Application of a Novel Proteomic Microarray Reveals High Exposure to Diarrhoeagenic Escherichia coli among Children in Zambia Participating in a Phase I Clinical Trial.

Diarrhoeagenic E. coli (DEC) significantly contributes to the burden of diarrhoea among children. Currently, there is no approved vaccine against DEC, but several vaccines against the enterotoxigenic E. coli (ETEC) pathotype are in advanced clinical trial stages, including the ETVAX® vaccine, undergoing evaluation in Zambia. This study reports on the reactivity of antibodies from ETVAX® vaccine and placebo recipients in a phase I clinical trial to proteins derived from (DEC) other than ETEC. Plasma samples collected at two time points (prior to any vaccination and post-third dose vaccination) from 16 vaccinated and 4 placebo participants in a phase 1 clinical trial examining the safety, tolerability, and immunogenicity of ETVAX® with dmLT adjuvant were evaluated for IgG response to E. coli antigens other than ETEC using the Pan-DEC protein microarray. This was the first field application of the novel pan-DEC array as a new tool in assessing the antigenic breadth of antibody responses induced by the ETVAX vaccine, as well as to assess early life exposure to DEC pathotypes and other bacterial enteric pathogens. We observed that plasma obtained from ETVAX® and placebo recipients had high antibody reactivity to Ipa, SseC and EspB proteins. These findings suggest that there is high exposure early in life to DEC pathogens, like EPEC, EHEC, EAEC and EIEC in addition to ETEC, in the Zambian population. These immunological observations are consistent with the results of recent epidemiological studies assessing the etiology of diarrheal disease among infants and young children in Zambia.

Prevalence of Diarrhoeagenic Escherichia coli among Children Aged between 0-36 Months in Peri-Urban Areas of Lusaka.

Diarrhoea is a major contributor to childhood morbidity and mortality in developing countries, with diarrhoeagenic Escherichia coli being among the top aetiological agents. We sought to investigate the burden and describe the diarrhoeagenic E. coli pathotypes causing diarrhoea among children in peri-urban areas of Lusaka, Zambia. This was a facility-based surveillance study conducted over an 8-month period from 2020 to 2021. Stool samples were collected from children aged 0-3 years presenting with diarrhoea at five peri-urban health facilities in Lusaka. Stool samples were tested for diarrhoeagenic E. coli using the Novodiag bacterial GE+® panel, a platform utilising real-time PCR and microarray technology to detect bacterial pathogens. Of the 590 samples tested, diarrhoeagenic E. coli were detected in 471 (76.1%). The top three pathogens were enteropathogenic E. coli 45.4% (n = 268), enteroaggregative E. coli 39.5% (n = 233), and enterotoxigenic E. coli 29.7% (n = 176). Our results revealed that 50.1% of the diarrhoeagenic E. coli positive samples comprised multiple pathotypes of varying virulence gene combinations. Our study demonstrates a high prevalence of diarrhoeagenic E. coli in childhood diarrhoea and the early exposure (<12 months) of children to enteric pathogens. This calls for the early implementation of preventive interventions for paediatric diarrhoea.

Leafy greens as a potential source of multidrug-resistant diarrhoeagenic Escherichia coli and Salmonella.

A continued rise in leafy green-linked outbreaks of disease caused by pathogenic Escherichia coli or Salmonella, particularly strains exhibiting multidrug resistance (MDR), has emerged as a major threat to human health and food safety worldwide. Thus, the present study was conducted to examine antimicrobial resistance, including MDR, in diarrhoeagenic E. coli (DEC) and Salmonella isolates obtained from leafy greens from rural and urban areas of India. Of the collected samples (830), 14.1 and 6.5% yielded 117 E. coli (40 DEC and 77 non-DEC) and 54 Salmonella isolates, respectively. Among the DEC pathotypes, enteroaggregative E. coli was the most prevalent (10.2 %), followed by enteropathogenic E. coli (9.4 %), enteroinvasive E. coli (7.6 %) and enterohemorrhagic E. coli (6.8 %). Antimicrobial susceptibility testing of all bacterial isolates with respect to drugs categorized as critically or highly important in both human and veterinary medicine revealed moderate to high (30-90%) resistance for amoxicillin/clavulanic acid, ampicillin, gentamycin and colistin, but relatively low resistance (>30 %) for ciprofloxacin, trimethoprim/sulfamethoxazole and fosfomycin. Notably, all DEC and more than 90% non-DEC or Salmonella isolates were found to be multidrug-resistant to drugs of both human and animal importance. Overall, the results of the present study suggest that leafy greens are potential reservoirs or sources of multidrug-resistant DEC and Salmonella strains in the rural or urban areas of India.

