Can the high levels of human verocytotoxigenic Escherichia coli O157 infection in rural areas of NE Scotland be explained by consumption of contaminated meat?

AIMS: To determine if contamination levels of Escherichia coli O157 and generic E. coli in retail-minced meat products are greater in rural shops compared with urban shops in Grampian, NE Scotland. We also investigated whether meat from supermarkets and meat from local butcher shops had a similar bacteriological quality. METHODS AND RESULTS: Minced beef and minced lamb were tested from November 2004 to August 2006. Escheichia coli O157 was found at low levels in four samples out of 530 tested samples (0.75%). Generic E. coli were present in 11% of the samples tested, of which 67% came from supermarkets. We observed no significant difference in the prevalence of generic E. coli between rural and urban areas. CONCLUSIONS: Low levels of contamination with E. coli O157 and generic E. coli in retail meat suggest that meat is not a major route of infection in NE Scotland. SIGNIFICANCE AND IMPACT OF THE STUDY: The study does not suggest that the high incidence of E. coli O157 human infection in the rural areas of Grampian is because of meat consumption--this provides further evidence of contact with animals or water being the routes of infection. Hence, risk mitigation should be focussed more on environmental pathways of infection.     

Prevalence and potential link between E. coli O157:H7 isolated from drinking water, meat and vegetables and stools of diarrhoeic confirmed and non-confirmed HIV/AIDS patients in the Amathole District - South Africa

Aim:  The current study investigated the prevalence and molecular relatedness between Escherichia coli O157:H7 isolated from water, meat and meat products and vegetables and from stools of confirmed and non-confirmed Human Immune Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS) patients with diarrhoea.
Methods and Results:  Culture-based and polymerase chain reaction techniques were used to identify E . coli O157:H7. Thirty-five per cent of meat products, 25·5% of water, 21·7% of vegetables as well as 56·5% and 43·5% of stools of confirmed and non-confirmed HIV/AIDS patients, respectively, were presumptively positive with E . coli O157. Molecular results indicated that 10·3%, 8·6% and 7·8% of the vegetables, water and meat products examined carried E . coli O157:H7, which had homologous fliC _H7 , rfbE _O157 and eaeA genetic loci to the genes of some E . coli O157:H7 isolated from 12·2% and 8·8% of the stools of confirmed and non-confirmed HIV/AIDS patients, respectively.
Conclusions:  Water, meat and meat products and vegetables are potential sources of E . coli O157:H7 that are potentially capable of causing diarrhoea in humans especially HIV/AIDS patients.
Significance and Impact of the Study:  Great care should be exercised to ensure that water and foods consumed by HIV/AIDS patients are safe, as contaminated water and foods can cause secondary infections in these patients.     

Oxidative stress involved in the antibacterial action of different antibiotics

Staphylococcus aureus and Escherichia coli sensitive to chloramphenicol incubated with this antibiotic suffered oxidative stress with increase of anion superoxide (O2-). This reactive species of oxygen was detected by chemiluminescence with lucigenin. S. aureus, E. coli, and Enterococcus faecalis sensitive to ciprofloxacin exhibited oxidative stress when they were incubated with this antibiotic while resistant strains did not show stimuli of O2-. Other bacteria investigated was Pseudomonas aeruginosa, strains sensitive to ceftazidime and piperacillin presented oxidative stress in presence of these antibiotics while resistant strains were not stressed. Higher antibiotic concentration was necessary to augment O2- in P. aeruginosa biofilm than in suspension, moreover old biofilms were resistant to oxidative stress caused by antibiotics. A ceftazidime-sensitive mutant of P. aeruginosa, coming from a resistant strain, exhibited higher production of O2- than wild type in presence of this antibiotic. There was relation between antibiotic susceptibility and production of oxidative stress.     

