نشریه میکروب شناسی پزشکی ایران
سال شانزدهم شماره 5 (مهر و آبان 1401)

At the time of the coevolution of humans and microorganisms, the human digestive tract was colonized by thousands of species of bacteria. Mostly, intestine-borne microbes amount to the overall number of cells in the body tissue. The latest metagenomics study of the human intestinal microbiota confirmed the existence of some 3.3 million genes relative to only 23,000 genes found in tissue cells in the human body. There is increasing evidence for multiple beneficial functions of the gut microbiota in human health and illness. The best-described plant prebiotics is fructans and inulin. The best-known prebiotic carbohydrates comprise many plants, roots and tubers, and fruit crops, whereas prebiotic-richer grain crops contain maize, chickpea, lentil, lupin, and wheat. Some prebiotic enriched crop germplasm were documented in maize, chickpea, lentil, wheat, and yacon. Intestinal microbiota perturbation may contribute to persistent diseases such as autoimmune diseases, bowel cancers, stomach ulcers, colon disorders, and malnutrition. This can be impossible to recover the intestinal microbiome, but the usage of probiotics has contributed to a positive effect in a significant number of very well-designed (clinical) trials. Microbiomics has prompted a significant growth of interest in probiotics and prebiotics as potential mediators for the administration and regulation of gut microbiota in medicine, industry, and the general public. Developing prebiotic-rich healthy plants can mitigate the prevalent malnutrition challenge and facilitate worldwide global health. Bioinformatics and genomics tools may help to create mechanistic associations between gut microflora, a person's health status, and the outcomes of plant prebiotic drug treatments.

 Despite significant progress in Hepatitis B Virus (HBV) treatment, the emergence of drug resistance mutations is a main challenging health threat. The data is lacking regarding circulation mutant strains in northeastern Iran; therefore, the present study was conducted to investigate HBV reverse transcriptase (RT) inhibitors drug resistance mutations in a group of treatment naïve patients in Mashhad, Iran.

 In this study, 25 patients were included. The genomic DNA was extracted from serum samples, and the RT gene of HBV was amplified using specific primers. The PCR products were then subjected to gel electrophoresis and were next sent for sequencing. Finally, the sequences were analyzed using the HBVseq database, mutation list analysis software supported by Stanford University (https://hivdb.stanford.edu).

The mean age of the patients was 42.7±16.5. Among the patients, 56% were men. Among 23 cases (92%), no resistance mutation was observed, while 2 cases showed mutations outside the YMDD motif of viral reverse transcriptase causing Lamivudine or Entecavir resistance. The detected mutations included: rt T184A, S202I, S202H, V180I, I169L, and V173L. All sequenced samples were identified as genotype D.

 Lamivudine/Entecavir resistant variants are circulating in a minority of treatment naïve patients, which may indicate transmission of mutated stains to these patients or may be due to prolonged replication of the virus. This finding might be considered an alarm for increasing circulating mutant variants.

 Breast cancer is one of the most common types of cancer among Iranian women. To date, the usual cancer treatments have not been entirely effective. Therefore, creating anticancer products is of great importance. The aim of this study was to evaluate the cytotoxic, anticancer, and induction effects of Bacillus coagulans probiotic bacterial supernatant on SKBR3 cells.

 The anticancer potential and cytotoxic effect of different concentrations of probiotic bacterial supernatants (1, 2, 3, 4, 5, 6 and 7 mg/mL) were evaluated on SKBR3 cells for 24, 48, and 72 h by MTT technique. QRT-RCR was used to assess the expression of bax, bcl2, casp3, and casp9 genes, and flow cytometry was used to evaluate apoptosis in cancer cells.

The inhibitory effect of dose- and time-dependent B. coagulans supernatant showed that the supernatant of this probiotic bacterium had a cytotoxic effect on SKBR3 cancer cells. On the other hand, analysis of flow cytometry results and increased expression of bax, casp3, and casp9 pro-apoptotic genes and decreased bcl2 expression in cancer cells showed induction of apoptosis.

 The anticancer and cytotoxic effect of B. coagulans probiotic bacterial supernatant on SKBR3 cancer cells shows that with further research, this probiotic bacterium can be used as a new strategy for the possible treatment of breast cancer.

 Leptospirosis is one of the most common zoonotic diseases worldwide, occurring mostly in tropical, subtropical, temperate, and humid regions with heavy rainfall. It is important to diagnose this condition correctly and promptly. Loa22 is an outer membrane protein exposed to the surface in some Leptospira serovars. The purpose of this study was to determine the presence of the gene encoding Loa22 protein in Leptospira interrogans serovars.

