Growth of Stressed Strains of Four Non-O157 Shiga Toxin–Producing Escherichia coli Serogroups in Five Enrichment Broths

2015 ◽  
Vol 78 (11) ◽  
pp. 1960-1966 ◽  
Author(s):  
BAVO VERHAEGEN ◽  
KOEN DE REU ◽  
MARC HEYNDRICKX ◽  
INGE VAN DAMME ◽  
LIEVEN DE ZUTTER
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Growth Dynamics ◽  
Maximum Growth ◽  
Maximum Growth Rate ◽  
Lag Phase ◽  
Sodium Pyruvate ◽  
Peptone Water ◽  
Pure Cultures ◽  
Freeze Stress

The purpose of this study was to evaluate (i) the behavior of several strains of non-O157 Shiga toxin–producing Escherichia coli (STEC) serogroups (O26, O103, O111, and O145) exposed to different stress conditions and (ii) the growth dynamics of stressed and nonstressed non-O157 STEC cells in five enrichment media. STEC strains were exposed to acid, cold, and freeze stresses. Lethal and sublethal injuries were determined by plating in parallel on selective and nonselective agar media. Freeze stress (8 days, −20°C) caused the most lethal (95.3% ± 2.5%) injury, as well as the most sublethal (89.1% ± 8.8%) injury in the surviving population. Growth of stressed and nonstressed pure cultures of non-O157 STEC on modified tryptic soy broth, buffered peptone water (BPW), BPW with sodium pyruvate, Brila, and STEC enrichment broth (SEB) was determined using total viable counts. To compare growth capacities, growth after 7 and 24 h of enrichment was measured; lag phases and maximum growth rates were also calculated. In general, growth on BPW resulted in a short lag phase followed by a high maximum growth rate during the enrichment of all tested strains when using all three stress types. Furthermore, BPW ensured the highest STEC count after 7 h of growth. Supplementing the medium with sodium pyruvate did not improve the growth dynamics. The two selective media, Brila and SEB, were less efficient than BPW, but Brila's enrichment performance was remarkably better than that of SEB. This study shows that irrespective of the effect of background flora, BPW is still recommended for resuscitation of non-O157 STEC.

Comparison of Enrichment Broths for the Recovery of Healthy and Heat-Injured Salmonella Typhimurium on Raw Duck Wings

2013 ◽  
Vol 76 (11) ◽  
pp. 1963-1968 ◽  
Author(s):  
QIANWANG ZHENG ◽  
CAROLINE BUSTANDI ◽  
YISHAN YANG ◽  
KEITH R. SCHNEIDER ◽  
HYUN-GYUN YUK
Keyword(s):  
Growth Rate ◽  
Salmonella Typhimurium ◽  
Doubling Time ◽  
Growth Parameters ◽  
False Negative ◽  
Maximum Growth ◽  
Maximum Growth Rate ◽  
Detection Methods ◽  
Lag Phase ◽  
Peptone Water

This study was performed to optimize Salmonella Typhimurium recovery from raw duck wings with five nonselective broths (buffered peptone water, tryptic soy broth, lactose broth, universal preenrichment broth, nutrient broth) and four selective broths (selenite broth, BAX System MP media [MP], Salmonella AD media [AD], ONE broth-Salmonella [OB]). Healthy or heat-injured (50 and 85% injury) cells were inoculated at a level of 102, 101, or 100 CFU/25 g on raw duck wings. Growth was modeled using DMfit with four growth parameters: lag-phase duration, maximum growth rate, doubling time, and maximum population density. Most enrichments were able to recover Salmonella Typhimurium to greater than 6 log CFU/ml. AD, MP, and OB had significantly (P < 0.05) higher maximum growth rate (0.9 to 1.0/h) and lower doubling time (0.7 to 0.8 h). Buffered peptone water, AD, MP, and OB recovered healthy and 50%-injured cells at low inoculum levels to more than 6.0 log CFU/ml; OB achieved the greatest recovery (7.6 and 7.9 log CFU/ml), following 24 h of incubation. The 85%-injured cells at 100 and 101 CFU/25 g, however, were only recovered in OB, reaching 7.3 and 7.5 log CFU/ml, respectively. These results suggest that OB may be an appropriate enrichment broth for the recovery of Salmonella Typhimurium from raw duck wings in standard diagnostic tests or other rapid detection methods, to avoid false-negative results.

