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1 Department of Internal Medicine, Seoul National University College of Medicine, Seoul 110-744, Republic of Korea
2 National Institute of Health, Korea Center for Diseases Control and Prevention, Seoul 122-701, Republic of Korea
Correspondence
Hong Bin Kim
hbkimmd{at}snu.ac.kr
Received 7 June 2006
Accepted 25 June 2007
Abbreviations: HUVEC, human umbilical vein endothelial cell; IQR, interquartile range.
Present address: Department of Internal Medicine, Chonnam National University Hospital, Gwangju 501-757, Republic of Korea. 
Present address: Department of Internal Medicine, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-707, Republic of Korea.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTSDISCUSSIONREFERENCES |
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Although little is known about the pathogenesis of metastatic complications in S. aureus bacteraemia, it is thought that interaction between vascular endothelial cells and circulating bacteria is a first step in the extravasation of S. aureus (Lowy, 1998). Many studies have shown that S. aureus bacteria attach avidly to and invade vascular endothelial cells (Vercellotti et al., 1984; Ogawa et al., 1985). After invasion, the infected endothelial cells express Fc receptors and adhesion molecules, and then release interleukin-1, interleukin-6 and interleukin-8. This sequence of events facilitates the inflammatory process and contributes to the establishment of metastatic infection (Lowy, 1998). However, to the best of our knowledge, no study has demonstrated the clinical relevance of invasion of vascular endothelial cells to the development of metastatic infection in patients with S. aureus bacteraemia.
Internalized S. aureus can either survive for several days in the cytoplasm (Balwit et al., 1994) or induce the death of endothelial cells by apoptosis (Menzies & Kourteva, 1998). Clinical isolates with increased cytotoxic activity produced higher lethality and more dissemination in a mouse septicaemia model (Krut et al., 2003). However, the clinical significance of the cytotoxicity of intracellular S. aureus remains unclear.
The purpose of the present study was to determine whether (1) the ability of S. aureus to invade vascular endothelial cells or (2) the cytotoxicity of intracellular S. aureus for vascular endothelial cells were determinants of metastatic infection in patients with S. aureus bacteraemia.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The portal of entry was defined as the localized focus of S. aureus infection preceding bacteraemia, and any other foci were considered to represent metastatic infection (Lautenschlager et al., 1993). Community-acquired S. aureus bacteraemia was defined as the presence of a positive blood culture within 48 h of admission. A simple bacteraemic patient was defined as one with defervescence within 3 days of the onset of antibiotic treatment, and no evidence of metastatic complications.
Bacterial storage, growth and harvest. The clinical isolates and the S. aureus ATCC 29213 reference strain which had been stored in skimmed milk at –70 °C were subcultured on blood agar. Fresh colonies were inoculated into brain heart infusion broth and grown overnight at 37 °C. Two hours before the experiment, overnight cultures were resuspended in fresh brain heart infusion broth and grown to mid-exponential phase. The bacteria were collected by centrifugation, washed three times in PBS, and resuspended in antibiotic-free EGM-2 medium (Cambrex). The suspensions were centrifuged at 75 g for 5 min to remove variable-sized aggregates (Van Belkum et al., 2002), and bacterial concentrations were measured spectrophotometrically at 620 nm and counted by plating serial dilutions on agar. Antibiotic susceptibility was determined by the disc diffusion method, as recommended by the CLSI (2005). PFGE was performed as described previously (Kim et al., 2006).
Preparation of endothelial cells. Human umbilical vein endothelial cells (HUVECs) were used in these experiments. They were grown to confluence in 24-well plates at 37 °C in a humidified incubator with 5 % CO2, in antibiotic-free EGM-2 medium containing 5 % fetal bovine serum and growth factors. All experiments were performed with cells that had been passaged between four and eight times.
Invasion assay. The invasion assay was performed to determine numbers of internalized bacteria, as described by Ogawa et al. (1985). Each well containing HUVECs was washed with antibiotic-free EGM-2 medium prior to inoculation with bacteria. Bacterial suspension (1 ml), adjusted by optical density to 5x107 c.f.u. ml–1 (m.o.i. 250), was added to each well and incubated for 1 h at 37 °C in 5 % CO2. The complementary study showed that the number of intracellular S. aureus was linearly correlated with the m.o.i., and that the difference in invasiveness between isolates was similar irrespective of m.o.i. under our experimental conditions (data not shown).
The plates were then washed twice with antibiotic-free EGM-2 medium and incubated at 37 °C for 20 min with antibiotic-free EGM-2 medium, which contained 10 µg lysostaphin ml–1 (Sigma) to lyse extracellular staphylococci. The HUVECs were then released with trypsin, disrupted in hypotonic solution, and serial dilutions were plated on mannitol salt agar. Results are expressed as means±SEM of three independent experiments performed in duplicate.
Cytotoxicity assay. Prior to infection, HUVECs were washed with EGM-2 medium without antibiotics, and a total of 107 S. aureus were added per well (m.o.i. 50). Determination of the m.o.i. was based on our pilot study and a previous study (Krut et al., 2003). After 1 h, the HUVECs were washed with antibiotic-free EGM-2 medium and incubated at 37 °C for 24 h with 10 µg lysostaphin ml–1. The supernatants of the infected cultures were then carefully removed to preserve any detached cells, and adherent cells were harvested by standard trypsin treatment and combined with the supernatants. Viable cells were then counted by the trypan blue (Sigma) exclusion method (Bonifacio, 2000), and cytotoxicity was expressed as the percentage of dead cells. Three independent experiments were performed.
