H7 Isolates Research Articles

Genetic Characterization of Novel H7Nx Low Pathogenic Avian Influenza Viruses from Wild Birds in South Korea during the Winter of 2020-2021.

Zoonotic infection with avian influenza viruses (AIVs) of subtype H7, such as H7N9 and H7N4, has raised concerns worldwide. During the winter of 2020–2021, five novel H7 low pathogenic AIVs (LPAIVs) containing different neuraminidase (NA) subtypes, including two H7N3, an H7N8, and two H7N9, were detected in wild bird feces in South Korea. Complete genome sequencing and phylogenetic analysis showed that the novel H7Nx AIVs were reassortants containing two gene segments (hemagglutinin (HA) and matrix) that were related to the zoonotic Jiangsu–Cambodian H7 viruses causing zoonotic infection and six gene segments originating from LPAIVs circulating in migratory birds in Eurasia. A genomic constellation analysis demonstrated that all H7 isolates contained a mix of gene segments from different viruses, indicating that multiple reassortment occurred. The well-known mammalian adaptive substitution (E627K and D701N) in PB2 was not detected in any of these isolates. The detection of multiple reassortant H7Nx AIVs in wild birds highlights the need for intensive surveillance in both wild birds and poultry in Eurasia.

Prevalence and antibiotic resistance of Escherichia coli O157:H7 in beef at a commercial slaughterhouse in Moro, Kwara State, Nigeria.

BackgroundGastroenteritis due to foodborne disease is a leading cause of death in developing countries. In Nigeria, there is an increasing demand for beef. Yet, there is no surveillance for Escherichia coli O157:H7 contamination of raw beef and little is known about the carriage of this pathogen in Nigeria’s livestock.MethodsA total of 415 samples, including 180 cow carcass swabs, 180 caecal content samples, 16 water samples, 25 hand swabs and 14 knife swabs were collected at a large abattoir in the Moro region of Kwara State, Nigeria. The samples were enriched in modified tryptone broth containing novobiocine, and plated onto Sorbitol–MacConkey agar (Oxoid SR0172E) supplemented with 0.05 mg l−1 cefixime and 2.5 mg l−1 potassium tellurite (Oxoid) (CT-SMAC). Indole-producing isolates were confirmed serologically by serotyping with antisera specific for the O157 and H7 antigens. The E. coli O157:H7 isolates were further tested for their susceptibility to antibiotic agents using the disc diffusion method. Commercially available Gram-negative multi-discs (Oxoid) comprising nitrofurantoin (30 µg), ampicillin (5 µg), ceftazidime (30 µg), gentamicin (10 µg), ciprofloxacin (5 µg), augmentin (30 µg), ofloxacin (5 µg) and cefuroxime (30 µg) were tested.ResultsOverall, 16 (3.9 %) samples were contaminated with E. coli O157:H7, of which 10 (5.6 %) were isolated from carcass swabs, 4 (2.2 %) from caecal content samples and 2 (12.5 %) from water. All isolates were multidrug-resistant (MDR), with resistance to ampicillin, ceftazidime and cefuroxime being the most common.ConclusionThis study provides evidence to suggest that E. coli O157:H7 exists in the beef production chain. The pathogen reveals a high frequency of multidrug resistance, suggesting that consumers and handlers of such meat are at risk of contracting antibiotic-resistant E. coli O157:H7-associated foodborne disease. Routine monitoring of antibiotic resistance is critical to uncovering novel therapeutic strategies that will help inform clinical practice guidelines.

Genomic analysis of shiga toxin-containing Escherichia coli O157:H7 isolated from Argentinean cattle.

