- Email: [email protected]
Molecular surveillance of methicillin-resistant Staphylococcus aureus by multiple-locus variable number tandem repeat fingerprinting (formerly multiple-locus variable number tandem repeat analysis) and spa typing in a hierarchic approach
Available online at www.sciencedirect.com
Diagnostic Microbiology and Infectious Disease 62 (2008) 255 – 262 www.elsevier.com/locate/diagmicrobio
Molecular surveillance of methicillin-resistant Staphylococcus aureus by multiple-locus variable number tandem repeat fingerprinting (formerly multiple-locus variable number tandem repeat analysis) and spa typing in a hierarchic approach Michal Karynski, Artur J. Sabat, Joanna Empel, Waleria Hryniewicz⁎ National Medicines Institute, Division of Clinical Microbiology and Infection Prevention, 00-725 Warsaw, Poland Received 17 August 2007; accepted 25 June 2008
Abstract In this study, clonal relatedness of 202 Staphylococcus aureus (mostly methicillin-resistant) isolates recovered in 29 Polish hospitals was investigated by multiple-locus variable number tandem repeat fingerprinting (MLVF) and spa typing. Our analysis yielded 69 MLVF patterns and 34 spa types. Almost all isolates (97.4%) identical by MLVF were also indistinguishable by spa typing. Therefore, the MLVF method can be a cheap and fast screen before spa typing. Moreover, results obtained by MLVF suggest a set of simple criteria for grouping of spa types. The proposed algorithm groups isolates into the same cluster when spa sequences differ by a single mutation event: i) a single deletion or insertion of repeat unit(s) at the X region of the protein A gene or ii) a single nucleotide polymorphism within a repeat sequence. The combined use of these 2 methods, MLVF in local laboratories and spa typing of selected isolates in reference centers, can improve the monitoring of hospital-to-hospital strain transmission events and public health interventions on a huge scale. © 2008 Elsevier Inc. All rights reserved. Keywords: MLVF; MLVA; MRSA; spa typing; Surveillance
1. Introduction Molecular typing systems came into widespread use in the 1990s. Among these techniques, pulsed-field gel electrophoresis (PFGE) has been considered to be the “gold standard” in typing of a variety of bacteria, including Staphylococcus aureus. Unfortunately, PFGE also has disadvantages, and the major drawback is the time needed for final results. Traditional polymerase chain reaction (PCR)-based methods are faster, more straightforward to perform, and less costly than PFGE. Unfortunately, no PCRbased method has full portability of data. The methods that use DNA sequencing, including multilocus sequence typing (MLST) (Enright et al., 2000) and spa typing (Harmsen et al., 2003; Koreen et al., 2004), combine a good discriminatory power with high portability of data. However, MLST is too expensive to use at a local laboratory level, and it is rather an evolutionary approach. Cost of spa typing can ⁎ Corresponding author. Tel.: +48-22-841-33-67; fax: +48-22-841-29-49. E-mail address: [email protected] (W. Hryniewicz). 0732-8893/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2008.06.019
be a limiting factor for many laboratories as well. It can be used in a limited number of specialized laboratories in 1 country; therefore, it is impossible to analyze all isolates of S. aureus recovered from a large country by spa typing. None of the currently available typing methods satisfy the needs of epidemiologic surveillance at both local and national level. Therefore, a combination of different methods may be preferable. As a 1st step, the isolates of S. aureus should be characterized by an easy, cheap, and fast method, which could be used in the laboratories with the necessary equipment, trained personnel, and reagents available. Such a method seems to be a multiplex PCR method for multiplelocus variable number tandem repeat analysis (MLVA) (Sabat et al., 2003). Criteria for clustering MLVA patterns have been proposed (Malachowa et al., 2005). Isolates classified into the same MLVA cluster differing by up to 6 DNA fragments differ by at most 6 bands in the PFGE analysis. Moreover, MLVA has shown excellent reproducibility, high discriminatory power, and good concordance with several other typing methods (Malachowa et al., 2005; Sabat et al., 2006a). Recently, this method has been renamed
256
M. Karynski et al. / Diagnostic Microbiology and Infectious Disease 62 (2008) 255–262
multiple-locus variable number tandem repeat fingerprinting (MLVF) (Sabat et al., 2006a, 2006b). In our previous publications, we have concluded that MLVF appears to be a useful and reliable typing method for short-term epidemiologic investigations of S. aureus (Malachowa et al., 2005; Sabat et al., 2006a). Being PCR based, the MLVF method may also be a convenient screening tool for the identification of genetically related isolates for larger strain collections, after which, the representative isolates can be selected for further characterization by other typing methods in a hierarchic approach. The aim of the present study was to establish if, for large-scale surveillance, the MLVF method has the potential of a reliable screening tool before spa typing approach. (This study was presented in part at the 12th International Symposium on Staphylococci and Staphylococcal Infections in Maastricht, The Netherlands, September 3–6, 2006.)
Isolates were derived from a variety of human, mostly invasive, infections and originated from 29 hospitals (Fig. 1) participating in the National Surveillance Network, which is coordinated by the National Medicines Institute in Warsaw, Poland. The aim of the National Surveillance Network is to monitor the occurrence and spread of antimicrobial resistant bacterial nosocomial pathogens in Poland. In addition, 1 reference MRSA strain (MIKROBANK, reference no. MICRO-PL 037682) and 1 reference MSSA strain (MIKROBANK, reference no. MICRO-PL 036873) were included in each PCR run (Fig. 2). The reference strains are always used in our laboratory to monitor the amplification efficiency of the MLVF method. 2.2. Preparation of total DNA for PCR
2. Materials and methods
Total DNA of the isolates was purified using the Genomic DNA Prep Plus kit (A&A Biotechnology, Gdynia, Poland) as previously described (Sabat et al., 2003).
2.1. Bacterial isolates
2.3. MLVF typing
A random sample of 196 methicillin-resistant S. aureus (MRSA) and 6 methicillin-susceptible S. aureus (MSSA) isolates from 2001 to 2006 was used in the current study.
MLVF typing was performed as described previously (Sabat et al., 2003). The MLVF approach is based on the analysis of the number of the tandem repeats in the variable
Fig. 1. Map of Poland with the locations of hospitals from which 1 or more isolates of S. aureus were obtained. Bydgoszcz and Warszawa were represented each by 3 hospitals and the remaining towns by a single hospital. The number of isolates from a location is indicated in parentheses.
M. Karynski et al. / Diagnostic Microbiology and Infectious Disease 62 (2008) 255–262
257
Fig. 2. MLVF patterns identified in the study. Lanes Lad, 100-bp ladder; lane MR, MRSA control strain; lane MS, MSSA control strain.
number tandem repeat (VNTR) regions of 7 individual genes (sspA, spa, sdrC, sdrD, sdrE, clfA, and clfB). Five of them (sspA, spa, sdrC, clfA, and clfB) were detected so far in all investigated S. aureus strains (Malachowa et al., 2005; Sabat et al., 2003). Most S. aureus strains possessed both sdrD and sdrE or one of them (Sabat et al., 2006b). However, the subset of S. aureus strains had only a single gene in the sdr locus (presence of only sdrC) (Sabat et al., 2006b). Therefore, the MLVF patterns usually consist of 6 or 7 bands, more rarely of 5 ones. A difference between the MLVF patterns of 7 and more bands was interpreted as a different type, with types being indicated by a capital letter. If patterns differed by up to 6 bands, they were classified as subtypes of a type, with subtypes being indicated by a numeral suffix (Malachowa et al., 2005). Only identical patterns were regarded as the same subtype. Initially, all MLVF types and subtypes were discerned by a visual interpretation. Then, the MLVF patterns were reanalyzed using GelCompar software (Applied Maths, Kortrijk, Belgium) using the following parameters: optimization, 0.5%, and position tolerance, 1.25%. Pairwise similarity coefficients were calculated using the Dice formula, and dendrograms were created using the unweighted pair-group method using geometric averages (UPGMA).
