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Multiclonality of Staphylococcus aureus in atopic patients as revealed by
spa gene DGGE-PCR
Andreas Matussek1,2*, Sara Melin1, Sofia Nygren1, Olaf Dienus1, Sture Löfgren1 and
Per-Eric Lindgren1,3
1Division
of Laboratory Medicine, Department of Clinical Microbiology, County
Hospital Ryhov, Jönköping, Sweden, 2Present address: Unilabs, Laboratory
Services, Göteborg, Sweden, 3Division of Medical Microbiology, Department of
Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
Key words: DGGE, spa, Staphylococcus aureus, multiclonality, atopic patients
Running title: Multiclonality of Staphylococcus aureus
*Corresponding
author:
Andreas Matussek
Unilabs
Laboratory services
SE-413 10 Göteborg
Sweden
Phone: 0046-31-636565
Fax: 0046-31-636560
email: [email protected]
ABSTRACT
Staphylococcus aureus is a gram-positive bacterium infections causing a wide range
of diseases, including skin infections, soft tissue infections, endocarditis, and
septicaemia. Bacteraemic infections often develop from the resident S. aureus flora
located in the anterior nares of the patient. The understanding of the biological nature
of S. aureus colonization is still limited, and most studies of the nasal carriage
presume that individuals are colonized by a single strain.
Recently simultaneous nasal carriage of multiple strains of S. aureus was shown
using mathematical modelling, and the model predicted that 6.6% of individuals carry
>1 strain. However, that study had limitations due to culture, and typing of only three
isolates of S. aureus per patient. The issue of clonality is clinically important in
carriage as well as infections since polyclonal infections may be induced by an
antibiotic-susceptible strain together with a second, more resistant strain, which may
lead to failure of antibiotic treatment.
In this study we therefore developed a powerful culture independent method to
characterize polyclonal bacterial communities of S. aureus. Using PCR – Denaturing
Gradient Gel Electrophoresis (DGGE), spa genes from S. aureus differing with only a
single base substitution can be separated, and carriage and infections by more than
one isolate can be characterized.
INTRODUCTION
Staphylococcus aureus is the leading cause of hospital-acquired infections and is
responsible for a wide range of diseases, including skin infections, soft tissue
infections, endocarditis, and septicaemia. In healthy individuals S. aureus is a
transient or persisting part of the residential flora in up to 30-70% of the population
(ref). However, atopic patients are colonized to a higher extent and exhibit an altered
resistence pattern comparing strains from lesional and nonlesional skin (ref).
Bacteraemic infections often develop from the resident S. aureus flora if the
protective barriers are broken and a major part of septicaemic isolates are recruited
from the anterior nares (ref). The understanding simultaneous colonization of
different S. aureus populations is still limited, and virtually all studies of the nasal
carriage presume that individuals are colonized by a single strain (ref).
In a recent study, the frequency of simultaneous nasal carriage of multiple strains of
S. aureus was investigated based on picking three bacterial colonies from plates
derived from each colonized subject (ref). The frequency of multiclonality was then
calculated with a mathematical model, predicting that 6.6% of them carry >1 strain.
The issue of multiclonality is clinically important in order to determine the virulent
disease-causing strain(s). The conventional laboratory methods for detection and
identification of S. aureus infections are mainly based on cultivation techniques
including picking only one colony from an agar plate. Screening of a single isolate
from patients infected with S. aureus may identify an antibiotic-susceptible strain,
belonging to the normal flora, rather than a second virulent and eventual more
resistant strain, which may impair the antibiotic treatment. Furthermore, most
previous epidemiological studies of S. aureus carriage and transmission are based
on investigations of a single colony derived from each patient, which may have
biased the conclusions. However, for coagulase negative staphylococci (CoNS),
infections caused by several different strains are often considered (ref). Up to date,
the lack of sufficient tools to characterize a multiclonal community of S. aureus may
have hampered proper treatment.
