MOlECUlAR CHARACTERIZATION OF ...

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lates were agr type 1, four isolates were agr 3 and one isolate was agr 4 (Fig. 5). SCCmec Typing. Both methicillin resistant IE isolates harbored. mecA gene in a ...
MOlECUlAR CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS STRAINS ISOlATED FROM INFECTIVE ENDOCARDITIS Mihaela Oprea1*, David Sebastian Patriche2, Monica Strãuþ1, Felicia Antohe3

¹Cantacuzino national Institute of Research-Development for microbiology and Immunology, 103 Splaiul Independenþei, 050096, Bucharest, Romania; 2Cantacuzino national Institute of Research-Development for microbiology and Immunology, 103 Splaiul Independenþei, 050096, Bucharest, Romania (internship); 3 nicolae Simionescu Institute of Cellular Biology and Pathology, 8 BP Haşdeu, 050568, Bucharest, Romania

ABSTRACT Infective endocarditis (IE) is an infection of the heart endothelium and valves and is frequently a consequence of a sanguine flow turbulence and injury of endocardium. Recent studies revealed an increase of Staphylococcus aureus strains involved in IE, but no evident correlations between the genetic background of this bacterium and IE involvement of certain strains have been found yet. In this study we analyzed the virulence profile, including adhesins, exotoxins, superantigens and biofilm determinants, along with agr type detection, for S. aureus strains isolated from IE, versus non-IE originating strains. We performed also bacterial typing (SCCmec typing, spa-typing and MLST typing), in order to compare our strains with international databases repositories. Although the study was carried out on a reduced number of isolates, our observations confirm the previous works, showing that no major differences were observed between the genetic backgrounds of the two groups of strains analyzed. Notably, the added value of this study was optimization of two new multiplex PCR protocols, and the enrichment of international databases with three new spa-types, three new MLST alleles and four new MLST sequence types.

REZUMAT Endocardita infecţioasă (EI) reprezintă o infecţie a endoteliului inimii şi a valvelor şi apare, frecvent, ca o consecinţă a turbulenţei fluxului sanguin şi a traumelor endocardului. Studiile recente au arătat o creştere a numărului tulpinilor de Staphylococcus aureus implicate în EI, dar încă nu s-au evidenţiat corelaţii între genomul acestei specii şi implicarea anumitor tulpini în EI. În acest studiu, am analizat profilul de virulenţă, incluzând adezinele, exotoxinele, superantigenele şi determinanţii de biofilme, alături de detectarea tipului agr, pentru tulpini de S. aureus izolate din EI, comparativ cu tulpini izolate în alte tipuri de bacteriemii. De asemenea, am utilizat tehnici de tipizare moleculară (tipizare SCCmec, spa şi MLST) pentru a compara tulpinile noastre cu cele aflate în bazele de date internaţionale. Chiar dacă acest studiu a fost realizat pe un număr redus de tulpini, observaţiile noastre confirmă studiile anterioare, nefiind semnalate diferenţe majore între informaţia genetică a tulpinilor din cele două grupuri studiate. Ca un câştig al acestui studiu, au fost optimizate doua noi protocoale PCR multiplex şi au fost îmbogăţite bazele de date internaţionale cu trei tipuri spa noi, trei noi allele MLST şi patru tipuri noi MLST.

Keywords: S. aureus, molecular characterization, endocarditis

IntroductIon

Infective endocarditis (IE) is an infection of the heart endothelium and valves and is frequently a consequence of a sanguine turbulence and an injury of endocardium. Initially, a sterile vegetation can occur, but it can be infected by bacteria accidentally entering the bloodstream [1].

