Changing Perspectives On Contaminants

Microbiology and genetics are in the midst of unprecedented historical change. Changes occurring that affect the way that microbial "contaminants" are viewed include (i) the recent explosion of knowledge in microbial genetics that has brought about the wholesale change in microbial classificationm as evidenced by the on-going retooling of classification from historical phenotypic to genotypic based approaches, (ii) the realization that most organisms remain unculturable by standard methods (iii) the discovery of emerging microbial pathogens in the form of genetic insertions (free of associated microbes) into human and animal genomes, and (iv) the discovery of a previously inconceivable form of infectious disease causing agent: the prion.

Historically, bacteria and other microbes have been classified by "what they do" (i.e., ferment various sugars, retain crystal violet in the Gram stain, etc.) but are now beginning to be reclassified by "what they are" or "who they are" (i.e., their genetic relatedness). The most widely used genetic classification system as proposed by Woese (78-80) can be briefly described as centered around the similarity or dissimilarity in ribosomal RNA (rRNA) sequences, which are conserved genetically across species barriers (and significantly in all life forms). The use of the rRNA avoids a caveat that exists in the characterization of genomes in that they contain errant or wandering sequences (horizontal transmission) associated with insertions from plasmids, phage, pieces of phage, etc. that may confound

'Embedded Refs. 116,117.

mNote that classification does not equal identification (78).

attempts toward classification, whereas rRNA is not shared. As an example, the 16S rRNAs for E. coli and P. aeruginosa are members of Proteobacteria and differ by about 15% whereas E. coli differs from B. subtilis by about 23% (81).

The realization that most of Nature's microbes (by some estimates 99%) cannot be cultivated by standard methods has supported genomics-based reclassification efforts. Genetic methods have allowed for the classification of unknown organisms that cannot be cultured and have the ability to place such organisms between known species within a genetic-based continuum. Amann et al. point out that the 5,000 known species of Bacteria and Archaea must represent a tiny fraction of species existing in nature (82). They note that there are 800,000 species of insects and each insect harbors millions to billions of bacteria and "thus, consideration of insect symbionts alone could increase the number of extant bacterial species by several orders of magnitude" (82). While the relevance to parenteral manufacturing is not known, it supports the contention that there are most certainly forms of contamination that are invisible to current methods of detection, particularly in water, air, and naturally sourced raw materials and culture media. Somen maintain that: "The Petri dish and traditional tissue stains have been supplanted by nucleic acid amplification technology and in situ oligonucleotide hybridization for 'growing' and 'seeing' some microorganisms."

The lines of disease causation have become blurred at the genetic level by the discovery of microbe-induced disease processes not originally associated with microbial causes and only recently identified by genotypic approaches. The latter include viral-induced cancerso (83-85), schizophrenia (86), and diabetes mellitus (87). Borrelia burgdorferi DNA incorporated in the genome of arthritic mice (88) [and detected in humans (89)] and a list of organisms referenced by Relman (87) have been found using genotypic approaches to detect microbial genes inserted into the genome of man and animals and therefore associated with specific diseases. These include: Helicobacter pylori (peptic ulcer disease), hepatitis C virus (non-A, non-B hepatitis), bartonella henselae (Bacillary angiomatosis), Tropheryma whippelii (Whipple's disease), sin nombre virus (Hantavirus pulmonary syndrome), and Kaposi's sarcoma-associated herpes virus (Kaposi sarcoma). In this context Fredricks and Relman have called for the modernization of Koch's postulates of disease causation.

The discovery of emerging pathogens brings with it the implication of precluding organisms that may be only vaguely associated with disease and that are very difficult to detect and cultivate. Relman maintains that the human intestinal tract harbors Archaea but there are no known pathogens from this group: "in vitro cultivation methods for many Archaea are unavailable, so how would we know if archaeal pathogens existed?" (87). Archaea represent an entire domain as defined by Woese (the other two being Bacteria and Eucarya). The limitations of microbial sampling have not been lost on some in the parenteral industry: "Our industry has conventionally defined sterility in aseptic processing only in terms nsee Chapter 6, p. 112.

oThe discovery of SV40 and subsequent detection in polio vaccines administered to an estimated 100 million people (1953-1960) is an interesting detective story. The vaccines were made from viruses grown in Rhesus monkey kidney cells that harbored SV40 and researchers now wonder if SV40 infection in man originated from those early polio inoculations to now cause specific cancers (brain, bone, lymphomas, and mesotheliomas) that mirror those occurring in hamsters infected with SV40 (83).

of bacteria, yeasts, and molds because of technical limitations in detection, growth and measurement rather than scientific realities" (90).

