible Host

Phage therapy is now reconsidered with interest in the treatment of bacterial infections. A major piece of information for this application is the definition of the molecular targets exploited by phages to infect bacteria. Here, the genetic basis of resistance to the lytic phage φBO1E by its susceptible host Klebsiella pneumoniae KKBO-1 has been investigated. KKBO-1 phage-resistant mutants were obtained by infection at high multiplicity. One mutant, designated BO-FR-1, was selected for subsequent experiments, including virulence assessment in a Galleria mellonella infection model and characterization by whole-genome sequencing. Infection with BO-FR-1 was associated with a significantly lower mortality when compared to that of the parental strain. The BO-FR-1 genome differed from KKBO-1 by a single nonsense mutation into the wbaP gene, which encodes a glycosyltransferase involved in the first step of the biosynthesis of the capsular polysaccharide (CPS). Phage susceptibility was restored when BO-FR-1 was complemented with the constitutive wbaP gene. Our results demonstrated that φBO1E infects KKBO-1 targeting the bacterial CPS. Interestingly, BO-FR-1 was less virulent than the parental strain, suggesting that in the context of the interplay among phage, bacterial pathogen and host, the emergence of phage resistance may be beneficial for the host.

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.

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 Interactions between the Prophage 919TP and Its Vibrio cholerae Host: Implications of gmd Mutation for Phage Resistance, Cell Auto-Aggregation, and Motility

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2342
Author(s):  
Na Li ◽  
Yigang Zeng ◽  
Bijie Hu ◽  
Tongyu Zhu ◽  
Sine Lo Svenningsen ◽  
...  
Keyword(s):  
Bacterial Pathogen ◽  
Genomic Analysis ◽  
Resistant Mutant ◽  
Resistance Mechanisms ◽  
Phage Resistance ◽  
Comparative Genomic ◽  
Lytic Phage ◽  
O Antigen ◽  
Trade Offs ◽  
Base Pair Deletion

Prophage 919TP is widely distributed among Vibrio cholera and is induced to produce free φ919TP phage particles. However, the interactions between prophage φ919TP, the induced phage particle, and its host remain unknown. In particular, phage resistance mechanisms and potential fitness trade-offs, resulting from phage resistance, are unresolved. In this study, we examined a prophage 919TP-deleted variant of V. cholerae and its interaction with a modified lytic variant of the induced prophage (φ919TP cI-). Specifically, the phage-resistant mutant was isolated by challenging a prophage-deleted variant with lytic phage φ919TP cI-. Further, the comparative genomic analysis of wild-type and φ919TP cI--resistant mutant predicted that phage φ919TP cI- selects for phage-resistant mutants harboring a mutation in key steps of lipopolysaccharide (LPS) O-antigen biosynthesis, causing a single-base-pair deletion in gene gmd. Our study showed that the gmd-mediated O-antigen defect can cause pleiotropic phenotypes, e.g., cell autoaggregation and reduced swarming motility, emphasizing the role of phage-driven diversification in V. cholerae. The developed approach assists in the identification of genetic determinants of host specificity and is used to explore the molecular mechanism underlying phage-host interactions. Our findings contribute to the understanding of prophage-facilitated horizontal gene transfer and emphasize the potential for developing new strategies to optimize the use of phages in bacterial pathogen control.

scholarly journals The antimicrobial activity of silver acetate against Acinetobacter baumannii in a Galleria mellonella infection model

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11196
Author(s):  
Eden Mannix-Fisher ◽  
Samantha McLean
Keyword(s):  
Acinetobacter Baumannii ◽  
Bacterial Infections ◽  
Galleria Mellonella ◽  
World Health Organisation ◽  
World Health ◽  
Infection Model ◽  
Silver Acetate ◽  
Selective Toxicity ◽  
Carbapenem Resistant

Background The increasing prevalence of bacterial infections that are resistant to antibiotic treatment has caused the scientific and medical communities to look for alternate remedies aimed at prevention and treatment. In addition to researching novel antimicrobials, there has also been much interest in revisiting some of the earliest therapies used by man. One such antimicrobial is silver; its use stretches back to the ancient Greeks but interest in its medicinal properties has increased in recent years due to the rise in antibiotic resistance. Currently antimicrobial silver is found in everything from lunch boxes to medical device implants. Though much is claimed about the antimicrobial efficacy of silver salts the research in this area is mixed. Methods Herein we investigated the efficacy of silver acetate against a carbapenem resistant strain of Acinetobacter baumannii to determine the in vitro activity of this silver salt against a World Health Organisation designated category I critical pathogen. Furthermore, we use the Galleria mellonella larvae model to assess toxicity of the compound and its efficacy in treating infections in a live host. Results We found that silver acetate can be delivered safely to Galleria at medically relevant and antimicrobial levels without detriment to the larvae and that administration of silver acetate to an infection model significantly improved survival. This demonstrates the selective toxicity of silver acetate for bacterial pathogens but also highlights the need for administration of well-defined doses of the antimicrobial to provide an efficacious treatment.

scholarly journals Identifying virulence determinants of multidrug-resistant Klebsiella pneumoniae in Galleria mellonella

