Use of bacteriophages as biocontrol agents to control Salmonella associated with seed sprouts

Two Salmonella bacteriophages (SSP5 and SSP6) were isolated and characterized based on their morphology and host range, and evaluated for their potential to control Salmonella Oranienburg in vitro and on experimentally contaminated alfalfa seeds. Phages SSP5 and SSP6 were classified as members of the Myoviridae and Siphoviridae families, respectively. Both phages had a broad host range of over 65% of the 41 Salmonella strains tested. During in vitro trials, the phages resulted in incomplete lysis of Salmonella cultures, in spite of high levels of phage remaining in the system. Phage SSP5 was more effective in reducing Salmonella populations. Addition of phage SSP6 to alfalfa seeds previously contaminated with S. Oranienburg caused an approximately 1 log(10) CFU g(-1) reduction of viable Salmonella, which was achieved 3 h after phage application. Thereafter the phage had no inhibitory effect on Salmonella population growth. A second addition of the same (SSP6) or different (SSP5) phage to a Salmonella culture treated with phage SSP6, did not affect Salmonella populations. It was further shown that development of Salmonella permanently resistant to phage was not evident in either seed or in vitro challenge trials, suggesting the existence of a temporary, acquired, non-specific phage resistance phenomenon. These factors may complicate the use of phages for biocontrol.     

Application of a bacteriophage cocktail to reduce Salmonella Typhimurium U288 contamination on pig skin

Multidrug-resistant Salmonella Typhimurium U288 is a significant pathogen of pigs, accounting for over half of all outbreaks on UK pig production premises. The potential of this serovar, and other salmonellae, to enter the food chain during the slaughtering process requires that efforts be made to reduce the prevalence of these bacteria at both the pre- and post-harvest stages of production. A bacteriophage cocktail (PC1) capable of lysing various Salmonella enterica serovars was designed using the broad host-range phage Felix 01, and three phages isolated from sewage. PC1 applied to pig skin experimentally-contaminated with U288 achieved significant reductions (P < 0.05) in Salmonella counts when stored at 4 °C over 96 h. Reductions of > 1 log₁₀ unit were observed when the ratio of phage applied was in excess of the bacterial concentration. The treatment was found to be effective at a multiplicity of infection (MOI) of 10 or above, with no significant reductions taking place when the MOI was less than 10. Under these conditions U288 counts of log₁₀ 4.1-4.3 CFU were reduced to undetectable levels following the application of PC1 to pig skin (> 99% reduction). These data suggest phage cocktails could be employed post-slaughter as a means to reduce Salmonella contamination of pig carcasses.     

Application of bacteriophages during depuration reduces the load of Salmonella Typhimurium in cockles

As bivalve molluscs are filter feeder, often consumed raw or lightly cooked and are frequently cultivated in contaminated waters, they are implicated in food-borne disease transmission to human. The present study investigated the potential application of bacteriophage (or phage) phSE-2, phage phSE-5 and phage cocktail phSE-2/phSE-5 to decrease the concentration of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) during the depuration of natural and artificially contaminated cockles (Cerastoderma edule). Cockles were artificially infected with 10⁵ and 10⁶ colony-forming units (CFU)/mL of S. Typhimurium in static seawater and infected group were treated with phages at four different MOI values: 0.1, 1, 10 and 100. Depuration in static seawater at multiplicity of infection (MOI) of 0.1 with single phage suspensions of phSE-2 and phSE-5 provided the best results, as it decreased by ~ 1.3 and 1.7 log CFU/g, respectively, the concentration of Salmonella spp. after a 4 h treatment. At a MOI of 0.1, the rate of inactivation with single phage suspensions was higher when compared with the results obtained using the phage cocktail. However, in naturally contaminated cockles treated in static seawater with single phage suspensions and phage cocktail phSE-2/phSE-5, similar decreases in cultivable bacteria concentration (~ 0.7–0.9 log CFU/g) were achieved after 6 h of treatment. When artificially contaminated cockles were depurated with phage phSE-5 in a recirculated seawater system (mimicking industrial depuration conditions), a 0.9 and 2.0 log CFU/g reduction of Salmonella spp. was reached after 4 and 6 h treatment. Once the depuration process was performed without phage, a 6 h treatment was needed to obtain a 1.1 log CFU/g reduction of Salmonella spp. Results indicated that combining phage biocontrol with depuration procedures enhance bivalve microbial safety for human consumption by improving decontamination efficiency, proving that this technology can be transposed to the bivalves industry. Moreover, this approach also displays the advantage of reducing the time required for depuration and consequently its associated costs.     

