Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding

Antimicrobial resistance in Gram-negative bacteria is one of the greatest threats to global health. New antibacterial strategies are urgently needed, and the development of antibiotic adjuvants that either neutralize resistance proteins or compromise the integrity of the cell envelope is of ever-growing interest. Most available adjuvants are only effective against specific resistance proteins. Here we demonstrate that disruption of cell envelope protein homeostasis simultaneously compromises several classes of resistance determinants. In particular, we find that impairing DsbA-mediated disulfide bond formation incapacitates diverse β-lactamases and destabilizes mobile colistin resistance enzymes. Furthermore, we show that chemical inhibition of DsbA sensitizes multidrug-resistant clinical isolates to existing antibiotics and that the absence of DsbA, in combination with antibiotic treatment, substantially increases the survival of Galleria mellonella larvae infected with multidrug-resistant Pseudomonas aeruginosa. This work lays the foundation for the development of novel antibiotic adjuvants that function as broad-acting resistance breakers.

Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding

Antimicrobial resistance in Gram-negative bacteria is one of the greatest threats to global health. New antibacterial strategies are urgently needed, and the development of antibiotic adjuvants that either neutralize resistance proteins or compromise the integrity of the cell envelope is of ever-growing interest. Most available adjuvants are only effective against specific resistance proteins. Here we demonstrate that disruption of cell envelope protein homeostasis simultaneously compromises several classes of resistance determinants. In particular, we find that impairing DsbA-mediated disulfide bond formation incapacitates diverse β-lactamases and destabilizes mobile colistin resistance enzymes. Furthermore, we show that chemical inhibition of DsbA sensitizes multidrug-resistant clinical isolates to existing antibiotics and that the absence of DsbA, in combination with antibiotic treatment, substantially increases the survival of Galleria mellonella larvae infected with multidrug-resistant Pseudomonas aeruginosa. This work lays the foundation for the development of novel antibiotic adjuvants that function as broad-acting resistance breakers.

Characterization of virulent Escherichia coli strains isolated from patients with urological infection

Objective. Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) affect 150 million people annually.Purpose: Characteristics of non-hospital strains of UPEC isolated from patients with UTI in Yaroslavl in 2016– 2017.Materials and methods. Susceptibility of UPEC strains (n = 20) to antibacterials was measured by the serial dilution method; the antibiotic resistance and virulence genes, phylogroups, O-serogroups and sequence types were identified by PCR and whole genome sequencing. The virulence of the strains was studied using the model of Galleria mellonella larvae.Results. UPEC strains were classified as resistant (n = 11) and multi-drug resistant (n = 9) pathogens. Betalactamase genes blaTEM (n = 10), blaCTX-M (n = 6), class 1 integrons (n = 8), and gene cassettes dfrA17-aadA5 (n = 2), dfrA1 (n = 1) and aacA4-cmlA1 (n = 1) were identified. UPEC-virulence genetic determinants coding adhesins fimH, papG, sfaS, focG, afa/draBC, csgA, siderophores iroN, fyuA, iutA, counteracting factors of host immunity ompT, traT, toxins hlyA, cnf1, usp, capsule transporter kpsMTII, colicin cvaC, and pathogenicity islands I536, II536, III536, IV536, IIJ96 и IICFT073 were detected. Highly virulent and slightly virulent for G. mellonella larvae UPEC strains were obtained with LD50 104–105 and 106–107 CFU, respectively. The phylogroups A, B1, B2, E and F, serogroups О2, О4, О6, O9, O11, О15, О18, О25, О75 and O89, known sequence types ST14, ST58, ST69, ST73, ST93, ST127, ST131, ST-141, ST165, ST297, ST457, ST537, ST744, ST1434 and novel ST9239 and ST10102 were revealed.Conclusions. The identified genetic diversity of non-hospital UPEC strains is consistent with the observed global trend in the spread of human pathogens, which are characterized with both high virulence and multiple drug resistance. This makes possible to assess prospectively the current epidemiological situation, give a forecast for its development in the future, as well as determine the optimal therapeutic options.

Carriage of distinct blaKPC-2 and blaOXA-48 plasmids in a single ST11 hypervirulent Klebsiella pneumoniae isolate in Egypt

Background Carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) causes serious infections with significant morbidity and mortality. However, the epidemiology and transmission mechanisms of CR-hvKP and the corresponding carbapenem-resistant plasmids require further investigation. Herein, we have characterized an ST11 K. pneumoniae strain EBSI041 from the blood sample encoding both hypervirulence and carbapenem resistance phenotypes from a patient in Egypt. Results K. pneumoniae strain EBSI041 showed multidrug-resistance phenotypes, where it was highly resistant to almost all tested antibiotics including carbapenems. And hypervirulence phenotypes of EBSI041 was confirmed by the model of Galleria mellonella infection. Whole-genome sequencing analysis showed that the hybrid plasmid pEBSI041-1 carried a set of virulence factors rmpA, rmpA2, iucABCD and iutA, and six resistance genes aph(3′)-VI, armA, msr(E), mph(E), qnrS, and sul2. Besides, blaOXA-48 and blaSHV-12 were harboured in a novel conjugative IncL-type plasmid pEBSI041-2. The blaKPC-2-carrying plasmid pEBSI041-3, a non-conjugative plasmid lacking the conjugative transfer genes, could be transferred with the help of pEBSI041-2, and the two plasmids could fuse into a new plasmid during co-transfer. Moreover, the emergence of the p16HN-263_KPC-like plasmids is likely due to the integration of pEBSI041-3 and pEBSI041-4 via IS26-mediated rearrangement. Conclusion To the best of our knowledge, this is the first report on the complete genome sequence of KPC-2- and OXA-48-coproducing hypervirulent K. pneumoniae from Egypt. These results give new insights into the adaptation and evolution of K. pneumoniae during nosocomial infections.

