Vol. 29, No. 2
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1991, p. 315-322
0095-1137/91/020315-08$02.00/0 Copyright © 1991, American Society for Microbiology
Application of Gas-Liquid Chromatographic Analysis of Cellular Fatty Acids for Species Identification and Typing of Coagulase-Negative Staphylococci PIRKKO KOTILAINEN,l.2* PENTTI HUOVINEN,l AND ERKKI EEROLA' Department of Medical Microbiology, Turku University,' and Department of Medicine, Turku University Central Hospital,2 Turku, Finland Received 21 August 1990/Accepted 7 November 1990 Gas-liquid chromatography (GLC) of bacterial cellular fatty acids was used to analyze 264 isolates of coagulase-negative staphylococci, of which 178 were Staphylococcus epidermidis. The presence and amounts of individual fatty acids were determined to generate fatty acid profiles for each of the seven coagulase-negative species tested. The fatty acid profiles were then analyzed by computerized correlation and cluster analysis to calculate mean correlation values between isolates belonging to the same or different species, as well as to establish cluster analysis dendrograms. These data ultimately allowed the clustering of individual samples into species-specific clusters. Species identification by the GLC clustering was highly consistent with species identification by biochemical assays; the results were similar in 92.4% of the cases. The GLC profile correlation analysis was further used to analyze multiple blood isolates from 60 patients in order to determine the usefulness of this methodology in establishing identity, as well as differences, between consecutive patient isolates. The correlation between those multiple S. epidermidis isolates determined to be identical by standard techniques (such as the antibiogram, biotype, and plasmid profile) was significantly (P < 0.001) higher than that between random isolates of the same species. The correlation coefficient was >97 for 40 (97.6%) of the 41 patients with multiple identical blood isolates, compared with 90% (1, 5, 6, 11, 12, 25). Recently, alternative methods have been introduced which identify different coagulase-negative staphylococcal species by the chemical compounds present in the bacterial cells. These include electrophoretic profiles of the total bacterial proteins as well as of the penicillin-binding proteins (26, 33). In addition, efforts have been made to apply bacterial cellular fatty acid composition for species identification of staphylococci (7, 21, 30). By gas-liquid chromatographic (GLC) analysis (8, 30), the fatty acid profiles of the isolates belonging to the genus Staphylococcus have been shown to be distinct from those of other bacterial species. The fatty acid profiles of different species of coagulase-negative staphylo*
cocci, on the other hand, proved qualitatively to be highly similar (7, 8, 21, 30). However, quantitative differences in the fatty acid composition between various species were obvious in these studies. Because of the ubiquitous nature of coagulase-negative staphylococci in the environment, effective methods are needed for identification of clinically significant strains. The most common approaches for epidemiological typing include the antimicrobial susceptibility pattern (antibiogram), biochemical characterization (biotyping), bacteriophage susceptibility pattern (phage typing), serological typing (serotyping), and detection of bacterial adherence and slime production by the tube adherence test (4, 22, 23, 25). In addition, molecular techniques, including plasmid profile analysis as well as DNA-DNA hybridization and restriction enzyme analysis of chromosomal and plasmid DNA, have been applied for strain characterization (9, 10, 22, 23, 28). Many of these assays (such as antibiograms, biotyping, the tube adherence test, and molecular analysis of plasmids) are readily available in most clinical laboratories. Others (such as phage typing, serotyping, restriction endonuclease mapping, and DNA-DNA hybridization) are predominantly research tools. A computer-driven GLC analysis method (8) has recently been developed in our laboratory for identification of bacterial strains. This is operationally accomplished by analysis of bacterial fatty acid profiles. The purpose of the present study was to analyze the fatty acid composition of different species of coagulase-negative staphylococci. The mean relative amount of each detected fatty acid in the isolates, belonging to seven coagulase-negative species, was examined to test whether the bacterial fatty acid profiles could be used for reliable speciation of coagulase-negative staphylococci. In
Corresponding author. 315
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addition, we tested the adequacy of this technique as a novel scheme for the typing of coagulase-negative staphylococci. To do so, we compared the degree of GLC fatty acid profile similarity in consecutive blood isolates previously designated as identical or nonidentical by conventional typing methods. MATERIALS AND METHODS Bacterial strains and identification. Four reference strains were obtained from the American Type Culture Collection (Rockville, Md.), including S. epidermidis ATCC 35983 (RP12), ATCC 35984 (RP62A), and ATCC 14990 and S. hominis ATCC 35981 (RP14). Three additional reference strains (S. epidermidis KH11, KH6, and V2) were provided by Georg Peters from the Institute of Hygiene, University of Cologne, Cologne, West Germany. A total of 312 coagulasenegative staphylococcal isolates were recovered from blood cultures at Turku University Central Hospital and Turku City Hospital during the years 1983 to 1989. These included 135 isolates from 60 patients who had more than one positive blood culture and 177 isolates from patients with single positive blood cultures. Stock cultures of the strains were maintained at -70°C in tryptone soy broth (Oxoid Ltd., Basingstoke, England) containing 20% glycerol. Staphylococci were identified by standard methods, including Gram staining as well as catalase and tube coagulase tests. All isolates were speciated by the API Staph-Trac system (Analytab Products, Plainview, N.Y.). The isolates not groupable by this system were identified to the species level by a new test system, the ATB 32 STAPH (API System, SA, La Balme-les-Grottes, Montalieu-Vercieu, France). This system contains 26 biochemical reactions (26). Identity between the multiple staphylococcal isolates recovered from a given patient was determined by using an antibiotic susceptibility profile, by biotyping using the biochemical assays described above, by positivity or negativity in the tube adherence test, and by using plasmid profile analysis. Plasmid profiles were determined as described by Etienne et al. (9). When multiple isolates from the same patient were determined to be identical by these techniques, they were defined, for the purposes of this study, to be the same strain. GLC analysis. The GLC analysis of the bacterial cellular fatty acids was performed as described previously (8, 19). The bacteria were cultured for GLC on Fastidious anaerobe agar (LAB M, Bury, England) for 18 h at 37°C, collected from the plates, saponified, methylated, and analyzed as described earlier (8). In brief, the collected bacteria were incubated for 30 min at 100°C in 15% (wt/vol) NaOH in 50% aqueous methanol, and they were acidified to pH 2 with 6 N aqueous HCl in CH30H. The methylated fatty acids were then extracted with ethyl ether and hexane. The GLC analysis was performed with an HP5890A gas chromatograph (Hewlett-Packard) and an Ultra 2, 004-11-09B fusedsilica capillary column (0.2 mm by 25 m; cross-linked 5% phenylmethyl silicone; Hewlett-Packard). Ultra-high-purity helium was used as the carrier gas. The GLC settings were as follows: injection port temperature, 250°C; detector temperature, 300°C; initial column temperature, 170°C (increasing at 5°C per min up to 270°C at 20 min); total analysis time, 25 min; sample volume, 1 ,u. The peak retention time and peak area values were recorded by an HP3392A integrator
(Hewlett-Packard). The individual fatty acids were identified by comparing their retention times to those of a bacterial fatty acid
J. CLIN. MICROBIOL.
standard (bacterial acid methyl ester mix CP, 4-7080; Supelco, Inc., Bellefonte, Pa.). The relative peak areas of these fatty acids in each isolate were calculated. Also, the mean relative peak areas of each of the fatty acids within each coagulase-negative staphylococcal species were calculated. Processing of the GLC data. The GLC results, including the retention time values and peak areas of all detected peaks in the chromatograph, were transferred to a computer. Correlation and cluster analyses of the data were performed as described earlier (8, 31, 32). All GLC profiles were compared as pairs to calculate similarity indices between individual sample pairs. These values were further subjected to cluster analysis and presented as dendrograms in order to determine the capacity, efficiency, and reliability of the GLC methodology in grouping staphylococcal isolates into species-specific clusters. In addition, mean values and standard deviations of the similarity indices within each species and between different species groups were calculated. Statistical analysis. Differences in the amounts of the fatty
acids between different coagulase-negative species were evaluated statistically by using the Student t test. This test was also used to analyze differences in the mean correlation values between multiple identical and nonidentical patient isolates.