Evaluation of stored lamb bio-preserved using a three-strain cocktail of Lactobacillus sakei

Commercially prepared lamb was stored at −1.5 °C after inoculation with a combination of three Lactobacillus sakei strains previously shown to inhibit spoilage and pathogenic bacteria of importance to the meat industry. Between 6 and 14 weeks storage samples were evaluated for growth of inoculated strains, production of fermentation end-products and sensory acceptance of the cooked product. All three L. sakei strains flourished during storage, formed consistently dominant populations and were associated with lower surface pH and increased levels of lactic and acetic acids. Inoculated samples were determined to be as equally acceptable for smell, acidity, rancidity and overall liking as un-inoculated controls.     

Evaluation of reference samples for the detection of Salmonella

Reference samples consisting of spray-dried milk artificially contaminated with Salmonella typhimurium II 505 contained in a gelatin capsule have been developed to evaluate the standard method for the detection of Salmonellae in foods and feeding stuffs. The present study was carried out to evaluate these reference samples. Salmonella isolations from reference samples in the absence of food were satisfactory in 4 out of 6 laboratories when Müller-Kauffmann tetrathionate broth prepared according to ISO-6579 (TBB-ISO) was used and in all laboratories when the secondary enrichment medium was the magnesium chloride-malachite green broth of Rappaport-Vassiliadis (RV). The proportion of Salmonella isolations from reference samples decreased from 80% to 47% with TBB-ISO and from 95% to 63% with RV in the presence of food. Most negative results were obtained with foods such as minced pork and minced beef, which had high aerobic and Enterobacteriaceae plate counts. It is concluded that the reference samples can be used to test the performance of media and the reproducibility of methods when it is used without the addition of food. The results obtained for Salmonella isolations in the presence of food questioned the role of numbers and types of competitive micro-organisms. In particular these latter results indicated that more research is needed to gain a better understanding of the Salmonella isolation procedure.     

Evaluation of stored lamb bio-preserved using a three-strain cocktail of Lactobacillus sakei

Commercially prepared lamb was stored at −1.5 °C after inoculation with a combination of three Lactobacillus sakei strains previously shown to inhibit spoilage and pathogenic bacteria of importance to the meat industry. Between 6 and 14 weeks storage samples were evaluated for growth of inoculated strains, production of fermentation end-products and sensory acceptance of the cooked product. All three L. sakei strains flourished during storage, formed consistently dominant populations and were associated with lower surface pH and increased levels of lactic and acetic acids. Inoculated samples were determined to be as equally acceptable for smell, acidity, rancidity and overall liking as un-inoculated controls.     

Prevalence of bacteriophages infecting Staphylococcus aureus in dairy samples and their potential as biocontrol agents

The prevalence of bacteriophages infecting Staphylococcus aureus in dairy samples was assessed. Fourteen Staph. aureus strains were used in enrichment cultures of 75 dairy samples. All samples grew specific Staph. aureus bacteriophages. According to the host range, 8 different phages were isolated. Three of them, phages ΦH5, ΦG7, and ΦA72, were found in 89% of the samples; all the isolated phages were temperate. Phages ΦH5 and ΦA72 were used in preliminary bacterial challenge tests against Staph. aureus in milk. A phage mixture (1:1) was more effective than each single phage, most likely by preventing the survival of lysogenized cells. Phages inhibited Staph. aureus in UHT and pasteurized whole-fat milk. However, the phages were less active in semi-skimmed raw milk and little inhibition was achieved in whole, raw milk. Killing of Staph. aureus was observed at room temperature and at 37°C, but not at refrigeration temperature.     

沙门氏菌和副溶血性弧菌裂解性噬菌体及其裂解酶研究进展

由微生物引发的食品安全问题给人类健康带来严重危害,其中沙门氏菌和副溶血性弧菌是最主要的2种微生物病原。噬菌体及其裂解酶的发现与应用为食源性致病菌的控制提供了新的技术,而因其具有安全、高效、特异性强、不易产生抗性等特点,在食品安全领域具有良好的应用前景。本文综述了沙门氏菌和副溶血性弧菌裂解性噬菌体及其裂解酶的研究与应用进展,沙门氏菌和副溶血性弧菌这2种致病菌的裂解性噬菌体在环境中广泛存在,对畜禽肉、海产品及其他食品中的沙门氏菌和副溶血性弧菌具有显著的减菌作用,其噬菌体裂解酶的研究还处于初级阶段,仅有几种裂解酶的研究报道,也显示了较好的溶菌活性。本文旨在为利用噬菌体及其裂解酶对食源性疾病的防控和人类的健康提供保障措施。     