Infection of Galleria mellonella (Lepidoptera) Larvae With the Entomopathogenic Fungus Conidiobolus coronatus (Entomophthorales) Induces Apoptosis of Hemocytes and Affects the Concentration of Eicosanoids in the Hemolymph

Apoptosis and autophagy, the mechanisms of programmed cell death, play critical roles in physiological and pathological processes in both vertebrates and invertebrates. Apoptosis is also known to play an important role in the immune response, particularly in the context of entomopathogenic infection. Of the factors influencing the apoptotic process during infection, two of the lesser known groups are caspases and eicosanoids. The aim of this study was to determine whether infection by the entomopathogenic soil fungus Conidiobolus coronatus is associated with apoptosis and changes in caspase activity in the hemocytes of Galleria mellonella larvae, and to confirm whether fungal infection may affect eicosanoid levels in the host. Larvae were exposed for 24 h to fully grown and sporulating fungus. Hemolymph was collected either immediately after termination of exposure (F24 group) or 24 h later (F48 group). Apoptosis/necrosis tests were performed in hemocytes using fluorescence microscopy and flow cytometry, while ELISA tests were used to measure eicosanoid levels. Apoptosis and necrosis occurred to the same degree in F24, but necrosis predominated in F48. Fungal infection resulted in caspase activation, increased PGE1, PGE2, PGA1, PGF2α, and 8-iso-PGF2α levels and decreased TXB2 levels, but had no effect on TXA2 or 11-dehydro-TXB2 concentrations. In addition, infected larvae demonstrated significantly increased PLA2 activity, known to be involved in eicosanoid biosynthesis. Our findings indicate that fungal infection simultaneously induces apoptosis in insects and stimulates general caspase activity, and this may be correlated with changes in the concentrations of eicosanoids.

Screening and Identification of Indigenous Entomopathogenic Fungal Isolates from Agricultural Farmland Soils in Nile Delta, Egypt

The compound negative impact of insect pests attacking agricultural ecosystems includes (i) direct yield losses from damaged crops, (ii) the economic cost of the attempt to prevent these losses and (iii) the negative short- and long-term hazard effects of chemical pesticides on human and environmental health. Entomopathogenic fungi (EMPF) are a group of microorganisms that represent the natural enemies of a number of crop pests, presenting an opportunity to harness their evolutionary fine-tuned relationship with their insect hosts as biocontrol agents in integrated pest management programs. The aim of this study was to establish an indigenous EMPF collection via the Galleria mellonella (greater wax moth) entrapment method from the soils of Nile Delta, Egypt. Obtained insect associated fungal isolates were bio-assayed for pathogenicity against the serious pest Spodoptera litura and Tenebrio molitor, and the seven outperforming isolates were selected for molecular identification and thermotolerance assay. Based on ITS sequence analysis and phylogeny, selected isolates were identified as Beauveria bassiana (four isolates), Metarhizium anisopliae (two isolates) and one isolate of Cordyceps javanica. The obtained results demonstrated (i) the efficacy of using insect baiting coupled with molecular identification and pathogenicity screening to isolate EMPF to control insect pests, and (ii) the availability of indigenous virulent EMPF in Nile Delta’s soil, which can be exploited for the development of sustainable crop protection strategies.

A multiwell-plate Caenorhabditis elegans assay for assessing the therapeutic potential of Bacteriophages against Clinical Pathogens

In order to establish phage therapy as a standard clinical treatment for bacterial infections, testing of every phage to ensure the suitability and safety of the biological compound is required. While some issues have been addressed over recent years, standard and easy-to-use animal models to test phages are still rare. Testing of phages in highly suitable mammalian models such as mice is subjected to strict ethical regulations, while insect larvae such as the Galleria mellonella model suffers from batch-to-batch variations and requires manual operator skills to inject bacteria, resulting in unreliable experimental outcomes. A much simpler model is the nematode Caenorhabditis elegans which feeds on bacteria, a fast growing and easy to handle organism which can be used in high-throughput screening. In this study, two clinical bacterial strains of Escherichia coli, one Klebsiella pneumoniae and one Enterobacter cloacae strain were tested on the model system together with lytic bacteriophages that we isolated previously. We developed a liquid-based assay, in which the efficiency of phage treatment was evaluated using a scoring system based on microscopy and counting of the nematodes, allowing increasing statistical significance compared to other assays such as larvae or mice. Our work demonstrates the potential to use Caenorhabditis elegans to test the virulence of strains of Klebsiella pneumoniae, Enterobacter cloacae and EHEC/ EPEC as well as the efficacy of bacteriophages to treat or prevent infections, allowing a more reliable evaluation for the clinical therapeutic potential of lytic phages.

Galleria mellonella as a Suitable Model of Bacterial Infection: Past, Present and Future

The increasing interest for Galleria mellonella larvae as an infection model is evidenced by the number of papers reporting its use, which increases exponentially since the early 2010s. This popularity was initially linked to limitation of conventional animal models due to financial, technical and ethical aspects. In comparison, alternative models (e.g. models using Caenorhabditis elegans, Drosophila melanogaster or G. mellonella) were cheap, simple to use and not limited by ethical regulation. Since then, similar results have been established with G. mellonella model comparatively to vertebrates, and it is more and more often used as a robust model per se, not only as an alternative to the murine model. This review attempts to summarize the current knowledge supporting the development of this model, both on immunological and microbiological aspects. For that, we focus on investigation of virulence and new therapies for the most important pathogenic bacteria. We also discuss points out directions for standardization, as well as recent advances and new perspectives for monitoring host-pathogen interactions.