Campylobacter and Salmonella contamination of unpasteurized cows' milk on sale to the public

Campylobacters and salmonellas were isolated respectively from 6 and 0.2% of samples of unpasteurized cow's milk on sale to the public. There was a significant association between the presence of Escherichia coli and that of Campylobacter jejuni. The mean E. coli count was also higher in campylobacter-positive samples. Enumeration of E. coli would seem to have value as an indicator of faecal contamination and thus potential hazard in raw milk.     

Fate of Escherichia coli O157:H7 in unpasteurized milk stored at 8 degrees C

Escherichia coli O157:H7 and verocytotoxins were not found in any of 100 unpasteurized milk samples obtained from the bulk tanks of eight dairy farms located in the Puglia and Basilicata areas. Seven E. coli O157:H7 (EHEC) strains were inoculated separately into raw milk samples and then examined periodically to determine the fate of EHEC as influenced by the storage temperature (8 degrees C) and time. There was essentially no change in the viable population of three EHEC strains for up to 14 d. The remaining four strains showed an increase in population from < 2 log to 3 log cfu ml-1 in a time period of between 9 and 17 d. The results indicate good survival or even multiplication of E. coli O157:H7 in raw milk when stored at 8 degrees C and reaffirm the need for pasteurization and holding the milk at < or = 5 degrees C.     

ASSESSMENT OF IMPEDANCE MICROBIOLOGICAL METHOD FOR THE DETECTION OF ESCHERICHIA COLI IN FOODS

This study was conducted to determine the sensitivity and specificity of the impedance-based microbiological method for the detection of Escherichia coli in foods within 24 h of testing. A Malthus Microbiological Analyzer system (Malthus System V, Malthus Instruments Ltd., Bury, United Kingdom), and a modified Malthus Coliform Broth Medium (MCBM), and an incubation temperature of 44C were used. The sensitivity of the impedance method was determined by testing E. coli-negative food samples spiked with different concentrations of E. coli. The specificity of the method was determined by testing E. coli -negative food samples spiked with Klebsiella pneumoniae, Enterobacter cloacae and Pseudomonas aeruginosa. The test results were compared with those obtained by the Most Probable Number (MPN) method. Milk, milk products, raw and ready-to-eat meats, and vegetables were tested for the presence of E. coli by both methods. The sensitivity of the impedance method and the MPN method for the detection of foods containing 10¹ CFU/g was 100% and 84.4%, respectively. Both methods had a specificity of 100% for food samples spiked with 10¹ CFU/g E. coli. The specificity of the impedance and the MPN methods for the detection of E. coli in naturally contaminated milk and meat samples was 100% and 95.7% respectively. E. coli was detected in foods by the impedance method within 4–24 h of testing at a detection limit of 1 CFU/mL. These results demonstrate that the impedance method can be used as a rapid and sensitive method for the detection of E. coli in foods.     

Incidence of toxigenic Escherichia coli in soft cheese made with raw or pasteurized milk

Soft cheeses made with raw (221 samples) or pasteurized (75) cow's milk were collected. Enterotoxigenic, verotoxigenic and necrotocigenic Escherichia coli strains were studied. Three raw milk cheeses were positive for toxigenic E. coli (1·4%) : the first with toxin CNF2 and serogroup O5 (40% of colonies studied with typical E. coli morphology) ; the second with VT and O2 (10%) ; and the third with LT and O51 (10%). Toxigenic E. coli of bovine origin can pass to the milk destined to make cheese, and survive. Soft cheese should be considered as a possible vehicle of infection in future investigations.     

Antibacterial activity of pepsin-digested lactoferrin on foodborne pathogens in buffered broth systems and ultra-high temperature milk with EDTA

AIMS: To evaluate the antimicrobial activity in peptone yeast extract glucose (PYG) broth and ultra-high temperature (UHT) milk of bovine lactoferrin hydrolysate (LFH) with pepsin against the foodborne pathogens Salmonella Stanley, Escherichia coli, Listeria monocytogenes and Staphylococcus aureus. METHODS AND RESULTS: The LFH was suspended in PYG and the minimum inhibitory concentration for each pathogen determined. The LFH was also suspended in UHT milk adjusted to pH 4 or 7, samples incubated at 4 or 35 degrees C and the change in bacterial cell population determined. Experiments in UHT milk were conducted using L. monocytogenes and E. coli O157:H7. At pH 4 LFH reduced the population of E. coli O157:H7 and L. monocytogenes by approx. 2 log; however, only E. coli O157:H7 was inhibited in samples adjusted to pH 7. The addition of EDTA (10 mg ml(-1)) to UHT milk supplemented with LFH did not markedly influence the growth of E. coli O157:H7 or L. monocytogenes. CONCLUSIONS: The results suggest that, under low pH and refrigeration conditions, LFH can limit the growth or reduce the population of pathogenic bacteria in a dairy product. SIGNIFICANCE AND IMPACT OF THE STUDY: Natural preservatives that are active against Gram-negative and Gram-positive bacteria are desirable to the food industry. This study demonstrates that LFH is effective in a complex food system. Moreover, the LFH used was not purified, making its use by industry more attractive.     

