Investigation of TLR2 and TLR4 Polymorphisms and Sepsis Susceptibility: Computational and Experimental Approaches.

Toll-like receptors (TLR) play an eminent role in the regulation of immune responses to invading pathogens during sepsis. TLR genetic variants might influence individual susceptibility to developing sepsis. The current study aimed to investigate the association of genetic polymorphisms of the TLR2 and TLR4 with the risk of developing sepsis with both a pilot study and in silico tools. Different in silico tools were used to predict the impact of our SNPs on protein structure, stability, and function. Furthermore, in our prospective study, all patients matching the inclusion criteria in the intensive care units (ICU) were included and followed up, and DNA samples were genotyped using real-time polymerase chain reaction (RT-PCR) technology. There was a significant association between TLR2 Arg753Gln polymorphisms and sepsis under the over-dominant model (p = 0.043). In contrast, we did not find a significant difference with the TLR4 Asp299Gly polymorphism with sepsis. However, there was a significant association between TLR4 Asp299Gly polymorphisms and Acinetobacter baumannii infection which is quite a virulent organism in ICU (p = 0.001) and post-surgical cohorts (p = 0.033). Our results conclude that the TLR2 genotype may be a risk factor for sepsis in adult patients.

Characterization of enterohemorrhagic Escherichia coli from diarrhoeic patients with particular reference to production of Shiga-like toxin.

Enterohemorrhagic Escherichia coli (EHEC) is a subtype of pathogenic E. coli that causes diarrhea or hemorrhagic colitis in humans, which can progresses to hemolytic uremic syndrome (HUS), a leading cause of acute renal failure in children, and morbidity and mortality in adults. Stool samples (n = 273) of patients (1 day-40 years old) suffered from bloody diarrhea and abdominal cramps, were examined bacteriologically and molecularly for the presence and pathogenicity of EHEC with phylogenetic analysis of the obtained stx1, stx2, and eaeA virulence genes' sequences. Overall, 71 (26.01%) E. coli isolates were identified as EHEC with the following serogroupes: O1:H11 (3), O128:H2 (9), O26:H11 (6), O157:H7 (3), O25:H2 (7), O145:H328 (2), O125:H6 (9), O86:H8 (5), O18:H15 (11) and untypable (16). The highest isolation rate were in samples belonged to infants below two years old (42.25%). Antimicrobial susceptibility testing showed that all isolates were highly sensitive to ciprofloxacin, nitrofurantoin, gentamycin, imipenem and vancomycin (100% each), however, they were resistant to ampicillin, cephalexin, penicillin and tetracycline (100% each). In-vitro pathogenicity testing of the isolates revealed that 67 (94.37%) isolates were positive for Congo red test, 47 (66.20%) isolates possessed P fimbriae (MRHA) and 17 (23.94%) possessed type 1 fimbriae (MSHA). Moreover, 46 (64.79%) isolates exhibited hemolysis and 42 (59.15%) isolates showed distinct cytopathic effect to Vero cells. Molecular detection of enterohemorrhagic E. coli (EHEC) pathotype virulence genes, confirmed the presence of stx1 gene in O157:H7 (MA2), O26:H11, O145:H328 and O125:H6 serogroups; stx2 gene in (O157:H7 (MA1), O128:H2 and O25:H2; while all serogroups except (O125:H6) carried the eaeA intimin virulence gene. A phylogenetic tree, based on the nucleotide sequences of toxin-encoding genes, demonstrates that Shiga toxin E. coli (STEC) isolates have considerable genetic variation and belong to various phylogenetic groupings.

Role of MBL2 Polymorphisms in Sepsis and Survival: A Pilot Study and In Silico Analysis.

Sepsis is a serious infection-induced syndrome with serious ramifications, especially in intensive care units. Global concern motivated the investigation of the role of related genes' polymorphism in predicting the liability to infection, sepsis, septic shock and survival. Among these genes is the gene encoding mannose-binding lectin (MBL), with its remarkable importance in the immune system. However, the previous studies showed conflicting results and ambiguity that urged us to engage with this issue in the Egyptian population. Prediction of functional and structural impacts of single nucleotide polymorphisms (SNPs) was done using in silico methods. A prospective observational study was conducted in intensive care units; one hundred and thirty patients were followed up. Genotyping was performed using real-time polymerase chain reaction (RT-PCR) technology. MBL SNPs showed a remarkable high frequency in our population, as well. No significant association was found between MBL2 genotypes and any of our analyses (sepsis, septic shock and survival). Only septic shock and age were independently associated with time of survival by Cox regression analysis. Our study may confirm the redundancy of MBL and the absence of significant impact on sepsis liability and mortality in adult patients.

Phenotypic characterization of NKT-like cells and evaluation of specifically related cytokines for the prediction of unexplained recurrent miscarriage.

