Stx2 Genes Research Articles

Application of Engineered Zinc Finger Proteins Immobilized on Paramagnetic Beads for Multiplexed Detection of Pathogenic DNA.

Micro-scale magnetic beads are widely used for isolation of proteins, DNA, and cells, leading to the development of in vitro diagnostics. Efficient isolation of target biomolecules is one of the keys to developing a simple and rapid point-of-care diagnostic. A zinc finger protein (ZFP) is a double-stranded (ds) DNA-binding domain, providing a useful scaffold for direct reading of the sequence information. Here, we utilized two engineered ZFPs (Stx2-268 and SEB-435) to detect the Shiga toxin (stx2) gene and the staphylococcal enterotoxin B (seb) gene present in foodborne pathogens, Escherichia coli O157 and Staphylococcus aureus, respectively. Engineered ZFPs are immobilized on a paramagnetic bead as a detection platform to efficiently isolate the target dsDNA-ZFP bound complex. The small paramagnetic beads provide a high surface area to volume ratio, allowing more ZFPs to be immobilized on the beads, which leads to increased target DNA detection. The fluorescence signal was measured upon ZFP binding to fluorophore-labeled target dsDNA. In this study, our system provided a detection limit of ≤ 60 fmol and demonstrated high specificity with multiplexing capability, suggesting a potential for development into a simple and reliable diagnostic for detecting multiple pathogens without target amplification.

Genetic and antimicrobial resistance profiles of non-O157 Shiga toxin-producing Escherichia coli from different sources in Egypt

BackgroundThe Shiga toxin-producing Escherichia coli (STEC) represented a great risk to public health. In this study, 60 STEC strains recovered from broiler and duck fecal samples, cow’s milk, cattle beef, human urine, and ear discharge were screened for 12 virulence genes, phenotypic and genotypic antimicrobial resistance, and multiple-locus variable-number tandem-repeat analysis (MLVA).ResultsThe majority of strains harbored Shiga toxin 1 (stx1) and stx1d, stx2 and stx2e, and ehxA genes, while a minority harbored stx2c subtype and eaeA. We identified 10 stx gene combinations; most of strains 31/60 (51.7%) exhibited four copies of stx genes, namely the stx1, stx1d, stx2, and stx2e, and the strains exhibited a high range of multiple antimicrobial resistance indices. The resistance genes blaCTX-M-1 and blaTEM were detected. For the oxytetracycline resistance genes, most of strains contained tetA, tetB, tetE, and tetG while the tetC was present at low frequency. MLVA genotyping resolved 26 unique genotypes; genotype 21 was highly prevalent. The six highly discriminatory loci DI = 0.9138 are suitable for the preliminary genotyping of STEC from animals and humans.ConclusionsThe STEC isolated from animals are virulent, resistant to antimicrobials, and genetically diverse, thus demands greater attention for the potential risk to human.

Occurrence of Virulence Genes and Antimicrobial Resistance of E. coli O157:H7 Isolated from the Beef Carcass of Bahir Dar City, Ethiopia.

E. coli O157:H7 is one of the most virulent foodborne pathogens. The aim of this study was to isolate E. coli O157:H7, determine virulence genes carried by the organism, and assess the antimicrobial susceptibility pattern of the isolates from beef carcass samples at Bahir Dar city. Swab samples (n = 280) were collected from the carcass of cattle slaughtered at the abattoir and processed using sorbitol MacConkey agar supplemented with cefixime telluride and confirmed with latex agglutination test. A polymerase chain reaction was performed on isolates for the detection of virulence genes stx1, stx2, hlyA, and eae. Antimicrobial susceptibility testing was performed using the disk diffusion method. Of 280 samples processed, 25 (8.9%) isolates were positive. Out of 25 isolates subjected for molecular detection, 8 (32%) and 14 (56%) isolates possessed stx1 and stx2 genes, respectively; from those, 5 (20%) isolates had both genes for the production of Shiga toxins. Compared from other virulent genes relatively higher proportion of 18 (72%) isolates carried the hlyA gene. Only 5 (2%) isolates were positive for eae. Resistance was detected in all 25 (100%) isolates and 3 (12%) against clindamycin and trimethoprim, respectively. This study result highlights the potential threat to public health. The abattoir workers need to be aware about the pathogen and should follow appropriate practices to prevent contamination of meat intended for human consumption.

