Development of a cost-effective quantitative in-house ELISA assay for screening anti-S1 IgG antibodies targeting SARS-CoV-2.

The RBD, S, and N proteins, the three main antigens of the SARS-CoV-2 virus, activate the host immune system and cause the formation of IgM and IgG antibodies. While IgM indicates an early, acute infection stage, IgG shows a past infection or persistent sickness. This study used an indirect ELISA assay that targets the S1 subunit of the SARS-CoV-2 S protein to create an in-house, qualitative serological test specific to COVID-19. A total of 60 serum samples were examined using ELISA for anti-SARS-CoV-2 IgG, and 50 of those results were positive. An additional 20 samples were taken from cases that occurred before the pandemic. For the in-house ELISA assay, a plasmid containing the gene coding for the S1 subunit was transformed into E. coli DH5ɑ bacterial cells and the protein was synthesized and purified. The purified protein was utilized to coat the ELISA plate, which was subsequently used to assess the levels of IgG among individuals with SARS-CoV-2 infection. The study found a significant association (p-value=0.01) between the in-house and the commercial anti-S1 subunit IgG antibodies kits. The in-house ELISA responded well, with a sensitivity and specificity of 75.0% and 88.89%, respectively. Furthermore, a library of SARS-CoV-2 recombinant S1 subunits was created by competent bacteria and may be employed for various tasks, such as creating diagnostic tools and scientific investigation. Overall, the in-house anti-SARS-CoV-2 human IgG-ELISA proved to be sensitive and specific for identifying IgG antibodies in patients exposed to SARS-CoV-2.

Effect of NLRP3 inflammasome genes polymorphism on disease susceptibility and response to TNF-α inhibitors in Iraqi patients with rheumatoid arthritis.

Background: Rheumatoid arthritis (RA) is a genetically predisposed, systemic, chronic, inflammatory disease. Immune system dysregulation and inherited susceptibility polymorphisms suggest that this type of variation is functional and may help predict disease susceptibility and develop new therapeutic strategies. Anti-TNF-alpha (TNF-α) drugs are highly effective RA treatments, but not all patients respond the same way. It's important to figure out whether RA risk alleles can identify and predict anti-TNF-α-responsiveness in RA patients. Aims of the study: Examine the function of the NLR family pyrin domain containing 3 (NLRP3) and caspase recruitment domain family member 8 (CARD8) genes polymorphisms and their morbid genotypes and alleles in RA patients and apparently healthy controls. In addition, their role in disease susceptibility, severity, and response to anti-TNF-α therapy. Also, examine how single nucleotide polymorphisms (SNPs) affect serum levels of pro-inflammatory cytokines like TNF-α and interleukin (IL)-1ß. Materials and methods: 100 RA patients (88 females, 12 males) and 100 apparently healthy people (86 females, 14 males) were examined. To measure serum TNF-α and IL-1ß, Elabscience sandwich ELISA kits were used. Iraq Biotech, Turkey DNA extraction kit was used to extract genomic DNA from whole blood. CARD8 (rs2043211) and NLRP3 (rs4612666) were genotyped using Agilent, AriaMx, USA, through Tri-Plex SYBR Green-based real-time PCR allelic discrimination assays. Geneious software, version 2019.2.2, used to design primers from published sequences (GenBank accession no. GCA 009914755.1). Primer specificity was determined by NCBI's BLAST. Results: Study found that there is association between cytokines serum level and 28-joints disease activity score (DAS-28). The level of TNF-α increases with the higher DAS-28 (r2 = 0.45, P < 0.0001). Also, IL- 1ß level increases with higher DAS-28 (r2 = 0.51, P < 0.0001). There were no statistically significant variations between patients with RA and the control group in the distribution of CARD8 SNP rs2043211 and NLRP3 SNP rs4612666 genotypes (P = 0.17 and 0.08 respectively) as well their alleles (P = 0.059 and 0.879 respectively). CARD8 (rs2043211) TT genotype was more frequent in patients with higher DAS-28 (P < 0.0001) and higher TNF-α and IL-1ß serum levels (P < 0.0001 for both). Also, NLRP3 (rs4612666) TT genotype was more frequent in patients with higher DAS-28 (P < 0.0001) and higher TNF-α and IL- 1ß serum levels (P < 0.0001 for both). Interestingly, this study revealed that CARD8 (rs2043211) and NLRP3 (rs4612666) variant genotypes are associated with lower response to anti-TNF-α drugs. Conclusions: Serum TNF-α and IL-1ß correlate with DAS-28 and disease activity. Non-responders have elevated TNF-α and IL-1ß. CARD8 rs2043211 and NLRP3 rs4612666 variant polymorphisms are associated with high serum TNF-α and IL-1ß, active disease course, poor disease outcomes, and low response to anti-TNF-α therapy.