 The present study was conducted on 23 pathogenic leptospira serovars and two non-pathogenic leptospira serovars. These serovars were prepared from the Reference Laboratory for Leptospira, Department of Microbiology, Razi Vaccine, and Serum Research Institute, Karaj, Iran. After genomic DNA extraction using the standard phenol-chloroform method, loa22 gene was amplified by specific primers.

PCR was performed on the loa22 gene by producing a 671-bp fragment. The results showed that the loa22 gene was present in all 23 pathogenic leptospira serovars but not in the non-pathogenic L. biflexa. The specificity of tested primers was confirmed as well.

 The loa22 gene is a specific gene for pathogenic leptospira serovars that is not found in saprophytic serovars, so it is suggested that this gene be used to detect leptospira pathogenic serovars.

 Early childhood caries is one of the most common chronic diseases in children, affecting both oral and general health. Oral microorganisms are the most important causative agents associated with dental caries in children. The aim of this study was to compare the antimicrobial activity of common Iranian and non-Iranian children's toothpaste on the growth of four standard bacteria strains, including Streptococcus mutans, Streptococcus sanguinis, Lactobacillus acidophilus, and Enterococcus faecalis.

 In this study, six types of the most common Iranian and non-Iranian children toothpaste produced by different companies were prepared. Different concentrations of toothpaste were prepared according to the Clinical and Laboratory Standards Institute (CLSI) standard. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of Iranian and non-Iranian children's toothpaste were measured by the microbroth dilution method at ten different concentrations.

For the S. mutans bacteria, the lowest MIC was found in Misswake, Vi-One, and 2080 toothpaste. In the case of S. sanguinis and L. acidophilus bacteria, the lowest MIC was related to Frice toothpaste, and for E. faecalis bacteria, the lowest MICs were found for Misswake and 2080 toothpaste. Mann-Whitney U test also revealed that the inhibitory and bactericidal activities of Iranian children's toothpaste on the studied bacteria were not significantly different from those of non-Iranian children's toothpaste.

 In general, the antimicrobial activity of Iranian children's toothpaste was higher than non-that of Iranian samples. In addition, the MIC of 2080 and Frice toothpaste in the four bacteria examined was lower than in other used toothpaste. To prevent early tooth decay in children use of these two kinds of toothpaste is recommended.

 Ornamental birds can serve as a reservoir for the virulent and antibiotic-resistant bacterium Escherichia coli. They can also play a role in transmitting these strains to humans. Therefore, obtaining information regarding drug resistance and virulence potential of the bacteria isolated from ornamental birds can contribute to disease treatment or prevention of pathogen transmission to humans. The present study was conducted to investigate the pattern of antibiotic resistance and virulence of Escherichia coli bacterium isolated from ornamental birds in Guilan province, Iran.

 Escherichia coli isolates were obtained from the feces of 80 apparently healthy ornamental birds in Rasht (Guilan, Iran) and were identified based on culture and biochemical tests. The antibiotic resistance pattern was determined using the disk diffusion method, and the frequency of virulence genes was investigated in test isolates using polymerase chain reaction.

Overall, 32 E. coli isolates were obtained from fresh feces of ornamental birds. In this study, 14 isolates (43.75%) had multiple drug resistance, and one extended-spectrum beta-lactamase-producing isolate was identified. Isolates were most sensitive to gentamicin (90%), and the highest resistance was associated with penicillin (90%). The frequency of iroN, ompT, hlyF, iss, and iutA genes in fecal isolates of ornamental birds was 28.12%, 34.37%, 40.62%, 30%, and 43.75%, respectively, and 25% of isolates were identified as avian pathogenic E. coli.

 The results of this study indicate the virulence potential and drug resistance in fecal E. coli isolates in ornamental birds in Rasht. The spread of these strains in the environment can endanger the health of owners and the whole society.

 The COVID-19 disease is an emerging infectious disease that appeared in December 2019 in Wuhan, China. An uncontrolled systemic inflammatory response is one of the primary mechanisms causing death in this disease. In this study, the expression levels of some inflammatory cytokines, vitamin D, and some hematological and biochemical parameters were compared in patients with severe COVID-19 and mild types.

 In this cross-sectional study, 60 blood samples were taken from 30 severe coronavirus patients and 30 mild coronavirus patients. The expression levels of cytokines such as IL (interleukin)-6, interferon (IFN)-α, IL-12, transforming growth factor (TGF) β, IL-8 and tumor necrosis factor (TNF)-α were evaluated using Real-time PCR. A T-test was used for Statistical Analysis.