Isolation of shiga toxin-producing Escherichia coli (STEC) from foods using EHEC agar

Enterohaemorrhagic Escherichia coli (EHEC) agar was evaluated for its ability to recover one isolate of each of three serotypes (O157:H7, O26 and O113:H21) of shiga toxin-producing E. coli (STEC) from raw mince, pasteurized milk and salami after enrichment. The method detected around one colony-forming unit (cfu) in 25 ml in milk, but was less sensitive with salami, requiring 10-1000 cfu 25 g-1 (depending on serotype) for detection. In raw minced beef any enterohaemolysin-producing colonies were outnumbered by other colonies and only one of 12 enrichments yielded the inoculum serotype. Additional tests were conducted on 15 retail meat products. One 25-g sample of each product was processed as purchased, while another was inoculated with 157-185 cfu of a cocktail of E. coli O157, O113 and O26 cultures. Recovery was easily achieved with cooked meat products and salami. Recovery from raw minced meat was again difficult, but sometimes possible. Testing more suspect colonies than were tested in this study would presumably increase the sensitivity of the method.     

Molecular characterization of Escherichia coli O157:H7 recovered from meat and meat products relevant to human health in Riyadh,Saudi Arabia

Raw meat can harbor pathogenic bacteria, potentially harmful to humans such as Escherichia coli O157:H7 causing diarrhea and hemolytic-uremic syndrome (HS). Therefore, the current study was carried out to evaluate the prevalence and the molecular detection characterization of E. coli serotype O157:H7 recovered from raw meat and meat products collected from Saudi Arabia. During the period of 25th January 2013 to 25th March 2014, 370 meat samples were collected from abattoirs and markets located in Riyadh, Saudi Arabia “200 raw meat samples and 170 meat products”. Bacteriological analysis of the meat samples and serotyping of the isolated E. coli revealed the isolation of 11 (2.97%) strains of E. coli O157:H7. Isolation of E. coli O157:H7 in raw beef, chicken and mutton were 2%, 2.5%, and 2.5%, respectively, however, there was no occurrence in raw turkey. The incidences of E. coli O157:H7 in ground beef, beef burgers, beef sausage, ground chicken and chicken burgers were 5%, 10%, 0.0%, 5% and 0.0%, respectively. The multiplex PCR assay revealed that 3 (27.27%) out of 11 E. coli O157:H7 isolates from raw beef, chicken and mutton had stx1, stx2, and eae while 5 (45.45%) E. coli O157:H7 isolates from ground beef, ground chicken, and raw beef had both stx1 and stx2. However, from beef burgers, only one E. coli O157:H7 isolate had stx1 while two were positive for hlyA gene. These results call for urgent attention toward appropriate controls and good hygienic practices in dealing with raw meat.     

Organization of the gene cluster for biosynthesis of penicillin in Penicillium nalgiovense and antibiotic production in cured dry sausages

Several fungal isolates obtained from two cured meat products from Spain were identified as Penicillium nalgiovense by their morphological features and by DNA fingerprinting. All P. nalgiovense isolates showed antibiotic activity in agar diffusion assays, and their penicillin production in liquid complex medium ranged from 6 to 38 microgram. ml-1. We constructed a restriction map of the penicillin gene cluster of P. nalgiovense and found that the organization of the penicillin biosynthetic genes (pcbAB, pcbC, and penDE) is the same as in Penicillium chrysogenum and Aspergillus nidulans. The pcbAB gene is located in an orientation opposite that of the pcbC and penDE genes in all three species. Significant amounts of penicillin were found in situ in the casing and the outer layer of salami meat during early stages of the curing process, coinciding with fungal colonization, but no penicillin was detected in the cured salami. The antibiotic produced in situ was sensitive to penicillinase.     

RAPID detection and quantification of E. coli O157/O26/O111 in minced beef by real-time PCR

