Reservoir Of Shiga Toxin-producing Escherichia Coli Research Articles

Characterization of Shiga Toxin-producing Escherichia coli Isolated from Cattle Around Ulaanbaatar City, Mongolia

Shiga toxin-producing Escherichia coli (STEC) are associated with severe infections including hemorrhagic colitis and hemolytic uremic syndrome in humans. Ruminants are known as reservoirs of STEC; however, no data are available on STEC in ruminants in Mongolia, where more than 5 million cattle and 25 million sheep are raised. To disclose the existence and characteristics of STEC in Mongolia, in this study, we isolated and characterized STEC from cattle in Mongolia. We collected 350 rectal swabs of cattle from 30 farms near Ulaanbaatar city and isolated 45 STEC from 21 farms. Rectal swabs were precultured with modified Escherichia coli broth and then inoculated to Cefixime-Tellurite Sorbitol MacConkey agar plate and/or CHROMagar STEC agar plate for the isolation of STEC. The isolation ratios in each farm were from 0% to 40%. Multiplex PCR for the estimation of O- and H-serotypes identified 12 O-genotypes (Og-types) and 11 H-genotypes (Hg-types) from 45 isolates; however, Og-types of 19 isolates could not be determined. Stx gene subtyping by PCR identified 2 stx1 subtypes (1a and 1c) and 4 stx2 subtypes (2a, 2c, 2d, and 2g). Forty-five isolates were divided into 21 different groups based on the Og- and Hg-types, stx gene subtypes and the existence of virulence factors, ehxA, eae, and saa, which includes several major serotypes associated with human illness such as O26:H11 and O157:H7. The most dominant isolate, OgUT:H19 [stx1a (+), stx2a (+), ehxA (+) and saa (+)], was isolated from eight farms. This is the first report on the characterization of STEC in cattle in Mongolia, and the results suggest the importance of further monitoring of STEC contamination in the food chains as well as STEC infection in humans.

Transcription levels of hes and their involvement in the biofilm formation of Shiga toxin-producing Escherichia coli O91.

Shiga toxin-producing Escherichia coli (STEC) are recognized as being responsible for many cases of foodborne diseases worldwide. Cattle are the main reservoir of STEC, shedding the microorganisms in their feces. The serogroup STEC O91 has been associated with hemorrhagic colitis and hemolytic uremic syndrome. Locus of Adhesion and Autoaggregation (LAA) and its hes gene are related to the pathogenicity of STEC and the ability to form biofilms. Considering the frequent isolation of STEC O91, the biofilm-forming ability, and the possible role of hes in the pathogenicity of STEC, we propose to evaluate the ability of STEC to form biofilms and to evaluate the expression of hes before and after of biofilm formation. All strains were classified as strong biofilm-forming. The hes expression showed variability between strains before and after biofilm formation, and this may be due to other genes carried by each strain. This study is the first to report the relationship between biofilm formation, and hes expression and proposes that the analysis and diagnosis of LAA, especially hes as STEC O91 virulence factors, could elucidate these unknown mechanisms. Considering that there is no specific treatment for HUS, only supportive care, it is necessary to know the survival and virulence mechanisms of STEC O91.

First report of the prevalence of Shiga toxinproducing Escherichia coli in ground beef in Quindío, Colombia.

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen associated with clinical cases of diarrhea in humans. Its main virulence factors are the Shiga toxins (Stx1 and Stx2). Cattle are the main reservoir of STEC, and many outbreaks in humans have been related to the consumption of undercooked ground beef contaminated with this pathogen. To determine the prevalence of STEC in ground beef commercialized in all the butcher shops of a township in the department of Quindío and to characterize the virulence genes of the strains found. Thirty ground beef samples were taken in three different times; stx genes and other STEC virulence factors (eae, ehxA, saa) were detected by multiplex PCR. The overall prevalence of STEC was 33.33 % (10/30 positive samples). We isolated eight non-O157 (LEE-negative) strains with four different genetic profiles: stx2 / stx2-ehxA-saa / stx1-stx2-ehxA-saa / stx1-saa. This is the first report on the prevalence of STEC in ground beef in a township in the department of Quindío.

