Characterization of antimicrobial susceptibility, extended-spectrum ß-lactamase genes and phylogenetic groups of Shigatoxin producing Escherichia coli isolated from patients with diarrhea in Iran.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) are among common foodborne bacterial pathogens and healthy livestock are the main source of this bacterium. Severe diseases attribute to two types of cytotoxin Stx1 and Stx2, which are also called Shiga toxin (Stx). Infection of humans with STEC may result in Acute diarrhea with or without bleeding, hemorrhagic colitis (HC) and the hemolytic uremic syndrome (HUS). As antibiotic resistance is increasingly being reported among STEC isolates obtained from livestock and patients worldwide, in this study the pattern of antibiotic resistance in clinical isolates was determined. METHODS: Stool samples were collected from patients with diarrhea. All samples were cultured and identified by biochemical and molecular tests. Antimicrobial susceptibility test and assessment of extended-spectrum ß-lactamase (ESBL)-related genes were conducted. Moreover, phylogenetic groups were analyzed using quadruplex PCR, and DNA analysis assessed multi-locus sequence types (MLST). RESULTS: Out of 340 E. coli samples, 174 were identified as STEC by PCR. Antimicrobial susceptibility test results showed that, 99.4%, 96% and 93.1% of isolates were susceptible to imipenem/ertapenem, piperacillin-tazobactam and amikacin, respectively. The highest resistance was towards ampicillin (68.4%), followed by trimethoprim-sulfamethoxazole (59.8%), and tetracycline (57.5%). A total of 106 (60.9%) isolates were multidrug resistance (MDR) and 40.8% of isolates were determined to be extended spectrum ß-lactamase producers. In 94.4% of isolates, genes responsible for ESBL production could be detected, and blaTEM was the most prevalent, followed by blaCTX-M9. Furthermore, phylogenetic grouping revealed that majority of STEC strains belonged to Group C, followed by Groups E, B2 and A. MLST unveiled diverse ST types. CONCLUSION: A periodical surveillance studies and thorough understanding of antibiotic resistant profiles in STEC isolates could help select effective antibiotic treatment for patients and develop strategies to effectively manage food contamination and human infections.

Anti-tumor activity of Escherichia coli Shiga toxin A subunit delivered by SF9 insect cells.

Cancer remains a major health problem around the world. A Shiga toxin is a bacterial toxin often produced by Shigella dysenteriae and Escherichia coli. A subunit of the Shiga toxin (StxA) is a cytotoxic agent which could be used to induce death in cancer cells. StxA expressed from baculovirus was evaluated in a pTriEx™ expression vector. The baculovirus vector was used for the A subunit delivery of StxA. StxA cell cytotoxicity was induced by the virus and assessed in the MCF7 and HeLa cell lines. In addition, the breast cancer cytotoxicity of the expressed StxA was also assessed in a cancer induced in mice. The cytotoxicity of the recombinant StxA baculovirus with different multiplicities of infection (MOI) was measured. The results showed that significant cytotoxicity can be induced on the mammalian epithelial breast cancer cell lines, MCF7 and HeLa cells with MOI ≥ 2. The results also showed that a malignant tumor induced by MCF7 could be inhibited in a mouse cancer model. Therefore, it can be concluded that StxA, expressed by baculovirus, could be used for in vitro and in vivo gene delivery. In this study StxA, delivered by the baculovirus inhibited cell proliferation, and eliminated HeLa and MCF7 cells, in vitro. In conclusion, this method can be used as a safe alternative for anticancer drug delivery inside cancer cells.

Transposons: the agents of antibiotic resistance in bacteria.

