H7 Cultures Research Articles

ELIMINASI BAKTERI Escherichia coli O157 H7 DAN Escherichia coli O157 PADA DAGING SAPI MENGGUNAKAN SINAR ULTRAVIOLET

Infection is a major problem mortality and morbidity in the world, a lot of an infection in the digestive tract is causes bacteria. The Escherichia coli O157 and Escherichia coli O157: H7 strains are bacteria that cause bloody diarrhea to death. Ultraviolet light has the potential to eliminate bacteria. The purpose is to know decontamination of E. coli O157 and E. coli O157: H7 on meat after ultraviolet exposure. The method is a factorial RBD with two factors (distance and time) with variations in distances (5, 10, 15, and 20 cm) and times (5, 10, 15, 20 dan 25 minutes). All treatments were repeated twice and obtained 160 samples. The quantitative data obtained were using univariant analysis followed by the Least Significant Difference with a significant level of 5%. The irradiation for E. coli O157 on meat with 5 cm for 25 minutes showed a significant reduction (88,62%), while the farthest distance 20 cm for 25 minutes decreased (73,42%). The nearest irradiation E. coli O157: H7 on meat 5 cm for 25 minutes show decrease (86,78%), while the farthest distance was 20 cm for 25 minutes (75,16%), there was an increase colonies in several treatment variations but overall decreased. In conclusion there was significant decrease number of bacterial colonies in meat given E. coli O157 culture, while there was a fluctuation decrease number of colonies in meats given E. coli O157: H7 culture.

Validation and Application of a Real-Time PCR Assay Based on the CRISPR Array for Serotype-Specific Detection and Quantification of Enterohemorrhagic Escherichia coli O157:H7 in Cattle Feces

Several real-time quantitative PCR (qPCR) assays have been developed for detection and quantification of Escherichia coli O157:H7 in complex matrices by targeting genes for serogroup-specific O-antigen ( rfbEO157), H7 antigen, and one or more major virulence factors (Shiga toxin and intimin). A major limitation of such assays is that coamplification of H7 and virulence genes in a sample does not signal association of those genes with the O157 serogroup. Clusters of regularly interspaced short palindromic repeats (CRISPR) polymorphisms are highly correlated with certain enterohemorrhagic E. coli (EHEC) serotypes, including O157:H7, and the presence of genes for Shiga toxin ( stx1 and stx2) and intimin ( eae). Our objectives were to develop and validate a qPCR assay targeting the CRISPR array for the detection and quantification of EHEC O157:H7 in cattle feces and to evaluate the applicability of the assay for detection of and comparison with a four-plex qPCR assay targeting rfbEO157, stx1, stx2, and eae genes and a culture method. Detection limits of the CRISPRO157:H7 qPCR assay for cattle feces spiked with pure cultures were 2.1 × 103 and 2.3 × 100 CFU/g before and after enrichment, respectively. Detection of E. coli O157 in feedlot cattle fecal samples ( n = 576) was compared among the CRISPRO157:H7 qPCR assay, culture method, and four-plex qPCR assay. The CRISPRO157:H7 qPCR detected 42.2% of the samples (243 of 576 samples) as positive for E. coli O157:H7, compared with 30.4% (175 samples) by the culture method. Nearly all samples (97.2%; 560 samples) were positive for rfbEO157 by the four-plex PCR, but 21.8% (122 of 560 samples) were negative for the stx and/or eae genes, making it unlikely that EHEC O157:H7 was present in these samples. Cohen's kappa statistic indicated a fair and poor agreement beyond that due to chance between the CRISPR assay and the culture method and four-plex assay, respectively. This novel qPCR assay can detect the EHEC O157:H7 serotype in cattle feces by targeting CRISPR polymorphisms.

