Department of Medical Microbiology & Infectious Diseases, Erasmus University Medical Center, Rotterdam,
The Netherlands
Received 23 December 1998/Returned for modification 27 February
1999/Accepted 14 July 1999
 |
INTRODUCTION |
Staphylococcus aureus
nasal carriage has been extensively studied in patients and healthy
individuals (22, 54). Cross-sectional surveys of S. aureus nasal carriage have designated individuals as either
carriers or noncarriers (10, 15, 17, 20, 21, 25, 29-32, 34, 35,
40, 55) (Table 1). In longitudinal studies, however, the carrier state has been shown to change over time
in some individuals. Three carriage patterns can be distinguished: persistent carriage, intermittent carriage, and noncarriage (1, 14, 15, 19, 20, 24, 28, 30, 31, 38). However, the criteria used
to identify these carriage patterns have varied from study to study
with respect to the number of nasal specimen cultures that are
performed, the follow-up period, and the interpretation of the culture
data that are obtained (Table
2). Despite this lack of
consistency, several studies have shown the importance of
distinguishing persistent from intermittent nasal carriage. The mean
number of CFU of S. aureus that can be isolated from the
anterior nares is higher in persistent carriers than in intermittent carriers (49, 52), resulting in more extensive dispersal of the staphylococci in the environment (50) and in an
increased risk of S. aureus infections (4, 5,
51). Moreover, the number of S. aureus phage types or
genotypes that are isolated in repeated cultures is significantly lower
for persistent carriers than for intermittent carriers (15, 38,
46), indicating that the basic determinants of persistent and
intermittent carriage may be different.
In the present study the S. aureus nasal carrier state was
determined for 91 healthy adults during a 12-week follow-up period. Eight years later, persistent carriers were reexamined to investigate their current S. aureus nasal carrier state. Different
genotyping techniques were applied to identify any genetic similarity
of the S. aureus strains isolated over this 8-year period.
The results provide unique evidence for the redefinition of the
persistent nasal carrier state.
| |
MATERIALS AND METHODS |
Study population.
In 1988 a screening for S. aureus nasal carriage was performed among 91 staff members of the
Departments of Bacteriology, Virology, Dermatology, Immunology, and
Epidemiology of the Erasmus University Medical Center, Rotterdam, The
Netherlands. All 51 male and 40 female participants were healthy
adults. Nasal swab specimen cultures were performed weekly for 10 to 12 weeks. For each person the S. aureus carrier index was
calculated. The carrier index was defined as the number of nasal swab
specimen cultures that grew S. aureus divided by the total
number of nasal swab specimen cultures performed for that person.
Persistent nasal carriers comprised those persons with carrier indices
of 0.80 or higher, intermittent carriers were those with carrier
indices between 0.1 and 0.70, and noncarriers were those with indices of zero. In 1995 single nasal swab specimens were obtained from 17 (52%) of the 33 persistent carriers that had been identified in 1988 and were available for renewed determination of their S. aureus nasal carrier state.
Nasal swabbing and isolation of S. aureus.
Nasal swab
specimens were obtained by using sterile dry cotton-wool swabs
(Transwab; Medical Wire & Equipment Co. Ltd., Corsham, United Kingdom).
Both the left and right anterior nares were swabbed by rubbing the swab
four times around the inside of each nostril while applying an even
pressure and rotating the swab without interruption. The swabs were
immediately placed in Stuart's transport medium (Transwab; Medical
Wire & Equipment Co. Ltd.) and kept at 4°C until inoculation.
In 1988 nasal swabs were inoculated within 24 h onto Columbia
blood agar plates (Becton-Dickinson B.V., Etten-Leur, The Netherlands) and phenol-red mannitol salt agar plates (Difco-Brunschwig B.V., Amsterdam, The Netherlands). The plates were incubated at 37°C for
48 h. Identification of S. aureus was based upon colony
morphology, a free coagulase (tube) test (Difco-Brunschwig B.V.), and a
bound coagulase (agar) test (47) applied to suspected
colonies after demonstration of catalase positivity. For persistent
carriers S. aureus isolates of the first, sixth, and last
nasal swab cultures were phage typed (National Institute for Public
Health and Environmental Hygiene, Bilthoven, The Netherlands). One
isolate per phage type was stored as a freeze-dried sample. In 1995, the identification of all isolates obtained in 1988 was confirmed by a
rapid S. aureus-specific latex agglutination test
(Staphaurex Plus; Murex Diagnostics Benelux B.V., Utrecht, The
Netherlands) (26).
