A novel antibacterial approach of Cecropin-B peptide loaded on chitosan nanoparticles against MDR Klebsiella pneumoniae isolates.

Egypt has witnessed the emergence of multidrug-resistant (MDR) Klebsiella pneumoniae, which has posed a serious healthcare challenge. The proper treatment choice for MDR-KP infections is not well determined which renders the problem more complicated, thus making the control of such infections a serious challenge for healthcare professionals. This study aims to encapsulate the cationic antimicrobial peptide; Cecropin-B (Cec-B), to increase its lifetime, drug targeting, and efficacy and study the antimicrobial effect of free and encapsulated recombinant rCec-B peptide on multidrug-resistant K. pneumoniae (MDR-KP) isolates. Fifty isolates were collected from different clinical departments at Theodore Bilharz Research Institute. Minimal inhibitory concentrations (MICs) of rCec-B against MDR-KP isolates were determined by the broth microdilution test. In addition, encapsulation of rCec-B peptide into chitosan nanoparticles and studying its bactericidal effect against MDR-KP isolates were also performed. The relative expression of efflux pump and porin coding genes (ArcrB, TolC, mtdK, and Ompk35) was detected by quantitative PCR in treated MDR-KP bacterial isolates compared to untreated isolates. Out of 60 clinical MDR isolates, 50 were MDR-KP. 60% of the isolates were XDR while 40% were MDR. rCec-B were bactericidal on 21 isolates, then these isolates were subjected to treatment using free nanocapsule in addition to the encapsulated peptide. Free capsules showed a mild cytotoxic effect on MDR-KP at the highest concentration. MIC of encapsulated rCec-B was higher than the free peptide. The expression level of genes encoding efflux and porin (ArcrB, TolC, mtdK, and Ompk35) was downregulated after treatment with encapsulated rCec-B. These findings indicate that encapsulated rCec-B is a promising candidate with potent antibacterial activities against drug-resistant K. pneumoniae.

Imipenem resistance associated with amino acid alterations of the OprD porin in Pseudomonas aeruginosa clinical isolates.

Globally, the spread of carbapenem-resistant strains has limited treatment options for multidrug-resistant (MDR) Pseudomonas aeruginosa infections. This study aimed to determine the role of point mutations as well as the expression level of the oprD gene in the emergence of imipenem-resistant P. aeruginosa strains isolated from patients referred to Ardabil hospitals. A total of 48 imipenem-resistant clinical isolates of P. aeruginosa collected between June 2019 and January 2022 were used in this study. Detection of the oprD gene and its amino acid alterations was performed using the polymerase chain reaction (PCR) and DNA sequencing techniques. The expression level of the oprD gene in imipenem-resistant strains was determined using the real-time quantitative reverse transcription PCR (RT-PCR) method. All imipenem-resistant P. aeruginosa strains were positive for the oprD gene based on the PCR results, and also five selected isolates indicated one or more amino acid alterations. Detected amino acid alterations in the OprD porin were Ala210Ile, Gln202Glu, Ala189Val, Ala186Pro, Leu170Phe, Leu127Val, Thr115Lys, and Ser103Thr. Based on the RT-PCR results, the oprD gene was downregulated in 79.1% of imipenem-resistant P. aeruginosa strains. However, 20.9% of strains showed overexpression of the oprD gene. Probably, resistance to imipenem in these strains is associated with the presence of carbapenemases, AmpC cephalosporinase, or efflux pumps. Owing to the high prevalence of imipenem-resistant P. aeruginosa strains due to various resistance mechanisms in Ardabil hospitals, the implementation of surveillance programs to reduce the spread of these resistant microorganisms along with rational selection and prescription of antibiotics is recommended.

Development of Resistance to Eravacycline by Klebsiella pneumoniae and Collateral Sensitivity-Guided Design of Combination Therapies.

