牦牛源产细菌素屎肠球菌的分离鉴定和益生特性

【背景】抗生素的滥用导致牦牛肠道常见病原菌耐药性增加,益生菌作为对抗耐药性细菌的新型武器,应用前景广阔。【目的】获取益生特性优良的牦牛源益生菌。【方法】将20份牦牛粪便样本在含0.5%CaCO3的MRS培养基上分离纯化,以大肠杆菌和金黄色葡萄球菌为指示菌,用牛津杯法筛选有抑菌活性的菌株;排除酸和过氧化氢后,经耐酸耐热试验和蛋白酶敏感试验筛选产细菌素菌株,用形态学和16S rRNA基因序列分析鉴定;通过对大肠杆菌、沙门氏菌等腹泻病原菌体外抑菌试验、耐模拟胃肠液、测定自聚集能力和疏水性及抗生素敏感试验分析益生特性。【结果】从20份牦牛粪便样本中共分离出11株产生溶钙圈的菌株,其中6株对大肠杆菌和金黄色葡萄球菌抑菌效果显著,经复筛得到2株产细菌素的乳酸菌SC6和SC9,经鉴定均为屎肠球菌(Enterococcus faecium)。其中SC9对腹泻病原菌抑菌效果明显,有良好的耐受性和肠道黏附能力,对5种常用抗生素均敏感。【结论】屎肠球菌SC9有一定的抗逆性和潜在的益生能力,具备作为益生菌的潜力。     

Effect of feeding sun-dried seaweed (Ascophyllum nodosum) on fecal shedding of Escherichia coli O157:H7 by feedlot cattle and on growth performance of lambs

Thirty-two steers orally inoculated with a four-strain mixture (10¹⁰ CFU) of nalidixic acid-resistant Escherichia coli O157:H7 had sun-dried Ascophyllum nodosum seaweed (Tasco-14™) added to their barley-based diet (860 g/kg barley grain and 90 g/kg whole crop barley silage, dry matter basis) to assess its effectiveness in reducing fecal shedding of the pathogen. Steers were housed in four groups of eight and received Tasco-14™ in the diet, in place of barley, at levels (as fed) of 10 g/kg for 14 days (T1-14), 20 g/kg for 7 days (T2-7), 20 g/kg for 14 days (T2-14), or not at all (i.e., control, CON). The dietary treatments commenced 7 days after E. coli O157:H7 inoculation and fecal shedding patterns were examined over 14 weeks. Water, water–trough interface, feed and fecal pat samples were also collected weekly and cultured for E. coli O157:H7. Detection of the pathogen in fecal samples was less frequent (P<0.05) in T1-14 (99/168) and T2-7 (84/168) versus CON (135/168) and T2-14 (115/168), and the concentrations of E. coli O157:H7 recovered in feces from T1-14 and T2-7 steers were lower (P<0.005) than from CON or T2-14 steers. Rates of decline in shedding of E. coli O157:H7 were similar among treatments, but final numbers of E. coli O157:H7 were lower (P<0.05) in T1-14 and T2-7 as compared to T2-14 and CON. Fecal volatile fatty acid concentrations and pH were similar among treatments, suggesting no fecal alterations that were antagonistic to survival. E. coli O157:H7 was present in 1 (from T2-7) of 56 cattle drinking water samples, 2 of 56 (T1-14, CON) feed samples and 32 of 56 fecal pats. A second experiment investigated effects of the dietary treatment on growth performance of non-inoculated sheep. Tasco-14™ was administered to 40 individually fed Canadian Arcott lambs beginning at day 56 of a 105-day finishing period. The lambs received Tasco-14™ at 0 g/kg (control, CON), at 10 g/kg for 14 days (T1-14), 20 g/kg for 14 days (T2-14), 10 g/kg for 28 days (T1-28) or at 20 g/kg for 7 days (T2-7) as a top-dress on their pelleted, barley grain-based diet (n = 8). E. coli O157:H7 was not isolated from fecal samples collected at 4-week intervals, but generic E. coli populations were lower (P<0.05) in T1-28 lambs than in other treatments. Average daily gain, feed intake, feed efficiency and carcass traits did not differ among treatments. Our challenge study supports past studies showing that Tasco-14™ decreases shedding of E. coli O157:H7 by cattle. The lamb study shows that this additive did not directly affect feed intake or animal growth.     

