(LC50) of Pathogenic Microorganisms and Their Antigens in Vaccine Development Saganuwan Alhaji Saganuwan*

Saganuwan BMC Res Notes (2020) 13:289 https://doi.org/10.1186/s13104-020-05126-x BMC Research Notes

RESEARCH NOTE Open Access Application of median lethal concentration (LC50) of pathogenic microorganisms and their antigens in vaccine development Saganuwan Alhaji Saganuwan*

Abstract Objective: Lack of ideal mathematical models to qualify and quantify both pathogenicity, and virulence is a dreadful setback in development of new antimicrobials and vaccines against resistance pathogenic microorganisms. Hence, the modifed arithmetical formula of Reed and Muench has been integrated with other formulas and used to deter- mine bacterial colony forming unit/viral concentration, virulence and immunogenicity. Results: Microorganisms’ antigens tested are Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa in mice and rat, Edwardsiella ictaluri, Aeromonas hydrophila, Aeromonas veronii in fsh, New Castle Disease virus in chicken, Sheep Pox virus, Foot-and-Mouth Disease virus and Hepatitis A virus in vitro, respectively. The LC 50s for the pathogens using diferent routes of administrations are 1.93 103(sheep poxvirus) and 1.75 1010 for Staphylococcus × × aureus (ATCC29213) in rat, respectively. Titer index (TI) equals N log10 LC 50 and provides protection against lethal dose in graded fashion which translates to protection index. N is the number of vaccine dose that could neutralize the LC 50. 3 11 Hence, parasite inoculum of 10 to 10 may be used as basis for determination of LC50 and median bacterial concentrations (BC50).Pathogenic dose for immune stimulation should be sought at concentration about LC 10. Keywords: Vaccine, Pathogenicity, Model, Arithmetic, Development, Colony forming unit

Introduction numbers of colony forming units of some pathogenic Many countries have renewed efort towards develop- bacteria, viruses and their antigens were determined, ment of vaccine against a number of infectious diseases, using median lethal concentrations (LC 50s) established such as mastitis caused by Staphylococcus aureus in in laboratories, with intent to calculating immunogenic bovine and human [1]. Capsular polysaccharide, virulent doses of various infectious agents. antigens [2, 3] using adhesive proteins [4] as immunogenic derivatives, deoxyribonucleic acid (DNA), autoly- Main text sin and protein-binding polysaccharides are also used to Methods stimulate immune system [5–7]. However, Saganuwan Reference was made to journal articles on development reported toxicological basis of antidote [8] and a number of vaccines against methicillin resistance Staphylococcus of vaccines presently being developed is based on modi- aureus and other pathogenic microorganisms that cause fed arithmetical method of Reed and Muench [9]. Hence diseases in human and animals. Median lethal concentrations (LC50s) of Staphylococcus aureus, Streptococ- cus pneumoniae, Pseudomonas aeruginosa in mice and *Correspondence: [email protected] rat, Edwardsiella ictaluri, Aeromonas hydrophila and Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Federal University of Agriculture, P.M.B. 2373, Aeromonas veronii in catfsh, New Zealand rabbit, fsh Makurdi, Benue, Nigeria and mice were translated to colony forming units. LC 50

© The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licen ses/by/4.0/ . The Creative Commons Public Domain Dedication waiver (http://creativeco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Saganuwan BMC Res Notes (2020) 13:289 Page 2 of 6

