Sishi 1 CHEN, Mengna JIANG1, Yingying 1 SUN, Yifei HE, Zuoyong ZHOU, Fangjun CHENG, Shimei LAN, Zhangcheng LI

Çin'de Kanatlı Hayvanlardan İzole Edilen Pasteurella multocida Sışlarının Moleküler Karakterizasyonu ve tonB Geni Açısından Genetik Analizi

Pasteurella multocida (P. multocida) kuşlarda kanatlı kolerasına neden olur ve hastalıkta virulans faktörleri hala tam olarak anlaşılamamıştır. TonB proteini Pasteurella da dahil pek çok Gram-negatif bakteride yaygın olarak bulunan periplazmik bir protein olup membran dışına ferrik demir komplekslerini taşımakta görevlidir ve bakterilerde besin sağlamaya yardım etmek suretiyle önemli bir virulans faktörü olarak rol oynar. Bu çalışmada, 172 klinik solunum hastalıklı ördek ve kazdan toplam 23 P. multocida izolatı elde edilerek %13.4'lük izolasyon oranı sağlandı. P. multocida Serogrup A 22 vakadan izole edilirken (%95.7), serogrup F 1 vakadan izole edildi (%4.3). Tüm izolatlar 15 antibiyotiğe karşı duyarlılıklarına ve virulans faktör olarak 18 genin mevcudiyetine göre analiz edildi. Duyarlılık profili, P. multocida'nın tetrasiklin, sefalosporin, enrofloksasin ve aminoglikozidlere karşı duyarlı olduğunu gösterdi. Ancak, elde edilen suşların %78.3'ü penisiline ve %52.2'si sulfisoksazola karşı dayanaklıydı. PCR sonuçları çoğu kanatlı P. multocida suşlarında ptfA, fimA, tonB, fur, hgbA, hgbB, sodA, sodC, pmHAS, nanH, nanB, plpB ve ompH genlerinin olduğunu gösterdi. toxA, tadD, hsf-1, pfhA ve ompA gibi virulans genlerinin her biri suşların.

Molecular Characteristics of Pasteurella multocida Strains Isolated from Poultry in China and Genetic Analysis of Strains in Terms of the tonB Gene

Pasteurella multocida (P. multocida) causes fowl cholera in birds and the pathogenesis and virulence factors involved are still poorly understood. TonB protein is a periplasmic protein prevalent in a large number of Gram-negative bacteria, as in Pasteurella, which is believed to be responsible for the import of ferric iron complexes across the outer membrane and plays a role as an important virulence factor to help bacteria to obtain nutrients. In this study, a total of 23 isolates of P. multocida were obtained from 172 cases of clinical respiratory disease in duck and geese, giving an isolation rate of 13.4%. P. multocida Serogroup A was isolated from 22 cases (95.7%), whereas serogroup F was from 1 case (4.3%). All isolates were analyzed for their susceptibility to 15 antibiotics and the presence of 18 genes for virulence factors. The susceptibility profiles suggested that Tetracycline, Cephalosporin, Enrofloxacin, and Aminoglycosides were the drugs most likely to be active against P. multocida. However, 78.3% and 52.2% of poultry strains were resistant to Penicillin and Sulfisoxazole, respectively. PCR results showed that ptfA, fimA, tonB, fur, hgbA, hgbB, sodA, sodC, pmHAS, nanH, nanB, plpB and ompH genes occur in most poultry strains of P. multocida. The virulence genes such as toxA, tadD, hsf-1, pfhA and ompA were each present in <73.9% of strains. The tonB gene was detected in all 23 clinical strains of P. multocida which were recovered from different hosts and harboring different serotypes. Phylogenetic analysis indicated that these tonB genes of P. multocida from different hosts and harboring different serotypes have close genetic relationship with a high similarity. Multiple sequence alignment demonstrated that TonB protein of Gram-negative bacteria exists in multiple conserved sites, such as -xSSGx-, -YP-, -LD- and xA[A/V]Lx motif. These findings provide clinical data into the epidemiological and molecular characteristics of avian P. multocida isolates and provide a reference for the researches of drug target in P. multocida.

