Occurrence of shigellosis in pediatric diarrheal patients in Chattogram, Bangladesh: A molecular based approach.

Shigellaa Gram-negative, non-motile bacillus, is the primary causative agent of the infectious disease shigellosis, which kills 1.1 million people worldwideevery year. The children under the age of five are primarily the victims of this disease. This study has been conducted to assess the prevalence of shigellosis through selective plating, biochemical test and conventional PCR assays, where the samples were collected from suspected diarrheoal patients. Invasive plasmid antigen H (ipaH) and O-antigenic rfc gene were used to identify Shigella spp. and S. flexneri respectively. For validation of these identification, PCR product of ipaH gene of a sample (Shigella flexneri MZS 191) has been sequenced and submitted to NCBI database (GenBank accession no- MW774908.1). Further this strain has been used as positive control. Out of 204, around 14.2% (n = 29)(P> 0.01) pediatric diarrheoal cases were screened as shigellosis. Another interesting finding was that most of shigellosis affected children were 7 months to 1 year (P> 0.01).The significance of this study lies in the analyses of the occurrenceand the molecular identification of Shigellaspp. and S. flexneri that can be utilized in improving the accurate identification and the treatment of the most severe and alarming shigellosis.

The Prevalence of Multi-Drug Resistant Salmonella typhi Isolated From Blood Sample.

Typhoid is a major public health concern. Even though antibiotics are usually used to treat typhoid fever, the spread of multi drug resistant Salmonella typhi is making antibiotics much less effective. This study was conducted to assess the prevalence of multidrug-resistant Salmonella typhi from the clinical samples. During this study, 154 blood samples of suspected typhoid patients were collected from the hospital and diagnostic center located in Chattogram City, Bangladesh. Isolation and identification of Salmonella typhi was done by both biochemical tests. PCR analysis was also done for the confirmation of biochemical result. Antimicrobial susceptibility test was performed according to the Kirby-Bauer disk diffusion method against ampicillin, chloramphenicol, cefepime, cotrimoxazole, ceptriaxone, ciprofloxacin, nalidixic acid, and azithtomycin. Out of 154, 21 (13.64%) isolates were identified as Salmonella typhi and the prevalence of typhoid in Chattogram, Bangladesh was 13.64% (n = 21). It was also found that children under the age of 5 are the more vulnerable target of Salmonella typhi infection. Antibiotic resistance profiling revealed 85% isolates were Multi-Drug Resistant (MDR) and highest resistance was found in case of Nalidixic acid. Although, most of the isolated Salmonella typhi were MDR, first generation antibiotics Co-trimoxazile, Chloramphenicol, and Ampicillin were found effective against Salmonella typhi.

Analysis of the Occurrence of Antibiotic Resistant Bacteria in the Hospital's Effluent and its Receiving Environment.

The use of antibiotics on a regular and excessive basis is a major factor in the spread of antibiotic-resistant bacteria. Patients discharge un-metabolized or relatively low doses of non-metabolized antibiotics through urine and stool, which might enter into the environment through sewage disposal and promote the emergence of antibiotic resistant bacteria. This study is designed to investigate how excessive use of antibiotics in the hospital sector and their release into hospital wastes contribute to the spread of antibiotic-resistant bacteria in different environmental settings. In this study, liquid hospital waste was collected from the sewage of Chittagong Medical College Hospital (CMCH), Bangladesh as well as from its distribution position in Chittagong city, Bangladesh. A total of 5 samples were collected from different positions in Chittagong city, including CMCH liquid waste. After collection, total bacteria and total cefixime resistant bacteria were counted by the total viable count (TVC) method. The result of bacteriological enumeration showed that a high magnitude of cefixime-resistant bacteria were available in all the hospital's associated waste samples. The highest proportion of cefixime resistant bacteria (23.35%) was found in sample 2, whereas 17.4%, 7.6%, 5%, and 1.32% were found in samples 1, 3, 4, and 5, respectively. The total number of cefixime-resistant bacteria decreased with the increase in distance between the sample collection site and the hospital drain. This means that resistant bacteria developed in the hospital effluent are transferred to the environmental distribution sites.

Antibiotic Resistance and Plasmid Profiling of Escherichia coli Isolated from Human Sewage Samples.