Simultaneous Enrichment of Shiga Toxin–Producing Escherichia coli O157 and O26 and Salmonella in Food Samples Using Universal Preenrichment Broth

2009 ◽  
Vol 72 (10) ◽  
pp. 2065-2070 ◽  
Author(s):  
MASASHI KANKI ◽  
KAZUKO SETO ◽  
JUNKO SAKATA ◽  
TETSUYA HARADA ◽  
YUKO KUMEDA
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Food Samples ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Significant Difference ◽  
Peptone Water ◽  
Radish Sprouts ◽  
Pork Samples ◽  
Better Than

Universal preenrichment broth (UPB) was compared with modified Escherichia coli broth with novobiocin (mEC+n) for enrichment of Shiga toxin–producing E. coli O157 and O26, and with buffered peptone water (BPW) for preenrichment of Salmonella enterica. Ten strains each of the three pathogens were inoculated into beef and radish sprouts following thermal, freezing, or no treatment. With regard to O157 and O26, UPB incubated at 42°C recovered significantly more cells from inoculated beef than UPB at 35°C and from radish sprout samples than UPB at 35°C and mEC+n. With regard to Salmonella, UPB incubated at 42°C was as effective as UPB at 35°C and BPW at recovering cells from beef and radish sprout samples. No significant difference was noted between the effectiveness of UPB at 42°C and UPB at 35°C or BPW in the recovery of Salmonella from 205 naturally contaminated poultry samples. By using UPB at 42°C, one O157:H7 strain was isolated from the mixed offal of 53 beef samples, 6 cattle offal samples, and 50 pork samples all contaminated naturally, with no pathogen inoculation. The present study found that UPB incubated at 42°C was as effective as, or better than, mEC+n for enrichment of O157 and O26 and comparable to BPW for preenrichment of Salmonella. These findings suggest that a great deal of labor, time, samples, and space may be saved if O157, O26, and Salmonella are enriched simultaneously with UPB at 42°C.

Evaluation of Three Commercially Available Enzyme-Linked Immunosorbent Assay Kits for Detection of Shiga Toxin

2009 ◽  
Vol 72 (4) ◽  
pp. 741-747 ◽  
Author(s):  
JOHN WILLFORD ◽  
KENNETH MILLS ◽  
LAWRENCE D. GOODRIDGE
Keyword(s):  
Escherichia Coli ◽  
Immunosorbent Assay ◽  
Shiga Toxin ◽  
Screening Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Microplate Assay ◽  
E Coli ◽  
Elisa Kit ◽  
Pure Cultures ◽  
Order Of Magnitude

Three commercially available Shiga toxin (Stx) enzyme-linked immunosorbent assay (ELISA) kits were evaluated for their ability to detect Stx in pure cultures of Stx-producing Escherichia coli (specificity). The detection limits (sensitivity) of each ELISA kit were also evaluated. Seventy-eight Stx-producing E. coli (STEC) isolates that produced Stx1, Stx2, or Stx1 and Stx2 variants were examined in this study. The specificities of the tests were comparable, and the sensitivities of two of the tests (Premier EHEC and rBiopharm Ridascreen Verotoxin Enzyme Immunoassay) were within the same order of magnitude. The ProSpecT Shiga Toxin E. coli Microplate Assay was approximately 10-fold less sensitive. The inability of all three tests to detect the Stx2d and Stx2e variants indicated that some STEC strains may not be detected by Stx ELISA. The ability of the Premier EHEC ELISA to detect toxin in artificially inoculated bovine fecal samples (following enrichment) indicated that this kit may be used to screen cattle for the presence of Stx as an indicator of the presence of STEC. In particular, such a screening method could be useful during the summer, when the number of STEC-positive animals and the number of STEC that they shed increase.