Cattle are the main reservoir of Enterohemorrhagic Escherichia coli (EHEC), with O157:H7 the distinctive serotype. EHEC is the main causative agent of a severe systemic disease, Hemolytic Uremic Syndrome (HUS). Argentina has the highest pediatric HUS incidence worldwide with 12-14 cases per 100,000 children. Herein, we assessed the genomes of EHEC O157:H7 isolates recovered from cattle in the humid Pampas of Argentina. According to phylogenetic studies, EHEC O157 can be divided into clades. Clade 8 strains that were classified as hypervirulent. Most of the strains of this clade have a Shiga toxin stx2a-stx2c genotype. To better understand the molecular bases related to virulence, pathogenicity and evolution of EHEC O157:H7, we performed a comparative genomic analysis of these isolates through whole genome sequencing. The isolates classified as clade 8 (four strains) and clade 6 (four strains) contained 13 to 16 lambdoid prophages per genome, and the observed variability of prophages was analysed. An inter strain comparison show that while some prophages are highly related and can be grouped into families, other are unique. Prophages encoding for stx2a were highly diverse, while those encoding for stx2c were conserved. A cluster of genes exclusively found in clade 8 contained 13 genes that mostly encoded for DNA binding proteins. In the studied strains, polymorphisms in Q antiterminator, the Q-stx2A intergenic region and the O and P γ alleles of prophage replication proteins are associated with different levels of Stx2a production. As expected, all strains had the pO157 plasmid that was highly conserved, although one strain displayed a transposon interruption in the protease EspP gene. This genomic analysis may contribute to the understanding of the genetic basis of the hypervirulence of EHEC O157:H7 strains circulating in Argentine cattle. This work aligns with other studies of O157 strain variation in other populations that shows key differences in Stx2a-encoding prophages.

Characterization of Escherichia coli from Edible Insect Species: Detection of Shiga Toxin-Producing Isolate.

Insects as novel foods are gaining popularity in Europe. Regulation (EU) 2015/2283 laid the framework for the application process to market food insects in member states, but potential hazards are still being evaluated. The aim of this study was to investigate samples of edible insect species for the presence of antimicrobial-resistant and Shiga toxin-producing Escherichia coli (STEC). Twenty-one E. coli isolates, recovered from samples of five different edible insect species, were subjected to antimicrobial susceptibility testing, PCR-based phylotyping, and macrorestriction analysis. The presence of genes associated with antimicrobial resistance or virulence, including stx1, stx2, and eae, was investigated by PCR. All isolates were subjected to genome sequencing, multilocus sequence typing, and serotype prediction. The isolates belonged either to phylogenetic group A, comprising mostly commensal E. coli, or group B1. One O178:H7 isolate, recovered from a Zophobas atratus sample, was identified as a STEC. A single isolate was resistant to tetracyclines and carried the tet(B) gene. Overall, this study shows that STEC can be present in edible insects, representing a potential health hazard. In contrast, the low resistance rate among the isolates indicates a low risk for the transmission of antimicrobial-resistant E. coli to consumers.

Prevalence and molecular characterization of multidrug‐resistant Escherichia coliO157: H7 from dairy milk in the Peshawar region of Pakistan

AbstractThis study analyzed raw (unpasteurized) dairy milk samples from farms, milk vendors, and shops in the Peshawar region of Pakistan for the prevalence of pathogenic multidrug‐resistant (MDR) Escherichia coli. During August 2018–September 2019, 100 cow′s milk samples from 10 subregions of the Town‐3 district were tested for the prevalence of E. coli and of Shiga toxin–producing serotype O157: H7 using various biochemical, morphological, and molecular tests. MDR was tested using the disc diffusion method, while Shiga toxin and antibiotic resistance genes were identified by polymerase chain reaction. Twenty eight samples contained E. coli, of which six were positive for O157: H7. The O157: H7 isolates had resistances of 16.7% to amoxicillin–clavulanic acid, 66.7% to ampicillin, 66.7% to ceftriaxone, 50.0% to kanamycin, 83.3% to streptomycin, 83.3% to trimethoprim sulfamethoxazole, and 83.3% to vancomycin. All O157: H7 isolates contained the blaCTXM resistance gene, three of which also contained the blaTEM1 and blaNDM1 resistance genes, and were resistant to six or seven out of the seven antibiotics tested in a disc diffusion assay with 10 μg or 30 μg of antibiotic. Our observations are of high concern to public health in Pakistan, so appropriate measures should be implemented to minimize E. coli O157: H7 cross‐contamination and infection, including screening for E. coli O157: H7 in foods, equipment, and storage vessels; testing of isolates for antibiotic resistance; implementation of rigorous hygiene protocols in milk production and storage; training of farmers and milk production workers; and education of the public about hygiene and food practices.

