ABSTRACT
Lampedusa, a picturesque Italian island in the Mediterranean, serves as a gateway for migrants from Africa and Asia to Europe. Despite populist rhetoric portraying migrants as carriers of disease, epidemiological data reveal very low levels of communicable diseases among migrants, challenging false narratives and xenophobic sentiments propagated by populist governments.
Subject(s)
Communicable Diseases , Transients and Migrants , Humans , Communicable Diseases/epidemiology , SicilyABSTRACT
Klebsiella pneumoniae, a prominent nosocomial pathogen, poses a critical global health threat due to its multidrug-resistant (MDR) and hypervirulent strains. This comprehensive review focuses into the complex approaches undertaken in the development of vaccines against K. pneumoniae. Traditional methods, such as whole-cell and ribosomal-based vaccines, are compared with modern strategies, including DNA and mRNA vaccines, and extracellular vesicles (EVs), among others. Each method presents unique advantages and challenges, emphasising the complexity of developing an effective vaccine against this pathogen. Significant advancements in computational tools and artificial intelligence (AI) have revolutionised antigen identification and vaccine design, enhancing the precision and efficiency of developing multiepitope-based vaccines. The review also highlights the potential of glycomics and immunoinformatics in identifying key antigenic components and elucidating immune evasion mechanisms employed by K. pneumoniae. Despite progress, challenges remain in ensuring the safety, efficacy, and manufacturability of these vaccines. Notably, EVs demonstrate promise due to their intrinsic adjuvant properties and ability to elicit robust immune responses, although concerns regarding inflammation and antigen variability persist. This review provides a critical overview of the current landscape of K. pneumoniae vaccine development, stressing the need for continued innovation and interdisciplinary collaboration to address this pressing public health issue. The integration of advanced computational methods and AI holds the potential to accelerate the development of effective immunotherapies, paving the way for novel vaccines against MDR K. pneumoniae.
Subject(s)
Bacterial Vaccines , Klebsiella Infections , Klebsiella pneumoniae , Vaccine Development , Klebsiella pneumoniae/immunology , Klebsiella pneumoniae/genetics , Humans , Bacterial Vaccines/immunology , Klebsiella Infections/prevention & control , Klebsiella Infections/immunology , Klebsiella Infections/microbiology , Animals , Vaccines, DNA/immunology , Artificial Intelligence , Extracellular Vesicles/immunology , Antigens, Bacterial/immunologyABSTRACT
INTRODUCTION: After receiving emergency approval during the COVID-19 pandemic, mRNA vaccines have taken center stage in the quest to enhance future vaccination strategies for both infectious diseases and cancer. Indeed, they have significantly overshadowed another facet of genetic vaccination, specifically DNA vaccines. Nevertheless, it is important to acknowledge that both types of genetic vaccines have distinct advantages and disadvantages that set them apart from each other. AREAS COVERED: In this work, we delve extensively into the history of genetic vaccines, their mechanisms of action, their strengths, and limitations, and ultimately highlight ongoing research in key areas for potential enhancement of both DNA and mRNA vaccines. EXPERT OPINION: Here, we assess the significance of the primary benefits and drawbacks associated with DNA and mRNA vaccination. We challenge the current lines of thought by highlighting that the existing drawbacks of DNA vaccination could potentially be more straightforward to address compared to those linked with mRNA vaccination. In our view, this suggests that DNA vaccines should remain viable contenders in the pursuit of the future of vaccination.
Subject(s)
Vaccines, DNA , Vaccines , Humans , Pandemics , Vaccination , DNA , RNA, Messenger/geneticsABSTRACT
Plasmodium sporozoites can block apoptotic pathways within host hepatocytes, ensuring the survival of the parasite. However, attenuated plasmodial sporozoites are unable to prevent apoptosis, which provides many parasite antigens to immune cells. This exposure leads to protection against Malaria in both human and animal models. If these hosts are later inoculated with infectious sporozoites, apoptosis of infected hepatocytes will occur, preventing parasite development. Considering that hydrogen peroxide can induce apoptosis, it is plausible that it plays a role in the mechanisms associated with the protection mediated by attenuated plasmodial sporozoites. Based on published results that describe the relationship between Plasmodium, hydrogen peroxide, and apoptosis, a rational explanation can be provided for this hypothesis.
