Production of slime polysaccharide by EHEC and STEC as well as the influence of culture conditions on slime production in Escherichia coli O157:H7

AIMS: To quantify the slime polysaccharide, composed of colanic acid (CA), produced by enterohaemorrhagic and Shiga toxin-producing Escherichia coli (EHEC and STEC) and to determine the influence of culture conditions on CA production in E. coli O157:H7. METHODS AND RESULTS: The study examined the amounts of CA produced by EHEC and STEC, and evaluated the production of CA in E. coli O157:H7 as influenced by medium pH and incubation temperatures. The results indicated that the amounts of CA produced by EHEC and STEC vary to a great extent and CA production in E. coli O157:H7 is influenced by the tested culture conditions. CONCLUSIONS: The abilities of EHEC and STEC to produce CA differ. Medium pH and incubation temperature are among the important factors affecting CA production in E. coli O157:H7. SIGNIFICANCE AND IMPACT OF THE STUDY: Slime polysaccharide can affect the abilities of E. coli O157:H7 cells to combat environmental stress. This study contributes to a better understanding of the physiological factors influencing slime polysaccharide production in EHEC and STEC.     

蚯蚓粪中乳酸菌的分离及其在大肠杆菌O157:H7中的抗菌活性

本研究利用SL培养基从蚯蚓粪中分离到54株具有产酸性能的菌株,并以E. coli O157:H7 (EDL933株)作为指示菌株,采用点种法检测分离菌株的抑菌活性.结果表明其中6个菌株对指示菌具有拮抗作用,通过形态特征,结合16S rDNA序列分析,初步鉴定该6个菌株分别为食物魏斯特菌(Listeria welshimeri)、乳酸片球菌(Pediococcus acidilactici)、短乳杆菌(Lactobacillus brevis)和格氏乳球菌(Lactococcus garvieae).分离到的乳酸菌对E. coli O157:H7 (EDL933株)具有显著的抑制作用,发酵温度和初始pH值影响发酵液的抑菌作用,优化环境因子可以促进拮抗菌对E. coli O157:H7的抑制作用.本研究为进一步分离抗菌产物用于人畜共患病的预防和治疗提供了理论依据.     

肠出血性大肠杆菌O157：H7监测及分析

为了了解长春地区动物和人感染肠出血性大肠杆菌O157H7状况,建立流行病学监测网.采集长春市动物养殖场动物粪便和腹泻病人便样进行监测.结果在牛粪和鸡粪中共检出2株O157H7大肠杆菌.可见,在长春地区存在肠出血性大肠杆菌O157H7菌潜在污染的威胁,需要加强监测力度.     

Survival of Escherichia coli O157:H7 in wastewater from dairy lagoons

AIM: To determine the survival of Escherichia coli O157:H7 in dairy wastewater from on-site holding lagoons equipped with or without circulating aerators. METHODS AND RESULTS: Survival was monitored in dairy lagoon microcosms equipped with or without scale-size circulators. Both laboratory strains of E. coli O157:H7 and an isolate of E. coli H7 from wastewater had poor survival rates and none proliferated in water from waste lagoons with or without circulators. Furthermore, the decline of E. coli O157:H7 was not enhanced in those microcosms equipped with circulators. Strain variation in survival was observed in both circulated and settling waters. The decline rate of E. coli O157:H7 Odwalla strain increased proportionately with the inoculum load. Escherichia coli failed to establish itself in wastewater even after four sequential inoculations simulating continuous faecal input into the lagoon. The native aerobic bacteria survived longer with a decimal reduction time of 21.3 days vs either introduced or native E. coli, which declined rapidly with decimal reduction time of 0.5-9.4 days. CONCLUSIONS: Escherichia coli O157:H7 failed to establish and proliferate in dairy wastewater microcosms equipped with or without circulating aerators. SIGNIFICANCE AND IMPACT OF THE STUDY: This study furthers our knowledge of pathogen survival in wastewater, and suggests that proper management of wastewater before its use in irrigation is essential to reduce pathogen transfer to crops.     