of in vitro cell cultures of hepatitis A virus and Foot and 5 NcxDf LC50 = Mouth Disease virus were translated to LC1, whereas N efective dose ffty (ED-50) for Newcastle Disease vaccines was translated to ED1 -in chickens [5–20]. Te method viii. Median bactericidal concentration (MC 50) formula of Reed and Muench [21] as modifed by Saganuwan [9] is determined as follows N N0 c = −BC was used for LC50 determination in various laboratories. 1+er(x 50) whereas N = Number of colonies for Protection index (PI) is equal to titration index = Nlog10 each plate. LD50, whereas N is number of titration using vaccine. ix. In vivo LD value can be replaced by tissue culture LD 50 50 No BC = (TCL 50). 50 2 Derivation of LD formula 50 Tus 2BC could replace MBC 50 1 BC = BC + lc(No− ) x. 1 50 r whereas r tangent slope i. Modifed formula of Reed and Muench = on infexion LD MLD+MSD No could estimate the bactericidal intensity [23] 50 = 2 whereas MLD Median lethal = xi. Since the rate of bacterial load depends on the con- dose; MSD = median survival dose [9]. centration of neutrophils. Exponent = (− kp + g) t, where k is the second-order rate constant for Derivation of LC50 formula bacterial killing, p neutrophil concentration; Conc. initial concentration of colony forming unit per = = g frst-order rate constant for bacterial growth; ml of sample x = = t time. When concentration is double fold, triple fold and = K 2 10−8 ml per neutrophil per min; g 8 10−3 tetra fold, they are represented as 2 x X, 3 x X and 4 x X, = × = × min respectively. P > g xii. When k = critical neutrophil concentration x+2x+3x+4x LC50 = x5 ii. Hence, 10 5 Te critical neutrophil concentration = 3–4 × 10 per ml, a value of 5 105 predisposes human to bacterial ≤ × infection [24]. All of the above formulas could be applied 10x in determination of lethal concentration of immunogenic LC50 = x5 10 and anti-immunogenic agents in various models of vaccine development. iii. LC50 = X × 5 x = initial concentration = colony forming unit; whereas Results LC 50 median lethal concentration that can kill = Te colony forming unit, LC 1-, median lethal concentra- 50% of test animals; x = initial concentration; mul- tion for each pathogenic microorganism, antigen, vac- tiplication factors for initial concentration = 10 cine, animal model and their routes of administrations iv. are presented in Table 1. Te most virulent microorgan- 10 LC50 ism is Sheep Pox virus with LC50 value of 1.93 10 cfu/ x = ×4 5 ml followed by Edwardsiella ictaluri (2.8 × 10 cfu/ml), Streptococcus pneumonia(104–107 cfu/ml) and Staph- v. Number of colony forming unit (NCFU) per unit ylococcus being the least virulent in rat with IC 50 of of sample [22] 10 1.75 × 10 cfu/ml, using intradermal, intraperitoneal, intravenous and intraperitoneal route of administra- NCFU = Nc × Df. tion, respectively. Sheep was most susceptible, followed by catfsh, mice and rat being the least susceptible in the Nc = Number of colonies; Df = Dilution factor of the present study (Table 1). plate counted CFU = NcxDf vi. Terefore N Discussion Substitute x for CFU in equation v Te median lethal concentration (1.1 108 CFU) for LC50 NcxDf × ∴ = 5 N plasmid cloned neomycin (PC1 = Neo) and plasmid LC50 N 5(Nc Df) cloned neomycin methicillin resistance Staphylococ- × = × 7 vii. cus aureus (PCl-Neo-MeccA) and 1 × 10 CFU for S. Saganuwan BMC Res Notes (2020) 13:289 Page 3 of 6

Table 1 The estimated colony forming unit and median lethal concentration (LD50) of pathogenic microorganisms’ antigens and vaccines

Pathogenic Animal model Antigen(s)/Strain Route CFU (LC 1) LC50 cfu/ml Comments Reference(s) microbes

