Occurrence of genes of putative fibrinogen binding proteins and hemolysins, as well as of their phenotypic correlates in isolates of S. lugdunensis of different origins

Background Staphylococcus lugdunensis is an important human pathogen that causes potentially fatal endocarditis, osteomyelitis and skin and soft tissue infections similar to diseases caused by Staphylococcus aureus. Nevertheless, in contrast to S. aureus, data on pathogenicity factors of S. lugdunensis is scarce. Two adhesins, a fibrinogen and a von Willebrand factor binding protein, and a S. lugdunensis synergistic hemolysin (SLUSH) have been previously described. Moreover, the newly sequenced genome of S. lugdunensis revealed genes of other putative fibrinogen binding adhesins and hemolysins. The aim of this study was to gain more insight into the occurrence of genes likely coding for fibrinogen binding adhesins and hemolysins using clinical strains of S. lugdunensis. Findings Most of the putative adhesin genes and hemolysin genes investigated in this study were highly prevalent, except for the SLUSH gene cluster. In contrast to previous reports, binding to fibrinogen was detected in 29.3% of the S. lugdunensis strains. In most strains, hemolysis on blood agar plates was weak after 24 h and distinct after 48 h of incubation. The fibrinogen binding and hemolysis phenotypes were also independent of the type of clinical specimen, from which the isolates were obtained. Conclusion In this study we described a pyrrolidonyl arylamidase negative S. lugdunensis isolate. Our data indicate that a matrix-assisted laser desorption ionisation time-of-flight MS-based identification of S. lugdunensis or species-specific PCR's should be performed in favour of pyrrolidonyl arylamidase testing. In contrast to the high occurrence of putative fibrinogen binding protein genes, 29.3% of the S. lugdunensis strains bound to fibrinogen. Putative hemolysin genes were also prevalent in most of the S. lugdunensis strains, irrespective of their hemolysis activity on Columbia blood agar plates. Similar to a previous report, hemolysis after 48 h of incubation is also indicative for S. lugdunensis. The SLUSH gene cluster was detected in an estimated 50% of the strains, indicating that this locus is different or non-prevalent in many strains.


Background
Staphylococcus lugdunensis is an important human pathogen that causes potentially fatal endocarditis, osteomyelitis and skin and soft tissue infections (SSTI) similar to diseases caused by S. aureus [1][2][3][4][5]. Fibrinogen and fibronectin binding adhesins have been discussed as a pathogenicity factor of S. aureus [6,7]. In S. lugdunensis, two adhesins, the fibrinogen binding protein (Fbl) [8][9][10] and the von Willebrand factor binding protein [11] have been described. A hemolysin, the S. lugdunensis synergistic hemolysin (SLUSH), has also been described [12,13]. The newly sequenced genome [14] of S. lugdunensis has revealed an additional gene (SLGD_01696) that has been annotated as a putative fibrinogen/fibronectin binding adhesin [15]. Nevertheless, data on the prevalence of S. lugdunensis adhesins and hemolysins, in contrast to S. aureus, is scarce. We therefore designed primers (Table 1) to characterize the occurrence of genes coding for putative fibrinogen binding proteins and supposed hemolysins.

Bacteria
Fifty-eight clinical strains of S. lugdunensis representing single patient isolates collected non-consecutively between 2003 and 2008 were included in this study ( Table 2). This collection represents both urban and rural settings from the Bochum area, as well as a variety of community and institutional facilities. S. lugdunenis was preliminary identified by typical characteristics, such as odor, and the GPI-card by the Vitek-2 automated identification system (bioMérieux, Marcy l'Etoile, France). In addition, the strains were tested for the presence of ornithine decarboxylase (ODC), an enzyme that catalyzes the decarboxylation of ornithine to form putrescine. Retrospectively, the presence of the pyrrolidonyl arylamidase (PYR), which hydrolyzes L-pyrrolidonyl-ßnaphtylamide to L-pyrrolidone and ß-naphtylamide, was also tested. The S. lugdunensis type strain DSM 4804 was used as a positive control in both tests. The species diagnosis was confirmed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) [16] and amplification of the tanA gene, as previously described [17]. A single isolate was also confirmed by sequencing of the sodA gene, as previously described [18] Three strains of S. epidermidis (n = 2) and S. hominis (n = 1) initially misidentified as S. lugdunensis were excluded from this study.

