Importation of Shiga Bacillus Dysentery into California—Clinical and Public Health Significance

Concurrent with the regional epidemic of classic Shiga dysentery in Central America during 1969 and 1970, a pronounced increase in the isolation of Shigella dysenteriae type 1 was noted in California. A retrospective study of 20 cases diagnosed in California in 1969 and 1970 revealed that 18 of the patients had traveled to Central America or Mexico during or immediately before the onset of symptoms. Sixteen were known to have been admitted to hospital; there was one death. Despite the concern that such importations might result in epidemics in this country among groups living in crowded, unsanitary settings, no definite secondary transmission was identified in this study. The problems of differential diagnosis, laboratory isolation of the agent, chemotherapy, and epidemic control are discussed.     

Is lack of susceptible recipients in the intestinal environment the limiting factor for transduction of Shiga toxin-encoding phages?

Aim: To determine whether a Shiga toxin 2 (Stx2)-encoding phage from Escherichia coli O157:H7 could be transmitted to commensal E. coli in a ruminant host without adding a specific recipient strain. Methods and Results: Sheep were inoculated with an E. coli O157:H7 strain containing an Stx2-encoding bacteriophage (Φ3538) in which a chloramphenicol-resistant gene, cat, is inserted into stx₂. A total of 149 faecal samples were sampled and analysed for detection and quantification of E. coli O157:H7 and presumptive transductants. Phage Φ3538 (Δstx₂::cat) was demonstrated to be transduced to an ovine E. coli O175:H16 at one occasion. Conclusions: The study demonstrates an in vivo transduction in sheep from an E. coli O157:H7 strain to an ovine E. coli O175:H16. A functional Stx2-encoding phage was incorporated into the host’s DNA. Significance and Impact of the Study: This is the first in vivo stx phage transduction study reported in which a recipient strain was not fed to the test animals. We suggest that the access to susceptible hosts is one main limiting factor for transduction to occur in the intestine.     

Spread and change in stress resistance of Shiga toxin-producing Escherichia coli?O157 on fungal colonies

To elucidate the effect of fungal hyphae on the behaviour of Shiga toxin-producing Escherichia coli (STEC) O157, the spread and change in stress resistance of the bacterium were evaluated after coculture with 11 species of food-related fungi including fermentation starters. Spread distances of STEC O157 varied depending on the co-cultured fungal species, and the motile bacterial strain spread for longer distances than the non-motile strain. The population of STEC O157 increased when co-cultured on colonies of nine fungal species but decreased on colonies of Emericella nidulans and Aspergillus ochraceus. Confocal scanning microscopy visualization of green fluorescent protein-tagged STEC O157 on fungal hyphae revealed that the bacterium colonized in the water film that existed on and between hyphae. To investigate the physiological changes in STEC O157 caused by co-culturing with fungi, the bacterium was harvested after 7 days of co-culturing and tested for acid resistance. After co-culture with eight fungal species, STEC O157 showed greater acid resistance compared to those cultured without fungi. Our results indicate that fungal hyphae can spread the contamination of STEC O157 and can also enhance the stress resistance of the bacteria.     

Genes from the exo–xis region of λ and Shiga toxin-converting bacteriophages influence lysogenization and prophage induction

The exo–xis region, present in genomes of lambdoid bacteriophages, contains highly conserved genes of largely unknown functions. In this report, using bacteriophage λ and Shiga toxin-converting bacteriophage ?24_Β, we demonstrate that the presence of this region on a multicopy plasmid results in impaired lysogenization of Escherichia coli and delayed, while more effective, induction of prophages following stimulation by various agents (mitomycin C, hydrogen peroxide, UV irradiation). Spontaneous induction of λ and ?24_Β prophages was also more efficient in bacteria carrying additional copies of the corresponding exo–xis region on plasmids. No significant effects of an increased copy number of genes located between exo and xis on both efficiency of adsorption on the host cells and lytic development inside the host cell of these bacteriophages were found. We conclude that genes from the exo–xis region of lambdoid bacteriophages participate in the regulation of lysogenization and prophage maintenance.     

Neuropsychological Outcome after Complicated Shiga Toxin–Producing Escherichia coli Infection

Background

The diarrhea associated hemolytic uremic syndrome (HUS) is a major cause of acute uremic failure in children, but not very common in adults. The enterohaemorrhagic Escherichia coli -epidemic in Germany in 2011 affected mostly young and healthy adults. While their immediate deficits have been published, not much is known about the time course and degree of recovery concerning cognitive and behavioral impairment.

