Stable Enterotoxin Research Articles

Evaluation for the Effect of Heat Stable Enterotoxin (a) Produced by Enterotoxigenic Escherichia coli on Different Cancer Cells In Vitro

This study was conducted for evaluating the cytotoxic effect of heat stable enterotoxin a (STa) produced by enterotoxigenic Escherichia coli on the proliferation of primary cancer cell cultures, obtained from tumor samples that were collected from (13) cancer patients and as follows: (five colon cancer patients, two bladder cancer patients, two breast cancer patients, two stomach cancer patients and two lung cancer patients), and on normal cell line (rat embryonic fibroblast / REF) (in vitro) with the use of different concentrations starting from (1) mg/ml and ending with (0.0002) mg/ml by making two fold serial dilutions by using the 96- well microtiter plate, and in comparison with negative (PBS) and positive (MMC, at concentration of 10 µg/ml) controls . Results showed that, after (24) hours of exposure to STa, the growth of all primary cancer cell cultures obtained from colon cancer patients was inhibited by STa treatment and this inhibition was concentration dependent. Also it was shown that the cytotoxic effect of the high concentration of STa was close to that seen after MMC treatment. While no differences were seen in the growth of all primary cancer cell cultures that were obtained from the other cancer patients, which mean that STa treatment neither inhibit nor enhanced their growth. At the same time STa did not show or has any cytotoxic effect on the normal cell line (REF).

Impact of the calcium concentration on the efficacy of phage phiIPLA-RODI, LysRODIΔAmi and nisin for the elimination of Staphylococcus aureus during lab-scale cheese production

Staphylococcus aureus is a Gram-positive human opportunistic pathogen that may also cause food poisoning because of the ability of some strains to produce heat stable enterotoxins that can persist in food even after the pathogen is successfully eliminated. In this context, biopreservation may be a forward-looking strategy to help eliminate staphylococcal contamination in dairy products by using natural compounds. However, these antimicrobials exhibit individual limitations that may be overcome by combining them. This work investigates the combination of a virulent bacteriophage, phiIPLA-RODI, a phage-derived engineered lytic protein, LysRODIΔAmi, and the bacteriocin nisin for the elimination of S. aureus during lab-scale cheese production at two CaCl2 concentrations (0.2 % and 0.02 %), and subsequent storage at two different temperatures (4 °C and 12 °C). In most of the assayed conditions, our results demonstrate that the combined action of the antimicrobials led to a greater reduction of the pathogen population than the compounds individually, albeit this effect was additive and not synergistic. However, our results did show synergy between the three antimicrobials for reducing the bacterial load after 14 days of storage at 12 °C, temperature at which there is growth of the S. aureus population. Additionally, we tested the impact of the calcium concentration on the activity of the combination treatment and observed that higher CaCl2 levels led to a notable increase in endolysin activity that allowed the utilization of approximately 10-times less protein to attain the same efficacy. Overall, our data show that the combination of LysRODIΔAmi with nisin and/or phage phiIPLA-RODI, and an increase in the calcium concentration are successful strategies to decrease the amount of protein required for the control of S. aureus contamination in the dairy sector with a low potential for resistance selection, thereby reducing costs.

