Comparison of toxin gene expression levels and molecular typing of Clostridioides difficile strains isolated from patients with diarrhea.

Aim: This study aimed to evaluate the expression of tcdA, tcdB, and binary toxin genes (cdtA and cdtB) by Real-Time PCR and molecular typing of Clostridioides difficile isolated from patient diarrhea samples from Hamadan Hospitals, west of Iran. Background: The concentration of C. difficile toxins (CDTs) is associated with the severity of the disease and the mortality rate. Measuring CDT levels could provide a reliable and objective means of determining the severity of C. difficile infection (CDI). Methods: From November 2018 to September 2019, 130 diarrhea samples were collected from hospitalized patients in three hospitals in Hamadan. C. difficle isolates were detected by culture and PCR. The presence of the genes encoding the toxin was identified by PCR, whereas the measurement of toxin expression was conducted using a relative Real-Time PCR technique. Genetic linkage of the isolates was also assessed by Ribotyping and Repetitive Extragenic Palindromic (rep-PCR) methods. Results: Among 130 diarrhea samples, 16 (12.3%) were positive for C. difficile. Genes encoding cdtA and tcdB were detected in all isolates, and 8 (50%) and 6 (37.5%) isolates were positive for the cdtA and cdtB genes. Real-time PCR results showed different expression levels of the toxin genes. A significant increase in the expression of the tcdA gene was observed compared with the control strain (P<0.05). Besides, more expression of cdtA gene was observed in the strains compared with cdtB gene. Ribotyping and rep-PCR results showed high genetic diversity of C. difficile among hospitals investigated. Conclusion: We encountered toxigenic C. difficile strains with various toxin expression levels, ribotypes, and rep types based on the findings of this study. This indicated that various clones from various sources circulate in the hospitals and among patients.

Effect of Mentha longifolia essential oil on oqxA efflux pump gene expression and biofilm formation in ciprofloxacin-resistant Klebsiella pneumoniae strains.

Background and Objectives: Today, medicinal plants and their derivatives are considered to reduce the prevalence of antibiotic resistance. The aim of this study was to investigate the effect of Mentha longifolia essential oil on oqxA efflux pump gene expression and biofilm formation in ciprofloxacin-resistant Klebsiella pneumoniae strains. Materials and Methods: A total of 50 clinical strains of K. pneumoniae resistant to ciprofloxacin were studied. The minimum inhibitory concentration (MIC) of M. longifolia essential oil and its synergistic effect with ciprofloxacin were determined using the microbroth dilution method and the fractional inhibitory concentration (FIC) method. Minimum biofilm inhibition concentration (MBIC) of M. longifolia essential oil was detected. The effect of essential oils on the expression level of the oqxA gene was detected by Real-time PCR. Results: M. longifolia essential oil showed inhibitory activity against ciprofloxacin-resistant strains of K. pneumoniae. When M. longifolia essential oil was combined with ciprofloxacin, the MIC was reduced 2-4 times. In 28% of the strains, M. longifolia with ciprofloxacin showed a synergistic effect. M. longifolia essential oil reduces the strength of biofilm formation and alters the biofilm phenotype. A significant decrease in oqxA gene expression was observed in all isolates after treatment with M. longifolia essential oil. Conclusion: Based on the results of this study, it was observed that supplementing M. longifolia essential oil can help reduce ciprofloxacin resistance and inhibit biofilm formation in fluoroquinolone-resistant K. pneumoniae strains.

Novel antimicrobial applications of copper oxide nanoparticles after combination with tissue conditioner used in complete prostheses.

BACKGROUND: Tissue conditioners are used for treating and improving the tissues supporting complete dentures. On the other hand, recent advances in nanotechnology have revolutionized various fields of science, including dentistry. The present study aimed to investigate novel antimicrobial applications of copper oxide nanoparticle-based tissue conditioner used in complete prostheses. METHODS: The present experimental study included 126 tissue conditioner samples with different concentrations of copper oxide nanoparticles (20%, 10%, 5%, 2.5%, 1.25%, 0.625%, and 0% w/w). The samples were incubated with Enterococcus faecalis, Pseudomonas aeruginosa, and Candida albicans in 24-well plates for 24 h. Then, samples from the wells were re-incubated for 24 h, and the microorganisms were counted. RESULTS: The culture media containing E. faecalis and P. aeruginosa showed significantly different growth between different nanoparticle concentrations following 24 h (P < 0.001), showing a reduction in bacterial growth with increased nanoparticle concentration. Both bacteria did not show any growth at the 20% concentration. However, C. albicans showed significant differences in growth between different nanoparticle concentrations following 48 h (P < 0.001), showing a reduction in growth with increased nanoparticle concentration. Also, the least growth was observed at the 20% concentration. CONCLUSIONS: In conclusion, the CuO nanoparticles were prepared using a green synthesis methon in the suitable sizes. Moreover, the tissue conditioners containing CuO nanoparticles showed acceptable antimicrobial properties against E. faecalis, P. aeruginosa, and C. albicans.

