Inhibition of the blaOXA-48 gene expression in Klebsiella pneumoniae by a plasmid carrying CRISPRi-Cas9 system.

Antibiotic resistance is an increasing concern that threatens the effectiveness of treating bacterial infections. The spread of carbapenem resistant Klebsiella pneumoniae poses a significant threat to global public health. To combat this issue, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system is being developed. This system includes a single guide RNA (sgRNA) and a nuclease dead Cas9 (dCas9), which work together to downregulate gene expression. Our project involved the use of the CRISPRi system to reduce gene expression of the beta-lactamase oxacillin-48 (blaOXA-48) gene in K. pneumoniae. We designed a sgRNA and cloned it into pJMP1363 plasmid harboring the CRISPRi system. The pJMP1363-sgRNA construct was transformed in K. pneumoniae harboring the blaOXA-48 gene. The MIC test was used to evaluate the antimicrobial resistance, and quantitative real-time RT-PCR was used to confirm the inhibition of the OXA-48 producing K. pneumoniae harboring the pJMP1363-sgRNA construct expression. The Galleria mellonella larvae model was also utilized for in vivo assay. Following the transformation, the MIC test indicated a 4-fold reduction in meropenem resistance, and qRT-PCR analysis revealed a 60-fold decrease in the mRNA OXA-48 harboring the pJMP1363-sgRNA construct expression. Additionally, G. mellonella larvae infected with OXA-48 producing K. pneumoniae harboring the pJMP1363-sgRNA showed higher survival rates. Based on the findings, it can be concluded that the CRISPR interference technique has successfully reduced antibiotic resistance and virulence in the K. pneumoniae harboring the blaOXA-48 gene.

Repurposing of Compounds from Streptomyces spp. as Potential Inhibitors of Aminoacyltransferase FemA: An Essential Drug Target against Drug-resistant Staphylococcus aureus.

BACKGROUND: Drug-resistant Staphylococcus aureus represents a substantial healthcare challenge worldwide, and its range of available therapeutic options continues to diminish progressively. Thus, this study aimed to identify potential inhibitors against FemA, a crucial protein involved in the cell wall biosynthesis of S. aureus. MATERIALS AND METHODS: The screening process involved a comprehensive structure-based virtual screening on the StreptomDB database to identify ligands with potential inhibitory effects on FemA using AutoDock Vina. The most desirable ligands with the highest binding affinity and pharmacokinetic properties were selected. Two ligands with the highest number of hydrogen bonds and hydrophobic interactions were further analyzed by molecular dynamics (MD) using the GROMACS version 2018 simulation package. RESULTS: Six H-donor conserved residues were selected as protein active sites, including Arg- 220, Tyr-38, Gln-154, Asn-73, Arg-74, and Thr-24. Through virtual screening, a total of nine compounds with the highest binding affinity to the FemA protein were identified. Frigocyclinone and C21H21N3O4 exhibited the highest binding affinity and demonstrated favorable pharmacokinetic properties. Molecular dynamics analysis of the FemA-ligand complexes further indicated desirable stability and reliability of complexes, reinforcing the potential efficacy of these ligands as inhibitors of FemA protein. CONCLUSION: Our findings suggest that Frigocyclinone and C21H21N3O4 are promising inhibitors of FemA in S. aureus. To further validate these computational results, experimental studies are planned to confirm the inhibitory effects of these compounds on various S. aureus strains. Combining computational screening with experimental validation contributes valuable insights to the field of drug discovery in comparison to the classical drug discovery approaches.

Polydopamine-based nano adjuvant as a promising vaccine carrier induces significant immune responses against Acinetobacter baumannii-associated pneumonia.

The objective of this study was to assess the effectiveness of polydopamine nanoparticles (PDANPs) as a delivery system for intranasal antigen administration to prevent Acinetobacter baumannii (A. baumannii)-associated pneumonia. In the in vitro phase, the conserved outer membrane protein 22 (Omp22)-encoding gene of A. baumannii was cloned, expressed, and purified, resulting in the production of recombinant Omp22 (rOmp22), which was verified using western blot. PDANPs were synthesized using dopamine monomers and loaded with rOmp22 through physical adsorption. The rOmp22-loaded PDANPs were characterized in terms of size, size distribution, zeta potential, field emission scanning electron microscopy (FESEM), loading capacity, Fourier transform infrared spectroscopy (FTIR), release profile, and cytotoxicity. In the in vivo phase, the adjuvant effect of rOmp22-loaded PDANPs was evaluated in terms of eliciting immune responses, including humoral and cytokine levels (IL-4, IL-17, and IFN-γ), as well as protection challenge. The rOmp22-loaded PDANPs were spherical with a size of 205 nm, a zeta potential of -14 mV, and a loading capacity of approximately 35.7 %. The released rOmp22 from nontoxic rOmp22-loaded PDANPs over 20 days was approximately 41.5 %, with preserved rOmp22 integrity. The IgG2a/IgG1 ratio and IFN-γ levels were significantly higher in immunized mice with rOmp22-loaded-PDANPs than in rOmp22-alum, naive Omp22, and control groups. Furthermore, rOmp22-loaded PDANPs induced effective protection against infection in the experimental challenge and showed more normal structures in the lung histopathology assay. The results of this study suggest the potential of PDANPs as a nano-adjuvant for inducing strong immune responses to combat A. baumannii.