In developing countries, the occurrence of antibiotic resistance is increasing day by day and antibiotic resistant microorganisms are being found in almost every environmental setting. Plasmids are considered as the main vector in the procurement and propagation of antibiotic resistance in many microorganisms such as Escherichia coli (E. coli). The goal of this study was to examine the antibiotic resistance and screening of plasmid in E. coli strains which were previously identified from human sewage samples. During this study antibiotic susceptibility of E. coli isolates were determined by Kirby-Bauer disk diffusion method against 5 antibiotics (ampicilin, ceftriaxone, amoxicillin, ciprofloxacin, azithromycin). Furthermore, plasmid extraction of each isolate was done according to the protocol of FavorPrepTMPlasmid Mini Kit and plasmid profiling was done by agarose gel electrophoresis. In antibiotic sensitivity test, all E. coli strains showed resistance to ampicilin, amoxicillin, and ceftriaxone. In the plasmid profiling, it was revealed that all the isolates of E. coli harbored plasmids. The plasmid sizes ranged from approximately 1.5 to 15 kb. The findings of this study prove the consequences of antibiotic resistance as well as relationship of plasmid with antibiotic resistance which necessitates proper surveillance on antibiotic usage in the developing countries.

Multiple regulatory mechanisms of the biological function of NRF3 (NFE2L3) control cancer cell proliferation.

Accumulated evidence suggests a physiological relationship between the transcription factor NRF3 (NFE2L3) and cancers. Under physiological conditions, NRF3 is repressed by its endoplasmic reticulum (ER) sequestration. In response to unidentified signals, NRF3 enters the nucleus and modulates gene expression. However, molecular mechanisms underlying the nuclear translocation of NRF3 and its target gene in cancer cells remain poorly understood. We herein report that multiple regulation of NRF3 activities controls cell proliferation. Our analyses reveal that under physiological conditions, NRF3 is rapidly degraded by the ER-associated degradation (ERAD) ubiquitin ligase HRD1 and valosin-containing protein (VCP) in the cytoplasm. Furthermore, NRF3 is also degraded by ß-TRCP, an adaptor for the Skp1-Cul1-F-box protein (SCF) ubiquitin ligase in the nucleus. The nuclear translocation of NRF3 from the ER requires the aspartic protease DNA-damage inducible 1 homolog 2 (DDI2) but does not require inhibition of its HRD1-VCP-mediated degradation. Finally, NRF3 mediates gene expression of the cell cycle regulator U2AF homology motif kinase 1 (UHMK1) for cell proliferation. Collectively, our study provides us many insights into the molecular regulation and biological function of NRF3 in cancer cells.

Possible roles of the transcription factor Nrf1 (NFE2L1) in neural homeostasis by regulating the gene expression of deubiquitinating enzymes.

The transcription factor Nrf1 (NFE2L1) maintains protein homeostasis (proteostasis) by regulating the gene expression of proteasome subunits in response to proteasome inhibition. The deletion of the Nrf1 gene in neural stem/progenitor cells causes severe neurodegeneration due to the accumulation of ubiquitinated proteins in Purkinje cells and motor neurons (Nrf1 NKO mice). However, the molecular mechanisms governing this neurodegenerative process remain unclear. We demonstrate herein that the loss of Nrf1 leads to the reduced gene expression of the deubiquitinating enzymes (DUBs) but not proteasome subunits in Nrf1 NKO mice between P7 and P18. First, we show that K48-linked polyubiquitinated proteins accumulate in Nrf1-deficient Purkinje cells and cerebral cortex neurons. Nevertheless, loss of Nrf1 does not alter the expression and proteolytic activity of proteasome. A significantly reduced expression of deubiquitinating enzymes was also demonstrated in Nrf1-deficient cerebellar tissue using microarray analysis. The genome database further reveals species-conserved ARE, a Nrf1 recognition element, in the regulatory region of certain DUB genes. Furthermore, we show that Nrf1 can activate Usp9x gene expression related to neurodegeneration. Altogether these findings suggest that neurodegeneration in Nrf1 NKO mice may stem from the dysfunction of the ubiquitin-mediated regulation of neuronal proteins.

USP15 stabilizes the transcription factor Nrf1 in the nucleus, promoting the proteasome gene expression.

The transcriptional factor Nrf1 (NF-E2-related factor 1) sustains protein homeostasis (proteostasis) by regulating the expression of proteasome genes. Under physiological conditions, the transcriptional activity of Nrf1 is repressed by its sequestration into the endoplasmic reticulum (ER) and furthermore by two independent ubiquitin-proteasome pathways, comprising Hrd1 and ß-TrCP in the cytoplasm and nucleus, respectively. However, the molecular mechanisms underlying Nrf1 activation remain unclear. Here, we report that USP15 (Ubiquitin-Specific Protease 15) activates Nrf1 in the nucleus by stabilizing it through deubiquitination. We first identified USP15 as an Nrf1-associated factor through proteome analysis. USP15 physically interacts with Nrf1, and it markedly stabilizes Nrf1 by removing its ubiquitin moieties. USP15 activates the Nrf1-mediated expression of a proteasome gene luciferase reporter and endogenous proteasome activity. The siRNA-mediated knockdown of USP15 diminishes the Nrf1-induced proteasome gene expression in response to proteasome inhibition. These results uncover a new regulatory mechanism that USP15 activates Nrf1 against the ß-TrCP inhibition to maintain proteostasis.