Subject(s)
Malaria Vaccines , Malaria , Plasmodium , Animals , Humans , Sporozoites , Hydrogen Peroxide/pharmacology , Malaria/prevention & control , Malaria/parasitology , Hepatocytes/parasitologyABSTRACT
Attenuated plasmodial sporozoite-induced immune response includes intrahepatic nitric oxide (NO) production, which promotes apoptosis of infected hepatocytes and consequent parasite clearance. NO in excess reacts with superoxide, forming peroxynitrite, a powerful cytotoxic agent. Here, I suggest that peroxynitrite proapoptotic action may contribute to the attenuated malarial sporozoite-mediated protection.
Subject(s)
Malaria Vaccines , Malaria , Plasmodium , Animals , Sporozoites , Peroxynitrous Acid , Malaria/prevention & control , Hepatocytes/parasitology , Nitric OxideABSTRACT
Historically, Klebsiella pneumoniae isolates were described either as hypervirulent or classical. While hypervirulent strains display a precise phenotype (thicker capsule, hypermucoviscosity, absence of antibiotic resistance markers, several siderophores, etc.), classical strains can relate to all other K. pneumoniae strains, including virulent multidrug-resistant clinical isolates. Recently, many surveillance studies reported virulent K. pneumoniae nosocomial strains resistant to all antibiotic classes which also contain genetic markers associated with hypervirulence. Due to their higher virulence and clinical importance, here it is proposed reclassify them as ultravirulent and as supervirulent, to distinguish them from each other and from those with either hypervirulent or virulent phenotypes.
Subject(s)
Klebsiella Infections , Klebsiella pneumoniae , Humans , Klebsiella pneumoniae/genetics , Phenotype , Virulence/genetics , Virulence Factors/genetics , Anti-Bacterial Agents/pharmacology , Klebsiella Infections/epidemiologyABSTRACT
Klebsiella pneumoniae is a Gram-negative pathogen of clinical relevance, which can provoke serious urinary and blood infections and pneumonia. This bacterium is a major public health threat due to its resistance to several antibiotic classes. Using a reverse vaccinology approach, 7 potential antigens were identified, of which 4 were present in most of the sequences of Italian carbapenem-resistant K. pneumoniae clinical isolates. Bioinformatics tools demonstrated the antigenic potential of these bacterial proteins and allowed for the identification of T and B cell epitopes. This led to a rational design and in silico characterization of a multiepitope vaccine against carbapenem-resistant K. pneumoniae strains. As adjuvant, the mycobacterial heparin-binding hemagglutinin adhesin (HBHA), which is a Toll-like receptor 4 (TLR-4) agonist, was included, to increase the immunogenicity of the construct. The multiepitope vaccine candidate was analyzed by bioinformatics tools to assess its antigenicity, solubility, allergenicity, toxicity, physical and chemical parameters, and secondary and tertiary structures. Molecular docking binding energies to TLR-2 and TLR-4, two important innate immunity receptors involved in the immune response against K. pneumoniae infections, and molecular dynamics simulations of such complexes supported active interactions. A codon optimized multiepitope sequence cloning strategy is proposed, for production of recombinant vaccine in classical bacterial vectors. Finally, a 3 dose-immunization simulation with the multiepitope construct induced both cellular and humoral immune responses. These results suggest that this multiepitope construct has potential as a vaccination strategy against carbapenem-resistant K. pneumoniae and deserves further validation.
