Unveiling promising immunogenic targets in Coxiella burnetii through in silico analysis: paving the way for novel vaccine strategies.

BACKGROUND: Coxiella burnetii, an intracellular pathogen, serves as the causative agent of zoonotic Q fever. This pathogen presents a significant threat due to its potential for airborne transmission, environmental persistence, and pathogenicity. The current whole-cell vaccine (WCV) utilized in Australia to combat Q fever exhibits notable limitations, including severe adverse reactions and limited regulatory approval for human use. This research employed the reverse vaccinology (RV) approach to uncover antigenic proteins and epitopes of C. burnetii, facilitating the development of more potent vaccine candidates. METHODS: The potential immunogenic proteins derived from C. burnetii RSA493/Nine Mile phase I (NMI) were extracted through manual, automated RV, and virulence factor database (VFDB) methods. Web tools and bioinformatics were used to evaluate physiochemical attributes, subcellular localization, antigenicity, allergenicity, human homology, B-cell epitopes, MHC I and II binding ratios, functional class scores, adhesion probabilities, protein-protein interactions, and molecular docking. RESULTS: Out of the 1850 proteins encoded by RSA493/NMI, a subset of 178 demonstrated the potential for surface or membrane localization. Following a series of analytical iterations, 14 putative immunogenic proteins emerged. This collection included nine proteins (57.1%) intricately involved in cell wall/membrane/envelope biogenesis processes (CBU_0197 (Q83EW1), CBU_0311 (Q83EK8), CBU_0489 (Q83E43), CBU_0939 (Q83D08), CBU_1190 (P39917), CBU_1829 (Q83AQ2), CBU_1412 (Q83BU0), CBU_1414 (Q83BT8), and CBU_1600 (Q83BB2)). The CBU_1627 (Q83B86 ) (7.1%) implicated in intracellular trafficking, secretion, and vesicular transport, and CBU_0092 (Q83F57) (7.1%) contributing to cell division. Additionally, three proteins (21.4%) displayed uncharacterized functions (CBU_0736 (Q83DJ4), CBU_1095 (Q83CL9), and CBU_2079 (Q83A32)). The congruent results obtained from molecular docking and immune response stimulation lend support to the inclusion of all 14 putative proteins as potential vaccine candidates. Notably, seven proteins with well-defined functions stand out among these candidates. CONCLUSIONS: The outcomes of this study introduce promising proteins and epitopes for the forthcoming formulation of subunit vaccines against Q fever, with a primary emphasis on cellular processes and the virulence factors of C. burnetii.

Statins, allies against antibiotic resistance?

Due to the ever-increasing rate of antibacterial resistance, the search for effective antibacterial agents has become imperative. Researchers have investigated the potential antimicrobial properties of various classes of nonantibiotic drugs. Statins are a group of antihyperlipidemic drugs with several cholesterol-independent effects, including anti-inflammatory, immune-modulating, antioxidant, and antibacterial effects. In vitro and in vivo studies have demonstrated the antibacterial properties of statins against various gram-positive and gram-negative bacteria. Simvastatin and atorvastatin are the most potent members of the family. Their antibacterial effect can be attributed to several direct and indirect mechanisms. Bacterial invasion, growth, and virulence are affected by statins. However, since in vitro minimum inhibitory concentrations (MICs) are significantly higher than serum concentrations at the lipid-lowering dosage, indirect mechanisms have been suggested to explain the positive clinical results, including reducing inflammation and improving immune response capacity. Further, statins have shown promising results when combined with antibiotics and other antibacterial agents, such as triazenes and silver nanoparticles. Despite this, the controversial aspects of statins have cast doubt on their efficacy as a possible solution for antibacterial resistance, and further research is required. Consequently, this review will examine in detail the current clinical and in vitro findings and controversies regarding statins' antibacterial properties and their relevance to antibacterial resistance.

CRISPR-Cas system as a promising player against bacterial infection and antibiotic resistance.

