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Journal of Clinical Microbiology, November 2008, p. 3778-3783, Vol. 46, No. 11
0095-1137/08/$08.00+0     doi:10.1128/JCM.02262-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Extreme Genetic Diversity of Methicillin-Resistant Staphylococcus epidermidis Strains Disseminated among Healthy Japanese Children

Tengku Zetty Maztura Tengku Jamaluddin,¹ Kyoko Kuwahara-Arai,^1,2 Ken Hisata,³ Masahiko Terasawa,⁴ Longzhu Cui,^1,2 Tadashi Baba,^1,2 Chie Sotozono,⁵ Shigeru Kinoshita,⁵ Teruyo Ito,^1,2 and Keiichi Hiramatsu^1,2*

Department of Infection Control Science,¹ Department of Bacteriology,² Department of Paediatrics, Juntendo University School of Medicine,³ Terasawa Children's Clinic, Sendai-city, Miyagi, Japan,⁴ Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan⁵

Received 22 November 2007/ Returned for modification 14 March 2008/ Accepted 19 September 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
For the past few years, we have been observing the dissemination of methicillin-resistant staphylococci in the community. From 2001 to 2003, an evaluation of nasal samples from 1,285 children in five day-care centers and two kindergartens in three districts in Japan revealed that methicillin-resistant coagulase-negative staphylococci (MRC-NS) have been widely disseminated in the Japanese community. Their prevalence is much greater than community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). Forty-nine children (3.81%) were colonized with MRSA, whereas 390 children (30.35%) were colonized with MRC-NS. These MRC-NS strains predominantly harbored a pair of cassette chromosome recombinase types A2 and B2 (ccrAB2). Of these, 40.8% harbored type IVa staphylococcal cassette chromosome mec (SCCmec) elements, a distinct/characteristic type of SCCmec in pandemic clones of CA-MRSA. Interestingly, there was also a high frequency of nontypeable strains which possessed atypical structures compared to previous SCCmec types. Among the MRC-NS, the majority of strains (63.59%) were methicillin-resistant Staphylococcus epidermidis (MRSE). Their genotypes, as judged from pulsed-field gel electrophoresis (PFGE), were highly diverse. They were so diverse that there was no sign of an immediate transmission of any MRSE clone among children in the same institutions. In a previous report, we expounded that a few CA-MRSA clones with distinct SCCmec types were disseminated among children in the same institutions. Au contraire, with the case of CA-MRSE, there was no single genotype of CA-MRSE disseminated among children even in the same institution or class.

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Staphylococcus aureus is not a stranger to pediatric patients; its ability to cause a wide spectrum of infections among this vulnerable group warrants its distribution to be observed periodically, more than ever with the emergence of methicillin-resistant Staphylococcus aureus (MRSA) in the community in the past decade (10, 11, 21, 23). In the past, MRSA was deemed to be a nosocomial pathogen and infrequently isolated in healthy individuals in the community. MRSA infections account for increased cost and mortality both locally and worldwide. Significant advances in MRSA epidemiology have been made since the discovery of the genetic mechanism by which each MRSA clone is generated, namely the integration of the staphylococcal cassette chromosome mec (SCCmec) element into the S. aureus chromosome (8, 9). For many years, it has been the focus of clinicians and researchers alike in determining and understanding the route of transmission of this clinically important mobile genetic element. However, the origin of SCCmec remains uncertain. There is no allelic equivalent of mecA in methicillin-susceptible Staphylococcus aureus. The common thought is that MRSA acquired SCCmec from other staphylococci species, most presumably from its coagulase-negative counterparts (1, 7, 13, 14, 16, 26).

Coagulase-negative staphylococci (C-NS) are commensal bacteria of human skin and nasal and oral mucosa. In the advent of increased invasive interventions and treatments, C-NS have been frequently detected as a cause of opportunistic infections (15). They are difficult to eradicate, as they possess the capacity to form biofilms on indwelling devices (6, 22, 26). Furthermore, medical and health-care workers pose as reservoirs of infection for susceptible patients (2). According to the National Nosocomial Infectious Surveillance of the United States of America, the rates of methicillin resistance have increased in the last two decades (17). Worldwide surveys revealed that 60 to 85% of clinical strains are resistant to methicillin (4, 17, 19) The detection of MRC-NS in health-care settings has never been more important due to the narrow therapeutic approaches available. Most alarmingly, the incidence of methicillin-resistant C-NS (MRC-NS) has also amplified in individuals without established risk factors in the community (4, 10, 23). For the past few years, we have been trying to comprehend the mode of dissemination of SCCmec among staphylococci in the community. Interestingly, we discovered that MRC-NS strains have been steadily disseminated in the Japanese community and are ominously more prevalent than CA-MRSA (K. Kuwahara-Arai, unpublished data) (10). These strains predominantly harbored type IV SCCmec elements, similarly to CA-MRSA strains disseminated in the world (5, 18, 21).