Outbreak of colistin and carbapenem-resistant Klebsiella pneumoniae ST16 co-producing NDM-1 and OXA-48 isolates in an Iranian hospital.

BACKGROUND: Colistin and carbapenem-resistant Klebsiella pneumoniae (Col-CRKP) represent a significant and constantly growing threat to global public health. We report here an outbreak of Col-CRKP infections during the fifth wave of COVID-19 pandemic. METHODS: The outbreak occurred in an intensive care unit with 22 beds at a teaching university hospital, Isfahan, Iran. We collected eight Col-CRKP strains from seven patients and characterized these strains for their antimicrobial susceptibility, determination of hypermucoviscous phenotype, capsular serotyping, molecular detection of virulence and resistance genes. Clonal relatedness of the isolates was performed using MLST. RESULTS: The COVID-19 patients were aged 24-75 years with at least 50% pulmonary involvement and were admitted to the intensive care unit. They all had superinfection caused by Col-CRKP, and poor responses to antibiotic treatment and died. With the exception of one isolate that belonged to the ST11, all seven representative Col-CRKP strains belonged to the ST16. Of these eight isolates, one ST16 isolate carried the iucA and ybtS genes was identified as serotype K20 hypervirulent Col-CRKP. The blaSHV and blaNDM-1 genes were the most prevalent resistance genes, followed by blaOXA-48 and blaCTX-M-15 and blaTEM genes. Mobilized colistin-resistance genes were not detected in the isolates. CONCLUSIONS: The continual emergence of ST16 Col-CRKP strains is a major threat to public health worldwide due to multidrug-resistant and highly transmissible characteristics. It seems that the potential dissemination of these clones highlights the importance of appropriate monitoring and strict infection control measures to prevent the spread of resistant bacteria in hospitals.

Echinococcus granulosus sheep strain (G1) as the predominant genotype in definitive host (dogs) isolates in northeastern Iran.

Echinococcus granulosus sensu lato (E. granulosus s.l.) is a zoonotic parasite, causing cystic echinococcosis in humans. In the present study, prevalence and genotypes of E. granulosus s.l. was assessed in stools collected from 244 dogs including 138 stray and 106 domestic animals using high resolution melting curve (HRM) method. Initially, to detect taeniid eggs in feces, all samples were examined using the formalin-ether techniques. Genomic DNA was extracted from the positive samples and E. granulosus s.l. was differentiated from other Taeniidae parasites using SSU-rDNA gene and E. granulosus s.l. was analyzed for genotyping using HRM based on the cox1 gene. In total, 12.7% (31/244) of the samples were positive for Taeniidae eggs. In addition, among the positive samples, 77.4% (24/31) were positive for E. granulosus s.l.. In details, 11.3% (12/106) of the domestic dogs and 8.7% (12/138) of the stray dogs were positive for E. granulosus s.l.. The results of HRM analysis showed that all E. granulosus s.l. isolates were G1 strain. Findings of the present study indicated a considerable prevalence of E. granulosus G1 among dogs in the northeast of Iran and imply a serious risk of transmitting to humans and livestock.

Characterization of class 1 integrons in metallo-ß-lactamase-producing Acinetobacter baumannii isolates from hospital environment.

BACKGROUND AND OBJECTIVE: The emergence and widespread dissemination of antibiotic resistance in A. baumannii, has become a globally challenge. The increasing hospital outbreaks by multi-drug resistant (MDR) A. baumannii strains, shows the necessity of continuous monitoring to find sources of resistant strains in hospitals. This study aimed to identify the presence of class 1 integrons and metallo-ß-lactamase (MBL) related genes in A. baumannii isolates from hospital environment. METHODS: In order to identify A. baumannii isolates, a total of 297 environmental samples were collected from burn wards and intensive care units (ICUs) of two university hospitals. Resistance to common antibiotics was studied by disk diffusion method and microbroth dilution assay was used to determine the minimum inhibitory concentrations (MICs) of imipenem, colistin and tigecycline. The A. baumannii isolates were studied by polymerase chain reaction (PCR) for the presence of class 1 integrons (intI1, intl CS) and metallo-ß-lactamases (MBLs) (blaIMP, blaVIM, blaNDM) genes. RESULTS: A. baumannii was identified in 68/297 (22.9%) of hospital environment. All A. baumannii strains were multidrug-resistant (MDR), but none of them were resistant to colistin, tigecycline and ampicillin-sulbactam. All (100%) and 38 (95.0%) of A. baumannii isolates from ICUs and burn wards were imipenem resistant respectively. Class 1 integrons was identified in 30/40 (75.0%) and 23/28 (82.1%) isolates from burn wards and ICUs respectively. Two different types of gene cassettes were identified, which included: arr-2, ereC, aadA1, cmlA5 and arr2, cmlA5. MBL genes including blaVIM and blaIMP were detected in 26/28 (92.8%), 27/28(96.4%) and 39/40 (97.5%) and 31/40 (77.5%) of the isolates from the ICUs and the burn wards respectively. None of the isolates contained the blaNDM-1 gene. CONCLUSION: The findings of the present study showed that the isolation rate of MBL producing carbapenem-resistant A. baumannii (CRAB) was relatively high in the environmental surface of burn wards and ICUs, which can be considered as a potential source of outbreaks in hospitalized patients.