The effect of two ribonucleases on the production of Shiga toxin and stx-bearing bacteriophages in Enterohaemorrhagic Escherichia coli

Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of intestinal pathogens responsible for a range of illnesses, including kidney failure and neurological compromise. EHEC produce critical virulence factors, Shiga toxin (Stx) 1 or 2, and the synthesis of Stx2 is associated with worse disease manifestations. Infected patients only receive supportive treatment because some conventional antibiotics enable toxin production. Shiga toxin 2 genes (stx2) are carried in λ-like bacteriophages (stx2-phages) inserted into the EHEC genome as prophages. Factors that cause DNA damage induce the lytic cycle of stx2-phages, leading to Stx2 production. The phage Q protein is critical for transcription antitermination of stx2 and phage lytic genes. This study reports that deficiency of two endoribonucleases (RNases), E and G, significantly delayed cell lysis and impaired production of both Stx2 and stx2-phages, unlike deficiency of either enzyme alone. Moreover, scarcity of both enzymes reduced the concentrations of Q and stx2 transcripts and slowed cell growth.

Inconsistent PCR detection of Shiga toxin-producing Escherichia coli: Insights from whole genome sequence analyses.

Shiga toxin-producing Escherichia coli (STEC) have been linked to food-borne disease outbreaks. As PCR is routinely used to screen foods for STEC, it is important that factors leading to inconsistent detection of STEC by PCR are understood. This study used whole genome sequencing (WGS) to investigate causes of inconsistent PCR detection of stx1, stx2, and serogroup-specific genes. Fifty strains isolated from Alberta feedlot cattle from three different studies were selected with inconsistent or consistent detection of stx and serogroup by PCR. All isolates were initially classified as STEC by PCR. Sequencing was performed using Illumina MiSeq® with sample library by Nextera XT. Virtual PCRs were performed using Geneious and bacteriophage content was determined using PHASTER. Sequencing coverage ranged from 47 to 102x, averaging 74x, with sequences deposited in the NCBI database. Eleven strains were confirmed by WGS as STEC having complete stxA and stxB subunits. However, truncated stx fragments occurred in twenty-two other isolates, some having multiple stx fragments in the genome. Isolates with complete stx by WGS had consistent stx1 and stx2 detection by PCR, although one also having a stx2 fragment had inconsistent stx2 PCR. For all STEC and 18/39 non-STEC, serogroups determined by PCR agreed with those determined by WGS. An additional three WGS serotypes were inconclusive and two isolates were Citrobacter spp. Results demonstrate that stx fragments associated with stx-carrying bacteriophages in the E. coli genome may contribute to inconsistent detection of stx1 and stx2 by PCR. Fourteen isolates had integrated stx bacteriophage but lacked complete or fragmentary stx possibly due to partial bacteriophage excision after sub-cultivation or other unclear mechanisms. The majority of STEC isolates (7/11) did not have identifiable bacteriophage DNA in the contig(s) where stx was located, likely increasing the stability of stx in the bacterial genome and its detection by PCR.

Multidrug resistance IncC plasmid carrying blaCMY-97 in Shiga toxin-producing Escherichia coli ST215-H54 of ovine origin.