Transposons are a group of mobile genetic elements that are defined as a DNA sequence. Transposons can jump into different places of the genome; for this reason, they are called jumping genes. However, some transposons are always kept at the insertion site in the genome. Most transposons are inactivated and as a result, cannot move. Transposons are divided into two main groups: retrotransposons (class І) and DNA transposons (class ІІ). Retrotransposons are often found in eukaryotes. DNA transposons can be found in both eukaryotes and prokaryotes. The bacterial transposons belong to the DNA transposons and the Tn family, which are usually the carrier of additional genes for antibiotic resistance. Transposons can transfer from a plasmid to other plasmids or from a DNA chromosome to plasmid and vice versa that cause the transmission of antibiotic resistance genes in bacteria. The treatment of bacterial infectious diseases is difficult because of existing antibiotic resistance that part of this antibiotic resistance is caused by transposons. Bacterial infectious diseases are responsible for the increasing rise in world mortality rate. In this review, transposons and their roles have been studied in bacterial antibiotic resistance, in detail.

In silico design, cloning, expression and immunologic evaluation of ED fusion protein of NT H. influenzae.

Infections due to nontypeable Haemophilus influenzae (NTHi) are important causes of child mortality throughout the world. Given the lack of effective vaccines for these strains and the spread and prevalence of these infections in the world, it is necessary to design novel vaccine candidates against these strains. D and E proteins are conserved membrane-specific lipoproteins among encapsulated and non-encapsulated H. influenza strains, which, according to the exposure surface and conservation degree between both strains, can be considered as vaccine candidates suitable for studies. This research was conducted to design a recombinant truncated fusion protein ED. Vaccination of BALB/c mice with recombinant truncated fusion protein ED showed high level of protective responses against NTHi. There were also strong responses of IgG and its subclasses (especially IgG1) as well as high titer levels of IL-4. A mixture of responses was observed considering IgG2a and INF-γ antibody titers, but the dominant response was toward Th2. According to the obtained results and the importance of humoral immunity in the immune system and vaccines production, it could be concluded that the produced recombinant construct can be used as a suitable vaccine candidate against NTHi or together with other carrier proteins.

DNA aptamer identification and characterization for E. coli O157 detection using cell based SELEX method.

Escherichia coli (E. coli) O157:H7 is a foodborne pathogen that causes symptoms in humans. Its rapid identification should be considered to avoid toxic effects of the pathogen. In this study, systematic evolution of ligands by exponential enrichment using whole cells (Cell-SELEX) method was used for recognizing E. coli strain, O157 by single-stranded DNA library of aptamer. Nine rounds of cell-selex procedure were applied using O157, as a whole-cell target, with O42, K12, Top10, DH5α E. coli cells, Shigella flexneri and Salmonella typhi as counterparts. The specific interaction between selected DNA aptamers and targeted cell was assessed. After applying six rounds of SELEX for selection of DNA aptamers, the candidate sequences were obtained. Finally, specific aptamer was selected as an ideal aptamer for detection and capturing of E. coli O157. Dissociation constant of the selected aptamer were calculated (107.6 ± 67.8 pM). In addition, the secondary structure prediction and cross reactivity assays were performed. The isolated aptamer efficiency was confirmed and it was shown that the new DNA aptamer sequence has the ability to use for detection. This specific O157:H7 aptamer have the potential for application as a diagnostic ligand and could be used for detection of the related food borne diseases.

Inhibition of the blaOXA-48 gene expression in Klebsiella pneumoniae by a plasmid carrying CRISPRi-Cas9 system.