2.4. spa typing Amplification of the spa gene X region was performed as described previously (Aires-de-Sousa et al., 2006; Shopsin et al., 1999), and amplicons were sequenced by using an ABI 310 sequencer (Applied Biosystems, Foster City, CA). The spa types and based upon repeat pattern (BURP) spa clonal complexes (spa-CCs) were determined with the Ridom StaphType software version 1.4.11 (Ridom GmbH, Würzburg, Germany) and the Ridom SpaServer (http://www. spaserver.ridom.de) (Harmsen et al., 2003). A cluster composed of 2 or more related spa types was regarded as a clonal complex. A singleton was defined as a spa type that was not grouped into a clonal complex. 3. Results 3.1. Multiple-locus VNTR fingerprinting Initially, an analysis conducted by a single person using 202 clinical S. aureus isolates from 29 hospitals in different parts of Poland (Fig. 1) generated 70 MLVF patterns (Table 1). Subsequently, the reproducibility of the MLVF method was assessed. The reproducibility was tested 3 times on the reextracted total DNA of the representative isolates of
258
M. Karynski et al. / Diagnostic Microbiology and Infectious Disease 62 (2008) 255–262
Table 1 Typing results obtained by MLVF and spa typing MLVF pattern (no. of isolates)
spa type Repeat profilea (no. of isolates)
A (8) B (1) C (3) D1 (8), D2 (1), D3 (1) E (2) F1 (1), F2 (1), F3 (1) F4 (1) G1 (1) G2 (1) G2 (3), G3 (1), G4 (1), G5 (1), G6 (1), G7 (2), G8 (1), G9 (1), G10 (6), G11 (1) G12 (1) G13 (1) G14 (1) G15 (1), G16 (1) G17 (1) G18 (1) H1 (2) H2 (19), H3 (1), H4 (24), H5 (2), H6 (11), H7 (1), H8 (9), H9 (2), H10 (4), H11 (1), H12 (1), H13 (3), H14 (1), H15 (1), H16 (1), H17 (1) H18 (2) H19 (1) H20 (1) H21 (3) H22 (1) I (1) J (1) K1 (2), K2 (6), K3 (1), K4 (1) K5 (26), K6 (6), K7 (1), K8 (2) K9 (1) L1 (1), M (1) L2 (1) N (1) O (1) P (1) R (1)
t002 (8) t010 (1) t041 (3) t003 (10) t053 (2) t283 (3) t1609 (1) t772 (1) t050 (1) t015 (18)
a
spa cluster spa-CC by by single BURP mutation event
Methicillin phenotype
r26 – – – r23r17r34r17r20r17r12r17r16 r26 – – – – – r17r34r17r20r17r12r17r16 r26r30r17r34r17r20r17r34r17r20r17r12r17r16 r26r17r20r17r12r17r17r16 r26r23r17r34r17r20r17r12r17r34 r26r23r17r13 – r17r20r17r12r17r12r17r34 r26r23r17r13r13r17r20r17r12r17r12r17r34 r08r16 – r02r16r34r34 – – – – r16r34 r08r16 – r02r16r34r34 – r17r34 – r16r34 r08r16 – r02r16r34r13 – r17r34 – r16r34
S1 S1 S1 S2 S3 S4 S4 S5 S5 S5
CC022 CC022 CC022 CC022 CC022 CC022 CC022 CC015 CC015 CC015
MRSA MRSA MRSA MRSA MRSA MRSA MRSA MRSA MRSA MRSA
t1608 (1) t1574 (1) t331 (1) t026 (2) t1078 (1) t069 (1) t421 (2) t037 (82)
r08r16r34r02r16r34r13 – r17r34 – r16r34 r08r16 – r02r16r34r13r13r17r34 – r16r34 r08r16 – – – r34r13 – r17r34 – r16r34 r08r16 – – – – – – – – – – r34 r08r16 – r02r16r34r13 – r17r34 – – r34 r08r16 – r02r16r34r13 – r17r34r16r16r34 r15r12r16r02 – r25r17 r15r12r16r02 – r25r17 – – r24
S5 S5 S5 S5 S5 S5 S6 S6
CC015 CC015 CC015 CC015 CC015 CC015 CC037 CC037
MSSA MRSA MRSA MRSA and MSSA MRSA MRSA MRSA MRSA and MSSA
t1152 (2) t1230 (1) t129 (1) t030 (3) t1493 (1) t012 (1) t018 (1) t008 (10) t052 (35) t024 (1) t127 (2) t177 (1) t032 (1) t077 (1) t084 (1) t1751 (1)
r15 – – – – r25r17 – – r24 r15r12r16r02r25r25r17 – – r24 r15r12 – – – – – – – r24 r15r12r16r02 – – – – r24r24 r15r12r16r02r24r24r02 – r24r24 r15r12r16r02r16r02r25r17r24r24 r15r12r16r02r16r02r25r17r24r24r24 r11r19r12r21r17r34r24r34r22r25 r11r21r12r21r17r34r24r34r22r25 r11 – r12r21r17r34r24r34r22r25 r07r23r21r16r34r33r13 r26r23r21r16r34r33r13 r26r23r23r13r23r31r29r17r31r29r17r25r17r25r16r28 r09r02r16r13r13r17r16r34 r07r23r12r34r34r12r12r23r02r12r23 r04r20r17r20r17r25r13
S6 S6 S6 S6 S6 S6 S6 S7 S7 S7 S8 S8 Sporadic Sporadic Sporadic Sporadic
CC037 CC037 CC037 CC037 CC037 CC037 CC037 CC052/008 CC052/008 CC052/008 CC127/177 CC127/177 Sporadic Sporadic Sporadic Sporadic
MRSA MRSA MRSA MRSA MRSA MSSA MSSA MRSA MRSA MRSA MRSA MRSA MRSA MRSA MSSA MRSA
Gaps within spa profiles indicate possible deletion/insertion events in the spa locus and were introduced in order to optimize the alignment of the profiles.