Methods and Findings

Twenty patients with Shiga toxin –producing Escherichia coli infection and neurological symptoms underwent comprehensive neuropsychological assessment 3 months and 1 year after the acute disease. Overall, there was an excellent recovery of cognitive functions. In a detailed neuropsychological analysis no significant deficits could be noticed 1 year after the infection in terms of cognitive function, alertness, executive functions and speech. Interestingly there were no correlations between different indicators for severity of disease (hemoglobin and creatinin levels, days of hospitalization, neurological symptoms and MRI changes) and neuropsychological outcome. However, there were a small number of patients with limitations in every day and professional life even one year after the acute disease.

Conclusions

Our study does not provide definitive answers regarding risk factors for these limitations. Still since Shiga toxin –producing Escherichia coli infection is a rare condition in adults, the information this study provides is important for the clinical practice. On one hand for consulting patients and on the other to raise the awareness of the physicians to possible long term complains and the consideration of neuropsychological assessment and supportive psychological treatment.     

Phenotypic and genotypic traits of Shiga toxin-producing Escherichia coli strains isolated from beef cattle from Paraná State, southern Brazil

AIMS: To investigate the prevalence and characteristics of Shiga toxin-producing Escherichia coli (STEC) in cattle from Paraná State, southern Brazil. METHODS AND RESULTS: One hundred and seven faeces cattle samples were cultured on Sorbitol-MacConkey agar. Escherichia coli colonies were tested for production of Shiga toxin using Vero-cell assay. A high prevalence (57%) of STEC was found. Sixty-four STEC were serotyped and examined for the presence of stx(1), stx(2), eae, ehxA and saa genes and stx(2) variants. The isolates belonged to 31 different serotypes, of which three (O152:H8, O175:H21 and O176:H18) had not previously been associated with STEC. A high prevalence of stx(2)-type genes was found (62 strains, 97%). Variant forms found were stx(2), stx(2c), stx(2vhb), stx(2vO111v/OX393) and a form nonclassifiable by PCR-RFLP. The commonest genotypes were stx(2)ehxA saa and stx(1)stx(2)ehxA saa. CONCLUSIONS: A high frequency of STEC was observed. Several strains belong to serotypes previously associated with human disease and carry stx(2) and other virulence factors, thus potentially representing a risk to human health. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study of STEC in Paraná State, and its findings emphasize the need for proper cattle handling to prevent human contamination.     

Production of double repeated B subunit of Shiga toxin?2e at high levels in transgenic lettuce plants as vaccine material for porcine edema disease

Pig edema disease is a bacterial disease caused by enterohemorrhagic Escherichia coli. E.?coli produces Shiga toxin?2e (Stx2e), which is composed of one A subunit (Stx2eA) and five B subunits (Stx2eB). We previously reported production of Stx2eB in lettuce plants as a potential edible vaccine (Matsui et?al. in Biosci Biotechnol Biochem 73:1628-1634, 2009). However, the accumulation level was very low, and it was necessary to improve expression of Stx2eB for potential use of this plant-based vaccine. Therefore, in this study, we optimized the Stx2eB expression cassette and found that a double repeated Stx2eB (2× Stx2eB) accumulates to higher levels than a single Stx2eB in cultured tobacco cells. Furthermore, a linker peptide between the two Stx2eB moieties played an important role in maximizing the effects of the double repeat. Finally, we generated transgenic lettuce plants expressing 2× Stx2eB with a suitable linker peptide that accumulate as much as 80?mg per 100?g fresh weight, a level that will allow us to use these transgenic lettuce plants practically to generate vaccine material.     

Virulence properties and antimicrobial susceptibility of Shiga toxin-producing Escherichia coli strains isolated from healthy cattle from Paraná State, Brazil

The presence of Shiga toxin-producing Escherichia coli (STEC) strains in feces samples of cattle was determined using the cytotoxicity assay on Vero cells and a screening PCR system to detect stx genes. The STEC isolates were serotyped, tested for antimicrobial susceptibility, and analyzed for virulence genes using multiplex PCR. The verocytotoxin-producing E. coli - reverse passive latex agglutination (VTEC-RPLA) assay was also used to detect Shiga toxin production. The frequency of cattle shedding STEC was 36%. The isolates belonged to 33 different serotypes, of which O10:H42, O98:H41, and O159:H21 had not previously been associated with STEC. The most frequent serotypes were ONT:H7 (10%), O22:H8 (7%), O22:H16 (7%), and ONT:H21 (7%). Most of the strains (96%) were susceptible to all antimicrobial agents tested. Shiga toxin was detected by the VTEC-RPLA assay in most (89%) of the STEC strains. The frequency of virulence markers was as follows: stx1, 10%; stx2, 43%; stx1 plus stx2, 47%; ehxA, 44%; eae, 1%; and saa, 38%. Several strains belong to serotypes associated with human disease, and most of them carried a stx2-type gene, suggesting that they represent a risk to human health. The screening PCR assay showed fewer false-negative results for STEC than the Vero-cell assay and is suitable for laboratory routine.     

