Identification of disease causing agent is a very important part clinical practice. The success of any treatment depends on the proper identification of the infecting microorganism. There are various methods through which bacteria and other microorganisms can be identified in a lab and these include gram staining, molecular identification, sensitivity testing and finally through biochemical tests. Molecular methods such as gene sequencing is the most expensive but offers more accurate identification compared to the other mentioned methods.
In recent times there has been a marked rise in the use of “gene sequencing for identification of bacteria recovered from environmental and clinical specimen” (Raoult 3, par. 2). This has specifically been facilitated by the fact that all bacteria have at least one copy of the 16S rNA gene, which contains highly conserved as well as hypervariable nucleic acid sequences (Mollet 4). This makes t possible for these sequences to be amplified and thus be used for bacteria identification.
This study sought to identify the species of bacteria that were present in a clinical sample. This was a virtual experiment in which the species was identified using DNA analysis in which the sequence was generated, amplified virtually and then submitted for comparison at the NCBI blast database.
Materials and Methods
The clinical specimen was inoculated on suitable solid media and the bacteria left to grow. The bacterial DNA was then extracted by; first transferring a bacterial colony from the plate into a tube containing a digestive buffer to dissolve the cell wall (Howard Hughes Medical Institute, par. 4 ). The supernatant containing the DNA was then collected after centrifugation and transferred to the thermocyler. The bacterial DNA was amplified using the Polymerase chain Reaction and purified using compact microfilters before being sequenced (Howard Hughes Medical Institute, par. 5 ).
The amplified and purified product from the previous step was then transferred to the “sequencing brew” consisting of buffers, primers (a different one in each tube), DNA polymerases, nucleotides, and fluorescence-tagged terminators in suitable proportions (Howard Hughes Medical Institute, par. 5 ). The reason for this step is to facilitate the production of many DNA copies of variable length. The tubes were then transferred to the sequencer that performed gel electrophoresis on the DNA contained in each tube to determine the nucleotide sequence (Howard Hughes Medical Institute, par. 6 ). The actual sequence of the bacterium was generated by the computer.
The sequence was then submitted to the Nucleotide Blast sequence data base for matching and identification of the bacteria present in the clinical specimen.
The species of the bacteria in the clinical specimen was identified as Bartonella henselae by NCBI blast.
Bartonella henselae is a small gram-negative baccilus (Euzeby 23). The organism is tiny aerobic bacteria and at times it appears curved. Its “optimal growing conditions are blood (5%), moist atmosphere with a rich CO2 count, and temperature at 35º C”. The bacteria is often identified “Warthin-Starry silver staining” (Euzeby 25, par. 4). It can also be accurately identified using 16s rRNA sequencing (Mollet 5).
The microorganism is associated with a number of disease conditions such as “bacillary epithelioid angiomatosis, bacillary peliosis hepatic, and bacteremia; in addition it is now it has also been associated with cat scratch fever” (Euzeby 24, par. 3 ). The cat scratch disease is often transmitted through a bite or scratch from a kitten. Older cats are usually not infected with the disease as their immune systems are well developed to keep the microorganism at bay. Thus the organism exists in the cats’ bodies but does not result in any symptoms in the older cats. The “organism is often transferred through the saliva of the cat or from under the cats’ claws” (Euzeby 26, par. 5). In human beings, the symptoms are usually seen in individuals aged seventeen years and below, many of them below the age of 12 years. The symptoms include “fever, malaise, headache, and within 3 to 10 days the lymph nodes nearest to the site of infection begin to swell” (Euzeby 26, par. 7). In most cases the disease usually resolves itself although these can take several months. Antibiotics can be used to treat the disease and in extreme cases the lymph nodes are drained to remove the bacteria. “Children and the immune compromised are the most susceptible to B. Henselae” (Euzeby 28, par. 7).
Euzeby, Joseph. Bartonella henselae. Veterinary Dictionary of Bacterilogy. Missouri : Medical Information , 2000.
Howard Hughes Medical Institute. “The Virtual Bacterial ID lab.” 2007. Web.
Mollet, Collins. “rpoB sequence analysis as a novel basis for bacterial identification.” Mol microbiol (2002): 26:1005-1011.
NCBI. “Basic Local Alignment Search Tool.” 2010. Web.
Raoult, Drancourt. “Sequence-Based Identification of New Bacteria: a Proposition for Creation of an Orphan Bacterium Repository.” journal of clinical Microbiology (2005): 43(9): 4311-4315.