Characterization of recombinant phytase of Klebsiella sp. and the influence of novel 3-phytase on mineral solubility in broiler diets under an in vitro digestion assay.

Phytate (inositol hexaphosphate) is the major storage form of phosphorus (P) in nature, and phytases catalyze the hydrolysis of P from phytate and the formation of inositol phosphate isomers. In this study, a bacterium that produces phytase was isolated in a phytase screening medium. The bacterium was identified as Klebsiella sp. using phenotypic and molecular techniques. The PhyK phytase gene was successfully amplified from the genome, inserted into the pET-21a (+) vector, and expressed as a recombinant protein in E. Coli BL21. The efficiency of a laboratory phytase (Lab-Ph, PhyK phytase) was determined and compared with a commercial phytase (Com-Ph, Quantum Blue 40P phytase, AB Vista) under an in vitro digestion assay. The native signal peptide effectively facilitated the translocation of the protein to the periplasmic space of E. Coli BL21, resulting in the proper folding of the protein and the manifestation of desirable enzyme activity. The Lab-Ph displayed the temperature and pH optima at 50 °C and 5 respectively. In addition, the Lab-Ph was inactivated at 80 °C. Under an in vitro digestion assay condition, Lab-Ph improved the P solubility coefficient in broiler diets. In comparison, the Com-Ph significantly increased the P solubility coefficient even when compared with the Lab-Ph. In summary, this study has shown that Lab-Ph possesses the necessary biochemical properties to be used in various industrial applications. However, Lab-Ph is extremely sensitive to heat treatment. The Lab-Ph and Com-Ph under an in vitro digestion assay improved the solubility coefficient of P in the broiler diet.

Staphopain mediated virulence and antibiotic resistance alteration in co-infection of Staphylococcus aureus and Pseudomonas aeruginosa: an animal model.

Polymicrobial communities lead to worsen the wound infections, due to mixed biofilms, increased antibiotic resistance, and altered virulence production. Promising approaches, including enzymes, may overcome the complicated condition of polymicrobial infections. Therefore, this study aimed to investigate Staphopain A-mediated virulence and resistance alteration in an animal model of Staphylococcus aureus and Pseudomonas aeruginosa co-infection. S. aureus and P. aeruginosa were co-cultured on the L-929 cell line and wound infection in an animal model. Then, recombinant staphopain A was purified and used to treat mono- and co-infections. Following the treatment, changes in virulence factors and resistance were investigated through phenotypic methods and RT-PCR. Staphopain A resulted in a notable reduction in the viability of S. aureus and P. aeruginosa. The biofilm formed in the wound infection in both animal model and cell culture was disrupted remarkably. Moreover, the biofilm-encoding genes, quorum sensing regulating genes, and virulence factors (hemolysin and pyocyanin) controlled by QS were down-regulated in both microorganisms. Furthermore, the resistance to vancomycin and doripenem decreased following treatment with staphopain A. According to this study, staphopain A might promote wound healing and cure co-infection. It seems to be a promising agent to combine with antibiotics to overcome hard-to-cure infections.

The healing effect of Pseudomonas Quinolone Signal (PQS) with co-infection of Staphylococcus aureus and Pseudomonas aeruginosa: A preclinical animal co-infection model.

BACKGROUND: Because of the rise in antibiotic resistance and the control of pathogenicity, polymicrobial bacterial biofilms exacerbate wound infections. Since bacterial quorum sensing (QS) signals can dysregulate biofilm development, they are interesting therapeutic treatments. In this study, Pseudomonas Quinolone Signal (PQS) was used to treat an animal model of a wound that had both Staphylococcus aureus and Pseudomonas aeruginosa co-infection. METHODS: S. aureus and P. aeruginosa mono- and co-infection models were developed in vitro on the L-929 cell line and in an animal model of wound infection. Moreover, PQS was extracted and purified using liquid chromatography. Then, the mono- and co-infection models were treated by PQS in vitro and in vivo. RT-PCR analysis was used to look into changes in biofilm, QS, tissue regeneration, and apoptosis genes after the treatment. RESULTS: PQS significantly disrupted established biofilm up to 90% in both in vitro and in vivo models. Moreover, a 93% reduction in the viability of S. aureus and P. aeruginosa was detected during the 10 days of treatment in comparison to control groups. In addition, the biofilm-encoding and QS-regulating genes were down-regulated to 75% in both microorganisms. Also, fewer epithelial cells died when treated with PQS compared to control groups in both mono- and co-infection groups. CONCLUSION: According to this study, PQS may facilitate wound healing by stimulating the immune system and reducing apoptosis. It seems to be a potential medication to use in conjunction with antibiotics to treat infections that are difficult to treat.

