Li et al. BMC Microbiology (2015) 15:77 DOI 10.1186/s12866-015-0414-8 RESEARCH ARTICLE Open Access Genetic characterization of Bacillus anthracis in Guizhou Province, Southwest of China Shijun Li1*†, Qing Ma1†, Hong Chen2, Dingming Wang1, Ying Liu1, Xiaoyu Wei1, Lv You1, Guanghai Yao1, Kecheng Tian1 and Guangpeng Tang1 Abstract Background: Bacillus (B.) anthracis is the pathogen that causes fatal anthrax. Guizhou Province is an old foci of anthrax in the southwest of China. Human anthrax has also been frequently reported in Guizhou in recent year. However, there is limited information on the genetic background of local B. anthracis isolates in Guizhou Province. Strain-specific detection of this bacterium using molecular approaches has enhanced our knowledge of microbial genetics. In the present study, we employed Multiple Locus Variable Number Tandem Repeats (VNTR) Analysis (MLVA) assay to analyze the genetic characteristics of B. anthracis strains isolated in Guizhou Province and their relationships to worldwide distributed isolates. Results: A total of 32 isolates of B. anthracis from soil, human, cattle, dog and water of different anthrax epidemics in Guizhou Province from 2006 to 2011 were confirmed with phage lysis test, penicillin inhibition test and PCR. MLVA-8 discriminated them into 28 unique MLVA types (MT G1 - G28), which were novel MTs compared with the previous reports. Cluster tree based on 32 isolates from Guizhou Province and 76 worldwide distributed isolates (30 MTs) showed they were divided into three clusters, designated A, B and C. All the 32 isolates were distributed in cluster A, which were further grouped into A1, A2, A3 and A4 sub-clusters. 32 isolates from Guizhou Province were closely grouped in each of the sub-clusters, respectively. Minimum Spanning Tree (MST) based on the MLVA data showed that the 28 MLVA profiles of isolates from Guizhou Province and 30 MLVA profiles of worldwide distributed isolates formed three clonal complexes (CCs) and ten singletons. Conclusions: 28 novel MTs of B. anthracis from Guizhou were revealed and their relationships to worldwide isolates were showed. The results will provide important information for prevention of anthrax and also enhances our understanding of genetic characteristics of B. anthracis in China. Keywords: Anthrax, Bacillus anthracis, MLVA, Genetic characterization Background handling infected animals or carcasses of animals that Anthrax is an often fatal bacterial infection that occurs have died of the disease, or meat, skins, hair, bones, etc. when Bacillus anthracis endospores enter the body from such animals [4]. Anthrax infection in humans oc- through abrasions in the skin or by inhalation or inges- curs by three major routes, the skin, the respiratory tion [1,2]. It is a zoonosis to which most mammals, es- tract or the gastro-intestinal tract, generating three dif- pecially grazing herbivores, are considered susceptible ferent primary forms of the disease, the cutaneous, the [2]. While anthrax currently affects mostly livestock and inhalational and the gastro-intestinal forms [2]. Cutane- wildlife around the world, it can and does kill humans ous anthrax, the most common form, occurs as a result [3]. Humans almost invariably contract anthrax from of contamination of skin with the bacterial spores, due to its mechanical abrasion or damage caused by insect * Correspondence: [email protected] bites [5]. Besides, the great current interest in anthrax is †Equal contributors due to its potential as a bioterrorism and biowarfare 1 Institute of communicable disease control and prevention, Guizhou agent [6,7]. Provincial Centre for Disease Control and Prevention, 101 Bageyan Road, Guiyang 550004, Guizhou, People’s Republic of China Ancient Chinese medical books suggest that an anthrax- Full list of author information is available at the end of the article like disease has been present in China for more than © 2015 Li et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li et al. BMC Microbiology (2015) 15:77 Page 2 of 8 5,000 years and the epidemiology and symptoms of an- Cellular debris was removed by centrifugation at 15,000 g thrax had been described [8]. An anthrax surveillance and for 5 min. The supernatant, containing DNA, was used as control project reported 72 outbreaks and 8,988 human the template for PCR amplification. cases in ten provinces (Sichuan, Tibet, Inner Mongolia, Xinjiang, Qinghai, Gansu, Guangxi, Guizhou, Yunnan and Hunan) in China in 1990–1994, which indicates a long MLVA experiments history of anthrax epidemics