Detection and recovery of sublethally-injured enterotoxigenic Staphylococcus aureus

Abstract

Aims: To determine whether sublethally-injured (acid-or heat-shocked) Staphylococcus aureus cells are recoverable using selective agar overlays. Methods and Results: Brain Heart Infusion (BHI) Agar overlaid with either Baird-Parker Agar (BPA) or GramPositive Agar (GPA) was compared in the ability to resuscitate heat-and acid-shocked enterotoxigenic Staph. aureus. BHI ⁄ BPA overlays allowed for greater recovery of both heat-and acid-shocked cells than BHI ⁄ GPA, although the former was not selective and allowed growth of bacteria other than Staph. aureus. No significant difference existed in percent recovery of heat-and acid-shocked cells between the two overlay approaches. Significant differences were noted in counts on BHI ⁄ GPA plates and straight selective GPA ⁄ GPA plates, however. Viability of heat-and acid-shocked Staph. aureus was also examined using fluorescence microscopy, the relative counts of which correlated well to the calculated percent recovery on selective agar overlays. Conclusions: This work has shown that an improved agar overlay technique increases the sensitivity of the standard plate count while enumerating sublethally-injured enterotoxigenic Staph. aureus compared with direct plating onto selective media. Significance and Impact of the Study: These data emphasize the need to develop practical and cost-effective methods that reliably detect and enumerate sublethally-injured pathogens such as Staph. aureus.

Modified Bacteriophage S16 Long Tail Fiber Proteins for Rapid and Specific Immobilization and Detection of Salmonella Cells

“…Salmonella cells in food matrices are typically injured, low in numbers, and unevenly dispersed in the presence of much higher numbers of other Enterobacteriaceae (51). Preenrichment is used to resuscitate damaged cells and increase their availability for detection (52). Lengthy preenrichments can be shortened with IMS to specifically isolate and concentrate low levels of bacteria into small volumes suitable for detection assays (8).…”

Salmonella Detection Methods for Food and Food Ingredients

“…The media may contain inhibitors in order to stop or delay the growth of non-target organisms, or particular substrates that only the target bacteria can degrade, or that confer a particular colour to the growing colonies (Manafi 2000). Cultural methods typically involve the enrichment of a portion of the food sample to recover sub-lethally injured cells due to heat, cold, acid, or osmotic shock (Sandel et al 2003;Gracias and McKillip 2004) in a non-selective pre-enrichment media, such as Buffered Peptone Water (BPW), and to increase the number of target cells as these are generally not uniformly distributed in foods, typically occur in low numbers, and may be present in a mixed microbial population. Next, primary enrichment cultures are typically inoculated into secondary selective enrichment broths, such as Selenite Cystine broth (SC), Rappaport Vasiliadis Soy broth (RVS), Tetrathionate Broth (TT), or Muller-Kauffmann TetrathionateNovobiocin broth (MKTTn) and incubated at elevated temperatures (37°C or 42°C for 18-24 hours) before being struck onto selective agars such as Xylose Lysine Deoxycholate agar (XLD agar), Bismuth Sulphite agar (BIS), Brilliant Green agar (BG) with or without the addition of sulfadiazine or sulfapyridine (BGS), modified semisolid Rappaport Vasiliadis (MSRV), Salmonella Shigella Agar, or Hektoen Enteric agar.…”

Enumeration of Sublethally Injured Escherichia coli O157:H7 ATCC 43895 and Escherichia coli Strain B-41560 Using Selective Agar Overlays versus Commercial Methods

“…After treatments, the majority of bacteria are killed, but a small population may be sublethally injured (10,11,(17)(18)(19)22). Traditional quality control measures during food processing could include either a nonselective enrichment step that may recover sublethally injured bacteria, or direct inoculation of selective media with a food sample.…”

“…Enteric bacteria may be detected and enumerated from foods, for example, by using pour plates of violet red bile agar (VRB) or inoculating appropriately selective Petrifilm (6,7,15). However, injured bacteria may not readily grow on selective media after being subjected to stressful conditions (10,15,17,18), leading to the underestimation of bacterial density for spoilers and pathogens (14,22). Moreover, nonselective enrichment steps preclude one from enumerating the original density in the suspect food.…”

“…One strategy to improve recovery of injured or stressed bacteria in foods is the use of a nonselective basal layer of media in which the bacteria can first undergo repair and resuscitation, followed by the application of a selective agar overlay to subsequently allow for growth and identification of pathogens (3,6,(17)(18)(19)22,24,26,27). This agar overlay method has been used to recover and enumerate a wide variety of pathogens and spoilers in the food industry, and it offers greater sensitivity compared to direct plating onto appropriate selective media (7).…”