Synergistic effects of nano curcumin mediated photodynamic inactivation and nano-silver@colistin against Pseudomonas aeruginosa biofilms.

BACKGROUND: Patients with burn injuries colonized by multidrug-resistant Pseudomonas aeruginosa face increased mortality risk. The efficacy of colistin, a last-resort treatment, is declining as resistance levels rise. P. aeruginosa's robust biofilm exacerbates antibiotic resistance. Photodynamic Inactivation (PDI) shows promise in fighting biofilm. MATERIALS AND METHODS: Nano curcumin (nCur) particles were synthesized, and their chemical characteristics were determined using zeta potential (ZP), dynamic light scattering analysis (DLS), energy-dispersive X-ray (EDX) analysis, and fourier transform infrared (FTIR). We conducted an MTT assay to assess the cytotoxicity of nCur-mediated PDI in combination with nanosilver colistin. The fractional biofilm inhibitory concentration (FBIC) of two P. aeruginosa clinical isolates and P. aeruginosa ATCC 27853 during nCur-mediated PDI@AgNPs@CL was determined using a 3-dimensional (3-D) checkerboard assay. To study the effect of nCur-mediated PDI@AgNPs@CL on lasI, lasR, rhlI, rhlR, pelA, and pslA gene expression, Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was conducted at each isolate's FBIC. The impact of treatments was also investigated using scanning electron microscopy (SEM). RESULTS: The ZP and mean DLS values of the nCur were 10.3 mV and 402.6 ± 24.6 nm, respectively. The distinct functional groups of nCur corresponded with the peaks of FTIR absorption. Moreover, the EDX analysis showed the ratios of different metals in nCur. Cell viability percentages of nCur-mediated PDI@AgNPs@CL at FBIC concentrations of clinical isolates Nos. 30, 354, and P. aeruginosa ATCC 27853 were 91.36 %, 83.20 %, and 92.48 %, respectively. nCur-mediated PDI@AgNPs@CL treatment showed synergistic effects in clinical isolates and P. aeruginosa ATCC 27853 in a 3-D checkerboard assay. All six of the investigated genes showed down-regulation after nCur-mediated PDI@AgNPs@CL treatment. The most suppressed gene during nCur-mediated PDI@AgNPs@CL treatment was the rhlR gene (-11.9-fold) of P. aeruginosa ATCC 27853. The SEM micrographs further proved the connecting cement reduction and biofilm mass mitigation following nCur-mediated PDI@AgNPs@CL treatments. CONCLUSIONS: The combined effect of nCur-mediated PDI and AgNPs@CL synergistically reduce the formation of biofilm in P. aeruginosa. This may be attributable to the suppression of the genes responsible for regulating the production of biofilms.

Circular RNAs in tuberculosis: From mechanism of action to potential diagnostic biomarker.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tuberculosis), continues to be a major global health concern. Understanding the molecular intricacies of TB pathogenesis is crucial for developing effective diagnostic and therapeutic approaches. Circular RNAs (circRNAs), a class of single-stranded RNA molecules characterized by covalently closed loops, have recently emerged as potential diagnostic biomarkers in various diseases. CircRNAs have been demonstrated to modulate the host's immunological responses against TB, specifically by reducing monocyte apoptosis, augmenting autophagy, and facilitating macrophage polarization. This review comprehensively explores the roles and mechanisms of circRNAs in TB pathogenesis. We also discuss the growing body of evidence supporting their utility as promising diagnostic biomarkers for TB. By bridging the gap between fundamental circRNA biology and TB diagnostics, this review offers insights into the exciting potential of circRNAs in combatting this infectious disease.

Approaches to determine the efficiency of novel 3-phytase from Klebsiella pneumoniae and commercial phytase in broilers from 1 to 14 d of age.