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Güler L, Gündüz K, Sarişahin AS: Capsular typing and antimicrobial susceptibility of Pasteurella multocida isolated from different hosts. Kafkas Univ Vet Fak Derg, 19 (5): 843-849, 2013. DOI: 10.9775/kvfd.2013.8936
Wilson BA, Ho M: Pasteurella multocida: From zoonosis to cellular microbiology. Clin Microbiol Rev, 26 (3): 631-655, 2013. DOI: 10.1128/ CMR.00024-13
Wilkie I W, Harper M, Boyce JD, Adler B: Pasteurella multocida: Diseases and pathogenesis. Curr Top Microbiol Immunol, 361, 1-22, 2012. DOI: 10.1007/82_2012_216
Carter GR: Studies on Pasteurella multocida. I. A hemagglutination test for the identification of serological types. Am J Vet Res, 16 (60): 481- , 1955.
Harper M, Cox AD, Adler B, Boyce JD: Pasteurella multocida lipopolysaccharide: The long and the short of it. Vet Microbiol, 153 (1): 115, 2011. DOI: 10.1016/j.vetmic.2011.05.022
Harper M, Boyce JD, Adler B: The key surface components of Pasteurella multocida: Capsule and lipopolysaccharide. Curr Top Microbiol Immunol, 361, 39-51, 2012. DOI: 10.1007/82_2012_202
Siegert P, Schmidt G, Papatheodorou P, Wieland T, Aktories K, Orth JH: Pasteurella multocida toxin prevents osteoblast differentiation by transactivation of the MAP-kinase cascade via the G? q/11-p63RhoGEF- RhoA Axis. Plos Pathog, 9 (5): e100338, 2013. DOI: 10.1371/journal. ppat.1003385
Schalk IJ, Guillon L: Fate of ferrisiderophores after import across bacterial outer membranes: Different iron release strategies are observed in the cytoplasm or periplasm depending on the siderophore pathways. Amino Acids 44 (5): 1267-1277, 2013. DOI: 10.1007/s00726-013-1468-2
Harper M, Boyce JD, Adler B: Pasteurella multocida pathogenesis: years after Pasteur. Fems Microbiol Lett, 265 (1): 1-10, 2006. DOI: 1111/j.1574-6968.2006.00442.x
Katoch S, Sharma M, Patil RD, Kumar S, Verma S: In vitro and in vivo pathogenicity studies of Pasteurella multocida strains harbouring different ompA. Vet Res Commun, 38 (3): 183-191, 2014. DOI: 10.1007/ s11259-014-9601-6
Hentze MW, Muckenthaler MU, Galy B, Camaschella C: Two to tango: Regulation of mammalian iron metabolism. Cell, 142 (1): 24-38, DOI: 10.1016/j.cell.2010.06.028
Schaible UE, Kaufmann SH: Iron and microbial infection. Nat Rev Microbiol, 2 (12): 946-953, 2004. DOI: 10.1038/nrmicro1046
Runyenjanecky LJ: Role and regulation of heme iron acquisition in gram-negative pathogens. Front Cell Infect Microbiol, 3 (3): 55, 2013. DOI: 10.3389/fcimb.2013.00055
Graham MR, Lo RY: Cloning and characterization of the exbB-exbD- tonB locus of Pasteurella haemolytica A1. Gene, 186 (2): 201-205, 1997. DOI: 10.1016/S0378-1119(96)00703-2
Nairz M, Schroll A, Sonnweber T, Weiss G: The struggle for iron-a metal at the host-pathogen interface. Cell Microbiol, 12 (12): 1691-1702, DOI: 10.1111/j.1462-5822.2010.01529.x
Wandersman C, Delepelaire P: Bacterial iron sources: From siderophores to hemophores. Annu Rev Microbiol, 58, 611-647, 2004. DOI: 1146/annurev.micro.58.030603.123811
Tong Y, Guo M: Bacterial heme-transport proteins and their heme- coordination modes. Arch Biochem Biophys, 481 (1): 1-15, 2009. DOI: 1016/j.abb.2008.10.013
Tullius M V, Harmston CA, Owens CP, Chim N, Morse RP, McMath LM, Horwitz MA: Discovery and characterization of a unique mycobacterial heme acquisition system. P Natl Acad Sci Usa, 108 (12): 5056, 2011. DOI: 10.1073/pnas.1009516108
Jarosik GP, Sanders JD, Cope LD, Muller-Eberhard U, Hansen EJ: A functional tonB gene is required for both utilization of heme and virulence expression by Haemophilus influenzae Type B. Infect Immun, (6): 2470-2477, 1994.
Nagai, S, Someno S, Yagihashi T: Differentiation of toxigenic from nontoxigenic isolates of Pasteurella multocida by PCR. J Clin Microbiol, (4): 1004-1010, 1994.
Townsend KM, Boyce JD, Chung JY, Frost AJ, Adler B: Genetic organization of Pasteurella multocida cap loci and development of a multiplex capsular PCR typing system. J Clin Microbiol, 39 (3): 924-929, DOI: 10.1128/JCM.39.3.924-929.2001
Ewers C, Lübke-Becker A, Bethe A, Kießling S, Filter M, Wieler LH: Virulence genotype of Pasteurella multocida strains isolated from different hosts with various disease status. Vet Microbiol, 114 (3): 304-317, DOI: 10.1016/j.vetmic.2005.12.012
