Development of a multiplexed PCR assay combined with propidium monoazide treatment for rapid and accurate detection and identification of three viable Salmonella enterica serovars

Salmonella is the leading pathogenic bacteria in food and contaminated water. The aim of this study was to develop a rapid and reliable technique for simultaneous detection of the main three serotypes (Salmonella enterica serovars Typhimurium, Paratyphi B and Typhi) of Salmonella. Primers were designed to amplify the genes specific to each of these three serotypes for simultaneous detection using polymerase chain reaction (PCR). To ensure the detection of only viable cells, propidium monoazide (PMA) was applied to selectively suppress the DNA signal from dead cells. Results showed that the PMA-multiplexed PCR (PMA-mPCR) assay always gave negative results for heat-killed Salmonella at concentrations up to 1 × 10⁶ CFU/ml in pure culture or 1 × 10⁶ CFU/g in spiked food products (tomato, chicken, beef and ham). Results showed that the detection limits of the PMA-mPCR assay were approximately 10² CFU/ml (4.3 × 10² CFU/ml for S. Typhimurium, 3.7 × 10² CFU/ml for S. Paratyphi B, 7.2 × 10² CFU/ml for S. Typhi) in pure culture and 10³ CFU/g (4.3 × 10³ CFU/g for S. Typhimurium, 3.7 × 10³ CFU/g for S. Paratyphi B, 7.2 × 10³ CFU/g for S. Typhi) in food produce. These results demonstrated that the PMA-mPCR assay can simultaneously detect and identify viable S. Typhimurium, Paratyphi B and Typhi in a short period of time, even in food produce.     

Large-volume immunomagnetic separation combined with multiplex PCR assay for simultaneous detection of Listeria monocytogenes and Listeria ivanovii in lettuce

Multiplex polymerase chain reaction (mPCR) has been widely used for the simultaneous detection of various target bacteria in vegetables. However, an enrichment period is necessary to improve the sensitivity of the mPCR method. In this paper, large-volume (10 mL) immunomagnetic separation (IMS) combined with mPCR for the rapid detection of Listeria monocytogenes and Listeria ivanovii in lettuce without further enrichment process is reported for the first time. Various parameters that affected the capture efficiency (CE) of IMS, including the amounts of streptavidin and biotinylated anti-Listeria monoclonal antibodies on the surface of magnetic nanobeads, the amount of immunomagnetic beads, immunoreaction time, and magnetic separation time, were systematically investigated. Moreover, the concentrations of primers, PCR conditions, and genomic DNA isolation for mPCR assay were optimized. Under optimum conditions, the CE of large-volume IMS for L. monocytogenes and L. ivanovii was greater than 90% when the concentration of target bacteria was less than 10⁶ CFU/mL in pure culture, and was more than 80% when the concentration was below 10⁵ CFU/mL in lettuce samples. The limit of detection of IMS combined with mPCR assay reached as low as 1.0 CFU/mL in pure culture and 10 CFU/g in lettuce. The overall assay time, including sample preparation, large-volume IMS, and mPCR assay, took less than 7 h. In summary, the developed large-volume IMS-based mPCR system exhibits great potential for routine screening detection of foodborne pathogenic bacteria for safety monitoring.     

NaCl-free processing,acidification, smoking and high pressure: Effects on growth of Listeria monocytogenes and Salmonella enterica in QDS processed® dry-cured ham

To evaluate the food safety effect of NaCl-free processing, acidification, smoking and high pressure in QDS processed^® dry-cured ham, 3 ham types (non-acidified smoked, acidified, and acidified smoked) were produced according to a standard (-S) and a new NaCl-free (-F) process. Slices were spiked with Listeria monocytogenes and Salmonella enterica (three strains each, <2 log CFU/g), dried by the QDS process^®, vacuum packed, high pressure treated at 600 MPa and stored under refrigeration for 112 days. Results of the challenge test showed that L. monocytogenes could only be eliminated from acidified smoked (AS) -S and -F processed ham slices at the end of storage, while S. enterica was present in all non-pressurized slices. The safest hams were those pressurized, especially AS-S hams, where L. monocytogenes was eliminated from 25 g of product immediately after HP treatment and S. enterica after 14 days. Compared with standard processing, NaCl-free processing showed lower levels of pathogens in non-pressurized slices but their elimination was delayed in pressurized ham slices.     