This study aimed to evaluate the effects of a laboratory 3-phytase (the expression of the phyK gene, Lab-Phy) and a commercial 6-phytase (Quantum Blue 40 P, Com-Phy) alone and in combination (corn-soy-based diets) in broilers. A total of 400, day-old Ross 308 male broilers were randomly assigned to 5 treatments with 10 replicate cages (8 chicks/cage) for a 14-day trial. Experimental treatments included the positive control (0.95% Ca and 0.48% nonphytate phosphorus (nPP), PC), negative control (0.90% Ca and 0.22% nPP, NC), and NC which was supplemented with Lab-Phy 250 FTU/kg and Com-Phy 250 FTU/kg alone or in combination of Lab-Phy 125 FTU/kg and Com-Phy 125 FTU/kg. The inclusion of Lab-Phy in the NC diet significantly improved the P and Ca content in the tibia compared to the NC group. Moreover, the inclusion of Com-Phy alone and in combination with Lab-Phy in the NC diet significantly increased the P and Ca content in the tibia compared to the Lab-Phy. The mRNA expression of NaPi-IIb was upregulated in the duodenum by the reduction of nPP and downregulated by the inclusion of any phytase, whereas other nutrient transporters were not influenced by the reduction of nPP or the addition of phytase in the small intestine mucosa. Broilers receiving the NC diet obtained the lowest body weight (BW) and body weight gain (BWG) at 8 to 14 and 1 to 14 d of age. The NC group showed the lowest villi height and surface area, Newcastle disease (ND) antibody titer, and digestibility of nutrients compared to the PC group at 14 d of age. Supplementing the NC diet with the Lab-Phy and Com-Phy individually, or in combination tended to improve BW, BWG, tibia characteristics, villi characteristics, ND, and retained CP and P, and apparent ileal digestibility of CP, P, methionine, and threonine. The present research indicated that the studied traits by the combination of phytases were slightly better than the average of the 2 individually, suggesting there might be some value in combining the laboratory and commercial phytases.

Bacterial Contamination of Collected Cockroaches and Determination Their Antibiotic Susceptibility in Khorramabad City, Iran.

Background: Cockroaches are one of the most important carriers of pathogenic microorganisms. Therefore, the presence of cockroaches in public places, especially in hospitals, homes, and restaurants, is dangerous, and threatens the health of society, people, and the environment. The aim of this study was evaluation of bacterial contamination of cockroaches and the sensitivity of these bacteria to various antibiotics, captured from Khorramabad City, Iran. Methods: This descriptive cross-sectional study was performed on 150 cockroaches collected from hospital environments, homes, and restaurants in Khorramabad. The outer surface of the cockroaches was washed with physiological saline. The suspension was centrifuged for 5 minutes at 2000rpm. Isolation and identification of bacteria was performed using phenotypic methods. Antibiotic susceptibility testing was performed by disk diffusion method according to Clinical and Laboratory Standard Institute (CLSI) guideline. Results: A total of 100 American cockroaches (66.66%), 28 B. germanica (18.66%) and 22 Blatta orientalis (14.66%) were identified. In total, 97.33% of the collected cockroaches were infected with bacteria. The most bacterial infection of the cockroaches was Escherichia coli, coagulase-negative Staphylococci and Bacillus respectively. The overall results of the antibiogram test showed that the identified bacteria were resistant to cephalothin, ampicillin, cefotaxime, and kanamycin antibiotics, semi-sensitive to ciprofloxacin and sensitive to tetracycline, gentamicin, nitrofurantoin, Trimethoprim/sulfamethoxazole, and Chloramphenicol. Conclusion: Infection of cockroaches with pathogenic bacterial agents in hospital, residential, and restaurant environments, as well as the observation of bacterial resistance to some common antibiotics is worrying.