Novel recombinant endolysin ointment with broad antimicrobial activity against methicillin-resistant Staphylococcus aureus isolated from wounds and burns.

The major problem in the management of burn wounds is infections. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of infection in burn wounds. Antibiotic-resistant bacteria around the world have become a major therapeutic challenge. Bacteriophages and their lysin are suggested as an antimicrobial alternative agent. The approach of this study was to evaluate the potential of recombinant phage lysin ointment efficacy in MRSA burn wound infection in vitro. Whole genome sequencing was performed to the three isolated bacteriophages by ABM, USA using Illumina next-generation sequencing (NGS) technology. De novo assembly and genetic analysis carried out. Expression of lysin genes was performed by cloning using Escherichia coli JM109. Lysin protein extraction and purification was performed before and after cloning using precipitation by ammonium sulfate, dialysis, and gel filtration chromatography. Dose-dependent assay and time-kill curve experiment was performed for 2 lysins showed that recombinant lysin 2 functions more than its non-recombinant lysins 2 with the same concentration of 0.5 µg/mL. Both lysins' ointment was prepared and compared with commercial ointments. 62 (78.4%) out of 79 wounds a burns swabs were detected as S. aureus and S methicillin-resistant S. aureus (MRSA) rate was determined to be 29 (46.8%) in total, while 33 isolates (53.2%) determined as methicillin-sensitive S. aureus (MSSA). According to the antibiotic susceptibility test results, all S. aureus isolates were identified as sensitive against vancomycin, ceftaroline, and linezolid. Results shows one lysogenic bacteriophage and three distinct lytic specific S. aureus bacteriophage were isolated from sewage. For each of the three samples, a single contig was possible to be obtained. Sample BP-SA2 had the best coverage, and the contig was slightly longer than the other bacteriophages. In addition, BLAST search identified Staphylococcus bacteriophage vB-SscM-1 (accession KX171212.1) as the closest match to the public database. Finally, the gene annotation was checked, and two potential lysin genes were identified. Besides the two ends, there are only 4 SNPs between the three genomes. It should be noted that the two lysin genes from the genomes have no SNPs, and are identical across the three genomes. It can be seen that the three bacteriophages (BP-SA1), (BP-SA 2), and (BP-SA3) form their own tight cluster. It can be seen that (BP-SA 2) is more closely related to Staphylococcus bacteriophage vB-SscM-1 genome and most noticeable 5' region of S5 and vB-SscM-1 are now located at 3' end of vB-Sau-Clo6. The investigation of the two lysin genes in (BP-SA 2) by whole genome sequencing showed that there is some homology with vB-SscM-1; although the first gene is annotated as hypothetical protein, the second gene is annotated as amidase. The same two lysin genes are identified in all three bacteriophage genomes by the RAST. The putative protein sequences of the discovered phage lysin was analyzed using protein search with UniProt/Swiss-Prot database, and all matches suggest that the putative protein of the discovered phage lysin is a real endolysin. The three samples of bacteriophage were harboring both (Lysin 1 and lysin 2) genes were amplified. Afterward, 2-lysin genes were cloned successfully; for the dose-dependent assay, the same incubation time of recombinant lysins and its two non-recombinant lysins with the bacteria for 30 min. It is found that the bactericidal activity of these groups increased in correlation with their concentrations. For the time-kill curve experiment, it showed that Recombinant lysin 2 functions more than its non-recombinant lysins 2 with the same concentration of 0.5 µg/mL. Both lysins' ointments have potential activity against S. aureus isolates more than mupirocin and have a similar activity with Fusidic acid through applying 10 µL from lysin 1 ointment, lysin 2 ointment, mupirocin ointment 2%, and Fusidic acid cream 2%. In vitro lytic spectrum analysis revealed that 100% (29/29) tested S. aureus were sensitive. One dose of lysin ointment resulted in a reduction of 3.3 log units in the number of bacteria (from an initial count of 2 × 105 CFU/mg) at 18 hours compared with one dose of mupirocin, PBS, or Aquaphor. Specifically, this study provides evidence that the application of lysin ointment has significant potential as an alternative strategy for MRSA infections.

Formation of therapeutic phage cocktail and endolysin to highly multi-drug resistant Acinetobacter baumannii: in vitro and in vivo study.

