A nested PCR method for the detection of Bacillus anthracis in environmental samples collected from former tannery sites

Beyer, Wolfgang; Glöckner, Petra; Otto, Julia; Böhm, Reinhard

doi:10.1016/s0944-5013(11)80054-6

Cited by 60 publications

(48 citation statements)

References 13 publications

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“…Since comparisons of the sequence sizes to the fragment size data revealed minor size discrepancies (1 bp for vrrA, 3 bp for vrrC 1 , and a previously reported 3-bp shift in the pXO1 size [P. Keim, personal communication] [6]), MLVA-8 fragment analysis of 88 diverse genotypes (13) and two Chadian isolates (isolate ChadA1, group VII, and isolate ChadA8, group VIII) was performed in the Keim genetics laboratory (Northern Arizona University) to ensure the comparability and accuracy of the raw VNTR scores from the Chadian strains with reference to those from the genotypes published by Keim et al in 2000 (13) (Fig. 1).…”

Section: Fig 1 Dendrogram Based On Mlva Of Eight Markers Of Chadianmentioning

confidence: 99%

Antibiotic Susceptibility and Molecular Diversity of Bacillus anthracis Strains in Chad: Detection of a New Phylogenetic Subgroup

et al. 2006

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We genotyped 15 Bacillus anthracis isolates from Chad, Africa, using multiple-locus variable-number tandem repeat analysis and three additional direct-repeat markers. We identified two unique genotypes that represent a novel genetic lineage in the A cluster. Chadian isolates were susceptible to 11 antibiotics and free of 94 antibiotic resistance genes.Bacillus anthracis, the etiological agent of anthrax, is a sporulating bacterium causing disease primarily in herbivores in many countries of Southern Europe, South America, Asia, and Africa (22). In Chad, anthrax is hyperendemic in cattle (22), and human cases of intestinal and cutaneous anthrax are reported each year (World Anthrax Data Site [http://www .vetmed.lsu.edu/whocc/]). However, studies of the genetic diversity and antibiotic susceptibilities of B. anthracis in this country have not been conducted. Here, we used a previously reported eight-marker multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) method (13) and three additional direct-repeat (DR) markers (15) to examine the genetic diversity of this pathogen in Chad. We also evaluated the Chadian B. anthracis isolates for antibiotic resistance by using physiological and genetic methods.Between 1996 and 2003, 174 blood samples were taken from the heart region or jugular vein of cattle carcasses in five southern prefectures in Chad. The samples were screened for B. anthracis by cultivation on tryptone soy agar plates containing 5% sheep blood. After incubation at 37°C for 48 h, cultures from 15 samples were identified as B. anthracis on the basis of colony morphology, absence of hemolysis, and susceptibility to B. anthracis-specific ␥ phage (2, 21). These cultures were sent to the Swiss Reference Centre (Bern, Switzerland) for further physiological and genetic testing.Template DNA for PCR was obtained by cell lysis followed by filtration (18). PCRs were performed using Taq DNA polymerase (Roche Diagnostics, Basel, Switzerland) and an annealing temperature of 55°C if not otherwise specified. We used PCR to screen the samples for the presence of B. anthracis chromosomal (Ba813) and plasmid (pXO1 and pXO2) sequences (see Table 1 for PCR targets and primer sequences). Eight VNTR markers, vrrA, vrrB 1 , vrrB 2 , vrrC 1 , vrrC 2 , CG3,

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Section: Fig 1 Dendrogram Based On Mlva Of Eight Markers Of Chadianmentioning

confidence: 99%

Antibiotic Susceptibility and Molecular Diversity of Bacillus anthracis Strains in Chad: Detection of a New Phylogenetic Subgroup

et al. 2006

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“…It is also associated with the waste from certain processing industries such as tanneries and can be found in the effluent from water processing plants (1). Cutaneous anthrax is associated with the handling of contaminated animal products and has a mortality rate of 5 to 20% when untreated.…”

