Inactivation of Escherichia coli O157:H7 on stainless steel upon exposure to Paenibacillus polymyxa biofilms

We investigated the potential use of biofilm formed by a competitive-exclusion (CE) microorganism to inactivate Escherichia coli O157:H7 on a stainless steel surface. Five microorganisms showing inhibitory activities against E. coli O157:H7 were isolated from vegetable seeds and sprouts. The microorganism with the greatest antimicrobial activity was identified as Paenibacillus polymyxa (strain T5). In tryptic soy broth (TSB), strain T5 reached a higher population at 25 °C than at 12 or 37 °C without losing inhibitory activity against E. coli O157:H7. When P. polymyxa (6 log CFU/mL) was co-cultured with E. coli O157:H7 (2, 3, 4, or 5 log CFU/mL) in TSB at 25 °C, the number of E. coli O157:H7 decreased significantly within 24 h. P. polymyxa formed a biofilm on stainless steel coupons (SSCs) in TSB at 25 °C within 24 h, and cells in biofilms, compared to attached cells without biofilm formation, showed significantly increased resistance to a dry environment (43% relative humidity [RH]). With the exception of an inoculum of 4 log CFU/coupon at 100% RH, upon exposure to biofilm formed by P. polymyxa on SSCs, populations of E. coli O157:H7 (2, 4, or 6 log CFU/coupon) were significantly reduced within 48 h. Most notably, when E. coli O157:H7 at 2 log CFU/coupon was applied to SSCs on which P. polymyxa biofilm had formed, it was inactivated within 1 h, regardless of RH. These results will be useful when developing strategies using biofilms produced by competitive exclusion microorganisms to inactivate foodborne pathogens in food processing environments.     

Effect of X-ray treatments on inoculated Escherichia coli O157: H7, Salmonella enterica, Shigella flexneri and Vibrio parahaemolyticus in ready-to-eat shrimp

This study was conducted to evaluate the inactivation effect of X-ray treatments on Escherichia coli O157: H7, Salmonella enteric (S. enterica), Shigella flexneri (S. flexneri) and Vibrio parahaemolyticus (V. parahaemolyticus) artificially inoculated in ready-to-eat (RTE) shrimp. A mixed culture of three strains of each tested pathogen was used to inoculate RTE shrimp. The shrimp samples were inoculated individually with selected pathogenic bacteria then aseptically placed in sterile plastic cups and air-dried at 22 °C for 30 min (to allow bacterial attachment) in the biosafety cabinet prior to X-ray treatments. The inoculated shrimp samples were then placed in sterilized bags and treated with 0.1, 0.2, 0.3, 0.5, 0.75, 1.0, 2.0, 3.0 and 4.0 kGy X-ray at ambient temperature (22 °C and 60% relative humidity). Surviving bacterial populations were evaluated using a non-selective medium (TSA) with the appropriate selective medium overlay for each bacterium; CT-SMAC agar for E. coli O157: H7, XLD for S. enterica and S. flexneri and TCBS for V. parahaemolyticus. More than a 6 log CFU reduction of E. coli O157: H7, S. enterica, S. flexneri and V. parahaemolyticus was achieved with 2.0, 4.0, 3.0 and 3.0 kGy X-ray, respectively. Furthermore, treatment with 0.75 kGy X-ray significantly reduced the initial microflora on RTE shrimp samples from 3.8 ± 0.2 log CFU g⁻¹ to less than detectable limit (<1.0 log CFU g⁻¹).     