In 1995 nasal swabs were inoculated within 24 h onto Columbia
blood agar plates (Becton-Dickinson B.V.) and phenol-red mannitol salt
agar plates (Difco-Brunschwig B.V.). After inoculation the swabs were
placed in brain heart infusion medium, incubated at 37°C for 24 h, and subsequently inoculated onto solid media. All solid media were
incubated at 37°C for 48 h. Identification of S. aureus was based upon colony morphology and a rapid S. aureus-specific latex agglutination test (Staphaurex Plus; Murex
Diagnostics Benelux B.V.) (26), which was applied to
suspected colonies after demonstration of catalase positivity. For each
carrier up to three colonies per colony morphotype per inoculated plate
were stored in glycerol stocks at 
80°C.
Genotyping. (i) RAPD.
Bacteria were grown overnight on
Columbia blood agar plates (Becton-Dickinson B.V.). Two to three
discrete colonies were resuspended in 150 µl of 25 mM Tris · HCl (pH 8.0)-10 mM EDTA-50 mM glucose. Lysostaphin (75 µl of a
100-µg/ml solution; Sigma Chemical Co., St. Louis, Mo.) was added,
and the mixture was incubated at 37°C for 1 h. DNA isolation was
performed by the method of Boom et al. (3). Stock solutions
of DNA were adjusted to a concentration of 0.5 ng/µl and were stored
at 20°C until use. PCR and subsequent electrophoresis of the
amplification products was performed essentially as described
previously (45). The primers used to discriminate S. aureus strains were RAPD1 (5'-GGTTGGGTGAGAATTGCACG-3'),
RAPD7(5'-GTGGATGCGA-3'), and ERIC2
(5'-AAGTAAGTGACTGGGGTGAGCG-3') (45, 48). Randomly amplified polymorphic DNA (RAPD) banding patterns were interpreted visually by two independent observers and were indexed with Roman numerals. Differences in band staining intensities and single band
differences were neglected. In this way all S. aureus
isolates were identified by a three-letter code.
(ii) PFGE.
Pulsed-field gel electrophoresis (PFGE) was
carried out on the basis of protocols previously described for S. aureus (6, 36). The bacteria were grown overnight on
Columbia blood agar plates (Becton-Dickinson B.V.). Two to three
discrete colonies were suspended in a 1:1 ratio in 1% InCert agarose
(FMC Bioproducts, Rockland, Maine). Agarose plugs were prepared with
Bio-Rad casting forms (Bio-Rad Inc., Veenendaal, The Netherlands) and
were incubated with lysostaphin (Sigma Chemical Co.). Spheroplasts were
lysed by incubating the plugs in buffer containing 1% sodium dodecyl sulfate and 1 mg of proteinase K (Boehringer Mannheim, Mannheim, Germany) per ml. The plugs were washed six times for 30 min each time
in 10 mM Tris · HCl (pH 8.0)-1 mM EDTA and were stored at 4°C. DNA was digested with the restriction enzyme SmaI
(Boehringer Mannheim), and PFGE was carried out in 1% SeaKem GTG
agarose gels (FMC Bioproducts) in a 0.5× TBE (Tris-borate-EDTA) buffer
at a temperature of 14°C. Electrophoresis was performed in a Bio-Rad CHEF Mapper. The running time was 22 h, with linear ramping from 2.16 to 44.69 s at an angle of 120°C (60°C and 60°C), at a
voltage of 6 V/cm. Banding patterns were interpreted by two independent observers according to the guidelines provided by Tenover et al. (44). Types were defined on the basis of the identity or
nonidentity of the banding patterns and were indexed with Roman numerals.
(iii) Protein A gene PCR.
Protein A gene polymorphisms were
determined by PCR as described previously (12). The
repetitive X region within the spa gene was amplified with
oligonucleotide primers with the following DNA sequences:
5'-TGTAAAACGACGGCCAGTGCTAAAAAGCTAAACGATGC-3' and 5'-CAGGAAACAGCTATGACCCCACCAAATACAGTTGTACC-3'. The PCR
product was digested with the restriction endonuclease RsaI
(Boehringer Mannheim), resulting in two fragments composed of 214 and
35 bases, respectively, and a third fragment containing the
variable-length repetitive DNA. The restriction fragment length
polymorphisms (RFLPs) of this third fragment were determined by
electrophoresis. RFLP patterns were visually interpreted by two
independent observers. The number of 24-bp repeats present was
determined in comparison with a 100-bp molecular length marker
(Pharmacia, Gouda, The Netherlands).
