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.     

Development of a quantitative real-time PCR assay for viable Salmonella spp. without enrichment

Propidium monoazide (PMA) combined with molecular quantitative real-time PCR (qPCR) has been widely used for only detection of viable bacteria. However, recent studies indicated PMA did not fully inhibit the detection of dead Salmonella. In this study, we developed a more effective PMA Taqman-based qPCR than previous studies to quantify viable Salmonella spp. in raw shrimp. This method has high specificity by using 60 strains belonging to 23 species. The optimization of the PMA concentration showed that 100 μM was considered optimal to effectively inhibit 10⁶ CFU/mL dead cells, while only 10³–10⁴ CFU/mL dead cells could be inhibited in previous reports. This assay could detect viable Salmonella spp. at as low as 36 CFU/mL in pure culture and 100 CFU/g in raw shrimp. By comparing with PMA-qPCR, qPCR and plate counting for quantifying Salmonella in samples, this PMA-qPCR was obviously superior to qPCR and had good agreement with plate counting. In conclusion, this effective method can be used as an available tool to quantify viable Salmonella spp. in raw shrimp.     

Development of an SD-PMA-mPCR assay with internal amplification control for rapid and sensitive detection of viable Salmonella spp., Shigella spp. and Staphylococcus aureus in food products

In this work, a specific, sensitive, and accurate technique was presented for simultaneous detection of Salmonella spp., Shigella spp., and Staphylococcus aureus in food products, three of the more frequent foodborne pathogens that were usually reported in a variety of food matrices. An internal amplification control (IAC) was added in a multiplex PCR (mPCR) reaction system as an indicator of false negative result that can come from the presence of PCR inhibitors in food products. In the presence of inhibitor, no signal would result for the target genes as well as the IAC which results in a positive signal, thereby, eliminating false negative results. To ensure detection of only the viable cells, the effects of sodium deoxycholate (SD) in combination with propidium monoazide (PMA) treatment in the presence of dead cells and viable cells were investigated. Results showed that PMA treatment alone could not effectively inhibit the detection of 10⁷ CFU/mL of dead Salmonella Typhimurium, Shigella sonnei, and S. aureus from PCR amplification. However, the SD in combination with PMA treatment gave negative results for PCR amplification of dead S. Typhimurium, S. sonnei, and S. aureus in pure culture and food products. When the developed SD-PMA-mPCR assay in combination with IAC was applied to detect the spiked food (milk, ground beef), the LOD of SD-PMA-mPCR assay for S. Typhimurium, S. sonnei, and S. aureus inoculated individually or inoculated simultaneously into milk or ground beef were 10¹ CFU/mL or 10¹ CFU/g after 15 h enrichment. The results suggested that the SD-PMA-mPCR assay in combination with IAC held promise for the detection of foodborne S. Typhimurium, S. sonnei, and S. aureus.     

Rapid detection of Salmonella using a redox cycling-based electrochemical method

An electrochemical method based on redox cycling combined with immunomagnetic separation and pre-concentration was developed for rapid and sensitive detection of Salmonella. Electrochemical methods for the detection of bacteria offer the advantages of instant quantification with minimal equipment. Unfortunately, the limits of detection are often poor compare to other transduction methods such as fluorescence and chemiliuminescence. We demonstrated an electrochemical method which is both rapid and has a low limit of detection. A two-step strategy, which included immunomagentic pre-concentration and redox cycling was used to amplify the signal. Magnetic beads modified with anti-Salmonella antibodies were used for separation and pre-concentration of Salmonella from phosphate buffered saline (PBS) and agricultural water. Then anti-Salmonella antibodies conjugated with alkaline phosphatase were employed for labeling the Salmonella which had been captured by magnetic beads. Alkaline phosphatase (ALP) catalyzed the substrate l-ascorbic acid 2-phosphate (AAP) to electroactive species l-ascorbic acid (AA) while tris(2-carboxyethyl)phosphine (TCEP) facilitated the regeneration of AA on the gold electrode to form redox cycling resulting in an amplified signal. Under the optimal conditions, the Salmonella in PBS buffer as well as in agricultural water were detected. The limit of detection of this approach was approximately 7.6 × 10² CFU/mL and 6.0 × 10² CFU/mL in PBS buffer and agricultural water, respectively, without pre-enrichment in 3 h. When the agricultural water has been pre-enriched for 4 h, the limit of detection was approximately 10 CFU/mL.     

