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Departments of Clinical Microbiology
Infectious Diseases,rebro University Hospital, SE-70185 rebro, Sweden
ABSTRACT
A protocol for multilocus sequence typing (MLST) of methicillin-resistant Staphylococcus aureus (MRSA) was adapted to real-time LightCycler System PCR for efficient and rapid amplification of seven housekeeping genes in the same PCR run and real-time detection of the products. The method was evaluated on a representative and well-characterized collection of clinical MRSA isolates (n = 57) obtained from an area of low endemicity. Twenty sequence types (STs) and nine clonal complexes were identified. Combining STs and the staphylococcal cassette chromosome mec (SCCmec) type identified 27 different genotypes, and type IV SCCmec was present in 11 different STs. The presence of the Panton Valentine leukocidin (PVL) genes was found in isolates of four different STs. Eleven different STs were found among the community-acquired as well as among the hospital-acquired MRSA. The genetic heterogeneity was also denoted by pulsed-field gel electrophoresis analysis that showed 24 different pulsotypes among the 57 MRSA isolates. The presence of more than one different type of SCCmec in the same ST indicates that the MRSA clones have arisen at several occasions in the same genetic background by independent acquisition of SCCmec into methicillin-sensitive strains. This circumstance shows the importance of combining MLST data with SCCmec-typing results when investigating the origins of MRSA.
INTRODUCTION
Methicillin-resistant Staphylococcus aureus (MRSA) is well known as a nosocomial pathogen that has spread worldwide since it was first described in the early 1960s (16). However, there has recently been a changing epidemiology and emerging problems with MRSA that are disseminating in the community (5, 23) among otherwise healthy people, including children (3). These isolates are described to harbor potential virulence factors, and reports from several parts of Europe describe the spread of clones that are producing the Panton-Valentine leukocidin (PVL) (2, 19, 28, 29).
Acquisition and expression of the gene mecA is the genetic event that makes S. aureus resistant to methicillin and all other -lactam antimicrobials. mecA encodes an altered penicillin-binding protein, PBP2a (or PBP2'), and it is always carried on the mobile staphylococcal cassette chromosomal mec (SCCmec) (15, 20). The SCCmec element is present in five (I to V) different allotypes and contains a characteristic combination of two essential genetic components, the mec gene complex and the ccr gene complex (14). The SCCmec elements are widely disseminated among coagulase-negative staphylococci, and studies have presented evidence for horizontal transfer between staphylococcal species, although this seems to be a rare event (12, 30).
There are various molecular typing methods available for S. aureus but pulsed-field gel electrophoresis (PFGE) is still the gold standard method for investigating outbreaks and local epidemiology. However, PFGE has major disadvantages, including difficulties in comparing the results of different laboratories (8, 25). Multilocus sequence typing (MLST) is used to identify clones within populations of pathogenic microorganisms and genetic variation is observed by assigning the alleles at each locus directly by nucleotide sequencing the internal fragments of seven housekeeping genes. The sequences of each gene are assigned as distinct alleles, and the sequence type (ST) or allelic profile of each isolate is defined by the alleles at each of the seven housekeeping loci. Isolates with the same allelic profile for six out of seven genes are assigned as members of the same clonal complex (8, 21). A major advantage of MLST is the ability to compare sequence data between laboratories via the MLST website on the Internet.
MLST of large numbers of MRSA from different geographic areas has revealed that there are five major clonal lineages that are internationally dominating (1, 4, 24). However, community-acquired MRSA are found to possess more diverse evolutional origins compared to nosocomial strains (7, 11, 20, 23, 24), and they are predominantly carrying type IV SCCmec.
The aim of the present study was to adapt the MLST protocol for S. aureus to real-time PCR amplification of the seven housekeeping genes and to employ the method on a well characterized collection of MRSA isolates from an area of low endemicity.
MATERIALS AND METHODS
Bacterial isolates. A selection (n = 57) of representative clinical MRSA isolates (n = 92), identified during 1987 to 2004 in rebro County (population approximately 280,000 inhabitants) were analyzed. Outbreak- or otherwise related isolates (e.g., intrafamily spread), confirmed by PFGE, were excluded. Fifty-one of the 57 isolates have previously been characterized by SCCmec typing, PFGE, and the presence of PVL genes (2). Contact tracing has been performed according to the Swedish Communicable Diseases Act since 2000 and essential epidemiological information has been collected and available in a county database since 1997. Isolates were stored at –70°C in preservation medium (yeast extract, horse serum, Trypticase soy broth) at the Department of Clinical Microbiology at rebro University Hospital.