CMY-type β-lactamases are the most reported plasmid-mediated AmpC (pAmpC), with the CMY-2-like group being the most clinically relevant described in Escherichia coli at human-animal-environment interface. Shiga toxin-producing E. coli (STEC) lineages are zoonotic pathogens commonly reported causing serious clinical conditions in humans, including severe diarrheagenic diseases. Therefore, this study aimed to investigate a multidrug-resistant (MDR) STEC isolate (A313) recovered from a healthy sheep and carrying mobile blaCMY-97, that encodes a pAmpC belonging to the CMY-2-like group. The A313 isolate exhibited a MDR profile to clinically relevant antimicrobials (i.e., cephalosporins, aminoglycosides, and fluoroquinolones), but reduced susceptibility to extended-spectrum cephalosporins and aztreonam. Besides, virulence genes (stx2, gad and iutA) were detected in A313, which belonged to ST215/CC10 and phylogenetic group A, whereas the fimH54 was identified. The blaCMY-97 gene and other antimicrobial resistance determinants [aph(6)-Id, aph(3″)-Ib, aac(3)-IId, aadA5, floR, tetA, sul1, and sul2], as well as genes encoding tolerance to mercury (merRTPCADE), were harbored by an IncC plasmid (named pA313-CMY-97, ~ 176kb). A novel genetic context of blaCMY-2-like, in which a 208-bp ISEcp1 was truncated by an IS26 in the opposite orientation upstream of the blaCMY-97 gene (IS26-∆ISEcp1-blaCMY-97-blc-sugE-encR), was also identified in pA313-CMY-97. To the best of our knowledge, this is the first report on the acquisition of blaCMY-97 into a plasmid. Therefore, we reported ovine as reservoir of clinically relevant MDR bacteria carrying mobile blaCMY-97 with potential for zoonotic transmission.

Molecular characterization of Escherichia coli isolated from milk samples with regard to virulence factors and antibiotic resistance

Background and Aim:Raw milk is considered an essential source of nutrition during all stages of human life because it offers a valuable supply of protein and minerals. Importantly, milk is considered a good media for the growth and contamination of many pathogenic bacteria, especially food-borne pathogens such as Escherichia coli. Thus, the objective of this study was to characterize E. coli and detect its virulence factors and antibiotic resistance from raw milk samples.Materials and Methods:Raw milk samples (n=100) were collected from different localities in Qena, Egypt, and investigated for the presence of E. coli using different biochemical tests, IMViC tests, serotyping to detect somatic antigen type, and molecularly by polymerase chain reaction (PCR) tests. The presence of different virulence and antimicrobial genes (hly, eae, stx1, stx2, blaTEM, tetA(A), and tetB genes) in E. coli isolates was evaluated using PCR.Results:The results demonstrated that 10 out of 100 milk samples were contaminated with E. coli. Depending on serology, the isolates were classified as O114 (one isolate), O27 (two isolates), O111 (one isolate), O125 (two isolates), and untypeable (five isolates) E. coli. The sequencing of partially amplified 16S rRNA of the untypeable isolates resulted in one isolate, which was initially misidentified as untypeable E. coli but later proved as Enterobacter hormaechei. Moreover, antibacterial susceptibility analysis revealed that nearly all isolates were resistant to more than 3 families of antibiotics, particularly to b-lactams, clindamycin, and rifampin. PCR results demonstrated that all E. coli isolates showed an accurate amplicon for the blaTEM and tetA(A) genes, four isolates harbored eae gene, other four harbored tetB gene, and only one isolate exhibited a positive stx2 gene.Conclusion:Our study explored vital methods for identifying E. coli as a harmful pathogen of raw milk using 16S rRNA sequencing, phylogenetic analysis, and detection of virulence factors and antibiotic-resistant genes.

Epidemiology and Characterization of Shiga Toxin-Producing Escherichia Coli of Hemolytic Uremic Syndrome in Argentina.

Argentina has one of the highest prevalence in Shiga toxin-producing Escherichia coli (STEC) and the high rate of hemolytic uremic syndrome (HUS) in the world. Though preventive steps such as food safety have been implemented as a way to reduce STEC infections, these have proven to be insufficient. STEC's pathogenesis, virulence factors, relationship with the environment, and emerging strains have been studied in the past few years in the country. Many factors that contribute to the morbidity and mortality of STEC infections include the expression of pathologic genes, alternative characteristics (inhibition of phagocytosis, invasion, cytotoxicity, and bacterial attachment), and host factors (age, immune status, treatments, medical history). However, research studies in combination with epidemiological data suggest trends of the prognosis, with the relationship between and genetic combinations of adherence, Shiga toxin (Stx) genes, and virulence genes, which significantly influence disease outcomes. This review explains the characteristics and epidemiology of STEC in Argentina. All these facts show that the application of molecular subtyping techniques in real-time is essential for detecting and controlling outbreaks. Applying molecular subtyping techniques in hemorrhagic diarrhea can avoid severe consequences caused by progression to HUS, and help the epidemiological analysis of the outbreak.

