Characterization of Antimicrobial Susceptibility, Extended-Spectrum β-Lactamase Genes and Phylogenetic Groups of Enteropathogenic Escherichia coli Isolated from Patients with Diarrhea

Objectives@#Infectious diarrhea is one of the most common causes of pediatric death worldwide and enteropathogenic Escherichia coli (EPEC) is one of the main causes. There are 2 subgroups of EPEC, typical and atypical, based on the presence or absence of bundle forming pili (bfp), of which atypical EPEC is considered less virulent, but not less pathogenic. Antimicrobial resistance towards atypical EPEC among children is growing and is considered a major problem. In this study the pattern of antibiotic resistance in clinical isolates was determined. @*Methods@#Using 130 isolates, antibiotic resistance patterns and phenotypes were assessed, and genotypic profiles of extended spectrum β-lactamase (ESBL) production using disc diffusion and PCR was carried out. Phylogenetic groups were analyzed using quadruplex PCR. @*Results@#There were 65 E. coli isolates identified as atypical EPEC by PCR, among which the highest antibiotic resistance was towards ampicillin, followed by trimethoprim-sulfamethoxazole, and tetracycline. Multidrug resistance was detected in 44.6% of atypical EPEC isolates. Around 33% of isolates were determined to be extended spectrum β-lactamase producers, and in 90% of isolates, genes responsible for ESBL production could be detected. Moreover, the majority of atypical EPEC strains belonged to Group E, followed by Groups B1, B2 and C. @*Conclusion@#High rates of multidrug resistance and ESBL production among atypical EPEC isolates warrant periodical surveillance studies to select effective antibiotic treatment for patients. It is considered a critical step to manage antibiotic resistance by avoiding unnecessary prescriptions for antibiotics.

Relationships between Virulence Factors and Antimicrobial Resistance among Escherichia coli Isolated from Urinary Tract Infections and Commensal Isolates in Tehran, Iran

OBJECTIVES: Uropathogenic Escherichia coli (UPEC) are the major cause of urinary tract infections (UTIs). Here, we determined whether sensitivity to antibiotics was related to the prevalence of iron scavenging genes, or to biofilm and hemolysis formation. METHODS: A total of 110 UPEC and 30 E coli isolates were collected from the urine of UTI patients and feces of healthy individuals without UTI, respectively. The presence of iron receptor genes and phenotypic properties were evaluated by polymerase chain reaction and phenotypic methods, respectively. Susceptibility to routine antibiotics was evaluated using the disc diffusion method. RESULTS: The prevalence of iron scavenging genes ranged from 21.8% (ireA) to 84.5% (chuA) in the UPEC. Resistance to ceftazidime and cefotaxime was significantly correlated with the presence of fyuA and iutA iron genes. Biofilm production was significantly associated with the prevalence of fyuA and hma iron genes. A higher degree of antibiotic resistance was exhibited by isolates that produced biofilms than by their non-biofilm producing counterparts. CONCLUSION: Our study clearly indicates that biofilm production is associated with antibiotic resistance, and that iron receptors and hemolysin production also contribute to reduced antibiotic sensitivity. These results further our understanding of the role that these virulence factors play during UPEC pathogenesis, which in turn may be valuable for the development of novel treatment strategies against UTIs.

Evaluation of prevalence, homology and immunogenicity of dispersin among enteroaggregative Escherichia coli isolates from Iran

Background: Diarrhea, caused by enteroaggregative Escherichia coli [EAEC], is an important infection leading toillness and death. Numerous virulent factors have been described in EAEC. However, their prevalence was highly variable among EAECs of distinct geographic locations. Studies have shown that dispersin [antiaggregation protein, aap] is one of the important and abundant virulent factors in EAEC. In this study, we aimed to determine the presence, conservation, and immunogenicity of aap gene in EAEC isolated from Iranian patients

Methods: PCR amplification of aap gene in the EAEC isolates was performed, and the aap gene was cloned in pBAD-gIIIA vector. The sequence of aap gene was analyzed using the ExPASy and BLAST tools. The expression of aap gene was performed in E. coli Top10, and expression confirmation was carried out by SDS-PAGE and Western-blot techniques. Rabbits were immunized with purified dispersin protein emulsified with Freund's adjuvant. Sera were collected and examined for antibody response. Finally, in vitro efficacy of dispersin and anti-dispersin was evaluated