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confidence: 99%

Bacillus Spore Inactivation Methods Affect Detection Assays

Dang

Heroux

Kearney

et al. 2001

Appl Environ Microbiol

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Detection of biological weapons is a primary concern in force protection, treaty verification, and safeguarding civilian populations against domestic terrorism. One great concern is the detection of Bacillus anthracis, the causative agent of anthrax. Assays for detection in the laboratory often employ inactivated preparations of spores or nonpathogenic simulants. This study uses several common biodetection platforms to detect B. anthracis spores that have been inactivated by two methods and compares those data to detection of spores that have not been inactivated. The data demonstrate that inactivation methods can affect the sensitivity of nucleic acid-and antibody-based assays for the detection of B. anthracis spores. These effects should be taken into consideration when comparing laboratory results to data collected and assayed during field deployment.Bacillus anthracis, the causative agent of anthrax, is an important zoonotic disease of domestic livestock. It is also associated with the waste from certain processing industries such as tanneries and can be found in the effluent from water processing plants (1). Cutaneous anthrax is associated with the handling of contaminated animal products and has a mortality rate of 5 to 20% when untreated. Inhalation of B. anthracis spores causes pneumonic anthrax that has a mortality rate approaching 100%. Antibiotic treatment is largely unsuccessful after the appearance of symptoms (11). From a military standpoint, B. anthracis has been described as the ultimate biological weapon because of its virulence and persistence on a battlefield when it is disseminated as a desiccated spore (13). Endospores are dormant forms of bacteria that are stable for great lengths of time and are resistant to inactivation from radiation and heat. Bacillus spores are so resistant and hardy that they have been revived from the abdominal cavity of an extinct bee entombed within Dominican amber 25 to 40 million years ago (2) and isolated from a brine inclusion dated at 250 million years old (14).Driven by the desire to develop and optimize detection devices to monitor and track the spores of B. anthracis, researchers face the need to handle moderate amounts of spore antigens. However, biosafety containment and occupational exposure are of great concern and necessitate inactivating the spores by gamma irradiation with a cobalt source or by thermal treatment such as autoclaving. B. anthracis has two major virulence factors encoded by plasmids pX01 (77 kb) and pX02 (95 kb). The plasmid pX01 codes for the lethal factor, the edema factor, and protective antigen that together form a tripartite protein exotoxin (4,7,9,12). Plasmid pX02 codes for the spore capsule (3). Laboratory detection assays often use inactivated spore preparations or nonpathogenic simulants to conduct development and testing of biosensors (6). The optimization of biodetection assays can necessitate handling moderate quantities of dangerous pathogens. Federal regulations limit the movement of pathogens and regulate safety controls...

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“…The nested PCR carried out with degenerate primers was also tested with nifH gene DNA fragments This method provided nifH gene fragments from pure cultures of 23 reference strains representing 14 Proteobacterial genera and two genera of Gram positive bacteria [24,25]. Using this technique, bands were found only at the control strain and at the samples from coniferous and beech vegetation zones.…”

Section: Resultsmentioning

confidence: 99%

Molecular identification of diazotroph microbial consortia in mountain soil

et al. 2010

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Nitrogen fixing microbial consortia from soil samples taken from five altitudinal vegetation zones (alpine, subalpine, coniferous, beech, Maleia flood plain) of Parâng Massif, Romania, were isolated and identified. Molecular characterisation of nitrogen fixing consortia was carried out by PCR and nested PCR with 7 primer sets specific to nifH genes. All nifH genes are specific to nitrogen fixation and are found within phylogenetically related organisms which have the nitrogenase enzyme complex. These molecular studies allowed the assessment of nifH gene diversity within these nitrogen fixing microbial consortia from different type of soils. At high altitude, a consortium of nitrogen fixing bacteria dominated by Azotobacter chroococcum and Azospirillum brasilense was found. Clostridium, Rhizobiales, Herbaspirillum, Frankia species were also found in different rations depending on the altitudinal vegetation zone.

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A nested PCR method for the detection of Bacillus anthracis in environmental samples collected from former tannery sites

Cited by 60 publications

References 13 publications

Antibiotic Susceptibility and Molecular Diversity of Bacillus anthracis Strains in Chad: Detection of a New Phylogenetic Subgroup

Antibiotic Susceptibility and Molecular Diversity of Bacillus anthracis Strains in Chad: Detection of a New Phylogenetic Subgroup

Bacillus Spore Inactivation Methods Affect Detection Assays

Molecular identification of diazotroph microbial consortia in mountain soil

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