The evolution of bacterial antibiotic resistance is exhausting the list of currently used antibiotics and endangers those in the pipeline. The combination of antibiotics is a promising strategy that may suppress resistance development and/or achieve synergistic therapeutic effects. Eravacycline is a newly approved antibiotic that is effective against a variety of multidrug-resistant (MDR) pathogens. However, the evolution of resistance to eravacycline and strategies to suppress the evolution remain unexplored. Here, we demonstrated that a carbapenem-resistant Klebsiella pneumoniae clinical isolate quickly developed resistance to eravacycline, which is mainly caused by mutations in the gene encoding the Lon protease. The evolved resistant mutants display collateral sensitivities to ß-lactam/ß-lactamase inhibitor (BLBLI) combinations aztreonam/avibactam and ceftazidime-avibactam. Proteomic analysis revealed upregulation of the multidrug efflux system AcrA-AcrB-TolC and porin proteins OmpA and OmpU, which contributed to the increased resistance to eravacycline and susceptibility to BLBLIs, respectively. The combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam suppresses resistance development. We further demonstrated that eravacycline-resistant mutants evolved from an NDM-1-containing K. pneumoniae strain display collateral sensitivity to aztreonam/avibactam, and the combination of eravacycline with aztreonam/avibactam suppresses resistance development. In addition, the combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam displayed synergistic therapeutic effects in a murine cutaneous abscess model. Overall, our results revealed mechanisms of resistance to eravacycline and collateral sensitivities to BLBLIs and provided promising antibiotic combinations in the treatment of multidrug-resistant K. pneumoniae infections. IMPORTANCE The increasing bacterial antibiotic resistance is a serious threat to global public health, which demands novel antimicrobial medicines and treatment strategies. Eravacycline is a newly approved antibiotic that belongs to the tetracycline antibiotics. Here, we found that a multidrug-resistant Klebsiella pneumoniae clinical isolate rapidly developed resistance to eravacycline and the evolved resistant mutants displayed collateral sensitivity to antibiotics aztreonam/avibactam and ceftazidime-avibactam. We demonstrated that the combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam repressed resistance development and improved the treatment efficacies. We also elucidated the mechanisms that contribute to the increased resistance to eravacycline and susceptibility to aztreonam/avibactam and ceftazidime-avibactam. This work demonstrated the mechanisms of antibiotic resistance and collateral sensitivity and provided a new therapeutically option for effective antibiotic combinations.

Genomic Surveillance of Clinical Pseudomonas aeruginosa Isolates Reveals an Additive Effect of Carbapenemase Production on Carbapenem Resistance.

Carbapenem resistance in Pseudomonas aeruginosa is increasing globally, and surveillance to define the mechanisms of such resistance in low- and middle-income countries is limited. This study establishes the genotypic mechanisms of ß-lactam resistance by whole-genome sequencing (WGS) in 142 P. aeruginosa clinical isolates recovered from three hospitals in Islamabad and Rawalpindi, Pakistan between 2016 and 2017. Isolates were subjected to antimicrobial susceptibility testing (AST) by Kirby-Bauer disk diffusion, and their genomes were assembled from Illumina sequencing data. ß-lactam resistance was high, with 46% of isolates resistant to piperacillin-tazobactam, 42% to cefepime, 48% to ceftolozane-tazobactam, and 65% to at least one carbapenem. Twenty-two percent of isolates were resistant to all ß-lactams tested. WGS revealed that carbapenem resistance was associated with the acquisition of metallo-ß-lactamases (MBLs) or extended-spectrum ß-lactamases (ESBLs) in the blaGES, blaVIM, and blaNDM families, and mutations in the porin gene oprD. These resistance determinants were found in globally distributed lineages, including ST235 and ST664, as well as multiple novel STs which have been described in a separate investigation. Analysis of AST results revealed that acquisition of MBLs/ESBLs on top of porin mutations had an additive effect on imipenem resistance, suggesting that there is a selective benefit for clinical isolates to encode multiple resistance determinants to the same drugs. The strong association of these resistance determinants with phylogenetic background displays the utility of WGS for monitoring carbapenem resistance in P. aeruginosa, while the presence of these determinants throughout the phylogenetic tree shows that knowledge of the local epidemiology is crucial for guiding potential treatment of multidrug-resistant P. aeruginosa infections. IMPORTANCE Pseudomonas aeruginosa is associated with serious infections, and treatment can be challenging. Because of this, carbapenems and ß-lactam/ß-lactamase inhibitor combinations have become critical tools in treating multidrug-resistant (MDR) P. aeruginosa infections, but increasing resistance threatens their efficacy. Here, we used WGS to study the genotypic and phylogenomic patterns of 142 P. aeruginosa isolates from the Potohar region of Pakistan. We sequenced both MDR and antimicrobial susceptible isolates and found that while genotypic and phenotypic patterns of antibiotic resistance correlated with phylogenomic background, populations of MDR P. aeruginosa were found in all major phylogroups. We also found that isolates possessing multiple resistance mechanisms had significantly higher levels of imipenem resistance compared to the isolates with a single resistance mechanism. This study demonstrates the utility of WGS for monitoring patterns of antibiotic resistance in P. aeruginosa and potentially guiding treatment choices based on the local spread of ß-lactamase genes.

