Abstract
Cellular fatty acids of Helicobacter pylori have taxonomic, physiological, and pathogenic implications. However, little is known about the fatty acid composition under various culture conditions. H. pylori is usually grown on blood-supplemented complex media, and the fatty acids in the blood may affect the fatty acids in the cells. In addition, frequently subcultivated laboratory-adapted strains may have properties different from those of fresh clinical isolates, which are culturable only for a limited number of passages. Therefore, the cellular fatty acid profiles of laboratory-adapted strains (LAS) and freshly isolated strains (FIS) were compared after growth on agar that was fatty acid free and growth on blood agar that contained fatty acids. LAS ATCC 43504, 51932, and 700392 and the FIS IMMi 88, 89, and 92, each with <10 subcultures, were cultured in parallel on a fatty acid-free agar (ISAF) and on 5% sheep blood agar (SBA), which contained oleic acid (18:1 9c), hexadecanoic acid (16:0), and octadecanoic acid (18:0). ISAF-grown cultures showed no 18:1 9c and no appreciable differences between the profiles of FIS and LAS. After culture on SBA, the strains showed 18:1 9c and increased 16:0 and 18:0 content combined with decreased tetradecanoic acid (14:0) content compared to ISAF-grown cells. The changes in the fatty acid profiles were much more pronounced in FIS than in LAS. LAS are obviously characterized by a lower uptake of the fatty acids from the growth medium than FIS. Furthermore, it could be shown that this LAS behavior is most likely a primary strain attribute that is favored under laboratory conditions. The pronounced uptake of fatty acids by strains with FIS behavior may be associated with the expression of virulence properties.
Helicobacter pylori exhibits a unique profile of cellular fatty acids which is markedly different from that of other enteropathogenic gram-negative bacteria (4-7, 11, 21). Tetradecanoic acid (14:0) and methyleneoctadecanoic acid (19:0 cyclo) are major fatty acids, whereas hexadecanoic acid (16:0) is represented in relatively small amounts, and hexadecenoic acid (16:1) cannot be detected at all (4-7, 11, 15). One property that is unusual but characteristic is the fatty acid distribution among the different lipid classes. The phospholipids are predominantly substituted with 14:0 and 19:0 cyclo fatty acids, whereas β-hydroxy or unsaturated fatty acids can only be detected, if at all, in small amounts, which may account for unusual membrane properties (4, 15, 18). The lipopolysaccharide (LPS) of H. pylori is predominantly different from those of other bacteria and has octadecanoic acid (18:0) and longer-chain 3-hydroxy fatty acids, like 3-hydroxy-hexadecanoic acid (3-OH 16:0) and 3-hydroxy-octadecanoic acid (3-OH 18:0), which may explain the low endotoxic and biological activities of H. pylori LPS (4, 13-15). Thus, the fatty acid substitution of H. pylori lipids may have physiological and pathogenic implications.
It is well known that environmental and physiological factors affect the fatty acid composition of bacteria (10, 12, 17). However, fatty acid analysis of H. pylori is usually performed with blood-supplemented growth media. Fatty acids in the blood may affect the cellular fatty acid profile (8), and easy-to-handle laboratory-adapted strains may have properties other than those of the often limited subculturable fresh clinical isolates (1). Therefore, the aim of the present investigation was to compare the cellular fatty acid profiles of laboratory-adapted strains (LAS) and freshly isolated strains (FIS) by using a fatty acid-free medium and a medium containing fatty acids. Furthermore, we examined whether fatty acid profiles of FIS and LAS change with multiple subcultivations and whether different fatty acid profiles can be found among FIS.
MATERIALS AND METHODS
Bacterial strains.
The H. pylori strains ATCC 43504, ATCC 51932, ATCC 700392, and IMMi 676 have a long subcultivation history and were therefore defined as LAS. H. pylori ATCC 43504 is identical to strain NCTC 11637 and is one of the most-investigated strains, particularly in fatty acid analysis (4-6, 14). Strain IMMi 676 was isolated from the stomach of a patient at the Universitätsklinikum Essen, Essen, Germany, in 1989, and since then has been subcultivated more than 100 times. The strains IMMi 27, 30, 88, 89, 92, 246, 389, 561, 562, 571, 576, 578, 580, 583, 589, 590, 597, 601, 605, and 606 are FIS. The strains were isolated and identified as described previously (1). After isolation from gastric biopsy specimens, the cultures were preserved in 10% mucin at −80°C (1). Recultivation was performed with sheep blood agar (SBA). Strains had less than 10 passages on SBA until fatty acid analysis.
Growth media.