PCR
Strains were molecularly characterized with regard to genes coding for putative fibrinogen binding proteins, such as fbl, SLGD 1696 (FbpA), a von Willebrand factor binding protein gene vwbl, and genes coding for putative hemolysins [14]. The published sequences were used [8,9] to design a primer pair for the detection of the fbl gene ( Table 1). The von Willebrand factor binding protein precursor gene (vwbl) sequences (AY530288) [11] and SLGD_02429 [14] were used to design a primer pair for the detection of the vwbl gene. The annotated putative fibrinogen/fibronectin binding protein (SLGD_01696) gene sequence [14] was used to design primers to amplify this gene ( Table 1). The SLUSH gene sequence (U73444.1) [12], the annotated putative beta-hemolysin (SLGD_00006) gene sequence [14] and the annotated putative hemolysin III (SLGD_00847) gene sequence [14] were used to design primers for the detection of the beta-hemolysin and hemolysin III genes (Table 1). Genomic DNA of S. lugdunensis was extracted using the QIAamp DNA Mini Kit (Quiagen, Hilden, Germany) suspending the bacteria in the recommended buffer and addition of 20 μg lysostaphin at the lysis step. The master mix contains dNTP's (dATP, dCTP, dGTP, and dTTP), 2 units of pure Taq DNA polymerase and reaction buffer. The reaction contained also 12.5 pmoles of each primer, 5 μl of 1:10 diluted template DNA, and water to 25 μl. All PCRs used an initial denaturation at 94°C for 5 min, followed by 35 cycles of an annealing step at 55°C for 30 s, an elongation step at 72°C for 60 s and denaturation step at 94°C for 30 s. The previously published fibrinogen binding protein gene (fbl) sequences [8,9], the von Willebrand factor binding protein precursor gene (vwbl) sequences (AY530288) [18] and SLGD_02429 [13], the putative fibrinogen/fibronectin binding protein (FbpA homologue SLGD_01696) gene sequence [13], the S. lugdunensis synergistic hemolysin (SLUSH) gene sequence (U73444.1) [11], the S. lugdunensis putative beta-hemolysin (SLGD_00006) gene sequence [11] and the S. lugdunensis putative hemolysin III (SLGD_00847) gene sequence [11] were used to design primer pairs (This study).

Stlu 37 wound swab
Fifty-eight clinical strains of S. lugdunensis representing single patient isolates were used in this study. The strains were characterized with regard to putative fibrinogen binding and hemolysin genes and compared to their phenotypic correlates. Strains with a complete hemolysis were interpreted as positive (+). Strains producing no hemolysis (-) were compared to the negative control S. carnosus TM 300. Strains with a strong hemolysis after 48 h similar to that of S. aureus Cowan I were interpreted as producing strong hemolysis (++); + = positive; -= negative; (+) = weakly positive; ++ = strongly positive; ±* = test result indeterminate; pyrrolidonyl arylamidase (PYR); ornithine decarboxylase (ODC).

Bacterial adherence to solid-phase fibrinogen
Binding of bacterial cells to immobilized fibrinogen was previously described [19]. Briefly, the strains were grown in 4 ml LB-medium for 18

Clumping factor and latex agglutination
All strains of the S. lugdunensis collection were tested for agglutination using the commercial S. aureus agglutination kit Slidex Staph Plus (bioMérieux). For detection of fibrinogen clumping a single colony of S. lugdunensis was stirred into 50 μl of human citrate plasma and then placed onto a glass slide. An agglutination within 60 s was interpreted as a positive result ( Table 2). The S. aureus Newman (ATCC 25904) was used as a positive control.

Results and Discussion
For S. aureus identification, agglutination-based kits are often used. These kits usually include antibodies against fibrinogen binding proteins. It has been described that some strains of S. lugdunensis react in these agglutination tests [20]. The correct identification of S. lugdunensis is thus difficult. For the identification of S. lugdunensis the determination of PYR and ODC has been described to be sufficient to distinguish S. lugdunensis from other Staphylococci [1,20]. Moreover, a new species, Staphylococcus pseudolugdunensis, has been recently described, which was also PYR and ODC positive [21]. In addition, PYR negative isolates of S. lugdunensis exist. Isolate Stlu 7 ( Table 2) was PYR negative and ODC positive. The identification was confirmed by MALDI-TOF MS [15], by sequencing of the sodA gene [17], and also by the detection of the species specific fbl and tanA genes as previously described. In two other isolates the PYR test was indeterminate ( Table 2). Since only 94.8% of S. lugdunensis isolates were PYR positive, an identification strategy based solely on the determination of the PYR activity could miss an estimated 5% of the S. lugdunensis isolates. In addition, the fibrinogen binding gene fbl has been used to establish a S. lugdunensis species-specific PCR [22,23]. In our strain collection all 58 strains were shown to be fbl positive using a primer set targeting the A-region of the fbl gene. This prevalence is similar to the high occurrence of the fbl previously described [22,23]. All S. lugdunensis strains were tanA positive (100%) and all were identified as S. lugdunensis by MALDI-TOF MS (100%). Our data suggest using MALDI-TOF MS technology or detection of the fbl or tanA gene for the identification of S. lugdunensis and argues against PYR testing for S. lugdunensis identification.