Staphylococcus Mice pC1-Neo Intraperitoneal 2.2 107 1.1 108 Less virulent [5] aureus pC1-Neo-MeccA × × Staphylococcus Mice Fibrinogen Intravenous 2 106 1 107* Less virulent [6] aureus Fibronectin × × Staphylococcus Mice Endotoxin-free PBS Intraperitoneal 1 108 5 108* Less virulent [10] aureus × × New Castle disease Chicken Lasota vaccine Oral 0.84–1.92 4.2–9.6/ml Very virulent [11] virus New Castle disease Chicken 12 vaccine Oral 1.14–1.92 5.7–9.6/ml Very virulent [11] virus Hepatitis A Virus In vitro HAV AM75/18F In vitro 2.8 106 1.4 107 Less virulent [12] × × Streptococcus pneu- Mice Pneumococcal sur- Subcut 9 104–106 4.5 104–106 Moderately virulent [13] moniae face protein A × × Streptococcus pneu- Mice PSPA1 and 2 bound Intravenous 2 103–2 106 104–107 Moderately virulent [14] moniae to Vi polysac- × × charide Foot and Mouth In vitro cell line Serotype A, 0 and Cell culture 2.8 108 1.4 109 Less virulent [15] Disease virus (hamster kidney SAT-2 × × 21 cell line) Staphylococcus Rat Strain (ATCC29213) Intraperitoneal 3.5 109 1.75 1010 Less virulent [16] aureus × × Pseudomonas Rat (ATCC27853) strain Intraperitoneal 6 107 3.0 108 Less virulent [16] aeruginosa × × Sheep pox virus Sheep SPPV strain (Hd Intradermal 3.86 102 1.93 103 Highly virulent [17] 2012) × × Edwardsiella ictaluri Catfsh Suspension of E. Intraperitoneal 5.6 103 2.8 104 Moderately virulent [18] ictaluri × × Aeromonas New Zealand rabbit, Glycoprotein Intradermal 1 109 5 109 Less virulent [19] hydrophila fsh based-vaccine × × 8 9 1 Aeromonas veronii Fish mice Bacteriovorax strain Oral 7.2 10 > 10 PFUg− Less virulent [20] × H2 CFU colony forming unit * sublethal dose; highly virulent statistically signifcant in relation to CFU/viral concentration; moderately virulent-statistically signifcant in relation to CFU/viral = = concentrations; Less virulent statistically not signifcant in relation to CFU/viral concentrations = aureus fbrinogen in mice show that the microorgan- ml), C30 strain (1.1–22/ml) and Villegas-Glisson Uni- ism is less virulent [5]. However, endotoxin-free phos- versity of Georgia (VG-VA) strain (0.3–16.2/ml), phate bufered-saline (PBS) did not show lethality at respectively [11]. But pneumococcal surface protein A 8 3+2 2+4 2+5 5 × 10 CFU [10]. Te fndings agree with the report (PspA ) is better than PspA and PspA vaccine indicating that active vaccination with a mixture of in respect of cross protection against pneumococcal recombinant penicillin binding protein 2a in rabbit infection [13]. Te conjugated α helical region of PspA (rPBP2a/r) autolysin reduced mortality in methicil- to Vi enhanced protective immune response and pro- lin resistant Staphylococcus aureus and protected mice vided protection against pneumococcal infection [14]. against infection [7]. Higher level of autolysin specifc Antibody elicited by PspA recombinant protein and antibodies has a predominant immune globulin G 1 DNA vaccine profer humoral response which is dif- (lgG1) indicating that S. aureus is opsonized in serum ferent from fragment crystallizable (Fc), (lgG1/lgG22 of immunized mouse and could increase phagocytic ratios) and fragment antigen-binding (Fab) epitopes killing [10]. But the lower concentration of New Cas- of the induced antibodies [22]. Te tissue culture tle Disease (NCD) Lasota (4.2–.6/ml) and 12 vaccine lethal dose 50 (TCLD50) determined by Cormier and (5.7–9.6/ml) that ofered protection against New Castle Janes showed that zeolite could be used against hepa- Disease may suggest robustness of the vaccines as com- titis A virus infection [12]. Foot and mouth disease pared to efective dose 50 (ED 50) of B1 strain (5.1–20.9/ (FMD) titer of serotype A, O and SAT-2 from the roller Saganuwan BMC Res Notes (2020) 13:289 Page 4 of 6

