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home:pathogenesis:microbiota:detecting [10.27.2018] – [Availability of tests influence which microbes are studied in chronic diseases] sallieq | home:pathogenesis:microbiota:detecting [09.14.2022] (current) – external edit 127.0.0.1 | ||
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- | It has been called one of the greatest false prophecies in the history of medicine. In 1821, Sir John Forbes concluded that the newly invented stethoscope was “ludicrous” and would never be generally adopted by physicians.(({{pubmed> | + | It has been called one of the greatest false prophecies in the history of medicine. In 1821, Sir John Forbes concluded that the newly invented stethoscope was “ludicrous” and would never be generally adopted by physicians.(({{pmid> |
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< | < | ||
- | Until very recently, efforts to detect and identify microorganisms have depended on //in vitro// studies – research in which bacteria were grown in culture in a laboratory setting. Because at least a fraction of microorganisms are not particular in their growth requirements, | + | Until very recently, efforts to detect and identify microorganisms have depended on //in vitro// studies – research in which bacteria were grown in culture in a laboratory setting. Because at least a fraction of microorganisms are not particular in their growth requirements, |
Koch's postulates is a set of ground rules to determine whether a given organism can cause a given disease. One of its dictates is that a bacterium must be shown to grow outside the body in culture in order to prove that the bacterium causes disease. For at least a century, Koch's postulates have held sway. | Koch's postulates is a set of ground rules to determine whether a given organism can cause a given disease. One of its dictates is that a bacterium must be shown to grow outside the body in culture in order to prove that the bacterium causes disease. For at least a century, Koch's postulates have held sway. | ||
- | According to Robert Koch, or at least the ideas since attributed to him, there were no cultivation-resistant microbes. Over the years, varied researchers had difficulty consistently culturing bacteria found in disease. As a result, many researchers began to assume that chronic diseases were not caused by microbes. The net effect of all this was that the understanding of pathogens in disease was driven by the study of well-known, easy-to-culture microbes(({{pubmed> | + | According to Robert Koch, or at least the ideas since attributed to him, there were no cultivation-resistant microbes. Over the years, varied researchers had difficulty consistently culturing bacteria found in disease. As a result, many researchers began to assume that chronic diseases were not caused by microbes. The net effect of all this was that the understanding of pathogens in disease was driven by the study of well-known, easy-to-culture microbes(({{pmid> |
===== A history of discounting culture-independent methods of microbe identification ===== | ===== A history of discounting culture-independent methods of microbe identification ===== | ||
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The case for uncultured organisms even existing let alone remaining uncultured has not been proven and repetitive uncontrolled observations do not serve the scientific community well and may mislead the unwary. | The case for uncultured organisms even existing let alone remaining uncultured has not been proven and repetitive uncontrolled observations do not serve the scientific community well and may mislead the unwary. | ||
- | // | + | // |
</ | </ | ||
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Researchers have long known that traditional methods for culturing bacteria are effective at identifying only a fraction of the bacteria in a given sample. One of the first reports of this came from Razumov((Razumov, | Researchers have long known that traditional methods for culturing bacteria are effective at identifying only a fraction of the bacteria in a given sample. One of the first reports of this came from Razumov((Razumov, | ||
- | In 1949, Winogradsky confirmed Razumov' | + | In 1949, Winogradsky confirmed Razumov' |
- | In 1985, Staley and Konopka pointed to Razumov' | + | In 1985, Staley and Konopka pointed to Razumov' |
Figure 1 shows the number of bacteria identified using a fluorescent dye, acridine orange. Acridine orange counts bacteria by interacting with bacterial DNA and RNA. | Figure 1 shows the number of bacteria identified using a fluorescent dye, acridine orange. Acridine orange counts bacteria by interacting with bacterial DNA and RNA. | ||
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As it is plainly evident, the genomic method for detecting bacteria (Figure 1) was orders of magnitude more sensitive than a method based on viability in a culture (Figure 2). The shortcomings of the cultivation method is striking and suggests that this traditional method for cultivation is only effective in identifying a fraction of all bacteria. | As it is plainly evident, the genomic method for detecting bacteria (Figure 1) was orders of magnitude more sensitive than a method based on viability in a culture (Figure 2). The shortcomings of the cultivation method is striking and suggests that this traditional method for cultivation is only effective in identifying a fraction of all bacteria. | ||