Characterization of Subtype H6 Avian Influenza A Viruses Isolated From Wild Birds in Poyang Lake, China.

Subtype H6 avian influenza A viruses (IAVs) are enzootic and genetically diverse in both domestic poultry and wild waterfowl and may cause spillovers in both pigs and humans. Thus, it is important to understand the genetic diversity of H6 IAVs in birds and their zoonotic potential. Compared with that in domestic poultry, the genetic diversity of H6 viruses in wild birds in China has not been well-understood. In this study, five H6 viruses were isolated from wild birds in Poyang Lake, China, and genetic analyses showed that these isolates are clustered into four genotypes associated with reassortments among avian IAVs from domestic poultry and wild birds in China and those from Eurasia and North America and that these viruses exhibited distinct phenotypes in growth kinetics analyses with avian and mammalian cells lines and in mouse challenge experiments. Of interest is that two H6 isolates from the Eurasian teal replicated effectively in the mouse lung without prior adaptation, whereas the other three did not. Our study suggested that there are variations in the mammalian viral replication efficiency phenotypic among genetically diverse H6 IAVs in wild birds and that both intra- and inter-continental movements of IAVs through wild bird migration may facilitate the emergence of novel H6 IAV reassortants with the potential for replicating in mammals, including humans. Continued surveillance to monitor the diversity of H6 IAVs in wild birds is necessary to increase our understanding of the natural history of IAVs.

Genetic features of verotoxigenic Escherichia coli O157:H7 isolated from clinical cases of Argentina and Chile

We aimed to compare the genetic diversity existing in VTEC O157:H7 strains isolated from cases of human disease from Argentina and Chile. For it, 76 strains were studied in relation to the distribution of genes encoding virulence factors and subtyped by lineage-specific polymorphisms (LSPA-6), and phylogroups assignment. Our results show the almost exclusive circulation of VTEC O157:H7 isolates belonging to lineage I/II, associated with hypervirulent strains, and to the phylogroup E and, on the other hand, genetic diversity present among Argentinean and Chilean strains analyzed, mainly in relation to putative virulence determinants and nle profiles.

The role of l-arabinose metabolism for Escherichia coli O157:H7 in edible plants.

Arabinose is a major plant aldopentose in the form of arabinans complexed in cell wall polysaccharides or glycoproteins (AGP), but comparatively rare as a monosaccharide. l-arabinose is an important bacterial metabolite, accessed by pectolytic micro-organisms such as Pectobacterium atrosepticum via pectin and hemicellulose degrading enzymes. However, not all plant-associated microbes encode cell-wall-degrading enzymes, yet can metabolize l-arabinose, raising questions about their use of and access to the glycan in plants. Therefore, we examined l-arabinose metabolism in the food-borne pathogen Escherichia coli O157:H7 (isolate Sakai) during its colonization of plants. l-arabinose metabolism (araBA) and transport (araF) genes were activated at 18 °C in vitro by l-arabinose and expressed over prolonged periods in planta. Although deletion of araBAD did not impact the colonization ability of E. coli O157:H7 (Sakai) on spinach and lettuce plants (both associated with STEC outbreaks), araA was induced on exposure to spinach cell-wall polysaccharides. Furthermore, debranched and arabinan oligosaccharides induced ara metabolism gene expression in vitro, and stimulated modest proliferation, while immobilized pectin did not. Thus, E. coli O157:H7 (Sakai) can utilize pectin/AGP-derived l-arabinose as a metabolite. Furthermore, it differs fundamentally in ara gene organization, transport and regulation from the related pectinolytic species P. atrosepticum, reflective of distinct plant-associated lifestyles.

