Soil-dwelling nematodes from Steinernematidae family are obligate parasites of insects and usually referredto as entomopathogenic nematodes (EPNs). These nematodes are symbiotically associated with entomopathogenic bacteria Xenorhabdus spp. The bacterial symbionts are carried monoxenically in a special vesicle in the infectivejuveniles (IJs). In the present study we report the isolation of two species of non-symbiotic bacteria from infectedinsect cadavers by the EPN, Steinernema feltiae. Galleria mellonella L. larvae were exposed to surface sterilize dinfective juveniles of S. feltiae and transferred to sterile Petri dishes for a further 24 hours. Hemolymph was collected and streaked onto both MacConkey and NBTA agar. Bacteria were identified using biochemical and phylogenetic analysis.16S-rRNA gene sequence based maximum parsimony, maximum likelihood and neighbourjoining phylogenetic analyses were conducted. Two non-symbiotic species including Citrobacter freundii and Staphylococcus succinus were identified and reported to be associated with S. feltiae. Our results provide further evidence for the existence of non-symbiotic bacteria associated with EPNs infection.

Introduction: Toxic heavy metal contamination of industrial water is a significant universal problem. They accumulate in living tissues throughout the food chain which has humans at its top. These toxic metals can cause accumulative poisoning, cancer, and brain damage. Uranium is one of the most serious heavy metals because of its high toxicity and radioactivity. Excessive amounts of uranium have found their way into the environment through the activities associated with the nuclear industry (1). Conventional methods for removing uranium from wastewater include precipitation, evaporation, ion exchange, membrane processing, and adsorption. Nevertheless, these methods have several disadvantages, such as high installation and operating costs, the requirement of preliminary treatment steps, the difficulty of treating the subsequently generated solid waste, and low efficiency at low metal concentration (2,3). Owing to an increase in environmental awareness, there has been an emphasis on the development of new environmentally friendly ways to decontaminate waters using low-cost methods and materials. In this study, microbial biomass has emerged as a complementary, economic, and eco-friendly device for controlling the mobility and bioavailability of metal ions (2,4). The present work evaluates the performance of the Citrobacter freundii biomass to remove uranium ions from aqueous solutions. The effect of pH, temperature, initial concentration, and sorbent dose on biosorption capacity is also studied.Materials and Methods: Materials: Citrobacter freundii bacteria used in this research with PTCC No. 1772 was purchased from the Scientific and Industrial Research Organization of Iran. Uranyl nitrate salt (UO2(NO3)2.6H2O) was obtained from the Research Institute of Nuclear Sciences and Technologies. Nutrient Broth culture medium, sulfuric acid, and sodium hydroxide and other materials used in this research were supplied from the Merck Company.Preparation of uranium solutions and biomass: A stock solution containing 1000 mg L-1 of U(VI) was prepared of UO2(NO3)4.6H2O. The working solutions were prepared daily from stock solutions. In this study, the biomass of Citrobacter freundii bacteria was heat treated in an autoclave at a temperature of 121°C for 15 minutes at a pressure of 1.5 atmospheres. Experimental design and batch biosorption studies: The design of the experiment was done using the response surface method by Design Expert software. Four variables, including initial uranium concentration (mg/l), temperature (°C), pH, and biosorbent dose (g/l), in five levels α-, -1, 0, +1, α+, 1 were used to design the experiment (Table 1). Therefore, 27 experiments were presented using a central composite design. The values of the variables and the obtained answers are shown in Table 2. Uranium biosorption experiments were performed by adding specified amounts of bacterial biomass in 20 ml Erlenmeyer flasks containing uranium solution with the concentration and pH corresponding to each experiment, with the specified temperature in the Shaker. After 90 minutes, each sample was centrifuged at 4500 rpm for 15 minutes at 4°C. Then, the remaining uranium in the solution was measured by ICP (Perkin Elmer/Optima 7300DV). The percentage of uranium removal (R) was calculated by equation 1:  Where C0 and Cf are the initial and the final concentrations of the metal ion solutions (mg/l), respectively.Table 1- Variables and Levels of the Central Composite Design MethodResults: By using the RSM-CCD method, the optimization of the biosorption process was carried out. Table 2 shows the experimental results based on each point of the experimental design. Then, using analysis of variance (ANOVA), the obtained results were evaluated.The equation obtained for the biosorption efficiency of uranium by Citrobacter freundii is expressed as follows:Removal= +68.97045-1.43160 * C (ppm)+12.81296 * pH+1.08935 * T (0C)+2.89856 * M (g/l)+0.55737 * C (ppm) * pH+0.011459* C (ppm)* T (0C)+0.014961* C (ppm)* M (g/l)-0.32111 * pH * T (0C)-0.62783 * pH * M (g/l)-0.037633* T (0C) * M (g/l)-6.93488E-003* C (ppm)2-4.06361 * pH2Table 2- Values of Variables and Experimental Responses in the Response Surface MethodDiscussion and Conclusion: The F-value and p-value of the proposed model are equal to 5.03 and 0.0027, respectively, reflecting the accuracy of the proposed model. This model with R2 equal to 0.81 shows that the proposed model can well predict the experimental values. The results showed that the factor of initial uranium concentration and pH statistically affect (p-value‹ 0.05) the uranium biosorption process. In contrast, temperature and sorbent dose factor (p-value› 0.05) have no statistically effect on uranium removal by Citrobacter freundii. With increasing uranium concentration from 10 mg/l to 77.5 mg/l, the removal increases from %66.5 to %99/92. Then, with increasing uranium concentration from 77.5 mg/l to 100 mg/l, the removal decreases to %97.34. On the other hand, one of the most important effective parameters in biosorption is the pH of the solution. With increasing the pH from 2 to 5, the removal decreased from %96.82 to %79.01 due to the formation of uranyl complexes (5). In this research, the results indicated that the pre-treated biomass under the conditions suggested by Design Expert software (19.84 g/l of biomass, temperature 28.92 OC, pH 2.89 and initial uranium concentration 53.71 mg/l) is able to remove approximately 99.99 percent of uranium from the contaminated area, which shows valuable potential Citrobacter freundii in bioremediation applications of uranium from contaminated wastewaters.