RESULTS Bacterial strains. Coagulase-negative staphylococcal blood isolates were grouped by the API Staph-Trac and ATB 32 STAPH procedures into seven different species: S. epidermidis, S. warneri, S. hominis, S. haemolyticus, S. capitis, S. simulans, and S. lugdunensis. All 177 single blood isolates were included in the mean fatty acid composition analysis of these seven coagulase-negative species. Characterization of the 135 multiple isolates from 60 patients revealed a subset of 96 isolates from 41 patients. In this latter group, two to four isolates from each patient were found to be identical by the antibiogram, biotype, tube adherence test result, and plasmid profile. Therefore, these two to four isolates were considered to be the same strain for each patient. The duplicate isolates were thus excluded from the fatty acid analysis. The remaining 39 multiple isolates from 19 patients proved to be nonidentical. From nine patients were recovered isolates belonging to different species. From the 10 remaining patients were recovered isolates of the same species, but with a different antibiotic susceptibility pattern and plasmid profile. All these isolates were included in the fatty acid analysis. Thus, the final strain collection consisted of 264 isolates of coagulase-negative staphylococci. These included the 177 single blood isolates, 80 of the multiple isolates (after exclusion of the multiple identical isolates), and finally, 7 reference strains. Of these isolates, 178 were grouped as S. epidermidis, 59 were grouped as S. warneri, 10 were grouped as S. hominis, 7 were grouped as S. haemolyticus, 6 were grouped as S. capitis, 2 were grouped as S. simulans, and 2 were grouped as S. lugdunensis. Bacterial fatty acid composition. The mean fatty acid compositions of the isolates of the seven coagulase-negative species studied are shown in Fig. 1, and the fatty acid compositions of five of these species, each represented by more than two isolates, are shown in Table 1. A total of 21 different fatty acids were detected in the chromatograms. Of these 21 fatty acids, 16 were identified by the fatty acid standard. The designations of the identified fatty acids are
VOL. 29, 1991
CHARACTERIZATION OF COAGULASE-NEGATIVE STAPHYLOCOCCI
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5 10 15 20 25 30 35 40 45 FIG. 1. Cellular fatty acid composition in seven species of coagulase-negative staphylococci. Mean relative peak areas ± standard deviation of the fatty acids are indicated on the vertical axes as percentages. The samples were analyzed for the presence of 46 fatty acids; numbers on the horizontal axis refer to the fatty acid elution order from the GLC column. The 21 fatty acids detected in the samples are listed in Table 1 under the same elution indicator numbers. Those 16 fatty acids identified by using the fatty acid standard are designated in Table 2.
given in Table 2. Essentially the same fatty acids were present in all analyzed coagulase-negative species. In general, the prevalence of any given fatty acid in the specimens was 100% if the relative amount of that fatty acid was greater than 1% of the total cellular fatty acid. Differences in the apparent prevalence were observed only in those fatty acids with the mean amount below 1% of the total content. The most abundant fatty acids of the staphylococci were as follows: Ci.15:0, Ca15:0, Ci-17:0, Ca-17:0, and C18:0. The relative amounts of the fatty acids Cji160, C16:0, C18:2912 ,
C18:19, and C20:0 and of three uncharacterized fatty acids, defined herein by the elution numbers 9, 14, and 35, were moderate or small. The relative amounts of the remaining eight fatty acids were insignificant. Only the S. epidermidis, S. warneri, S. hominis, S. haemolyticus, and S. capitis species (each represented by more than two isolates) were analyzed statistically. Statistical differences were calculated for those fatty acids with a mean amount of >1% of the total fatty acid content and a prevalence of 100%. Only the major differences in the predominant fatty acids are considered herein. Compared with the other species, S. epidermidis dis-
played a significantly larger (P < 0.010) amount of the fatty acid C18:0 and a smaller (P < 0.001) amount of the fatty acid Ca15:0. The amount of this same fatty acid Ca 15:0 was significantly (P < 0.001) larger in S. warneri, which in contrast contained a significantly (P < 0.002) smaller amount of C18:0. In S. hominis, the amount of the fatty acid Cai17:0 was significantly (P < 0.001) smaller than in the four other species; S. hominis contained a significantly (P < 0.050) larger amount of the fatty acid C20:0. The amount of the fatty acid Ca17:0 was significantly (P < 0.020) larger in S. haemolyticus than in the other species. Finally, the S. capitis species displayed a significantly (P < 0.001) larger amount of the fatty acid Ci 17:0 than did the others. There was considerable overlapping in the statistical differences between various species. For instance, the quantities of another uncharacterized fatty acid, defined by the elution number 14, were significantly (P < 0.001) larger in both S. epidermidis and S. hominis than in the other three species, the quantities of Ci 15:0 were significantly (P < 0.01) smaller in both S. warneri and S. haemolyticus than in the others, and the quantities of Cji17:0 were significantly (P < 0.020) smaller in both S. hominis and S. haemolyticus than in the others.