The prevalence and serovar diversity of Salmonella in various food products in Estonia

In this work the prevalence and serovar diversity of Salmonella in various food products including non-thermally processed food and ready-to-eat (RTE) food in Estonia in 2008–2012 are summarized. The findings demonstrate that the overall prevalence of Salmonella in these food categories was low. A total of 260 (0.54%) of 47,927 food samples were found to be positive for Salmonella, the overall prevalence in non-thermally processed food was 0.81% (256/31,576) and in RTE products only 0.02% (4/16,351). Salmonella was most often isolated from raw eggs and products thereof (2.17%, 5/230), followed by raw meat products (0.95%, 207/21,723), RTE mayonnaises (0.90%, 2/221) and raw meat (0.89%, 38/4252). In the raw meat category, Salmonella was most frequently isolated from turkey meat (6.96%, 11 positive samples out of 158), broiler chicken meat (4.00%, 7/175) and from layer hen meat (2.22%, 11/496). Salmonella was isolated in lesser extent from meat preparations (1.91%, 82/4292), minced meat and mechanically separated meat products (0.97%, 100/10,344) and from raw sausages (0.35%, 25/7087).Altogether 24 different serovars were identified among the 260 Salmonella positive samples. Salmonella Typhimurium was the most frequent serovar (26.90% of the positive samples) and it was isolated most commonly in raw food products. The next most frequent serovars were Salmonella Derby (17.50%), Salmonella Enteritidis (8.37%) and Salmonella Newport (7.57%). The only serovars isolated from the Salmonella positive RTE food samples were Salmonella Infantis (two isolates) and S. Enteritidis (two isolates).     

Use of a commercial mixture of volatile compounds from the fungus Muscodor to inhibit Salmonella in ground turkey and beef

Additional interventions to reduce the risk of Salmonella in ground meat products are needed in the industry. Fungi in the genus Muscodor produce an array of volatile compounds with antimicrobial activity. A commercial mixture of these volatile compounds (all considered to be GRAS), in proportions similar to that produced by the fungus, was assessed for its inhibitory activity against Salmonella in vitro. The minimal inhibitory concentration of the volatiles mixture for growth of Salmonella enterica in Mueller-Hinton broth was 0.5% (v/v). Exposure to the vapor phase of the volatile compounds similarly inhibited visible growth of Salmonella on agar (up to 6 cm zone of inhibition). Addition of the volatiles mixture (0.25%–1.0% v/w) inhibited Salmonella by 1.5 and 2.8 log₁₀ CFU in ground turkey (85% or 93% lean, respectively) and 2.2 and 1.7 log₁₀ CFU in ground beef (73% or 93% lean, respectively) during a 5 day period at 8 °C. Addition of the volatiles also inhibited growth of normal microflora on ground turkey at 8 °C by approximately 5.3 log₁₀ CFU. These findings indicate this mixture of volatile compounds retards growth of spoilage organisms and Salmonella in ground meat.     

Prevalence,molecular identification and antimicrobial resistance profile of Salmonella serovars isolated from retail beef products in Mansoura,Egypt