Molecular systematic and phylogenetic analysis of indigenous bacterial isolates with potential as bioremediation agent based on 16S rRNA gene analysis

Liquid biomedical waste is a form of medical waste from community health centers (Pusat Kesehatan Masyarakat or Puskesmas) with high levels of health hazardous organic contaminants. Bioremediation is an alternative way to eliminate toxic components in liquid waste. A bacteria community that can be used as component of organic waste bioremediation is indigenous hydrolytic and non-pathogenic to low-pathogenic bacteria. From previous studies, 4 hydrolytic indigenous bacterial isolates with such characteristics were obtained from liquid clinical wastes of two health centers in Semarang City, namely H1, H3, H5 (from Puskesmas Halmahera), and T3 (from Puskesmas Tlogosari Kulon). This study aimed to reveal the molecular identity and kinship these bacterial isolates to understand more of their properties as consortium of bioremediation agent. Molecular identification and phylogenetic tree construction works were carried out based on 16S rRNA gene sequences. Sequences of 16S rRNA gene sequences were obtained by isolation and gene amplification using the PCR method followed by sequencing. Based on the results of molecular identification, the four isolates studied were in the same class, namely Gammaproteobacter with Phylum Proteobacter. H1 bacterial isolates have 98.01% similarity with Acinetobacter schindleri. H3 and H5 isolates share the same genus, Stenotrophomonas, with 99.79% similarity with S. maltophiphila and 97.69% with S. acidaminiphila, respectively. T3 isolate had a similarity of 98.85% with Pararheinheimera aquatica species, which was widely known as a potent bioremediation agent. The phylogenetic tree design with the MEGA 6 program showed that the H3 and H5 isolates had the closest kinship compared to the other two isolates, while the T3 isolates had the farthest relationship with the 3 other isolates. This is in line with the fact that T3 isolate was originated from a different location separating it from 3 other isolates.

Prevalence of Multidrug Resistant Escherichia coli O157:H7 Causing Diarrhoea in Children at District Khairpur

Aim: Multidrug resistance (MDR) is a major public health problem worldwide. The aim of this study was to assess the prevalence, levels of antimicrobial susceptibility and extended-spectrum beta lactamase (ESBL) production by Escherichia coli O157: H7.
 Methodology: A cross sectional study was performed and a total of 116 stool samples were collected from children aged ≤ 5 years presenting diarrhoea from hospital located at district Khairpur. E. coli O157: H7 was isolated on Sorbitol MacConkey agar and isolates were initially characterized by Gram staining. Subsequently biochemical characteristics except oxidase test (which was performed manually) and drug sensitivity test was performed by using Automated MicroScan® walkaway machine. ESBL production by isolates was determined by disc diffusion method as per Clinical & Laboratory Standards Institute (CLSI) guidelines.
 Results: Out of 116 diarrheal samples tested, 16 (13.79%) samples were positive for E. coli O157:H7. High number of E. coli O157: H7 were isolated from children aged up to 1 year followed by 2-3 years aged. Isolates were frequently found in summer season (12 times) followed by winter (3 times) and Autumn (1 time). The isolates were found 100% resistant against Nalidixic acid, Imipenem, Amoxycillin /Clavulanic Acid, Norfloxacin, Chloramphenicol, Vancomycin, Doxycycline, and Ampicillin and 90% resistant to Cifixime. ESBL production was noted in 12 (75%) out of 16 E. coli O157:H7 isolates as confirmed by CLSI protocol.
 Conclusion: This study revealed that E. coli O157: H7 should be considered as major pathogenic bacterium causing diarrhea in children less than three years. The prevalence of ESBL producer MDR E. coli O157: H7 is of great concern, which requires monitoring of infection control measures through efficient antimicrobial management to ensure an uncompromised public health.

