Natural Background Flora Research Articles

A duplex real-time NASBA assay targeting a serotype-specific gene for rapid detection of viable Salmonella Paratyphi C in retail foods of animal origin.

Salmonella enterica serovar Paratyphi C is highly adapted to humans and can cause a typhoid-like disease with high mortality rates. In this study, three serovar-specific genes were identified by comparative genomics for Salmonella Paratyphi C, SPC_0871, SPC_0872, and SPC_0908. Based on the SPC_0908 and xcd genes for testing Salmonella spp., we developed a duplex real-time nucleic acid sequence-based amplification (real-time NASBA) with a molecular beacon approach for the simultaneous detection of viable cells of Salmonella spp. and serotype Paratyphi C. The test selectively and consistently detected 53 Salmonella spp. (representing 31 serotypes) and 18 non-Salmonella strains. Additionally, the method showed high resistance to interference from natural background flora in pork and chicken samples. The sensitivity of the established approach was determined to be 4.89 cfu/25 g in artificially contaminated pork and chicken samples after pre-enrichment. We propose this NASBA-based protocol as a potential detection method for Salmonella spp. and serotype Paratyphi C in foods of animal origin.

Comparison of Oxygen‐ and Nitrogen‐Enriched Atmospheres on the Growth of Listeria Monocytogenes Inoculated on Poultry Breast Fillets

AbstractThe growth behavior of Listeria monocytogenes in the presence of typical spoilage microorganisms on poultry breast fillets (Pseudomonas spp., Brochothrix thermosphacta, Enterobacteriaceae, Lactobacillus spp., total viable count) was investigated. The aim of the study was to compare the growth of L. monocytogenes under oxygen‐ and nitrogen‐enriched atmospheres and to figure out possible interactions between L. monocytogenes and typical spoilage microorganisms on skinless poultry breast fillets. Therefore, the natural meat surface was inoculated with L. monocytogenes and packed under two commonly used gas atmospheres (70% O2/30% CO2 and 70% N2/30% CO2). The atmosphere was monitored over the storage period and typical sensory attributes (color, odor, texture, drip loss and general appearance) were evaluated. The results show that the storage under 70% N2/30% CO2 favored the growth of L. monocytogenes in comparison with the 70% O2/30% CO2. Under nitrogen, L. monocytogenes reached a maximal bacterial count of 5.7 (log10 cfu/g) after approximately 500 h of storage, but no specific microorganism could be identified responsible for the favored growth. The growth of L. monocytogenes was suppressed in the oxygen‐enriched atmosphere, whereas B. thermosphacta dominated the spoilage flora. The results indicate that the combination of high oxygen with carbon dioxide and possible interactions with the spoilage background flora cause a delay of the L. monocytogenes growth under 70% O2/30% CO2.Practical ApplicationsFresh poultry meat has become an increasingly popular food during the last 10 years due to its beneficial nutritional composition. From the microbiological point of view, raw poultry meat is sensitive for microbial growth because of its physical and chemical properties. In this context, the prediction of the growth of foodborne pathogenic bacteria e.g., Listeria monocytogenes is of vital importance to ensure a safe poultry production. Therefore, packaging under modified atmosphere conditions is widely established to improve the quality, shelf life as well as safety aspects. Taking into account that the growth of L. monocytogenes is also influenced by the natural spoilage flora of poultry reflects that interactions between the microorganisms have to be considered while packaging poultry filets under modified atmosphere conditions. The present study shows how two commonly used gas mixtures for poultry influence the outgrowth of L. monocytogenes in the presence of the natural background flora.

Microbial background flora in small-scale cheese production facilities does not inhibit growth and surface attachment of Listeria monocytogenes

The background microbiota of 5 Norwegian small-scale cheese production sites was examined and the effect of the isolated strains on the growth and survival of Listeria monocytogenes was investigated. Samples were taken from the air, food contact surfaces (storage surfaces, cheese molds, and brine) and noncontact surfaces (floor, drains, and doors) and all isolates were identified by sequencing and morphology (mold). A total of 1,314 isolates were identified and found to belong to 55 bacterial genera, 1 species of yeast, and 6 species of mold. Lactococcus spp. (all of which were Lactococcus lactis), Staphylococcus spp., Microbacterium spp., and Psychrobacter sp. were isolated from all 5 sites and Rhodococcus spp. and Chryseobacterium spp. from 4 sites. Thirty-two genera were only found in 1 out of 5 facilities each. Great variations were observed in the microbial background flora both between the 5 producers, and also within the various production sites. The greatest diversity of bacteria was found in drains and on rubber seals of doors. The flora on cheese storage shelves and in salt brines was less varied. A total of 62 bacterial isolates and 1 yeast isolate were tested for antilisterial activity in an overlay assay and a spot-on-lawn assay, but none showed significant inhibitory effects. Listeria monocytogenes was also co-cultured on ceramic tiles with bacteria dominating in the cheese production plants: Lactococcus lactis, Pseudomonas putida, Staphylococcus equorum, Rhodococcus spp., or Psychrobacter spp. None of the tested isolates altered the survival of L. monocytogenes on ceramic tiles. The conclusion of the study was that no common background flora exists in cheese production environments. None of the tested isolates inhibited the growth of L. monocytogenes. Hence, this study does not support the hypothesis that the natural background flora in cheese production environments inhibits the growth or survival of L. monocytogenes.

