Arctic Dialogue in the Global World, Ulan-Ude, June 16-17, 2015 UDC 577.17:58(571.54+517.3) DOI: 10.18101/978-5-9793-0754-1-43-45
THE USE OF GAS CHROMATOGRAPHY-MASS SPECTROMETRY, MICROBIAL MARKERS IN THE STUDY OF THE MICROBIAL LANDSCAPE OF PATIENTS WITH DIFFERENT FORMS OF ACUTE PANCREATITIS ©Larisa Radnaeva1,2, Vladislav Saganov1, Vasilii Taraskin1,2*, Vladimir Khitrikheev1 1
Medical Institute, Buryat State University 36,Oktyabrskaya st., Ulan-Ude, Buryatia, Russia, 670002 2 Laboratory of Chemistry of Natural Systems, Baikal Institute of Nature Management SB RAS 6,Sakhaynova st., Ulan-Ude,Buryatia, Russia, 670047 *
[email protected]
Abstract. For the first time, to assess the state of microbial flora of patients with different forms of acute pancreatitis it was the method of gas chromatography-mass spectrometry, microbial markers. It is shown that the method can be used as a new method of diagnosing various forms of acute pancreatitis. Keywords: GC/MS,microbes-markers method, acute forms of pancreatitis. Recently, increasing attention is paid to developing new diagnostic methods of various diseases and improving the existing ones. Physical and physicochemical methods are becoming a frequent practice. One of the most promising methods in medicine is a hybrid method of gas chromatography-mass spectrometry (GC/MS), which combines sensitive, rapid and selective characteristics. The method applied to environmental, biotechnological and clinical problems was presented in a doctoral thesis of Prof. G.A. Osipov in 1996. In 2010 it was officially permitted to practice the new medical technology “Assessment of microecological status of a person by gas chromatography-mass spectrometry”. The method is highly accurate in determining the presence of molecular characteristics of microorganisms from the number of their cellular lipids — higher fatty acids, aldehydes, alcohols and sterols in the sample. The features of fatty acid composition are now used along with some other parameters in bacterial taxonomy and clinical bacterial diagnostics [1]. We have studied the microbial markers of the infected pancreatonecrosis using GC/MS. Existing standard methods of diagnosing the pancreatogenic infection delay in real time when operative therapy of patients with pancreatonecrosis becomes vital. It is well known that an indication for laparotomy surgery is an infection in the pancreas and retroperitoneum. For a variety of reasons the existing method of microbiological examination
of a patient in clinical laboratories includes analyzing only a dozen of species aerobic microorganisms among enterobacteria, aerobic cocci and pseudomonades [2] Material and methods Assay samples (blood) were collected in evacuated blood collection receptacles with heparin sodium. Samples preparation for analysis by GC/MS was performed according to the procedure [3]. Fatty acid methyl esters and trimethylsilyl ethers have been studied by GC/MS on gas chromatograph Agilent Packard HP 6890 with quadrupole mass-spectrometer HP MSD 5973N as a detector. The column has been used for chromatography — HP-5MS column with an inner diameter of 0.25 microns. The percentage of the mixture was calculated from the area of the gas-chromatographic peaks. The qualitative analysis is based on the comparison of retention time and total mass spectra of the corresponding pure compounds using the data library NIST08.L and standard mixtures (Bacterial Acid Methyl Esters (CP Mix, Supelco, Bellefonte, PA, USA), as well as by the number of inserted standard (deuteromethyl ether of tridecoic acid). Microflora of 12 patients with infected pancreatonecrosis and 18 apparently healthy people for comparing. Results Table 1 shows the dynamics of microorganisms during the process of treating the patients with infected pancreatonecrosis.
