Korean Lepr Bull.  2019 Dec;52(1):41-50. 10.33161/klb.2019.52.1.41.

Effect of temperature and medium on the viability of Mycobacterium leprae during long term-storage

• Park JH
• Kim YJ
• Kim JP

• KMID: 2466491
• DOI: http://doi.org/10.33161/klb.2019.52.1.41

Abstract

BACKGROUND
Mycobacterium leprae (M. leprae) is pathogenic bacterium with polymorphic, acid-fast properties and causes leprosy that it is called Hansen's disease. Leprosy can be completely cured using multidrug therapy (MDT), but it is not easy to eradicate leprosy and M. leprae on the planet. OBJECT: We still do not understand the exact pathogenesis mechanism of leprosy. The main reason is that we cannot grow bacteria in vitro. Therefore, quantitative measurement and damage-free storage of live M. leprae are very important.
METHODS
Here, we generated bacteria stocks of M. leprae using HBSS with 0.05% tween 80 or freezing solution 11 months ago and evaluated conditional survival of bacteria by Propidium monoazide (PMA) staining, real-time PCR.
RESULTS
There were assessed for bacteria viability under the conditions of each temperature or medium by delta-Ct level of real-time PCR. We also observed that frozen-stored M. leprae (2.14) compare to refrigerated-stored M. leprae (1.03) was significant decreased delta-Ct in HBSS (P<0.05). However, frozen-stored M. leprae (1.14) was not difference refrigerated-stored M. leprae (0.84) in freezing solution (P=NS). Real-time PCR with SYBR green method was reliable for results and statistical significance, but data for real-time PCR with probe method were unreliable.
CONCLUSIONS
Taken together, these results indicated that freezing solution regardless of temperature increase much more bacterial survival. In addition, if use not freezing solution, M. leprae must be stored frozen.

Keyword

Leprosy; Mycobacterium leprae; Propidium monoazide; Real-time PCR

MeSH Terms

Bacteria
Freezing
In Vitro Techniques
Leprosy
Methods
Microbial Viability
Mycobacterium leprae*
Mycobacterium*
Planets
Polysorbates
Propidium
Real-Time Polymerase Chain Reaction
Polysorbates
Propidium

Figure

• 

  Fig. 1 Real-time PCR for differences in stored media PMA-stained M. leprae was confirmed by real-time PCR of SYBR green method. A) ΔCt of Freezing solution group (Freezing sol., n=7) was significantly decreased at 4℃ compare to HBSS group (HBSS, n=7)(HBSS 2.14 vs Freezing sol. 1.03, *p<0.05). B) ΔCt of Freezing solution group (Freezing sol., n=7) was decreased at −20℃ compare to HBSS group (HBSS, n=7)(HBSS 1.14 vs Freezing sol. 0.84, NS). Samples were stored for 11 months in HBSS or freezing solution (Mean ± S.E.M; *p<0.05; NS, Not significant; each data point is represented on graphs).

• 

  Fig. 2 Real-time PCR for differences in stored temperature PMA-stained M. leprae was confirmed by real-time PCR of SYBR green method. A) ΔCt of refrigerated group (4℃, n=7) was significantly decreased in HBSS compare to frozen group (−20℃, n=7)(4℃ 2.14 vs −20℃ 1.14, *p<0.05). B) ΔCt of refrigerated group (4℃, n=7) was decreased in freezing solution compare to frozen group (−20℃, n=7)(4℃ 1.03 vs −20℃ 0.84, NS). Samples were stored for 11 months in HBSS or freezing solution. (Mean ± S.E.M; *p<0.05; NS, Not significant; each data point is represented on graphs).

• 

  Fig. 3 Real-time PCR for differences in stored media PMA-stained M. leprae was confirmed by real-time PCR of probe method. A) ΔCt of freezing solution group (Freezing sol., n=7) was increased at 4℃ compare to HBSS group (HBSS, n=7)(HBSS −0.03 vs Freezing sol. 0.31, NS). B) ΔCt of freezing solution group (Freezing sol., n=7) was increased at −20℃ compare to HBSS group (HBSS, n=7)(HBSS 0.41 vs Freezing sol. 0.6, NS). Samples were stored for 11 months in HBSS or freezing solution (Mean ± S.E.M; NS, Not significant; each data point is represented on graphs).

• 

  Fig. 4 Real-time PCR for differences in stored temperature PMA-stained M. leprae was confirmed by real-time PCR of probe method. A) ΔCt of refrigerated group (4℃, n=7) was increased in HBSS compare to frozen group (−20℃, n=7)(4℃ −0.03 vs −20℃ 0.41, NS). B) ΔCt of refrigerated group (4℃, n=7) was increased in freezing solution compare to frozen group (−20℃, n=7)(4℃ 0.31 vs −20℃ 0.6, NS). Samples were stored for 11 months in HBSS or freezing solution. (Mean ± S.E.M; *p<0.05; NS, Not significant; each data point is represented on graphs).

• 

  Fig. 5 Summary in this experiment PMA-stained M. leprae was confirmed by real-time PCR. The results of experiments using real-time PCR of SYBR green showed increase ΔCt deviation compare to real-time PCR of probe method. However, Contrary to SYBR green method, probe method results are unreliable because group of each observed ΔCt.

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