EMSL Analytical, Inc.

IAQ investigators often overlook Bacteria as the Root Cause of Occupant Complaints

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Courtesy of EMSL Analytical, Inc.

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Many investigators have been dealing with mold issues the last few years and may have been overlooking bacteria as the underlying microbial problem. Bacteria typically outnumber fungi in the indoor air and may also account for foul odor problems. Bacterial contamination of HVAC systems has also been linked to hypersensitivity pneumonitis in building occupants.

Bacteria are different than all other life forms on earth because they are singlecelled, prokaryotic organisms. 'Prokaryotic' refers to the lack of membrane-bound nuclei and organelles in their cellular make-up. Bacteria are free-living and can utilize a variety of carbon sources as food. The environment is literally teeming with bacteria, many of which are innocuous and not associated with human disease. Of concern to IAQ investigators are the potentially pathogenic (disease-causing) bacteria that can proliferate in homes and offices. This article will describe some of the microbiological tests that can be performed to help identify and enumerate pathogenic bacteria from environmental samples. Sampling techniques for different types of samples will also be described.

Laboratory identification

Microbiology laboratories initially place bacteria into specific groups based on Gram staining results. The Gram reaction is determined by the composition of the cell wall of the bacteria in question. During the staining process, the ability, or not, to hold the primary stain allows the bacterium to be placed into one of two groups: Gram-positive or -negative. Once the Gram staining status is determined, further tests are performed that allow for more specific identification of the organism. Traditional methods include examination of colony morphologies, performance of biochemical tests and assays of metabolic capabilities. A number of identification kits (BBL Crystal miniaturized identification systems and bioMerieux acute API strips) are available that allow for accurate identification of bacteria. Newer identification methods include analysis of substrate utilization patterns (BIOLOG), fatty acid methylesterase (FAME) analysis (MIDI Sherlock Identification Systems) or DNA-based methods (PCR). The traditional methods work well but require greater analyst experience to achieve accurate identifications; some of these tests require subjective analysis and interpretation. The newer methods use advanced technologies for greater accuracy; although interpretation of results is not as subjective as the more traditional methods, the microbiologist may require special training to learn how to use the instruments.

Environmental considerations

Bacteria require greater levels of available water than fungi in order to proliferate. While most mycelial fungi require water activities (Aw) between 0.80 and 0.90, most bacteria require water activities that are significantly higher. For example, Pseudomonas, Escherichia, Proteus, Shigella, Klebsiella, and Bacillus grow best at 0.95 Aw or higher, whereas Salmonella, Serratia, Lactobacillus, and Pediococcus prefer 0.91 Aw or higher. Moisture condensation on building materials promotes growth of bacteria and any standing water is a potential breeding ground for bacteria. Typical standing water is found in showers, baths, evaporative condensers, and humidifiers. Other areas of concern include poorly maintained or improperly functioning drain pans, drains, and cooling towers (particularly a problem with Legionella sp.).

Bacterial contaminants

It is not feasible to explain the health effects of the major bacterial pathogens in this article. For more specific information, refer to a good microbiology reference (see listing at end of article) and also consult with the microbiologist in the laboratory. Clinically-relevant bacteria from environmental sources can cause the following: diarrhea, vomiting, cutaneous infections (skin, eye) and nuisance symptoms (rashes, burning eyes, odor). Bacteria can cause significant respiratory disease in humans; the more common diseases include tuberculosis, nosocomial pneumonia and legionellosis. Tuberculosis (TB), caused by Mycobacterium tuberculosis, is still a major cause of death with nearly 3 million deaths worldwide. Many of these deaths are in developing countries but the 1999 census data still showed over 17,500 cases of TB in the United States with close to 5% mortality. Fatigue, general malaise, weight loss, fever, chest pain, and a cough with bloody sputum are some of the symptom of the disease.

Nosocomial pneumonia is a lung infection that arises shortly after being admitted to a hospital. Nosocomial pneumonia accounts for 15% of all hospital-acquired infections. Inhalation of bacteria occurs typically from contaminated hospital breathing equipment or alternatively from aerosolized propagules. Many bacteria are implicated in nosocomial pneumonia including E. coli, Klebsiella pneumoniae, Haemophilus influenzae and Pseudomonas aeruginosa.

