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Genome-Wide Diversity and Differentiation of Two Novel Multidrug-Resistant Populations of Pasteurella Multocida Type B:2 from Fo

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Genome-Wide Diversity and Differentiation of Two Novel Multidrug-Resistant Populations of Pasteurella Multocida Type B:2 from Fo bioRxiv preprint doi: https://doi.org/10.1101/2020.08.24.262618; this version posted August 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 Genome-wide diversity and differentiation of two novel multidrug-resistant populations of 2 Pasteurella multocida type B:2 from fowl cholera 3 Otun Saha1*, M. Rafiul Islam1*, M. Shaminur Rahman1*, M. Nazmul Hoque1,2, M. Anwar 4 Hossain1,3, Munawar Sultana1 5 1Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh 6 2Department of Gynecology, Obstetrics and Reproductive Health, Bangabandhu Sheikh Mujibur 7 Rahman Agricultural University, Gazipur-1706, Bangladesh 8 3Present address: Jashore University of Science and Technology, Jashore-7408, Bangladesh 9 10 *Equal contribution 11 12 13 Correspondence to: 14 Munawar Sultana, Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh 15 Email: [email protected] 16 17 18 Running title: Pasteurella multocida type B:2 causing fowl cholera 19 20 21 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.24.262618; this version posted August 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 22 ABSTRACT 23 Pasteurella multocida is the etiologic agent of fowl cholera (FC), a highly contagious and 24 severe disease in poultry with higher mortality and morbidity. Twenty-two P. multocida strains 25 isolated from the FC outbreaks were subjected to phenotypic and genotypic characterization. The 26 isolates were grouped into two distinct RAPD biotypes harboring a range of pathogenic genes; 27 exbB, ompH, ptfA, nanB, sodC, and hgbA. Among these strains, 90.90% and 36.37% were 28 multidrug-resistant and strong biofilm formers, respectively. Whole genome sequencing of the 29 two representative RAPD isolates confirmed as P. multocida type B:L2:ST122 harboring a 30 number of virulence factors, and antimicrobial resistance genes. Pan-genome analysis revealed 31 90 unique genes in these genomes associated with versatile metabolic functions, pathogenicity, 32 virulence, and antimicrobial resistance. This study for the first time reports the association of P. 33 multocida genotype B:L2:ST122 in the pathogenesis of FC, and provides a genetic context for 34 future researches on P. multocida strains. 35 36 Keywords: Fowl cholera, multidrug-resistance, biofilm former, Pasteurella multocida, genotype 37 B:2, pan-genome, unique genes, host-adaptation 38 39 1. Introduction 40 Poultry raring is one of the important sources of income in Bangladesh, including both 41 commercial broiler, and layer farming. This subsector of livestock has been noted as the largest 42 primary source of eggs, and meat since last decade, and contributes ~3.0% to the GDP of 43 Bangladesh [1]. However, this industry faces a number of constraints including limited feed 44 resource, and frequent outbreaks of infectious diseases. Emerging infectious diseases with 45 zoonotic potentials are appearing at an alarming rate, and pose a growing threat to global 46 biodiversity [2, 3]. With this backdrop, there are rising apprehensions among health care bioRxiv preprint doi: https://doi.org/10.1101/2020.08.24.262618; this version posted August 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 47 providers, veterinarians, policymakers, and the general public about human acquisition of 48 zoonotic infections from nearby encounters with domesticated pets, wild animals, and/or avian 49 species like commercial poultry [4-6]. 50 Pasteurella multocida is identified as a major bacterial etiologic agent for many 51 infectious diseases in a wide spectrum of domestic and wild animals such as poultry and wild 52 birds, pigs, cattle and buffaloes, rabbits, small ruminants, cats, dogs and other mammals [4, 7-9]. 53 P. multocida is a zoonotic Gram negative, and opportunistic bacterium [10], and causes FC or 54 pasteurellosis, an economically important disease to the poultry industry [8, 11]. Among the 55 bacterial diseases, FC is a major threat to the poultry industry causing 25-35% mortality [12]. 56 Different strains of P. multocida are associated with clinical manifestations of FC ranging from 57 depression, anorexia, lameness, swollen wattles, mucoid discharge from the mouth, ruffled 58 feathers, diarrhea, increased respiratory rate, and per acute or sudden asymptomatic death [12, 59 13]. The disease can range from acute septicemia to chronic and localized infections, and the 60 morbidity and mortality may be up to 100%. The route of infection is oral or nasal 61 with transmission via nasal exudate, faces, contaminated soil, equipment, and people [14]. 