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Growth Kinetics of Pasteurella multocida B: 2 in Iron Controlled Conditions in Broth Media

Rajat Varshney Vinod Chaturvedi Aditya Agrawal Bablu Kumar
Vol 8(5), 205-212
DOI- http://dx.doi.org/10.5455/ijlr.20171111072414

To investigate the role of iron on the bacterial biomass of Pasteurella multocida B: 2, the cultivation of P. multocida B: 2 was done in casein-sucrose-yeast (CSY) broth supplemented with varying amount of FeSO4 (iron rich conditions) or 2, 2’ bipyridyl (BP) (iron limited conditions). The preliminary studies showed substantial growth of P. multocida B: 2 in broth with CSY-3, CSY-3+ 100 µM FeSO4, CSY-3 + 100 µM BP media in the shaking and stationary culture on 18 hrs post inoculation. Hence, these media were selected for growth kinetics study of P. multocida B: 2 in both shaking and stationary incubation at 37 °C upto 18 hrs. The growth of P. multocida B: 2 was measured via spectrophotometer at OD600nm and observed that P. multocida B: 2 showed better bacterial biomass on iron rich media in comparison to iron limited media.


Keywords : Bacterial Biomass Growth Kinetics Pasteurella multocida B: 2

Introduction

Haemorrhagic septicaemia (HS), caused by Pasteurella multocida B: 2, is considered as the second most important disease of buffaloes and cattle in India during the last two decades as per NADRES (National Animal Diseases Referral Expert System) report and accounted for maximum number of deaths in livestock in the country (Rahman et al., 2012) which need to be controlled on a higher priority via early and rapid diagnosis for implementing the directed therapy and via effective vaccination. Although Molecular techniques such as P. multocida-specific polymerase chain reaction PCR (Pm-PCR), HSPmB-PCR (HS causing P. multocida type B-specific PCR) targeting 6b (Townsend et al., 1998), multiplex PCR,  HS causing P. multocida type B-specific LAMP (HSPmB-LAMP) targeting 6b gene (Varshney et al., 2017)  and P. multocida species specific LAMP (Pm-LAMP) targeting kmt1 gene (Sun et al., 2010) etc. have been developed for early diagnosis of HS, however treatment for HS is of limited value due to acute nature of disease. Vaccination is generally considered as the most effective way to limit HS. Diverse types of vaccines like alum precipitated vaccine (APV), aluminium hydroxide gel adsorbed, oil adjuvanted vaccine (OAV) have been reported in the scientific literature (Verma and Jaiswal, 1998). APV imparts immunity for 4-6 months only. Thick viscosity and difficulty in administration of OAV limit its use in the field. Thus, need for improvement of HS vaccine qualities in term of safety, potency, efficacy, duration of immune response, viscosity etc. has always been felt which in turn may vary due to media used for the growth of P. multocida B: 2 for differential biomass and outer membrane protein (OMP) profiling. Hence, there is need to devise some media where the bacterial growth may be obtained which may be more immunogenic with qualitative increase in biomass per unit of media.

Iron is needed for survival of all bacteria. P. multocida evolved many mechanisms to sequester iron in vitro or in vivo free form of iron or from the iron-rich host proteins such as haem, lactoferrin and transferrin via. siderophores. P. multocida grown in iron limiting conditions in presence of iron chelators such as 2, 2’ dipyridyl showed the diminution in capsular material (Jacques et al., 1994). Besides there is alteration in the OMPs expression of P. multocida according to the host environment (Boyce et al., 2006). Genes encoding iron binding and transport protein was found to be up-regulated while expression of genes involved in energy metabolism and electron transport generally decreased in response to iron limited conditions with respect to normal iron conditions as per gene analysis via whole genome analysis (Paustian et al., 2001). Iron Regulated Outer Membrane Proteins (IROMPs) of P. multocida type A has been found to be immunogenic not only against homologous challenges but also against heterologous challenge in mice, birds, rabbits and calves (Adler et al., 1996; Chawak et al., 2001; Confer et al., 2001). That why’s growth pattern and growth kinetic study of P. multocida in iron rich to iron limited media would guide standard growth conditions for P. multocida B: 2 and bacterial biomass estimation with respect to time which in turn help in bacterial biomass adjustment per dose of vaccine and better formulation of HS vaccine. Therefore, the growth pattern and growth kinetic study of P. multocida B: 2 (strain P52) under different iron and sucrose concentration in CSY media was undertaken and details of findings of the study are discussed hereunder in the present communication.