Carbapenemase genes distribution in clonal lineages of Acinetobacter baumannii: a comprehensive study on plasmids and chromosomes.

Background: The global spread of plasmids carrying carbapenemase genes within carbapenem resistant Acinetobacter baumannii (CRAB) strains poses a worldwide public health issue. In this study, we conducted a comprehensive genetic analysis of plasmids and chromosomes harboring the major carbapenemase genes (bla NDM, bla KPC, bla VIM, bla IMP, bla GES, bla OXA-58-like, bla OXA-24/40-like, bla OXA-143-like, and bla OXA-23-like) in CRAB strains using bioinformatic tools. Methods: We retrieved plasmids and chromosomes carrying the major carbapenemase genes from GenBank. The size, replicon type, and conjugal apparatus of the plasmids were also determined. Furthermore, allele types, co-existence of other antimicrobial resistance genes alongside carbapenemases in plasmids or chromosomes, co-occurrence of carbapenemase genes, gene repetition, and sequence types (ST) of whole genomes were characterized. Results: The database contained 113 plasmids and 38 chromosomes harboring carbapenemase genes. This investigation revealed that bla NDM and bla OXA-58-like were the predominant allele types in both the plasmids and chromosomes. Nine (7.96%) plasmids with bla NDM-1 were potentially conjugative. The most common replicon types of the plasmids were R3-T1, R3-T8, R3-T2, R3-T23, and RP-T1. The analysis revealed that bla NDM-1 and bla OXA-58-like genes possessed the highest variety of co-existence with other antibiotic resistance genes. The co-occurrence of dual carbapenemases was identified in 12 plasmids and 19 chromosomes. Carbapenemase gene repetitions were identified in 10 plasmids and one chromosome. Circular alignment revealed that the plasmids carrying the co-occurrence of bla NDM-1 and bla OXA-58 were more homogeneous. However, there was heterogeneity in certain regions of these plasmids. According to the minimum spanning tree (MST) results, the majority of the plasmids belonged to the genomes of ST2Pas, ST1Pas, ST422Pas, ST622Pas, and ST85Pas. Conclusion: A. baumannii appears to have a strong ability for genome plasticity to incorporate carbapenemase genes on its plasmids and chromosomes to develop resistance against carbapenems. Mobilizable plasmids harboring carbapenemases significantly contribute to the dissemination of these genes. The genetic structure of the plasmids revealed a strong associations of class I integrons, ISAba-like structures, Tn4401 elements, and aac (6')-Ib with carbapenemases. Furthermore, gene repetition may also be associated with carbapenem heteroresistance.

Design and fabrication of a vaccine candidate based on rOmpA from Klebsiella pneumoniae encapsulated in silk fibroin-sodium alginate nanoparticles against pneumonia infection.

The present study describes the design and fabrication of a novel vaccine candidate based on the outer membrane protein A (rOmpA) from Klebsiella pneumoniae (K. pneumoniae) encapsulated in silk fibroin-sodium alginate nanoparticles (SF-SANPs) against K. pneumoniae-mediated pneumonia. The physicochemical properties, toxicity, release profile, and in vivo potency of SF-SANPs encapsulated with rOmpA were evaluated. The spherical nano vaccine was created with an average particle size of 160 nm and an encapsulation efficiency of 80 %. Antigen release from SF-SANPs was 40 % after 22 days release assay. The SF-SANPs showed a zeta potential of -24.8 mV and had no toxic effect on the L929 cells in vitro. It was found that SF-SANPs in the vaccine formulation promoted systemic and mucosal antibodies and also stimulated cytokine responses, inducing both humoral (Th2) and cellular (Th1) immune responses, with a Th1-polarized response. The vaccine candidate was effective in protecting the mice lung against experimental pneumonia and reducing inflammation. These findings suggest that the rOmpA-based vaccine encapsulated in SF-SANPs could be a promising strategy for preventing pneumonia caused by K. pneumoniae.

Decoding the genetic structure of conjugative plasmids in international clones of Klebsiella pneumoniae: A deep dive into blaKPC, blaNDM, blaOXA-48, and blaGES genes.