all MLVF patterns (n = 70). The 1st 2 tests were conducted by the same person and the 3rd one by another operator. Reproducibility of 100% was observed when the isolates were tested by the same person. In the case of 2nd operator, a single isolate showed a slightly different banding pattern than that of previous tests, producing an extra band that was less intense than other bands in the pattern. However, the extra band, being less intense, also was the 8th band in the pattern, which allowed it to be excluded from the analysis. The MLVF method uses polymorphism of the VNTR regions in 7 individual genes; therefore, the MLVF pattern can consist of, at most, 7 bands. In addition, simplex PCR reactions confirmed that the extra band in the multiplex pattern was a background and was not found when the
primer pairs were used separately. Interpretation of the banding patterns between 2 operators differed slightly. The 2nd operator distinguished 69 MLVF patterns, classifying a pair of isolates as indistinguishable, which differed by 2 bands according to the 1st person. The remaining types and subtypes were identically determined by both investigators. Taken into account different banding patterns of a single isolate and different interpretations of relatedness for a pair of isolates, the reproducibility of the MLVF method obtained by both investigators was 99% (208/210). Finally, the 3rd person analyzed the banding patterns previously obtained by the 2 operators. Based on the results initially obtained on total collection of 202 isolates and those produced during testing of the reproducibility of the method, the 3rd person
M. Karynski et al. / Diagnostic Microbiology and Infectious Disease 62 (2008) 255–262
259
discerned 69 MLVF patterns (Fig. 2, Table 1). Twenty free patterns were shared by 2 or more isolates (156 isolates in total). Forty-six patterns were represented by a single isolate. One hundred ninety-four of the isolates were grouped into 9 types of related isolates (designated A, C, D, E, F, G, H, K, and L), whereas 8 types (B, I, J, M, N, O, P, and R) were represented by a unique isolate. To confirm the visual analysis of the MLVA method, we also determined the relatedness of the 71 patterns (including reference MRSA and MSSA strains) with GelCompar software (Fig. 3). When the cutoff value between the patterns was set up at the level of 69%, identical set of the MLVF clusters was distinguished by GelCompar to that obtained by visual analysis. 3.2. Comparison of the spa typing results with the MLVF data Our analysis yielded 34 spa types (20 were represented by a single isolate) among 202 S. aureus isolates, including 4 new types: t1493, t1608, t1609, and t1751 (Table 1). Almost all isolates (97.4%, 152/156) that were identical by MLVF (156 isolates) were also indistinguishable by spa typing (152 isolates). The exceptional case of greater discriminatory ability of spa typing in comparison to MLVF was confined to 1 group of 4 isolates that shared the same MLVF pattern G2 and were split into 2 spa types, t050 and t015. Nevertheless, types t015 and t050 were closely related because they were identical by a number of the repeats and differed only by a single point mutation. Almost all isolates (98.7%, 153/155) that were indistinguishable by spa typing (155 isolates) belonged to the same MLVF type (153 isolates). The only isolates defined as identical by spa typing and unrelated by MLVF were 2 isolates of the spa type t127 (with the MLVF patterns L1 and M). The largest spa type, t037, was differentiated into 16 subtypes by MLVF, as well as most other spa types comprising at least 2 isolates (t002, t003, t015, t026, t008, t052, t283) were subdivided into corresponding MLVF subtypes. 3.3. Criteria for defining spa clusters for short-term investigations (single mutation event rule) To establish criteria for clustering the spa profiles into similarity groups, we compared the spa typing results with those obtained by MLVF. Isolates classified into the same MLVF type differing by up to 6 DNA fragments were identical or differed mostly by only a single mutation event at the X region of the protein A gene. As a mutation event, we considered only deletion or insertion of repeat unit(s) that could occur on a single occasion as well as a single point mutation within a repeat sequence. Isolates that were not related by MLVF invariably differed by 2 or more mutation events at the X region (except for the isolates with the MLVF types L and M). A total of 8 spa clusters (S1–S8) that comprised 198 isolates were distinguished (Table 1). Six clusters (S1 and S4–S8) contained more than 1 spa type (clonal complexes). All spa types within a clonal complex had at least 1 other spa type that differed by only a single
Fig. 3. MLVF dendrogram of the study isolates generated by the UPGMA algorithm. Isolate clusters were delineated with 69% similarity cutoff value. Pattern MR, MRSA control strain; pattern MS, MSSA control strain.
260
M. Karynski et al. / Diagnostic Microbiology and Infectious Disease 62 (2008) 255–262
Fig. 3 (continued).
M. Karynski et al. / Diagnostic Microbiology and Infectious Disease 62 (2008) 255–262
mutation event. The remaining 2 clusters (S2 and S3) were represented by a single type (singletons). In all cases, S. aureus isolates of a given MLVF cluster were grouped together within the same spa cluster. MLVF clusters D, E, F, G, and K were identical to the spa clusters S2, S3, S4, S5, and S7, respectively. The remaining spa clusters (S1, S6, and S8) were split by MLVF into different clusters and singular isolates. The sporadic isolates recognized by single mutation event rule were also classified as unique isolates by the MLVF analysis. 3.4. Determination of the spa clusters by the BURP algorithm Using the Ridom StaphType software version 1.4.11 (Harmsen et al., 2003), 34 spa types were distributed by the BURP algorithm (with the calculated cost between members of a group ≤7) in 5 spa-CCs (002, 015, 037, 052/008, and 127/177), and 4 singletons represented each by a single isolate (Table 1). Two of the clonal complexes, spa-CC015 and spa-CC052/008, were identical to the MLVF types G and K, respectively. The remaining 3 CCs, spa-CC002, spaCC037, and spa-CC127/177, were split by MLVF into different types, from A to F, from H to J, and L and M, respectively. The sporadic isolates recognized by BURP were also classified as unique isolates by the MLVF analysis. 4. Discussion MLVF is a superior method to spa typing, which examines isolates locally. There are 3 reasons for that: i) MLVF is more discriminatory than spa typing (Malachowa et al., 2005); ii) results produced by multiplex PCR can be obtained faster than by sequencing analysis; and iii) costs of equipment and typing procedure for the DNA sequencing approach are higher. The MLVF method possesses adequate stability to be broadly used in an outbreak setting (Sabat et al., 2003). Being as highly discriminatory as PFGE (Malachowa et al., 2005), which is regarded as a “gold standard” for the epidemiologic typing of S. aureus, MLVF can be used to detect outbreaks as well as identify potential sources of transmission. In addition, comparison of the MLVF data for the epidemiologic clustering can be substantially facilitated by using BioNumerics software (GelCompar is a version of BioNumerics; Applied Maths) and a cutoff of ≥70% relatedness. In general, spa typing has also appropriate discriminatory power to determine whether the isolates are closely related or unique in a hospital setting. However, our study showed that MLVF was able to differentiate among the identical isolates by spa typing; for example, spa type t037 was divided into 16 subtypes by MLVF. This is very important because MLVF is able to show a person-to-person mode of strain transmission especially in hospitals with high rates of epidemic MRSA infections, thus, allowing the development of strategies to prevent further spread. In this situation, spa typing can suffer from uncertainties in showing direct transmission.
