Automated ribotyping

Related Terms

Automated ribotyping, bacterial taxonomy, contamination identification, DNA fingerprinting, DNA probes, determinative bacteriology, gel electrophoresis, manual ribotyping, microbial contamination, molecular epidemiology, phenotyping, polymerase chain reaction, RFLP, rRNA operon, restriction endonuclease, restriction fragment length polymorphism, RiboBank, Riboprinter? system, ribosomal DNA, ribosomal ribonucleic acid, Svedberg units.

Background

Genes are considered the building blocks of life because they provide instructions for all cells in the body. Genes, which are located inside of cells, control an organism's development and functions by instructing cells to make new molecules (usually proteins).
DNA is a long thread-like molecule made up of large numbers of nucleotide bases. The sequence of nucleotide bases in DNA serves as the carrier of genetic (hereditary) information. Nucleotides are molecules composed of a nitrogen-containing base, a 5-carbon sugar, and one or more phosphate groups. Long strands of nucleotides form nucleic acids.
RNA (ribonucleic acid) is a polymeric constituent of all living cells and many viruses. The structure and base sequence of RNA are determinants of protein synthesis and the transmission of genetic information.
Ribotyping is a highly sensitive and precise method for the identification, classification, observation, and tracking of various bacteria from the sample of interest. This helps to distinguish organisms within the same species, which might help in the identification of the disease-causing organism and finding an appropriate treatment for it. It can also determine if an infection is caused by a single strain or by multiple strains. In an outbreak of an infection in a large population, ribotyping can detect the source of infection, identify its mode of transmission, and monitor the distribution and occurrence of a particular strain of bacteria. In clinical studies, ribotyping can identify the role of an organism in the occurrence of the disease and monitor the treatment regimens.
Ribotyping involves detecting and identifying the different species and/or strains (subspecies) of microbial organisms. This is achieved by obtaining a genetic fingerprint of the genome that codes for ribosomal ribonucleic acid (rRNA) using restriction fragment length polymorphism (RFLP) analysis. RFLP is a laboratory technique in which organisms may be differentiated by analysis of patterns derived from cleavage (cutting) of their DNA. Ribosomal RNA (rRNA) is a molecular component of ribosomes, which are tiny cell organs made of complexes of RNA and protein.

Methods

Ribotyping is a highly sensitive and precise method used to identify, classify, observe, and track various bacteria from the sample of interest. Ribotyping may be done either manually or automatically.
Manual method: The initial step in manual ribotyping involves the extraction of genomic DNA from the bacterial colony or sample of interest (food, water, feces). The extracted DNA is then cut (cleaved) into smaller pieces by enzymes called restriction endonucleases. These enzymes cleave the DNA sequences at specific sites called recognition restriction sites, which results in characteristic fragments of DNA of different lengths and strand orientation. The fragmented DNA sequences are of different lengths and differ among species, which assists in identifying the organism. This is the key principle of restriction fragment length polymorphism (RFLP).
The fragmented DNA sequences are put on gel electrophoresis, a process that uses electricity to separate the DNA fragments based on their size and electric charges as they migrate through a gel matrix to form distinct bands.
The separated fragments of DNA are then transferred to a membrane matrix (southern blot transfer) and are immobilized or fixed to the membrane. The immobilized DNA fragments are paired with complementary sequences of DNA called probes in a process known as hybridization. The probes have specific sequences of nucleotides that are complementary to specific DNA fragments to which it gets hybridized. These special probes are tagged with either a radioactive dye or an enzyme to facilitate easier detection.
The DNA fragments hybridized with the probes give out distinct band patterns when visualized with autoradiography and recorded. This is called genetic fingerprinting. Autoradiography is a technique in which the DNA sequences tagged with radioactive molecules are exposed to X-rays and visualized.
The band patterns are compared with a database to identify the strain of the organism. The recognition of similarities and differences among the various samples becomes easier with the addition of more strains to the database. Hence, storage of the pattern helps in early and easy identification of the organism.
The conventional, manual method for ribotyping has limited usage as it is time consuming and labor-intensive, requiring sufficient experience and expertise. In addition, the lack of standardization and subjective interpretation leads to a lot of variation among laboratories and technicians, which decreases the reliability of the test. However, smaller fragments can be separated with greater resolution by manual methods than by automated processes. This may be due to the larger gel sizes, resulting in greater band separation. The manual ribotyping takes about 10-12 days for total processing of the samples.
PCR (polymerase chain reaction) is an enzymatic method for the repeated copying of the two strands of DNA of a particular gene sequence. It is widely used to amplify minute quantities of biologic material so as to provide adequate specimens for laboratory study. It may be used in combination with ribotyping, which reduces the time required to perform the tests. It also eliminates the problem of insufficiently isolated DNA. For example, in certain slow-growing bacteria that contain very few copies of the rRNA genes, PCR can amplify very small amounts of the genetic material to the levels required for RFLP analysis within a short period of time.
Automated: Automated ribotyping involves the use of systems such as the Riboprinter? system from DuPont Qualicon, Wilmington, Delaware. It eliminates manual intervention and provides standardized and consistent results within one day (eight hours). It also aids in matching the genetic snapshot of organisms with databases, thereby helping in recognition of the contamination with ease, speed, and precision. Even in rare cases, when the identification of the organism is not possible, it still helps in finding the source of contamination, which may facilitate the initiation of action against the organism. The electronic fingerprints obtained can be compared with other riboprinters and then incorporated into a common database.
There are several advantages of automated ribotyping. It is a very easily reproducible and standardized method for classification of organisms. Hence, this method is useful as a rapid screening method for analyzing genetic relationships among isolates and for identifying strains of particular regional relevance. Also, remote identification is possible through the Internet as electronic databases of important ribotypes may be constructed using this method. This may facilitate the tracking of the organisms from anywhere in the world. This technique is not affected by bacterial culture conditions or growth stages of the organism; therefore, it produces accurate and reliable results.

