Non-contrast spiral computed tomography diagnosis of urolithiasis and associated features: hospital based study

Background: Urolithiasis is prevalent across the world and affects a diverse group of people, irrespective of culture, race or geographic location. Non-contrast computed tomography (CT), has been considered as gold standard for the initial as well as follow-up assessment of patients with suspected urolithiasis. Present study describes the findings on non-contrast spiral computed tomography in clinically suspected patients of urolithiasis visiting radiodiagnosis department of a tertiary care hospital.Methods: It is a descriptive observational study done at Department of Radiodiagnosis and Imaging at Shri Chhatrapati Shivaji Maharaj General Hospital in Solapur district of Maharashtra state in India. Study duration was Jan 2005 to Oct 2006. 120 patients who presented with symptoms and signs of urolithiasis for diagnosis and treatment in Department of Surgery and Medicine, including the referrals from other hospitals and institutes and referred to Department of Radiodiagnosis and Imaging of the institute for computerised tomography (CT) were enrolled. Detailed history and physical examination was done. The description of findings on non-contrast spiral CT study was done with respect to size and CT attenuation value of the calculus, secondary signs of obstruction, CT diagnosis of urolithiasis, genitourinary or other diseases.Results: In hundred patients diagnosed as urolithiasis on NCSCT, 140 calculi were found. The mean calculus size (breadth) was 4.65 mm ± 7.03 with a range of 1 to 70 mm. The mean calculus size (length) was 11.1±12.87 mm with a range of 2 to 110 mm. The range of CT attenuation value of calculus was from 60 to 1100 with median value of 311 HU.  Among the 100 patients of urolithiasis, hydronephrosis (84%) and hydroureter (82%) were the most common secondary signs of obstruction. Out of 120 patients suspected clinically with diagnosis of urolithiasis, 99 (82.5%) had obstruction with or without urolithiasis. In 86 (71.7%) patients, obstruction with urolithiasis was present. In 13(10.8%) patients, obstruction because of cause other than urolithiasis was present. We have observed additional diagnosis related to genito-urinary tract in 16 (13.5%) cases. We have observed additional diagnosis not related to genito-urinary tract in 6 (0.5%) cases.Conclusions: Non contrast spiral CT scan evaluation helped in diagnosis of urolithiasis and secondary obstruction. It also provided very useful information regarding genitourinary as well as other than genitourinary pathology

A novel prokaryotic vector for identification and selection of recombinants: Direct use of the vector for expression studies in E. coli

<p>Abstract</p> <p>Background</p> <p>The selection of bacterial recombinants that harbour a desired insert, has been a key factor in molecular cloning and a series of screening procedures need to be performed for selection of clones carrying the genes of interest. The conventional cloning techniques are reported to have problems such as screening high number of colonies, generation of false positives, setting up of control ligation mix with vector alone etc.</p> <p>Results</p> <p>We describe the development of a novel dual cloning/expression vector, which enables to screen the recombinants directly and expression of the gene of interest. The vector contains Green fluorescence protein (GFP) as the reporter gene and is constructed in such a way that the <it>E. coli </it>cells upon transformation with this vector does not show any fluorescence, but readily fluoresce upon insertion of a foreign gene of interest. The same construct could be easily used for screening of the clones and expression studies by mere switching to specific hosts.</p> <p>Conclusions</p> <p>This is the first vector reported that takes the property of colour or fluorescence to be achieved only upon cloning while all the other vectors available commercially show loss of colour or loss of fluorescence upon cloning. As the fluorescence of GFP depends on the solubility of the protein, the intensity of the fluorescence would also indicate the extent of solubility of the expressed target protein.</p

Molecular characterization of typing and subtyping of Staphylococcal cassette chromosome SCCmec types I to V in methicillin-resistant Staphylococcus aureus from clinical isolates from COVID-19 patients

