Occurrence of Escherichia coli O157:H7 in faeces, skin and carcasses from sheep and goats in Ethiopia

Aims:  To determine the occurrence and proportion of Escherichia coli O157:H7 in faeces, skin swabs and carcasses before and after washing, from sheep and goats in Ethiopia.
Method and Results:  Individual samples were enriched in modified tryptic soy broth with novobiocin, concentrated using immunomagnetic separation (IMS) and plated onto cefixime-tellurite containing sorbitol MacConkey agar. Presumptive colonies were confirmed by biochemical tests and subjected to latex agglutination tests. A PCR was performed on isolates for the detection of stx ₁, stx ₂ and eae genes. Escherichia coli O157:H7 was isolated from faeces (4·7%), skin swabs (8·7%) and carcasses before washing (8·1%) and after washing (8·7%) and on water samples (4·2%). The proportion of carcasses contaminated with E. coli O157:H7 was strongly associated with those recovered from faecal and skin samples. Both stx ₁ and stx ₂ genes were identified from one E. coli O157:H7 isolate from a goat carcass.
Conclusions:  Even though the numbers of samples examined in this study were limited to one abattoir, sheep and goats can be potential sources of E. coli O157:H7 for human infection in the country. Control measures to reduce the public health risks arising from E.   coli O157:H7 in reservoir animals need to be addressed at abattoir levels by reducing skin and faecal sources and carcass contaminations at different stages of slaughter operations.
Significance and Impact of the Study:  Escherichia coli O157:H7 was detected from carcasses before and after washing during slaughtering operations, and one O157 isolate was positive for verotoxins.     

Evaluation of a PCR detection method for Escherichia coli O157:H7/H- bovine faecal samples

AIMS: Combinations of PCR primer sets were evaluated to establish a multiplex PCR method to specifically detect Escherichia coli O157:H7 genes in bovine faecal samples. METHODS AND RESULTS: A multiplex PCR method combining three primer sets for the E. coli O157:H7 genes rfbE, uidA and E. coli H7 fliC was developed and tested for sensitivity and specificity with pure cultures of 27 E. coli serotype O157 strains, 88 non-O157 E. coli strains, predominantly bovine in origin and five bacterial strains other than E. coli. The PCR method was very specific in the detection of E. coli O157:H7 and O157:H- strains, and the detection limit in seeded bovine faecal samples was <10 CFU g(-1) faeces, following an 18-h enrichment at 37 degrees C, and could be performed using crude DNA extracts as template. CONCLUSIONS: A new multiplex PCR method was developed to detect E. coli O157:H7 and O157:H-, and was shown to be highly specific and sensitive for these strains both in pure culture and in crude DNA extracts prepared from inoculated bovine faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex PCR method is suitable for the rapid detection of E. coli O157:H7 and O157:H- genes in ruminant faecal samples.     

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.     

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.     

Detection and quantification of Escherichia coli O157:H7 in environmental samples by real-time PCR

AIMS: To apply the real-time Polymerase chain reaction (PCR) method to detect and quantify Escherichia coli O157:H7 in soil, manure, faeces and dairy waste washwater. METHODS AND RESULTS: Soil samples were spiked with E. coli O157:H7 and subjected to a single enrichment step prior to multiplex PCR. Other environmental samples suspected of harbouring E.coli O157:H7 were also analysed. The sensitivity of the primers was confirmed with DNA from E.coli O157:H7 strain 3081 spiked into soil by multiplex PCR assay. A linear relationship was measured between the fluorescence threshold cycle (C T ) value and colony counts (CFU ml(-1)) in spiked soil and other environmental samples. The detection limit for E.coli O157:H7 in the real-time PCR assay was 3.5 x 10(3) CFU ml(-1) in pure culture and 2.6 x 10(4) CFU g(-1) in the environmental samples. Use of a 16-h enrichment step for spiked samples enabled detection of <10 CFU g(-1) soil. E. coli colony counts as determined by the real-time PCR assay, were in the range of 2.0 x 10(2) to 6.0 x 10(5) CFU PCR (-1) in manure, faeces and waste washwater. CONCLUSIONS: The real-time PCR-based assay enabled sensitive and rapid quantification of E. coli O157:H7 in soil and other environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to quantitatively determine cell counts of E.coli O157:H7 in large numbers of environmental samples, represents considerable advancement in the area of pathogen quantification for risk assessment and transport studies.     