cultivation system provided protection at 2 weeks post- interaction [37], during the late stage of new antibiotic 10 vaccination [15]. Te LC 50 of S. aureus (1.75 10 cfu/ development. Tis can help pharmaceutical companies 8 × ml) and P. aeruginosa (3.0 × 10 cfu/ml) show that the that have limited resources to discover and develop microorganisms are less virulent [16]. Te pathogenic- new antibiotics [38] for emerging and rare diseases that ity is based on clinical signs, survivability and post- may need orphan drugs [39]. mortem changes of the infected animal. Terefore, the Determination of pathogenicity using a revised arith- 3 LC50 of 1.93 × 10 shows that the intradermal Roma- metical method of Reed and Munch [9] is an application nian SPPV is a potent vaccine for control and preven- of computational biology, which is the science of using tion of sheep pox in a disease-free or endemic country biology to develop algorithms or models for under- [17]. Edwardsiella ictaluri is moderately pathogenic in standing biological relationship [40] that involves data 4 Pangasionodon hypophthalamus with LC50 of 2.8 × 10 analysis and interpretation [41]. Using heterogeneity of cfu/ml and caused necrosis of liver and haemolysis animal models in the present study and the data gener- [18]. Vaccination against A. hydrophila using glycopro- ated, pose a special challenge [42], which could be sum- 9 teins (5 × 10 cfu/ml) with ginseng, provided reliable marized by expanding the computation that would fnd immunity in fsh and rabbit [19], though the immunity a range of value, which would serve as basis for deter- may not be strong. Bacteriovorax strain H2 is relatively mination of one or more biological parameters [43]. In safe in mammalian bio system including snakehead and the present study, the LC 50 of pathogenic microorgan- could be used as a probiotic agent for the bio control isms, antigens and titrated antibodies should be sought 3 10 of A. veronii infection in snakehead [20]. As a number between 1.93 × 10 and 1.75 × 10 CFU/ml depending of promising protein-based and whole cell vaccines are on the in vitro or in vivo test models, route of inocu- currently undergoing diferent phases of development lation and pathogenicity of the test pathogen, antigen [29], microorganisms and antigens with lower LC50 and titrated antibody [44]. Computational immunology values are more pathogenic and may require higher may translate to the possibility of all mammals having doses of vaccines. More so, diferent bacteria have dif- homogeneity of immunogenes from evolution [45]. ferent incubation periods and mixed infection decrease Data derived from complex processes driven by evolu- incubatory period and longevity of the host [22]. Path- tion [46], and deep learning methods as complicated ogenicity is multifactorial with genetic regions asso- by powerful programmed machine with improved soft- ciated with virulence and resistance determinants. ware infrastructures, may not provide ultimate solution Although pathogenicity islands (PAIs) and resistance for the feld of computational biology [47], making the islands (RIs) play great role in bacterial infection [25]. present study very relevant. Pathogenicity Island (150-kb) encodes several genes for Diversity of quasispecies predicts a limit between pathogenesis and antibiotic resistance [26]. Terefore mutation rate, population dynamics and pathogene- pathogenicity is qualitative whereas virulence is quan- sis [48] via mathematical modeling, that may produce titative [27]. Pathogenicity islands are acquired by hori- results similar to hypothetical and real experiments zontal gene transfer that promote genetic variability [49]. Te locus that determines pathogenicity may be described as evolution quantum leaps involving large involved in lipopolysaccharide biosynthesis [50]. Also, amounts of DNA [28]. Mechanisms of pathogenicity pathogenicity of a microbe varies with the genetic are via lysis of cell wall, toxin, adhesins and invasion background of mouse strain [32]. Te strategies used of host cell [29]. Application of monitoring programs, by pathogenic bacteria to cause pathogenicity are via prudent use of guidelines and campaigns could mini- cell wall, toxins, adhesins, invasion, intracellular life- mize the transmission and spread resistant bacteria styles, regulation of virulence factor, evolution of bacte- [30, 31]. Pathogenic potential of microbes is a continu- rial pathogen, antibacterial resistance, pathogen-innate ous phenomenon [32] that is related to infective dose immune system interaction and viability of complete and virulence [33]. Hence, host–pathogen parameters genome sequences [29]. But the evolution of patho- give progression of infection and may lead to survival genicity is based on traits that ensure survival of micro- or death [34]. But sometimes cell lines are used and organisms in their habitats [51]. Diferent pathogenic the information related to intercellular mechanism is microbes isolated from host species have diferent lacking [35], making it difcult to predict ideal patho- incubation period. But when there is mixed infection, genicity/virulence, most especially in in vitro-in vivo the incubation period decreases [22]. Te pathogenicity translation. However, molecular basis of pathogens index of 100µ per 10 6 cfu may be applied for screening has made possible, identifcation of many therapeutic of P. multocida [52]. Infuenza virus can afect coloni- interventions [36], as evidenced by disease-gene-drug zation of S. pneumoniae, S. aureus, N. meningitidis, M. tuberculosis, and S. pyogenes, RSV, Rhinovirus and Saganuwan BMC Res Notes (2020) 13:289 Page 5 of 6

