Survival and growth of Cronobacter species (Enterobacter sakazakii) in wheat-based infant follow-on formulas

Aims:  To determine the survival and growth characteristics of Cronobacter species ( Enterobacter sakazakii ) in infant wheat-based formulas reconstituted with water, milk, grape juice or apple juice during storage.
Methods and Results:  Infant wheat-based formulas were reconstituted with water, ultra high temperature milk, pasteurized grape or apple juices. The reconstituted formulas were inoculated with Cronobacter sakazakii and Cronobacter muytjensii and stored at 4, 25 or 37°C for up to 24 h. At 25 and 37°C, Cronobacter grew more (>5 log₁₀) in formulas reconstituted with water or milk than those prepared with grape or apple juices ( c. 2–3 log₁₀). The organism persisted, but did not grow in any formulas stored at 4°C. Formulas reconstituted with water and milk decreased from pH 6·0 to 4·8–5·0 after 24 h, whereas the pH of the formulas reconstituted with fruit juices remained at their initial pH values, c. pH 4·8–5·0.
Conclusions:  Cronobacter sakazakii and C. muytjensii can grow in reconstituted wheat-based formulas. If not immediately consumed, these formulas should be stored at refrigeration temperatures to reduce the risk of infant infection.
Significance and Impact of the Study:  The results of this study will be of use to regulatory agencies and infant formula producers to recommend storage conditions that reduce the growth of Cronobacter in infant wheat-based formulas.     

Decreased time for detection and quantification of virulent Bacillus anthracis and Yersinia pestis using a BioNanoPore (BNPTM) membrane technology

Many aspects of biodefense research require quantitative growth assessments of the test agent. This study evaluated the BioNanoPore (BNP™) technology to quantitate Bacillus anthracis and Yersinia pestis faster than traditional plate counting methods. The BNP™ technology enabled quantification of B. anthracis and Y. pestis in phosphate-buffered saline and naïve rabbit blood at 6 and 24 h, respectively. After 6 h of growth, counts for B. anthracis ranged from 6·19–6·45 log₁₀ CFU ml⁻¹ on BNP™, while counts after 24 h on tryptic soy agar (TSA) ranged from 6·51–6·58 log₁₀ CFU ml⁻¹. For Y. pestis , counts on BNP™ at 24 h ranged from 6·31–6·41 log₁₀ CFU ml⁻¹ on BNP™ and ranged from 6·44–6·89 log₁₀ CFU ml⁻¹ on TSA at 48 h. This study demonstrates that the BNP™ technology provides a more rapid detection of B. anthracis and Y. pestis , which could aid in the evaluation of potential medical countermeasures and treatments as well as other biological defense applications such as surface sampling or decontamination efficacy.     

Heat resistance of Cronobacter species (Enterobacter sakazakii) in milk and special feeding formula

Aim:  To determine D - and z -values of Cronobacter species ( Enterobacter sakazakii ) in different reconstituted milk and special feeding formula and the effect of reconstitution of powdered milk and special feeding formula with hot water on the survival of the micro-organism.
Methods and Results:  Five Cronobacter species (four C. sakazakii isolates and C. muytjensii ) were heated in reconstituted milk or feeding formula pre-equilibrated at 52–58°C for various times or mixed with powdered milk or feeding formula prior to reconstitution with water at 60–100°C. The D -values of Cronobacter at 52–58°C were significantly higher in whole milk (22·10–0·68 min) than in low fat (15·87–0·62 min) or skim milk (15·30–0·51 min) and significantly higher in lactose-free formula (19·57–0·66 min) than in soy protein formula (17·22–0·63 min). The z -values of Cronobacter in reconstituted milk or feeding formula ranged from 4·01°C to 4·39°C. Water heated to ≥70°C and added to powdered milk and formula resulted in a > 4 log₁₀ reduction of Cronobacter .
Conclusions:  The heat resistance of Cronobacter should not allow the survival of the pathogen during normal pasteurization treatment. The use of hot water (≥70°C) during reconstitution appears to be an effective means to reduce the risk of Cronobacter in these products.
Significance and Impact of the Study:  This study supports existing data available to regulatory agencies and milk producers that recommended heat treatments are sufficient to substantially reduce risk from Cronobacter which may be present in these products.     