AIM: To develop a real-time PCR detection procedure for Escherichia coli O111, O26 and O157 from minced meat. METHODS AND RESULTS: Strains (n = 8) of each of E. coli O26, E. coli O111 and E. coli O157 were inoculated at ca 10-20 CFU g(-1) into minced retail meat and enriched for 6 h at 41.5 degrees C as follows: E. coli O26 in tryptone soya broth (TSB) supplemented with cefixime (50 microg l(-1)), vancomycin (40 mg l(-1)) and potassium tellurite (2.5 mg l(-1)); E. coli O111 in TSB supplemented with cefixime (50 microg l(-1)) and vancomycin (40 mg l(-1)); E. coli O157 in E. coli broth supplemented with novobiocin (20 mg l(-1)). DNA was extracted from the enriched cultures, and detected and quantified by real-time PCR using verotoxin (vt1 and vt2) and serogroup (O157 per gene; O26 fliC-fliA genes and O111 wzy gene) specific primers. CONCLUSIONS: The methods outlined were found to be sensitive and specific for the routine detection of E. coli O111, O26 and O157 in minced beef. SIGNIFICANCE AND IMPACT OF THE STUDY: The enrichment, isolation and detection procedures used in this study provide a rapid routine-based molecular method for the detection and differentiation of E. coli O26, O111 and O157 from minced meat.     

Pandemic Serotypes of Vibrio cholerae Isolated from Ships’ Ballast Tanks and Coastal Waters: Assessment of Antibiotic Resistance and Virulence Genes (tcpA and ctxA)

There is concern that ships’ ballasting operations may disseminate Vibrio cholerae to ports throughout the world. Given evidence that the bacterium is indeed transported by ships, we isolated pandemic serotypes O1 and O139 from ballast tanks and characterized them with respect to antibiotic resistance and virulence genes ctxA and tcpA. We carried out concurrent studies with V. cholerae isolated from coastal waters. Of 284 isolates, 30 were serotype O1 and 59 were serotype O139. These serotypes were overrepresented in ballast tanks relative to the coastal waters sampled. All locations, whether coastal waters or ballast tanks, yielded samples from which serotype O1, O139, or both were isolated. There were three groups among the 62 isolates for which antibiotic characterization was conclusive: those exhibiting β-lactamase activity and resistance to at least one of the 12 antibiotics tested; those negative for β-lactamase but having antibiotic resistance; those negative for β-lactamase and registering no antibiotic resistance. When present, antibiotic resistance in nearly all cases was to ampicillin; resistance to multiple antibiotics was uncommon. PCR assays revealed that none of the isolates contained the ctxA gene and only two isolates, one O139 and one O1, contained the tcpA gene; both isolates originated from ballast water. These results support the bacteriological regulations proposed by the International Maritime Association for discharged ballast water.     

High prevalence of Yersinia enterocolitica 4:O3 on pig offal in southern Germany: a slaughtering technique problem

Prevalence and contamination routes of pathogenic Yersinia enterocolitica were studied in Southern Germany. Tonsil and faeces samples of 50 fattening pigs, 140 offal samples and 120 minced meat samples were examined. Pig and offal samples were collected from a slaughterhouse approved by the European Union, and minced meat samples from two large meat factories. Yersinia enterocolitica was isolated using direct plating, overnight enrichment and selective enrichment in MRB and ITC broth. The isolates were bio- and serotyped, and pathogenicity was studied using two plasmid-encoded virulence markers: calcium dependence and Congo red absorption. The genotypes were studied with pulsed-field gel electrophoresis using NotI enzyme. Prevalence of pathogenic Y. enterocolitica 4:O3 was 60% and 10% in tonsils and faeces of fattening pigs, respectively. Besides tonsils, prevalence of pathogenic Y. enterocolitica 4:O3 was also high in other pluck set samples, including tongues, lungs, hearts, diaphragms and livers. However, the highest isolation rate was obtained from the tonsils. Kidneys, which were not attached to the pluck set and did not hang together with tonsils on the rack, had the lowest isolation rate. Yersinia enterocolitica 4:O3 was isolated from 12% of minced meat samples. A total of 25 NotI profiles were obtained from porcine samples. The most common genotype, NBI, found in tonsils was also the most common type recovered from offal and minced meat samples. The high contamination rate of tonsils, and the indistinguishable NotI profiles obtained from tonsils and offal indicate that the tonsils contaminate offal when they are removed and hung on the rack together. When the head, with the tonsils and tongue, is not removed prior to evisceration and is not handled and inspected separately, it is difficult to control the spread of Y. enterocolitica 4:O3 from tonsils to the carcass, and subsequently, to meat.     