STUDY OF PATHOGENESIS, DIAGNOSIS AND VARIOUS TREATMENT APPROACHES FOR DIARRHOEA IN INFANTS

Diarrhea is common in infants (children less than 2 years of age), usually acute, and, if chronic, commonly caused by allergies and occasionally by infectious agents. Congenital diarrheas and enteropathies (CODEs) are rare causes of devastating chronic diarrhea in infants. Evaluation of CODEs is a lengthy process and infrequently leads to a clear diagnosis. However, genomic analyses and the development of model systems have increased our understanding of CODE pathogenesis.
 The European Society for Paediatric Gastroenterology, Hepatology and Nutrition and the European Society of Paediatric Infectious Diseases guide­lines make a stronger recommendation for the use of probiotics for the management of acute gastroenteritis, particularly those with documented efficacy such as Lactobacillus rhamnosus GG, Lactobacillus reuteri, and Saccharomyces boulardii.
 Studies of microbial pathogens and the toxins they produce are important for determining the mechanisms by which they cause disease and spread throughout a population. Some bacteria produce secretory enterotoxins (such as cholera toxin or the heat-labile or stable enterotoxins produced by Escherichia coli) that invade cells directly. Others invade cells or produce cytotoxins (such as those produced by Shigella, enteroinvasive E coli, or Clostridium difficile) that damage cells or trigger host responses that cause small or large bowel diseases (such as enteroaggregative or enteropathogenic E coli or Salmonella). Viruses (such as noroviruses and rotaviruses) and protozoa (such as Cryptosporidium, Giardia, or Entamoeba histolytica) disrupt cell functions and cause short- or longterm disease.
 KEYWORDS: Diagnosis & Treatment, Diarrhoea, Acute infective gastroenteritis, Oral rehydration solution, Vomiting

Prevalence, Molecular Characterization, Antibiotics Resistance of Staphylococcus aureus Isolated from Egyptian Soft Cheese, and Effect of Rosemary Essential Oil on its Viability

Staphylococcus aureus has the ability to yield heat stable enterotoxins resulting in food intoxication in human with variable intensity. So, the objectives of this research are to investigate the incidence of Staphylococcus aureus in Egyptian Soft Cheese (Talaga cheese), molecular identification of nuc and mecA genes in isolated strains by Multiplex PCR, assess the antimicrobial resistance profile using 16 different antibiotics as well as evaluate the Antimicrobial activity of rosemary oil on the viability of S. aureus. Sixty random samples of Talaga soft cheese were collected from different market places and dairy shops in various districts of Mansoura town, Dakahlia governorate. Egypt. S. aureus was discovered in 30% (18/60) from examined samples where 4 isolates (22.2%) were proved to be enterotoxigenic strains. All S. aureus isolates contained on nuc gene, however the mecA gene was found only in 5 S. aureus isolates (27.7%). S. aureus isolates showed a higher multiple antibiotic resistance (MAR) where the average MAR index was 0.479 and this leads to an increase in the public health hazard. The antimicrobial properties of rosemary essential oil as a natural antimicrobial agent were evaluated against S. aureus strain (ATCC 25923). It could be concluded that using rosemary essential oil with 2% concentration was highly effective in reducing and inhibiting S. aureus.

Isolation and molecular detection of enterotoxigenic Staphylococcus aureus from raw milk of cows

Staphylococcus aureus is a microbe associated with human’s food poisoning. It is caused by isolates producing different heat stable enterotoxins which act as one of the most spread worldwide gastroenteritis causes. The goal of the current study was to assess the incidence of S. aureus (enterotoxigenic) in the raw milk samples of cows which were collected from various places of Baghdad city. Isolation and identification of S. aureus were done by conventional laboratory method and the diagnoses were confirmed by using molecular method RT-PCR to detect the specific gene of S. aureus nuc gene. In addition, we investigate the occurrence of some of Staphylococcal enterotoxins genes such as SEA, SEB, SEC, SED, and SEE. We found that 12/50 (24%) of the isolates were S. aureus and these isolates carried one or more of the enterotoxin genes such as enterotoxin type SEC 12/12 (100) followed by enterotoxin type SEA 7/12 (58.33%) and only 1/12 (8.33%) was type SED, while type SEB and SEE were negative 0%. Some of the isolates have genes that coded for two enterotoxins patterns such as SEA and SEC 7/12 (58.33%) and SEC and SED 1/12 (8.33%). Fewer of the isolates expressed only one genotype of enterotoxin gene like SEC 4/12 (33.3%). This study has proven that there was a high chance of occurrence for the enterotoxigenic S. aureus at the raw milk of cows in Baghdad city.

