The typing data resulting from those studies can also be used to enlarge our knowledge of spa-MLST mappings, which is extremely useful for the daily routine typing of S. aureus, in which the BURP algorithm together with “reference spa types” enables the classification of isolates into particular clonal lin- eages. This work, as well as previous studies, has shown that, in general, the typing concordance between spa typing-BURP analysis and alternative methods is high; however, the occur- rence of “group violations” (12) associated with particular BURP groups and clonal lineages was also demonstrated. Some of these misclassifications (in BURP groups A, E, and G) are due to related spa repeat successions in isolates of different clonal lineages, possibly caused by recombination events in the spa locus (reflected by insufficiently branched parts of the phylogenetic tree). The highest degree of misclas- sification and, at the same time, the epidemiologically most relevant misclassification was found in BURP group A, which included isolates of clonal lineages CC1, ST80, ST7, ST15, and ST97 (see also reference 12). Since isolates of CC1 and ST80 represent important caMRSA clones, their unambiguous rec- ognition is of particular importance to prevent their spread in the community and their introduction into hospitals. Isolates of ST398 (spa types t034 and t011 in BURP group E) constitute another clonal lineage of special interest that is falsely grouped. Isolates of this clonal lineage are increasingly isolated from animals throughout Europe and were also found in hos- pital patients; thus, these isolates possibly possess some zoo- notic potential (45) and it is crucial that they be identified in a timely manner. To reduce this type of misclassification, Mell- mann et al. (22) suggested that the parameter “costs” (costs 聿 4) for the definition of BURP groups should be reduced, and FIG. 4. Epidemiological investigation of clusters of infection. The genetic relatedness of the isolates is demonstrated by the spa type as well as by SmaI macrorestriction analysis. In hospital A, four identical isolates and one additional isolate were detected. In hospital B, all isolates were identical, as demonstrated by both methods applied. ICU, intensive care unit.
Pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments has been used by the Belgian Reference Laboratory for Staphylococci for national hospital surveys of methicillin-resistant Staphylococcus aureus since 1992. The sequencing of the polymorphic X region of the protein A gene (spa typing) offers significant advantages over PFGE in terms of speed, ease of interpretation, and exportability. To validate its potential use for national surveillance, we evaluated the robustness of spa typing compared with that of PFGE based on a collection of 217 S. aureus strains representative of the Belgian S. aureus epidemiology during the last 13 years. spa typing and PFGE both showed high discriminatory power (discriminatory indexes of 0.98 and 0.96, respectively) and achieved high concordance (95.9%) in type classification. Both methods also showed good concordance with multilocus sequence typing (MLST) (95.5%). However, we observed occasional “viola- tions” of MLST clonal complex assignment by spa typing. Our results suggest that both PFGE and spa typing are reliable methods for long-term, nationwide epidemiological surveillance studies. We suggest that spa typing, which is a single-locus-based method, should preferably be used in combination with additional markers, such as staphylococcal cassette chromosome mec typing or resistance or virulence gene detection.
Each MRSA isolate underwent characterization by PFGE, using SmaI as a restriction endonuclease, at the Minnesota Department of Health (3). Patterns were compared by using Dice coefficients, and those that were within 80% similarity were considered part of the same pulsotype and characterized according to USA group (3). Those isolates with a banding pattern that was a 100% match were considered indistinguishable. Other genotyping methods were performed at the University of Chicago MRSA Research Center. These methods included MLST (12) and detection of the PVL genes (13), arcA, and opp3 (11) by PCR, as described previously. In addition, the SCCmec type was determined by a series of PCR assays as described previously (14), and each isolate underwent spa typing as de- scribed previously (15).