ABSTRACT
Chronic hepatitis C virus (HCV) infection is the principal etiology of cirrhosis and, ultimately, hepatocellular carcinoma (HCC). At present, approximately 71 million people are chronically infected with HCV, and 10%-20% of these are expected to develop severe liver complications throughout their lifetime. Scientific evidence has clearly shown the causal association between miRNAs, HCV infection and HCC. Although it is not completely clear whether miRNA dysregulation in HCC is the cause or the consequence of its development, variations in miRNA patterns have been described in different liver diseases, including HCC. Many studies have analyzed the importance of circulating miRNAs and their effect on cell proliferation and apoptosis. In this Review, we aim to summarize current knowledge on the association between miRNA, HCV and HCC from a diagnostic point of view, and also the potential implications for therapeutic approaches.
Subject(s)
Carcinoma, Hepatocellular , Hepatitis C, Chronic , Hepatitis C , Liver Neoplasms , MicroRNAs , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/therapy , Hepacivirus/genetics , Hepatitis C/complications , Hepatitis C/genetics , Hepatitis C/pathology , Hepatitis C, Chronic/complications , Hepatitis C, Chronic/drug therapy , Hepatitis C, Chronic/pathology , Humans , Liver Neoplasms/genetics , Liver Neoplasms/therapy , MicroRNAs/genetics , MicroRNAs/therapeutic useABSTRACT
In this work, we assessed and characterized the epidemiological scenario of carbapenem-resistant Klebsiella pneumoniae strains (CR-Kp) at IRCCS-ISMETT, a transplantation hospital in Palermo, Italy, from 2008 to 2017. A total of 288 K. pneumoniae clinical isolates were selected based on their resistance to carbapenems. Molecular characterization was also done in terms of the presence of virulence and resistance genes. All patients were inpatients from our facility and clinical isolates were collected from several sources, either from infection or colonization cases. We observed that, in agreement with the Italian epidemiological scenario, initially only ST258 and ST512 clade II (but not from clade I) were identified from 2008 to 2011. From 2012 onwards, other STs have been observed, including the clinically relevant ST101 and ST307, but also others not previously observed in other Italian health settings, such as ST220 and ST753. The presence of genes involved in resistance and virulence was confirmed, and a heterogeneous genetic resistance profile throughout the years was observed. Our work highlights that resistance genes are rapidly disseminating between different and novel K. pneumoniae clones which, combined with resistance to multiple antibiotics, can derive into more aggressive and pathogenic multidrug-resistant strains of clinical importance. Our results stress the importance of continuous surveillance of CR Enterobacterales in health facilities so that novel STs carrying resistance and virulence genes that may become increasingly pathogenic can be identified and adequate therapies to adopted to avoid their dissemination and derived pathologies.
Subject(s)
Klebsiella Infections , Klebsiella pneumoniae , Bacterial Proteins , Hospitals , Humans , Klebsiella Infections/epidemiology , Klebsiella pneumoniae/genetics , Retrospective Studies , Sicily , beta-LactamasesABSTRACT
BACKGROUND: Spontaneous bacterial peritonitis (SBP) is a severe and often fatal infection in patients with decompensated cirrhosis and ascites. The only cure for SBP is antibiotic therapy, but the emerging problem of bacterial resistance requires novel therapeutic strategies. Human amniotic mesenchymal stromal cells (hA-MSCs) possess immunomodulatory and anti-inflammatory properties that can be harnessed as a therapy in such a context. METHODS: An in vitro applications of hA-MSCs in ascitic fluid (AF) of cirrhotic patients, subsequently infected with carbapenem-resistant Enterobacterales, was performed. We evaluated the effects of hA-MSCs on bacterial load, innate immunity factors, and macrophage phenotypic expression. RESULTS: hA-MSCs added to AF significantly reduce the proliferation of both bacterial strains at 24 h and diversely affect M1 and M2 polarization, C3a complement protein, and ficolin 3 concentrations during the course of infection, in a bacterial strain-dependent fashion. CONCLUSION: This study shows the potential usefulness of hA-MSC in treating ascites infected with carbapenem-resistant bacteria and lays the foundation to further investigate antibacterial and anti-inflammatory roles of hA-MSC in in vivo models.