IL-6, IFN-α, IL-12, TGF-β, IL-8, and TNF-α cytokines in the peripheral blood of severe patients, were positive in 28/30 (93.33%), 27/30 (90%), 24/30 (80%), 25/30 (83.33%), 26/30 (86.66%), and 27/30 (90%) respectively. The positive rate of these cytokines in the mild patients were 20/30 (66.67%), 21/30 (70%), 18/30 (60%), 17/30 (56.67%), 19/30 (63.33%), 18/30 (60%), respectively. There was a statistically significant difference between these two groups in terms of cytokines biomarkers. A significant difference was found between both groups in terms of the serum level of lactate dehydrogenase (LDH), the mean number of lymphocytes and neutrophils as well as the mean percentage of neutrophils/ lymphocytes ratio (NLR).

 The expression of cytokine genes and their release into the peripheral blood was increased in both severe and mild patients with COVID-19. However, they were more intense in patients with severe symptoms than those with mild symptoms and can cause inflammatory and even destructive reactions. Vitamin D deficiency plays no role in causing severe COVID-19 in patients without risk factors. Severe COVID-19 is characterized by elevated serum levels of LDH and NLR≥3.45.

 Currently, HIV, HBV, and HIV/HBV co-infection are important global health issues. It has been estimated that 2.7 million people are co-infected with HIV and HBV worldwide. In recent years, miRNAs have shown promising results as a research area. The current study aimed to investigate the expression level of miRNA-122, miRNA-149, miRNA-199a, and miRNA-let7 in HIV, HBV, HIV/HBV co-infected patients, and healthy controls.

 In the current study, miRNA expression levels were assessed in PBMC obtained from patients by real-time PCR.

The miRNA-122 was significantly upregulated in HIV/HBV co-infection patients compared with other groups (P<0.001). Also, miRNA-199a and miRNA-let7 were significantly down-regulated in all patient groups compared to the healthy controls (P< 0.001). Correlation analysis demonstrated significant negative correlations between the expression level of miR- 149 and miR-199a with HIV viral load (P<0.001) and HBV viral load, respectively (P<0.01).

 This preliminary study could reflect a limited perspective on the field of the miRNAs in HBV and HIV infection. The study suggested further investigation to understand better the role of the miRNA in HIV and HBV infections.

 Cutaneous leishmaniasis is a significant public health issue worldwide. Cutaneous leishmaniasis is the most prevalent in the world among the different types of leishmaniasis. Currently, available medications have had no discernible influence on the disease's progression. Up to now, there has been no approved cutaneous leishmaniasis vaccine. New developments in vaccination might be a potential way to come up with a vaccination that is successful for the treatment of cutaneous leishmaniasis.

 This research was conducted to learn more about an effective vaccine for Leishmania major, the ailment's primary cause of CL, which was designed using computational methods. Thus, a multiepitope protein was designed by utilizing potential immune system epitopes, including predicted MHC class I, MHC class II, Cytotoxic T lymphocytes, B-cell, and Interferon-gamma epitopes of Cysteine protease b (CPB), Leishmania homologue of activated C kinase (LACK), and Kinetoplastid membrane protein-11 (KMP-11) antigenic proteins. In order to enhance vaccine immunogenicity, two resuscitation-promoting factors of Mycobacterium tuberculosis were used as adjuvants. Final epitopes were matched with suitable linkers to construct the recombinant structure. The physicochemical and immune-based characteristics of the designed vaccine have been forecasted by using different tools. Moreover, homogeneity modeling was performed to obtain a high-quality 3D structure, followed by refinement and validation. Finally, the codon optimization based on E. coli resulted in a higher CAI value and optimal GC content, followed by combining it in the pET-14b cloning vector.

Evaluation of the various characteristics of the designed vaccine showed that it is an immunogenic and non-allergenic antigen that can induce immune responses against Leishmania major infection, which could be promising for cutaneous leishmaniasis.

 Research shows that a recombinant vaccine can be an effective candidate against cutaneous leishmaniasis.

 Hospital-acquired infections are considered a major health concern worldwide because of the increasing morbidity and mortality rate. Hence, selecting the most efficient disinfectants in a clinic is crucial. This study evaluated the antibacterial efficacy of seven mostly used disinfectants against clinically isolated Escherichia coli.

 For this purpose, minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of Aniosyme DD1 0.5%, Steranios 2%, Aniospray 29 and Surfanios 0.5% at Hazrat Valiasr hospital and PROSEPT® Floor 0.75%, PROSEPT® Instru 0.05% and PROSEPT® Med used at Ayatollah Mousavi hospital were determined both in the presence and absence of bovine serum albumin (BSA) using microbroth dilution assay.

