Switchgrass extractives to mitigate Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium contamination of romaine lettuce at pre- and postharvest.

The antimicrobial potential of switchgrass extractives (SE) was evaluated on cut lettuce leaves and romaine lettuce in planta, using rifampicin-resistant Escherichia coli O157:H7 and Salmonella Typhimurium strain LT2 as model pathogens. Cut lettuce leaves were swabbed with E. coli O157:H7 or S. Typhimurium followed by surface treatment with 0.8% SE, 0.6% sodium hypochlorite, or water for 1 to 45 min. For in planta studies, SE was swabbed on demarcated leaf surfaces either prior to or after inoculation of greenhouse-grown lettuce with E. coli O157:H7 or S. Typhimurium; the leaf samples were collected after 0, 24, and 48 h of treatment. Bacteria from inoculated leaves were enumerated on tryptic soy agar plates (and also on MacConkey's and XLT4 agar plates), and the recovered counts were statistically analyzed. Cut lettuce leaves showed E. coli O157:H7 reduction between 3.25 and 6.17 log CFU/leaf, whereas S. Typhimurium reductions were between 2.94 log CFU/leaf and 5.47 log CFU/leaf depending on the SE treatment durations, from initial levels of ∼7 log CFU/leaf. SE treatment of lettuce in planta, before bacterial inoculation, reduced E. coli O157:H7 and S. Typhimurium populations by 1.88 and 2.49 log CFU after 24 h and 3 h, respectively. However, SE treatment after bacterial inoculation of lettuce plants decreased E. coli O157:H7 populations by 3.04 log CFU (after 0 h) with negligible reduction of S. Typhimurium populations. Our findings demonstrate the potential of SE as a plant-based method for decontaminating E. coli O157:H7 on lettuce during pre- and postharvest stages in hurdle approaches.

Propidium monoazide for viable Salmonella enterica detection by PCR and LAMP assays in comparison to RNA-based RT-PCR, RT-LAMP, and culture-based assays.

Rapid and sensitive detection of live/infectious foodborne pathogens is urgently needed in order to prevent outbreaks and food recalls. This study aimed to (1) evaluate the incorporation of propidium monoazide (PMA) into PCR or LAMP assays to selectively detect viable Salmonella Enteritidis following sublethal heat or UV treatment, and autoclave sterilization; and (2) compare the detection of PMA-PCR and PMA-LAMP to DNA-based PCR and LAMP (without PMA), RNA-based RT-PCR and RT-LAMP, and culture-based methods. Nucleic acids (DNA or RNA) from 1-mL S. Enteritidis samples were used for PCR, RT-PCR, LAMP, and RT-LAMP assays. Serially diluted samples were plated on Xylose Lysine Tergitol-4 agar for cultural enumeration. Comparable detection of overnight cultured S. Enteritidis was obtained by PMA-PCR, PCR, and RT-PCR, though 1 to 2 log less sensitive than cultural assays. PMA-LAMP and RT-LAMP showed similar detection of overnight cultures, being 1 to 2 log less sensitive than the LAMP assay, and ∼4 log less than culture-based detection. Autoclaved S. Enteritidis did not test positive by RNA-based methods or PMA-PCR, but PMA-LAMP showed detection of 1 log CFU/mL. PMA-PCR and RT-PCR showed comparable detection of sublethal heat-treated cells to cultural assays, while PMA-LAMP showed 1 to 2 log less detection. Our results suggest that PMA-PCR and PMA-LAMP assays are not suitable for selective viable cell detection after UV treatment. While PMA-LAMP assay needs optimization, PMA-PCR shows promise for live/viable S. Enteritidis detection. PMA-PCR shows potential for routine testing in the food industry with results within 1-day, albeit depending on the inactivation method employed.

High Intensity Ultrasound for Salmonella Enteritidis Inactivation in Culture and Liquid Whole Eggs.