The present study was undertaken to determine the prevalence of Salmonella in 270 raw meat samples (90 each of fresh beef, ground beef, and beef burger) purchased on nine occasions from various supermarkets and butchers' shops in Mansoura city, Egypt. Using conventional biochemical identification, Salmonella species were recovered from 23.3% (21/90), 20% (18/90), and 12.2% (11/90) of fresh beef, ground beef and beef burger samples, respectively with an overall prevalence of 18.5% (50/270) among all the meat products examined. Higher prevalence were obtained based on molecular identification, by detecting gyrB and invA genes, which verified the presence of Salmonella species in 30.0% (27/90), 26.7% (24/90), and 16.7% (15/90) of fresh beef, ground beef, and beef burger samples, respectively with an overall prevalence of 24.4% (66/270) among all the meat products tested. Of the 2635 presumptive colonies tested, 228 were biochemically identified as Salmonella, while 272 were molecularly identified as Salmonella, which were all positive for the enterotoxin (stn) virulent gene. Of the 272 serologically tested strains, 266 were serologically identified into six Salmonella serovars, while 6 strains were untypable. Salmonella Typhimurium and Salmonella Enteritidis were the most prevalent serovars with an incidence of 38.2% (104/272) and 34.6% (94/272), respectively. The other four serovars identified were Salmonella Haifa, Salmonella Muenster, Salmonella Virchow, and Salmonella Anatum were detected at lower prevalences of 11% (30/272), 7.4% (20/272), 4% (11/272) and 2.6% (7/272), respectively. Interestingly, the antimicrobial susceptibility testing indicated that all of the 100 Salmonella serovars tested were multidrug resistant (resistant to three or more antibiotics). Our findings demonstrated that the retail beef products tested were widely contaminated with multidrug-resistant Salmonella and such contamination may constitute a major public health concern.     

Inactivation of Salmonella spp. in ground chicken using high pressure processing

High Pressure Processing (HPP) is a safe and effective process for improving the microbial safety and shelf-life of foods. Salmonella is a common contaminant in poultry meat and is frequently responsible for foodborne illness associated with contaminated poultry meat. In this study the inactivation of a five-isolate cocktail of Salmonella spp. in ground chicken (95% lean) using HPP at refrigeration temperature (4–6 °C) was studied. More than 5-log CFU/g inactivation was achieved at 450 MPa for10 min. In contrast, HPP treatment at 250 MPa or 350 MPa (single-cycle, 15 min) inactivated 0.5 log or 1.7 log CFU/g, respectively. The multiple-cycle HPP mode at 250 or 350 MPa (3-cycle with 5 min/cycle) showed higher cell reduction at 1.3 or 3.3 log CFU/g, respectively. HPP at 550 MPa for 10 min may reduce the cell counts, initially at 8.5 log CFU/g, to below the detection limit (1.0 log CFU/g) in current study. The images (electron microscopy) of the HPP shocked cells were examined for structural damage, which demonstrated that Salmonella cells may still look intact (with damages on rough/irregular surface at 450 MPa stress) under Scanning Electron Microscopy (SEM), but have significant damage internally (voids and uneven mass distribution patterns) under Transmission Electron Microscopy (TEM).     

Rapid and simultaneous detection of Salmonella,Shigella, and Staphylococcus aureus in fresh pork using a multiplex real-time PCR assay based on immunomagnetic separation

The incidence of foodborne infections caused by Salmonella sp., Shigella sp. and Staphylococcus (S.) aureus in fresh pork is increasing each year, which poses a great potential threat to public health. In this study, a rapid and simultaneous detection for these three pathogens from fresh pork samples was developed by combining immunomagnetic separation (IMS) with multiplex real-time PCR (RT-PCR). Magnetic beads coated with specific antibodies were used to capture and purify the pathogens from 250 mL matrix prepared by both spiked and commercial samples, followed by DNA extraction. Then, multiplex RT-PCR was applied with three sets of specific primers and probes. The limit of detections were evaluated in 67 spiked pork samples and were 2.0 CFU/g for Salmonella, 6.8 CFU/g for Shigella, and 9.6 CFU/g for S. aureus. The sensitivity, specificity, and accuracy of IMS-multiplex RT-PCR method were 99.2%, 100%, and 99.5%, respectively. One hundred fifty-one samples were tested using the IMS-multiplex RT-PCR and culture methods, and a comparison of the results showed that the former was a potentially reliable method for rapid and effective detection of Salmonella sp., Shigella sp., and S. aureus in fresh pork.     

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.     

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.     