Abstract PO-033: Bacterial cytotoxin therapy limits tumor growth for pancreatic ductal adenocarcinoma

Abstract Treating pancreatic ductal adenocarcinoma (PDAC) with systemic chemotherapeutic drugs has remained a challenge, due in part to the hypovascularized and poorly perfused nature of PDAC tumors. Limited blood flow within the tumor tissue creates an extremely hypoxic microenvironment and impedes the accumulation of drugs. Moreover, local immunosuppression in PDAC has so far limited the efficacy of immunotherapy approaches. However, these very features that have interfered with systemic therapy in PDAC (hypoperfusion, hypoxia, and immunosuppression) are potential advantages for the use of bacterial therapies. Bacteria have been used as a directed cancer therapy for over 100 years, starting with Dr. William Coley’s use of heat killed bacteria (Coley’s Toxin) against sarcomas in 1893. Recent developments in the field of synthetic biology have made it possible to engineer complex logical circuits into bacteria, enabling the manufacture of anticancer therapies directly within the tumor parenchyma. Bacteria can actively migrate through tissues, they can thrive in hypoxic microenvironments, and they benefit from the local immune suppression. We have therefore worked to develop novel bacterial strains for targeting PDAC. We began by testing a range of bacteria-derived toxins that could be used as a payload to target PDAC. These toxins were produced by an engineered strain of a non-toxic, probiotic E. coli Nissle 1917. We identified four pore-forming toxins that significantly reduced viability of the cells compared to bacterially produced GFP: hemolysin E, heat stable enterotoxin, magainin, and theta toxin. We then performed a secondary screen using a novel PDAC explant model system developed in our lab, in which thick slices of murine or human PDAC are culture intact for up to 7 days. Consistent with the monolayer screen, two of the candidate compounds, heat stable enterotoxin and theta toxin, significantly increased tissue death compared to non-toxic GFP-producing bacteria. Finally, we delivered live bacteria producing either toxins or GFP into KPC mouse tumors through intratumoral injection. While GFP-producing strains did not induce a change in tumor growth kinetics, theta toxin treatment demonstrated an immediate, prolonged stabilization of tumor volume for more than a month. Histological analyses of treated tumors demonstrated that diffuse populations of bacteria co-localized with regions of tumor necrosis and cell death. Most interestingly, while there was minimal spread of bacteria to healthy non-target tissues, translocation of the bacteria did occur to regions of liver metastases and secondary papilloma tumors, suggesting a mechanism for diffuse treatment of known and unknown metastases following the initial treatment of the primary tumor. Together these studies demonstrate that cytotoxic bacterial therapy is an effective candidate strategy to circumvent the difficulties in systemic treatment of PDAC. Citation Format: Amanda R. Decker, Tetsuhiro Harimoto, Steve A. Sastra, Tal Danino, Kenneth P. Olive. Bacterial cytotoxin therapy limits tumor growth for pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-033.

In Silico Prediction, Molecular Docking and Dynamics Studies of Steroidal Alkaloids of Holarrhena pubescens Wall. ex G. Don to Guanylyl Cyclase C: Implications in Designing of Novel Antidiarrheal Therapeutic Strategies.

The binding of heat stable enterotoxin (STa) secreted by enterotoxigenic Escherichia coli (ETEC) to the extracellular domain of guanylyl cyclase c (ECDGC-C) causes activation of a signaling cascade, which ultimately results in watery diarrhea. We carried out this study with the objective of finding ligands that would interfere with the binding of STa on ECDGC-C. With this view in mind, we tested the biological activity of a alkaloid rich fraction of Holarrhena pubescens against ETEC under in vitro conditions. Since this fraction showed significant antibacterial activity against ETEC, we decided to test the screen binding affinity of nine compounds of steroidal alkaloid type from Holarrhena pubescens against extracellular domain (ECD) by molecular docking and identified three compounds with significant binding energy. Molecular dynamics simulations were performed for all the three lead compounds to establish the stability of their interaction with the target protein. Pharmacokinetics and toxicity profiling of these leads demonstrated that they possessed good drug-like properties. Furthermore, the ability of these leads to inhibit the binding of STa to ECD was evaluated. This was first done by identifying amino acid residues of ECDGC-C binding to STa by protein–protein docking. The results were matched with our molecular docking results. We report here that holadysenterine, one of the lead compounds that showed a strong affinity for the amino acid residues on ECDGC-C, also binds to STa. This suggests that holadysenterine has the potential to inhibit binding of STa on ECD and can be considered for future study, involving its validation through in vitro assays and animal model studies.