Sextuplex PCR combined with immunomagnetic separation and PMA treatment for rapid detection and specific identification of viable Salmonella spp., Salmonella enterica serovars Paratyphi B,Salmonella Typhimurium,and Salmonella Enteritidis in raw meat

Salmonella is one of the most common pathogens that cause foodborne diseases in humans, resulting in high medical and economical costs worldwide. The aim of this study was to develop a rapid and accurate approach for simultaneous detection of viable Salmonella spp. in raw meat, including Salmonella enterica serovars Paratyphi B, S. Typhimurium, and S. Enteritidis. To reduce detection time and improve sensitivity, immunomagnetic separation (IMS) was used as a pre-concentration and separation method. In addition, false positive and false negative results were removed by combining propidium monoazide (PMA) treatment with internal amplification control. Results showed that the detection limit of IMS-PMA combined with sextuplex polymerase chain reaction (PCR) assay reached as low as 10¹ CFU/mL in pure culture and 10² CFU/g in spiked raw meat (ground pork and ground beef), and the total assay time took less than 6 h. Thus, the novel IMS-PMA-mPCR assay developed in this study holds promise for routine screening of viable Salmonella serovars in meat and other samples.     

Balsamic vinegar from Modena: An easy and effective approach to reduce Listeria monocytogenes from lettuce

The microbiological safety of fresh produce is a significant concern of consumers and industry. After applying at an inoculated level (6–7 log CFU/mL) of Listeria monocytogenes on Iceberg lettuce, the antilisterial properties of balsamic vinegar from Modena, white wine vinegar and acetic acid solutions were investigated.Different proportions of the vinegars, acetic acid (58.7 g/L), and deionized water were evaluated to determine the role of those solutions at the stage of washing Iceberg lettuce to remove L. monocytogenes. The maximum observed log reduction of L. monocytogenes was 2.15 ± 0.04 for balsamic vinegar (50% (v/v)), 1.18 ± 0.06 for white wine vinegar ((50% (v/v)) and 1.13 ± 0.06 for acetic acid ((50% (v/v)). Washing with water only reduces 0.05 ± 0.04 log CFU/mL of L. monocytogenes numbers.Listeria reductions observed for balsamic vinegar are similar or higher than those of chlorine-based sanitizers evaluated in other studies with lettuce. In the case of balsamic vinegar solutions, Listeria inhibition followed a linear reduction according to the model: Log (N/N₀) = −4.09 × balsamic vinegar proportion % (v/v) − 0.13; R² = 0.95. Balsamic vinegar washings may be a promising method for reducing other foodborne pathogens present in produce or other foods, at home and retail environments.     

Effect of hydrogen peroxide vapor treatment for inactivating Salmonella Typhimurium,Escherichia coli O157:H7 and Listeria monocytogenes on organic fresh lettuce

In this study, the efficacy of hydrogen peroxide vapor (HPV) for reducing Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes on lettuce was investigated as well as its effect on lettuce quality. Lettuce was inoculated with a cocktail containing three strains of each pathogen then treated with vaporized hydrogen peroxide for 0, 2, 4, 6, 8 and 10 min. The concentrations of hydrogen peroxide used were 0, 1, 3, 5 and 10%. With increasing treatment time and hydrogen peroxide concentration, HPV treatment showed significant (P < 0.05) reduction compared to the control (0%, treated with vaporized distilled water). In particular, vaporized 10% hydrogen peroxide treatment for 10 min was the most effective combination for reducing the three pathogens on lettuce. The reduction levels of S. Typhimurium, E. coli O157:H7 and L. monocytogenes on lettuce were 3.12, 3.15 and 2.95 log₁₀ CFU/g, respectively. Furthermore, there were no significant (P > 0.05) quality changes (color and texture) of lettuce among all tested samples, and hydrogen peroxide residues were not detected after 36 h storage time in any of the treated samples. These results suggest that HPV treatment could be an alternative method for reducing S. Typhimurium, E. coli O157:H7 and L. monocytogenes on fresh produce.     