Subject(s)
Amnion/cytology , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/metabolism , Peritoneal Fibrosis/etiology , Peritoneal Fibrosis/therapy , Bacterial Load , Biomarkers , Carbapenems/pharmacology , Complement Activation/immunology , Complement System Proteins/immunology , Complement System Proteins/metabolism , Disease Susceptibility , Enterobacter/drug effects , Enterobacter/genetics , Enterobacteriaceae Infections/complications , Enterobacteriaceae Infections/microbiology , Humans , Immunomodulation , Inflammation Mediators , Macrophages , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/cytology , Peritoneal Fibrosis/metabolism , Peritonitis/complications , Peritonitis/microbiology , Phagocytosis , Receptors, Pattern Recognition/metabolism , Treatment Outcome , beta-Lactam ResistanceABSTRACT
[This corrects the article DOI: 10.1016/j.omtm.2021.05.002.].
ABSTRACT
Glioblastoma (GBM) is the most aggressive among the neurological tumors. At present, no chemotherapy or radiotherapy regimen is associated with a positive long-term outcome. In the majority of cases, the tumor recurs within 32-36 weeks of initial treatment. The recent discovery that Zika virus (ZIKV) has an oncolytic action against GBM has brought hope for the development of new therapeutic approaches. ZIKV is an arbovirus of the Flaviviridae family, and its infection during development has been associated with central nervous system (CNS) malformations, including microcephaly, through the targeting of neural stem/progenitor cells (NSCs/NPCs). This finding has led various groups to evaluate ZIKV's effects against glioblastoma stem cells (GSCs), supposedly responsible for GBM onset, progression, and therapy resistance. While preliminary data support ZIKV tropism toward GSCs, a more accurate study of ZIKV mechanisms of action is fundamental in order to launch ZIKV-based clinical trials for GBM patients.
Subject(s)
Brain Neoplasms/therapy , Glioblastoma/therapy , Oncolytic Virotherapy/methods , Zika Virus/genetics , Brain Neoplasms/pathology , Glioblastoma/pathology , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Neoplastic Stem Cells/cytology , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/virology , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neural Stem Cells/virology , Zika Virus/physiologyABSTRACT
Endotoxin content is a critical factor that affects the safety of biological pharmaceutical products. International pharmacopoeias describe several reference methods to determine endotoxin levels in advanced therapy medicinal product (ATMP) preparations. Administration of ATMPs must be done as rapidly as possible to ensure complete viability and potency of the cellular product. To evaluate the endotoxin content in the shortest time possible, we chose to validate an alternative method based on the use of the Charles River Portable Testing System (PTS) and FDA-approved cartridges, compliant with the requirements of the European Pharmacopoeia and providing results in <20 min. Here, we describe a unique and complete validation approach for instrument, personnel, and analytical method for assessment of endotoxins in ATMP matrices. The PTS system provides high sensitivity and fast quantitative results and uses less raw material and accessories compared with compendial methods. It is also less time consuming and less prone to operator variability. Our validation approach is suitable for a validated laboratory with trained personnel capable of conducting the ATMP release tests, and with very low intra-laboratory variability, and meets the criteria required for an alternative approach to endotoxin detection for in-process and product-release testing of ATMPs.