HPIV. Tis has been proven by various mathematical References 1. Fattom AI, Horwith G, Fuller S, Propst M, Naso R. Development models of microbial pathogenicity [53]. of StaphVAX, a polysaccharide conjugate vaccine against S. aureus infection: from the lab bench to phase III clinical trials. Vaccine. Limitations 2004;22:880–7. https://doi.org/10.1016/j.vaccine.2003.11.034. 2. Shinefeld H, Black S, Fattom A, Horwith G, Rasgon S, Ordonez J, Yeoh H, Law D, Robbins JB, Schneerson R, Muenz L, Fuller S, Johnson J, Fireman • Te study was based on data generated in various B, Alcorn H, Naso R. Use of a Staphylococcus aureus conjugate vaccine in laboratories; hence standard operating procedure patients receiving hemodialysis. N Engl J Med. 2002;346:491–6. 3. Weichhart T, Horky M, Sollner J, Gang S, Henics T, Naggy E, Meinke A, von (SOP) and general lab practice (GLP) may afect Gabain A, Fraser CM, Gill SR, Hafner M, von Ahsen U. Functional selection our fndings. of vaccine candidate peptides from Staphylococcus aureus whole-genom • Diferences in formulas may also afect the data expression libraries in vitro. Infect Immun. 2003;71(8):4633–41. 4. Kalyvaja R. Staphylococcus aureus and Lactobacillus crispatus. Adhesive generated. characteristics of two gram-positive bacterial species. Ph.D. Microbiology, • Routes of administration, animal models and vari- University of Helsinki. 2006. ation in pathogenic molecules may afect the data 5. Senna JPM, Roth DM, Oliveira JS, Machado DC, Santos DS. Protec- tive immune response against methicillin resistant Staphylococcus generated. aureus in ma murine model using a DNA vaccine approach. Vaccine. 2003;21:2661–6. 6. Gaudreau MC, Lecasse P, Talbot BG. Protective immune response to a multi-gene DNA vaccine against Staphylococcus aureus. Vaccine. 2007;25:814–24. 7. Haghighat S, Siyadat SD, Sorkhabadi SMR, Sepahi AA, Mahdavi M. Clon- Abbreviations nig, expression and purifcation of autolysin from methicillin-resistant LC50: Median lethal concentration; LD50: Median lethal dose; BC50: Median Staphylococcus aureus: potency and challenge in Balb/c mice. Mol Immu- bacterial concentration; N: Number of vaccine dose; T1: Titre index; LC10: Lethal nol. 2017;8:10–8. concentration 10; x: Initial concentration; MLD: Median lethal dose; MSD: 8. Saganuwan SA. Toxicity: the basis for development of antidotes. Toxicol Median survival dose; NCFU: Number of colonies forming unit; Nc: Number of Open Access. 2015;1(1):1–2. colony; Df: Dilution factor; e: Exponent; r: Tangent slope on infexion; k: Second 9. Saganuwan SA. A modifed arithmetical method of Reed and Muench order rate constant; p: Neutrophil concentrations; g: First order constant for for determination of median lethal dose (LD50) Afr. J Pharm Pharmacol. bacterial growth; t: Time taken to grow; SPPV: Sheep Pox virus; HAV: Hepatitis 2011;5(11):1543–6. A virus; PBS: phosphate bufered-saline; pCl-neo: Plasmid cloned neomycin; 10. Haghighat S, Siadat SV, Sorkhabadi SMR, Sepahi AA, Sadat SM, Yazdi pCl-neo-Mecca: Plasmid cloned neomycin methicillin Staphylococcus aureus; MH, Mahdavi M. Recombination PBP2G as a vaccine candidate against IgG1: Immunoglobulin G 1; rPBP2a/r: Recombinant penicillin binding protein methicillin-resistant Staphylococcus aureus: immunogenicity and protec- 2a in rabbit; NCD: New Castle disease; ED50: Efective dose 50; TCLD50: Tissue tivity. Microbiol Pathol. 