Carbapanem-resistant Klebsiella pneumoniae is a globally healthcare crisis. The distribution of plasmids carrying carbapenemase genes among K. pneumoniae poses a serious threat in clinical settings. Here, we characterized the genetic structure of plasmids harboring major carbapenemases (e.g. blaKPC, blaNDM, blaOXA-48-like, and blaGES) from K. pneumoniae using bioinformatics tools. The plasmids carrying at least one major carbapenemase gene were retrieved from the GenBank database. The DNA length, Inc type, and conjugal apparatus of these plasmids were detected. Additionally, allele types, co-existence, co-occurrence of carbapenemase genes, gene repetition, and sequence types of isolates, were characterized. There were 2254 plasmids harboring carbapenemase genes in the database. This study revealed that blaKPC-2, blaNDM-1, blaOXA-48, and blaGES-5 were the most prevalent allele types. Out of 1140 (50%) plasmids were potentially conjugative. IncFII, IncR, IncX3, and IncL replicon types were predominant. The co-existence analysis revealed that the most prevalent of other resistance genes were blaTEM-1 (related to blaKPC), blaOXA-232 (related to blaOXA-48), bleMBL (related to blaNDM), and aac (6')-Ib4 (related to blaGES). The co-occurrence of carbapenemases was detected in 42 plasmids while 15 plasmids contained carbapenemase gene repetitions. Sequence alignments highlighted that plasmids carrying blaKPC and blaOXA-48-like were more homogeneous whereas the plasmids carrying blaNDM were divergent. It seems that K. pneumoniae utilizes diversity of genetic flexibility and recombination for resistance against carbapenems. The genetic structure of the plasmids showed that class I and III, Tn3 family, Tn5403 family derivatives, and Tn7-like elements were strongly associated with carbapenemases. The mobilizable plasmids carrying carbapenemases play an important role in the spread of these genes. In addition, gene repetition maybe is related to carbapenem heteroresistance. According to MST (minimum spanning tree) results, the majority of plasmids belonged to sequence type (ST) 11, ST14, and ST12. These international clones have a high capacity to acquire the carbapenemase-containing plasmids.

Various arrangements of mobile genetic elements among CC147 subpopulations of Klebsiella pneumoniae harboring blaNDM-1: a comparative genomic analysis of carbapenem resistant strains.

BACKGROUND: Certain clonal complexes (CCs) of Klebsiella pneumoniae such as CC147 (ST147 and ST392) are major drivers of blaNDM dissemination across the world. ST147 has repeatedly reported from our geographical region, but its population dynamics and evolutionary trajectories need to be further studied. METHODS: Comparative genomic analysis of 51 carbapenem-nonsusceptible strains as well as three hypervirulent K. pneumoniae (hvKp) recovered during 16-months of surveillance was performed using various bioinformatics tools. We investigated the genetic proximity of our ST147 strains with publicly available corresponding genomes deposited globally and from neighbor countries in our geographic region. RESULTS: While IncL/M plasmid harboring blaOXA-48 was distributed among divergent clones, blaNDM-1 was circulated by twenty of the 25 CC147 dominant clone and were mostly recovered from the ICU. The NDM-1 core structure was bracketed by a single isoform of mobile genetic elements (MGEs) [ΔISKpn26-NDM-TnAs3-ΔIS3000-Tn5403] and was located on Col440I plasmid in 68.7% of ST392. However, various arrangements of MGEs including MITESen1/MITESen1 composite transposon or combination of MITESen1/ISSen4/IS903B/IS5/ISEhe3 on IncFIb (pB171) were identified in ST147. It seems that ST392 circulated blaNDM-1 in 2018 before being gradually replaced by ST147 from the middle to the end of sample collection in 2019. ST147 strains possessed the highest number of resistance markers and showed high genetic similarity with four public genomes that harbored blaNDM-1 on the same replicon type. Mainly, there was a convergence between clusters and isolated neighboring countries in the minimum-spanning tree. A conserved arrangement of resistance markers/MGEs was linked to methyltransferase armA which was embedded in class 1 integron in 8 isolates of ST147/ST48 high-risk clones. CONCLUSION: Our findings highlight the dynamic nature of blaNDM-1 transmission among K. pneumoniae in Iran that occurs both clonally and horizontally via various combinations of MGEs. This is the first analysis of Iranian ST147/NDM + clone in the global context.

Defeating a superbug: A breakthrough in vaccine design against multidrug-resistant Pseudomonas aeruginosa using reverse vaccinology.