Application of an optimized 18-h method involving one step culturing and single primer-based PCR assay for detection of Salmonella spp. in foods

A polymerase chain reaction (PCR) using 20-mer oligonucleotide single primer (named primer 3) randomly designed on the basis of Salmonella Typhimurium gatD gene encoding galactitol-1-phosphate dehydrogenase could produce the specific DNA product of approximate 770-bp in all 38 Salmonella strains used. No 770-bp DNA band was amplified from any DNA samples of 20 non-Salmonella bacteria. The DNA band was detected by 1% agarose gel electrophoresis and ethidium bromide staining. The sensitivity of the RAPD–PCR assay for detection of pure genomic DNA from S. Typhimurium ATCC 13311 and Salmonella Enteritidis DMST 15676 was as few as 0.01 ng. When simple boiling in TE buffer for 5 min was used for extraction both Salmonella spp. DNA, the detection limits were at least 230 and 320 cells, respectively. By using the RAPD–PCR assay following at least 14 h pre-enrichment in nutrient broth (NB), as few as 1 CFU of S. Typhimurium ATCC 13311 or S. Enteritidis DMST 15676 per 25 g of autoclaved chicken meat was detected. When the optimized 18-h method involving pre-enrichment in NB and DNA extraction by boiling protocol followed by RAPD–PCR using primer 3 was evaluated in comparison with the conventional method on 195 possibly naturally-contaminated food samples, 36 samples were found positive by both methods. In addition, the results of the developed RAPD–PCR-based assay proved to be identical to those by the conventional method. The optimized 18-h method was simple, rapid and sensitive, achieved the same detection limit as the conventional method and produced a zero level of false-negative results.     

Effect of controlled-release chlorine dioxide on the quality and safety of cherry/grape tomatoes

The effect of controlled-release chlorine dioxide (ClO₂) gas on the food safety and quality of cherry/grape tomatoes was investigated. Whole grape tomatoes artificially inoculated with either Escherichia coli or Alternaria alternata, or whole cherry tomatoes inoculated with Salmonella enterica Newport, were packed in 1-lb clamshells, and stored at 20 °C for 14 days. ClO₂ pouches were attached under the lids with the following four dosages/release rates: single dose slow-release (S), single dose fast-release (F), fast/slow-release combination (FS), and double dose fast-release (FF). The corresponding equilibrium ClO₂ concentration in the headspace was about 2, 4, 6 and 8 ppm, respectively. Treatment with F reduced populations of E. coli and A. alternata by 2.9–4.7, and 1.6 to 4.0 log CFU/g, respectively, within 14 days storage at 20 °C. FS and FF treatments showed little benefit over F. The F and FF treatments reduced population of S. enterica for inoculated cherry tomatoes by 3.28 and 3.80 log CFU/g, respectively, compared to control after 14 days’ storage at 20 °C. ClO₂-treated grape tomatoes retained higher firmness and had less weight loss compared to the control. The results indicate that 2 ppm of ClO₂ (S) in the clamshells did not adequately control microbial populations; the minimum effective concentration of ClO₂ was 4 ppm (F). Higher concentrations provided a small but incremental improvement in ability to control microorganisms. ClO₂ released into packages of cherry tomatoes during storage reduced weight loss, while maintaining firmness.     

Effects of weed management practices on enteric pathogen transfer into lettuce (Lactuca sativa var. capitata)

We evaluated the effect of using farmyard manure (FYM) in combination with four weed control techniques on the hygienic quality of lettuce. Treatments with six replications included the application of fresh FYM on the soil combined with hand-hoeing (assumed high-risk) and fresh FYM incorporation combined with plastic mulch (assumed low risk). The experiment was repeated twice per season over two years at Bonn University, Germany. The amounts of enteric bacteria in all treatments were within the acceptable limits. Enterococcus was detected in the majority of the samples in concentrations up to 3.7 log₁₀ CFU/g. The Escherichia coli contents of lettuce ranged between n.d. and 10³ CFU/g. There was no consistent effect suggesting a specific risk linked to one of the experimental treatments. We conclude that weeding techniques did not increase the risk of exposure to E. coli when FYM is used for fertility in organic production systems.     