Antibiotic resistance is an increasing concern that threatens the effectiveness of treating bacterial infections. The spread of carbapenem resistant Klebsiella pneumoniae poses a significant threat to global public health. To combat this issue, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system is being developed. This system includes a single guide RNA (sgRNA) and a nuclease dead Cas9 (dCas9), which work together to downregulate gene expression. Our project involved the use of the CRISPRi system to reduce gene expression of the beta-lactamase oxacillin-48 (blaOXA-48) gene in K. pneumoniae. We designed a sgRNA and cloned it into pJMP1363 plasmid harboring the CRISPRi system. The pJMP1363-sgRNA construct was transformed in K. pneumoniae harboring the blaOXA-48 gene. The MIC test was used to evaluate the antimicrobial resistance, and quantitative real-time RT-PCR was used to confirm the inhibition of the OXA-48 producing K. pneumoniae harboring the pJMP1363-sgRNA construct expression. The Galleria mellonella larvae model was also utilized for in vivo assay. Following the transformation, the MIC test indicated a 4-fold reduction in meropenem resistance, and qRT-PCR analysis revealed a 60-fold decrease in the mRNA OXA-48 harboring the pJMP1363-sgRNA construct expression. Additionally, G. mellonella larvae infected with OXA-48 producing K. pneumoniae harboring the pJMP1363-sgRNA showed higher survival rates. Based on the findings, it can be concluded that the CRISPR interference technique has successfully reduced antibiotic resistance and virulence in the K. pneumoniae harboring the blaOXA-48 gene.

Sequence-subtype association of multi-drug-resistant diarrheagenic Escherichia coli.

Background and Objectives: Multi-drug-resistant pathogens pose a significant threat as they can rapidly spread, leading to severe healthcare-associated invasive infections. In developing countries, diarrheagenic Escherichia coli (DEC) is a major bacterial pathogen responsible for causing diarrhea. However, the outbreak of resistant strains has made the treatment of DEC infections much more challenging. This study aimed to investigate the relationship between antibiotic resistance genes and other virulence categories in E. coli strains that cause diarrhea, particularly DEC. Materials and Methods: The phylogenetic grouping was defined using PCR and multi-locus sequence type (MLST) methods. Results: Among the isolates analyzed, 14 were identified as resistant and were classified into eight distinct sequence types: ST3, ST53, ST77, ST483, ST512, ST636, ST833, and ST774, indicating genetic diversity among the resistant strains. Certain sequence types, notably ST512 and ST636, were found to be associated with multiple antibiotic resistance in DEC. Regarding antibiotic susceptibility, strains showed the highest resistance to amoxicillin, suggesting that this antibiotic may not be effective in treating DEC infections. On the other hand, the isolates demonstrated susceptibility to amikacin and chloramphenicol, implying that these antibiotics could be more suitable treatment options for DEC infections. Conclusion: The findings underscore the importance of promptly identifying antibiotic resistance patterns and their correlation with specific pathogenic virulence categories, as this knowledge can aid in selecting the most appropriate antibiotics for treating DEC infections. Considering the antibiotic resistance profiles and associated resistance genes is crucial in managing and containing diarrheal outbreaks and in selecting effective antibiotic therapies for DEC infections.

Inhibition of Mir-21-5p Affects the Expression of LNCRNA X-Inactive Specific Transcript and Induces Apoptosis in MCF-7 Breast Cancer Cells.

Background: We aimed to investigate miR-21-5p inhibition effect on lncRNA-XIST expression and apoptosis status of MCF-7 cells. Methods: The MCF-7 cells were cultured and transfected by the anti-miR-21-5p oligonucleotide and expression of miR-21-5p, lncRNA-XIST, apoptosis-associated genes (bax and p53) and one miR-21-5p-unrelated lncRNA (BC200) was assessed by RT-qPCR. Furthermore, cell viability checked by MTT assay and apoptosis and cell cycle in transfected cells were detected by flow cytometry. Also, bioinformatics analysis on the transcriptome data confirmed that the lncRNA XIST might have a critical role in breast cancer (BC) cell apoptosis through ceRNAs mechanism and possible regulatory interactions with miR-21-5p. Results: Expression of miR-21-5p and lncRNA-XIST was significantly down- and up-regulated respectively (P<0.05). However, there was no significant change in lncRNA-BC200 expression. Also, the expression of bax and p53 upraised significantly (P<0.05). In transfected cells, MTT and flow cytometry assays reported a highly significant decrease and increase in viability and apoptosis respectively. Conclusion: Inhibition of miR-21-5p resulted in significant upregulation of lncRNA-XIST and apoptosis-associated genes bax and p53, which led to the induction of apoptosis in MCF-7 cells. Therefore, more investigations may provide a valuable target for studies on molecular therapies for BC.