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TABLE 1. Mean composition of cellular fatty acids in five species of coagulase-negative staphylococcia
No.b
S. epidermidis (n = 178)
Fatty acid designationc
Peak area
Prev
8 9 14 16 18 19 20 21 24 25 26 28 29 31 35 36 37 39
44 45 46
C12:0 Cn Cn C14:0
0.00 1.23 1.86 0.61 7.37 40.59 0.00 0.20 2.06 0.03 2.88 6.49 15.76 0.36 1.63 1.56 1.61 9.98 0.61 0.82 4.35
0.6 99.4 100.0 100.0 100.0 100.0 2.9 55.2 100.0 11.6 100.0 100.0 100.0 97.1 100.0 100.0 100.0 100.0 99.4 95.3 100.0
Ci15:0 Ca-15:0
Cn C15:0
Cj16:0
C16:19 C16:0
Ci17:0
Ca17:0 C17:0 Cn C18:29'12 C18:19 C18:0
C19:0
Cn C20:0
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
S. hominis (n = 10)
S. warneri (n = 59)
Prev
0.04 0.32 0.34 0.22 1.17 2.39 0.03 0.22 0.33 0.07 0.59 1.00 2.31 0.12 0.32 0.69 0.62 1.67 0.20 0.44 1.34
0.0 100.0 100.0 53.4 100.0 100.0 1.7 50.0 100.0 1.7 100.0 100.0 100.0 94.8 100.0 100.0 91.4 100.0 84.5 98.3 100.0
Peak area
1.35 0.82 0.13 5.11 48.27 0.00 0.14 1.20 0.00 2.10 6.45 22.03 0.35 0.75 2.01 0.95 5.29 0.25 0.99 1.81
± ± ± ± ± ± ± ±
± ±
± ± ±
± ± ± ±
± ± ±
0.31 0.21 0.14 0.95 1.70 0.02 0.16 0.22 0.02 0.44 1.01 2.14 0.18 0.20 0.56 0.43 1.13 0.16 0.40 0.77
Prev
0.0 100.0 100.0 100.0 100.0 100.0 0.0 10.0 100.0 0.0 100.0
100.0 100.0 50.0 100.0 100.0 70.0 100.0 100.0 100.0 100.0
Peak area
2.05 2.06 0.49 8.23 45.50
± 0.42 ± 0.72 ± 0.21
± 1.07 + 2.51
0.03 ± 0.08 1.47 ± 0.39 1.91 5.63 13.12 0.16 1.93 1.04 0.82 8.52 0.68 1.09 5.28
± 0.32
± 0.73 + 2.93 ± 0.16
± 0.68 ± 0.08 ± 0.70 ± 1.20
± 0.19 ± 0.38 ± 1.55
S. haemolyticus (n = 7) Prev
0.0 100.0 100.0 57.1 100.0 100.0 0.0 42.9 100.0 0.0 100.0 100.0 100.0 85.7 100.0 100.0 71.4 100.0 100.0 85.7 100.0
Peak area
1.55 0.68 0.17 5.56 45.41
± 0.44 ± 0.23 ± 0.16
± 1.24 + 1.36
0.17 + 0.22 0.85 ± 0.28 2.15 5.47 24.09 0.28 0.53 1.04 0.60 8.28 0.32 0.60 2.23
± 0.44
± 0.99 ± 1.74 ± 0.15 ± 0.14 ± 0.22 ± 0.42 ± 1.23 ± 0.10 ± 0.42 ± 0.44
S. capitis (n = 6)
Prev
0.0 100.0 100.0 66.7 100.0 100.0 0.0 16.7 100.0 0.0 100.0 100.0 100.0 66.7 100.0 100.0 100.0 100.0 83.3 83.3 100.0
Peak area
1.05 ± 0.30 ± 0.19 ± 0.16 ± 0.86
0.60 0.17 7.76 45.48
± 1.32
0.08 ± 0.17 0.72 ± 0.18 1.73 7.96 19.62 0.19 0.57 2.26 1.30 6.90 0.20 0.90 2.52
± ± ± ±
± ± ± ±
± ± ±
0.51 1.00 2.36 0.19 0.11 0.53 0.31 1.15 0.14 0.47 0.26
a Prevalence (Prev) indicates the percentage of isolates with detectable fatty acid within each species. Peak area is expressed as the mean + standard deviation. The samples were analyzed for the presence of 46 fatty acids; No. refers to the elution order from the GLC column. These indicator numbers are used to define the detected fatty acids in the text, in Fig. 1 (horizontal axes), and in Table 2. c Of the 46 analyzed fatty acids, only the 21 fatty acids detected in the samples are listed. C. indicates an uncharacterized fatty acid. The fatty acids, identified according to the fatty acid standard, are designated in Table 2. b
Furthermore, all five coagulase-negative species differed significantly from each other in the quantities of the fatty
acid Ca17:0 detected. Correlation analysis. TABLE 2.
No.b 8 16 18 19 21 24 25 26 28 29 31 36 37 39 44 46
Similarity indices were calculated by
Fatty acids isolated from coagulasenegative staphylococcia
Designationc
C12:0 C14:0 Cji15:0 Ca-15:0 C15:0 Cji16:0 C16:19 C16:0 Cji17:0 Ca17:0 C17:0 C18:29'12 C18:1 9 C18:0 C19:0 C20:0
Name
Dodecanoate Tetradecanoate
13-Methyltetradecanoate 12-Methyltetradecanoate Pentadecanoate 14-Methylpentadecanoate
cis-9-Hexadecenoate Hexadecanoate
15-Methylhexadecanoate 14-Methylhexadecanoate Heptadecanoate
cis-9,12-Octadecadienoate
cis-9-Octadecenoate Octadecanoate Nonadecanoate Eicosanoate
a Staphylococci were analyzed for the presence of 46 fatty acids. Listed are the 16 fatty acids detected in the test samples and identified according to the fatty acid standard. b Numbering refers to the elution order from the GLC column. These indicator numbers are used to define the detected fatty acids in the text, in Fig. 1 (horizontal axes), and in Table 1. c The number before the colon refers to the number of carbon atoms, the number after the colon refers to the number of double bonds, i indicates a branched-chain acid with the branched methyl group at the iso position, and a indicates a branched-chain acid with the branched methyl group at the
anteiso position.
comparing the isolates within the same species as pairs and also by comparing the isolates of one species to the isolates of a different species. The means of similarity indices were then calculated within each species and between different species. Isolates belonging to the same species resembled each other more closely than isolates belonging to different species: the mean correlation of the fatty acid profiles of the isolates of the same species varied from 85.54 +7.68 in S. hominis to 95.65 in S. simulans and the mean correlation of the isolates of different species varied from 62.28 ± 5.87 between S. haemolyticus and S. simulans to 86.76 ± 4.56 between S. haemolyticus and S. warneri (Table 3). The GLC fatty acid profile similarities between the seven coagulasenegative species studied are presented in Fig. 2 as a dendrogram after cluster analysis of the mean correlations between these species. S. epidermidis and S. hominis formed one loosely related cluster, S. warneri, S. haemolyticus, S. capitis, and S. Iugdunensis formed another, and S. simulans was grouped in a separate cluster by itself. The individual isolates were also analyzed by a weightedpair cluster analysis. Results are presented as a dendrogram in Fig. 3. Isolates belonging to the same species formed separate clusters. Of the 178 S. epidermidis isolates, 168 formed a defined cluster. Also included in this cluster were five S. hominis and two S. warneri isolates. Another cluster was formed by 55 of the 59 S. warneri, 1 S. haemolyticus, and 3 S. epidermidis isolates. Additionally, a subcluster of six of the seven S. haemolyticus isolates was grouped into this cluster. Still other distinct clusters were formed by 6 S. capitis, by 2 S. lugdunensis, by 2 S. simulans, and by 5 of the 10 S. hominis isolates. Finally, seven S. epidermidis and two S. warneri isolates were adjusted to fall outside all clusters. When the species identification of staphylococci by the biochemical assays (API Staph-Trac and ATB 32 STAPH)
CHARACTERIZATION OF COAGULASE-NEGATIVE STAPHYLOCOCCI
VOL. 29, 1991
Species
S. S. S. S. S. S. S.
epid
TABLE 3. Cellular fatty acid GLC correlation between seven different species of coagulase-negative staphylococcia Mean correlation coefficient ± SD between species S. epid (n = 178)
S. war (n = 59)
S. hom (n = 10)
S. haem (n = 7)
S. cap (n = 6)
S. sim (n = 2)
S. lugd (n = 2)
87.27 ± 8.04
70.00 ± 11.27 91.24 ± 4.59
76.60 ± 8.49 63.93 ± 9.79 85.54 ± 7.68
73.12 86.76 67.82 91.42
77.43 82.26 75.05 82.54 89.10
66.28 ± 5.74 70.16 ± 4.66 65.43 ± 6.82 62.28 ± 5.87 68.62 ± 5.14 95.65
71.46 ± 9.58 79.15 ± 4.27 67.61 ± 8.32 81.30 ± 3.15 74.80 ± 2.68 66.41 ± 3.26 91.84
war hom haem
cap Sim lugd
± 11.1 ± 4.56 ± 7.47 ± 3.82
± ± ± ± ±
9.43 5.32 9.18 5.65 5.27
a Abbreviations: S. epid, S. epidermidis; S. war, S. warneri; S. hom, S. hominis; S. haem, S. haemolyticus; S. cap, S. capitis; S. sim, S. simulans; S. S. lugdunensis.
