Typing of ônh ôstaphylococcus epidermidis ôns ô Colonizing in Human Nares by Pulsed-Field Gel Electrophoresis

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1 Microbiol. Immunol., 39(5), , 1995 Typing of ônh ôstaphylococcus epidermidis ôns ô Colonizing in Human Nares by Pulsed-Field Gel Electrophoresis Lan Hu*, Akiko Umeda, and Kazunobu Amako Department of Bacteriology, Faculty of Medicine, Kyushu University, Fukuoka, Fukuoka 812, Japan Received October 7, Accepted February 1, 1995 ônh ô Abstract ôns ô: From the nares of 11 healthy adults, 253 strains of coagulase negative staphylococcus were iso latedand 88% of them were identified as ônh ôstaphylococcus epidermidis ôns ô using the API STAPH system. Chromosomal DNA fingerprinting of the isolated strains revealed that each person carried multiple types of ônh ôs. epidermidis ôns ô in his or her pares. The colonization of the strains was not stable; the types of the isolates changed in the first and the second examinations 5 months apart. The results contrasted with previous findings in which only one strain of ônh ôs. aureus ôns ô colonized persistently in the nares of healthy adults. ônh ô Key words ôns ô: ônh ôstaphylococcus epidermidis ôns ô, Typing, Chromosomal DNA, Fingerprinting pattern, Pulsed-field gel electrophoresis Coagulase-negative staphylococci (CNS), commen salorganisms of human skin or nasal cavity, are the principal cause of infections from prosthetic devices and indwelling catheters (7, 11, 14). Among the vari ousspecies of coagulase-negative staphylococci, Staphylococcus epidermidis is the most frequently encountered species in these cases (2, 13). In contrast to S. aureus, which are isolated from about 30% of the nares of healthy adults, CNS can be isolated from almost all healthy adults (6). The generic DNA profile by pulsed-field gel elec trophoresis(pfge) has recently been utilized for the typing of staphylococcus. Consequently, a large degree of genomic diversity in staphylococci has been report ed(1, 4, 9, 10, 12, 14). In spite of such diversity of the strains, we previously demonstrated that only one strain of S. aureus persistently colonized in human nares while the infection of different types of S. aureus only seldom occurs (6). This study was conducted to determine the carriage state of S. epidermidis, so we typed the strains of S. epidermidis in the nares of healthy adults with DNA fingerprinting and showed that multiple types of the organism persistently colonized the nares of healthy adults. Materials and Methods Isolation of S. epidermidis. Bacteria were isolated *Address correspondence to Dr. Lan Hu, Department of Bac teriology,faculty of Medicine, Kyushu University, Maidashi, Higashi-ku, Fukuoka, Fukuoka 812, Japan. from the right nares of healthy adults using sterile wet cotton swabs (Eiken Kagaku Co., Ltd., Tokyo) on December 25, 1993 and May 15, Bacteria on the cotton swab were directly spread on a plate of staphylococcus selective medium No.110 (Eiken Kagaku Co., Ltd.). After incubating the plate at 37C for 48hr, the suspected colonies of staphylococcus other than S. aureus on the plate were selected and stored at nutrient agar. For the preparation of chromo somaldna, the bacteria was cultured in a nutrient broth at 37C with aeration. Identification of species. The identification of the isolated bacteria species was done using the API STAPH system (Bio merieux, S.A., Marcy I'Etoile, France) according to the manufacturer's instructions (3, 5). Preparation of chromosomal DNA. Chromosomal DNA of S. epidermidis was prepared by the method of Weil and McClelland (15). The bacteria in the late log phase were collected by centrifugation (3,500 ~g, 20min, washed with TE buffer (100mM tris-hcl and 1mM EDTA, ph 7.6) and suspended in 100mM EDTA buffer (ph 8.0) resulting in a concentration of 1 ~109 cells/ml. The suspension was then mixed with melted low-melting-point agarose L (Wako Pure Chemical Industries, Ltd., Osaka, Japan). After solidification of the molten mixture, the block was treated with lysozyme (1mg/ml) and lysostaphin (0.5mg/ml) Abbreviations: CNS, coagulase-negative staphylococcus; EDTA, ethylenediaminetetraacetic acid; PFGE, pulsed-field gel electrophoresis; TE buffer, tris (hydroxymethyl) amino methane-edta. 315

