Metagenomic analysis reveals the dissemination mechanisms and risks of resistance genes in plateau lakes.

Antibiotic resistance genes (ARGs) are emerging as environmental pollutants that can persist and disseminate in aquatic environments. Lakes, as important sources of freshwater, also serve as potential natural reservoirs of ARGs. In this study, we analyzed the distribution and potential risks of resistance genes in five typical freshwater lakes on the Yunnan-Guizhou Plateau. Our findings revealed that multidrug and MLS ARGs dominated in the studied lakes. Notably, while Lugu Lake exhibited higher abundance of ARGs, mobile genetic elements (MGEs), and metal resistance genes (MRGs), a greater resistome risk was observed in the eutrophic Xingyun Lake. The dissemination processes of ARGs and MRGs are primarily driven by microbial communities and the horizontal gene transfer via MGEs. Limnohabitans, Flavobacterium, and Acinetobacter were identified as key players in the dissemination of ARGs. Our study highlights the persistence of ARGs and provides valuable baseline data and risk assessment of ARGs in plateau freshwater lakes.

New findings about comparing the effects of antibiotic therapy and phage therapy on memory and hippocampal pyramidal cells in rats.

BACKGROUND: Pseudomonas aeruginosa is a significant cause of infection in burn wounds. Antibiotics are widely used to treat infectious diseases, and alongside their therapeutic benefits, they can damage host cells. Significant side effects, such as nephrotoxicity and neurotoxicity, are observed in 60% of patients treated with colistin. Therefore, using a suitable alternative instead of antibiotics is paramount. This study aimed to investigate the effects of phage therapy and antibiotic therapy on memory function in rats with P. aeruginosa infected burn wounds. METHODS: Adult male rats were divided into three groups: (1) infected without treatment (control), (2) infected and treated with colistin antibiotic (3,000,000 international units/kg/day), and (3) infected and treated with 100 µL of phage suspension (approximately 109 PFU/mL). In all animals, after anesthesia, a third-degree burn was created in the back area. One hour later, treatment was performed for seven consecutive days. Passive avoidance test, novel object recognition test, locomotion activity, hippocampal neuron count, and oxidative stress measurement in blood serum were performed. RESULTS: In antibiotic-treated group memory recall, recognition index, number of healthy neurons in CA1, CA2, and CA3 hippocampus areas and the amounts of MDA, and FRAP significantly decreased compared with the control group. The phage-treated group was not shown any harmful effect on the memory process, number of healthy hippocampal neurons, and showed more positive effects in blood serum examinations compared with the antibiotic group. CONCLUSIONS: Phage therapy could be a safe and effective alternative to antibiotics in the treatment of burn-related infections.

Developing erythromycin resistance gene by heavy metals, Pb, Zn, and Co, in aquatic ecosystems.

Industrial development is the main cause of environmental pollution with various substances such as antibiotics and heavy metals. Many heavy metals with antimicrobial properties could contribute to antibiotic resistance and the emergence of antibiotic resistance genes due to the co-selection phenomenon. The aim of this study was to investigate the concurrent presence and correlation between several heavy metals and the erythromycin resistance genes in six aquatic ecosystems of Iran. Distribution and assessment of 11 erythromycin resistance genes were investigated using specific primers and online enrichment and triple-quadrupole LC-MS/MS. The concentration of heavy metals was measured using inductively coupled plasma atomic emission spectroscopy by Thermo electron corporation. Principal component analysis was performed to globally compare and to determine the similarities and differences among different aquatic ecosystems in different parts of the world in terms of the concentration of zinc and lead in their water. The results of the simple logistic regression analysis for the correlation between erythromycin resistance genes and heavy metals concentrations revealed the most significant correlation between erythromycin resistance genes and Pb concentration, followed by Co and Zn concentrations.

Detection of Staphylococcus aureus and their toxin genes inhabit on the scorpions surface.