Circulating LncRNAs landscape as potential biomarkers in breast cancer.

BACKGROUND: In Iran, the delay in diagnosis and treatment of breast cancer results in low survival rates. AIM: It is essential to characterize new therapeutic targets and prognostic breast cancer biomarkers. The rising evidence suggested that long non-coding RNAs (lncRNAs) expression levels are deregulated in human cancers and can use as biomarkers for the rapid diagnosis of breast cancer. METHODS: In the present study, a quantitative real-time polymerase chain reaction (qRT-PCR) technique was used to measure 20 oncogenic and tumor suppressor lncRNAs expression levels in whole blood samples of female breast cancer patients and healthy women. Receiver operating characteristic curve (ROC) was used to assess the diagnostic value of each selected lncRNA as a biomarker. RESULTS: The results revealed that some circulating lncRNAs (MEG3, NBAT1, NKILA, GAS5, EPB41L4A-AS2, Z38, and BC040587) were significantly down-regulated in breast cancer patients compared to healthy women. In contrast, other circulating lncRNAs (H19, SPRY4-IT1, XIST, UCA1, AC026904.1, CCAT1, CCAT2, ITGB2-AS, and AK058003) were significantly up-regulated in breast cancer patients compared to controls. It was shown that the expression levels of NKILA, and NBAT1 lncRNAs were related to tumor size, and BC040587 expression level related to age, node metastasis, tumor size, and grade (p < .05). The association between H19 and SPRY4-IT1 lncRNAs with HER-2 was confirmed statistically (p < .05). ROC curves illustrated that the blood levels of SPRY4-IT1, XIST, and H19 lncRNAs have excellent potential in discriminating breast cancer from the healthy controls, showing an AUC of 1.0 (95% CI 1.0-1.0, p = .00), 0.898 (95% CI 0.815-0.981, p = .00), and 0.848 (95% CI 0.701-0.995, p = .01), respectively. CONCLUSION: In conclusion, the expression levels of circulating H19 and SPRY4-IT1 lncRNAs in breast cancer patients could consider as the prognostic biomarkers and therapeutic targets in breast cancer, because of their excellent power in discriminating breast cancer from healthy individuals and the significant correlation of H19, and SPRY4-IT1 lncRNAs with clinicopathological traits. We also suggest the possible application of BC040587 lncRNA as a diagnostic and prognostic indicator to assess tumor progression in case of verification in larger patients' cohorts.

Antibacterial Properties and Efficacy of LL-37 Fragment GF-17D3 and Scolopendin A2 Peptides Against Resistant Clinical Strains of Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii In Vitro and In Vivo Model Studies.

Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii have emerged as major clinical threats owing to the increasing prevalence of ventilator-associated pneumonia caused by multidrug-resistant or extensively drug-resistant strains. The present study aimed to assess the antibacterial effects and efficacy of LL-37 fragment GF-17D3 and synthetic Scolopendin A2 peptides against resistant clinical strains in vitro and in vivo models. P. aeruginosa, S. aureus, and A. baumannii were isolated from clinical infections. Their antibiotic resistance and minimum inhibitory concentration were assessed. LL-37 fragment GF-17D3 peptide was selected from available databases. Scolopendin A2 peptide's 6th amino acid (proline) was substituted with lysine and peptides and MICs were determined. The biofilm inhibitory activity was quantified at sub MIC concentrations. Synergetic effects of Scolopendin A2 and imipenem were assessed by checkerboard. After mice nasal infection with P. aeruginosa, peptides LD50 was determined. Isolates harbored complete resistance toward the majority of antibiotics and MIC values ranged between 1 and > 512 µg/ml. The majority of isolates exhibited strong biofilm activity. Synthetic peptides showed lower MIC values than antibiotic agents and the lowest MIC values were obtained for synthetic peptides in combination with antibiotics. The Synergisms effect of Scolopendin A2 with imipenem was also determined. Scolopendin A2 was found to have antibacterial efficacy against P. aeruginosa, S. aureus, and A. baumannii with MIC 64 µg/ml, 8 µg/ml, and 16 µg/ml, respectively, and LL37 showed antibacterial efficacy against P. aeruginosa, S. aureus, and A. baumannii with MIC 128 µg/ml, 32 µg/ml, and 32 µg/ml, respectively. Both AMPs decreased biofilms by ≥ 96% at 1 × MIC. The biofilm inhibitory activity was measured at sub MIC concentrations of the peptides and the results demonstrated that Scolopendin A2 exhibited anti-biofilm activity at 1/4 × MIC and 1/2 × MIC concentrations was 47.9 to 63.8%, although LL37 among 1/4 × MIC and 1/2 × MIC concentrations was 21.3 to 49.6% against three pathogens. The combination of Scolopendin A2 and antibiotics demonstrated synergistic activity-resistant strains with FIC values ≤ 0.5 for three pathogens, while LL37 and antibiotics showed synergistic activity FIC values ≤ 0.5 for only P. aeruginosa. Infection model Scolopendin A2 with Imipenem (2 × MIC) was efficacious in vivo, with a 100% survival rate following treatment at 2 × MIC after 120 h. The mRNA expression of biofilm-related genes was decreased for both peptides. Synthesis Scolopendin A2 decreased the expression of biofilm formation genes compared to the control group. Synthetic Scolopendin A2 exhibits antimicrobial activity without causing toxicity on the human epithelial cell line. Based on our findings, it seems that synthetic Scolopendin A2 is an appropriate antimicrobial source. That could be a promising option in combination with antibiotics for a topical medication and in the prevention of acute and chronic infections caused by multidrug-resistant bacteria. Nevertheless, additional experiments are required to assess another potential of this novel AMP.

Attempts to express the A1-GMCSF immunotoxin in the baculovirus expression vector system.

Immunotoxins are fusion proteins consisting of two elements, a targeting and a toxin moiety, and are designed for specific elimination of tumor cells. Previously we expressed a recombinant fusion protein consisting of the toxic fragment of Shiga toxin (A1) and GMCSF (A1-GMCSF) in Escherichia coli, and evaluated its cytotoxic properties in acute myeloid leukemia and colon carcinoma cell lines. In view of the specific cytotoxic effects of this immunotoxin, further detailed in-vitro and preclinical studies were undertaken. Large amounts of the recombinant protein of high purity and free of unwanted side products, such as lipopolysaccharides (LPS), were required. Since GMCSF is of mammalian origin and it requires proper disulfide bond formation, we intended to use the baculovirus expression vector system (BEVS) for the expression of the recombinant fusion protein. However, despite previous reports on the expression of several other immunotoxins by this system, the A1 derived fusion proteins revealed an inhibitory effect on baculoviral particle formation and even caused cell death in insect cells. This observation was further pursued and confirmed by the use of other baculoviral specific promoters. The salient features of this finding are described below.

Quantitative CK19 biomarker detection in breast cancer cell lines.

Cytokeratin19 (CK19) was detected as the most related marker for circulating tumor cells, which was assessed in specific cell lines. MCF7, SKBR3, T47D, and MDA-MB-231, and HeLa cell line as negative control were used. CK19 expression was confirmed by using mouse monoclonal anti-human CK19 antibody. CK19 detection in MDA-MB-231 was not observed. CK19 marker expression was compared in T47D, MCF7, and SKBR3 cell lines. T47D and MCF7 belonged to the luminal subtype of breast cancer (BC) that CK19 expression regulated with an ER marker. SKBR3 belonged to the HER2 positive subtype of BC. However, MDA-MB-231 belonged to the claudin-low subtype of BC that lack of CK19 expression strongly is related to negative ER, PR, and HER2. Therefore, there are not only quantitative differences in CK19 expression, but its expression could also link to the other markers of BC that should be considered in the molecular classification of breast carcinoma. Different expression levels related to cell classification could be useful in the prognosis and treatment of cancers with epithelial origins.