Research

General: Ribotyping technique has been used in various areas to identify disease-causing organisms. The information generated by ribotyping is stored in libraries for future reference because it helps in the early detection of disease-causing organisms. The new ribotypes obtained can be compared to other known ribotypes in the library, thereby assisting in the recognition of the organism with ease, speed, and precision. The recognition of similarities and differences among the various isolates becomes easier with the addition of more strains to the database. Currently more than 600 patterns are in the database, and this database is continuously updated. Databases for Listeria (80 pattern types), Salmonella (97 pattern types), Escherichia (65 pattern types), and Staphylococcus (252 pattern types) have been established. These databases continue to grow as more bacteria are ribotyped.
Researchers, diagnostic investigators, and epidemiologists may use the database for various reasons. Investigators may use the database to identify specific disease-causing organisms, which may help treat individuals affected by this pathogen. Epidemiologists, who study the frequency and distribution of diseases within the human population and environment, may use the database to identify the commonly occurring pathogens in the environment. This may help in developing strategies for the management of disease outbreaks due to particular strains of organism. These databases may be accessed through the Internet. Certain organizations maintain databases for specific organisms. For instance, the World Health Organization has a ribotype database for Corynebacterium diphtheriae. To provide a platform for online comparison of bacterial fingerprints, RiboBank was created and is maintained under the GENE project (Genetic Epidemiology Network for Europe) with support from the European Union Fifth Framework Programme. This facilitates the comparison of the ribotypes from any part of the world.
Implant-associated infections: Implant is a medical device that is used to replace a missing part of the body. However, implants are associated with increased risk of infection. Staphylococcus epidermis is a major pathogen, or disease-causing agent, that causes implant-associated infections. Several microorganisms such as Staphylococcus epidermis may enter the surgical wound, adhere to the implant, and cause infection. By using ribotyping technique, researchers have identified different bacterial strains responsible for causing the infection. This also helps researchers to understand the development of multiresistant strains, bacteria that are resistant to multiple antibiotics, thereby assists in finding a suitable treatment against the organisms.
Genetic relatedness: Ribotyping has also been used to study the gene responsible for the development of multidrug-resistant Acinetobacter baumannii strains. Acinetobacter baumannii bacteria commonly cause infections in individuals who have compromised immune systems, such as those with AIDS. Further research is required to determine whether the multi-drug resistant strains from different geographic regions are genetically similar. This may assist in developing treatments that target a particular disease-causing gene.
Isolation from surface water: Studies have been conducted to identify enterococcal species in surface water using automated ribotyping. Ribotyping may be an effective tool for the identification of different bacterial strains. Because surface water consists of many organisms, this method has the potential to identify the exact source of contamination in surface water, thereby helping to develop ways to remove water contamination. Enterococci are the bacteria usually found in the intestine of humans and animals. However, they may cause certain serious infections such as infections acquired as a result of treatment in a hospital.

Implications

General: Ribotyping may be used to accurately identify or differentiate species and/or strains (subspecies) of microbial organisms and thus helps to find the cause of infection. This information may help identify ways of preventing or treating the infection or disease. Ribotyping has widespread applications as explained below.
Animal farms: Consumption of contaminated feed causes a number of diseases in animal farms that may lead to food-borne outbreaks such as listeriosis, which is caused by eating food contaminated with Listeria monocytogenes. Ribotyping may provide useful information by identifying the contaminated feed source and how the disease-causing bacteria are being spread. Ribotyping can thereby assist in the elimination of the contaminated source, reduce the food-borne outbreaks, and help maintain food safety in the animal farms.
Nosocomial infection: Patients may acquire infections in hospitals from a hospital source (nosocomial) such as medical equipment or from another patient. Ribotyping helps identify the source and route of infections so that individuals may take precautions to prevent further infections. Because ribotyping can identify the disease-causing organism, physicians can prescribe the appropriate medicine, which facilitates quicker recovery in patients.
Environmental pollution: Bacterial contamination impairs water quality due to human activity. Both human and animal fecal pollution poses a major health hazard due to the disease-causing bacteria. Ribotyping may be used to identify sources of potential and actual disease-causing bacteria, thereby preventing the outbreak of several illnesses.
Food industry: Fermented foods are subjected to the action of microorganisms or enzymes, which causes modification in the foods. This process may make fermented foods more digestible, nutritious, safer, or tastier. The use of ribotyping for the detection and identification of the microbial strains and subtypes in the fermentation food industry helps ensure the safe fermentation of foods under controlled conditions. Hence, identification of the disease-causing strain using ribotyping may help in preventing infections.