Genome engineering of Stx1-and Stx2-converting bacteriophages unveils the virulence of the dairy isolate Escherichia coli O174:H2 strain UC4224.

The past decade witnessed the emergence in Shiga toxin-producing Escherichia coli (STEC) infections linked to the consumption of unpasteurized milk and raw milk cheese. The virulence of STEC is primarily attributed to the presence of Shiga toxin genes (stx1 and stx2) carried by Stx-converting bacteriophages, along with the intimin gene eae. Most of the available information pertains to the "Top 7" serotypes associated with STEC infections. The objectives of this study were to characterize and investigate the pathogenicity potential of E. coli UC4224, a STEC O174:H2 strain isolated from semi-hard raw milk cheese and to develop surrogate strains with reduced virulence for use in food-related studies. Complete genome sequence analysis of E. coli UC4224 unveiled the presence of a Stx1a bacteriophage, a Stx2a bacteriophage, the Locus of Adhesion and Autoaggregation (LAA) pathogenicity island, plasmid-encoded virulence genes, and other colonization facilitators. In the Galleria mellonella animal model, E. coli UC4224 demonstrated high pathogenicity potential with an LD50 of 6 CFU/10 μL. Upon engineering E. coli UC4224 to generate single and double mutant derivatives by inactivating stx1a and/or stx2a genes, the LD50 increased by approximately 1 Log-dose in the single mutants and 2 Log-doses in the double mutants. However, infectivity was not completely abolished, suggesting the involvement of other virulence factors contributing to the pathogenicity of STEC O174:H2. Considering the possibility of raw milk cheese serving as a reservoir for STEC, cheesemaking model was developed to evaluate the survival of UC4224 and the adequacy of the respective mutants as reduced-virulence surrogates. All tested strains exhibited the ability to survive the curd cooking step at 48°C and multiplied (3.4 Log CFU) in cheese within the subsequent 24 h. These findings indicate that genomic engineering did not exert any unintended effect on the double stx1-stx2 mutant behaviour, making it as a suitable less-virulent surrogate for conducting studies during food processing.

Detection and analysis of Shiga toxin producing and enteropathogenic Escherichia coli in cattle from Tierra del Fuego, Argentina.

Shiga toxin producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) are pathovars that affect mainly infants' health. Cattle are the main reservoir of STEC. Uremic hemolytic syndrome and diarrheas can be found at high rates in Tierra del Fuego (TDF). This study aimed to establish the prevalence of STEC and EPEC in cattle at slaughterhouses in TDF and to analyze the isolated strains. Out of 194 samples from two slaughterhouses, STEC prevalence was 15%, and EPEC prevalence was 5%. Twenty-seven STEC strains and one EPEC were isolated. The most prevalent STEC serotypes were O185:H19 (7), O185:H7 (6), and O178:H19 (5). There were no STEC eae + strains (AE-STEC) or serogroup O157 detected in this study. The prevalent genotype was stx2c (10/27) followed by stx1a/stx2hb (4/27). Fourteen percent of the strains presented at least one stx non-typeable subtype (4/27). Shiga toxin production was detected in 25/27 STEC strains. The prevalent module for the Locus of Adhesion and Autoaggregation (LAA) island was module III (7/27). EPEC strain was categorized as atypical and with the ability to cause A/E lesion. The ehxA gene was present in 16/28 strains, 12 of which were capable of producing hemolysis. No hybrid strains were detected in this work. Antimicrobial susceptibility tests showed that all strains were resistant to ampicillin and 20/28 were resistant to aminoglycosides. No statistical differences could be seen in the detection of STEC or EPEC either by slaughterhouse location or by production system (extensive grass or feedlot). The rate of STEC detection was lower than the one reported for the rest of Argentina. STEC/EPEC relation was 3 to 1. This is the first study on cattle from TDF as reservoir for strains that are potentially pathogenic to humans.