The change in microbial classification and new microbial-host disease associations comes at a unique time in microbiological history concurrent with a new type of infectious agent that is being elucidated: the prion. Dr. Prusiner proposed the existence of prions, or proteinecious infectious agents in 1997 for which he received the Nobel Prize in Medicine (57). These agents of disease are not alive; indeed they do not contain DNA or RNA, but propagate within living hosts (with resulting neurological damage) by a domino effect of altering the three-dimensional protein conformation of the normal prion protein (PrPc) in the neurological systems of several mammals including humans, sheep, cattlep, mink, deer, elk, and catsq (91). The body can break down the normal form of PrPc but not the abnormal form (PrPSc) (92). The prion concept as elucidated by Prusiner, demonstrates how prion-generated disease may be manifested by spontaneous mutation, heredity, as well as infection (by ingestion, injection, transfusion, and transplantation1) (93). The existence of prions has affected the parenteral manufacturing industry by necessitating the exclusion of certain animal-sourced raw materials and requiring additional testing for those that cannot be replaced. Furthermore, traditional methods of detections and decontamination have little or no effect on prions, which have been described as virtually indestructiblet by heat, chemical treatment, or desiccation. Iatrogenic (medically induced) passage of prions has been documented in several instances and point to the tenacity of the prion molecule:

An electrode that had been inserted into the cortex of an unrecognized CreutzfeldtJakob Disease (CJD) patient was subjected to a decontamination procedure involving treatment with benzene, 70% ethanol, and formaldehyde vapor. It was then used in succession on two young patients and cleaned as explained earlier in this chapter after each use. Within two years, both patients came down with CJD. After these events, the tip of the electrode was implanted into the brain of a chimpanzee where it too caused lethal spongiform encephalopathy, proving that the electrode had retained infectious prions over several years and despite repeated attempts at sterilization (94).

Lastly, relevant to paradigm changes in the view of contaminants, consider current speculation that the prion concept of infection may apply to other disease processes:

Ongoing research may also help determine whether prions consisting of other proteins play a part in more common neurodegenerative conditions, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. There are some marked similarities in all these disorders. As is true of the known prion diseases, the more widespread ills mostly occur sporadically but sometimes "run" in families. All pAnd other ruminants in UK zoos between 1986-1992: bison, nyala, gemsbok, oryx, greater kudu, and eland (93).

qIncluding puma, cheetah, ocelot, and a tiger in the same zoos and period noted earlier (93). rThe normal prion protein is coded by mammalian genomes and occurs predominately in white blood cells and brain cells.

sRNA/DNA methods cannot be used since they contain no nucleic acid and infectivity assays are costly and inexact.

t"... sheep were imported from Belgium and the Netherlands and may have consumed tainted feed. The sheep were euthanized and their carcasses dissolved in boiling lye. Barn surfaces and implements were disinfected with sodium hypochlorite or incinerated, and the pastures have been put off limits for five years to allow residual infectivity to diminish" (95).

are also usually diseases of middle to later life and are marked by similar pathology: neurons degenerate, protein deposits can accumulate as plaques, and glial cells (which support and nourish nerve cells) grow larger in reaction to do damage to neurons. Strikingly, in none of these disorders do white blood cells—those ever present warriors of the immune system—infiltrate the brain. If a virus were involved in these illnesses, white cells would be expected to appear (57).

This begs the question: Will discoveries follow of additional infectious proteins and, if so, how might this be relevant to the use of transgenics, given that the crossover of pathogenic contaminants has in the past gone unrecognized"? The degree of similarity or dissimilarity in the mammalian gene that encodes the PrP has been found to explain the mechanism of barrier between animals that can and cannot contract the disease in terms of protein conformation similarity (and susceptibility to being converted) relative to the gene that encodes it (the PrP genes of cows, sheep, and humans are very similar). It is not known at what levels of concentration prions are infective or the cause(s) of variability in the time of onset of symptoms. Governments around the world have enacted precautions in food, medical (including blood collection and handling), and drug regulation to contain the spread of known prion diseases (95-97).

It is frightening to see that CJD does occur on a "normal" basis and somewhat reassuring that the CDC is monitoring any suspected outbreaks to determine if they may be due to vCJD, such as the 14 deaths related to CJD that occurred at a New Jersey racetrack over the course of approximately 9 years:

In 2001, Garden State Racetrack was closed permanently. The number and ages of all persons visiting or dining at the racetrack is unknown, however, according to New Jersey Racing Commission records, attendance at the racetrack during 1988-1992 was approximately 4.1 million. Based on an annual CJD rate of 3.4 cases per 1 million persons (CDC, unpublished data, 2004) and an overall death rate from all causes of 2.9% for persons aged >50 years, the occurrence over approximately 9.25 years (1995-2004) of at least 14 CJD related deaths among as few as 300,000 persons aged > 50 years would not be unusual. This number is within the estimated range of the number of persons attending (98).

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