Antibiotic resistance in strains of Listeria spp. from meat products

The susceptibility or resistance to 26 antimicrobial agents was determined for 64 strains of Listeria monocytogenes and 102 strains of L. innocua isolated from Italian meat products. Some strains of L. monocytogenes were found to be resistant to tetracycline, erythromycin, co-trimoxazole and clindamycin. No plasmids were found in any L. monocytogenes strain. Five strains of L. innocua contained a 7.9 kbp plasmid, but these isolates were not resistant to any antibiotic in common and treatment with curing agents could not eliminate resistance to antibiotics. These results suggest that antibiotic resistance was not likely to be plasmid mediated in our strains.     

Comparison of Genotypes and Antibiotic Resistances of Campylobacter jejuni and Campylobacter coli on Chicken Retail Meat and at Slaughter

Multilocus sequence typing (MLST) and antibiotic resistance patterns of Campylobacter jejuni and Campylobacter coli from retail chicken meat showed high overlap with isolates collected at slaughterhouses, indicating little selection along the production chain. They also showed significant common sequence types with human clinical isolates, revealing chicken meat as a likely source for human infection.     

Anisakis simplex (s.s.) larvae in wild Alaska salmon: no indication of post-mortem migration from viscera into flesh

The prevalence, mean intensity and distribution of Anisakis nematode third-stage larvae (L3) in the muscle and viscera of wild-caught chum salmon Oncorhynchus keta, pink salmon O. gorbuscha and sockeye salmon O. nerka were compared immediately after catch. Salmon were collected during the fishing season in July 2007 in Bristol Bay and Prince William Sound close to Cordova, Alaska (USA). All fish were infected, and more than 90% of the nematode larvae were found in the edible muscle meat. The isolated anisakid L3 were genetically identified as A. simplex (s.s.). The distribution of nematodes in the muscle meat of fresh-caught salmon was examined in 49 O. keta, 50 O. nerka and 12 O. gorbuscha from Cordova. Most of the larvae were detected in the muscle parts around the body cavity, but nematodes were also found in the tail meat and epaxial muscle (loins). The mean intensity of Anisakis larvae in the edible part was 21 individuals for O. gorbuscha, 62 individuals for O. keta and 63 individuals for O. nerka. No difference in the intensity of Anisakis larvae in the hypaxial muscle was found between fresh-caught and immediately gutted salmon and individuals stored ungutted for 24 h either on ice or in refrigerated sea water.     

Exposure to non-acid fresh meat decontamination washing fluids sensitizes Escherichia coli O157:H7 to organic acids

AIMS: To investigate whether Escherichia coli O157:H7 maintains acid tolerance in water meat decontamination washing fluids. METHODS AND RESULTS: A rifampicin-resistant derivative of E. coli O157:H7 strain ATCC 43895 was inoculated (10(5) cfu ml(-1)) in spray-washings from meat sprayed with cold (10 degrees C) or hot (85 degrees C) water, stored at 10 degrees C for up to 14 days, and its acid tolerance was assessed at 2 and 8 days by exposure to broth or new washings adjusted to pH 3.5 or 3.7 with lactic or acetic acid. The pathogen survived in the water washings, but it was outgrown by the natural, Pseudomonas-like flora, and it was sensitized to acid. CONCLUSIONS: The acid tolerance of E. coli O157:H7 decreases following exposure to non-acid, but otherwise stressful, conditions prevailing in water meat washings at 10 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that the more intense use of water-based technologies should be included in meat decontamination strategies because they may contribute to enhanced meat safety by inducing acid sensitization in E. coli O157:H7.     

Determination of the biological activity of antibiotics by using dry nutrient media

The possibility of using available dry nutrient media Nos. 5 to 12 in assays of antibiotic biological activity with the agar diffusions method was studied with respect to benzylpenicillin, gramicidin S, kanamycin sulfate, kanamycin B, oleandomycin, novobiocin, tetracycline and erythromycin. The dry media were used instead of the respective media prepared with meat hydrolyzate. Optimal conditions of the assays on such media were determined.     