During the last decade several molecular methods, including denaturing gradient gel
electrophoresis (DGGE), using PCR products derived from amplification of selected
key genes have been developed. Using DGGE DNA fragments, differing in only a
single base substitution can be separated, which enables simultaneous detection of a
large number of different bacterial populations in a single sample with or without prior
culture. In a recent study, the total bacterial community in venous leg ulcers was
characterized using a 16S rRNA gene-DGGE approach (ref). The S. aureus protein A
(spa) gene has previously been shown to possess the needed discriminatory power
for epidemiological subtyping (ref). The aim of this study was to develop a species
specific DGGE PCR –method for S. aureus using the spa gene to characterize
multiclonal colonization of atopic patients in order to facilitate proper treatment.
According to our knowledge this is the first published successful use of spa-DGGE
for similar purposes.
MATERIALS AND METHODS
Bacterial strains and culture conditions
Ten S. aureus isolates of known spa types were acquired from the Microbiology
laboratory, Ryhov County Hospital, Jönköping, Sweden. The isolates were cultivated
at 37 ºC on blood agar plates. The number of spa repeats in the isolates varied from
2 to 16, as shown in Table 1. Repeat numbers 13 and 34 differ by only one point
mutation similar as repeat nos. 5 and 21.
Collection of S. aureus from patient samples
Blood agar contact plates from affected and non affected skin, and swabs from the
anterior nares (Copan, Brescia, Italy) from eight patients with atopic dermatitis were
received from the Dermatology Department at the County Hospital Ryhov in
Jönköping, Sweden (etikansökan dnr). The contact plates were incubated at 37 ºC
over and visually examined for growth of S. aureus after 24 and 48 h. The swabs
were inoculated on BD BBL™ CHROMagar™ Staph aureus plates (Becton
Dickinson, Stockholm, Sweden), and then subjected to DNA extraction (see below).
The plates were then incubated for 48 h. Potential S. aureus colonies were picked
and confirmation was performed by DNase testing. Up to 10 confirmed S. aureus
isolates from each contact plate were stored in skimmed milk at -80 ºC until further
analysis. All bacteria grown on each contact plate were then harvested in 700 μl
0.9% NaCl, of which 100 μl was used for DNA extraction, and the remaining part was
frozen at -80 ºC in skimmed milk.
(One additional patient from the Intensive Care Unit, County Hospital Ryhov,
Jönköping, Sweden, was included after revealing double colonization of S. aureus.)
DNA extraction and purification
DNA from the S. aureus isolates with defined spa types, and bacteria from contact
plates, were purified using the protocol “Purification of DNA from bacterial culture
samples using the EZ1 DNA Tissue Kit and the EZ1 DNA Bacteria card” (Qiagen,
Hilden, Germany). Briefly, bacterial cell pellets, obtained after centrifugation at 5000
× g for 5 minutes of 100 µl of the bacterial suspensions from the harvested contact
plates, and approximately 10 colonies of S. aureus, grown on blood agar at 37 ºC
overnight, were resuspended in 180 μl Buffer G2 (Qiagen). Thereafter 20 μl lysozyme
(50 mg/ml, företag?) was added and the suspensions were incubated at 37 ºC for 30
minutes. The swabs were submerged into a mixture of 290 μl Buffer G2 and 10 μl of
proteinase K, vortexed and incubated at 56 ºC for 15 minutes.
200 μl of these pretreated samples (isolates with defined spa types, isolates from
contact plates and swabs, bacterial suspensions) were then subjected to DNA
purification on the EZ1 BioRobot® (Qiagen) using the method “Purification of
bacterial DNA from primary samples using the EZ1 DNA Tissue Kit and the EZ1 DNA
Bacteria Card” and eluted in 100 μl. DNA concentration was measured using the
NanoDrop (Thermo Scientific, Wilmington, USA).