Assessment of bacterial biofilm on stainless steel by hyperspectral fluorescence imaging

Hyperspectral fluorescence imaging techniques were investigated for detection of two genera of microbial biofilms on stainless steel material which is commonly used to manufacture food processing equipment. Stainless steel coupons were deposited in nonpathogenic E. coli O157:H7 and Salmonella cultures, prepared using M9 minimal medium with casamino acids (M9C), for 6 days at 37 °C. Hyperspectral fluorescence emission images of the biofilm formations on the stainless coupons were acquired from 416 to 700 nm with the use of ultraviolet-A (320–400 nm) excitation. In general, emission peaks for both bacteria were observed in the blue region at approximately 480 nm and thus provided the highest contrast between the biofilms and background stainless steel coupons. A simple thresholding of the 480 nm image showed significantly larger biofilm regions for E. coli O157:H7 than for Salmonella. Viable cell counts suggested that Salmonella formed significantly higher density biofilm regions than E. coli O157:H7 in M9C medium. On the basis of principal component analysis (PCA) of the hyperspectral fluorescence images, the second principal component image exhibited the most distinguishable morphological differences for the concentrated biofilm formations between E. coli and Salmonella. E. coli formed granular aggregates of biofilms above the medium on stainless steel while Salmonella formed dense biofilm in the medium-air interface region (pellicle). This investigation demonstrated the feasibility of implementing fluorescence imaging techniques to rapidly screen large surface areas of food processing equipment for bacterial contamination. Company and product names are used for clarity and do not imply any endorsement by USDA to the exclusion of other comparable products.     

Temperature and NaCl Affect Growth and Survival of Escherichia coli 0157:H7 in Poultry-Based and Laboratory Media

In tryptic soy broth (TSB) and a poultry extract broth (PB) with 0 to 10% (w/v) NaCl incubated at 37°C, growth of E. coli 0157:H7 was inhibited at 28% NaCl whereas at 10°C, growth was inhibited at 24% NaCl in TSB and at 26% NaCl in PB. The bacterium did not grow at 4°C. Increased NaCl-sensitivity observed at 10°C was a bacteriostatic effect that was ineffective with increasing incubation temperature. At 10°C, E. coli 0157:H7 was more salt-tolerant in PB than in TSB, although PB growth rates were lower. Findings suggest that PB may be a more suitable medium for testing E. coli 0157:H7 in poultry products. Cells of E. coli 0157:H7 that were exposed to refrigeration (4°C) and/or NaCl for 24 days did not grow on MacConkey agar with 1% sorbitol.     

Stress resistance of biofilm and planktonic Lactobacillus plantarum subsp. plantarum JCM 1149

The purpose of this study was to investigate the change in resistance of biofilm and planktonic food spoilage lactic acid bacteria (LAB) to environmental stresses, which strongly inhibit bacterial growth and are important in food preservation or in disinfection. The stress responses of biofilm and planktonic cells of Lactobacillus plantarum subsp. plantarum JCM 1149, which was used as a model spoilage bacterium, in various organic acids (namely, acetic acid, citric acid, lactic acid, and malic acid), ethanol, and sodium hypochlorite, were investigated using survival tests. The bacterial cells in biofilms showed greater resistance to all treatments than the planktonic bacterial cells in either the stationary or logarithmic phase. The planktonic bacterial cells showed reduced resistance to acetic acid after the cell suspension was diluted; however, intriguingly, the bacterial cells in biofilms maintained their resistance to acetic acid even after they were suspended or the cell suspension was diluted. These findings suggested the risk for food spoilage due to LAB derived from biofilms and suspended or diluted in foods, and demonstrated the importance of controlling biofilms of LAB in the food industry.     

Behavior of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium in teewurst, a raw spreadable sausage

The fate of Listeria monocytogenes, Salmonella Typhimurium, or Escherichia coli O157:H7 were separately monitored both in and on teewurst, a traditional raw and spreadable sausage of Germanic origin. Multi-strain cocktails of each pathogen (ca. 5.0 log CFU/g) were used to separately inoculate teewurst that was subsequently stored at 1.5, 4, 10, and 21 °C. When inoculated into commercially-prepared batter just prior to stuffing, in general, the higher the storage temperature, the greater the lethality. Depending on the storage temperature, pathogen levels in the batter decreased by 2.3 to 3.4, ca. 3.8, and 2.2 to 3.6 log CFU/g for E. coli O157:H7, S. Typhimurium, and L. monocytogenes, respectively, during storage for 30 days. When inoculated onto both the top and bottom faces of sliced commercially-prepared finished product, the results for all four temperatures showed a decrease of 0.9 to 1.4, 1.4 to 1.8, and 2.2 to 3.0 log CFU/g for E. coli O157:H7, S. Typhimurium, and L. monocytogenes, respectively, over the course of 21 days. With the possible exceptions for salt and carbohydrate levels, chemical analyses of teewurst purchased from five commercial manufacturers revealed only subtle differences in proximate composition for this product type. Our data establish that teewurst does not provide a favourable environment for the survival of E. coli O157:H7, S. Typhimurium, or L. monocytogenes inoculated either into or onto the product.     