Prevalence, characteristics and antibiogram profile of Escherichia coli O157:H7 isolated from raw and fermented (nono) milk in Benin City, Nigeria

Background: Most Escherichia coli strains are harmless commensals, but some serotypes can cause serious food poisoning in their hosts, and are infrequently responsible for product recalls due to food contamination. The present study was carried out to determine the occurrence of E. coli O157:H7 and other E. coli strains from raw and fermented (nono) milk in Benin City, Nigeria.Methodology: A total of 66 (33 raw and 33 nono) milk samples were obtained from retailers from 3 different stations in Aduwawa market, Benin City, Nigeria between January and June, 2017. Samples were analysed by cultural methods for faecal coliforms using M-Fc agar, E. coli using Chromocult coliform agar, and E. coli O157:H7 using sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Presumptive E. coli andE. coli O157:H7 isolates were confirmed by polymerase chain reaction (PCR) assay using specific primers. Antimicrobial susceptibility profile of confirmed isolates was performed using the Kirby-Bauer disk diffusion method, with zones of inhibition interpreted according to the guidelines of Clinical and Laboratory Standards Institute (CLSI). Data were analysed using the SPSS version 21.0.Results: From the 66 nono and raw milk samples assessed in this study, all (100%) were phenotypically positive for E. coli O157:H7. A total of 19 E. coli O157:H7 and 41 other strains of E. coli were confirmed by PCR. The resistance profile of the 19 E. coli O157:H7 isolates showed 100% (19/19) resistance to penicillin G and ampicillin; 94.7% (18/19) to chloramphenicol; 89.5% (17/19) to erythromycin; and 78.9% (15/19) to sulfamethoxazole and oxytetracycline, while the sensitivity profile showed that 100% (19/19) E. coli O157:H7 isolates were sensitive to gentamicin and ofloxacin. The resistance profile of other 41 E. coli isolates showed 100% (41/41) resistance to penicillin G and ampicillin; 97.6% (40/41) to chloramphenicol; and 92.7% (38/41) to erythromycin, while 97.6% (40/41) were sensitive to gentamicin and kanamycin. Ten E. coli O157:H7 isolates (52.6%) showed extensive drug resistance pattern to 11 antibiotics in 7 antimicrobial classes with multiple antibiotic resistance (MAR) index of 0.46.Conclusion: Findings from the present study clearly indicated that the safety and quality of fresh and fermented milk were not satisfactory and could be of public health concern.
 Key words: Nono, Escherichia coli; Pathotypes, Resistance index, Public health, Milk

Occurrence and Antibiogram of Escherichia coli O157 : H7 in Raw Beef and Hygienic Practices in Abattoir and Retailer Shops in Ambo Town, Ethiopia.

Foodborne infections are widespread and growing public health problems in the world. Shiga toxin-producing Escherichia coli O157 : H7 is one of the most significant foodborne pathogens. This study was conducted to assess the occurrence and antibiogram of E. coli O157 : H7 from raw beef as well as hygienic and sanitary practices of meat handling in abattoir and retailer shops. Systematic random sampling technique and census methods were used to collect samples from abattoir and retailer shops, respectively. All tryptone soya broth preenriched carcass samples were subcultured onto MacConkey agar. Then, the bacterium confirmed as Escherichia coli using biochemical tests was streaked onto Sorbitol-MacConkey agar and incubated at 37°C for 24 hrs. Escherichia coli O157 : H7 was confirmed by latex agglutination kit. In vitro antimicrobial susceptibility test of Escherichia coli O157 : H7 isolates was done against 13 antimicrobials. Hygiene and sanitation data were collected using a pretested structured questionnaire and observational checklist. Pearson Chi-square and Fisher's exact two-tailed tests were performed and differences were considered significant at P ≤ 0.05. Out of 197 meat samples, 23.4% (95% confidence interval (CI): 17.6–29.9%) and 9.1% (95% CI: 5.5–14.1%) were contaminated with Escherichia coli and Escherichia coli O157 : H7, respectively. There was a significant variation in the occurrence of Escherichia coli O157 : H7 between retailer shops (19.1%) and abattoir (7.2%) (P = 0.03). The study revealed that the municipal abattoir and retailer shops in Ambo town did not adhere to the required sanitation and hygienic standards. All Escherichia coli O157 : H7 isolates were susceptible to norfloxacin, sulfamethoxazole-trimethoprim, chloramphenicol, and ceftazidime. However, all isolates were resistant to amoxicillin. Multidrug resistance was widespread and was found in 66.3% of Escherichia coli O157 : H7 isolates. The occurrence of Escherichia coli O157 : H7 was high. Therefore, fulfilling national and international meat safety requirements, training and monitoring of meat handlers, and rational use of antimicrobials are recommended

Escherichia coli O15 : H7 from Food of Animal Origin in Arsi: Occurrence at Catering Establishments and Antimicrobial Susceptibility Profile.