Prevalence of Salmonella infecting bacteriophages associated with Ontario pig farms and the holding area of a high capacity pork processing facility

BACKGROUND: There is interest in applying bacteriophages to control Salmonella in pig production and pork processing. The following reports on the prevalence of Salmonella infecting bacteriophages within Ontario pig farms and associated with the holding area of a pork slaughterhouse. RESULTS: Salmonella infecting bacteriophages were present in 30 and 28 of the effluent manure samples collected from 36 farms using S. Typhimurium DT104 or S. Heidelberg as host cell respectively. Bacteriophages were recovered in 95–100% of the 48 samples taken from holding pens within a high capacity slaughterhouse over a 12 month period. Bacteriophages isolated from farms exhibited similar host ranges which differed to that of slaughterhouse isolates. Salmonella (n = 21) from the slaughterhouse were susceptible to the endogenous bacteriophages. Despite being susceptible to the resident phages, the Salmonella populations were found to be genetically stable with the same genotypes being recovered over successive visits. Salmonella isolated from the farms were frequently resistant to the endogenous phages. CONCLUSIONS: Bacteriophages are prevalent in the pig slaughterhouse environment although they do not have a significant impact on the genetic structure of Salmonella populations. However, there was evidence that the Salmonella population structure on farms is influenced by the presence of infecting phages. Copyright © 2010 Society of Chemical Industry     

Evaluation of the Premi Test Salmonella, a commercial low-density DNA microarray system intended for routine identification and typing of Salmonella enterica

A new commercial system based on genetic profiling and aimed at identifying Salmonella enterica serovars was evaluated by comparing its performance with classical serotyping on 443 strains. Within 62 serovars represented, 60 gave unique genetic profiles while 2 were undistinguishable. Results were obtained within 8 h, were reproducible and clear-cut. The system allowed single-tube processing of the samples and required no peculiar technical skill. It showed interesting potential for routine laboratory testing.     

Effect of high hydrostatic pressure and pressure cycling on a pathogenic Salmonella enterica serovar cocktail inoculated into creamy peanut butter

The ability of Salmonella enterica serovars to survive in high fat content, low water activity foods like peanut butter has been demonstrated by large foodborne illness outbreaks in recent years. This study investigates the potential of high hydrostatic pressure processing, including pressure cycling, to inactivate Salmonella inoculated into creamy peanut butter. A cocktail of pathogenic strains of Salmonella Enteritidis PT30, Salmonella Tennessee, Salmonella Oranienburg, Salmonella Anatum, Salmonella Enteritidis PT 9c, and Salmonella Montevideo obtained from peanut butter- and nut-related outbreaks was inoculated (10(6) to 10(7) CFU/g) into creamy peanut butter and high pressure processed under five different sets of conditions, which varied from 400 to 600 MPa and from 4 to 18 min. The log CFU reductions achieved varied from 1.6 to 1.9. Control experiments in which Salmonella was inoculated (10(9) CFU/g) into 0.1% peptone buffer and high pressure processed at 600 MPa for 18 min showed inactivation to below the detection limit of 100 CFU/g, confirming that high pressure processing is effective at destroying Salmonella in high-moisture environments. Pressure cycling under three sets of conditions consisting of pressures from 400 to 600 MPa, 3 to 10 pressure cycles, and hold times of 6 min for each cycle showed reductions similar to those seen in noncycling experiments. The results of our experiments suggest that the peanut butter food matrix facilitates the survival of Salmonella when exposed to high hydrostatic pressure processing.     