Food safety and microbiological quality aspects of QDS process® and high pressure treatment of fermented fish sausages

A new fermented fish sausage product, based on monkfish, was developed by using an accelerated drying process, the QDS process^®. To evaluate food safety, a challenge test was performed, in which the raw materials were inoculated with low levels of Listeria monocytogenes and Salmonella enterica (≤150 CFU/g). The product was manufactured, fermented, QDS dried, and half of the samples were pressurized (600 MPa, 5 min, 13 °C). Pathogens, technological microbiota, spoilage indicator bacteria from fish (hydrogen sulphite producing bacteria, coliforms and Escherichia coli) and physicochemical parameters were monitored during manufacturing and after 6, 13, 20 and 27 days of refrigerated storage at 4 and 8 °C. Results showed that in the finished product, pathogens and spoilage indicator bacteria could not grow but decreased and E. coli was not detected during storage. Pressurization had an important reducing effect on technological microbiota, and eliminated L. monocytogenes, S. enterica, hydrogen sulphite producing bacteria and coliforms immediately after production and during refrigerated storage.     

Inactivation of Cronobacter sakazakii in head lettuce by using a combination of ultrasound and sodium hypochlorite

The present study investigated the synergistic effects of combined ultrasound (37 kHz, 380 W for 5–100 min) and sodium hypochlorite (NaOCl) (50–200 ppm) on reducing Cronobacter sakazakii KCTC 2949 in head lettuce. Ultrasound was not enough to inactivate C. sakazakii (0.01–0.58 log-reduction), whereas NaOCl significantly (P < 0.05) reduced C. sakazakii (0.58–2.77 log-reduction). Despite the significant reduction in C. sakazakii with NaOCl treatment (200 ppm), the combination of 100 min ultrasound and 200 ppm NaOCl resulted in an additional 1.67 log-reduction of C. sakazakii (4.44 log-reduction = 2.77 + 1.67 log). Synergistic reductions on C. sakazakii were observed in most combined treatments, although the most synergistic reduction values were <1.0 log₁₀ CFU/g. The highest synergistic value of reduction in head lettuce was 1.08 log₁₀ CFU/g when treated with a combination of 100 min ultrasound and 200 ppm NaOCl. Moreover, the pH and °Brix after combined treatment with 5–100 min ultrasound and 50–200 ppm NaOCl did not significantly differ from any single treated lettuce. These results indicate that combined treatment with 100 min ultrasound and 200 ppm NaOCl could be a potential approach to reduce C. sakazakii on post-harvest leaf vegetable during processing and enhance the shelf-life during distribution and storage.     

Increasing the microbial inactivation of Staphylococcus aureus in sauces by a combination of enterocin AS-48 and 2-nitropropanol, and mild heat treatments

Staphylococcus aureus was challenged with the cyclic bacteriocin AS-48 in four commercial sauces singly and in combination with sublethal heat treatments (50-60 °C, for 5 min) and with 2-nitro-1-propanol (2NPOH). Heating at 60 °C in combination with AS-48 (25 μg/ml) reduced initial counts (4.1-4.5 log CFU/ml) below detection limit (1.0 log CFU/ml), while a combined treatment at 55 °C inhibited proliferation of staphylococci in sauces for 24 h at 22 °C. In BHI broth, enterocin AS-48 (0-15 μg/ml) acted synergistically with 2NPOH (0-220 mM), reducing the concentrations of both antimicrobials required for inactivation of S. aureus. In sauces, enterocin AS-48 (25 μg/ml) plus 2NPOH (25 mM) reduced initial cell concentrations (4.1-5.0 log CFU/ml) below detectable levels within 8-24 h, depending on the sauce. The combinations of enterocin AS-48 and 2NPOH or sublethal heat are interesting for testing as part of hurdle technology aimed at reducing the risks for proliferation of S. aureus in sauces.     