Molecular Detection of Enterotoxin Genes of Multiresistant Staphylococcus aureus Isolates from Different Sources of Food

Foodborne diseases are a major risk for human health. Millions of people become sick as a result of eating contaminated food with microorganisms that cause diseases. S. aureus is considered as one of the most important pathogenic bacteria, having the ability to activate certain genes that encode for heat stable enterotoxins and cause Staphylococcal food poisoning. Thus, this study aimed to determine the prevalence of multi resistant Staphylococcus aureus that produce enterotoxins in different sources of food . Forty nine isolates were identified as S.aureus, according to morphological and biochemical tests. They were isolated from 387 different food samples from several randomly covered restaurants and supermarkets in different regions of Baghdad. Molecular diagnosis of S. aureus using specific primers for the 16S rRNA gene was carried out by Polymerase Chain Reaction (PCR ) technique . Susceptibility of 43 isolates of S.aureus was tested against 15 antimicrobial agents. The results revealed that all the isolates were resistant (100%) to mecillinam, highly resistant to vancomycine and meropenemin (74.4 %) and moderately resistant to Oxacillin, Erythromycin Cefotaxime, and Cefiximein (67.4, 60.4, 62.8, , 60.5 %, respectively), while they showed low resistance to Gentamicin (34.8%). In addition, all of these isolates were susceptible to Tigecycline and Amoxicillin/ clavulanic acid and Cefoxitin-Cloxacilin. High percentages of oxicillin resistant S. aureus were isolated from cooked food samples, followed by meat products, and with less percentage from pastry products. Molecular detection of enterotoxins A and B of Staphylococcus aureus isolates was performed using specific primers based on PCR. The results revealed that S. aureus isolated from cooked food had the highest percentage of the isolates producing the enterotoxins A and B. Type A enterotoxin gene showed a higher prevalence than type B gene among cooked food , dairy products and pastry. In conclusion, the results revealed a high prevalence of some classical enterotoxin genes in multi-drug resistant S.aureus isolated from different sources of food, which can cause food-poisoning and, consequently, a potential serious problem for public health.

Comparison of BioFire FilmArray gastrointestinal panel versus Luminex xTAG Gastrointestinal Pathogen Panel (xTAG GPP) for diarrheal pathogen detection in China

ObjectivesTo compare the performance of two syndromic panels: Luminex xTAG Gastrointestinal Pathogen Panel (GPP) and FilmArray Gastrointestinal (GI) panel. MethodsA total of 243 diarrhea specimens were detected by two panels in parallel, and the inconsistent results were analyzed by real-time PCR or reverse transcription PCR (RT-PCR). The target concentration in specimens was examined by comparing the crossing point values of FilmArray, the median fluorescence intensity of xTAG and the cycle threshold values in any discrepancies. ResultsFor pathogens detected by both panels, the positive rates of FilmArray GI and xTAG GPP were 65.0% and 48.6%, respectively. The two panels showed high consistency (kappa ≥0.74) in detecting norovirus, rotavirus and Campylobacter, while there was low consistency (kappa ≤0.40) in detecting Cryptosporidium, Salmonella, Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic Escherichia coli (ETEC). Samples with low concentration targets were more often detected by FilmArray than with xTAG GPP. The xTAG GPP was more likely to be affected by amplification inhibitors. Several defects of xTAG GPP were found in detecting ETEC. ConclusionsFilmArray was more sensitive. For specimens with low target concentrations or containing ETEC heat stable enterotoxin, the false negatives of xTAG GPP need to be considered.

Plasmid profile analysis for enterotoxigenic Escherichia coli and detection of heat stable enterotoxin I (STI) gene by polymerase chain reaction

Enterotoxigenic Escherichia coli (ETEC) is one of the food pathogens which may cause diarrhea in humans. The enterotoxins produced by ETEC are heat labile toxins (LT) and heat stable toxins (ST). These pathogenic factors are usually encoded on the plasmid DNA of E. coli cells. In this study, E. coli plasmid DNA was isolated and subjected to 0.7% agarose gel electrophoretic analysis and the STI gene was detected by polymerase chain reaction (PCR) using new primers. It was found that most of the plasmids from ETEC cells showed molecular weights in the range of 2-22 kb and the PCR primers used specifically amplified a 203 bp fragment.