261
As a reference typing system, the spa typing offers significant advantages over MLVF in terms of the following: i) ease of results of interpretation for huge numbers of genetically diverse isolates; ii) better reproducibility; iii) appropriate stability of the X region of the protein A gene for long-term epidemiologic surveillance studies; and, most importantly, iv) exportability of the data. The aim of this study was to check if the MLVF method could be a reliable screening tool for the identification of genetically identical and closely related isolates. Isolates that were indistinguishable from each other by MLVF were almost always (97.4%) indistinguishable by spa typing. Therefore, because spa typing is unable to differentiate among the identical isolates by MLVF, only selected representatives of the MLVF patterns can be sent from local laboratories to the reference ones for characterization by spa typing. If a local laboratory that decides itself to monitor epidemiologic situation and the spa typing approach is too costly to use for a larger scale, only selected representatives of the MLVF types can be further characterized. In all cases, the isolates of a given MLVF type were grouped together within the same spa-CC (Table 1). The most appropriate candidate for characterization by spa typing is an isolate with the MLVF pattern, which is the most often encountered within a type, for example, for the type G, the pattern G10; for the type H, the pattern H4; and for the type K, the pattern K5. The selected representatives on the basis of MLVF analysis were also founders of the corresponding spa-CCs (t015, t037, and t052, respectively). The events of discrepancy between the results produced by both methods used in this study usually could be explained by the higher discriminatory power of MLVF. However, spa typing, being a single locus typing approach, is sensitive to the chromosomal replacements, which can lead to grouping of genetically unrelated isolates. Such a situation was found for the spa-CC037 in which isolates belonging to the distinct MLVF types had very similar spa profiles. Relevant situation was previously described by Strommenger et al. (2006), where isolates of spa types t030 and t037 were grouped together with isolates of the types t012 and t018, although they belonged to different clonal complexes by MLST, CC8 and CC30, respectively. The analysis of Robinson and Enright (2004) revealed that a chromosomal replacement, encompassing the spa locus, from ST30 to ST8, which resulted in the ST239 (CC8) mosaic chromosome, was responsible for the observed incongruence. Most of the isolates were grouped together in the same manner by 2 approaches of clustering spa types used in this study (Table 1). However, clusters S1, S2, S3, and S4 obtained on the basis of single mutation event rule were grouped together after applying the BURP algorithm. The difference observed between the 2 clustering approaches resulted from different interpretation criteria. The BURP clustering criteria allow for identifying broader similarity groups that are useful in phylogenetic studies. However, epidemiologic studies aimed mostly at outbreak analyses and
262
M. Karynski et al. / Diagnostic Microbiology and Infectious Disease 62 (2008) 255–262
monitoring shorter-time interhospital spread of S. aureus strains usually require more stringent criteria. In the previous investigations (Malachowa et al., 2005; Sabat et al., 2006a), we compared the data produced by MLVF (formerly known as MLVA) and spa typing. Previously, we assigned any 2 spa types that had a majority of identical repeats or differed in a single deletion or duplication of the nucleotide sequence into the same cluster. Those criteria for clustering spa types were less stringent but very similar to that of the single mutation event rule applied in this study. Like in the previous investigations (Malachowa et al., 2005; Sabat et al., 2006a), all isolates of S. aureus of a MLVF cluster were grouped within the same spa cluster. However, this time, the spa clusters were identical more often (5 of 8) to the MLVF clusters, whereas previously, almost each one was split by MLVF into several clusters and singular isolates. Moreover, previously, isolates within a defined cluster were usually indistinguishable by spa typing. This was not the case in this investigation (the MLVF clusters: F, G, H, K, and L consisted of 2, 9, 7, 3, and 2 spa types, respectively, whereas only 4 MLVF clusters shared the same spa type: A, C, D, and E). The differences obtained in this study and in the previous ones (Malachowa et al., 2005; Sabat et al., 2006a) can be explained by the larger number of isolates included into the present study. During this study, we analyzed 202 isolates, whereas previously, only 59 isolates. Within a cluster, not all isolates appeared to be genetically related (difference more than 6 bands between the patterns); for example, the isolates of the MLVF subtypes F3 and F4 were unrelated. However, they were linked by the isolates of the MLVF subtypes F1 and F2. During characterization of a much smaller number of most often epidemiologically unrelated isolates (202/59 = 3.4), we could miss the isolates of the subtypes F1 and F2, and then the isolates of the subtypes F3 and F4 would not be within the same MLVF cluster but still a part of the same spa cluster. We could show that by increasing the overall number of isolates, we were more likely to detect heterogeneity with respect to the spa types. The biggest MLVF clusters G and H were composed of 27 and 92 isolates, respectively. However, 15 isolates (55% of total, the subtypes G3–G11) and 81 isolates (89% of total, the subtypes H2–H17) were indistinguishable by spa typing within the MLVF clusters G and H, respectively. The MLVF method is a good and speedy tool in local laboratories for the identification of S. aureus strains in outbreak clusters and chains of transmissions. Further information on epidemic potential of individual isolates can be obtained by using spa typing. Therefore, the combined use of MLVF and spa typing can benefit in monitoring hospital-to-hospital S. aureus strain transmission events and public health interventions in a real-time and on a huge scale.
Acknowledgments The authors thank the members of National Surveillance Network (OPTY) for participating in the study. They are also grateful to Hajo Grundmann (National Institute of Public Health and the Environment, Bilthoven, The Netherlands) for helpful discussion and thoughtful review of the manuscript. These studies were supported by grant 2 P05A 052 30 (to A.S. and W.H.) from the Committee of Scientific Research (KBN, Poland). References Aires-de-Sousa M, Boye K, de Lencastre H, Deplano A, Enright MC, Etienne J, Friedrich A, Harmsen D, Holmes A, Huijsdens XW, Kearns AM, Mellmann A, Meugnier H, Rasheed JK, Spalburg E, Strommenger B, Struelens MJ, Tenover FC, Thomas J, Vogel U, Westh H, Xu J, Witte W (2006) High interlaboratory reproducibility of DNA sequence-based typing of bacteria in a multicenter study. J Clin Microbiol 44:619–621. Enright MC, Day NP, Davies CE, Peacock SJ, Spratt BG (2000) Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J Clin Microbiol 38:1008–1015. Harmsen D, Claus H, Witte W, Rothganger J, Claus H, Turnwald D, Vogel U (2003) Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management. J Clin Microbiol 41: 5442–5448. Koreen L, Ramaswamy SV, Graviss EA, Naidich S, Musser JM, Kreiswirth BN (2004) spa typing method for discriminating among Staphylococcus aureus isolates: implications for use of a single marker to detect genetic micro- and macrovariation. J Clin Microbiol 42:792–799. Malachowa N, Sabat A, Gniadkowski M, Krzyszton-Russjan J, Empel J, Miedzobrodzki J, Kosowska-Shick K, Appelbaum PC, Hryniewicz W (2005) Comparison of multiple-locus variable-number tandem repeat analysis with pulsed-field gel electrophoresis, spa typing, and multilocus sequence typing for clonal characterization of Staphylococcus aureus isolates. J Clin Microbiol 43:3095–3100. Robinson DA, Enright MC (2004) Evolution of Staphylococcus aureus by large chromosomal replacements. J Bacteriol 186:1060–1064. Sabat A, Krzyszton-Russjan J, Strzalka W, Filipek R, Kosowska K, Hryniewicz W, Travis J, Potempa J (2003) New method for typing Staphylococcus aureus strains: multiple-locus variable-number tandem repeat analysis of polymorphism and genetic relationships of clinical isolates. J Clin Microbiol 41:1801–1804. Sabat A, Malachowa N, Miedzobrodzki J, Hryniewicz W (2006a) Comparison of PCR-based methods for typing Staphylococcus aureus isolates. J Clin Microbiol 44:3804–3807. Sabat A, Melles DC, Martirosian G, Grundmann H, van Belkum A, Hryniewicz W (2006b) Distribution of the serine–aspartate repeat protein-encoding sdr genes among nasal-carriage and invasive Staphylococcus aureus strains. J Clin Microbiol 44:1135–1138. Shopsin B, Gomez M, Montgomery SO, Smith DH, Waddington M, Dodge DE, Bost DA, Riehman M, Naidich S, Kreiswirth BN (1999) Evaluation of protein A gene polymorphic region DNA sequencing for typing of Staphylococcus aureus strains. J Clin Microbiol 37:3556–3563. Strommenger B, Kettlitz C, Weniger T, Harmsen D, Friedrich AW, Witte W (2006) Assignment of Staphylococcus isolates to groups by spa typing, SmaI macrorestriction analysis, and multilocus sequence typing. J Clin Microbiol 44:2533–2540.
