Isolation & characterization of Brucella melitensis isolated from patients suspected for human brucellosis in India

Brucellosis, a zoonosis caused by the genus Brucella is responsible for considerable human morbidity and causes enormous economic losses due to abortion and infertility in livestock1. Brucellosis in humans occurs as an acute, sub-acute or chronic illness which develops from direct or indirect contact with animals or by consumption of unpasteurized milk, milk products or meat of infected animals. Six classical species of Brucella that differ in their host preference are B. abortus which preferentially infects cattle; B. melitensis infects sheep and goats; B. suis infects pigs; B. canis the dog; B. ovis, sheep and goats; and B. neotomae infects the desert wood rat2. Later on, new species isolated from marine mammals namely B. ceti and B. pinnipedialis have been described3. Two more species namely, B. microti (common vole; seals)456 and B. inopinata from a human breast implant infection were also isolated and characterized as a new member of genus Brucella7. A novel Brucella strain originating from wild native rodent species in North Queensland, Australia, and a Brucella isolate from two cases of stillbirth in non-human primates have also been described but not yet included in the genus Brucella28.

The low infection dose and easy transmission to humans via aerosols make Brucella species a potential biological warfare agent9,10. In the absence of reliable, reproducible and validated molecular tests for confirmation, culture isolation is still considered as gold standard for brucellosis. A combination of biochemical, serological and molecular methods is needed for reliable identification and biotyping of Brucella. Biochemical tests like CO₂ requirement, H₂S production, dye sensitivity, urease, oxidase, catalase tests are also used for identification of Brucella species11. In addition, biotyping method based on agglutination with mono-specific A and M antiserum is also helpful in characterization of isolates11. Several serological tests like serum tube agglutination test (STAT), Rose Bengal plate test (RBPT), 2-mercaptoethanol (2ME), complement fixation test (CFT) and enzyme linked immunosorbent assay (ELISA) are used for the diagnosis of human brucellosis12. The major disadvantages with these serological tests include the presence of background antibodies from endemic areas and no differentiation of acute cases from previous exposure. Numerous PCR based assays have been developed and evaluated ranging from identification of the genus Brucella (Genus-Specific PCR assay) to single unique genetic locus that is highly conserved in Brucella species (e.g. 43 kDa omp, BCSP31, IS6501/711 or 16S rRNA genes)131415. In the present study an attempt was made to characterize the isolates of Brucella obtained from patients suspected for brucellosis using biochemical, serological and molecular methods.

Material & Methods

This study was conducted in the Microbiology Division, Defence Research and Development Establishment (DRDE), Gwalior, on the isolates collected at the department of Microbiology, BLDEU's Shri B.M. Patil Medical College, Bijapur, Karnataka, India. A total of 496 febrile patients from endemic region of Bijapur were selected consecutively during the period 2010-2012. Of these, 102 high risk patients (5-55 yr) were identified based on the history given by them that included animal exposure, raw milk ingestion and clinical symptoms such as fever, headache, nausea, vomiting, backache, joint pains. Blood samples (15 ml) were collected aseptically from these 102 patients. Five ml of blood in duplicate was immediately used for blood culture and the remaining was saved for separation of serum for serological testing of brucellosis. All serological tests and isolation were performed at Shri B.M. Patil Medical College, Bijapur, Karnataka, and serum samples and culture were sent to DRDE for further biochemical and molecular confirmation of isolates.

Serological tests with patients’ serum samples: The serum sample collected was subjected to conventional serological tests (STAT, 2ME and RBPT) to check for the presence of anti-Brucella antibodies. In STAT, patient serum was serial diluted from 1:10 to 1:1280 with 0.5 per cent phenol saline, Brucella antigen (Division of Biological products, IVRI, Izatnagar, India) was added and incubated at 37°C for 24 h. The maximum dilution that exhibited 50 per cent agglutination was considered as the end point of serum activity and recorded as the titre of antibodies present in the individual against Brucella. RBPT and 2ME tests were done as per the protocol described earlier161718.