Coagulase-negative staphylococci and viridans streptococci were considered the predominant cause of IE, but recent studies revealed an increasing number of Staphylococcus aureus strains involved in IE. A study conducted by “International Collaboration on Endocarditis” between 2000 and 2003 revealed that 31.4% of 1779 IE cases were due to S. aureus [2]. Another work, published in 2012, studying cases

*Corresponding author: Mihaela Oprea, Cantacuzino National Institute of Research-Development for Microbiology and Immunology, 103 Splaiul Independenţei, 050096, Bucharest, Romania, e-mail [email protected], tel. +40213069223

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Molecular characterization of Staphylococcus aureus strains isolated from infective endocarditis

from 1999 to 2008 reported a proportion of 46% out of 87300 cases [3] as well. This may be due to a changing epidemiology of IE, with an increase of medical care involvement (prosthetic devices, gastrointestinal surgery, dental medicine, etc.), as S. aureus is an important nosocomial agent. Immunocompromised subjects, with a predisposition for IE, can be easily infected with nosocomial S. aureus isolates, sometimes with a multidrug resistance phenotype. Also, S. aureus is the predominant species found in narcotic consumers with IE. But S. aureus may be also community-acquired, entering the bloodstream following a superficial lesion and producing IE, especially in those patients with an affected endocardium [1]. The IE produced by S. aureus is often very severe due to characteristics of this versatile bacterium. A number of studies were conducted in order to reveal if there is any difference between the isolates producing IE and the other isolates circulating in the bloodstream but not producing IE. The results did not demonstrate any striking genetic or phenotypic characteristics of IE strains analyzed so far [4-6]. In addition, when the IE strains belonged predominantly to some clonal complexes (CC), the results from different studies did not overlap. Nienaber et al. [7] suggested that CC30 is frequently associated with IE comparing with other staphylococcal infections. Instead, Nethercott et al. [8] showed that CC12 and CC20 are predominantly associated with IE, while CC45 could not be detected in this disease. The latter also reported a t160 predominant spa-type for IE strains. On the contrary, other researchers did not find any evident relation between IE strains and spa-types [4-5]. The involvement of some adhesins in IE progression was already described. It is now known that clumping factor A and B (ClfA and ClfB), which are adhesion proteins for fibrinogen, fibronectin binding proteins (FnbPA and FnbPB), serine-rich adhesin for platelets (SraP) or collagen adhesion (Cna) are important factors in the development of IE vegetations. These proteins are present in the majority of S. aureus strains. However, FnbPB, ClfB and Cna seem to be associated more frequently to IE strains [7, 9-10]. Some studies also reported correlations between IE and superantigens bearing strains or producing extracellular fibrinogen binding protein (Efb) and a subunit compound of leukocidin/hemolysin family [6]. In this study, we analyzed the virulence profile, including adhesins, exotoxins, superantigens and

biofilm determinants detection, along with agr type detection for S. aureus strains isolated from IE, versus non-IE derived strains. Also, the methicillin sensitivity was determined, as this issue is very important for treatment options. We performed also bacterial typing, in order to compare our strains with the strains from international database repositories. MATERIAL AND METHOD

Strains Collection We characterized the genetic background of seven S. aureus strains isolated from patients with IE, along with nine isolates from other blood infections and one reference S. aureus strain, namely S. aureus MW2 (GenBank accession no. BA000033.2). The IE S. aureus strains were isolated from patients with definite endocarditis from three different hospitals and were labeled from 1 to 7. The nine nonIE strains were labeled from 8 to 16 and were collected from patients with other bloodstream infections than IE and hospitalized in a single unit, at the same time period as IE isolates. The reference strain S. aureus MW2 was labeled in this study with no. 17.

DNA Extraction DNA extraction was performed with NucleoSpin Tissue kit (Macheray Nagel, Germany), following producer’s instructions.

