Survival of bacterial enteric pathogens in traditional fermented foods

The survival of strains of bacterial enteric pathogens was investigated in two traditional fermented foods (mahewu and sour porridge) and in unfermented porridge. The foods were inoculated with cell suspensions of Salmonella, Shigella, Campylobacter, Aeromonas species and pathogenic Escherichia coli which had a final concentration of 10⁶-10⁷ cfu/ml of food. None of the strains of Aeromonas and Campylobacter were detected in mahewu and sour porridge 20 min after inoculation. The salmonellas were not found 4 h after inoculation in either fermented foods but the shigellas and pathogenic E. coli strains were more tolerant to the low pH of the fermented foods. Some of the shigellas and pathogenic E. coli strains survived for 24 h after inoculation but showed a sharp decrease in numbers. All the strains of the enteric pathogens survived for 24 h in the unfermented porridge and increased in the numbers except for campylobacters, the numbers of which declined. These results suggest that the traditional fermented foods have bacteriostatic and bactericidal properties and are unlikely to play a major role in the transmission of bacterial enteric pathogens.     

Antimicrobial effect of fermented Ghanaian maize dough

Unhygienic conditions of a typical rural community in a developing country were simulated in the laboratory by inoculating fermented maize dough porridge with Shigella flexneri and enterotoxigenic Escherichia coli (ETEC). The antimicrobial effects of the different processes involved in the preparation of fermented maize dough porridge were assessed. The soaking process reduced the pH but no antimicrobial effect against shigella and ETEC was noted. Unfermented maize dough did not inhibit any of the test strains. When the fermentation process had become established, half of the strains tested were inhibited by the fermented maize dough when examined 8 h after inoculation. Cooking the fermented maize dough into porridge reduced the antimicrobial effect but there was still significant inhibition of pathogens. This suggests that the antimicrobial effect of fermented maize dough is not due to pH per se. Fermentation of maize dough appears to be a useful strategy for reducing contamination of weaning foods by Sh. flexneri and ETEC. The possible nature of the antimicrobial agent(s) produced during the fermentation of maize dough is discussed.     

Antimicrobial effect of fermented Ghanaian maize dough

Unhygienic conditions of a typical rural community in a developing country were simulated in the laboratory by inoculating fermented maize dough porridge with Shigella flexneri and enterotoxigenic Escherichia coli (ETEC). The antimicrobial effects of the different processes involved in the preparation of fermented maize dough porridge were assessed. The soaking process reduced the pH but no antimicrobial effect against shigella and ETEC was noted. Unfermented maize dough did not inhibit any of the test strains. When the fermentation process had become established, half of the strains tested were inhibited by the fermented maize dough when examined 8 h after inoculation. Cooking the fermented maize dough into porridge reduced the antimicrobial effect but there was still significant inhibition of pathogens. This suggests that the antimicrobial effect of fermented maize dough is not due to pH per se. Fermentation of maize dough appears to be a useful strategy for reducing contamination of weaning foods by Sh. flexneri and ETEC. The possible nature of the antimicrobial agent(s) produced during the fermentation of maize dough is discussed.     

Fate of pathogenic and non-pathogenic Escherichia coli strains in two fermented milk products

The growth and survival of pathogenic and non-pathogenic strains of Escherichia coli was determined in traditionally fermented pasteurized and unpasteurized milk and in Lacto, an industrially fermented milk. Each milk treatment was incubated at 20 degrees C for 24 h and then stored at either 20 degrees C or 5 degrees C for 96 h. Lacto inhibited all the three E. coli strains. Two strains could not be recovered and the third survived only in very low numbers after 24 h storage of Lacto at both 20 degrees C and 5 degrees C. All three E. coli strains survived and multiplied to maximum cell numbers in the range 10(7)-10(9)/ml during traditional fermentation of unpasteurized milk. Cell numbers decreased to 10(3)-10(6) and 10(2)-10(5) during storage of the fermented product at 20 degrees C and 5 degrees C respectively. Higher maximum numbers, 10(9)-10(10), of the three strains of E. coli were attained during traditional fermentation of pasteurized milk. The numbers decreased to 10(5)-10(8) and 10(4)-10(7) during storage of the fermented product at 20 degrees C and 5 degrees C respectively. Generally, fewer E. coli survived when the fermented milk products were stored at refrigeration temperature.     