Table 1. Microflora of patients with infected pancreatonecrosis during treatment (n=12) (М±σ) № 1 2 3 4 5 6
Microorganisms, cells/gх10*5 Streptococcus (oral)
1. Healthy
114,8±26.1 [50-275]2 Eubacterium lentum 56,3±37,7 (group А) [95-74] 2,3,4,5,6 Bacillus cereus 27,5±6,9 [0-64] 2,3,4,5 Nocardia, 14:1d11 252,6±56,2 [102-326] Peptostrep-tococcus 1,6±1,5 anaerobius [0-14] 2,3,5 Moraxella/Acinetoba 3,0±1,6 c-ter [2-10] 2,3,4,5,6
2. On admission 55,7±28,2 [0-350] 319,3±66,9 [0-953] 6,3±3,3 [0-28] 171,7±47,1 [30-539] 197,3±170,8 [0-2069] 20,2±7,6 [0-82]
3.The 5th day 133,4±41,1 [0-350] 398,3±166,9 [0-1840] 179,9±106,4 [0-1091] 172,5±37,1 [51-343] 171,7±47,1 [0-1539] 53,6±18,4 [0-209]
4.The 10th day 131,4±41,1 [0-350] 310,7±53,3 [10-442] 128,5±99,0 [0-621] 176,0±27,7 [107-243] 4,1±3,9 [0-24] 27,7±10,9 [1-61]
5.The 15th day 6.The 20th day 83,9±48,9 [0-350] 257,1±47,6 [0-358] 6,7±3,3 [0-46] 204,7±62,0 [30-539] 298,6±295,1 [0-2069] 14,6±6,5 [0-46]
108,2±50,6 [0-262] 217,3±57,0 [18-408] 30,2±19,2 [0-99] 188,7±77,1 [29-545] 4,3±4,2 [0-21] 37,9±20,5 [0-134]
Norm 249 68 23 262 0 0
43
Arctic Dialogue in the Global World, Ulan-Ude, June 16-17, 2015 7
Pseudomonas aeruginosa Clostridium propionicum Actinomycetales
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
24 25
26 27 28 29 30 31
32
33 34 35
36 44
2,4±1,9 [2-20] 2,3,4,5,6 84,4±32,9 [0-220] 36,6±4,9 [15-59] Pseudono-cardia 24,2±5,4 [6-60] Streptomyces 49,7±13,7 [6-133] 2 Clostridium 2234,3±230,4 ramosum [1051-3241] 3,4 Fusobacte-rium/Hae3,7±2,1 mophylus [0-18] 2,3,4,5,6 Alcaligenes 31,7±4,4 2,3,4,5,6 [6-46] Rhodococcus 283,7±45,4 [63-512] 2,3 Staphylo-coccus 355,1±72,9 intermedius [119-778] 2 Corineform CDC125,9±22,5 group XX [32-250] Lactobacillus 5661,5±1868,3 [965-16220] Campylo-bacter 44,8±16,8 mucosalis [0-142] 2,3,4,5,6 Mycobacterium/Can 106,8±25,3 dida [0-239] Family Enterobacte0,11±0,01 riaceae (E. coli ипр.) [0-0,02] Eubacterium 0 moniliforme sbsp Cl. difficile 80,2± 12,8 [43-155] 2,3,4 Prevotella 0
47,8±10,4 [0-101] 14,1±13,9 [0-168] 33,2±6,5 [8-64] 39,9±10,4 [5-101] 92,7±31,1 [0-342] 3348,5±712,2 [1088-10177] 35,4±8,1 [7-92] 130,1±24,2 5 [28-257] 389,4±40,8 [228-571] 161,0±89,3 [161-4626] 252,7±51,2 [65-689] 4690,2±923,4 [1454-12203] 236,8±41,8 [79-462] 466,4±74,7 [157-993] 0,13±0,03 [0-0,2] 0,13±0,03 [0-0,2] 502,3±78,3 [152-737] 5
68,4±28,7 [0-298] 0
30,5±26,7 [14-81] 0
25,9±11,5 [0-78] 0
39,6±18,4 [0-90] 0
27,9±6,0 25,8±6,0 33,1±7,8 38,3±6,8 [7-68] [7-68] [9-61] [12-54] 43,5±13,5 51,0±23,3 53,0±13,1 54,8±10,9 [7-167] [20-167] [14-101] [14-94] 86,4±31,1 54,8±13,5 71,6±46,3 73,5±33,5 [0-342] [14-125] [0-342] [0-187] 3211,5±359,4 4211,5±359,4 2622,6±456,4 2044,5±617,7 [990-5142] [950-5542] [920-4228] [7-4622] 18,9±3,5 26,3±13,3 17,2±6,9 27,7±9,1 [9-42] [9-92] [0-45] [5-55] 74,6±12,9 90,5±14,9 63,3±23,3 79,7±24,0 [33-155] [38-357] [6-156] [6-156] 377,5±41,3 318,5±21,3 324,9±57,7 332,0±62,9 [143-571] [232-471] [124-565] [124-516] 790,1±176,9 806,1±276,9 803,6±271,3 1109,7±220,6 [332-2268] [416-868] [161-2268] [571-1882] 221,3±63,9 181,7±37,7 243,0±77,1 304,8±79,3 [57-689] [97-338] [97-689] [87-605] 4809,3±845,3 4152,7±822,1 5182,7±1221,1 5363,5±578,6 [472-5673] [3001-12203] [3197-6829] [926-12203] 243,7±52,4 280,5±72,9 219,7±34,3 250,5±17,6 [100-632] [122-632] [94-341] [212-305] 483,5±43,8 528,5±72,6 494,0±64,8 686,7±220,7 [169-705] [316-746] [296-705] [169-1392] 0 0 0 0