Stimulation of biomethanation by Clostridium sp. PXYL1 in coculture with a Methanosarcina strain PMET1 at psychrophilic temperatures

Aim:  Bioaugumentation of low temperature biogas production was attempted by addition of cold-adapted Clostridium and a methanogen.
Methods and Results:  A psychrotrophic xylanolytic acetogenic strain Clostridium sp. PXYL1 growing optimally at 20°C and pH 5·3 and a Methanosarcina strain, PMET1, growing optimally on acetate and producing methane at 15°C were isolated from a cattle manure digester. Anaerobic conversion of xylose at 15°C with the coculture of the two strains was performed, and batch culture methane production characteristics indicated that methanogenesis occurred via acetate through 'acetoclastic' pathway. Stimulation studies were also undertaken to evaluate the effect of exogenous addition of the coculture on biogas yields at 15°C. Addition of 3 ml of PXYL1 at the rate of 12 × 10² CFU ml⁻¹ increased the biogas 1·7-fold (33 l per kg cowdung) when compared to control (19·3 l per kg cowdung) as well as increased the volatile fatty acid (VFA) levels to 3210 mg l⁻¹ when compared to 1140 mg l⁻¹ in controls. Exogenous of addition of 10 ml PMET1 inoculum at the rate of 6·8 ± 10² CFU ml⁻¹ in addition to PXYL1 served to further improve the biogas yields to 46 l kg⁻¹ as well as significantly brought down the VFA levels to 1350 mg l⁻¹.
Conclusions:  Our results suggest that the rate-limiting methanogenic step at low temperatures could be overcome and that biogas yields improved by manipulating the population of the acetoclastic methanogens.
Significance and Impact of the Study:  Stimulation of biomethanation at low temperature by coculture.     

Survival of Escherichia coli O157 : H7 in yoghurt during preparation and storage at 4°C

Cow's milk was inoculated with ca 10³ and 10⁷ cfu ml⁻¹ Escherichia coli O157 : H7. After fermentation at 42°C for 0–5 h, the yoghurt was stored at 4°C. Two kinds of yoghurt were used : traditional yoghurt (TY), made with Streptococcus thermophilus and Lactobacillus bulgaricus starter cultures, and 'bifido' yoghurt (BY), made with the two starter cultures plus Bifidobacterium bifidum . After 7 d E. coli O157 : H7 decreased from 3·52 to 2·72 log₁₀ cfu ml⁻¹ and from 7·08 to 5·32 log₁₀ cfu ml⁻¹ in TY, and from 3·49 to 2·73 log₁₀ cfu ml⁻¹ and from 7·38 to 5·41 log₁₀ cfu ml⁻¹ in BY. The pH values of yoghurt dropped from 6·6 to 4·5 and 4·4 in TY (for low and high pathogen inocula, respectively), and from 6·6 to 4·6 and 4·5 in BY (for low and high pathogen inocula, respectively).     

Automated concentration and recovery of micro-organisms from drinking water using dead-end ultrafiltration