The good agreement observed between MLVA and PFGE on one hand and spa typing and MLST on the other indicates a low level of genetic recombination in S. aureus. Its clonal population structure arises mostly from point mutations (10), which explains the presence of a phylogenetic signal within the S. aureus population (14). However, long-term evolution may be also influenced by chromosomal replacements that compli- cate the view of the relationships between distantly related isolates. Clusters revealed by spa typing and MLST have not always corresponded in our study. The largest MLST clonal complex CC8 was also the most diverse with six different STs, including ST8 and ST239. This cluster was split into two spa clusters, S1 and S2 (with ST8 and ST239 isolates, respectively), of which cluster S2 was also identified in another MLST group, CC30 (with ST30). Isolates of ST8 and ST239 were considered closely related because they were single-locus variants of each other, differing only in the arcC locus. However, analysis of Robinson and Enright, which included spa and seven surface protein-encoding genes sas, revealed that a large ( ⬃ 557-kb) chromosomal fragment containing all of these genes and arcC was in ST239 identical to that of ST30 and not that of ST8. Therefore, a chromosomal replacement between the ST30 par- ent and the ST8 parent which resulted in the ST239 mosaic chromosome (23) was most likely responsible for the observed incongruence. The second case was found for the spa cluster S3 and the MLST complex CC1 and cluster ST80. The CC1 iso- late 3502 was indistinguishable from isolate BN4 by MLST but different by spa type. On the other hand, its spa type t386 was closely related to the type t044, which is characteristic for three ST80 isolates (cluster S3). There are no detailed data on ge- nome structure of these clones; therefore, we suggest that a chromosomal replacement encompassing the spa locus, fol- lowed by its mutational changes, could have also occurred in the evolution of S. aureus CC1 (ST1) clone.
The strong congruence of spa typing’s genotyping results with multiple techniques implies that the single-nucleotide polymorphisms within the repeat region occurred only once, as recombination and repeated mutation would obliterate any compatibility (27). Occurrence of neutral (as evidenced by the dS/dN analysis) mutations due to purifying selection just once at this locus, probably resulting from functional biological con- straints placed on the protein A gene (as opposed to a gene under positive selection or nonexpressed intergenic regions, pseudogenes, etc., under no constraints), along with a clonal population structure within the species, explains how the spa locus alone can accurately identify strains throughout the breadth of the species and then correctly assign them to lin- eages. These factors, along with the higher rate of nucleotide substitutions and greater variation in repeat number compared with the larger-sized coa repeats, are criteria that give spa typing the capability of reflecting extensive genome-wide vari- ation within S. aureus. Using these characteristics as guidelines, we are currently studying single-locus VNTRs for dual use in local and global epidemiologic analyses of other bacteria, such as E. faecalis and Acinetobacter baumannii, and we predict that in species with known heterogenous clonal population struc- tures, such as Escherichia coli (18), suitable single-locus mark- ers should be successful. The relatively new ability to analyze and search genomes for polymorphic repeat regions combined with the unique ability of certain minisatellite VNTRs to dis- play two different levels of genotypic information will provide many attractive candidates for use for a variety of strain typing goals and should be studied for all types of microorganisms.
Previous studies have shown that there is a fairly good cor- relation between clonal groupings of MRSA isolates obtained by spa typing and other typing techniques (15, 22, 29, 36). The broader application of spa typing revealed a considerable de- gree of spa gene repeat polymorphism within particular clonal groups and clonal lineages of MRSA isolates, as defined by MLST and eBURST, indicating a higher discriminatory power for this method. However, in daily infection control, an unam- biguous and quick attribution of newly arising spa types to known clonal complexes and clonal lineages is essential be- cause of their differential dynamics of emergence and spread (33). This is exemplified by the occurrence of cMRSA isolates, most often containing the lukS-lukF determinant coding for Panton-Valentine leukocidin. They may emerge as (i) clonal lineages not previously reported (40), (ii) derivatives of clonal lineages which have already been known as nosocomial patho- gens in the “pre-MRSA era” (23, 26), and (iii) clones belong- ing to the same clonal lineages as nosocomial MRSA strains and containing both the mecA gene and the lukS-lukF deter- minant (17).