in China [8]. More recent For the MLVA test, we utilized primers that flank the eight reports showed the sporadic epidemics or outbreaks of an- VNTR regions (vrrA, vrrB1, vrrB2, vrrC1, vrrC2, CG3, thrax in many provinces of China, such as, Mongolia, pXO1, pXO2) as described by previous study [3,15]. Am- Liaoning and Jiangsu [9-11]. plifications were performed in 50 μl total volumes of PCR Guizhou Province, with nearly 50 million people, is an reaction system contained approximately 25 μl of PreMix old foci of anthrax in the southwest of China. For ex- Taq (TaKaRa, Otsu, Japan), 2 μl of forward and reverse ample, a total of 17,975 cases of anthrax in Guizhou primers with concentrations of 10 pmol/μl, 2 μl of DNA, Province from 1957 to 1999 had been reported, and the 19 μl of deionized water, respectively. Amplification was epidemics covered all of the nine prefectures, including performed on an Biometra TProfession thermocycler Guiyang, Qianxinan, Qiandongnan, Qiannan, Anshun, (Biometra, Goettingen, Germany) using amplification Bijie, Liupanshui, Tongren and Zunyi, in Guizhou parameters included an initial denaturation at 94°C for Province [12]. Human anthrax has also been frequently 5 minutes, followed by 34 cycles of 94°C for 20 seconds, reported in Guizhou in recent year. For instance, there 60°C for 20 seconds, 65°C for 20 seconds, then 65°C for were 32 outbreaks of anthrax occurred in Guzhou 5 minutes. PCR products were detected by electrophor- Province [13]. An more recent outbreak of cutaneous esis of 1 μl of each reaction on a 1.2% agarose gel for anthrax including seven cases has been reported in a 30 min at 100 V, and were sequenced by ABI PRISM village of Wangmo County in Guizhou Province in 2010 377 DNA sequencer. The full length of each locus were [14]. However, there is limited information on the genetic obtained by assembling of forward and reverse sequences background of local B. anthracis isolates in Guizhou using ContigExpress (Invitrogen Life Science, Carlsbad, Province. In this study, we applied MLVA-8 to analyze CA), on which the forward sequence and the reverse com- the genetic characteristics of B. anthracis strains iso- plement sequence of reverse primer were marked to de- lated from Guizhou Province. termine the actual length of amplification. The actual length were calibrated to the nearest sizes for the corre- sponding VNTR in the report of Keim et al. [3]. Methods Bacterial isolates and confirmation A total of 32 of isolates were used for analysis in this study Data analysis (Table 1). All of these isolates were come from the local Each unique profile based on allelic combination was des- epidemics of cutaneous anthrax in Guizhou Province from ignated a unique MLVA type (MT). The Bionumerics soft- 2006–2011, which included isolates from human and ware package, version 4.0 (Applied Maths, Belgium) was epidemiologic related animal (dog and cattle), soil and used for UPGMA clustering analysis based on categorical water in each epidemic. The localities covered Prefecture coefficient, and minimum spanning tree algorithm was Qianxinan, Qiannan, Anshun, Tongren, and Bijie. All the used to construct a minimum spanning tree (MST) to de- isolates used in this study were confirmed with PCR kit termine phylogenetic pattern. The MLVA data of isolates (Takara, Janpan) and conventional methods including of other provinces in China and worldwide isolates were phage lysis test, penicillin inhibition test for anthrax come from B. anthracis data base (http://mlva.u-psud.fr/ diagnosis. MLVAnet/spip.php?article123). DNA templates preparation DNA templates for PCR were prepared directly from Ethic statements and biosafety containment bacterial colonies by the boiling method [3]. Briefly, B. All experiments involving B. anthracis including isolates anthracis cells were streaked onto blood agar plates and from human, animal and environments were performed then incubated at 37°C overnight. A single colony from according to the General Requirements for Bio-safety (GB each plate was transferred into a microcentrifuge tube 19489–2008) and approved by the Biosafety Committee of containing 100 μl of DNA extraction liquid (Liferiver, Guizhou Provincial Centre for Disease Control and Pre- China). The colony was resuspended by vortexing or re- vention, and were also approved by the Ethics Review petitive pipetting. The cellular suspension was heated to Committee of Guizhou Provincial Centre for Disease 95°C for 20 min and then cooled to room temperature. Control and Prevention. Li et al. BMC Microbiology