Fate of pathogenic and non-pathogenic Escherichia coli strains in two fermented milk products

F eresu , S. & N yati , H. 1990. Fate of pathogenic and non-pathogenic Escherichia coli strains in two fermented milk products. Journal of Applied Bacteriology 69 , 814–821.
The growth and survival of pathogenic and non-pathogenic strains of Escherichia coli was determined in traditionally fermented pasteurized and unpasteurized milk and in Lacto, an industrially fermented milk. Each milk treatment was incubated at 20C for 24 h and then stored at either 20C or 5C for 96 h.
Lacto inhibited all the three E. coli strains. Two strains could not be recovered and the third survived only in very low numbers after 24 h storage of Lacto at both 20C and 5C. All three E. coli strains survived and multiplied to maximum cell numbers in the range 10⁷-10⁹/ml during traditional fermentation of unpasteurized milk. Cell numbers decreased to 10³-10⁶ and 10²-10⁵ during storage of the fermented product at 20C and 5C respectively. Higher maximum numbers, 10⁹-10¹⁰, of the three strains of E. coli were attained during traditional fermentation of pasteurized milk. The numbers decreased to 10⁵-10⁸ and 10⁴-10⁷ during storage of the fermented product at 20C and 5C respectively. Generally, fewer E. coli survived when the fermented milk products were stored at refrigeration temperature.     

Inhibition of Salmonella typhimurium and Escherichia coli by fermented flour of finger millet (Eleusine coracana)

The inhibitory effect on Salmonella typhimurium and Escherichia coli of prolonged incubation (0–48h) by finger millet flour fermented for varying time periods (0, 12, 18, 24 and 48h) was tested. S. typhimurium was completely inhibited (100%) in 12h by the 24 and 48h and after 48h by the 12 and 18h fermented samples. Escherichia coli was less inhibited than S. typhimurium, as the 48h fermented sample showed about 50% inhibition in 12h and 100% at 48h. Inhibition of both pathogens was more effective after a longer period of fermentation, suggesting that metabolites produced by the fermenting microbes play a role in this effect. The unfermented millet sample (0 h) also inhibited the pathogens on prolonged incubation for 48 h.     

Mitogenic response and probiotic characteristics of lactic acid bacteria isolated from indigenously pickled vegetables and fermented beverages

Lactic acid bacteria from indigenous pickled vegetables and fermented beverages (fermented rice and Madhuca longifolia flowers) were isolated and investigated for their functional characteristics in vitro as potential new probiotic strains. Four isolates (all Lactobacillus spp.) selected on the basis of high tolerance to bile (0.2%) were identified by standard and molecular methods (16S rDNA) as L. helveticus, L. casei, L. delbrueckii and L. bulgaricus from pickled vegetables and fermented beverages respectively. These selected strains had antibiotic resistance, tolerance to artificial gastric juice and phenol (0.4%), enzymatic profile, and antagonistic activity against enteric pathogens (Enterobacter sakazakii, Salmonella typhimurium, Shigella flexneri 2a, Listeria monocytogenes, Yersinia enterocolitica and Aeromonas hydrophila). All strains survived well in artificial gastric juice at low pH (3.0) values for 4 h, possessed bile salt hydrolase activity and were susceptible to most antibiotics including vancomycin. Additionally, the isolates exhibited high tolerance to phenol, high cell surface hydrophobicity (>60%) and induced proliferation of murine splenocytes. All the four strains of present study suppressed the Con A-stimulated proliferation of the mouse spleen cells, although L. casei had the strongest suppressive effect. The results of this study suggest a potential application of the strains (following human clinical trials), for developing probiotic foods.     

Specificity of catecholamine-induced growth in Escherichia coli O157:H7, Salmonella enterica and Yersinia enterocolitica

The present study demonstrates that catecholamine responsiveness in Yersinia enterocolitica, a bacterial pathogen whose infectious spectrum is principally limited to the gut, is limited to norepinephrine and dopamine, and not epinephrine; this behavior contrasts with observations for two pathogens with a wider extra-gastrointestinal spectrum, Escherichia coli O157:H7 and Salmonella enterica, which respond to all three catecholamines. Epinephrine showed lower potency than norepinephrine and dopamine in inducing growth of E. coli and S. enterica, and was a potent antagonist of norepinephrine and dopamine growth responsiveness in Y. enterocolitica. Given that only norepinephrine and dopamine and not epinephrine-containing neurons are found with the enteric nervous system, the results suggest that certain of the more exclusive enteric pathogens may have developed response systems preferentially for those neuroendocrine hormones that are produced by the enteric nervous system as host-derived signals by which to sense the environment and initiate pathogenic processes.     

