Detection microbial fatty acid markers in liquor and blood of meningitis patients by using in situ Gas Chromatography - Mass Spectrometry measurements

Sample preparation

Liquor (0.5ml) were subjected to lipid extraction procedure in water-chloroform-methanol mixture (Folch procedure) followed by acid methanolysis of dry lipid residue in 0,4ml 1N HCl / methanol by heating to 80oC for 1hr. Phospholipid fatty acid methyl esters (FAME), lipopolysaccharide (LPS) hydroxy FAME were formed as a result and extracted with hexane. The hexane fraction were dried, and dry residue was silylated in 20mkl N,O-bis-trimethylsilyl-trifluoroacetamide (BSTFA) by heating at 80 oC for 15min.

Whole blood (0.05 ml) were dried with equal quantity of methanol at 80OC and methanolysed in 0,4ml 1N HCl / methanol by heating to 80oC for 1hr. Fatty acid methyl and trimethylsilyl derivatization procedure was the same as liquor case.

Gas chromatography - mass spectrometry (GC-MS) analysis.

Measurements were performed on a Shimadzu QP-2000 system equipped with a cross-linked methyl silicone capillary column (Ultra-1). Analysis of fatty acids was done at the oven temperature was started 120OC and then programmed to 320OC at 5OC/min. 1mkl of derivatized sample were injected to gas chromatograph at 2800C. Fatty acids and other lipid components after separation in GC column were ionized by electron impact and analyzed in the selected ion monitoring (SIM) mode using appropriate ions for thirty four substances distributed in five separate ion groups throughout analytical range from decanoic acid to heaviest cholesterol metabolites. For instance, ion 87 was used for nonhydroxy and ion 175 for hydroxy FA. Each substance was confirmed by another specific ion, molecular or fragmentous, which is characteristic in mass spectrum. Another confirmations were achieved by measuring the specific retention times and ratio of chromatographic peak areas for selected ions of single substance. The analysis of lipid moiety by GC-MS-SIM technique provides a convenient way of obtaining quantitative data in spite of surpassing background caused by blood cells components. This is because the strongest ions of blood cells were not included in SIM and time program. Known quantity of CD3- tridecanoate was added as internal standard.

Example 1.

Gas Chromatography - Mass Spectrometry investigation microbial markers in liquor. Sample LT-064. Patient M.A.
Fungal meningitis.

Fatty acid concentrations were considered in balance equations with partial include of single micro-organisms proposed as mixt infection members. Computerised solution appears to be effective in discovery of genera-species composition of symbiotic and invasive microflora and follow-up infection and disbiosis in body fluids with their 104 background lipid prevalence.

The resulting virtual concentration of micro-organisms in locus's, wich have contact with liquor is as follows:

No.

Genera, species

Sample content*

Normal level**

1

Clostridium perfringens

123

10

2

Bacteroides fragilis

0

2

3

Bacillus cereus

1838

83

4

Staphylococcus haemolyticus

0

13

5

Peptostreptococcus anaerobius

5289

19

6

Propionibacterium

243

71

7

Chlamidia trachomatis

0

1

8

Enterococcus

0

50

9

Enterobacteriaceae

1011

56

10

Fusobacterium/Haemophylus

0

21

11

Staphylococcus epidermidis

2296

125

12

Neisseria/Moraxella

0

0

13

Mycobacteria (TSA)

0

114

14

Candida albicans

860

136

15

Acinetobacter

0

8

16

Bacillus (Streptomyces)

3599

1800

17

Prevotela melaninogenica

0

0

18

Corinebacterium

0

0

19

Pseudomonas

0

96

20

Flavobacterium

0

0

21

Enterobacteriaceae (Serratia)

0

863

22

Klebsiella

0

397

23

Streptococcus

0

0

24

Eubacterium

0

526

25

Brucella, Francisella

1801

0

26

Streptococcus pneumonia

0

27

27

P.aeruginosa

0

84

28

Diphteroids

0

512

29

Cholestendiol (herpes)