Evaluation of the antimicrobial efficacy of neutral electrolyzed water on pork products and the formation of viable but nonculturable (VBNC) pathogens

The goals of this study were to evaluate the antimicrobial efficacy of Neutral electrolyzed oxidizing (NEO) water on E. coli O157:H7, Salmonella Enteritidis and Yersinia enterocolitica in both pure culture and on inoculated pork chops and skin samples, and to investigate the formation of viable but nonculturable (VBNC) pathogens after treatments. Both the plate count method and flow cytometry were used to evaluate antimicrobial efficacy on pure cultures. Different concentrations of NEO water were prepared by diluting the original NEO water (100%) with sterilized deionized water. The antimicrobial efficacy increased as the concentrations of NEO water increased. The flow cytometry results showed that treating with diluted NEO water led to the formation of VBNC cells. No VBNC cells formed when treating pure cultures with 50% or 100% NEO water. Yersinia cultures were found to be more resistant to NEO treatments than Salmonella and E. coli O157:H7 cultures, with Yersinia cultures showing lower reductions and higher levels of VBNC cells after treatments. The antimicrobial efficacy of NEO water was significantly better on skin samples than on pork chops. The differences in protein content and structure between pork chops and skin samples serve as major factors impacting the NEO water's efficacy.

Development of a Sandwich ELISA for EHEC O157:H7 Intimin γ1.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen of worldwide importance that causes foodborne infections in humans. Intimin gamma 1 (intimin γ1) is one of the most important outer membrane proteins required for EHEC’s intimate adhesion to epithelial cells. Here, we generated a polyclonal antibody (pAb) and a monoclonal antibody (mAb) against intimin γ1 to develop a double antibody sandwich ELISA (DAS-ELISA) with increased sensitivity and specificity for measuring EHEC O157:H7. To achieve this goal, a rabbit pAb was used as a capture antibody, and a mouse mAb was a detection antibody. No cross-reactivity was observed with the other genera of pathogenic bacteria tested with the DAS-ELISA, which included Salmonella enteritidis, Shigella flexneri type 2, Listeria monocytogenes, Streptococcus suis type 2, and other 18 serotype E. coli. Detection limits of the DAS-ELISA were 1 × 103 CFU/mL for EHEC O157:H7 cultures, 1 × 104 CFU/g before enrichment, and 1 × 102 CFU/g after enrichment of contaminated samples. Field samples (n = 498) were tested using a previously established duplex-PCR method and compared to our DAS-ELISA. The DAS-ELISA had a specificity of 94.4%, a sensitivity of 91.5% and accuracy of 94.0% compared with duplex-PCR. The DAS-ELISA developed here can be applied to EHEC O157:H7 quantification in food, animal, and environmental samples.

Stx1prophage excision inEscherichia colistrain PA20 confers strong curli and biofilm formation by restoring nativemlrA

Prophage insertions in Escherichia coli O157:H7 mlrA contribute to the low expression of curli fimbriae and biofilm observed in many clinical isolates. Varying levels of CsgD-dependent curli/biofilm expression are restored to strains bearing prophage insertions in mlrA by mutation of regulatory genes affecting csgD Our previous study identified strong biofilm- and curli-producing variants in O157:H7 cultures that had lost the mlrA-imbedded prophage characteristic of the parent population, suggesting prophage excision as a mechanism for restoring biofilm properties. In this study, we compared genomic, transcriptomic and phenotypic properties of parent strain PA20 (stx1, stx2) and its prophage-cured variant, 20R2R (stx2), and confirmed the mechanism underlying the differences in biofilm formation.

Design of an internal amplification control for a duplex PCR used in the detection of Shiga toxin producing Escherichia coli in pediatric feces

A conventional PCR targeted directly to the detection of Shiga toxin-producing Escherichia coli (STEC) in diarrheal stools of symptomatic patients may require the introduction of internal controls to detect false negative results. In the present study, we designed a competitive internal amplification control (IAC) to be included in a well-known PCR protocol used to amplify the stx1and stx2 genes from STEC isolates. The IAC was introduced in the PCR reaction and amplified when E. coli O157:H7 cultures and contaminated pediatric feces were assayed. When STEC concentration was 103 CFU ml−1 in pure culture and 104 CFU g−1 in contaminated stools, the IAC at concentration of 0.143 pg μl−1 in the PCR reaction mixture was co-amplified with the stx2 sequence, producing bands of 279 and 349 bp, respectively. These STEC values were considered the detection limits of the duplex PCR. The specific detection of STEC by duplex PCR including IAC might be achieved directly on pediatric feces when the pathogen load reaches concentrations of at least 104 CFU g−1.

