Selenite Cystine Agar for Enumeration of Inoculated Salmonella Serovars Recovered from Stressful Conditions during Antimicrobial Validation Studies

Abstract

Process validation studies often require the inoculation of select foodborne pathogens into targeted foods to determine the lethality of the process or antimicrobial ingredients, and quantitative recovery of surviving inoculum bacteria helps to make those assessments. Such processes introduce various stressors on the inoculated challenge microorganisms whereby traditional selective media are too harsh to enumerate the remaining viable and injured population quantitatively. Innate antibiotic resistance of challenge organisms has often been used to establish simple selective media (i.e., Tryptic Soy Agar/TSA + antibiotics) for recovering inoculated strains, but sometimes antibiotic resistant background microorganisms are higher than desired. Salmonella Thompson 120, Salmonella Heidelberg F5038BG1, Salmonella Hadar MF60404, Salmonella Enteritidis H3527, and Salmonella Typhimurium H3380 were characterized for antibiotic resistance and acid adaptation in Tryptic Soy Broth containing 0%, 0.25%, or 1.0% glucose. Sodium pyruvate was evaluated for recovery after stress but no enhancing effect was observed, possibly because the strains were acid-adapted. Selenite Cystine Broth, traditionally used as a selective enrichment broth, was used as the basis for Selenite Cystine Agar (SCA) in combination with three antibiotics to which our Salmonella are resistant. Serovars of Salmonella, both individually and in mixtures, were enumerated on TSA, SCA, Xylose Lysine Desoxycholate (XLD), and Hektoen Enteric (HE) selective agars (all containing the same antibiotics) after conditions of nutrient starvation, desiccation, acid stress, and thermal stress. The data show that quantitative enumeration of our Salmonella serovars on SCA was not significantly different (p > 0.05) than those achieved on TSA for all tested stress categories. Levels of Salmonella enumerated on XLD and/or HE were significantly different (p < 0.05) than on TSA and SCA and often more than 1–2-log lower, consistent with the inhibition of injured cells. These data confirm that SCA (+ antibiotics) is a suitable selective medium for enumeration of these acid-adapted Salmonella serovars as challenge organisms recovered from various conditions of stress.

Quantification of Process Lethality (5-Log Reduction) of Salmonella and Salt Concentration during Sodium Replacement in Biltong Marinade

“…Given the concerns for high sodium in our diets, questions have developed on whether alternative salts such as KCl or CaCl 2 can provide an equivalent 5-log reduction of Salmonella while reducing the amount of sodium in the product. Using acid-adapted cultures to reduce the acid sensitivity of Salmonella [23], a >5-log reduction of Salmonella was obtained in 7-days using 2.2% NaCl and 4% vinegar as observed in this paper (Figure 2) and as described previously [24]. By replacing NaCl with either CaCl 2 or KCl in the marinade formulation, we still achieved a >5-log reduction of Salmonella in 6 and 8 days, respectively (Figure 2).…”

“…Rather, it is dried at moderate temperatures (i.e., 23.9 • C/75 • F) and relative humidity (~55% RH) and relies on salt, spices, and vinegar, along with extended drying (4-10 days) relative to jerky, to achieve USDA-FSIS recommended ≥ 5-log reduction of Salmonella. The use of acid-adapted Salmonella cultures ensures that the Salmonella inoculum is not easily inhibited by acid treatment with vinegar and/or other antimicrobials that are allowed during biltong processing [23]. The vinegar/acid treatment provided by a short term antimicrobial dip treatment (30-60 s) or meat marinade (i.e., 30 min) is effective as a microbial inhibitor during the short time the surface pH is lowered by vinegar or acid treatment.…”

“…enterica serotype Enteritidis H3527 (phage type 13a, clinical isolate). These strains have been described in numerous research publications involving antimicrobial processing interventions against Salmonella spp [19][20][21][22][23][24].…”

“…Acid adaptation of these Salmonella serovars was carried out with cultures inoculated into TSB augmented with 1% glucose according to Wilde et al and others [23][24][25][26]. Individual cultures were harvested by centrifugation, resuspended with 0.1% BPW (BD Difco), and held at refrigerated temperature until use (5 • C).…”

“…After addition of 100 mL of 1% neutralizing Buffered Peptone Water (nBPW, Criterion, Hardy Diagnostics, Santa Maria, CA, USA), samples were stomached for 90 s in a Masticator paddle-blender (IUL Instruments, Barcelona, Spain). Serial dilutions were surface plated on Selenite Cystine Agar (SCA) containing spectinomycin (5 µg/mL), clindamycin (5 µg/mL) and novobiocin (50 µg/mL) as described previously by Karolenko et al [23]. The filter bag dilution was considered the 10 0 dilution.…”