|Year : 2002 | Volume
| Issue : 4 | Page : 174-177
Uncultivable bacteria: Implications and recent trends towards identification
S Bhattacharya, N Vijayalakshmi, SC Parija
Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education & Research, Pondicherry - 605 006, India
Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education & Research, Pondicherry - 605 006, India
Diseases due to uncultivable bacteria could represent emerging infectious diseases. However, the growing importance of these pathogens remains ill understood and undefined. Non-culture based approaches, especially molecular genetic methods are evolving as the most important tool in our understanding of these enigmatic pathogens. This article attempts to discuss the scientific implications of the evolution of uncultivable bacteria, review the recent trends in identification, and highlight their relevance in clinical medicine.
|How to cite this article:|
Bhattacharya S, Vijayalakshmi N, Parija S C. Uncultivable bacteria: Implications and recent trends towards identification. Indian J Med Microbiol 2002;20:174-7
|How to cite this URL:|
Bhattacharya S, Vijayalakshmi N, Parija S C. Uncultivable bacteria: Implications and recent trends towards identification. Indian J Med Microbiol [serial online] 2002 [cited 2014 Mar 11];20:174-7. Available from: http://www.ijmm.org/text.asp?2002/20/4/174/6951
Isolation and identification of bacteria in vitro in pure culture in an artificial medium, is one of the fundamental Koch's postulates. Apart from necessity of fulfilment of the Koch's postulate, cultivation of bacteria serves several important functions. These include: 1) isolation of bacteria in pure culture, 2) identification of bacteria by colony morphology and biochemical reactions, 3) testing for viability, 4) study of basic biology, 5) antibiotic sensitivity testing, 6) production of antigens, and 7) production of vaccines. Colony purification and preparation of limiting dilutions of liquid culture media have provided at least two benefits: amplification of microbial material and purification of single organisms along with their direct descendants. Therefore, when the cultivation of bacteria, which is the sine qua non of microbiological practice, becomes difficult or 'impossible', these essential functions, become unachievable. Uncultivable bacteria in the history of microbiology are not new. Mycobacterium leprae and Treponema pallidum, two of the oldest pathogens of man are still uncultivable in artificial media. In recent years Bartonella henselae, the causative agent of bacillary angiomatosis and cat-scratch disease, and Tropheryma whippelii the causative agent of Whipple's disease have emerged as newer uncultivable pathogens.
| ~ Factors responsible for bacterial uncultivability|| |
Cultivation of bacteria in vitro in an artificial medium needs to be objectively evaluated. Objective evaluation of bacterial cultivation becomes important when trying to assess the cultivability of fastidious or slow growing organisms, and hitherto regarded as “uncultivable” bacteria. This may be done by three biochemical parameters viz., increase in bacterial ATP, increase in bacterial DNA, and tritiated-thymidine uptake, besides the detection of increase in bacterial numbers. But does bacterial uncultivability represent an intrinsic property of bacteria or does it reflect the deficiency of our knowledge about bacterial growth requirements? All possible indicators seem to suggest that the answer is a combination of both of the above factors. The primary cause of bacterial uncultivability in artificial media seems to be the absence of key metabolic pathways, which prevent the bacteria from utilizing essential nutrients in vitro, which are readily available in vivo. Recent molecular biologic analysis of M.leprae has suggested that its genome is considerably smaller than that of other bacteria including M.tuberculosis implying the absence of critical enzymatic pathways that necessitates reliance on host parasitism. In addition to it, one should not forget the role of intracellular protective mechanisms, which shield the bacteria from many deleterious influences, viz., the role of oxygen and reactive oxygen species. In the absence of these in vivo intracellular protective mechanisms the bacteria become vulnerable to toxic chemicals in vitro and hence any increase in the number may be negated by overwhelming bacterial death in extra cellular in vitro environments. This seems to be the case in attempts of in vitro cultivation of Treponema pallidum.