Exploring the nature of interaction between shiga toxin producing Escherichia coli (STEC) and free-living amoeba - Acanthamoeba sp

Free-living amoebae (FLA) are widely distributed protozoa in nature, known to cause severe eye infections and central nervous system disorders. There is growing attention to the potential role that these protozoa could act as reservoirs of pathogenic bacteria and, consequently, to the possibility that, the persistence and spread of the latter may be facilitated, by exploiting internalization into amoebae. Shiga toxin-producing strains of Escherichia coli (STEC) are zoonotic agents capable of causing serious diseases, such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Cattle represent the main natural reservoir of STEC, which are frequently found also in other domestic and wild ruminants, often without causing any evident symptoms of disease. The aspects related to the ecology of STEC strains in animal reservoirs and the environment are poorly known, including the persistence of these microorganisms within niches unfavorable to survival, such as soils or waters. In this study we investigated the interaction between STEC strains of serotype O157: H7 with different virulence gene profiles, and a genus of a wild free-living amoeba, Acanthamoeba sp. Our results confirm the ability of STEC strains to survive up to 20 days within a wild Acanthamoeba sp., in a quiescent state persisting in a non-cultivable form, until they reactivate following some stimulus of an unknown nature. Furthermore, our findings show that during their internalization, the E. coli O157 kept the set of the main virulence genes intact, preserving their pathogenetic potential. These observations suggest that the internalization in free-living amoebae may represent a means for STEC to resist in environments with non-permissive growth conditions. Moreover, by staying within the protozoa, STEC could escape their detection in the vehicles of infections and resist to the treatments used for the disinfection of the livestock environment.

Molecular surveillance of non-O157 Shiga toxigenic Escherichia coli in selected chicken abattoirs and retail outlets in Osogbo, Osun State, Nigeria

Three selected chicken abattoirs and two retail locations were studied to determine the frequency of occurrence and profile for Shiga toxin-producing Escherichia coli (STEC) strains present in abattoirs and retail (frozen) chicken carcasses in Osun state, Nigeria. Samples were plated on Eosin Methylene Blue agar for the presence of E. coli. Furthermore, the isolates were confirmed serologically as non-O157 STEC using latex agglutination serotyping kit. Multiplex PCR was used to check for specific virulence factors in the isolated E. coli strains. The mean colony count results showed that effluent water samples from the Ikirun slaughter slab type abattoir were the highest at 25 cfu/ml. A post hoc comparison showed that this value was significantly higher than that of the slaughtering table at Oluode-1 (P = 0.04) and retail chicken meat samples at Igbona (P = 0.01). The results show that chicken abattoirs are poor reservoirs of STEC. Moreover, the results from this study showing that the stx2-producing strains that are more prone to cause hemolytic uremic syndrome is the predominant strain in the study area is worrisome. These results underscore the improper hygiene practices of the abattoir workers combined with inadequate waste management and biological waste disposal systems. It is recommended that regulatory bodies in this locality should focus on ensuring the upgrade of biological waste disposal from these abattoirs in order to limit spread of potentially virulent pathogens into the runoff and ground water.

A Validation System for Selection of Bacteriophages against Shiga Toxin-Producing Escherichia coli Contamination

Shiga toxin-producing Escherichia coli (STEC) can cause severe infections in humans, leading to serious diseases and dangerous complications, such as hemolytic-uremic syndrome. Although cattle are a major reservoir of STEC, the most commonly occurring source of human infections are food products (e.g., vegetables) contaminated with cow feces (often due to the use of natural fertilizers in agriculture). Since the use of antibiotics against STEC is controversial, other methods for protection of food against contaminations by these bacteria are required. Here, we propose a validation system for selection of bacteriophages against STEC contamination. As a model system, we have employed a STEC-specific bacteriophage vB_Eco4M-7 and the E. coli O157:H7 strain no. 86-24, bearing Shiga toxin-converting prophage ST2-8624 (Δstx2::cat gfp). When these bacteria were administered on the surface of sliced cucumber (as a model vegetable), significant decrease in number viable E. coli cells was observed after 6 h of incubation. No toxicity of vB_Eco4M-7 against mammalian cells (using the Balb/3T3 cell line as a model) was detected. A rapid decrease of optical density of STEC culture was demonstrated following addition of a vB_Eco4M-7 lysate. However, longer incubation of susceptible bacteria with this bacteriophage resulted in the appearance of phage-resistant cells which predominated in the culture after 24 h incubation. Interestingly, efficiency of selection of bacteria resistant to vB_Eco4M-7 was higher at higher multiplicity of infection (MOI); the highest efficiency was evident at MOI 10, while the lowest occurred at MOI 0.001. A similar phenomenon of selection of the phage-resistant bacteria was also observed in the experiment with the STEC-contaminated cucumber after 24 h incubation with phage lysate. On the other hand, bacteriophage vB_Eco4M-7 could efficiently develop in host bacterial cells, giving plaques at similar efficiency of plating at 37, 25 and 12 °C, indicating that it can destroy STEC cells at the range of temperatures commonly used for vegetable short-term storage. These results indicate that bacteriophage vB_Eco4M-7 may be considered for its use in food protection against STEC contamination; however, caution should be taken due to the phenomenon of the appearance of phage-resistant bacteria.