Escherichia coli O157 : H7 (E. coli O157 : H7) has been found to be the major cause of food-borne diseases and a serious public health problem in the world, with an increasing concern for the emergence and spread of antimicrobial-resistant strains. Hitherto, little is known about the carriage of E. coli O157 : H7 and its antimicrobial susceptibility profile in the food of animal origin in Ethiopia. This study aimed to determine the occurrence and multidrug resistance profile of E. coli O157 : H7 from food of animal origin at different catering establishments in the selected study settings of Arsi Zone. One hundred ninety-two animal origin food items, namely, raw/minced meat (locally known as “Kitfo,” “Kurt,” and “Dulet”), raw milk, egg sandwich, and cream cake samples were collected and processed for microbiological detection of E. coli O157 : H7. Out of 192 samples, 2.1% (4/192) were positive for E. coli O157 : H7. Two E. coli O157 : H7 isolates were obtained from “Dulet” (6.3%) followed by “Kurt” (3.1%, 1/32) and raw milk (3.1%, 1/32), whereas no isolate was obtained from “Kitfo,” egg sandwich, and cream cake samples. Of the 4 E. coli O157 : H7 isolates subjected to 10 panels of antimicrobial discs, 3 (75%) were highly resistant to kanamycin, streptomycin, and nitrofurantoin. Besides, all the isolates displayed multidrug resistance phenotypes, 3 to 5 antimicrobial resistance, amid kanamycin, streptomycin, nitrofurantoin, tetracycline, and chloramphenicol. The occurrence of multidrug-resistant E. coli O157 : H7 isolates from foods of animal origin sampled from different catering establishments reveals that the general sanitary condition of the catering establishments, utensils used, and personnel hygienic practices did not comply with the recommended standards. Thus, this finding calls for urgent attention toward appropriate controls and good hygienic practices in different catering establishments dealing with consuming raw/undercooked foods of animal origin.

Genome Sequences of 104 Escherichia coli O157:H7 Isolates from Pigs, Cattle, and Pork Production Environments in Alberta, Canada

Genome sequences of Escherichia coli O157:H7 originating from pigs are limited in the public databases. We sequenced 104 E. coli O157:H7 isolates from pig and cattle feces and pork production environments in Alberta, Canada. The information will aid studies investigating sources of E. coli O157:H7 contaminating pork and the associated environments.

Molecular Characterization of the Enterohemolysin Gene (ehxA) in Clinical Shiga Toxin-Producing Escherichia coli Isolates.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is an important foodborne pathogen with the ability to cause bloody diarrhea (BD) and hemolytic uremic syndrome (HUS). Little is known about enterohemolysin-encoded by ehxA. Here we investigated the prevalence and diversity of ehxA in 239 STEC isolates from human clinical samples. In total, 199 out of 239 isolates (83.26%) were ehxA positive, and ehxA was significantly overrepresented in isolates carrying stx2a + stx2c (p < 0.001) and eae (p < 0.001). The presence of ehxA was significantly associated with BD and serotype O157:H7. Five ehxA subtypes were identified, among which, ehxA subtypes B, C, and F were overrepresented in eae-positive isolates. All O157:H7 isolates carried ehxA subtype B, which was related to BD and HUS. Three ehxA groups were observed in the phylogenetic analysis, namely, group Ⅰ (ehxA subtype A), group Ⅱ (ehxA subtype B, C, and F), and group Ⅲ (ehxA subtype D). Most BD- and HUS-associated isolates were clustered into ehxA group Ⅱ, while ehxA group Ⅰ was associated with non-bloody stool and individuals ≥10 years of age. The presence of ehxA + eae and ehxA + eae + stx2 was significantly associated with HUS and O157:H7 isolates. In summary, this study showed a high prevalence and the considerable genetic diversity of ehxA among clinical STEC isolates. The ehxA genotypes (subtype B and phylogenetic group Ⅱ) could be used as risk predictors, as they were associated with severe clinical symptoms, such as BD and HUS. Furthermore, ehxA, together with stx and eae, can be used as a risk predictor for HUS in STEC infections.