Results: The results of PCR showed the presence of aap gene in all of the EAEC isolates with significant homology. Finally, the significant difference between the levels of IgG response in dispersin-injected rabbits and control group was observed

Conclusion: Our results were in accordance with other studies that reported the presence of dispersin in the EAEC isolates with high conservation and immunogenicity. Hence, dispersin could be a promising candidate for any probable prevention against EAEC infections

In Silico Signature Prediction Modeling in Cytolethal Distending Toxin-Producing Escherichia coli Strains

In this study, cytolethal distending toxin (CDT) producer isolates genome were compared with genome of pathogenic and commensal Escherichia coli strains. Conserved genomic signatures among different types of CDT producer E. coli strains were assessed. It was shown that they could be used as biomarkers for research purposes and clinical diagnosis by polymerase chain reaction, or in vaccine development. cdt genes and several other genetic biomarkers were identified as signature sequences in CDT producer strains. The identified signatures include several individual phage proteins (holins, nucleases, and terminases, and transferases) and multiple members of different protein families (the lambda family, phage-integrase family, phage-tail tape protein family, putative membrane proteins, regulatory proteins, restriction-modification system proteins, tail fiber-assembly proteins, base plate-assembly proteins, and other prophage tail-related proteins). In this study, a sporadic phylogenic pattern was demonstrated in the CDT-producing strains. In conclusion, conserved signature proteins in a wide range of pathogenic bacterial strains can potentially be used in modern vaccine-design strategies.

In silico analysis of Brucella abortus Omp2b and in vitro expression of SOmp2b

PURPOSE: At present, there is no vaccine available for the prevention of human brucellosis. Brucella outer membrane protein 2b (Omp2b) is a 36 kD porin existed in common Brucella pathogens and it is considered as priority antigen for designing a new subunit vaccine. MATERIALS AND METHODS: In the current study, we aimed to predict and analyze the secondary and tertiary structures of the Brucella abortus Omp2b protein, and to predict T-cell and B-cell epitopes with the help of bioinformatics tools. Subsequently, cloning and expression of the short form of Omp2b (SOmp2b) was performed using pET28a expression vector and Escherichia coli BL21 host, respectively. The recombinant SOmp2b (rSOmp2b) was purified with Ni-NTA column. RESULTS: The recombinant protein was successfully expressed in E. coli host and purified under denaturation conditions. The yield of the purified rSOmp2b was estimated by Bradford method and found to be 220 microg/mL of the culture. CONCLUSION: Our results indicate that Omp2b protein has a potential to induce both B-cell- and T-cell-mediated immune responses and it can be evaluated as a new subunit vaccine candidate against brucellosis.

Non-coding CK19 RNA in peripheral blood and tissue of breast cancer patients

Breast carcinoma is the major cause of cancer-related death in women. The incidence of this carcinoma is rising and there are many attempts to decrease this problem. The aim of this study was detection of full-length cytokeratin 19 [CK19] mRNA, in peripheral blood and tissue of breast cancer patients in early stage of cancer. In this study, RT-PCR [reverse transcriptase-polymerase chain reaction] technique was used for detection of CK19 mRNA in peripheral blood and tissue of breast cancer patients. Primers were established to amplify the CK19 as a tumor marker. Moreover, CYFRA 21-1 subunit of CK19 protein was measured in the serum of patients. CK19 mRNA was detected and sequenced. It is shown that the most released CK19 mRNAs in blood and tissue of cancer patients are non-coding RNA. The mutated forms of mRNA are the incomplete transcripts of protein-coding gene as a long non-coding RNA [lncRNA] that could regulate gene expression. Moreover, small non-coding RNA [ncRNA] as fragments of CK19 is mostly observed in this experiment. They may play a role in tumorogenesis and their biologic exact function in breast cancer should be further elucidated

Enteroaggregative Escherichia coli, a heterogenous, underestimated and under-diagnosed E. coli pathotype in Iran

The main features of enteroaggregative Escherichia coli [EAEC] pathogenesis include attachment of bacteria to the intestinal mucosa, production of various toxins and cytotoxins, and stimulation of mucosal inflammation. 'Virulence' genes encode these features. Comparison of different EAEC isolates has shown that the virulence gene content of these isolates varies considerably. The heterogeneity of EAEC strains was concluded from the results obtained from the volunteer as well as other studies. Although the underlying mechanism behind the apparent increase in O104:H4 virulence is not known, several bacterial factors have been implicated. In this review, the known virulence factors involved in pathogenesis of EAEC pathotype are summarized