Relationship between antibiotic resistance with class 1 integron and SmeDEF efflux pump encoding genes in clinical isolates of Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is an emerging multidrug-resistant organism with an increasing frequency of hospital-acquired infections predominantly in developing countries. The purpose of this study was to determine the antibiotic resistance and frequency of the smeD, class 1 integron, and sul1 genes in clinical isolates of S. maltophilia in two Iranian provinces. From January 2020 to September 2021, 38 clinical isolates of S. maltophilia were collected from patients in hospitals in Tabriz and Sanandaj provinces of Iran. S. maltophilia isolates were confirmed by standard bacteriological tests and 16S rRNA gene PCR. Disk diffusion and the MIC test strip methods were used to determine the antibiotic resistance patterns. PCR was performed to investigate the presence of smeD, class 1 integron, and sul1 genes. The antimicrobial test for the isolated S. maltophilia showed a high level of sensitivity against most of the antibiotics used. Maximum sensitivity was recorded for ciprofloxacin (100% (38/38)) and levofloxacin 100% (38/38), followed by ceftazidime (97.36% (37/38)), trimethoprim-sulfamethoxazole (81.57% (31/38)), ticarcillin-clavulanate (60.52% (23/38)), and piperacillin-tazobactam (55.26% (21/38)). We observed a high prevalence of smeD (100% (38/38)) and class 1 integron (94.73% (36/38)) genes in the isolates, and none of the isolates carried the sul1 gene. The findings from this study indicate that resistance to trimethoprim-sulfamethoxazole was not observed, and still, trimethoprim-sulfamethoxazole is the best drug with desirable antimicrobial effect in the treatment of nosocomial infections caused by S. maltophilia strains. Despite the observation of a high number of class 1 integron, the sul1 gene was not observed, which indicates the role of this gene in high-level trimethoprim-sulfamethoxazole resistance and not having a role in low-level resistance. Based on our results, clinical microbiology laboratories need continuous surveillance of resistance rates to trimethoprim-sulfamethoxazole, because of the possibility of S. maltophilia acquiring trimethoprim-sulfamethoxazole-resistance by mobile gen elements.

Molecular Epidemiology of Stenotrophomonas maltophilia Strains Isolated from Bacteremia in Hospitalized Children.

Objectives: The aim of this study was to determine the phenotypic and genotypic characteristics of Stenotrophomonas maltophilia isolates obtained from blood culture samples of pediatric patients hospitalized in Borujerd and Hamadan hospitals in western Iran. Methods: Oxidase-negative isolates were collected from the blood cultures of pediatric patients. S. maltophilia isolates were identified and confirmed by routine microbiological and molecular testing. Antibiotic susceptibility of the isolates was determined. The phenotypic and genotypic biofilm-forming ability of the isolates were investigated. Molecular typing of all isolates was performed by repetitive element sequence-based polymerase chain reaction. Results: Out of 450 oxidase-negative bacilli, 72 (16.0%) were identified as S. maltophilia isolates. Biofilm assay results showed strong biofilm formation in 19 (26.4%) isolates, moderate in 38 (52.8%), weak in 10 (13.9%), and no biofilm formation in five (6.9%) isolates. Biofilm-associated genes rmlA, rpfF, and spgM were detected respectively in 59 (81.9%), 54 (75.0%), and 72 (100%) of isolates. Antimicrobial susceptibility testing showed that 67 (93.1%) isolates were sensitive to trimethoprim-sulfamethoxazole. All isolates were sensitive to levofloxacin and resistant to ceftazidime. The S. maltophilia isolates were grouped into 14 different types of repetitive sequence by repetitive element sequence-based polymerase chain reaction analysis. Conclusions: The results of this study indicate that S. maltophilia should be considered an important opportunistic pathogen in pediatric units. Different genotypes of S. maltophilia with the ability to form a biofilm (an important virulence factor) were circulating in the hospitals investigated. Levofloxacin and trimethoprim-sulfamethoxazole are recommended to treat S. maltophilia infections.

Enhanced antibacterial activity of cadmium telluride nanocrystals in combination with 940-nm laser diode on anaerobic bacteria P. gingivalis: an in vitro study.

Periodontal disease is one of the most common chronic diseases in the oral cavity that causes tooth loss. Root scaling and leveling cannot eliminate all periodontal pathogens, and the use of antibacterial agents or lasers can increase the efficiency of mechanical methods. The aim of this study was to evaluate and compare the antibacterial activity of cadmium telluride nanocrystals in combination with 940-nm laser diode. Cadmium telluride nanocrystals were prepared by a green route of synthesis in aqueous medium. The results of this study showed that cadmium telluride nanocrystals significantly inhibit the growth of P. gingivalis. The antibacterial property of this nanocrystal increases with increasing its concentration, laser diode 940-nm irradiation and with increasing the time. It was shown that the antibacterial activity of combination of 940-nm laser diode and cadmium telluride nanocrystals is greater than the effect of either alone and can have a similar effect with its long-term presence of microorganisms. This is very important because it is not possible to use these nanocrystals in the mouth and in the periodontal bag for a long time.

Antimicrobial photodynamic therapy: modern technology in the treatment of wound infections in patients with burns.