Treatment of Escherichia coli O157:H7 with lactic acid, neutralized electrolyzed oxidizing water and chlorine dioxide followed by growth under sub-optimal conditions of temperature, pH and modified atmosphere

The utilization of sub-lethal decontamination treatments gains more and more interest due to the increased consumers' demand for fresh, minimally processed and convenient food products. These products rely on cold chain and hurdle (combination) technology to provide microbiological safety and quality during their shelf life. To investigate the ability of surviving cells to resuscitate and grow in a food simulating environment, sub-lethal decontamination treatments were coupled with subsequent storage under sub-optimal growth conditions. For this purpose chlorine dioxide (ClO₂) and neutralized electrolyzed oxidizing water (NEW)-treated cultures of Escherichia coli O157:H7 were inoculated in TSB-YE of pH 5.8 and a_w 0.99, and stored at 10 °C, 12.5 °C and 15 °C, under four different atmospheres (0%, 30% and 60% CO₂ balanced with N₂, and air). Due to the severity of injury, lactic acid-treated cells were inoculated in TSB-YE pH 7.0. Data obtained reveal that the fraction of sub-lethally injured E. coli O157:H7 undergoes an additional inhibitory effect during the storage period under of sub-optimal conditions. Observed extension in the lag growth phase was a direct consequence prior sub-lethal injury. The effects of liquid ClO₂ and NEW were less pronounced in comparison to lactic acid. The current study signifies the potential utilization of appropriate combination of different extrinsic and intrinsic factors in the elimination or growth inhibition of food-borne pathogens.     

Production of biofilm and quorum sensing by Escherichia coli O157:H7 and its transfer from contact surfaces to meat,poultry, ready-to-eat deli,and produce products

Multistate outbreaks of Escherichia coli O157:H7 infections through consumption of contaminated foods including produce products have brought a great safety concern. The objectives of this study were to determine the effect of biofilm and quorum sensing production on the attachment of E. coli O157:H7 on food contact surfaces and to evaluate the transfer of the pathogen from the food contact to various food products. E. coli O157:H7 produced maximum levels of AI-2 signals in 12 h of incubation in tested meat, poultry, and produce broths and subsequently formed strong biofilm in 24 h of incubation. In general, E. coli O157:H7 formed stronger biofilm on stainless steel than glass. Furthermore, E. coli O157:H7 that had attached on the surface of stainless steel was able to transfer to meat, poultry, ready-to-eat deli, and produce products. Strong attachment of the transferred pathogen on produce products (cantaloupe, lettuce, carrot, and spinach) was detected (>10³ CFU/cm²) even after washing these products with water. Our findings suggest that biofilm formation by E. coli O157:H7 on food contact surfaces can be a concern for efficient control of the pathogen particularly in produce products that require no heating or cooking prior to consumption.     