Identification, Shiga toxin subtypes and prevalence of minor serogroups of Shiga toxin-producing Escherichia coli in feedlot cattle feces

Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that cause illnesses in humans ranging from mild to hemorrhagic enteritis with complications of hemolytic uremic syndrome and even death. Cattle are a major reservoir of STEC, which reside in the hindgut and are shed in the feces, a major source of food and water contaminations. Seven serogroups, O26, O45, O103, O111, O121, O145 and O157, called ‘top-7’, are responsible for the majority of human STEC infections in North America. Additionally, 151 serogroups of E. coli are known to carry Shiga toxin genes (stx). Not much is known about fecal shedding and prevalence and virulence potential of STEC other than the top-7. Our primary objectives were to identify serogroups of STEC strains, other than the top-7, isolated from cattle feces and subtype stx genes to assess their virulence potential. Additional objective was to develop and validate a novel multiplex PCR assay to detect and determine prevalence of six serogroups, O2, O74, O109, O131, O168, and O171, in cattle feces. A total of 351 strains, positive for stx gene and negative for the top-7 serogroups, isolated from feedlot cattle feces were used in the study. Of the 351 strains, 291 belonged to 16 serogroups and 60 could not be serogrouped. Among the 351 strains, 63 (17.9%) carried stx1 gene and 300 (82.1%) carried stx2, including 12 strains positive for both. The majority of the stx1 and stx2 were of stx1a (47/63; 74.6%) and stx2a subtypes (234/300; 78%), respectively, which are often associated with human infections. A novel multiplex PCR assay developed and validated to detect six serogroups, O2, O74, O109, O131, O168, and O171, which accounted for 86.9% of the STEC strains identified, was utilized to determine their prevalence in fecal samples (n = 576) collected from a commercial feedlot. Four serogroups, O2, O109, O168, and O171 were identified as the dominant serogroups prevalent in cattle feces. In conclusion, cattle shed in the feces a number of STEC serogroups, other than the top-7, and the majority of the strains isolated possessed stx2, particularly of the subtype 2a, suggesting their potential risk to cause human infections.

Abundance and Expression of Shiga Toxin Genes in Escherichia coli at the Recto-Anal Junction Relates to Host Immune Genes

Shiga toxin (Stx) is the main virulence factor of Shiga toxin-producing Escherichia coli (STEC), and ruminants are the main reservoir of STEC. This study assessed the abundance and expression of Stx genes and the expression of host immune genes, aiming to determine factors affecting these measures and potential gene markers to differentiate Stx gene expression in the recto-anal junction of feedlot beef cattle. Rectal tissue and content samples were collected from 143 feedlot steers of three breeds (Angus, Charolais, and Kinsella Composite) over 2 consecutive years 2014 (n=71) and 2015 (n=72). The abundance and expression of stx1 and stx2 were quantified using qPCR and reverse-transcription-qPCR (RT-qPCR), respectively. Four immune genes (MS4A1, CCL21, CD19, and LTB), previously reported to be down-regulated in super-shedder cattle (i.e., > 104 CFU g-1) were selected, and their expression was evaluated using RT-qPCR. The stx1 gene abundance was only detected in tissue samples collected in year 2 and did not differ among breeds. The stx2 gene was detected in STEC from all samples collected in both years and did not vary among breeds. The abundance of stx1 and stx2 differed (P < 0.001) in content samples collected across breeds (stx1:AN>CH>KC, stx2: AN=CH>KC) in year 1, but not in year 2. Expression of stx2 was detected in 13 RAJ tissue samples (2014: n=6, 2015: n=7), while expression of stx1 was not detected. Correlation analysis showed that the expression of stx2 was negatively correlated with the expression of MS4A1 (R=-0.56, P=0.05) and positively correlated with the expression of LTB (R=0.60, P=0.05). The random forest model and Boruta method revealed that expression of selected immune genes could be predictive indicators of stx2 expression with prediction accuracy of MS4A1 >LTB >CCL21 >CD19. Our results indicate that the abundance of Stx could be affected by cattle breed and sampling year, suggesting that host genetics and environment may influence STEC colonization of the recto-anal junction of feedlot cattle. Additionally, the identified relationship between expressions of host immune genes and stx2 suggests that the host animal may regulate stx2 expression in colonizing STEC through immune functions.