A real-time PCR method for the detection of Salmonella enterica from food using a target sequence identified by comparative genomic analysis

A 5′-nuclease real-time PCR assay using a minor groove binding probe was developed for the detection of Salmonella enterica from artificially contaminated foods. S. enterica-specific sequences were identified by a comparative genomic approach. Several species-specific target sequences were evaluated for specificity. A real-time PCR assay was developed targeting a nucleotide sequence within the putative type III secretion ATP synthase gene ( ssaN). An internal amplification control (IAC) probe was designed by randomly shuffling the target probe sequence and a single-stranded oligonucleotide was synthesized to serve as an IAC. The assay demonstrated 100% inclusivity for the 40 Salmonella strains tested and 100% exclusivity for 24 non- Salmonella strains. The detection limit of the real-time PCR assay was 41.2 fg/PCR with Salmonella Typhimurium genomic DNA and 18.6 fg/PCR using Salmonella Enteritidis genomic DNA; 8 and 4 genome equivalents, respectively. In the presence of a natural background flora derived from chicken meat enrichment cultures, the sample preparation and PCR method were capable of detecting as few as 130 Salmonella cfu/mL. Using the developed real-time PCR method to detect Salmonella in artificially contaminated chicken, liquid egg and peanut butter samples, as few as 1 cfu/10 g of sample was detectable after a brief (6 h) non-selective culture enrichment.

A novel packaging method with a dissolving CO 2 headspace combined with organic acids prolongs the shelf life of fresh salmon

The aim of this study was to evaluate a novel packaging method for fresh fish and to determine its effect on the bacterial growth in fresh salmon. Fresh salmon was packed with a small amount of 100% CO 2 (gas/product ratio 0.2/1.0 v/v) and a brine solution containing various combinations of citric acid (3% w/w, pH 5), acetic acid (1% w/w, pH 5) and cinnamaldehyde (200 μg/ml). Total bacterial counts, counts of sulphur reducing bacteria, lactic acid bacteria and Enterobacteriaceae as well as the bacterial composition in the product after storage were determined. The combination of CO 2 and organic acids completely inhibited bacterial growth during 14 days of storage at 4 °C both in inoculation experiments and in experiments on salmon with natural background flora. CO 2, acetic acid and citric acid alone each inhibited the growth of total bacterial counts, lactic acid bacteria, sulphur reducing bacteria and Enterobacteriaceae, but effects were enhanced in combinations. The addition of cinnamaldehyde did not influence bacterial growth. Analysis of the bacterial flora of salmon inoculated with different spoilage bacteria showed that Photobacterium phosphoreum and Carnobacterium maltaromaticum remained the dominating species after inoculation while Yersinia aldovae, Aeromonas salmonicida and Shewanella putrefaciens were outcompeted by other species. In addition, lactic acid bacteria from the natural background flora grew to high numbers. Combinations of CO 2 and acetic acid reduced the relative abundance of P. phosphoreum. All CO 2 dissolved in the product, thereby creating a product with the outer appearance of a vacuum package. Further work is needed to determine consumer acceptability of acid concentrations and to implement the packaging method for industrial purposes. However, implication of this packaging method in the industry may lead to a new packaging technology, combining the advantages of vacuum packaging (low space requirement) and modified atmosphere packaging (antimicrobial effect of CO 2).

Effect of infrared heating on quality and microbial decontamination in paprika powder

Infrared radiation (IR) was explored as a technique for decontaminating paprika powder. The effect of water activity ( a w) and IR heat flux on paprika temperature and water loss were measured during near- or medium-IR heating. Paprika was evaluated in terms of colour, a w, natural flora, and inoculated Bacillus cereus spores. Surface temperatures were considerably higher than temperatures inside the powder, especially at low a w; greater differences were observed with medium- than with near-IR. Surface darkening was observed, though the overall colour was not considerably affected. IR effectively removed water from paprika, especially at a w 0.5 and 0.8, resulting in unsatisfactory spore reduction. However, at a w 0.8, the load of the natural flora was reduced ( P < 0.05). In a w 0.96 powder, areas with high remaining a w displayed a reduction >6 log 10 CFU/g for B. cereus ( P < 0.05). In addition, no microbial counts of the natural background flora were observed in the paprika.