The results indicate that PROSEPT® Med had the lowest MIC and MBC values, followed by Aniospray 29 and PROSEPT® Instro, and the presence of BSA reduced antibacterial activities of disinfectants.

 The disinfectants applied at Ayatollah Mousavi hospital generally had higher antibacterial activities. Due to the importance of nosocomial pathogens at healthcare centers, selecting the most potent, fast-acting, and efficient disinfectants to prevent hospital-acquired infections is essential.

 Bartonella quintana is an aerobic, gram-negative, rod-shaped, and polar bacterium. Detection of this bacterium is done through blood culture in an agar medium, and the longtime of detection by culture has made molecular methods such as PCR important for more accurate and faster detection.

 For this reason, 100 cultured negative endocarditis specimens were collected in this study. DNA extraction was performed from B. quintana, and the concentration and quality of the obtained DNA were measured. PCR reaction was performed on the genome of negative control samples. To clone a portion of the amplified gene in PUC 18 plasmid, the PCR product was first purified. After ligation, JM107 E. coli susceptible to calcium chloride was used. Transformed bacteria were cultured on LB Broth medium containing Ampicillin antibiotic. Then 2 to 3 white colonies were selected, and PCR was performed. Plasmid extraction was performed after confirming the presence of recombinant and inserted plasmids.

The last dilution of PUC18 plasmid for B. quintana with an initial concentration of 780 ng/µL, which formed a detectable band on the gel, was calculated to be 10-7, and the minimum number of detectable copies in a 25 μL PCR reaction equal to 24 copies. . In quantitative DNA analysis, its amount was calculated between 1.69 and 1.8.

 The collected samples were then examined for the presence of B. quintana in patients. Of the 60 samples collected, none were positive.

 Urinary tract infections (UTIs) that influence millions of humans yearly are still among the most common infections in the community and hospitals. This study aimed to investigate the prevalence of UTIs and their antibiotic resistance susceptibility in patients in Qazvin province, northwest Iran, from 2017 to 2019.

 This study was conducted on 3521 urine samples of patients referred to a non-hospital medical laboratory (Mehr) between April 2017 and January 2019. Samples were collected and processed immediately for laboratory analysis. Biochemical tests were conducted the bacteria identification, and the antibiotic susceptibility test of the bacterial isolates was determined using Kirby-Bauer disc diffusion.

347 of the 3521 urine samples had significant bacteriuria, with a prevalence of 9.9 %. Escherichia coli occurred more frequently (54.4 %), while Pseudomonas aeruginosa had the lowest frequency of occurrence (1.4 %) in the samples. Nitrofurantoin and Amikacin were the most effective antibiotics against gram-negative and positive, respectively. This study revealed that bacterial resistance in UTIs continues to be a great problem and needs drug resistance surveillance periodically.

 Vancomycin resistant Enterococci (VRE) and high level aminoglycoside resistant (HLAR) Enterococci have complicated the available treatment modalities for Enterococci worldwide. The existing study was planned to evaluate the occurrence of HLAR and VRE strains in a tertiary care center in India and to study the association of HLAR with vancomycin sensitive Enterococci (VSE) and VRE.

 A total of 50 enterococcal isolates from various clinical specimens were incorporated in the study. Speciation was done on the basis of standard biochemical tests. HLAR was tested by the disc diffusion method using 150µg gentamicin disc and 200 µg streptomycin discs. Vancomycin susceptibility patterns were reported using vancomycin disc and agar dilution methods.

Pus samples comprised of the majority for the isolation of enterococcal strains (40%). 54% isolated strains were HLGR, 32% were HLSR and 14% isolates were positive for both HLGR and HLSR. 61.7% of Enterococcus faecium isolates demonstrated resistance for high-level gentamicin (HLGR) and 43.75% Enterococcus faecalis isolates were resistant to high-level streptomycin (HLSR). When VRE was compared to VSE, the rate of HLSR was detected to be 4.64% in VRE, while it was 32.55% in VSE; the rate of HLGR was noted to be 11.62% in VRE and it was 41.87% in VSE. The association of HLGR with HLSR (HLAR) was 2.32% in VRE and 13.95% in VSE strains. Enterococci strains are showing an increase in their antimicrobial resistance patterns. The increment of such strains in health care settings has to be reserved and controlled to avert complicated infections.

