Bacteriology
Prevalence of genes encoding for members of the staphylococcal leukotoxin family among clinical isolates of Staphylococcus aureus

https://doi.org/10.1016/j.diagmicrobio.2004.03.009Get rights and content

Abstract

Well-characterized Staphylococcus aureus nasal and blood isolates (N = 429) were tested by polymerase chain reaction for the prevalence of genes that encode leukocidal toxins. The leukotoxin genes lukE+lukD were found at high prevalence, significantly more so in blood (82%) than in nasal isolates (60.5%). Although almost all isolates were positive for the γ-hemolysin gene, none was positive for lukM. Genes encoding Panton-Valentine leukocidin (PVL) components were very rare in either nasal or blood isolates. The lukE+lukD-negative isolates were significantly more likely to be positive for the staphylococcal enterotoxin gene combination seg/sei (89.5%) and the toxic shock syndrome toxin-1 gene (39.3%) than lukE+lukD-positive isolates (41.7% and 12.7%, respectively). The lukE+lukD-negative isolates were also more likely to show positivity for the accessory gene regulatory locus agr III, but less likely to be positive for the agr II locus. The co-possession of different virulence factors and their probable synergy should receive more attention in order to better understand their role in pathogenicity.

Introduction

In recent years, Staphylococcus aureus has emerged as major cause of severe nosocomial and community-acquired infections (Lowy, 1998). Its carriage in the anterior nares, considered to be the ecologic niche, plays a key role in the epidemiology and pathogenesis of infection that is due to this pathogen (Kluytmans et al., 1995, Study Group et al., 2001).

Nearly all S. aureus isolates produce numerous toxins and enzymes with contributory roles in pathogenesis. Several exotoxins are clearly associated to clinical syndromes, such as toxic shock syndrome (TSS), staphylococcal scalded skin syndrome (SSSS) or staphylococcal food poisoning (SFP), however, the vast majority of severe S. aureus infections cannot be explained by the action of a single virulence determinant. Cumulative effects that are due to different factors with diverse mechanisms of action during the infective process may contribute to staphylococcal disease pathogenesis (Peacock et al., 2002).

Leukocidal (synergohymenotropic) toxins, including the Panton-Valentine leukocidin (PVL), are cytotoxins produced by staphylococci (S. aureus and S. intermedius) and constitute a family of pore-forming toxins that are composed of 2 distinct components. The toxic effect depends on the synergistic action of both class S (slow eluted)-related and class F (fast eluted)-related proteins on human polymorphonuclear cells or erythrocytes (e.g., LukF-PV, LukM and γ-hemolysin (Hlg) B belong to class F, and LukS-PV, HlgA, and HlgC belong to class S-related proteins) (Prevost et al., 1995b, Prevost et al., 2001).

More recently, a new member of the staphylococcal bicomponent leukotoxin family, LukE (58% to 68% identical with class S proteins) and LukD (71% to 77% identical with class F proteins of the family) has been characterized (Gravet et al., 1998). Cell membranes appear to be a primary target for triggering the lysis of phagocytic cells caused by staphylococcal leukocidal toxins.

The frequent recovery of staphylococcal isolates that produce leukocidal toxins from patients with necrotic skin lesions, furuncles, and osteomyelitis suggest that these toxins have a role in the virulence of staphylococci, at least in skin and soft tissue infections (Couppie et al., 1994, Lina et al., 1999, Prevost et al., 1995a). Recently, an association was detected between S. aureus isolates that carry the gene for PVL and highly lethal hemorrhagic, necrotizing pneumonia in young immunocompetent patients (Gillet et al., 2002). However, despite the presumed importance of leukocidal toxins in S. aureus virulence, very few data are available on their prevalence among isolates recovered from deep-seated infections compared with carrier isolates cultivated from nasal specimens.

The expression of most S. aureus virulence factors is controlled by the accessory gene regulator (agr) locus, which is characterized by a polymorphism of its autoinducing peptide that divides S. aureus isolates into 4 major groups (Jarraud et al., 2002). Links between the possession of one of the agr alleles and staphylococcal toxin-mediated syndromes that are due to staphylococcal pyrogenic superantigen toxins (PTSAgs) and exfoliative toxins (ETs) have been previously reported (Jarraud et al., 2000, Jarraud et al., 2002). In contrast, little is known about the relationship between leukotoxins and agr alleles. Furthermore, a possible synergy between leukotoxins and other staphylococcal virulence factors in the impairment of host defenses and the promotion of bacterial growth and spreading is debated (Prevost et al., 2001).

The aims of this study were, therefore, (1) to examine the prevalence of genes that code for the members of the staphylococcal leukotoxin family, (2) to compare the prevalence in well-characterized strain populations (blood isolates versus isolates from nasal carriers), (3) to study a possible relationship to agr groups, and (4) to analyze any coexistence to other exotoxin genes that encode ETs (eta and etb) and members of the PTSAg gene family comprising the toxic shock syndrome-1 toxin (TSST-1), the classic staphylococcal enterotoxin (SE) genes (sea-see), and their newly described members (seg-sej).

Section snippets

Bacterial isolates

S. aureus isolates (N = 429) collected during the course of a German multicenter study comprising 32 university and community hospitals (von Eiff et al., 2001) were tested by polymerase chain reaction (PCR) for the prevalence of genes encoding LukE-LukD (lukE + lukD), LukM (lukM), PVL components S and F (lukS-PV + lukF-PV), and γ-hemolysin (hlgC-hlgB). The results were compared with the analyses of agr alleles and the possession of ET and PTSAg genes as previously reported (Becker et al., 2003,

Presence of leukotoxins

The results of testing blood and nasal S. aureus isolates for the presence of leukotoxins are given in Table 1. Apart from 1 blood isolate, all isolates were tested PCR-positive for the γ-hemolysin gene. Fragments of LukM gene were not amplified in any isolate. The genes coding for leukotoxins LukE-LukD were detected in a high prevalence (n = 180; 82%) with significantly more positive isolates among blood isolates (p < 0.013) than were found for nasal isolates (n = 127; 60.5%). Except for 1

Discussion

As members of the group of the so-called membrane-damaging toxins (MDTs), leukocidal toxins disrupt membranes of host defense cells and erythrocytes by the synergistic action of 2 nonassociated classes of secretory proteins, designated S and F (König et al., 1995, Prevost et al., 1995b, Prevost et al., 2001). Any combination of a given class S protein with a class F protein may be toxic and was reported to generate an intense inflammatory response in animal models (Siqueira et al., 1997).

Acknowledgements

The authors are grateful to Michaela Brück, Brigitte Schuhen, Martina Schulte, Angela Terliesner, and Susanne Weber for excellent technical assistance and P. Cullen for a careful review of the manuscript. We thank J. Etienne, Lyon, France for the kind provision of the S. aureus FRI-913.

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