was compared to the clustering by the automated GLC analysis, identical results were obtained in 244 of 264 cases (92.4%). Within the individual species, the agreement between the methods was as follows: S. epidermidis, 168 of 178 (94.4%); S. warneri, 55 of 59 (93.2%); S. hominis, 5 of 10 (50.0%); S. haemolyticus, 6 of 7 (85.7%); S. capitis, 6 of 6 (100.0%); S. simulans, 2 of 2 (100.0%); and S. lugdunensis, 2 of 2 (100.0%). Identical results were obtained for all seven reference strains. A number of incongruently positioned samples in the various clusters was apparent. In the S. epidermidis cluster, 7 of 175 (4%) fell into this category. In the S. warneri cluster, 10 of 65 (15.4%) of the tested samples proved to be disparately positioned. This particular cluster, as stated above, contained a subcluster of six S. haemolyticus samples. Alternatively, if this cluster was reorganized to include a combined S. warneri-S. haemolyticus cluster, the incidence of variability in the positions of the samples would decrease to 4 of 65 (6.15%). For the S. capitis (n = 6), the S. simulans (n = 2), and the S. hominis (n = 5) clusters, none of the organisms were disparately positioned. As stated above, seven S. epidermidis and two S. warneri samples remained outside all clusters. Correlation analysis of the multiple isolates from a given patient. Similarity indices between consecutive patient isolates were calculated for all 60 patients from whom multiple blood isolates were recovered. The purpose was to determine whether the GLC profile correlation analysis could establish identity between those isolates which, in fact, were representative of the same bacterial strain. Those multiple isolates from a given patient, determined to be identical by the above-mentioned standard techniques, were operationally defined as the same strain. The correlation coefficients
S.epidermidis S.hominis S.warneri S.haemolyticus S.capitis S.lugdunensis S.simulans 0
319
50
100
FIG. 2. A dendrogram showing the GLC fatty acid profile similarities between seven species of coagulase-negative staphylococci. The dendrogram was established by cluster analysis of the mean values of similarity indices. Interspecies similarity indices are indicated on the horizontal axis.
lugd,
of the fatty acid profiles between the multiple patient isolates are shown in Table 4. The correlation coefficients of the multiple identical blood isolates from 41 patients varied from 92.79 to 99.01. In 90.2% (37 of 41) of the cases, the correlation value was >97; in 97.6% (40 of 41) of the cases, it was >95. The correlation values of the multiple, nonidentical blood isolates from 19 patients varied from 59.97 to 94.90. When the isolates were of the same species, the correlation value varied from 84.22 to 94.90 (mean, 89.99 + 3.87). The mean value of correlation between multiple nonidentical S. epidermidis blood isolates (from 8 patients) was 89.79 ± 4.04, which was quite comparable to the mean correlation value of 87.27 + 8.04 (Table 3) between random S. epidermis isolates. In contrast, a significantly (P < 0.001) higher mean correlation value between consecutive isolates, 97.65 + 1.16, was detected in those 33 patients who had multiple identical S. epidermidis blood isolates. DISCUSSION
Different species of coagulase-negative staphylococci can be unequivocally distinguished when stringent DNA-DNA hybridization conditions are used (29). This methodology, however, is highly specialized and used primarily as a research tool. At present, no completely reliable system practical as a routine diagnostic tool exists for identification of coagulase-negative staphylococci to the species level. Commercially available panels, which identify on the basis of functional differences in the metabolic pathways, do not allow species identification with certainty (25). Thus, alternative methods are being developed for speciation. Among the most interesting is whole-cell protein analysis; sodium dodecyl sulfate-polyacrylamide gel electrophoresis has provided species identification with a high degree of accuracy. By using this technique and immunoblotting, ThomsonCarter and Pennington (33) were able to characterize nine coagulase-negative species. According to Pierre et al. (26), the penicillin-binding protein profiles provide an even greater degree of species identification accuracy than does examination of the total solubilized proteins. Another interesting approach to species identification, the GLC analysis of cellular fatty acids, has indicated distinct quantitative differences between various coagulase-negative species, despite qualitative similarities (7, 21, 30). The purpose of this study was to further evaluate the applicability of the GLC fatty acid analysis for species identification of coagulase-negative staphylococci. Consistent with the previous findings (7, 8, 21, 30), no obvious qualitative differences in the fatty acid compositions were observed between the different coagulase-negative Staphy-
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-S.
haemolyticus
-S. warneri
C S .capitis -S.lugdunensis
-S.epidermidis
simulans hominis 50
0
100
the GLC fatty acid profile simicoagulase-negative staphylococci. The isolates were identified by the API Staph-Trac and ATB 32 STAPH procedures as seven different species: S. epidermidis, S. warneri, S. hominis, S. haemolyticus, S. capitis, S. simulans, and S. Iugdunensis. A weighted-pair cluster analysis grouped the isolates into species-specific clusters; the compositions of the seven clusters shown in the figure are explained in detail in the text. The fatty acid FIG.
3.
A
dendrogram showing
larities between 264 isolates of
profile
correlation between individual
horizontal axis.