0 288 77 70 62 2000 0 48 423 756 605 6613 99 549 0
0
0
0
0
0
402,3±48,3 [155-537]
299,2±39,3 [158-384]
232,4±24,9 [143-312]
276,8±54,6 [143-529]
385
9,4±4,1 0 0 0,3±0,2 38 [0-38] [0-1] Eubacterium monili- 6589,8±1385,7 18143,6±3456,6 15234±2573,0 13426,7±217 8260,5±1851,9 9626,8±2082,5 6912 forme, 7,2 [7344- [1252-16626] [3192-16717] [1049-12508] [1628-45228] 5,6 [1628-37657] 2,3,4,5,6 E. nodatum, E. sabu20479] reum Staphylo-coccus 123,0±19,3 271,6±42,4 164,4±17,0 169,2±29,3 198,7±25,1 234,4±41,8 120 [57-220] 2,6 [122-456] [45-253] [43-252] [149-332] [150-384] Bifidobac-terium 2395,4±453,5 4411,9±745,2 4337,3±447,3 4319,8±844,2 2904,9±672,9 2539,0±611,9 5067 [355-3973] [1132-8025] 1 [2327-6635] [1507-6828] [718-4838] [2327-6828] Helicobacter pylori, 12,0±2,5 88,3±17,9 1512,3±592,7 14 75,8±14,7 119,3±20,5 87,1±27,6 h18 [0-22] 2,3,4,5,6 [29-240] 6 [29-3697] [14-179] 6 [72-179] 6 [28-240] 6 Clostridium 19,6±3,4 34,4±8,9 34,8±6,4 43,4±5,7 36,9±10,5 44,2±10,8 12 perfringens [4-35] 2,3,4,5,6 [10-80] 6 [12-81] [36-66] [12-80] [12-81] Enterococcus 1,9±1,3 74,4±26,4 89,3±28,7 43,8±9,2 59,0±20,3 45,7±15,9 290 [0-11] 2,3,4,5,6 [0-290] [5-290] [5-69] [0-151] [0-100] Propionibacterium 2033,4±339,1 3149,8±612,9 2717,9±347,9 2324,5±364,0 1935,4±170,9 2369,5±624,4 4480 spp. (P. freudenrei[770-4480] [747-3305] [1560-7827] [916-4480] [1819-4121] [1198-2537] chii) Streptococcus 253,7± 1553,4±889,1 539,4± 611,0± 524,3± 566,0± 229 mutans (anaerobic) 45,8 66,5 48,8 97,9 102,9 [284-11268] 3,4,5 [89-525] 2,3,4,5,6 [229-983] [404-725] [273-1010] [229-983] Herpes 60,4±12,4 332,9±78,9 408,6±99,1 430,2±97,8 219,9±86,5 277,0±102,7 59 [19-112] 2,3,4,5,6 [18-906] [49-1180] [70-673] [21-578] [21-578] 99,9±25,7 288,4±83,9 113,1±31,3 92,6±33,6 115,4±38,6 842 Microfungi,campeste 130,1±27,5 [0-212] [0-842] [0-216] [0-213] [0-213] rol [24-293] 3,4 Nocardia asteroides 287,3±115,2 714,7±150,9 493,4±63,6 512,0±98,7 669,6±225,8 790,8±241,2 274 [184-1194] 2,4 [205-1932] [274-1058] [305-897] [305-1932] [426-1932] Cytomegalovirus
30,2±3,7
0
65,2±15,2
126,3±21,7
99,2±25,2
55,9±18,3
67,7±21,2
166
Arctic Dialogue in the Global World, Ulan-Ude, June 16-17, 2015
37 38 39 40 41
Microfungi, sitosterol Ruminicoccus Actinomycetes 10Me14 E. lentum 7741 (group В) Actinomyces viscosus
[15-42] 2,3,4,5,6 130,1±27,5 [24-293] 4 372,6±50,1 [119-547] 2,3,4,5 165,4±20,0 [95-260] 6,0±20,1 [0-190] 2,3,4,5,6 503,4±84,6 [165-855] 2
[0-120] 4 145,1±42,7 [0-459] 1446,3±442,1 [0-5071] 163,1±56,5 [29-749] 138,1±45,7 [19-576] 1131,6±209,7 [250-2521]
[0-254] [45-216] [0-120] 179,6±41,3 103,0±27,4 96,5±46,4 [0-412] [41-216] [0-302] 1120,8±161,9 1440,4±299,9 1475,1±603,8 [443-2226] [698-2167] [598-5071] 131,8±30,9 109,6±44,6 110,0±25,1 [29-309] [30-264] [29-185] 75,9±19,9 96,2±12,3 73,6±28,7 [0-214] [70-153] [15-236] 1016,3±60,4 940,2±86,6 956,6±204,2 [629-1255] [732-1191] [588-2122]
[0-120] 173,9±75,1 [0-446] 2071,0±640,5 [940-5071] 108,0±19,2 [52-166] 84,5±32,3 [19-236] 986,3±241,0 [532-2122]
384 640 309 0 1190
Note: 2,3,4,5,6 — statistical significance (p