Community-acquired infection was defined as identification of MRSA in the outpatient setting or a culture positive for MRSA within 48 h after hospital admission. In addition, the patient had no medical history of MRSA infection or colonization, and no medical history in the last year of hospitalization, admission to a nursing home, dialysis or surgery. The patient had no permanent indwelling catheters or medical devices that pass through the skin. Twenty-nine of 57 (50.9%) MRSA were categorized as community-acquired MRSA, 24 (42.1%) were hospital-acquired MRSA, and four (7.0%) could not be classified due to lack of epidemiological information. Isolates were derived from skin (n = 46), nares (n = 9), blood (n = 1), and urine (n = 1).
DNA isolation. All isolates were cultured on blood agar (Columbia II agar, 4.25% wt/vol; horse blood, defibrinized, SVA, 6% wt/vol) and incubated overnight at 37°C. DNA isolation of the reference strain was made with Dynabeads DNA DIRECT system I kit (Dynal Biotech A.S, Oslo, Norway) for higher purity and longer stability. Genomic DNA of the clinical isolates was extracted by denaturating a few colonies suspended in sterile water at 98°C for 15 min and then centrifuged at 13,000 x g for 30 seconds. The supernatant was used as template for amplification in the real-time PCR.
MLST. The conventional procedure for performing MLST in S. aureus described by Enright et al. (8) was adapted and optimized for use in the real-time LightCycler System PCR and for amplifying the seven genes in the same PCR program, but in different reactions for each gene. An alternative primer described by Crisostomo et al. (4) was used for amplification of carbamate kinase (arcCF2) in one isolate. Primers used in the present study were synthesized by Scandinavian Gene Synthesis (SGS), Kping, Sweden. The strain NCTC 8325 with a characterized allelic profile (ST8 [3-3-1-1-4-4-3]) was used as the reference during optimization of the protocols and was included as a standard DNA control for all amplifications.
The LightCycler FastStart DNA Master SYBR Green I (FastStart Taq DNA polymerase, SYBR Green I, deoxynucleoside triphosphates with dUTP instead of dTTP, reaction buffer, 10 mM MgCl2) (Roche Diagnostics, Mannheim, Germany) was used. The reactions were performed in glass capillaries at a volume of 20 μl and the cycling conditions were optimized for LightCycler System PCR. These included titration of MgCl2 and primer concentrations as well as optimizing annealing temperatures and extension times. Each reaction contained 2 μl LightCycler FastStart DNA Master SYBR Green I, 0.5 μM of each primer, 3 mM MgCl2 and 2 μl DNA template (3 ng). Samples were preincubated for 10 min at 95°C and then subjected to 30 cycles of amplification run according to the following schedule: denaturation at 95°C for 10 s, annealing at 55°C for 5 s, and extension at 72°C for 24 s.
Characterization of the amplification products was performed by melting curve analysis subsequent to the amplification run. The melting program consisted of one cycle of 95°C for 0 s, 65°C for 15 s and then the temperature was increased at a transition rate of 0.1°C/s to 95°C during continuous fluorescence monitoring. The samples were then cooled at 40°C during 30 s. Melting peaks were derived from the initial melting curves (fluorescence [F1] versus temperature [T]) by plotting the negative derivative of fluorescence over temperature versus temperature (-dF1/dT versus T). The specific Tm obtained from all runs of the analyzed isolates (n = 57) and the reference strain was used to calculate the mean Tm of each gene. Intra-assay variations were tested by comparing the calculated Tm of reference strain NCTC 8325, (3 ng), in five different capillaries during the same run. Interassay variations were tested with NCTC 8325 (3 ng) by comparing the given Tm in five different runs.
Prior to sequencing, the PCR product was purified by High Pure PCR Purification Kit (Roche Diagnostics) and sequenced with ABI PRISM BigDye Terminator version 3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Stockholm, Sweden). Sequencing was performed in 96-well plates and each reaction (10 μl) consisted of 1 μl of purified DNA (2 to 50 ng DNA), and 2 μl of Terminator Ready Reaction mix (A, BigDye terminator labeled with dichloro [R6G], C, BigDye terminator labeled with dichloro, G, BigDye terminator labeled with dichloro[R110], T, BigDye terminator labeled with dichloro, dATP, dCTP, dITP, dUTP, AmpliTaq DNA polymerase [FS] with rTth pyrophosphatase, MgCl2, Tris-HCl buffer [pH 9.0]), and 1.6 pmol primer.