Phenotypic Characterization, Antimicrobial Susceptibility Patterns Profile and Risk Factors of Escherichia Colio157:H7 Isolated from Cattle Meat at Jimma Ethiopia

Escherichia coli O157:H7 is considered the most prevalent food borne pathogen that has gained increasing attention worldwide in recent years. A cross sectional study was undertaken from January 2018 to September 2018 on carcass swab and utensil at slaughter house as well as meat and utensil obtained from butcher shop at Jimma town to isolate identify and assess association risk factor of Escherichia coli O157:H7 and its antimicrobial susceptibility patterns. From total of 288 samples 51 of them were biochemically positive for Escherichia coli. From those isolated by biochemical tests four of them were found to be positive for Escherichia coli O157:H7 strain as confirmed by Biolog incubation manual. Among the samples (144) from slaughter house examined 3 of the E. coli O157:H7 isolates were found to be positive for E. coli O157:H7. Two of the isolates were identified from carcass and one from hanging up wire at slaughter house while the rest one was from meat at butcher shop. The antimicrobial susceptibility investigation of 4 E. coli O157:H7 isolates using 12 commonly marketable antimicrobial discs revealed that the isolates were susceptible to eight antimicrobials from 75% to 100%. Results of the present study indicated that an overall resistance of 100%, 75%, 50% and 25% was observed to Ampicillin, Neomycin, Cefoxitin and Oxytetracycline respectively. Multi drug resistance to three drugs was detected in 1 (25%) of the isolates. This study indicates the possible risk of E. coli O157:H7 particularly for the consumers who have the habit of eating raw or undercooked meat. These findings stress the need for implementation of E. coli O157:H7 prevention and control strategies from slaughter house to butcher shop and consumption of meat.

Isolation, Molecular Detection and Antimicrobial Susceptibility Profile of ‎Escherichia coli O157:H7 in Household - reared Small Ruminants in Zaria ‎Metropolis, Kaduna State, Nigeria

Escherichia coli O157:H7 is a zoonotic enteric pathogen of public health significance worldwide. A cross-sectional study was carried out during which 384 faecal samples of household-reared small ruminants and water used in the various houses where the animals are reared were collected. The samples were enriched on tryptone soya broth and cultured on EMB and CT-SMAC to isolate E. coli and E. coli O157:H7 respectively; subjected to conventional biochemical tests and E. coliO157:H7 was confirmed using Wellcolex latex agglutination test kit. E. coli O157:H7 isolates were subjected to antimicrobial susceptibility test and multiplex PCR was carried out to detect the presence of virulence genes stx1, stx2, eaeA and hlyA. The results of the isolation showed isolation rate of E. coli O157:H7 of 4.69% (9/192), 0.52% (1/192) which were obtained from faeces and water samples respectively. The results of the characterisation showed that one of the E. coli O157:H7 isolated harboured the eaeA and hlyA genes but was negative for stx1 and stx2 genes. The highest number of isolates showed resistance to erythromycin (90.9%) while the least was to gentamicin (6.3%). About 97.7% (43/44) of the isolates had multiple antibiotic resistance index greater than 0.2. In conclusion, household-reared small ruminants in the study area were found to be reservoirs of E. coli O157:H7 and humans living within these households are at risk of infection. The multiple antibioticresistance recorded in this study suggests widespread use of antimicrobial drugs in the study area.