Fatty acid-free agar (ISAF) was composed of Isosensitestagar (CM 471; Oxoid, Basingstoke, United Kingdom) supplemented with 0.5% fatty acid-free bovine serum albumin (A 6003; Sigma, St. Louis, Mo.). SBA was composed of blood agar base (CM 55; Oxoid) supplemented with 5% sheep blood (FSR 1055; Oxoid), 6 mg of vancomycin per ml (E. Lilly, Giessen, Germany), and 5 mg of amphotericin B per ml (Squibb-van-Heyden, Munich, Germany). The growth media ISAF and SBA (0.7 to 0.8 g) were processed for gas-liquid chromatography (GLC).
Influence of growth medium on the fatty acid compositions of FIS and LAS.
The ATCC 43504, ATCC 51932, and ATCC 700392 strains were used as LAS, and the IMMi 88, 89, and 92 strains were used as FIS. Parallel cultures of each strain on ISAF and SBA were performed at 37°C for 5 days in a microaerobic atmosphere (Anaerocult C; E. Merck, Darmstadt, Germany) with GasPak jars (BBL, Heidelberg, Germany). Cells were then harvested and processed for GLC.
Influence of repeated subcultivation on the fatty acid composition of FIS and LAS.
ATCC 43504 and IMMi 676 were used as LAS; IMMi 246 and 389 were used as FIS. Strains were cultured on SBA up to 25 passages. Cells of every second passage were harvested and processed for GLC.
Comparison of fatty acid profiles of FIS on SBA.
Five-day-old cultures of IMMi 27, 30, 561, 562, 571, 576, 578, 580, 583, 589, 590, 597, 601, 605, and 606 were performed on SBA and compared with a 5-day-old culture of ATCC 43504 on SBA. Cells were harvested and processed for GLC.
Preparation of FAMEs.
Fatty acid methyl esters (FAMEs) were prepared—including saponification, methylation, and extraction—according to the instructions of the MIDI system (M. Sasser, MIDI technical note 101. 1990. MIDI, Inc., Newark, Del.). Approximately 100 mg (wet weight) of culture material was processed.
GLC.
The FAMEs were analyzed with the following Hewlett-Packard (Avondale, Pa.) gas chromatographic system: a 5890 series II gas chromatograph equipped with a split inlet, a flame ionization detector, automatic sampler 6890, and a fused-silica capillary column (Ultra 2, HP 19091 B-102; 25 m by 0.2 mm) with 5% cross-linked phenylmethyl silicone (film thickness, 0.33 μm) as the stationary phase. The instrument was coupled with a Vectra XU 5/90C computer loaded with 3365 series II Chemstation (version 3.34) software. The chromatography parameters followed the instructions of the MIDI system (Microbial Identification System, operating manual, version 5.0, 1995. MIDI, Inc.): hydrogen as the carrier gas, sample volume of 2 μl, split ratio of 1/100, injector temperature of 250°C, detector temperature of 300°C, and column temperatures ramping from 170°C to 260°C at 5°C/min and from 260°C to 310°C at 40°C/min following an isotherm phase at 310°C for 4 min. The analysis time amounted to 25 min. FAME standard no. 1200-A (MIDI) was used for calibration of the system and as a quality control.
Processing of GLC data.
FAME peaks were identified, FAMEs were quantified, FAME profiles were compared, and dendrograms were created with system software (part 1300), microbial databases (parts 1301 and 1302), and library generation software (part 1303) of the Sherlock software package of MIDI. The dendrogram program was used to determine the relationship between fatty acid profiles. The program calculates the Euclidian distance (ED) as a resemblance coefficient.
Statistical methods.
Differences between the percentages of fatty acids were determined by using the Student's t test. The level of significance was P = 0.05.
RESULTS
GLC analysis of the fatty acids of the growth media ISAF and SBA showed that ISAF was free of fatty acids, whereas SBA contained several fatty acids. The three main fatty acids of SBA were oleic acid (18:1 9c) at 45% and octadecanoic acid (18:0) and hexadecanoic acid (16:0), both at 15%. The fatty acids in SBA derived from the supplementation with sheep blood that contained the same fatty acids, whereas the blood agar base was free of fatty acids.