BACKGROUND: Multidrug-resistant Pseudomonas aeruginosa has become a major cause of severe infections. Due to the lack of approved vaccines, this study has presented putative vaccine candidates against it. METHODS: P. aeruginosa 24Pae112 as a reference strain was retrieved from GenBank database. The surface-exposed, antigenic, non-allergenic, and non-homologous human proteins were selected. The conserved domains of selected proteins were evaluated, and the prevalence of proteins was assessed among 395 genomes. Next, linear and conformational B-cell epitopes, and human MHC II binding sites were determined. Finally, five conserved and highly antigenic B-cell epitopes from OMPs were implanted on the three platforms as multi-epitope vaccines, including FliC, the bacteriophage T7 tail, and the cell wall-associated transporter proteins. The immunoreactivity was investigated using molecular docking and immune simulation. Furthermore, molecular dynamics simulation was done to refine the chimeric cell-wall-associated transporter-TLR4 complex as the best interaction. RESULTS: Among 6494 total proteins of P. aeruginosa 24Pae112, 16 proteins (seven OMPs and nine secreted) were ideal according to the defined criteria. These proteins had a molecular weight of 110 kDa and were prevalent in ≥ 75% of P. aeruginosa genomes. Among the presented multi-epitope vaccines, the chimeric cell-wall-associated transporter had the strongest interaction with TLR4. Moreover, the immune simulation response revealed that the bacteriophage T7 tail chimeric protein had the strongest ability to stimulate the immune system. In addition, molecular docking and molecular dynamic simulation indicated the proper and stable interactions between the chimeric cell-wall-associated transporter and TLR4. CONCLUSION: This study proposed 16 shortlisted proteins as promising immunogenic targets. Two novel platforms (e.g. cell-wall-associated transporter and bacteriophage T7 tail proteins) for designing of multi-epitope vaccines (MEVs), showed the better performance compared to FliC. In our future studies, these two MEVs will receive more scrutiny to evaluate their immunoreactivity.

Introduction of Novel Drug Targets against Staphylococcus aureus and Proposing Putative Inhibitors against Adenine N1 (m1A22)-tRNA Methyltransferase (TrmK) using Computer-aided Drug Discovery.

BACKGROUND: Nowadays, the emergence of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) strains has dramatically restricted the treatment options against this microorganism. AIM: In this study, we aimed to discover new drug targets and inhibitors against S. aureus. METHODS: This study consists of two major sections. In the upstream evaluation, after a comprehensive coreproteome analysis, essential cytoplasmic proteins with no similarity to the human proteome were selected. Then the S. aureus metabolome-specific proteins were selected, and novel drug targets were identified using the DrugBank database. In the downstream analysis, a structure-based virtual screening approach was performed to reveal potential hit compounds against adenine N1 (m(m1A22)-tRNA methyltransferase (TrmK) using the StreptomeDB library and AutoDock Vina software. The compounds with a binding affinity > -9 kcal/mol were analyzed based on ADMET properties. Finally, the hit compounds were selected based on Lipinski's rule of five (RO5). RESULTS: Three proteins, including glycine glycosyltransferase (FemA), TrmK, and heptaprenyl pyrophosphate synthase subunit A (HepS1), were selected as feasible and promising drug targets based on PDB file availability and their essential role in the survival of the S. aureus. Finally, seven hit compounds, including Nocardioazine_ A, Geninthiocin_D, Citreamicin_delta, Quinaldopeptin, Rachelmycin, Di-AFN_A1 and Naphthomycin_ K were introduced against the binding cavity of TrmK, as a feasible drug target. CONCLUSION: The results of this study provided three feasible drug targets against S. aureus. In the following, seven hit compounds were introduced as potential inhibitors of TrmK, and Geninthiocin_D was identified as the most desirable agent. However, in vivo and in vitro investigations are needed to confirm the inhibitory effect of these agents on S. aureus.

Clonal relatedness of carbapenem-resistant Acinetobacter baumannii: high prevalence of ST136pas in a burn center.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a global health crisis. This study aimed to determine the clonal relatedness of antibiotic-resistant A. baumannii isolates in hospitalized patients who suffered from burn wound infection. METHODS: One hundred and six A. baumannii isolates from 562 patients with burn wound infections, were identified and examined for antimicrobial susceptibility. Detection and characterization of carbapenem-hydrolyzing class D OXA-type beta-lactamases (CHDLs) were performed by PCR assays. The clonal relatedness of A. baumannii isolates was determined by multilocus sequence typing (MLST) according to the Pasteur scheme, dual-sequence typing of blaOXA-51-like and ampC genes, and RAPD-PCR method. RESULTS: All isolates were carbapenem-resistant while susceptible to colistin, minocycline, doxycycline, and ampicillin-sulbactam. The intrinsic blaOXA-51-like was detected in all isolates, and blaOXA-23-like was identified in 92.5% of isolates. However, blaOXA-143-like and blaOXA-58-like genes were not detected among isolates. Four distinct blaOXA-51-like alleles were determined as follows: blaOXA-317 (67.0%), blaOXA-90 (9.4%), blaOXA-69 (17.0%), and blaOXA-64 (6.6%) and four ampC (blaADC) allele types including ampC-25 (6.6%), ampC-39 (9.4%), ampC-1 (17.0%), and blaADC-88 (67.0%) were identified. MLST (Pasteur scheme) analysis revealed four ST types including ST136 (singleton), ST1 (CC1), ST25 (CC25), and ST78 (singleton) in 71, 18, 7, and 10 of A. baumannii strains, respectively. Five RAPD clusters including A (1.9%), B (26.4%), C (57.5%), D (7.5%), and E (1.9%) were characterized and 5 (4.7%) strains were found to be singletons. CONCLUSION: The present study demonstrated that there was a high prevalence of blaOXA-23-like producing CRAB in the clinical setting. The majority of isolates belonged to ST136 (singleton). However, blaOXA-23-like producing multi-drug resistant international clones including ST1, and emerging lineages (e.g. ST25 and ST78) were also identified. Interestingly, in this study ST2 was not detected.

