The recovery of heat-stressed Escherichia coli in lake water microcosms

Heat stress has been used as a method of killing bacteria for many years, and is one approach promulgated by federal regulations to reduce pathogens in biosolids (40 CFR 503). However, recent studies have suggested that heat stressed organisms may be able to recover and re-grow after thermal treatment. The purpose of this study was to examine bacterial response to heat stress over time and to evaluate the ability of bacteria to recover and grow. Washed Cultures of E. coli were placed into bottles containing sterile buffer and buffer amended with 1% nutrient broth. The bottles were then subjected to heat at 55°C for 4, 6, and 24 hr. Contents of bottles were assayed over 10 days for growth on non-selective agar plates using the spread plate method. All plates were negative for growth immediately after heat stress, however in the samples heated for 4 hr. and 6 hr., recovery of E. coli was seen in buffer with nutrient broth after 24 hr. Recovery was also seen in buffer alone after 72 hr. Samples heated for 24 hr. were able to recover only in the presence of nutrients. Further work is being conducted using lower initial densities of E. coli to assess the difference between recovery and re-growth. A similar study was performed using a biosolids sample from a full-scale process that utilized pre-pasteurization followed by mesophilic digestion. Samples after digestion had no measurable densities of E. coli, however, after storage of the samples, detectable densities of E. coli were measured over a period of several days.

Section: Introductionmentioning

confidence: 73%

Section: Full-scale Results After Holding Time Researchers Have Showmentioning

confidence: 99%

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

Boczek¹,

Hoelle-Schwabach²,

Johnson³

et al. 2010

Section: Introductionmentioning

confidence: 88%

Recovery of Bacteria Following Heat Treatment

Boczek¹,

Hoelle-Schwabach²,

Johnson³

et al. 2011

Heat stress has been used as a method of killing bacteria for many years, and is the basis for federal regulations promulgated to reduce pathogens in biosolids (40 CFR 503). However, recent studies have suggested that heat stressed organisms may be able to reactivate and re-grow. The purpose of this study was to examine bacterial response to heat stress over time and to evaluate the ability of bacteria to recover and grow. Washed Cultures of E. coli were placed into bottles containing sterile buffer and buffer amended with 1% nutrient broth. The bottles were then subjected to heat at 55°C for 4, 6, and 24 hours. Contents of bottles were assayed over 10 days for growth on non-selective agar plates using the spread plate method along with the multiple tube fermentation test (MPN). All procedures were negative for growth immediately after heat stress; however in the samples heated for 4 hrs and 6 hrs, recovery of E. coli was seen in buffer with nutrient broth after 24 hrs. Recovery was also seen in buffer alone after 72 hrs. Samples heated for 24 hrs did not show the ability to recover.

“…Researchers have shown that thermally inactivated E. coli could be resuscitated if they were held at lower temperatures, about 15 °C, for a period of time before enumerating using culturing methods (Lim and Flint, 1995). For example, E. coli became nonculturable in selective media after thermal treatment at 65 °C for 6 hrs, and were below detection.…”

Section: Increased Holding Timementioning

confidence: 99%

“…For example, E. coli became nonculturable in selective media after thermal treatment at 65 °C for 6 hrs, and were below detection. However, storing the thermophilically treated samples at 15 °C for several days led to full recovery of the population of bacteria (Lim and Flint, 1995). To test the effect of holding time, a full-scale sample was evaluated from a plant that utilized pre-pasteurization followed by mesophilic digestion and centrifuge dewatering.…”

Section: Increased Holding Timementioning

confidence: 99%

Development of a Laboratory Based Method for Enumeration of Non-Culturable Indicator Fecal Coliform and E. coli in Thermophilic Anaerobically Digested Biosolids

Higgins¹,

Beightol²,

Murthy³

et al. 2009

Recent research has shown that a substantial, sudden increase in the indicator bacteria density, fecal coliform and E. coli, could occur immediately after centrifuge dewatering of thermally treated biosolids. One possible explanation is that the EPA promulgated MPN method used for enumeration (Method 1680) is underestimating the potentially viable bacteria in the sample prior to dewatering, and the dewatering process reactivates the bacteria such that they become culturable. The objectives of this research were to investigate alternative methods for enumerating indicator bacteria in thermally treated biosolids samples. The research approach used methods proposed in the literature for resuscitating bacteria that have become nonculturable after exposure to stress. For thermophilically treated biosolids samples that meet the EPA time-temperature requirements for Class A biosolids, the densities measured by the EPA 1680 Method were typically non-detect with detection limits less than 10 MPN/g DS. In comparison, the alternative or enhanced methods often had E. coli densities in the range of 1-4 log 10 per gram dry solids. The results support the non-culturable hypothesis, however, additional work is needed to further verify the results and assure that the target organisms are being enumerated.