Assessment of the microbiological safety of salad vegetables and sauces from kebab take-away restaurants in the United Kingdom

The purpose of this study was to establish the microbiological safety of salad vegetables and sauces served in kebab take-away restaurants. Comparison with published microbiological guidelines revealed that 4.7% of 1213 salad vegetable samples were of unsatisfactory microbiological quality due to Escherichia coli and/or Staphylococcus aureus levels at ≥10² cfu g⁻¹. Another 0.3% of salad samples were of unacceptable quality due to S. aureus at ≥10⁴ cfu g⁻¹ (2 samples) or the presence of Salmonella Kentucky (1 sample). Cucumber was the most contaminated salad vegetable with regards to unsatisfactory levels of E. coli (6.0%) or S. aureus (4.5%). Five percent of 1208 sauce samples were of unsatisfactory microbiological quality due to E. coli, S. aureus at ≥10² cfu g⁻¹ and/or Bacillus cereus and other Bacillus spp. at ≥10⁴ cfu g⁻¹. A further 0.6% of sauce samples were of unacceptable quality due to Bacillus spp. (Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis) at ≥10⁵ cfu g⁻¹ or the presence of Salmonella Agbeni (1 sample). More samples of chilli sauce (8.7%) were of unsatisfactory or unacceptable microbiological quality than any other sauce types. The results emphasize the need for good hygiene practices in kebab take-away restaurants handling these types of ready-to-eat products.     

Radio frequency electric fields processing of orange juice

The non-thermal process of radio frequency electric fields (RFEF) has been shown to inactivate bacteria in apple juice at moderately low temperatures, but has yet to be extended to inactivate bacteria in orange juice. An 80 kW RFEF pasteurizer was used to process pulp-free orange juice at flow rates of 1.0 and 1.4 l/min. Escherichia coli K12 in orange juice was exposed to electric field strengths of 15 and 20 kV/cm at frequencies of 21, 30, and 40 kHz. Ascorbic acid (Vitamin C) content and color of the juice before and after treatment were analyzed. Electrical energy costs were calculated using the measured voltage and current. An energy balance was performed using the inlet and outlet temperatures. Processing at an outlet temperature of 65 °C reduced the population of E. coli by 3.3 log relative to the control. Increasing the treatment time and temperature and decreasing the frequency enhanced the level of inactivation. Varying the electric field strength over the range of conditions used had no effect on the inactivation. No loss in ascorbic acid or enzymatic browning was observed due to RFEF processing. The electrical energy determined using the voltage and current was 180 J/ml. This was in good agreement with the energy calculated using the temperature data. The electrical cost was $0.0026/l of orange juice. The results provided the first evidence that the RFEF process inactivates bacteria in orange juice at moderately low temperatures.

Industrial relevance

The RFEF process has been shown to inactivate E. coli in apple juice at moderately low temperatures, but has yet to be extended to inactivate bacteria in orange juice. An RFEF pilot plant pasteurizer was used to process orange juice at rates of up to 1.4 l/min. RFEF processing reduced the population of E. coli by 99.3% at 60 °C and a hold time of 3 s, whereas conventional heating at the same conditions had no effect on the E. coli. This work demonstrated that the non-thermal RFEF process can be extended to inactivate bacteria in orange juice.     

Effect of growing bacteria isolated from food on staphylococcal growth and thermonuclease activity

The effect of growing Bacillus subtilis, Streptococcus faecalis var. liquefaciens, Escherichia coli and Lactobacillus plantarum on staphylococcal growth and thermonuclease (TNase) activity was investigated in liquid media and in foods. Growth of S. aureus at 37°C for 24 h under aerobic conditions was not inhibited by the four test strains. However, staphylococcal TNase activity decreased by 70 and 80% in the presence of B. subtilis and S. faecalis var. liquefaciens respectively. Staphylococcal growth and TNase activity were strongly inhibited by L. plantarum under anaerobic conditions at pH 5.5 but not at pH 7.0. Furthermore, optimal TNase production by S. aureus occurred in cooked meat medium containing 0.5 to 5.0% NaCl. TNase production significantly decreased at higher concentrations of NaCl. In the presence of B. subtilis and S. faecalis var. liquefaciens. TNase activity decreased at NaCl levels of 0.5 to 5.0% but not at NaCl concentrations>5.0%. TNase activity was also inhibited by growing B. subtilis and S. faecalis var. Liquefaciens at pH 5.0 to 7.0. The rate of inhibition increased with increasing pH. TNase activity was not inhibited after 48 h incubation at 20° in the presence of B. subtilis and S. faecalis var. liquefaciens but significant inactivation could be demonstrated at 25° to 37°C. The results obtained with artificially contaminated, sterile food samples were similar to those obtained with brain-heart infusion broth, but the degree of decrease in TNase activity in food was much lower than that in brain-heart infusion broth.     