Reverse vaccinology approaches to introduce promising immunogenic and drug targets against antibiotic-resistant Neisseria gonorrhoeae: Thinking outside the box in current prevention and treatment.

Gonorrhea is an urgent antimicrobial resistance threat and its therapeutic options are continuously getting restricted. Moreover, no vaccine has been approved against it so far. Hence, the present study aimed to introduce novel immunogenic and drug targets against antibiotic-resistant Neisseria gonorrhoeae strains. In the first step, the core proteins of 79 complete genomes of N. gonorrhoeae were retrieved. Next, the surface-exposed proteins were evaluated from different aspects such as antigenicity, allergenicity, conservancy, and B-cell and T-cell epitopes to introduce promising immunogenic candidates. Then, the interactions with human Toll-like receptors (TLR-1, 2, and 4), and immunoreactivity to elicit humoral and cellular immune responses were simulated. On the other hand, to identify novel broad-spectrum drug targets, the cytoplasmic and essential proteins were detected. Then, the N. gonorrhoeae metabolome-specific proteins were compared to the drug targets of the DrugBank, and novel drug targets were retrieved. Finally, the protein data bank (PDB) file availability and prevalence among the ESKAPE group and common sexually transmitted infection (STI) agents were assessed. Our analyses resulted in the recognition of ten novel and putative immunogenic targets including murein transglycosylase A, PBP1A, Opa, NlpD, Azurin, MtrE, RmpM, LptD, NspA, and TamA. Moreover, four potential and broad-spectrum drug targets were identified including UMP kinase, GlyQ, HU family DNA-binding protein, and IF-1. Some of the shortlisted immunogenic and drug targets have confirmed roles in adhesion, immune evasion, and antibiotic resistance that can induce bactericidal antibodies. Other immunogenic and drug targets might be associated with the virulence of N. gonorrhoeae as well. Thus, further experimental studies and site-directed mutations are recommended to investigate the role of potential vaccine and drug targets in the pathogenesis of N. gonorrhoeae. It seems that the efforts for proposing novel vaccines and drug targets appear to be paving the way for a prevention-treatment strategy against this bacterium. Additionally, a combination of bactericidal monoclonal antibodies and antibiotics is a promising approach to curing N. gonorrhoeae.

Antibiotic Susceptibility, Biofilm-Forming Ability, and Prevalence of Extended-Spectrum Beta-Lactamase (ESBL)- and Biofilm-Associated Genes Among Klebsiella pneumoniae Isolates from Hospitalized Patients in Northwest of Iran.

Klebsiella pneumoniae is an opportunistic bacterium, which is globally recognized for its high prevalence and antimicrobial resistance (AMR). Biofilm-forming capability, susceptibility testing, and phenotypic confirmatory test for extended-spectrum beta-lactamase (ESBL)-producing isolate recognition of 104 K. pneumoniae isolates were performed according to the Clinical Laboratory Standard Institute (CLSI) guidelines. The prevalence of ESBL-associated genes bla-VIM, bla-NDM, and bla-OXA-48, as well as biofilm-associated genes luxS, fimH1, wza, and mrkD, was determined by multiplex PCR. The highest resistance rate was against ampicillin (100.0%). Among the 104 K. pneumoniae isolates, 52 (50.0%) and 31 (29.8%) isolates were determined as multi- and extensively drug resistant (MDR, XDR), respectively. Moreover, 21 (40.4%) isolates were determined as ESBL producing. Among 50 biofilm-producing K. pneumoniae isolates, 7 (14.0%), 15 (30.0%), and 28 (56.0%) isolates exhibited high, moderate, and weak levels of biofilm formation, respectively. A number of 41 (78.8%) isolates were susceptible to colistin, and 10 (19.2%) were resistant. AMR was significantly higher (P < 0.05) in the biofilm-forming isolates compared with non-biofilm formers.

Identification of Putative Drug Targets in Highly Resistant Gram-Negative Bacteria; and Drug Discovery Against Glycyl-tRNA Synthetase as a New Target.