Investigation of Shiga-like toxigenic Escherichia coli in meat products by quantitative PCR

The aim of this study was to investigate and evaluate effectiveness of quantitative PCR (qPCR) in quantification of Shiga-like toxigenic E. coli (STEC) in artificially contaminated deli meat slices compared to plate count technique. Experiment was designed to investigate capability of qPCR using selected pair of primers amplifying rpoB household gene and serial decimal dilution of an overnight suspension of E. coli O157:H7 culture. Subsequently, slices of smoked pork loin were contaminated by respective decimally diluted dose of STEC. DNA was extracted from the samples and qPCR was run in triplicate. Mean Ct values of amplified rpoB gene were compared to each inoculum dose and standard curves were generated. Results clearly showed that the lowest detectable level of STEC in pork loin slices using qPCR was 6.8×101 CFU/g. Further optimization of method should be done in order to resolve, if possible, discrepancies at the levels of contamination less than 100 CFU/g.

Microbial quality and associated health risks of raw milk marketed in the Tanga region of Tanzania

Objective To evaluate microbial quality and associated health risks of raw milk marketed in the Tanga region of Tanzania. Methods A microbial quality assessment of marketed raw milk was undertaken by evaluating 59 samples of milk from selling points (collecting centres =15), bicycle boys (12) and kiosks/restaurants (32) in Tanga city during April-May 2005. Quality and milk-borne hazards were assessed using a combination of tests in order to quantify the occurrence of Brucellosis (milk ring test), Escherichia coli ( E. coli) O157:H7 (culture), the coliform bacteria as well as standard plate count (SPC). Specific gravity (SG) determination was used as an indicator of adulteration. Results The mean coliform plate count (c.f.u/mL) of milk handled by bicycle boys (4.2 × 10 6) was significantly higher than that handled by collecting centres (3.0 × 10 6) and kiosk/restaurants (1.4 × 10 6), respectively ( P < 0.05). Of the 59 milk samples collected, 33 (56%) were Brucella milk ring test (MRT)-positive and 78% and 17% of the samples graded satisfactorily based on SG and coliform plate counts as prescribed by East African Community standards for raw milk. There was no verocytotoxigenic E. coli (VTEC) O157: H7 in any of the milk samples collected and analysed during the present study. Conclusions It can be concluded that raw market milk in the study area is of poor bacteriological quality and hazardous for human consumption. This highlights the need to implement good hygiene practices and effective monitoring from production through the delivery chain to the consumer. Further studies are needed for detection of toxins that are produced by E. coli, other pathogenic spore forming bacteria ( Bacillus spp. and Clostridium spp.) and other harmful microorganisms.

Development and characterization of a magnetic bead-quantum dot nanoparticles based assay capable of Escherichia coli O157:H7 quantification

The development and characterization of a magnetic bead (MB)-quantum dot (QD) nanoparticles based assay capable of quantifying pathogenic bacteria is presented here. The MB-QD assay operates by having a capturing probe DNA selectively linked to the signaling probe DNA via the target genomic DNA (gDNA) during DNA hybridization. The signaling probe DNA is labeled with fluorescent QD 565 which serves as a reporter. The capturing probe DNA is conjugated simultaneously to a MB and another QD 655, which serve as a carrier and an internal standard, respectively. Successfully captured target gDNA is separated using a magnetic field and is quantified via a spectrofluorometer. The use of QDs (i.e., QD 565/QD 655) as both a fluorescence label and an internal standard increased the sensitivity of the assay. The passivation effect and the molar ratio between QD and DNA were optimized. The MB-QD assay demonstrated a detection limit of 890 zeptomolar (i.e., 10 −21 mol L −1) concentration for the linear single stranded DNA (ssDNA). It also demonstrated a detection limit of 87 gene copies for double stranded DNA (dsDNA) eaeA gene extracted from pure Escherichia coli ( E. coli) O157:H7 culture. Its corresponding dynamic range, sensitivity, and selectivity were also presented. Finally, the bacterial gDNA of E. coli O157:H7 was used to highlight the MB-QD assay's ability to detect below the minimum infective dose (i.e., 100 organisms) of E. coli O157:H7 in water environment.