Occurrence of airborne enteric bacteria in Mexico city

Summary An investigation of microbial air quality in the area of the National Autonomous University of Mexico, located in the southern part of Mexico City, was conducted for one year. Ambient outdoor concentrations and size distribution of airborne bacteria were measured, 130 samples were taken at noon, using an Andersen 6 stage sampler, located 2 m above ground level. Concentration ranges and colony-forming units per cubic meter of air (CFU m^–3) found, were as follows:14 to 12999 for total bacteria, No growth (NG) to 55 for coliform bacteria, NG to 11 for fecal coliform and NG to 10 for fecal Streptococci.Bacteria associated with the potentially respirable fraction (0.65 to 4.7 µm) averaged 37% and 9% for total bacteria and coliform bacteria respectively. In 23% of the samples, coliform bacteria were recovered, with higher incidences during dry season. The most common of these were:Escherichia coli (15%), followed bySerratia (13%) andEnterobacter (10%),The total bacteria correlated significatively (p<0.05) with the following parameters: particulate matter smaller than 10 µm (PM₁₀) (r=0.40), total suspended particulates (TSP) (r=0.26), daily variation of temperature (r=0.18), and vapor pressure (r=–0.16). These relationships indicate that fecal soil pollution could affect air quality with potential health risks.     

Survival of bifidobacteria administered to calves

Twenty-five bifidobacteria were isolated from feces of calves. Isolates were identified, and their functional properties and antimicrobial activity were determined. From 10 strains with suitable properties rifampicin-resistant mutants (RRBs) were prepared and mixture of RRBs was administered to 2-d-old calves. These strains were identified by sequencing as Bifidobacterium animalis ssp. animalis (6 strains), B. thermophilum (2 strains), B. choerinum (1 strain) and B. longum ssp. suis (1 strain). The control group was without probiotic treatment. Survival ability of administered bifidobacteria was monitored in fecal samples by cultivation on modified TPY agar supplemented with mupirocin, acetic acid, and rifampicin. Administered bifidobacteria survived in gastrointestinal tract of calves for at least 60 d. Other bacteria were also determined after cultivation using fluorescence in situ hybridization (FISH). Bifidobacteria and lactobacilli dominated in fecal microflora. Significantly lower amounts of E. coli and higher amounts of bifidobacteria and total anaerobes were found in the treated group relative to the control group.     

Bacterial aerosol emission rates from municipal wastewater aeration tanks.

In this report we describe the results of a study conducted to determine the rates of bacterial aerosol emission from the surfaces of the aeration tanks of the Metropolitan Water Reclamation District of Greater Chicago John E. Egan Water Reclamation Plant. This study was accomplished by conducting test runs in which Andersen six-stage viable samplers were used to collect bacterial aerosol samples inside a walled tower positioned above an aeration tank liquid surface at the John E. Egan Water Reclamation Plant. The samples were analyzed for standard plate counts (SPC), total coliforms (TC), fecal coliforms, and fecal streptococci. Two methods of calculation were used to estimate the bacterial emission rate. The first method was a conventional stack emission rate calculation method in which the measured air concentration of bacteria was multiplied by the air flow rate emanating from the aeration tanks. The second method was a more empirical method in which an attempt was made to measure all of the bacteria emanating from an isolated area (0.37 m2) of the aeration tank surface over time. The data from six test runs were used to determine bacterial emission rates by both calculation methods. As determined by the conventional calculation method, the average SPC emission rate was 1.61 SPC/m2/s (range, 0.66 to 2.65 SPC/m2/s). As determined by the empirical calculation method, the average SPC emission rate was 2.18 SPC/m2/s (range, 1.25 to 2.66 SPC/m2/s). For TC, the average emission rate was 0.20 TC/m2/s (range, 0.02 to 0.40 TC/m2/s) when the conventional calculation method was used and 0.27 TC/m2/s (range, 0.04 to 0.53 TC/m2/s) when the empirical calculation method was used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sources of Variation in the Ampicillin-Resistant Escherichia coli Concentration in the Feces of Organic Broiler Chickens