Background: Gram-negative bacterial infections are on the rise due to the high prevalence of multidrug-resistant bacteria, and efforts must be made to identify novel drug targets and then new antibiotics. Methods: In the upstream part, we retrieved the genome sequences of 4 highly resistant Gram-negative bacteria (e.g., Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Enterobacter cloacae). The core proteins were assessed to find common, cytoplasmic, and essential proteins with no similarity to the human proteome. Novel drug targets were identified using DrugBank, and their sequence conservancy was evaluated. Protein Data Bank files and STRING interaction networks were assessed. Finally, the aminoacylation cavity of glycyl-tRNA synthetase (GlyQ) was virtually screened to identify novel inhibitors using AutoDock Vina and the StreptomeDB library. Ligands with high binding affinity were clustered, and then the pharmacokinetics properties of therapeutic agents were investigated. Results: A total of 6 common proteins (e.g., RP-L28, RP-L30, RP-S20, RP-S21, Rnt, and GlyQ) were selected as novel and widespread drug targets against highly resistant Gram-negative superbugs based on different criteria. In the downstream analysis, virtual screening revealed that Rimocidin, Flavofungin, Chaxamycin, 11,11'-O-dimethyl-14'-deethyl-14'-methylelaiophylin, and Platensimycin were promising hit compounds against GlyQ protein. Finally, 11,11'-O-dimethyl-14'-deethyl-14'-methylelaiophylin was identified as the best potential inhibitor of GlyQ protein. This compound showed high absorption capacity in the human intestine. Conclusion: The results of this study provide 6 common putative new drug targets against 4 highly resistant and Gram-negative bacteria. Moreover, we presented 5 different hit compounds against GlyQ protein as a novel therapeutic target. However, further in vitro and in vivo studies are needed to explore the bactericidal effects of proposed hit compounds against these superbugs.

Enhanced anti-biofilm activity of the minocycline-and-gallium-nitrate using niosome wrapping against Acinetobacter baumannii in C57/BL6 mouse pneumonia model.

Acinetobacter baumannii is a worldwide health issue in terms of its high antibiotic resistance and ability to form biofilms. Nanoparticles (NPs) with high biocompatibility, high penetrating ability, and low medication dose can successfully treat the antibiotic-resistant infections. In this research, the anti-biofilm activity of niosomes containing minocycline and gallium nitrate (GaN) against A. baumannii biofilm was determined. In order to improve their anti-biofilm properties, minocycline and GaN were encapsulated in niosomes as biocompatible drug carriers. The niosomes' size, zeta potential, shape, stability, drug entrapment efficacy, drug release pattern and antibacterial activity were assessed. Several clinical samples were isolated from the lungs of patients hospitalized at Loghman hospital, Tehran, Iran. The biofilm formation of most lethal clinical isolates of A. baumannii was analyzed. The pneumonia model was generated by intranasally administering A. baumannii suspension to anesthetized mice whose immune systems was compromised twice by cyclophosphamide. Lung infection of the mouse with A. baumannii was confirmed using PCR. After treatment, the lungs were excised under sterile conditions and stained with hematoxylin and eosin (H&E) to determine histological symptoms, inflammation and intercellular secretions. The niosomes contained minocycline and GaN had an average size of 230 nm and a zeta potential of -40 mV, respectively. The percentage of drug entrapment and delayed drug release was both high in niosomal formulations. Niosomes containing minocycline and GaN dispersed 1, 3 and 5 day old biofilms. The mice given the combination of two compounds required less time to be treated than the animals given the single medication (minocycline). The minocycline& GaN-loaded niosomes could be considered as promising candidates to treat the infections caused by A. baumannii biofilm.

Detection of multidrug-resistant Acinetobacter baumannii from burn patients and healthcare workers in Iran.

Multidrug-resistant (MDR) Acinetobacter baumannii is a serious global health threat. Burn patients are at high risk to acquire A. baumannii infections from endogenous sources. This study evaluated carbapenem resistance and clonal relatedness of A. baumannii isolated from burn patients and healthcare workers (HCWs).The study was performed in 100 non-duplicated A. baumannii isolates from nasal and hand samples of hospitalized burn patients and HCWs in two hospitals of Iran from June 2020 to August 2021. Antimicrobial susceptibility testing was performed and carbapenemase genes were detected by PCR. Clonal relatedness of A. baumannii isolates was determined by two single-locus sequence-based typing of blaOXA-51-like and ampC and by multilocus sequence typing (MLST).All A. baumannii isolates were found to be MDR while susceptible to colistin. The intI1, conserved segments of class 1 integron (intI1 CS), blaIMP, blaVIM, blaOXA-51-like, and blaOXA-23-like, genes were detected in 32.5%, 29.1%, 36%, 95.3%, 100%, 100%; and 14.3%, 14.3%, 21.4%, 92.9%, 100%, and 85.7% of isolates from patients and from healthcare workers, respectively. The blaOXA-58, and blaOXA-143 were not detected among the isolates. Using dual-locus blaOXA-51-like and ampC sequence-based typing (SBT), the isolates obtained from nasal samples of burn patients were grouped into 3 clusters including blaOXA-317, blaADC-88 (72.1%); blaOXA-64, ampC-25 (18.6%); and blaOXA-69, ampC-1 (9.3%). While only allele type blaOXA-317, blaADC-88 was determined among isolates from HCWs. MLST results showed A. baumannii ST136, ST25, and ST1 from burn patients. However, A. baumannii strains from HCWs belonged to ST136. Our findings indicate high prevalence of globally spreading of MDR A. baumannii ST136 carrying blaOXA-23-like from nasal and hand samples of burn patients and HCWs.