However, one should not be oblivious of the common secondary causes of culture negativity before declaring bacteria as uncultivable. These reasons are often common and include deficiency of essential nutrients, deficiency of atmospheric requirements, inappropriate pH, inappropriate temperature of incubation, insufficient time of incubation, prior treatment with antibiotics, and presence of other growth inhibitors like selective agents in the culture medium. One fundamental question that remained unanswered for a long time was why M.leprae does not multiply under the cell-free conditions? The intracellular ATP content in cells of M.leprae increased consistently in the medium containing adenosine after 4-6 weeks of cultivation and decreased thereafter. This was not a result of deterioration in the culture medium during cultivation but was found to be a result of the characteristic property of M.leprae cell wall, i.e., fragility. Several reports have appeared in the literature from time to time claiming successful cultivation of M.leprae in artificial media., However, in 1977 Pattyn surveyed the mycobacterial strains isolated in the last 10 years from leprosy lesions and concluded that those strains belonged to taxonomically different species and could not be considered to be M.leprae. It was apparent that increased knowledge about the intracellular environment and the metabolic activities of this organism was essential and that could only be achieved by the application of modern biochemical and histochemical techniques. Moreover this author proposed criteria to help evaluate papers appearing in the literature claiming successful cultivation of M.leprae.
| ~ Implications of bacterial uncultivability|| |
The implications of bacterial uncultivability are manifold. At the theoretical plane it exposed the limitations of the Koch's postulate, stimulated its reassessment, recommended a set of molecular guidelines, culminating in the development of the molecular Koch's postulates. In the practical plane it led to the development of cell lines (McCoy cells for the cultivation of Chlamydia trachomatis), use of embryonated eggs (cultivation of rickettsia and chlamydia in the yolk sac) and laboratory animals (use of guinea pigs and mice for the cultivation of rickettsia, foot pad of mice for M.leprae, and rabbit testes for the maintenance of T.pallidum ), and developments of novel methods of cultivation, e.g. co-cultivation. Last but not the least, it stimulated search for unknown pathogens in diseases hitherto thought to be non-infective. These include chronic ailments like sarcoidosis, inflammatory bowel disorders like Crohn's disease, rheumatoid arthritis, systemic lupus erythematosus, Wegener's granulomatosis, diabetes mellitus, primary biliary cirrhosis, tropical sprue, and Kawasaki disease.
| ~ Recent trends towards identification of uncultivable bacteria|| |
Notwithstanding the great scientific debate about the significance of uncultivable pathogens, the cardinal question that remains from a practical point of view is “how are uncultivable pathogens identified?” Uncultivable bacteria are detected by non-culture based methods. These include direct microscopy, immunological methods based on antigen and antibody detection, chromatographic methods like gas-liquid chromatography and high performance liquid chromatography, protein profile electrophoresis, ATP bioluminescence, and finally molecular genetic methods. The non-genetic methods have already been tried, tested and standardized for diseases like leprosy and syphilis. However, they have been found to be inadequate when dealing with unknown pathogens and deducing the phylogeny of these organisms.
Molecular genetics opens up some new vistas in the exploration and identification of uncultivable pathogens. Culture-independent identification procedures like comparative rRNA sequence analysis based on non-amplification based molecular methods like nucleic acid hybridization, and amplification techniques are reliable methods for taxonomic studies and phylogenetic analysis. Sequence-based molecular methods provide an alternative approach for microbial identification directly from host specimens. The rapid expansion of genome sequence databases (many of which are accessible through the world wide web), and advances in biotechnology present unforeseen opportunities as well as challenges. Three fundamental approaches appear to be evolving in the quest for identification of unknown and uncultivable pathogens. These include 1) the identification of consensus sequences from which reliable and specific phylogenetic information can be deduced for all taxonomic groups of pathogens, 2) broad-range pathogen identification on the basis of virulence-associated gene families, and finally 3) the use of expression response profiles of the host genes as specific signatures of microbial infection. Conserved genomic sequences of the uncultivable organism might be used to discover the evolutionary origin and be amplified directly from natural sites of infection. But it is important to understand the essential features of a genetic sequence that makes it useful for identifying uncharacterized microorganisms. First, the sequence should be conserved among a relatively large number of known microorganisms. Second, its rate of change should be constant over long periods of time. Third, the sequence should not have been shared among different microorganisms by horizontal transmission. Finally, the sequence should be amenable to broad-range amplification or detection.
