[1] Clarridge JE 3rd, Raich TJ, Pirwani D, Simon B, Tsai L, Rodriguez-Barradas MC, Regnery R, Zollo A, Jones DC, Rambo C. Strategy to detect and identify ...
Chemotaxonomy of Bartonella Species Using Cellular Fatty Acids Profiles Li Dong Mei1§, Miao Zhigang 1, 2§, Song Xiuping1, Liu Qiyong1 1 Department of Vector Biology and Control, State Key Laboratory for Infectious Diseases Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 102206, China 2 Shandong University, Jinan, Shandong, 250100, China
ICDC China CDC
§ These two authors contribute equally to this work.
Introduction ► As early as 1963, Abel et al. and Kaneda, respectively, presented evidences to suggest that cellular fatty acids (CFAs) analysed by gas chromatography could be successfully used to identify bacteria. Other early studies helped make CFAs analysis an established and widely accepted method for bacterial identification and classification. ►Previous studies demonstrated that cellular fatty acids (CFAs) analysis was a useful tool for identifying unknown strains of Bartonella because Bartonella species as a group have a unique and characteristic CFAs profiles. ►For establishing taxonomic relationships between the species, gas chromatography (GC) was utilized to investigate CFAs composition of the isolates from different animal hosts including cats, dogs, monkeys, rats, mice and voles, and the ATCC reference strains of Bartonella spp.
The Fatty Acids Composition of Several Bartonella spp. in References
WELCH ET AL. 1992
Methods ► For developing a standardized method to determine the CFAs of Bartonella spp., the following factors that might influent the qualities and quantities of CFAs compositions were investigated. Harvesting time; Culture conditions (medium, temperature, blood & subculrue) ► The whole cell fatty acid methyl esters (FAMEs) were obtained by aponification, methylation and extraction followed with analysis using a standardized Microbial Identification System (MIS).
Results ►The different base medium and concentration of blood, subculture, growth phase and temperature of incubation had a more significant impact on the relative proportions and components of minor CFAs than those of major CFAs. The most stable and reproducible CFAs profiles is achieved by carefully regulating and standardizing the growth conditions. ►ALL ten reference strains of Bartonella spp. are quite similar in major CFAs profiles, which include 18:1ω7c, 16:0 and 18:0, accounting for more than 85% of the total CFAs. ►The components and proportions of CFAs are different among the reference strains of Bartonella species. There are distinct difference in quantities of 18:0, 16:0, 14:0 and 18:1ω9c, which were selected from the components of significant difference including 12:03OH, 14:0, 16:0, 16:03OH, 18:1ω9c, 18:1ω5c, 18:0, 19:0 and 20:1ω7c between B. henselae and B. grahamii based on nonparametric tests and discriminant analysis. ► The dendrogram of CFAs from the wild isolates from cats which were B. henselae and the reference strains revealed a approximately consistent with the hierarchical structure of molecular phylogenetic systematics.
Comparison of the Effects of Different Medium on CFAs of B. henselae 60
9 8 7 6 5 4 3 2 1 0
TSA CBA SIM 30 15
Contents(%)
Contents (%)
45
0 16:0
18:1 w7c
TSA
CBA
SIM
11:0 12:0 14:0 16:1 17:0 16:0 18:2 18:1 18:1 20:1 ISO 3OH w7c 3OH w6,9c w9c w5c w7c 3OH CFAs
18:0
CFAs
Liquid culture by Schneider’s Insect Medium 2.4
Comparison of the Effects of Different Temperature of Culture on CFAs of B. henselae 70 37 °C Content s（%）
50
35 °C
40 30 20 10 0
37 °C
Bd 1.6
2.00
OD 600 nm
35 °C
1.00
0.00 16:0
18:1 w7c CFAs
11:0 ISO 3OH
18:0
12:0 3OH
14:0
16:1 w7c
17:0
5th Generation
16:0 3OH
18:2 w6,9c
18:1 w9c
0.4
6th Generation
30 15
4th Generation
4 4 4 4 4
Bvv
5
Bvb
5
Bq
5
Bh
5
0
5th Generation
4 Content (%)
Content (%)
45
Bva
1.2
CFAs
5
4th Generation
Bt
0.8
Comparison of the Effects of Subculture on CFAs of B. henselae 60
Bg
2
3.00
60 Contents (%)
Be
Harvesting time (Day)
0
6th Generation
1
3
2
3
4
5
Time (Days)
2
6
7
8
9
Dendrogram Based on Cellular Fatty Acid compositions of Bartonella Strains
1 Rotated Component Matrixa
0
0 16:0
18:1 w7c
11:0 ISO 3OH
18:0
CFAs
12:0 3OH
14:0
16:1 w7c
17:0
18:2 w6,9c
18:1 w9c
CFAs
18:1 w5c
3.5 5%
50 40 30 20
1.5 1
0
0 18:1 w7c
10%
2
0.5 16:0
5%
2.5
10
12:0 14:0 16:1 17:1 17:0 16:0 18:2 18:1 18:1 19:0 20:1 3OH w7c w6c 3OH w6,9c w9c w5c w7c
18:0
Component
CFAs
2
1
-.186
C12:13OH
.079
C12:03OH
.095
-.037
2 -.130
.303
-.140
.387
C14:0
1.344
.762
C16:1w7c
2.723
.673
C16:0
6.720
C17:0anteiso
-5.726
C17:0
.304
C16:03OH
3 Content (%)
Content (%)
10%
Component
C11:03OH
Comparison of the Effects of Different Concentration of Blood in the Medium on CFAs of B. henselae 60
Rescaled
1
CFAs
70
Raw
5.120
.781
.595
-.946 .276
.144
-.547 -.131
.131
• Differentiation of Bartonella spp. from A. tumefaciens, E. coli, S. aureus by plotting the ratios of the components of CFAs in the table- Rotated Component Matrix.