Occurrence, virulence genes and antibiotic resistance of Escherichia coli O157 isolated from raw bovine, caprine and ovine milk in Greece

The examination of 2005 raw bovine (n = 950), caprine (n = 460) and ovine (n = 595) bulk milk samples collected throughout several regions in Greece for the presence of Escherichia coli serogroup O157 resulted in the isolation of 29 strains (1.4%) of which 21 were isolated from bovine (2.2%), 3 from caprine (0.7%) and 5 from ovine (0.8%) milk. Out of the 29 E. coli O157 isolates, only 12 (41.4%) could be classified as Shiga-toxigenic based on immunoassay and PCR results. All 12 Shiga-toxigenic E. coli serogroup O157 isolates belonged to the E. coli O157:H7 serotype. All except one of the 12 Shiga-toxin positive isolates were stx₂-positive, five of which were also stx₁-positive. The remaining isolate was positive only for the stx₁ gene. All stx-positive isolates (whether positive for stx₁, stx₂ or stx₁ and stx₂) were also PCR-positive for the eae and ehxA genes. The remaining 17 E. coli O157 isolates (58.6%) were negative for the presence of the H7 flagellar gene by PCR, tested negative for Shiga-toxin production both by immunoassay and PCR, and among these, only four and three strains were PCR-positive for the eae and ehxA genes, respectively. All 29 E. coli O157 isolates displayed resistance to a wide range of antimicrobials, with the stx-positive isolates being, on average, resistant to a higher number of antibiotics than those which were stx-negative.     

Radiation sensitivity of planktonic and biofilm‐associated Shigella spp. and Aeromonas spp. on food and food‐contact surfaces

Although ionising radiation has been shown to kill human pathogens Shigella spp. and Aeromonas spp. on various food products, there is lack of information regarding the relative efficacy of gamma radiation against their free‐living planktonic and biofilm‐associated cells. The radiation sensitivity (D₁₀ values) of planktonic, glass‐ and carrot‐associated biofilm cells of Shigella spp. and Aeromonas spp. was determined by forming biofilms on sterile glass and carrot surfaces, incubated at 37 °C (Shigella spp.) and 30 °C (Aeromonas spp.) for 48 h. No significant difference in the D₁₀ values of planktonic and glass‐associated biofilm cells of Shigella spp. and Aeromonas spp. was observed. However, significant increase in the D₁₀ values of carrot‐associated biofilm cells as compared to planktonic and glass‐associated biofilm cells of Shigella spp. and A. hydrophila A331 was observed, whereas A. salmonicida Y567 showed insignificant difference. SEM analysis further validated the formation of biofilm on the carrot and glass surfaces. The antimicrobial effectiveness of ionising radiation against both Shigella spp. and Aeromonas spp. is affected by growth form, strain and nature of attachment surface.     

Non-thermal pasteurization of fruit juices by combining high-intensity pulsed electric fields with natural antimicrobials

The effect of high-intensity pulsed electric fields (HIPEF) on the Salmonella Enteritidis and Escherichia coli O157:H7 populations inoculated in apple, pear, orange and strawberry juices as influenced by treatment time and pulse frequency was investigated. Combinations of HIPEF (35 kV/cm, 4 μs pulse length in bipolar mode without exceeding 40 °C) with citric acid or cinnamon bark oil against these pathogenic microorganisms in fruit juices were also evaluated. Treatment time was the more influential factor on the microbial reduction in all the fruit juices analyzed. S. Enteritidis and E. coli O157:H7 were reduced by more than 5.0 log₁₀ units in orange juice treated by only HIPEF; whereas strawberry, apple and pear juices were pasteurized when HIPEF was combined with citric acid at 0.5, 1.5, 1.5%, respectively, or cinnamon bark oil at 0.05, 0.1 and 0.1%, respectively. Synergistic and additive killing effects against S. Enteritidis and E. coli O157:H7 in fruit juices by combining treatments were observed.