Cycle sequencing was carried out on the GeneAmp PCR System 2700 (Applied Biosystems, Warrington, England) and the same primers initially used for LightCycler System PCR (4, 8). Sequences were confirmed on both strands, by 25 cycles consisting of 96°C denaturation for 10 min, 55°C of annealing for 5 s, 60°C of extension for 4 min, and then cooling at 4°C.
Reactions were precipitated by using 3 M sodium acetate, pH 4.6 and 95% ethanol with a final ethanol concentration of 65%, washed in 70% ethanol, and resuspended in 10 μl formamide (Applied Biosystems, Stockholm, Sweden) before separation on ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). The nucleotide sequences of the seven loci were analyzed using ABI PRISM AutoAssembler DNA Sequence Assembly 1.4.0 software and were compared to allelic profiles and associated epidemiological data via the MLST website (http://www.mlst.net). The allelic profiles were compared by using the program eBURST v.2. (based upon related sequence types), developed by Feil et al. (10) and available on the MLST website.
Determination of SCCmec types. SCCmec typing was performed by real-time LightCycler System PCR to detect the essential genetic components mecA, mecR1, IS1272, ccrA, and ccrB according to Berglund et al. (2).
Detection of lukS-PV and lukF-PV. The LightCycler System PCR and SYBR Green I dye were used to detect PVL genes as described previously (17).
Pulsed-field gel electrophoresis. The 57 MRSA isolates were characterized by pulsed-field gel electrophoresis (PFGE) of chromosomal SmaI digests, prepared with the GenePath group 1 reagent kit (Bio-Rad Laboratories, Hercules, CA) and PFGE patterns were obtained with a contour-clamped homogeneous electric field apparatus (GenePath System; Bio-Rad) as described (2).
RESULTS
MLST was successfully performed in all (n = 57) isolates by sequencing the seven housekeeping genes following amplification with real-time LightCycler System PCR. Single peaks were observed for all gene products and the mean Tm and intra- as well as interassay variations are given in Table 1.
Twenty STs were identified by MLST and these are summarized in Table 2. The evolutionary origins were examined by the algorithm eBURST, in order to group the received allelic profiles into clonal complexes. Grouping was based on the similarity between sequence types in six of seven loci and singleton STs are defined as not sharing six out of seven loci with any other STs in the data set. Four groups of related genotypes were identified and in addition 10 singletons. Comparison with all S. aureus isolates in the database at mlst.net revealed the presence of nine clonal complexes (CC1, -5, -8, -22, -30, -45, -59, -78, and -80) and one singleton (ST140). Eleven STs (ST22, -39, -46, -111, -125, -140, -150, -154, -241, -247, and -375) were represented by a single isolate, while ST45 (n = 13) and ST80 (n = 12) were the most common profiles.
Five sequence types (ST1, ST5, ST8, ST30, and ST45) were represented by at least two isolates each that has been assigned different SCCmec types (or different subtypes of type IV SCCmec). Combining ST and the SCCmec type, 27 different genotypes were identified in our area. Type IV SCCmec was present in 11 different STs, 1, 5, 8, 22, 30, 45, 46, 80, 125, 140, and 375. Those were clustered into three clonal complexes, and the remaining six STs did not correspond to any group and were defined as singleton STs. If compared to all S. aureus isolates in the mlst.net database, one of the profiles (ST140) was defined as a singleton.
Type I SCCmec was found in three different STs (111, 228, and 247), type II in two STs (5 and 39) as well as type III (STs 239 and 241), and isolates with untypeable SCCmec were present in six different STs (1, 5, 45, 150, 152, and 154). In contrast, ST80 (n = 12) was associated only with type IVc SCCmec, ST228 (n = 2) with type I SCCmec, and isolates with ST239 (n = 5) all carried type III SCCmec. ST80 and type IVc SCCmec were predominant among the 30 community-acquired isolates, but there were 10 additional STs represented by a single or few community-acquired MRSA carrying either type II, type III, type IV, or unknown types of SCCmec. The hospital-acquired isolates (n = 24) were represented by 11 STs, and ST45 and ST239 were the most frequent. The isolates (n = 4) that it was not possible to classify as community-acquired or hospital-acquired MRSA were represented by three STs.
The genes lukS-PV and lukF-PV were found among isolates in four different STs (8, 80, 152, and 154) whereas ST8 and ST80 all had the type IVc SCCmec but ST152 and ST154 both included isolates that were untypeable for type I to IV SCCmec. These four STs are assigned to different origins according to the database www.mlst.net.