After culture on the ISAF, seven fatty acids with area percentages of >1% were regularly found in LAS as well as in FIS (Table 1): Tetradecanoic acid (14:0) and methyleneoctadecanoic acid (19:0 cyclo) were the main fatty acids, followed by octadecanoic acid (18:0), 3-hydroxyoctadecanoic acid (3-OH 18:0), hexadecanoic acid (16:0), 3-hydroxyhexadecanoic acid (3-OH 16:0), and the 18:1 complex, a fatty acid fraction of either cis-octadec-11-enoic, trans-octadec-9-enoic, or trans-octadec-6-enoic acid, which could not be discriminated by this method. Oleic acid (18:1 9c) was regularly absent. The average distribution of the fatty acids showed no significant differences between LAS and FIS. After culture on SBA, the same seven fatty acids were detected as with ISAF-grown cells. Additionally, 18:1 9c was regularly present (Table 1). However, the profile of fatty acids showed significant differences between LAS and FIS, particularly regarding 14:0, 16:0, 18:0, and 18:1 9c (Table 1). The differences between LAS and FIS after growth on SBA were also corroborated by dendrogram analysis (Fig. 1). The LAS formed a cluster linked at an ED of about 13, and the FIS formed a separated cluster linked at an ED of about 17. The two clusters are linked at an ED of about 24. The dendrogram of LAS and FIS grown on ISAF revealed no corresponding formation of a LAS cluster and a FIS cluster (dendrogram not shown).
TABLE 1.
Average cellular fatty acid composition of LAS and FIS of H. pylori after culture on ISAF and SBA
| Fatty acid | % (± SD) of total fatty acidsa
|
|||
|---|---|---|---|---|
| ISAF
|
SBA
|
|||
| LAS | FIS | LAS | FIS | |
| 14:0 | 47.7 ± 5.3 | 44.5 ± 10.8 | 45.0 ± 3.1 | 26.1 ± 3.7c |
| 16:0 | 1.4 ± 0.3 | 1.5 ± 0.7 | 2.4 ± 1.0 | 4.4 ± 0.5b,c |
| 3-OH 16:0 | 2.2 ± 0.8 | 3.1 ± 1.0 | 2.1 ± 0.4 | 2.9 ± 0.71 |
| 18:1 9c | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.9 ± 0.3b | 1.5 ± 0.25b,c |
| 18:1 complex | 6.3 ± 3.2 | 10.9 ± 5.8 | 6.4 ± 3.1 | 11.0 ± 4.4 |
| 18:0 | 5.0 ± 1.2 | 5.1 ± 1.6 | 7.3 ± 1.1 | 15.9 ± 3.0b,c |
| 19:0 cyclo | 29.7 ± 3.7 | 28.0 ± 6.2 | 29.2 ± 4.3 | 30.2 ± 5.7 |
| 3-OH 18:0 | 3.9 ± 0.8 | 4.7 ± 1.1 | 4.0 ± 1.0 | 6.0 ± 1.3 |
| Not identified | 3.8 ± 2.0 | 2.2 ± 0.6 | 2.7 ± 1.8 | 2.0 ± 1.8 |
LAS comprised ATCC 43504, ATCC 51932, and ATCC 700392, and FIS comprised IMMi 88, IMMi 89, and IMMi 92.
Significant difference compared to ISAF-grown cultures (P < 0.05).
Significant difference compared to LAS (P < 0.05).
FIG. 1.
Clustering of the fatty acid profiles of LAS (ATCC 43504, ATCC 700392, and ATCC 51932) and FIS (IMMi 88, 89, and 92) of H. pylori after culture on sheep blood agar for 5 days. The letters a and b mark parallel cultures of the same strain. Strains of the two main clusters differ significantly in their amounts of 16:0, 18:1 9c, 18:0, and 14:0 (P < 0.05).
The formation of a LAS cluster and a FIS cluster after culture on SBA, in contrast to culture on ISAF, was due to a growth medium-dependent change of the fatty acid distribution in FIS rather than in LAS (Table 1). When cultured for 5 days on SBA or ISAF, the fatty acid profiles of LAS showed no significant difference. Only the occurrence of traces of 18:1 9c in SBA-grown cells was obvious. The FIS, however, exhibited major changes in the fatty acid profile. The increase in 16:0, 18:1 9c, and 18:0 was statistically significant (Table 1). The increases in fatty acids 16:0 and 18:0 as well as the fatty acid 18:1 9c, which occurred only in SBA-grown cells, belong to the main fatty acids of the growth medium SBA.
Repeated subculture of two FIS and two LAS on SBA after 5 days of incubation showed no significant change in the fatty acid distribution during 25 passages. The FIS retained their higher content of 16:0, 18:1 9c, and 18:0 compared to LAS. Cluster analysis revealed two clusters linked at an ED of about 20 separating FIS from LAS (dendrogram not shown).
Cluster analysis of 15 FIS compared with one LAS revealed two clusters linked at an ED of about 13 (Fig. 2). One cluster contained two FIS and the LAS linked at an ED of about 6. The other FIS formed a cluster linked at an ED of about 7. In a comparison of the two FIS in the LAS cluster with the other FIS, a significant larger amount of 14:0 (average of 37.8% versus 27.3%) and significantly smaller amounts of 16:0 (average of 4.1% versus 5.8%), 18:1 9c (average of 1.8% versus 2.9%), and 18:0 (average of 13% versus 18%) were found, indicating a LAS profile of these FIS.