Shiga Toxin–Producing Escherichia coli in Feces of Finisher Pigs: Isolation, Identification, and Public Health Implications of Major and Minor Serogroups

Shiga toxin-producing Escherichia coli (STEC) are major foodborne human pathogens that cause mild to hemorrhagic colitis, which could lead to complications of hemolytic uremic syndrome. Seven serogroups, O26, O45, O103, O111, O121, O145, and O157, account for the majority of the STEC illnesses in the United States. Shiga toxins 1 and 2, encoded by stx1 and stx2, respectively, and intimin, encoded by eae gene, are major virulence factors. Cattle are a major reservoir of STEC, but swine also harbor them in the hindgut and shed STEC in the feces. Our objectives were to use a culture method to isolate and identify major and minor serogroups of STEC in finisher pig feces. Shiga toxin genes were subtyped to assess public health implications of STEC. Fecal samples (n = 598) from finisher pigs, collected from 10 pig flows, were enriched in E. coli broth and tested for stx1, stx2, and eae by a multiplex PCR (mPCR) assay. Samples positive for stx1 or stx2 gene were subjected to culture methods, with or without immunomagnetic separation and plating on selective or nonselective media, for isolation and identification of stx-positive isolates. The culture method yielded a total of 178 isolates belonging to 23 serogroups. The three predominant serogroups were O8, O86, and O121. The 178 STEC strains included 26 strains with stx1a and 152 strains with stx2e subtypes. Strains with stx1a, particularly in association with eae (O26 and O103), have the potential to cause severe human infections. All stx2-positive isolates carried the subtype stx2e, a subtype that causes edema disease in swine, but is rarely involved in human infections. Several strains were also positive for genes that encode for enterotoxins, which are involved in neonatal and postweaning diarrhea in swine. In conclusion, our study showed that healthy finisher pigs harbored and shed several serogroups of E. coli carrying virulence genes involved in neonatal diarrhea, postweaning diarrhea, and edema disease, but prevalence of STEC of public health importance was low.

Differential induction of Shiga toxin in environmental Escherichia coli O145:H28 strains carrying the same genotype as the outbreak strains

Shiga toxin-producing Escherichia coli (STEC) O145 is a major serotype associated with severe human disease. Production of Shiga toxins (Stxs), especially Stx2a, is thought to be correlated with STEC virulence. Since stx genes are located in prophages genomes, induction of prophages is required for effective Stxs production. Here, we investigated the production of Stxs in 12 environmental STEC O145:H28 strains under stresses STEC encounter in natural habitats and performed comparative analysis with two O145:H28 clinical strains, one linked to a 2010 U.S. lettuce-associated outbreak (RM13514) and the other linked to a 2007 Belgium ice cream-associated outbreak (RM13516). Similar to the outbreak strains, all environmental strains belong to Sequence Type (ST)-78 using the EcMLST typing scheme. Although all Stx1a-prophages were grouped together, variations in Stx1a production were observed prior to or following the inductions. Among all stx2a positive environmental strains, only the Stx2a-prophage in cattle isolate RM9154-C1 was clustered with the Stx2a-prophages in RM13514, the Stx2a-phage induced from a STEC O104:H4 strain linked to the 2011 outbreak of enterohemorrhagic infection in Germany, and the Stx2a-prophage in STEC O157:H7 strain EDL933, a prototype of enterohemorrhagic E. coli. Furthermore, the Stx2a-prophage in RM9154-C1 shared the same chromosomal insertion site and carried the same antiterminator Q gene and the late promoter PR' as the Stx2a-prophage in RM13514. Following mitomycin C or enrofloxacin treatment, the production of Stx2a in RM9154-C1 was the highest among all environmental strains tested. In contrast, following acid challenge and recovery, the production of Stx2a in RM9154-C1 was the lowest among all the environmental strains tested, at a level comparable to the clinical strains. A significant increase in Stx2a production was detected in all strains when exposed to H2O2, although the induction fold was much lower than those by other inducers. This low-efficiency induction of Stx-prophages by H2O2, a natural inducer of Stx-prophages, supports the hypothesis of bacterial altruism in controlling Stxs production, a strategy that assures the survival of the STEC population as a whole by sacrificing a small fraction of cells for Stxs production and release. Differential induction of Stxs among strains carrying nearly identical Stx-prophages suggests a role of host bacteria in regulating Stxs production. Our study revealed diverse Stx-prophages in STEC O145:H28 strains that were genotypically indistinguishable. Identification of a cattle isolate harboring a Stx2a-prophage associated with high virulence supports the premise that cattle, a natural reservoir of STEC, serve as a source of hypervirulent STEC strains.