isolates is indicated on the
lococcus species. Admittedly, a few fatty acids which produced very low mean peak areas were not detected in all coagulase-negative species studied. Additionally, differences in the apparent fatty acid prevalences were observed between individual isolates of those species in which the relative mean amount of that particular fatty acid was less than 1%. It should be noted here that in such cases, the observed differences most probably reflected a function of the detection limit of the method rather than any true dissimilarity. Although qualitative differences were not observed, various species differed significantly from each other in the relative amounts of several individual fatty acids. Some of the species-specific characteristics were quite distinctive. For example, S. epidermidis was characterized by a relatively large amount of the fatty acid C18:0, S. warneri was characterized by a large amount of the fatty acid Ca-15:0, 5 haemolyticus was characterized by a large amount of the fatty acid Cai17:0, and S. capitis was characterized by a large amount of the fatty acid Cji17:0. Still, there was too much overlapping in the quantitative differences between various species to permit reliable species identification on the basis of the quantity of any single fatty acid. The computerized fatty acid pattern correlation analysis did, however, separate the staphylococcal isolates into species-specific clusters, thereby allowing species identification. Two main clusters were formed by S. epidermidis and S. warneri, the species constituting the bulk of our strain collection. Although the number of isolates belonging to the five other species was few, even these isolates were divided into species-specific clusters. Species identification by GLC clustering was quite consistent with species identification by biochemical analysis; the results were similar in 92.4% of the cases. Moreover, correlation analysis demonstrated that the intraspecies similarities were higher than were the interspecies similarities. On the basis of the correlation values, the most distinct species were S. epidermidis, S. hominis, and S. simulans; the interspecies similarity indices between these and all other species were
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1991, p. 315-322
0095-1137/91/020315-08$02.00/0 Copyright © 1991, American Society for Microbiology
Application of Gas-Liquid Chromatographic Analysis of Cellular Fatty Acids for Species Identification and Typing of Coagulase-Negative Staphylococci PIRKKO KOTILAINEN,l.2* PENTTI HUOVINEN,l AND ERKKI EEROLA' Department of Medical Microbiology, Turku University,' and Department of Medicine, Turku University Central Hospital,2 Turku, Finland Received 21 August 1990/Accepted 7 November 1990 Gas-liquid chromatography (GLC) of bacterial cellular fatty acids was used to analyze 264 isolates of coagulase-negative staphylococci, of which 178 were Staphylococcus epidermidis. The presence and amounts of individual fatty acids were determined to generate fatty acid profiles for each of the seven coagulase-negative species tested. The fatty acid profiles were then analyzed by computerized correlation and cluster analysis to calculate mean correlation values between isolates belonging to the same or different species, as well as to establish cluster analysis dendrograms. These data ultimately allowed the clustering of individual samples into species-specific clusters. Species identification by the GLC clustering was highly consistent with species identification by biochemical assays; the results were similar in 92.4% of the cases. The GLC profile correlation analysis was further used to analyze multiple blood isolates from 60 patients in order to determine the usefulness of this methodology in establishing identity, as well as differences, between consecutive patient isolates. The correlation between those multiple S. epidermidis isolates determined to be identical by standard techniques (such as the antibiogram, biotype, and plasmid profile) was significantly (P < 0.001) higher than that between random isolates of the same species. The correlation coefficient was >97 for 40 (97.6%) of the 41 patients with multiple identical blood isolates, compared with 90% (1, 5, 6, 11, 12, 25). Recently, alternative methods have been introduced which identify different coagulase-negative staphylococcal species by the chemical compounds present in the bacterial cells. These include electrophoretic profiles of the total bacterial proteins as well as of the penicillin-binding proteins (26, 33). In addition, efforts have been made to apply bacterial cellular fatty acid composition for species identification of staphylococci (7, 21, 30). By gas-liquid chromatographic (GLC) analysis (8, 30), the fatty acid profiles of the isolates belonging to the genus Staphylococcus have been shown to be distinct from those of other bacterial species. The fatty acid profiles of different species of coagulase-negative staphylo*
cocci, on the other hand, proved qualitatively to be highly similar (7, 8, 21, 30). However, quantitative differences in the fatty acid composition between various species were obvious in these studies. Because of the ubiquitous nature of coagulase-negative staphylococci in the environment, effective methods are needed for identification of clinically significant strains. The most common approaches for epidemiological typing include the antimicrobial susceptibility pattern (antibiogram), biochemical characterization (biotyping), bacteriophage susceptibility pattern (phage typing), serological typing (serotyping), and detection of bacterial adherence and slime production by the tube adherence test (4, 22, 23, 25). In addition, molecular techniques, including plasmid profile analysis as well as DNA-DNA hybridization and restriction enzyme analysis of chromosomal and plasmid DNA, have been applied for strain characterization (9, 10, 22, 23, 28). Many of these assays (such as antibiograms, biotyping, the tube adherence test, and molecular analysis of plasmids) are readily available in most clinical laboratories. Others (such as phage typing, serotyping, restriction endonuclease mapping, and DNA-DNA hybridization) are predominantly research tools. A computer-driven GLC analysis method (8) has recently been developed in our laboratory for identification of bacterial strains. This is operationally accomplished by analysis of bacterial fatty acid profiles. The purpose of the present study was to analyze the fatty acid composition of different species of coagulase-negative staphylococci. The mean relative amount of each detected fatty acid in the isolates, belonging to seven coagulase-negative species, was examined to test whether the bacterial fatty acid profiles could be used for reliable speciation of coagulase-negative staphylococci. In
Corresponding author. 315
316
KOTILAINEN ET AL.
addition, we tested the adequacy of this technique as a novel scheme for the typing of coagulase-negative staphylococci. To do so, we compared the degree of GLC fatty acid profile similarity in consecutive blood isolates previously designated as identical or nonidentical by conventional typing methods. MATERIALS AND METHODS Bacterial strains and identification. Four reference strains were obtained from the American Type Culture Collection (Rockville, Md.), including S. epidermidis ATCC 35983 (RP12), ATCC 35984 (RP62A), and ATCC 14990 and S. hominis ATCC 35981 (RP14). Three additional reference strains (S. epidermidis KH11, KH6, and V2) were provided by Georg Peters from the Institute of Hygiene, University of Cologne, Cologne, West Germany. A total of 312 coagulasenegative staphylococcal isolates were recovered from blood cultures at Turku University Central Hospital and Turku City Hospital during the years 1983 to 1989. These included 135 isolates from 60 patients who had more than one positive blood culture and 177 isolates from patients with single positive blood cultures. Stock cultures of the strains were maintained at -70°C in tryptone soy broth (Oxoid Ltd., Basingstoke, England) containing 20% glycerol. Staphylococci were identified by standard methods, including Gram staining as well as catalase and tube coagulase tests. All isolates were speciated by the API Staph-Trac system (Analytab Products, Plainview, N.Y.). The isolates not groupable by this system were identified to the species level by a new test system, the ATB 32 STAPH (API System, SA, La Balme-les-Grottes, Montalieu-Vercieu, France). This system contains 26 biochemical reactions (26). Identity between the multiple staphylococcal isolates recovered from a given patient was determined by using an antibiotic susceptibility profile, by biotyping using the biochemical assays described above, by positivity or negativity in the tube adherence test, and by using plasmid profile analysis. Plasmid profiles were determined as described by Etienne et al. (9). When multiple isolates from the same patient were determined to be identical by these techniques, they were defined, for the purposes of this study, to be the same strain. GLC analysis. The GLC analysis of the bacterial cellular fatty acids was performed as described previously (8, 19). The bacteria were cultured for GLC on Fastidious anaerobe agar (LAB M, Bury, England) for 18 h at 37°C, collected from the plates, saponified, methylated, and analyzed as described earlier (8). In brief, the collected bacteria were incubated for 30 min at 100°C in 15% (wt/vol) NaOH in 50% aqueous methanol, and they were acidified to pH 2 with 6 N aqueous HCl in CH30H. The methylated fatty acids were then extracted with ethyl ether and hexane. The GLC analysis was performed with an HP5890A gas chromatograph (Hewlett-Packard) and an Ultra 2, 004-11-09B fusedsilica capillary column (0.2 mm by 25 m; cross-linked 5% phenylmethyl silicone; Hewlett-Packard). Ultra-high-purity helium was used as the carrier gas. The GLC settings were as follows: injection port temperature, 250°C; detector temperature, 300°C; initial column temperature, 170°C (increasing at 5°C per min up to 270°C at 20 min); total analysis time, 25 min; sample volume, 1 ,u. The peak retention time and peak area values were recorded by an HP3392A integrator
(Hewlett-Packard). The individual fatty acids were identified by comparing their retention times to those of a bacterial fatty acid
J. CLIN. MICROBIOL.
standard (bacterial acid methyl ester mix CP, 4-7080; Supelco, Inc., Bellefonte, Pa.). The relative peak areas of these fatty acids in each isolate were calculated. Also, the mean relative peak areas of each of the fatty acids within each coagulase-negative staphylococcal species were calculated. Processing of the GLC data. The GLC results, including the retention time values and peak areas of all detected peaks in the chromatograph, were transferred to a computer. Correlation and cluster analyses of the data were performed as described earlier (8, 31, 32). All GLC profiles were compared as pairs to calculate similarity indices between individual sample pairs. These values were further subjected to cluster analysis and presented as dendrograms in order to determine the capacity, efficiency, and reliability of the GLC methodology in grouping staphylococcal isolates into species-specific clusters. In addition, mean values and standard deviations of the similarity indices within each species and between different species groups were calculated. Statistical analysis. Differences in the amounts of the fatty
acids between different coagulase-negative species were evaluated statistically by using the Student t test. This test was also used to analyze differences in the mean correlation values between multiple identical and nonidentical patient isolates.