Statistical analysis. The Mann–Whitney U test and Fisher's exact test were used for comparison of continuous and categorical variables, respectively. P <0.05 was considered to be statistically significant. Statistical analysis was carried out using the SPSS software package.
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RESULTS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Eleven patients were selected as the control group, as described in Methods. The median age was 55.5 years (range 24–70) in the case group and 51 years (range 23–76) in the control group (P=0.70). Sixty per cent of the case patients and 55 % of the control patients were male (P=1.00). The portal of entry was not identified in nine (82 %) of the patients with metastatic infection and in two (18 %) of the patients without metastatic infection (P=0.01). An isolate from one patient in the case group was not available; therefore, 10 isolates from the case group and 11 from the control group were used in this study. All were meticillin-susceptible and PFGE did not show any evidence of outbreak in the community. Five patients in the case group and four in the control group were treated with anti-staphylococcal penicillin, and five patients in the case group and seven in the control group were treated with cephalosporin (P=0.67).
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). The median invasiveness, relative to the reference strain ATCC 29213, was 145 % [interquartile range (IQR) 103–160] in the cases with metastatic infection and 153 % (IQR 111–173) in the control patients without metastatic infection (Fig. 1). There was no significant difference in invasiveness between the case and control patients (P=0.44).
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). The median cytotoxicity was 59 % (IQR 47–68) in the case group and 65 % (IQR 50–74) in the control group. No statistically significant differences between the two groups were observed (P=0.44).
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DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These data strongly suggest that some factor(s) other than those that permit the organism to invade and kill endothelial cells is needed for the development of a metastatic infection. Possibly, S. aureus contains either a molecule permitting adhesion to specific tissue sites (Marriott et al., 2005) or a toxin that can cause tissue injury (Fowler et al., 2005), or possesses a mechanism for evading the host immune system (Lowy, 1998). It is also possible that S. aureus extravasates via the intercellular space. Palmqvist et al. (2005) have shown that a gene knockout of S. aureus LS-1 which lacks fibronectin-binding protein gives rise to spontaneous arthritis, despite the fact that the fibronectin-binding protein is indispensable for invasion of endothelial cells (Peacock et al., 1999). It seems likely, therefore, that metastatic infection can develop without direct invasion of vascular endothelial cells. We suggest that host factors or host–pathogen interactions may be more important for the development of metastatic infection than are strictly microbial factors, because in a mouse bacteraemia model, we did not detect any difference in dissemination to internal organs in clinical isolates from patients with and without metastatic infection (data not shown).
Host factors that increase rates of metastatic seeding during S. aureus bacteraemia include: (1) an unknown primary focus; (2) community acquisition; and (3) S. aureus bacteraemia not associated with removable foci (Lautenschlager et al., 1993; Finkelstein et al., 1984; Hedstrom & Christensson, 1983; Fowler et al., 2005). The present study also revealed an association between patients with metastatic infection and unknown primary focus. Perhaps S. aureus specifically responsible for community-acquired infection may have an unknown virulence factor that facilitates metastatic infection, given that patients from the community display relatively intact immunity in the usual clinical setting. Alternatively, community-acquired bacteraemia may be associated with persistent bacteraemia. The above-cited clinical studies support the hypothesis that bacteraemia of long duration is linked to metastatic infection. However, in this study, we were unable to examine this factor because the duration of bacteraemia prior to admission could not be established.
We confined the study population to patients with community-acquired S. aureus bacteraemia because: (1) patients with nosocomial S. aureus bacteraemia usually have severe immune defects that override other pathogenic virulence factors in the development of metastatic infection; and (2) community-acquired S. aureus may possess as-yet-unknown virulence factors that can promote the invasion of vascular endothelial cells.
The variable frequency of metastatic infection in earlier studies may have been due to variation in the methods of detection; for instance, the use of echocardiography to detect endocarditis in some cases (Ringberg et al., 2000). In the present study, to minimize the possibility of overlooking subclinical metastatic infection in the control group, we excluded those patients whose fever lasted >3 days after the initiation of antibiotic treatment, because a delay in response suggests the existence of undetected metastatic foci.
There are many methods for counting intracellular organisms, such as fluorescence assays and direct visual methods (Beekhuizen et al., 1997; Krut et al., 2003). In the present study, we chose the approach of plating and colony counting because it yields numbers of live organisms, and dead or dying organisms do not cause metastatic infection.
This study has several limitations. First of all, conditions of flow in vivo may influence the interaction of S. aureus with vascular endothelial cells, and endothelial receptors may show differential adhesion under shear flow conditions, with rolling and static receptors (McCormick et al., 1997). With respect to in vitro experiments and in vivo infection, there may be marked differences in expression of genes associated with virulence, such as that encoding fibronectin-binding protein, a major mediator of non-professional phagocyte invasion (Menzies, 2003). Finally, the number of clinical strains used in this study may not be sufficient to detect any subtle differences between each group. To prove the difference of invasiveness in 80 % power and 0.05 alpha error, the isolates from the case group should have been >30 % more invasive than those from the control group.
In summary, clinical S. aureus isolates from patients with simple bacteraemia were as effective at invasion of vascular endothelial cells and induction of cell death as were isolates from patients with metastatic infection. This finding suggests the importance of host factors or as-yet-unidentified virulence factors other than invasiveness or cytotoxicity of S. aureus for vascular endothelial cells, which result in metastatic complications in patients with S. aureus bacteraemia.
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ACKNOWLEDGEMENTS |
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