Characteristics of new pKMR-plasmids detected in natural strains of Enterobacteriaceae

pKMR-plasmids controlling the antibiotic resistance and adhesive properties were isolated from clinical strains of E. coli O26 and O124, and Sh. sonnei. Two of them, i.e. pKMR 207 and pKMR 208 were conjugative. On conjugation they jointly transferred the features of the antibiotic resistance and capacity for production of the colonization antigen. The studies on transformation of E. coli K 12 802 with the plasmid DNA of E. coli O124 showed that the antibiotic resistance and colonization properties in E. coli O124 were controlled by the nonconjugative plasmid pKMR 209. It was found that plasmids pKMR 207 and pKMR 208 had the fi(-)-phenotype. None of the plasmids allotted the host cells sensitivity to the donor specific phages of the incompatibility groups F, N, P, W, and I. Probably, the plasmids did not belong to these incompatibility groups. When the cells of E. coli K 12 802 were transformed with the plasmid DNA of the wild strain to the hemolytic strain of S. typhimurium with multiple antibiotic resistance, 3 pKMR 210 plasmids with different markers of the antibiotic resistance were detected in the transformants. One of the plasmids controlled both the drug resistance and the capacity for production of hemolysin. The ability of the detected pKMR plasmids to inhibit fertility and relation to the donor specific phages was studied.     

Comparison of meat and carcass quality in organically reared and conventionally reared pasture-fed lambs

The 'Organic' product label guarantees a production process that avoids the use of synthetic fertilisers, pesticides and hormones and minimises recourse to pharmaceuticals or veterinary drugs; however, the product's quality remains an issue that needs to be addressed in response to consumer demand. Consequently, this study was conducted to compare the sensory and nutritional qualities of meat and carcasses from pasture-fed lambs reared organically (O) or conventionally (C). Mean lamb growth profile was kept similar between the two treatments to avoid confounding effects with lamb age or weight at slaughter. The experiment was conducted over 3 years (2005 to 2007) with 12 O and 12 C lambs each year. The O and C treatments differed in the level of on-pasture mineral N fertilisation inducing a higher proportion of white clover in the organic pasture than the conventional pasture. Lambs were slaughtered when they attained a fat class of 2 to 3, and carcass and meat quality were evaluated. Lambs were slaughtered at an average weight and age of 35.3 kg and 156 days in the O treatment, respectively, and 35.2 kg and 155 days in the C treatment, respectively. Sensory evaluation indicated that loin chops from the O treatment had a higher level of abnormal fat odour compared with the C treatment. Carcasses from the O treatment had a softer subcutaneous fat one among 3 years (2007) compared to the C treatment. These results are probably due to a higher proportion of white clover in the diet. Organically reared lambs did offer the slight advantage of muscle fatty acid containing a higher level of stearic acid, which may have positive effects in the prevention of cardiovascular disease in humans. This may be the result of a higher rumen bio-hydrogenation of C18:3n-3 due to differences in the botanical composition between the O and the C pasture. Production system had no effect on the colour characteristics of the meat and subcutaneous fat, except lightness of subcutaneous dorsal fat, which was slightly higher in the O lambs. There were no differences between O and C lambs in terms of colour stability and lipid oxidation of the meat during the 6-day refrigerated storage under gas-permeable film.     

Antimicrobial susceptibility of enterococci recovered from commercial swine carcasses: effect of feed additives

Enterococcus faecium is an important nosocomial pathogen often displaying multiple antibiotic resistance. The increase in clinical isolates can be attributed in part to hospital practices in antibiotic usage, but there is concern that antibiotic-resistant strains might also originate in animals fed rations containing antibiotic growth promoters. Ingestion of meat from carcasses contaminated with faecal enterococci might then result in human colonization or resistance gene transfer to human enterococci. Because there are few comparisons of bacteria isolated from matched animals that have, or have not, been fed a diet containing antibiotic, two such groups of pig carcasses were sampled at a commercial abattoir. Forty isolates from each group of pigs were tested for their resistance to avilamycin and tylosin. Although a modest number of pigs was examined, and the number of strains of E. faecium tested was small, there was no evidence that the feeding of a growth promoter caused selection of enterococci resistant to tylosin or avilamycin.     