P53 but not cyclin E acts in a negative regulatory loop to control HER-2 expression in MCF-7 breast carcinoma cell line

Cyclin E, HER-2 and p53, are considered as major prognostic markers in breast cancer. As they are related in patho-clinical level, we aimed to check if they have any direct interaction on expression of each other. To study the effect of cyclin E on HER-2 expression, cell lines stably overexpressing cyclin E or its low molecular weight [LMW] isoforms were generated. To understand the results of p53 silencing either alone or in combination with cyclin E overexpression, we created three different p53 stably knocked down cell lines. Protein expression was analyzed by western blot, HER-2 expression in the established cell lines were determined using SYBR green real time PCR and data analyzed by REST software. Results indicate that HER-2 expression is only downregulated following p53 silencing and none of cyclin E isoforms can alter its expression. The presence of cyclin E isoforms in p53 silenced clones also does not altered HER-2 expression. Given the fact that p53 degradation is increased by HER-2 overexpression, these data can draw a regulatory loop in which a non-mutated functional p53 and HER-2 can bidirectionally regulate the expression of these two genes. This study improves our understandings of these pathways and these proteins can be introduced either as a marker or as a target in cancer treatment.

Functional Recombinant Extra Membrane Loop of Human CD20, an Alternative of the Full Length CD20 Antigen

Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length transmembrane protein CD20. In this study, we expressed the extra membrane loop of hCD20 [exCD20] consisting of a non-glycosylated 47-amino acids region. The exCD20 coding sequence was amplified by PCR and cloned in pET32a[+] expression vector. The desired protein was expressed in fusion with thioredoxin and 6 His tag in E. coll Origami strain. ELISA and Western-blotting data were performed to indicate the functionality of this protein. We have obtained the exCD20 recombinant protein which can be detected in ELISA and Western-blot experiments. This recombinant fusion protein was soluble and stable without aggregation and misfolding problems. Conclusion: The recombinant extra membrane loop of human CD20 protein in fusion with thioredoxin [exCD20] can be used in function assays and some applications such as ELISA, immuneblotting, affinity purification, immunization, screening, and development of anti-CD20 antibodies

In vivo characterization of fusion protein comprising of al subunit of shiga toxin and human GM-CSF: assessment of its immunogenicity and toxicity

Most cancer cells become resistant to anti-cancer agents. In the last few years, a new approach for targeted therapy of human cancer has been developed using immunotoxins which comprise both the cell targeting and the cell killing moieties. In the present study, the recombinant Shiga toxin Al subunit fused to human granulocyte-macrophage colony stimulating factor [Al-GM-CSF], previously produced in E. coll, was further characterized. The recombinant protein could cause 50% cytotoxicity and induced apoptosis in cells bearing GM-CSF receptors. The non-specific toxicity of the fusion protein was assessed in C57BL/6 and BALB/c mice. No mortality was observed in either group of mice, with different concentration of fusion protein. The lymphocyte proliferation assay, induction of specific IgG response and a mixed [Thl/Th2] response were observed only in BALB/c mice. The mixed response in BALB/c mice [Thl/Th2] could be explained on the basis of the two components of the fusion protein i.e. Al and GM-CSF

Immune responses of mice immunized with active recombinant shiga toxin and its derivatives

Bacterial protein toxins have been exploited as therapeutic agents and as vaccines. An issue of deserving interest is development of new generations of vaccines and immune adjuvants. In this study an active assembled recombinant Shiga toxin of Escherichia coli [rStx1] and its derivatives, recombinant A and B subunits [Stx1-A and Stx1-B], were used to immunize mice. The elicited antibody responses were compared with and without using adjuvant. Protection against intraperitoneal lethal dose of rStx challenge was observed by immunization with sublethal dose of rStx1, rStx-A and rStx-B subunits. The immunological studies on toxin subunits can be used for immunization against systemic shiga toxin mediated disease and also subunits as a vector for antigen presentation in immunotherapeutic approaches. In our experiment, while stimulation of the immune system by A and B subunits were different, both subunits produced neutralizing antibodies. Regarding B subunit the amount of specific IgG1/IgG2a antibody ratio was higher than A subunit. In addition B subunit stimulated proliferation of immune cells with IFNê production the same as rStx1, suggesting that B subunit can be used as an immunomodulator to stimulate the immune response in conjunction with other recombinant proteins