The effects of environmental factors on the prevalence and diversity of bacteriophages lytic against the top six non‐O157 Shiga toxin‐producing Escherichia coli on an organic farm

AbstractBacteriophages (or phages) specific to Shiga toxin‐producing Escherichia coli (STEC) strains are frequently isolated from animal‐associated environments primarily because ruminant animals are the natural reservoir of STEC. However, little is known about these phages in produce‐growing environments. The objective of this study was to determine the effects of environmental factors on the prevalence of the phages lytic against O157 and the top six non‐O157 STEC on an organic farm. A total of 370 samples were collected from an organic farm, containing animal‐active and produce‐growing areas, for 1 year. A bacterial cocktail, including nonpathogenic and pathogenic E. coli strains, was used for phage isolation. Meanwhile, culture methods and PCR were used to isolate STEC strains. Weather information was also collected for each sampling trip. Twenty‐eight samples contained phages lytic against STEC (or STEC‐infecting phages), of which 26 were collected from the animal‐active area. Moreover, the winter season had a higher phage prevalence than other seasons, likely due to high rain precipitation. The phages belonging to the Myoviridae family and those lytic against STEC O103 were the most prevalent. One E. coli O103:H2 was isolated from a water sample where no STEC O103‐infecting phages were found. Additionally, no STEC O103 was present in the samples containing STEC O103‐infecting phages. The findings indicate that animal is the primary factor contributing to the prevalence of the STEC‐infecting phages in the surrounding environment of the organic farm, and the presence of these phages contributes to a negative correlation with their STEC hosts.

Draft Genome Sequences of 12 Shiga Toxin-Producing Escherichia coli Strains Isolated from Dairy Cattle in Portugal.

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen transmitted from animals to humans through contaminated food. Cattle are the main reservoir of STEC, but their genetic diversity is still poorly characterized, especially regarding strains isolated in Portugal. We therefore present the draft genomic sequences of 12 STEC strains isolated from cattle in the north of Portugal.

Recent Updates on Outbreaks of Shiga Toxin-Producing Escherichia coli and Its Potential Reservoirs.

Following infection with certain strains of Shiga toxin-producing Escherichia coli (STEC), particularly enterohemorrhagic ones, patients are at elevated risk for developing life-threatening extraintestinal complications, such as acute renal failure. Hence, these bacteria represent a public health concern in both developed and developing countries. Shiga toxins (Stxs) expressed by STEC are highly cytotoxic class II ribosome-inactivating proteins and primary virulence factors responsible for major clinical signs of Stx-mediated pathogenesis, including bloody diarrhea, hemolytic uremic syndrome (HUS), and neurological complications. Ruminant animals are thought to serve as critical environmental reservoirs of Stx-producing Escherichia coli (STEC), but other emerging or arising reservoirs of the toxin-producing bacteria have been overlooked. In particular, a number of new animal species from wildlife and aquaculture industries have recently been identified as unexpected reservoir or spillover hosts of STEC. Here, we summarize recent findings about reservoirs of STEC and review outbreaks of these bacteria both within and outside the United States. A better understanding of environmental transmission to humans will facilitate the development of novel strategies for preventing zoonotic STEC infection.