Detection of Virulence Factors Genes The presence of some virulence factors genes was determined by simplex or multiplex PCR reactions, using 1.5 U of GoTaq Polymerase (Promega Corporation, Madison, WI, USA), 200 µM each dNTPs (Promega Corporation, Madison, WI, USA) and GeneAmp PCR system 2700 thermocycler (Applied Biosystems CA, USA). l Triplex PCR for nuc, lukF/S and mecA genes amplification An already published PCR protocol was used to amplify in a single reaction three genes: nuc (coding for thermonuclease, as internal positive control), lukF/S (coding for Panton-Valentine leukocidin, a potent virulence factor) and mecA gene (the main determinant of methicillin resistance) [11]. Primers used are listed in Table 1 and the amplification program was: 95ºC – 4 min., 35 cycles with 94ºC – 1 min., annealing at 55ºC – 1 min., extension at 72ºC – 1,5 min., and final extension at 72ºC – 4 min. 75

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table 1. Primers for nuc, lukS/F and mecA amplification

table 2. Primers for efb, clfA and clfB genes amplification

table 3. Primers for cna, eno and ebpS genes amplification

Triplex PCR for efb, clfA and clfB genes amplification Fragments of genes coding for proteins binding to fibrinogen (Efb, ClfA, ClfB) were amplified in a single reaction, optimized in this study. For a 50 µl reaction, 1.5 units of enzyme were used. Primers are listed in Table 2 and concentrations used were: 0.14 µM for efb and 0.08 µM for clfA and clfB. The same amplification program as above was used.

l

Triplex PCR for cna, eno and ebpS genes amplification For amplification of cna (coding for adhesin to collagen), eno (coding for enolase, a protein binding to laminin) and ebpS (coding for protein binding to elastin) a triplex PCR was optimized, using 0.08 µM primers for cna, 0.04 µM primers for eno and 0.26 µM primers for ebpS (Table 3), 1.5 units of enzyme, in a volume of 50 μl and the same amplification program as above was used. l

l

PCR for amplification of genes coding for adhesins (fnbA, fnbB, sraP - gene coding for serine-rich adhesin for platelets, map/eap – coding for MHC class II analog protein), enterotoxins A, B, C, D, H, I, J, M (sea, seb, sec, sed, seh, sei, sej, sem), exfoliatins A, B, D (eta, etb, etd) and toxic shock syndrome toxin (tst).

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Several simplex PCR were performed in order to identify the corresponding genes. The amplification reaction conditions for previous triplex reactions runs were used with slight modifications: 0.2 µM was used for each primer and the annealing temperature was decreased to 50ºC. Primers used for these reactions are listed in Table 4. In this study, primers for sraP were designed with GeneRunner software (www.generunner.net/). l PCR for amplification of icaA gene, a member of ica locus For identification of icaA gene (coding for N-acetylglucosamine transferase, responsible for polysaccharide intercellular adhesin synthesis), primers ica3F and ica4R (Table 5) were used, and amplification program was: 94ºC – 5min., 25 cycles with 94ºC – 1min., 52ºC – 45 sec., 72ºC – 1min. and final extension 2 min. at 72ºC [20]. agr Type Identification agr locus, the main and the most studied system involved in the control of virulence factors expression, was identified by PCR, using 5 primers: a universal primer, specific for all five agr types and one primer for each distinct agr type (Table 6). The parameters of the reaction were described by Holtfreter et al. [18].

Molecular characterization of Staphylococcus aureus strains isolated from infective endocarditis

table 4. Primers for simplex Pcrs

table 5. Primers for icaA gene amplification

Detection of SCCmec Type for mecA Positive Strains Detection of SCCmec type can be used as a rapid typing method and also can give information about strains traceability [21]. Until now, 11 SCCmec types are known, but SCCmec I to V are the predominant types (http://www.sccmec.org/Pages/ SCC_ClassificationEN.html). In this study types I to V were identified in a 25 µl reaction with parameters described by Boye et al., 2007 [22]. Primers are listed in Table 7. The results were interpreted as presented in Table 8.

spa-Typing For amplification of a fragment spanning spa gene, SeqNet protocol was followed (http://www.seqnet.org/downloads.html; [25]). For better results, FastStart High Fidelity PCR system, dNTPack kit (Roche) was used. Amplicons were purified with NucleoSpin Extract II (Macheray Nagel, Germany), directly from the amplification products. The same SeqNet protocol was followed for sequencing, using ABI Prism BigDye Terminator v3.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystems CA, USA). Reaction products were purified with DyeEx 2.0 Spin Kit (Qiagen) and ABI 3130 Avant Genetic 77

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Analyzer (Applied Biosystems CA, USA) was used for sequencing. The resulting sequences were analyzed with Ridom StaphType software (Ridom GmbH, Germany) [25] and synchronized with Ridom spa server (http://www.spaserver.ridom.de/).