2017;108:32–9. culture median lethal dose; PSPA: Pneumococcal surface protein A; Fc: Frag- 11. Boumart Z, Hamdi J, Daouam S, Elakarm A, Tadlaoui KO, El Harrak N. Ther- ment crystallizable; Fab: Fragment anti-gen binding; VG-VA: Villegas-Glisson, mal stability study of fve New Castle disease attenuated vaccine strains. University of Georgia; FMD: Foot-and-Mouth Disease. Avian Dis. 2016;60:779–83. 12. Cormier J, Janes N. Concentration and detection of Hepatitis A virus Acknowledgements and its indicator from artifcial seawater using zeolite. J Virol Method. I sincerely thank Williams Yusuf of Federal University of Agriculture Makurdi 2016;235:1–8. and Kehinde Ola Emmanuel of National Open University all in Nigeria for 13. Piao Z, Akeda T, Takeuchi O, Ishii KJ, Ubukata K, Briles DE, Tomono K, typing the work. Chishi K. Protective properties of a fusion pneumococcal surface protein A (PSPA) Vaccine against pneumococcal challenge by fve diferent PSPA Authors’ contributions clades in mice. Vaccine. 2014;32:5607–13. SAS designed and carried out the study, analyzed the data, wrote and proof 14. Kothari N, Kothari S, Choi TY, Dry A, Briles DE, Rhee DK, Carbish R. A biva- read the manuscript. The author read and approved the fnal manuscript. lent conjugate vaccine containing PSPA families 1 and 2 has the potential to protect against a wide range of Streptococcus pnuemoniae strains and Funding Salmonella typhi. Vaccine. 2015;33:783–8. The study was carried out using my monthly emoluments. 15. Al Hassan. Efect of diferent culture systems on the production of Foot- and-Mouth Disease trivalent vaccine. Vet World. 2016;9(1):32–7. Availability of data and materials 16. Jankie S, Lenelle J, Suepaul R, Pereira LP, Akpaka P, Adebayo AS, Pillai G. All data generated or analyzed during this study are included in this published Determination of the infective disease of Staphylococcus aureus (ATCC article. 27853) and Pseudomonas aeruginosa (ATCC 27853) when injected intra- peritoneally in Sprague dawley rats. Br J Pharm Res. 2016;14(1):1–11. Ethics approval and consent to participate 17. Boumart Z, Daouawm S, Belkourati I, Raf L, Tuppurainen E, Tadkoli KO, El Not applicable, because neither animals nor humans were used for the study; Harrak M. Comparative innocuity and efciency of live and inactivated the data were generated from laboratories. sheep vaccines. BMC Vet Res. 2016;12(133):1–6. 18. Susanti W, Indrawati A, Pasaribu FH. Kajiampatogenisitasbakteri Edwards Consent to publish iellaictaluri Padaikan Patin Pangosion odonhypothalamus. J Akuakult Not applicable. Indonesia. 2016;15(2):99–107. 19. Ciftci A, Onuk EE, Ciftci G, Findik A, Sogut MU, Didinen BI, Aksou A, Ustu- Competing interests nakin K, Gulhan T, Balta F, Altun S. Development and validation of glyco- The author declares that he has no competing interests. protein protein-based native-subunit vaccine for fsh against Aeromonas hydrophila. J Fish Dis. 2016;39:981–92. Received: 10 February 2020 Accepted: 4 June 2020 20. Cao H, Hou S, Lu L, Yang X. Identifcation of a bacteriovovax sp. isolate as a potential biocontrol bacterium against snakehead fsh pathogen Aeromonas Veronii. J Fish Dis. 2014;37:283–9. 21. Reed LJ, Muench H. A simple method of estimating ffty percent end- point. Am J Epidemiol. 1938;27:493–7. Saganuwan BMC Res Notes (2020) 13:289 Page 6 of 6

