Molecular Identification of Shiga-Toxin Producing and Enteropathogenic <i>Escherichia coli</i>(STEC and EPEC) in Diarrheic and Healthy Young Alpacas

Isolation and biochemical and molecular identification of 303 strains of Escherichia coli obtained from diarrheic and healthy young alpacas of Puno-Peru, were realized. PCR amplification for 7 virulence factor genes associated with STEC, STEC O157:H7, EPEC: sxt1, sxt2, rfbO157, fliCH7, hlyA, eae y bfp were determined. A total of 39 strains (12.88%) showed amplification for one or more of these genes. Twenty three strains (59%) were classified as STEC and 16 strains (41%) as EPEC. An 88.18% (34/39) of STEC and EPEC strains were obtained from healthy alpacas and only 11.82% (5/39) from diarrheic alpacas considering this specie as potential zoonotic reservoir of STEC and EPEC.

Antibacterial efficacy of five medicinal plants against multidrug-resistant enteropathogenic bacteria infecting under-5 hospitalized children

OBJECTIVE： To evaluate in vitro antibacterial effectiveness of five medicinal plants used by an Indian aborigine, against 8 multidrug-resistant （MDR） enteropathogenic bacteria isolated from clinical samples of under-5 hospitalized children. METHODS： Antibiotic sensitivity patterns of eight clinically isolated strains of enteropathogenic bacteria, Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae, Salmonella paratyphi, S. typhi, Shigella dysenteriae, S. sonnei and Vibrio cholerae were assessed by disc-diffusion method. Antibacterial activities of 8 solvent-extracts of leaves and bark of five medicinal plants were monitored by the agar-well diffusion method. The microbroth dilution method was used to assess minimum inhibitory concentration （MIC） and minimum bactericidal concentration （MBC）. Qualitative phytochemical analyses of active plant extracts were carried out. RESULTS： Ethanol, ethyl acetate and methanol extracts of Holarrhena antidysenterica leaf tissue were most effective against 8 MDR pathogens in vitro. Similarly, acetone, ethanol and methanol extracts of Terminalia alata leaf tissue; chloroform, ethyl acetate and methanol extracts of Terminalia aquna leaf tissue and ethyl acetate, ethanol and methanol extracts of Paederia foetida leaf tissue were most effective in inhibiting in vitro growth of the 8 MDR enteropathogens. Ethyl acetate and methanol extracts of H. antidysenterica bark tissue; acetone, ethanol and methanol extracts of T. alata bark tissue and acetone, ethanol and methanol extracts of T. arjuna bark tissue were most effective in controlling enteropathogen growth. The minimum inhibitory concentration and minimum bactericidal concentration values of the 3 most antimicrobial leaf and bark extracts from the five plants were in the range of 1.56 to 50 mg/mL. CONCLUSION： These 5 plants exhibited in vitro control over a cohort of 8 enteropathogenic bacteria strains isolated from clinical samples.

Diarrheic Escherichia coli: A Predominant Etiological Agent of Gastroenteritis, a Case Study in Douala, Cameroon