Quantitative study of cross-contamination with Escherichia coli,E. coli O157, MS2 phage and murine norovirus in a simulated fresh-cut lettuce wash process

The water management in fresh-cut produce processing is an important factor affecting the microbial quality and safety of fresh-cut produce. For this study, a commercial lettuce washing process was simulated using two subsequent washing baths (WB). The worst case scenario, when no sanitizers are used and still common in some European countries, was investigated to fully understand the potential for cross-contamination and to obtain baseline data for further risk assessment. The two cross-contamination processes (from lettuce to water and from water to lettuce) were included in the simulation study and the transfer of Escherichia coli, E. coli O157, MS2 phage and murine norovirus was quantified. The mean reduction of initially contaminated lettuce through the washing in two successive WB was limited: 0.3 ± 0.1 log CFU E. coli/g after washing in WB1 and an additional reduction of 0.2 ± 0.1 log after WB2. The microbial load of the water in the washing baths, initially starting off with potable water, increased rapidly during the washing process of the contaminated lettuce. Furthermore to quantify the transfer of the four implicated micro-organisms from contaminated water to the lettuce, the first washing bath was inoculated with either approximately 3.0, 4.0 and 5.0 log CFU E. coli/100 ml or 4.8, 5.6 or 6.7 log CFU E. coli O157/100 ml, 4.0, 5.1 or 6.5 log PFU MS2 phages/100 ml or 6.5 log PFU/100 ml norovirus surrogate MNV-1. The contamination of the subsequently washed lettuce portions resulted in levels of ca. 1.0 up to 1.9 log CFU E. coli/g after passing the two washing steps. In addition, after a rapid initial increase, due to spill over of water from WB 1 to WB2, the contamination of WB2 further augmented during the washing process to approximately 1.0 to 0.5 log below the inoculation level of WB1. Transfer of E. coli O157, MS2 phages or MNV-1 from the water to the lettuce was respectively 0.9% ± 0.3%, 0.5% ± 0.2% and 0.5 ± 0.1% after WB1 and resulted in a contamination level for the highest inoculum level of WB1 of respectively ca. 2.9 ± 0.1 log CFU/g, 3.7 ± 0.1 log PFU/g and 4.4 ± 0.1 log PFU/g lettuce. The quantitative data of lettuce contamination and transfers established in this study further highlight the vulnerability of fresh produce to cross-contamination during the washing stage and stresses that notwithstanding the use of initial potable water and partial refreshment of water but without the use of sanitizers, microbial pathogens (or indicator organisms) may easily be introduced and reside for prolonged periods in washing baths enabling cross-contamination to the final fresh-cut product.     

Dynamics of low (1–4 cells) vs high populations of Listeria monocytogenes and Salmonella Typhimurium in fresh-cut salads and their sterile liquid or solidified extracts