Currently, there are limited published data for the population dynamics of antimicrobial-resistant commensal bacteria. This study was designed to evaluate both the proportions of the Escherichia coli populations that are resistant to ampicillin at the level of the individual chicken on commercial broiler farms and the feasibility of obtaining repeated measures of fecal E. coli concentrations. Short-term temporal variation in the concentration of fecal E. coli was investigated, and a preliminary assessment was made of potential factors involved in the shedding of high numbers of ampicillin-resistant E. coli by growing birds in the absence of the use of antimicrobial drugs. Multilevel linear regression modeling revealed that the largest component of random variation in log-transformed fecal E. coli concentrations was seen between sampling occasions for individual birds. The incorporation of fixed effects into the model demonstrated that the older, heavier birds in the study were significantly more likely (P = 0.0003) to shed higher numbers of ampicillin-resistant E. coli. This association between increasing weight and high shedding was not seen for the total fecal E. coli population (P = 0.71). This implies that, in the absence of the administration of antimicrobial drugs, the proportion of fecal E. coli that was resistant to ampicillin increased as the birds grew. This study has shown that it is possible to collect quantitative microbiological data on broiler farms and that such data could make valuable contributions to risk assessments concerning the transfer of resistant bacteria between animal and human populations.     

Occurrence and antimicrobial drug susceptibility patterns of commensal and diarrheagenic <Emphasis Type="Italic">Escherichia coli</Emphasis> in fecal microbiota from children with and without acute diarrhea

Acute diarrhea is a public health problem and an important cause of morbidity and mortality, especially in developing countries. The etiology is varied, and the diarrheagenic Escherichia coli pathotypes are among the most important. Our objectives were to determine the occurrence of commensal and diarrheagenic E. coli strains in fecal samples from children under five years old and their drug susceptibility patterns. E. coli were isolated from 141 fresh fecal samples; 84 were obtained from clinically injured donors with acute diarrhea (AD) and 57 from clinically healthy donors without diarrhea (WD). Presumptive phenotypic species identification was carried out and confirmed by amplification of specific 16S ribosomal RNA encoding DNA. Multiplex PCR was performed to characterize the diarrheagenic E. coli strains. Drug susceptibility patterns were determined by the disc-diffusion method. In total, 220 strains were recovered from the fecal specimens (61.8% from AD and 38.2% from WD). Diarrheagenic E. coli was identified at a rate of 36.8% (n=50) in diarrheic feces and 29.8% (n=25) in non-diarrheic feces. Enteroaggregative E. coli was the most frequently identified pathotype in the AD group (16.2%) and the only pathotype identified in the WD group (30.9%). Enteropathogenic E. coli was the second most isolated pathotype (10.3%), followed by Shiga toxin-producing E. coli (7.4%) and enterotoxigenic E. coli (2.9%). No enteroinvasive E. coli strains were recovered. The isolates showed high resistance rates against ampicillin, tetracycline, and sulfamethoxazole-trimethoprim. The most effective drugs were ceftazidime, ceftriaxone, imipenem and piperacillin-tazobactam, for which no resistance was observed. Differentiation between the diarrheagenic E. coli pathotypes is of great importance since they are involved in acute diarrheal diseases and may require specific antimicrobial chemotherapy. The high antimicrobial resistance observed in our study raises a broad discussion on the indiscriminate or improper use of antimicrobials, besides the risks of self-medication.     

Isolation of microbial pathogens of subclinical mastitis from raw sheep's milk of Epirus (Greece) and their role in its hygiene

The natural raw milk microflora is a factor that expresses its sensorial characteristics. The microbial charge into the mammary gland of healthy animal is low and the application of right and healthy conditions during milking and cheese making procedure, prevents from contaminating as well as maintains the natural microflora in order to lend the particular characteristics of milk.The purpose of the present project was the study of the Total Viable Count (T.V.C.) and the count of total psychrotropic bacteria of raw sheep milk from Boutsiko and Karamaniko breeds, collected from healthy animals, as well as the isolation, identification and enumeration of pathogenic bacteria related with the hygiene and the quality of raw sheep milk (with a particular interest in bacteria that may cause human infection).During the experiment we examined two hundred forty (240) samples of raw sheep milk. In these samples a) Staphylococcus aureus, Salmonella sp., Escherichia coli, Clostridium perfringens (vegetative cells and spores) and Bacillus sp. were isolated and identified b) the Total Viable Count and the total number of psychrotropic bacteria were also specified. The sampling, the preparation of samples and decimal dilutions were based on international methods. The Total viable count was determined using the standard methods of the American Public Health Association, 2002. The total number of psychrotropic bacteria was determined using APHA 1976, 1978 rules. The identification of the bacteria was carried out according to the Bergey’s manual. Microscopic examination of Gram stained cells, catalase, oxidase and biochemical tests were performed when necessary to further identify.From the 240 milk samples tested, only 5% were E. coli positive, with mean counts ranged from 2 × 10³ to 2.4 × 10⁴ cfu/ml. S. aureus was isolated from 24% of the samples and the mean count per ml was ranged from <10 to 3.4 × 10². Meanwhile, Bacillus spp. was also detected in 29% samples. Vegetative forms and spores of C. perfringens were detected in 13% and 63% of the samples respectively. However, microbiological analyses revealed the presence of a small number of selected pathogens in milk samples such as Salmonella, which was only detected in 5% of the samples. Listeria sp., Pseudomonas sp. and Vibrio cholerae were never found.From the experimental results, the Total Viable Count from raw sheep milk samples, fulfils the microbiological criteria of EU Legislation in a percentage of approximately 97%.     