The phenomenon of antibiotic resistance (AR) and its increasing global trends and destructive waves concerns patients and the healthcare system. In order to combat AR, it is necessary to explore new strategies when the current antibiotics fail to be effective. Thus, knowing the resistance mechanisms and appropriate diagnosis of bacterial infections may help enhance the sensitivity and specificity of novel strategies. On the other hand, resistance to antimicrobial compounds can spread from resistant populations to susceptible ones. Antimicrobial resistance genes (ARGs) significantly disseminate AR via horizontal and vertical gene transfer. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is a member of the bacterial immune system with the ability to remove the ARGs; therefore, it can be introduced as an effective and innovative strategy in the battle against AR. Here, we reviewed CRISPR-based bacterial diagnosis technologies. Moreover, the strategies to battle AR based on targeting bacterial chromosomes and resistance plasmids using the CRISPR-Cas system have been explained. Besides, we have presented the limitations of CRISPR delivery and potential solutions to help improve the future development of CRISPR-based platforms.

Colistin resistance mechanisms in Gram-negative bacteria: a Focus on Escherichia coli.

Multidrug-resistant (MDR) Escherichia coli strains have rapidly increased worldwide, and effective antibiotic therapeutic options are becoming more restricted. As a polymyxin antibiotic, colistin has a long history of usage, and it is used as a final line of treatment for severe infections by Gram-negative bacteria (GNB) with high-level resistance. However, its application has been challenged by the emergence of E. coli colistin resistance. Hence, determining the mechanism that confers colistin resistance is crucial for monitoring and controlling the dissemination of colistin-resistant E. coli strains. This comprehensive review summarizes colistin resistance mechanisms in E. coli strains and concentrates on the history, mode of action, and therapeutic implications of colistin. We have mainly focused on the fundamental mechanisms of colistin resistance that are mediated by chromosomal or plasmid elements and discussed major mutations in the two-component systems (TCSs) genes and plasmids that transmit the mobilized colistin resistance resistant genes in E. coli strains.

In silico analysis of the substitution mutations and evolutionary trends of the SARS-CoV-2 structural proteins in Asia.

Objectives: To address a highly mutable pathogen, mutations must be evaluated. SARS-CoV-2 involves changing infectivity, mortality, and treatment and vaccination susceptibility resulting from mutations. Materials and Methods: We investigated the Asian and worldwide samples of amino-acid sequences (AASs) for envelope (E), membrane (M), nucleocapsid (N), and spike (S) proteins from the announcement of the new coronavirus 2019 (COVID-19) up to January 2022. Sequence alignment to the Wuhan-2019 virus permits tracking mutations in Asian and global samples. Furthermore, we explored the evolutionary tendencies of structural protein mutations and compared the results between Asia and the globe. Results: The mutation analyses indicated that 5.81%, 70.63%, 26.59%, and 3.36% of Asian S, E, M, and N samples did not display any mutation. Additionally, the most relative mutations among the S, E, M, and N AASs occurred in the regions of 508 to 635 AA, 7 to 14 AA, 66 to 88 AA, and 164 to 205 AA in both Asian and total samples. D614G, T9I, I82T, and R203M were inferred as the most frequent mutations in S, E, M, and N AASs. Timeline research showed that substitution mutation in the location of 614 among Asian and total S AASs was detected from January 2020. Conclusion: N protein was the most non-conserved protein, and the most prevalent mutations in S, E, M, and N AASs were D614G, T9I, I82T, and R203M. Screening structural protein mutations is a robust approach for developing drugs, vaccines, and more specific diagnostic tools.

Mutations in SARS-CoV-2 structural proteins: a global analysis.