Antagonistic Action of Lactobacillus delbrueckii ssp. lactis RM2-5 Toward Spoilage Organisms in Cottage Cheese

Cells of Lactobacillus delbrueckii ssp. lactis RM2-5 were added to cottage cheese stored at 7°C in different amounts to determine if they would inhibit the growth of Pseudomonas fluorescens, also inoculated into the cheese samples. In addition, experiments were conducted in which no spoilage organisms were added to determine the effect of the lactobacilli on the natural background flora in the cottage cheese. For most experiments, as the numbers of lactobacilli increased, the numbers of spoilage organisms were lower than in the control on any given day of storage. In cheese inoculated with P. fluorescens, the numbers of spoilage organisms in the control had increased 5 log cycles by d 7, whereas the treatment containing the highest level of L. delbrueckii ssp. lactis RM2-5 (1.0×109 cfu/g) had not, and did not, increase over the course of the 21-d study. In the experiments where no spoilage organisms were added, lactobacilli significantly retarded the growth of gram-negative bacteria in the cheese. However, in these experiments, mold growth on the samples became a limiting factor during extended storage. The results of these experiments indicate that lactobacilli could be effective at helping control gram-negative spoilage bacteria in cottage cheese, thus potentially extending its shelf life.

Rapid quantification of Yersinia enterocolitica in pork samples by a novel sample preparation method, flotation, prior to real-time PCR.

The development of real-time PCR thermal cycles in the late 1990s has opened up the possibility of accurate quantification of microorganisms in clinical, environmental, and food samples. However, a lack of suitable sample preparation methods that allow rapid quantification of the nucleic acids, remove PCR inhibitors, and prevent false-positive results due to DNA originating from dead cells has limited the use of quantitative PCR. We have used for the first time a new variant of density gradient centrifugation, called flotation, as a user-friendly sample preparation method prior to PCR. This paper describes the use of this sample preparation method, without DNA purification, for direct detection and quantification of Yersinia enterocolitica in PCR-inhibitory meat juice from pork. Flotation combined with qPCR could overcome PCR interference in juice from pork, as was shown by amplification efficiencies of 1.006 +/- 0.021 and 1.007 +/- 0.025, which are comparable to the amplification efficiency obtained for purified DNA samples (1.005 +/- 0.059). Applying flotation to meat juice samples containing natural background flora and spiked with different levels of Y. enterocolitica showed that direct quantification of Y. enterocolitica was possible down to a level of at least 4.2 x 10(3) CFU per ml of meat juice, even in the presence of 10(6) CFU of background flora per ml. Finally, the results showed that samples containing large amounts of Y. enterocolitica DNA did not result in a positive PCR signal. This indicates that the risk of false-positive results due to detection of DNA originating from dead cells can be greatly reduced by using flotation prior to PCR.

High levels of background flora inhibits growth of Escherichia coli O157:H7 in ground beef

The influence of natural background flora under aerobic and anaerobic incubation on the growth of Escherichia coli O157:H7 in ground beef was investigated. The background flora from eight different commercial ground beef were added to ground beef spiked with E. coli O157:H7 and stored either aerobically or anaerobically at 12°C. The results showed that the presence of a large number of background bacteria in the ground meat inhibited the growth of E. coli O157:H7 both aerobically and anaerobically. Inhibition was more pronounced under anaerobic conditions. The background floras consisted mainly of lactic acid bacteria of which approximately 80% were Lactobacillus sakei. These results show the importance of the natural background flora in meat for inhibition of growth of E. coli O157:H7.

Influence of raw meat natural background flora on growth of Escherichia coli O157: H7 in ground beef

Escherichia coli O157:H7 is a foodborne pathogen of increasing importance. It has been involved in several threatening outbreaks, most of them associated with meat products. In this study, the influence of some bacteria from the natural background flora of raw meat over E.coli O157:H7 in ground beef stored under refrigeration and at room temperature was evaluated. Different levels of E.coli O157:H7 (101-102, 103-104 and 106-107 CFU/g), inoculated in ground beef samples, were challenged with strains of non-pathogenic E.coli, Pseudomonas putida or Leuconostoc sp. Growth of the pathogen was monitored using standard cultural methods and an ELISA-type rapid method. Non-pathogenic E.coli, Pseudomonas putida and Leuconostoc sp. did not affect growth of E.coli O157:H7 in ground beef, both under refrigeration and at room temperature. Based on these findings, the low occurrence of E.coli O157:H7 in raw meat may not be attributed to antagonistic effects of bacteria from the natural background flora.