The 57 MRSA isolates were clustered into 24 pulsotypes (Fig. 1) that were indistinguishable, closely related, or possibly related (27). Isolates with ST1, ST8, ST45, ST80, and ST239 were clustered into more than one pulsotype, while two pulsotypes included isolates that were assigned to different STs.
DISCUSSION
In this study, we adapted an existing protocol for MLST (8) to the real-time LightCycler System PCR and amplification of the seven housekeeping genes in the same PCR run. The method was evaluated on a representative material of clinical MRSA (n = 57) that was well characterized by SCCmec typing, PFGE, and the presence of PVL genes. MLST was highly discriminatory and identified 20 STs among the 57 isolates, although a much higher diversity was seen when combining MLST data with associated types of SCCmec.
The isolates analyzed could be assigned to nine clonal complexes based on the similarity between sequence types in six of seven loci. CC5 (n = 7), CC45 (n = 14), and CC80 (n = 12) were the predominant clonal complexes represented in rebro County, Sweden, which was expected since those are widely disseminated international clones (7, 28). ST111 is a single-locus variant of ST228, and those STs were grouped but not defined as a clonal complex. However, by using the less stringent group definition (i.e., five of seven shared alleles) ST228 and ST111 are assigned to CC5. ST152 and ST154 were assigned to different groups, and only one singleton ST (ST140) was identified. There were equal numbers (n = 11) of STs among the community-acquired isolates and the hospital-acquired MRSA, which indicates a high diversity of genotypes and sporadic cases in the hospital environment. Only three STs (5, 45, and 239) were represented by MRSA that were isolated both in the community and among the nosocomial isolates. The different genetic backgrounds of community-acquired MRSA and hospital-acquired MRSA suggest that they are not related and that community-acquired MRSA are not nosocomial isolates that have spread outside the hospital environment.
Enright et al. (7) strongly recommend characterization of MRSA with both MLST and SCCmec typing since isolates with the same ST may carry different SCCmec. By using this approach, four of the five major pandemic clones were recognized in our area. The Brazilian-Hungarian clone (ST239, type III SCCmec) was represented by five isolates, the New York-Japan clone (ST5, type II SCCmec) by two isolates, the pediatric clone (ST5, type IV SCCmec) by three isolates, clone V (ST8, type IV SCCmec) by two isolates, and the Iberian clone (ST247, type I SCCmec) by one isolate.
Interestingly, one community-acquired MRSA with type II SCCmec was isolated in rebro and is assigned to ST39, an allelic profile that, along with ST30 and the widespread MRSA clone ST36, belongs to CC30. However, all MRSA isolates within this complex have been assigned to ST36, while ST39 has been described as a successful clone of methicillin-susceptible S. aureus (9). MRSA with type II SCCmec are described as mainly nosocomial and as carrying multiple resistance genes for non--lactam antibiotics (20, 22) and these circumstances makes our community-acquired isolate unique since it has a non-multiresistant nature and was manifested by a clinical significant infection.
The presence of more than one different type of SCCmec in the same ST indicates, in congruence with other studies (1, 7), that the MRSA clones arose at several occasions in the same genetic background by independent acquisition of SCCmec into methicillin-sensitive strains. This circumstance denotes the importance of combining MLST data with the SCCmec typing results when trying to understand the origins of MRSA. About one third of the isolates in our county were not typeable for type I to IV SCCmec (2), and those may represent novel types and are therefore taken in consideration when discussing possible origins of our MRSA. The presence of unknown types of SCCmec in as many as six unrelated STs depicted by eBURST gives rise to suspicion that these elements represent either more than one novel type of SCCmec or perhaps one new type with an enhanced mobility that has generated several clones of MRSA.
A previous study (23) describes nontypeable SCCmec (i.e., not type I to IV) in community-acquired MRSA with ST45 that originated from Australia, and those were found to contain the class C2 mec complex, an essential part of SCCmec that is commonly found among coagulase-negative staphylococci (18). The isolates with ST45 and nontypeable SCCmec in our county might carry the same mec gene complex, recently described as a part of the type V SCCmec (14). The presence of more than two different types of SCCmec in our isolates with ST45 may indicate that this clone of S. aureus has acquired SCCmec on more than one occasion, and it is therefore possible that this ST carries novel types of SCCmec.
The PVL genes are found among isolates in four different STs, two of which (ST8 and ST80) contain type IVc SCCmec and the other two of which (ST152 and ST154) were untypeable for type I to IV SCCmec. The ST80 strains with type IVc SCCmec, fusidic acid resistance, and PVL locus are disseminating in several parts of Europe (28, 31) and this ST appears to be a predominant clone of community-acquired MRSA in rebro.