Most of the studies pertaining to community-acquired staphylococci mainly concentrated on S. aureus. Our literature review also revealed that a wide proportion of studies on the Staphylococcus epidermidis subspecies have been conducted exclusively on clinical strains, i.e., hospital-acquired methicillin-resistant Staphylococcus epidermidis (MRSE). No research has been published specifically with respect to community strains.

In this study, we aimed at investigating the molecular epidemiology of MRC-NS strains disseminated in healthy Japanese children and to describe their attributes and potential as a reservoir for interspecies genetic element transfer.

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Isolation of MRC-NS from the nasal swabs of healthy children. To determine the prevalence of methicillin-resistant staphylococci in the population, nasal swabs were obtained from healthy children in five day-care centers and two kindergartens in three different prefectures, Miyagi, Kyoto, and Saga, from northern to southern Japan.

To understand the stability of colonization and to determine the carriage rate of MRC-NS, samples were obtained at several intervals where possible. In Miyagi, sampling was done twice over an 8-month interval. The initial sampling in July 2001 totaled 362, and the consecutive sampling totaled 292. A total of 236 children were resampled at the second Miyagi study. In Kyoto, sampling was done over a 5-month interval. In the second study in Kyoto in 2003, another institution was incorporated into the study. The initial sampling in December 2002 totaled 150, and the second sampling totaled 231. Of these, a total of 103 children were resampled at the second Kyoto study. In Saga, 250 children were sampled in December 2002. No resampling was undertaken in Saga. Absentees and those whose parents did not consent to the sampling were not included in this study. In all, 1,285 samples were obtained.

Sterile, dry cotton swabs were used to obtain specimens from both nares that were directly inoculated onto mannitol-salt agar (Eiken Chemical Co., Ltd., Tokyo, Japan) with or without 10 mg/liter of ceftizoxime (Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan) (25). The plates were incubated at 37°C for 48 h. To distinguish other staphylococcus species from S. aureus, a Staphylo LA test kit (Denka Seiken Co., Ltd., Niigata, Japan) was used to test the production of clumping factor and protein A from yellow colonies. Further determination of species that showed a negative reaction to the Staphylo LA test was done by using an identification kit (Staphyogram; Wako Pure Chemical Industries, Ltd., Osaka, Japan).

Antimicrobial susceptibility testing. Minimal growth inhibitory concentrations (MICs) of isolates against 11 antibiotics were determined by the agar dilution method recommended by CLSI (formerly the National Committee for Clinical Laboratory Standards) (3). The antibiotics tested were obtained from the following companies: oxacillin, ampicillin, vancomycin, and gentamicin from Sigma Chemical Co., Ltd., St. Louis, MO; ceftizoxime from Fujisawa Pharmaceutical Co., Ltd.; imipenem from Banyu Pharmaceutical Co., Ltd., Tokyo, Japan; erythromycin and tetracycline from Wako Pure Chemical Industries, Ltd.; tobramycin from Shinogi Co., Ltd., Osaka, Japan; kanamycin from Meiji Seika Kaisha, Ltd., Tokyo, Japan; and norfloxacin from Kyorin Pharmaceutical Co., Ltd., Tokyo, Japan.

Identification of SCCmec elements. Chromosomal DNA was extracted from cells by phenol-chloroform extraction (21) and subjected to PCR using the existing SCCmec primers (12) and protocol as described previously (21). PCR amplification was performed in a 50-µl reaction mixture consisting of 2 U of Ex Taq (Takara Shuzo Co., Ltd., Kyoto, Japan), 10 pmol of each primer, 0.2 mM deoxynucleoside triphosphate mixture, 10 ng of chromosomal DNA, 1x reaction buffer (Takara Shuzo Co., Ltd.), and H₂O. Thermal cycling was set at 30 cycles (30 s for denaturation at 94°C, 1 min for annealing at 50°C, and 2 min for elongation at 72°C) and was performed with a Gene Amp PCR system 9600 (Perkin-Elmer, Wellesley, MA).