Industrial relevance

The use of high-intensity pulsed electric fields treatment as a non-thermal pasteurization method in combination with organic acids or essential oils is an effective process for eliminating S. Enteritidis and E. coli O157:H7 populations in fruit juices upper 5.0 log₁₀ reductions. Therefore, combinations of those treatments may help to ensure the microbiological safety in juice products, and to reduce the risk of food-borne illness caused by the consumption of these kinds of foods.     

Tunisian Salvia officinalis L. and Schinus molle L. essential oils: their chemical compositions and their preservative effects against Salmonella inoculated in minced beef meat

The essential oils (EOs) extracted from the aerial parts of cultivated Salvia officinalis L. and the berries of Schinus molle L. were analysed by gas chromatography–mass spectrometry (GC–MS) and 68 and 67 constituents were identified, respectively. The major constituents were 1,8-cineole (33.27%), β-thujone (18.40%), α-thujone (13.45%), borneol (7.39%) in S. officinalis oil and α-phellandrene (35.86%), β-phellandrene (29.3%), β-pinene (15.68%), p-cymene (5.43%) and α-pinene (5.22%) in S. molle oil.In its second part, the present study was conducted to evaluate the in vitro antimicrobial activity of both studied EOs. For this purpose, paper disc-diffusion method and broth microdilution test were used. The disc-diffusion method showed significant zone of lysis against all the pathogens studied (gram-negative and gram-positive bacteria, yeast). These activities remained stable after six months, and decreased approximately by 20% after one year of storage of the EOs at 4 to 7 °C. On comparing the efficiency of both EOs, S. officinalis EO exhibited higher antibacterial activity against the majority of strains and especially against Candida albicans (two fold more active according to the inhibition zones values). The minimal inhibitory concentrations (MICs) were reported between 4.5 mg/ml and 72 mg/ml on nutrient broth. The particular chemotype of each EO may be involved in its specific antimicrobial behaviour.Furthermore, the inhibitory effect of these EOs were evaluated against two foodborne pathogens belonging to Salmonella genus, experimentally inoculated (10³ CFU/g) in minced beef meat, which was mixed with different concentrations of the EO and stored at 4 to 7 °C for 15 days. Although the antibacterial activities of both EOs in minced beef meat were clearly evident, their addition had notable effects on the flavour and taste of the meat at concentrations more than 2% for S. molle and 1.5% for S. officinalis. One solution to the above-mentioned problem may be the use of combinations of different food preservation systems. In this context, each of the EOs has been used along with low water activity (addition of NaCl) in addition to low refrigeration temperatures. Results on the Salmonella growth showed that some combinations could be recommended to eliminate germs from minced raw beef. By using this method, a stable and, from a microbiological point of view, safe meat can be produced without substantial loss in sensory quality.Results obtained herein, may suggest that the EOs of S. officinalis and S. molle possess antimicrobial activity, and therefore, they can be used in biotechnological fields as natural preservative ingredients in food and/or pharmaceutical industry.     

Biofilm formation and exopolysaccharide (EPS) production by Cronobacter sakazakii depending on environmental conditions

