A Microbial Biorealm page on the genus Brucella melitensis
Higher order taxa
Class: Alpha Proteobacteria
Description and significance
Brucella sp. is a small, Gram-negative coccobacillus, which can be grown slowly in vitro, and causes brucellosis. Brucellosis can be described as a "zoonotic disease that causes systemic symptoms and can involve many organs and tissues." (10). This disease is not very common in the United States, but has plagued many countries where there is a lack of good standardized and effective public health systems. Countries currently high at risk include the Mediterrean Basin area, South and Central America, Eastern Europe, Asia, Africa, and the Middle East. The Brucella species acts as a pathogen that can induce "abortion and sterility in domestic mammals and chronic infections in humans known as Malta fever." (13). These infections could easily be contracted through "consumption of unpasteurized dairy products and occupational contact." (10). The Brucella species may also be used as agricultural, civilian, or military bioterrorism agents.
The genome of Brucella melitensis stain 16M was found to have two circular chromosomes in which 3,294,931 base pairs were distributed. Of those two chromosomes, 3918 ORFs (open reading frames) were predicted. It was also discovered that on both chromosomes, there resided genes that encoded for DNA replication, protein synthesis, core metabolism and cell-wall biosynthesis (all of which were considered "housekeeping genes"). (3).
After sequencing the genomes of Brucella sp., it was discovered that Brucella abortus, Brucella suis 1330, and Brucella melitensis 16M had a genetic content and gene organization that were remarkably similar. On the contrary, a number of insertion and deletion events were also identified in the genomes. The similarity of B. suis, B. melitensis and B. abortus was evident due to these insertion and deletion events, which led to the discovery of several fragments of unique sequences that were present in all three species. Therefore, further analysis of the genome sequence of Brucella abortus can give exquisite information about these bacteria. (7).
Cell structure and metabolism
Brucella is a Gram-negative coccobacilli pathogenic bacteria that adapts to an intracellular lifestyle, is non-spore-forming and is non-motile. These organisms are mainly aerobic but some may require an atmosphere containing about 5-10% of carbon dioxide. The growth of the Brucella species is slow, sometimes taking as long as 2-3 days and an enriched medium is needed for optimal growth at 37°C. Experiments suggested that in Brucella melitensis strains, the expression of a fatty tissue called O-polysaccharides (OPS) on the outer membrane of the bacterium controls whether the bacterium will look smooth or round. (5). The absence of these O-polysaccharide chains turns the organism into a rough variant. This layer is important in identifying whether a pattern of species-specific flagellar gene inactivations and flagellum gene clusters exist, because this would give a better understanding of host specificity and virulence. The need for these species to survive in a species-specific environment provides an explanation that the adaptation of the Brucella species requires an "intracellular life-style in a protected and more stable local environment or niche that provides a constant supply of nutrients." (2). Currently, little is known about their chromosomal exchange, and there is no evidence of plasmids or bacteriophages in these species.
Brucella melitensis mainly interacts with animals such as goat and sheep in domestic or wild animal reservoirs. Infectious food-borne diseases usually result in humans when contaminated or poorly pasteurized or unpasteurized milk and cheese products are consumed because of the ability for the organism to colonize in the udder of animals. These organisms thrive in the phagocytic cells of its host. However, the main sources of infection and the routes of contamination are variable because each of the Brucella species has distinctive factors controlling the presence or absence of a disease, which adds complexity when trying to identify interactions between the organism, its environment, and its host. Also, because of the rapidly changing environment in different aspects, whether it be social, cultural, or agricultural, new Brucella strains may emerge. (6).
Brucella melitensis is one of the six species that causes Brucellosis, which can be described as a fatal zoonotic disease that affects multiple body systems. B. melitensis was originally found as a pathogen that mainly affected goats and sheep, which caused a decrease in fertility, loss of young, and a decrease in milk production. However, there has been more recent cases where this species has also been a highly pathogenic cause of human brucellosis. Brucellosis is common in many parts of the world, but is rare in the United States. This disease is mainly caused by the transfer of bacteria from farm animals to humans, usually through unpasteurized, contaminated goat milk, in which the bacteria can localize itself intracellularly once inside a host. (9). People can become infected through ingestion, inhalation, or direct contact.