Edible Apple Film Wraps Containing Plant Antimicrobials Inactivate Foodborne Pathogens on Meat and Poultry Products

Apple-based edible films containing plant antimicrobials were evaluated for their activity against pathogenic bacteria on meat and poultry products. Salmonella enterica or E. coli O157:H7 (10(7) CFU/g) cultures were surface inoculated on chicken breasts and Listeria monocytogenes (10(6) CFU/g) on ham. The inoculated products were then wrapped with edible films containing 3 concentrations (0.5%, 1.5%, and 3%) of cinnamaldehyde or carvacrol. Following incubation at either 23 or 4 degrees C for 72 h, samples were stomached in buffered peptone water, diluted, and plated for enumeration of survivors. The antimicrobial films exhibited concentration-dependent activities against the pathogens tested. At 23 degrees C on chicken breasts, films with 3% antimicrobials showed the highest reductions (4.3 to 6.8 log CFU/g) of both S. enterica and E. coli O157:H7. Films with 1.5% and 0.5% antimicrobials showed 2.4 to 4.3 and 1.6 to 2.8 log reductions, respectively. At 4 degrees C, carvacrol exhibited greater activity than did cinnamaldehyde. Films with 3%, 1.5%, and 0.5% carvacrol reduced the bacterial populations by about 3, 1.6 to 3, and 0.8 to 1 logs, respectively. Films with 3% and 1.5% cinnamaldehyde induced 1.2 to 2.8 and 1.2 to 1.3 log reductions, respectively. For L. monocytogenes on ham, carvacrol films induced greater reductions than did cinnamaldehyde films at all concentrations tested. In general, the reduction of L. monocytogenes on ham at 23 degrees C was greater than at 4 degrees C. Added antimicrobials had minor effects on physical properties of the films. The results suggest that the food industry and consumers could use these films as wrappings to control surface contamination by foodborne pathogenic microorganisms.

Inactivation of Escherichia coli O157:H7 by cinnamon extracts in carrot-kinnow mandarin blends.

We investigated the antimicrobial efficacy of cinnamon extracts in laboratory prepared Kinnow-mandarin carrot blends challenged with Escerichia coli O157:H7. Freshly squeezed carrot and kinnow-mandarin juices were mixed to obtain a typical blend, inoculated with E. coli O157:H7 cultures at 10(2) CFU/mL with and without cinnamon extracts (0.3%) and stored at 4, 8 and 28°C for up to 10 hours. Counts on tryptic soy agar (TSA) selective medium (Mac conkey sorbitol agar) and thin agar layer (TAL) were determined at every 2 hour. The TAL method was used for recovery of sublethally injured cells. Inactivation of E. coli O157:H7 in blends containing 0.3% cinnamon extracts were observed, with killing effects being more pronounced at 28°C, similar trends were evident with blends stored under refrigeration conditions (4 and 8°C).The decrease in counts were attributed to several factors namely, pH, storage temperature and addition of cinnamon. The results of our study indicate that cinnamon extracts could be used as an effective, natural antimicrobial for assuring consumer safety at the point of preparation of carrot-kinnow mandarin blends, which is a popular, nutritional beverage consumed in India.