Detection of Escherichia Coli Labile and Stable Enterotoxins from Children Suffering from Diarrhea

Detection of Escherichia Coli Labile and Stable Enterotoxins from Children Suffering from Diarrhea

Antimicrobial usage in pig production: Effects on Escherichia coli virulence profiles and antimicrobial resistance.

Antimicrobials (AM) are used for growth promotion and therapy in pig production. Its misuse has led to the development of resistant organisms. We evaluated Escherichia coli virulence genes, and compared phenotypic–genotypic antimicrobial resistance (AMR) patterns of faecal E. coli from pigs receiving routine farm treatment without antimicrobial agents against pigs treated routinely with AM over 70 days. Recovered E. coli were tested for AMR using disk diffusion and polymerase chain reaction. Virulence genes were detected in 24.8% of isolates from antimicrobial group and 43.5% from non-antimicrobial group (p = 0.002). The proportion of virulence genes heat-stable enterotoxins a & b (STa, STb), enteroaggregative heat stable enterotoxin 1 [EAST1] and Shiga toxin type 2e [Stx2e]) were 18.1%, 0.0%, 78.7% and 3.0% for antimicrobial group and 14.8%, 8.5%, 85.1% and 12.7% for non-antimicrobial groups, respectively. Resistance to oxytetracycline was most common (p = 0.03) in samples collected between days 10 and 21. Resistance shifted to amoxicillin on days 56–70, and trimethoprim resistance was observed throughout. Seventeen phenotypic AMR combinations were observed and eight were multidrug resistant. At least one tetracycline resistance gene was found in 63.9% of the isolates. tet (A) (23.3%) was most common in the antimicrobial group, whereas tet (B) (43.5%) was prevalent in the non-antimicrobial group. Usage or non-usage of antimicrobial agents in growing pigs does not preclude virulence genes development and other complex factors may be involved as previously described. Heavily used AM correspond to the degree of resistance and tetracycline resistance genes were detected during the growth phase.

Molecular characterisation and pathogenicity evaluation of Aeromonas hydrophila strains isolated from cultured tilapia Oreochromis niloticus in Egypt

The objectives of the present study were to characterise Aeromonas hydrophila isolated from diseased tilapia Oreochromis niloticus cultured in Kafrelsheikh farms and to evaluate virulence and pathogenecity. About 200 adult fish exhibiting the onset of clinical signs were bacteriologically examined using Aeromonas isolation medium. The isolates were identified by 16S rRNA gene sequence analysis using species specific primers and then tested for ten virulence genes employing specific primers. Experimental challenge studies were also conducted to investigate the pathogenicity of the recovered A. hydrophilastrains. Thirty five isolates were identified as A. hydrophila based on 16S rRNA gene sequence analysis,which were also positive for presence of four virulence genes (aerolysin, lipase, cytotonic heat stable enterotoxin and haemolysin). Results showed that majority of the examined strains carried one or more virulence genes.

The Role of Regulatory Mechanisms and Environmental Parameters in Staphylococcal Food Poisoning and Resulting Challenges to Risk Assessment.