ABSTRACT
Currently, Klebsiella pneumoniae is a pathogen of clinical relevance due to its plastic ability of acquiring resistance genes to multiple antibiotics. During K. pneumoniae infections, lipopolysaccharides (LPS) play an ambiguous role as they both activate immune responses but can also play a role in immune evasion. The LPS O2a and LPS O2afg serotypes are prevalent in most multidrug resistant K. pneumoniae strains. Thus, we sought to understand if those two particular LPS serotypes were involved in a mechanism of immune evasion. We have extracted LPS (serotypes O1, O2a and O2afg) from K. pneumoniae strains and, using human monocytes ex vivo, we assessed the ability of those LPS antigens to induce the production of pro-inflammatory cytokines and chemokines. We observed that, when human monocytes are incubated with LPS serotypes O1, O2a or O2afg strains, O2afg and, to a lesser extent, O2a but not O1 failed to elicit the production of pro-inflammatory cytokines and chemokines, which suggests a role in immune evasion. Our preliminary data also shows that nuclear translocation of NF-κB, a process which regulates an immune response against infections, occurs in monocytes incubated with LPS O1 and, to a smaller extent, with LPS O2a, but not with the LPS serotype O2afg. Our results indicate that multidrug resistant K. pneumoniae expressing LPS O2afg serotypes avoid an initial inflammatory immune response and, consequently, are able to systematically spread inside the host unharmed, which results in the several pathologies associated with this bacterium.
ABSTRACT
Endoscopes are medical instruments that are used routinely in health structures. Due to their invasive nature and contact with many patients, they may cause hospital-acquired infections if not disinfected correctly. To ensure a high-level disinfection procedure or reprocessing, since the methods currently adopted in our institute are adequate, we evaluated retrospectively the presence of microorganisms in our endoscopes after reprocessing. Microbiological surveillance was performed from January 2016 to December 2019 in the instruments in use in our endoscopic room after reprocessing. In total, 35 endoscopes (3 duodenoscopes, 3 echoendoscopes, 12 bronchoscopes, 5 colonoscopes, and 12 gastroscopes) were evaluated for the presence of microorganisms, including multidrug-resistant pathogens and indicator microorganisms (IMOs). Our procedures were in agreement with an internal protocol based on Italian, international, and the Center for Disease Control and Prevention (CDC) recommendations. Of a total of 811 samples, 799 (98.5%) complied with the regulatory guidelines, while 9 (1.1%) were positive for IMOs, and 3 (0.4%) displayed more than 10 colony-forming units (CFU) of environmental and commensal pathogens. Our results show that the internal reprocessing protocol is very efficient, leading to a very low number of observed contaminations, and it could be easily implemented by other health facilities that face a huge number of hospital-acquired infections due to incorrectly disinfected endoscopes.
Subject(s)
Endoscopes , Equipment Reuse , Disinfection , Equipment Contamination , Hospitals , Humans , Italy/epidemiology , Retrospective StudiesABSTRACT
Effective detection of microbiological contaminations present in medicinal cellular products is a crucial step to ensure patients' safety. In recent decades, several rapid microbiological methods have been developed and validated, but variabilities linked to the use of different resources have led to discordant validation of methods and performance results. Considering this, while developing an in-house BacT/Alert-based method, we evaluated all of the materials used in its validation. Of particular importance, we noticed that the syringe gauge used to inject the samples into the bottles was crucial to obtain robust results. We chose to conduct a comparative test between the BacT/Alert system and the compendial method described in the European Pharmacopoeia, using five dilutions of nine reference microorganism strains and 21G or 27G needles. Our results confirmed that the BacT/Alert system is a valid and faster alternative method to assess sterility of clinical cell therapy products, and that the use of 27G needles increases its sensitivity to detect reference anaerobe microorganisms.