Isolation of bacterial culture: The isolation of bacteria from patient's blood was performed using conventional Castaneda culture technique19. Five ml of blood was inoculated aseptically into 50 ml broth phase of Castaneda biphasic medium consisting of brain heart infusion (BHI) agar and broth (Hi-media, Mumbai) in duplicate. The contents were mixed well and tapped daily to allow the blood-broth mixture to flow over the agar slant. The bottles were then incubated in upright position in incubator at 37°C, one with and another without CO₂ atmosphere and examined daily for growth19. If no colonies were observed, the bottles were tilted and re-incubated and the cycle was repeated for a minimum of 30 days. If still no growth was observed after 30 days, the samples were considered negative and discarded.

Microbiological and biochemical identification of isolates: All isolates were grown on Brucella selective medium (Hi-media, Mumbai) and studied for colony morphology, culture characteristics like smooth or rough colony by examination under inverted microscope. The cultures were subjected to Gram staining. The isolates were characterized by biochemical tests such as oxidase, catalase, urease, CO₂ requirement, H₂S production, methyl red, indole and sensitivity to thionin, basic fuchsin and safranin dyes20. B. melitensis biotype I expresses M antigen21; therefore, all isolates were also reacted with monospecific A and M agglutinating antiserum (Veterinary laboratories agency, Addlestone, UK) for culture identification.

The standard bacterial strains of Brucella species used in this study for comparison were B. melitensis 16M and B. abortus S19. Both these species were maintained in 30 per cent glycerol stocks at -80°C as well as in liquid nitrogen.

DNA isolation: DNA of all the isolates and standard strains were isolated using conventional phenol: chloroform: isoamylalcohol method22. Purity and the concentration of DNA were checked spectrophotometrically at an absorbance 260/280 nm and visualized under UV on 0.8 per cent agarose gel electrophoresis.

Molecular characterization

PCR amplification of 16S rRNA and sequence analysis: PCR amplification of 16S rRNA gene of Brucella species was done in two fragments using the primers as described earlier23. The PCR reactions were standardized in 25 µl reaction mixture of 200 µM each dNTPs, 1.5 mM of MgCl₂, 10 pmol of each primers, 1 U of Taq polymerase and 50 ng of DNA in 1x PCR buffer. All the PCR reagents used in the study were procured from MBI, Fermentas, USA. The PCR cycling conditions were standardized as initial denaturation of 95°C for 4 min followed by 30 cycles of denaturation at 95°C for 50 sec, annealing at 58°C for 50 sec and extension of 72°C for 1 min and then final extension at 72°C for 10 min. The PCR products were run on 1.2 per cent agarose gel, stained with ethidium bromide and visualized under gel documentation system (Alpha innotech, USA). The PCR products were purified using QIAGEN gel extraction kit, USA and the purified PCR products were sequenced from both ends using forward and reverse primers in separate reaction by dideoxy chain termination method24 in ABI 3730 sequencer (Applied biosystem, USA). The alignment of DNA sequences was performed by using software DNA star 9.0 version (www.dnastar.com) along with the reference sequence of B. melitensis 16M (GenBank accession no. DQ845343). The BLAST analysis of the aligned sequences was also done to compare with other reported sequences of Brucella species25. The 16S rRNA sequence of 18 isolates were submitted to GenBank and assigned accession numbers JF939172 to JF939189.

PCR amplification of omp2a and restriction fragment length polymorphism (RFLP) analysis: The 1233 bp omp2a gene was amplified and PCR reaction was standardized using the primers and the protocol as described above for 16S rRNA23. The Brucella omp2a gene was PCR amplified, purified using QIAGEN gel extraction kit and digested with Hinf I, Alu I, Ban I and Pst I restriction enzymes. The restriction digestion reaction was standardized in 30 µl reaction mixture of 10 µl of purified omp2a PCR product, 3 µl of 10x buffer, and one unit of restriction enzyme (Fast digest, Fermentas). The reaction mixtures was incubated at 37°C for one h and digested products were electrophoresed in 2 per cent agarose gel, stained with ethidium bromide and visualized under gel UV in documentation system (Alpha innotech, USA).