spa typing
The polymorphic X region of the spa gene was amplified by PCR using the primers
spa-1113f (5'-TAAAGACGATCCTTCGGTGAGC-3') and spa-1514r (5'CAGCAGTAGTGCCGTTTGCTT-3'), according to Ridom GmbH (Würzburg,
Germany)(ref). Both strands were sequenced using a CEQ 8800 capillary sequencer
(Beckman Coulter, Fullerton, CA). By the use of Ridom StaphType software (Ridom
GmbH), spa types were assigned each isolate as described previously
Primer design and optimization of the PCR reaction for DGGE analysis
spa primers, previously described by Harmsen et al., were modified for DGGE
analysis by the attachment of a GC clamp. The PCR amplification was carried out
using either the forward or reverse primer with an attached GC clamp, in combination
with the corresponding primer without GC clamp. PCR conditions were tested and
the optimal annealing temperature 63 ºC, was determined by a temperature gradient
PCR ranging from 58 to 72 ºC.
Extracted DNA was amplified in 25 μl reactions. PCR-program.
DGGE optimization and evaluation
PCR products were analysed using a DCode universal mutation detection system
(Bio-Rad Laboratories Inc.,Hercules, USA). Polyacrylamide gradient gels (160 x 160
x 1 mm) composed of 37.5:1 acrylamide:bisacrylamide (7%) and 1 x TAE (40mM
Tris-HCl; 20mM sodium acetate; 1mM EDTA, pH 7.4/8.3) with 15–50% denaturant
were cast with the aid of a gradient maker (GM-40, C.B.S. Scientific Company Inc.,
Del Mar, USA). The composition of 100% denaturant was defined as 7M urea and
40% (vol/vol) formamide (Muyzer et al.,1993). The gels were polymerised with 6.9 ml
of TEMED and 110.5 ml of 10% ammonium persulphate. Ten µl of the PCR-products
(240 ng DNA) were loaded to each well on the DGGE gel. The gels were run in 1 x
TAE buffer at 62˚C for 13.5 h at 130 V. After electrophoresis, the gels were stained in
1×TAE buffer containing SYBRGold (Invitrogen) for 40 minutes and visualized in UV-
transillumination using a CCD camera (LAS-3000, FujiFilm, Tokyo, Japan), following
the manufacturers instructions (Blue light, 460 nm EPI, filter Y515-Di).
To evaluate the discriminatory power of the DGGE method, the spa-PCR products
from up to 14 different strains were mixed and subsequently analyzed in a single
well. In addition, PCR was performed on suspensions containing up to six different S.
aureus isolates to evaluate if a number of different strains could be amplified
simultaneously and separated by DGGE.
RESULTS
DGGE optimization and evaluation
Initially the two different primer pairs were evaluated, and best results were achieved
when the GC clamp (marked in bold) was attached to the reverse primer spa-1514rGC (5’CgCCCgCCgCgCCCCgCgCCCgTCCCgCCgCCCCCgCCCgCAgCAgTAgTgCCgTT
TgCTT-3’) (TIB MOLBIOL, Berlin, Germany). The detection limit of the PCR assay,
using DNA from one strain each, was 10 gene copies/reaction as evaluated for three
different S. aureus isolates containing 7, 10 and 12 spa repeats, respectively (data
not shown).
Mixes containing 1000 gene copies of up to seven different spa types (Table 1,
isolates 1041, 1139, Q30, N32, 1100, 1144, O29) were amplified and analyzed by
DGGE. The method allowed discrimination of all types, even though three of them
had the same product length and just differed by a single point mutation. In a mixture
containing amplified spa gene DNA of 14 different spa types run in one well, 13
bands could be distinguished (Figure 1). Five of the strains were of the same product
length differing only in sequence.
Mixes containing different ratios of DNA from isolates of different spa types revealed
a dynamic range of 1:1000 (Figure 2).
spa DGGE analysis and spa typing from conventional isolated S. aureus from
atopic patients
DNA from affected and non affected skin and anterior nares were subjected to spa
DGGE analysis (Figure 3). The results showed that X of the patients exhibited two
bands in DGGE, indicating presence of two S. aureus populations. Sequencing of
these bands revealed spa types ty and tz of the first patient and ty and tz of the
second patient, respectively. By spa typing of the isolated colonies picked from agar
plates, up to 10 per plate, we were able to confirm these data. Indeed these patients
carried two different S. aureus isolates. Cultures from all other patients did
exclusively reveal carriage of a single isolate both by the spa DGGE method and by
spa typing of up to 10 different S. aureus from conventional bacterial cultivation
(Table 2).