62 Currently, the strains of P. multocida are divided into 5 serotypes such as A (hyaD- 63 hyaC), B (bcbD), D (dcbF), E (ecbJ), and F (fcbD based on capsular typing [7, 8]. Among these 64 serotypes, capsular serogroups A and F cause the majority of FC, whereas serogroups B and E 65 are predominantly associated with hemorrhagic septicemia (HS) in cattle and wild ruminants [4, 66 15]. In addition to capsular serogroups, P. multocida strains are currently classified into 16 67 Heddleston lipopolysaccharide (LPS) serovars [16, 17]. Different serotypes of the P. multocida 68 exhibit varying degrees of virulence in different host [7, 8]. Various strains of P. multocida 69 harbor a wide arsenal virulence factors-associated genes (VFGs), and of them, genes involved in bioRxiv preprint doi: https://doi.org/10.1101/2020.08.24.262618; this version posted August 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 70 the capsule formation, LPS, fimbriae and adhesins, toxins, iron regulation and acquisition 71 proteins, sialic acid metabolism, hyaluronidase, and outer membrane proteins (OMPs) are the 72 key components of regulating the pathogenesis [4, 7, 18, 19]. Though not well studied, many of 73 these VFGs might play a substantial role in the pathogenesis of FC, and survival in the complex 74 host environment [7, 18, 19]. Previous reports showed that there is a clear correlation between 75 certain VFGs, and capsular types or biovars [20, 21]. Therefore, identification of prevalent VFGs 76 is important to predict the pathogenic nature of the bacterium, and select potential vaccine 77 candidates. 78 Till to date, there is lack of effective coverage in multi-serotype vaccines, and thus, 79 antibiotics remain as the most commonly employed treatment strategies for prevention and 80 control of FC despite their increased correlation to drug resistance, and food safety risks due to 81 residual effects [4, 8, 22]. Furthermore, the infrequent, and unnecessary overuse of antibiotics in 82 the poultry farms, livestock, fisheries, and agriculture is believed to have augmented the 83 emergence of MDR pathogenic bacterial species like P. multocida posing a serious threat to 84 public health and livestock [4]. 85 Bio-molecular techniques such as polymerase chain reaction (PCR), ribotyping, random 86 amplification of polymorphic DNA (RAPD)-PCR, multilocus sequence type (MLST), and 16S 87 rRNA gene sequencing analysis enabled the studies of molecular epidemiology, and genetic 88 diversity of P. multocida [18, 23-25]. In addition to these molecular techniques, whole-genome 89 sequencing (WGS) is an affordable, convenient, and rapid technique for outbreak investigations, 90 diagnostics, and epidemiological surveillance [26, 27]. The state of the art WGS technology 91 might enable us to study the underlying genetic mechanisms associated with pathogenicity, 92 virulence fitness, and host adaptability of P. multocida in multiple hosts [4, 28, 29]. However, bioRxiv preprint doi: https://doi.org/10.1101/2020.08.24.262618; this version posted August 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 93 there is limited information on the molecular epidemiology of P. multocida circulating in the 94 global poultry industry with particular reference to Bangladesh. Therefore, in this study, we’ve 95 employed different bio-molecular techniques along with the WGS of two representative MDR, 96 and SBF P. multocida isolates to investigate the phenotypic and genotypic diversity, and 97 underlying genetic contents such as VFGs, antimicrobial resistance genes (ARGs), and metabolic 98 functional potentials to be associated with the pathogenesis of FC in laying hens in Bangladesh. 99 Moreover, herein this study, we first time report the phenotypic and genotypic traits, and 100 associated genetic factors of highly pathogenic strains of P. multocida type B:2 causing FC in 101 chickens of Bangladesh. 102 103 2. Methods 104 2.1. Sample collection, bacterial strain isolation, and genomic DNA extraction 105 We collected fifty-seven samples (n=57) of suspected FC including 36 suspected live 106 birds, and 21 dead laying hens from six commercial layer farms located in Narsingdi (23.9193° 107 N, 90.7176° E), Narayangonj (3.6238° N, 90.5000° E), and Manikgonj (23.8617° N, 90.0003° E) 108 districts of Bangladesh during August 2017 to November 2017. Diseased birds were diagnosed 109 with FC by observing clinical signs and symptoms including sudden death, swollen wattle and 110 combs, lameness, respiratory rales, and diarrhea by practicing veterinarians.
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