Development of a quantitative PCR method to differentiate between viable and nonviable bacteria in environmental water samples

Ethidium monoazide bromide (EMA) treatment of pure culture and environmental waters at low concentrations (1.0–7.5 μg/ml) indicated effective enumeration of viable and viable but nonculturable Escherichia coli in pure cultures, creek waters, and secondary activated sludge effluent samples by quantitative polymerase chain reaction (qPCR) amplification of the uidA and fliC gene targets at turbidity values <10 NTU. However, EMA treatment was not effective in primary clarifier and secondary trickling filter effluents where turbidities were ≥10 NTU. In viable pure cultures, rapidly dividing and senescent cells were most affected by increasing EMA concentrations. Amplification of heat-killed pure bacterial cultures decreased 4 to 6 logs depending on EMA concentration and culture age. The greatest difference was observed in 5-h cultures using 7.5 μg/ml EMA. Turbidity (≥100 NTU) in environmental samples inhibited EMA effectiveness on viability discrimination. Enumeration of E. coli in certain wastewaters using EMA-qPCR was similar to culture suggesting that EMA treatment could be incorporated into qPCR assays for the quantification of viable bacteria increasing assay time no more than 30 min. Our results indicate that EMA can be used in routine qPCR assays, but optimum conditions for exposure must be identified for each sample type due to sample matrix effects such as turbidity.     

Impact of Dexamethasone-Induced Immunosuppression on the Duration and Level of Shedding of Escherichia coli O157:H7 in Calves

The goal of this study was to determine whether immunosuppression plays a role in the level and duration of fecal shedding of Escherichia coli O157. Immunosuppression was induced in calves by administering dexamethasone. Six 1-week-old Holstein bull calves were injected intramuscularly with dexamethasone and orally inoculated with 10⁹ CFU of a mixture of three nalidixic-acid resistant strains of E. coli O157:H7. Five 1-week-old Holstein bull calves that were given the same oral inoculation of E. coli O157:H7, but not the dexamethasone injections, served as controls. All calves were examined daily and fecal samples were collected three times a week for detection and enumeration of the nalidixic-acid resistant E. coli O157. Four weeks after the last calf stopped shedding, all calves were necropsied and samples from the gastrointestinal tract were taken for the detection of the nalidixic-acid resistant E. coli O157. Dexamethasone-injected calves shed at higher levels (P = 0.04) on days 4 and 7 postinoculation, but not thereafter. None of the samples collected at necropsy were positive for E. coli O157. Data from this study suggest that there may be a time-dependent relationship between dexamethasone immunosuppression and the fecal concentration of E. coli O157 but that transient immunosuppression does not appear to prolong shedding of E. coli O157.     

Biocontrol of Escherichia coli O157 with O157-Specific Bacteriophages

Escherichia coli O157 antigen-specific bacteriophages were isolated and tested to determine their ability to lyse laboratory cultures of Escherichia coli O157:H7. A total of 53 bovine or ovine fecal samples were enriched for phage, and 5 of these samples were found to contain lytic phages that grow on E. coli O157:H7. Three bacteriophages, designated KH1, KH4, and KH5, were evaluated. At 37 or 4°C, a mixture of these three O157-specific phages lysed all of the E. coli O157 cultures tested and none of the non-O157 E. coli or non-E. coli cultures tested. These results required culture aeration and a high multiplicity of infection. Without aeration, complete lysis of the bacterial cells occurred only after 5 days of incubation and only at 4°C. Phage infection and plaque formation were influenced by the nature of the host cell O157 lipopolysaccharide (LPS). Strains that did not express the O157 antigen or expressed a truncated LPS were not susceptible to plaque formation or lysis by phage. In addition, strains that expressed abundant mid-range-molecular-weight LPS did not support plaque formation but were lysed in liquid culture. Virulent O157 antigen-specific phages could play a role in biocontrol of E. coli O157:H7 in animals and fresh foods without compromising the viability of other normal flora or food quality.     