A simple quantum dot-based fluoroimmunoassay method for selective capturing and rapid detection of Salmonella Enteritidis on eggs

The aim of this paper was to demonstrate a rapid and selective fluorescence-linked immunoassay method based on quantum dots as the fluorescent marker for the detection of Salmonella Enteritidis on eggshell. Highly-fluorescent and water-soluble CdTe quantum dots were prepared by using thioglycolic acid and 1-thioglycerol as ligands and were then conjugated with anti-Salmonella antibodies. As a result of specific interaction, Salmonella Enteritidis were specifically captured by bioconjugated CdTe quantum dots which led to the detection of a fluorescent signal. The bacterial cell images were obtained using fluorescence microscopy. Under optimal conditions, the quenched fluorescence intensity increased linearly with the log total count of Salmonella Enteritidis ranging from 3 × 10² to 3 × 10⁷ CFU/mL within 1–2 h. The low detection limit was 1 × 10² CFU/mL without sample enrichment. This method was satisfactorily applied to the analysis of egg samples, which was demonstrated as a simple scheme for quick and selective detection of Salmonella Enteritidis on eggshell.     

Presence,distribution, serotypes and antimicrobial resistance profiles of Salmonella among pigs,chickens and goats in South Africa

Salmonellosis is an infectious zoonotic disease of socio-economic importance worldwide. Food animals with subclinical infection as well as farm effluents are usually the sources of contaminated meat, eggs and milk, which cause diarrhoea and systemic infections in humans. The indiscriminate use of antibiotics to curb salmonellosis in both animals and humans has contributed to the emergence and spread of drug-resistant bacteria among both pathogenic and commensal organisms. The aim of the study was therefore to determine the presence, serovar distribution and antimicrobial resistance profiles of Salmonella isolated from domestic livestock species in South Africa. For this purpose,1069 rectal and cloacal swabs were collected from pigs (n = 322), chickens (n = 286) and goats (n = 461) from smallholder farms in Limpopo, Eastern Cape, Northern Cape, North West and KwaZulu Natal provinces of South Africa. The frequency of occurrence of Salmonella per animal species was highest in pigs (5.90%; n = 19), followed by chickens (3.15%; n = 9) and goats had the lowest proportion of 0.43% (n = 2). Nine Salmonella serovars were obtained including S. Techimani, a serovar that was not previously observed in South African animals. Six isolates were assigned to Salmonella II. Some of the Salmonella were untypable (n = 6). All Salmonella isolates were sensitive to cefotaxime, enrofloxacin, florphenicol and polymyxin B. Most of the Salmonella isolates were resistant to at least one antimicrobial (n = 20; 66.7%) and resistance was predominant towards trimethoprim (n = 11; 36.7%), followed by ampicillin (n = 5; 16.7%), oxytetracycline (n = 3; 10%), and kanamycin (n = 1; 3.3%). The results illustrate the presence of diverse and rare Salmonella serovars that were not previously isolated from animals in South Africa. The pattern of development of antibiotic resistance should be monitored and followed-up. The occurrence of elevated trimethoprim resistant Salmonella in South African food animals could lead to the emergence and distribution of drug resistant salmonellosis in human beings.     

Development of a multiplex real-time PCR method for simultaneous detection of Vibrio parahaemolyticus,Listeria monocytogenes and Salmonella spp. in raw shrimp

Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella spp. are important pathogens contaminating seafood in China. In this study, we developed an efficient multiplex real-time PCR for the simultaneous detection of V. parahaemolyticus, L. monocytogenes and Salmonella spp. in raw shrimp without a prior enrichment step. In a test using 28 target and non-target strains only the targets were detected and two calibration curves, for pure cultures and artificially contaminated samples, were used to evaluate the efficiencies of this method. Amplification efficiencies of this multiplex real-time PCR were excellent in pure cultures and artificially contaminated shrimps. The limits of detection in artificially contaminated shrimps were 112 CFU/g for V. parahaemolyticus, 158 CFU/g for L. monocytogenes and 103 CFU/g for Salmonella spp. We validated this multiplex real-time PCR method on 48 commercial samples and the results were comparable to standard culture methods. This efficient multiplex real-time PCR, where each test takes only 50 min after DNA extraction, is a useful tool for high-throughput surveillance of V. parahaemolyticus, L. monocytogenes and Salmonella spp. in seafood products.     