The growth variability of low or high populations of Listeria monocytogenes and Salmonella Typhimurium in fresh-cut lettuce and cabbage was studied. The salads were inoculated with a few (1–4) or 1000 cells/sample of the above organisms and stored at 8 °C. Their liquid or solidified sterile extracts were also inoculated with the same cell numbers to evaluate the behavior of pathogens in the presence or absence of the epiphytic flora and illustrate the effect of the micro-environment structure, where growth may occur. The sterile extracts were stored at 8–10 °C simulating marginal temperature fluctuations. Inoculum of 1000 cells/sample increased with limited variation (SD < 0.5 log CFU/g) on vegetable salads, as opposed to the great variability (<0.7–3.4 log CFU/g) in the growth from 1 to 4 cells/sample. Total logarithmic increase of 1000 cells/sample of the pathogens on the salads ranged from 1.8 to 2.1 log CFU/g, contrary to 1–4 cells/sample, which exhibited higher increase (2.7–3.4 log CFU/g). The latter suggests that “fail-dangerous” implications may derive from challenge tests with unrealistic high inocula. Different batches of vegetables used for preparation of sterile extracts, introduced high variability in the growth of 1–4 cells/sample, suggesting nutrient-dependent effect on growth of pathogens. Low inoculum of L. monocytogenes did not increase in sterile cabbage extracts, whereas they increased from 1 to 3.5 logs in cabbage salad, probably due to the stimulatory effect of indigenous flora. In contrast, 1–4 cells/sample of S. Typhimurium grew only on the solidified extract of cabbage but not on the salad, indicating competitive activity of the indigenous microflora against the pathogen. Salmonella showed no growth at 8 °C but increased 4 logs at 10 °C, illustrating the impact of boundary storage conditions. Monte Carlo simulation of bacterial growth based on broth data overestimated growth of L. monocytogenes on lettuce, while remarkably underestimated the actual increase in cabbage. Significant deviation between model and data is likely when extrapolating broth-based simulations of growth from low populations in foods, due to the various factors affecting the microbial growth on a real food, which are (inevitably) ignored by broth-based models.     

Fat removal with hydrolyzed corn starch for real-time qPCR detection of Salmonella enterica in ground beef in 4.5 hours without enrichment

The rapid detection of low number of Salmonella in ground beef ideally requires an effective and economic molecular assay. The molecular analysis for the detection of Salmonella in ground beef by the polymerase chain reaction requires efficient methodology for extraction of targeted cells and effective removal of PCR inhibitors from the sample. The efficacy of hydrolyzed corn starch for the removal of fat along with the use of activated charcoal coated with milk proteins to remove PCR inhibitors were assessed. Salmonella enterica ser. Enteritidis was detected by real-time quantitative PCR at a level of 1 CFU/g of ground beef in 25 g samples containing 7%, 15% or 27% fat without enrichment. This study documents that partially hydrolyzed corn starch functions as effectively as beta-cyclodextrin for selective removal of fat.     

Inactivation of Listeria monocytogenes and Salmonella enterica serovar Senftenberg 775W inoculated into fruit juice by means of ultra high pressure homogenisation

The inactivation of Listeria monocytogenes and Salmonella enterica serovar Senftenberg 775 W by ultra high pressure homogenisation (UHPH) was evaluated in grape and orange juices inoculated at a concentration of approximately 7 log CFU/ml. The fluid inlet temperature used was 6 °C and the pressure levels assayed were 200, 300 and 400 MPa. Viable and injured bacterial counts were obtained 2 h after the UHPH treatments and after 5, 8, and 15 days of storage at 4 °C. Pressure level had a significant impact on the lethal effect of UHPH and complete inactivation of S. enterica serovar Senftenberg 775 W was achieved at 400 MPa. L. monocytogenes showed more resistance than S. enterica serovar Senftenberg 775 W to the UHPH treatments and no significant differences were observed between 300 and 400 MPa treatments in both juices. Sublethal injuries were not detected in any case. During the storage at 4 °C viable counts of both strains showed a decreasing trend. L. monocytogenes viable counts became undetectable in UHPH treated and also in control samples of grape juice which could be attributed to the presence of natural compounds with antilisterial effect.     

Fiber optic and light scattering sensors: Complimentary approaches to rapid detection of Salmonella enterica in food samples

Salmonella-related foodborne infections present a major public health problem worldwide despite more stringent regulations. Salmonella enterica serovars Enteritidis and Typhimurium are the two most frequent causes of poultry related outbreaks; therefore, their rapid and accurate detection would improve Salmonella control at the farm, processing plant, and at retail. In this study, we investigated if a fiber optic immunosensor and light scattering sensor, BARDOT (bacterial rapid detection using optical scattering technology) could facilitate the detection of these two serovars in naturally contaminated poultry (n = 50). The fiber optic sensor with a detection limit of 10³ CFU/ml identified S. enterica in selective enrichment broth in less than 12 h. The colonies (1.0 ± 0.2 mm) produced by plating the enriched samples on selective XLT4 agar for 13–15 h were scanned using BARDOT and S. enterica was identified after matching individual colony scatter patterns to the scatter image library with a sample-to-answer time of about 24 h. Both sensors identified 4 positive samples (8%), which corresponded to the results of the USDA-FSIS protocol, PCR, and lateral flow immunoassays. The colony scatter patterns identified all natural isolates as S. Enteritidis, which was further verified by serovar-specific PCR. The sensors used individually or in combination demonstrate potential for accurate and rapid detection of S. enterica in poultry.     

