Evaluation by molecular tools of Brucella persistence in soil

Abstract number: 1733_1358

Koncan R., del Campo R., Bianchi S., Amendola A., Mazzariol A., Cantón R., Baquero F., Cornaglia G.

Background: Persistence in soil of pathogens such as Brucella has never been explored with the newly available molecular tools. The aim of this work was to investigate the biological risk in the waste pit of a vaccine-producing factory, in which a number of vials originally containing viable Brucella were being buried over 30 years and subsequently disrupted and spilt in the soil during the reclamation procedures.

Methods: The contents of the unbroken vials found in the waste pit were inoculated in Brain-Heart broth, and incubated for 7 days. Positive cultures were sub-cultivated onto Columbia agar plates, and a morphological analysis of the resulting colonies was performed. For bacterial identification, DNA was extracted from the different colonies and amplified by PCR with general primers based on conservative region of the 16S ribosomal. Positive amplicons were sequenced and compared by Clustal analysis with the Genbank data. Specific primers for Brucella spp. were also used with the DNA extracted from the vials' content.

Total soil DNA was extracted by the FastDNA® SPIN Kit for soil (Qbiogene, Carlsabd, CA, USA). Qualitative bacteriological analysis of the soil was performed by DGGE experiments, using clinical isolates of Brucella as the positive controls. Dot-blot assays were also performed with both the total DNA from soil and the bacteria grown on Brain-Heart.

Results: A total of sixty-nine unbroken vials were studied, six of them labelled as containing Brucella. Positive cultures were obtained in twenty-nine vials (42%), and they yielded in all cases contaminant environmental bacteria, namely Paenibacillus barengoltzii, Paenibacillus dendritiformis Bacillus thuringensis, Bacillus licheniformis, Bacillus benzoeorarus, and Ochrobactrum spp. Positive amplification with the specific primers for Brucella was not successful in any case. This result was also confirmed by the dot blot experiments. In the DGGE experiments with the DNA extracted from soil, no band corresponding to the Brucella size was detected.

Conclusion: Modern molecular tools might help to carry out a thorough reclamation of putative contaminated areas whilst minimising the potential biological risks. In particular, they allow assessment of possible soil contamination by highly infectious bacterial pathogens such as Brucella, avoiding risk exposure related to the classical culture techniques.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Location: ICC, Munich, Germany
Presentation type:
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