Use of bioluminescent Escherichia coli O157:H7 to study intra-protozoan survival of bacteria within Tetrahymena pyriformis

A method was developed that enabled real-time monitoring of the uptake and survival of bioluminescent Escherichia coli O157 within the freshwater ciliate Tetrahymena pyriformis. Constitutively bioluminescent E. coli O157 pLITE27 was cocultured with T. pyriformis in nutrient-deficient (Chalkley's) and in nutrient-rich (proteose peptone, yeast extract) media. Non-internalised bacteria were inactivated by addition of colistin, indicated by a decline in bioluminescence. Protozoa were subsequently lysed with Triton X-100 which lead to a further drop in bioluminescence, consistent with release of live internal bacteria from T. pyriformis into the colistin-containing environment. Bioluminescence measurements for non-lysed cultures indicated that internalised E. coli O157 pLITE27 cells were only slowly digested by T. pyriformis, in both media, over the time period studied. The results suggest that bioluminescent bacteria are useful tools in the study of bacterial intra-protozoan survival.     

大肠杆菌O157:H7核酸探针检测方法的建立

目的：应用核酸探针方法快速检测大肠杆菌O157：H7。方法：通过使用吖啶酯标记的特异DNA探针方法检测大肠杆菌O157：H7,对此种方法的特异性、敏感性、准确性进行研究,比较该方法与传统国标法的检测结果。结果：核酸探针方法检测大肠杆菌O157：H7特异性以及敏感性强,检出大肠杆菌O157：H7菌液浓度最低限约为106cfu/ml,检测大肠杆菌O157：H7的结果与国标法相一致;对O157：H7鉴定时间仅需30min,简便快捷。结论：核酸探针方法可用于大肠杆菌O157：H7的快速检测。     

Rapid sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef

AIM: To develop an improved, rapid and sensitive sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef. METHODS AND RESULTS: Fresh ground beef samples were experimentally inoculated with varying concentrations of E. coli O157:H7. PCR inhibitors were removed and bacterial cells were concentrated by filtration and centrifugation, and lysed using enzymatic digestion and successive freeze/thaw cycles. DNA was purified and concentrated via phenol/chloroform extraction and the Shiga toxin 1 gene (stx1) was amplified using PCR to evaluate the sample preparation method. Without prior enrichment of cells in broth media, the detection limit was 103 CFU g-1 beef. When a 6 h enrichment step was incorporated, the detection limit was 1 CFU g-1 beef. The total time required from beginning to end of the procedure was 12 h. CONCLUSIONS: The sample preparation method developed here enabled substantially improved sensitivity in the PCR-based detection of E. coli O157:H7 in ground beef, as compared to previous reports. SIGNIFICANCE AND IMPACT OF THE STUDY: Superb sensitivity, coupled with quick turn-around time, relative ease of use and cost-effectiveness, makes this a useful method for detecting E. coli O157:H7 in ground beef.     

Antibacterial activities of zinc oxide nanoparticles against Escherichia coli O157:H7

Aims:  To investigate antibacterial activities of zinc oxide nanoparticles (ZnO NP) and their mode of action against an important foodborne pathogen, Escherichia coli O157:H7.
Methods and Results:  ZnO NP with sizes of 70 nm and concentrations of 0, 3, 6 and 12 mmol l⁻¹ and NP-free solutions were used in antimicrobial tests against E. coli O157:H7. ZnO NP showed increasing inhibitory effects on the growth of E. coli O157:H7 as the concentrations of ZnO NP increased. A complete inhibition of microbial growth was achieved at the concentration level of 12 mmol l⁻¹ or higher. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Raman spectroscopy were used to characterize the changes of morphology and cellular compositions of bacterial cells treated with ZnO NP and study the mode of action of ZnO NP against E. coli O157:H7. The intensity of lipid and protein bands in the Raman spectra of bacterial cells increased after exposure to ZnO NP, while no significant changes in nucleic acid bands were observed.
Conclusions:  ZnO NP were found to have antibacterial activity against E. coli O157:H7. The inhibitory effects increase as the concentration of ZnO NP increased. Results indicate that ZnO NP may distort and damage bacterial cell membrane, resulting in a leakage of intracellular contents and eventually the death of bacterial cells.
Significance and Impact of the Study:  These results suggest that ZnO NP could potentially be used as an effective antibacterial agent to protect agricultural and food safety.     