ABSTRACT
To evaluate and validate the efficacy of disinfectants used in our cleaning procedure, in order to reduce pharmaceutical hospital surfaces' contaminations, we tested the action of three commercial disinfectants on small representative samples of the surfaces present in our hospital cleanrooms. These samples (or coupons) were contaminated with selected microorganisms for the validation of the disinfectants. The coupons were sampled before and after disinfection and the microbial load was assessed to calculate the Log10 reduction index. Subsequently, we developed and validated a disinfection procedure on real surfaces inside the cleanrooms intentionally contaminated with microorganisms, using approximately 107-108 total colony forming units per coupon. Our results showed a bactericidal, fungicidal, and sporicidal efficacy coherent to the acceptance criteria suggested by United States Pharmacopeia 35 <1072>. The correct implementation of our cleaning and disinfection procedure, respecting stipulated concentrations and contact times, led to a reduction of at least 6 Log10 for all microorganisms used. The proposed disinfection procedure reduced the pharmaceutical hospital surfaces' contaminations, limited the propagation of microorganisms in points adjacent to the disinfected area, and ensured high disinfection and safety levels for operators, patients, and treated surfaces.
Subject(s)
Disinfectants , Pharmaceutical Preparations , Disinfection , Hospitals , HumansABSTRACT
Klebsiella pneumoniae is a Gram-negative bacterium of clinical importance, due to its resistance to several antibiotic classes. We have identified 4 clinical isolates of K. pneumoniae sequence type (ST) 392 KPC-3-producing strains from patients at the Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione (IRCCS-ISMETT), a Southern Italian transplantation health facility, during a routine surveillance for carbapenemase-producing Enterobacterales from in-house clinical samples. Since those were among, to the best of our knowledge, the first KPC-producing K. pneumoniae ST392 isolated in Europe, we assessed their virulence potential, to understand if this particular ST can become an endemic clinical threat. ST392 isolates were investigated to assess their virulence potential, namely resistance to human sera, formation of abiotic biofilms, adhesion to biotic surfaces, exopolysaccharide production and in vivo pathogenesis in the wax moth Galleria mellonella animal model. ST392-belonging strains were highly resistant to human sera. These strains also have a high capacity to form abiotic biofilms and high levels of adhesion to the human epithelial colorectal adenocarcinoma HT-29 cell line. An increase of transcriptional levels of genes involved in serum resistance (aroE and traT) and adhesion (pgaA) was observed when compared with the Klebsiella quasipneumoniae subsp. similipneumoniae strain ATCC 700603 reference strain. Infection of G. mellonella larvae with ST392 clinical isolates showed that the latter were not highly pathogenic in this model. Together, our results indicate that ST392 isolates have the potential to become a strain of clinical relevance, especially in health settings where patients are immunosuppressed, e.g., transplant recipients.
Subject(s)
Bacterial Proteins/metabolism , Klebsiella pneumoniae/isolation & purification , Klebsiella pneumoniae/metabolism , Phenotype , beta-Lactamases/metabolism , Animals , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Biofilms , Cross Infection/microbiology , Drug Resistance, Multiple, Bacterial/genetics , HT29 Cells , Humans , Italy , Klebsiella , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/genetics , Larva , Microbial Sensitivity Tests , Polysaccharides, Bacterial/metabolism , Virulence , beta-Lactamases/geneticsABSTRACT
Advanced therapy medicinal products (ATMP) are required to maintain their quality and safety throughout the production cycle, and they must be free of microbial contaminations. Among them, mycoplasma contaminations are difficult to detect and undesirable in ATMP, especially for immunosuppressed patients. Mycoplasma detection tests suggested by European Pharmacopoeia are the "culture method" and "indicator cell culture method" which, despite their effectiveness, are time consuming and laborious. Alternative methods are accepted, provided they are adequate and their results are comparable with those of the standard methods. To validate a novel in-house method, we performed and optimized, a real time PCR protocol, using a commercial kit and an automatic extraction system, in which we tested different volumes of matrix, maximizing the detection sensitivity. The results were compared with those obtained with the gold standard methods. From a volume of 10 ml, we were able to recognize all the mycoplasmas specified by the European Pharmacopoeia, defined as genomic copies per colony forming unit ratio (GC/CFU). Our strategy allows to achieve faster and reproducible results when compared with conventional methods and meets the sensitivity and robustness criteria required for an alternative approach to mycoplasmas detection for in-process and product-release testing of ATMP.