BACKGROUND: Emergence of new variants mainly variants of concerns (VOC) is caused by mutations in main structural proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, we aimed to investigate the mutations among structural proteins of SARS-CoV-2 globally. METHODS: We analyzed samples of amino-acid sequences (AASs) for envelope (E), membrane (M), nucleocapsid (N), and spike (S) proteins from the declaration of the coronavirus 2019 (COVID-19) as pandemic to January 2022. The presence and location of mutations were then investigated by aligning the sequences to the reference sequence and categorizing them based on frequency and continent. Finally, the related human genes with the viral structural genes were discovered, and their interactions were reported. RESULTS: The results indicated that the most relative mutations among the E, M, N, and S AASs occurred in the regions of 7 to 14, 66 to 88, 164 to 205, and 508 to 635 AAs, respectively. The most frequent mutations in E, M, N, and S proteins were T9I, I82T, R203M/R203K, and D614G. D614G was the most frequent mutation in all six geographical areas. Following D614G, L18F, A222V, E484K, and N501Y, respectively, were ranked as the most frequent mutations in S protein globally. Besides, A-kinase Anchoring Protein 8 Like (AKAP8L) was shown as the linkage unit between M, E, and E cluster genes. CONCLUSION: Screening the structural protein mutations can help scientists introduce better drug and vaccine development strategies.

Relationships between Efflux Pumps and Emergence of Heteroresistance: A Comprehensive Study on the Current Findings.

Heteroresiatnce (HR) is the type of resistance toward one or more antibiotics appearing as a population of the bacterial load consisting of one or more subpopulations with lower antibiotic susceptibility levels than others. Due to the lack of appropriate diagnosis of HR isolates and their importance in resistance emergence to antibiotics, investigating the origins, emergence factors, and HR inhibitors is critical in combating antibiotic resistance. Efflux pumps (EPs) are bacterial systems that own an influential role in acquiring resistance toward anti-bacterial compounds. Studies on EPs revealed that they can affect HR emergence mechanisms and are competent to be introduced as a suitable bacterial target for diagnostic and therapeutic development in combating HR isolates. This review will consider the relations between EPs and the emergence of HR isolates and discuss their importance in confronting this type of antibiotic resistance.

Bismuth nanoparticles against microbial infections.

The destructive effect of infectious diseases on human life and the emergence of antibiotic-resistant strains highlight the importance of developing new and appropriate treatment strategies, one of which is the use of metals as therapeutic agents. Bismuth nanoparticles are an example of prominent metal-containing drugs. The therapeutic effects of bismuth-based drugs in the treatment of wounds have been proven. Various laboratory studies have confirmed the antimicrobial effects of bismuth nanoparticles, including the clinical treatment of ulcers caused by Helicobacter pylori. Therefore, considering the performance of this nanoparticle and its potent effect on infectious agents and its therapeutic dimensions, the present study fully investigated the properties and performance of this metal-based nanoparticle.

Molecular characterization of pathogenicity locus (PaLoc) and tcdC genetic diversity among tcdA+B+Clostridioides difficile clinical isolates in Tehran, Iran.

Clostridioides difficile is the main cause of healthcare-associated diarrhea worldwide. It is proposed that certain C. difficile toxinotypes with distinct pathogenicity locus (PaLoc) variants are associated with disease severity and outcomes. Additionally, few studies have described the common C. difficile toxinotypes, and also little is known about the tcdC variants in Iranian isolates. We characterized the toxinotypes and the tcdC genotypes from a collection of Iranian clinical C. difficile tcdA+B+ isolates with known ribotypes (RTs). Fifty C. difficile isolates with known RTs and carrying the tcdA and tcdB toxin genes were analyzed. Toxinotyping was carried out based on a PCR-RFLP analysis of a 19.6 kb region encompassing the PaLoc. Genetic diversity of the tcdC gene was determined by the sequencing of the gene. Of the 50 C. difficile isolates investigated, five distinct toxinotypes were recognized. Toxinotypes 0 (33/50, 66%) and V (11/50, 22%) were the most frequently found. C. difficile isolates of the toxinotype 0 mostly belonged to RT 001 (12/33, 36.4%), whereas toxinotype V consisted of RT 126 (9/11, 81.8%). The tcdC sequencing showed six variants (35/50, 70%); tcdC-sc3 (24%), tcdC-A (22%), tcdC-sc9 (18%), tcdC-B (2%), tcdC-sc14 (2%), and tcdC-sc15 (2%). The remaining isolates were wild-types (15/50, 30%) in the tcdC gene. The present study demonstrates that the majority of clinical tcdA+B+ isolates of C. difficile frequently harbor tcdC genetic variants. We also found that the RT 001/toxinotype 0 and the RT 126/toxinotype V are the most common types among Iranian isolates. Further studies are needed to investigate the putative association of various tcdC genotypes with CDI severity and its recurrence.