Limitations

The manual method for ribotyping is time consuming, labor intensive, and not as discriminating as pulsed field gel electrophoresis (PFGE). However, the development of automated ribotyping systems has increased the usefulness of this technique for the analysis of a large number of isolates.
The interpretation of the subtyping (identification of different strains of the organism) data by ribotyping includes a subjective component and depends on the training of the technician.
The ability to distinguish organisms is limited in that the polymorphism of variations in DNA sequences is confined to a small number of bands (3-4). The identification of the different strains of the organisms, therefore, becomes difficult.
It may be difficult to distinguish between several ribopatterns, the distinct protein band patterns derived from ribotyping, because there are only slight differences in the size of the bands.

Future research

Molecular fingerprinting with ribotyping has helped to identify multiple routes of Salmonella contamination in seafood and has also established a genetic association among different groups of microorganisms based on their cell surface antigens. This helps in the identification of genetic variation in the different strains of Salmonella and their possible route of entry into the seafood. Identification helps to inhibit the disease-causing organism before contamination occurs, thereby preventing the infection.
Patients may acquire infection in hospitals either from a hospital source such as medical equipment or from another patient. Such infections caused by Clostridium difficile is a major cause for concern. Hence, identification of the disease-causing strains of the organism using ribotyping may help to prevent the disease. It may also make possible the development of drugs for the disease-causing organism. Researchers are conducting a study to examine the efficacy of the antibiotic oritavancin against Clostridium difficile for the treatment of such hospital infections.
With the help of ribotyping from the roots of certain teeth that have undergone root canal Researchers have identified a new bacterial strain, Endo-EH, which belongs to the Vagococcus species. Root canal treatment is a process in which the dentist treats the infected pulp blood or nerve supply of the tooth. Further research is needed to understand the mechanism underlying this disease-causing organism and possible treatment strategies.

Author information

This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).

Bibliography

Al-Ahmad A, Pelz K, Schirrmeister JF, et al. Characterization of the first oral vagococcus isolate from a root-filled tooth with periradicular lesions. Curr Microbiol. 2008 Jun 17.
Bouchet V, Huot H, Goldstein R. Molecular genetic basis of ribotyping. Clin Microbiol Rev. 2008 Apr;21(2):262-73.
Centers for Disease Control and Prevention. .
. .
Campoccia D, Montanaro L, von Eiff C, et al. Cluster analysis of ribotyping profiles of Staphylococcus epidermidis isolates recovered from foreign body-associated orthopedic infections. J Biomed Mater Res A. 2008 May 21.
Clark CG, Kruk TM, Bryden L, et al. Subtyping of Salmonella enterica serotype enteritidis strains by manual and automated PstI-SphI ribotyping. J Clin Microbiol. 2003 Jan;41(1):27-33.
D?az Ruiz G, Wacher Rodarte C. [Methods for the study of microbial communities in fermented foods] Rev Latinoam Microbiol. 2003 Jan-Jun;45(1-2):30-40.
Kumar R, Surendran PK, Thampuran N. Molecular Fingerprinting of Salmonella enterica subsp. enterica Typhimurium and Salmonella enterica subsp. enterica Derby Isolated from Tropical Seafood in South India. Mol Biotechnol. 2008 May 15.
Nandi S, Ganguly NK, Kumar R, et al. Genotyping of group A streptococcus by various molecular methods. Indian J Med Res. 2008 Jan;127(1):71-7.
Natural Standard Research Collaboration: The Authority on Integrative Medicine. .
O'Connor R, Baines SD, Freeman J, et al. In vitro susceptibility of genotypically distinct and clonal Clostridium difficile strains to oritavancin. 2008 Jul 7.
Okatani TA, Ishikawa M, Yoshida S, et al. Automated ribotyping, a rapid typing method for analysis of Erysipelothrix spp. strains. J Vet Med Sci. 2004 Jun;66(6):729-33.
Svec P, Sedl?cek I. Characterization of Lactococcus lactis subsp. lactis isolated from surface waters. Folia Microbiol (Praha). 2008;53(1):53-6.
United States Department of Agriculture (USDA). .