The transmission of infectious agents by arthropods is of particular importance. Every year, many people are bitten by scorpions around the world. Staphylococcus aureus is of the most important infectious bacteria. This study aimed to investigate the distribution of S. aureus in scorpion specimens and the presence of some toxin genes in these species. The fauna of scorpions in the Kuhdasht region was studied for one year. Then, S. aureus was identified on the body surface of scorpions by biochemical and molecular methods, and the presence of Sea, Seb, Sec, Sed, See, Pvl, Tsst1, Eta, Etb, and mecA genes was examined by the PCR method. The pattern of antibiotic resistance was determined by the use disk diffusion method. MRSA isolates were identified using genotypic and phenotypic methods. Of 75 studied scorpion specimens, Hottentotta saulcyi was the most abundant species. Sixteen (21.3%) isolates of S. aureus were identified from all samples. The highest and lowest antibiotic resistance levels belonged to penicillin and clindamycin, respectively. MRSA was observed in 50% of the isolates. Thirteen out of 16 isolates possessed at least one of the toxin genes. Due to the presence of S. aureus on the body surface of scorpions, it should always be expected that an infection may occur after the bite. Moreover, the presence of toxin genes in the studied isolates showed that infection with these bacteria would seriously threaten one's health.

Involvement of caspase-3 in apoptosis of human lymphocytes exposed to cadmium chloride.

BACKGROUND: Lymphocytes are a group of white blood cells with a variety of roles their integrity is crucial for the body's immune responses. Cadmium, a heavy metal and environmental pollutant, is known as a toxicant to exert its adverse effects on some sort of cells including blood cells. RESEARCH DESIGN: In this study, human lymphocytes were divided into 3 groups: (1) lymphocytes at 0-h, (2) lymphocytes at 24 h (control), (3) lymphocytes treated with cadmium chloride (15 µM). Lymphocyte viability and plasma membrane integrity were assessed in these groups. In addition, the occurrence of apoptosis was investigated by assessment of nucleus diameter and flow cytometry. Activation of caspase-3 was also detected by immunocytochemistry. RESULTS: Result showed that lymphocyte's viability and plasma membrane integrity decreased in lymphocytes treated with cadmium as compared with the control group. Decreased nucleus diameter and result of flow cytometry demonstrated cadmium-induced apoptosis in human lymphocytes. Furthermore, lymphocytes treated with cadmium displayed intensely activated caspase-3 immunoreactivity in their cytoplasm. CONCLUSION: In conclusion, cadmium not only negatively effect on viability and plasma membrane, but also induces caspase-dependent apoptosis in human lymphocytes.

Isolation of a lytic bacteriophage against extensively drug-resistant Acinetobacter baumannii infections and its dramatic effect in rat model of burn infection.

OBJECTIVES: Acinetobacter Baumannii is an opportunistic nosocomial pathogen belonging to the Moraxellaceae family. The emergence of multidrug resistant strains of this pathogen caused many problems for hospitals and patients. The aim of the current study was to isolate, identify, and morphologically, physiologically, and in vivo analyze a new lytic bacteriophage targeting extensively drug-resistant (XDR) A. baumannii. MATERIALS AND METHODS: Different wastewater samples were tested for isolation of lytic bacteriophage against 19 A. baumannii isolates obtained from patients hospitalized in a hospital in Arak, Iran, from January 2019 to March 2019. The phenotypic and genotypic characteristics of A. baumannii strains (resistance genes including: adeA, adeB, adeC, adeR, adeS, ISAba1, blaOXA-23, blaOXA-24) were analyzed. The isolated phage characteristics including adsorption time, pH and thermal stability, host range, one-step growth rate, electron microscopy examination, and therapeutic efficacy of the phage were also investigated. Therapeutic efficacy of the phage was evaluated in a rat model with burn infection of XDR A. baumannii. The lesion image was taken on different days after burning and infection induction and was compared with phage untreated lesions. RESULTS: The results showed unique characteristics of the isolated phage (vB-AbauM-Arak1) including high specificity for Acinetobacter baumannii, stability at a relatively wide range of temperatures and pH values, short adsorption time, short latent period, and large burst size. In relation to the therapeutic efficacy of the phage, the lesion area decreased in phage-treated groups over 14 days than in those untreated, significantly (p < 0.05). CONCLUSION: Our findings demonstrated that isolated lytic phage was able to eliminate burn infections caused by XDR A. baumannii in a rat model. So, it may be recommended as alternative options toward to developing a treatment for extensively drug resistant Acinetobacter infections.

Designing PCR Primers for the Amplification-Refractory Mutation System.