Prevention, prediction, control, and handling of bacterial foodborne diseases – an ongoing, serious, and costly concern worldwide – are continually facing a wide array of difficulties. Not the least due to that food matrices, highly variable and complex, can impact virulence expression in diverse and unpredictable ways. This review aims to present a comprehensive overview of challenges related to the presence of enterotoxigenic Staphylococcus aureus in the food production chain. It focuses on characteristics, expression, and regulation of the highly stable staphylococcal enterotoxins and in particular staphylococcal enterotoxin A (SEA). Together with the robustness of the pathogen under diverse environmental conditions and the range of possible entry routes into the food chain, this poses some of the biggest challenges in the control of SFP. Furthermore, the emergence of new enterotoxins, found to be connected with SFP, brings new questions around their regulatory mechanisms and expression in different food environments. The appearance of increasing amounts of antibiotic resistant strains found in food is also highlighted. Finally, potentials and limitations of implementing existing risk assessment models are discussed. Various quantitative microbial risk assessment approaches have attempted to quantify the growth of the bacterium and production of disease causing levels of toxin under various food chain and domestic food handling scenarios. This requires employment of predictive modeling tools, quantifying the spatiotemporal population dynamics of S. aureus in response to intrinsic and extrinsic food properties. In this context, the armory of predictive modeling employs both kinetic and probabilistic models to estimate the levels that potentiate toxin production, the time needed to reach that levels, and overall, the likelihood of toxin production. Following risk assessment, the main challenge to mitigate the risk of S. aureus intoxication is first to prevent growth of the organism and then to hamper the production of enterotoxins, or at least prevent the accumulation of high levels (e.g., >10–20 ng) in food. The necessity for continued studies indeed becomes apparent based on the challenges to understand, control, and predict enterotoxin production in relation to the food environment. Different types of food, preservatives, processing, and packaging conditions; regulatory networks; and different staphylococcal enterotoxin-producing S. aureus strains need to be further explored to obtain more complete knowledge about the virulence of this intriguing pathogen.

34 – E.Coli Heat Stable Enterotoxin (ST) Inhibition of Nhe3 Occurs by Different Signaling Pathways Downstream of Cyclic Gmp in Murine Jejunum and Caco-2 Cells: A Warning About Assumed Equivalence

34 – E.Coli Heat Stable Enterotoxin (ST) Inhibition of Nhe3 Occurs by Different Signaling Pathways Downstream of Cyclic Gmp in Murine Jejunum and Caco-2 Cells: A Warning About Assumed Equivalence

Su1012 – Mechanical Stimuli Affect Enterotoxigenic E. Coli (ETEC) Heat Stable Enterotoxin (ST)-Cyclic Nucleotide Signaling by Altering Mrp4 Expression in a Human Enteroid Monolayer Model of Etec Diarrhea

Su1012 – Mechanical Stimuli Affect Enterotoxigenic E. Coli (ETEC) Heat Stable Enterotoxin (ST)-Cyclic Nucleotide Signaling by Altering Mrp4 Expression in a Human Enteroid Monolayer Model of Etec Diarrhea

Staphylococcus aureus Enterotoxin Production in Raw and Pasteurized Milk: The Effect of Selected Different Storage Durations and Temperatures.

Introduction:S. aureus is one of the most prevalent potential pathogenic bacteria found in DHM. Some strains produce heat stable enterotoxins that are able to survive pasteurization. These enterotoxins have been associated with gastritis and potentially necrotizing enterocolitis in preterm infants. The aim of this study is to assess the impact of different storage temperatures on Staphylococcus aureus growth and enterotoxin A and B production in raw and Holder pasteurized donor human milk (DHM). Materials and Methods: Raw and pasteurized DHM were inoculated with enterotoxin A and B producing S. aureus. Samples were incubated at 4°C (10 days), 21°C, and 37°C (18 hours). Bacterial growth and enterotoxin A and B were quantified. Results:S. aureus count increased in pasteurized DHM. Bacterial count decreased in the raw milk when incubated at 21°C and 4°C and slightly increased when incubated at 37°C. Enterotoxins A and B were only detected in pasteurized DHM at 37°C from 9 hours onward. Conclusion: This study showed that raw milk is capable of suppressing S. aureus growth compared to pasteurized DHM. It also provides a measure of assurance of the safety of raw and pasteurized DHM when refrigerated or left at room temperature for a few hours.