Protein Kinase Signaling by Shiga Toxin Subunits.

Background: Escherichia coli produces Shiga toxin (Stx), a pentamer composed of one A subunit and four B subunits. The B subunit of Stx (StxB) mediated the attachment of the holotoxin to the cell surface while the A subunit (StxA) has N-glycosidase activity, resulting in protein synthesis and cell death inhibition. Stx-induced cytotoxicity and apoptosis have been observed in various cell lines, although the signaling effectors are not precisely defined. Activated by protein kinases (PK), the signaling pathway in human tumors plays an oncogenic role. Tumor proliferation, survival, and metastasis are promoted by kinase receptors. In this regard, PK regulatory effects on the cellular constituents of the tumor microenvironment can affect immunosuppressive purposes. Methods: In this study, kinase inhibitors were used to evaluate the influence of Stx and its subunits on HeLa and Vero cells. Selective inhibitors of protein kinase C (PKC), CaM kinase (calmodulin kinase), protein kinase A (PKA), and protein kinase G (PKG) were used to compare the signaling activity of each subunit. Results: The ribotoxic activity in the target cells will lead to rapid protein synthesis inhibition and cell death in the mammalian host. The expression of Bcl2 family members was also assessed. Protein kinase signaling by Stx and its A and B subunits was induced by PKA, PKG, and PKC in HeLa cells. CaM kinase induction was significant in Vero cells. StxB significantly induced the pro-apoptotic Bax signaling factor in HeLa cells. Conclusion: The assessment of different signaling pathways utilized by Stx and its subunits could help in a better understanding of various cell death responses. The use of inhibitors can block cell damage and disease progression and create therapeutic compounds for targeted cancer therapy. Inhibition of these pathways is the primary clinical goal.

Bacterial DNA Detection in the Blood of Healthy Subjects.

Background: The presence of microbiome in the blood samples of healthy individuals has been addressed. However, no information can be found on the healthy human blood microbiome of Iranian subjects. The current study is thus aimed to investigate the existence of bacteria or bacterial DNA in healthy individuals. Methods: Blood samples of healthy subjects were incubated in BHI broth at 37 °C for 72 h. The 16S rRNA PCR and sequencing were performed to analyze bacterial isolates. The 16S rRNA PCR was directly carried out on DNA samples extracted from the blood of healthy individuals. Next generation sequencing (NGS) was conducted on blood samples with culture-positive results. Results: Fifty blood samples were tested, and six samples were positive by culture as confirmed by Gram staining and microscopy. The obtained 16S rRNA sequences of cultured bacterial isolates revealed the presence of Bacilli and Staphylococcus species by clustering in the GeneBank database (≥97% identity). The 16S rRNA gene sequencing results of one non-cultured blood specimen showed the presence of Burkholderia. NGS results illustrated the presence of Romboutsia, Lactobacillus, Streptococcus, Bacteroides, and Staphylococcus in the blood samples of positive cultures. Conclusion: The dormant blood microbiome of healthy individuals may give the idea that the steady transfer of bacteria into the blood does not necessarily lead to sepsis. However, the origins and identities of blood-associated bacterial rDNA sequences need more evaluation in the healthy population.

Computational evaluation of modified peptides from human neutrophil peptide 1 (HNP-1).