MLST Typing MLST typing consists of sequencing fragments spanning seven housekeeping genes and the recommended protocol and primers were published by Enright et al., 2000 [26]. DNA sequences obtained with ABI 3130 Avant Genetic Analyzer, were edited with BioEdit Sequence Alignment Editor v7.0.9 software (http://www.mbio.ncsu.edu/bioedit/bioedit.html) [27] and analyzed on http://saureus.mlst.net/ site for identification of every single allele and, finally, sequence type (ST) for each strain.

RESULTS

Methicillin Resistance Distribution Only for two S. aureus IE isolates mecA gene PCR amplification yielded a positive result (strains 6 and 7). Also, for three out of nine S. aureus nonIE isolates a specific 532 bp amplicon was obtained (strains 9, 13 and 16). The internal control was positive for all the reactions and the MW2 strain methicillin resistance was also confirmed (Fig. 1).

Virulence Factors Distribution Only two non-IE isolates harbored gene for Panton-Valentine leukocidin, along with the reference strain (Fig. 1). All the tested isolates were positive for genes coding for proteins binding to elastin and laminin. Two out of 7 IE isolates and one out of 9 non-IE isolates do not bear cna gene (Fig. 2).

table 6. Primers for agr type identification

table 7. Primers for Sccmec typing

table 8. Interpretation panel for Sccmec typing

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table 9. spa-types distribution

Amplification of clfA and clfB genes was positive for all strains. Three IE and two non-IE isolates did not exhibit gene efb (Fig. 3). Performing PCR for other adhesins and secreted virulence factors revealed that some genes were present in all the isolates (fnbPA, sraP and eap/map), and other genes (eta, etb and etd) in none of the isolates. tst gene was present only in two IE isolates. Three IE isolates and two non-IE isolates did not harbor fnbPB gene.

Regarding the enterotoxin genes presence, three IE isolates were positive for enterotoxin M, whereas zero was positive in non-IE group. No isolates were found positive for enterotoxin B (bioterrorism agent) and enterotoxin J. All the isolates were positive for icaA gene. A complete distribution of tested virulence factors is presented in Fig. 4.

agr Type Identification In IE group, four isolates were agr 3, two were agr 2 and one was agr 4. In non-IE group, four isolates were agr type 1, four isolates were agr 3 and one isolate was agr 4 (Fig. 5).

SCCmec Typing Both methicillin resistant IE isolates harbored mecA gene in a type IV SCCmec element. Also, one non-IE methicillin resistant strain harbored a SCCmec IV type and other three non-IE isolates revealed the presence of SCCmec type III. For MW2 strain, type IV SCCmec was confirmed.

Fig. 1. triplex Pcr for nuc, lukS/F and mecA genes Legend: 1 – isolate no. 8, 2 – isolate no. 3, 3 - isolate no, 4 – molecular weight (MW) marker 100 bp, 5 – isolate no. 14, 6 – S. aureus MW2, 7 – negative control

Fig. 2. triplex Pcr for cna, eno and ebpS genes Legend: 1 – MW marker 100 bp, 2 – isolate no. 1, 3 – isolate no 2., 4 - isolate no 4., 5 – isolate no.8, 6 – isolate no. 11, 7 – MW marker 100 bp

spa-Typing Submitting the sequences obtained for spa gene hyper-variable region to the Ridom spa server, spa types were assigned, as follows:

Fig. 3. triplex Pcr for efb, clfA and clfB genes Legend: 1 – isolate no. 8, 2 - MW marker 100 bp, 3 – isolate no. 9, 4 - isolate no. 10, 5 – negative control 79