Material and Methods

Phase 1 culture of P.  multocida B: 2 (strain P52), used for the growth kinetic study under different iron limited to rich conditions, was procured from the Type-culture Laboratory, Division of Standardization, IVRI, Izatnagar under freeze-dried condition. The culture was revived on blood agar and BHI agar plates at 37 °C overnight (O/N). The purity and identity of isolated bacterial colony was done by morphological, cultural, biochemical and molecular characterization of bacteria via P. multocida species specific PCR and multiplex PCR as per Townsend et al. (1998) and Townsend et al. (2001), respectively.

A well characterized, isolated colony of P. multocida B: 2 from blood agar plate was inoculated in 3 ml CSY-broth in 15 ml tube and incubated in stationary incubator at 37 °C for 16hr. One ml of this grown culture was further inoculated into 100 ml CSY broth and incubated at 37 °C in stationary incubator for 16hr for preparing downstream inoculum. The purity of culture was tested at every step.

Growth of P. multocida B: 2 in Iron Regulated Conditions

Two sets of each broth tube containing 5ml CSY broth with varying concentration of sucrose (CSY broth having 0.1%, 0.2%, 0.3%, 0. 4% and 0.5% sucrose conc. designated as CSY-1, CSY-2, CSY-3, CSY-4 and CSY-5, respectively) and supplemented with varying amount of iron (FeSO4) (i.e. 0 µM, 50µM, 100 µM, 150 µM, 300 µM, 500 µM, 1000 µM and 100 mM) were inoculated with 0.05 ml (1%) standardized inoculum (16 hr broth culture) of P. multocida B:2. Simultaneously, the broth tubes containing CSY-3 broth added with varying concentration of BP (i.e. 0 µM, 100 µM, 200 µM, 300 µM and 400 µM) were inoculated with 0.05 ml (1%) standardized inoculum (16 hr broth culture) of P. multocida B: 2. One set of each inoculated broth tube was incubated at 37 oC in stationary incubator for 18 hr and another set of inoculated broth tube was incubated in orbital shaker at 120 rpm for 18 hr. Un-inoculated control broth tubes were used to normalize the media’s colour value and to check the sterility of media. The bacterial biomass was estimated in terms of turbidity with Brown’s opacity tubes. The purity of culture was tested at every step.

Growth Kinetic Study of P. multocida B: 2 in Iron Regulated Conditions

CSY-3 broth, CSY-3 +100 µM BP (iron limiting media) and CSY-3 + 100 µM FeSO4 (iron rich media were selected for the growth kinetic study of P. multocida B:2 as P. multocida B:2 grew substantially in these media in the preliminary study. The time intervals viz. 3, 6, 9, 12, 15 and 18hr were taken into account for the growth kinetic study. Out of three sets of 5 ml of each above mentioned broth for each time interval, two sets were inoculated with 0.05 ml of 16 hr broth culture of P. multocida B: 2 and one set was kept un-inoculated in order to set as blank or normalize media’s colour value. One set of inoculated broth tubes along with un-inoculated broth tubes were incubated in stationary incubator at 37 °C and another set of inoculated broth tubes were incubated at 37 °C in orbital shaker at 120 rpm for each time interval 3, 6, 9,12, 15 and 18 hr.  The bacterial biomass was estimated via spectrophotometer (SpectraMax M5) at OD600nm. The experiment was replicated three times for each culture condition. The purity of culture was tested at every step.

