Structural Pasteurization reduces viable bacteria in indoor spaces

Efficacy of Structural Pasteurization for Reduction of Viable Bacterial Levels in Indoor Environments

Sean P. Abbott^1,*, Larry Chase² and M. Chance Villines¹

¹Natural Link Mold Lab, Inc., 4900 Mill Street, Suite 3, Reno, Nevada 89502, USA

²Precision Environmental, 180 Cañada Larga Road, Ventura, California 93001, USA

^*Corresponding email: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

SUMMARY

This study examines the efficacy of high temperature pasteurization of buildings for reducing levels of viable bacteria in indoor environments employing both laboratory and field data. Laboratory heat chamber testing using four species of environmental bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus) at a range of temperatures and durations was performed to determine efficacy of thermal application. Bacteria exposed to high temperature/short duration (60-75°C for 0.25-4 hr) demonstrated that thermal death occurs rapidly at temperatures of 60°C or higher. When exposed to low temperature/long duration (45-50°C for 4-120 hr), time required to reach the thermal death point was significantly extended. Laboratory-prepared samples inoculated with E. coli were also subjected to the thermal sanitization process in field situations to confirm effectiveness at temperatures and durations routinely used for sewage remediation projects. Results demonstrated mortality of E. coli at 60°C for 2 hours.

IMPLICATIONS

Structural pasteurization employs engineer-controlled convective dry heat to sanitize building materials in situ, and is typically used in conjunction with structural drying and traditional microbial remediation processes. Building pasteurization allows for sanitization of entire structures and can provide significant hygiene benefits by reducing overall levels of microorganisms in indoor environments.

KEYWORDS

Microbial Contamination, Thermal Death Point, Heat, Sewage, Remediation

INTRODUCTION

Pasteurization is a process to eliminate human pathogens and reduce the overall concentration of microorganisms in food and industrial processing (Stetzenbach and Yates, 2003). Louis Pasteur developed heat preservation processes between 1854 and 1864 when he determined that bacteria were causing wine to spoil. Through experimentation, he discovered that when heated to a certain threshold and held for specific period of time, the bacteria could be killed without damaging the wine. Later, applications for pasteurization included other liquids such as milk and fruit juices, and heat has become an important tool for reducing biological contamination and a routine method of preservation throughout the food industry. Pasteurization is distinct from sterilization, which uses extreme physical or chemical means to eliminate all biological agents, but can adversely affect the food or other materials being sterilized (Black, 1999; Doyle et al. 2001).

In recent years, indoor environmental technicians have honed the engineered application of dry heat to a whole building, a portion of the structure, or its contents, with the purpose of killing targeted organisms. The principle of this process, termed structural pasteurization, is analogous to food pasteurization in that microorganisms are susceptible to heat and that overall microbial levels can be significantly reduced. Each organism has a specific thermal death point, and many microbes inhabiting built structures fall within the range of efficacy for this process. The thermal death point for organisms, including species of insects, fungi, bacteria and viruses, is a function of temperature and duration coupled with biomass and environmental factors. Death rates at high temperatures for short durations may be equivalent to lower temperatures for longer times.

While thermal death points for bacteria in liquid and solid foods have been thoroughly investigated (e.g., Doyle et al. 2001), there has been minimal research regarding temperatures and times required for reduction of viable bacteria in environmental situations. Some studies have examined efficacy of steam heat treatment for greenhouse soils (Bollen, 1969), thermal disinfection of bacterial pathogens in building water systems (Stout et al. 1986), or survival of bacteria in self-heating green compost or composted sewage (Jones and Martin, 2003; Wiley and Westerberg, 1969). Prior studies of thermal death points for E. coli and other environmental bacteria have documented effectiveness of 60°C for 20 to 60 minutes (Jones and Martin, 2003; Padhye and Doyle, 1992).

Bacterial contamination in buildings may be a result of proliferation due to water damage, sewage loss, or human habitation (IICRC, 2006). A wide variety of genera and species of bacteria may occur in indoor environments. Of primary concern are potentially pathogenic bacteria that occur in fecal waste including Escherichia coli and other fecal coliform bacteria such as Klebsiella pneumoniae. Pseudomonas aeruginosa is a potentially pathogenic gram-negative bacterium frequently isolated from water samples. Other pathogens, such as Staphylococcus aureus, may be disseminated in indoor environments from human occupants. To test the efficacy of the structural pasteurization process, these representative gram-negative and gram-positive bacteria were chosen for thermal death studies in laboratory and field situations.

METHODS

Test Organisms

Four species of bacteria isolated from environmental sources were chosen for the thermal death studies. All strains are maintained by Natural Link Mold Lab, Inc. as NLML 1B70 Escherichia coli, NLML 1B73 Klebsiella pneumoniae, NLML 2B33 Pseudomonas aeruginosa, NLML 1B48, 1B76 Staphylococcus aureus.