Background and Objectives: Methicillin resistance is acquired by the bacterium due to mecA gene which codes for penicillin-binding protein (PBP2a) having low affinity for β-lactam antibiotics. mecA gene is located on a mobile genetic element called staphylococcal cassette chromosome mec (SCCmec). SCCmec genomic island comprises two site-specific recombinase genes namely ccrA and ccrB [cassette chromosome recombinase] accountable for mobility. Currently, SCCmec elements are classified into types I, II, III, IV and V based on the nature of the mec and ccr gene complexes and are further classified into subtypes according to variances in their J region DNA. SSCmec type IV has been found in community-acquired isolates with various genetic backgrounds. The present study was undertaken to categorize the types of SCCmec types and subtypes I, II, III, IVa, b, c, d, and V and PVL genes among clinical MRSA isolates from COVID-19 confirmed cases. Materials and Methods: Based on the Microbiological and Molecular (mecA gene PCR amplification) confirmation of MRSA isolated from 500 MRSA SCCmec clinical samples, 144 cultures were selected for multiplex analysis. The multiplex PCR method developed by Zhang et al. was adapted with some experimental alterations to determine the specific type of these isolates. Results: Of the total 500 MRSA, 144 MRSA (60 were CA-MRSA and 84 were HA-MRSA) were selected for characterization of novel multiplex PCR assay for SSCmec Types I to V in MRSA. Molecular characterization of multiplex PCR analysis revealed results compare to the phenotypic results. Of the 60 CA-MRSA; in 56 MRSA strains type IVa was found and significantly defined as CA-MRSA while 4 strains showed mixed gens subtypes. Type II, III, IA, and V were present in overall 84 HA-MRSA. Molecular subtyping was significantly correlated to define molecularly as CA-MRSA and HA-MRSA however 15 (10%) strains showed mixed genes which indicates the alarming finding of changing epidemiology of CA-MRSA and HA-MRSA as well. Conclusion: We have all witnessed of COVID-19 pandemic, and its mortality was mostly associated with co-morbid conditions and secondary infections of MDR pathogens. Rapid detections of causative agents of these superbugs with their changing epidemiology by investing in typing and subtyping clones are obligatory. We have described an assay designed for targeting SSCmec types and subtypes I, II, III, IVa,V according to the current updated SCCmec typing system. Changing patterns of molecular epidemiology has been observed by this newly described assay

Serratia odorifera a midgut inhabitant of Aedes aegypti mosquito enhances its susceptibility to dengue-2 virus.

Mosquito midgut plays a crucial role in its vector susceptibility and pathogen interaction. Identification of the sustainable microflora of the midgut environment can therefore help in evaluating its contribution in mosquito-pathogen interaction and in turn vector competence. To understand the bacterial diversity in the midgut of Aedes aegypti mosquitoes, we conducted a screening study of the gut microbes of these mosquitoes which were either collected from fields or reared in the laboratory "culture-dependent" approach. This work demonstrated that the microbial flora of larvae and adult Ae. aegypti midgut is complex and is dominated by gram negative proteobacteria. Serratia odorifera was found to be stably associated in the midguts of field collected and laboratory reared larvae and adult females. The potential influence of this sustainable gut microbe on DENV-2 susceptibility of this vector was evaluated by co-feeding S. odorifera with DENV-2 to adult Ae. aegypti females (free of gut flora). The observations revealed that the viral susceptibility of these Aedes females enhanced significantly as compared to solely dengue-2 fed and another gut inhabitant, Microbacterium oxydans co-fed females. Based on the results of this study we proposed that the enhancement in the DENV-2 susceptibility of Ae. aegypti females was due to blocking of prohibitin molecule present on the midgut surface of these females by the polypeptide of gut inhabitant S. odorifera

Serratia odorifera mediated enhancement in susceptibility of Aedes aegypti for chikungunya virus

Background & objectives: The susceptibility of the mosquito to the invading pathogen is predominantly dictated by the complex interactions between the mosquito midgut and the surface proteins of the invading pathogen. It is well documented that the midgut microbiota plays an important role in determining the susceptibility of the mosquito to the pathogen. In the present study, we investigated the influence of Serratia odorifera, an endogenous cultivable midgut inhabitant of Aedes aegypti on the chikungunya virus (CHIKV) susceptibility to this mosquito. Methods: Ae. aegypti females free of gutflora were co-fed with CHIKV and either of the two midgut inhabitants namely, S. odorifeara and Microbacterium oxydans. CHIKV dissemination was checked on 10 th day post feeding (DPF) using indirect immunoflurescence assay and plaque assay. CHIKV interacting proteins of the mosquito midgut were identified using virus overlay protein binding assay and MALDI TOF/TOF analysis. Results: The observations revealed that co-feeding of S. odorifera with CHIKV significantly enhanced the CHIKV susceptibility in adult Ae. aegypti, as compared to the mosquitoes fed with CHIKV alone and CHIKV co-fed with another midgut inhabitant, M. oxydans. Virus overlay protein binding assay (VOPBA) results revealed that porin and heat shock protein (HSP60) of Ae. aegypti midgut brush border membrane fraction interacted with CHIKV. Interpretation & conclusions: The results of this study indicated that the enhancement in the CHIKV susceptibility of Ae. aegypti females was due to the suppression of immune response of Ae. aegypti as a result of the interaction between S. odorifera P40 protein and porin on the gut membrane

P40 localization in the <i>Ae. aegypti</i> gut.