An Automated Fluorescent PCR Method for Detection of Shiga Toxin-Producing Escherichia coli in Foods

1998 ◽  
Vol 64 (11) ◽  
pp. 4210-4216 ◽  
Author(s):  
Shu Chen ◽  
Renlin Xu ◽  
Arlene Yee ◽  
Kai Yuan Wu ◽  
Chang-Ning Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Raw Milk ◽  
Clinical Samples ◽  
Pcr Assay ◽  
System A ◽  
Pure Cultures ◽  
Pcr Method ◽  
Meat Samples ◽  
Shiga Toxin Genes

ABSTRACT An automated fluorescence-based PCR system (a model AG-9600 AmpliSensor analyzer) was investigated to determine whether it could detect Shiga toxin-producing Escherichia coli (STEC). The AmpliSensor PCR assay involves amplification-mediated disruption of a fluorogenic DNA signal duplex (AmpliSensor) that is homologous to conserved target sequences in a 323-bp amplified fragment of Shiga toxin genes stx 1, stx 2, and stx e. Using the Amplisensor assay, we detected 113 strains of STEC belonging to 50 different serotypes, while 18 strains of non-Shiga-toxin-producing E. coli and 68 strains of other bacteria were not detected. The detection limits of the assay were less than 1 to 5 CFU per PCR mixture when pure cultures of five reference strains were used and 3 CFU per 25 g of food when spiked ground beef samples that were preenriched overnight were used. The performance of the assay was also evaluated by using 53 naturally contaminated meat samples and 48 raw milk samples. Thirty-two STEC-positive samples that were confirmed to be positive by the culture assay were found to be positive when the AmpliSensor assay was used. Nine samples that were found to be positive when the PCR assay was used were culture negative. The system described here is an automated PCR-based system that can be used for detection of all serotypes of STEC in food or clinical samples.

Pooling of Immunomagnetic Separation Beads Does Not Affect Detection Sensitivity of Six Major Serogroups of Shiga Toxin–Producing Escherichia coli in Cattle Feces†

2016 ◽  
Vol 79 (1) ◽  
pp. 59-65 ◽  
Author(s):  
LANCE W. NOLL ◽  
WILLIAM C. BAUMGARTNER ◽  
PRAGATHI B. SHRIDHAR ◽  
CHARLEY A. CULL ◽  
DIANA M. DEWSBURY ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Foodborne Pathogens ◽  
Shiga Toxin ◽  
Fecal Sample ◽  
Food Product ◽  
Immunomagnetic Separation ◽  
Detection Sensitivity ◽  
Fecal Samples ◽  
Affect Detection ◽  
Pure Cultures

ABSTRACT Shiga toxin–producing Escherichia coli (STEC) of the serogroups O26, O45, O103, O111, O121, and O145, often called non-O157 STEC, are foodborne pathogens. Cattle are asymptomatic reservoirs for STEC; the organisms reside in the hindgut and are shed in the feces, which serve as the source of food product contaminations. Culture-based detection of non-O157 STEC involves an immunomagnetic separation (IMS) step to capture the specific serogroups in complex matrices, such as feces. The IMS procedure is time consuming and labor intensive because of the need to subject each fecal sample to six individual beads. Therefore, our objective was to evaluate whether pooling of IMS beads affects sensitivity of non-O157 STEC detection compared with using individual IMS beads. The evaluation was done by comparing detection of serogroups in feces spiked with pure cultures (experiments 1 and 2) and from feces (n = 384) of naturally shedding cattle (experiment 3). In spiked fecal samples, detection with pools of three, four, six, or seven beads was similar to, or at times higher than, detection with individual IMS beads. In experiment 3, the proportions of fecal samples that tested positive for the six serogroups as detected by individual or pooled beads were similar. Based on noninferiority tests, detection with pooled beads was not substantially inferior to detection with individual beads (P < 0.05). In conclusion, the pooling of IMS beads is a better option for detection of STEC serogroups in fecal samples compared with individual beads because the procedure saves time and labor and has the prospect of a higher throughput.