Sensitivity of Direct Plating for Detection of High Levels of <Emphasis Type="Italic">E. coli</Emphasis> O157:H7 in Bovine Fecal Samples

To assess the sensitivity of direct plating of bovine fecal samples for detection of Escherichia coli O157:H7, calves (n = 28) were orally inoculated with 10⁹ colony-forming units (cfu) per calf of a mixture of three strains of nalidixic acid-resistant E. coli O157:H7, and fecal samples were collected for analysis. One-gram samples from inoculated calves were mixed with 9 mL of Gram-negative broth with vancomycin, cefixime, and cefsoludin. From this suspension, serial dilutions were made (10⁻¹ to 10⁻⁴) and spread plated in triplicate on Sorbitol MacConkey agar with nalidixic acid for enumeration of E. coli O157:H7 in fecal samples. Direct plating samples were streaked for isolation on Sorbitol MacConkey agar with cefixime, and tellurite (SMACct). After incubation overnight at 37°C, morphologically typical colonies from direct streak plates were plated onto blood agar and incubated overnight at 37°C; then an indole test was performed on each colony. Indole-positive colonies were confirmed by O157 agglutination and were then plated on SMAC agar with 20 μg/mL nalidixic acid (SMACnal) to confirm nalidixic acid resistance. Overall sensitivity of detection was 32.5% (110/338 samples). Sensitivity to detect fecal samples shedding at above 5 × 10⁴ cfu/g was 83% (71/86 samples). Based on these data, direct plating of fecal samples might be an effective way to identify cattle that are likely to be shedding E. coli O157 at high levels.     

Expression of leucine genes from an extremely thermophilic bacterium in Escherichia coli

Summary The organisation of the leucine genes in Thermus thermophilus HB8 was analysed by examining the ability of recombinant DNAs to complement Escherichia coli mutations. The arrangement of the genes is different from that in the mesophilic bacteria E. coli and Salmonella typhimurium. The promoter responsible for the expression of the leuB, leuC and leuD genes of Thermus HB8 in E. coli was identified. The sequence of Thermus DNA containing this promoter revealed structural similarities to the promoter and attenuator regions of the E. coli leucine operon.     

Microbiological quality of walnut kernels and apple juice concentrate

In the present study, we have evaluated the microbiological quality of walnut kernels and pasteurized apple juice concentrate and the application of PCR for quality control of these important horticultural products. PCR assays for the detection of Bacillus cereus, Salmonella, Escherichia coli and E. coli O157:H7 were standardized using minimum time for each step of the reaction. The protocols were effective for their detection in these products after pre-enrichment for 6–12 h. 2, 68 and 30% of the samples of walnut kernels were respectively found satisfactory, acceptable and unsatisfactory on the basis of their viable count. Only 15% of the samples of pasteurized apple juice concentrate were found to possess the desired viable count of less than 100 c.f.u./ml. The predominant contaminants of walnut kernels were found to be the species of Bacillus, Klebsiella, Enterobacter and Staphylococcus. Samples of apple juice concentrate were predominantly found contaminated with species of Bacillus, Staphylococcus and Micrococcus. However, B. cereus, Salmonella and E. coli were also isolated from some of the samples of walnut kernels. Bacillus cereus was also obtained from some of the samples of pasteurized apple juice concentrate in high numbers. Among the moulds Penicillium, Aspergillus, Cladosporium, Rhizopus and Mucor were isolated from these products.     

The specific effect of gallic acid on Escherichia coli biofilm formation by regulating pgaABCD genes expression