High intensity ultrasound (HIU) continues to be researched as a nonthermal inactivation technology of appeal to food manufacturers. The advantages of HIU include maintenance of product quality, freshness, product homogenization, along with simultaneous inactivation of pathogens. Besides, it is simple, relatively inexpensive, and easily adaptable to most processing environments. This study investigated the effect of HIU for Salmonella Enteritidis inactivation in culture and liquid whole eggs (LWEs) to decrease egg-associated outbreaks. Overnight S. Enteritidis cultures and spiked LWE (both at 8 log CFU/mL) were treated with 20-kHz HIU for 0, 1, 5, 10, and 30 min (n = 6) in a temperature-controlled system, not to exceed 20 °C, and replicated thrice. At each time point, samples were enumerated on XLT4 agar and morphologically analyzed using scanning electron microscopy, with measurements of color and rheological properties. Our results revealed significant reduction of healthy S. Enteritidis cells up to 3.6 log CFU/mL and 2.3 log CFU/25 mL after HIU treatment of merely 10 min of overnight culture and 30 min in LWE, respectively (P < 0.05). After 5 and 10-min HIU treatment, significant reduction of 1.4-log CFU/25 mL healthy S. Enteritidis in LWE was obtained (P < 0.05). Even at 1-min exposure time, HIU showed significant 1.9 log CFU/mL reduction of cultures (P < 0.05); however, no log-reduction was observed in LWE after 1 min. Scanning electron micrographs showed increased cell structural damage using longer HIU exposure. For product color changes, lower redness and yellowness of LWE were observed visually and instrumentally after 5-min HIU treatment (P < 0.05). The rheological properties of LWE measured at 0 to 200 s-1 shear rate, showed that shear stress of HIU-treated LWEs decreased after 5-min HIU exposure, but increased after 30-min treatment. This study demonstrated that HIU shows promise for rapid Salmonella control in LWE and other liquid foods, as an alternative inactivation method for use in hurdle approaches.

Antimicrobial Resistance of Staphylococcus aureus Isolates from Dairy Cows and Genetic Diversity of Resistant Isolates.

Staphylococcus aureus is a frequent and major contagious mastitis bacterial pathogen. The antibiotic treatment cure rates vary considerably from 4% to 92%. Staphylococcus aureus readily becomes resistant to antibiotics, resulting in persistent noncurable intramammary infection that usually results in culling of infected animals. Because of its notorious ability to acquire resistance to the commonly used as well as last resort antimicrobials such as methicillin and vancomycin and the development of multidrug-resistant strains, antimicrobial resistance (AMR) in S. aureus is of paramount importance in human medicine. The objective of this study was to evaluate the prevalence of AMR and genetic diversity of S. aureus isolates from milk of dairy cattle. Staphylococcus aureus isolates (n = 239) from 33 dairy farms in Tennessee were tested against 10 antimicrobials by broth microdilution method using the Sensititer system. Genetic diversity of resistant isolates was evaluated by pulsed-field gel electrophoresis (PFGE). Overall, AMR of the S. aureus isolates varied from as low as 1.3% for ceftiofur to as high as 25% for sulfadimethoxine. Out of 239 S. aureus isolates, 82 (34.3%) of them were resistant to at least 1 of the 10 antimicrobials. The AMR isolates belonged to two major PFGE types, indicating the presence of dominant clonal patterns among the resistant isolates. In general, there was a variation of prevalence of AMR within and among farms over time, with an increasing trend in tetracycline resistance. Judicious use of antimicrobials in dairy cattle farms can reduce the development of antimicrobial-resistant S. aureus.

Survival and inactivation of human norovirus surrogates in blueberry juice by high-pressure homogenization.

Human noroviruses (HNoV) have been implicated in gastrointestinal outbreaks associated with fresh produce, juices, and ready-to-eat foods. In order to determine the risk of HNoV transmission by contaminated blueberry juice, survival characteristics of cultivable HNoV surrogates (murine norovirus, MNV-1; feline calicivirus, FCV-F9; and bacteriophage MS2) in blueberry juice (pH = 2.77) after 0, 1, 2, 7, 14, and 21 days at refrigeration temperatures (4°C) were studied. High-pressure homogenization (HPH) was studied as a novel processing method for noroviral surrogate inactivation in blueberry juice. Blueberry juice or phosphate-buffered saline (PBS; pH 7.2 as control) was inoculated with each virus, stored over 21 days at 4°C or subjected to HPH, and plaque assayed. FCV-F9 (∼5 log(10) PFU/mL) was undetectable after 1 day in blueberry juice at 4°C. MNV-1 (∼4 log(10) PFU/ml) showed minimal reduction (1 log(10) PFU/mL) after 14 days, with greater reduction (1.95 log(10) PFU/mL; p < 0.05) after 21 days in blueberry juice at 4°C. Bacteriophage MS2 (∼6 log(10) PFU/mL) showed significant reduction (1.93 log(10) PFU/mL; p < 0.05) after 2 days and was undetectable after 7 days in blueberry juice at 4°C. FCV-F9 remained viable in PBS for up to 21 days (2.28 log(10) PFU/mL reduction), while MNV-1 and MS2 survived after 21 days (1.08 and 0.56 log(10) PFU/mL reduction, respectively). Intriguingly, FCV-F9 and bacteriophage MS2 showed reduction after minimal homogenization pressures in blueberry juice (pH = 2.77), possibly due to the combination of juice pH, juice components, and mechanical effects. MNV-1 in blueberry juice was only slightly reduced at 250 (0.33 log(10) PFU/mL) and 300 MPa (0.71 log(10) PFU/mL). Virus surrogate survival in blueberry juice at 4°C correlates well with the ease of HNoV transmission via juices. HPH for viral inactivation in juices is dependent on virus type, and higher homogenization pressures may be needed for MNV-1 inactivation.

Human norovirus surrogate reduction in milk and juice blends by high pressure homogenization.

Novel processing technologies such as high pressure homogenization (HPH) for the inactivation of foodborne viruses in fluids that retain nutritional attributes are in high demand. The objectives of this research were (i) to determine the effects of HPH alone or with an emulsifier (lecithin) on human norovirus surrogates-murine norovirus (MNV-1) and feline calicivirus (FCV-F9)-in skim milk and orange juice, and (ii) to determine HPH effects on FCV-F9 and MNV-1 in orange and pomegranate juice blends. Experiments were conducted in duplicate at 0, 100, 200, 250, and 300 MPa for <2 s and plaque was assayed in duplicate. In milk, FCV-F9 was reduced by ≥4 and ∼1.3 log PFU/ml at 300 and 250 MPa, respectively, and ≥4- and ∼1-log PFU/ml reductions were obtained in orange juice at 300 and 250 MPa, respectively. In orange juice or milk combined with lecithin, FCV-F9 was reduced to nondetectable levels at 300 MPa, and by 1.77 and 0.78 log PFU/ml at 250 MPa. MNV-1 in milk was reduced by ∼1.3 log PFU/ml only at 300 MPa, and by ∼0.8 and ∼0.4 log PFU/ml in orange juice at 300 and 250 MPa, respectively. MNV-1 in milk or orange juice containing lecithin at 300 MPa showed 1.32- and 2.5-log PFU/ml reductions, respectively. In the pomegranate-orange juice blend, FCV-F9 was completely reduced, and MNV-1 was reduced by 1.04 and 1.78 log PFU/ml at 250 and 300 MPa, respectively. These results show that HPH has potential for commercial use to inactivate foodborne virus surrogates in juices.

Reverse-transcriptase loop-mediated isothermal amplification as a rapid screening/monitoring tool for Salmonella enterica detection in liquid whole eggs.

Reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) is a novel molecular detection method that is specific, fast, and simple. It is based on reverse transcription followed by DNA amplification using the Bst DNA polymerase large fragment requiring one temperature and a simple waterbath, without the need for any expensive equipment. Detection is by turbidity or agarose gel electrophoresis. Our objective was to apply this LAMP-based technology to rapidly and sensitively detect Salmonella enterica serovar Enteritidis in liquid whole eggs (LWEs) within 1 d. LWE were inoculated with S. Enteritidis and stomached in tetrathionate broth (TTB), and spread-plated on Xylose lysine tergitol 4 agar either immediately or after 6, 12, or 16-h enrichment. RNA was extracted from 5-mL TTB and the RT-LAMP assay was carried out using invA primers. After 16 and 12-h enrichment, improved Salmonella detection up to 10° to 10¹ and 104 CFU/25 mL LWE, respectively was obtained. Without enrichment, Salmonella could be detected at 107 CFU/25 mL; however, after 6-h enrichment a 1-log improvement to 106 CFU/25 mL was obtained. This RT-LAMP assay appears to be suitable as a potential screening/monitoring tool for Salmonella enterica from LWE products in routine settings with results obtainable within 24-h, which is significantly faster than traditional cultural assays.

Survival of human norovirus surrogates in milk, orange, and pomegranate juice, and juice blends at refrigeration (4 °C).

Fresh fruits, juices, and beverages have been implicated in human noroviral and hepatitis A virus outbreaks. The purpose of this study was to determine the survival of human norovirus surrogates (murine norovirus, MNV-1; feline calicivirus, FCV-F9; and bacteriophage MS2) in juices (orange and pomegranate juices), juice blends (pomegranate and orange juice) and milk over 0, 1, 2, 7, 14, and 21 days at refrigeration (4 °C). Juices, juice blends, and milk were inoculated with each virus over 21 days, serially diluted in cell culture media, and plaque assayed. MNV-1 showed no reduction in titer after 21 days in orange juice and milk, but moderate reduction (1.4 log) in pomegranate juice from a titer of 5 log(10) PFU/ml. However, MNV-1 was completely reduced after 7 days in the orange and pomegranate juice blend. FCV-F9 from a titer of 6 log(10) PFU/ml was completely reduced after 14 days in orange as well as pomegranate juice and by ∼ 3 logs after 21 days in milk at 4 °C. Interestingly, FCV-F9 was completely reduced after 1 day in the orange and pomegranate juice blend at 4 °C. MS2 was reduced by ∼ 1.28 log after 21 days in orange juice from a titer of 6 log(10) PFU/ml, and <1 log after 21 days in milk or pomegranate juice, with juice blends showing minimal reduction (<1 log) after 21 days at 4 °C. These results show the survival pattern of noroviruses that aid in the transmission of foodborne viral outbreaks. The data obtained can be used in quantitative viral risk assessment studies and to develop improved measures to prevent virus survival towards controlling outbreaks.

Optimization of rapid Salmonella enterica detection in liquid whole eggs by SYBR green I-based real-time reverse transcriptase-polymerase chain reaction.

Eggs and egg products have a high risk of Salmonella enterica serovar Enteritidis contamination leading to gastroenteritis outbreaks in humans. Thus, a rapid screening tool for viable Salmonella Enteritidis cells in the egg industry is needed. Our objective was to rapidly and sensitively detect viable Salmonella Enteritidis from spiked liquid whole eggs (LWEs) within 24 h using SYBR green I-based real-time reverse transcriptase-polymerase chain reaction (PCR) targeting the Salmonella specific invA gene along with an internal amplification control in a Bio-Rad iCycler. LWE was inoculated with Salmonella Enteritidis and mixed with tetrathionate broth, and 100 µL of serially diluted portions in phosphate-buffered saline was plated on Xylose Lysine Tergitol 4 agar or 5 mL were used for RNA extraction by the TRIzol method immediately or after enrichment of 6, 12, or 16 h at 37 °C. The real-time reverse transcriptase-PCR assay was carried out using previously described Salmonella invA gene primers. Melt temperature analysis of the PCR product was included to determine specific invA amplification. Without enrichment, the assay detection limit was 10(7) colony forming units (CFU)/25 mL LWE. After enrichment for 6 and 12 h, Salmonella Enteritidis could be detected from LWE up to 10(4) and 10(2) CFU/25 mL, respectively. Improved Salmonella Enteritidis detection up to 10(0) CFU/25 mL was obtained after 16-h enrichment. Even with 16-h enrichment, the results could be still be obtained within 24 h, which is much faster than by traditional cultural detection that takes several days. Therefore, this assay appears suitable for routine detection of Salmonella enterica contamination by the egg industry to help prevent the transmission of egg-associated Salmonella outbreaks and timely recall of contaminated products.

Comparison of reverse transcriptase PCR, reverse transcriptase loop-mediated isothermal amplification, and culture-based assays for Salmonella detection from pork processing environments.

Novel rapid Salmonella detection assays without the need for sophisticated equipment or labor remain in high demand. Real-time reverse transcriptase PCR (RT-PCR) assays, though rapid and sensitive, require expensive thermocyclers, while a novel RT loop-mediated isothermal amplification (RT-LAMP) method requires only a simple water bath. Our objective was to compare the detection sensitivity of Salmonella Typhimurium from the pork processing environment by RT-LAMP, RT-PCR, and culture-based assays. Carcass and surface swabs and carcass rinses were obtained from a local processing plant. Autoclaved carcass rinses (500 ml) were spiked with Salmonella Typhimurium and filtered. Filters were placed in stomacher bags containing tetrathionate broth (TTB) and analyzed with or without 10-h enrichment at 37 °C. Natural swabs were stomached with buffered peptone water, and natural carcass rinses were filtered, preenriched, and further enriched in TTB. Serially-diluted enriched samples were enumerated by spread plating on xylose lysine Tergitol 4 agar. RNA was extracted from 5 ml of enriched TTB with TRIzol. RT-LAMP assay using previously described invA primers was conducted at 62 °C for 90 min in a water bath with visual detection and by gel electrophoresis. SYBR Green I-based-real-time RT-PCR was carried out with invA primers followed by melt temperature analysis. The results of RT-LAMP detection for spiked carcass rinses were comparable to those of RT-PCR and cultural plating, with detection limits of 1 log CFU/ml, although they were obtained significantly faster, within 24 h including preenrichment and enrichment. RT-LAMP showed 4 of 12 rinse samples positive, while RT-PCR showed 1 of 12 rinse samples positive. For swabs, 6 of 27 samples positive by RT-LAMP and 5 of 27 by RT-PCR were obtained. This 1-day RT-LAMP assay shows promise for routine Salmonella screening by the pork industry.