Characterization of biofilm formation and virulence factors of Staphylococcus aureus isolates from paediatric patients in Tehran, Iran.

OBJECTIVES: Staphylococcus aureus can cause several infections. Its capability to form biofilm has been reported to be a vital property involved in the bacteria's pathogenesis. Various genes contributing to biofilm formation have not yet been completely clarified. This study was designed to evaluate the factors influencing adherence and biofilm formation in S. aureus isolated from paediatric patients. MATERIALS AND METHODS: One hundred and ninety-seven S. aureus isolates were obtained from pediatric patients and confirmed with phenotypic and molecular examinations. Antimicrobial susceptibility testing and biofilm formation were evaluated using standard methods. The genes encoding adhesion and virulence factors were investigated by the PCR method. RESULTS: The most efficient antibiotics against S. aureus isolates were vancomycin and linezolid. Approximately, 54.2% of MSSA and 85.6% of MRSA isolates were biofilm producers according to the microtiter test. Our analysis indicated that MRSA isolates are better able to form biofilm compared with MSSA isolates. All isolates harbored clfA, fnbpA, icaA, icaB, icaC, and icaD, while clfB, fnbB, hlg, and pvl were detected in 99.5%, 42.1%, 97.5%, and 5.6% of isolates, respectively. In addition, a significant difference was found in fnhB gene and biofilm formation. CONCLUSION: Our findings showed a significant correlation between mecA and pvl genes and MRSA and biofilm formation in S. aureus isolates. Additionally, this study indicated the significant role of the fnhB gene as a major marker for S. aureus biofilm formation. Therefore, further experiments are warranted to exactly elucidate the function of the fnhB gene in the formation of biofilm.

Chronic Suppurative Otitis Media: A Case Report.

BACKGROUND CONTEXT: Chronic Suppurative Otitis Media (CSOM) is a common cause of hearing impairment and disability. CSOM caused by Pseudomonas aeruginosa is usually treated with topical ciprofloxacin and resistance to ciprofloxacin in CSOM isolates has rarely been reported. CASE PRESENTATION: A 24-year-old male patient with CSOM due to p. aeruginosa was reported. CSOM was prolonged for ten years and physician prescribed topical ciprofloxacin drops, pus suctioning and ear pH alteration. The treatment wasn't effective and the patient came back to the clinic with relapse of suppurative otitis media. P. aeruginosa was isolated as the cause of CSOM and the isolate was resistant to ciprofloxacin, aztreonam, imipenem, gentamicin, doripenem, cefepime, levofloxacin, amikacin and susceptible to colistin and ceftazidime. There were two mutations in gyrA and eight mutations were observed in nfxB genes. Finally, tympanomastoidectomy was done. CONCLUSION: Usually topical antibiotics, especially ciprofloxacin, is effective against ear infections but our case was different and the P. aeruginosa isolated from CSOM was resistant to most of the antibiotics. One reason for CSOM recurrence might be surgery failure. The routine and primary treatment for CSOM did not seem sufficient and tympanomastoidectomy is suggested to be the best treatment approach for these patients.

Multiple Myeloma or Brucellosis: A Case Report.