2021 ◽  
Author(s):  
Sebastian Bruchmann ◽  
Theresa Feltwell ◽  
Julian Parkhill ◽  
Francesca L Short
Keyword(s):  
Klebsiella Pneumoniae ◽  
Large Scale ◽  
Galleria Mellonella ◽  
Fitness Landscape ◽  
Dominant Role ◽  
Bacterial Pathogens ◽  
Model Organism ◽  
Multidrug Resistant ◽  
Infection Model ◽  
Virulence Determinants

Abstract Infections caused by Klebsiella pneumoniae are a major public health threat. Extensively drug-resistant and even pan-resistant strains have been reported. Understanding K. pneumoniae pathogenesis is hampered by the fact that murine models of infection offer limited resolution for non-hypervirulent strains which cause the majority of infections. The insect Galleria mellonella larva is a widely used alternative model organism for bacterial pathogens. We have performed genome-scale fitness profiling of a multidrug-resistant K. pneumoniae ST258 strain during infection of G. mellonella, to determine if this model is suitable for large-scale virulence factor discovery in this pathogen. Our results demonstrated a dominant role for surface polysaccharides in infection, with contributions from siderophores, cell envelope proteins, purine biosynthesis genes and additional genes of unknown function. Comparison with a hypervirulent strain, ATCC 43816, revealed substantial overlap in important infection-related genes, as well as additional putative virulence factors specific to ST258, reflecting strain-dependent fitness effects. Our analysis also identified a role for the metalloregulatory protein NfeR (YqjI) in virulence. Overall, this study offers new insight into the infection fitness landscape of K. pneumoniae, and provides a framework for using the highly flexible and easily scalable G. mellonella infection model to dissect molecular virulence mechanisms of bacterial pathogens.

scholarly journals Identifying virulence determinants of multidrug-resistant Klebsiella pneumoniae in Galleria mellonella

2020 ◽  
Author(s):  
Sebastian Bruchmann ◽  
Theresa Feltwell ◽  
Julian Parkhill ◽  
Francesca L. Short
Keyword(s):  
Klebsiella Pneumoniae ◽  
Large Scale ◽  
Galleria Mellonella ◽  
Fitness Landscape ◽  
Cell Envelope ◽  
Dominant Role ◽  
Multidrug Resistant ◽  
Infection Model ◽  
Virulence Determinants ◽  
Genome Scale

AbstractInfections caused by Klebsiella pneumoniae are a major public health threat. Extensively drug-resistant and even pan-resistant strains have been reported. Understanding K. pneumoniae pathogenesis is hampered by the fact that murine models of infection offer limited resolution for the non-hypervirulent strains which cause the majority of infections. We have performed genome-scale fitness profiling of a multidrug-resistant K. pneumoniae ST258 strain during infection of the insect Galleria mellonella, with the aim to determine if this model is suitable for large-scale virulence factor discovery in this pathogen. Our results demonstrated a dominant role for surface polysaccharides in infection, with contributions from siderophores, cell envelope proteins, purine biosynthesis genes and additional genes of unknown function. Comparison with a hypervirulent strain, ATCC 43816, revealed substantial overlap in important infection-related genes, as well as additional putative virulence factors that may be specific to ST258. Our analysis also identified a role for the metalloregulatory protein NfeR (also called YqjI) in virulence. Overall, this study offers new insight into the infection fitness landscape of K. pneumoniae ST258, and provides a framework for using the highly flexible, scalable G. mellonella infection model to dissect the molecular virulence mechanisms of K. pneumoniae and other bacterial pathogens.

Evolution of hypervirulence in carbapenem-resistant Klebsiella pneumoniae in China: a multicentre, molecular epidemiological analysis

2019 ◽  
Vol 75 (2) ◽  
pp. 327-336 ◽  
Author(s):  
Yawei Zhang ◽  
Longyang Jin ◽  
Pengwen Ouyang ◽  
Qi Wang ◽  
Ruobing Wang ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Virulence Genes ◽  
Galleria Mellonella ◽  
Infection Model ◽  
Epidemiological Analysis ◽  
Virulence Plasmids ◽  
Carbapenem Resistant ◽  
Resistance Plasmid ◽  
Serum Killing ◽  
Evolution Of Resistance