Legionella pneumophila can causes a life-threatening pneumonia known as Legionnaires´ Disease. Symptoms include chills, fever, general malaise, headache, diarrhea, cough, and chest pains. It is usually the inhalation of aerosolized bacteria in fine water droplets that exposes the individual. Legionella can potentially contaminate any aquatic environment including cooling towers, humidifiers, fountains, misting machines, water taps and evaporative condensers.

Although bacteria are not typically considered large contributors to allergenic hypersensitivity, they may elicit hypersensitivity in sensitive individuals. Bacteria have been implicated in exacerbating asthma. Thermophilic Actinomycetes, specifically, have been linked to hypersensitivity pneumonitis and have been found growing in poorly maintained heating, ventilation and air-conditioning (HVAC) systems. Gram-negative bacteria contain components in their cell wall known as endotoxin. Endotoxin exposure has been linked to the severity of asthma, tightness in the chest, itchy eyes, nose and throat, as well as flu-like symptoms.

There has been recent media publicity on the occurrence of Mycobacterium infections from individuals having foot spa treatments. The infection manifests as persistent boils on the individuall´s lower legs. The bacteria proliferate and grow on the hair and skin debris that collects in the poorly cleaned suction inlets of the salon´s foot baths.

Sampling and analysis of bacteria

Swabs and bulk cultures
One of the simplest sampling methods is to swab the area of contamination. Most microbiology laboratories can provide sterile swabs suitable for this use. After swabbing the suspect area, the swab can be placed in a specialized transport vessel containing a small amount of stabilizing medium. The sample should be delivered immediately to the laboratory on ice, as time and elevated heat conditions could degrade the bacteria in your sample. Bulk material can also be submitted for bacterial analysis. If possible, enclose a small piece of material (generally weighing at least 0.1 grams) in a clean container such as a plastic zipper bag. Liquids can also be submitted for bacterial analysis using a sterile container that does not leak.

Air Samples
Air samples from Andersen collectors can also be submitted to the laboratory for analysis. Although microbiologists have at their disposal hundreds of different media to identify bacterial isolates in the laboratory, media requirements for collection of air samples are quite simple. Most environmental isolates can be collected on tryptic soy agar (TSA), a general-purpose isolation and culture medium. Some laboratories may recommend using TSA supplemented with 5% sheep blood, which allows the hemolyticcactivity of some organisms to be characterized. If Gram-negative bacterial contamination is suspected (e.g. in some hospital environments), additional sampling on MacConkey agar is also recommended. MacConkey media alone should never be used as the only source, as some Gram-negative bacteria do not grow on MacConkey media. Collection of air samples is traditionally performed with Andersen samplers. Attach the Andersen collector to a pump at a flow rate of 28.3 liters per minute. Clean the stage with alcohol before placing the first plate of media on it. Also, always clean the stage between samples to prevent cross-contamination. Collect samples for 3-5 minutes, using the lower sampling time for areas with potentially higher levels of contamination. Secure the lid on the media plate using tape or Parafilm® and deliver to the laboratory as quickly as possible. Should sampling occur in a very warm environment, make sure the plates are kept cool so as not to melt the media. On the other hand, plates should be acclimatized (brought to room temperature) if the have been stored in a refrigerator or cooler before sampling to reduce the likelihood of stressing the sampled microbes.

For standard bacterial culture and identification, most laboratories will identify and enumerate the bacteria in your sample to the genus, and in some cases, the species level. Knowing the species information may be critical to solving your situation. For example, most Staphylococcus species isolated from environmental samples are unlikely to cause significant human disease. However, one species, Staphylococcus aureus, is a serious potential pathogen that can cause skin, eye and gastrointestinal infections. It is critical to communicate with your laboratory before sampling or submitting samples for bacterial analysis if you suspect contamination by unusual or unique bacteria (e.g. Mycobacterium. Some bacteria require special media or incubation temperatures for growth.