MOLECULAR IDENTIFICATION OF Aeromonas hydrophila STRAINS RECOVERED FROM KAFRELSHEIKH FISH FARMS

The aim of this study was to recover Aeromonas hydrophila from Kafrelsheikh tilapia (Oreochromis niloticus) farms and to study its virulence. Adult fish (n = 100) exhibiting the onset of clinical signs were bacteriologically examined using aeromonas isolation base medium. The isolates were identified by PCR using A. hydrophila-specific 16S rRNA primers and the virulence was determined using specific primers for ten virulence genes targeting hidrolipase (Lip), elastase (ahyB), lipase (pla/lip), aerolysin (aer), cytotonic enterotoxin (alt), cytotoxic enterotoxin (act), temperature sensitive protease (eprCAI), serine protease (Ahp), haemolysin (hlyA), and cytotonic heat stable enterotoxin (ast). Molecular screening revealed presence of 35 isolates positive for A. hydrophila-specific 16S rRNA and 4 virulence genes (aer, pla/lip, ast, and hlyA). It also showed that the majority of the examined strains carried one or more virulence genes. These data indicate higher virulence for A. hydrophila in infected O. niloticus in Kafrelsheikh fish farms.Key words: PCR; A. hydrophila; virulence genes

Study the ability of some gram positive bacillus species isolated from powdered infant milk to produce toxin

Forty samples of different types of powdered infant milk had been collected including (celia 1-2 , biomil 1-2 , lery 1-2 , dialak 1-2 , albadee 2 ,nido 2 , kikos 2 , nictalia 1-2 , cerelak ). Some of the milk cans were opened directly at the beginning of test while others had been opened three days or more before testing. The results showed that Bacillus species were isolated and identified at different rates as Bacillus cereus has the highest rate (12.5%) then B.circulance and B. laterosporous at (5%) for each of them respectively, while B.megaterium was isolated at a rate of (2.5%). The ability of the isolated Bacillus species to produce enterotoxin was detected by the use of Rapid and delayed permeability test for the detection of the heat stable enterotoxin and the results were recorded by the appearance of blue non necrotic lesions after injection of the rabbit with the cell free culture supernated (CFCS) and Lysate supernatant (LS) of the isolated Bacillus species, and for the detection of heat labile enterotoxin, the paw oedema test was also used , the increase in thickness of fore and hind arms appeared. The ability of the Bacillus species to produce the enzyme lecithinase and lyse red blood cells were also studied and the results revealed that only Bacillus cereus produce the enzyme lecithinase. in addition all the isolates showed positive hemolytic activity.

Characterization of a novel plasmid encoding F4-like fimbriae present in a Shiga-toxin producing enterotoxigenic Escherichia coli isolated during the investigation on a case of hemolytic-uremic syndrome

In February 2017 a case of Hemolytic-Uremic Syndrome (HUS) was reported to the National Registry of HUS in an adult living in Northern Italy. Stool specimens from the patient and his family contacts were collected and the analyses led to the isolation of a Locus of Enterocyte Effacement (LEE)-negative Shiga toxin 2 (Stx2)-producing Escherichia coli. The epidemiological investigations performed brought to collect fecal samples from the animals reared in a farm held by the case’s family and a mixture of bovine and swine feces proved positive for Shiga toxin-producing E. coli (STEC) and yielded the isolation of a LEE-negative stx2-positive E. coli strain. Further characterization by whole genome sequencing led to identify the isolates as two identical O2:H27 hybrid Enterotoxigenic Shiga toxin-producing E. coli (ETEC-STEC). Sequencing of a high molecular weight plasmid present in the human isolate disclosed a peculiar plasmid harboring virulence genes characteristic for both pathotypes, including the enterohemolysin-coding gene and sta1, encoding the heat stable enterotoxin. Moreover, a complete fae locus encoding the ETEC F4 fimbriae could be identified, including a novel variant of faeG gene responsible for the production of the main structural subunit of the fimbriae. This novel faeG showed great diversity in the nucleotidic sequence when compared with the reference genes encoding the swine F4 allelic variants, whereas at the amino acid sequence level the predicted protein sequence showed some similarity with FaeG from E. coli strains of bovine origin. Further investigation on the plasmid region harboring the newly identified faeG allelic variant allowed to identify similar plasmids in NCBI sequence database, as part of the genome of other previously uncharacterized ETEC-STEC strains of bovine origin, suggesting that the novel F4-like fimbriae may play a role in bovine host specificity.