Rapid procedure for detecting enterohemorrhagic Escherichia coli O157:H7 in food.

A sensitive, specific procedure was developed for detecting Escherichia coli O157:H7 in food in less than 20 h. The procedure involves enrichment of 25 g of food in 225 ml of a selective enrichment medium for 16 to 18 h at 37 degrees C with agitation (150 rpm). The enrichment culture is applied to a sandwich enzyme-linked immunosorbent assay (ELISA) with a polyclonal antibody specific for E. coli O157 antigen as the capture antibody and a monoclonal antibody specific for enterohemorrhagic E. coli of serotypes O157:H7 and O26:H11 as the detection antibody. The ELISA can be completed within 3 h. The sensitivity of the procedure, determined by using E. coli O157:H7-inoculated ground beef and dairy products, including different varieties of cheese, was 0.2 to 0.9 cell per g of food. A survey of retail fresh ground beef and farm raw milk samples with this procedure revealed that 3 (2.8%) of 107 ground beef samples and 11 (10%) of 115 raw milk samples were positive for E. coli O157:H7. Most-probable-number determinations revealed E. coli O157:H7 populations of 0.4 to 1.5 cells per g in the three ground beef samples. In addition to being highly specific, sensitive, and rapid, this procedure is easy to perform and is amenable to use by laboratories performing routine microbiological testing.     

Rapid procedure for detecting enterohemorrhagic Escherichia coli O157:H7 in food.

A sensitive, specific procedure was developed for detecting Escherichia coli O157:H7 in food in less than 20 h. The procedure involves enrichment of 25 g of food in 225 ml of a selective enrichment medium for 16 to 18 h at 37 degrees C with agitation (150 rpm). The enrichment culture is applied to a sandwich enzyme-linked immunosorbent assay (ELISA) with a polyclonal antibody specific for E. coli O157 antigen as the capture antibody and a monoclonal antibody specific for enterohemorrhagic E. coli of serotypes O157:H7 and O26:H11 as the detection antibody. The ELISA can be completed within 3 h. The sensitivity of the procedure, determined by using E. coli O157:H7-inoculated ground beef and dairy products, including different varieties of cheese, was 0.2 to 0.9 cell per g of food. A survey of retail fresh ground beef and farm raw milk samples with this procedure revealed that 3 (2.8%) of 107 ground beef samples and 11 (10%) of 115 raw milk samples were positive for E. coli O157:H7. Most-probable-number determinations revealed E. coli O157:H7 populations of 0.4 to 1.5 cells per g in the three ground beef samples. In addition to being highly specific, sensitive, and rapid, this procedure is easy to perform and is amenable to use by laboratories performing routine microbiological testing.     

原核表达MurA蛋白并制备其多克隆抗体用于免疫磁珠快速检测单增李斯特菌

【目的】制备MurA多抗,结合免疫磁珠与选择平板进行单增李斯特菌的快速检测,建立单增李斯特菌的免疫磁珠快速检测方法。【方法】构建MurA的原核表达载体,转化大肠杆菌进行优化表达。镍柱纯化表达产物,质谱鉴定重组蛋白,再免疫小鼠,制备其多克隆抗体。用所获多抗制备免疫磁珠,建立单增李斯特菌免疫磁珠-选择性培养基检测方法,并对人工污染牛奶样品进行检测。【结果】在大肠杆菌中高效表达了分子量约为72 kD的可溶性融合蛋白,质谱鉴定其为MurA蛋白;免疫小鼠获得的抗血清效价达1:10 000,与伤寒沙门氏菌、副溶血弧菌、大肠杆菌及属内其它病原菌均无交叉;所建立的免疫磁珠-选择性培养基检测法可检出浓度为103 CFU/mL及以上的单增李斯特菌,仅与英诺克李斯特菌存在一定交叉反应;牛奶样品单次仅需9 h增菌就能被检出,较常规增菌时间缩短39 h;检测限为0.4 CFU/mL。【结论】表达并纯化得到高纯度的单增李斯特菌MurA蛋白,制备的鼠源多克隆抗体亲和力高,特异性好;建立了快速检测单增李斯特菌的免疫磁珠联合选择性培养基法,在灵敏度不变的情况下,实现24 h内成功对牛奶样品的检测,较国标法减少42 h以上。     