PFGE. The chromosomal DNA of SCCmec type IVa MRSE isolates was digested with SmaI and was separated by pulsed-field gel electrophoresis (PFGE) with a Gene Path system. Settings for the PFGE were as follows: initial switch time, 5.0 s; final switch time, 40.0 s; included angle, 120°; current, 200 V; and run time, 22 h. The buffer temperature was maintained at 14°C. BioNumerics software (version 2.5; Applied Maths, Kortrijk, Belgium) was used to analyze the correlations of the banding patterns. A similarity index was determined for each pair of strains by using the Dice coefficient with 0.5% band tolerance. Clustering correlation coefficients were calculated with the unweighted-pair group method of arithmetic averages (UPGMA). Isolates were rendered "potentially genetically related" if macrorestriction DNA patterns differed by less than seven bands (24).

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Carriage of MRC-NS by healthy Japanese children. From the years 2001 to 2003, a total of 1,285 nasal smear samples were isolated from healthy Japanese children in five day-care centers and two kindergartens from three localities in Japan: Miyagi, Kyoto, and Saga. To determine the continuous carriage rates of resistant strains, sampling was done twice over an 8-month interval at four institutions in Miyagi, namely, Miyagi 2001 for the first study in July 2001, Miyagi 2002 for the first-timers sampled in the second study in March 2002, and Miyagi 2002-R for children sampled in both consecutive studies. The Kyoto study was done over a 5-month interval, the first in December 2002 and the second in May 2003. The continuous carriage rate was not determined in the Saga study.

Table 1 summarizes the carriage of MRC-NS and MRSA in healthy children in Japan. Interestingly, we found that the prevalence of MRC-NS strains was higher than that of MRSA strains in healthy Japanese children, with a mean percentage of 29.73% and a mean ratio of 9.1. The percentage of children carrying MRC-NS strains was relatively higher in the locality of Miyagi in both years the study was conducted and in the second year in Kyoto (Miyagi 2001, Miyagi 2002, and Kyoto 2003, from highest to lowest). The mean ratios of the MRC-NS and MRSA carriage rates were also relatively higher in the Miyagi 2002 sampling, which had a mean ratio of 10, and the Kyoto 2003 sampling, which had a mean ratio of 21.85, than in the other samplings.

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TABLE 1. Geographical distribution of methicillin-resistant staphylococci by species

A majority of the resistant strains in all localities were S. epidermidis strains, amounting to 63.59% of all MRC-NS strains isolated (Table 2). The greatest frequency was in the Miyagi 2002 sampling (85.0%), followed by Miyagi 2002-R (82.0%), Miyagi 2001 (79.86%), Saga 2002 (47.37%), Kyoto 2003 (42.2%), and Kyoto 2002 (37.93%). This shows an upward trend of the MRSE carriage rates in all localities except Saga, where the sampling was done only once.

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TABLE 2. Geographical distribution of MRC-NS by staphylococcal speciesa

SCCmec typing of MRC-NS strains by geographical distribution. To determine the clonal compositions of MRC-NS strains in healthy Japanese children, all 390 MRC-NS strains were then subjected to SCCmec typing via PCR using the methods described previously. We found that a high percentage (57.0%) of healthy children in Japan asymptomatically harbored MRC-NS with type IV SCCmec elements (Table 3). They were predominantly type IVa SCCmec elements (40.8%). Of the type IV SCCmec strains, there was a sizeable amount (9.08%) that carried unknown subtypes of the type IV SCCmec elements so far documented.

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TABLE 3. Geographical distribution of MRC-NS by SCCmec typing

As for the type II SCCmec strains, type IIa was distinct in the Kyoto 2002 sampling, whereas type IIb was found in the Miyagi 2001, Kyoto 2003, and Saga 2002 samplings. There was a distinct geographical distribution of type I SCCmec strains in Saga and type V SCCmec strains in Kyoto. There was also a notable amount (23.51%) of nontypeable strains whose structures did not correspond to any existing SCCmec elements tested so far.

Antibiotic susceptibilities. The resistance of strains was further scrutinized, and we found that the MICs of oxacillin in the Saga and Kyoto strains were relatively lower compared to those in the strains in Miyagi. However, the MICs of erythromycin were extremely high in all constituencies. Most strains were susceptible to the other antimicrobials tested.

Mode of dissemination and colonization of MRSE strains in healthy Japanese children. Out of the total of 248 MRSE strains, 161 strains harbored SCCmec type IVa elements. These type IVa strains were subjected to PFGE and their pulsotypes determined. Surprisingly, we found that the type IVa MRSE strains were extensively heterogenous, regardless of being isolated from the same facility or class. These features differed greatly from the CA-MRSA strains in this study, which showed more homogeneity. CA-MRSA strains isolated from children belonging to the same class had distinctive pulsotypes and SCCmec types (10).