0,0

0

30

Virus 366/382

7

0,3

31

Treponema

0

0,21

32

Ergosterol (fungi)

0

0

33

Brucella, Francisella

2630

0

*- calculated relative units which proportional to cells number
**- statistical level for blood in donor was taken as norm

The same data in diagram form:
Brown sticks - norm, blue sticks - patient

The content of Candida albicans marker (17:1, heptadecenoic acid) overestimate normal level substantially. As well as markes of Clostridium perfringens (10h18, 10-hydroxystearic acid), members of Enterobacteriaceae family (cyclo-heptadecanoic acid), Peptostreptococcus anaerobius (prevalent one) also Staphylococcus epidermidis and Bacillus are in excess of norm.

Cholesterol metabolites referred to viruses (item 30) were discovered.

The interesting finding are Francisella (Brucella) double marker - hydroxyhexadecanoic and hydroxyoctadecanoic acids. We choose Francisella filomiragia because of seven cases (Medline search results) confirmed this microbe as meningitis agent. More of it, F.filomiragia was found in CSF by CDC staff collaborators and confirmed by GC FAME's profile ( D.G.Hollis, ..., C.W.Moss, et al., J.Clin.Microbiol. 27(7), 1601-08, 1989).

Microbial markers were analyzed in blood of the same patient, and calculated microorganisms presented in the next table:

Gas Chromatography - Mass Spectrometry investigation the composition of microbial lipid markers in blood
Sample NT-064. Patient A.M.
Fungal meningitis.

Genera, species

Sample content*

Normal level**

1

Clostridium perfringens

23

10

2

Bacteroides fragilis

0

2

3

Bacillus cereus

286

83

4

Staphylococcus haemolyticus

30

13

5

Peptostr. anaerobius

368

19

6

Propionibacterium

82

71

7

Chlamidia trachomatis

0

1

8

Enterococcus

357

50

9

Enterobacteriaceae

614

56

10

Fusobacterium/Haemophylus

0

21

11

Staphylococcus epidermidis

0

125

12

Neisseria/Moraxella

0

0

13

Mycobacteria (TSA)

0

114

14

Candida albicans

1234

136

15

Acinetobacter

0

8

16

Bacillus (Streptomyces)

3614

1800

17

Prevotela melaninogenica

0

0

18

Corinebacterium

0

0

19

Pseudomonas

0

96

20

Flavobacterium

0

0

21

Enterobacteriaceae(Serratia)

0

863

22

Klebsiella

721

397

23

Streptococcus

0

0

24

Eubacterium

0

526

25

Bacteroides urealyticum

231

816

26

Streptococcus pneumonia

0

27

27

P.aeruginosa

0

84

28

Diphteroids

108

512

29

Cholestendiol (herpes)

1,7

0

30

Virus 366/382

2

0,3

31

Treponema

0

0,21

32

Ergosterol (fungi)

0

0

33

Helicobacter pylory

310

369

The same data in diagram form:
Brown sticks - norm, blue sticks - patient

There are Candida albicans (main microbe), bacteria of Enterobacteriaceae family, Enterococcus, Clostridium perfringens, anaerobic Peptostreptococcus anaerobius, and member of Bacillus which markers exceeds the normal level in blood of healthy people. Herpes virus cholestendiol is detectable.
The data does not mean, this bacteria are present in blood.
This microorganisms could be localised in various organs of human body, not really in blood.
They come through in blood as a products of phagocytosis in that organs, and treated in blood stream like other exogenous particles and substances.
Most of them come from liquor region. That ones which markers simultaneously appear in liquor. But Enterococcus and Klebsiella originate from another part of body because of their absence in liquor.

Example 2.

Gas Chromatography - Mass Spectrometry investigation microbial markers in liquor. Sample LT-065. Patient B.
Bacterial and Fungal meningitis/encephalitis.

Measurements before and after treating.