22. Sharma P, Sihag RC. Pathogenicity test of bacterial and fungal fsh response elicited by immunization with pneumococcal surface protein pathogens in Cirrihinus madrigals infected with EUS disease. Pak J BiolSci. A (PspA) as recombinant protein or DNA with Streptococcus pneumonia. 2013;16(20):1204–7. Microb Pathol. 2012;53:243–9. 23. Liu YQ, Zhang YZ, Gao PJ. Novel concentration killing curve method for 41. Saganuwan SA. The new algorithm for determination of median lethal estimation of bactericidal potency of antibiotics in an invitro dynamic dose ffty (LD50)and efective dose ffty(ED50) for snake venom and model. Antimicrob Agent Chemother. 2004;48(10):3884–91. antivenom in mice. Int J Vet Sci Med. 2015;4(3):1–4. 24. Li Y, Katlin A, Like JD, Silverstem SC. A critical concentration of neutrophils 42. Loman N, Watson M. Do you want to be a computational biologist? Nat is required for efective bacterial killing in suspension. Proc Natl Acad Sci. Biotechnol. 2013;37(11):996–8. 2002;99(12):8289–94. 43. Nusinov R, Bonhoefor S, Papin JA, Sporns O. From, “what is” to “what 25. Yoon SH, Park Y-K, Kim JF. Exploration and analysis of pathogenicity and isn’t?” computational biology. PLoS Comput Biol. 2015;11(7):1–3. resistance islands. Nucleic Acid Res. 2015;43:624–30. 44. Markowety F. All biology is computational biology. PLoS Biol. 26. McBride SM, Coburn PS, Baghdayan AS, Williams RJL, Grand MJ, Shankar 2015;15(3):1–4. N, Gilmore MS. Genetic variation and evolution of the pathogenicity 45. Berger B, Daniel NM, Yu YW. Algorithm advances take advantages of island of Enterococcus faecalis. J Bacteriol. 2009;191(10):3392–402. the structure of massive biological data landscape. Commun Can. 27. Shapeiro-Ian D, Fuxa JR, Lacey LA, Onstad DW, Kaya HK. Defnitions of 2016;59(8):71–8. pathogenicity and virulence in invertebrate pathology. J Invert Pathol. 46. Angermueller C, Parnamae T, Parts L, Stegle O. Deep learning of compu- 2005;88:1–7. tational biology. MolSyst Biol. 2016;12(878):1–16. 28. Hentschel U, Hacker J. Pathogenicity islands: the tip of iceberg. Microb 47. Feldman C, Anderson R. Review: current and new generation pneumo- Infect. 2001;3:545–8. coccal vaccines. J Infect. 2014;69:309–25. 29. Wilson JW, Schurr MJ, LeBlanc CL, Ramamurthy R, Buchanan KL, 48. Vignuzzi M, Stone JK, Arnold JJ, Cameron CE, Andino R. Quasi species Nickerson CA. Mechanisms of bacterial pathogenicity. Postgrad Med J. diversity determines pathogenesis through cooperative interactions 2002;78:216–24. within a viral population. Nature. 2006;439(7074):344–8. 30. Saga T, Yamaguchi K. History of antimicrobial agents and resistance 49. Garcia J, Shea J, Alvarez-Vasquez F, Qureshi A, Luberto C, Voit EO, Poeta bacteria. JMAJ. 2009;52(2):103–8. MD. Mathematical modelling of pathogenicity of Cryptococcus neorfor- 31. Mctwen SA, Fedorka-Cray PJ. Antimicrobial use and resistance in animals. mans. MolSystBiol. 