Discovery of previously unrecognized microbial pathogens by nucleic acid amplification and sequencing has been used with great success in the identification of several pathogens. These include the detection of Hanta virus by gene amplification and sequence analysis in patients and deer mouse, the construction of random-primed complementary DNA library for the detection of hepatitis C virus, and the use of representational difference analysis for the identification of Kaposi's sarcoma associated herpes virus in AIDS patients. However, not all genetic sequences are the ideal targets for phylogenetic analysis because they lack many of the attributes described above. The sequence of the small subunit ribosomal RNA or DNA (ssu rRNA or ssu rDNA) meets these criteria. Ssu rRNA and ssu rDNA are the most reliable genetic sequences in the study of the evolutionary origin of prokaryotes and eukaryotes. The ssu rDNA and ssu rRNA is highly conserved and provides the ideal sites for broad-range polymerase chain reaction (PCR). They were used by Relman and colleagues in the discovery of the uncultivable bacteria of bacillary angiomatosis in 1990 and of Whipple's disease in 1992., A similar approach using sequence analysis of 18S ssu rDNA, and PCR of the 18S rRNA gene has shown Rhinosporidium seeberi to be a protistan parasite and not an aquatic fungus., Therefore, molecular genetic techniques such as in situ hybridization, PCR, and representational difference analysis not only reveal previously uncharacterized fastidious or uncultivable, microbial pathogens that resist the application of Koch's original postulates, but they also provide new approaches for proving disease causation by microbial pathogens.
Serologic approaches have been useful in providing important leads. For example, the first clues of a possible chlamydial etiology for coronary atherosclerosis were serologic findings. Molecular and other in situ methods were then used to substantiate the findings.
| ~ Conclusions|| |
Before we conclude it must be emphasized that the development of techniques suitable for the recognition of uncultivable pathogens is an endeavour worthy of investment. There remain many diseases in the tropics, as in any other part of the world, where a microbial etiology is strongly suspected from clinical features but a pathogen or an etiologic agent is never identified. Many of these diseases have been clubbed together and prefixed as so called “idiopathic” or “cryptic” diseases. The medical scientists need to ask themselves “Are the so called idiopathic/primary/essential/cryptic diseases really primary?” or do they represent the primary inability of a practitioner to go beyond the confines of his/her knowledge to seek for innovative solutions from the greener pastures of unexplored territories. The discovery of microbial etiology for disease like Whipple's disease, coronary atherosclerosis, and peptic ulcer have reinforced the belief that many of the 'idiopathic' diseases may have microbes as the primary or the precipitating cause. It has been estimated that out of the total number of bacteria present in the world only about 0.4% have been identified. It is also known that 40% of the bacteria present in the oral cavity and 99% in the environment are uncultivable. Out of the tripartite tree of life consisting of the prokaryotes, eukaryotes and the archaebacteria, only the first two have been associated with disease and hence considered pathogenic. Why should the archaebacteria, which are as abundant as anything else, be confined to hydrothermal vents and not also isolated from diseased tissues and degenerated organs is beyond our present capability of comprehension. It needs to be investigated whether the “sterile culture” reports are a true picture of diseased tissue or a reflection of the inadequacy of our methods. The answer to these difficult questions may be the key to our understanding about the pathogenesis, diagnosis, treatment and prevention of diseases, which seem to defy conventional approaches.
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