The recent development in genetic research indicates that there exists intraspecific genetic variability in many organism groups. These variations, which result in a variety of genotypes and phenotypes within a population, are called polymorphism. Mutations in different ways can alter the organism's phenotype and affect its fitness, for example, by altering disease susceptibility or resistance. Therefore, the detection of point mutations in different genes of a population is of particular importance. The amplification-refractory mutation system technique is a PCR-based method to detect single nucleotide polymorphisms in the genome. The high repeatability, low cost, high accessibility, and no need for sophisticated technology are the main advantages of the ARMS-PCR technique, compared with other available methods such as PCR-RFLP. This chapter describes the design and analysis method of primers for the ARMS-PCR technique.

Association of Herpes simplex virus I&II infections with rs187084 SNP of TLR9 and male infertility.

Since TLR9 recognises unmethylated CpG motifs in viral DNA, its polymorphisms may contribute to the susceptibility to Herpes simplex virus I&II infection. In the present study, to evaluate the role of rs187084 SNP (single nucleotide polymorphism) of TLR9 in Herpes simplex virus I&II infection and male infertility, 103 infertile and 27 fertile blood and semen samples were analysed. We assessed the micro and macro properties of semen specimens and the presence of HSV immunoglobulins. Tetra-primer ARMS PCR was used to detect SNP and to investigate the genotype distribution of TLR9-rs187084 SNPs, and the correlation between polymorphisms of TLR9 gene and male infertility. Moreover, the odds ratio (OR) and 95% confidence intervals were used to estimate the strength of the association. Based on our finding, a significant correlation was observed between HSV infection, agglutination and polymorphism (TT) under dominant (OR = 1.28, 95% CI = 0.94-1.75) and recessive (OR = 0.44, 95% CI = 0.21-0.94) models for the data, which was complied with Hardy-Weinberg equilibrium (HWE) (OR = 2.91, 95% CI = 1.02-8.30). The result showed a significant association between HSV IgM and agglutination in HSV infection (p < .001), and in addition, there were associations between alleles so that rs187084 SNP might be considered as a risk factor for the incidence of HSV infection.

Investigation into antibacterial and wound healing properties of platelets lysate against Acinetobacter baumannii and Klebsiella pneumoniae burn wound infections.

BACKGROUND AND AIM: Treatment of burn wound infections has become a global challenge due to the spread of multidrug-resistant bacteria; therefore, the development of new treatment options for the mentioned infections is essential. Platelets have drawn much attention for this purpose because they are a safe and cost-effective source of different antimicrobial peptides and growth factors. The present study evaluated antibacterial effects and wound healing properties of Platelet-derived Biomaterial (PdB) against Acinetobacter baumannii and Klebsiella pneumoniae burn wound infections. METHODS: PdB was prepared through the freezing and thawing process and then, in vitro antibacterial effect was determined by disk diffusion and broth microdilution methods. Afterward, burn wound was inflicted on 56 rats, infected with both bacteria, and topical administration was performed to evaluate antibacterial effects and wound healing properties of PdB. RESULTS: In vitro results showed that PdB inhibited the growth of A. baumannii in the highest dose (0.5), while we did not detect any inhibitory effects against K. pneumoniae. By contrast, PdB significantly inhibited the growth of bacteria in treated animal wounds compared to the control groups (P value < 0.05). Macroscopic assessments pointed to the significant enhancement of wound closure in the treated animals. In addition, histopathological examination demonstrated that treatment of rats with PdB led to a considerable increase in re-epithelialization and attenuated the formation of granulation tissue (P value < 0.05). CONCLUSION: The use of topical PdB is an attractive strategy for treating A. baumannii and K. pneumoniae burn wound infections because it inhibits bacterial growth and promotes wound healing properties.

Heavy metal pollution promotes antibiotic resistance potential in the aquatic environment.