Background & Aims: The inappropriate use of antibiotics has led to antibiotic resistance in microorganisms of Enterobacteriaceae family, especially in carbapenems. The aim of this study was to identify the carbapenemase producing Citrobacter frundii and Citrobacter koseri isolated from clinical specimens.Methods: One hundred Citrobacter isolates from various patient samples in Imam Reza Hospital, Kermanshah, Iran, were identified using the microbiologic differential tests and API-E20 Kit. After antibiotic susceptibility testing with disc, the isolates resisted to carbapenems were screened using MHT (Modified Hodge Test) for the presence of carbapenemases. Then, carbapenemase genes coded Verona integrin-encoded metallo-beta-lactamase (VIM), Klebsiella pneumoniae carbapenemase (KPC), Imipenemase (IMP), New Delhi metallo-beta-lactamase-1 (NDM) were tested using specific primers via polymerase chain reaction (PCR) method.Results: From 100 isolates, 11 cases were carbapenem resistant. In the phenotypic screening test, 2 isolates were positive. PCR on isolates resistant to carbapenams confirmed VIM gene in 5 isolates: 3 (3.9%) of Citrobacter frundii and 2 (15.4%) of Citrobacter koseri. But the genes of blaKPC, blaVIM, blaIMP and blaNDM were not found in isolates. The highest and lowest antibiotic susceptibility were for meropenem (93%) and cefazolin (1%), respectively.Conclusion: It seems that the prevalence of carbapenemase genes in Citrobacter kosari and Citrobacter frundii was low in Kermanshah; however, VIM gene in these two species is probably more prevalent. This may suggest that most genes have not been high prevalent in this area yet. But, there may be other genes for resistance to carbapenems in our area which need further investigations. The results indicate that carbapenems are still effective antibiotics against Citrobacter species.

Background: The aim of this study was to assess associations of Citrobacter freundii foodborne outbreaks with environmental factors in various regions of Yazd Province, Iran, 2012– 2016. Methods: The public health surveillance data were used for one period of five years reported foodborne disease outbreaks in various regions of the Province. Multilevel regression statistical method was used to analyze associa-tions of climatic and demographic variables with outbreaks. Significant associations were tested using likelihood ratio tests. Results: Results showed a significant association between C. freundii outbreaks and air dust conditions, age groups and various regional cities. Conclusion: The current study revealed necessity of etiologic agent identification for use in foodborne disease outbreak guidance in future outbreaks. Systemic surveillance schemes can help prevent and control similar sce-narios using reports of environmental effects on foodborne disease outbreaks.

Background and Objectives: Plasmid-mediated quinolone resistance (PMQR) plays an essential role in developing resistance to quinolones. This study aimed to investigate the presence and effect of PMQR genes in clinical isolates of Citrobacter from urinary tract infections. Methods: Fifty-one citrobacter isolates were found in urinary samples of patients referred to Imam Reza Hospital in Kermanshah City, Iran, in 2019. The susceptibility to antibiotics was determined by disk diffusion and broth microdilution methods. The qnrA, qnrB, qnrS, aac (6 ')-Ib, and qepA genes of isolates were detected by the polymerase chain reaction (PCR) method. Then, the PCR-RFLP (restriction fragment length polymorphism) method was used to differentiate the aac (6')-Ib gene from its aac (6')-Ib-cr variant. The obtained data were analyzed using the chi-square and Mann-Whitney statistical methods. Results: The highest resistance of isolates was seen against cefazolin (74.5%), ciprofloxacin (41.2%), and cotrimoxazole (35.3%). More than 80% of isolates were susceptible to carbapenems, gentamicin, and piperacillin/tazobactam. The frequency of qnr genes in isolates was 41.5%. The qnrB gene was the most frequent (43.2%), followed by qnrS (5.9%) and qnrA (1.9%). The aac (6') -Ib gene was found in 27.5% of the isolates; all were aac (6')-Ib-cr. The qepA gene was not found in any isolate. Conclusion: The results of this study indicate the importance of Citrobacter freundii as an agent of urinary tract infection and its high resistance to ciprofloxacin. PMQR genes are highly prevalent in Citrobacter isolates, and there is a significant correlation between the qnrB and qnrS genes with the increased minimum inhibitory concentration of ciprofloxacin. Therefore, antibiotic susceptibility should be performed before treating Citrobacter urinary tract infections.