Evaluation of a polymerase chain reaction-based test for detecting Salmonella spp. in food samples: Probelia Salmonella spp

A commercially available polymerase chain reaction (PCR) kit was evaluated for the detection of Salmonella spp. in food samples. The test combines PCR amplification and sandwich hybridization of the amplified DNA in microtiter plates. The sensitivity and specificity was evaluated with 52 Salmonella strains and 51 non-Salmonella strains and showed that the test was entirely reliable. The threshold sensitivity was 10(2) CFU/ml. The limit of detection of dead cells that determines the minimum detection level of dead cells in food samples was superior to 10(6) CFU/25 g, a level rarely achieved in naturally contaminated samples. After an 18-h pre-enrichment step, the test could detect viable Salmonella in artificially contaminated food samples, even for the lower contamination level (3 CFU/25 g). There was complete agreement between the PCR test and the ISO 6579 bacteriological reference method with artificially contaminated samples. Regarding the accuracy of the results obtained from 253 naturally or noncontaminated foods and from 32 artificially contaminated foods, the agreement percentage was 99.6%. The fidelity of the technique was evaluated in a collaborative study with eight European laboratories and showed a correlation of 98.4%.     

Evaluation of a capillary immunoassay system for detection of Salmonella typhimurium in poultry products

A capillary immunoassay system was constructed and optimized for detection of Salmonella Typhimurium. The system consisted of a capillary bioseparator-bioreactor and a flow-injection electrochemical detector. Three methods were compared for immobilizing antibodies on the inner surface of silica capillary columns; these methods were based on the use of a homobifunctional cross-linker glutaraldehyde, a heterobifunctional cross-linker N-succinimidyl-4-maleimidobutyrate, and biotin-streptavidin chemistry, respectively. The glutaraldehyde method gave the best reproducibility with a relative standard deviation of 1 to 6% for detection of Salmonella Typhimurium. The optimized immunoassay system could detect Salmonella Typhimurium in chicken breast and ground turkey meats with a detection limit of 2.4 x 10(3) and 2.4 x 10(4) CFU/ml, respectively. The total detection time was less than 2.5 h without any preenrichment. When stored at 4 degrees C, the immunocolumns could retain their activities for at least 3 months.     

3种沙门氏菌检测方法能力验证

目的比较国标法、VETIK和荧光定量PCR法3种沙门氏菌检测方法的检测效果。方法 10份盲样经GB4789.4-2010《食品安全国家标准食品微生物学检验沙门氏菌检验》、实时荧光定量PCR方法和VIDAS仪器检测,生化部分用VITEK2 compact执行。结果国标法在10个盲样中检出3种血清型沙门氏菌,分别为5号样品检出阿贡纳沙门氏菌、7号样品检出蒙得维的亚沙门氏菌和肯塔基沙门氏菌。VETIK和荧光定量PCR法检出5号和7号呈沙门氏菌阳性,其他为阴性。结论 VIDAS和实时荧光PCR检测方法快速、可靠、灵敏度高,可作为传统检测方法有效补充。     

Evaluation of a two-step protocol for rapid detection of Salmonella in ice-cream and Cheddar cheese

A two-step protocol for repair, multiplication and rapid detection of Salmonella species in ice-cream and Cheddar cheese was evaluated. The first step involves selective preenrichment using lactose broth supplemented with sodium pyruvate and yeast extract for the repair of injured cells and brilliant green for repression of the competing flora (LBPYEBG). This enrichment is incubated for 7 h at 40°C. The second step involves direct plating of 7 h selective preenrichment onto XLD agar and simultaneous inoculations into Salmonella 1–2 Test^®for both isolation and presumptive identification of Salmonella the next day. The two-step protocol requires only 26±2 h for isolation and presumptive identification of Salmonella. This protocol was successfully used in detecting as few as 2 colony forming units (cfu) of non-stressed S. enteritidis per ml in 250 ml of enrichment. Initial inocula of 3 cfu ml⁻¹of freeze-thaw-injured S. enteritidis inoculated into various ice-creams grew to significantly (p≤0·05) higher numbers after 6 and 7 h in the LBPYEBG enrichment than in the conventional lactose broth. This was also found to be true with a naturally contaminated ice-cream (MPN of 0·09 g⁻¹of S. enteritidis).S. typhimurium HF artificially incorporated into Cheddar cheese grew more rapidly in the LBPYEBG enrichment than in the conventional lactose broth. This indicates the usefulness of this protocol for rapid detection of Salmonella spp. from ice-cream and Cheddar cheese.     