PROBLEM: Immune system dysregulation is a major cause of unexplained recurrent miscarriage (URM). Women with URM need screening for their pregnancy microenvironment and immune regulators, to prevent spontaneous abortion. METHOD OF STUDY: In this study we evaluated NKT-like cell subsets in peripheral venous blood of women with URM using flow cytometry. The expression levels of specifically related Th1 cytokines (IFN-γ and IL-2), Th2 cytokine (IL-4), and Th17 cytokines (IL-17), were measured using enzyme-linked immunosorbent assay. RESULTS: The percentage of CD16+CD56+NKT-like (Double Positive NKT-like; DPNKT-like) cell subset, and the levels of IL-2 and IFN-γ were significantly elevated in blood of non-pregnant and pregnant patients with URM compared with the healthy control groups, and these parameters were significantly increased after pregnancy in the same patients with URM. Based on the prevalence of the candidate immunological factors in patients with URM, the prognostic significance of the NKT-like cell subsets, IFN-γ and IL-2 profiles were evaluated as potential predictors of URM. A cut-off point of 2.55% for DPNKT-like cell subset in the blood and cut-off values of 39.5 and 20.5 pg/ml for the levels of IFN-γ and IL-2, respectively could be used for the prediction of the risk of spontaneous abortion. To the best of our knowledge, this is the first study that described the prognostic significance of the aforementioned immunological parameters before and after pregnancy, and highlighted the correlation of NKT-like cells and the candidate Th1 cytokines with pregnancy loss in women with URM. CONCLUSIONS: DPNKT-like cells, IFN-γ and IL-2 patient profiles could be used as markers to predict the risk of miscarriage in patients with URM.

Emerging MDR-Mycobacterium avium subsp. avium in house-reared domestic birds as the first report in Egypt.

BACKGROUND: Avian tuberculosis is a chronic and zoonotic disease that affects a wide variety of birds, mammals, and humans. This study aimed to estimate the frequency of Mycobacterium avium subsp. avium in some domestic birds based on molecular diagnosis, antibiogram profile, and PCR-based detection of inhA, rpoB, rpsL, and otrB antibiotic resistance-related genes. METHODS: A total of 120 fecal samples were collected from small flocks of house-reared domestic birds at Ismailia Governorate, Egypt. The collected samples were processed and subjected to the bacteriological examination. The antimicrobial susceptibility testing of the recovered isolates was performed using the broth microdilution method for the detection of minimum inhibitory concentrations (MICs). The genetic detection of the IS901confirmatory gene, inhA, rpoB, rpsL, and otrB genes was carried out using PCR. RESULTS: The frequency of M. avium subsp. avium was 4.1% (5/120); 10% (4/40) in ducks, and 2.5% (1/10) in geese. The identification of the recovered isolates was confirmed using PCR, where all the tested isolates were positive for IS901confirmatory gene. The results of the broth microdilution method revealed that most of the recovered isolates exhibited multidrug resistance (MDR) to isoniazid, rifampicin, streptomycin, oxytetracycline, and doxycycline, and harbored the inhA, rpoB, rpsL, and otrB genes. CONCLUSION: In brief, to the best of our knowledge this is the first report that emphasized the emergence of avian tuberculosis in house-reared domestic birds in Egypt. The emergence of MDR- M. avium subsp. avium is considered a public health threat. Emerging MDR-M. avium subsp. avium in domestic birds are commonly harbored the IS901, inhA, rpoB, rpsL, and otrB genes. Azithromycin and clofazimine revealed a promising in-vitro antibacterial activity against M. avium subsp. avium.

atpD gene sequencing, multidrug resistance traits, virulence-determinants, and antimicrobial resistance genes of emerging XDR and MDR-Proteus mirabilis.

Proteus mirabilis is a common opportunistic pathogen causing severe illness in humans and animals. To determine the prevalence, antibiogram, biofilm-formation, screening of virulence, and antimicrobial resistance genes in P. mirabilis isolates from ducks; 240 samples were obtained from apparently healthy and diseased ducks from private farms in Port-Said Province, Egypt. The collected samples were examined bacteriologically, and then the recovered isolates were tested for atpD gene sequencing, antimicrobial susceptibility, biofilm-formation, PCR detection of virulence, and antimicrobial resistance genes. The prevalence of P. mirabilis in the examined samples was 14.6% (35/240). The identification of the recovered isolates was confirmed by the atpD gene sequencing, where the tested isolates shared a common ancestor. Besides, 94.3% of P. mirabilis isolates were biofilm producers. The recovered isolates were resistant to penicillins, sulfonamides, ß-Lactam-ß-lactamase-inhibitor-combinations, tetracyclines, cephalosporins, macrolides, and quinolones. Using PCR, the retrieved strains harbored atpD, ureC, rsbA, and zapA virulence genes with a prevalence of 100%, 100%, 94.3%, and 91.4%, respectively. Moreover, 31.4% (11/35) of the recovered strains were XDR to 8 antimicrobial classes that harbored blaTEM, blaOXA-1, blaCTX-M, tetA, and sul1 genes. Besides, 22.8% (8/35) of the tested strains were MDR to 3 antimicrobial classes and possessed blaTEM, tetA, and sul1genes. Furthermore, 17.1% (6/35) of the tested strains were MDR to 7 antimicrobial classes and harbored blaTEM, blaOXA-1, blaCTX-M, tetA, and sul1 genes. Alarmingly, three strains were carbapenem-resistant that exhibited PDR to all the tested 10 antimicrobial classes and shared blaTEM, blaOXA-1, blaCTX-M, tetA, and sul1 genes. Of them, two strains harbored the blaNDM-1 gene, and one strain carried the blaKPC gene. In brief, to the best of our knowledge, this is the first study demonstrating the emergence of XDR and MDR-P.mirabilis in ducks. Norfloxacin exhibited promising antibacterial activity against the recovered XDR and MDR-P. mirabilis. The emergence of PDR, XDR, and MDR-strains constitutes a threat alarm that indicates the complicated treatment of the infections caused by these superbugs.