BACKGROUND: Staphylococcus aureus is one of the most frequently isolated microorganisms from burn wounds. Antimicrobial photodynamic therapy (aPDT) is a new strategy that may improve antimicrobial treatment. METHOD: This study evaluated three meticillin-resistant Staphylococcus aureus (MRSA) and three meticillin-sensitive Staphylococcus aureus (MSSA) clinical isolates, which produced a biofilm with 0.1mg/ml Toluidine Blue O (TBO) (Sigma-Aldrich, Germany) with an energy density of 45J/cm2 and 90J/cm2, for MRSA and MSSA, respectively. The antibiofilm potential of aPDT with TBO was analysed using crystal violet assays and scanning electron microscopy. RESULTS: TBO-aPDT significantly degraded the biofilm formed by MRSA and MSSA clinical isolates (p<0.05). CONCLUSION: Our results indicated that aPDT is an effective approach to combat bacterial biofilms associated with burn wound infection. aPDT could provide a supplemental to the treatment of wound and tissue infection, and patients with burns may benefit from combined treatments.

Investigation of antimicrobial resistance patterns and molecular typing of Pseudomonas aeruginosa isolates among Coronavirus disease-19 patients.

BACKGROUND: Pseudomonas aeruginosa is a common co-infecting pathogen recognized among COVID-19 patients. We aimed to investigate the antimicrobial resistance patterns and molecular typing of Pseudomonas aeruginosa isolates among Coronavirus disease-19 patients. METHODS: Between December 2020 and July 2021, 15 Pseudomonas aeruginosa were isolated from COVID-19 patients in the intensive care unit at Sina Hospital in Hamadan, west of Iran. The antimicrobial resistance of the isolates was determined by disk diffusion and broth microdilution methods. The double-disk synergy method, Modified Hodge test, and polymerase chain reaction were utilized to detect Pseudomonas aeruginosa extended spectrum beta-lactamase and carbapenemase producers. Microtiter plate assay was performed to evaluate the biofilm formation ability of the isolates. The isolates phylogenetic relatedness was revealed using the multilocus variable-number tandem-repeat analysis method. RESULTS: The results showed Pseudomonas aeruginosa isolates had the most elevated resistance to imipenem (93.3%), trimethoprim-sulfamethoxazole (93.3%), ceftriaxone (80%), ceftazidime (80%), gentamicin (60%), levofloxacin (60%), ciprofloxacin (60%), and cefepime (60%). In the broth microdilution method, 100%, 100%, 20%, and 13.3% of isolates showed resistance to imipenem, meropenem, polymyxin B, and colistin, respectively. Ten (66.6%) isolates were identified as multiple drug resistance. Carbapenemase enzymes and extended spectrum beta-lactamases were identified in 66.6% and 20% of the isolates, respectively and the biofilm formation was detected in 100% of the isolates. The blaOXA-48, blaTEM, blaIMP, blaSPM, blaPER, blaVEB, blaNDM, blaSHV, and blaCTX-M genes were detected in 100%, 86.6%, 86.6%, 40%, 20%, 20%, 13.3%, 6.6%, and 6.6% of the isolates, respectively. The blaVIM, blaGIM, blaGES, and blaMCR-1 genes were not identified in any of the isolates. The MLVA typing technique showed 11 types and seven main clusters and most isolates belong to cluster I, V and VII. CONCLUSION: Due to the high rate of antimicrobial resistance, as well as the genetic diversity of Pseudomonas aeruginosa isolates from COVID-19 patients, it is indispensable to monitor the antimicrobial resistance pattern and epidemiology of the isolates on a regular basis.

Polymorphisms in vitamin D receptor genes and its relation with susceptibility to brucellosis: a case-control study.

OBJECTIVE: One of the systemic infections is Brucellosis which is caused by facultative intracellular bacteria of the genus Brucella. Vitamin D is a fat-soluble prohormone, that metabolizes enzymes and its intracellular receptor creates the active hormone and also mediate in responses of immune system. METHODS: Current research consists of 102 patients with brucellosis who were selected based on culture, PCR results serology, and clinical symptoms. The control group composed of 102 healthy people. The polymorphism of genes (Bsm I, Fok I, Taq I, Apa I) encoding Vitamin D receptor (VDR) were assessed by the PCR-RFLP method. RESULTS: The results showed that ff, tt, aa, and bb genotypes in Fok I, ApaI, TaqI, and BsmI were significant in case/control groups (P-value ≤ 0.0001). The genotype frequency AA in the control group is higher than that of the study group, while genotype frequency aa in the study group is more than the control. The odds ratio for brucellosis in individuals with ff genotype is 37 times higher than that of Ff genotype. Also, the odds ratio of brucellosis in individuals with genotype tt, aa, and bb was 12, 53, and 6 times higher than those of the Aa, Bb, and Tt genotypes. CONCLUSION: The genotypes aa and ff in the positions of the ApaI and FokI are of higher importance. The brucellosis risk in individuals accompanied aa genotype at Apa I is 53 times higher than that of the genotype AA, in other words, AA and BB, TT and FF genotypes are protective against the disease.