Synergistic Activity of Imipenem in Combination with Ceftazidime/Avibactam or Avibactam against Non-MBL-Producing Extensively Drug-Resistant Pseudomonas aeruginosa.

Extensively drug-resistant Pseudomonas aeruginosa (XDRPA) infection is a significant public health threat due to a lack of effective therapeutic options. New ß-lactam-ß-lactamase inhibitor combinations, including ceftazidime-avibactam (CZA), have shown a high resistance rate to XDRPA. This study was therefore conducted to describe the underlying genomic mechanism of resistance for CZA nonsusceptible XDRPA strains that are non-metallo-ß-lactamase (MBL) producers as well as to examine synergism of CZA and other antipseudomonal agents. Furthermore, the synergistic antibacterial activity of the most effective antimicrobial combination against non-MBL-producing XDRPA was evaluated through in vitro experiments. The resistance profiles of 15 CZA-resistant XDRPA strains isolated from clinical specimens in China-Japan Friendship Hospital between January 2017 to December 2020 were obtained by whole-genome sequencing (WGS) analysis. MBL genes blaIMP-1 and blaIMP-45 were found in 2 isolates (2/15, 13.3%); the other underlying CZA-resistance mechanisms involved the decreased OprD porin (13/13), blaAmpC overexpression (8/13) or mutation (13/13), and upregulated efflux pumps (13/13). CZA-imipenem (CZA-IPM) combination was identified to be the most effective against non-MBL-producing XDRPA according to the results of WGS analysis and combined antimicrobial susceptibility tests, with an approximately 16.62-fold reduction in MICs compared to CZA alone. Furthermore, the results of checkerboard analysis and growth curve displayed the synergistic antimicrobial activity of CZA and IPM against non-MBL-producing XDRPA. Electron microscopy also revealed that CZA-IPM combination might lead to more cellular structural alterations than CZA or IPM alone. This study suggested that the CZA-IPM combination has potential for non-MBL-producing XDRPA with blaAmpC overexpression or mutation, decreased OprD porin, and upregulated efflux pumps. IMPORTANCE Handling the infections by extensively drug-resistant Pseudomonas aeruginosa (XDRPA) strains is challenging due to their complicated antibiotic resistance mechanisms in immunosuppressed patients with pulmonary diseases (e.g., cystic fibrosis, chronic obstructive pulmonary disease, and lung transplant), ventilator-associated pneumonia, and bloodstream infections. The current study suggested the potentiality of the ceftazidime-avibactam-imipenem combination against XDRPA with blaAmpC overexpression or mutation, decreased OprD porin, and/or upregulated efflux pumps. Our findings indicate the necessity of combined drug sensitivity tests against XDRPA and also lay a foundation for the development of prevention, control, and treatment strategies in XDRPA infections.

Evaluation of the Leptospira interrogans Outer Membrane Protein OmpL37 as a Vaccine Candidate.