Tang X, Zhao Z, Hu J, Wu B, Cai X, He Q, Chen H: Isolation, antimicrobial resistance, and virulence genes of Pasteurella multocida strains from swine in China. J Clin Microbiol, 47 (4): 951-958, 2009. DOI: 1128/JCM.02029-08
Khamesipour F, Momtaz H, Mamoreh MA: Occurrence of virulence factors and antimicrobial resistance in Pasteurella multocida strains isolated from slaughter cattle in Iran. Front Microbiol, 5, 536, 2014. DOI: 3389/fmicb.2014.00536
Clinical and Laboratory Standards Institute: Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Animals. Third edn., Approved Standard M31-A3. CLSI, Wayne, PA, USA, 2008.
Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Paterson DL: Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infec, (3): 268-281, 2012. DOI: 10.1111/j.1469-0691.2011.03570.x
Kumar S, Stecher G, Tamura K: MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol, 33 (7): 1874, 2016. DOI: 10.1093/molbev/msw054
Talavera G, Castresana J: Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst Biol, 56 (4): 564-577, 2007. DOI: 10.1080/
Darriba D, Taboada GL, Doallo R, Posada D: jModelTest 2: More models, new heuristics and parallel computing. Nat Methods, 9 (8): 772, DOI: 10.1038/nmeth.2109
Eddy, S R: Accelerated profile HMM searches. Plos Comput Biol, 7 (10): e1002195, 2011. DOI: 10.1371/journal.pcbi.1002195
Katoh K, Standley DM: MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol Biol Evol, 30 (4): 772-780, 2013. DOI: 10.1093/molbev/mst010
Yachdav G, Kloppmann E, Kajan L, Hecht M, Goldberg T, Hamp T, Richter L: PredictProtein-an open resource for online prediction of protein structural and functional features. Nucleic Acids Res, 42 (Web Server issue): W337-W343, 2014. DOI: 10.1093/nar/gku366
Furian TQ, Borges KA, Laviniki V, Rocha SL, de Almeida CN, do Nascimento VP, Moraes HL: Virulence genes and antimicrobial resistance of Pasteurella multocida isolated from poultry and swine. Braz J Microbiol, 47 (1): 210-216, 2016. DOI: 10.1016/j.bjm.2015.11.014
Christensen JP, Bisgaard M: Fowl cholera. Rev Sci Tech, 19 (2): 626- , 2000.
Strugnell BW, Dagleish MP, Bayne CW, Brown M, Ainsworth HL, Nicholas RAJ, Hodgson JC: Investigations into an outbreak of corvid respiratory disease associated with Pasteurella multocida. Avian Pathol, 40 (3): 329-336, 2011. DOI: 10.1080/03079457.2011.571659
Davies RL, MacCorquodale R, Baillie S, Caffrey B: Characterization and comparison of Pasteurella multocida strains associated with porcine pneumonia and atrophic rhinitis. J Med Microbiol, 52 (1): 59-67, 2003. DOI: 1099/jmm.0.05019-0
Katsuda K, Hoshinoo K, Ueno Y, Kohmoto M, Mikami O: Virulence genes and antimicrobial susceptibility in Pasteurella multocida isolates from calves. Vet Microbiol, 167 (3): 737-741, 2013. DOI: 10.1016/j. vetmic.2013.09.029
Bosch M, Garrido E, Llagostera M, Pérez de Rozas AM, Badiola I, Barbé J: Pasteurella multocida exbB, exbD and tonB genes are physically linked but independently transcribed. Fems Microbiol Lett, 210 (2): 201- , 2002. DOI: 10.1111/j.1574-6968.2002.tb11181.x
Chu BC, Peacock RS, Vogel HJ: Bioinformatic analysis of the TonB protein family. Biometals, 20 (3-4): 467-483, 2007. DOI: 10.1007/s10534- 9049-4
Postle K: TonB protein and energy transduction between membranes. J Bioenerg Biomembr, 25 (6): 591-601, 1993.
Peacock RS, Weljie AM, Howard SP, Price FD, Vogel HJ: The solution structure of the C-terminal domain of TonB and interaction studies with TonB box peptides. J Mol Biol, 345 (5): 1185-1197, 2005. DOI: 10.1016/j. jmb.2004.11.026
Lee J, Kim YB, Kwon M: Outer membrane protein H for protective immunity against Pasteurella multocida. J Microbiol, 45 (2): 179-184, 2007.
Shultis DD, Purdy MD, Banchs CN, Wiener MC: Outer membrane active transport: Structure of the BtuB: TonB complex. Science, 312 (5778): 1396-1399, 2006. DOI: 10.1126/science.1127694
Pawelek PD, Croteau N, Ng-Thow-Hing C, Khursigara CM, Moiseeva N, Allaire M, Coulton JW: Structure of TonB in complex with FhuA, E. coli outer membrane receptor. Science, 312 (5778): 1399-1402, DOI: 10.1126/science.1128057