Water Tests
Specialized tests to ensure safety of water can also be performed, whether from drinking water (potable) or other sources such as ponds (non-potable). These tests identify and in some cases enumerate specific subsets of bacteria that are indicators of fecal or other contamination. These include tests for total and fecal coliforms, fecal Streptococcus and Enterococcus, E. coli, Pseudomonas, Staphylococci and Legionella. In some cases (e.g. potable water samples for coliform analysis), only the presence of absence of bacteria needs to be verified, as 1 colony forming unit (CFU)/100 ml is sufficient to fail the water sample. In other cases, it may be important to know the level of contamination. The laboratory can identify and enumerate bacteria using membrane-filtration techniques, in which dilutions of the sample are filtered and the filter is inoculated onto special media appropriate for a particular test. Presumptive colonies are further tested using various media and the bacteria are confirmed and counted.

Coliforms are Gram-negative rods that ferment lactose on laboratory media and that produce gas and acid at 35°C. This group includes E. coli and members of the Enterobacter, Klebsiella and Citrobacter families. Although this group includes bacteria that are associated with fecal contamination that can potentially cause diarrheal and vomiting illness, some ubiquitous environmental bacteria not associated with these diseases are also coliforms. Therefore, it may be useful to also analyze for fecal coliforms in your water sample. Fecal coliforms are thermotolerant -- able to ferment lactose and produce gas and acid at higher laboratory temperatures. These are a special subset of the coliform group that are considered to be better indicators of fecal contamination. This group includes E. coli and members of the Klebsiella and Citrobacter families. In potable water samples, a test indicating the presence or absence of coliforms and/or E. coli can be ordered.

Although fecal Streptococcus and Enterococcus are Gram-positive cocci that do not specifically cause diarrhea and vomiting, they do serve as indicators of fecal contamination as they are present in the gastrointestinal tracts of mammals. The Enterococcus group, a subgroup of Streptococcus that have stricter growth requirements in the laboratory, are useful indicators of human fecal contamination.

Pseudomonas, a Gram-negative rod, is ubiquitous and is commonly isolated in non-potable water samples. Many Pseudomonas species are not associated with disease. Certain isolates however, such as Pseudomonas aeruginosa, are strongly associated with human disease. Pseudomonas aeruginosa can cause serious skin and eye infections.

Staphylococcus is a Gram-positive coccus. Though most species of Staphylococcus are not associated with gastrointestinal disease, some species (e.g. Staphylococcus aureus) can cause infections of the skin, eye and ear. Common sources of contamination are swimming pools.

Legionella is a fastidious Gram- negative rod that is commonly found in many outdoor water environments. Many species of Legionella are not associated with disease. However, some species (namely, Legionella pneumophila), usually isolated from showers, baths, evaporative condensers, and humidifiers, drain pans, drains, and cooling towers, cause severe pneumonial disease known as Legionnaires´ Disease or a milder ailment known as Pontiac Fever. A Legionella outbreak in 1976 at an American Legion convention killed 34 people; it is estimated that 8,000 to 18,000 people get Legionnaires' disease in the United States each year.

To collect water samples, first obtain appropriate sterile sampling containers from the laboratory. If the water sample is chlorinated, sodium thiosulfate (Na2S2O3) should be added to as a neutralizer. Also, if heavy metal contamination is suspected, EDTA should be added to the sample. To collect potable (drinking) water samples, wipe down the faucet with bleach then allow the water (cold) to run for at least 5 minutes. For each test ordered, collect 100 ml in a sterile container and transport to the laboratory as soon as possible. It is critical to keep the samples cold (preferably on ice). For non-potable samples (for example, from a pond or river), collect 100 ml of water from representative areas, taking care not to contact the bank or bed. Holding times vary for different tests and source samples and may vary from state to state. Some tests even require the sample at the lab within 6 hours to begin processing. Check with the laboratory to determine appropriate holding time for samples. Each state also mandates acceptable levels in the potable and non-potable categories.

This article contains guidelines for sampling and submitting samples to a microbiology laboratory. For more complete information on bacteriology, good sources of information include references such as ASM´s Manual of Clinical Microbiology (8th Edition, ISBN 1-55581-255-4), ASMs Manual of Environmental Microbiology (2nd Edition, ISBN 1-55581-199-X). An excellent source for additional information on sampling and testing of water samples is Standard Methods of Water and Wastewater Testing (20th Edition, ISBN 0-87553-235-7).

Questions? - please contact Jason Dobranic, PhD at: 800-220-3675.

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