Genome-Wide Subtraction Analysis and Reverse Vaccinology to Detect Novel Drug Targets and Potential Vaccine Candidates Against Ehrlichia chaffeensis.

Human monocytotropic ehrlichiosis is an emerging tick-borne infection caused by the obligate intracellular pathogen, Ehrlichia chaffeensis. The non-specific symptoms can range from a self-limiting fever to a fatal septic-like syndrome and may be misdiagnosed. The limited treatment choices including doxycycline are effective only in the initiation phase of the infection. It seems that novel therapeutic targets and new vaccine strategies could be effective to control this pathogen. This study is comprised of two major phases. First, the common proteins retrieved through subtractive analysis and potential drug targets were evaluated by subcellular localization, homology prediction, metabolic pathways, druggability, essentiality, protein-protein interaction networks, and protein data bank availability. In the second phase, surface-exposed proteins were assessed based on antigenicity, allergenicity, physiochemical properties, B cell and T cell epitopes, conserved domains, and protein-protein interaction networks. A multi-epitope vaccine was designed and characterized using molecular dockings and immune simulation analysis. Six proteins including WP_011452818.1, WP_011452723.1, WP_006010413.1, WP_006010278.1, WP_011452938.1, and WP_006010644.1 were detected. They belong to unique metabolic pathways of E. chaffeensis that are considered as new essential drug targets. Based on the reverse vaccinology, WP_011452702.1, WP_044193405.1, WP_044170604.1, and WP_006010191.1 proteins were potential vaccine candidates. Finally, four B cell epitopes, including SINNQDRNC, FESVSSYNI, SGKKEISVQSN, and QSSAKRKST, were used to generate the multi-epitope vaccine based on LCL platform. The vaccine showed strong interactions with toll-like receptors and acceptable immune-reactivity by immune simulation analysis. The findings of this study may represent a turning point in developing an effective drug and vaccine against E. chaffeensis. However, further experimental analyses have remained.

Immunization with recombinant DcaP-like protein and AbOmpA revealed protections against sepsis infection of multi-drug resistant Acinetobacter baumannii ST2Pas in a C57BL/6 mouse model.

BACKGROUNDS: The prevalence of infections associated with multi-drug resistant (MDR) Acinetobacter baumannii is increasing worldwide. Therefore, the introduction of effective vaccines against this bacterium seems necessary. METHODS: AbOmpA and DcaP-like protein were selected as promising and putative immunogenic candidates based on previous in silico studies. Three formulations including AbOmpA, DcaP-like protein, and AbOmpA + DcaP-like protein were injected into C57BL/6 mice three times with Alum adjuvant. The specific production of IgG antibodies (e.g. total IgG, IgG1 and IgG2c) and cytokines (e.g. IL-4, IL-6, and IL-17A), were evaluated. LD50% of MDR A. baumannii ST2Pas was measured using Probit's method. After the challenge with bacteria, a decrease in bacterial loads (DLs) in the lung and spleen of mice was measured. Then serum bactericidal assay was performed to determine the function of antibodies on day 42. In addition, histopathological examinations of the spleen and lung, the number of macrophage and neutrophil, as well as the rate of lymphocyte infiltration were assessed. RESULTS: The highest level of total IgG was reported in the group immunized with DcaP-like protein on day 42. The survival rate of mice was 80% in the AbOmpA immunized group and 100% for the rest of two groups. DLs in the spleen of mice immunized with AbOmpA, DcaP-like protein, and combination form were 3.5, 3, and 3.4 Log10 (CFU/g), respectively. While in the lung, the DLs were 7.5 Log10 (CFU/g) for the AbOmpA group and 5 for the rest of two groups. The levels of IL-6, IL-4, and IL-17A were significantly decreased in all immunized groups after the bacterial challenge (except for IL-17A in the group of AbOmpA). The bactericidal effect of antibodies against DcaP-like protein was more effective. No histopathological damage was observed in the combination immunized group. The DcaP-like protein was more effective in neutrophil and macrophage deployment and decreased lymphocyte infiltration. CONCLUSION: The results of immunization with AbOmpA + DcaP-like protein induced a protective reaction against the sepsis infection of MDR A. baumannii. It seems that in the future, these proteins can be considered as promising components in the development of the A. baumannii vaccine.