A SIMPLE AND RAPID DETECTION BY PCR OF ENTEROPATHOGENIC ESCHERICHIA COLI IN NATURALLY CONTAMINATED VEGETABLES

Contaminated vegetables have been identified as one of the principal sources of foodborne illnesses. Escherichia coli is one of the bacteria that can contaminate vegetables and cause serious foodborne disease. The development of simple and rapid assays for detection of E. coli would enable official agencies and food industries to identify contaminated foodstuffs in a timelier manner. In this work, we detected E. coli contamination in four types of vegetables using a 24 h procedure. This method is very specific, rapid and simple to use in the laboratory. Indeed, the enrichment, DNA isolation and polymerase chain reaction (PCR) amplification procedures described here can also be used for detection of E. coli in other foods.

PRACTICAL APPLICATIONS

Foodborne disease remains an important public health threat worldwide; one of the most important food safety hazards is associated with raw vegetables. Several studies have found that products can support the growth of enteric bacterial pathogens such as Salmonella , Shigella and Escherichia coli . Culture techniques are universally recognized as the standard method for detecting pathogenic bacteria in foods. Bacteria are detected and subsequently identified by growth on solid selective culture media and by analysis of metabolic properties or serotyping. This process is lengthy and may last 5–10 days or more. In our investigation, the polymerase chain reaction (PCR) detection of E. coli in vegetables was realized within 24 h. The pre-enrichment step used in this study did not have any inhibiting effect on the PCR. Therefore, it became possible to rapidly and directly detect E. coli in raw vegetables by the PCR technique just by heating samples.     

2009~2012年黑龙江省细菌性腹泻症候群病原菌检测结果分析

为了解黑龙江省细菌性腹泻病原谱构成,探讨主要病原体的变异和流行变迁规律,提高监测实验室腹泻病原实验室诊断、监测预警、突发细菌性腹泻疫情的处置能力,并为进一步防治工作提供科学依据。收集哨点医院肠道门诊的细菌感染性腹泻患者粪便,采用分离培养、生化鉴定和血清分型的方法,检测沙门氏菌、志贺氏菌、致泻大肠杆菌等肠道致病菌。检测细菌性腹泻症候群患者标本537份,检出率为36.69%,其中,主要致病菌为致泻性大肠杆菌、志贺氏菌、副溶血弧菌、沙门氏菌、类志贺邻单胞菌、嗜水气单胞菌、小肠结肠炎耶尔森菌、霍乱弧菌、空肠弯曲菌、大肠埃希氏菌、检出率分别为9.31%、6.89%、1.30%、3.35%、0.56%、0.19%、0.37%、0.74%、5.40%、16.95%;性别差异不显著,各年龄组病原菌检出率和各月病原菌检出率差异显著,病原菌检出率存在季节性差异,其中6、7、8、9月份病原菌检出率高,检出率之和占总检出率的93.40%,0～10岁年龄组患病人数和检出率均高于其他年龄组。 分析发现:黑龙江省夏秋两季是细菌感染性腹泻高发季节,主要致病菌为大肠杆菌埃希氏菌、志贺氏菌、沙门氏菌、空肠弯曲菌。     

嗜酸乳杆菌在模拟胃肠环境中抗性的研究

采用MRS培养基,模拟胃肠环境,即低pH值（1.5～4.5）。高胆汁盐（0.1％～0.4％）对嗜酸乳杆菌抗性进行了研究。同时对肠道中致病性大肠杆菌、金黄色葡萄球菌的拮抗特性以及服用抗生素后嗜酸乳杆菌的耐药性进行了研究。结果表明,嗜酸乳杆菌在pH2.5～4.5时具有较强的生存能力,6h活菌数仍达 10⁷cfu/mL以上,pH1.5条件下仍有部分存活。在0.1％～0.3％胆汁盐条件下4h活菌数仍达106cfu/mL以上,且能在0.4％胆汁盐中存活。同时,对致病性大肠杆菌和金黄色葡萄球菌具     

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.     