Water pollution is one of the main challenges and water crises, which has caused the existing water resources to be unusable due to contamination. To understand the determinants of the distribution and abundance of antibiotic resistance genes (ARGs), we examined the distribution of 22 ARGs in relation to habitat type, heavy metal pollution and antibiotics concentration across six lakes and wetlands of Iran. The concentration of 13 heavy metals was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES) by Thermo Electron Corporation, and five antibiotics by online enrichment and triple-quadrupole LC-MS/MS were investigated. We further performed a global meta-analysis to evaluate the distribution of ARGs across global lakes compared with our studied lakes. While habitat type effect was negligible, we found a strong correlation between waste discharge into the lakes and the abundance of ARGs. The ARGs abundance showed stronger correlation with the concentration of heavy metals, such as Vanadium, than with that of antibiotics. Our meta-analysis also confirmed that overuse of antibiotics and discharge of heavy metals in the studied lakes. These data point to an increase in the distribution of ARGs among bacteria and their increasing resistance to various antibiotics, implying the susceptibility of aquatic environment to industrial pollution.

Characterization of a novel bullet-shaped lytic bacteriophage against extensively drug-resistant Pseudomonas aeruginosa isolated from human and domestic sources.

Global spread and emergence of the extensively drug-resistant (XDR) strains of P. aeruginosa have become a concern, thus, searching for new alternative treatment approaches are required. This study was aimed to isolate and characterize a novel lytic phage against P. aeruginosa. Seventy XDR isolates of P. aeruginosa were collected from May to September 2018. Wastewater samples were used for isolation of lytic phage against XDR P. aeruginosa isolates. Host range, thermal and pH stability, adsorption rate, latent period, burst size and morphology of phage were determined following the standard protocols. Morphological characteristics of the phage revealed that it belonged to Podoviridae family and it was named vB-PaeP-007. Although the phage had a narrow host range, 47 out of 70 XDR isolates were susceptible to it. The adsorption rate, latent period and burst size of vB-PaeP-007 were approximately 89.80% in 8 min, 10 min and 93 phages per cell, respectively. Its lysis activity remained at a wide range of pH (4 up to 12) and temperature (- 20.00 up to 70.00 ˚C). Regarding the physiological features and host range of the vB-PaeP-007 phage, it could be a promising candidate for phage therapy and bio-controlling of infections from XDR isolates of P. aeruginosa in human and livestock storage centers.

A New Phage Cocktail Against Multidrug, ESBL-Producer Isolates of Shigella sonnei and Shigella flexneri with Highly Efficient Bacteriolytic Activity.

The globally increasing incidence of antibiotic resistance in pathogenic microorganisms such as Shigella, a cause of human acute gastrointestinal infections, calls for developing effective alternatives. In this study, the antibiotic resistance pattern, extended-spectrum ß-lactamase (ESBL)-production, and molecular characteristics of 70 multidrug-resistant isolates belong to the two most frequent species of Shigella genus, that is, Shigella sonnei (44 isolates) and Shigella flexneri (26 isolates) were investigated. These isolates were used to evaluate both specificity and activity of Shigella-specific bacteriophages, vB_SflS-ISF001, vB_SsoS-ISF002, and a cocktail of both. Twelve out of the 21 tested resistance genes were detected in the isolates. About 59% of S. sonnei and 46% of S. flexneri isolates were identified as ESBL producers. The bacteriophages showed a high efficiency of plating (EOP ≥0.5) in about 75% of the isolates. Moreover, the growth of >85% of the isolates was inhibited by the phage cocktail of vB_SflS-ISF001 and vB_SsoS-ISF002. The phage cocktail was effective against a wide range of ESBL-positive and -negative isolates of S. sonnei and S. flexneri. Therefore, this phage cocktail has the potential to inhibit or significantly decrease the spread of drug-resistant Shigella in humans, food chains, and water/wastewater sanitation systems.

Isolation, characterization, and PCR-based molecular identification of a siphoviridae phage infecting Shigella dysenteriae.