-.490
C18:2w69c
.097
.206
-.279
C18:1w9c
.129
C18:1w7c
13.832
-12.130
.751
-.659
C18:0
1.608
-1.513
.274
-.257
.209
• Principal component analysis and factor analysis show that PC1 were C17:0, C12:03OH and C18:1w9c and PC2 were C14:0, C16:1w7c and C16:03OH which could be contributed to differentiate Barotnella spp . from the other Bacteria .
CFAs
The Comparison of Cellular Fatty Acids Components of Bartonella species Strains
11:0 3OH 12:0 3OH
Bd Bva Bt Bvb Be Bq Bvv Bc Bh Bg At
E.coil S.aureus
3.65 7.11 2.5 3.47 1.8 2.51 1.11 3.6 2.72 4.78 2.23 2.64 2.41
0.26 0.38 0.61 0 0.33 0.2 0.33 0.62 0.16 0.58 0 0 0
14:0
16:1w7c
16:0
17:0
0.37 0.51 0.75 0.43 0.69 0.45 0.6 0.45 0.25 0.16 0.15 7.1 0.17
2.21 1.63 1.8 2.8 1.78 1.23 2.02 1.15 0.73 0.89 3.14 16.49 0
19.91 22.07 32.21 21.71 27.86 26.14 24.2 14.45 11.08 21.67 9.39 24.3 1.41
3.4 5.24 0.2 0 1.09 0 6.26 0.86 3.26 0.89 0 3.32 0.19
18:2w69c 18:1w9c 0.29 1.26 0.56 0.5 0.53 0.44 0.77 1.38 0.56 1.29 0 0 0.23
0.42 2.03 0.78 0 0.94 0 0.67 1.5 0.84 0 0 0 0
18:1w7c
18:0
62.84 44.27 44.43 62.35 51.53 56.5 54.15 55.31 59.27 54.98 62.69 18.96 0.64
7.39 10.26 12.45 6.64 12.33 11.05 8.18 16.75 22.24 13.56 0.75 0.43 4.47
Discussions
The more data of the wild Bartonella strains from the different hosts should be supplemented to validate the discriminant ability of CFAs at Bartonella species level. The detailed interpretation of CFAs profiles in terms of Bartonella taxonomy must be viewed with caution until our knowledge of the quantitative and qualitative distribution of fatty acids over a wide variety of taxa and the effects of growth conditions on fatty acid profiles is more extensive.
References [1] Clarridge JE 3rd, Raich TJ, Pirwani D, Simon B, Tsai L, Rodriguez-Barradas MC, Regnery R, Zollo A, Jones DC, Rambo C. Strategy to detect and identify Bartonella species in routine clinical laboratory yields Bartonella henselae from human immunodeficiency virus-positive patient and unique Bartonella strain from his cat. J Clin Microbiol. 1995. 33: 2107-2113. [2] Welch DF, Pickett DA, Slater LN, Steigerwalt AG, Brenner DJ. Rochalimaea henselae sp. nov., a cause of septicemia, bacillary angiomatosis, and parenchymal bacillary peliosis. J Clin Microbiol. 1992. 30: 275-280. [3] Daly, J. S., M. G. Worthington, D. J. Brenner, C. W. Moss, D. G. Hollis, R. S. Weyant, A. G. Steigerwalt, R. E. Weaver, M. I. Daneshvar, and S. P. O’Connor. Rochalimaea elizabethae sp. nov. isolated from a patient with endocarditis. J. Clin. Microbiol. 1993. 31:872-881.
中国疾病预防控制中心,传染病预防控制所,媒介生物控制室 Raleigh, NC, USA, April 27, 2012