2008;183:1–13. CID. 2002;34(3):93–106. 50. Dow JM, Osbourn AE, Wilson TJG, Daniels MJ. A locus determining patho- 32. Casadevall A. The pathogenic potential of a microbe. Msphere. genicity of Xanthomonas campestris is involved in lipopolysaccharide 2017;2(1):1–7. biosynthesis. MPMI. 1995;8(5):766–77. 33. Legget HC, Cornwallis CK, West SA. Mechanisms of pathogenesis, infec- 51. Van Baarlen P, van Belkum A, Summerbell RC, Crous PW, Thomma PHJ. tive dose and virulence in human parasites. PLoS Pathog. 2012;8(2):1–5. Molecular mechanisms of pathogenicity: How do pathogenic micro- 34. Duneau D, Ferry J-B, Rerah J, Kondof H, Ortiz GA, Lazaro BP, Bouchon N. organisms develop cross-kingdom host jumps? FEMS Microbiol Rev. Stochastic variation in the initial phase of bacterial infection predicts the 2007;31:239–77. probability of survival in D. melanogaster. Life. 2017;6:e28298. 52. Pilatti RM, Furian TQ, Lima DA, Finkler F, Brit BG, Salle CTP, Morae HLS. 35. Law GL, Tisoncik-Go J, North MJ, Katie MG. Drug repurposing: a Establishment of a pathogenicity index of one-day-old broilers to Pas- better approach for infectious drug discovery? Curr Opin Immunol. teurella multocida strains isolated. Braz J Poult Sci. 2013;37(11):344–8. 2013;25(5):585–92. 53. Opatowski L, Baguelin M, Eggo RM. Infuenza interaction with circulating 36. Groft SC. Rare diseases research expanding collaborative translational pathogens and its impact on surveillance, pathogenesis and epidemic research opportunities. CHFST. 2013;144(1):16–23. profle: a key role for mathematical modeling. PLoS Pathog. 2017. https:// 37. Tasleem M, Ishrat R, Islam A, Ahmad F, Hassan MI. Human disease insight: doi.org/10.1101/203265. an integrated knowledge-based platform for disease-gene-drug information. J Infect Publ Health. 2016;9:331–8. 38. Fernandes P, Martens E. Antibiotics in late clinical development. Biochem Publisher’s Note Pharmacol. 2017;133:152–63. Springer Nature remains neutral with regard to jurisdictional claims in pub- 39. Aronson JK. Rare diseases and orphan drugs. Br J Clin Pharmacol. lished maps and institutional afliations. 2006;61(3):243–5. 40. Vadesitho CFM, Ferreira DN, Moreno AT, Chavez-Olortegui G, de Machado Avila RA, Oliveira MLS, Ho PL, Miyaji EN. Characterization of the antibody

Ready to submit your research ? Choose BMC and benefit from:

• fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations • maximum visibility for your research: over 100M website views per year

At BMC, research is always in progress.

Learn more biomedcentral.com/submissions