Jundishapur Journal of Microbiology
Volume:16 Issue: 6, Jun 2023

 Pulmonary tuberculosis is currently diagnosed using traditional techniques, such as smear production from sputum samples, to detect acid-fast bacilli (AFB) and bacteriological culture. The detection of Mycobacterium tuberculosis DNA in peripheral blood samples could potentially aid in tuberculosis diagnosis.

 This study aimed to compare the effectiveness of polymerase chain reaction (PCR) assay on peripheral blood mononuclear cells (PBMCs) with established diagnostic techniques for detecting M. tuberculosis.

 We collected peripheral blood and sputum samples from 45 patients with smear-positive pulmonary tuberculosis. Standard microscopy and culture techniques were performed on both sputum and PBMC samples. The PCR was conducted on PBMC and sputum specimens using primers specific for the M. tuberculosis complex insertion sequence IS6110.

 Thirty-nine sputum samples and 2 PBMC samples were determined to contain M. tuberculosis based on bacterial culture and biochemical tests. PCR results were positive for 32 (82%) sputum samples and 29 (75%) PBMC samples. None of the PBMCs tested positive through AFB staining.

 The M. tuberculosis PCR assay on PBMCs using IS6110 primers demonstrated high sensitivity and specificity in detecting M. tuberculosis DNA. However, the implementation of real-time PCR with a specific probe may further enhance the detection of M. tuberculosis DNA in peripheral blood.

SARS-CoV-2 is a single-stranded RNA virus and a member of a large family of Coronaviruses that are important human pathogens. This virus caused severe acute respiratory syndrome and was initially identified to be transmitted between humans on November 17, 2019.

To investigate the lineage, mutational patterns, variants, and serotypes of SARS-CoV-2 viruses circulating in the Duhok governorate population and to compare them with those identified in travelers crossing the border from Turkey in order to trace the epidemiological patterns.

Nasopharyngeal swabs were collected from 700 individuals living in Duhok and 700 travelers crossing the border to Duhok-Iraq from Turkey. The subjects were recruited by random sampling and questioned about demographic features and symptoms of upper or lower respiratory tract infections. Exclusion criteria included vaccination with COVID-19 vaccines of any approved previous infection. Samples were subjected to RT-PCR, and 30 positive samples with the highest viral load (lowest Ct values) were chosen for sequencing of the complete S gene by next-generation sequencing (NGS). Three platforms of Nextstrain, GISAID, and PANGO were used to identify variants, clades, and lineages and analyze sequences.

Out of 1400 participants, 353 (25.21%) positive samples were identified by RT-PCR, of which 30 representative positive samples (15 from each group: Patients and travelers) were sent for complete sequencing of the S spike gene using NGS. Nineteen samples were successfully sequenced and retrieved, including nine samples from Duhok residents and ten samples from travelers. Nextclade results revealed that 12 samples belonged to the delta strain (Pango lineages: B1.617.2.78, B1.617.2, B1.617.126, and B.1.617.121) distributed among the two groups while 5 omicron (BA.1.1) and 2 alpha (B.1.1.7) strains were found among travelers. A total of 76 mutations, including 52 non-synonymous, 16 synonymous, and 8 deletions, were detected without identifying a unique mutation. Sequencing results were submitted to GISAID, and accession numbers were obtained. A phylogenetic tree was constructed using the sequences obtained from Iraqi and non-Iraqi variants from GISAID.

The present research presents a description and observation of the genetic and epigenetic status of SARS-CoV-2 in Iraq based on sequencing results. The study revealed the impact of travels in introducing new variants to the country, including those with mutations in the S1 domain of the spike protein that can enhance viral attachment to receptors.

 Colorectal cancer (CRC) is the third most common cancer worldwide, and its development is influenced by genetic and environmental factors, including the gut microbiota. Recent studies have reported an association between Fusobacterium nucleatum abundance and CRC.

 This study aimed to investigate the abundance of F. nucleatum in CRC and polyp patients and its association with the expression of Chemokine ligand -3(CCL3), Vascular endothelial growth factor (VEGF), and Nuclear factor-kappa B (NF-KB11) genes and the presence of deoxyribonucleic acid (DNA) mutations and polymorphisms in the Kirsten rat sarcoma viral oncogene homolog (KRAS) gene.

 A total of 80 biopsy samples were collected from CRC, polyp, and colitis patients. Moreover, F. nucleatum abundance was measured by quantitative polymerase chain reaction (qPCR). The expression of CCL3, VEGF, and NF-KB11 genes was measured by reverse transcription polymerase chain reaction (RT-PCR). Additionally, KRAS gene mutations and polymorphisms were detected by the Mutation Surveyor software (V5.1.2).

 The results showed that F. nucleatum abundance was significantly higher in CRC and polyp patients than in colitis patients (P < 0.05). The expression of CCL3 and VEGF genes was also significantly higher in F. nucleatum-positive samples (P < 0.05). However, NF-KB11 gene expression was non-significant. F. nucleatum-positive biopsy samples had a higher frequency of KRAS gene mutations and polymorphisms than F. nucleatum-negative CRC patients (odds ratio = 3). Most of the mutations observed in the positive samples were (6144A>AT,31E>E) at exon 2 of the KRAS gene.

 The study findings suggest that F. nucleatum might play a role in CRC and polyp development and contribute to KRAS gene mutations. Therefore, targeting F. nucleatum in the gut microbiota could be a potential therapeutic strategy for preventing CRC and polyp development.