Molecular typing and prevalence of extended-spectrum ß-lactamase genes in diarrhoeagenic Escherichia coli strains isolated from foods and humans in Mashhad, Iran.

AIMS: Present study was aimed to determine ESBL-encoding genes distribution in Diarrhoeagenic Escherichia coli (DEC) isolated from animal-source food products and human clinical samples in Mashhad, Iran. The strains were also further studied to analyse genotypic diversity and find genetic relationships between them. METHODS AND RESULTS: The number of 85 DEC strains including 52 and 33 strains isolated from 300 food and 520 human stool samples, respectively. Randomly amplified polymorphic DNA (RAPD), and repetitive extragenic palindromic-PCR (rep-PCR) typing methods were used to track their genetic relationships. The ESBL-encoding genes prevalence was approximately 70% in both groups of isolates. The blaTEM , blaCTX-M and blaSHV were prevalent in 67·1, 20 and 10·6% of isolates, respectively. The ESBL-positives showed significantly higher resistance rates to gentamicin, co-trimoxazole, tetracycline, aztreonam and chloramphenicol (P < 0·05). Fingerprinting patterns-based dendrograms divided DEC strains into separate clusters irrespective of their sources and pathotypes. In typing field, rep-PCR provided more discriminatory power (Simpson's index of diversity (SID) = 0·925) than RAPD (SID = 0·812). CONCLUSION: Molecular similarity between certain animal-sourced food products and clinical sample strains supported food-borne transmission routes for genotypic elements such as ESBL-encoding genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Findings emphasize the importance of resistance issues, the need to improve treatment guidelines and routine surveillance of hygienic measures during food processing.

Behaviour of Non-O157 STEC and Atypical EPEC during the Manufacturing and Ripening of Raw Milk Cheese.

This study was carried out to assess the survival of Shiga toxin-producing E. coli (STEC) and atypical enteropathogenic Escherichia coli (aEPEC) during the traditional manufacturing and ripening of Spanish hard cheese from raw cow's milk. Milk samples were spiked with up to 3.1-3.5 log cfu/mL of one strain of STEC (O140:H32 serotype) and one of aEPEC (serotype O25:H2). The first steps of cheesemaking allow for a STEC and aEPEC increase of more than 1 log cfu/mL (up to 4.74 log cfu/g and 4.55 log cfu/g, respectively). After cheese pressing, a steady reduction of both populations was observed, with the STEC strain being more sensitive. The studied pathogenic E. coli populations decreased by 1.32 log cfu/g in STEC and 0.59 log cfu/g in aEPEC in cheese ripened during a minimum period of 60 d. Therefore, a moderate contamination by these diarrhoeagenic E. coli pathotypes, in particular, with aEPEC, on cheese manufactured from raw milk may not be totally controlled through the cheesemaking process and during a maturation of 90 d. These findings remark the importance of improvement in bacteriological quality of raw milk and cross-contamination prevention with diarrhoeagenic E. coli in the dairy industry.

Mixed Aetiology of Diarrhoea in Infants Attending Clinics in the North-West Province of South Africa: Potential for Sub-Optimal Treatment.