Context: Gastroenteritis remains an infectious disease with high morbidity and mortality particularly in low incomes countries, where the capacity to search all etiological agents, especially pathogenic Escherichia coli, is very limited. We investigated the contribution of pathogenic Escherichia coli and their antibiotic resistance profiles in cases of gastroenteritis. Methods: A cross-sectional study was carried out on human stool samples from October 2021 to June 2022 at Laquintinie Hospital. Samples were received from patients of all age groups and screened for bacteriological and parasitological identification by microscopy, bacterial culture, biochemical identification, and antimicrobial susceptibility tests. Results: A total of 296 patients with gastroenteritis complaints, were enrolled in the study with ages ranging from 5 months to 90 years old (Median = 35.5;SD = 20.8). Among the samples analyzed, 1.7% (n = 5/296) were positive for parasites and 27% (n = 80/296) were positive for bacterial pathogens. Parasites were found in mono parasitism, mainly Entamoeba histolytica (60%;n = 3/5), followed by Trichomonas intestinalis (20%;n = 1/5), and Giardia intestinalis (20%;n = 1/5). Three species of bacterial pathogens were identified with no co-infection: diarrheic Escherichia coli (DEC), Salmonella sp, and Shigella sp with respective proportions of 90% (n = 72/80), 6.3% (n = 5/80), and 3.7% (n = 3/80). For antibiotic resistance profiles (ARPs) of the 72 isolates of DEC, high levels of resistance were observed globally with amoxicillin (93.1%;n = 67/72), followed by ciprofloxacin (75%;n = 54/72), and to trimethoprim + sulfamethazole (73.6%;n = 53/72). In contrast, DEC showed low resistance rates with nitrofurans (6.9%;n = 5/72) and imipenem (2.8%;n = 2/72). The strains had 56 distinct ARPs, of which 88.9% (n = 64/72) were MDR. Salmonella sp and Shigella sp showed high levels of resistance to amoxicillin and trimethoprim + sulfamethazole. Conclusion: These results emphasize the need to consider DEC as the main cause of consultation in cases of gastroenteritis and reiterate the urgent need to rationalize antibiotic use in Cameroon.

A Multiplex PCR Assay for the Detection of Pathogenic Genes of EPEC,ETEC and EIEC

A multiplex polymerase chain reaction （PCR） was developed to detect three pathogenic genes of enteropathogenic, enterotocigenic and enteroinvasive Escherichia coli In this study three different sets of oligonucleotide primer were simultaneously used, and in this way, specific fragments of 880, 600, 150 bp for EPEC eaeA, EIEC ipaH and ETEC ST genes were amplified, respectively. The best condition of the multiplex PCR was： after an initial heat denaturation step at 95℃for 5 min, followed by 30 cycles of denaturation at 94 ℃ for 40 s, primer annealing at 51.3℃ for 40 s and extension at 72 ℃ for 1 min, final extension at 72 ℃ for 10 min. The detection limit of the eaeA, ipaH and ST primers was 38.7423, 3.60519, 29.9448 ng·mL^-1 （4.3×10^4, 1.5×10^3, 2.6×10^4 CFU·mL^-1）, respectively. It may be a good way for the detection and identification of Diarrhea-causing E. coli.

Occurrence of Escherichia coli virulence genes in feces of wild birds from Central Italy

Objective: To investigate the potential role of wild birds as fecal spreaders of enteropathogenic,enterohemorrhagic and Shiga-toxins producing Escherichia coli(E. coli),enteropathogenic E. coli,enterohemorrhagic E. coli and Shiga toxin-producing E. coli strains. Methods: Fecal samples collected from 121 wild birds of different orders and species were submitted to molecular analyses. In particular,eaeA encoding intimin,hlyA encoding for hemolysin,stx1 and stx2 genes encoding Shiga-toxins 1 and 2,respectively,were investigated. Results: Overall,21(17.35%) fecal samples resulted positive for at least one of the investigated genes. In detail,12(9.91%) samples were positive for eaeA,10(8.26%) for stx1,4(3.31%) for hylA and 1(0.83%) for stx2. An owl(Athene noctua) positive for the four investigated genes suggesting that it harbored a STEC strain. However,virulence genes characterizing EPEC,and EHEC strains were mainly found among seagulls,waterfowl and feral pigeons. Conclusions: Seagulls,waterfowl and feral pigeons,which frequently reach and contaminate rural,urban and peri-urban areas with their droppings,may be important sources of E. coli infection for other animals and humans.