In Miyagi, we successfully obtained consecutive samplings from 236 children. Among the resampled children (Miyagi 2002-R), 106 children were previously colonized with MRSE, of which 70 had MRSE SCCmec type IVa. However, only 41 children had nasal colonization of MRSE in the second sampling. Among these children, 29 were newly positive for MRSE, of which 15 children harbored MRSE SCCmec type IVa (Table 4). Only 12 children (17.1%) carried MRSE SCCmec type IVa in both consecutive samplings and were considered potential continuous carriers. These strains were separately assessed from the full MRSE collection via PFGE. We found that out of the 12 children, 5 had strains which were identical to the initial isolates and 1 child had a one-band difference between the strains. The other strains were rendered "genetically unrelated" as judged by Tenover's criteria (24).

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TABLE 4. Geographical distribution of MRSE by SCCmec typing

In Kyoto, consecutive samplings were successfully obtained from 103 children. Of these, 17 children were found to be newly positive for MRSE (Table 4). Five out of seven children previously positive were negative at the second screening. Only two children had nasal colonization of MRSE SCCmec type IVa in both screenings. The two children were from separate classes. PFGE revealed that one child harbored the same strain at both screenings. However, the other child harbored a totally different strain at the second screening (24).

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This study is an extension of the study done by Hisata et al. (10) on the dissemination of methicillin-resistant staphylococci among healthy Japanese children. The purpose of this study was to further explore and describe the degree of diversity of CA-MRCNS, carried by Japanese healthy children.

Dissemination of MRSE in the healthy Japanese community. Miyagi is situated 300 kilometers northeast of Tokyo, at a longitude of 141°E and a latitude of 38°N. The study in Miyagi was done in Sendai, from July 2001 to March 2002. It is well known that the rainy season in Sendai starts around the end of June to early July, and the cold wind from the Okhotsk air mass called Yamase blows in this season. Being in the north, Miyagi averages a temperature of 25°C and precipitation of 140 to 145 mm in the summer. In March, the temperature averages around 8 to 10°C, and the average precipitation is 60 to 65 mm.

The survey conducted in Saga and Kyoto was done in December 2002 and May 2003. Both cities being located southerly, the average temperature in winter is 10 to 12°C and the average precipitation is 45 to 48 mm (Allmetsat [http://en.allmetsat.com/index.html]).

The climate in the Miyagi prefecture is damp and has a lower annual average temperature than the Saga or Kyoto prefectures. The first Miyagi study was done during the hot and rainy season, which might have been the contributing factor to the high rate of colonization and transmission of microorganisms, i.e., staphylococci, in this area. It is documented that physical contact and sweating contribute to increased transmission of and infection with CA-MRSA (20). Children attending day-care centers and kindergarten, in this respect, are very vulnerable, especially in the summertime when they tend to sweat and are outdoors more, to share small portable pools in schoolyards, and to go to the public wading pools.

Variation of SCCmec elements in the healthy Japanese community. The extensive diversity of CA-MRSE revealed by this study of healthy children further expounds that within the S. epidermidis species, there is a high degree of genetic diversity. The presence of the same SCCmec types in the CA-MRCNS and CA-MRSA collection suggests that there is a high possibility of interspecies and intraspecies transfer of SCCmec elements. Hisata et al. previously reported that CA-MRSA disseminated in healthy Japanese children were predominantly SCCmec type IIb, followed by types IIa, IVnt, and IVa (10). However, our data shows that the majority of CA-MRSE strains carried by healthy Japanese children were SCCmec type IVa. From this finding in the Japanese community, we could further hypothesize that there is a higher interspecies transmissibility rate of the ccrAB2 element, which constructs the structure of both SCCmec type II and IV.

This further supports the hypothesis that there is a degree of genetic transfer of SCCmec elements between staphylococcal species. The existence of a large proportion of nontypeable SCCmec elements in this study further suggests a higher diversity of the SCCmec cassettes harbored by MRSE compared to that of the cassettes harbored by MRSA.