Routine diagnostic methods for the aetiologic agents of diarrhoea in most developing countries are usually not sensitive enough, leading to under-diagnosis. Thus, this study investigated possible mixed diarrhoeal aetiology by using cultures and real-time polymerase chain reactions (PCR) in children younger than four years old in the Northwest Province, South Africa. In total, 505 stool samples were collected from symptomatic and asymptomatic children who were attending three clinics and the Brits hospital in Madibeng District, between September 2016 and December 2017. Rotavirus, norovirus, Campylobacter, Arcobacter, and diarrhoeagenic Escherichia coli (DEC) were targeted. Campylobacter spp. (24.6%), Arcobacter (15.8%) and DEC (19.6%) were detected using PCR; only Campylobacter spp. (29.7%) and DEC (26.9%) were detected through the culture. Campylobacter jejuni (36%), Campylobacter coli (28%), Campylobacter upsalensis (12%), and Arcobacter butzleri (15.8%) were the only spp. of Campylobacter and Arcobacter identified. The eaeA gene (31.4%) of enteropathogenic E. coli/enterohaemorrhagic E. coli (EPEC/EHEC) was the most prevalent DEC virulence gene (VG) identified. Rotavirus and norovirus were detected at 23.4% and 20%, respectively. Mixed viral aetiology (7.3%) and the co-infection of A. butzleri and Campylobacter (49%) were recorded. A mixed bacterial-viral aetiology was observed in 0.6% of the specimens. Sensitive diagnostic procedures like PCR should be considered to provide the best treatment to children experiencing diarrhoea.

The First Isolation and Molecular Characterization of Shiga Toxin-Producing Virulent Multi-Drug Resistant Atypical Enteropathogenic Escherichia coli O177 Serogroup From South African Cattle.

Atypical enteropathogenic E. coli (aEPEC) is a group of diarrhoeagenic Escherichia coli with high diversity of serogroups, which lack the bundle-forming pili (BFP) and genes encoding for shiga toxins. The aim of this study was to isolate, identify and determine virulence and antibiotic resistance profiles of aEPEC O177 strains from cattle feces. A total of 780 samples were collected from beef and dairy cattle and analyzed for the presence of E. coli O177. One thousand two hundred and seventy-two (1272) presumptive isolates were obtained and 915 were confirmed as E. coli species. Three hundred and seventy-six isolates were positively confirmed as E. coli O177 through amplification of rmlB and wzy gene sequences using multiplex PCR. None of these isolates harbored bfpA gene. A larger proportion (12.74%) of the isolates harbored hlyA gene while 11.20, 9.07, 7.25, 2.60, and 0.63% possessed stx2, stx1, eaeA, stx2a , and stx2d , respectively. Most of E. coli O177 isolates carried stx2/hlyA (9.74%). Furthermore, 7.40% of the isolates harbored stx1/stx2 while 7.09% possessed stx1/stx2/hlyA genes. Only one isolate harbored stx1/stx2/hly/eaeA/stx2a/stx2d while 5.11% of the isolates harbored all the four major virulence genes stx1/stx2/hlyA/eaeA, simultaneously. Further analysis revealed that the isolates displayed varied antimicrobial resistance to erythromycin (63.84%), ampicillin (21.54%), tetracycline (13.37%), streptomycin (17.01%), kanamycin (2.42%), chloramphenicol (1.97%), and norfloxacin (1.40%). Moreover, 20.7% of the isolates exhibited different phenotypic multi-drug resistance patterns. All 73 isolates harbored at least one antimicrobial resistance gene. The aadA, streA, streB, erm, and tetA resistance genes were detected separately and/or concurrently. In conclusion, our findings indicate that environmental isolates of aEPEC O177 strains obtained from cattle in South Africa harbored virulence and antimicrobial resistance gene determinants similar to those reported in other shiga-toxin producing E. coli strains and suggest that these determinants may contribute to the virulence of the isolates.

Locals get travellers' diarrhoea too: risk factors for diarrhoeal illness and pathogenic Escherichia coli infection across an urban-rural gradient in Ecuador.