Priming hepatitis B surface [HBsAg]- and core antigen [HBcAg]- specific immune responses by chimeric, HBcAg with a HBsAg 'a' determinant

We developed an immunogen to stimulate multivalent immunity against hepatitis B surface antigen [HBsAg] and hepatitis B core antigens [HBcAg]. Immune responses specific for both HBsAg and HBcAg play an important role in controlling the infection. HBsAg-specific antibodies mediate elimination of virions at an early stage of infection and prevent the spread of virus. The immunogen was constructed by inserting the immunodominant, antibody-binding 'a' determinant [aa 111-149] of HBsAg [with or without a poly-glycine [PG] linker] into the e2 epitope of HBcAg. Only the constructs in which the HBsAg 'a' determinant was inserted into HBcAg, flanked by PG linkers, expressed a chimeric protein in human embryonic kidney cells with HBsAg and HBcAg antigenicity. Both glycosylated and non-glycosylated forms of the chimeric protein were immunoprecipitated from cell lysate. Intramuscular DNA vaccination of mice with plasmids expressing chimeric HBcAg primed antibody responses against well-defined serologically-defined determinants of both, native HBcAg, and native HBsAg. In addition, CD8[+] T cell responses against HBcAg epitopes were primed by this chimeric HBV antigen. The e2 sequence of HBcAg can thus be used to present heterologous epitopes without loss of immunogenicity of the HBcAg protein

Expression of a Chimeric protein containing the catalytic domain of Shiga-like toxin and Human Granulocyte macrophage colony-stimulating Factor [hGM-CSF] in Escherichia coli and Its recognition by reciprocal antibodies

Fusion of two genes at DNA level produces a single protein, known as a chimeric protein. Immunotoxins are chimeric proteins composed of specific cell targeting and cell killing moieties. Bacterial or plant toxins are commonly used as the killing moieties of the chimeric immunotoxins. In this investigation, the catalytic domain of Shiga-like toxin [A1] was fused to human granulocyte macrophage colony stimulating factor [hGM-CSF] gene and the fused gene was then expressed using an expression vector containing arabinose promoter. The protein thus obtained could be recognized by two different ELISA system designed for detection of hGM-CSF and Shiga-toxin and reconfirmed by Western-blot. The recognition of the chimeric protein by specific antibodies could be indicative of the proper form of the protein, which justifies further steps to be taken to evaluate the potential effects of the chimeric protein

Construction of hybrid gene of hepatitis B surface antigen carrying heat-stable enterotoxin of escherichia coli and its expression in mammalian cell line

Hepatitis B surface antigen is the first genetically engineered vaccine licensed for human use. Various strategies have been proposed to obtain a vaccine that would bypass the need for injection. In this study, a non-toxic portion of heat-stable enterotoxin of Escherichia coli that is capable of adhering to epithelial cells was inserted at amino acid position 112 of hepatitis surface antigen. The construct was used for transfection of human embryonic kidney cells in order to assess the expression of the hybrid protein. The data obtained showed a very low level of expression. In vivo antibody production and cytotoxic T lymphocyte response in B6 mice were assessed using DNA immunization. Three out of five injected mice responded with titers 10 mIU/ml anti-HBsAg and cytotoxic T-lymphocyte response was much higher with construct encoding the chimeric protein. Although this study proves that the chimeric protein is capable of eliciting both humoral and cellular responses, but further work is required to fully explore the feasibility of combining the properties of the two proteins

PCR-mediated expression of the human GM-CSF gene in escherichia coli

Four exons of the human genomic GM-CSF gene were assembled together using gene splicing by overlap extension [SOE] method. The resulting nucleotide sequence was cloned in the pET23a [+] expression vector under the control of strong bacteriophage T7 transcription and translation signals. The construct obtained was Transferred into the E. coli strain, BL21 [DE3] pLysS and IPTG was used for induction of GM-CSF gene and production of the target protein, one mg of protein per liter of cell culture, was obtained as revealed by ELISA

Cloning and expression of thermus aquaticus DNA polymerase gene, using a thermo - inducible experession vector

DNA polymerase gene from Thermus aquaticus strain YT1 was amplified using VENTTH DNA polymerase and cloned under the control of lambda pr promoter and expression was induced by a shift in temperature. The culture was then sonicated, and after centrifugation the lysate was treated with polyethyleneimine followed by a salting-out step. Finally the protein was precipitated with ammonium sulfate and fractionated by gel filtration. The resulting enzyme preparation was stable and active