BACKGROUND: Brucellosis, a major health problem in developing countries, is a multisystem infection with a broad spectrum of clinical manifestations. Hematological complications, ranging from an intravascular coagulopathy to mild homeostasis disorders (such as gammopathy), have been reported in brucella infection. These signs and symptoms may lead to misdiagnosis of brucellosis with other hematological diseases. CASE: A 65-year-old male whose occupation was shepherding was referred to our hospital as a known case of multiple myeloma with continuous fever, muscle weakness, and night sweating after taking 2 courses of chemotherapy. The laboratory diagnosis of multiple myeloma had been based on the observation of a high percent of plasma cells in the bone marrow aspiration. At follow- up, the result of patient's fever workup, with 2 sets of blood cultures, was positive for Brucella melitensis. Isolated brucella was confirmed as B. melitensis by 16S rRNA sequencing. Brucellosis serologic test was performed by agglutination test and positive results were obtained. The patient was discharged with the cessation of fever and general improvement after the end of the parental treatment phase of brucella bacteremia. CONCLUSIONS: Brucella infection may cause a severe disease, mimicking a primary hematological disease, which could complicate the correct diagnosis. In brucellosis cases, due to the wide range of symptoms, in addition to cultivation and serological methods, molecular methods should also be used to prevent inappropriate diagnosis and additional costs.

Coexistence of Clostridioides difficile and Staphylococcus aureus in gut of Iranian outpatients with underlying inflammatory bowel disease.

Clostridioides difficile and Staphylococcus aureus are two well-known pathogens both causing hospital- and community-acquired infections. However, their intestinal coexistence was not well investigated in inflammatory bowel disease (IBD). Herein, we explored the prevalence of C. difficile, S. aureus and their coexistence in the gut of Iranian patients with IBD. Fecal and colon specimens were obtained from 70 outpatients with underlying IBD, and investigated for the presence of C. difficile and S. aureus. C. difficile isolates were characterised by CE-ribotyping. PCR was used for detection of toxin-encoding genes of C. difficile and S. aureus isolates. The antimicrobial susceptibility testing of C. difficile and S. aureus isolates were examined by agar dilution and Kirby-Bauer disk diffusion methods, respectively. Totally, C. difficile and S. aureus were detected in only 5.7% and 15.8% of IBD flares. Coexistence of C. difficile and S. aureus was detected in 5.7% of IBD flares. Two different C. difficile ribotypes including RT 126 and RT 017 were identified showing toxin profiles of tcdA+B+/cdtA+B+ and tcdA+B+, respectively. In S. aureus isolates, only positivity for the presence of sea enterotoxin was detected. C. difficile isolates were susceptible to metronidazole, ceftazidime and fidaxomicin. The highest resistance of S. aureus isolates was observed against penicillin (92.3%), following amoxicillin-clavulanate (38.5%) and amikacin (30.8%). Our findings demonstrated that patients with IBD flare are more sensitive to acquire coinfection of C. difficile and S. aureus than remission. However, more robust data is required to study the crosstalk between these enteric infections and their clinical relevance in patients with IBD flare.

Molecular analysis of accessory gene regulator functionality and virulence genes in Staphylococcus aureus derived from pediatric wound infections.

Staphylococcus aureus is a major human pathogen causing infections with high morbidity and mortality in both healthcare and community settings. The accessory gene regulator (Agr) is a key genetic element controlling the expression of numerous virulence factors in S. aureus. The significance of a functional Agr system in clinical S. aureus isolates derived from pediatric wound infections is still unclear. Therefore, the present study was conducted to identify virulence genes and determine Agr functionality from this cohort of patients. A total of 48 S. aureus wound isolates were collected from patients referred to Tehran Children's Medical Center Hospital from April 2017 to April 2018. In addition, in vitro antimicrobial susceptibility of the isolates was assessed using the disk diffusion and E-test methods. Conventional PCR was performed for the detection of toxins (tsst-1, hla, hlb, hld, eta, etb, etd, edin-A, edin-B, edin-C) and Agr typing (agrI, agrII, agrIII, agrIV). Agr functionality was assessed by quantitative reverse transcriptase real-time PCR (qRT-PCR). All S. aureus isolates were found to be susceptible to linezolid and vancomycin. The most frequently detected toxin gene was eta (100%), and the most prevalent Agr type was agrIII (56.3%). Importantly, qRT-PCR revealed that Agr was functional in 28 (58%) of wound isolates. Consequently, our data suggests that a functional Agr system may not be required for the development of S. aureus wound infections.