 Most Acinetobacter baumannii species have become resistant to all common antibiotics. Many antimicrobial peptides (AMPs) have been identified with efficient functions in infection management. E50-52 (UniProtKB: P85148) and Ib-AMP4 (UniProtKB: O24006) AMPs have shown marked antibacterial functions.

 This investigation was designed to produce E50-52 and Ib-AMP4 AMPs through recombinant protein production. Subsequently, the synergistic antimicrobial functions of these two peptides were assessed under in vitro and in vivo circumstances on multidrug-resistant A. baumannii to investigate the antimicrobial effects of Ib-AMP4 and E50-52 AMPs on drug-resistant Acinetobacter.

 The gene sequence of E50-52 and Ib-AMP4 AMPs were codon optimized and separately inserted into the pET-32α vector. The recombinant structures were expressed in host bacteria. The antibacterial functions of the individual and combined application of the purified refolded E50-52 and Ib-AMP4 AMPs against multidrug-resistant A. baumannii were evaluated through the time-kill, minimum inhibitory concentration (MIC), growth kinetic assays, and in vivo (mouse body) systemic infection.

 The minimum concentrations of the produced refolded E50-52 and Ib-AMP4 AMPs against A. baumannii were 0.325 and 0.0625 mg/mL, respectively. Moreover, the checkerboard procedure confirmed the synergic effects of the produced AMPs. The use of E50-52 and Ib-AMP4 AMPs in combination resulted in an over five times reduction in log10 CFU/mL of alive cells during the first 240-min exposure. The antibacterial efficiency of the produced AMPs was confirmed by growth kinetic assays, scanning electron microscopy (SEM) results, and in vivo evaluation tests. The in vivo assay on rats confirmed the significant antibacterial functions of the produced recombinant proteins on A. baumannii systemic infection.

 The results proved the considerable synergistic antibacterial functions of the produced recombinant Ib-AMP4 and E50-52 AMPs to treat A. baumannii systemic infection effectively.

 Patients undergoing orthopedic surgery are at risk of nosocomial infections, and antibiotic resistance is known to increase the risk of such infections.

 We aimed to determine the rate of antibiotic resistance in patients admitted to orthopedic wards in one of the largest referral hospitals in Iran. We also ascertained responsible antibiotic-resistant microorganisms in patients with bone and joint infections.

 The present cross-sectional investigation was concluded over a period of five years, from March 2018 to February 2023, at a great referral hospital in Tehran. Laboratory data, including the organisms isolated and their antibiotic resistance patterns, were collected by reviewing the hospital information system.

 In total, 2650 specimens obtained from patients with suspected bacterial infections were transferred to the hospital’s laboratory, 880 (33.2%) of which were positive for bacterial infections. The maximum antibiotic resistance rate against an antibiotic was observed to be 58% for Staphylococcus aureus (erythromycin), 75% for Klebsiella pneumonia (ampicillin/sulbactam), 64.5% for Escherichia coli (imipenem), 76.2% for coagulase-negative Staphylococcus (vancomycin), 100% for Acinetobacter baumannii (imipenem), 52% for S. epidermidis (erythromycin), 85.9% for Enterobacter species (gentamycin), and 65.6% for Pseudomonas aeruginosa (ampicillin/sulbactam). The overall rate of multi-drug resistance was obtained as 27.6%.

 A high rate of resistance of various bacterial strains to common antibiotics, especially erythromycin, ampicillin, imipenem, vancomycin, and gentamycin, was denoted in orthopedic wards. Also, a high rate of multi-antibiotic resistance was encountered in these wards, where more than a quarter of the bacterial strains showed such resistance.

 Candida is the main causative agent of severe mucosal and invasive candidiasis. Different species of Candida have shown varying levels of resistance to antifungal treatments. It is estimated that each 12-hour delay in antifungal treatment is associated with a significant increase in patient mortality and treatment costs. The culture method is regarded as the gold standard for identifying Candida species, but its time-consuming process is a clear disadvantage.

 This study established a method using membrane technology combined with dual-target melting analysis for rapid cultivation and identification of common Candida species. This method is expected to preserve the advantages of the conventional culture method and improve upon its weaknesses while also evaluating the practical application of the method.

 A microfiltration membrane-based culture followed by a color indicator method was established to rapidly cultivate Candida cultures. The 5.8S ribosomal DNA region and internal transcribed spacer 2 (ITS2) region were used as target gene regions, for which two sets of primers were employed. Melting analysis following dual-target real-time polymerase chain reaction (PCR) was conducted to distinguish among Candida albicans, C. tropicalis, C. glabrata, and C. krusei. To evaluate its practical application, the method was tested with 72 clinical isolates, and the results were compared with those obtained using the chromogenic culture method and DNA sequencing.

 Distinctive melting temperatures in the two gene targets were detected among the four common Candida species. The entire process, from cultivation to identification, was completed within 12 hours, about 50% less time than the gold-standard method. The minimum detection limit of Candida species was 10 femtograms. The results of the identification of the clinical isolates were consistent with those of DNA sequencing.

 The short-term membrane-based cultivation combined with dual-target melting analysis can be used to rapidly, easily, and accurately identify common Candida species, thus reducing the time needed to initiate targeted treatment for patients with severe candidiasis.