RESULTS Bacterial strains. Coagulase-negative staphylococcal blood isolates were grouped by the API Staph-Trac and ATB 32 STAPH procedures into seven different species: S. epidermidis, S. warneri, S. hominis, S. haemolyticus, S. capitis, S. simulans, and S. lugdunensis. All 177 single blood isolates were included in the mean fatty acid composition analysis of these seven coagulase-negative species. Characterization of the 135 multiple isolates from 60 patients revealed a subset of 96 isolates from 41 patients. In this latter group, two to four isolates from each patient were found to be identical by the antibiogram, biotype, tube adherence test result, and plasmid profile. Therefore, these two to four isolates were considered to be the same strain for each patient. The duplicate isolates were thus excluded from the fatty acid analysis. The remaining 39 multiple isolates from 19 patients proved to be nonidentical. From nine patients were recovered isolates belonging to different species. From the 10 remaining patients were recovered isolates of the same species, but with a different antibiotic susceptibility pattern and plasmid profile. All these isolates were included in the fatty acid analysis. Thus, the final strain collection consisted of 264 isolates of coagulase-negative staphylococci. These included the 177 single blood isolates, 80 of the multiple isolates (after exclusion of the multiple identical isolates), and finally, 7 reference strains. Of these isolates, 178 were grouped as S. epidermidis, 59 were grouped as S. warneri, 10 were grouped as S. hominis, 7 were grouped as S. haemolyticus, 6 were grouped as S. capitis, 2 were grouped as S. simulans, and 2 were grouped as S. lugdunensis. Bacterial fatty acid composition. The mean fatty acid compositions of the isolates of the seven coagulase-negative species studied are shown in Fig. 1, and the fatty acid compositions of five of these species, each represented by more than two isolates, are shown in Table 1. A total of 21 different fatty acids were detected in the chromatograms. Of these 21 fatty acids, 16 were identified by the fatty acid standard. The designations of the identified fatty acids are
VOL. 29, 1991
CHARACTERIZATION OF COAGULASE-NEGATIVE STAPHYLOCOCCI
50r S.epidermidis
50 - S. worneri
40
40 -
30
30 20-
20
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40
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50 40 30 20 10
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5 10 15 20 25 30 35 40 45 FIG. 1. Cellular fatty acid composition in seven species of coagulase-negative staphylococci. Mean relative peak areas ± standard deviation of the fatty acids are indicated on the vertical axes as percentages. The samples were analyzed for the presence of 46 fatty acids; numbers on the horizontal axis refer to the fatty acid elution order from the GLC column. The 21 fatty acids detected in the samples are listed in Table 1 under the same elution indicator numbers. Those 16 fatty acids identified by using the fatty acid standard are designated in Table 2.
given in Table 2. Essentially the same fatty acids were present in all analyzed coagulase-negative species. In general, the prevalence of any given fatty acid in the specimens was 100% if the relative amount of that fatty acid was greater than 1% of the total cellular fatty acid. Differences in the apparent prevalence were observed only in those fatty acids with the mean amount below 1% of the total content. The most abundant fatty acids of the staphylococci were as follows: Ci.15:0, Ca15:0, Ci-17:0, Ca-17:0, and C18:0. The relative amounts of the fatty acids Cji160, C16:0, C18:2912 ,
C18:19, and C20:0 and of three uncharacterized fatty acids, defined herein by the elution numbers 9, 14, and 35, were moderate or small. The relative amounts of the remaining eight fatty acids were insignificant. Only the S. epidermidis, S. warneri, S. hominis, S. haemolyticus, and S. capitis species (each represented by more than two isolates) were analyzed statistically. Statistical differences were calculated for those fatty acids with a mean amount of >1% of the total fatty acid content and a prevalence of 100%. Only the major differences in the predominant fatty acids are considered herein. Compared with the other species, S. epidermidis dis-
played a significantly larger (P < 0.010) amount of the fatty acid C18:0 and a smaller (P < 0.001) amount of the fatty acid Ca15:0. The amount of this same fatty acid Ca 15:0 was significantly (P < 0.001) larger in S. warneri, which in contrast contained a significantly (P < 0.002) smaller amount of C18:0. In S. hominis, the amount of the fatty acid Cai17:0 was significantly (P < 0.001) smaller than in the four other species; S. hominis contained a significantly (P < 0.050) larger amount of the fatty acid C20:0. The amount of the fatty acid Ca17:0 was significantly (P < 0.020) larger in S. haemolyticus than in the other species. Finally, the S. capitis species displayed a significantly (P < 0.001) larger amount of the fatty acid Ci 17:0 than did the others. There was considerable overlapping in the statistical differences between various species. For instance, the quantities of another uncharacterized fatty acid, defined by the elution number 14, were significantly (P < 0.001) larger in both S. epidermidis and S. hominis than in the other three species, the quantities of Ci 15:0 were significantly (P < 0.01) smaller in both S. warneri and S. haemolyticus than in the others, and the quantities of Cji17:0 were significantly (P < 0.020) smaller in both S. hominis and S. haemolyticus than in the others.