The efficacy of disinfectant misting in the lairage of a pig abattoir to reduce Salmonella and Enterobacteriaceae on pigs prior to slaughter

Water misting/showers are used in abattoir lairages to improve meat quality, and to cool and calm pigs after transport and during hot weather. One novel approach, which has not been investigated to date, is to add a disinfectant to the misting water as a means of topically reducing Salmonella on pigs prior to slaughter, thereby potentially controlling this organism in the abattoir. The objective of this study was therefore to evaluate misting with water or with Virkon^® S (an approved disinfectant for use in the presence of animals), for their ability to topically reduce Salmonella on high seroprevalence pig herds before stunning and to reduce Enterobacteriaceae.Three experimental groups were investigated: control group (i.e., no misting); water group (misting with cold, 15–17 °C, water, herein referred to as water); and a disinfectant group (misting with 0.5% Virkon^® S). As pigs entered the abattoir, each animal was swabbed along its back before being allocated to its experimental group. Each group was randomly assigned to one of 3 lairage pens that were separated by non-trial pens. After 30 min of misting with water or disinfectant, pigs were moved to the stunning area, where each pig was again swabbed, as above. Swabs were analyzed for the presence of Salmonella and enumeration of Enterobacteriaceae.Before misting, Salmonella prevalence on the pigs was 79.0%, 72.1% and 83.6% for the control, water and disinfectant groups, respectively. After misting, Salmonella prevalence increased to 94.3% in the water group; whereas for the disinfectant group, the prevalence increased marginally to 85.9%. No change in Salmonella prevalence was detected for the control group. In line with the Salmonella results, no significant differences were observed in Enterobacteriaceae counts in the control group at either time point (4.37 and 5.01 log₁₀ CFU/cm², respectively) or in the disinfectant group before and after misting (4.02 and 4.26 log₁₀ CFU/cm², respectively). However, a 2.3 log₁₀ CFU/cm² increase in Enterobacteriaceae was recorded for the water group after misting as compared to before misting (p < 0.05).Since misting with water alone increased topical Salmonella contamination on pigs before slaughter, a risk assessment based on known Salmonella data, meat quality and welfare is recommended to determine whether its use is justifiable. On the other hand, the findings from this study suggest that misting with Virkon^® S at 0.5% could have a role in topical antisepsis of pigs contaminated with Salmonella prior to slaughter and as such this warrants further investigation.     

Prevalence,genetic characterization and antimicrobial resistance of Salmonella isolated from fresh pork sausages in Porto Alegre,Brazil

A total of 336 samples of fresh pork sausage randomly obtained from supermarkets and butcher shops in Porto Alegre, Brazil, were examined for the presence of Salmonella serovars. Salmonella enterica was detected in 82 (24.4%) of the samples, with a most probable number count ranging from 0.03 MPN g^?1 to 460 MPN g^?1. Strains belonging to the most isolated S. enterica serovars (Brandenburg, Panama, Derby and Typhimurium) were further characterized by XbaI-macrorestriction, resulting in a total of 17 profiles. Resistance to tetracycline was the most prevalent among the Salmonella isolates. S. panama and S. typhimurium presented the greatest number of resistance phenotypes.     

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.     

Prevalence of Salmonella in raw minced meat,raw fresh sausages and raw burger patties from retail outlets in Gaborone,Botswana

The prevalence of Salmonella in raw minced meat, raw burger patties and raw fresh sausages was determined by analysing 122 minced meat, 120 sausages and 58 burger patties obtained from retail outlets in Gaborone, Botswana. The prevalence rate was 20%. The most prevalent serogroups were B, followed by C and E/G. The Salmonella enterica serovars isolated were S. Typhi, S. Enteritidis, S. Anatum, S. Reading, S. Melagridis, S. Typhimurium, S. Paratyphi B, S. Newport, S. Bovis-morbificans, S. Braenderup, S. Infantis, S. Tennessee and S. Montevideo. The presence of S. Typhi and Paratyphi in meat products indicate human origin and therefore poor personal hygiene during handling of the meat products. Multidrug resistance patterns involving sulphatriad, sulphafurazole, tetracycline and cotrimoxazole were observed. Isolates in serogroups B and C were resistant to a greater number of antibiotics than isolates from other serogroups.     