Diversity of CA-MRSE in the healthy Japanese community. Unlike CA-MRSA strains which are highly transmissible from human to human, the mode of transmission of CA-MRSE seemed to be complex. It is common to both MRSA and MRSE that their carriage is transient, and only a few children persistently carried the same methicillin-resistant strains at the second sampling held after an interval of 5 months in Kyoto and 8 months in Miyagi. However, the MRSE genotypes were extremely diverse, and there was no single clone with a fixed pulsotype disseminating among multiple children. There are some clusters of similar pulsotypes observed in certain institutions (Fig. 1, shown as clusters 1, 2, and 3). Therefore, it seems that there are several clones that were disseminated among children presumably through close physical contact. However, there was no single identical pulsotype observable among any of the clusters (Fig. 1).

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FIG. 1. Unweighted-pair group method of arithmetic averages dendrogram of the PFGE banding patterns of SCCmec type IVa MRSE strains. This dendrogram shows a selection of 84 strains of type IVa MRSE. Three clusters were identified in this assessment. The strains are coded according to prefecture, institution, class, and year of study, e.g., Miyagi-S-1-2001. In Miyagi, there were four institutions with multiple classes. The isolates from each class of the four institutions are indicated with the following abbreviations: S (1 to 5), F (1 to 7), I (1 and 2), and Y (1 to 4). The isolates from each class in an institution in Saga are indicated as S (1 and 2), and those from two institutions in Kyoto are indicated as I (1 to 6) and W (1 to 6), respectively. *, prefecture (Pref.), institution (Inst.), class, and year of sampling conducted.

This difference between MRSA and MRSE may indicate the higher flexibility of the genome of S. epidermidis than that of S. aureus, and the transmitted MRSE might change its genotype in the course of establishing itself as a new cohabitant of the host. Alternatively, the samples we obtained from each child might have represented the S. epidermidis stably habituated on the child. If the latter were the case, a part of the resident S. epidermidis cells of each child had at least temporarily acquired SCCmec from a certain microorganism that transiently colonized the child. The most likely culprit carrying SCCmec, in this case, would be a CA-MRSA or a CA-MRSE strain from another child/individual. A well-designed future study is required to test these hypotheses for final understanding of the MRSE epidemiology in the community.

Variation of PFGE macrorestriction patterns of MRSE SCCmec type IVa. Having observed such a large diversity of pulsotypes of CA-MRSE strains across Japan, they must have originated from a huge divergent genetic background. These findings also denote that there is frequent acquisition and may also be a loss of SCCmec elements amid CA-MRSE. Again, this highlights another major difference between CA-MRSE and CA-MRSA. This unexpected observation may further signify that SCCmec is now acknowledged as one of the important genetic resources that are persistently maintained in the genomes of human natural flora.

These observations were also reported by Miragaia et al. for clinical isolates of MRSA and MRSE. Unlike HA-MRSA with pandemic clones responsible for nosocomial MRSA infections, hospital-acquired MRSE have an extensive genetic diversity (19). Variation in the PFGE macrorestriction pattern has been detected in collections originating from a single intensive care unit and even from a single infection site (19). Strikingly, the strains with different PFGE types often harbored the same SCCmec element, type IV (19).

Conclusion. We can construe that the human skin and nasal nares serve as a reservoir for both resident organisms, such as S. epidermidis, and many commensal pathogens, such as S. aureus, with a high carriage turnover rate. The ineffective continuous carriage ability (or high turnover rate) and a diversified pool of microorganisms colonized at a given time period might contribute to an efficient selection of preresident S. epidermidis transmitted with SCCmec from commensal methicillin-resistant staphylococci.

The most intriguing observation of this study was the absence of a highly transmissible CA-MRSE clone with a single stable genotype. Nevertheless, the majority of CA-MRSE strains harbored type IVa SCCmec. The exact genetic events contributing to this apparent discrepancy are unknown. Conversely, it is highly suggestive that our resident S. epidermidis plays an important part in partaking as an efficient recipient and/or carrier of the SCCmec elements, be it transient or not. Further studies are warranted to comprehend the frequent genetic change of S. epidermidis and its high receptivity of the SCCmec elements.

 
This work was supported by a grant-in-aid for 21st-century COE research and a grant-in-aid for scientific research on priority areas (grant 13226114) from the Ministry of Education, Science, Sports, Culture and Technology of Japan.

 
* Corresponding author. Mailing address: Department of Bacteriology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81-3-5802-1040. Fax: 81-3-5684-7830. E-mail: khiram06{at}med.juntendo.ac.jp

Published ahead of print on 1 October 2008.

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Journal of Clinical Microbiology, November 2008, p. 3778-3783, Vol. 46, No. 11
0095-1137/08/$08.00+0     doi:10.1128/JCM.02262-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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