 Brucellosis is a zoonotic disease which has become endemic in Iran. Contaminated milk with Brucella bacteria is the main way of transmission of this disease in humans. Therefore, the aim of this study was to investigate the prevalence of Brucella spp. and B. abortus in raw milk samples collected from farms belongs to six geographical areas of Lorestan province.

 In the present study, 100 raw milk samples of cows which were less than 4 years, 4 to 6 years and over 6 years old were randomly collected. The isolates were identified by PCR method using specific bcsp31 and IS711 primers for Brucella spp. and B. abortus, respectively.

The results showed that 26 of the samples were infected with Brucella bacteria, of which 19 samples (73%) were B. abortus. Most of the brucellosis infection in cows belonged to cows less than 4 years (31.4%) and 4 to 6 years (31.4%) categories, 11 samples in both groups, so that in cows older than 6 years this rate was 13% (4 samples). The east of the province with 12 samples and the northwest of the province with one sample showed the highest and lowest levels of Brucella infection, respectively. The findings demonstrated that there is a significant difference between the frequency of Brucella contamination in milk in the eastern regions of Lorestan province with other regions (p<0.05). It should be noted that the possibility of transmitting brucellosis to humans through consumption of contaminated raw milk and dairy products in Lorestan province is high.

For the first, Omicron variants (B. 1.1. 529) of SARS-CoV-2 emerged in Botswana and South Africa which probably compromise vaccine effectiveness and the protective capability of antibodies released via infection of former variants. Public concerns raised due to abundant mutations in the spike protein and elsewhere on Omicron variant resulting in escape from vaccine elicited immunity. Less than three month ago, the Iranian national media reported that the Omicron variant had been identified in a patient who had traveled to the UAE. This news caused a huge amount of concern for the people and even the medical staff. After the Delta variant caused widespread deaths and large numbers of hospitalized patients to the point where hospitals were unable to accept new patients and the health system was paralyzed, it is now the turn of the Omicron variant to bring many patients to the brink of death. It is presented that social stressors are linked to enhance inflammatory cytokines, which are responsible for the production of central nervous system signals for neurological and behavioral changes along with psychiatric symptoms. Therefore, inflammatory cytokines have been reported to be key factors in psychological disorder. In this way, in this article, we offer suggestions to help people in this critical situation where people are mentally disturbed and the virus is spreading.

Severe acute respiratory syndrome coronavirus-2 (SARS‑CoV‑2) is a strain of coronavirus that causes COVID-19 that was first identified in Wuhan of, China, on 2019 (Coronaviridae Study Group of the International Committee on Taxonomy of Viruses, 2020). Omicron variant is one of the SARS-CoV-2 variants that was first informed to WHO (World Health Organization) via South Africa (Network for Genomics Surveillance) on 24 November 2021 (Quarleri et al., 2020). The omicron was first detected in Botswana, and then the variant has spread to become omicron's predominant variant in circulation worldwide (Gowrisankar et al., 2022). Behind the original BA.1 variant (the first dominant omicron variant), several subvariants of omicron, such as BA.2, BA.3, BA.4, and BA.5 (Yao et al., 2022). Omicron subvariants BA.4 and BA.5 were classified as V-22APR-03 and V-22APR-04 by the VTG. BA.4 was designated based on potentially biologically significant mutations in spike. The BA.4 mutations are NSP4: L438 (WT, wild type); S: 69/70 deletion, L452R, F486V, Q493 (WT); ORF 7b: L11F; N: P151S and the S gene 69/70 deletion. The BA.5 has similar mutations/deletions with BA.4 except M: D3N; ORF 6: D61 (WT); ORF 7b: L11 (WT); N: P151 (WT); synonymous SNPs: A27038G, and C27889T (UK health security agency, 2022). Some people believe that the currently used vaccines are ineffective against new subvariants (Ba.4 and BA.5), which is why they do not get vaccinated. This study aimed to investigate the Ba.4 and BA.5 mutations by Bioinformatic analysis.Since some currently used vaccines target the RBD (Receptor Binding Domain) of the S protein of SARS-CoV-2, in this research, the mutations of RBD have been investigated. The results obtained from the IEDB analyzer showed that there are a minimum of five highest score B cell epitopes that remained unchanged (Figure 1). T-Cell epitopes analysis indicated that there are still a lot of unchanged (lots of HLA-A, HLA-B, and HLA-C) and common epitopes (for different MHCI) between the original variant (Wuhan) and BA. 4/5 variants. The result of our analysis indicates that the currently used vaccine can stimulate humoral and cellular immunity and the vaccines help protect against severe illness, hospitalization, and death.