2 316 L. HU ET AL (Sigma Chemical Co., St. Louis, Mo., U.S.A.) in 100mm EDTA buffer for 6-8 hr at 37 C and then treated with proteinase K (1 mg/ml) (Sigma Chemical Co.) in 250mm EDTA and 1% sodium dodecyl sulfate buffer for 12 hr at 50 C. Pulsed field gel electrophoresis. The agarose block prepared as described above was digested with endonuclease Smal or Sall (Toyobo Co., Ltd., Osaka, Japan). The blocks were then loaded in the wells of a 1% agarose gel for electrophoresis. Electrophoresis was carried out with an apparatus for PFGE (CHEF- DR II, Bio-Rad Laboratory, Richmond, Calif., U.S.A.). The pulse time linearly increased during the initial 20 hr from 20 sec to 35 sec and from 40 sec to 70 sec in the following 7 hr. A lambda DNA ladder (Bio-Rad Laboratory) was used as a DNA size marker. Results The Carriage State of S. epidermidis in Healthy Adults All 11 persons tested carried CNS in their nares. In the first isolation on December 25, 1993, 126 suspected colonies suspected to be Staphylococcus but not S. aureus were isolated from the nares of 11 persons and 113 strains of these were identified as S. epidermidis by the API STAPH system. Four strains were S. capitis, while the other species were nontypable strains. In the second isolation on May 15, 1994, 127 strains were isolated, 111 of which were identified as S. epidermidis, 2 strains were S. capitis, while the other species were nontypable strains. These findings indicated that S. epidermidis is the most frequently encountered species of staphylococcus in human pares. Typing of S. epidermidis Strains with the API STAPH System Using the API STAPH system, the S. epidermidis strains were categorized into 6 biological types and then they were identified by Arabic numbers with 7 digits. As shown in Table 1, the predominant types of S. epidermidis isolated from the nares were No , No , and No ; these comprised 86% of the isolated S. epidermidis. These findings indicate that multiple types of strains might colonize in the nares of healthy human adults. This was further supported by typing the strains with the chromosomal DNA fingerprinting pattern (see next section). Typing by Chromosome DNA Fingerprinting Figure 1A shows the chromosomal DNA patterns digested with Smal of 20 strains isolated from the nares of two persons (L10 and L5) on the same day (December 25, 1993). The strains isolated from L5 were grouped into four DNA types while those of L10 were isolated into two types. Sall digestion gave Table 1. Typing of S. epidermidis by the API STAPH system Fig. 1. Chromosomal DNA fingerprinting of 20 strains isolated from healthy adults L5 and L10 on December 25, (A) the pattern by Smal digestion and (B) by Sall digestion. The molecular sizes of the DNA are shown to the right of the figure.

3 COLONIZATION OF S. EPIDERMIDIS IN HUMAN NARES 317 Fig. 2. The types of chromosomal DNA fingerprinting by SmaI digestion of 110 strains isolated from 11 healthy adults. The molecular sizes of the DNA are shown to the right of the figure. results similar to SmaI (Fig. 1B). However, no significant relationship between the DNA patterns and the biological types was found. For example, the strains from L10, which belong to the same biological type, showed a different DNA pattern. We extended this experiment to 110 strains of S. epidermidis isolated from 11 persons in the first isolation. The results are shown in Fig. 2. As can be seen in Fig. 1, the DNA patterns of the strains from 11 persons were diverse. One to four DNA patterns were found in the isolates from each person. For example, in Ll and L6 we recognized 3 different types but only one type in L9. This indicates that in the nares of one person 1 to 4 different types of S. epidermidis were colonized. Stability of the Strains Colonized in the Nares We have reported that only one strain of S. aureus demonstrated a stable colonization in the nares of each person (6). To see whether S. epidermidis could also show a stable colonization or not, we determined the DNA type of the strains isolated from the same person on two different days at an interval of 5 months. Figure 3 shows the DNA patterns of three randomly chosen cases (L1, L6, and L9). Similar results were also obtained in 8 other cases. In case Ll, we identified three DNA types in the first isolation (December 25, 1993), while in the second isolation 5 months later (May 15, 1994) we identified 5 types. In addition, the major type in the first isolation (8 strain) became a minor type in the second isolation (1 strain) (Fig. 3A, panels I and II). In the case of L6 we obtained similar results, but the number of types in the first isolation (3 types) decreased in the second isolation (2 types) (Fig. 3B, panels I and II). Case L9 was rather unique among the 11 cases. The DNA types of 10 strains in each isolation were identical. No change of type was found in this case (Fig. 3C, panels I and II). These results indicate that a multiple type of S. epidermidis was able to colonize in the nares of healthy adults, but not in a stable fashion. Discussion We isolated CNS from the nares of all 11 healthy adults, and 88% of them were identified as S. epidermidis using the API STAPH system. The typing of these strains by chromosomal DNA fingerprinting showed a great diversity of the types of this species and 30 types were identified. There were some minor differences in the patterns of DNA which are difficult to categorize as the same pattern. The interpretation of these minor differences is difficult since we do not yet know their underlying meaning. The presence of a shift in the size of one or two bands could be due to minor chromosome rearrangements. Large plasmids, if present, that could be linearized by digestion with the enzymes may also lead to an incorrect interpretation. Staphylococcal strains frequently harbor phage DNA which has the ability to either integrate or be excised from the chromosome, thereby creating a difference in the PFGE patterns (8). In this study, Smal digestion and PFGE were used to examine the chromosomal polymorphism of the human isolates of S. epidermidis. The experiment revealed that multiple types of strains colonized the nares of every person. In addition, the colonization by S. epidermidis does not seem to be stable. The isolates from the nares on two separate days about 5 months apart showed differences in DNA patterns. These results are in direct contrast to the colonization by S. aureus. The