ST8 is an ancestral genotype that has created two major clones of MRSA with either type II or type IV SCCmec (6). ST8 is represented by two community-acquired MRSA in our area, with either a type IVa SCCmec or a type IVc and the PVL locus. ST1 and ST30 are known as community-acquired MRSA and as carrying the PVL locus (23, 26), however, our ST1 and ST30 isolates were not found to carry the PVL genes. The presence of the PVL genes in four genetic backgrounds among our isolates is, in addition, an indication of the genetic diversity of community-acquired MRSA. Previously, the PVL toxin has only been associated with MRSA carrying the type IV SCCmec (19, 23). Therefore, the isolates with untypeable SCCmec and PVL genes may represent altered type IV SCCmec that are not detectable with the primers used, or they may represent the acquisition of a novel SCCmec into PVL-producing lineages of S. aureus.
The association between ST type and SCCmec is more obvious in hospital isolates with a more multiresistant nature and type I to III SCCmec, whereas the type IV SCCmec is a promiscuous element found in widely divergent genotypes. Type IV SCCmec is considered to have an enhanced ability for horizontal transfer into distantly related clones of S. aureus (24), probably due to its smaller size and functional recombinases, which are responsible for site-specific excision and integration of the element (13).
Isolates that are considered not related by PFGE but are assigned to the same genetic background by MLST are well demonstrated by ST45 and ST239, which were clustered into four different pulsotypes. All but one isolate in ST80 were clustered in one pulsotype, which demonstrates a younger or maybe more stable clone of MRSA compared to ST45 and ST239. On the other hand, ST154, which has a totally different allelic profile, is clustered together with ST45 in the same pulsotype. PFGE reflects genetic events that have occurred during a short time period and is an unsurpassed method for analyzing outbreak situations, but long-term epidemiology is better understood by MLST.
LightCycler System PCR for MLST offers the advantage to perform efficient and rapid amplification, and real-time detection of the seven housekeeping genes in the same PCR run. Melting-curve analysis of the products is performed subsequent to every run to increase the specificity of SYBR Green I detection and thus there is no need for time-consuming gel electrophoresis that is often applied when using a conventional protocol. In addition, amplification and detection are performed in a closed system to eliminate carry over contamination. With the LightCycler System PCR thermal cycling is performed using air instead of thermal blocks in a capillary system to ensure efficient heat transfer to the samples, which decreases each PCR cycle to approximately 15 to 20 seconds.
The gene arcC was not amplified in one isolate using primers described by Enright et al. (8), possibly due to alterations in the binding site of the primers. Amplification of this isolate (00T-231) was possible using the alternative primer arcCF2 described by Crisostomo et al. (4). Another advantage of using the real-time LightCycler PCR is that such failure in amplification is observed during the run and is noticed at an early stage of the MLST analysis. Single peaks were seen in all runs and the melting points were specific for each of the seven genes. The PCR conditions were stable and showed minor intra-assay variations as well as interassay variations for all seven genes. The ability to perform amplification of all genes in one PCR run is convenient when a small number of isolates are investigated. MRSA are still uncommon in Sweden (0.8% of the invasive S. aureus in 2003 [SWEDRES 2003, a report on Swedish antibiotic utilization and resistance in human medicine]), but increasing, and this method is suitable for a laboratory operating in a low-prevalence area.
To conclude, this protocol for MLST of S. aureus adapted to the real-time LightCycler System PCR offers an efficient and rapid amplification of seven housekeeping genes in the same PCR run and real-time detection of the products without gel electrophoresis. This application is suitable and convenient to perform when a small number of isolates of MRSA will be investigated, particularly in an area of low endemicity as in Sweden. MLST of 57 representative clinical MRSA isolates from such an area showed a high heterogeneity, with 20 STs identified and nine clonal complexes. Combining ST and SCCmec types distinguished 27 different genotypes. The presence of various SCCmec types in the same ST indicates that the MRSA clones occurred on several occasions in the same genetic background by independent insertions of SCCmec into methicillin-sensitive strains. This circumstance shows the importance of analyzing MLST data together with the SCCmec typing results when investigating the origins of MRSA.
ACKNOWLEDGMENTS
We thank Susanne Jacobsson for technical support throughout the sequencing and Birgitta Dragsten for performing the PFGE.
This study was supported by grants from the rebro County Council Research committee and the Foundation for Medical Research at rebro University Hospital, Sweden.
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