PCR amplification of IS711 gene, sequence and RFLP analysis: The species level confirmation of the isolates was done by PCR amplification of the 733 bp B. melitensis-specific DNA fragment of IS711 repetitive genetic element. The amplification of 733 bp B. melitensis-specific DNA fragment was carried out using specific primers23. The PCR conditions were optimized as in the case of 16S rRNA except annealing at 68°C. The amplified products were run on 1.2 per cent agarose gel, stained with ethidium bromide and visualized under gel documentation system. Sequencing of IS711 gene was also performed as per protocol described above. The BLAST analysis of the final sequences obtained for each isolates was done and compared with other reported sequences of Brucella species in NCBI database (www.ncbi.nlm.nih.gov).

The 733 bp B. melitensis-specific IS711 gene was also used for PCR-RFLP analysis for confirmation of isolates as B. melitensis. The IS711 gene was PCR amplified and purified using QIAGEN gel extraction kit and digested with Hinf I and Tru I restriction enzymes (Table). The restriction digestion reaction was performed in a 30 μl reaction volume containing 10 μl purified IS711 PCR product, 3 μl of 10x buffer, and 1U of restriction enzyme (Fast digest, Fermentas). The reaction mixture was incubated at 37°C for 1 h and the digested products were electrophoresed in 2 per cent agarose gel stained with ethidium bromide and visualized under UV in gel documentation system (Alpha innotech, USA). The sequences of IS711 gene of all 18 isolates were submitted to GenBank and assigned accession numbers JF939142 to JF939150, JF939153 to JF939157 and JF939161 to JF939169. Phylogenetic analysis of isolates was done based on IS711 sequence, closest match of the deduced sequences of IS711 isolates was determined by BLAST search and a phylogenetic tree were constructed by neighbor-joining method26 using Mega software version 5 with 1000 bootstrap (www.megasoftware.net).

Table Restriction fragment length polymorphism analysis (www.neb.com)

Results

Isolation of Brucella and biotyping: Of the 102 samples subjected to blood culture, 18 isolates were obtained with an overall isolation rate of 17.64 per cent. These 18 cultures were initially identified as Brucella species based on Gram staining and colony morphology. The colonies grown on Brucella selective medium were round, convex, with smooth margin, translucent, honey-coloured, glistening and Gram-negative coccobacilli. These isolates were positive for catalase, oxidase, and urease tests and negative for methyl red, indole, CO₂ requirement and H₂S production. The biochemical tests profile of these 18 isolates was identical and matched with that of standard strain B. melitensis 16M. The isolates were positive with Brucella anti-M monospecific serum confirming that all 18 isolates belonged to B. melitensis biotype 1. The isolates were also able to grow in presence of dyes namely thionin, basic fuchsin and safranin.

Serological characterization: The serum samples of 18 patients who were positive for culture isolation also showed significant titre with STAT (≥160 IU), 2ME (≥80 IU) and were positive by RBPT, confirming the presence of anti-Brucella antibody in these patients.