Selective Killing of Shiga Toxin-Producing Escherichia coli with Antibody-Conjugated Chitosan Nanoparticles in the Gastrointestinal Tract.

Shiga toxin-producing Escherichia coli (STEC) are critical foodborne pathogens, which cause serious human health issues, including hemolytic uremic syndrome. Illnesses caused by STEC lack effective treatments that target the elimination of these bacteria from the gastrointestinal tract without causing an adverse effect. Reducing this pathogen from a reservoir of STEC is an effective strategy, but the challenges remain due to the lack of efficient, selective antimicrobial agents. We developed specific antibody-conjugated chitosan nanoparticles (CNs) to selectively target and treat STEC in the gastrointestinal tract. Given the great broad-spectrum antimicrobial activity of CN, we conjugated antibodies to CN. Antibodies were raised and purified from egg yolks after immunization of hens with seven different O-side-chain antigens isolated from STEC (O26, O45, O103, O111, O121, O145, and O157). We prepared CN-immunoglobulin Y (IgY) conjugates by forming amide bonds at different ratios of CN:IgY (10:1, 10:2, and 10:4). The CN-IgY conjugated at a 10:2 ratio demonstrated significantly enhanced antimicrobial activity against E. coli O157:H7. Conjugates of CN and anti-STEC IgY antibodies killed corresponding STEC serotypes specifically and selectively, while showing no significant impact on nontargeted bacteria, including Salmonella enterica and Lactobacillus plantarum. The enhanced antimicrobial activity of CN-IgY against STEC was also confirmed in synthetic intestinal fluid, as well as an in vivo animal model of Caenorhabditis elegans. These results suggest that the CN-IgY conjugates have strong and specific antimicrobial activity and that they are also great candidates to eliminate pathogens selectively in the gastrointestinal tract without inhibiting beneficial bacteria.

Feces of Finisher Pigs Have a Low Prevalence of Shiga Toxin-Producing Escherichia coli that are of Public Health Importance

Shiga toxin-producing Escherichia coli (STEC) are major food pathogens that cause mild to bloody diarrhea, including complications of kidney damage and even death, particularly in children and elderly. Seven serogroups of STEC, O26, O45, O103, O111, O121, O145, and O157, called top-7 STEC, are responsible for the majority of STEC infections in the US. Shiga toxins, which are proteins secreted by the bacteria, are major virulence factors contributing to the disease. There are two Shiga toxin types, 1 and 2, encoded stx1 and stx2 gene, respectively and each type has several subtypes. Another major virulence factor, intimin, a protein on the bacterial cell surface encoded by the eae gene, mediates attachment of the bacterial cell to the intestinal epithelial cells. The severity of STEC infections in humans is dependent on the Shiga toxin type and subtype. Cattle are a major reservoir of STEC and carry the bacteria in the hindgut and shed them in the feces, which is a source of contamination of food and water. Swine have also been shown to harbor STEC in the gut and shed in the feces, and a few outbreaks of STEC infections in humans have been linked to pork and pork products. The STEC does not cause infections in cattle, but in swine, particularly in weaned piglets, it causes edema disease. We conducted a study that utilized molecular (polymerase chain reaction [PCR]) and culture methods to determine prevalence and characteristics of top-7 STEC in the feces of finisher pigs collected from ten pig flows in eight states. A total of 598 fecal samples were collected and analyzed. The overall preva­lence of Shiga toxin genes, stx1 or stx2, was 70.1%, and eae was detected in 66.7% of the samples. Based on the PCR method, among the top-7 STEC, O26 (14.4%), O121 (22.9%) and O157 (18.5%) were the predominant serogroups detected. None of the E. coli O157 isolated, a serogroup implicated in pork-linked outbreaks, contained Shiga toxin genes. Although a number of fecal samples were positive for the top-7 STEC serogroups, culture method identified one strain each of stx1-positive O26 (0.2%) and O103 (0.2%), and 23 strains of stx2-positive O121 (3.9%). Serogroups O26 and O103 possessed stx1a subtype and eae, which have the potential to cause serious infections in humans. Serogroup O121 carried the stx2e subtype, which is involved in causing edema disease in swine and rarely implicated in human infections. Our results indicated that

Shiga toxin-producing Escherichia coli in the animal reservoir and food in Brazil.