1. Before treating

Genera, species

Sample content*

Normal level**

1

Clostridium perfringens

59

10

2

Bacteroides fragilis

0

2

3

Bacillus cereus

861

83

4

Staphylococcus haemolyticus

62

13

5

Peptostr. anaerobius

1830

19

6

Propionibacterium

642

71

7

Chlamidia trachomatis

0

1

8

Enterococcus

123

50

9

Enterobacteriaceae

0

56

10

Fusobacterium/Haemophylus

111

21

11

Staphylococcus epidermidis

2238

125

12

Neisseria/Moraxella

0

0

13

Mycobacteria (TSA)

49

114

14

Candida albicans

817

136

15

Acinetobacter

0

8

16

Bacillus (Streptomyces)

4564

1800

17

Prevotela melaninogenica

0

0

18

Corinebacterium

0

0

19

Pseudomonas

0

96

20

Flavobacterium

861

0

21

Enterobacteriaceae(Serratia)

3126

863

22

Klebsiella

984

397

23

Streptococcus

0

0

24

Eubacterium

0

526

25

Bacteroides urealyticum

1394

816

26

Streptococcus pneumonia

763

27

27

P.aeruginosa

0

84

28

Diphteroids

295

512

29

Cholestendiol (herpes)

0,0

0

30

Virus 366/382

0

0,3

31

Treponema

0

0,21

32

Ergosterol (fungi)

0

0

33

Helicobacter, Francisella

1441

369

The same data in diagram form:
Brown sticks - norm, blue sticks - patient

BEFORE TREATING

Microflora composition differ against normal in excess of Bacillus, Staphylococcus epidermidis, Peptostreptococcus anaerobius, Streptococcus pneumonia, Enterobacteriaceae sps., Propionibacterium, Clostridium perfringens, Candida albicans and others, and also occurance of Flavobacterium. Exceed of hydroxyhexadecanoic and hydroxyoctadecanoic acids could be assessed also to Bacteroides and Helicobacter (items 24,33) as compared to Example 1. But Francisella could be suspected too.

2. After treating with diflucan

Genera, species

Sample content*

Normal level**

1

Clostridium perfringens

61

10

2

Bacteroides fragilis

0

2

3

Bacillus cereus

0

83

4

Staphylococcus haemolyticus

248

13

5

Peptostr. anaerobius

1491

19

6

Propionibacterium

389

71

7

Chlamidia trachomatis

0

1

8

Enterococcus

0

50

9

Enterobacteriaceae

0

56

10

Fusobacterium/Haemophylus

139

21

11

Staphylococcus epidermidis

2296

125

12

Neisseria/Moraxella

153

0

13

Mycobacteria (TSA)

0

114

14

Candida albicans

232

136

15

Acinetobacter

0

8

16

Bacillus (Streptomyces)

3017

1800

17

Prevotela melaninogenica

0

0

18

Corinebacterium

0

0

19

Pseudomonas

0

96

20

Flavobacterium

0

0

21

Enterobacteriaceae(Serratia)

885

863

22

Klebsiella

496

397

23

Streptococcus

0

0

24

Eubacterium

0

526

25

Bacteroides urealyticum

1202

816

26

Streptococcus pneumonia

114

27

27

P.aeruginosa

64

84

28

Diphteroids

646

512

29

Cholestendiol (herpes)

0,0

0

30

Virus 366/382

0

0,3

31

Treponema

0

0,21

32

Ergosterol (fungi)

0

0

33

Helicobacter, Francisella

1962

369

The same data in diagram form:

AFTER TREATING

As compared to previous analysis quantity of Enterobacteriaceae sps., , Candida albicans lowered to normal level. Flavobacterium was eradicated. Streptococcus pneumonia and Bacillus decreased, but still exceed norm., S.epidermidis, , Peptostreptococcus, Propionibacterium, Clostridium perfringens and Francisella remain prevalent.

Unfortunately, new pathogen, Moraxella was appeared.