Isolation of Shiga toxin-producing Escherichia coli O103 from sheep using automated immunomagnetic separation (AIMS) and AIMS-ELISA: sheep as the source of a clinical E. coli O103 case?

AIMS: To investigate whether a sheep flock was the original reservoir of a Shiga toxin-producing Escherichia coli (STEC) O103 strain causing a clinical human case and to compare the two diagnostic methods automated immunomagnetic separation (AIMS) and AIMS-ELISA. METHODS AND RESULTS : AIMS detected Escherichia coli O103 in 36.5% of the samples and AIMS-ELISA detected E. coli O103 in 52.1% of the samples. Polymerase chain reaction detected stx1 and eae in three of 109 E. coli O103 isolates. Pulsed field gel electrophoresis showed that the sheep and human STEC O103 were characterized by distinctly different profiles. CONCLUSIONS: The sheep flock was shown to carry STEC O103, although an association between the sheep flock and the clinical human case could neither be proven nor eliminated. Substantial agreement was found between AIMS and AIMS-ELISA, but AIMS-ELISA was less time consuming and resulted in a higher recovery of E. coli O103. SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows that sheep may be carriers of STEC that are associated with human disease and that the methods described can be used to increase the sensitivity of STEC detection.     

产Shiga毒素噬菌体φ297在大肠杆菌K12染色体上整合位点的定位研究

鉴定大肠杆菌O15 7产生Shiga毒素 (Stx)的噬菌体 φ2 97在大肠杆菌K12染色体上的插入位点。采用加接头PCR方法从与噬菌体 933W的整合酶基因int同源的核苷酸开始向未知区域进行步移测序 ,寻找目的基因。将获得的DNA序列与核苷酸数据库进行比较 ,确定了细菌性噬菌体 φ2 97的attL、attR和中心序列位于大肠杆菌K12的yecE基因内。噬菌体 φ2 97在宿主细胞E .coliK12染色体上的整合位点是yecE基因。     

产Shiga毒素噬菌体ψ297在大肠杆菌K12染色体上整合位点的定位研究

鉴定大肠杆菌O157产生Shiga毒素(Stx)的噬菌体ψ297在大肠杆菌K12染色体上的插入位点.采用加接头PCR方法从与噬菌体933W的整合酶基因int同源的核苷酸开始向未知区域进行步移测序,寻找目的基因.将获得的DNA序列与核苷酸数据库进行比较,确定了细菌性噬菌体ψ297的attL、attR和中心序列位于大肠杆菌K12的yecE基因内.噬菌体ψ297在宿主细胞E.coliK12染色体上的整合位点是yecE基因.     

产Shiga毒素噬菌体φ297在大肠杆菌K 12染色体上整合位点的定位研究

鉴定大肠杆菌O157产生Shiga 毒素 (Stx)的噬菌体φ297在大肠杆菌K12染色体上的插入位点。采用加接头PCR方法从与噬菌体933W的整合酶基因int同源的核苷酸开始向未知区域进行步移测序,寻找目的基因。将获得的DNA序列与核苷酸数据库进行比较,确定了细菌性噬菌体φ297的attL 、attR和中心序列位于大肠杆菌K12的yecE基因内。噬菌体φ297在宿主细胞E. coli K12染色体上的整合位点是yecE基因。     

Detection of the Emerging Shiga Toxin-Producing Escherichia coli O26:H11/H? Sequence Type 29 (ST29) Clone in Human Patients and Healthy Cattle in Switzerland

Shiga toxin-producing Escherichia coli O26:H11/H⁻ strains showing the characteristics of the emerging human-pathogenic ST29 clone (stx_2a⁺ only, eae⁺, plasmid gene profile hlyA⁺ etpD⁺) were detected from human patients and healthy cattle, indicating a possible reservoir. Sheep also appear to shed strains related to the new pathogenic clone O26:H11/H⁻ (ST29, stx_1a⁺ only, eae⁺, plasmid gene profile hlyA⁺ etpD⁺).     