Diagnostic Microbiology and Infectious Disease 62 (2008) 255 – 262 www.elsevier.com/locate/diagmicrobio
Molecular surveillance of methicillin-resistant Staphylococcus aureus by multiple-locus variable number tandem repeat fingerprinting (formerly multiple-locus variable number tandem repeat analysis) and spa typing in a hierarchic approach Michal Karynski, Artur J. Sabat, Joanna Empel, Waleria Hryniewicz⁎ National Medicines Institute, Division of Clinical Microbiology and Infection Prevention, 00-725 Warsaw, Poland Received 17 August 2007; accepted 25 June 2008
Abstract In this study, clonal relatedness of 202 Staphylococcus aureus (mostly methicillin-resistant) isolates recovered in 29 Polish hospitals was investigated by multiple-locus variable number tandem repeat fingerprinting (MLVF) and spa typing. Our analysis yielded 69 MLVF patterns and 34 spa types. Almost all isolates (97.4%) identical by MLVF were also indistinguishable by spa typing. Therefore, the MLVF method can be a cheap and fast screen before spa typing. Moreover, results obtained by MLVF suggest a set of simple criteria for grouping of spa types. The proposed algorithm groups isolates into the same cluster when spa sequences differ by a single mutation event: i) a single deletion or insertion of repeat unit(s) at the X region of the protein A gene or ii) a single nucleotide polymorphism within a repeat sequence. The combined use of these 2 methods, MLVF in local laboratories and spa typing of selected isolates in reference centers, can improve the monitoring of hospital-to-hospital strain transmission events and public health interventions on a huge scale. © 2008 Elsevier Inc. All rights reserved. Keywords: MLVF; MLVA; MRSA; spa typing; Surveillance
1. Introduction Molecular typing systems came into widespread use in the 1990s. Among these techniques, pulsed-field gel electrophoresis (PFGE) has been considered to be the “gold standard” in typing of a variety of bacteria, including Staphylococcus aureus. Unfortunately, PFGE also has disadvantages, and the major drawback is the time needed for final results. Traditional polymerase chain reaction (PCR)-based methods are faster, more straightforward to perform, and less costly than PFGE. Unfortunately, no PCRbased method has full portability of data. The methods that use DNA sequencing, including multilocus sequence typing (MLST) (Enright et al., 2000) and spa typing (Harmsen et al., 2003; Koreen et al., 2004), combine a good discriminatory power with high portability of data. However, MLST is too expensive to use at a local laboratory level, and it is rather an evolutionary approach. Cost of spa typing can ⁎ Corresponding author. Tel.: +48-22-841-33-67; fax: +48-22-841-29-49. E-mail address: [email protected] (W. Hryniewicz). 0732-8893/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2008.06.019
be a limiting factor for many laboratories as well. It can be used in a limited number of specialized laboratories in 1 country; therefore, it is impossible to analyze all isolates of S. aureus recovered from a large country by spa typing. None of the currently available typing methods satisfy the needs of epidemiologic surveillance at both local and national level. Therefore, a combination of different methods may be preferable. As a 1st step, the isolates of S. aureus should be characterized by an easy, cheap, and fast method, which could be used in the laboratories with the necessary equipment, trained personnel, and reagents available. Such a method seems to be a multiplex PCR method for multiplelocus variable number tandem repeat analysis (MLVA) (Sabat et al., 2003). Criteria for clustering MLVA patterns have been proposed (Malachowa et al., 2005). Isolates classified into the same MLVA cluster differing by up to 6 DNA fragments differ by at most 6 bands in the PFGE analysis. Moreover, MLVA has shown excellent reproducibility, high discriminatory power, and good concordance with several other typing methods (Malachowa et al., 2005; Sabat et al., 2006a). Recently, this method has been renamed
256
M. Karynski et al. / Diagnostic Microbiology and Infectious Disease 62 (2008) 255–262
multiple-locus variable number tandem repeat fingerprinting (MLVF) (Sabat et al., 2006a, 2006b). In our previous publications, we have concluded that MLVF appears to be a useful and reliable typing method for short-term epidemiologic investigations of S. aureus (Malachowa et al., 2005; Sabat et al., 2006a). Being PCR based, the MLVF method may also be a convenient screening tool for the identification of genetically related isolates for larger strain collections, after which, the representative isolates can be selected for further characterization by other typing methods in a hierarchic approach. The aim of the present study was to establish if, for large-scale surveillance, the MLVF method has the potential of a reliable screening tool before spa typing approach. (This study was presented in part at the 12th International Symposium on Staphylococci and Staphylococcal Infections in Maastricht, The Netherlands, September 3–6, 2006.)
Isolates were derived from a variety of human, mostly invasive, infections and originated from 29 hospitals (Fig. 1) participating in the National Surveillance Network, which is coordinated by the National Medicines Institute in Warsaw, Poland. The aim of the National Surveillance Network is to monitor the occurrence and spread of antimicrobial resistant bacterial nosocomial pathogens in Poland. In addition, 1 reference MRSA strain (MIKROBANK, reference no. MICRO-PL 037682) and 1 reference MSSA strain (MIKROBANK, reference no. MICRO-PL 036873) were included in each PCR run (Fig. 2). The reference strains are always used in our laboratory to monitor the amplification efficiency of the MLVF method. 2.2. Preparation of total DNA for PCR
2. Materials and methods
Total DNA of the isolates was purified using the Genomic DNA Prep Plus kit (A&A Biotechnology, Gdynia, Poland) as previously described (Sabat et al., 2003).
2.1. Bacterial isolates
2.3. MLVF typing
A random sample of 196 methicillin-resistant S. aureus (MRSA) and 6 methicillin-susceptible S. aureus (MSSA) isolates from 2001 to 2006 was used in the current study.
MLVF typing was performed as described previously (Sabat et al., 2003). The MLVF approach is based on the analysis of the number of the tandem repeats in the variable
Fig. 1. Map of Poland with the locations of hospitals from which 1 or more isolates of S. aureus were obtained. Bydgoszcz and Warszawa were represented each by 3 hospitals and the remaining towns by a single hospital. The number of isolates from a location is indicated in parentheses.
M. Karynski et al. / Diagnostic Microbiology and Infectious Disease 62 (2008) 255–262
257
Fig. 2. MLVF patterns identified in the study. Lanes Lad, 100-bp ladder; lane MR, MRSA control strain; lane MS, MSSA control strain.
number tandem repeat (VNTR) regions of 7 individual genes (sspA, spa, sdrC, sdrD, sdrE, clfA, and clfB). Five of them (sspA, spa, sdrC, clfA, and clfB) were detected so far in all investigated S. aureus strains (Malachowa et al., 2005; Sabat et al., 2003). Most S. aureus strains possessed both sdrD and sdrE or one of them (Sabat et al., 2006b). However, the subset of S. aureus strains had only a single gene in the sdr locus (presence of only sdrC) (Sabat et al., 2006b). Therefore, the MLVF patterns usually consist of 6 or 7 bands, more rarely of 5 ones. A difference between the MLVF patterns of 7 and more bands was interpreted as a different type, with types being indicated by a capital letter. If patterns differed by up to 6 bands, they were classified as subtypes of a type, with subtypes being indicated by a numeral suffix (Malachowa et al., 2005). Only identical patterns were regarded as the same subtype. Initially, all MLVF types and subtypes were discerned by a visual interpretation. Then, the MLVF patterns were reanalyzed using GelCompar software (Applied Maths, Kortrijk, Belgium) using the following parameters: optimization, 0.5%, and position tolerance, 1.25%. Pairwise similarity coefficients were calculated using the Dice formula, and dendrograms were created using the unweighted pair-group method using geometric averages (UPGMA).