Multiplex PCR assay for the detection and quantification of Campylobacter spp., Escherichia coli O157:H7, and Salmonella serotypes in water samples

Three pathogens, Campylobacter, Salmonella, and Shiga-toxin-producing Escherichia coli, are leading causes of bacterial gastroenteritis in the United States and worldwide. Although these three bacteria are typically considered food-borne pathogens, outbreaks have been reported due to contaminated drinking water and irrigation water. The aim of this research was to develop two types of PCR assays that could detect and quantify three pathogens, Campylobacter spp., E. coli O157:H7, and Salmonella spp., in watershed samples. In conventional PCR, three target strains were detected by multiplex PCR (m-PCR) using each specific primer pair simultaneously. Under optimized m-PCR conditions, the assay produced a 90-bp product for Campylobacter jejuni, a 150-bp product for E. coli O157:H7, and a 262-bp product for Salmonella Typhimurium, and the limitation of detection was approximately 700 copies for all three bacteria. In addition, real-time PCR was performed to quantify the three pathogens using SYBR green fluorescence. The assay was designed so that each target had a different melting temperature [C. jejuni (80.1 °C), E. coli O157:H7 (83.3 °C), and S. Typhimurium (85.9 °C)]. Therefore, this system could quantify and distinguish three pathogens simultaneously in a single reaction.     

Microbiological quality of blue mussels (Mytilus edulis) in Nunavik, Quebec: a pilot study

This pilot study was aimed at documenting the presence of fecal indicators and enteric pathogens in blue mussels (Mytilus edulis) from 6 communities in Nunavik, Quebec. One to four 2?kg samples of mussels were collected at low tide in each community. Samples were investigated by enumeration methods for the fecal indicators enterococci, Escherichia coli, F-specific coliphages, Clostridium perfringens, and by molecular identification for the pathogens norovirus, Salmonella spp., Campylobacter jejuni, Campylobacter coli, and Campylobacter lari, verocytotoxin-producing E.?coli (particularly serovar O157:H7), Shigella spp., and Yersinia enterocolitica. In 5 communities, the presence of Giardia duodenalis and Cryptosporidium spp. was also tested by microscopy and molecular methods and that of Toxoplasma gondii was tested by molecular methods. Apart from small quantities of Clostridium perfringens in 2 samples, no bacterial or viral pathogens were detected in the mussels. Toxoplasma gondii was also not detected. However, G.?duodenalis and Cryptosporidium spp. were present in 18% and 73% of the samples investigated for these pathogens, respectively. When considering the indicators and the viral and bacterial pathogens investigated, the mussels examined were of good microbiological quality, but considering the presence of potentially zoonotic protozoa, it should be recommended that consumers cook the molluscs well before eating them.     

A laboratory-based survey of Campylobacter infections in Prahova County

In developing countries, as well as in many western countries, members of the genus Campylobacter are recognized as one of the most common cause of acute bacterial enteritis. Campylobacter jejuni and Campylobacter coli isolation rates have been shown to be equal to, and sometimes higher than those of other enteric pathogens. The Microbiology Laboratory of the local Public Health Authority in Prahova County conducted a one and a half-year laboratory-based survey of Campylobacter infections in patients suffering from gastrointestinal symptoms. From a total of 3284 stool samples screened, the culture-positive ones confirmed the bacterial etiology for 551 diarrhea cases. Campylobacter was found in 345 specimens, being the most frequently isolated enteropathogen. C. jejuni outnumbered C. coli species (239 vs. 106 isolates). Salmonella isolates were the second local cause of diarrhea. The highest isolation rate of Campylobacter was found in children 5 years of age (262 strains). The prevalence of campylobacteriosis declined with age. The isolation rate of Campylobacter (10.5%), the unimodal age-specific distribution of cases, as well as the identification of polymicrobial infections among the screened population were epidemiological aspects resembling reports on campylobacteriosis in developing countries. The susceptibility of Campylobacter isolates to various antimicrobial agents, including macrolides and fluoroquinolones was also assessed. Among the screened isolates, Erythromycin retained a good activity, while an increased ciprofloxacin resistance was observed. The information gathered through this local study sustains the importance of Campylobacter in the etiology of autochthonous infectious diarrhea. A development of a national surveillance program regarding the most important foodborne pathogens would be beneficial for improving prevention and controlling measures.     