Shiga toxin-producing Escherichia coli O100:H⁻: stx2e in drinking water contaminated by waste water in Finland

In November 2007, 450 m³ of treated wastewater leaked into the drinking water distribution system contaminating the drinking water of over 10,000 inhabitants of Nokia, Southern Finland. Nearly 1,000 people visited the health centre because of gastroenteritis during the following 5 weeks. A wide range of enteric pathogens was found in the patients. The authors used the 16-plex PCR to investigate whether the five major diarrheagenic Escherichia coli pathotypes (EPEC, ETEC, STEC, EIEC or EAEC) were present in the contaminated drinking water and in the patients’ stool samples. The contaminated drinking water was positive for genes characteristic of various E. coli pathotypes: pic, invE, hlyA, ent, escV, eae, aggR, stx ₂ , estIa and astA. These genes, except stx ₂ , hlyA and invE, were also detected in the stool samples of the patients linked to this outbreak. A sorbitol positive, streptomycin resistant STEC strain was isolated from the drinking water, and belonged to the serotype O100:H ^– , produced Stx2 toxin (titre 1:8 by reversed-passive latex agglutination method), and carried the genes stx _2e, estIa and irp2.     

Shiga Toxin-Producing Escherichia coli O157 Is More Likely to Lead to Hospitalization and Death than Non-O157 Serogroups – Except O104

The clinical spectrum following infection with Shiga toxin-producing Escherichia coli (STEC) is wide ranging and includes hemorrhagic colitis and life-threatening hemolytic uremic syndrome (HUS). Severity of STEC illness depends on patients'' age and strongly on the infecting strains'' virulence. Serogroup O157 is often assumed to be more virulent than others. Age-adjusted population-based data supporting this view are lacking thus far.We conducted a large retrospective cohort study among patients of community-acquired gastroenteritis or HUS diagnosed with STEC infection, reported in Germany January 2004 through December 2011. Age-adjusted risks for reported hospitalization and death, as proxies for disease severity, were estimated for STEC serogroups separately, and compared with STEC O157 (reference group) using Poisson regression models with robust error estimation.A total of 8,400 case-patients were included in the analysis; for 2,454 (29%) and 30 (0.4%) hospitalization and death was reported, respectively. Highest risks for hospitalization, adjusted for age and region of residence, were estimated for STEC O104 (68%; risk ratio [RR], 1.33; 95% confidence interval [CI], 1.19–1.45), followed by STEC O157 (46%). Hospitalization risks for the most prevalent non-O157 serogroups (O26, O103, O91, O145, O128, O111) were consistently and markedly lower than for O157, with the highest RR for O145 (0.54; 95% CI, 0.41–0.70) and the lowest for O103 (0.27; 95% CI, 0.20–0.35). Mortality risk of O104 was similar to O157 (1.2% each), but the group of all other non-O157 STEC had only 1/10 the risk (RR, 0.09; 95% CI, 0.02–0.32) compared to O157.The study provides population-based and age-adjusted evidence for the exceptional high virulence of STEC O157 in relation to non-O157 STEC other than O104. Timely diagnosis and surveillance of STEC infections should prioritize HUS-associated E. coli, of which STEC O157 is the most important serogroup.     

A comparative analysis of karyotypes of three species of Macleaya—producers of a complex of isoquinoline alkaloids

Using a set of methods (C-banding, DAPI-staining, fluorescence hybridization in situ (FISH) with probes of 26S and 5S rDNA, and analysis of meiosis), the first comparative cytogenetic study of three species of Macleaya, producers of complex isoquinoline alkaloids, cordate Macleaya cordata (Willd.) R. Br. (2n = 20), small-fruited Macleaya microcarpa (Maxim.) Fedde (2n = 20) and Macleaya kewensis Turrill (2n = 20), was first carried out. On the basis of morphometric analysis, formulas of karyotypes were made for each species. Species ideograms for M. cordata, M. microcarpa, and M. kewensis were constructed taking into account the polymorphic variants of the C-banding patterns and indicating the location of 26S and 5S rDNA sites. A comparative study revealed that the karyotypes of M. microcarpa and M. kewensis have more in common with each other than with M. cordata. Analysis of meiotic chromosomes suggests of genetic stability of Macleaya genomes. The results of chromosome analysis were used to confirm the close relationship of Macleaya and to clarify their phylogenetic relationships.     

Effect of γ-Irradiation on Development of Lachrymator of Onion

A thermosensitive uracil requiring mutant of Bacillus subtilis Marburg 168 thy trp₂ ts42 was examined as to the colony forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in the modified Woese’s (MW) medium. However, the cells retained viability when sodium succinate or potassium chloride was added to the medium at that temperature although uracil deficiency was unchanged. A little but significant incorporation of adenine-8-¹⁴C into RNA still continued even after the incorporation of N-acetyl-³H-d-glucosamine into acid insoluble fraction of the cells terminated in the MW medium at 48°C. Both incorporations as well as increase of absorbance were slowed down in the presence of sodium succinate at 48°C. This mutant, ts42, was more sensitive to deoxycholate (DOC) than the parent strain. The restoration of colony forming ability after the temperature shift back from 48 to 37°C was suppressed by the addition of DOC to the medium. However, the cell became resistant to DOC when uracil was added to the medium prior to the temperature shift.