General patterns of background microbiota and selected bacterial pathogens during production of fermented sausages in Serbia

The presence of Salmonella spp. or Escherichia coli O157 and background microbiota, pH and a_w were determined in raw fermented sausages produced from pork or beef and without lactic acid bacteria starters. The investigation was conducted at five meat processing plants, and the sampling was done at five steps of the production process at each plant. In meat trimmings, total viable count (TVC) ranged around 6 log CFU/g and around 5–6 log CFU/g in the pork and the beef sausages, respectively. Enterobacteriaceae count (EBC) ranged in the vicinity of 3–4 log CFU/g, whilst E. coli count (ECC) ranges were comparably lower (by 1–2 logs). During chopping of both the pork and the beef trimmings, the levels of TVC, EBC and ECC increased by 1–1.5 logs. After the additives and the spices were added, background microbiota tended to slightly decrease, generally more noticeably in pork sausages and with ECC. During the fermentation-drying stage, in both pork and beef sausages, initial TVC levels (6–7 log CFU/g) increased by the mid-process (by approximately 1.5–2 logs) and remained at those levels in finished products. During the same period, lactic acid bacteria (LAB) increased from initial levels of 5.5–6 log CFU/g to around 7–8 log CFU/g in pork and around 8–9 log CFU/g in beef sausages, and became the predominant microbial group. Salmonella spp. was found in the first three stages of the production process (trimmings, trimmings chopping, mixing with additives/spices), in two of three meat processing plants, but not at later stages of the production process. E. coli O157 was found only in one sample of chopped trimmings in one meat processing plant. The background microbiota patterns and levels were, generally, similar to those commonly reported for raw fermented sausages in other published studies. The initial presence of foodborne pathogens in raw fermented sausage production may be considered as a potential meat safety risk, because in the case of high initial pathogen counts, their total elimination cannot be assumed.     

Bacterial assessment of phage magnetoelastic sensors for Salmonella enterica Typhimurium detection in chicken meat

Salmonella is one of the most common pathogens associated with foodborne illness in chickens. Food outbreaks from this pathogen haven’t declined in the past 15 years according to the data from Centers for Disease Control and Prevention. It is our goal to improve food safety monitoring in this area by developing a real time Salmonella detection sensor on food surfaces. Previously, we demonstrated the use of phage C4-22 immobilized onto a rapid magnetoelastic (ME) biosensor for use as a front-line detection ligand to detect all Salmonella enterica serotypes in Tris Buffer Saline (TBS). In this study, by using fluorescent imaging, the phage peptide binding to Salmonella enterica serotype Typhimurium cells is again confirmed. Moreover, we constructed two detection models to evaluate the detection of Salmonella on/in chicken meat using the phage coated ME sensors.In the chicken surface detection method, phage C4-22 sensors demonstrated more than 12 times higher Salmonella binding capacity than the control sensors with no phage for the Salmonella spiked at the concentration of 7.86 × 10⁵ cfu/mm². In the second model, phage sensors were placed at different depths inside the chicken breast (0.1 cm; 0.5 cm; 1.0 cm below the meat surface) after surface inoculation of Salmonella. The second detection system showed that 23.27%–33% of the inoculated Salmonella cells absorbed inside the chicken breast fillets below 0.1 cm of the surface.The data for direct detection on chicken showed that phage C4-22 ME biosensors bind ultimately when there are high concentrations of Salmonella on the chicken surface. The results also suggest that the phage sensors can detect Salmonella effectively when the bacterial contaminants are absorbed into the chicken, and are not detectable by the surface detection method.