Symptoms in Human Brucellosis include:
- Back pain
- Headache (cephalgia)
- Muscle pain (myalgia)
- Weight loss
- Feeling of general discomfort or uneasiness (often one of the first indications of an infection or disease)
B. melitensis increases its survival and replication in phagocytic cells by minimizing the activation of the defense system of the host. This bacteria is also known for its macrophage infections. Macrophage function is responsible for many pathways important in apoptosis. Because apoptosis kills off infected cells to prevent the spread of infection, disruption in these pathways allows for pathogen survival. (9).
To eliminate the infection in hosts, hygiene, vaccine, and pasteurization of dairy products should be implemented. Hygienic precautions are especially important because contraction of Brucellosis may be induced by skin contact of infected materials. Vaccines in humans "have had limited efficacy and have been associated with serious medical reactions. Vaccines developed to prevent and control livestock infection are effective in reducing the incidence of human brucellosis." (11).
The virulence factors of B. melitensis are variable. The best known factors include the Type IV secretion system (responsible for signal delivery into host eukaryotic cells), flagella, and other regulatory elements.
Treatment of Brucellosis depends on the age of the patient and whether the patient is pregnant or not. Various treatments include the combination of antibiotics such as Rifampicin, Trimethoprim, Doxycycline, Gentamicin, and Ciprofloxacin.
Application to Biotechnology
There is currently no biotechnological use of this organism.
One of the most recent studies is involved with developing PCR assays for use in diagnosing human brucellosis. In this experiment, a sequence-specific hybridization probe detection on an instrument called the LightCycler was used on three assays, which were then evaluated and compared to the "16S-23S internal transcribed spacer region (ITS) and the genes encoding for omp25 and omp31." (8). This was to test the analytical sensitivity and specificity of real-time PCR assays. Whole blood and tissue specimen were used for this laboratory diagnosis because of the intracellular pathogenic characteristic of the Brucella sp. Results indicated that a positive amplification signal was achieved in all 28 Brucella for omp25, omp31 and ITS assays. Controls used all had negative signals. However, it appeared that the omp25 and omp31 assays had a poor performance compared to the ITS assay, which limited the detection of the Brucella sp. in tissue samples. The amount of human genomic DNA used is important because it may improve the sensitivity of these assays; however, sensitivity could also decrease if too much human genomic DNA is used because that increases the level of potential inhibitors. (8). The use of PCR diagnosis of human brucellosis remains to be determined.
Another study was conducted in which there was a development of a vaccine against brucellosis. A chimera with "the scaffold protein BLS decorated with 10 copies of a known protective epitope derived from an outer membrane protein of 31 kDa from Brucella sp." was engineered. (1). This recombinant protein subunit turned out to an effective vaccine against the Brucella sp., providing the best level of protection due to a strong B cell response in immunized mice. Results revealed that a strong immune response was elicited when antigens residing on microbial surfaces had an epitope spacing of 50–100 Å. Researchers concluded that the BLS protein on the Omp31 gene (BLSOmp31) "could be a useful candidate for the development of subunit vaccines against brucellosis since it elicits humoral, Th and CTL responses and protection against Brucella". (1).
Researchers also conducted random gene inactivations to test and identify important cellular functions involved in the virulence and survival of the Brucella species. It was discovered that these random gene inactivations depended on the heavy use of G/C base pairs with the transposon Tn5 integrated in these base pairs. A transposable element, Himar1, was also delivered into the B. melitensis genome. It was found that while "this concept is useful when attempting to restrict the number of mutants and to target functions or mechanisms that are specific for survival of the organism, it may overlook the significance of gene products that sustain the organism to survive and replicate within the professional phagocytic cells of the host." (12). Therefore, researchers concluded that using a random mutagenesis approach for identifying the virulence of Brucella did not yet fully give specifics on intracellular survival but remains a viable option.