The detection of novel spa types is probably due to limited pre- vious studies of ovine isolates from broad geographical origins. There is a greater diversity, and therefore discriminatory ability, of spa compared with MLST typing (Eriksson et al., 2013; Porrero et al., 2012), and there was a close relationship between spa types and MLST CCs; only one spa type was present in more than one CC. However spa typing did permit the discrimination of isolates with identical STs. Isolates from Australia (all ST133) were split into four spa types; two were novel, one (t3042) has been detected in Danish sheep populations (Eriksson et al., 2013), and one (t998) was de- tected in France (unknown host: http://spa.ridom.de/spa- t998.shtml, last accessed 7th August 2012). This adds to the evi- dence of a direct connection between European and Australian ovine isolates, possibly because of the introduction of sheep from Europe into Australia. Three isolates collected from a single sheep at two time-points [37_008, 37_010, 37_125; Supplementary data- set 1] were included in our study; spa typing reveals that two clo- sely-related strains were present during this infection.
To determine the diversity of the MRSA population in Aus- tria, isolates from diverse regions and hospitals were typed. Because of the high level of polymorphism within the X region of the protein A gene and the fact that spa typing is associated with speed, low costs, and comparability of data, spa typing was used in this study for typing of MRSA isolates. Forty-six dif- ferent spa types were found, of which the majority, 82%, be- long to four spa types only. However, as SSR are generally described as unstable entities that undergo frequent DNA se- quence variation (19, 33, 39, 40, 41), these 46 spa types were grouped into spa complexes with respect to SSR unit compo- sition and organization (34). This system of classification of related types had already been used to group MLSTs into clonal complexes (8). The grouping of related types (spa, MLST, or PFGE) is a valuable tool for strain analysis and supports a hospital epidemiologist’s work by simplifying the identification of epidemiologically related strains. However, molecular subtyping on its own is not able to elucidate whether distinct subtypes have evolved from one another or whether they represent single introductions from outside. Without ad- ditional epidemiological information, it is, for instance, impos- sible to say whether spa type t008 in Vienna is the precursor of the most frequent spa type, t190, or whether these spa types occurred independently. Moreover, without epidemiological data it is even impossible to postulate a correlation between TABLE 5. spa complex V
MLST and spa typing. MLST was performed as previously described by En- right et al. (8). Internal fragments of the seven housekeeping genes for carbam- ate kinase (arcC), shikimate dehydrogenase (aroE), glycerol kinase (glpF), guan- ylate kinase (gmK), phosphate acetyltransferase (pta), triosephosphate isomerase (tpi), and acetyl coenzyme A acetyltransferase (yqiL) were amplified by PCR with the specified primers, and the PCR products were purified (QiaQuick PCR purification kit; QIAGEN GmbH, Germany) and sequenced using an ABI Prism 377 DNA sequencer. Consensus sequences were assembled from both orienta- tions. spa typing was performed according to the procedure of Shopsin et al. (22) using the forward primer 5⬘-GAACAACGTAACGGCTTCATCC-3⬘ and re- verse primer 5 ⬘ -CAGCAGTAGTGCCGTTTG-3 ⬘ , and consensus sequences were assembled from both forward and reverse sequences (12, 22).
tages have been described by Maiden et al. (13), who devel- oped the multilocus sequence typing (MLST) approach. This technique combines sequence information from several house- keeping genes to compare strains in a manner similar to mul- tilocus enzyme electrophoresis (MLEE) (22). Recently, DNA sequencing of the polymorphic X, or short sequence repeat (SSR), region (32) of the protein A gene (spa) has been pro- posed as an alternative to current techniques for the typing of S. aureus (8). The polymorphic X region (Fig. 1) consists of a variable number of 24-bp repeats and is located immediately upstream of the region encoding the C-terminal cell wall at- tachment sequence (9, 21, 29). The diversity of the SSR region seems to arise from deletion and duplication of the repetitive units and also by point mutation (4). While the biological function is not known, the protein A domain encoded by the X region may serve to extend the N-terminal immunoglobulin G binding portion of the protein through the cell wall (34). The existence of well-conserved regions flanking the X region cod- ing sequence in spa allows the use of primers for PCR ampli- fication and direct sequence typing (Fig. 1). The sequencing of the spa SSR region combines many of the advantages of a sequencing-based system such as MLST but may be more rapid and convenient for outbreak investigation in the hospital set- ting since spa typing involves a single locus. Inasmuch as the protein A X region has a high degree of polymorphism, it may have a variation rate (or clock speed) that provides suitable discrimination for outbreak investigation.
ST22-methicillin-resistant Staphylococcus aureus type IV (ST22-MRSA-IV) is endemic in Irish hospitals and is designated antibiogram-resistogram type-pulsed-field group (AR-PFG) 06-01. Isolates of this highly clonal strain exhibit limited numbers of pulsed-field gel electrophoresis (PFGE) patterns and spa types. This study investigated whether combining PFGE and spa typing with DNA sequencing of the staphylococcal cassette chromosome mec element (SCCmec)-associated direct repeat unit (dru typing) would improve isolate discrim- ination. A total of 173 MRSA isolates recovered in one Irish hospital during periods in 2007 and 2008 were investigated using antibiogram-resistogram (AR), PFGE, spa, dru, and SCCmec typing. Isolates representative of each of the 17 pulsed-field group 01 (PFG-01) spa types identified underwent multilocus sequence typing, and all isolates were ST22. Ninety-seven percent of isolates (168 of 173) exhibited AR-PFG 06-01 or closely related AR patterns, and 163 of these isolates harbored SCCmec type IVh. The combination of PFGE, spa, and dru typing methods significantly improved discrimination of the 168 PFG-01 isolates, yielding 65 type combinations with a Simpson’s index of diversity (SID) of 96.53, compared to (i) pairwise combinations of spa and dru typing, spa and PFGE typing, and dru and PFGE typing, which yielded 37, 44, and 43 type combinations with SIDs of 90.84, 91.00, and 93.57, respectively, or (ii) individual spa, dru, and PFGE typing methods, which yielded 17, 17, and 21 types with SIDs of 66.9, 77.83, and 81.34, respectively. Analysis of epidemiological information for a subset of PFG-01 isolates validated the relationships inferred using combined PFGE, spa, and dru typing data. This approach significantly enhances discrimination of ST22-MRSA-IV isolates and could be applied to epidemiological investigations of other highly clonal MRSA strains.
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ered from the NICU were not associated with infections. All ST772-MRSA-V isolates identified in this study exhibited the same multiantibiotic-resistant AR pattern, which was distinctly different from that exhibited by the most frequently occurring MRSA clone in Irish hospitals (ST22-MRSA-IV) (3). Based on spa typing, all of the isolates apart from one were assigned to spa type t657. The remaining isolate was assigned to spa type t345, which differs in one repeat unit only from spa type t657. DNA microar- ray analysis and MLST assigned the isolates as ST772-MRSA-V and showed that they harbored similar virulence and antimicro- bial resistance genes to each other and to previously reported ST772-MRSA-V isolates. However, unlike some of the recently reported ST772-MRSA-V isolates, the isolates in the present study lacked the antimicrobial resistance genes erm(C) and tet(K) (24) and the enterotoxin genes sek and seq (34). The absence of DNA microarray signals corresponding to the presence of either the disrupted or complete beta-hemolysin gene hlb in the ST772- MRSA-V isolates in this and previous studies (24, 34) is interest- ing. Most human S. aureus isolates, including MRSA, harbor a disrupted hlb gene due to insertional inactivation during lysogeni- zation by hlb-converting bacteriophages (8, 9). These findings in- dicate the possible presence of mutations in the primer or probe binding sites within hlb used with the DNA microarray system in isolates of this strain. The presence of the IEC gene scn, which is carried on bacteriophages that integrate within hlb, suggests that hlb is present in a truncated form in these isolates. Additional studies are under way to investigate this further.
A spa typing application has been developed to be freely available to the academic purposes. It ﬁnds sequence types, new types, and new repeats in the Staphylococcus aureus sequences. Furthermore, it has a rich and easy- to-use GUI environment which allows users to deal with as many as they want of ﬁles managed in projects. It also supports the forward-reverse sequence alignment for sequence assembling task. For portability and installa- tion easiness, the system is built in Java and on top of the NetBeans Platform; therefore, it can run on diﬀerent machines with diﬀerent platforms.