改良环介导等温扩增技术快速检测肉类中的大肠杆菌O157: H7

针对大肠杆菌O157:H7(Escherichia coli O157:H7,E.coli O157:H7)传统检测方法检测周期长的问题,建立了肉类中的E.coli O157:H7的改良环介导等温扩增(LAMP)快速检测方法。以E.coli O157:H7的O157特异性抗原rfbE基因、鞭毛H7特异性抗原fliC基因序列作为靶序列,分别设计2套增加了环引物的改良LAMP引物序列,单管同时检测,通过肉眼观察白色沉淀,判断检测结果。采用36株细菌验证了该改良LAMP引物的特异性。热裂解法提取的DNA经改良LAMP体系扩增20 min,检测E.coli O157:H7的灵敏度为1.4 CFU/mL,人工污染肉中的E.coli O157:H7检出限为1.8 CFU/g。137份实样中,检测出1份E.coli O157:H7假阳性,与行业标准SNT0973-2000符合率达到99.3%。     

Changes in Escherichia coli O157:H7 numbers during holding on excised lean, fascia and fat beef surfaces at different temperatures

Aim:  To investigate changes in Escherichia coli O157:H7 numbers on excised beef carcass surfaces over 72 h at different temperatures.
Methods and Results:  Excised lean meat, fascia and fat were inoculated with E. coli O157:H7 and held in an environmental chamber for 72 h, at air speed 0·5 m s⁻¹, relative humidity (RH) 90%, and temperatures 4, 8 and 12°C. On lean, pathogen counts increased significantly at 12°C. On fascia, significant reductions in counts occurred at 4 and 8°C. Pathogen numbers were significantly reduced on fat at 4, 8 and 12°C (64 h). Counts on fat were significantly less at all temperatures, compared to lean or fascia and surface water activity, a_w, decreased significantly over time on fat at 4°C. Significant decreases in surface pH values were recorded on all meat substrates.
Conclusions:  The survival of E. coli O157:H7 varied in relation to the meat substrate and the holding temperature. Reductions in counts on fat surfaces appeared to be related to low surface a_w values. No relationship between pathogen survival and surface pH was established.
Significance and Impact of the Study:  The use of excised meat pieces in an environmental cabinet offers a more flexible approach to determining the use of different chilling regimes in the production of safe meat.     

Molecular and serotyping characterization of shiga toxogenic Escherichia coli associated with food collected from Saudi Arabia

Shiga toxin-producing Escherichia coli (STEC) strains are considered as one of the major food-borne disease agents in humans worldwide. STEC strains, also called verotoxin-producing E. coli strains. The objectives of the present study were serotyping and molecular characterization of shiga toxigenic E. coli associated with raw meat and milk samples collected from Riyadh, Saudi Arabia. A total of 540 milk samples were collected from 5 dairy farms and 150 raw meat samples were collected from different abattoirs located in Riyadh, Saudi Arabia. E. coli were recovered from 86 milk samples (15.93%), serotyping of E. coli isolates revealed, 26 (4.81%) strains O157: H7, 23 (4.26%) strains O111, 20 (3.70%) strains O113: H21, 10 (1.85%) strains O22: H8 and 7 (1.3%) strains O172: H21. Meanwhile, 17 (11.33%) strains of E. coli were recovered from raw meat samples, serotyping of E. coli isolates revealed, 6 (4%) strains O157: H7, 5 (3.33%) strains O111 and 4 (2.67%) strains O174: H2 and only two (1.33%) strains were identified as O22: H8. Shiga toxin2 was detected in 58 (67.44%) serotypes of E. coli recovered from milk samples and 16 (94.12%) serotypes of E. coli recovered from meat samples, while intimin gene was detected in 38 (44.186%) serotypes of E. coli recovered from milk samples and in 10 (58.82%) serotypes of E. coli recovered from meat samples. The results of this study revealed the efficiency of combination between serotyping and molecular typing of E. coli isolates recovered from food of animal origin for rapid detection and characterization of STEC.