Abstract Objectives Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) have been increasingly reported in China. Here, a multicentre, longitudinal surveillance study on CR-hvKP is described. Methods We retrospectively investigated carbapenem-resistant K. pneumoniae (CRKP) in 56 centres across China during 2015–17 and screened the virulence genes (iucA, iroN, rmpA and rmpA2) for the presence of virulence plasmids. Hypermucoviscosity, serum killing and Galleria mellonella lethality experiments were conducted to identify CR-hvKP among strains with all four virulence genes. Capsule typing, fitness and plasmid features of CR-hvKP were also investigated. Results A total of 1052 CRKP were collected. Among these, 34.2% (360/1052) carried virulence genes and 72 of them had all four of the virulence genes tested. Fifty-five (76.4%) were considered to be CR-hvKP using the G. mellonella infection model, with KPC-2-producing K64-ST11 being the most common type (80%, 44/55). Prevalence of CR-hvKP differed greatly between regions, with the highest in Henan (25.4%, 17/67) and Shandong (25.8%, 25/97). A significant increase in CR-hvKP among KPC-2-producing ST11 strains was observed, from 2.1% (3/141) in 2015 to 7.0% (23/329) in 2017 (P=0.045). Alarmingly, compared with classic CRKP, no difference in growth was found among CR-hvKP (P=0.7028), suggesting a potential risk for dissemination. The hybrid virulence and resistance-encoding plasmid evolved from pLVPK and the resistance plasmid harbouring blaKPC-2, indicating evolution existed between the hypervirulence and hyper-resistance plasmid. Conclusions CR-hvKP were more frequently detected than previously assumed, especially among KPC-2-producing ST11. Dissemination of hypervirulence could be extremely rapid due to limited fitness cost. Also, the evolution of resistance genes into hypervirulence plasmids was identified, presenting significant challenges for public health and infection control.

scholarly journals 1952. Bacteriophage Treatment Improves Survival of Mice Infected with Carbapenem-Resistant Klebsiella pneumoniae

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S60-S60
Author(s):  
Shayla Hesse ◽  
Natalia Malachowa ◽  
Adeline Porter ◽  
Brett Freedman ◽  
Scott Kobayashi ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Bacterial Infections ◽  
Phage Therapy ◽  
Control Group ◽  
Lytic Phage ◽  
Mouse Blood ◽  
Carbapenem Resistant ◽  
In Vitro Data

Abstract Background Bacteriophage (phage) therapy is being considered as a treatment option for patients with multi-drug-resistant bacterial infections. However, there is a dearth of controlled clinical data to support therapeutic phage efficacy. As a first step toward addressing this deficiency, we tested the ability of two well-characterized phages, alone and in combination, to kill carbapenem-resistant Klebsiella pneumoniae (ST258) in blood in vitro and rescue mice from lethal ST258 infection. Methods Wild-type C57BL/6J mice were infected with a lethal inoculum of ST258 by intra-peritoneal (IP) injection followed 1 hour later by IP administration of lytic phage P1, P2, or P1+P2 at a multiplicity of infection (MOI) estimated at 1. Survival of each group of mice was tracked for 10 days. In separate experiments, mice were sacrificed at 1 hour, 24 hours, and 48 hours post-phage treatment. Mouse blood and tissues were collected at each timepoint for enumeration of bacteria and phage, screening for phage resistance, and histopathology. Results ST258 survival in mouse blood in vitro was significantly less after 1 hour of incubation with P1 or P1+P2 (MOI 1) compared with the control group (no phage). Consistent with the in vitro data, none of the mice (0/15) in the control group (no phage) survived to 10 days post-infection, whereas 12/15, 14/15, and 15/15 mice survived in the P2, P1, and P1+P2-treated groups, respectively (P < 0.0001). Conclusion Prompt, systemic administration of lytic bacteriophages rescued mice from lethal ST258 infection. These data support the potential of phage therapy to effectively treat infections caused by ST258. It will be important to assess whether, for other phage-bacteria combinations, in vitro lysis in blood correlates with in vivo treatment efficacy and therefore may have predictive utility. Disclosures All Authors: No reported Disclosures.

Complex interactions of Klebsiella pneumoniae with the host immune system in a Galleria mellonella infection model

2013 ◽  
Vol 62 (12) ◽  
pp. 1790-1798 ◽  
Author(s):  
Matthew E. Wand ◽  
James W. I. McCowen ◽  
Philip G. Nugent ◽  
J. Mark Sutton
Keyword(s):  
Immune System ◽  
Klebsiella Pneumoniae ◽  
Galleria Mellonella ◽  
Cell Damage ◽  
Opportunistic Pathogen ◽  
Infection Model ◽  
Host Immune System ◽  
Special Importance ◽  
Human Pathogens ◽  
Complex Interactions

Worldwide, Klebsiella pneumoniae is an increasingly problematic opportunistic pathogen, with the emergence of carbapenem-resistant isolates of special importance. The mechanisms of virulence are poorly understood, and the current study utilized the invertebrate model Galleria mellonella to investigate facets of the virulence process. A range of UK clinical isolates and reference strains was assessed in Galleria by measuring survival as an end point. The clinical strains showed a range of virulence, with the majority of strains (68 %) causing greater than 50 % mortality at a challenge dose of 1×105 c.f.u. Three additional intermediate read-outs were developed to allow the mechanisms of virulence of Klebsiella to be dissected further. The release of lactate dehydrogenase as a marker of cell damage was the best predictor of virulence. Melanization as a marker of the insect innate immune system and ability to proliferate within Galleria as a marker of immune evasion also broadly correlated with survival but with some notable exceptions. No direct correlation was observed between virulence and either K1 or other defined capsular types, the carriage of defined virulence factors or particular functional phenotypes. Overall, the study showed that Galleria can provide significant insights into the mechanisms of virulence, and that this can be applied to the study of opportunistic human pathogens.

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