A rapid most-probable-number-based enzyme-linked immunosorbent assay for the detection and enumeration of Salmonella Typhimurium in poultry wastewater

The rapid and accurate detection and enumeration of low levels of Salmonella Typhimurium in food processing facilities are critical components of an effective hazard analysis critical control point program. The objective of this study was to develop a rapid (8 h) most probable number (MPN)-enzyme-linked immunosorbent assay (ELISA) for the detection and enumeration of Salmonella Typhimurium in wastewater. The specific objectives were to (i) characterize poly- and monoclonal Salmonella Typhimurium-specific antibodies in order to select the most specific and sensitive antibody for Salmonella Typhimurium detection, and (ii) validate the MPN assay through a correlation between the 8-h MPN-ELISA and the traditional 48-h Salmonella Typhimurium MPN method in poultry scald water. Poultry scald water samples were spiked with 10 and 50 CFU/ml of Salmonella Typhimurium. The traditional MPN method used a 48-h enrichment period followed by an analysis, while the MPN-ELISA used a 5-h enrichment period followed by a 3-h ELISA analysis. No differences (P < 0.05) were found between the traditional MPN and the MPN-ELISA, indicating the promise of the MPN-ELISA for the rapid detection and enumeration of Salmonella Typhimurium within an 8-h shift. This abbreviated assay will permit increased product sampling and more rapid movement of food between production and processing, resulting in reduced spoilage and quality losses.     

Evaluation of the BAX gel and fluorometric systems for the detection of foodborne Salmonella

The present study compared the sensitivity of the BAX automated fluorometric and the recently discontinued BAX gel electrophoresis systems with a standard culture method to detect Salmonella in 333 high-moisture and 171 low-moisture foods. A total of 95 naturally contaminated foods, including 63 high-moisture and 32 low-moisture foods, were detected by the standard culture method. No contaminated samples were identified exclusively by the BAX systems. By means of the analytical protocol stipulated by the manufacturer, the BAX fluorometric system detected 36 (57.1%) and 29 (90.6%) of the contaminated high- and low-moisture foods, respectively. Similar results were obtained with the BAX gel electrophoresis system, which identified 40 (63.5%) and 26 (81.3%) of the contaminated high- and low-moisture foods. The rate of false-positive reactions with the BAX systems was low. Our results indicate that the low sensitivity of the BAX systems with high-moisture foods, notably raw meats and poultry products, was serovar-independent. The high levels of background microflora that commonly occur in raw meat and on fresh fruit and vegetable products, and the high successive dilutions of test materials for PCR analysis, suggestively undermined the sensitivity of the gel and the fluorometric BAX assays. The potential benefits of immunomagnetic separation of Salmonella in preenrichment cultures, of selective broth enrichment following preenrichment to markedly reduce levels of background microflora in PCR test materials, and the use of larger portions of test materials in PCR analyses should be investigated.     

Evaluation of overhead spray-applied sanitizers for the reduction of Salmonella on tomato surfaces

Abstract: Efficacy of sanitizers in an overhead spray and brush roller system was examined for reducing Salmonella on unwaxed, mature green tomatoes. Surface inoculated tomatoes were treated in the overhead spray system for 5, 15, 30, and 60 s. A sodium hypochlorite (NaOCl) study tested NaOCl (25, 50, and 100 mg/L) against a water control. A sanitizer study examined NaOCl (100 mg/L), chlorine dioxide (ClO₂; 5 mg/L), peroxyacetic acid (PAA; 80 mg/L), and water. The overhead spray system was also compared to a scale‐model flume. All NaOCl concentrations were significantly more effective at removing Salmonella than water and achieved at least a 3‐log₁₀ CFU/mL reduction at different treatment times (P < 0.05). NaOCl (100 mg/L) achieved a 4 ± 1.8 log₁₀ CFU/mL reduction at 15 s. In the sanitizer study, NaOCl, ClO₂, and PAA achieved at least a 3‐log₁₀ CFU/mL reduction at 15 s and between 3.9 and 5.5 log₁₀ CFU/mL reductions at 30 to 60 s. NaOCl (100 mg/L) in the overhead spray system significantly reduced more Salmonella than in the flume at 15 to 60 s. NaOCl flume treatment only reached a 1.3 ± 1.1 log₁₀ CFU/mL reduction at 15 s. Results of this study demonstrate the ability of sanitizers in the laboratory model overhead spray system to reduce Salmonella on tomato surfaces. An overhead spray system could be implemented instead of flumes to achieve higher pathogen reduction with less water and sanitizer use, thereby lowering packing costs. Practical Application: The use of a non‐recirculating, overhead spray brush roller system could offer a cost effective and efficacious way of washing tomatoes. The use large communal dump tanks in tomato processing has been suspected as a source of contamination in the tomato processing process. If effective, the brush roller system could augment or possible replace currently used dump tanks.