(iv) Coagulase gene PCR.
Coagulase gene polymorphism was
determined by PCR as described previously (42). The primers
used for amplification of the coagulase gene were COAG2
(5'-CGAGACCAAGATTCAACAAG-3') and COAG3 (5'-AAAGAAAACCACTCACATCA-3') (13). The PCR
product (10 µl) was digested with the restriction endonuclease
AluI (Boehringer Mannheim). RFLP patterns were visually
interpreted by two independent observers and were indexed with Roman numerals.
Statistical analysis.
Differences in the distributions of
continuous and categorical variables between groups were tested by
one-way analysis of variance and the chi-square test, respectively.
| |
RESULTS |
The carrier indices for the 91 persons screened in 1988 are
presented in Fig. 1. According to our
1988 definitions for the nasal carrier state (see Materials and
Methods), 43 (47%) noncarriers, 15 (17%) intermittent carriers, and
33 (36%) persistent carriers were identified. Population
characteristics are shown in Table 3.
Sex, age, and department of employment were not statistically significantly different for the three carrier states.
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FIG. 1.
Distribution of S. aureus nasal carrier
indices among 91 healthy adults repeatedly cultured over a 10- to
12-week period in 1988. The carrier index is defined as the proportion
of cultures yielding S. aureus for a given individual.
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TABLE 3.
Distribution of sex, age, and department of employment by
S. aureus nasal carrier state among 91 healthy adults
repeatedly sampled for culture over a 10- to 12-week period in 1988
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Seventeen (52%) of the 33 persistent carriers in the 1988 screening
were available for nasal swab specimen culture in 1995. The
distributions of sex, age, and department of employment in 1988 for
individuals available for reexamination were comparable to those for
individuals not available for reexamination (data not shown). In 12 (71%) of these 1988 carriers, S. aureus was again isolated
from a single nasal swab specimen. S. aureus isolation in
1995 was significantly more frequent in carriers with 1988 carrier
indices of 1.0 (100%) than in carriers with 1988 indices below 1.0 (50%) (P = 0.04). The isolation rate for the latter group was significantly lower than the expected isolation rate of 85%
(95% confidence interval, 78 to 92%) on the basis of the previous
carrier indices.
All S. aureus strains isolated in 1988 and 1995 were
genotyped by RAPD analysis and PFGE and were screened for polymorphisms in the protein A and coagulase genes (Table
4; Fig. 2 to
5). The results of the two whole-genome typing methods, RAPD analysis and
PFGE, were quite concordant, with the methods detecting 15 and 16 different genotypes, respectively. RAPD analysis and PFGE revealed the
genetic similarity of the 1988 and 1995 S. aureus isolates
in four and three individuals, respectively. PFGE types G and K,
isolated from carrier 11 in 1995, and types A and N, isolated from
carrier 13 in 1995, differed by two and three bands, respectively.
According to the guidelines provided by Tenover et al. (44),
these isolates should be considered closely related, indicating that
the genomes of persistent S. aureus strains may slowly
evolve in an individual over time. The numbers of protein A repeat
units in the 1988 and 1995 isolates were identical in six carriers, but
identical numbers of protein A repeat units were detected in isolates
from only two of the three individuals for whom the genetic similarity
of the S. aureus isolates was identified by RAPD analysis
and PFGE, indicating variability within the protein A gene of S. aureus strains that otherwise showed constant overall genotypic
characteristics. The RFLP patterns of the coagulase genes of the 1988 and 1995 isolates were identical in all three individuals in which the
genetic similarity of the S. aureus isolates was identified
by RAPD analysis and PFGE. In one additional carrier (carrier 15) the
coagulase gene typing patterns of the 1988 and 1995 isolates were
identical, but this result could not be confirmed by RAPD analysis or
PFGE. This observation can be explained by the limited resolution of
coagulase gene typing, which is illustrated by the data in Table 4.
Only 7 coagulase gene RFLP patterns were observed; this is in contrast
to the 16 different genotypes that were detected by PFGE. When RAPD
analysis and PFGE, the typing techniques with the highest
discriminatory powers, were combined, three carriers of S. aureus isolates with genetic similarity in 1988 and 1995 were
identified. Genetic similarity was seen only for isolates from carriers
with carrier indices of 1.0 in the original screening in 1988. Renewed
isolation of S. aureus strains in 1995 did not seem to be
associated with the number of protein A repeats or with the coagulase
gene RFLP pattern, as the patterns observed for these strains were also
found for strains that were isolated only in 1988 (Table 4).
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FIG. 2.