The issue beyond resistance: Methicillin-resistant Staphylococcus epidermidis biofilm formation is induced by subinhibitory concentrations of cloxacillin, cefazolin, and clindamycin.

Staphylococcus epidermis is one of the most frequent causes of device-associated infections due to biofilm formation. Current reports noted that subinhibitory concentrations of antibiotics induce biofilm production in some bacteria. Accordingly, we evaluated the effect of exposure of different subinhibitory concentrations of cloxacillin, cefazolin, clindamycin, and vancomycin on the biofilm formation of methicillin-resistant S. epidermidis (MRSE). Antimicrobial susceptibility testing and minimum inhibitory/bactericidal concentration of antimicrobial agents were determined. MRSE isolates were selected, and their biofilm formation ability was evaluated. The effect of subinhibitory concentrations of cloxacillin, cefazolin, clindamycin, and vancomycin, antibiotics selected among common choices in the clinic, on MRSE biofilm formation was determined by the microtitre method. Besides, the effect of subinhibitory concentrations of cloxacillin, cefazolin, clindamycin, and vancomycin on the expression of the biofilm-associated genes icaA and atlE was evaluated by Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR). Antimicrobial susceptibility patterns of MRSE strains showed a high level of resistance as follows: 80%, 53.3%, 33.3%, 33.3%, and 26.6%, for erythromycin, trimethoprim-sulfamethoxazole, tetracycline, clindamycin, and gentamicin, respectively. Besides, 73.3% of S. epidermidis strains were Multidrug-resistant (MDR). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were in the range of 0.5 to512 µg/mL and 1 to1024 µg/mL for cloxacillin, 0.125 to256 µg/mL and 1 to512 µg/mL for cefazolin, 0.125 to64 µg/mL and 4 to>1024 µg/mL for clindamycin, and 2 to32 µg/mL and 4 to32 µg/mL for vancomycin, respectively. The findings showed that subinhibitory concentrations of cloxacillin, cefazolin, and clindamycin induce biofilm production in MRSE strains. In particular, the OD values of strains were in the range of 0.09-0.95, 0.05-0.86, and 0.06-1 toward cloxacillin, cefazolin, and clindamycin, respectively. On the other hand, exposure to subinhibitory vancomycin concentrations did not increase the biofilm formation in MRSE strains. The findings also demonstrated that sub-MIC of antibiotics up-regulated biofilm-associated genes. In particular, atlE and icaA were up-regulated 0.062 to 1.16 and 0.078 to 1.48 folds, respectively, for cloxacillin, 0.11 to 0.8, and 0.1 to 1.3 folds for cefazolin, 0.18 to 0.98, and 0.19 to 1.4 folds, respectively, for clindamycin. In contrast, the results showed that sub-MIC of vancomycin did not increase the biofilm-associated genes. These findings overall show that exposure to sub-MIC of traditional antibiotics can cause biofilm induction in MRSE, thereby increasing the survival and persistence on various surfaces that worsen the condition of comorbid infections.

Optimization and development of high-resolution melting curve analysis (HRMA) assay for detection of New Delhi metallo-ß-lactamase (NDM) producing Pseudomonas aeruginosa.

New Delhi metallo-ß-lactamase-1 (NDM-1) producing Pseudomonas aeruginosa strain detection plays a vital role in confirming bacterial disease diagnosis and following the source of an outbreak for public health. However, the standard method for NDM-1 determination, which relies on the features of the colony of the bacteria cultured from the patient's specimen, is time-consuming and lacks accuracy and sensitivity. This study aimed to standardize a high-resolution melting curve analysis (HRMA) assay to detect NDM producing P. aeruginosa. For optimization and development of the HRMA method, a reference strain of P. aeruginosa was used. For evaluating the broad range PCR data, ABI Step One-Plus Manager Software version 3.2 and Precision Melt Analysis Software 3.02 (Applied Biosystems) were used. Based on the results, expected results were obtained for all tested strains, with high analytical sensitivity and specificity. Temperature melting analyses of the HRMA time PCR assays showed the Tm at 89.57 °C, 76.92 °C and 82.97 °C for N-1, N-2 and N-3 genes, respectively. Also, melting point temperatures of the bla VIM, bla SPM and bla SIM amplicons for isolates identified as MBL strains were 84.56 °C, 85.35 °C and 86.62 °C, respectively. The amplification results using negative control genomes as templates were negative, showing the specificity of the designed assays. Our study's data indicated that the sensitivity and specificity of the HRMA method are linked to the primer length and the fluorescent dye. We can further identify antibiotic resistance in NDMproducing P. aeruginosa by software analysis and melting curve analysis.