Aims:  Concentration of pathogens diluted in large volumes of water is necessary for their detection. An automated concentration system placed online in drinking water distribution systems would facilitate detection and mitigate the risk to public health.
Methods and Results:  A prototype concentrator based on dead-end hollow fibre ultrafiltration was used to concentrate Bacillus atrophaeus spores directly from tap water. Backflush was used to recover accumulated particulates for analysis. In field tests conducted on a water utility distribution system, 3·2 × 10⁴–1·4 × 10⁶ CFU ml⁻¹ (6·1 × 10⁶–3·0 × 10⁸ CFU) were recovered from the filter when 2·9 × 10⁷–1·0 × 10⁹ CFU were spiked into the system. Per cent recovery ranged from 21% to 68% for flow volumes of 15–21 l. Tests using spore influent levels <10 CFU l⁻¹ (spike < 1000 CFU) yielded 23–40% recovery for volumes >100 l.
Conclusions:  B. atrophaeus spores at levels <10 CFU l⁻¹ were concentrated directly from tap water using an automated dead-end hollow-fibre ultrafiltration system.
Significance and Impact of the Study:  The prototype concentrator represents a critical step towards an autonomous system that could be installed in drinking water distribution lines or other critical water lines to facilitate monitoring. Recovered samples can be analysed using standard or rapid biosensor methods.     

Multiplex real-time PCR for exhaustive detection of diarrhoeagenic Escherichia coli

Aims:  The source and routes of diarrhoeagenic Escherichia coli (DEC) have not been clarified because it is difficult to detect these organisms in samples with numerous coliform bacteria. We have developed multiplex real-time PCR assays for exhaustive detection of DEC.
Methods and Results:  Primers and TaqMan probes were designed to amplify and quantify one gene ( eae , stx1 , stx2 , elt , est , virB , aggR , astA, and afaB ) from each of seven pathotypes of DEC, in duplex or triplex reactions under the same PCR cycling conditions. Specificity was confirmed using 860 strains including 88 DEC strains. The fluorescence threshold cycle and DNA concentrations correlated with decision coefficients of more than 0·99. Subsequently, meat samples and enrichment broths were spiked with DEC and the assays used to detect the genes. The detection limits varied from 7·1 × 10² to 1·1 × 10⁴ CFU ml⁻¹, depending on the target genes. All meat samples spiked with a variety of DEC (more than 10 CFU 10 g⁻¹) were found to be positive by the method.
Conclusions:  The present system allows for the efficient and simultaneous determination of various DEC pathotypes.
Significance and Impact of the Study:  This system makes epidemiological investigations for DEC sensitive and quick, and is a useful tool to clarify the source and routes of DEC.     

Use of flow cytometry for enumeration of Mycoplasma mycoides subsp. mycoides large-colony type in broth medium

Aims:  The potential of using flow cytometry (FC) in combination with a fluorescent dye (SYBR green-I) for rapidly estimating Mycoplasma mycoides subSPS. mycoides large-colony type (MmmLC) in broth culture was examined.
Methods and Results:  The FC analysis was performed by staining the MmmLC cells with a fluorescent dye, SYBR green-I (SYBR), and the results were compared with plate count method (colony forming units, – CFUs). There was a good correlation (linear regression, r ² = 0·93) between mycoplasma counts determined by FC (cells ml⁻¹) and by traditional plate count method (CFU ml⁻¹). The lowest bacterial concentration detected by FC and traditional plate count was of the order of 10⁴ cells ml⁻¹ and 10³ CFU ml⁻¹, respectively. FC method allowed results in 20–30 min, whereas at least 24 h were necessary to obtain results with the traditional plate count method (CFU).
Conclusion:  Growth rates of MmmLC in broth medium determined by FC were highly reproducible and correlated well with mycoplasma counts assessed by the plate count method.
Significance and Impact of the Study:  These findings suggest that FC could be a good alternative to replace other time-consuming techniques that are currently used to enumerate mycoplasma in broth medium, such as plate count method (CFU).     