Adhesion and immunomodulatory effects of Bifidobacterium lactis HN019 on intestinal epithelial cells INT-407

AIM:To elucidate the adherence and immunomodulatory properties of a probiotic strain Bifidobacterium lactis(B.lactis) HN019.METHODS:Adhesion assays of B.lactis HN019 and Salmonella typhimurium(S.typhimurium) ATCC 14028 to INT-407 cells were carried out by detecting copies of species-specific genes with real-time polymerase chain reaction.Morphological study was further conducted by transmission electron microscopy.Interleukin-1β(IL-1β),interleukin-8,and tumor necrosis factor-α(TNF-α) gene expression were assessed while enzyme linked immunosorbent assay was used to detect IL-8 protein secretion.RESULTS:The attachment of S.typhimurium ATCC 14028 to INT407 intestinal epithelial cells was inhibited significantly by B.lactis HN019.B.lactis HN019 could be internalized into the INT-407 cells and attenuated IL-8 mRNA level at both baseline and S.typhimuriuminduced pro-inflammatory responses.IL-8 secretion was reduced while IL-1β and TNF-α mRNA expression level remained unchanged at baseline after treated with B.lactis HN019.CONCLUSION:B.lactis HN019 does not up-regulate the intestinal epithelium expressed pro-inflammatory cytokine,it showed the potential to protect enterocytes from an acute inflammatory response induced by enteropathogen.

Antimicrobial usage and resistance in beef production

Antimicrobials are critical to contemporary high-intensity beef production. Many different antimicrobials are approved for beef cattle, and are used judiciously for animal welfare, and controversially, to promote growth and feed efficiency. Antimicrobial administration provides a powerful selective pressure that acts on the microbial community, selecting for resistance gene determinants and antimicrobial-resistant bacteria resident in the bovine flora. The bovine microbiota includes many harmless bacteria, but also opportunistic pathogens that may acquire and propagate resistance genes within the microbial community via horizontal gene transfer. Antimicrobial-resistant bovine pathogens can also complicate the prevention and treatment of infectious diseases in beef feedlots,threatening the efficiency of the beef production system. Likewise, the transmission of antimicrobial resistance genes to bovine-associated human pathogens is a potential public health concern. This review outlines current antimicrobial use practices pertaining to beef production, and explores the frequency of antimicrobial resistance in major bovine pathogens. The effect of antimicrobials on the composition of the bovine microbiota is examined, as are the effects on the beef production resistome. Antimicrobial resistance is further explored within the context of the wider beef production continuum, with emphasis on antimicrobial resistance genes in the food chain, and risk to the human population.

Prevalence of enteric pathogen-associated community gastroenteritis among kindergarten children in Gaza

Gastroenteritis is considered as one of the leading causes of illness and death in children under 5 years age, especially in developing countries. It is one of the major public health problems among childhood in Gaza strip, Palestine. This study was conducted to determine the prevalence of enteric pathogen-associated community gastro- enteritis among kindergarten children in Gaza. A total of 150 stool samples were collected and investigated for parasitic, viral and bacterial pathogens at Al Azhar microbiology laboratories by using standard microbiological and serological procedures. Out of the 150 study samples, the overall percentage of positive stool samples with a known enteric pathogen was 60.6%. The prevalence of different enteric pathogens causing community gastroenteritis among symptomatic cases （88.5%） was significantly higher than the prevalence in asymptomatic carriage （11.1%）. The most prevalent isolated enteric pathogens were Entamoeba histolytica （28.0%） and Giardia lamblia （26.7%）. Rotavirus was found in 3.1% of symptomatic cases but not detected in asymptomatic carriage. However, adenovirus type 40 and 41 were not detected in any of the study samples. The bacterial enteric pathogens Shigella and Enterohemorrhagic Escherichia coli O157：H7 （EHEC） have comparable occurrence as rotavirus （3.1%）, meanwhile, Salmonella was not isolated. Mixed infection with more than 1 pathogen was found （11.4%） only among symptomatic cases. Children aged 3-year-old showed the highest prevalence of community gastroenteritis. This study demonstrates a high prevalence of parasitic enteropathogens and a relatively low prevalence of bacterial and viral enteropathogens among kindergarten children living in Gaza city, moreover, children aged 3 years old showed the highest prevalence of isolated enteropathogens.