The development of bacterial resistance toward antibiotics has been led to pay attention to the antimicrobial peptides (AMPs). The common mechanism of AMPs is disrupting the integrity of the bacterial membrane. One of the most accessible targets for α-defensins human neutrophil peptide-1 (HNP-1) is lipid II. In the present study, we performed homology modeling and geometrical validation of human neutrophil defensin 1. Then, the conformational and physicochemical properties of HNP-1 derived peptides 2Abz14S29, 2Abz23S29, and HNP1ΔC18A, as well as their interaction with lipid II were studied computationally. The overall quality of the predicted model of full protein was -5.14, where over 90% of residues were in the most favored and allowed regions in the Ramachandran plot. Although HNP-1 and HNP1ΔC18A were classified as unstable peptides, 2Abz14S29 and 2Abz23S29 were stable, based on the instability index values. Molecular docking showed similar interaction pattern of peptides and HNP-1 to lipid II. Molecular dynamic simulations revealed the overall stability of conformations, though the fluctuations of amino acids in the modified peptides were relatively higher than HNP-1. Further, the binding affinity constant (Kd) of HNP-1 and 2Abz23S29 in complex with lipid II was 10 times stronger than 2Abz14S29 and HNP1ΔC18A. Overall, computational studies of conformational and interaction patterns have signified how derived peptides could have displayed relatively similar antimicrobial results compared to HNP-1 in the reported experimental studies. Chemical modifications not only have improved the physicochemical properties of derived peptides compared to HNP-1, but also they have retained the similar pattern and binding affinity of peptides. Communicated by Ramaswamy H. Sarma.

Emergence of K1 ST23 and K2 ST65 hypervirulent klebsiella pneumoniae as true pathogens with specific virulence genes in cryptogenic pyogenic liver abscesses Shiraz Iran.

Hypervirulent Klebsiella pneumoniae (hvKp) pathotype is emerging worldwide in pyogenic liver abscesses (PLAs). However, the role of virulence factors in pathogenicity remains unclear. On the other hand, the epidemiology of PLAs in Iran is unknown. From July 2020 to April 2022, bacterial species were isolated and identified from the drainage samples of 54 patients with PLAs. K. pneumoniae as the most common pathogen of pyogenic liver abscesses was identified in 20 (37%) of the 54 patients. We analyzed the clinical and microbiological characteristics of K. pneumoniae-related pyogenic liver abscesses. Antibiotic susceptibility testes and string test were performed. 16S rRNA, antibiotic resistance, and virulence genes were determined by polymerase chain reaction amplification. Clonal relatedness of isolates was identified by multilocus sequence typing. Virulence levels were assessed in the Galleria mellonella larval infection model. Four hvKp isolates (K1/K2) were found to be responsible for cryptogenic PLAs, and 16 classical K. pneumoniae isolates (non-K1/K2) were associated with non-cryptogenic PLAs. Three capsular serotype K1 strains belonged to sequence type 23 (ST23) and one K2 strain to ST65. Meanwhile, the non-K1/K2 strains belonged to other STs. ST231 was the most common strain among the classical K. pneumoniae strains. Compared with the non-K1/K2 strains, capsular serotypes K1/K2 strains were less resistant to antibiotics, had positive string test results, and had more virulence genes. In Galleria mellonella, a concentration of 106 colony-forming units of the K1 hvKp strain resulted in 100% death at 24 hours, confirming the higher virulence of the hvKp strain compared with cKp. K. pneumoniae isolates represented that the acquisition of any plasmid or chromosomal virulence genes contributes to pathogenicity and high prevalence in PLAs. Meanwhile, hvKp isolates with a specific genetic background were detected in cryptogenic PLAs.

Assessment of immune response of the B subunit of Shiga toxin fused to AAF adhesin of enteroaggregative Escherichia coli.