High-Pressure Resistance Variation of Escherichia coli O157:H7 Strains and Salmonella Serovars in Tryptic Soy Broth, Distilled Water, and Fruit Juice

The effect of high pressure on the log reduction of six strains of Escherichia coli O157:H7 and five serovars of Salmonella enterica was investigated in tryptic soy broth, sterile distilled water, and commercially sterile orange juice (for Salmonella) and apple cider (for E. coli). Samples were subjected to high-pressure processing treatment at 300 and 550 MPa for 2 min at 6°C. Samples were plated onto tryptic soy agar directly after pressurization and after being held for 24 h at 4°C. At 300 MPa, little effect was seen on E. coli O157:H7 strains, while Salmonella serovars varied in resistance, showing reductions between 0.26 and 3.95 log CFU/ml. At 550 MPa, E. coli O157:H7 strains exhibited a range of reductions (0.28 to 4.39 log CFU/ml), while most Salmonella populations decreased beyond the detection limit (>5-log CFU/ml reduction). The most resistant strains tested were E. coli E009 and Salmonella Agona. Generally, bacterial populations in fruit juices showed larger decreases than did populations in tryptic soy broth and distilled water. E. coli O157:H7 cultures held for 24 h at 4°C after treatment at 550 MPa showed a significant log decrease as compared with cultures directly after treatment (P ≤ 0.05), while Salmonella serovars did not show this significant decrease (P > 0.05). All Salmonella serovars tested in orange juice treated at 550 MPa for 2 min at 6°C and held for 24 h showed a >5-log decrease, while E. coli O157:H7 strains require a higher pressure, higher temperature, longer pressurization, or a chemical additive to achieve a 5-log decrease.

Thermal Inactivation of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in Breaded Pork Patties

Decimal reduction times (D-values) and thermal resistance constants (z-values) for 3 foodborne pathogenic bacteria in formulated ready-to-eat breaded pork patties were determined with thermal inactivation studies. Meat samples, inoculated with Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes cultures or uninoculated controls, were packaged in sterile bags, immersed in circulated water bath, and held at 55, 57.5, 60, 62.5, 65, 67.5, and 70 degrees C for different durations of time. The D- and z-values were determined by using a linear regression model. Average calculated D-values for E. coli O157:H7, Salmonella, and L. monocytogenes at a temperature range of 55 to 70 degrees C were 32.11 to 0.08 min, 69.48 to 0.29 min, and 150.46 to 0.43 min, respectively. Calculated z-values for E. coli O157:H7, Salmonella, and L. monocytogenes were 5.4, 6.2, and 5.9 degrees C, respectively. The results of this study will be useful to food processors to validate thermal lethality of the studied foodborne pathogens in ready-to-eat breaded pork patties.

Effects on Escherichia coli O157:H7 and meat starter cultures of bovine lactoferrin in broth and microencapsulated lactoferrin in dry sausage batters

The effects of lactoferrin (LF) alone or with various chelating agents on the growth of 5 strains of Escherichia coli O157:H7 and 7 meat starter cultures were evaluated. E.coli O157:H7 and starter cultures were grown at 13 or 26 °C in Lauria (LB) or All Purpose Tween (APT) broths, respectively, with both broths being supplemented with 2.9% NaCl. LF alone prevented the growth of E. coli O157:H7 strains 0627 and 0628 but other strains grew. The antimicrobial effectiveness of LF was enhanced by EDTA but LF alone did not affect the growth of meat starter cultures in broth. However, when LF plus EDTA and sodium bicarbonate (SB) were used the growth of all meat starter cultures except Lactobacillus curvatus was reduced. During dry sausage manufacture with L. curvatus and Staphylococcus carnosus starter cultures the effects of LF, unencapsulated or microencapsulated in paste-like and dried powder forms, in sausage batters with or without EDTA and SB, on the viability of E. coli O157:H7 were examined. The reduction of E. coli O157:H7 during sausage manufacture was significantly enhanced ( p < 0.05) by all LF treatments. The largest reduction (4.2 log units) was obtained with unencapsulated LF. However, some of the apparent reduction in E.coli O157:H7 numbers with all treatments was due to cell injury rather than lethality, since significantly greater numbers were recovered on APT agar overlaid with the selective medium cefixime-tellurite Sorbitol McConkey agar (ct-SMAC) than on ct-SMAC alone. The narrow spectrum of LF activity and induction of injury rather than inactivation of E. coli O157:H7 limit the effectiveness of this agent against the pathogen in fermented meats.