Molecular and HPLC-based approaches for detection of aflatoxin B1 and ochratoxin A released from toxigenic Aspergillus species in processed meat.

BACKGROUND: Meat-products are considered an enriched media for mycotoxins. This study aimed to investigate the prevalence of toxigenic Aspergillus species in processed meat samples, HPLC-quantitative measurement of aflatoxin B1 and ochratoxin A residues, and molecular sequencing of aflR1 and pks genes. One hundred and twenty processed beef meat specimens (basterma, sausage, and minced meat; n = 40 for each) were collected from Ismailia Province, Egypt. Samples were prepared for total mold count, isolation, and identification of Aspergillus species. All samples were analyzed for the production of both Aflatoxin B1 and Ochratoxin A mycotoxins by HPLC. Molecular identification of Aspergillus flavus and Aspergillus ochraceus was performed using PCR amplification of the internal transcribed spacer (ITS) region; furthermore, the aflR1 and pks genes were sequenced. RESULTS: The total mold count obtained from sausage samples was the highest one, followed by minced meat samples. The prevalence of A. flavus was (15%), (7.5%), and (10%), while the prevalence of A. ochraceus was (2.5%), (10%), and (0%) in the examined basterma, sausage, and minced meat samples, respectively. Using PCR, the ITS region was successfully amplified in all the tested A. flavus and A. ochraceus strains. Aflatoxin B1 was detected in six basterma samples (15%). Moreover, the ochratoxin A was detected only in four sausage samples (10%). The aflR1 and pks genes were amplified and sequenced successfully and deposited in the GenBank with accession numbers MF694264 and MF694264, respectively. CONCLUSIONS: To the best of our knowledge, this is the first report concerning the HPLC-Molecular-based approaches for the detection of aflatoxin B1 and ochratoxin A in processed beef meat in Egypt. The production of aflatoxin B1 and ochratoxin A in processed meat constitutes a public health threat. Aflatoxin B1 is commonly associated with basterma samples. Moreover, ochratoxin A was detected frequently in sausage samples. The routine inspection of mycotoxins in processed meat products is essential to protect human consumers.

Virulence-determinants and antibiotic-resistance genes of MDR-E. coli isolated from secondary infections following FMD-outbreak in cattle.

This study aimed to evaluate the prevalence, multidrug-resistance traits, PCR-detection of virulence, and antibiotic-resistance genes of E. coli isolated from secondary infections following FMD-outbreak in cattle. A total of 160 random samples were gathered from private dairy farms in Damietta Province, Egypt. The specimens were subjected to bacteriological examination, serotyping, congo-red binding assay, antibiogram-testing, and PCR-monitoring of virulence-determinant genes (tsh, phoA, hly, eaeA, sta, and lt) as well as the antibiotic-resistance genes (blaTEM, blaKPC, and blaCTX). The prevalence of E. coli was 30% (n = 48) distributed in 8 serogroups (40/48, 83.3%), while 8 isolates (8/48, 16.6%) were untypable. Besides, 83.3% of the examined isolates were positive for CR-binding. The tested strains harbored the virulence genes phoA, hly, tsh, eaeA, sta, and lt with a prevalence of 100% and 50%, 45.8%, 25%, 8.4%, and 6.2%, respectively. Furthermore, 50% of the recovered strains were multidrug-resistant (MDR) to penicillins, cephalosporins, and carbapenems, and are harboring the blaTEM, blaCTX, and blaKPC genes. Moreover, 25% of the examined strains are resistant to penicillins, and cephalosporins, and are harboring the blaTEM and blaCTX genes. To the best of our knowledge, this is the first report concerning the E. coli secondary bacterial infections following the FMD-outbreak. The emergence of MDR strains is considered a public health threat and indicates complicated treatment and bad prognosis of infections caused by such strains. Colistin sulfate and levofloxacin have a promising in vitro activity against MDR-E. coli.