Comparison of a Miniaturized Cassette PCR System with a Commercially Available Platform for Detecting Escherichia coli in Beef Carcass Swabs.

Detection sensitivity of cassette PCR was compared with a commercial BAX® PCR system for detection of eae and stx genes in Escherichia coli from 806 beef carcass swabs. Cassette PCR detects multiple genetic markers on multiple samples using PCR and melt curve analysis. Conventional PCR served as a gold standard. Overall, for positive and negative concordance, cassette PCR was 98.6% concordant with conventional PCR, and BAX PCR was 65.4% concordant. Of 806 beef carcass swabs, 339 by cassette PCR and 84 by BAX PCR harbored eae + stx+ E. coli. For BAX PCR reactions, 84% of eae+ swabs, 79% of stx+ swabs, and 86% of eae + stx+ swabs were also detected by cassette PCR. For cassette PCR reactions, 457 swabs were eae+ with only 117 scored as eae+ using BAX PCR for 26% positive concordance. For stx primers, cassette PCR scored 480 samples as stx+ but only 215 samples were stx+ by BAX PCR, giving 45% positive concordance. Importantly, cassette PCR scored 339 swabs as harboring eae + stx+ E. coli, but BAX PCR detected only 71 positives giving only 21% positive concordance, with many false negatives. Cassette PCR is a highly sensitive method for detection of STEC genes in E. coli found in carcass swabs.

Diversity of Non-O157 Shiga Toxin-Producing Escherichia coli Isolated from Cattle from Central and Southern Chile.

Cattle are the main reservoir of Shiga toxin-producing Escherichia coli (STEC), one of the world's most important foodborne pathogens. The pathogen causes severe human diseases and outbreaks. This study aimed to identify and characterize non-O157 STEC isolated from cattle feces from central and southern Chile. We analyzed 446 cattle fecal samples and isolated non-O157 STEC from 12.6% (56/446); a total of 93 different isolates were recovered. Most isolates displayed β-glucuronidase activity (96.8%; 90/93) and fermented sorbitol (86.0%; 80/93), whereas only 39.8% (37/93) were resistant to tellurite. A subgroup of 30 representative non-O157 STEC isolates was selected for whole-genome sequencing and bioinformatics analysis. In silico analysis showed that they grouped into 16 different sequence types and 17 serotypes; the serotypes most frequently identified were O116:H21 and O168:H8 (13% each). A single isolate of serotype O26:H11 was recovered. One isolate was resistant to tetracycline and carried resistance genes tet(A) and tet(R); no other isolate displayed antimicrobial resistance or carried antimicrobial resistance genes. The intimin gene (eae) was identified in 13.3% (4/30) of the genomes and 90% (27/30) carried the stx2 gene. A phylogenetic reconstruction demonstrated that the isolates clustered based on serotypes, independent of geographical origin. These results indicate that cattle in Chile carry a wide diversity of STEC potentially pathogenic for humans based on the presence of critical virulence genes.

Isolation and Identification of Escherichia coli O157:H7 Lytic Bacteriophage from Environment Sewage

Escherichia coli O157:H7 is one of the pathogenic bacteria causing foodborne disease. The use of lytic bacteriophages can be a good solution to overcome the disease. This study is aimed at isolating lytic bacteriophages from environmental sewage with E. coli O157:H7 bacterial cells. The sample used in this study was eight bacteriophages, and the technique used in identifying E. coli O157:H7 carriers of the stx1 and stx2 genes was PCR. The double layer plaque technique was used to classify bacteriophages. Plaque morphology, host specificity, and electron micrograph were used to identify the bacteriophages. The result obtained plaque morphology as a clear zone with the largest diameter size of 3.5 mm. Lytic bacteriophage could infect E. coli O157:H7 at the highest titer of 10 × 108 PFU/mL. Bacteriophages have been identified as Siphoviridae and Myoviridae. Phage 3, phage 4, and phage 8 could infect Atypical Diarrheagenic E. coli 1 (aDEC1) due to their host specificity. The Friedman statistical tests indicate that lytic bacteriophage can significantly lyse E. coli O157:H7 (p = 0.012). The lysis of E. coli O157:H7 by phage 1, phage 2, phage 3, and phage 5 bacteriophages was statistically significant, according to Conover's posthoc test (p < 0.05). The conclusion obtained from this study is that lytic bacteriophages from environmental sewage could lyse E. coli O157:H7. Therefore, it could be an alternative biocontrol agent against E. coli O157:H7 that contaminates food causing foodborne disease.