1. Cassataro J., Pasquevich K.A., Estein S.M., Laplagne D.A., Velikovsky C.A., de la Barrera S., Bowden R., Fossati C.A., Giambartolomei G.H., Goldbaum F.A. 2007. "A recombinant subunit vaccine based on the insertion of 27 amino acids from Omp31 to the N-terminus of BLS induced a similar degree of protection against B. ovis than Rev.1 vaccination." Vaccine. Volume 25, Issue 22, 30 May 2007, Pages 4437-4446.
2. Chain P.S., Comerci D.J., Tolmasky M.E., Larimer F.W., Malfatti S.A., Verquez L.M., Aquero F., Land M.L., Ugalde R.A., Garcia E. 2005. "Whole-genome analyses of speciation events in pathogenic Brucellae". Infection and Immunity. 2005 December, 73(12): 8353-8361.
3. DelVecchio V., Kapatral P., Elzer G., Patra C.V., Mujer. 2002. "The genome of Brucella melitensis". Veterinary Microbiology. Volume 90, Issues 1-4, 20 December 2002, Pages 587-592.
4. Detilleux P.G., Deyoe B.L., Cheville N.F. 1990. "Penetration and intracellular growth of Brucella abortus in nonphagocytic cells in vitro". Infection and Immunity. Volume 8, No. 7, 1990 July; Pages 2320-2328.
5. Fernandez-Prada C.M., Zelazowska E.B., Bhattacharjee A.K., Nikolich M.P., Hoover D.L. 2006. "Identification of smooth and rough forms in cultures of Brucella melitensis strains by flow cytometry". Journal Immunology Methods. 2006 August 31 315(1-2):162-70.
6. Godfroid J., Cloeckaert A., Liautard J.P., Kohler S., Fretin D., Walravens K., Garin-Bastuji B., Letesson J.J. 2005. "From the discovery of the Malta fever's agent to the discovery of a marine mammal reservoir, brucellosis has continuously been a re-emerging zoonosis." Veterinary Research. 2005 May-June; 36(3): 313-26.
7. Halling S.M., Peterson-Burch, Bricker B.J., Zuerner R.L., Qing Z., Li L.L., Kapur V., Alt D.P., Olsen S.C. 2005. "Completion of the genome sequence of Brucella abortus and comparison to the highly similar genomes of Brucella melitensis and Brucella suis." Journal of Bacteriology. 2005 April; 187(8): 2715-2726.
8. Kattar M., Zalloua P.A., Samaha-Kfoury J., Shbaklo H., Kanj S.K., Khalife S., Deeb M. 2007. "Development and evaluation of real-time polymerase chain reaction assays on whole blood and paraffin-embedded tissues for rapid diagnosis of human brucellosis." Diagnostic Microbiology and Infectious Disease. 2007 May 25.
9. Rajashekara G., Eskra L., Mathison A., Petersen E., Yu Q., Harms J., Splitter G. 2006. "Brucella: functional genomics and host-pathogen interactions". Cambridge Journals. Volume 7, Issue 1-2, 2006 December.
10. Sakran W., Chazan B., Koren A. 2006. "Brucellosis: clinical presentation, diagnosis, complications, and therapeutic options." Harefuah. 2006 November; 145(11): 836-40, 860.
11. Sauret J.M., Vilissova N. 2002. "Human Brucellosis". Journal of the American Board of Family Medicine. Volume 15 No. 5, 2002 September-October.
12. Wu Q., Pei J., Turse C., Ficht T.A. 2006. "Mariner mutagenesis of Brucella melitensis reveals genes with previously uncharacterized roles in virulence and survival." BMC Microbiology. 2006 December; 6:102.
13. Zygmunt M.S., Hagius S.D., Walker J.V., Elzer P.H. 2006 "Identification of Brucella melitensis 16M genes required for bacterial survival in the caprine host." Microbes and Infection. Volume 8, Issues 14-15, 2006 November-December, Pages 2849-2954.
Edited by Kathleen Wong KMG