We present a novel tool for rapid determination of spa repeats in S. aureus. An important feature of this software is automated data submission via the Internet. Thus, the server can be used to collate and harmonize data from various geo- graphic regions. Furthermore, DNA sequences are automati- cally subjected to quality control. This feature greatly facili- tates the implementation of centralized servers since data need not to be checked by a curator. The software is designed in a way that it can be adapted to single-locus typing schemes relevant to other genetically variable pathogens responsible for nosocomial infections, e.g., vancomycin-resistant enterococci, for which MLST proved to be highly discriminatory (16, 21). In order to simplify spa type nomenclature, a numerical repeat code was established in this study. This approach was chosen despite the current existence of an alpha-numerical repeat nomenclature because numerical codes are now widely used for MLST and because Ridom StaphType is the first Internet- based tool available for assignment of spa types. This tool now provides the opportunity to harmonize spa type designations. It has long been established that spa typing is less discrimi- natory than PFGE (26, 29). Tang et al. showed that 20 strains with the same spa type that were collected during an outbreak that lasted 107 weeks exhibited several related but distinguish- able PFGE patterns. This finding corresponds to the PFGE analysis performed in this study. The significance of subtle changes of one band in the PFGE patterns of related strains, e.g., with allele spa-3, may be a matter of debate. In N. men- ingitidis, subtle changes in PFGE patterns could be identified if the nasopharyngeal and clonally identical blood isolates of a patient were compared or if the isolate of a patient and that of the clonally identical one of the closest contact were compared (30; U. Vogel, H. Claus, and M. Frosch, Letter, N. Engl. J. Med. 342:219–220, 2000). In a study on S. aureus carriage, several nasal isolates did not differ from clonally identical iso- FIG. 3. Dendrogram deduced from the cluster analysis of PFGE
Results: Total of 159 S. aureus isolates were collected from two tertiary hospitals in Shiraz. Isolates were identified by biochemical tests. Antimicrobial susceptibility tests were performed by standard disk diffusion method and then genetic analysis of bacteria was performed using SCCmec and spa typing. In this study 31.4% of the isolates were methicillin‑resistant S. aureus. The majority of isolates were SSCmec type III. Spa type t030 was the most prominent type among MRSA strains. For the first time in Iran, spa003, t386, t1877, t314, t186, t1816, t304, t325, t345 were reported in this study. It was shown that there is a possibility that these spa types are native to this region. Our find‑ ings showed that SCCmec II, III and IV disseminate from hospital to community and vice versa. Thus, effective monitor‑ ing of MRSA in hospital and community is necessary.
nome sequencing) as laboratory approaches for reconstructing the molecular epidemiology of MRSA strains, with special atten- tion to their ability to discriminate subclonal strain outbreaks. MLVA of MRSA isolates demonstrated higher discriminatory power than did spa typing, as evidenced by its identification of 17 groups compared to the 12 groups resolved by spa typing (Fig. 1A). In contrast, each MRSA isolate was uniquely identified by whole-genome sequencing, and higher-resolution relationships among MRSA strains were ascertained (Fig. 1A and B). The quan- titation of the underlying genomic variants that distinguish be- tween populations identified as genetically identical by either spa typing or MLVA revealed that substantial numbers of genomic variants may be present among isolates within different groups, and surprisingly, among members of the same typed group (Ta- bles 2 and 3). These values help define the number of absolute genomic differences that are reflected by the group partitions as defined by spa and MLVA typing. We conclude that MLVA and spa typing are relatively insensitive to underlying genomic poly- morphisms and may therefore classify strains together when they are actually only indirectly related. As a consequence of this insuf- ficiently high level of genetic resolution, false identification of out- breaks may occur, with the risk of unnecessary follow-up investi- gations and clinical responsive actions. As a case in point, MLVA was able in this study to resolve a potential USA300 outbreak group from other USA300 strains, whereas spa typing was not.