Identification of novel fibronectin-binding proteins by 2D-far Western blot in atypical enteropathogenic Escherichia coli serotype O55:H7

Atypical enteropathogenic Escherichia coli (aEPEC) is a subgroup of EPEC, which is one of the major pathogens responsible for fatal diarrhoea in children. Compared with typical EPEC (tEPEC), aEPEC lack an EAF (EPEC adherence factor) plasmid (pEAF), which encodes a series of virulence-associated genes. The extracellular matrix (ECM) component of human cells has been reported to be an important element in the interaction between host and bacterial pathogens. In this research, a 2D-Far Western blot method was performed to identifiy the bacterial proteins that could bind to fibronectin, one of the most common constituents of ECM. A total of 17 protein spots were identified, including 4 outer membrane proteins (OMPs), namely, OmpC, OmpD, OmpX and LamB. In vitro studies were used to determine whether these OMPs were involved in the adherence process. Through indirect immunofluorescence assays, four OMPs could be observed on the surfaces of host cells. After incubating the cells with the recombinant proteins, the adhesion rate of the O55:H7 isolate was decreased. Furthermore, the deletion of OmpX and LamB can also decrease the adhesion rate of WT. Taken together, a high-throughput screening method for host ECM-binding proteins based on 2D Far-Western blot was established, and four outer membrane proteins identified by this method were found to be involved in the adherence process.

Evolution and pathogenicity of H6 avian influenza viruses isolated from Southern China during 2011 to 2017 in mice and chickens

H6 subtype avian influenza viruses spread widely in birds and pose potential threats to poultry and mammals, even to human beings. In this study, the evolution and pathogenicity of H6 AIVs isolated in live poultry markets from 2011 to 2017 were investigated. These H6 isolates were reassortant with other subtypes of influenza virus with increasing genomic diversity. However, no predominant genotype was found during this period. All of the H6N2 and most of the H6N6 isolates replicated efficiently in lungs of inoculated mice without prior adaptation. All of the H6N2 and two H6N6 isolates replicated efficiently in nasal turbinates of inoculated mice, which suggested the H6N2 viruses were more adaptive to the upper respiratory tract of mice than the H6N6 viruses. One of H6N2 virus caused systemic infection in one out of three inoculated mice, which indicated that H6 avian influenza virus, especially the H6N2 viruses posed a potential threat to mammals. Five H6 strains selected from different genotypes caused no clinical signs to inoculated chickens, and their replication were limited in chickens since the viruses have been detected only from a few tissues or swabs at low titers. Our study strongly suggests that the H6 avian influenza virus isolated from live poultry markets pose potential threat to mammals.

A conserved multi-epitope-based vaccine designed by targeting hemagglutinin protein of highly pathogenic avian H5 influenza viruses.

The highly pathogenic avian H5N1 influenza viruses have been recognized as a potential pandemic threat to humans, and to the poultry industry since 1997. H5 viruses consist of a high mutation rate, so universal vaccine designing is very challenging. Here, we describe a vaccinomics approach to design a novel multi-epitope influenza vaccine, based on the highly conserved regions of surface glycoprotein, Hemagglutinin (HA). Initially, the HA protein sequences from Bangladeshi origin were retrieved and aligned by ClustalW. The sequences of 100% conserved regions extracted and analyzed to select the highest potential T-cell and B-cell epitope. The HTL and CTL analyses using IEDB tools showed that DVWTYNAELLVLMEN possesses the highest affinity with MHC class I and II alleles, and it has the highest population coverage. The docking simulation study suggests that this epitope has the potential to interact with both MHC class I and MHC class II. The B-cell epitope prediction provides a potential peptide, GAIAGFIEGGWQGM. We further retrieved HA sequences of 3950 avian and 250 human H5 isolates from several populations of the world, where H5 was an epidemic. Surprisingly, these epitopes are more than 98% conserved in those regions which indicate their potentiality as a conserved vaccine. We have proposed a multi-epitope vaccine using these sequences and assess its stability and potentiality to induce B-cell immunity. In vivo study is necessary to corroborate this epitope as a vaccine, however, setting forth groundwork for wet-lab studies essential to mitigate pandemic threats and provide cross-protection of both avian and humans against H5 influenza viruses.Electronic supplementary materialThe online version of this article (10.1007/s13205-020-02544-3) contains supplementary material, which is available to authorized users.