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Fig. 4. distribution of virulence factors genes: a) adhesins; b) exoenzymes; c) enterotoxins

Fig. 5. agr type identification Legend: 1 – isolate no. 9, 2 – isolate no. 10, 3 - isolate no. 8, 4 – isolate no. 13, 5 – isolate no. 14, 6 – isolate no. 11, 7 – MW marker100 bp, 8 – isolate no. 12, 9 – isolate no. 1, 10 – isolate no. 16, 11 – negative control. 80

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table 10. St distribution

spa-types t5891, t5890 and t9057, detected in IE isolates, were considered, at the time of synchronization with Ridom spa server, new types, and a spa type number was assigned for each allele. Also, the strains with these new spa alleles were entered into the server database.

MLST Typing Following the sequencing and questioning of S. aureus MLST database, we identified a new arrangement of the seven alleles and the new ST was designated as ST 2959. Also, three new alleles were found and a unique number was assigned by the database curator for each allele, respectively each new ST: tpi gene – allele 320 (ST 2958), aroE gene – allele 435 (ST2980) and tpi gene – allele 323 (ST2981). ST distribution in the two groups is shown in Table 10. DISCUSSION

No major differences were observed between IE and non-IE isolates in terms of genetic background. The methicillin resistant IE isolates, harboring SCCmec IV cassette, seems to be a community acquisition, as this type of cassette is predominantly found in community strains [28]. It is not surprising that gene for Panton-Valentine leukocidin was not detected in IE strains, since this virulence factor was reported to be rarely present in bloodstream isolated strains [29]. However, two non-IE strains were positive for this gene, but the medical history of patients suggested a nosocomial acquisition. Adhesins distribution was uniform between the examined groups, therefore we cannot conclude that some of the adhesins are favoring the attachment of bacteria to vegetation. We can remark two strains with potential for toxic shock syndrome, and also four strains enterotoxin M producing in IE group, in contrast to non-

IE isolates where the corresponding genes were not detected. Toxic shock syndrome protein and enterotoxin M are potent superantigens, enhancing the virulence potential of these strains. Jarraud et al., [18] showed that agr 1 type presence could be positively correlated with IE. In contrast with these findings, we identified mostly types 2 and 3 in IE isolates and agr types 1 and 4 in nonIE isolates. The spa and MLST types identified in this study revealed a great diversity of isolates, without an epidemiological or causal correlation. Notably, we identified three new spa-types for IE isolates, methicillin-sensitive, suggesting that these strains are not bearing virulence determinants that can give the status of successful clones. Some authors suggested that particular CC are prevalent in IE [7-8], but those findings seem to be related to circulating strains characteristics for the respective area or to the study design, because the results do not overlap entirely. The four new ST identified in this study lead us to the same conclusion, despite the limited statistical data about the circulating ST in Romania. Maybe a better knowledge of local MLST types is required in the future. Following the molecular analysis of IE versus non-IE isolates, we cannot point out major differences between the two groups or that the genetic background alone can influence the course of IE. However, the IE isolates tested in this study seem more likely to be community acquired, entered the bloodstream as a result of quotidian activities, than nosocomial strains, as a results of medical procedures. Despite the fact that the study was performed on a reduced number of isolates, our observations confirm the previous studies [4-6]. Taken together, these data support the conclusion that there is not an evident correlation between pathogenic potential of particular strains of S. aureus involvement in IE. 81

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However, local and micro-environmental favoring conditions should be taken into consideration when evaluating the causal-effect relation between S. aureus and IE co-existence. Regardless of the number of studied strains, as a gain of this study, we optimized two new multiplex PCRs protocols that can reduce the time and the costs for further studies. Also, we identified three new spa-types, three new MLST alleles and four new ST and, thus, we contributed to the enrichment of available databases repositories. AcKnoWLEdGEMEntS: This study was financially supported by Ministry of National Education - PARTENERIATE 42-119/2008 and PN 09-22 01 01. rEFErEncES

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