Growth and Survival ofEscherichia coliO157:H7 During Manufacture and Storage of Indian Cheese (Paneer)

The growth and survival of Escherichia coli O157:H7 during manufacture and storage of Indian cheese or paneer was investigated. Pasteurized and raw milk that had been inoculated with 10(4) CFU/mL of E. coli O157:H7 cultures were used for manufacturing paneer samples which were vacuum-packaged and stored at 4 degrees C, 8 degrees C, and 28 degrees C. Survival and growth of E. coli O157:H7 in paneer samples was determined after every 4 h for up to 48 h. Escherichia coli O157:H7 could survive the manufacturing process of paneer and were present at the end of the storage period (at 28 degrees C) in significantly (p < 0.05) greater numbers than the initial inoculum. No significant difference in survival and growth were noticeable in paneer samples manufactured from raw milk. Though refrigeration (4 degrees C or 8 degrees C) effectively inhibited the growth of E. coli O157:H7, this pathogen could survive over a period of 48 h. Our observations suggest that unpasteurized or improperly pasteurized milk could be an important source for transmission of E. coli O157:H7 through paneer, and appropriate steps must be taken by government agencies to ensure consumer safety for this dairy product.

Impact of inoculum preparation and storage conditions on the response of Escherichia coli O157:H7 populations to undercooking and simulated exposure to gastric fluid.

This study evaluated the impact of inoculum preparation and storage conditions on the response of Escherichia coli O157:H7 exposed to consumer-induced stresses simulating undercooking and digestion. Lean beef tissue samples were inoculated with E. coli O157:H7 cultures prepared in tryptic soy broth or meat decontamination runoff fluids (WASH) or detached from moist biofilms or dried biofilms formed on stainless steel coupons immersed in inoculated WASH. After inoculation, the samples were left untreated or dipped for 30 s each in hot (75 degrees C) water followed by lactic acid (2%, 55 degrees C), vacuum packaged, stored at 4 (28 days) or 12 degrees C (16 days), and periodically transferred to aerobic storage (7 degrees C for 5 days). During storage, samples were exposed to sequential heat (55 degrees C; 20 min) and simulated gastric fluid (adjusted to pH 1.0 with HCl; 90 min) stresses simulating consumption of undercooked beef. Under the conditions of this study, cells originating from inocula of planktonic cells were, in general, more resistant to heat and acid than cells from cultures grown as biofilms and detached prior to meat inoculation. Heat and acid tolerance of cells on meat stored at 4 degrees C was lower than that of cells on nondecontaminated meat stored at 12 degrees C, where growth occurred during storage. Decontamination of fresh beef resulted in injury that inhibited subsequent growth of surviving cells at 12 degrees C, as well as in decreases in resistance to subsequent heat and acid stresses. The shift of pathogen cells on beef stored under vacuum at 4 degrees C to aerobic storage did not affect cell populations or subsequent survival after sequential exposure to heat and simulated gastric fluid. However, the transfer of meat stored under vacuum at 12 degrees C to aerobic storage resulted in reduction in pathogen counts during aerobic storage and sensitization of survivors to the effects of sequential heat and acid exposure.

The influence of an extracellular polysaccharide, comprised of colanic acid, on the fate of Escherichia coli O157:H7 during processing and storage of stirred yogurt

The goal of this study was to determine the role of an extracellular polysaccharide (EPS), comprised of colanic acid (CA), in assisting the cells of Escherichia coli O157:H7 in surviving the processing and storage of stirred yogurt. A wild-type E. coli O157:H7 strain W6–13, its CA-deficient mutant M4020, and a mixture of the two strains (1:1; WM mixture) were inoculated into pasteurized, homogenized whole milk prior to fermentation or into fermented yogurt before stirring. The yogurts made from the inoculated milk and un-inoculated controls were assayed twice a week for total plate counts, as well as the populations of E. coli O157:H7 and starter cultures on tryptic soy agar (TSA), sorbitol MacConkey agar (SMAC), as well as MRS and M17 agar, respectively, during a three-week storage period at 4 °C or 15 °C. The results indicated that the survival of W6–13 vs. M4020, W6–13 vs. the WM mixture, and M4020 vs. the WM mixture in stirred yogurt as influenced by processing and storage conditions were not statistically different ( P > 0.05 ). The stirring step during yogurt processing might have detached the EPS from the surfaces of E. coli O157:H7 cells. The protective effect of the EPS towards the cells of E. coli O15:H7 during subsequent processing and storage was therefore, not observed.