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Data analysis and PFGE epidemic type assignment. PFGE data were analyzed using BioNumerics software (version 5.1; Applied Maths), and PFGE epidemic types were assigned based on guidelines published by the National Microbiology Laboratory (NML; [13, 26]). Briefly, isolates genotyped using PFGE were classified as CMRSA7/USA400 if their PFGE fingerprint profile did not differ from that of the NML reference strain by more than seven fingerprint bands. Sequencing results from spa typing were imported and analyzed using BioNumerics and submitted to the online Ridomspa database (http://spaserver.ridom.de/), which was devel- oped by Ridom GmbH and is curated by SeqNet.org (http://www.SeqNet .org), for Ridom spa type designation (27). Ridom repeat successions were also assigned by the Ridom spa database. For isolates genotyped usingspa, SCCmec, and PVL typing, the Alberta MRSA typing database (unpub- lished data) was used to assign PFGE epidemic types based on correlations
Sequence-based methods for typing Staphylococcus aureus, such as multilocus sequence typing (MLST) and spa typing, have increased interlaboratory reproducibility, portability, and speed in obtaining results, but pulsed-field gel electrophoresis (PFGE), remains the method of choice in many laboratories due to the extensive experience with this methodology and the large body of data accumulated using the technique. Comparisons between typing methods have been overwhelmingly based on a qualitative assessment of the overall agreement of results and the relative discriminatory indexes. In this study, we quantitatively assess the congruence of the major typing methods for S. aureus, using a diverse collection of 198 S. aureus strains previously characterized by PFGE, spa typing, MLST, and, in the case of methicillin-resistant S. aureus (MRSA), SCCmec typing in order to establish the quantitative congruence between the typing methods. The results of most typing methods agree in that MRSA and methicillin-susceptible S. aureus (MSSA) differ in terms of diversity of genetic backgrounds, with MSSA being more diverse. Our results show that spa typing has a very good predictive power over the clonal relationships defined by eBURST, while PFGE is less accurate for that purpose but nevertheless provides better typeability and discriminatory power. The combination of PFGE and spa typing provided even better results. Based on these observations, we suggest the use of the conjugation of spa typing and PFGE typing for epidemiological surveillance studies, since this combination provides the ability to infer long-term relationships while maintaining the discriminatory power and typeability needed in short-term studies.
Since their emergence in the early 1980s, EMRSA-15 and EMRSA-16 have become the most prevalent nosocomial epi- demic MRSA clones in the United Kingdom. More recently, community-acquired MRSA strains have started to replace hospital-acquired MRSA strains in some health care settings (5). Understanding the epidemiology of MRSA strains has become a major challenge for health care institutions. A wide variety of molecular techniques are currently used to type MRSA strains, including PFGE, MLST, and spa and SCCmec typing. Because PFGE, MLST, and spa typing are relatively time-consuming and require specialist equipment and exper- tise, their use is often beyond the resources of routine clinical microbiology laboratories. To overcome these limitations, we have developed a typing technique with a discriminatory power that is similar to that of PFGE, but which is rapid and suitable for use in routine laboratories. Like PFGE, SMT is based around SNPs in discriminatory SmaI sites, and the presence or absence of a PCR product therefore reflects the presence or absence of a SNP in a specific SmaI restriction site: the size of the resulting product identifies the targeted SmaI site irrespec- FIG. 5. SmaI-multiplex PCR profiles of S. aureus strains isolated during two periods of increased MRSA incidence. Cluster 1 consisted of 11 S. aureus isolates from the screening of patients and staff on a surgical ward. Cluster 2 consisted of five screening samples and one blood sample from a baby care unit.
PFGE is still considering the gold standard in molecular typing, owing to its excellent discriminatory ability , but the main disadvantages of this technique are related to the technical demands, the costs of the equip- ment, the time and labor required. Accordingly, there is a need for a rapid, inexpensive, and reliable method in routine epidemiological surveillance. Several PCR-based methods have been applied for molecular typing, for example PCR-RFLP, AP-PCR, MLST, SCCmec, coa typing and spa typing based on sequencing. In this study we used spa typing, due to its high degree of polymorphism in X region, for discrimination of different S. au- reus isolated from patient, healthy carriers and between MRSA and MSSA.