Laboratory Thermal Death Studies

To examine the efficacy of a thermal antimicrobial process for reduction of levels of viable bacteria, over 500 test samples were prepared and exposed within a laboratory heat cabinet. This allowed us to determine the effects of dry heat treatments at various temperatures on the thermal death of selected species of bacteria over given periods of time.

Preparation of bacterial suspensions and test samples: Bacterial stock cultures were grown for 24 hr on Tryptic Soy Agar (TSA; Hardy Diagnostics, Santa Maria, CA) plates. These were used to prepare the primary suspensions in 7.0 mL sterile H₂O for each of the organisms being challenged. The suspensions were prepared to a minimum of 2 MacFarland cell density and were used to prepare the inoculated test samples. 200 mL of primary suspension was inoculated onto sterile cotton-tipped applicator sticks (Premiere, Hardy Diagnostics, Santa Maria, CA), and each prepared applicator stick was placed into a sterile glass test tube.

Heat chamber exposure: The laboratory heat chamber (560,000 cm³volume, Hot Food Boxes, Inc., Chicago, IL) was set to desired temperature 24 hours prior to testing, and monitored with a thermal probe thermometer (VWR Traceable digital thermometer, Radnor, PA) to ensure temperature stability within +/-1°C. All inoculated test samples were placed into the heat chamber, caps on test tubes were removed and left open for exposure to dry heat within the chamber.

High Temperature/Short Duration (HTSD): Samples of each organism were exposed to 60, 65, and 70°C over a four-hour period and removed at 15, 30, 45, 60, 75, 90, 105, 120 and 240 minute intervals (356 total samples). E. coli was also tested at 75°C. Each test sample was placed into 1 mL of Tryptone Soy broth (Hardy Diagnostics, Santa Maria, CA), and incubated at 35°C for 48 hours to monitor for growth. Incubated broth tubes were examined visually and microscopically to assess viability and confirm that bacterial growth recovered was the appropriate challenge organism. Each test sample was recorded as ‘Growth’ or ‘No Growth.’ Additionally, 180 quantitative HTSD tests were performed using E. coli at 60 and 65°C under the same duration parameters as described above. TSA dilution plates were prepared and enumerated after 48 hours incubation at 35°C. The concentration of viable bacterial cells was averaged for all test replicates at each temperature/duration.

Low Temperature/Long Duration (LTLD): Although the HTSD exposures defined here are typical for the structural pasteurization process, the effects of less extreme heat over greater periods of time were examined in order to compare effectiveness. Heat parameters included testing at 45 and 50°C at intervals over a 5 day period. Samples of each organism were removed at 4, 24, 48, 72, 96 and 120 hours. Methodology as described above for HTSD trials.

Field Thermal Death Studies

Sterile culture swabs (StarPlex, Labsco, Louisville, KY) were inoculated with the primary suspension of E. coli (see above) and sealed. Test and control samples were transported to the field site. Test samples were placed into representative areas of the buildings and subjected to the structural pasteurization process. Temperatures were elevated within the structure to 60°C and temperature was maintained for two to four hours. Samples were removed at three time intervals during the process. Time 0 samples were removed after initial heating period (i.e., removed as soon as target temperature of 60°C was reached). Time 1 samples were removed after an exposure period of 0.5-1 hours and Time 2 samples were removed at 2-4 hours exposure at 60°C. Field control samples were transported to the field site, but not subjected to the thermal treatment. All samples were returned to the laboratory via overnight courier following field-testing. In the laboratory, swab samples were inoculated into Colisure (IDEXX, Westbrook, ME) broth culture and incubated at 35°C for 24 hours to determine survival of E. coli. Each test and control sample was recorded as ‘Growth’ or ‘No Growth.’

RESULTS

Laboratory Thermal Death Studies

Results of HTSD thermal death studies at 60°C are presented in Table 1. Increasing temperature correlated with more rapid achievement of complete thermal death in the samples of bacteria species tested. Results at 65 and 70°C (data not shown) demonstrated survival of all organisms at 0.5 hr exposure, but complete mortality was achieved within 45 to 75 minutes. One additional round of testing using E. coli at 75°C indicated complete mortality within 15 minutes. Quantitative results of HTSD testing using E. coli at 60 and 65°C are presented in Table 2. Under LTLD exposure conditions, longer exposure duration was required to kill the bacteria, but patterns of bacterial susceptibility were similar to HTSD exposure data. LTLD thermal death results are presented in Table 3.

Table 1. Bacterial survival under laboratory HTSD exposure conditions of 60°C over a 4 hour period.

Duration (hr)

# Test Replicates

E. coli

K. pneumoniae

P. aeruginosa

S. aureus

0.25

0.5

0.75

1.0

1.25

1.5

1.75

2.0

4.0

Control

V (60%)

V (80%)

V (20%)

V (80%)

V (40%)

V (80%)

V (60%)

V (20%)

V (70%)

V (60%)

V (50%)

V (60%)

V (40%)

V (10%)

(G=growth survival, NG=no growth complete mortality, V=variable survival/mortality with percent survival in parentheses).

