September 2005

Volume 3 | Issue 9

I hope you're doing well and will find the attached articles on sewage contamination by Mark Wallin and on Acremonium by Dr. Srivandana Kilambi informative, interesting, and helpful.

With best wishes,
Dave Gallup
Chairman

Sewage Contamination
By: Mark Wallin

Why should I test for Sewage Contamination?

Living spaces can be contaminated with sewage or wastewater through a variety of sources, including raw sewage overflows, severe flooding and leaking sewer lines or septic tanks. Exposure to sewage increases the risk of contracting gastrointestinal and other related illnesses. If you suspect that a source of water might be from a leaking sewer line, or if you want to determine the amount of contamination from a backed up toilet, drain or other water source, then testing for sewage contamination may be helpful.

Testing for sewage contamination generally involves analysis for organisms that are called "indicators". These organisms are considered indicators because they are unique to sewage or, more specifically, fecal contamination and are found in high numbers in fecal material. Historically, a number of organisms have been used as indicators but many of these have fallen out of favor because they can be found naturally in the environment, even in the absence of fecal contamination. These organisms are included in categories such as Total Coliforms, and the misnomers Fecal Coliforms and Fecal Streptococci. The best indicators to test for fecal contamination are E. coli and Enterococci because they are generated in high numbers only in the lower intestines of warm-blooded animals.

Typically, two types of analyses are available. One is qualitative, giving a present or absent result whle the other is quantitative, giving a "how many" result. There are pros and cons for each analysis. The pros of the qualitative (present/absent) test are that the results from a rush sample can usually be obtained within 24 hours and the test is less expensive. The cons are that a positive result gives no indication of the extent of the contamination and only one organism, E. coli, is used as the indicator. The pros of the quantitative analysis are that the results give an indication of the extent of the contamination and that both E. coli and Enterococcus species are used, making the analysis more robust. The con is a longer TAT (3 days). The presence/absence method should be used after remediation, when an individual wants to know if the cleaning steps effectively removed the fecal contamination and/or quick results are required, especially if occupants want to reenter a living space. The quantitative method should be used when the extent of the contamination needs to be known (i.e. there is a question as to whether intruding water contains fecal contamination and how much contamination is present) and/or a "before and after" analysis is being performed to determine the effectiveness of a cleaning method.

Since presence/absence tests are generally used after remediation, the best sampling method is to take a swab sample of the cleaned area. The amount of area to swab should be determined by the loading of material on the swab. If there is a large amount of background debris on the surface, then a smaller area should be sampled so as not to overload the swabs with potentially interfering substances. Bulks, waters and soils also can be tested using presence/absence tests, but for soils the TAT will be increased due to the fact that another methodology will have to be used.

For quantitative assessments, just about any material can be sampled including wallboard, baseboards, carpet, and flooring. For both potable and non-potable waters, collect water in a sterile wide mouthed bottle. For potable water samples, let the water run for a minute before collecting the water. Insure that the materials used to collect samples are clean to avoid cross contamination. Bacterial testing is time sensitive so samples should be sent to the laboratory within 24-30 hours of sample collection and shipped with an ice pack. These samples are potentially hazardous so gloves should be worn at all times.

A positive presence/absence test indicates that at least one viable E. coli was present in the sample. E. coli can be found in the lower intestines of all warm blooded animals and therefore, a positive result is not necessarily indicative of human fecal contamination. E. coli can be introduced into an environment through a variety of routes other than a major contamination event including, but not limited to, contamination of an area by an individual with poor sanitary practices (especially children), tracked in on the soles of pets and footwear, and contaminated sampling devices. Therefore, it is possible that a positive result was due to a source other than the original contamination event. A negative Coliform Screen indicates that no viable E. coli were detected in the sample.

