Clearance | Microorganism of the Month: Flavobacterium
By Dr. Harriet Burge, EMLab P&K Chief Aerobiologist and Director of Scientific Advisory Board
In the indoor air community clearance has several different meanings. When correcting a problem where published health-based standards exist, clearance means that the environment now meets those standards. Thus, if there is a carbon monoxide problem, one needs to reduce concentrations to less than the published limit, and, in most environments, to non-detect levels.
For microorganisms, no health-based standards exist, and, in fact, no environmental guidelines are widely accepted. It is for this reason that hypothesis-based investigations are essential. Such investigations allow the setting of clearance standards for each investigation, and the standards can be agreed upon by the investigator and the client ahead of time and in writing.
There are at least 5 types of clearance that could be used.
None of a particular organism is to be present. This is the standard for biological warfare agents and for other extremely virulent pathogens. Unfortunately, as we have discussed many times, proving the negative case is not possible. Hence, one decides on a detection limit that cannot be exceeded. Examples of this could be 1 Bacillus anthracis (the bacteria that causes anthrax) colony forming unit (cfu)/100 surface samples (meaning that if you found 1 culturable Bacillus anthracis cell you would fail clearance). Obviously, if you don't find any in 100 samples you pass clearance, but you have not proven that there is no risk. Note that this is the only health-based standard.
Concentrations are to be reduced by a specific amount. This is the standard for testing biocides. Usually reductions of 3-5 orders of magnitude are considered acceptable. In this case if you start with moderate concentrations (say 1,000 cfu/m3 of air) you would reduce the number to about 1 using the 3 orders of magnitude rule. On the other hand, if you start with 10,000,000 cfu/m3, you still have 10,000 cfu/m3 after a reduction of 3 orders of magnitude, and you would want to use the 5 orders of magnitude rule so that concentrations would be 100 cfu/m3. This approach is not commonly used for indoor air investigations.
Concentrations are to be reduced to below a specific level. This approach is widely used in the indoor air community. The long-lasting 1,000 spore/m3 "guideline" originally proposed by the ACGIH is an example of this approach. One could use a concentration guideline if the hypothesis being tested is appropriate. For example, for an air-conditioned building where the outdoor aerosol has relatively little effect, you could arbitrarily specify that total spore concentration will be below 500 spores/m3 and that the aerosol is mixed with respect to spore types. The latter part is necessary because you probably would not want to clear a space following a Penicillium infestation if the entire aerosol following remediation is composed of Penicillium. This approach requires a good knowledge of the aerobiology of uncontaminated environments.
Conditions are returned to pre-infestation conditions. This is actually an excellent approach, but it requires information on the pre-infestation conditions, which is generally not available. One use of this method is to rely on visual observation and information provided by the client. For example, if a particular wall had no signs of water or mold growth before a specific water event, then a water event led to mold growth, then the water and the mold is removed and the damaged materials replaced, then the observation that the job was done should be sufficient. Another approach is to assume that your database tells you what the average interior of the type being studied looks like aerobiologically, and use that data to establish your clearance criteria. For example, in a large database assume that the median (50th percentile) total spore value is 350 for office environments. Knowing this, you can select this value as the upper limit for clearance. This could also be done by individual species. Note that this is very similar to the previous approach.
Finally, indoor concentrations are to be less than those outdoors. Of course, the big questions are "How much less?" and "What constitutes a representative outdoor sample?" A major problem with this approach is the fact that, as outdoor concentrations increase, the indoor/outdoor ratio tends to decrease. Thus, if you have 200 spores/m3 outdoors, you would have to have fewer than 200 indoors, which would probably be unusual for many indoor environments. In many parts of the world where snow covers the ground in winter, outdoor concentrations are likely to be near, or below, the detection limit. It is obviously impossible to use indoor/outdoor ratios under these circumstances and one of the other approaches will be needed. You could also have a very low ratio (meaning that the indoor concentration is much lower than that outdoors) and still have significant mold growth indoors. Let's say you have 10,000 spores/m3 outdoors, and 500 indoors. The indoor/outdoor ratio in this case would be 0.05. However, what if the 500 indoor counts are all Stachybotrys? So, you need to take into account the kinds of spores present indoors and out. This sounds straightforward. However, consider the following scenario:
|Spore Type||Indoors (spores/m3)||Outdoors (spores/m3)||Inside to Outside Ratio|
The overall ratio is considerably less than 1 indicating that the outdoor concentrations overall are higher than those indoors. If your goal is to document that fact, then you are in good shape. If your goal is to document that you have removed sources of mold and residual spores, then what does the table tell you? You have Penicillium and Alternaria ratios greater than one. Given the detection limit is 7 spores/m3, you have a raw count of one Stachybotrys spore. You have unusually high levels of ascospores indoors. Are you clear or not? This complexity was the driving force for development of a numerical rule of thumb called MoldSCORE™. The intent of MoldSCORE™ was to try to improve on the inside to outside ratio by taking into account additional factors such as which spores are capable of colonizing indoor building materials, how common different spore types are in the outside air, what typical outdoor concentrations are, and the distribution of spore types. This, then, provides a score that is unbiased and not influenced by emotions, or what happened in the last investigation. It could be used as a guideline in clearance samples. For example, in the initial study design you can specify that the MoldSCORE™ will be less than 150 or whatever you think might be appropriate for the type of interior you are working on.