Fibrinogen binding
Fibrinogen binding ability is usually targeted in S. aureus identification assays such as the Slidex Staph plus (bioMérieux) and the clumping factor test. It has been previously described that some strains of S. lugdunensis could be misidentified as S. aureus using these tests [20]. In our strains, 17 out of 58 (29.3%) were positive in the clumping factor test and 17 out of 58 (29.3%) strains were also positive in the S. aureus identification kit Slidex Staph plus. Expectedly, all of the strains that reacted in the clumping factor bound to solid phase fibrinogen, too. One isolate (Stlu 20) reacted in the Slidex Staph plus test but did not bind to soluble and solid phase fibrinogen. In another isolate (Stlu 1), binding to soluble and solid phase fibrinogen was detected but no agglutination was observed in the Slidex Staph plus test (Table 2). It has been recently reported that S. aureus virulence in sepsis is facilitated by the multiple repeats within an fibrinogen and fibronectin binding adhesin [24]. In the subgroup of blood culture samples 43.7% of the strains bound to fibrinogen compared to 26.5% in the subgroup of wound swabs. Nevertheless, the difference was statistically not significant as tested by Fischer's exact test. Differences in fibrinogen binding may be explained by different expression of the fibrinogen binding proteins. Moreover, the production of an extra cellular matrix, such as a capsule, which has been described for S. lugdunensis [2] could also mask the accession of an adhesin. Similar results has been described for S. aureus: fibrinogen binding greatly varies within the strains [25]. In contrast to previous reports, where 64.7% [26] and 83.7% [27] had been described as clumping factor positive 29.3% of our strains would be misidentified as S. aureus in the clumping factor and in the Slidex Staph plus agglutination test. In all strains, the genes for the FbpA-homologue (SLGD_01696) and fbl were present and, therefore we cannot judge if FbpA is involved in fibrinogen binding. Whether fbl or other fibrinogen binding proteins react in these assays warrants confirmation in future studies. The occurrence of the vwbl gene was also high (98.2%), similar to a previous report [18].
No statistical difference was observed in our study with regard to the type of clinical specimen from which the isolate was obtained and the fibrinogen binding phenotype. A cluster analysis was performed, but also did not show any correlation of bacterial phenotype and sample source (data not shown).

Beta-hemolysis
The ability to induce beta-hemolysis on Columbia blood agar plates (bioMérieux) after 24 h incubation and 48 h incubation was investigated. The beta-hemolysis of most strains of S. lugdunensis was weaker compared to S. aureus. Only a hemolysin similar to delta hemolysin of S. aureus has been described for S. lugdunensis [28]. In addition, synergistic hemolysins have also been described [11,12,29]. Similar to findings by others, virtually all strains showed hemolysis after 48 h of incubation [30]. The hemolysis of red blood cells is an important pathogenicity factor. Expectedly, the iron level is an important regulator for hemolysin expression in many bacteria such as Serratia marcescence [31] and E. coli [32]. Fifty-four of 58 strains (93.1%) showed no hemolysis after 24 h. After 48 h three strains also showed no hemolysis, two strains showed a weak hemolysis, 49 showed distinct hemolysis and four showed a strong hemolysis, similar to the hemolysis induced by S. aureus after 48 h. In all strains the gene for hemolysin III was found. Fifty-six out of 58 strains were also positive for the beta-hemolysin gene. Interestingly, 30 out of 58 strains (only 51.7%) were negative for the PCR detecting the SLUSH gene cluster. The initially described [11] SLUSH gene cluster (SLUSH A to C) was annotated as methylated-DNA protein-cysteine methyltransferase (SLGD_00440), and has an identity of 89% in the newly sequenced S. lugdunensis genome [13]. The gene SLGD_00441 was annotated as SLUSH B with an identity of 92% to the previously described gene cluster [11]. The following gene SLGD_00442 was also annotated as "SLUSH B" with an identity of 87% to the previously described gene cluster [11]. Interestingly, three out of four strains that were strongly positive with regard to phenotypic hemolysis were negative in the SLUSH gene specific PCR (Table 2). Notably, SLUSH gene-derived proteins have also been described as phenol soluble modulins [33]. This indicates that the SLUSH gene locus could be different or non-prevalent in an approximately 50% of the strains and therefore the SLUSH gene cluster and the basis of hemolysis should be characterized in further detail. No statistical difference was observed in our study with regard to the type of clinical specimen from which the isolate was obtained and the hemolysis phenotype.

Conclusion
In this study we described a PYR negative isolate of S. lugdunensis. Our data indicate that a matrix-assisted laser desorption ionisation time-of-flight MS-based identification of S. lugdunensis or species-specific PCR's, detecting the fbl or the tanA gene, should be used in favour of a PYR testing. In addition, this study provides a first look into the occurrence of putative fibrinogen-binding proteins and suspected hemolysins of S. lugdunensis and their phenotypic correlates. The fbl, the FbpA-homologue (SLGD 1696) and the vwbl are highly prevalent in S. lugdunensis strains. Notably, this high occurrence was independent of the fibrinogen binding ability of the strains. In contrast to previous reports only 29.3% of the strains bound to fibrinogen. Such strains could be misidentified as S. aureus in fibrinogen binding-based agglutination tests. Genes coding for hemolysins (hemolysin III [SLGD_00847] and beta-hemolysin [SLGD_00006]) are also highly prevalent in the strains except for the SLUSH gene cluster, which was detected in only an approximately 50% of the strains. This indicates that the SLUSH gene locus could be different or non-prevalent among many strains. The fibrinogen binding and hemolysis phenotypes were independent of the type of clinical specimen from which the isolates were obtained.