Prevalence of Escherichia coli O157 and O157:H7-infecting bacteriophages in feedlot cattle feces

AIM: To estimate the distribution and prevalence of both Escherichia coli O157 and O157:H7-infecting bacteriophages within a 50,000 head commercial beef feedlot. METHODS AND RESULTS: Escherichia coli O157 was detected in approximately 27% of the individual samples, distributed across seven of the 10 pens screened. In a simple initial screen to detect O157:H7-infecting phages, none were detected in any pen or individual sample. In contrast, after a series of enrichment procedures O157:H7-infecting phages were detected in every pen and in the majority of the samples from most pens; virulent bacteriophages active against E. coli O157:H7 were detected post-enrichment from 39/60 (65%) of the feedlot samples, and 58/60 (approximately 97%) contained phage that infected E. coli B or O157:H7. CONCLUSIONS: The data we present here indicates that we may be grossly underestimating the prevalence of O157:H7-infecting phages in livestock if we simply screen samples and that enrichment screening is required to truly determine the presence of phages in these ecosystems. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest that O157:H7-infecting phages may play a role in the ecology and transient colonization of cattle by E. coli O157:H7. Further, this and previous data suggest that before starting in vivo pathogen eradication studies using phage or any other regime, test animals should be enrichment screened for phage to avoid erroneous results.     

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.     

肉类中大肠杆菌O157:H7多重PCR检测方法的建立

以编码大肠杆菌 O157 抗原的 rfbE 基因、编码 H7 抗原的 fliC 基因以及编码毒力因子的eaeA 基因为靶基因,选择3对引物,建立并优化了检测大肠杆菌 O157:H7 的多重 PCR 体系,扩增产物分别为291 bp、625 bp,368 bp,采用30株细菌验证了该多重 PCR 具有特异性.PCR 检测的灵敏度在 DNA 水平上达到91.35 Pg;在存在干扰菌鼠伤寒沙门氏(Salmonella typhimurium)的情况下,当起始污染量为1.4 CFU/mL时,37℃培养6 h即可检出.在30份肉类样品中,有3份检出了大肠杆菌 O157:H7.本研究建立的多重 PCR 方法可特异、灵敏地实现对大肠杆菌 O157:H7 的检测.     

肉类中大肠杆菌O157:H7多重PCR检测方法的建立

以编码大肠杆菌O157抗原的rfbE基因、 编码H7抗原的fliC基因以及编码毒力因子的eaeA基因为靶基因, 选择3对引物, 建立并优化了检测大肠杆菌O157:H7的多重PCR体系, 扩增产物分别为291 bp、625 bp、368 bp, 采用30株细菌验证了该多重PCR具有特异性。PCR检测的灵敏度在DNA水平上达到91.35 pg; 在存在干扰菌鼠伤寒沙门氏菌(Salmonella?typhimurium)的情况下, 当起始污染量为1.4 CFU/mL时, 37 ℃培养6 h 即可检出。在30份肉类样品中, 有3份检出了大肠杆菌O157:H7。本研究建立的多重PCR方法可特异、灵敏地实现对大肠杆菌O157:H7的检测。     

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.     

Attachment and biofilm formation on stainless steel by Escherichia coli O157:H7 as affected by curli production

AIMS: The aim of this study was to determine the role of curli in attachment and biofilm formation by Escherichia coli O157:H7 on stainless steel. METHODS AND RESULTS: Three curli-deficient strains (43895-, 43894- and E0018-) and three curli over-producing strains (43895+, 43894+ and E0018+) of E. coli O157:H7 were studied. Stainless steel coupons (SSC) were immersed in cell suspensions of each strain for 24 h at 4 degrees C. The number of cells attached to SSC was determined. To determine the ability of attached cells to form biofilm, SSC were immersed in 10% of tryptic soya broth up to 6 days at 22 degrees C. Curli-deficient and curli-producing strains did not differ in their ability to attach to SSC, but only curli-producing strains formed biofilms. CONCLUSIONS: Curli production by E. coli O157:H7 does not affect attachment of cells on stainless steel but curli-producing strains are better able to form biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: Curli production by E. coli O157:H7 enhances its ability to form biofilm on stainless steel, thereby potentially resulting in increased difficulty in removing or killing cells by routine cleaning and sanitizing procedures used in food-processing plants.     