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TABLE 1. Mean composition of cellular fatty acids in five species of coagulase-negative staphylococcia
No.b
S. epidermidis (n = 178)
Fatty acid designationc
Peak area
Prev
8 9 14 16 18 19 20 21 24 25 26 28 29 31 35 36 37 39
44 45 46
C12:0 Cn Cn C14:0
0.00 1.23 1.86 0.61 7.37 40.59 0.00 0.20 2.06 0.03 2.88 6.49 15.76 0.36 1.63 1.56 1.61 9.98 0.61 0.82 4.35
0.6 99.4 100.0 100.0 100.0 100.0 2.9 55.2 100.0 11.6 100.0 100.0 100.0 97.1 100.0 100.0 100.0 100.0 99.4 95.3 100.0
Ci15:0 Ca-15:0
Cn C15:0
Cj16:0
C16:19 C16:0
Ci17:0
Ca17:0 C17:0 Cn C18:29'12 C18:19 C18:0
C19:0
Cn C20:0
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
S. hominis (n = 10)
S. warneri (n = 59)
Prev
0.04 0.32 0.34 0.22 1.17 2.39 0.03 0.22 0.33 0.07 0.59 1.00 2.31 0.12 0.32 0.69 0.62 1.67 0.20 0.44 1.34
0.0 100.0 100.0 53.4 100.0 100.0 1.7 50.0 100.0 1.7 100.0 100.0 100.0 94.8 100.0 100.0 91.4 100.0 84.5 98.3 100.0
Peak area
1.35 0.82 0.13 5.11 48.27 0.00 0.14 1.20 0.00 2.10 6.45 22.03 0.35 0.75 2.01 0.95 5.29 0.25 0.99 1.81
± ± ± ± ± ± ± ±
± ±
± ± ±
± ± ± ±
± ± ±
0.31 0.21 0.14 0.95 1.70 0.02 0.16 0.22 0.02 0.44 1.01 2.14 0.18 0.20 0.56 0.43 1.13 0.16 0.40 0.77
Prev
0.0 100.0 100.0 100.0 100.0 100.0 0.0 10.0 100.0 0.0 100.0
100.0 100.0 50.0 100.0 100.0 70.0 100.0 100.0 100.0 100.0
Peak area
2.05 2.06 0.49 8.23 45.50
± 0.42 ± 0.72 ± 0.21
± 1.07 + 2.51
0.03 ± 0.08 1.47 ± 0.39 1.91 5.63 13.12 0.16 1.93 1.04 0.82 8.52 0.68 1.09 5.28
± 0.32
± 0.73 + 2.93 ± 0.16
± 0.68 ± 0.08 ± 0.70 ± 1.20
± 0.19 ± 0.38 ± 1.55
S. haemolyticus (n = 7) Prev
0.0 100.0 100.0 57.1 100.0 100.0 0.0 42.9 100.0 0.0 100.0 100.0 100.0 85.7 100.0 100.0 71.4 100.0 100.0 85.7 100.0
Peak area
1.55 0.68 0.17 5.56 45.41
± 0.44 ± 0.23 ± 0.16
± 1.24 + 1.36
0.17 + 0.22 0.85 ± 0.28 2.15 5.47 24.09 0.28 0.53 1.04 0.60 8.28 0.32 0.60 2.23
± 0.44
± 0.99 ± 1.74 ± 0.15 ± 0.14 ± 0.22 ± 0.42 ± 1.23 ± 0.10 ± 0.42 ± 0.44
S. capitis (n = 6)
Prev
0.0 100.0 100.0 66.7 100.0 100.0 0.0 16.7 100.0 0.0 100.0 100.0 100.0 66.7 100.0 100.0 100.0 100.0 83.3 83.3 100.0
Peak area
1.05 ± 0.30 ± 0.19 ± 0.16 ± 0.86
0.60 0.17 7.76 45.48
± 1.32
0.08 ± 0.17 0.72 ± 0.18 1.73 7.96 19.62 0.19 0.57 2.26 1.30 6.90 0.20 0.90 2.52
± ± ± ±
± ± ± ±
± ± ±
0.51 1.00 2.36 0.19 0.11 0.53 0.31 1.15 0.14 0.47 0.26
a Prevalence (Prev) indicates the percentage of isolates with detectable fatty acid within each species. Peak area is expressed as the mean + standard deviation. The samples were analyzed for the presence of 46 fatty acids; No. refers to the elution order from the GLC column. These indicator numbers are used to define the detected fatty acids in the text, in Fig. 1 (horizontal axes), and in Table 2. c Of the 46 analyzed fatty acids, only the 21 fatty acids detected in the samples are listed. C. indicates an uncharacterized fatty acid. The fatty acids, identified according to the fatty acid standard, are designated in Table 2. b
Furthermore, all five coagulase-negative species differed significantly from each other in the quantities of the fatty
acid Ca17:0 detected. Correlation analysis. TABLE 2.
No.b 8 16 18 19 21 24 25 26 28 29 31 36 37 39 44 46
Similarity indices were calculated by
Fatty acids isolated from coagulasenegative staphylococcia
Designationc
C12:0 C14:0 Cji15:0 Ca-15:0 C15:0 Cji16:0 C16:19 C16:0 Cji17:0 Ca17:0 C17:0 C18:29'12 C18:1 9 C18:0 C19:0 C20:0
Name
Dodecanoate Tetradecanoate
13-Methyltetradecanoate 12-Methyltetradecanoate Pentadecanoate 14-Methylpentadecanoate
cis-9-Hexadecenoate Hexadecanoate
15-Methylhexadecanoate 14-Methylhexadecanoate Heptadecanoate
cis-9,12-Octadecadienoate
cis-9-Octadecenoate Octadecanoate Nonadecanoate Eicosanoate
a Staphylococci were analyzed for the presence of 46 fatty acids. Listed are the 16 fatty acids detected in the test samples and identified according to the fatty acid standard. b Numbering refers to the elution order from the GLC column. These indicator numbers are used to define the detected fatty acids in the text, in Fig. 1 (horizontal axes), and in Table 1. c The number before the colon refers to the number of carbon atoms, the number after the colon refers to the number of double bonds, i indicates a branched-chain acid with the branched methyl group at the iso position, and a indicates a branched-chain acid with the branched methyl group at the
anteiso position.
comparing the isolates within the same species as pairs and also by comparing the isolates of one species to the isolates of a different species. The means of similarity indices were then calculated within each species and between different species. Isolates belonging to the same species resembled each other more closely than isolates belonging to different species: the mean correlation of the fatty acid profiles of the isolates of the same species varied from 85.54 +7.68 in S. hominis to 95.65 in S. simulans and the mean correlation of the isolates of different species varied from 62.28 ± 5.87 between S. haemolyticus and S. simulans to 86.76 ± 4.56 between S. haemolyticus and S. warneri (Table 3). The GLC fatty acid profile similarities between the seven coagulasenegative species studied are presented in Fig. 2 as a dendrogram after cluster analysis of the mean correlations between these species. S. epidermidis and S. hominis formed one loosely related cluster, S. warneri, S. haemolyticus, S. capitis, and S. Iugdunensis formed another, and S. simulans was grouped in a separate cluster by itself. The individual isolates were also analyzed by a weightedpair cluster analysis. Results are presented as a dendrogram in Fig. 3. Isolates belonging to the same species formed separate clusters. Of the 178 S. epidermidis isolates, 168 formed a defined cluster. Also included in this cluster were five S. hominis and two S. warneri isolates. Another cluster was formed by 55 of the 59 S. warneri, 1 S. haemolyticus, and 3 S. epidermidis isolates. Additionally, a subcluster of six of the seven S. haemolyticus isolates was grouped into this cluster. Still other distinct clusters were formed by 6 S. capitis, by 2 S. lugdunensis, by 2 S. simulans, and by 5 of the 10 S. hominis isolates. Finally, seven S. epidermidis and two S. warneri isolates were adjusted to fall outside all clusters. When the species identification of staphylococci by the biochemical assays (API Staph-Trac and ATB 32 STAPH)
CHARACTERIZATION OF COAGULASE-NEGATIVE STAPHYLOCOCCI
VOL. 29, 1991
Species
S. S. S. S. S. S. S.
epid
TABLE 3. Cellular fatty acid GLC correlation between seven different species of coagulase-negative staphylococcia Mean correlation coefficient ± SD between species S. epid (n = 178)
S. war (n = 59)
S. hom (n = 10)
S. haem (n = 7)
S. cap (n = 6)
S. sim (n = 2)
S. lugd (n = 2)
87.27 ± 8.04
70.00 ± 11.27 91.24 ± 4.59
76.60 ± 8.49 63.93 ± 9.79 85.54 ± 7.68
73.12 86.76 67.82 91.42
77.43 82.26 75.05 82.54 89.10
66.28 ± 5.74 70.16 ± 4.66 65.43 ± 6.82 62.28 ± 5.87 68.62 ± 5.14 95.65
71.46 ± 9.58 79.15 ± 4.27 67.61 ± 8.32 81.30 ± 3.15 74.80 ± 2.68 66.41 ± 3.26 91.84
war hom haem
cap Sim lugd
± 11.1 ± 4.56 ± 7.47 ± 3.82
± ± ± ± ±
9.43 5.32 9.18 5.65 5.27
a Abbreviations: S. epid, S. epidermidis; S. war, S. warneri; S. hom, S. hominis; S. haem, S. haemolyticus; S. cap, S. capitis; S. sim, S. simulans; S. S. lugdunensis.