With quantitative testing, high numbers of either E. coli or Enterococcus species generally indicate the presence of significant fecal contamination. How high is high? From Standard Methods for the Examination of Water and Wastewater, 20th ed., typical concentrations for coliforms include:

• 10² to 10⁵ CFU/ml in raw sewage
• 10¹ to 10³ CFU/ml in chlorinated sewage
• 10¹ to 10⁴ CFU/ml in river water

Because the natural habitat of E. coli and Enterococci is the lower intestines of warm-blooded animals, they will eventually die when exposed to the outside environment. The rate at which they die is dependent on many factors such as moisture levels, competition from environmental organisms, availability of nutrients, exposure to biocides, etc. Therefore, the longer after a contamination event testing occurs, the lower the expected concentrations of these organisms. Lower numbers (1-10 CFU/unit) are difficult to interpret. In some instances one organism will be detected and in others not. The detection limit for these analyses is <1 CFU per unit of measurement. Results at the lower detection limit indicate that the organisms were not found in the samples.

Fungus of the month: Acremonium species
By: Dr. Srivandana Kilambi

Acremonium, a filamentous and delicate deuteromycete (fungi that do not have sexually produced spores as a part of their life cycle or, their sexual life cycle is unknown) is cosmopolitan in nature. It is mainly isolated from plant debris, soil, rotting mushrooms, hay, foodstuffs and indoor building materials such as the acoustic and thermal fiber glass insulation used in heating ventilation and air conditioning systems. Acremonium has a high water affinity (Aw 0.90-0.98) and is often isolated from cooling coils, drain pans, window seals, and water from humidifiers. Some species are parasitic toward other fungal organisms (mycoparasites).

The genus Acremonium was first described by Link ex Fries in 1809. Cephalosporium (Corda, 1839) is an obsolete synonym for this genus. Gams described this fungus as being both hyaline (colorless) and dematiaceous (brown pigmented) and, traditionally, both types were included under the genus Acremonium. Dark species are now classified under the genus Gliomastix (Corda) Hughes. Acremonium currently contains 100 species, most of which are saprophytic (leaving on dead organic matter) in nature.

Colonies are moderately fast growing on all laboratory media at room temperature, but do not tend to grow well at 37° C and are therefore, not generally considered thermophilic (heat loving). To the naked eye, colonies often appear compact and flat, occasionally raised at the centre, moist at first, becoming powdery or floccose with age and may be whitish, yellowish or pinkish in color. Sometimes the microconidia of Acremonium may be confused with those produced by Fusarium species. Macroscopically, Fusarium colonies may be confused with Acremonium as well, but the former usually grow faster and have colonies with a characteristic fluffy appearance.

Fig. 1: Microscopic photo of an Acremonium species.

Microscopic features of Acremonium include fine, hyaline hyphae, long awl shaped (long pointed) and weakly branched structures (phialides) giving rise to conidia (spores). Conidia are single-celled, cylindrical and mostly aggregate in wet clusters at the apex of each philalide. The wet spores are disseminated mechanically by insects or water droplets. Occasionally, spores from old growth are wind disseminated.

Acremonium is often found growing with Stachybotrys and, similar to Stachybotrys, the spores of this fungus are produced in a slimy mass, causing aerosolization to be limited. Acremonium is not easily identified on spore traps because the spores are very small, non-distinctive, and colorless. Some spores are so small they may be easily obscured by background debris. Other spores within this genus may be counted as "other colorless". Culturable air sampling is a better way to recover and identify airborne spores of this genus. Acremonium is easily identified on direct examination because it is possible to see distinctive chains of spores or the slimy heads of conidia.

A number of species within the genus Acremonium have been identified as opportunistic pathogens of humans and animals. Conversely, some species of the genus are a source of a group of antibiotics called cephalosporins. Cephalosporins are effective against both gram-positive and gram-negative bacteria. The antibacterial properties of these cephalosporins are similar to those of semi synthetic penicillins. They are considered effective therapeutically and have a low toxicity level.

References:
1) www.ttuhsc.edu
2) www.mycology.adelaide.edu.au

The data and other information contained in this newsletter are provided for informational purposes only and should not be relied upon for any other purpose. Environmental Microbiology Laboratory, Inc. hereby disclaims any liability for any and all direct, indirect, punitive, incidental, special or consequential damages arising out of the use or interpretation of the data or other information contained in, or any actions taken or omitted in reliance upon, this newsletter.

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