BACKGROUND: Shigella dysenteriae is one of the members of Shigella genus which was the main responsible of different Shigellosis outbreaks worldwide. The increasing consumption of antibiotics has led to the emergence and spreading of antibiotic-resistant strains. Therefore, finding new alternatives for infection control is essential, one of which is using bacteriophages. MATERIALS AND METHODS: Lytic bacteriophage against Shigella dysenteriae was isolated from petroleum refinery wastewater. Phage morphological and genetic characteristics were studied using TEM, and sequencing, respectively. In addition, the genome size was estimated, and phage resistance to different temperatures and pH, host range, adsorption rate, and one-step growth were investigated. RESULTS: According to the morphology and genetic results, this phage was named vB-SdyS-ISF003. Sequencing of the PCR products revealed that the vB-SdyS-ISF003 phage belongs to the species T1virus, subfamily Tunavirinae of family Siphoviridae. This was the first detected bacteriophage against S. dysenteriae, which belongs to the family Siphoviridae. In addition, its host range was limited to S. dysenteriae. The genome size was about 62 kb. vB-SdyS-ISF003 phage has a number of desirable characteristics including the limited host range to S. dysenteriae, very short connection time, a relatively wide range of temperature tolerance -20 to 50 °C, pH tolerance of 7-9 without significant reduction in the phage titer. CONCLUSION: vB-SdyS-ISF003 is a novel virulent T1virus phage and has the appropriate potential for being used in bio controlling of S. dysenteriae in different condition.

Genotypes of John Cunningham (JC) Virus Urinary Excretion in Pregnant and Non-Pregnant Women in Isfahan, Iran.

Objective: To evaluate presence of different subtypes and genetic variations of JC virus in different geographical areas is a useful tool for reconstructing of the genetic information and understanding of the evolution of the virus and also in tracing of the last and present history of human immigration. Materials and methods: This study aimed to investigate the reactivation of different genotypes of JC virus in kidney and its excretion in the urine of the 50 pregnant and 50 non-pregnant women. Phenol-chloroform method was used to extract DNA. Oligo 7 and MEGA 7 software were used for designing nested PCR specific primers based on vp1 capsid gene, and construction of phylogenetic tree, respectively. Fisher's exact test was used for statistical analyses. Results: All of the positive samples were sequenced and according to them, genotypes 1 and 3 of the virus were detected for the first time in pregnant and non-pregnant women in Asia. The frequency of genotypes 1 and 3 were 14.28% and 85.71% respectively. Conclusion: For the first time genotype 3 was reported as the frequent genotype in pregnant women in Asia. Confirming these needs more studies particularly with a higher number of cases and full genome sequencing of isolated JCVs.

Prevalence of cytomegalovirus infection and its role in total immunoglobulin pattern in Iranian patients with different subtypes of multiple sclerosis.

Multiple sclerosis (MS) is the most common autoimmune disease characterized by multifocal areas of inflammatory demyelination within the central nervous system. Cytomegalovirus (CMV) has a complex pathobiology and in most cases is simply asymptomatic. There is some recent controversy over the role of CMV in the pathology of MS. The aim of this study was to evaluate active CMV infection and its effect on the humoral immunity in patients with MS. Serum, plasma, peripheral blood mononuclear cells (PBMCs), saliva and urine collected from MS patients (n=78) and healthy subjects (n=123) were screened for the presence of anti-CMV antibodies and CMV-DNA by nephelometric and PCR methods. Concentrations of total antibodies in MS subtypes were measured using both nephelometric and enzyme linked fluorescent assay (ELFA) techniques. The results extend the observation of an increased frequency of CMV-DNA in patients, in contrast with controls (p<0.001). Furthermore, systemic CMV infections were found in 25.5% of patients and only 3.2% of controls (p<0.001). There was significant difference in the titers of anti-CMV IgG and total IgE in patient and controls (P<0.001). These results support the hypothesis that CMV may contribute to MS thought to establish systemic infection process and induce immune response.

Torque teno mini virus infection and multiple sclerosis.

Multiple sclerosis (MS) is a disease of young adults which is characterized by autoimmune demyelination of the central nervous system. Interaction of genetics and environmental factors are required to cause MS. Among the proposed environmental factors for MS, viral infections are thought to play a role in the pathogenesis of the disease. Torque teno mini virus (TTMV), which has recently been shown to infect humans, is a member of circoviridae, and has a circular DNA with 2860 nucleotides. Since there are a few data about the pathogenicity of this virus, this study sought to investigate the presence of TTMV in sera from MS patients and healthy individuals. We studied 149 serum samples from MS patients and 150 sera of healthy individuals. Serum DNA was extracted using phenol-chloroform and was subjected to nested polymerase chain reaction. TTMV-DNA was detected in 24 (16%) sera of the healthy blood donors and in 21 (14.1%) samples of the MS patients, where the difference did not reach significance (p > .05). The result of this study could not establish an association between TTMV infection and MS.