RAPD analysis with primer ERIC2 of S. aureus
strains isolated in 1988 and 1995 from carriers 2, 11, 12, and 14. The
arrows on the left and right identify the molecular length marker that
is 600 bp long in the 100-bp ladder. The other fragments differ in size
by multiples of 100 bp in length. Neg., negative control.
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FIG. 3.
PFGE of S. aureus strains isolated in 1988 and 1995 from carriers 2, 11, 12, and 14. The lane marked lambda
contains bacteriophage lambda concatemers that differ in size by
multiples of 50 kbp.
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FIG. 4.
Protein A gene PCR of S. aureus strains
isolated in 1988 and 1995 from carriers 2, 11, 12, and 14. The arrows
on the left and right identify the molecular length marker that is 600 bp long in the 100-bp ladder. The other fragments differ by multiples
of 100 bp in length.
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FIG. 5.
Coagulase gene PCR of S. aureus strains
isolated in 1988 and 1995 from carriers 2, 11, 12, and 14. The arrows
on the left and right identify the molecular length marker that is 600 bp in the 100-bp ladder. The other fragments differ by multiples of 100 bp in length. Neg., negative control.
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| |
DISCUSSION |
In the initial 12-week screening of 91 healthy adults we
identified 33 (36%) persons with S. aureus carrier indices
of 0.80 or higher, 15 (17%) with carrier indices between 0.1 and 0.7, and 43 (47%) with carrier indices of zero. Eight years later, reexamination of carriers with indices of 0.8 or higher demonstrated that S. aureus was still present in the nares of each
individual with a carrier index of 1.0 during the initial 12-week
screening but in only half of those with 1988 indices below 1.0. Moreover, only the 1988 and 1995 S. aureus isolates observed
in carriers with indices of 1.0 were found to be genetically similar.
The anterior nares have proven to be the primary reservoir of S. aureus in humans (7, 33, 53), and S. aureus
nasal carriage has been established as a major risk factor for the
development of both community-acquired and nosocomial infections
(8, 9, 23, 27). S. aureus nasal carriage has been
extensively studied in patients and healthy individuals (22,
54). Cross-sectional surveys of healthy adult populations have
reported S. aureus nasal carriage rates between 20 and 55%
(10, 15, 17, 20, 21, 25, 29-32, 34, 35, 40, 55).
Longitudinal studies, however, indicated that carriage patterns differ
between individuals, and that 10 to 35% of individuals carry S. aureus persistently, 20 to 75% carry S. aureus
intermittently, and 5 to 70% are persistently free of S. aureus (noncarriers) (1, 14, 15, 19, 20, 24, 28, 30, 31,
38). The variation in reported rates results, at least partly,
from differences in study populations, sampling and culture techniques,
and criteria for the definition of persistent or intermittent carriage.
First, many studies of S. aureus nasal carriage rates in
healthy adults have been performed in selected populations, including
medical students, hospital personnel, job applicants, and blood donors
(Tables 1 and 2); the reported rates may therefore differ from those
for unselected populations (34). Second, differences in the
procedures of nasal swabbing and isolation of S. aureus may
account for some variation in carriage rates. It has been documented
that swabs from the anterior nares (i.e., the vestibulum nasi) yield
higher carriage rates than swabs taken from sites beyond this region
(21, 33). A recent study has shown that the number of
carriers in a given population is also dependent on the swab material,
the transport medium, the medium for cultivation, and the incubation
period (37). However, this was not true for the
identification of persistent carriers, which was confirmed by our
finding that broth enrichment of the 1995 nasal swabs did not result in
the detection of additional carriers. Thus, noncarriage in some
individuals was not due to the presence of only small numbers of
bacteria in the anterior nares. Third, and perhaps most important,
differences in reported carriage rates are also due to the various
definitions used to assign the persistent and intermittent carrier
state. The criteria used to assign an individual to either carriage
pattern have varied from study to study in terms of both the interval
and the number of cultures performed and the required proportion of
cultures that grow S. aureus (Table 2). Persistent carriage
rates as well as noncarriage rates tend to decrease with increasing
follow-up periods and decreasing culture intervals, indicating that
intermittent carriers may be misclassified as either persistent
carriers or noncarriers if the follow-up period is short or when only a
few specimens are cultured. Obviously, persistent carriage rates
decrease if the required proportion of cultures that grow S. aureus, i.e., the carrier index, increases. In the present
follow-up of S. aureus carriers with 1988 carrier indices of
0.8, the observed S. aureus carriage rate in 1995 was
significantly lower than that expected for individuals with initial
carrier indices of 0.8 or 0.9. In carriers with 1988 indices of 1.0, however, S. aureus was again isolated from all individuals,