2.4. spa typing Amplification of the spa gene X region was performed as described previously (Aires-de-Sousa et al., 2006; Shopsin et al., 1999), and amplicons were sequenced by using an ABI 310 sequencer (Applied Biosystems, Foster City, CA). The spa types and based upon repeat pattern (BURP) spa clonal complexes (spa-CCs) were determined with the Ridom StaphType software version 1.4.11 (Ridom GmbH, Würzburg, Germany) and the Ridom SpaServer (http://www. spaserver.ridom.de) (Harmsen et al., 2003). A cluster composed of 2 or more related spa types was regarded as a clonal complex. A singleton was defined as a spa type that was not grouped into a clonal complex. 3. Results 3.1. Multiple-locus VNTR fingerprinting Initially, an analysis conducted by a single person using 202 clinical S. aureus isolates from 29 hospitals in different parts of Poland (Fig. 1) generated 70 MLVF patterns (Table 1). Subsequently, the reproducibility of the MLVF method was assessed. The reproducibility was tested 3 times on the reextracted total DNA of the representative isolates of
258
M. Karynski et al. / Diagnostic Microbiology and Infectious Disease 62 (2008) 255–262
Table 1 Typing results obtained by MLVF and spa typing MLVF pattern (no. of isolates)
spa type Repeat profilea (no. of isolates)
A (8) B (1) C (3) D1 (8), D2 (1), D3 (1) E (2) F1 (1), F2 (1), F3 (1) F4 (1) G1 (1) G2 (1) G2 (3), G3 (1), G4 (1), G5 (1), G6 (1), G7 (2), G8 (1), G9 (1), G10 (6), G11 (1) G12 (1) G13 (1) G14 (1) G15 (1), G16 (1) G17 (1) G18 (1) H1 (2) H2 (19), H3 (1), H4 (24), H5 (2), H6 (11), H7 (1), H8 (9), H9 (2), H10 (4), H11 (1), H12 (1), H13 (3), H14 (1), H15 (1), H16 (1), H17 (1) H18 (2) H19 (1) H20 (1) H21 (3) H22 (1) I (1) J (1) K1 (2), K2 (6), K3 (1), K4 (1) K5 (26), K6 (6), K7 (1), K8 (2) K9 (1) L1 (1), M (1) L2 (1) N (1) O (1) P (1) R (1)
t002 (8) t010 (1) t041 (3) t003 (10) t053 (2) t283 (3) t1609 (1) t772 (1) t050 (1) t015 (18)
a
spa cluster spa-CC by by single BURP mutation event
Methicillin phenotype
r26 – – – r23r17r34r17r20r17r12r17r16 r26 – – – – – r17r34r17r20r17r12r17r16 r26r30r17r34r17r20r17r34r17r20r17r12r17r16 r26r17r20r17r12r17r17r16 r26r23r17r34r17r20r17r12r17r34 r26r23r17r13 – r17r20r17r12r17r12r17r34 r26r23r17r13r13r17r20r17r12r17r12r17r34 r08r16 – r02r16r34r34 – – – – r16r34 r08r16 – r02r16r34r34 – r17r34 – r16r34 r08r16 – r02r16r34r13 – r17r34 – r16r34
S1 S1 S1 S2 S3 S4 S4 S5 S5 S5
CC022 CC022 CC022 CC022 CC022 CC022 CC022 CC015 CC015 CC015
MRSA MRSA MRSA MRSA MRSA MRSA MRSA MRSA MRSA MRSA
t1608 (1) t1574 (1) t331 (1) t026 (2) t1078 (1) t069 (1) t421 (2) t037 (82)
r08r16r34r02r16r34r13 – r17r34 – r16r34 r08r16 – r02r16r34r13r13r17r34 – r16r34 r08r16 – – – r34r13 – r17r34 – r16r34 r08r16 – – – – – – – – – – r34 r08r16 – r02r16r34r13 – r17r34 – – r34 r08r16 – r02r16r34r13 – r17r34r16r16r34 r15r12r16r02 – r25r17 r15r12r16r02 – r25r17 – – r24
S5 S5 S5 S5 S5 S5 S6 S6
CC015 CC015 CC015 CC015 CC015 CC015 CC037 CC037
MSSA MRSA MRSA MRSA and MSSA MRSA MRSA MRSA MRSA and MSSA
t1152 (2) t1230 (1) t129 (1) t030 (3) t1493 (1) t012 (1) t018 (1) t008 (10) t052 (35) t024 (1) t127 (2) t177 (1) t032 (1) t077 (1) t084 (1) t1751 (1)
r15 – – – – r25r17 – – r24 r15r12r16r02r25r25r17 – – r24 r15r12 – – – – – – – r24 r15r12r16r02 – – – – r24r24 r15r12r16r02r24r24r02 – r24r24 r15r12r16r02r16r02r25r17r24r24 r15r12r16r02r16r02r25r17r24r24r24 r11r19r12r21r17r34r24r34r22r25 r11r21r12r21r17r34r24r34r22r25 r11 – r12r21r17r34r24r34r22r25 r07r23r21r16r34r33r13 r26r23r21r16r34r33r13 r26r23r23r13r23r31r29r17r31r29r17r25r17r25r16r28 r09r02r16r13r13r17r16r34 r07r23r12r34r34r12r12r23r02r12r23 r04r20r17r20r17r25r13
S6 S6 S6 S6 S6 S6 S6 S7 S7 S7 S8 S8 Sporadic Sporadic Sporadic Sporadic
CC037 CC037 CC037 CC037 CC037 CC037 CC037 CC052/008 CC052/008 CC052/008 CC127/177 CC127/177 Sporadic Sporadic Sporadic Sporadic
MRSA MRSA MRSA MRSA MRSA MSSA MSSA MRSA MRSA MRSA MRSA MRSA MRSA MRSA MSSA MRSA
Gaps within spa profiles indicate possible deletion/insertion events in the spa locus and were introduced in order to optimize the alignment of the profiles.