Probiotic inhibits the cytopathic effect induced by Escherichia coli O157:H7 in Vero cell line model

Aims: The effectiveness of four strains of Bifidobacteria against enterohemorrhagic Escherichiacoli O157:H7 infection was studied using a Vero cell model. Methods and results: E. coli O157 was inoculated on the Vero cell line before and after treatment with probiotic. The cytopathic effect (CPE) was evaluated during 24 h of incubation. The results indicated that Shiga toxin activity was inhibited by the probiotic. To prevent a Stx2 CPE, the probiotic needs one log more than the Stx1. Conclusion: The Vero cell assay, in particular, is a good model to evaluate the effect of Bifidobacteria inhibiting bacterial attachment because of soluble substances and the competitive aspect and could be used in a variety of foods like milk and yoghurt to protect pathogen bacteria. Significance and Impact of the Study: Probiotics could control pathogenic bacteria and Vero cell introduce as a model for evaluation of probiotics against pathogen bacteria.     

Isolation of Escherichia coli O157:H7 from dung beetles Catharsius molossus

In an epidemiological survey, Escherichia coli O157:H7 was isolated from the intestine 4 of 113 dung beetle Catharsius molossus captured below ground at Tongshan County, Jiangsu Province of China. In parallel, 10 strains of E. coli O157:H7 were isolated from fecal samples of 383 diarrhea patients from the same region. Most importantly, using pulsed field gel electrophoresis (PFGE) of chromosomal DNA restriction fragments and PCR method, we found that the PFGE pattern and virulence genes of beetle isolates were identical to those of the human isolates, such as Shiga-toxins (stx) and enterohemorrhagic Escherichia coli hemolysin A (EHEC-hlyA). Therefore, dung beetle might acquire pathogenic E. coli O157:H7 through contact with feces of domestic animals.     

The prevalence and spread of Escherichia coli O157:H7 at a commercial beef abattoir

AIMS: To investigate the prevalence and virulence characteristics of Escherichia coli O157:H7 after a number of beef process operations at a commercial Irish abattoir. METHODS AND RESULTS: Two 12-month studies were carried out. The first study (study 1) examined the prevalence of E. coli O157:H7 at up to six sites on carcasses at eight stages of the dressing, washing, chilling and boning process. The second study (study 2) examined the prevalence of E. coli O157:H7 in bovine faeces and rumen contents post-slaughter and on dressed, washed carcasses. Isolates from both studies were phage-typed and the presence of genes encoding verocytotoxin, enterohaemolysin and intimin production was determined. E. coli O157:H7 was isolated from four of 36 carcasses in study 1. E. coli O157:H7 was detected during hide removal and was detected at multiple carcass sites and multiple process stages, including boning. On two carcasses, contamination was first detected at the bung following its freeing and tying. All isolates from study 1 were phage type (PT) 2, eaeAO157 and ehlyA positive, but were verocytotoxin 1 (VT1) and verocytotoxin 2 (VT2) negative. In study 2, E. coli O157:H7 was isolated from 2.4% of faecal, 0.8% of rumen and 3.2% of carcass samples. In some cases, isolates recovered from the faeces of a particular animal, the resulting carcass and adjacent carcasses on the line had the same phage typing and virulence characteristic profile patterns. All isolates from study 2 were eaeAO157 and ehlyA positive and only one isolate was VT1 and VT2 negative. Most isolates were PT 32. A higher frequency of positive isolations was noted from samples taken during spring and late summer. CONCLUSION: These studies show that in a typical Irish beef abattoir, carcass contamination with E. coli O157:H7 can occur during hide removal and bung tying and this contamination can remain on the carcass during subsequent processing. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data that is necessary for the understanding of how E. coli O157:H7 contamination of beef occurs.     

Earthworms as vectors of Escherichia coli O157:H7 in soil and vermicomposts

Survival and movement of Escherichia coli O157:H7 in both soil and vermicompost is of concern with regards to human health. Whilst it is accepted that E. coli O157:H7 can persist for considerable periods in soils, it is not expected to survive thermophilic composting processes. However, the natural behavior of earthworms is increasingly utilized for composting (vermicomposting), and the extent to which earthworms promote the survival and dispersal of the bacterium within such systems is unknown. The faecal material produced by earthworms provides a ready supply of labile organic substrates to surrounding microbes within soil and compost, thus promoting microbial activity. Earthworms can also cause significant movement of organisms through the channels they form. Survival and dispersal of E. coli O157:H7 were monitored in contaminated soil and farmyard manure subjected to earthworm digestion over 21 days. Our findings lead to the conclusion that anecic earthworms such as Lumbricus terrestris may significantly aid vertical movement of E. coli O157 in soil, whereas epigeic earthworms such as Dendrobaena veneta significantly aid lateral movement within compost. Although the presence of earthworms in soil and compost may aid proliferation of E. coli O157 in early stages of contamination, long-term persistence of the pathogen appears to be unaffected.     