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathotype associated with human gastrointestinal disease that may progress to severe complications. Ruminants, especially cattle, are the main reservoirs of STEC contaminating the environment and foods of animal or vegetable origin. Besides Shiga toxin, other virulence factors are involved in STEC virulence. O157:H7 remains the most frequent serotype associated with disease. In Brazil, the prevalence of STEC reaches values as high as 90% in cattle and 20% in meat products which may impact the Brazilian food export trade. However, only few reports are related to human disease. The stx1 gene prevails in cattle, whereas the stx2 gene is more frequent in food. Several STEC serotypes have been isolated from cattle and food in Brazil, including the O157:H7, O111:NT, NT:H19 as well as O26 and O103 serogroups. O113: H21 STEC strains are frequent in ruminants and foods but with no report in human disease. The virulence profile of Brazilian STEC strains from cattle and food suggests a pathogenic potential to humans, although some differences with clinical strains have been detected. Further studies, employing recent and more discriminative techniques are in need to better clarify their virulence potential.

Identification of pathogenic Escherichia coli strain from river and sewage water in Bangladesh

The study was conducted to isolate and identify the presence of Shiga toxin producing Escherichia coli (STEC) strains in water samples of the Old Brahmaputra River and Sewage water at Mymensingh Municipality. A total of 20 water samples were collected for this experiment. Samples were cultured on EMB agar, stained and PCR was done to detect the pathogenic E. coli. The Old Brahmaputra River is used as a sink of all types of municipal sewage, agricultural wastes, domestic wastes and religious ritual wastes through unplanned sewerage to the river water body. Low quality sanitation system and open defecation are also considerable problems to deteriorate river water quality. Due to accumulation of municipal untreated wastes to river body, it is possible to contain various pathogens. After investigation and identification, fourteen isolates of E. coli was found to contain stx-1 gene with none of stx-2 gene among twenty isolates which indicate pathogenic STEC. There may present major health risk to human and animal due to STEC. Several human diseases like mild diarrhea, bloody diarrhea or even severe hemolytic-uremic syndrome (HUS) may occur for drinking of untreated river water. Domestic ruminants can act as a reservoir for STEC and play a significant role in the epidemiology of human infections.

Shiga toxin-producing Escherichia coli (STEC) isolated from fecal samples of African dromedary camels

Shiga toxin-producing Escherichia coli (STEC) cause gastrointestinal illnesses including non-bloody or bloody diarrhoea, haemorrhagic colitis (HC), and the haemolytic uremic syndrome (HUS). To investigate the occurrence of STEC among grazing dromedaries from Kenya, E. coli isolated from fecal matter collected from 163 dromedaries on a large ranch were screened for the presence of stx1 and stx2. STEC strains were isolated and serotyped. Isolates were subjected to PCR for the subtyping of stx genes and for the detection of eae and ehx. In addition, whole genome sequencing (WGS) was carried out to detect further virulence genes and to determine the multilocus sequence types (MLST). Antimicrobial resistance profiles were determined by disk diffusion.STEC was isolated from 20 (12.3%) of the fecal samples. Thereof, nine (45%) isolates were STEC O156:H25, three (15%) isolates typed STEC O43:H2. The remaining isolates occurred as single serotypes or were O non-typeable. Eleven (55%) of the isolates harboured stx2a, nine (45%) eae, and 14 (70%) ehx, respectively. WGS revealed the presence of iss in 16 (80%), subAB in four (20%) and astA in two (10%) of the isolates, Furthermore, espA, tccP, nleA, nleB, tccP, and tir were found exclusively among STEC O156:H25.Eleven different sequence types (ST) were detected. The most prominent was ST300/ST5343, which comprised STEC O156:H25. All STEC isolates were pan susceptible to a panel of 16 antimicrobial agents. Overall, the results indicate that dromedary camels in Kenya may be reservoirs of STEC, including serotypes possessing virulence markers associated to disease in humans, such as STEC O156:H25. STEC in camels may represent a health hazard for humans with close contact to camels or to consumers of camel derived foodstuffs, such as unpasteurised camel milk.