4 318 L.HUETAL isolation rate of S. aureus from the nares is about 30% (6). The remaining 70% of the persons are non-carriers. Moreover, as we have examined the carrier state for a year, only one strain colonizes persistently in the nares, and replacement of the strain seldom occurs. It is difficult to interpret the differences in the colonization between these two species of staphylococci at present. The staphylococcal species colonizing the nares are mostly S. epidermidis while the other species colonizing seems to be rare. Therefore it is reasonable to assume that the nares are occupied predominantly by S. epidermidis and that the space for the other species is very limited. Regarding the great diversity of the DNA types of S. epidermidis, as discussed previously, we have to consider the presence of a plasmid or phage in these bacteria. It is less likely that the bacteria rapidly change their chromosomal DNA patterns during multiplication in the nares because, after culturing the two different types of strains in the same medium for more than 3 days, we were not able to find any changes in the DNA patterns of these strains. A more detailed analysis such as culturing several strains in the same medium for a longer time and some induced experiments are thus still needed before any definitive conclusions can be reached. References Fig. 3. Chromosomal DNA fingerprinting of 20 strains isolated from healthy adults Ll, L6, L9 on two separate days (I. December 25, 1993 and II. May 15, 1994). (A) L1 case. (B) L6 case. (C) L9 case. The molecular sizes of the DNA are shown to the right of the figure. 1) Etienne, J., Charpin, B., Grando, J., Brun, Y, Bes, M., and Fleurette, J Characterization of clinically significant isolates of Staphylococcus epidermidis from patients with cerebrospinal fluid shunt infections. Epidemiol. Infect. 106: ) Gemmell, C.G Coagulase-negative staphylococci. J. Med. Microbiol. 22: ) Gemmell, C.G., and Dawson, J.E Identification of coagulase-negative staphylococci with the API STAPH system. J. Clin. Microbiol. 16: ) George, C.G., and Moos, WE Comparison of the Smal-digested chromosomes of Staphylococcus epidermidis and the closely related species Staphylococcus capitis and Staphylococcus caprae. Int. J. Syst. Bacteriol. 44: ) Giger, 0., Charilaou, C.C., and Cundy, K.R Comparison of the Staph-ident and DMS Staph-Trac systems with conventional methods used for the identification of coagulase-negative Staphylococci. J. Clin. Microbiol. 19: ) Hu, L., Umeda, A., Kondo, S., and Amako, K Typing of Staphylococcus aureus colonizing in human nares by pulsed-field gel electrophoresis. J. Med. Microbiol. 42: ) Leeming, J.P., Holland, K.T., and Cunliffe, W.J The microbial ecology of pilosebaceous units isolated from human skin. J. Gen. Microbiol. 130: ) Lina, B., Bes, M., Vandenesch, F., Greenland, T., Etienne,

5 COLONIZATION OF S. EPIDERMIDIS IN HUMAN NARES 319 J., and Fleurette, J Role of bacteriophages in generic variability of related coagulase-negative staphylococci. FEMS Microbiol. Lett. 109: ) Lina, B., Vandenesch, F., Etienne, J., Kreiswirth, B., and Fleurette, J Comparison of coagulase-negative staphylococci by pulsed-field gel electrophoresis. FEMS Microbiol. Lett. 92: ) Linhardt, F., Ziebuhr, W., Meyer, P., Witte, W., and Hacker, J Pulsed-field gel electrophoresis of genomic restric tionfragments as a tool for the epidemiological analysis of Staphylococcus aureus and coagulase-negative staphylo cocci.fems Microbiol. Lett. 95: ) Noble, W.C Skin microbiology: coming of age. J. Med. Microbiol. 17: ) Parisi, J.T Coagulase-negative staphylococci and the epidemiological typing of Staphylococcus epidermidis. Microbiol. Rev. 49: ) Roth, R.R., and James, W.D Microbial ecology of the skin. Ann. Rev. Microbiol. 42: ) Vandenesch, F., Lina, B., Lebeau, Ch., Greenland, T.B., and Etienne, J Epidemiological markers of coagu lase-negative staphylococci. J. Intensive Care Med. 19: ) Weil, M.D., and McClelland, M Enzymatic cleav ageof a bacterial genome at a 10-base-pair recognition site. Proc. Natl. Acad. Sci. U.S.A. 86: