Michael F. Slavik

Spraying chicken skin with selected chemicals to reduce attached Salmonella typhimurium.

Aqueous solutions of 5% and 10% trisodium phosphate (TSP), 0.1% and 0.5% cetylpyridinium chloride (CPC), 1% and 2% lactic acid (LA), and 0.1% and 0.5% grapefruit seed extract (DF-100) were evaluated in prechill spraying for reducing Salmonella typhimurium attached on chicken skins. Chicken skins were inoculated with S. typhimurium and then sprayed with the selected chemical solutions for 30 sec at 206 kPa and 20 degrees C. After chemical spraying, the skins were rinsed by spraying tap water for 30 sec. Each skin was stomached in buffered peptone water (BPW) for 1 min. The stomaching water was then diluted serially, inoculated onto both xylose lysine tergitol (XLT4) agar and Aerobic Plate Count (APC) Petrifilm, and incubated for 24 hr at 37 degrees C. The results showed that the numbers of Salmonella on the chicken skins after the chemical spraying were significantly lower than those without spray (P < 0.05). The CPC reduced Salmonella by 1.5 to 1.9 log10. TSP resulted in a 2.1 to 2.2 log10 reduction of Salmonella and DF-100 produced a 1.6 to 1.8 log10 reduction of Salmonella. The LA had a number of Salmonella with a 2.2 log10 reduction. The 0.5% CPC resulted a significantly greater reduction in Salmonella than 0.1% CPC. There were no significant differences in Salmonella reduction between different concentrations of the other three chemicals.

Cetylpyridinium Chloride (CPC) Treatment on Poultry Skin To Reduce Attached Salmonella

Cetylpyridinium chloride (1-hexadecylpyridinium chloride, CPC) was evaluated for its effectiveness in removing or killing salmonellae attached to poultry skin. Two different treatment methods were used: (i) spraying 0.1% CPC solution at 15 degrees C or 50 degrees C against inoculated skin surface for I min at 138 kPa, and (ii) immersing inoculated skin surface in 0.1% CPC solution at room temperature for either 1 min, 1 min plus 2 min holding without CPC, or 3 min. After rinsing, cells on the skins were enumerated by conventional plating as well as direct counting from scanning electron microscopy (SEM). Compared with controls, CPC spraying reduced the numbers of salmonellae by 0.9 to 1.7 log units (87 to 98%) assayed by the plating method (P < 0.05). SEM gave results similar to plating. Generally 50 degrees C CPC spraying showed greater reduction than 15 degrees C CPC spraying; however, the differences were not always significant. Water spraying at either temperature did not show any reduction compared to nonsprayed skins. In the immersion test, significant differences also were noticed among the control and the three other CPC-immersed groups (P < 0.05) as assayed by plating, ranging from 1.0 to 1.6 log units, which were similar to the CPC spraying results. However, no difference was noticed among the three CPC-immersed groups. Direct counting from SEM was not a suitable method for recovering cells in CPC immersion tests because dead cells were still attached to the skin while retaining their intact morphology. On the basis of the amount of CPC used, immersion appears to be more cost-effective than spraying CPC on poultry skin.

Use of Antimicrobial Spray Applied with an Inside–Outside Birdwasher To Reduce Bacterial Contamination on Prechilled Chicken Carcasses

Antimicrobial sprays applied using a modified inside-outside birdwasher to reduce Salmonella typhimurium and total aerobic bacteria on prechilled chicken carcasses were evaluated in a poultry processing pilot plant. Four chemicals, including trisodium phosphate (TSP, 10%), lactic acid (LAC, 2%), cetylpyridinium chloride (CPC, 0.5%), and sodium bisulfate (SBS, 5%) were selected to be tested as antimicrobial agents. Each chicken carcass was inoculated by spraying the outside and inside of each carcass with S. typhimurium at 10(5) CFU per carcass. The inoculated carcasses then were passed through the birdwasher and sprayed with selected chemicals at 35 degrees C at a pressure of 413 kPa for 17 s. After a 60-s setting time on a shackle line, the carcasses were sprayed with tap water to rinse off chemical residue. All the chemical treatments reduced Salmonella on the chicken carcasses by approximately 2 log10 CFU per carcass. Total aerobes on the chicken carcasses, however, were reduced by 2.16, 1.66, 1.03, and 0.74 log10 CFU per carcass after spraying with 0.5% CPC, 5% SBS, 2% LAC, or 10% TSP, respectively. Spray treatments of both SBS and LAC caused slight discoloration in part of the chicken skin. The most effective antimicrobial spray treatment for reducing both Salmonella and total aerobes on prechilled chicken carcasses was 0.5% CPC.

Isolation, partial purification and characterization of a bacteriocin produced by a newly isolated Bacillus subtilis strain

A wild type micro‐organism producing antibacterial substances has been isolated from a Chinese fermented soybean seasoning and identified as Bacillus subtilis. A crude antibacterial preparation (CABP) was obtained by ammonium sulphate precipitation. Isoelectric focusing assay revealed at least four antimicrobial components in the CABP. However, in SDS‐PAGE analysis, only one peptide band displayed antimicrobial activity against pathogenic Bacillus cereus and Listeria monocytogenes. This inhibitory peptide had a molecular weight of approximately 3·4 kDa and a pI value of approximately 4·7. Results of this study suggest that at least one antimicrobial substance produced by this wild type strain of B. subtilis may be a new bacteriocin. Its sensitivity to gastric peptidases and activity against the food‐borne pathogens make this bacteriocin potentially useful as an antimicrobial agent in foods.

Rapid, sensitive, and simultaneous detection of three foodborne pathogens using magnetic nanobead-based immunoseparation and quantum dot-based multiplex immunoassay.

Losses caused by foodborne diseases are enormous in terms of human life, illness, medical costs, and food product recalls. Rapid detection of multiple bacterial pathogens in foods is extremely important to ensure food safety. The objective of this research was to develop a multiplex immunoassay by integrating magnetic nanobeads (MNBs) for immunoseparation with quantum dots (QDs) as fluorescent labels for rapid, sensitive, and simultaneous detection of three major pathogenic bacteria, Salmonella Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes, in food products. In this research, both streptavidin-conjugated MNBs (30- and 150-nm diameter) and QDs (530-, 580-, and 620-nm emission wavelength) were separately coated with biotinylated anti-Salmonella, anti-E. coli, and anti-Listeria antibodies. The immuno-MNBs were mixed with a food sample to capture the three target bacteria. After being magnetically separated from the sample, the MNB-cell conjugates were mixed with the immuno-QDs to form the MNB-cell-QD complexes, and unattached QDs were removed. The fluorescence intensity of the MNB-cell-QD complexes was measured at wavelengths of 530, 580, and 620 nm to determine the populations of Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes, respectively. This multiplex immunoassay simultaneously detected Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes at levels as low as 20 to 50 CFU/ml in food samples in less than 2 h without enrichment. The change in fluorescence intensity was linearly correlated (R(2) > 0.96) with the logarithmic value of bacterial level in the range of 10 to 10(3) CFU/ml. More than 85% of the three target pathogens could be simultaneously separated from food samples. The multiplex immunoassay could be expanded to detect more target pathogens, depending on the availability of specific antibodies and QDs with different emission wavelengths.

Evaluation of a PCR based assay for specific detection of Campylobacter jejuni in chicken washes

An assay for Campylobacter jejuni based on the polymerase chain reaction was developed in our laboratory and shown to be a sensitive and specific method to identify this bacterium in pure culture. This assay was evaluated as a method to rapidly detect C. jejuni attached to chicken carcasses. Chicken carcasses were sampled for PCR using three methods including pre-enrichment of the washes, direct plating of the washes and differential centrifugation of the washes prior to testing. It was found that plating the wash solutions on Campy Cefex plates prior to performing PCR was the most specific and reliable of the three treatment methods evaluated.

Effect of trisodium phosphate on Campylobacter attached to post-chill chicken carcasses

Trisodium phosphate (TSP) was evaluated as a means to reduce Campylobacter on chicken carcasses. Post-chill chicken carcasses were dipped into a 10% TSP solution at 50°C for 15 s. After storing the TSP-treated carcasses for 0, 1 or 6 days at 4°C, the carcasses were subjected to the recovery of Campylobacter . The incidence and reduction of Campylobacter attached to the carcasses were measured using a nitrocellulose (NC) membrane lift, conventional culture method, and a most probable number (MPN) technique. In trials 1 and 2, the incidence of Campylobacter was measured. For 1 day-stored groups, Campylobacter was present on 96 and 100% of control carcasses and present on 24 and 28% of TSP-treated carcasses as measured by NC membrane lift method. The reduction was less (4 to 36%) when measured by culture method. For carcasses immediately subjected for the recovery of cells after treatment, there was no difference between TSP-treated and control carcasses by either NC membrane or culture method. In trial 3, the reduction levels of Campylobacter were quantified by using a MPN method. The levels of Campylobacter on carcasses were decreased by 1.5 and 1.2 logs in 1- and 6-day stored, TSP-treated carcasses, respectively (p < 0.05). However, TSP treatment at 10°C reduced the level of Campylobacter only by 0.16 log (p > 0.10).

Trisodium Phosphate and Cetylpyridinium Chloride Spraying on Chicken Skin to Reduce Attached Salmonella typhimurium

Spraying treatments with trisodium phosphate (TSP) and cetylpyridinium chloride (CPC) were evaluated for their effectiveness in reducing Salmonella typhimurium attached to chicken skins. Chicken skins with an area of 38.5 cm2 were cut from the breast areas of pre-chill chicken carcasses, mounted in a plastic holder, and inoculated with S. typhimurium . The inoculated skins were sprayed with tap water, 10% (wt/vol) TSP, or 0.1 % CPC solutions at 10, 35, or 60°C and 206.8, 413.7, 620.5, 827.4, or 1034.2 kPa for 30 s. After spraying, each skin was rinsed with tap water, transferred to a plastic bag containing 50 ml buffered peptone water, and stomached for 1 min. The stomaching water was collected, diluted serially, plated on xylose lysine tergitol 4 (XLT4) agar and Petrifilm aerobic count plates, and incubated for 18 to 24 h at 37°C. The results showed that tap water spraying reduced S. typhimurium by 0.7 to 1.6 log, while the reduction ranges for TSP and CPC spraying treatments were 1.6 to 2.3 and 1.5 to 2.5 log, respectively. Greater reductions in the numbers of S. typhimurium were obtained in TSP spraying treatments in the high pressure range (620.5 to 1034.2 kPa) and in CPC spraying treatments at 10°C.

Campylobacter jejuni as a secondary colonizer of poultry biofilms.

Aims:  The objective of this study was to determine if survival of culturable Campylobacter jejuni outside the host was increased by entrapment in pre‐established biofilms.

Rapid Detection of Salmonella Typhimurium in Chicken Carcass Wash Water Using an Immunoelectrochemical Method

An immunoelectrochemical method coupled with immunomagnetic separation was developed for rapid detection of Salmonella Typhimurium in chicken carcass wash water. Samples of chicken carcass wash water were inoculated with Salmonella Typhimurium at different cell numbers. Possible nonspecified inhibitors in the wash water were minimized by filtration and centrifugation. An approximately 9.4% loss of Salmonella cells was found after filtration (P < 0.01). The samples were mixed with anti-Salmonella-coated magnetic beads (ASCMB) and alkaline phosphatase-labeled anti-Salmonella (APLAS) to form ASCMB-Salmonella-APLAS conjugates. The conjugates were separated from the solution using a magnetic separator and then incubated with phenylphosphate substrate to produce phenol. The number of Salmonella was determined by measuring the phenol concentration using an amperometric tyrosinase carbon paste electrode in a flow injection analysis system. Under optimized parameters (1 mM MgCl2, 0.2 microg/ml APLAS, and 1 mM phenylphosphate in pH 7.0 Tris buffer solution), Salmonella Typhimurium in chicken carcass wash water could be identified and enumerated within 2.5 h with a detection limit of 5 x 10(3) CFU/ml. A linear relationship on a log-log scale was found between Salmonella cell number and the peak current ratio for Salmonella concentrations ranging from 10(3) to 10(7) CFU/ml (R2 = 0.963). The peak currents of multibacteria samples, containing Salmonella Typhimurium, Listeria monocytogenes, and Campylobacter jejuni, were not significantly different from Salmonella-only samples (P > 0.01).