all MLVF patterns (n = 70). The 1st 2 tests were conducted by the same person and the 3rd one by another operator. Reproducibility of 100% was observed when the isolates were tested by the same person. In the case of 2nd operator, a single isolate showed a slightly different banding pattern than that of previous tests, producing an extra band that was less intense than other bands in the pattern. However, the extra band, being less intense, also was the 8th band in the pattern, which allowed it to be excluded from the analysis. The MLVF method uses polymorphism of the VNTR regions in 7 individual genes; therefore, the MLVF pattern can consist of, at most, 7 bands. In addition, simplex PCR reactions confirmed that the extra band in the multiplex pattern was a background and was not found when the
primer pairs were used separately. Interpretation of the banding patterns between 2 operators differed slightly. The 2nd operator distinguished 69 MLVF patterns, classifying a pair of isolates as indistinguishable, which differed by 2 bands according to the 1st person. The remaining types and subtypes were identically determined by both investigators. Taken into account different banding patterns of a single isolate and different interpretations of relatedness for a pair of isolates, the reproducibility of the MLVF method obtained by both investigators was 99% (208/210). Finally, the 3rd person analyzed the banding patterns previously obtained by the 2 operators. Based on the results initially obtained on total collection of 202 isolates and those produced during testing of the reproducibility of the method, the 3rd person
M. Karynski et al. / Diagnostic Microbiology and Infectious Disease 62 (2008) 255–262
259
discerned 69 MLVF patterns (Fig. 2, Table 1). Twenty free patterns were shared by 2 or more isolates (156 isolates in total). Forty-six patterns were represented by a single isolate. One hundred ninety-four of the isolates were grouped into 9 types of related isolates (designated A, C, D, E, F, G, H, K, and L), whereas 8 types (B, I, J, M, N, O, P, and R) were represented by a unique isolate. To confirm the visual analysis of the MLVA method, we also determined the relatedness of the 71 patterns (including reference MRSA and MSSA strains) with GelCompar software (Fig. 3). When the cutoff value between the patterns was set up at the level of 69%, identical set of the MLVF clusters was distinguished by GelCompar to that obtained by visual analysis. 3.2. Comparison of the spa typing results with the MLVF data Our analysis yielded 34 spa types (20 were represented by a single isolate) among 202 S. aureus isolates, including 4 new types: t1493, t1608, t1609, and t1751 (Table 1). Almost all isolates (97.4%, 152/156) that were identical by MLVF (156 isolates) were also indistinguishable by spa typing (152 isolates). The exceptional case of greater discriminatory ability of spa typing in comparison to MLVF was confined to 1 group of 4 isolates that shared the same MLVF pattern G2 and were split into 2 spa types, t050 and t015. Nevertheless, types t015 and t050 were closely related because they were identical by a number of the repeats and differed only by a single point mutation. Almost all isolates (98.7%, 153/155) that were indistinguishable by spa typing (155 isolates) belonged to the same MLVF type (153 isolates). The only isolates defined as identical by spa typing and unrelated by MLVF were 2 isolates of the spa type t127 (with the MLVF patterns L1 and M). The largest spa type, t037, was differentiated into 16 subtypes by MLVF, as well as most other spa types comprising at least 2 isolates (t002, t003, t015, t026, t008, t052, t283) were subdivided into corresponding MLVF subtypes. 3.3. Criteria for defining spa clusters for short-term investigations (single mutation event rule) To establish criteria for clustering the spa profiles into similarity groups, we compared the spa typing results with those obtained by MLVF. Isolates classified into the same MLVF type differing by up to 6 DNA fragments were identical or differed mostly by only a single mutation event at the X region of the protein A gene. As a mutation event, we considered only deletion or insertion of repeat unit(s) that could occur on a single occasion as well as a single point mutation within a repeat sequence. Isolates that were not related by MLVF invariably differed by 2 or more mutation events at the X region (except for the isolates with the MLVF types L and M). A total of 8 spa clusters (S1–S8) that comprised 198 isolates were distinguished (Table 1). Six clusters (S1 and S4–S8) contained more than 1 spa type (clonal complexes). All spa types within a clonal complex had at least 1 other spa type that differed by only a single
Fig. 3. MLVF dendrogram of the study isolates generated by the UPGMA algorithm. Isolate clusters were delineated with 69% similarity cutoff value. Pattern MR, MRSA control strain; pattern MS, MSSA control strain.
260
M. Karynski et al. / Diagnostic Microbiology and Infectious Disease 62 (2008) 255–262
Fig. 3 (continued).
M. Karynski et al. / Diagnostic Microbiology and Infectious Disease 62 (2008) 255–262
mutation event. The remaining 2 clusters (S2 and S3) were represented by a single type (singletons). In all cases, S. aureus isolates of a given MLVF cluster were grouped together within the same spa cluster. MLVF clusters D, E, F, G, and K were identical to the spa clusters S2, S3, S4, S5, and S7, respectively. The remaining spa clusters (S1, S6, and S8) were split by MLVF into different clusters and singular isolates. The sporadic isolates recognized by single mutation event rule were also classified as unique isolates by the MLVF analysis. 3.4. Determination of the spa clusters by the BURP algorithm Using the Ridom StaphType software version 1.4.11 (Harmsen et al., 2003), 34 spa types were distributed by the BURP algorithm (with the calculated cost between members of a group ≤7) in 5 spa-CCs (002, 015, 037, 052/008, and 127/177), and 4 singletons represented each by a single isolate (Table 1). Two of the clonal complexes, spa-CC015 and spa-CC052/008, were identical to the MLVF types G and K, respectively. The remaining 3 CCs, spa-CC002, spaCC037, and spa-CC127/177, were split by MLVF into different types, from A to F, from H to J, and L and M, respectively. The sporadic isolates recognized by BURP were also classified as unique isolates by the MLVF analysis. 4. Discussion MLVF is a superior method to spa typing, which examines isolates locally. There are 3 reasons for that: i) MLVF is more discriminatory than spa typing (Malachowa et al., 2005); ii) results produced by multiplex PCR can be obtained faster than by sequencing analysis; and iii) costs of equipment and typing procedure for the DNA sequencing approach are higher. The MLVF method possesses adequate stability to be broadly used in an outbreak setting (Sabat et al., 2003). Being as highly discriminatory as PFGE (Malachowa et al., 2005), which is regarded as a “gold standard” for the epidemiologic typing of S. aureus, MLVF can be used to detect outbreaks as well as identify potential sources of transmission. In addition, comparison of the MLVF data for the epidemiologic clustering can be substantially facilitated by using BioNumerics software (GelCompar is a version of BioNumerics; Applied Maths) and a cutoff of ≥70% relatedness. In general, spa typing has also appropriate discriminatory power to determine whether the isolates are closely related or unique in a hospital setting. However, our study showed that MLVF was able to differentiate among the identical isolates by spa typing; for example, spa type t037 was divided into 16 subtypes by MLVF. This is very important because MLVF is able to show a person-to-person mode of strain transmission especially in hospitals with high rates of epidemic MRSA infections, thus, allowing the development of strategies to prevent further spread. In this situation, spa typing can suffer from uncertainties in showing direct transmission.