Benefits and effectiveness of high pressure processing on cheese: a ricotta case study

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Roberta Stefanini ◽  
Anna Ronzano ◽  
Giulia Borghesi ◽  
Giuseppe Vignali
Keyword(s):  
High Pressure ◽  
Microbial Growth ◽  
Maximum Growth ◽  
Maximum Growth Rate ◽  
High Pressure Processing ◽  
Lag Phase ◽  
Starting Point ◽  
Volatile Organic ◽  
Parmigiano Reggiano

Abstract Today High Pressure Processing (HPP) is receiving interest thanks to its ability to stabilize foods preserving nutritional and sensorial characteristics. This work applies HPP on nutrient ricottas created in the Parmigiano Reggiano area and demonstrates not only its benefits, but also disadvantages, testing different pressures and packaging. Moreover, the ability of HPP to prolong the lag phase and reduce the maximum growth rate of bacteria is illustrated with a mathematical model. Results show the influence of HPP parameters on microbial growth, volatile organic compounds, syneresis, softness and colour, and demonstrate that not all packaging are suitable for the treatment. Obtained data highlight the effectiveness of HPP, which results the best stabilization method to sell safe and nutritive ricottas on the market with a long shelf life. Of course, the work can be a starting point for food companies who want to test an innovative and promising non-thermal technology.

scholarly journals Expression of a Temperature-Sensitive Esterase in a Novel Chaperone-Based Escherichia coli Strain

2004 ◽  
Vol 70 (8) ◽  
pp. 4499-4504 ◽  
Author(s):  
Manuel Ferrer ◽  
Tatyana N. Chernikova ◽  
Kenneth N. Timmis ◽  
Peter N. Golyshin
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Growth Temperature ◽  
Specific Activity ◽  
Maximum Growth ◽  
Maximum Growth Rate ◽  
Coli Strain ◽  
Temperature Sensitive ◽  
Psychrophilic Bacterium ◽  
E Coli

ABSTRACT A new principle for expression of heat-sensitive recombinant proteins in Escherichia coli at temperatures close to 4°C was experimentally evaluated. This principle was based on simultaneous expression of the target protein with chaperones (Cpn60 and Cpn10) from a psychrophilic bacterium, Oleispira antarctica RB8T, that allow E. coli to grow at high rates at 4°C (maximum growth rate, 0.28 h−1) (M. Ferrer, T. N. Chernikova, M. Yakimov, P. N. Golyshin, and K. N. Timmis, Nat. Biotechnol. 21:1266-1267, 2003). The expression of a temperature-sensitive esterase in this host at 4 to 10°C yielded enzyme specific activity that was 180-fold higher than the activity purified from the non-chaperonin-producing E. coli strain grown at 37°C (32,380 versus 190 μmol min−1 g−1). We present evidence that the increased specific activity was not due to the low growth temperature per se but was due to the fact that low temperature was beneficial to folding, with or without chaperones. This is the first report of successful use of a chaperone-based E. coli strain to express heat-labile recombinant proteins at temperatures below the theoretical minimum growth temperature of a common E. coli strain (7.5°C).

Growth of a Methane-Utilizing Mixed Culture HD6T on Methanol and Poly-β-Hydroxybutyrate Biosynthesis

2010 ◽  
Vol 160-162 ◽  
pp. 171-175 ◽  
Author(s):  
Jing Dong ◽  
Jia Ying Xin ◽  
Ying Xin Zhang ◽  
Lin Lin Chen ◽  
Hong Ye Liang ◽  
...  
Keyword(s):  
Mixed Culture ◽  
Mineral Salt Medium ◽  
Biomass Concentration ◽  
Dry Weight ◽  
Maximum Growth ◽  
Maximum Growth Rate ◽  
Production Costs ◽  
Lag Phase ◽  
Phase Duration ◽  
Phb Production

Methane-utilizing mixed culture HD6T was successfully cultivated in a brief non-sterile process using methanol as a sole carbon and energy source for the production of poly-β-hydroxybutyrate(PHB). Shake-flask experiments showed HD6T could grow well in the mineral salt medium with the addition of methanol exposed to the air directly. This non-sterile process and the use of cheap substrates (methanol) can reduce the production costs of PHB. It was found that HD6T grew better and PHB production in a more effective way with an initial liquid methanol concentration of 0.15%(v/v).The lag phase duration, the maximum growth rate, the biomass concentration and the PHB yield, for the optimal conditions were, respectively, 12.03h, 0.04h-1(OD600), 1.54g/l(dry weight), 0.424g/l(dry weight). Methane-utilizing mixed culture HD6T appears to be a promising organism for PHB production.