Sources of Escherichia coli O157 and experiences over the past 15 years in Sheffield, UK

In the first documented outbreak of HC caused by Escherichia coli O157, which occurred in the North-west USA in 1982, there was a strong association between infection and prior consumption of ground beef from a chain of fast food restaurants. Foods of bovine origin, including beef, milk and dairy products, have since been implicated in many outbreaks of infection world-wide. Investigations during the course of outbreaks, or at random, have shown that cattle are a major reservoir of E. coli O157. E. coli O157 was isolated from cattle at slaughter in Sheffield in 1987, this being the first isolation from cattle in the UK. Following a cluster of cases in May/June 1992, an abattoir study showed the organism to be present in 4% of cattle at slaughter and on up to a third of carcasses from rectal swab-positive animals. E. coli O157 was isolated from a food source (unpasteurized milk), for the first time in the UK, in Sheffield in May 1993. During surveillance in 1995-6, E. coli O157 was isolated from 15.7% of cattle, with a monthly prevalence which varied from 5 to 37%. E. coli O157 was also isolated from 2.2% of sheep. During surveillance in 1996, E. coli O157 was isolated from 5.9% of samples of lamb products and from 1.5% of samples of beef products, despite the prevalence in cattle being much higher than in sheep. Work is in progress to try to explain this higher prevalence in lamb products. During 1997 in Sheffield, the only cases of E. coli O157 for which a confirmed source was established were associated with direct animal contact on farm visits. During on-farm investigations of these cases, E. coli O157 was isolated from faecal samples from adult cattle, calves, three different breeds of sheep, two different breeds of pigs, goats and a pony.     

Survey of retail cheeses, dairy processing environments and raw milk for Escherichia coli O157 : H7

Escherichia coli was isolated from 58% (11/19) of retail soft and semi-soft cheeses tested, but E. coli O157 : H7 was not detected. The presence of E. coli in retail cheeses and the lack of baseline data on the prevalence of serotype O157 : H7 strains prompted us to survey ingredients and the environment in 15 cheese and dairy plants. Escherichia coli O157 : H7 was not detected in any of the 1104 samples tested, including 42 raw milk samples. These results suggest that serotype O157 : H7 is not prevalent within dairy product ingredients and processing environments.     

Bacteriological studies on Iraqi milk products.

Samples from different types of domestic milk products including cheese, kishfa and gaymer were assessed for bacteriological quality over a 4-month period. A total of 400 samples were randomly selected across Mosul city and tested for faecal coliform counts, enteropathogenic Escherichia coli (EPEC) and enterotoxigenic E. coli (ETEC). Faecal coliforms were present at levels greater than 10(2) cfu/g in 72.5% and less than 10(2) cfu/g in 27.5% of samples. Of the 430 E. coli strains isolated from the 400 samples of milk products, 138 were serotypes of EPEC. These were found in 81 (40.5%) samples of cheese, 35 (23.8%) of kishfa and 22 (29.7%) of gaymer. During this period, 26 strains of ETEC were also isolated, all of which demonstrated heat-labile or heat-stable toxins. The high proportion of strains of three groups of E. coli showing resistance to antibiotics is discussed in relation to widespread use of antibiotics and the possible public health implication.     

Comparison of the sensitivity of manual and automated immunomagnetic separation methods for detection of Shiga toxin-producing Escherichia coli O157:H7 in milk

AIM: To determine the sensitivity of methods for detection of injured and uninjured Escherichia coli O157:H7 (E. coli O157) in raw and pasteurized milk. METHODS AND RESULTS: Raw milk, pasteurized milk with 1.5% fat content and pasteurized milk with 3.5% fat content were spiked with E. coli O157 at low levels. The samples were enriched in modified tryptone soya broth with novobiocin (mTSBn) at 37 degrees C. Aliquots of the enriched culture were analysed either by manual immunomagnetic separation (MIMS) and culturing on sorbitol MacConkey agar with or without cefixime and potassium tellurite (SMACct or SMAC), or by automated immunomagnetic separation and integrated ELISA (EiaFosstrade mark). Uninjured E. coli O157 organisms were detected in milk by both methods at 1 cfu 10 ml-1 sample). Injured organisms were detected at levels of about 4 cfu 10 ml-1 sample. Direct enrichment in mTSBn (22 h incubation) showed better sensitivity for injured cells than enrichment in buffered peptone water (BPW, 22 h incubation), or in a two-step enrichment consisting of BPW (6 h, 37 degrees C) and mTSBn (16 h, 37 degrees C), successively. CONCLUSIONS: The methods showed equal sensitivity in that they were both able to detect 1 cfu 10 ml-1 milk sample. Injured organisms can be detected and isolated at a level almost as low as this. A resuscitation step is not recommended for the detection and isolation of injured and non-injured E. coli O157 from milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the dilution of contamination in the bulk tank, analysis of milk for the presence of E. coli O157 requires a very sensitive method. Both methods described here are useful for such analysis.     