​A Study on Alteration in Serum Biochemical Parameters in Colibacillosis Affected Diarrhoeic Cattle Calves

Background: The objective of the present study was to evaluate the changes in serum biochemical parameters in colibacillosis affected diarrhoeic cattle calves below one month of age. Methods: The study was undertaken at Veterinary Clinical Complex, College of Veterinary and Animal Science, Rajasthan University of Veterinary and Animal Sciences, Bikaner, Rajasthan, India, during the period from September 2018 to March 2019. A total of 32 calves affected with colibacillosis confirmed on the basis of Multiplex PCR (for presence of K99, Stx1 and eaeA genes of E. coli) and eight clinically normal healthy calves (as control) were taken to investigate the serum biochemical profiles. Result: Analysis of serum samples of calves affected with colibacillosis revealed significant increase in the values of total protein, albumin, globulin, A/G ratio, blood urea nitogen, creatinine, potassium, total oxidant status andoxidative stress index while there is a significant decrease in the values of glucose, sodium, chloride, bicarbonate, IgG, IgM and total antioxidant status. There were no significant changes observed in the values of ALP, AST and ALT in affected calves as compared to healthy control group of calves.

Molecular detection of Shiga toxin-producing Escherichia coli (STEC) O157 in sheep, goats, cows and buffaloes.

Shiga toxin-producing E. coli (STEC) are important foodborne pathogens that causing serious public health consequences worldwide. The present study aimed to estimate the prevalence ratio and to identify the zoonotic potential of E. coli O157 isolates in slaughtered adult sheep, goats, cows and buffaloes. A total of 400 Recto-anal samples were collected from two targeted sites Rawalpindi and Islamabad. Among them, 200 samples were collected from the slaughterhouse of Rawalpindi included sheep (n = 75) and goats (n = 125). While, 200 samples were collected from the slaughterhouse of Islamabad included cows (n = 120) and buffalos (n = 80). All samples were initially processed in buffered peptone water and then amplified by conventional PCR. Samples positive for E. coli O157 were then streaked onto SMAC media plates. From each positive sample, six different Sorbitol fermented pink-colored colonies were isolated and analyzed again via conventional PCR to confirm the presence of rfbE O157 gene. Isolates positive for rfbE O157 gene were then further analyzed by multiplex PCR for the presence of STEC other virulent genes (sxt1, stx2, eae and ehlyA) simultaneously. Of 400 RAJ samples only 2 (0.5%) showed positive results for E. coli O157 gene, included sheep 1/75 (1.33%) and buffalo 1/80 (1.25%). However, goats (n = 125) and cows (n = 120) found negative for E. coli O157. Only 2 isolates from each positive sample of sheep (1/6) and buffalo (1/6) harbored rfbE O157 genes, while five isolates could not. The rfbE O157 isolate (01) of sheep sample did not carry any of STEC genes, while the rfbE O157 isolate (01) of buffalo sample carried sxt1, stx2, eae and ehlyA genes simultaneously. It was concluded that healthy adult sheep and buffalo are possibly essential carriers of STEC O157. However, rfbE O157 isolate of buffalo RAJ sample carried 4 STEC virulent genes, hence considered an important source of STEC infection to humans and environment which should need to devise proper control systems.

The Prevalence of Shiga Toxin-1 in Non-Shigella Dysenteriae Isolates Collected from Diarrhea Samples in Patients, Ahvaz, Iran.