OBJECTIVES: Phage therapy is a potential alternative treatment for infections caused by Acinetobacter baumannii, a significant nosocomial pathogen, which has evolved resistance to almost all conventional antimicrobial drugs in poor hygiene and conflicts areas such as Iraq. MATERIALS AND METHODS: Bacteriophages were isolated to highly resistant isolates of A. baumannii to form therapeutic phage cocktail, and to extract and evaluate native endolysin activity. Bacterial samples were collected in Al-Imamein Al-kadhimein Medical City Hospital. Phages were isolated from different regions in Baghdad city including (soil, sewage, irrigation channels). Phage endolysin was extracted from highly lytic phages that produced halo-like appearance around inhibition zone. RESULTS: Up to 23 isolates of extensive- and pan- drug resistant (XDR, PDR) A. baumannii were isolated from patients with various infections, and 136 lytic phages specific to A. baumannii were isolated. Each bacterial isolate was sensitive to at least one lytic phage. Accordingly, a phage cocktail was formulated which remarkably minimized bacterial resistance to lysis by phages when compared to individual lytic phages. And, the phage cocktail succeeded in treating and saving life of all bacteremic mice with A. baumannii versus the non-treated group. In addition, the endolysin native activity to A. baumannii was evaluated in this study; endolysin revealed a potent antibacterial activity (> 1 log) reduction of bacterial density in just one hour of endolysin treatment. CONCLUSION: The phage therapy assessed in this study showed an ability to efficiently solve the problems of "superbug" bacteria by lysing effectively most XDR, PDR bacteria in vitro and in vivo. And, phage cocktail was shown to be superior over single-phage preparations in treating A. baumannii with much less resistance rate to therapeutic phages. Furthermore, intrinsic activity of native endolysin revealed promising results to tackling superbug pathogens.

The potential of bacteriophage cocktail in eliminating Methicillin-resistant Staphylococcus aureus biofilms in terms of different extracellular matrices expressed by PIA, ciaA-D and FnBPA genes.

BACKGROUND: This study assessed novel approach of using highly lytic phages against methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) biofilms with and without biofilm extracellular matrix- disrupting chemical. METHOD: The resultant phage-based control was assessed in relation to the type of biofilm extracellular matrix namely, polysaccharide intercellular adhesion (PIA) or proteinacious fibronectin-binding protein A (FnBPA). The biofilms were formed in vitro by 24 h incubation of bacteria in 96 wells microtiter plates at room temperature. The formed biofilms were assessed by tissue culture plate (TCP). Moreover, the nature of the biofilm was assessed by scanning electron microscopy (SEM) and PCR assay for detecting PIA genes, ciaA-D and FnBPA genes. RESULTS: this study showed that applied phages with 0.08 % benezenthonium chloride, for PIA biofilms, and 0.06 % ethanol, for proteinacious FnBPA biofilms, exerted 100 % eradication for MSSA biofilms and about 78 % of MRSA biofilms. The phage-based control of biofilms with chemical adjuvant showed significantly higher efficiency than that without adjuvant (P < 0.05). Moreover, FnBPA biofilms were more common in MRSA than in MSSA while PIA biofilms were more common in MSSA than in MRSA. And the most resistant type of biofilms to phage-based control was FnBPA in MRSA where 50 % of biofilms were reduced but not eradicated completely. CONCLUSIONS: It is concluded that PIA-disturbing agent and protein denaturing alcohol can increase the efficiency of attacking phages in accessing host cell walls and lysing them which in turn lead to much more efficient MRSA and MSSA biofilm treatment and prevention.

Novel approach of using a cocktail of designed bacteriophages against gut pathogenic E. coli for bacterial load biocontrol.

BACKGROUND: This study was conducted to explore new approaches of animal biocontrol via biological control feed. METHOD: White rats were subjected to 140 highly lytic designed phages specific against E. coli. Phages were fed via drinking water, oral injection, and vegetable capsules. Phage feeding was applied by 24 h feeding with 11 d monitoring and 20 d phage feeding and monitoring. Group of rats received external pathogenic E. coli and another group did not, namely groups A and B. RESULTS: Phage feeding for 20 d via vegetable capsules yielded the highest reduction of fecal E. coli, 3.02 and 4.62 log, in rats group A and B respectively. Second best, feeding for 20 d via drinking water with alkali yielded 2.78 and 4.08 log in rats groups A and B respectively. The peak reduction in E. coli output was 5-10 d after phage feeding. Phage control declined after 10th day of feeding. CONCLUSIONS: The use of cocktail of designed phages succeeded in suppressing flora or external E. coli. The phage feed biocontrol is efficient in controlling E. coli at the pre-harvest period, precisely at the 6th-8th day of phage feeding when the lowest E. coli output found.