Molecular characterization: The PCR results for 16S rRNA amplification showed amplification of 839 bp product with first primer set and 699 bp with second primer set. PCR amplification of Omp2a gene amplified 1233 bp products, and 733 bp for B. melitensis specific IS711 gene along with the reference strain of B. melitensis 16M. For further confirmation of isolates, PCR-RFLP of omp2a locus and IS711 was performed. Omp2a gene was digested with Pst I, Ban I and Hinf I restriction enzymes revealed similar restriction profiles in all isolates and also matched with B. melitensis 16M (Table). The restriction profile of all isolates did not match with B. abortus S19 reference strain. Similarly, the restriction pattern generated for IS711 gene when digested with Hinf I and Tru I restriction enzymes was similar for all isolates and matched with B. melitensis 16M reference strain (Table). Sequencing analysis of 16S rRNA and IS711 gene was done for 18 isolates using dideoxy chain termination and the obtained sequences were aligned and matched with B. melitensis 16M reference. The BLAST analysis of deduced sequences of 16S rRNA revealed 100 per cent similarity with Brucella species, whereas IS711 BLAST analysis revealed nearest match with B. melitensis species thereby confirming that all the isolates belonged to B. melitensis 16M (biotype1).

Phylogenetic analysis: The phylogenetic tree based on 733 bp sequence of IS711 repetitive genetic element for all isolates was constructed. IS711 gene sequencing and phylogenetic analysis revealed that all 18 isolates closely matched with B. melitensis 16M standard strain and were of clonal origin. One of the isolates from Israel also showed 100 per cent similarity to the isolates of present study in clade 1 (Figure). The isolates of B. melitensis from other countries are placed into different clades along with the other species of Brucella and showed dissimilarity with the present isolates (Figure).

Close

Figure

Neighbor joining tree based on IS711 gene sequences of B. melitensis isolates and related reference strains constructed with 1000 bootstrap using Mega software version 5. The bootstrap values are given at the nodes.

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Discussion

Among all the Brucella species, B. melitensis is mostly responsible for human brucellosis and prevalent in Southeast Asian countries including India27. Isolation and genotyping of the bacterial species is essential to know the exact incidence of a disease in a region and also to plan epidemiological studies, control and eradication programmes. The identification of isolates from culture is the gold standard in diagnosis of brucellosis both in humans and animals. Molecular confirmation by PCR utilizing different gene targets has become the most common approach for confirmation of human and animal isolates2829. In this study, we reported the isolation of B. melitensis from human patients suspected for human brucellosis and characterized the isolates by conventional biochemical and serological methods. The molecular confirmation was done by PCR, PCR-RFLP and gene sequencing analysis. Sequence analysis of 16S rRNA gene is extensively used for molecular detection or taxonomic analysis of different bacterial species; 16SrRNA gene sequence among Brucella species is significantly conserved and it has been reported that 16S rRNA gene sequencing is a reliable tool for rapid genus level identification of Brucella30. The omp2a and omp2b genes encoding 36 kDa outer membrane proteins (OMPs) of Brucellae are reported to be highly diverse among Brucella spp, biovars and strains31. The present study also showed that PCR-RFLP at omp2a locus was an important tool in identification and biotyping of Brucella species. The amplification of 733bp repetitive genetic element of IS711 and sequencing are highly suitable and practically applicable for molecular characterization of B. melitensis isolates from human patients. The successful isolation protocol followed by the conventional biochemical tests and confirmation by PCR, PCR-RFLP and sequence analysis of 16S rRNA and IS711 genes followed in this study could be a practical approach for not only disease diagnosis but also as a model for surveillance of human brucellosis in India.

In India, only a few studies3233 have addressed the importance of brucellosis as a public health problem. The disease may be overlooked and misdiagnosed because of the difficult diagnosis and lack of experience with the laboratory testing. It has also been estimated that the true incidence of brucellosis may be 25 times higher than the reported incidence due to misdiagnosis and underreporting31. Our results indicate that human brucellosis can be a common public health problem in many of the States of India. The disease is normally associated with persons who are in close contact with animals like shepherds and veterinarian and those who have the habit of consumption of raw milk of sheep and goat. More studies from different geographical regions of the country on isolation of this important bacterial species are needed. In addition, facilities and expertise required for isolation and characterization should be established in endemic areas so as to help in prompt diagnosis and treatment as well as to understand the actual incidence of human brucellosis in the country.