Molecular physiology and pathophysiology of tight junctions V. assault of the tight junction by enteric pathogens

Studies of the impact of enteric pathogens and their virulence factors on the proteins comprising the tight junction and zonula adherens offer a novel approach to dissection of tight junctional complex regulation. Most studies to date provide only tantalizing clues that select pathogens may indeed assault the tight junctional complex. Information on critical human pathogens such as Campylobacter jejuni and Shigella and Salmonella subspecies is lacking. Mechanistic studies are currently sparse, but available results on pathogenic Escherichia coli and specific virulence factors such as the Rho-modifying and protease bacterial toxins indicate four major mechanisms by which these pathogens may act: 1) direct cleavage of tight junctional structural proteins; 2) modification of the actin cytoskeleton; 3) activation of cellular signal transduction; and 4) triggering transmigration of polymorphonuclear cells across the epithelial cell barrier. New therapeutics may evolve from detailed studies of these pathogens and the cellular processes and proteins they disrupt.     

In vitro studies on the inhibition of the growth of Shigella sonnei by Lactobacillus casei and Lact. acidophilus

The inhibitory effect of lactobacilli on growth of Shigella sonnei was studied. The effect was not due to pH alone, as addition of hydrochloric, lactic or acetic acids to culture media did not inhibit the normal growth of the shigellas. The degree of inhibition was measured by disc assay and showed that the inhibitory substance(s) can be extracellular and diffusible, varying the degrees of inhibition depending on the media tested.
When broth was inoculated with mixed cultures of Lactobacillus and Shigella strains, the inhibition began at 6 h and the death phase at 9 h. The higher inhibition was produced by the mixture of lactobacilli (35.5 ± 2.5% at 6 h culture, 57.4 ± 1.9% at 9 h and 91.2 ± 1.2% at 14 h). The degree of inhibition was higher when the relationship pathogen: lactobacilli was 1: 10³. The specific growth rate of lactobacilli and shigella was different in pure or mixed cultures. When the lactobacillus alone was grown for 12 h and the shigellas then added, the numbers of shigellas began to decrease immediately at 37°C. This work shows that the Lactobacillus strains employed in fermented milk can be used to inhibit the growth of Sh. sonnei.     

A universal protocol for PCR detection of 13 species of foodborne pathogens in foods

A universal protocol for PCR detection of 13 species of foodborne pathogens in foods wasdeveloped. The protocol used a universal culture medium and the same PCR conditions with 13sets of specific primers. The 13 species of foodborne pathogens examined were Escherichiacoli, E. coli- ETEC, E. coli -O157:H7, Shigella spp. , Salmonella spp. , Yersinia enterocolitica, Y. pseudotuberculosis, Vibrio cholerae, V.parahaemolyticus, V. vulnificus , Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus . No interference was observed using the PCR assay when foodsample was artificially inoculated with each individual bacterial species. Twelve different seafoodsamples and two soft cheese samples without artificial inoculation were examined by thisprotocol. Vibrio vulnificus, Salmonella spp. , E. coli,Listeria monocytogenes and Bacillus cereus were detected in some foods.Internal probe hybridization and nested PCR procedures were used to confirm the above findings.     

High-resolution genomic fingerprinting of Campylobacter jejuni and Campylobacter coli by analysis of amplified fragment length polymorphisms

A method for high-resolution genomic fingerprinting of the enteric pathogens Campylobacter jejuni and Campylobacter coli, based on the determination of amplified fragment length polymorphism, is described. The potential of this method for molecular epidemiological studies of these species is evaluated with 50 type, reference, and well-characterised field strains. Amplified fragment length polymorphism fingerprints comprised over 60 bands detected in the size range 35-500 bp. Groups of outbreak strains, replicate subcultures, and 'genetically identical' strains from humans, poultry and cattle, proved indistinguishable by amplified fragment length polymorphism fingerprinting, but were differentiated from unrelated isolates. Previously unknown relationships between three hippurate-negative C. jejuni strains, and two C. coli var. hyoilei strains, were identified. These relationships corresponded to available epidemiological data. We conclude that this amplified fragment length polymorphism fingerprinting method may be a highly effective tool for molecular epidemiological studies of Campylobacter spp.