<p>The midgut sections (10 µm) of <i>Ae. aegypt</i>i fourth instar larvae (a) adult female (b) and slit opened gut of adult females (c) were incubated with <i>S. odorifera</i> cell lysate and control midgut sections were incubated with PBS (pH 7.4). P40 interaction with the midgut epithelium was detected using mouse anti-P40 antibody and with a Cy3 conjugated rabbit anti mouse IgG secondary antibody. The signal was detected using a Zeiss microscope equipped with the Axiovesion detection system.</p

<i>Serratia odorifera</i> and <i>Ae. aegypti</i> interaction.

<p>a. Significance of <i>S. odorifera</i>’s presence in the blood meal on the DENV-2 susceptibility of <i>Ae. aegypti</i>: Adult females were fed with Blood + DENV-2, Blood + DENV-2+ <i>M. oxydans</i> (Blood + DENV-2+ <i>S. odorifera</i> via blood meal. DENV-2 dissemination was detected in the head squashes on 14 post infection days by IFA. The presence of <i>S. odorifera</i> in the blood meal significantly enhanced the dengue virus susceptibility (Mann-Whitney U test; P<0.05) compared to <i>M. oxydans</i> (Mann-Whitney U test; P>0.05). Post feeding virus titers in the blood meal were determined by plaque assay (1.8× 10⁵ PFU/mL of blood). b. Overlay assay with <i>S. odorifera</i> and <i>M. oxydans</i> cell lysates: The bacterial cell lysates of <i>S. odorifera</i> (So1 and So2) and <i>M. oxydans</i> (Mo1 and Mo2) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was overlaid with BBMF of <i>Ae. aegypti.</i> The putative binding proteins were detected by mouse anti-BBMF antibody and HRP labeled-secondary antibody. c. Expression of P40 in cell lysates and cell supernatants under different temperature conditions: <i>S. odorifera</i> cell lysate (S1 and S3 30°C, S2 41°C), <i>M. oxydans</i> cell lysate (M1 and M3 30°C, M2 41°C), culture filtrate of <i>S. odorifeara</i> (S4 and S6 30°C, S5 41°C) and culture filtrate of <i>M. oxydans</i> (M4 and M6 30°C, M5 41°C) were separated by SDS-PAGE and transferred to Hybond-C membranes. The membranes were incubated with the anti P40 mouse IgG (lanes S1, S2, S4, S5, M1, M2, M4 and M5) and with PBS pH 7.4 (lanes S3, S6, M3 and M6). Presence of P40 was detected by incubating the membranes with the secondary antibody (peroxidase-conjugated goat anti mouse IgG). Reaction was developed using H₂O₂ and DABT.d. Protein-protein interaction between BBMF and <i>S. odorifera</i> cell lysate: The membrane proteins of <i>Ae. aegypti</i> midgut (Lanes L1, L2 ) were separated by SDS–PAGE and transferred to Hybond-C membranes. The membranes were incubated with <i>S. odorifera</i> cell lysate (L1) and PBS pH 7.4 (L2) at 37°C. The putative P40 binding proteins were visible after incubation with anti P40 mouse antibody and the secondary antibody (peroxidase-conjugated goat anti mouse IgG). The reaction was developed using H₂O₂ and DABT. The molecular weights of DENV-2 binding proteins are shown on the left side of the blot.</p

Virus overlay protein binding assay (VOPBA).

<p><i>Serratia odorifera</i> cell lysate (lanes L1 and L2) were subjected to SDS-PAGE and transferred to Hybond-C membranes. Lane L1 was incubated with 5×10⁵ plaque-forming units of DENV-2, and lane L2 with PBS (pH 7.4) at 37°C. The putative DENV-2 binding proteins were visible after incubation with a rabbit polyclonal antibody to DENV-2 and a secondary antibody (peroxidase-conjugated goat anti-rabbit IgG). The reaction was developed using H₂O₂ and DABT. The molecular weights of dengue-2 binding proteins are shown on the left side of the blot.</p