Quantitative relationships for specific growth rates and macromolecular compositions of Mycobacterium tuberculosis, Streptomyces coelicolor A3(2) and Escherichia coli B/r: an integrative theoretical approach

Microbiology ◽  
2004 ◽  
Vol 150 (5) ◽  
pp. 1413-1426 ◽  
Author(s):  
Robert A. Cox
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Mycobacterium Tuberculosis ◽  
Growth Rates ◽  
Specific Growth ◽  
Streptomyces Coelicolor ◽  
Maximum Growth ◽  
Maximum Growth Rate ◽  
E Coli ◽  
Specific Growth Rates

Further understanding of the physiological states of Mycobacterium tuberculosis and other mycobacteria was sought through comparisons with the genomic properties and macromolecular compositions of Streptomyces coelicolor A3(2), grown at 30 °C, and Escherichia coli B/r, grown at 37 °C. A frame of reference was established based on quantitative relationships observed between specific growth rates (μ) of cells and their macromolecular compositions. The concept of a schematic cell based on transcription/translation coupling, average genes and average proteins was developed to provide an instantaneous view of macromolecular synthesis carried out by cells growing at their maximum rate. It was inferred that the ultra-fast growth of E. coli results from its ability to increase the average number of rRNA (rrn) operons per cell through polyploidy, thereby increasing its capacity for ribosome synthesis. The maximum growth rate of E. coli was deduced to be limited by the rate of uptake and consumption of nutrients providing energy. Three characteristic properties of S. coelicolor A3(2) growing optimally (μ=0·30 h−1) were identified. First, the rate of DNA replication was found to approach the rate reported for E. coli (μ=1·73 h−1); secondly, all rrn operons were calculated to be fully engaged in precursor-rRNA synthesis; thirdly, compared with E. coli, protein synthesis was found to depend on higher concentrations of ribosomes and lower concentrations of aminoacyl-tRNA and EF-Tu. An equation was derived for E. coli B/r relating μ to the number of rrn operons per genome. Values of μ=0·69 h−1 and μ=1·00 h−1 were obtained respectively for cells with one or two rrn operons per genome. Using the author's equation relating the number of rrn operons per genome to maximum growth rate, it is expected that M. tuberculosis with one rrn operon should be capable of growing much faster than it actually does. Therefore, it is suggested that the high number of insertion sequences in this species attenuates growth rate to still lower values.

Limitations of Immunomagnetic Separation for Detection of the Top Seven Serogroups of Shiga Toxin–Producing Escherichia coli

2017 ◽  
Vol 80 (4) ◽  
pp. 598-603 ◽  
Author(s):  
J. Hallewell ◽  
T. Alexander ◽  
T. Reuter ◽  
K. Stanford
Keyword(s):  
Escherichia Coli ◽  
Quantitative Pcr ◽  
Foodborne Pathogens ◽  
Shiga Toxin ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Conventional Pcr ◽  
E Coli ◽  
Pure Cultures

ABSTRACT Shiga toxin–producing Escherichia coli (STEC) strains are foodborne pathogens that negatively impact human health and compromise food safety. Serogroup O157 is the most frequently isolated and studied STEC serogroup, but six others (O26, O45, O103, O111, O121, and O145) have also been identified as significant sources of human disease and collectively have been referred to as the “top six” pathogenic serogroups. Because detection methods for non-O157 serogroups are not yet refined, the objective of this study was to compare the effectiveness of immunomagnetic separation (IMS) for recovery of serogroup O157 isolates with that for each of the top six E. coli serogroups in pure and mixed cultures of STEC at 103 to 107 CFU/mL. After serogroup-specific IMS, DNA was extracted from cultured isolates to analyze the specificity of each IMS assay using conventional and quantitative PCR. In pure cultures, DNA copy number obtained after IMS was lower for O111 and O157 (P < 0.01) than for other serogroups. Based on quantitative PCR (qPCR) analyses, specificity was reduced for all IMS assays when STEC isolates were mixed at 7 log CFU/mL, although the O157 IMS assays recovered only O157 over a wider range of concentrations than did assays for non-O157 serogroups. At the lowest dilution tested, conventional PCR was specific for all serogroups except O121 and O145. For these two serogroups, no dilution tested recovered only O121 or O145 when evaluated with conventional PCR. Refinements to IMS assays, development of selective media, and determination of optimal enrichment times to reduce background microflora or competition among serogroups would be especially beneficial for recovery of O111, O121, and O145 serogroups to improve STEC detection and isolation.

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