Anti-Helicobacter pylori activity of Lactobacillus delbrueckii subsp. bulgaricus strains: preliminary report

Aims:  To evaluate the activities of six Lactobacillus delbrueckii subsp. bulgaricus (LB) strains against 30 Helicobacter pylori strains by agar-well diffusion method.
Methods and Results:  LB cultures [4 × 10⁸–4 × 10⁹ CFU ml⁻¹) either were prepared in milk at their native pH, 3·8–5·0, or were adjusted to pH 6·4–7·7. At low and neutralized pH, LB strains inhibited the growth by 40–86·7% and 16·7–66·7% of H. pylori strains, respectively. LB activity was strain-dependent. At low and neutralized pH, one and five H. pylori strains, respectively, were not inhibited by any LB strain. LB2 and LB3, taken together, were active against most metronidazole and clarithromycin resistant strains.
Conclusions:  All LB strains inhibited a number of H. pylori strains, including also antibiotic resistant strains. LB activity was strain-dependent and better at low pH. At low pH values, the most active LB strains were LB1, LB2 and LB3, inhibiting 86·7% of H. pylori strains, while at neutralized pH values, the most active LB strains were LB2 and LB3, inhibiting 53·3 and 66·7% of H. pylori strains, respectively.
Significance and Impact of the Study:  LB could be utilized in the treatment or prophylaxis of H. pylori infection and warrants clinical investigations.     

Rapid and sensitive detection of Vibrio cholerae by loop-mediated isothermal amplification targeted to the gene of outer membrane protein ompW

Aims:  The present study was aimed to develop a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Vibrio cholerae .
Methods and Results:  A set of five designed primers that recognized specifically the V. cholerae ompW gene was used. The optimized time and temperature conditions for the LAMP assay were 75 min at 65°C, respectively. The LAMP method accurately identified 16 isolates of V. cholerae but did not detect 28 non- cholerae Vibrio isolates and 37 non- Vibrio bacterial isolates. The sensitivity of LAMP for V. cholerae detection in pure cultures was 2·2 × 10³ CFU ml⁻¹ or equivalent to 8 CFU per reaction. In the case of spiked shrimp samples without enrichment, the detection limit for V. cholerae was 2·2 × 10⁴ CFU g⁻¹ or equivalent to 20 CFU per reaction, while that of PCR was 100 CFU per reaction.
Conclusion:  The developed LAMP assay targeting ompW gene was rapid, specific and sensitive for V. cholerae detection.
Significant and Impact of the study:  The developed LAMP assay appears to be precise, accurate and a valuable tool for detection of V. cholerae . This assay can replace laborious biochemical tests for the identification of V. cholerae in contaminated food sample.     

Synbiotics growth optimization of Bifidobacterium pseudocatenulatum G4 with prebiotics using a statistical methodology

Aims:  This study demonstrated the optimum growth of Bifidobacterium pseudocatenulatum G4 with prebiotics via statistical model.
Methods and Results:  Commercial prebiotics [inulin and fructooligosaccharide (FOS)], together with sorbitol, arabinan and inoculum rate, were tested by fractional factorial design to determine their impact on growth of Bif. pseudocatenulatum G4 in skim milk. At 48 h incubation, bacterial growth was mainly influenced by FOS and inoculum rate. Growth reduction was observed in all samples incubated for 72 h. Central composite design (CCD) was adopted using FOS and inoculum rate at 48 h incubation to develop the statistical model for optimization. The model predicted that 2·461 log CFU ml⁻¹ produced the optimum growth increase of Bif. pseudocatenulatum G4. The combination that produced the optimum point was 2·86% FOS (g/v) and 0·67% inoculum rate (v/v).
Conclusion:  At optimum combination of inoculum rate and FOS, validation experiments recorded 2·40 ± 10·02 log CFU ml⁻¹. The application in 1-l bioreactor for 24 h showed higher growth increase of 2·95 log CFU ml⁻¹.
Significant and Impact of the Study:  Response surface methodology approach is useful to develop optimum synbiotics combination for strain G4 with FOS.     