DISCUSSION
In the present study we have developed a method, spa-DGGE, to simultaneously
detect multiple carriage and/or infection of S. aureus strains. Previous studies
addressing this question are biased due to using conventional cultivation of one or a
limited number of S. aureus colonies picked from agar plates. However, this new
molecular method has the ability to detect a large number of different strains in one
clinical sample, and to detect one bacterial cell in a background of 104 cfu (Table ). In
words this means in traditional bacterial culture one would need to pick 10000
colonies to find the spa type of the minor population.
So far very few studies have focused on this issue. In one study by Cespedes et al.
the authors have shown that in nasal carriage less than 10% are colonized by more
than one strain. Their approach, cultivation of three colonies from each sample, is
time consuming, and will only reveal the major clones. Thus, the risk of
underestimation of the diversity of S. aureus is obvious both regarding the number of
clones in one individual as well as the numbers with polyclonal colonization.
We here describe the development of a method that can discriminate between at
least 15 different spa types in a single well, when adding PCR products. The number
of clones detected in a single sample is dependent on the gene copy number of each
clone, and when mixing 1000 gene copies of six different spa types in a single
sample we were able to detect them all. Furthermore, we detected colonisation of
patients with >1 strain in samples from different locations of carriage as well as
infections. These data indicate that infections, not just carriage can be induced by
multiple S. aureus isolates.
In contrast to CoNS, where polyclonal infections and bacteremias are established
facts (ref), for S. aureus it is still unclear, however polyclonal infections very likely
occur (ref). Such infections would indeed affect the selection of antimicrobial therapy
with different antibiograms for different isolates, thus hampering the treatment
outcome of serious infections. (ref).
Fallbeskrivning om det finns kring bristande AB terapi (ref).
The spa-DGGE methodology opens up the possibility to characterize the S. aureus
community structure, which will enable a better understanding of S. aureus
colonization and infection. Furthermore, tracing the reservoirs for transmission within
hospital settings will be facilitated. Recently, one study described that newborns were
colonized to one third from the parents, one third from the staff, and the remaining of
unknown origin (Matussek et al. 2007). Inclusion of spa-DGGE methodology may add
new dimensions to determine the origin of unknown transmissions.
Recently, Johnson et al. described sharing of fecal E. coli between household
members, and that one single individual carried 1 to 6 (median, 2) clones (ref).
However, the authors indicated limitations making these findings only a minimum
estimate of the extent of within-household strain sharing, and clone diversity in
individuals.
We have developed a culture independent method for typing of S. aureus
communities, which may shed new light into the issue of the epidemiology. The spaDGGE method offers the potential to be developed into a valuable tool to investigate
complicated and/or recurrent infections.
ACKNOWLEDGEMENTS
We would like to acknowledge. This study was in part financed by FORSS.
Table 1. Staphylococcus aureus strains with different spa types, number of repeats
and repeat succession.
Strain
1144
L1
1100
1139
1041
N32
1110
Q30
930
O29
spa type
t233
t037
t630
t015
t050
t064
t008
t355
t166
t032
Repeat succession
15-12
15-12-16-02-25-17-24
08-16-02-16-34-17-34-16-34
08-16-02-16-34-13-17-34-16-34
08-16-02-16-34-34-17-34-16-34
11-19-12-05-17-34-24-34-22-25
11-19-12-21-17-34-24-34-22-25
07-56-12-17-16-16-33-31-57-12
04-44-33-31-12-16-34-16-12-25-22-34
26-23-23-13-23-31-29-17-31-29-17-25-17-25-16-28
REFERENCES