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。     

Prevalence of Escherichia coli O157:H7 on hides and faeces of ruminants at slaughter in two major abattoirs in Nigeria

Aim: To determine the occurrence of Escherichia coli O157: H7 in hides and faeces of slaughtered ruminants in Nigeria. Methods and Results: A total number of 320 animals were sampled from January to December covering the wet and harmattan seasons. Samples were obtained from the hides and faeces of animals at slaughter. The ISO (ISO 16654:2001, Microbiology of food and animal feedingstuffs – horizontal method for the detection of Escherichia coli O157) method for enrichment and isolation of E. coli O157 incorporating selective enrichment using modified tryptone soya broth with novobiocin (mTSBn),immunomagnetic separation and plating on sorbitol‐MacConkey agar with cefixime tellurite (CT‐SMAC) was used. Overall cattle had a prevalence rate of 49·4% followed by sheep and goats with rates of 6·3% and 2·5%, respectively. There was a significant difference in carriage of E. coli O157 among two different cattle breeds. Conclusions: The prevalence of E. coli O157: H7 is substantial from two abattoirs in the country. The carriage and shedding of E. coli O157: H7 did not differ with season but differed among groups of ruminants and among breeds of cattle in a tropical country. Significance and Impact of the Study: This is the first study on E. coli O157: H7 from abattoir operations in Nigeria. The study emphasizes the risk of E. coli O157: H7 along the meat chain and the need for concerted effort to limit it through best hygiene practices.     

Direct PCR detection of Escherichia coli O157:H7

AIMS: This paper reports a simple, rapid approach for the detection of Shiga toxin (Stx)-producing Escherichia coli (STEC). METHODS AND RESULTS: Direct PCR (DPCR) obviates the need for the recovery of cells from the sample or DNA extraction prior to PCR. Primers specific for Stx-encoding genes stx1 and stx2 were used in DPCR for the detection of E. coli O157:H7 added to environmental water samples and milk. CONCLUSIONS: PCR reactions containing one cell yielded a DPCR product. SIGNIFICANCE AND IMPACT OF THE STUDY: This should provide an improved method to assess contamination of environmental and other samples by STEC and other pathogens.     

Sequence analysis of Escherichia coli O157:H7 bacteriophage ΦV10 and identification of a phage-encoded immunity protein that modifies the O157 antigen

Bacteriophage ΦV10 is a temperate phage, which specifically infects Escherichia coli O157:H7. The nucleotide sequence of the ΦV10 genome is 39 104 bp long and contains 55 predicted genes. ΦV10 is closely related to two previously sequenced phages, the Salmonella enterica serovar Anatum (Group E1) phage ɛ15 and a prophage from E. coli APEC O1. The attachment site of ΦV10, like those of its two closest relatives, overlaps the 3' end of guaA in the host chromosome. ΦV10 encodes an O -acetyltransferase, which modifies the O157 antigen. This modification is sufficient to block ΦV10 superinfection, indicating that the O157 antigen is most likely the ΦV10 receptor.     

可视化抗体阵列快速联合检测大肠杆菌O157:H7和鼠伤寒沙门氏菌

【目的】建立一种同时快速检测大肠杆菌O157:H7(E.coli O157:H7)和鼠伤寒沙门氏菌(Salmonella typhimurium)的可视化抗体阵列技术。【方法】将免疫学技术与蛋白芯片技术相结合,基于双抗体夹心法检测原理利用蛋白质芯片技术的高通量,结合可视化结果判定技术,用一份样品,同步检测大肠杆菌O157:H7和鼠伤寒沙门氏菌两种病原。【结果】检测结果肉眼可见,检测周期短至90 min,纯菌液检测灵敏度达105 CFU/mL,模拟带菌检测灵敏度为106 CFU/mL,与常规的ELISA灵敏度等同且具有良好的特异性和重复性。【结论】该可视化抗体阵列检测结果肉眼可见,检测通量高,无需大型设备,操作简便,检测成本低廉,同时为快速检测致病菌提供一种新途径。     