The incidence of Shiga toxin-producing Escherichia coli in cattle with mastitis in Brazil

AIMS: To determine the prevalence and molecular characteristics of Shiga toxin-producing Escherichia coli (STEC) isolates from bovine mastitic milk in Brazil. METHODS AND RESULTS: A total of 2144 milk samples from dairy cattle showing mastitis were screened for the presence of E. coli. A total of 182 E. coli isolates were selected and examined. All were subjected to dot blot analysis using the CVD419 probe for the detection of the enterohaemolysin (hly) gene, and to a multiplex PCR for the detection of stx1, stx2 and eaeA genes. STEC were isolated from 22 (12.08%) milk samples. All the STEC isolates were tested for sensibility to 10 antimicrobials; the resistances most commonly observed were to cephalothin (86.3%), tetracycline (63.6%) and doxycycline (63.6%). CONCLUSION: STEC isolates were found in bovine mastitic milk in Brazil. SIGNIFICANCE AND IMPACT OF THE STUDY: STEC isolates from mastitic milk were potentially pathogenic for human in that they belonged to serogroups associated with diarrhoea and haemolytic-uraemic syndrome, some of them were stx2, eaeA and hly positive.     

COLI-AEROGENES BACTERIA OF PASTEURIZED MILKS, WITH SPECIAL REFERENCE TO PATHOGENIC SEROTYPES OF ESCHERICHIA COLI

SUMMARY: A total of 375 samples of retail pasteurized milk in bottles was examined from January to the end of August 1955, for coli-aerogenes bacteria and especially for pathogenic serotypes of Escherichia coli. These serotypes were not found, though 101 of the samples contained bacteria of the coli-aerogenes group. During the summer months, June-August, the percentage of contaminated samples was about 40% and 10% of the samples contained E. coli I or II. The epidemiological significance of these findings is discussed.     

Immunomagnetic-electrochemiluminescent detection of Escherichia coli O157 and Salmonella typhimurium in foods and environmental water samples.

Hemorrhagic Escherichia coli O157:H7 strains and other virulent enteric pathogens can pose a serious health threat in tainted meats, poultry, and even drinking water. Traditional culture-based methods for assay of enteric pathogens in foods and water sources are relatively slow, and results can be ambiguous. Immunomagnetic separation (IMS) and detection methods have been investigated and appear promising for rapid bacterial assay of foods and environmental samples. In this work, a commercial sensor which combines IMS with electrochemiluminescence (ECL) detection is evaluated for detection of E. coli O157 and Salmonella typhimurium in foods and fomites. Results indicate that detection limits are in the range of 100 to 1,000 bacteria per ml in pristine buffer for E. coli O157 and S. typhimurium, respectively, or 1,000 to 2,000 bacteria per ml in food samples (depending on the sample) and that total processing and assay time is rapid (< 1 h) even in food samples. An immunologic "hook" or high-antigen-concentration prozone effect was observed above 10(4) and 10(5) bacteria per ml for E. coli O157 and S. typhimurium, respectively. IMS was accomplished in milk, juices, serum, supernatant fluids from ground beef, finely minced chicken, and fish suspensions as well as several freshwater sources and followed by ECL assay. Some samples, especially fish, gave unexpectedly high background ECL. Conversely, low ECL intensity was observed in nonfat and 2% fat milk samples, which appeared to be related to binding or entrapment of the antibody-coated magnetic beads by particulates in the milk, as revealed by microscopy. Results of this evaluation suggest the feasibility of immunomagnetic-ECL methodology for rapid, sensitive, and facile preliminary screening of various foods and fomites for the presence of virulent enteric pathogens.     

Bacteriological studies on Iraqi milk products

F. ABBAR AND H. KH. KADDAR. 1991. Samples from different types of domestic milk products including cheese, kishfa and gaymer were assessed for bacteriological quality over a 4-month period. A total of 400 samples were randomly selected across Mosul city and tested for faecal coliform counts, enteropathogenic Escherichia coli (EPEC) and enterotoxigenic E. coli (ETEC). Faecal coliforms were present at levels greater than 10² cfu/g in 72.5% and less than 10² cfu/g in 27.5% of samples. Of the 430 E. coli strains isolated from the 400 samples of milk products, 138 were serotypes of EPEC. These were found in 81 (40.5%) samples of cheese, 35 (23.8%) of kishfa and 22 (29.7%) of gaymer. During this period, 26 strains of ETEC were also isolated, all of which demonstrated heat-labile or heat-stable toxins. The high proportion of strains of three groups of E. coli showing resistance to antibiotics is discussed in relation to widespread use of antibiotics and the possible public health implication.     