Legionella pollution in cooling tower water of air-conditioning systems in Shanghai, China

Aims:  To determine Legionella pollution prevalence, describe the amount of Legionellae with respect to temperature in Shanghai cooling tower water (CTWs) in various types of public sites.
Methods and Results:  Six urban districts were selected as the study fields, adopting multiple-phase sampling methods. Routine culture was used to identify Legionellae. Of the samples, 58·9% (189/321) were observed to be positive, 19·9% were isolated over 100 CFU ml⁻¹. Legionella pneumophila serogroup 1 was the most frequently isolated species (155/189, 82·0%), followed by Leg. micdadei that was at the second place (44/189, 23·3%). The mean CFU ml⁻¹ of Legionellae in CTWs reached its peak from July to September. Over all 15·4% of the samples exceeding 100 CFU ml⁻¹ were observed in a hospital setting.
Conclusions:  The prevalence of Legionella pollution in CTWs, especially in CTWs of subway stations and hospitals, is worrying, and the positive rate and CFU ml⁻¹ of Legionellae in CTWs have a close relationship with air temperature.
Significance and Impact of the Study:  The study demonstrates pollution prevalence rates in different types of sites and various seasons, and provides a proportion of different serogroups of Legionellae . It illuminates an urgent need for dealing with the potential risk of legionellosis in Shanghai, through improved control and prevention strategies.     

Diarrhoeal enterotoxin production by psychrotrophic Bacillus cereus present in reconstituted milk-based infant formulae (MIF)

One hundred reconstituted milk-based infant formulae (MIF) representative of 10 leading brands available in many European Economic Community countries were examined for psychrotrophic Bacillus cereus and for the presence of diarrhoeal enterotoxin. Of the 38 B. cereus isolates recovered from MIF, one, four and 16 strains grew at 4, 6 and 8 °C after 15 d. One (2·6%), two (5·3%) and six (15·8%) of the isolates were identified as potential psychrotrophic food poisoning strains as they were both enterotoxigenic and exhibited good growth at 4, 6 and 8 °C, respectively. Enterotoxin was not detected in MIF in which less than 5·36 log₁₀ cfu of B. cereus ml⁻¹ had grown. While psychrotrophic enterotoxigenic B. cereus strains occur occasionally in MIF, brief storage of reconstituted MIF at the recommended refrigeration temperature of 4 °C will allow this product to remain safe for consumption.     

Integration and decontamination of Bacillus cereus in Pseudomonas fluorescens biofilms

Aims:  The hypothesis that surrogate planktonic pathogens ( Bacillus cereus and polystyrene microspheres) could be integrated in biofilms and protected from decontamination was tested.
Methods and Results:  Pseudomonas fluorescens biofilms were grown on polyvinyl chloride coupons in annular reactors under low nutrient conditions. After biofilm growth, B. cereus spores and polystyrene microspheres (an abiotic control) were introduced separately. Shear stress at the biofilm surface was varied between 0·15 and 1·5 N m⁻². The amount of surrogate pathogens introduced ranged from approximately 10⁵ CFU ml⁻¹ to 10¹⁰ spheres ml⁻¹. The quantity of surrogate pathogens integrated in the biofilm was proportional to the amount introduced. In 14 of the 16 cases, 0·4–3·0% of the spores or spheres introduced were measured in the biofilms. The other two cases had 10% and 21% of the spores detected. Data suggested that the spores germinated in the system. The amount of surrogate pathogens detected in the biofilms was higher in the mid-shear range. Chlorine treatment reduced the quantity of both surrogate pathogens and biofilm organisms. In one experiment, the biofilms and B. cereus recovered when the chlorine treatment was terminated.
Conclusions:  Planktonic surrogate pathogens can be integrated in biofilms and protected from chlorination decontamination.
Significance and Impact of the Study:  This knowledge assists in understanding the impact of biofilms on harbouring potential pathogens in drinking-water systems and protecting the pathogens from decontamination.     