In all of these cases, the more samples you collect the more likely you are to make good decisions with respect to whether or not an environment should be "cleared."
What do all of these criteria miss? They neglect the fact that the sampling data is just one clue. It is only part of the investigation, and not even the most important part. The most important part, more important than the laboratory data, is the knowledge and expertise of the onsite investigator, trained (hopefully) to do these types of investigations and who understands the real world constraints and what is and has happened on site. While this is often an uncomfortable and difficult position to be in, it is also what makes indoor air quality investigations interesting and thought provoking and not a mere 'turning of the crank.'
By Murali Putty, EMLab P&K Analyst
Flavobacterium is a genus of gram-negative, nonmotile, aerobic or facultatively anaerobic, rod-shaped bacteria, characterized by production of a yellow pigment. The organisms occur widely in soil and water, and are opportunistic pathogens in humans. Flavobacterium has been isolated from surface and air samples in the indoor environment, reported from air conditioning cooling coils and within building HVAC systems.
Flavobacterium are also found in plants, foodstuffs and most water systems including distilled water lines and dentistry units. They are also found in raw meats, milk and other foods, in hospital environments and in human clinical settings. They can also survive in intravenous anesthetics, eyes, urine, and stool samples. In hospitals they are found in incubators, water baths, drinking fountains, and cold humidifiers. Flavobacterium occur in 0.1-1.0% of all blood cultures and 0.01% of all urine cultures. In soils, Flavobacterium are concentrated in the rhizosphere, the soil immediately surrounding roots.
To date, there are three hundred and twelve strains of Flavobacterium. Two hundred and fifty of these strains are acid producing. The acid producing strains include F. meningosepticum, the strain which causes the disease meningitis. All but one species produces indole, which is a unique characteristic among non-fermenters. They are chemoorganotrophs, which have respiratory metabolism. Some strains are halotolerant (able to live in conditions of high salinity). Flavobacterium are fastidious microbes and some strains are pathogenic to humans and animals. Flavobacterium colonies may be yellow, orange, and red to brown pigmented with a fruity odor. The colonies are 2-5mm long, 0.3-0.5mm wide, often circular, smooth and shiny. The hue and intensity of pigmentation varies considerably and may be affected by the growth medium, temperature and incubation period. When being cultured they can thrive in everything from blood agar medium to basal medium (a medium which does not require any special nutrients supplements). The temperatures for growth of environmental isolates are usually between 5°C-30°C, but clinical isolates will grow at 37°C. Some strains of F. meningosepticum will also grow at 42°C.
Members of Flavobacterium can cause infection in premature infants and immunocompromised individuals. F. meningosepticum, a pathogenic species that is a major cause of hospital-acquired infections is the causal agent for meningitis and septicemia. In adults, it causes a mild bacteremia (bacterial presence in blood), yet in premature and newborn infants, this disease has a high fatality rate. Flavobacterium odoratum is another pathogenic species recovered from infections of wounds and the urinary tract. Vancomycin is one of the bactericidal antibiotics used in treating Flavobacterium infection.
Several species are known to cause diseases in freshwater fish. Flavobacterium columnare causes "cotton-wool" disease. F. branchiophilum causes "bacterial gill disease" (BGD) in trout, F. psychrophilum causes "bacterial cold water disease" (BCWD) in salmonids and "rainbow trout fry disease" (RTFS) in rainbow trout. Infections caused by these bacteria are responsible for heavy mortalities and considerable economic losses in fish farms worldwide. Flavobacterium psychrophilum is considered to be one of the most prevalent and troublesome infections of hatchery salmonids in the Pacific Northwest region. Prudent fish culture management practices along with rapid treatment with U.S. Food and Drug Administration approved compounds has been found to be effective in treating these diseases. It has been suggested that development of mass immunization along with selective breeding for disease resistance could be a good long-term strategy.
1. Holmes B, Owen R.J and McMeekin T.A: Genus Flavobacterium Bergey, Harrison, Breed, Hammer and Huntoon 1923, 97 Al In: Kreig N.R., Holt J.G. (eds): Bergey's Manual of Systematic Bacteriology, first edition, Volume 1, The Williams and Wilkins Co, Baltimore, 1984, pp. 353-360.
2. Wikipedia: Flavobacterium
3. Encyclopedia.com: Sick-building syndrome and building-related illness
4. Virginia Tech, Soil Microbiology: Flavobacterium
5. Two Indoor Air Quality Investigations - Oceans Apart
6. SolAir: March 2000 (764kb PDF)
7. Flavobacterium 2007 Workshop (1MB PDF)
This article was originally published on October 2007.