A 20–24 H MICROCOLONY‐IMMUNOBLOT TECHNIQUE to DIRECTLY DETECT and ENUMERATE LISTERIA MONOCYTOGENES INOCULATED INTO FOODS

A microcolony‐immunoblot technique (MCIBI) was developed to directly enumerate, in less than 24 h, very low numbers of Listeria monocytogenes (8–12 colony forming units: CFU/g or mL) inoculated into foods. Four meat and poultry and two dairy products were artificially inoculated with L. monocytogenes V7 diluted and plated on Oxford agar medium. Each plate was overlaid with an Immobilon‐P membrane and incubated for 18–20 h at 37C. Blot‐transferred colonies on these membranes were probed with C11E9 monoclonal antibody (MAb) and developed using peroxidase conjugated goat antimo use Ig G and a water insoluble substrate (3,3‐diaminobenzidin tetrahydmchloride; (DAB‐HCI), Nickel chloride and H2O2). the MCIBT gave L. monocytogenes counts that were not significantly lower than direct colony counts on selective agars. This technique allowed the recovery of 94–100% of L. monocytogenes cells inoculated into foods containing natural background flora counts of 3 × 104 to 8 × 106 CFU/g or mL. Using a 2 h resuscitation period on nonselective agar before overlay with Oxford media, the MCIBT allowed detection of sublethally heat injured cells of strain V7.

Incidence of Salmonellae in Clams, Oysters, Crabs and Mullet

Sixty samples each of oysters (Crassostrea virginica), clams (Mercenaria mercenaria), striped mullet (Mugil cephalus), and blue crab (Callinectes sapidus) were analyzed for the presence of salmonellae within 4 h of harvesting from an east and west coast location in Florida. Mullet was the only seafood from which salmonellae were not recovered. Clams from the west coast location had the highest incidence (43%) of salmonellae. Eleven serotypes of Salmonella were recovered and identified from the samples. In quantitative MPN studies, the number of salmonellae present in west coast oysters was 2.2 organisms per 100 g of oyster meats. The variety of serotypes isolated, coupled with consistent recoveries throughout the year, suggests that salmonellae may represent a portion of the natural background flora in these seafoods rather than recent single source contamination.

Interaction of salt, pH, and temperature on the growth and survival of salmonellae in ground pork.

The interaction of temperature, pH, and NaCl concentration on the growth and survival of several strains of salmonellae has been determined in broth and ground pork. Growth of 23 strains occurred in broth at 30 C over a wide range of pH-NaCl combinations; at 10 C, growth was limited to only a few combinations. Cultures which would not grow at 10 C because of the pH-NaCl effect survived for long periods, however. In contrast, cultures which would not grow at 30 C remained viable for only a short time. Results in fresh ground pork were in close agreement with the broth studies. Salmonellae would not grow in ground pork stored at 4 C but would grow in pork containing 3.5% salt stored at 10 C. Salmonellae grew competitively with the natural background flora at 10 C even when the salmonellae constituted less than 5% of the initial flora, and the background flora would grow at a lower temperature than the salmonellae. The data show that, whereas decreasing temperatures increase the inhibitory effects of pH and NaCl, they decrease the lethal effects.

Interaction of Salt, pH, and Temperature on the Growth and Survival of Salmonellae in Ground Pork

The interaction of temperature, pH, and NaCl concentration on the growth and survival of several strains of salmonellae has been determined in broth and ground pork. Growth of 23 strains occurred in broth at 30 C over a wide range of pH-NaCl combinations; at 10 C, growth was limited to only a few combinations. Cultures which would not grow at 10 C because of the pH-NaCl effect survived for long periods, however. In contrast, cultures which would not grow at 30 C remained viable for only a short time. Results in fresh ground pork were in close agreement with the broth studies. Salmonellae would not grow in ground pork stored at 4 C but would grow in pork containing 3.5% salt stored at 10 C. Salmonellae grew competitively with the natural background flora at 10 C even when the salmonellae constituted less than 5% of the initial flora, and the background flora would grow at a lower temperature than the salmonellae. The data show that, whereas decreasing temperatures increase the inhibitory effects of pH and NaCl, they decrease the lethal effects.