Escherichia coli (E. coli) is associated with an array of health-threatening contaminations, some of which are related to biofilm states. The pgaABCD-encoded poly-beta-1,6-N-acetyl-D-glucosamine (PGA) polymer plays an important role in biofilm formation. This study was conducted to determine the inhibitory effect of gallic acid (GA) against E. coli biofilm formation. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of GA against planktonic E. coli were 0.5 and 4 mg/mL, and minimal biofilm inhibitory concentration and minimal biofilm eradication concentration values of GA against E. coli in biofilms were 2 and 8 mg/mL, respectively. Quantitative crystal violet staining of biofilms and ESEM images clearly indicate that GA effectively, dose-dependently inhibited biofilm formation. CFU counting and confocal laser scanning microscopy measurements showed that GA significantly reduced viable bacteria in the biofilm. The contents of polysaccharide slime, protein, and DNA in the E. coli biofilm also decreased. qRT-PCR data showed that at the sub-MIC level of GA (0.25 mg/mL) and expression of pgaABC genes was downregulated, while pgaD gene expression was upregulated. The sub-MBC level of GA (2 mg/mL) significantly suppressed the pgaABCD genes. Our results altogether demonstrate that GA inhibited viable bacteria and E. coli biofilm formation, marking a novel approach to the prevention and treatment of biofilm-related infections in the food industry.

     

白花丹素对E.coli藻酸盐合成的抑制作用

细菌分泌的胞外多糖在生物被膜的形成和发展过程中发挥着重要作用。通过测定白花丹素对大肠埃希菌10389菌株(E.coli 10389)藻酸盐合成的影响及其对rse A和rpo E基因表达量的影响,探讨白花丹素对大肠埃希菌生物被膜(biofilm,BF)形成的抑制作用及机制。研究结果显示,白花丹素能抑制E.coli 10389生物被膜的形成,其抑杀E.coli 10389的最低抑菌浓度(minimum inhibitory concentration,MIC)和最低杀菌浓度(minimal bactericidal concentration,MBC)分别为16和64μg/mL。白花丹素对成熟BF内的细菌也有抑制和杀灭作用,其抑杀E.coli 10389成熟BF内细菌的MIC和MBC分别为64和128μg/mL。白花丹素能够抑制E.coli 10389藻酸盐的合成,其中1/2MIC的白花丹素作用E.coli 10389 24 h后,与对照组比,藻酸盐的合成量降低了34.83%(P0.01)。白花丹素可显著影响E.coli 10389 rse A和rpo E基因的相对表达量,其中1/2MIC的白花丹素作用E.coli 10389 24 h后,与对照组相比,rse A的表达量上调了17.43%,rpo E的表达量降低了12.8%(P0.05)。结果表明,白花丹素能够抑制E.coli 10389 BF的形成,其作用机制可通过影响rse A和rpo E的基因表达量,进而抑制藻酸盐的合成来抑制大肠埃希菌生物被膜的形成。     

Molecular monitoring of Escherichia coli O157: H7 sterilization rate using qPCR and propidium monoazide treatment

Propidium monoazide is a DNA‐intercalating dye. PMA‐qPCR has been reported as a novel method to detect live bacteria in complex samples. In this study, this method was used to monitor the sterilization effects of UHP, ultrasound and high PEF on Escherichia coli O157:H7. Our results showed that all three sterilization techniques are successful to kill viable E. coli O157:H7 cells under their appropriate conditions. PMA‐qPCR can effectively monitor the amount of DNA released from viable E. coli O157:H7 cells, and the results from PMA‐qPCR were highly consistent with those from plate counting after treatment with UHP, ultrasound and high PEF. The maximal ΔC_t between PMA‐qPCR and qPCR obtained in this study was 10·39 for UHP, 5·76 for ultrasound and 2·30 for high PEF. The maximal sterilization rates monitored by PMA‐qPCR were 99·92% for UHP, 99·99% for ultrasound and 100% for high PEF. Thus, PMA‐qPCR can be used to detect the sterilization effect on food and water supplies after treatment with UHP, ultrasound and high PEF.

Significance and Impact of the Study

The reliable detection of viable foodborne pathogenic bacteria in water and food is of great importance in our daily life. However, the traditional bacteria cultivation‐based methods are time‐consuming and difficult to monitor all viable bacteria because of the limitation of cultivation conditions. This study demonstrated that PMA‐qPCR technique is very effective to monitor viable E. coli O157:H7 after sterilization and will help to monitor the viable bacteria in food and water.     

Escherichia coli-induced productions of pro-inflammatory cytokines are regulated by MAP kinases and G-protein but not by Akt: Relationship with phylogenetic groups and resistance patterns

Introduction

We investigated the role of PI3-K, MAP kinases, and heterotrimeric G proteins in inducing cytokines production in human whole blood cultures stimulated by viable Escherichia coli (E. coli) clinical strains.

