Resuscitation and quantification of stressed Escherichia coli K12 NCTC8797 in water samples

Abstract

The aim of this study was to investigate the impact on numbers of using different media for the enumeration of Escherichia coli subjected to stress, and to evaluate the use of different resuscitation methods on bacterial numbers. E. coli was subjected to heat stress by exposure to 55 degrees C for 1h or to light-induced oxidative stress by exposure to artificial light for up to 8h in the presence of methylene blue. In both cases, the bacterial counts on selective media were below the limits of detection whereas on non-selective media colonies were still produced. After resuscitation in non-selective media, using a multi-well MPN resuscitation method or resuscitation on membrane filters, the bacterial counts on selective media matched those on non-selective media. Heat and light stress can affect the ability of E. coli to grow on selective media essential for the enumeration as indicator bacteria. A resuscitation method is essential for the recovery of these stressed bacteria in order to avoid underestimation of indicator bacteria numbers in water. There was no difference in resuscitation efficiency using the membrane filter and multi-well MPN methods. This study emphasises the need to use a resuscitation method if the numbers of indicator bacteria in water samples are not to be underestimated. False-negative results in the analysis of drinking water or natural bathing waters could have profound health effects.

Self-reinoculation with fecal flora changes microbiota density and composition leading to an altered bile-acid profile in the mouse small intestine

“…For the pilot study (Additional file 1: Figure S4A), the MPN assays (adapted from [140][141][142][143][144]) were performed on each GIT section (stomach, three sub-sections of the small intestine, cecum, and colon) from five mice fitted with functional tail cups and five control mice. The growth medium was brain heart infusion broth (Bacto BHI, #237500, Becton Dickinson, Franklin Lakes, NJ, USA), prepared in ultrapure water (Milli-Q), sterilized by autoclaving, allowed to cool to room temperature, and supplemented with 1.0 mg/L vitamin K 1 (#L10575, Alfa Aesar, Haverhill, MA, USA), 5 mg/L hematin (#H3281, Sigma-Aldrich St. Louis, MO, USA), and 0.25 g/L L-cysteine (#168149, Sigma-Aldrich).…”

I'm Not Dead Yet: Bacterial Tales from the Crypt and Survival after Heat Treatment

“…Higgins et al, (2007) suggested that the increase in densities was due to the resuscitation or reactivation of bacteria that had become temporarily nonculturable. This phenomena of reactivation has also been documented in matrixes such as water (Lim 1995, Momba 2002,Ozkanca 2009 These studies suggest that bacteria subjected to heat stress enter into a protective state where they are not able to grow on standard microbiological media. However, the bacteria are able to recover from this state over time as conditions become favorable for growth.…”

Detection of Vibrio spp., Salmonella spp., and Shigella spp. among the frozen food samples employing enrichment culture technique

“…Gram negative pathogens such as Salmonella and other Enterobacteriaceae are sensitive to freezing injury and there is also some mortality of mesophilic Vibrios (30). Various stress factors may lead to the generation of a dormant or viable but non-culturable state in the bacterial cells which direct bacteria to be unable to produce observable colonies when cultured on selective and non-selective agar media (31). Despite this condition, these bacteria possess the ability to cause disease in suitable conditions (32,33,34).…”