Biofilm matrices are formed largely of extracellular polymeric substance (EPS). This study was conducted to investigate biofilm formation and EPS production by Cronobacter sakazakii under various conditions (media, nutrition, and relative humidity (RH)) by quantification of EPS and cell populations, Field Emission Scanning Electron Microscope (FE-SEM), and colony observation. Various agar media conditions (TSA without dextrose (W/D), M9 minimum salt medium (MSM) agar, and M9 MSM agar with 3% glucose, 3% NaCl, 3% Tween 80, 3% sucrose, and adjusted to pH 5 with HCl) were prepared. C. sakazakii biofilm formed on the surface of stainless steel coupons (SSCs) immersed in TSB W/D and M9 MSM with or without 0, 1, 3, and 5% sucrose and subsequently exposed to various RH levels (23, 43, 68, 85, and 100%). EPS production by C. sakazakii on TSA W/D was significantly higher than that on other media after 1 and 2 days. However, C. sakazakii ATCC 12868 produced the highest levels of EPS (209.18 ± 16.13 and 207.22 ± 4.14 μg/mL after 1 and 2 days, respectively) on M9 MSM agar with 3% sucrose. Regarding C. sakazakii ATCC 12868 biofilm formed on the surface of SSCs immersed in M9 MSM with 0, 1, 3, and 5% sucrose and subsequently exposed to various RHs, populations were significantly different among the various RHs and sucrose concentrations, and EPS production was significantly higher (4.69 mg/L) compared to other sucrose concentrations (0%:0.71 mg/L and 1%:0.98 mg/L), except for M9 MSM with 3% sucrose (2.97 mg/L) (P ≤ 0.05). From these results, biofilm formation and EPS production by C. sakazakii differed depending on the nutrient or environmental conditions provided to the cells.     

Characterization of Escherichia coli from raw poultry in Belgium and impact on the detection of Campylobacter jejuni using Bolton broth

A comparative study examining Bolton broth and Preston broth for enrichment and reliable detection of Campylobacter jejuni (both healthy and freeze stressed cells) was performed. Tested as pure cultures, Bolton broth enabled faster resuscitation and growth of C. jejuni compared to Preston broth. When C. jejuni was co-incubated with extended-spectrum-beta-lactamase (ESBL) producing Escherichia coli isolated from Belgian poultry meat preparations, the latter dominated in the Bolton enrichment broth and crowded the mCCDA plates. This resulted in the inability to recover C. jejuni by ISO 10272-1:2006 standard method. Preston broth did not support the growth of the ESBL E. coli isolates, but showed longer detection time of C. jejuni compared to Bolton broth. The use of the same antibiotic (sodium cefoperazone) in Bolton broth and in mCCDA plates may explain the problems encountered for detection of C. jejuni, as high numbers of ESBL E. coli present after enrichment in Bolton broth, also caused overgrowth and masked the few C. jejuni colonies present on the mCCDA plates. The use of Campylobacter spp. specific real-time PCR circumvented these problems and enabled rapid detection of the pathogen after 24 h enrichment in both Bolton and Preston broth, for both healthy and freeze stressed cells.     

Aggregation profile, preparation and nutritional characterization of African yam bean (Sphenostylis stenocarpa) acid and salt protein isolates

Aggregation tendencies of African yam bean (Sphenostylis stenocarpa) protein in the various aqueous extraction media with Ca²⁺, Mg²⁺ or at pI (isoelectric point) were determined. Water extractable S. stenocarpa protein aggregates more with addition of either Ca²⁺ or Mg²⁺ than at pI. In alkaline extractants considered (except at pH 10), aggregation tendency of the protein is in the order: pI > Ca²⁺ > Mg²⁺. The protein aggregation trend in salt media is a function of the salt type, aggregation in NaCl solution is of order: Ca²⁺ = pI > Mg²⁺, while in Na₂SO₄, we had pI > Mg²⁺ > Ca²⁺. S. stenocarpa protein aggregation was significantly (P < 0.05) more in Na₂SO₄ than NaCl. The amount of Ca²⁺ required for maximum precipitation of S. stenocarpa from alkaline (water-pH10) extractant was higher than that of Na₂SO₄ and more Ca-proteinate was obtained from the alkaline aliquot. The crude protein of the Ca-proteinates and isoelectric protein isolates obtained from salt and alkaline extract were in the range 71.7–91.8% (dry basis). Protein isolate from alkaline extract had significantly (P < 0.05) higher fat content than the one from salt extract. Isoelectrically precipitated isolates had lower ash content than Ca-proteinates. The percentage ratio of essential to non-essential amino acid was in the range 45–47%. With reference to FAO/WHO standard, the chemical score showed that most of the essential amino acid were in excess, thus, the amino acid distribution of S. stenocarpa protein isolates showed that it can fulfill the essential amino acid requirement of human especially the acid proteins and can be a good protein supplement in food and a wide range of new food products.     