261
As a reference typing system, the spa typing offers significant advantages over MLVF in terms of the following: i) ease of results of interpretation for huge numbers of genetically diverse isolates; ii) better reproducibility; iii) appropriate stability of the X region of the protein A gene for long-term epidemiologic surveillance studies; and, most importantly, iv) exportability of the data. The aim of this study was to check if the MLVF method could be a reliable screening tool for the identification of genetically identical and closely related isolates. Isolates that were indistinguishable from each other by MLVF were almost always (97.4%) indistinguishable by spa typing. Therefore, because spa typing is unable to differentiate among the identical isolates by MLVF, only selected representatives of the MLVF patterns can be sent from local laboratories to the reference ones for characterization by spa typing. If a local laboratory that decides itself to monitor epidemiologic situation and the spa typing approach is too costly to use for a larger scale, only selected representatives of the MLVF types can be further characterized. In all cases, the isolates of a given MLVF type were grouped together within the same spa-CC (Table 1). The most appropriate candidate for characterization by spa typing is an isolate with the MLVF pattern, which is the most often encountered within a type, for example, for the type G, the pattern G10; for the type H, the pattern H4; and for the type K, the pattern K5. The selected representatives on the basis of MLVF analysis were also founders of the corresponding spa-CCs (t015, t037, and t052, respectively). The events of discrepancy between the results produced by both methods used in this study usually could be explained by the higher discriminatory power of MLVF. However, spa typing, being a single locus typing approach, is sensitive to the chromosomal replacements, which can lead to grouping of genetically unrelated isolates. Such a situation was found for the spa-CC037 in which isolates belonging to the distinct MLVF types had very similar spa profiles. Relevant situation was previously described by Strommenger et al. (2006), where isolates of spa types t030 and t037 were grouped together with isolates of the types t012 and t018, although they belonged to different clonal complexes by MLST, CC8 and CC30, respectively. The analysis of Robinson and Enright (2004) revealed that a chromosomal replacement, encompassing the spa locus, from ST30 to ST8, which resulted in the ST239 (CC8) mosaic chromosome, was responsible for the observed incongruence. Most of the isolates were grouped together in the same manner by 2 approaches of clustering spa types used in this study (Table 1). However, clusters S1, S2, S3, and S4 obtained on the basis of single mutation event rule were grouped together after applying the BURP algorithm. The difference observed between the 2 clustering approaches resulted from different interpretation criteria. The BURP clustering criteria allow for identifying broader similarity groups that are useful in phylogenetic studies. However, epidemiologic studies aimed mostly at outbreak analyses and
262
M. Karynski et al. / Diagnostic Microbiology and Infectious Disease 62 (2008) 255–262
monitoring shorter-time interhospital spread of S. aureus strains usually require more stringent criteria. In the previous investigations (Malachowa et al., 2005; Sabat et al., 2006a), we compared the data produced by MLVF (formerly known as MLVA) and spa typing. Previously, we assigned any 2 spa types that had a majority of identical repeats or differed in a single deletion or duplication of the nucleotide sequence into the same cluster. Those criteria for clustering spa types were less stringent but very similar to that of the single mutation event rule applied in this study. Like in the previous investigations (Malachowa et al., 2005; Sabat et al., 2006a), all isolates of S. aureus of a MLVF cluster were grouped within the same spa cluster. However, this time, the spa clusters were identical more often (5 of 8) to the MLVF clusters, whereas previously, almost each one was split by MLVF into several clusters and singular isolates. Moreover, previously, isolates within a defined cluster were usually indistinguishable by spa typing. This was not the case in this investigation (the MLVF clusters: F, G, H, K, and L consisted of 2, 9, 7, 3, and 2 spa types, respectively, whereas only 4 MLVF clusters shared the same spa type: A, C, D, and E). The differences obtained in this study and in the previous ones (Malachowa et al., 2005; Sabat et al., 2006a) can be explained by the larger number of isolates included into the present study. During this study, we analyzed 202 isolates, whereas previously, only 59 isolates. Within a cluster, not all isolates appeared to be genetically related (difference more than 6 bands between the patterns); for example, the isolates of the MLVF subtypes F3 and F4 were unrelated. However, they were linked by the isolates of the MLVF subtypes F1 and F2. During characterization of a much smaller number of most often epidemiologically unrelated isolates (202/59 = 3.4), we could miss the isolates of the subtypes F1 and F2, and then the isolates of the subtypes F3 and F4 would not be within the same MLVF cluster but still a part of the same spa cluster. We could show that by increasing the overall number of isolates, we were more likely to detect heterogeneity with respect to the spa types. The biggest MLVF clusters G and H were composed of 27 and 92 isolates, respectively. However, 15 isolates (55% of total, the subtypes G3–G11) and 81 isolates (89% of total, the subtypes H2–H17) were indistinguishable by spa typing within the MLVF clusters G and H, respectively. The MLVF method is a good and speedy tool in local laboratories for the identification of S. aureus strains in outbreak clusters and chains of transmissions. Further information on epidemic potential of individual isolates can be obtained by using spa typing. Therefore, the combined use of MLVF and spa typing can benefit in monitoring hospital-to-hospital S. aureus strain transmission events and public health interventions in a real-time and on a huge scale.
Acknowledgments The authors thank the members of National Surveillance Network (OPTY) for participating in the study. They are also grateful to Hajo Grundmann (National Institute of Public Health and the Environment, Bilthoven, The Netherlands) for helpful discussion and thoughtful review of the manuscript. These studies were supported by grant 2 P05A 052 30 (to A.S. and W.H.) from the Committee of Scientific Research (KBN, Poland). References Aires-de-Sousa M, Boye K, de Lencastre H, Deplano A, Enright MC, Etienne J, Friedrich A, Harmsen D, Holmes A, Huijsdens XW, Kearns AM, Mellmann A, Meugnier H, Rasheed JK, Spalburg E, Strommenger B, Struelens MJ, Tenover FC, Thomas J, Vogel U, Westh H, Xu J, Witte W (2006) High interlaboratory reproducibility of DNA sequence-based typing of bacteria in a multicenter study. J Clin Microbiol 44:619–621. Enright MC, Day NP, Davies CE, Peacock SJ, Spratt BG (2000) Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J Clin Microbiol 38:1008–1015. Harmsen D, Claus H, Witte W, Rothganger J, Claus H, Turnwald D, Vogel U (2003) Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management. J Clin Microbiol 41: 5442–5448. Koreen L, Ramaswamy SV, Graviss EA, Naidich S, Musser JM, Kreiswirth BN (2004) spa typing method for discriminating among Staphylococcus aureus isolates: implications for use of a single marker to detect genetic micro- and macrovariation. J Clin Microbiol 42:792–799. Malachowa N, Sabat A, Gniadkowski M, Krzyszton-Russjan J, Empel J, Miedzobrodzki J, Kosowska-Shick K, Appelbaum PC, Hryniewicz W (2005) Comparison of multiple-locus variable-number tandem repeat analysis with pulsed-field gel electrophoresis, spa typing, and multilocus sequence typing for clonal characterization of Staphylococcus aureus isolates. J Clin Microbiol 43:3095–3100. Robinson DA, Enright MC (2004) Evolution of Staphylococcus aureus by large chromosomal replacements. J Bacteriol 186:1060–1064. Sabat A, Krzyszton-Russjan J, Strzalka W, Filipek R, Kosowska K, Hryniewicz W, Travis J, Potempa J (2003) New method for typing Staphylococcus aureus strains: multiple-locus variable-number tandem repeat analysis of polymorphism and genetic relationships of clinical isolates. J Clin Microbiol 41:1801–1804. Sabat A, Malachowa N, Miedzobrodzki J, Hryniewicz W (2006a) Comparison of PCR-based methods for typing Staphylococcus aureus isolates. J Clin Microbiol 44:3804–3807. Sabat A, Melles DC, Martirosian G, Grundmann H, van Belkum A, Hryniewicz W (2006b) Distribution of the serine–aspartate repeat protein-encoding sdr genes among nasal-carriage and invasive Staphylococcus aureus strains. J Clin Microbiol 44:1135–1138. Shopsin B, Gomez M, Montgomery SO, Smith DH, Waddington M, Dodge DE, Bost DA, Riehman M, Naidich S, Kreiswirth BN (1999) Evaluation of protein A gene polymorphic region DNA sequencing for typing of Staphylococcus aureus strains. J Clin Microbiol 37:3556–3563. Strommenger B, Kettlitz C, Weniger T, Harmsen D, Friedrich AW, Witte W (2006) Assignment of Staphylococcus isolates to groups by spa typing, SmaI macrorestriction analysis, and multilocus sequence typing. J Clin Microbiol 44:2533–2540.