Efficacy of oregano oil in the inactivation of Salmonella typhimurium on lettuce

This study was conducted to determine the efficacy of oregano oil in the inactivation of Salmonella typhimurium inoculated onto iceberg lettuce. The effect of washing with oregano oil (Oreganum onites), typical of Turkey, at three different concentrations (25, 40 and 75 ppm) and four different treatment times (5, 10, 15 and 20 min) on survival of S. typhimurium inoculated to fresh cut iceberg lettuce were determined at 20 °C and compared with a 50 ppm chlorine wash at the same conditions. The spot and dip inoculation methods for applying inoculum to iceberg and romaine type lettuce were evaluated and the effects of selective and nonselective media on the recovery of S. typhimurium were also examined. Populations of S. typhimurium recovered from spot and dip inoculated lettuce were not significantly different (p > 0.05). No significant differences were found between two different media, and two different lettuce types (p > 0.05). Reductions of S. typhimurium by washing with oregano did not exceed 1.92 logarithmic units regardless of the washing times and concentrations. The effectiveness of washing lettuce with 75 ppm oregano oil on inactivation of S. typhimurium was comparable with that affected by 50 ppm chlorine. The results suggested that oregano oil might be a suitable decontamination alternative to chlorine for lettuce.     

Potential of high-throughput sequencing for broad-range detection of pathogenic bacteria in spices and herbs

A broad-range culture-independent method was developed and evaluated regarding its sensitivity of detection of pathogenic bacteria in spices and herbs, with focus on paprika powder. The method involved DNA extraction using cetyltrimethylammonium bromide (CTAB), 16S rDNA amplification using universal bacterial polymerase chain reaction, and high-throughput sequencing on Illumina MiSeq platform. The sensitivity of the method was evaluated with series of model samples contaminated at different levels with Salmonella enterica and Escherichia coli (as representatives of Gram-negative bacteria) and Staphylococcus aureus (as a representative of Gram-positive bacteria). For spices (paprika, black pepper), the method had a screening-level sensitivity with limits of detection in the range of 10⁴–10⁵ CFU/g, and a semi-quantitative response. Low sensitivity (LOD ≥10⁷ CFU/g) was observed with herbs (oregano, parsley). The developed method demonstrated a good potential for microbiological screening of spices, with a prospect of further improvement of sensitivity based on progress in high-throughput sequencing technology.     

Attachment of foodborne pathogens to banana (Musa sp.) leaves

Fresh produce, and in particular minimally processed leafy green vegetables, have been recognized as a source of transmission for foodborne pathogens of animal origin. In South-East Asian and other cultures the leaves of the banana plant (Musa sp.) are widely used as food wrappings or as serving plates because of their waxy surfaces and represent a largely uninvestigated leafy green product. This study was undertaken to quantify the attachment of two strains of each of the three bacterial foodborne pathogens Staphylococcus aureus, Salmonella enterica and Escherichia coli to the top side and underside of banana leaf surfaces. All bacteria tested attached to banana leaves at levels in the range of 3.5–4.5 cfu/cm². Differences in attachment to leaves between strains were apparent. Most notably both Salmonella strains attached to the top side of leaf surfaces in significantly (p < 0.05) lower numbers than two (one E. coli and one S. aureus) of the other four strains. Furthermore, the two S. aureus strains attached to the undersides of leaves in significantly (p < 0.05) lower numbers than to the top side of leaves. Despite the waxy nature of the banana leaf surfaces the bacteria tested were capable of attaching to them in numbers equivalent to the attachment of bacterial pathogens to other leafy green produce.