OBJECTIVES: Diarrhoea is a common and well-studied cause of illness afflicting international travellers. However, traveller's diarrhoea can also result from travel between high and low disease transmission regions within a country, which is the focus of this study. METHODS: We recruited participants for a case-control study of diarrhoea at four sites along an urban-rural gradient in Northern Ecuador: Quito, Esmeraldas, Borbón and rural communities outside of Borbón. At each of these sites, approximately 100 subjects with diarrhoea (cases) were recruited from Ministry of Health clinics and were age-matched with subjects visiting the same clinics for other complaints (controls). RESULTS: Travellers to urban destinations had higher risk of diarrhoea and diarrhoeagenic Escherichia coli (DEC) infections. Travel to Quito was associated with diarrhoea (aOR = 2.01, 95% CI = 1.10-3.68) and travel to Guayaquil (another urban centre in Ecuador) was associated with Diffuse Adherent E. coli infection (OR = 2.09, 95% CI = 1.01-4.33). Compared to those not travelling, urban origins were also associated with greater risk of diarrhoea in Esmeraldas (aOR = 2.28, 95% CI = 1.20-4.41), and with higher risk of diarrhoeagenic E. coli infections in Quito (aOR = 2.61, 95% CI = 1.16-5.86), with >50% of travel from Quito and Esmeraldas specified to another urban destination. CONCLUSIONS: This study suggests that individuals travelling from lower-transmission regions (rural areas) to higher transmission regions (urban centres) within a single country are at a greater risk of acquiring a diarrhoea-related illness. Investments to improve water, sanitation and hygiene conditions in urban areas could have impacts on outlying rural areas within a given country.

Polyacrylonitrile (PAN) nanofibres spun with copper nanoparticles: an anti-Escherichia coli membrane for water treatment.

Copper nanoparticles (CNPs) were mixed with polyacrylonitrile (PAN) and electrospun into nanofibres (CuPAN nanofibres). PAN nanofibres containing 1.0, 3.0 and 5.0% copper (w/v) displayed beads-on-string morphology with protrusions of copper particles. The diameter of the CuPAN nanofibres differed according to the copper content, ranging from 386 nm (1.0%, w/v, copper) to 922 nm (5.0%, w/v, copper). No chemical interaction of copper with PAN was observed when studied with X-ray diffraction, ATR-FTIR (attenuated total reflection-Fourier transform infrared) spectroscopy and TGA (thermogravimetric analysis). None of the CuPAN nanofibres showed signs of degradation after 7 days in water. Bacteria suspended in random mobility buffer and filtered through a 3% CuPAN nanofibre membrane (25 mm diameter, 75-80 µm thickness), at a filtration rate of 20 ml min-1, reduced the cell numbers of enterotoxigenic Escherichia coli (ETEC) from 3.3 × to 2.1 × 106 cfu ml-1 and methicillin-resistant Staphylococcus aureus (MRSA) from 1.2 × 10 to 1.3 × 103 cfu ml-1. Membranes produced with 1.0, 3.0 and 5.0% (w/v) CuPAN inhibited the growth of enteroaggregative E. coli (EAEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), ETEC and MRSA, as shown with LIVE/DEAD™ BacLight™ staining. Real-time bactericidal activity of CuPAN membranes was recorded by staining the cells with SYTO 9 and PI, followed by flow cytometry. Filter membranes made from CuPAN fibres may be used to reduce pathogenic E. coli cell numbers in potable water.

Diarrheagenic Escherichia coli infections among the children of Andaman Islands with special reference to pathotype distribution and clinical profile.