Result and Discussion

Haemorrhagic septicaemia, a major peril to buffaloes and cattle in India and other Asian countries needs to be controlled on a higher precedence via early and correct diagnosis of the disease and effective vaccination in order to minimize the economic losses due to mortality of animals. Bacterial biomass is imperative factor for effective formulation of HS vaccine via dose adjustment which in turn depends upon in which media bacteria are grown. Almost iron is required for the growth of all bacteria that’s why iron concentration in media or in vivo environment regulates bacterial biomass, toxin production, pathogencity and differential OMP profiling of bacteria. On account of these facts, growth kinetic study of P. multocida B: 2 in iron rich to restricted media are needed in order to explore out the effect of iron on the growth of bacteria.

  1. multocida B: 2 strain P52 (vaccine strain) had typical characteristics of P. multocida with an amplicon product of expected size ~460 bp of Pm-PCR targeting kmt1 gene corresponds to species specificity and amplified products of expected size ~460 bp and ~760 bp of multiplex PCR targeting kmt1 gene and all capsular genes corresponds to species specificity and capsular type B specificity respectively as shown in Fig. 1. The similar observations were also reported by Townsend et al. (1998) and Townsend et al. (2001).

Fig. 1: Agarose gel electrophoresis of Pm-PCR product targeting only kmt1 gene and multiplex PCR products targeting capsular and kmt1 gene [Lane #M, 1 Kb DNA ladder; #2, 460 bp Pm-PCR product; #3, negative control, #4, 460 bp and 760 bp amplified multiplex PCR]

On preliminary study in respect to sucrose and iron concentration, it was found that the growth of P. multocida B: 2 was fairly better in CSY-3 media supplemented with 100 µM FeSO4. Iron supported the growth of bacteria upto a extent of 500 µM FeSO4 but further addition of  iron in media had negative effect on growth of bacteria and drastic reduction in growth at 100 mM FeSO4 as shown in Table 1 which is  in concurrence  with the earlier  findings where substantial growth of  P. multocida A:1 was observed  in BHI media supplemented with low iron concentration (i.e. 100 µM FeCl3) but high iron supplementation  (i.e. 100 mM FeCl3) in the grown culture media inactivated P. multocida A:1 (Herath et al., 2010). However, insignificant growth of P. multocida B: 2 were observed in CSY media supplemented with 100 mM FeSO4 which may be due to different media (CSY), iron supplementation (FeSO4) and mode of iron supplementation  and bacterial strain (P. multocida B: 2 strain P52) used in the present study.

Table 1: Effect of sucrose and iron concentration on the growth of P. multocida B: 2 in stationary and shaker culture

Medim Concentration of FeSO4
0 µM 50µM 100 µM 150µM 300µM 500 µM 750µM 1000 µM 100 mM
S NS S NS S NS S NS S NS S NS S NS S NS S NS
CSY-1 3 ≤ 2 3 2 3 ≥2 3 ≤ 3 3 ≥2 3 ≤ 2 3 ≥ 1 3 < 1 1 < 1
CSY-2 3 ≤ 2 3 2 3 2 4>x>3 2 4>x>3 3>y>2 4>x>3 2 3 ≥ 1 3 1 1 < 1
CSY-3 3 ≥2 3 ≥2 4>x>3 3 4>x>3 3>y>2 4>x>3 ≤ 3 3 ≤ 2 3 ≥ 1 3 1 1 < 1
CSY-4 3 ≤ 3 4>x>3 3 4>x>3 3 4>x>3 ≤ 3 4>x>3 3 3 ≤ 3 3 2 3 >1 1 < 1
CSY-5 4>x>3 3 4>x>3 3 4>x>3 3 4>x>3 3 4>x>3 ≥ 3 3 ≤ 3 3 < 2 3 >1 1 < 1