Autoinducer-2 activity of gram-negative foodborne pathogenic bacteria and its influence on biofilm formation

ABSTRACT:  This study evaluated whether autoinducer-2 (AI-2) activity would be associated with biofilm formation by Salmonella and Escherichia coli O157:H7 strains on food contact surfaces. In study I, a Salmonella Typhimurium DT104 strain and an E . coli O157:H7 strain, both AI-2 positive, were individually inoculated into 50 mL of Luria–Bertani (LB) or LB + 0.5% glucose (LBG) broth, without or with stainless steel or polypropylene ( Salmonella ) coupons. At 0, 14 ( Salmonella ), 24, 48, and 72 h of storage (25 °C), cells in suspension and detached cells from the coupons, obtained by vortexing, were enumerated on tryptic soy agar. In study II, a Salmonella Thompson AI-2-positive strain and an AI-2-negative strain, and an E . coli O157:H7 AI-2-positive strain and an AI-2-negative strain were inoculated into LB broth with stainless steel coupons. Cells were enumerated as in study I. In both studies, AI-2 activity was determined in cell-free supernatants. Cell numbers of S . Typhimurium DT104 on biofilms were higher ( P < 0.05) in LB than those in LBG, while the E . coli O157:H7 strain showed no difference ( P ≥ 0.05) in biofilm cell counts between LB and LBG after storage for 72 h. Both S . Typhimurium DT104 and E . coli O157:H7 strains produced higher ( P < 0.05) AI-2 activity in LBG than LB cell suspensions. Cell counts of AI-2-positive and-negative S . Thompson and E . coli O157:H7 strains were not different ( P ≥ 0.05) within suspensions or coupons (study II). The results indicated that, under the conditions of this study, AI-2 activity of the pathogen strains tested may not have a major influence on biofilm formation on food contact surfaces, which was similar between AI-2-positive and -negative strains.     

Evaluation of currently employed food preservation conditions to tackle biofilm forming food pathogens

Controlling biofilm forming, pathogenic bacteria is an emerging challenge in the food industry, and the present study aims to test the efficacy of existing food preservation methods against these bacteria. We isolated 10 different biofilm forming pathogenic bacteria viz. Klebsiella quasipneumoniae, Bacillus cereus, Bacillus amyloliquefaciens, Bacillus anthracis, and Myroides odoratimimus from spoiled milk, biscuits, and cakes. K. quasipneumonia and B. amyloliquifaciens formed strong biofilm (OD₅₅₀ > 0.85), whereas B. cereus, B. anthracis, and M. odoratimimus made moderate biofilm (OD₅₅₀₅ > 0.6) in 72 hr. The isolates, B. amyloliquifaciencs, B. anthracis, and M. odoratimimus were found positive for protease activity. The isolates showed significant tolerance to preservation conditions: pH (4–10), salt (up to 5%), temperature (up to 50°C) and sodium benzoate (up to 0.1%). The isolates also exhibited high antibiotic resistance, maximum by M. odoratimimus (MIC ≤ 256 µg/ml) against chloramphenicol which can have serious implications especially in tackling food borne illness caused by the biofilm forming food pathogens.

Practical applications

The present study discloses the presence of biofilm forming pathogenic bacterial strains in common food stuff and their tolerance of preservation conditions and resistance to antibiotics. The occurrence of pathogens tolerant to common preservation conditions and their resistance to antibiotic should be addressed very cautiously. The significant findings of this study warrant the need for more stringent preservation methods to control food pathogens, especially the biofilm forming type. Moreover, findings of the present study will help to design novel preservation techniques such as incorporating antibiofilm coatings in food processing units and packaging materials, which will ensure food safety and public health.