Materials and methods

We used eight E. coli strains that belong to different phylogenetic groups and presented by different antibiotic resistance patterns. Whole blood from healthy volunteers was incubated at 37 °C for 150 min, with lipopolysaccharide (LPS) from E. coli O111:B4 or selected viable E. coli clinical strains, with or without SB202190 (p38 inhibitor), PD98059 (ERK inhibitor), PTX (pertussis toxin; heterotrimeric G proteins inhibitor), wortmaninn (PI3-K inhibitor). The TNF-α, IL-1β, IL-10 and IFN-γ concentrations were measured in culture supernatants (ELISA).

Results

IL-10 and IFN-γ were not detectable. Susceptible strains induced higher TNF-α and IL-1β productions than β-lactam resistant strains (p < 0.05), with no difference between phylogenetic groups. A transformed strain carrying a plasmid-mediated AmpC-β-lactamase gene (CMY-2) induced lower TNF-α and IL-1β production than the parent wild type strain (p < 0.05). SB202190 (p38 inhibitor) and PD98059 (ERK inhibitor) reduced TNF-α concentrations by, respectively, 80% (p < 0.05) and 50% (p < 0.05). Wortmaninn (PI3-K inhibitor) had no significant effect. PTX (heterotrimeric G proteins inhibitor) altered TNF-α production after viable bacteria stimulation (1.7-fold increase; p < 0.05) but not after LPS (TLR-4) stimulation. Regarding IL-1β, wortmaninn, SB202190 and PTX had no significant effect whereas PD98059 significantly decreased production in whole cell cultures (p < 0.05).

Conclusion

Susceptible strains induce greater TNF-α and IL-1β productions than resistant strains. ERK kinase plays a major role in viable E. coli strains inducing TNF-α and IL-1β production. E. coli exerts an effect on the pertussis toxin-sensitive G-protein through a TLR-4-independent mechanism.     

A biomarker for the identification of swine fecal pollution in water,using the STII toxin gene from enterotoxigenic<Emphasis Type="Italic"> Escherichia coli</Emphasis>

This research developed a PCR method to identify swine fecal pollution in water, using a portion of the STII toxin gene from enterotoxigenic Escherichia coli as the target sequence. This method showed the gene to have a wide-spread geographical distribution and temporal stability; and the primers demonstrated high specificity, sensitivity, and reliability. A total of 110 DNA extracts from different animal fecal and human sewage samples were screened using the primers and no positives resulted. Centrifugation and filtration methods for concentrating E. coli seeded into stream, ocean, secondary effluent, and dairy lagoon waters resulted in detection limits at the femtogram and attogram levels. E. coli with the biomarker seeded into stream, ocean, and secondary effluent waters remained stable for approximately 2 weeks for all water types. Of the farm lagoon and waste samples tested, 94% were positive for the STII trait, regardless of the number of E. coli screened and 100% were positive when 35 E. coli isolates were screened. As the PCR product of the target sequence yielded a single band, the method is applicable to dot blot detection methodology, yielding great accuracy in determining the presence of swine fecal sources.     

High-Throughput and Quantitative Procedure for Determining Sources of Escherichia coli in Waterways by Using Host-Specific DNA Marker Genes

Escherichia coli is currently used as an indicator of fecal pollution and to assess water quality. While several genotypic techniques have been used to determine potential sources of fecal bacteria impacting waterways and beaches, they do not allow for the rapid analysis of a large number of samples in a relatively short period of time. Here we report that gene probes identified by Hamilton and colleagues (M. J. Hamilton, T. Yan, and M. J. Sadowsky, Appl. Environ. Microbiol. 72:4012-4019, 2006) were useful for the development of a high-throughput and quantitative macroarray hybridization system to determine numbers of E. coli bacteria originating from geese/ducks. The procedure we developed, using a QBot robot for picking and arraying of colonies, allowed us to simultaneously analyze up to 20,736 E. coli colonies from water samples, with minimal time and human input. Statistically significant results were obtained by analyzing 700 E. coli colonies per water sample, allowing for the analysis of approximately 30 sites per macroarray. Macroarray hybridization studies done on E. coli collected from water samples obtained from two urban Minnesota lakes and one rural South Carolina lake indicated that geese/ducks contributed up to 51% of the fecal bacteria in the urban lake water samples, and the level was below the detection limit in the rural lake water sample. This technique, coupled with the use of other host source-specific gene probes, holds great promise as a new quantitative microbial source tracking tool to rapidly determine the origins of E. coli in waterways and on beaches.