Different morphologies of Leishmania major amastigotes with no molecular diversity in a neglected endemic area of zoonotic cutaneous leishmaniasis in Iran

Molecular diversity of Leishmania major and its morphological changes have become a controversial issue among researchers. Some aspects of polymorphic shapes of amastigotes in clinical manifestations along with molecular variation were evaluated among suspected patients of some exceptional zoonotic cutaneous leishmaniasis locations in Northern Khuzestan, Southwestern Iran. Suspected patients [n = 165] were sampled in zoonotic cutaneous leishmaniasis foci over two consecutive years during 2012- 2014. Prepared smears were stained, scaled and measured by ocular micrometer. DNA was extracted from smears; ITS-rDNA and Cytochrome b [Cyt b] markers were amplified, and PCR products were digested by BsuR1 restriction enzyme. Then the RFLP and sequencing were employed. Only L. major was identified in patients containing regular amastigotes' shapes [oval or round] with a size of 2-4 microm in each of classical wet, dry, mixed lesions. Meanwhile, irregular shapes [spindle, pear, or cigarette] were observed separately in non-classical wet lesions with more than 4 microm. Interestingly, a few amastigotes with an external flagellum were observed in some lesions. All sequenced ITS-rDNA and Cyt b genes of L. major did not show any molecular variation [chi [2] P > 0.05], including only one common haplotype [GenBank access no. EF413075]. Findings proved that unlike other endemic foci, there is not a meaningful correlation between phenotypic and genotypic features of L. major isolates. This study is considered as the first comprehensive report to incriminate morphometric shapes of L. major amastigotes, which enhances our knowledge concerning their relevance with various clinical appearances and genotypic traits

[Determination of abundance of tellurite-resistant bacteria and the ability of remove of them from Qom province industrial Wastewater, Qom, Iran]

Accumulation of toxic oxyanion of potassium tellurite, which has increased in the environment due to industrial activities, can cause complications in human, such as skin irritation, dermatitis, anorexia, tremor, nausea, vomiting, nervous system stimulation, convulsion, and respiratory arrest. The purpose of this research was to isolate tellurite-resistant bacteria, determine the minimum inhibitory concentration [MIC], and evaluate bioreduction of tellurite in strains. MIC was measured by agar dilution method in 84 resistant strains isolated from wastewater. QWTm[6] strain with tolerance of tellurite concentration of 6684 microg/ml was selected as superior strain. Toleration of this level of tellurite has not been reported yet. According to biochemical, phenotypic, and physiological characteristics, QWTm[6] was initially classified into Staphylococcus genus. Using the spectrophotometric technique and DDTC reagent [A[340nm] and sodium diethyldithiocarbamate trihydrate], maximum elimination was seen in 0.4mM concentration of potassium tellurite in 24 hours. The strain showed high ability in the elimination of toxic oxyanion of potassium tellurite under a wide range of factors such as pH=[5-11], temperature [15-50°C], blender speed [50, 100, 150, and 200rpm], various oxyanion concentrations [0.04-1mM], and different percentages of NaCl [0-20%]. This strain was also resistant to penicillin, cefixime, tetracycline, chloramphenicol, streptomycin, neomycin, erythromycin, gentamicin, kanamycin, norfloxacin, and ciprofloxacin. The results of this study showed that QWTm[6] could be introduced in the international societies as an acceptable candidate for bioremediation, because of its high ability in removal and reduction of potassium tellurite

Design of an improved multiplex PCR method for diagnosis of enterohaemoraghic E.coli and enteropathogic E.coli pathotypes

We aimed to develop a multiplex PCR assay for specific detection of EPEC and EHEC pathotypes based on specific marker genes. About 2.5 million infant's morbidity in developing countries occurs by E.coli pathotypes because of diarrhea and intestinal diseases. The traditional phenotypic methods are time consuming and sometimes detection and differentiation of the pathotypes are not done easily. Multiplex PCR technology is used as a sensitive, specific and rapid molecular method for detection of various pathogens. PCR reactions were performed with primers which targeted the virulence genes selected for each category [stx[1], stx[2] genes for EHEC and bfpA for EPEC].For preparation of a positive control, the PCR products were cloned in pTZ57R/T plasmid. The same PCR reactions were done but in presence of genomes of various negative control bacteria for evaluation of test specificity. As expected, gel agarose electrophoresis of PCR products of the stx1, stx2 and bfpA, showed 329bp, 586bp and459bp bands respectively. Result of amplification using negative control genomes as template was negative. The multiplex PCR assay followed by capillary electrophoresis presented in the present paper provides a simple, reliable, and rapid procedure that in a single reaction identifies the four main pathotypes of E. coli. This assay will replace the previous molecular genetics methods used in our laboratory and work as an important supplement to the more time consuming phenotypic assays