Acute diarrhea is a major public health problem, particularly in developing countries. Shigellosis is one of the substantial causative agents of microbial dysentery and still has a remarkable prevalence, particularly in areas with poor hygienic infrastructures. The probable existence of the deadly Shiga toxin (Stx) protein in some Shigella strains would manifest life-threatening clinical symptoms of the infection. The aim of this study was to determine the presence of Shigella toxin 1 (Stx1) in isolated from patients with diarrhea. Totally, 227 Shigella species, including 60 S. flexneri, 157 S. sonnei, and 10 S. boydii were collected from diarrheal patients in the tropical infectious diseases research center of Ahvaz, Iran, during 2013-2015. The isolates were collected mostly from the intensive care unit, infectious disease, and surgery settings. The isolates were identified, and the polymerase chain reaction (PCR) was performed to detect the stx gene. The results indicated that none of them encode the stx1 gene. Isolates of this study were not capable of stx1 encoding. Future investigations should consider the relations between other Shigella species and Shigella toxin in Iran.

High Occurrence of Shiga Toxin-Producing Escherichia coli in Raw Meat-Based Diets for Companion Animals-A Public Health Issue.

Feeding pets raw meat-based diets (RMBDs) is becoming increasingly popular but comes with a risk of pathogenic bacteria, including Shiga toxin-producing Escherichia coli (STEC). In humans, STEC may cause gastrointestinal illnesses, including diarrhea, hemorrhagic colitis (HC), and the hemolytic uremic syndrome (HUS). The aim of this study was to evaluate commercially available RMBDs with regard to the occurrence of STEC. Of 59 RMBD samples, 59% tested positive by real-time PCR for the presence of Shiga toxin genes stx1 and/or stx2. STECs were recovered from 41% of the 59 samples, and strains were subjected to serotyping and virulence gene profiling, using whole genome sequencing (WGS)-based methods. Of 28 strains, 29% carried stx2a or stx2d, which are linked to STEC with high pathogenic potential. Twenty different serotypes were identified, including STEC O26:H11, O91:H10, O91:H14, O145:H28, O146:H21, and O146:H28, which are within the most common non-O157 serogroups associated with human STEC-related illnesses worldwide. Considering the low infectious dose and potential severity of disease manifestations, the high occurrence of STEC in RMBDs poses an important health risk for persons handling raw pet food and persons with close contact to pets fed on RMBDs, and is of concern in the field of public health.

RETRACTED ARTICLE: Molecular detection of pathogenic Escherichia coli strains and their antibiogram associated with risk factors from diarrheic calves in Jimma Ethiopia

Diarrheagenic Escherichia coli are a number of pathogenic E. coli strains that cause diarrheal infection both in animal and human hosts due to their virulence factors. A cross sectional study was conducted between November, 2016 and April, 2017 to isolate and molecularly detect pathogenic E. coli from diarrheic calves to determine the pathogenic strains, antibiogram and associated risk factors in Jimma town. Purposive sampling technique was used to collect 112 fecal samples from diarrheic calves. Conventional culture and biochemical methods were conducted to isolate E. coli isolates. Molecular method was followed to identify virulence factors of pathogenic E. coli strains. Antimicrobial sensitivity patterns of the isolates were tested using the Kirby–Bauer disk diffusion method. A structured questionnaire was also used to collect information from dairy farms and socio-demographic data. The overall isolation rate of E. coli in calves was 51.8% (58/112) (95% CI 42.0–61.0). The occurrence of the bacterium differed significantly by age, colostrum feeding time, amount of milk given per time and navel treatment (P < 0.05). Multivariable analysis revealed that the odds of being infected was significantly highest in calves which fed 1–1.5 L amount of milk per a time (OR 5.38, 95% CI 1.66–17.45, P = 0.005). The overall virulence genes detection rate was 53.5% (95% CI 40.0–67.0). Eleven (19.6%) of eaeA, 6 (10.7%) of Stx1 and 13 (23.2%) of Stx2 genes were detected from calves isolates. Except ciprofloxacillin, all isolates were resistant to at least one drug. Multi drug resistance was recorded in 68.0% (38/56) of calves isolates. Neomycin, 83.3% (25/30), followed by amoxicillin, 53.3% (16/30) were the highest resisted virulence genes. The study demonstrated considerable isolation rate, multiple antimicrobial resistant isolates and high resistant virulent genes in diarrheic calves. It also indicated that the potential importance of calves as source of pathogenic E. coli strains and resistant genes for human diarrhea infection. Improving the hygienic practice of farms and wise use of antimicrobials could help to reduce the occurrence of pathogenic E. coli in farms. Hence, further studies are needed to describe all virulent factors and serotypes associated with the emergence of drug resistant pathogenic E. coli strains in calves.