The anti-inflammatory drug Diclofenac retains anti-listerial activity in vivo

Aims:  The interactions between nonsteroidal anti-inflammatory drugs (NSAID) and Listeria monocytogenes have not been sufficiently documented to date. The aim of this study was to investigate the possible effects of Diclofenac (Dc) in a murine listerial infection model.
Methods and Results:  Dc was administered orally at 2·5 μg g⁻¹ to female albino strain of laboratory mouse (BALB/c) thrice postinfection (1 × 10⁸ CFU ml⁻¹ oral challenge with L. monocytogenes ATCC 51774), which resulted in significantly ( P  < 0·01) reduced bacterial counts in liver and spleen, decreased (10-fold, P  < 0·05) hepatic colonization and necrosis, and caused up-regulation of the expression of inflammatory cytokines (interferon-γ, interleukin-1β, tumour necrosis factor-α), compared with drug-free control.
Conclusions:  Dc may be useful as a promising adjuvant to the existing therapies in controlling systemic listerial infection. Further, quantitative structure–activity relationship studies might contribute in manipulating it as a lead compound for the synthesis of new, more effective nonantibiotics, perhaps, devoid of side-effects that could be recommended as a compassionate therapy for listeriosis.
Significance and Impact of the study:  This is the first in vivo study designed to evaluate the antilisterial effect of the NSAID Dc with special emphasis on the immunological mechanism of action of the drug.     

Virulence of luminescent and non-luminescent isogenic vibrios towards gnotobiotic Artemia franciscana larvae and specific pathogen-free Litopenaeus vannamei shrimp

Aims:  This study was conducted to test the virulence of luminescent (L) and non-luminescent (NL) isogenic strains of Vibrio campbellii LMG21363, Vibrio harveyi BB120 (wild type) and quorum-sensing mutant strains derived from the wild type such as Vibrio harveyi BB152, BB170, MM30 and BB886.
Methods and Results:  The NL strains could be obtained by culturing rifampicin-resistant luminescent strains in the dark under static condition. The virulence of the L and NL strains was tested in gnotobiotic Artemia franciscana larvae challenged with 10⁴ CFU ml⁻¹ of bacteria. All luminescent isogenic tested strains showed higher virulence compared to the NL strains. The virulence of L and NL V. campbellii and V. harveyi BB120 was also tested in specific pathogen-free juvenile shrimp upon intramuscular injection with 10⁶ CFU of bacteria. In contrast with Artemia , there was no significant difference in mortality between the groups challenged with L and NL strains ( P  > 0·05). The non-luminescent strains were not able to revert back to the luminescent state and quorum sensing did not influence this phenotypic shift.
Conclusions:  Luminescent Vibrio strains can switch to a non-luminescent state by culturing them in static conditions. The NL strains become less virulent as verified in Artemia .
Significance and Impact of the Study:  The luminescent state of Vibrio cells in a culture needs to be verified in order to assure maintenance of virulence.     

Effect of high-temperature, short-time (HTST) pasteurization on milk containing low numbers of Mycobacterium paratuberculosis

The efficacy of high-temperature, short-time (HTST) pasteurization (72 °C/15 s) when low numbers (≤ 10³ cfu ml ⁻¹ ) of Mycobacterium paratuberculosis are present in milk was investigated. Raw cows' milk spiked with Myco. paratuberculosis (10³ cfu ml⁻¹, 10² cfu ml⁻¹, 10 cfu ml⁻¹, and 10 cfu 50 ml⁻¹) was subjected to HTST pasteurization using laboratory pasteurizing units. Ten bovine strains of Myco. paratuberculosis were tested in triplicate. Culture in BACTEC Middlebrook 12B radiometric medium detected acid-fast survivors in 14·8% and 10% of HTST-pasteurized milk samples at the 10³ and 10² cfu ml⁻¹ inoculum levels, respectively, whereas conventional culture on Herrold's egg yolk medium containing mycobactin J detected acid-fast survivors in only 3·7% and 6·7% of the same milk samples. IS900-based PCR confirmed that these acid-fast survivors were Myco. paratuberculosis . No viable Myco. paratuberculosis were isolated from HTST-pasteurized milk initially containing either 10 cfu ml⁻¹ or 10 cfu 50 ml⁻¹.     