Detection of Escherichia coli O157 in French food samples using an immunomagnetic separation method and the VIDASTME. coli O157

C. VERNOZY-ROZAND, C. MAZUY, S. RAY-GUENIOT, S. BOUTRAND-LOEï, A. MEYRAND AND y. richard. 1997. Two commercially available screening methods, an automated enzyme-linked fluorescent immunoassay (VIDAS^TM E. coli O157) and an immunomagnetic separation followed by culture onto cefixime tellurite sorbitol MacConkey agar (CT-SMAC), were compared for detection of Escherichia coli O157 in naturally and artificially contaminated food samples. A total of 250 naturally contaminated food samples, including raw milk cheeses, poultry, raw sausages and ground beef retail samples, were examined. Four poultry, one raw sausage and one ground beef sample were found to be positive for E. coli O157 by both methods. Of the six positive samples, five were shown to contain sorbitol-positive, O157-positive, H7-negative, motile and non-verotoxin-producing E. coli .     

E. coli proteolytic activity in milk and casein breakdown

Previous studies have focused on both LPS and E. coli experimental mastitis and underlined the respective roles of endogenous proteolysis (including plasmin from the blood stream and other proteases from milk leukocytes), as well as the presence of E. coli in a more intricate system. The aim of this study was to assess the role of E. coli in milk proteolysis and especially that of its proteases in casein breakdown. The first part consisted in the incubation of 104 cfu.mL(-1) of the E. coli strain in raw milk at 37 degrees C for 24 h; the same milk was also incubated with 0.04% sodium azide. Several parameters were evaluated: CFU, plasmin activity, gelatinase activity and pH 4.6 insoluble peptides, including the proportion of gamma-CN. The profile of gelatinase activity was determined by zymography and identified by immunoblotting. In the second part of the study, we examined the profile of CN (alphas-, beta- and kappa-CN) breakdown by E. coli lysate. The results suggest that E. coli proteases have a direct effect on CN, and the increase of gamma-CN in inoculated milk may be generated by both plasmin and the gelatinase. Moreover, the gelatinase activity in the inoculated milk was higher after 24 h of incubation.     

PCR-ELISA detection of Escherichia coli in milk

AIMS: The purpose of this study was to develop a reliable molecular procedure for the detection of Escherichia coli in milk. METHODS AND RESULTS: Robust and expeditious DNA extraction and PCR techniques were evaluated using Enzyme-Linked Immunosorbent Assay (ELISA) detection of biotin-labelled amplicons to facilitate optimal detection of E. coli DNA. CONCLUSIONS: It was found that 5 E. coli colony-forming units (cfu) could be detected per PCR reaction using the PCR-ELISA system, equating to a sensitivity of detection of 100 E. coli cfu ml(-1) pasteurized milk. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach should facilitate evaluation of milk contamination and enable rapid detection of E. coli mastitis, leading to correct deployment of relevant antibiotic therapy and improved animal welfare.     

Escherichia coli infections in newborn puppies--clinical and epidemiological investigations

Epidemiological analyses were performed in five breeding kennels with Escherichia coli infections in newborn pups using pulsed field gel electrophoresis (PFGE). Previous reports demonstrated the high discriminatory power of this method and its usefulness for detecting epidemiologically related isolates. A total of 113 E. coli strains were isolated from vagina, faeces, oral cavity, milk and organs from 19 adult dogs, and 57 diseased or dead pups from 12 litters. Restriction enzyme analyses were performed using XbaI and BlnI digests and the resulting 91 DNA patterns were aligned for comparison. The results showed that a total of 60% of E. coli strains from progeny were also found in vaginal samples of the mothers. Another bacterial source was the faeces found within the kennels. One instance of milk and oral cavity isolates of the mother was found to be identical with strains isolated from the pups. The results indicate that for repeated cases of E. coli infections in neonates, diagnostic procedures of vaginal and faecal swabs from dams result in isolation of the responsible bacteria with high probability and further suggest that preterm treatment could help to control bacterial diseases and losses in pups. In addition, the observation that two canine strains were found to be identical with an E. coli strain isolated from a human case of diarrhoea strongly supports the canine reservoir hypothesis.