Virulence determinants and antimicrobial resistance of E. coli isolated from bovine clinical mastitis in some selected dairy farms of Bangladesh.

E. coli is one of the major significant pathogens causing mastitis, the most complex and costly diseases in the dairy industry worldwide. Present study was undertaken to isolate, detect the virulence factors, phylogroup, antimicrobial susceptibility and antimicrobial resistance genes in E. coli from cows with clinical mastitis. A total of 68 milk samples comprising 53 from clinical mastitis and 15 from apparently healthy cattle were collected from four different established dairy farms in Bangladesh. E. coli was isolated from the milk samples and identified by PCR targeting malB gene and sequencing of 16S rRNA gene. E. coli isolates were screened by PCR for the detection of major virulence genes (stx, eae and cdt) of diarrheagenic E. coli followed by phylogenetic grouping. Antimicrobial susceptibility of the E. coli isolates was determined by disk diffusion test and E. coli showing resistance was further screened for the presence of antimicrobial resistance genes. E. coli was isolated from 35.8% of the mastitis milk samples but none from the apparently healthy cattle milk. All the E. coli isolates were negative for stx, eae and cdt genes and belonged to the phylogenetic groups A and B1 which comprising of commensal E. coli. Antibiotic sensitivity testing revealed 84.2% (16/19) of the isolates as multidrug resistant. Highest resistance was observed against amoxicillin (94.5%) followed by ampicillin (89.5%) and tetracycline (89.5%). E. coli were found resistant against all the classes of antimicrobials used at the farm level. Tetracycline resistance gene (tetA) was detected in 100% of the tetracycline resistant E. coli and blaTEM-1 was present in 38.9% of the E. coli isolates. Findings of this study indicate a potential threat of developing antimicrobial resistance in commensal E. coli and their association with clinical mastitis. Occurrence of multidrug resistant E. coli might be responsible for the failure of antibiotic therapies in clinical mastitis as well as pose potential threat of transmitting and development of antibiotic resistance in human.

Zoonotic Potential of Escherichia Coli in Poultry Intestinal Contents in Ismailia City, Egypt with Special Reference to Shiga Toxin-Producing (STEC) Strains

Avian colibacillosis can affect birds of all ages. It is now one of the major causes of economic losses in the poultry industry, as well as a public health risk all over the world.This study aimed to determine the occurrence of E. coli in market-age poultry and the risk of its zoonotic infections in Ismailia city, Egypt. A total of 350 samples were collected from the intestinal contents of the slaughtered poultry (100 farm chickens, 100 backyard chickens, 50 ducks, 50 geese, and 50 turkeys) in Ismailia City. On the other hand, a total of 200 samples were collected from humans at outpatient clinics (100 stool samples and 100 urine samples) from the same investigated area. Samples were examined using bacteriological and molecular examination for genus-specific and virulence gene detection. Results revealed that the isolation rates of E. coli were 70% in farm chickens and turkeys, 88% in backyard chickens, 86% in domestic ducks, 72% in geese. Of avian E. coli isolates, the rates of Shiga toxin-producing E. coli (STEC) strains were 91.4%, 96.6%, 90.9%, 41.7%, and 51.4%% in farm chickens, backyard chickens, ducks, geese, and turkey respectively. In humans, the isolation rate of E. coli was 42% in urine samples and 66% in stool samples. The human STEC isolates were higher in stool samples (26%) than in urine samples (6%). The isolation rates of E. coli were significantly higher in persons who were in contact with poultry than in persons who were not in contact with poultry (p ≤ 0.01). In conclusion, the high isolation rates of STEC and detection with similarities of some E. coli virulence genes (It, St, eaeA, Stx1, and Stx2 genes) from poultry intestinal contents and human samples indicated a significant risk of zoonotic transmission of E. coli via food chain in the investigated area.

