Presence of mycobacterial L-forms in human blood: Challenge of BCG vaccination

Figures & data

Figure 1. “Non-cultivable elements” from blood isolates (No1; 34; 37, 48; 91; 96). Light microscopy of: (A, B, C) spherical and filamentous forms found in semisolid agar during the first phase/week of cultivation; (D, E, F) initiation of growth from filamentous elements during the second phase/week of cultivation. Magnification: 200x

Figure 2. Development of L-form growths in semisolid agar during the second phase/week of cultivation. Light microscopy of blood isolates (No1; 34; 37, 48; 91; 96): typical “fried eggs” colonies of different size, consistence and density (A, B, C, D); formation of biofilm with gliding motility at the periphery (E, F). Magnification: 400x

Figure 3. SEM of huge filamentous and membranous L-type formations with many small granular, oval or coccoid cells fit together (A-E) and tightly packed rods (F) from blood isolate No1 grown in semisolid agar during the second phase/week of cultivation. Bars: 100 μm (D); 10 μm (A, B, E, F).

Figure 4. SEM of L-form cells from blood isolate No1 grown in semisolid agar during the second phase/week of cultivation. (A) Cluster of granular forms; (B, C, F) groups of polymorphic granular, oval, rod and elongated filamentous forms; (D, E) typical coccoid and large spherical L-form bodies of different size. Bars: 10 μm (A, B,C, D, E); 1 μm (F).

Figure 5. TEM of L-form cells from blood isolate No1 grown in semisolid agar: (A-C) cell wall deficient cells observed during the first phase/week of cultivation; (D-F) heterogeneous population of reverting bacteria with partially or fully recovered cell walls found during the second phase/week of cultivation.

Figure 6. 16S rRNA PCR of blood L-form cultures (L) grown during the second phase/week of cultivation in semisolid agar. Legend: 1, DNA ladder 100 bp; 2,Water; 3, L1; 4, L6; 5, L14 6, L47; 7, L58; 8, L60; 9, L62; 10, L64; 11, L65; 12, L70; 13, L96; 14, L16; 15, L19; 16, L20; 17, L23; 18, L25; 19, L26; 20, L34; 21, L37; 22, L41; 23, L52; 24, L53; 25, L91; 26, L93; 27, M. tuberculosis H37Rv 10ˆ-5; 28, M. tuberculosis H37Rv 10ˆ-3; 29, DNA ladder 100 bp.

Table 1. IS6110 Real Time PCR of blood L-form cultures grown during the second phase/week of cultivation in semisolid agar.

Name	Type	Ct
M. tub. H37Rv	Positive Control	11.76
MG water	NTC	—
1	Unknown	39.26
6	Unknown	42.94
14	Unknown	42.37
16	Unknown	44.57
19	Unknown	43.53
20	Unknown	41.09
23	Unknown	39.89
25	Unknown	41.07
26	Unknown	45.07
34	Unknown	45.30
37	Unknown	43.87
38	Unknown	48.95
41	Unknown	47.53
47	Unknown	43.99
52	Unknown	43.55
53	Unknown	39.55
58	Unknown	42.94
60	Unknown	43.19
62	Unknown	41.10
64	Unknown	42.41
65	Unknown	39.60
70	Unknown	39.32
91	Unknown	41.56
93	Unknown	48.14
96	Unknown	40.02

MG-molecular grate water; NTC – non-template control.

Table 2. Spoligotyping profiles of blood L-form cultures grown during the second phase/week of cultivation in semisolid agar.

Sample	Spacers (1–43)
1	1111111111111101111101111110000000011111010
2	0001000100100000000010000000000010001011001
3	0000000010000000000000001001111000000000000
5	0100000000010000000001100110100000000001000
6	0111110000011000001101111010001000100000011
7	0000001000000000000001010000000011110100000
9	1100000010000000000001000000000111000000000
10	0011010010001001100000101000010000000000011
14	1111000011111000000001100000000000000000011
16	1111110011111001111101111111111010000101111
17	0000001000100000100111010000110000000000000
18	0100010000010000000000010111000000000001000
20	0111111111111101111101111111111111111101011
23	0010110010011100000101111011011100000000001
25	0010010010000000000000100010000000000000001
34	0011010011111000000101100001111100001000111
36	0000000000011101100000101111001001001000001
37	1111010010111101111101101001111100101101111
38	0000000000000000000110010001111000001000000
40	0000010000000000000001101001101000000100000
43	0111111110001001111001010011111100111001011
44	0000101000000000000000000110010010000000000
45	0000100000000000000000001110000100000100100
46	0000000010000000000000010110111010001100000
47	0011111111111100111101111111111111111101111
48	0001100000000000000100001110000000000000000
51	0000000010010100101000011111101010000101111
53	1101111010110000000000110111000010011100000
54	1101101101001000001110110011110010111100101
55	0111110110100001100000101011101100000111001
57	0001000000000000000001000110101001000100000
58	0000010010000000100001001000000000000000011
59	1101111000100000100001111000111111101000100
60	1111111011111101111101111111111011101101011
62	1111010010111101111101101111111111101101011
64	1111111111111101111101111111111011111101011
65	1111110011111101111101111111110001101101111
70	1111111011111100111101111111111011101101111
96	0011110001111101111101111111111111101100010
M. bovis BCG	1101111101111111111111111111111111111100000
M. tub. H37Rv	1111111111110011111111111111111100001111111
M. tub. H37Rvˆ10–6	0100001111110000011001111111111100001111111

(1) –presence of spacer (0) absence of spacer.