Antibacterial Activity of Gold Nanoparticles Conjugated by Aminoglycosides Against A. baumannii Isolates from Burn Patients.

BACKGROUND AND OBJECTIVE: Today, resistance to multiple classes of antibiotics, and notably to the ß-lactam and aminoglycosides in A. baumannii is becoming a great problem and it necessitates to make a new approach to combat with multidrug-resistant (MDR), extensive drug-resistance (XDR) or Pandrug-resistant (PDR) isolates. In this case, a new strategy and ways should be designed and introduced against such infections. Therefore the aim of the present study was the evaluation of antibacterial activity of nanoconjugate gentamicin and amikacin with gold against clinical isolates of A. baumannii that were collected from burn wound infection. There are some patents of gold nanoparticles that are conjugated with antibiotics (WO2017161296A1, US20090181101A1). METHODS: Eighteen A. baumannii were collected from burn wound infections. For confirmation and detection of aminoglycoside-resistant genes, PCR was carried out. Gold nanoparticles and nanoconjugates were prepared according to the protocol. For the evaluation of the nanoconjugate, Dynamic light cattering, Transmission electron microscopy and FTIR Analysis were carried out. Then, the antibacterial activity of nanoconjugates was conducted by using micro broth dilution method. RESULT: Prevalence of aminoglycoside-resistant genes was aacC1, aphA6, aadA1, aadB genes 55.5%, 22.2%, 38.8% and 22.2% respectively. Synthesis of bare nanoconjugates resulted in nanoparticle in a size of 10 nm. Amikacin bound to Gnps showed excellent antibacterial activity (94.5%) and just one isolate showed intermediate resistance. Also, gentamicin bound to Gnps had a good antimicrobial effect (50%) in contrast to gentamicin alone. CONCLUSION: Our study showed that a combination of amikacin and gentamicin with Gnps has a significant antibacterial efficiency against clinical isolates of A. baumannii. Gnps can be used as extraordinary molecular carriers for targeting, and delivery of the antibiotic molecules to the specific infection.

Evidence of Interruption of the comM Gene in a Large Series of Clinical Isolates of Multidrug-Resistant Acinetobacter baumannii.

Recent studies have recognized the ATPase-encoding comM gene as a hot spot for the integration of Acinetobacter baumannii resistance islands (RIs). Despite the circulation of high numbers of multidrug-resistant A. baumannii (MDR-AB) isolates in Middle East countries, no information is available about the interruption of comM and subsequent transposition into comM in isolates belonging to the global clones (GC) GC1, GC2, or GC3. In this study 401 A. baumannii isolates from hospitals in Tehran, Iran, were included. The resistance profile was determined by disc diffusion against 22 antibiotics. PCR was used to assess the GC type, presence of the comM gene, and the boundary junctions (J1 and J2) of RIs. Most of the MDR-AB isolates (384 of 388; 98%) and more than half of the susceptible A. baumannii isolates (9 of 13; 69%) had interrupted comM gene-carrying integrative elements. Among the isolates tested, 57 belonged to GC1, 86 to GC2, and 8 to GC3. A set of 250 isolates showed distinct patterns of allele-specific PCR for ompA, csuE, and blaOXA-51-like genes. All but 2 of the GC1 isolates and 2 of the GC2 isolates contained interrupted comM genes. Four A. baumannii isolates harbored intact comM, but were multiply resistant to antibiotics. This study demonstrated that the comM gene is targeted by transposons in Iranian MDR-AB isolates belonging to different GCs. The data also showed that the carriage of interrupted comM is not exclusive to MDR isolates of A. baumannii.