- | The author' | + | The author' |
< | < | ||
As the figures illustrate, only approximately 0.1-1.0% of the total bacteria can be enumerated by the plating procedure. Indeed, as a general rule we have found that the maximum recovery of heterotrophic bacteria [bacteria that don't use photosynthesis] is 1% of the total direct count using plating procedures or other viable enumeration methods.... From a microbiological perspective, | As the figures illustrate, only approximately 0.1-1.0% of the total bacteria can be enumerated by the plating procedure. Indeed, as a general rule we have found that the maximum recovery of heterotrophic bacteria [bacteria that don't use photosynthesis] is 1% of the total direct count using plating procedures or other viable enumeration methods.... From a microbiological perspective, | ||
- | //**James T. Staley** and **Allan Konopka, PhD**// | + | //**James T. Staley** and **Allan Konopka, PhD**// |
Over the years, researchers have pointed out two reasons why the majority of bacteria that comprise the human microbiome do not culture: | Over the years, researchers have pointed out two reasons why the majority of bacteria that comprise the human microbiome do not culture: | ||
- | * Some forms and kinds of bacteria only grow in specific conditions offered by the human body including a very narrow pH, the right nutrient availability, | + | * Some forms and kinds of bacteria only grow in specific conditions offered by the human body including a very narrow pH, the right nutrient availability, |
* Certain bacteria only grow in the presence of certain other species of bacteria. | * Certain bacteria only grow in the presence of certain other species of bacteria. | ||
- | By David Relman' | + | By David Relman' |
- | According to a 2009 review by James D. Oliver, "The debate over whether a VBNC [viable but not culturable] state truly exists has largely been put to rest, largely as a result of numerous molecular studies reported in recent." | + | According to a 2009 review by James D. Oliver, "The debate over whether a VBNC [viable but not culturable] state truly exists has largely been put to rest, largely as a result of numerous molecular studies reported in recent." |
* nutrient starvation | * nutrient starvation | ||
* incubation outside the normal temperature range of growth | * incubation outside the normal temperature range of growth | ||
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* exposure to white light | * exposure to white light | ||
- | The switch to the VBNC stage has been described and documented for at least the following bacterial species: //Vibrio spp. (cholerae, vulnificus and other species), Escherichia coli (including EHEC), Campylobacter jejuni, Helicobacter pylori, Salmonella spp., Listeria monocytogenes, | + | The switch to the VBNC stage has been described and documented for at least the following bacterial species: //Vibrio spp. (cholerae, vulnificus and other species), Escherichia coli (including EHEC), Campylobacter jejuni, Helicobacter pylori, Salmonella spp., Listeria monocytogenes, |
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As an alternative to traditional methods for culturing, various //in silico// methods for DNA and RNA sequencing have been developed. The most commonly used DNA and RNA amplification techniques is polymerase chain reaction (PCR), a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of a particular DNA sequence | As an alternative to traditional methods for culturing, various //in silico// methods for DNA and RNA sequencing have been developed. The most commonly used DNA and RNA amplification techniques is polymerase chain reaction (PCR), a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of a particular DNA sequence | ||
- | One commonly used practice is to look at a bacterial species' | + | One commonly used practice is to look at a bacterial species' |
- | Currently, the conventional 16S rDNA PCR technique must involve three steps. First, a step that corresponds to the amplification and revelation of amplified products on agarose gels. Second, a step in which the amplified product is sequenced. Third, a step in which the obtained sequence is analyzed and compared with that given in a database, mainly the GenBank, containing all known bacterial sequences to allow an accurate identification. A sequence similarity of less than 97% of the 16S rRNA sequence is the criterion used to define a potentially new bacterial species.(({{pubmed> | + | Currently, the conventional 16S rDNA PCR technique must involve three steps. First, a step that corresponds to the amplification and revelation of amplified products on agarose gels. Second, a step in which the amplified product is sequenced. Third, a step in which the obtained sequence is analyzed and compared with that given in a database, mainly the GenBank, containing all known bacterial sequences to allow an accurate identification. A sequence similarity of less than 97% of the 16S rRNA sequence is the criterion used to define a potentially new bacterial species.(({{pmid> |