WLIP, a lipodepsipeptide of Pseudomonas 'reactans', as inhibitor of the symptoms of the brown blotch disease of Agaricus bisporus

A cell-free crude extract containing the white line inducing principle (WLIP), a lipodepsipeptide produced by Pseudomonas 'reactans' , could inhibit browning of mushrooms caused by Pseudomonas tolaasii . Mushrooms inoculated with Ps. tolaasii at concentrations of 2·7 × 10⁶ cfu ml⁻¹ or higher showed the symptoms of the disease after 2 d of incubation. Mushroom caps treated with various concentrations of a crude WLIP preparation, and later inoculated with bacterial concentrations higher than the threshold value, did not develop the symptoms of the disease. One milligram of a crude WLIP preparation could block 50% of the symptoms caused by 1·2 × 10⁷ cfu. The inhibition of browning was effective when incubating at low temperatures for 4 d. A suspension containing 1·6 mg ml⁻¹ of pure WLIP was also able to inhibit the symptoms of brown blotch disease induced by 7·6 × 10⁶ cfu ml⁻¹ of Ps. tolaasii .     

Combined effect of organic acids and supercritical carbon dioxide treatments against nonpathogenic Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and E. coli O157:H7 in fresh pork

Aims:  To evaluate the effectiveness of organic acids and supercritical carbon dioxide (SC-CO₂) treatments as well as their combined effect for the reduction of nonpathogenic Escherichia coli and three pathogenic bacteria in fresh pork.
Methods and Results:  The different treatment conditions were as follows: (i) treatment with acetic (1%, 2% or 3%) or lactic acid (1%, 2% or 3%) only, (ii) treatment with SC-CO₂ at 12 MPa and 35°C for 30 min only and (iii) treatment with 3% acetic or lactic acid followed by treatment with SC-CO₂. Within the same organic acid concentration, the lactic and acetic acid treatments had similar reductions. For the combined treatment of lactic acid and SC-CO₂, micro-organism levels were maximally reduced, ranging from 2·10 to 2·60 log CFU cm⁻² ( E. coli , 2·58 log CFU cm⁻²; Listeria monocytogenes , 2·60 log CFU cm⁻²; Salmonella typhimurium , 2·33 log CFU cm⁻²; E. coli O157:H7, 2·10 log CFU cm⁻²).
Conclusions:  The results of this study indicate that the combined treatments of SC-CO₂ and organic acids were more effective at destroying foodborne pathogens than the treatments of SC-CO₂ or organic acids alone.
Significance and Impact of the Study:  The combination treatment of SC-CO₂ and organic acids may be useful in the meat industry to help increase microbial safety.     

The use of a surface adhesion immunofluorescent (SAIF) method for the rapid detection of Yersinia enterocolitica serotype O:3 in meat

This study examined the attachment kinetics of Yersinia enterocolitica serotype O:3 to determine the optimum conditions for its isolation from meat enrichment systems using a novel surface adhesion technique. Minced beef was inoculated with Y. enterocolitica at an initial level of 10 cfu g⁻¹ and incubated at 25 °C in an enrichment broth. Yersinia was recovered from enriched samples on polycarbonate membranes by surface adhesion and enumerated using immunofluorescence microscopy. The surface adhesion immunofluorescence technique (SAIF) had a minimum detection limit of approximately 4·0–4·5 log₁₀ cfu ml⁻¹ and provided good correlation between the estimation of the numbers of Yersinia in the enrichment broth derived from plate counts on Yersinia Selective agar (CIN) and those determined by SAIF ( r ² ₌ 0·94; rsd ₌ ± 0·21). A derived regression equation of the SAIF count vs plate counts was used to predict Y. enterocolitica numbers in spiked meat samples stored at 0 °C for up to 20 d. The numbers as predicted by the SAIF method showed good correlation with counts derived by plating techniques ( r ² ₌ 0·78; rsd ₌ ± 0·42). The application of the SAIF technique for the rapid detection of Y. enterocolitica serotype O:3 from meat is discussed.