Diarrhoeagenic E. coli (DEC) is one of the most common causes of diarrhoeal death in children less than five years globally. It is responsible for 30%-40% of all diarrhoeal episodes in developing countries. It is estimated that 0.12million children died of diarrhoea caused by DEC in 2011 globally. There is no baseline data on the occurrence of DEC diarrhoea in Andaman Islands, the remote islands of India. The study is particularly important as these strains are the emerging enteric pathogen in both developed and developing countries. DEC was screened from E. coli isolates obtained from diarrhoeal stool samples by multiplex PCR with specific primers using stasndard protocols. During the study period, among the 1394 stool samples collected, 95 (6.82%) patients were found infected with DEC. Of the 97 isolates from 95 patients, 68 (70.1%) were EAEC, 19 (19.6%) were EPEC and 10 (10.3%) were ETEC. Of the 19 EPEC isolates, 63.2% were atypical EPEC which is the emerging enteric pathogen among the children in developing as well as developed countries. More than 80% of the patients had watery diarrhoea and 6% of them had invasive diarrhoea. Persistent diarrhoea was also found in three infected children. This study documents the occurrence and type of DEC diarrhoea in Andaman Islands first time and highlights the significant proportions of E. coli diarrhoea being caused by EAEC and atypical EPEC strains.

Virulence determinants in enteroaggregative Escherichia coli from North India and their interaction in in vitro organ culture system.

Enteroaggregative Escherichia coli (EAEC) is an important diarrhoeal pathogen causing diseases in multiple epidemiological and clinical settings. In developing countries like India, diarrhoeal diseases are one of the major killers among paediatric population and oddly, few studies are available from Indian paediatric population on the variability of EAEC virulence genes. In this study, we examined the distribution of plasmid and chromosomal-encoded virulence determinants in EAEC isolates, and analysed cytokines response generated against EAEC with specific aggregative adherence fimbriae (AAF) type in duodenal biopsies using in vitro organ culture (IVOC) mimicking in vivo conditions. Different virulence marker combinations among strains were reflected as a function of specific adhesins signifying EAEC heterogeneity. fis gene emerged as an important genetic marker apart from aggA and aap Further, EAEC infection in IVOC showed upregulation of IL-8, IL-1ß, IL-6, TNF-α and TLR-5 expression. EAEC with AAFII induced significant TLR-5 and IL-8 response, conceivably owing to more pathogenicity markers. This study sheds light on the pattern of EAEC pathotypes prevalent in North Indian paediatric population and highlights the presence of unique virulence combinations in pathogenic strains. Thus, evident diversity in EAEC virulence and multifaceted bacteria-host crosstalk can provide useful insights for the strategic management of diarrhoeal diseases in India, where diarrhoeal outbreaks are more frequent.

Occurrence of diarrhoeagenic Escherichia coli virulence genes in water and bed sediments of a river used by communities in Gauteng, South Africa.

In most developing countries, especially in Southern Africa, little is known about the presence of diarrhoeagenic Escherichia coli (DEC) pathotypes in riverbed sediments. The present study sought to investigate the presence of DEC virulence genes in riverbed sediments of the Apies River, a river used by many communities in Gauteng, South Africa. Water and sediment samples were collected from the river between July and August 2013 (dry season) and also between January and February 2014 (wet season) following standard procedures. Isolation of E. coli was done using the Colilert®-18 Quanti-Tray® 2000 system. DNA was extracted from E. coli isolates using the InstaGene™ matrix from Bio-Rad and used as template DNA for real-time PCR. Water pH, temperature, dissolved oxygen, electrical conductivity and turbidity were measured in situ. Over 59 % of 180 samples analysed were positive for at least one of the seven DEC virulence genes investigated. The eaeA gene was the most isolated gene (29.44 %) while the ipaH gene the least isolated (8.33 %). The ipaH gene (p = 0.012) and the ST gene (stIa, p = 0.0001, and stIb, p = 0.019) were positively correlated with temperature. The detection of diarrhoeagenic E. coli virulence genes in the sediments of the Apies River shows that the sediments of this river might not only be a reservoir of faecal indicator bacteria like E. coli but also pathogenic strains of this bacterium. These organisms could represent a public health risk for poor communities relying on this water source for various purposes such as drinking and recreational use. There is therefore an urgent need to monitor these DEC pathotypes especially in areas without adequate water supplies.