Highly Synergistic Effects of Melittin With Vancomycin and Rifampin Against Vancomycin and Rifampin Resistant Staphylococcus epidermidis.

Methicillin-resistant Staphylococcus epidermidis (MRSE) strains are increasingly emerging as serious pathogens because they can be resistant to many antibiotics called multidrug resistance (MDR) that limit the therapeutic options. In the case of vancomycin- and rifampin-resistant MDR-MRSE, the physicians are not allowed to increase the doses of antibiotics because of severe toxicity. Accordingly, we investigated the synergistic activity of melittin antimicrobial peptide with vancomycin and rifampin against vancomycin-resistant, and rifampin-resistant MDR-MRSE isolates. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), fractional inhibitory concentration index (FICi), and fractional bactericidal concentration index (FBCi) of antimicrobial agents against isolates were determined. Coagulate activities and serum and salt stability as well as melittin cytotoxicity on the human embryonic kidney (HEK) 293 cells and human red blood cells (RBCs) at their synergistic concentrations. MIC and MBC values for melittin were in the range of 0.312-2.5 and 0.312-5, respectively. Results also showed that the interaction of melittin with drugs was highly synergistic in which the geometric means of FICi and FBCi were < 0.5. Induced synergism led to a decrease in melittin, rifampin, and vancomycin concentrations by 8-1,020, 2-16, and 4-16-folds, respectively. This phenomenon caused a reduction in melittin toxicity by which the synergistic concentration of melittin needed to kill bacteria did not show cytotoxicity and hemolytic activity. Besides, no coagulation activity was found for the synergistic and alone concentrations of melittin in both Prothrombin Time (PT) and Partial Thromboplastin Time (PTT). Interestingly, the antibacterial activity of melittin in Mueller Hinton Broth (MHB) containing human serum did no significant differences between MIC and MBC values of melittin in MHB and MHB containing 10% human serum. The present findings showed that the therapeutic index of melittin was improved by 32.08- and 12.82-folds when combined with vancomycin and rifampin, respectively. Taken together, the obtained data show that melittin alone was effective against MDR-MRSE isolates and this antimicrobial peptide showed highly synergistic effects with vancomycin and rifampin without causing toxicity. Therefore, the combination of melittin and traditional antibiotics could be a promising strategy for the treatment of infections caused by MDR-MRSE.

Molecular Detection of blaOXA -type Carbapenemase Genes and Antimicrobial Resistance Patterns among Clinical Isolates of Acinetobacter baumannii.

Acinetobacter baumannii is a bacterium found in most places, especially in clinics and hospitals, and an important agent of nosocomial infections. The presence of class D enzymes such as OXA-type carbapenemases in A. baumannii is proven to have a key function in resistance to carbapenem. The aim of the current study is to determine the blaOXA -type carbapenemase genes and antimicrobial resistance among clinically isolated samples of A. baumannii. We assessed 100 clinically isolated specimens of A. baumannii from patients in intensive care units of educational hospitals of Hamadan, West of Iran. The A. baumannii isolates' susceptibility to antibiotics was performed employing disk diffusion method. Multiplex polymerase chain reaction was used to identify the bla OXA-24-like , bla OXA-23-like , bla OXA-58-like , and bla OXA-51-like genes. The bla OXA-23-like , bla OXA-24-like , and bla OXA-58-like genes' prevalence were found to be 84, 58, and 3%, respectively. The highest coexistence of the genes was for bla OXA-51/23 (84%) followed by bla OXA-51/24-like (58%). The bla OXA-51/23- like pattern of genes is a sort of dominant gene in resistance in A. baumannii from Hamadan hospitals. The highest resistance to piperacillin (83%) and ciprofloxacin (81%) has been observed in positive isolates of bla OXA-23-like . The A. baumannii isolates with bla OXA-58-like genes did not show much resistance to antibiotics. Based on the results of the phylogenetic tree analysis, all isolates have shown a high degree of similarity. This study showed the high frequency of OXA -type carbapenemase genes among A. baumannii isolates from Hamadan hospitals, Iran. Thus, applying an appropriate strategy to limit the spreading of these strains and also performing new treatment regimens are necessary.