S=shaking incubation; NS=non-shaking (stationary) incubation; x=bacterial turbidity; Arabic numbers are indicative of Brown opacity tube’s number

Sucrose though known to promote the bacterial growth but upto limited extent. However, iron showed enormous impact on growth of bacteria that’s why sucrose concentration in CSY media were kept constant (i.e. 0.3%) with varying concentration of 2, 2’ bipyridyl (BP), an iron chelator, in order to rule out iron limitation effect on the growth of P. multocida B: 2 and to select the concentration of BP in CSY-3 media in which appreciable growth of P. multocida B: 2 can be observed which in turn help in up scaling the vaccine production.P. multocida B: 2 showed substantial growth (turbidity matched with brown opacity tube no. 3 in shaking and ≥ 2 in stationary incubation) in CSY-3 supplemented with 100 µM BP while poor growth (turbidity matched with brown opacity tube no. ≤ 1 in shaking and stationary incubation) in concentration of BP (200 µM BP to 500 µM BP) .However the present observation are slightly divergent with the observation of Prasannavadhana et al. (2014) who observed appreciable growth of P. multocida B: 2 in BHI media supplemented with 200 µM BP which may be due to difference in available iron content in media and growth conditions. Further kinetic study of P. multocida B: 2 in CSY-3 medium supplemented with FeSO4 or BP (i.e. 100 µM) was done and the growth profiles in terms of bacterial density are shown in Table 2 and Fig. 1 and 2. The major findings of the study were that the CSY-3 supplemented with 100 µM FeSO4 can be beneficially used for quality production of bacterial biomass for vaccine production. However, further studies are needed to unearth the role of such biomass in immunity against pasteurellosis in cattle.

 

Fig. 2: Effect of iron on the growth of P. multocida B:2 in shaker culture

Table 2: Iron controlled media were inoculated with P. multocida B: 2 and incubated in orbital shaker and stationary incubator at 37 °C for different time interval

Media Time Interval
3hr 6hr 9hr 12hr 15 hr 18hr
S NS S NS S NS S NS S NS S NS
CSY-3+ 100 µM BP 0.0827

±0.0007

0.0823

±0.0006

0.1034

±0.0024

0.0962

±0.0009

0.1162

±0.0016

0.1023

±0.0012

0.123

±0.0025

0.1162

±0.0013

0.1653

±0.0039

0.1284

±0.0013

0.1809 ±0.0042 0.1365 ±0.0010
CSY-3 0.0936

±0.0006

0.0932

±0.0006

0.1261

±0.0021

0.1025

±0.0011

0.1367

±0.0032

0.1151

±0.0010

0.1811

±0.0033

0.1336

±0.0021

0.1929

±0.0031

0.1583

±0.0016

0.2728 ±0.0041 0.1769 ±0.0021
CSY-3+ 100 µM FeSO4 0.0946

±0.0009

0.0941

±0.0007

0.1697

±0.0088

0.1269

±0.0012

0.2033

±0.0033

0.1326

±0.0012

0.2183

±0.0054

0.1557

±0.0013

0.2473

±0.0048

0.1698 ±0.0017 0.2904 ±0.0043 0.2019 ±0.0024

Bacterial biomass in form of turbidity measured via spectrometer at OD 600 nm  

S=shaking incubation; NS=non-shaking (stationary) incubation

Fig. 3: Effect of iron on the growth of P. multocida B: 2 in stationary culture

 

Conclusion

HS, major threat to livestock, needs to be resolved via implementation of effective vaccination programme. With the improvement in vaccines and reduction in cold chain requirement will contribute to better standards of animal wellbeing and farming opulence which can be achieved with high magnitude and prolonged immune status which primarily depend on the media and growth conditions used for the growth of P. multocida B: 2 and can be attained via adjustment of iron content in media.

Acknowledgment

The authors are thankful to ICAR-IVRI for providing necessary facilities for research work.

References

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