检测大肠杆菌(Escherichia coli) O157:H7免疫磁珠的制备、保存与应用

【背景】大肠杆菌(Escherichia coli) O157:H7是导致肠出血性大肠杆菌食源性疾病暴发的主要血清型,免疫磁珠(Immunomagnetic beads,IMBS)在E. coli O157的检测中发挥着重要作用,而免疫磁珠的稳定性、特异性、广谱性等性能指标关系着在实际应用中的使用效果。【目的】制备高效、稳定且具有广谱性的免疫磁珠,联合分子检测技术如环介导恒温扩增 (Loop-mediated isothermal amplification,LAMP)技术、PCR等,提高目标菌的检出率。【方法】采用新型的磁珠活化剂MIX&GO制备E. coli O157免疫磁珠,并进行广谱性以及特异性检测;针对6种试剂牛血清白蛋白(Bovine serum albumin,BSA)、酪蛋白(Casein)、海藻糖(Trehalose)、聚乙烯吡咯烷酮(Polyvinyl pyrrolidone,PVP)、抗坏血酸(Vitamin C)和防腐剂ProClin 300,利用正交试验L18(37)优化免疫磁珠保存液组分;采用IMBS-LAMP、IMBS-PCR、IMBS-生化、菌液-LAMP、菌液-PCR、显色平板-生化鉴定6种方式对20份生猪肉样品进行检测。【结果】利用MIX&GO活化剂制备的免疫磁珠捕获率最高达到81.5%±1.3%;免疫磁珠保存液最优配方为：牛血清白蛋白15.0 g/L,酪蛋白10.0 g/L,海藻糖10.0 g/L,PVP 2.0 g/L,抗坏血酸5.0 g/L,ProClin 300 2.5 g/L,保存6个月后免疫磁珠捕获率为75.5%;在20份生猪肉样品的检测中,自制磁珠和商品化磁珠与LAMP联用均检出9例阳性样品;IMBS-LAMP在6种检测方式中具有最高的检测灵敏度,但检出的样品会因磁珠抗体的差异而有所不同。【结论】与商品化磁珠相比,实验制备的免疫磁珠具有良好的特异性和广谱性,免疫磁珠-LAMP联用提高了目标菌的检出率,是一种高灵敏度、具有应用前景的检测方法。     

Survival of Escherichia coli O157:H7 and Salmonella typhimurium in cow manure and cow manure slurry

An exponential linear destruction was observed for Escherichia coli O157:H7 and Salmonella typhimurium in cattle manure and manure slurry stored at 4, 20 or 37 degrees C. The resulting decimal reduction times ranged from 6 days to 3 weeks in manure and from 2 days to 5 weeks in manure slurry. The main effects of time as well as temperature were pronounced with the most rapid destruction at 37 degrees C. The ammonia concentration in manure increased slightly during storage but did not exceed 0.1%. pH values in the deeper layers of manure remained constant except at 37 degrees C when the pH increased by 1 unit in 60 days. In the surface layers of manure, pH increased by 1.5-2 units, the oxidation-reduction potential of the manure declined rapidly to values below -200 mV. These changes do not seem to be reflected in changing rates of bacterial destruction. The observed order of destruction makes it possible to predict storage conditions (temperature and time) that will lead to a predetermined level of reduction of the two pathogens.     

The coexistence of Escherichia coli serotype O157:H7 and its specific bacteriophage in continuous culture

For the development of phage therapy, systematic understanding mechanisms of bacteriophage resistance will be required. We describe a new strain of Escherichia coli O157:H7, named Mu(L), which stably co-exists with the O157:H7-specific lytic bacteriophage PP01. Chemostat cultures of E. coli O157:H7 infected with PP01 showed unchanging cell concentration, but phage concentrations which increased by approximately 10(8) PFU mL(-1). However, the latent period, burst size, and growth rate of Mu(L) were the same as in a PP01-susceptible strain. The binding rate of PP01 to the cell surface was diminished 8.5-fold in Mu(L). By observation of the binding of fluorescently labeled O157:H7-specific phage to individual Mu(L) cells, we found that clonal Mu(L) cultures were heterogeneous in their ability to bind bacteriophage. 15% of the Mu(L) population was completely resistant to PP01 infection. Mu(L) also co-existed with bacteriophages unrelated to PP01. Broad-range phage resistance by clonal heterogeneity represents a new class of bacteria-phage interactions.