Clonal turnover of enterohemorrhagic Escherichia coli O157:H7 in experimentally infected cattle

A total of 401 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates from two experimentally infected calves were analyzed using molecular biological methods. Genetic differences detected by pulsed-field gel electrophoresis were observed between the inoculated and recovered strains as early as 1 day post inoculation. The loss of the inoculated clone was observed in one calf. Replication and dissemination of the EHEC O157:H7 strains that mutated in cattle may result in the diversification of this organism among cattle populations.     

Escherichia coli O157:H7 colonization in small domestic ruminants

Enterohaemorrhagic Escherichia coli O157:H7 was first implicated in human disease in the early 1980s, with ruminants cited as the primary reservoirs. Preliminary studies indicated cattle to be the sole source of E. coli O157:H7 outbreaks in humans; however, further epidemiological studies soon demonstrated that E. coli O157:H7 was widespread in other food sources and that a number of transmission routes existed. More recently, small domestic ruminants (sheep and goats) have emerged as important sources of E. coli O157:H7 human infection, particularly with the widespread popularity of petting farms and the increased use of sheep and goat food products, including unpasteurized cheeses. Although the colonization and persistence characteristics of E. coli O157:H7 in the bovine host have been studied intensively, this is not the case for small ruminants. Despite many similarities to the bovine host, the pathobiology of E. coli O157:H7 in small domestic ruminants does appear to differ significantly from that described in cattle. This review aims to critically review the current knowledge regarding colonization and persistence of E. coli O157:H7 in small domestic ruminants, including comparisons with the bovine host where appropriate.     

Fate of Escherichia coli O157:H7 during the manufacture of Mozzarella cheese

AIMS: The fate of Escherichia coli O157:H7 was investigated during the manufacture of Mozzarella cheese. METHODS AND RESULTS: The Mozzarella cheese was made from unpasteurized milk which was inoculated to contain ca 10(5) cfu ml(-1)E. coli O157:H7. Two different heating temperatures (70 and 80 degrees C), commonly used during curd stretching, were investigated to determine their effects on the viability of E. coli O157:H7 in Mozzarella cheese. Stretching at 80 degrees C for 5 min resulted in the loss of culturability of E. coli O157:H7 strains, whereas stretching at 70 degrees C reduced the number of culturable E. coli O157:H7 by a factor of 10. CONCLUSIONS: The results show that stretching curd at 80 degrees C for 5 min is effective in controlling E. coli O157:H7 during the production of Mozzarella cheese. Brining and storage at 4 degrees C for 12 h was less effective than the stretching. Significance and Impact of the Study: Mozzarella cheese should be free of E. coli O157:H7 only if temperatures higher than or equal to 80 degrees C are used during milk processing.     

Highly diverse variable number tandem repeat loci in the E. coli O157:H7 and O55:H7 genomes for high-resolution molecular typing

AIM: Evaluation of the Escherichia coli genome for variable number tandem repeat (VNTR) loci in order to provide a subtyping tool with greater discrimination and more efficient capacity. METHODS AND RESULTS: Twenty-nine putative VNTR loci were identified from the E. coli genomic sequence. Their variability was validated by characterizing the number of repeats at each locus in a set of 56 E. coli O157:H7/HN and O55:H7 isolates. An optimized multiplex assay system was developed to facility high capacity analysis. Locus diversity values ranged from 0.23 to 0.95 while the number of alleles ranged from two to 29. This multiple-locus VNTR analysis (MLVA) data was used to describe genetic relationships among these isolates and was compared with PFGE (pulse field gel electrophoresis) data from a subset of the same strains. Genetic similarity values were highly correlated between the two approaches, through MLVA was capable of discrimination amongst closely related isolates when PFGE similar values were equal to 1.0. CONCLUSIONS: Highly variable VNTR loci exist in the E. coli O157:H7 genome and are excellent estimators of genetic relationships, in particular for closely related isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli O157:H7 MLVA offers a complimentary analysis to the more traditional PFGE approach. Application of MLVA to an outbreak cluster could generate superior molecular epidemiology and result in a more effective public health response.