Shiga toxin is a member of AB toxin family and is composed of an A subunit which mediated toxicity and a homopentameric protein responsible for toxin binding and internalization into target cells. Another group of diarrheagenic Escherichia coli, enteroaggregative E. coli (EAEC) is a group of E. coli with aggregative adherence to epithelial cells, which play an important role in its pathogenesis. In the present investigation, the immune response of recombinant hybrid peptide composed of B subunit of Shiga toxin (StxB) and Aggregative Adherence Fimbriae (AAF) of EAEC (B-AAF/I, B-AAF/II) that elicited protective response was further characterized. The assessment of IgG subclasses (IgG1 and IgG2a) and cytokine production by these peptides indicated that although the hybrid peptides could induce immune response, but two adhesins behave differently in this regard. Lymphocyte proliferation assay and IFN-γ production were highly significant for B-AAF/II. Overall, based on the data obtained from this study it seems that mixed population of Th1-Th2 type of immune responses were induced by these hybrid peptides, which probably lead to observed protective response. In the present study, it is shown that the two hybrid peptides i.e. B-AAF/I and B-AAF/II, could be a promising strategy to make more effective and powerful vaccine.

LncRNAs AK058003 and MVIH Overexpression in the Blood Samples of Iranian Breast Cancer Patients.

Background: Long non-coding RNAs (LncRNAs) are considered as novel biological regulators and potential cancer biomarkers. LncRNAs microvascular invasion in hepatocellular carcinoma (HCC; microvascular invasion [MVIH]) and AK058003 are associated with MVIH in HCC. In breast cancer (BC), upregulated MVIH and AK058003 expression levels have been shown to promote cell proliferation, though LncRNA-AK058003 acts as a tumor suppressor in HCC. Methods: Blood samples were collected from 30 healthy women and 30 female BC patients. RNA was extracted from the blood of both groups, and cDNA was then synthesized. A real-time PCR technique was conducted to measure the expression level of LncRNA-AK058003 and MVIH. Results: The expression level of two LncRNAs in the blood samples of BC patients increased significantly compared with healthy individuals. The levels of AK058003 and MVIH were not associated with lymph node metastasis (p = 0.402 and p = 0.39), tumor size (p = 0.76 and p = 0.461), and tumor size; lymph nodes, metastasis stage (TNM; p = 0.574 and p = 0.711), respectively. Conclusion: As per our findings, LncRNA-AK058003 could serve as a suitable indicator for low stage of BC. In addition, the increased level of LncRNA-MVIH could be considered as a biomarker for BC, which needs more evaluation in the future.

Circulating Tumor Cells Detection in Patients with Early Breast Cancer Using MACS Immunomagnetic Flow Cytometry.

BACKGROUND: Circulating Tumor Cells (CTCs) detection in peripheral blood of epithelial cancer patients is an indicator of the presence of primary tumors and metastasis. The CTC phenotype detection uses epithelial markers in defining, detecting, and isolating CTCs. Circulating cell-separation technologies, with the epithelial origin, can be identified by epithelial biomarkers, with different techniques such as flow cytometry. The purpose of this study was to evaluate the expression of molecular Cytokeratins (CKs), CK7, CK8, CK18, CK19 (Pan-CK) and Epithelial Cell Adhesion Molecule (EpCAM) markers for CTC detection. METHODS: The Magnetic Activated Cell Sorting (MACS) was used to identify CTCs in the blood of patients. Specific antibodies to EpCAM and Pan-CK were used and analyzed by flow cytometry. In this study, 35 blood samples of patients with breast cancer were assessed before any treatment and 35 healthy blood samples as the control were evaluated. RESULTS: Expression of CK markers in the peripheral blood of breast cancer patients was statistically significant with p≤0.05, specifically at stages II-IV, but it was not significant in patients at stage I and healthy controls. Biomarkers expression in the blood of patients and healthy controls was assessed along with the pathologic characteristics of patients. CONCLUSION: CTC assessment by flow cytometry in patients with breast cancer could not only be used for detection but also can be considered as a source of specific and subjective evaluation for monitoring the therapy. Besides, the sensitivity and specificity of CTC detection were shown that could be enhanced by specific CK markers.