Comparative study on epidemiological and etiological characteristics of patients with acute diarrhea with febrile or non-febrile symptoms in China

Background Acute diarrhea with fever can potentially represent a more severe form of the disease compared to non-febrile diarrhea. This study was to investigate the epidemiological characteristics and enteric pathogen composition of febrile-diarrheal patients, and to explore factors including pathogens associated with fever by age group.Methods A nationwide surveillance study of acute diarrheal patients of all ages was conducted in 217 sentinel hospitals from 31 provinces (autonomous regions or municipalities) in China between 2011 and 2020. Seventeen diarrhea-related pathogens, including seven viruses and ten bacteria, were investigated and their association with occurrence of fever symptoms was assessed using multivariate logistic analysis.Results A total of 146,296 patients with acute diarrhea (18.6% with fever) were tested. Th diarrheal children below 5 years had the highest frequency of fever (24.2%), and related to significantly higher prevalence of viral enteropathogens (40.2%) as compared with other age groups (P < 0.001). Within each age group, the febrile-diarrheal patients were associated with a significantly higher prevalence of bacterial pathogens than afebrile-diarrheal patients (allP < 0.01). There was discrepancy when each pathogen was compared, i.e., nontyphoidalSalmonella (NTS) was over-represented in febrile vs non-febrile patients of all age groups, while the febrile vs non-febrile difference for diarrheagenicEscherichia coli (DEC) was only significant for adult groups. The multivariate analysis revealed significant association between fever and infection with rotavirus A among children [odds ratio (OR) = 1.60], for DEC in adult groups (OR = 1.64), for NTS in both children (OR = 2.95) and adults (OR = 3.59).Conclusions There are significant discrepancy of the infected enteric pathogens in patients with acute diarrhea with fever between age groups, and it is valuable for priority detection of NTS and rotavirus A in patients with children < 5 years old and NTS and DEC in adult patients. The results may be useful in identifying dominant pathogen candidates for the application of diagnostic assays and prevention control.

Sensing bacterial infections by NAIP receptors in NLRC4 inflammasome activation

The inflammasome is an emerging new pathway in innate immune defense against microbial infection or endogenous danger signals.The inflammasome stimulates activation of inflammatory caspases,mainly caspase-1.Caspase-1 activation is responsible for processing and secretion of IL-1βand IL-18 as well as for inducing macrophage pyroptotic death.Assembly of the large cytoplasmic inflammasome complex is thought to be mediated by members of NOD-like receptor(NLR)family.While functions of most of the NLR proteins remain to be defined,several NLR proteins including NLRC4 have been shown to assemble distinct inflammasome complexes.These inflammasome pathways,particularly the NLRC4 inflammasome,play a critical role in sensing and restricting diverse types of bacterial infections.Here we review recent advances in defining the exact bacterial ligands and the ligand-binding receptors involved in NLRC4 inflammasome activation.Implications of the discovery of the NAIP family of inflammasome receptors for bacterial flagellin and type III secretion apparatus on future inflammasome and bacterial infection studies are also discussed.

Animals as sources of food-borne pathogens: A review

Food-producing animals are the major reservoirs for many foodborne pathogens such as Campylobacter species, non-Typhi serotypes of Salmonella enterica, Shiga toxin-producing strains of Escherichia coli, and Listeria monocytogenes. The zoonotic potential of foodborne pathogens and their ability to produce toxins causing diseases or even death are sufficient to recognize the seriousness of the situation. This manuscript reviews the evidence that links animals as vehicles of the foodborne pathogens Salmonella,Campylobacter, Shiga toxigenic E. coli, and L. monocytogenes, their impact, and their current status. We conclude that these pathogenic bacteria will continue causing outbreaks and deaths throughout the world, because no effective interventions have eliminated them from animals and food.