was compared to the clustering by the automated GLC analysis, identical results were obtained in 244 of 264 cases (92.4%). Within the individual species, the agreement between the methods was as follows: S. epidermidis, 168 of 178 (94.4%); S. warneri, 55 of 59 (93.2%); S. hominis, 5 of 10 (50.0%); S. haemolyticus, 6 of 7 (85.7%); S. capitis, 6 of 6 (100.0%); S. simulans, 2 of 2 (100.0%); and S. lugdunensis, 2 of 2 (100.0%). Identical results were obtained for all seven reference strains. A number of incongruently positioned samples in the various clusters was apparent. In the S. epidermidis cluster, 7 of 175 (4%) fell into this category. In the S. warneri cluster, 10 of 65 (15.4%) of the tested samples proved to be disparately positioned. This particular cluster, as stated above, contained a subcluster of six S. haemolyticus samples. Alternatively, if this cluster was reorganized to include a combined S. warneri-S. haemolyticus cluster, the incidence of variability in the positions of the samples would decrease to 4 of 65 (6.15%). For the S. capitis (n = 6), the S. simulans (n = 2), and the S. hominis (n = 5) clusters, none of the organisms were disparately positioned. As stated above, seven S. epidermidis and two S. warneri samples remained outside all clusters. Correlation analysis of the multiple isolates from a given patient. Similarity indices between consecutive patient isolates were calculated for all 60 patients from whom multiple blood isolates were recovered. The purpose was to determine whether the GLC profile correlation analysis could establish identity between those isolates which, in fact, were representative of the same bacterial strain. Those multiple isolates from a given patient, determined to be identical by the above-mentioned standard techniques, were operationally defined as the same strain. The correlation coefficients
S.epidermidis S.hominis S.warneri S.haemolyticus S.capitis S.lugdunensis S.simulans 0
319
50
100
FIG. 2. A dendrogram showing the GLC fatty acid profile similarities between seven species of coagulase-negative staphylococci. The dendrogram was established by cluster analysis of the mean values of similarity indices. Interspecies similarity indices are indicated on the horizontal axis.
lugd,
of the fatty acid profiles between the multiple patient isolates are shown in Table 4. The correlation coefficients of the multiple identical blood isolates from 41 patients varied from 92.79 to 99.01. In 90.2% (37 of 41) of the cases, the correlation value was >97; in 97.6% (40 of 41) of the cases, it was >95. The correlation values of the multiple, nonidentical blood isolates from 19 patients varied from 59.97 to 94.90. When the isolates were of the same species, the correlation value varied from 84.22 to 94.90 (mean, 89.99 + 3.87). The mean value of correlation between multiple nonidentical S. epidermidis blood isolates (from 8 patients) was 89.79 ± 4.04, which was quite comparable to the mean correlation value of 87.27 + 8.04 (Table 3) between random S. epidermis isolates. In contrast, a significantly (P < 0.001) higher mean correlation value between consecutive isolates, 97.65 + 1.16, was detected in those 33 patients who had multiple identical S. epidermidis blood isolates. DISCUSSION
Different species of coagulase-negative staphylococci can be unequivocally distinguished when stringent DNA-DNA hybridization conditions are used (29). This methodology, however, is highly specialized and used primarily as a research tool. At present, no completely reliable system practical as a routine diagnostic tool exists for identification of coagulase-negative staphylococci to the species level. Commercially available panels, which identify on the basis of functional differences in the metabolic pathways, do not allow species identification with certainty (25). Thus, alternative methods are being developed for speciation. Among the most interesting is whole-cell protein analysis; sodium dodecyl sulfate-polyacrylamide gel electrophoresis has provided species identification with a high degree of accuracy. By using this technique and immunoblotting, ThomsonCarter and Pennington (33) were able to characterize nine coagulase-negative species. According to Pierre et al. (26), the penicillin-binding protein profiles provide an even greater degree of species identification accuracy than does examination of the total solubilized proteins. Another interesting approach to species identification, the GLC analysis of cellular fatty acids, has indicated distinct quantitative differences between various coagulase-negative species, despite qualitative similarities (7, 21, 30). The purpose of this study was to further evaluate the applicability of the GLC fatty acid analysis for species identification of coagulase-negative staphylococci. Consistent with the previous findings (7, 8, 21, 30), no obvious qualitative differences in the fatty acid compositions were observed between the different coagulase-negative Staphy-
320
KOTILAINEN ET AL.
J. CLIN. MICROBIOL.
-S.
haemolyticus
-S. warneri
C S .capitis -S.lugdunensis
-S.epidermidis
simulans hominis 50
0
100
the GLC fatty acid profile simicoagulase-negative staphylococci. The isolates were identified by the API Staph-Trac and ATB 32 STAPH procedures as seven different species: S. epidermidis, S. warneri, S. hominis, S. haemolyticus, S. capitis, S. simulans, and S. Iugdunensis. A weighted-pair cluster analysis grouped the isolates into species-specific clusters; the compositions of the seven clusters shown in the figure are explained in detail in the text. The fatty acid FIG.
3.
A
dendrogram showing
larities between 264 isolates of
profile
correlation between individual
horizontal axis.
isolates is indicated on the
lococcus species. Admittedly, a few fatty acids which produced very low mean peak areas were not detected in all coagulase-negative species studied. Additionally, differences in the apparent fatty acid prevalences were observed between individual isolates of those species in which the relative mean amount of that particular fatty acid was less than 1%. It should be noted here that in such cases, the observed differences most probably reflected a function of the detection limit of the method rather than any true dissimilarity. Although qualitative differences were not observed, various species differed significantly from each other in the relative amounts of several individual fatty acids. Some of the species-specific characteristics were quite distinctive. For example, S. epidermidis was characterized by a relatively large amount of the fatty acid C18:0, S. warneri was characterized by a large amount of the fatty acid Ca-15:0, 5 haemolyticus was characterized by a large amount of the fatty acid Cai17:0, and S. capitis was characterized by a large amount of the fatty acid Cji17:0. Still, there was too much overlapping in the quantitative differences between various species to permit reliable species identification on the basis of the quantity of any single fatty acid. The computerized fatty acid pattern correlation analysis did, however, separate the staphylococcal isolates into species-specific clusters, thereby allowing species identification. Two main clusters were formed by S. epidermidis and S. warneri, the species constituting the bulk of our strain collection. Although the number of isolates belonging to the five other species was few, even these isolates were divided into species-specific clusters. Species identification by GLC clustering was quite consistent with species identification by biochemical analysis; the results were similar in 92.4% of the cases. Moreover, correlation analysis demonstrated that the intraspecies similarities were higher than were the interspecies similarities. On the basis of the correlation values, the most distinct species were S. epidermidis, S. hominis, and S. simulans; the interspecies similarity indices between these and all other species were