Bloody Diarrhea and Shiga Toxin–Producing Escherichia coli Hemolytic Uremic Syndrome in Children: Data from the ItalKid-HUS Network

To analyze the results of an enhanced laboratory-surveillance protocol for bloody diarrhea aimed at identifying children with Shiga toxin-producing Escherichia coli (STEC) infection early in the course of the disease toward the early identification and management of patients with hemolytic uremic syndrome (HUS). The study (2010-2019) involved a referral population of 2.3 million children. Stool samples of patients with bloody diarrhea were screened for Shiga toxin (Stx) genes. Positive patients were rehydrated and monitored for hemoglobinuria until diarrhea resolved or STEC-HUS was diagnosed. A total of 4767 children were screened; 214 (4.5%) were positive for either Stx1 (29.0%) or Stx2 (45.3%) or both Stx1+2 (25.7%); 34 patients (15.9%) developed STEC-HUS (0.71% of bloody diarrheas). Hemoglobinuria was present in all patients with HUS. Patients with Stx2 alone showed a greater risk of STEC-HUS (23.7% vs 12.7%) and none of the patients with Stx1 alone developed HUS. During the same period of time, 95 other patients were diagnosed STEC-HUS but were not captured by the screening program (26 had nonbloody diarrhea, 11 came from areas not covered by the screening program, and 58 had not been referred to the screening program, although they did meet the inclusion criteria). At HUS presentation, serum creatinine of patients identified by screening was significantly lower compared with that of the remaining patients (median 0.9 vs 1.51mg/dL). Nearly 1% of children with bloody diarrhea developed STEC-HUS, and its diagnosis was anticipated by the screening program for Stx. The screening of bloody diarrhea for Stx is recommended, and monitoring patients carrying Stx2 with urine dipstick for hemoglobinuria is suggested to identify the renal complication as early as possible.

Shiga Toxin-Producing Escherichia coli in Wheat Grains: Detection and Isolation by Polymerase Chain Reaction and Culture Methods.

Shiga toxin-producing Escherichia coli (STEC) are major foodborne pathogens and seven serogroups, O26, O45, O103, O111, O121, O145, and O157, often called top-7 STEC, account for the majority of the STEC-associated human illnesses in the United States. Two Shiga toxins, Shiga toxins 1 and 2, encoded by stx1 and stx2 genes, are major virulence factors that are involved in STEC infections. Foodborne STEC infections have been linked to a variety of foods of both animal and plant origin, including products derived from cereal grains. In recent years, a few STEC outbreaks have been linked to contaminated wheat flour. The microbiological quality of the wheat grains is a major contributor to the safety of wheat flour. The objective of the study was to utilize polymerase chain reaction (PCR)- and culture-based methods to detect and isolate STEC in wheat grains. Wheat grain samples (n = 625), collected from different regions of the United States, were enriched in modified buffered peptone water with pyruvate (mBPWp) or E. coli (EC) broth, and they were then subjected to PCR- and culture-based methods to detect and isolate STEC. Wheat grains enriched in EC broth yielded more samples positive for stx genes (1.6% vs. 0.32%) and STEC serogroups (5.8% vs. 2.4%) than mBPWp. The four serogroups of top-7 detected and isolated were O26, O45, O103, and O157 and none of the isolates was positive for the Shiga toxin genes. A total of five isolates that carried the stx2 gene were isolated and identified as serogroups O8 (0.6%) and O130 (0.2%). The EC broth was a better medium to enrich wheat grains than mBPWp for the detection and isolation of STEC. The overall prevalence of virulence genes and STEC serogroups in wheat grains was low. The stx2-positive serogroups isolated, O8 and O130, are not major STEC pathogens and have only been implicated in sporadic infections in animals and humans.