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Polymerase chain reaction is a relatively specific and sensitive test, however: | Polymerase chain reaction is a relatively specific and sensitive test, however: | ||
- | < | + | < |
On resurgence to an acute-phase pathogen, they get those genes back, by methods not really fully understood. | On resurgence to an acute-phase pathogen, they get those genes back, by methods not really fully understood. | ||
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//**Trevor Marshall, PhD**// </ | //**Trevor Marshall, PhD**// </ | ||
- | Another problem is that PCR primers may miss half of rRNA microbial diversity(({{pubmed> | + | Another problem is that PCR primers may miss half of rRNA microbial diversity(({{pmid> |
[{{ : | [{{ : | ||
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===== Existence of microbes in healthy tissue supposed to be sterile ===== | ===== Existence of microbes in healthy tissue supposed to be sterile ===== | ||
- | * **blood** – The presence of microbes in the blood is known as bacteremia. While many doctors believe it to be rare, a significant blood microbiome is ubiquitous. The Relman Lab at Stanford used real time PCR to show that there is a substantial and " | + | * **blood** – The presence of microbes in the blood is known as bacteremia. While many doctors believe it to be rare, a significant blood microbiome is ubiquitous. The Relman Lab at Stanford used real time PCR to show that there is a substantial and " |
- | * **lungs** – Erb-Downward found bacterial 16S sequences in the lung tissues of all subjects in a 2011 based study of healthy patients as well as those with chronic obstructive pulmonary disease (COPD).(({{pubmed> | + | * **lungs** – Erb-Downward found bacterial 16S sequences in the lung tissues of all subjects in a 2011 based study of healthy patients as well as those with chronic obstructive pulmonary disease (COPD).(({{pmid> |
===== Role of cultivation-resistant microbes in disease ===== | ===== Role of cultivation-resistant microbes in disease ===== | ||
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Regardless of the role that the VBNC [viable but not culturable] state plays, it is clear that a large number of non-spore-forming bacteria, most notably a large number of human pathogens, are capable of entering this state, maintaining cellular structure and biology and continuing significant gene expression while otherwise nonculturable by " | Regardless of the role that the VBNC [viable but not culturable] state plays, it is clear that a large number of non-spore-forming bacteria, most notably a large number of human pathogens, are capable of entering this state, maintaining cellular structure and biology and continuing significant gene expression while otherwise nonculturable by " | ||
- | //**James D. Oliver**// (({{pubmed> | + | //**James D. Oliver**// (({{pmid> |
Recent experiments using PCR have offered compelling proof that traditional cultivation methods offer only a limited glimpse into the full extent of the human microbiota and that those microbes found play a role in disease. | Recent experiments using PCR have offered compelling proof that traditional cultivation methods offer only a limited glimpse into the full extent of the human microbiota and that those microbes found play a role in disease. | ||
- | * **amniotic fluid** – One study compared the rate of amniotic fluid infection with pre-term delivery among pregnant women (see right).(({{pubmed> | + | * **amniotic fluid** – One study compared the rate of amniotic fluid infection with pre-term delivery among pregnant women (see right).(({{pmid> |
- | * **chronic wounds** – Another study examined the bacteria present in three types of chronic wounds: diabetic foot ulcers, venous leg ulcers, and pressure ulcers.(({{pubmed> | + | * **chronic wounds** – Another study examined the bacteria present in three types of chronic wounds: diabetic foot ulcers, venous leg ulcers, and pressure ulcers.(({{pmid> |
* **urinary tract infections** – | * **urinary tract infections** – | ||
< | < | ||
- | // | + | // |
- | * **atherosclerosis** – Sometimes novel uses of culture-based techniques can confirm the presence of additional microbes. Using an array of new culture-based techniques, Kozarov //et al.// recovered isolates from // | + | * **atherosclerosis** – Sometimes novel uses of culture-based techniques can confirm the presence of additional microbes. Using an array of new culture-based techniques, Kozarov //et al.// recovered isolates from // |
===== Availability of tests influence which microbes are studied in chronic diseases ===== | ===== Availability of tests influence which microbes are studied in chronic diseases ===== | ||
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{{tag> | {{tag> | ||
+ | < | ||
===== Notes and comments ===== | ===== Notes and comments ===== | ||
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< | < | ||
- | http:// | + | https:// |
</ | </ | ||
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- | ===== References ===== | + | ===== References =====</ |