The identification of potential vaccine candidates against leptospirosis remains a challenge. However, one such candidate is OmpL37, a potentially surface-exposed antigen that has the highest elastin-binding ability described to date, suggesting that it plays an important role in host colonization. In order to evaluate OmpL37's ability to induce a protective immune response, prime-boost, DNA and subunit vaccine strategies were tested in the hamster model of lethal leptospirosis. The humoral immune response was evaluated using an indirect ELISA test, and the cytokine profile in whole blood was determined by quantitative real-time PCR. Unlike the DNA vaccine, the administration of recombinant OmpL37 induced a strong IgG antibody response. When individually administrated, both formulations stimulated a TNF-α mediated inflammatory response. However, none of the OmpL37 formulations or vaccination strategies induced protective immunity. Further studies are required towards the identification of new vaccine targets against leptospirosis.

Beyond conventional antibiotics for the future treatment of methicillin-resistant Staphylococcus aureus infections: two novel alternatives.

The majority of antibiotics currently used to treat methicillin-resistant Staphylococus aureus (MRSA) infections target bacterial cell wall synthesis or protein synthesis. Only daptomycin has a novel mode of action. Reliance on limited targets for MRSA chemotherapy, has contributed to antimicrobial resistance. Two alternative approaches to the treatment of S. aureus infection, particularly those caused by MRSA, that have alternative mechanisms of action and that address the challenge of antimicrobial resistance are cationic host defence peptides and agents that target S. aureus virulence. Cationic host defence peptides have multiple mechanisms of action and are less likely than conventional agents to select resistant mutants. They are amenable to modifications that improve their stability, effectiveness and selectivity. Some cationic defence peptides such as bactenecin, mucroporin and imcroporin have potent in vitro bactericidal activity against MRSA. Antipathogenic agents also have potential to limit the pathogenesis of S aureus. These are generally small molecules that inhibit virulence targets in S. aureus without killing the bacterium and therefore have limited capacity to promote resistance development. Potential antipathogenic targets include the sortase enzyme system, the accessory gene regulator (agr) and the carotenoid biosynthetic pathway. Inhibitors of these targets have been identified and these may have potential for further development.

Anti-porin antibodies prevent excitotoxic and ischemic damage to brain tissue.

The mitochondrial permeability transition (MPT) is a converging event for different molecular routes leading to cellular death after excitotoxic/oxidative stress, and is considered to represent the opening of a pore in the mitochondrial membrane. There is evidence that the outer mitochondrial membrane protein porin is involved in the MPT and apoptosis. We present here a proof-of-principle study to address the hypothesis that anti-porin antibodies can prevent excitotoxic/ischemia-induced cell death. We generated anti-porin antibodies and show that the F(ab)(2) fragments penetrate living cells, reduce Ca(2+)-induced mitochondrial swelling as other MPT blockers do, and decrease neuronal death in dissociated and organotypic brain slice cultures exposed to excitotoxic and ischemic episodes. These observations present direct evidence that anti-porin antibody fragments prevent cell damage in brain tissue, that porin is a crucial protein involved in mitochondrial and cell dysfunction, and that it is conceivable that antibodies can be used as therapeutic agents.

Protection of immunocompromised mice against lethal infection with Pseudomonas aeruginosa by active or passive immunization with recombinant P. aeruginosa outer membrane protein F and outer membrane protein I fusion proteins.

Recombinant outer membrane proteins (Oprs) of Pseudomonas aeruginosa were expressed in Escherichia coli as glutathione S-transferase (GST)-linked fusion proteins. GST-linked Oprs F and I (GST-OprF190-350 [GST linked to OprF spanning amino acids 190 to 350] and GST-OprI21-83, respectively) and recombinant hybrid Oprs (GST-OprF190-342-OprI21-83 and GST-OprI21-83-OprF190-350) were isolated and tested for their efficacy as vaccines in immunodeficient mice. GST-OprF-OprI protected the mice against a 975-fold 50% lethal dose of P. aeruginosa. Expression of GST-unfused OprF-OprI failed in E. coli, although this hybrid protein has been expressed without a fusion part in Saccharomyces cerevisiae and used for immunizing rabbits. The immune rabbit sera protected severe combined deficient (SCID) mice against a 1,000-fold 50% lethal dose of P. aeruginosa. Evidence is provided to show that the most C-terminal part of OprF (i.e., amino acids 332 to 350) carries an important protective epitope. Opr-based hybrid proteins may have implications for a clinical vaccine against P. aeruginosa.