Table 2. Quantitative reduction of viable E. coli exposed to HTSD.

Duration (minutes)

# Test Replicates

60°C (# viable cells)

65°C (# viable cells)

Control, Before

120

Control, After

2.8 x 10⁸

5.5 x 10⁶

3.8 x 10⁶

5.1 x 10⁴

5.4 x 10³

1.0 x 10³

2.5 x 10⁸

6.1 x 10⁸

3.1 x 10⁷

6.7 x 10⁵

N/A

6.0 x 10⁸

Table 3. Bacterial survival under laboratory LTLD exposure conditions of 45-50°C over a 5 day period.

Duration (hr)

# Test Replicates

E. coli

K. pneumoniae

P. aeruginosa

S. aureus

0.5

120

Control

V (67%)

V (20%)

V (67%)

V (40%)

V (20%)

V (80%)

V (50%)

V (30%)

V (20%)

(G=growth survival, NG=no growth complete mortality, V=variable survival/mortality with percent survival in parentheses).

Field Thermal Death Studies

Results from 177 field projects are presented in Table 4. These data show complete mortality of E. coli in 192 of the 204 samples after exposure to 60°C for 2-4 hours under field conditions. All control samples exhibited survival of E. coli.

Table 4. Survival of E. coli under field structural pasteurization conditions.

Exposure Duration

# Test Replicates

# Field Projects Tested

Survival (%)

Time 0 (preheat only)

Time 1 (0.5-1 hr)

Time 2 (2-4 hr)

Control

204

180

177

13.3

5.9

100

DISCUSSION

The HTSD exposure parameters used in this study were sufficient to provide significant and rapid reduction of bacterial viability under both laboratory and field conditions. Similar pasteurization efficacy of lower temperatures for longer durations has been well documented in the food pasteurization industry (e.g., Black, 1999; Grant et al., 2005), and the LTLD testing as defined here confirmed a similar end result.

While all bacteria tested in this study demonstrated rapid mortality at temperatures of 60°C, consistent differences in susceptibility were noted. In all cases, the gram-negative, water inhabiting P. aeruginosa was killed the most quickly. The two species of fecal coliform bacteria, E. coli and K. pneumoniae, demonstrated a similar pattern of survival at various temperatures. The gram-positive S. aureus was most resistant to the thermal treatments and frequently demonstrated survival for longer periods than the other bacteria. The two strains of S. aureus used for testing exhibited minor differences in thermal death points that suggest strain-to-strain variability in thermal tolerance may also be a factor.

Some variability in thermal death times is expected under field conditions of structural pasteurization due to the many environmental factors encountered when trying to uniformly heat a structure. Sources of variation may include size and composition of the structure and its contents, external temperature and climatic conditions, geography, and numbers or types of heating devices employed. Variability in sample placement, uneven heat distribution within the structure and distance from heat source also should be expected. Bacteria in the building may be more or less exposed to convective heat or may be insulated within building materials, contents or debris. In field thermal death studies, 5.9% of samples survived the structural pasteurization process. It is probable that these samples did not reach 60°C or were not maintained at this temperature for 2 hours.

In another field efficacy study (Abbott, unpublished data), bathroom surfaces were tested before and after structural pasteurization treatments to assess reduction of naturally occurring bacteria in the buildings. The results of 16 paired samples from 12 buildings showed an order of magnitude reduction from 293.56 CFU/cm² before to 22.68 CFU/cm² following a minimum exposure of 2 hours at 60°C. Many of the surviving colonies were gram-positive bacteria, including endospore-formers such as Bacillus. Bacteria that produce resistant endospores (including anthrax, B. anthracis) are not expected to be killed by the structural pasteurization process, but instead require temperatures and times used in autoclave sterilization (121°C for 15 min at 15 psi).

The application of thermal sanitization processes to biological problems in structures has proved successful in a number of situations, including reduction or elimination of viable bacteria in sewage-contaminated buildings. In all cases where structural pasteurization treatments are employed, the process is used in conjunction with traditional remediation methods that rely on physical removal of microbial growth and damaged building materials, and control of aerosols through the use of HEPA filters (IICRC, 2006; IICRC 2008).

CONCLUSIONS

This study demonstrates that structural pasteurization can be an effective process to reduce environmental bacteria that are known to accompany damp structures. Included as a part of the restorative drying process, structural pasteurization can be an effective sanitizer and provides improved levels of cleanliness.

ACKNOWLEDGEMENT

Primary funding for this project was provided by E-Therm, Inc. and TPE Associates (Ventura, CA). Other financial and technical support was received through collaborative field projects with IAQ Consult, Certified Disaster Cleaning and Mitigation, Inc., SFCS Environmental, Inc., and Precision Environmental, Inc.

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