Loop-mediated isothermal amplification (LAMP) for the rapid and sensitive detection of Salmonella Typhimurium from pork.

UNLABELLED: Loop-mediated isothermal amplification (LAMP) is a novel molecular detection method that is more rapid and simpler than PCR. Products can be detected by turbidity using one temperature without the need for expensive PCR equipment. Our objective was to sensitively detect Salmonella Typhimurium from pork products within 1 d using the LAMP assay. Pork chop and pork sausage samples (25 g) were inoculated with high (10(8) to 10(6) CFU) and low (10(5) to 10(0) CFU) inocula of S. Typhimurium. Serial dilutions in phosphate buffered saline were plated on XLT4 agar either immediately or after selective preenrichment in tetrathionate broth (225 mL) for 10-h at 37 degrees C. Nucleic acid was extracted using the TRIzol method from 1-mL samples. The LAMP assay using 6 specific invA gene primers and Bst DNA polymerase reaction mix was carried out at 62 degrees C for 90 min in a waterbath. Turbid products were detected visually and by agarose gel electrophoresis. Improved Salmonella detection at 10(2) CFU/25 g for both pork chop and sausage was obtained after 10-h enrichment and 10(6) CFU/25 g without enrichment for both products. This assay can detect Salmonella from pork within 1 d, significantly faster than traditional methods that take >5 d. This method shows tremendous potential for routine diagnostics and monitoring of Salmonella by the pork industry. PRACTICAL APPLICATION: The novel loop-mediated isothermal amplification (LAMP) assay is a rapid, specific, and sensitive method that has potential application for routine diagnostics of Salmonella from pork products. The isothermal method does not require expensive equipment such as a PCR thermocycler but only a simple waterbath for amplification within 90 min. Detection is even simpler by visual eye or turbidimeters that are less expensive than fluorescent spectrophotometers or real-time PCR machines. All these advantages make it a practical approach for routine use by processing industries to rapidly detect Salmonella in their environment and to implement appropriate control strategies. To improve detection sensitivities, preenrichment followed by selective enrichment may be necessary. Even so, the entire assay can be completed at the most within two 8-h working shifts.

Real-time reverse transcriptase PCR for the rapid and sensitive detection of Salmonella typhimurium from pork.

Reverse transcriptase PCR (RT-PCR) detects the presence of mRNA and has a greater potential for detecting viable pathogens than do DNA-based PCR assays, with improved speed and sensitivity compared with traditional methods. Our objective was to rapidly and sensitively detect Salmonella Typhimurium from pork within two 8-h work shifts using a SYBR Green I real-time RT-PCR (rt-RT-PCR) assay. Pork chop and sausage samples (25 g) were inoculated with 10(8) to 10(0) CFU of Salmonella Typhimurium and stomached in 225 ml of tetrathionate broth. Serial dilutions were spread plated on xylose lysine Tergitol 4 agar either immediately or after 10 h of selective preenrichment or preenrichment followed by 12 h of selective enrichment (for stressed cells) at 37 degrees C for standard cultural enumeration. RNA was extracted using the TRIzol method. The rt-RT-PCR assay was carried out in a Bio-Rad iCycler using a SYBR Green I one-step RT-PCR kit and Salmonella specific invA gene primers with an internal amplification control (IAC). The PCR was followed by melting temperature (T(m)) analysis to determine specific Salmonella invA (T(m) = 87.5 degrees C) and IAC (T(m) = 82 degrees C) products. Improved Salmonella detection up to 10(1) CFU/25 g of pork and 10(0) CFU/25 g of sausages was obtained after 10 h of enrichment within approximately 24 h. Even without enrichment, Salmonella could be detected from both pork chop and sausage at 10(6) CFU/25 g within 1 day. This robust rt-RT-PCR detects and confirms Salmonella in pork within approximately 24 h and thus is significantly faster than traditional methods that take >/=1 week. This assay shows promise for routine testing and monitoring of Salmonella by the pork industry.