FIG. 2.
Clustering of the fatty acid profiles of 15 FIS (IMMi 27, 30, 88, 89, 92, 246, 389, 561, 562, 571, 576, 578, 580, 583, 589, 590, 597, 601, 605, and 606) and 1 LAS (ATCC 43504) after culture on SBA for 5 days. Strains of the two main clusters differ significantly in their amounts of 16:0, 18:1 9c, 18:0, and 14:0 (P < 0.05).
Compilation of the data gave the following results. (i) H. pylori cells cultured on fatty acid-free medium ISAF contained no 18:1 9c. (ii) Culture on SBA, which contained particularly 18:1 9c, 16:0, and 18:0, yielded cells with a higher content of these fatty acids than ISAF-cultured cells. (iii) FIS differed from LAS by a more pronounced increase of 18:1 9c, 16:0, and 18:0 during culture on SBA. (iv) FIS keep their pronounced fatty acid uptake from the blood-supplemented growth medium compared to LAS under multiple passages. (v) Comparison of the fatty acid profiles of FIS on SBA showed that few strains exhibit a LAS-like fatty acid profile.
DISCUSSION
The unsaturated fatty acid 18:1 9c has been repeatedly found as a constituent of H. pylori lipids (4, 20), although this fatty acid is typical for eukaryotic rather than bacterial cells (16, 19). The present investigation shows that 18:1 9c occurs when H. pylori is grown on the 18:1 9c-containing SBA, but not when grown on the fatty acid-free ISAF. Obviously, 18:1 9c is not produced by the H. pylori cells, but is taken up from the growth medium. The saturated fatty acids 16:0 and 18:0 are produced by the cells, as their presence in ISAF cultures shows. Their presence in larger amounts in SBA cultures, however, indicates that 16:0 and 18:0 are additionally taken up from the growth medium containing 16:0 and 18:0.
The degree of the uptake of fatty acids is obviously strain dependent. The FIS differ from the LAS by a more intense uptake of 18:1 9c, 16:0, and 18:0 combined with a more pronounced decrease in 14:0. Although the fatty acid profiles of H. pylori have been described in many publications (4-7, 11), this differential strain uptake has not been described before. This study's investigations of fatty acid profiles of FIS and their stability under prolonged subcultivation showed that this different behavior seems to be a primary rather than laboratory-acquired attribute of strains. After multiple passages, the two FIS examined kept their typical FIS-like profile with a pronounced uptake of 16:0, 18:1 9c, and 18:0, whereas among 15 FIS, 2 strains could be found to exhibit an LAS-like profile with smaller amounts of 16:0, 18:0, and 18:1 9c and a larger amount of 14:0 compared to the typical FIS profile.
In conclusion, this study has some implications for laboratory practice and further investigations: It is known that unsaturated fatty acids are toxic for bacterial cells (3, 8, 9). For H. pylori, it has been shown that 18:1 9c has dose-dependent toxic effects, leading to disruption of cell membranes and cell lysis (8). Therefore, the increased uptake of 18:1 9c by FIS may explain the difficulties that have been described in subcultivating clinical isolates repeatedly (1). The majority of isolates did not survive more than 10 subcultures on 10% SBA (1). This observation can be explained, because the present study shows that the majority of fresh clinical isolates have a typical FIS profile of their fatty acid distribution, indicating a higher uptake of fatty acids from blood-supplemented growth media and thus have a higher susceptibility to potentially toxic effects of these fatty acids. It can be assumed that LAS-profiled strains are generally characterized by a higher tolerance to fatty acids in the growth medium and thus can cope better with laboratory conditions. Consequently, the so-called LAS are laboratory-selected rather than laboratory-adapted strains. Therefore, it is advisable to use fatty acid-free media like ISAF for subcultivation of fresh isolates in order to avoid the loss of strains with higher uptake of and lower tolerance to fatty acids, respectively.
Recently two colony variants, S and L, were described in a fresh clinical isolate grown on blood agar (2, 20). These colony variants were found to exhibit different fatty acid profiles, with S variants showing amounts of 14:0 half those and amounts of 16:0 and 18:0 double those of L variants. These differences very much resemble the differences between FIS and LAS found in the present investigation. Moreover, these two colony variants differed in their expression of virulence factors. S variants with a FIS-like profile released vacuolating cytotoxin (Vac A) and urease and adhered to or invaded epithelial cells, whereas L variants with a LAS-like profile retained Vac A and urease and did not invade epithelial cells (2). However, further experiments are necessary to elucidate whether the different degree of fatty acid uptake is connected with other properties of the FIS and LAS.
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