The criterion for selecting effective phage for Escherichia coli O157:H7 control

The bacteriophages SP15 and PP17, which showed strong virulency against Escherichia coli O157:H7, were isolated from activated sludge at a wastewater treatment plant and from pig feces, respectively. In liquid culture, the mixture of these phages delayed the appearance of phage-resistant E. coli O157:H7 cells longer than the addition of either phage alone. According to analysis of phage-resistant cells isolated from E. coli O157:H7 cultures infected with SP15 or PP17, it seemed that in the mixture, each phage attacked cells resistant to the other phage in complimentary manner. Phage-resistant cells existed in E. coli O157:H7 cultures at a certain ratio even before exposure to phage. This ratio, named R / T ratio (CFU-resistant/CFU-total), was determined as (2.7±0.7)×10 −7 for SP15 and (3.6±0.8)×10 −5 for PP17. The R / T ratio for the mixture of SP15 and PP17 was less than 2.0×10 −9 , vastly reduced compared to administration of a single phage, thus quantitatively demonstrating the increased antibacterial potential of phage mixtures relative to single phages. For these reasons, the R / T ratio represents a useful criterion in selecting effective phages for phage therapy.

The criterion for selecting effective phage for Escherichia coli O157:H7 control

The bacteriophages SP15 and PP17, which showed strong virulency against Escherichia coli O157:H7, were isolated from activated sludge at a wastewater treatment plant and from pig feces, respectively. In liquid culture, the mixture of these phages delayed the appearance of phage-resistant E. coli O157:H7 cells longer than the addition of either phage alone. According to analysis of phage-resistant cells isolated from E. coli O157:H7 cultures infected with SP15 or PP17, it seemed that in the mixture, each phage attacked cells resistant to the other phage in complimentary manner. Phage-resistant cells existed in E. coli O157:H7 cultures at a certain ratio even before exposure to phage. This ratio, named R/ T ratio (CFU-resistant/CFU-total), was determined as (2.7±0.7)×10 −7 for SP15 and (3.6±0.8)×10 −5 for PP17. The R/ T ratio for the mixture of SP15 and PP17 was less than 2.0×10 −9, vastly reduced compared to administration of a single phage, thus quantitatively demonstrating the increased antibacterial potential of phage mixtures relative to single phages. For these reasons, the R/ T ratio represents a useful criterion in selecting effective phages for phage therapy.

Survival of Escherichia coli O157:H7 in Set Yogurt as Influenced by the Production of an Exopolysaccharide, Colanic Acid

Previous studies conducted in our laboratory revealed that Escherichia coli O157:H7 cells capable of producing colanic acid (CA), the acidic polysaccharide of mucoid slime, had increased tolerance to sublethal heat and the extreme pH of microbiological culture media. This study was undertaken to determine the effect of CA on the fate of E. coli O157:H7 during the processing and storage of an acid food: yogurt. Pasteurized and homogenized whole milk was inoculated with a wild-type E. coli O157:H7, its CA-deficient mutant, or a mixture (1:1) of the two strains. Set yogurt was processed from the contaminated milk and stored at 4° and 15°C for 3 weeks. Samples of milk and yogurt were withdrawn during processing and storage and analyzed for total plate counts and populations of E. coli O157:H7 and starter cultures. The results showed that E. coli O157: H7 survived longer in yogurt stored at 15°C than at 4°C. Cells of E. coli O157:H7 deficient in CA production died off more rapidly than those of the parent strain. This suggests that CA plays a role in protecting cells of E. coli O157:H7 from stress during the processing and storage of set yogurt.