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This case study reports culture and acid-fast bacteria direct counts from 99 MWF samples received for microbiological testing at Warren, Mich.-based Biosan Laboratories between December 2006 and September 2007. The objective of the study was to determine whether the presence of gramnegative bacteria affected the presence of mycobacteria in MWFs. Data from the MWFs tested during this investigation demonstrated that there is a statistically significant relationship between mycobacteria recovery and non-mycobacteria recovery in MWFs.

BACKGROUND

Mycobacterium immunogenum is a relatively fast-growing acid- fast bacterium that has been recovered from some MWF systems nearby where workers contracted the respiratory disease hypersensitivity pneumonitis (HP). Since the sentinel HP outbreak at a metalworking plant in 1991, there have been fewer than a dozen HP clusters reported in metalworking facilities (1). In total, fewer than 200 cases of HP have been reported among workers exposed to MWFs. These statistics don't diminish the seriousness of the HP issue.

Rather, they highlight the difficulty of assessing cause-andeffect relationships. Further complicating diagnostic efforts is the fact that most of the HP clusters included four to 10 individuals at plants where 100-500 workers were exposed to MWF aerosols routinely. In their survey of HP outbreaks, Kreiss and Cox-Ganser noted that mycobacteria had been recovered at five of eight plants where HP had been reported between 1991 and 1996.

Despite the absence of mycobacteria from a significant number of the facilities at which HP clusters were reported and the fact that a number of gram-negative bacteria and fungi commonly recovered from MWFs are known to cause HP (2), a number of investigators argued that M. immunogenum was the organism responsible for HP at MWF facilities. The apparent inverse relationship between mycobacteria and non-mycobacteria colony count data in MWF samples was used in support of this theory. Watt argued that the use of certain formaldehyde condensate microbicides selectively inhibited the growth of gram-negative bacteria and thereby created conditions particularly favorable for the proliferation of mycobacteria in MWFs (3).

There was one major problem with all of the early data. Before 1998 the medium used to enumerate mycobacteria in MWFs did not contain antibiotics to inhibit the growth of non-mycobacteria. Consequently, when non-mycobacteria were present, they would overgrow the culture medium before mycobacteria colonies could be seen. Colonies don't become visible to the naked eye until they contain approximately two billion cells. Assuming that each colony is formed from a single cell, it takes 30 generations (cell division cycles) to get from one cell to a billion. The time required for a cell to divide is called the generation time. A bacterium with a 0.5h generation time will form a visible colony in 15h. One with a generation time of 6h takes 7.5 days to form a visible colony. Many of the commonly recovered gram-negative bacteria have generation times of 0.5 to 1.5h. Fast-growing mycobacteria (non-tuberculosis mycobacteria-NTM) have generation times of 6 to 10h (other mycobacteria grow substantially slower than NTM). Colonies continue to grow (diameter increases) after they become visible so that colonies that are close to one another eventually merge. Confluent growth occurs when all of the colonies on a growth medium merge to form a uniform lawn of growth over the medium's surface. This can occur within three to four days when the culture medium has more than 100 colonies on its surface.

To illustrate the effect this phenomenon has on the ability to detect mycobacteria in a MWF also contaminated with non-mycobacteria, we use an analogy. Imagine yourself in a helicopter over Times Square on New Year's Eve. Now try to count the number of children under the age of five who are standing in the crowd. Any child who is not on an adult's shoulders is eclipsed by the surrounding adults. They can't be counted. Similarly, colonies of mycobacteria may be present but are eclipsed by the confluent lawn of fast-growing colonies. Once appropriate antibiotics were incorporated into the mycobacteria enumeration media, mycobacteria were recovered with increased frequency from MWFs that also supported substantial populations of non-mycobacteria. The apparent inverse relationship seemed to be an artifact of earlier test methodology. Despite anecdotal reports of the relative frequency of mycobacteria recovery, there were no published reports of the relationship between NTM and non-mycobacteria recoveries in MWFs.

To address this information gap, the authors decided to test the next MWF samples received by the laboratory until we had data for approximately 100 samples. All samples would be tested for culturable bacteria (non-mycobacteria), culturable fungi, culturable NTM and other acid-fast bacteria. The latter test is a direct count method.

METHODOLOGY

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Dr. Frederick Passman, PhD is a Certified Metalworking Fluids Specialist with over 35 years experience in Environmental & Industrial Microbiology. His company, Biodeterioration Control Associates, Inc. (BCA) provides clients with unparalleled expertise in Microbial Contamination Control.

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