suggesting that the starting point in the identification of persistent
S. aureus nasal carriage during a relatively short follow-up
period should be the isolation of S. aureus in 100% of the
nasal swab specimen cultures. The correct separation of the population
into persons who are true persistent carriers versus those who carry
S. aureus only intermittently may be highly relevant, since
it allows studies into the molecular and genetic basis of S. aureus nasal carriage to become better focused. Moreover, it may
have a direct clinical impact when one is designing intervention
strategies because of the risks of infection associated with S. aureus nasal carriage. As the risks of S. aureus nasal
carriage differ for intermittent and persistent carriers, the
distinction between intermittent and persistent carriage enables the
differential application of elimination strategies, which reduces costs
and diminishes the risk of the development of antibiotic resistance. To
our knowledge no data on the long-term persistence of S. aureus nasal carriage in healthy persons are available. Follow-up
periods in longitudinal studies of S. aureus nasal carriage in healthy individuals have varied from 6 weeks to 5 years (1, 14,
15, 19, 20, 24, 28, 31, 38) (Table 2). In the present study, 71%
of persistent S. aureus nasal carriers, as defined in the
initial 12-week screening in 1988, were again identified as nasal
carriers 8 years later. The finding of genetic similarity of strains
isolated over such a long time frame in one-third of these carriers
suggests that nasal carriage may persist for years, although
intermittent colonization with these S. aureus strains over
the 8-year period cannot be excluded. The persistence of single
S. aureus clones in some of the carriers confirms previous reports on the exchange of S. aureus strains over time in
nasal carriers (15, 28, 38, 46). A recent study noted that
the S. aureus exchange rate was significantly higher in
intermittent carriers than in persistent carriers (46),
which would agree with our finding that the persistence of single
S. aureus clones occurred only in carriers with carrier
indices of 1.0.
Many different typing methods have been used to study clonal
relatedness between S. aureus strains (43). PFGE
and RAPD analysis are considered to be among the most reliable and
reproducible whole-genome typing procedures (43, 45), with
even increased resolution when combined analyses are performed
(45). RFLP analyses of the genes encoding S. aureus proteins, such as protein A and coagulase, have also been
applied as genotyping techniques (11-13, 18, 41-43).
Although it has been suggested that protein A genotyping may be an
important tool in the clonal analysis of methicillin-resistant S. aureus isolates (11), our data confirm the findings of
recent studies that reported striking heterogeneity in the number of protein A gene repeat units in otherwise genetically highly related S. aureus isolates (18, 46), indicating that the
protein A gene behaves in a hypervariable, unstable manner that is
unrelated to the overall evolution of the S. aureus genome.
Coagulase gene typing has been successful in the identification of
outbreak-related strains (13, 18, 42, 43); however,
unrelated strains may share identical AluI RFLP patterns
(41, 43), suggesting that it should not be used as the sole
method for the typing of S. aureus. In this study only 7 coagulase genotypes were observed, whereas 15 were observed by RAPD
analysis and 16 were observed by PFGE, confirming the lower
discriminatory power of coagulase gene typing compared to those of RAPD
analysis and PFGE.
Protein A and coagulase, S. aureus proteins that protrude
from the bacterial surface, have been suggested to play a role in the
process of adherence of S. aureus to host cell structures (2, 16) and the epidemic behavior of S. aureus
(12, 39). As polymorphisms in the genes that encode these
proteins might be related to the efficiency of S. aureus
adherence, we studied whether there was a relation between the
composition of the coagulase genes and/or protein A genes of S. aureus strains and the likelihood of isolation of these specific
strains after 8 years. Confirming recent data (46), the
results of this study do not provide evidence of such a relation.
In summary, the lack of consistency in defining the S. aureus nasal carrier states has resulted in striking variations in reported carriage rates. Major factors that should be considered when
comparing carriage rates are the population studied, the sampling and
culture techniques applied, the follow-up period, the number of
cultures available, and the criteria used to distinguish intermittent
from persistent carriers. The results of this study indicate that
persistent carriage of S. aureus is a unique characteristic of a fraction of the population and that the attribute "persistent" should be confined to those individuals for whom serial nasal swab
specimen cultures uniformly and consistently yield S. aureus.
We thank Erwin Panken and Miranda Boers for excellent technical
assistance with the genotyping.
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Abstract
Introduction
Present address: Regional Public Health Laboratory, Haarlem, The Netherlands.
