Panton-Valentine leukocidin gene detected in Staphylococcus aureus strain isolated from a knee arthroprosthesis infection.

This report focuses on the molecular characterization of a Staphylococcus aureus strain isolated from a knee arthroprosthesis infection and recognized retrospectively as a carrier of the Panton-Valentine leukocidin gene. The stored microbiological isolate, which belonged to the strain collection of the Research Unit on Implant Infections of the Rizzoli Orthopaedic Institute, was retrieved for molecular analysis. Genotyping was carried out, revealing an interesting profile. In addition to the positivity for the Panton-Valentine toxin gene, the results indicated that the isolate belonged to the agr III group and was endowed with bbp and cna genes, both encoding for staphylococcal adhesins that bind bone proteins. The strain had the mecA gene for methicillin resistance, even though it was unable to resist any of the beta-lactam or other antibiotics. Its gene configuration matched that of other community-acquired methicillin-resistant and methicillin-susceptible Staphylococcus aureus(CA-MRSA and CA-MSSA) strains which have recently been reported worldwide. As far as we know,this is the first report on a PVL-positive S. aureus strain associated with an orthopedic implant (knee arthroprosthesis) infection.

[Cohort study of the incidence of heel pressure sores in patients with leg casts at the Rizzoli Orthopedic Hospital and of the associated risk factors]. / Studio di coorte sull'incidenza dei decubiti al calcagno in pazienti immobilizzati da gesso agli arti inferiori presso l'Istituto Ortopedico Rizzoli e dei possibili fattori di rischio.

INTRODUCTION: Pressure sores, especially at the heel, are a side effect of the cast. AIM: To assess the incidence of late skin complications (heel pressure sores) of a cast and determine risk factors. METHODS: All consecutive patients treated with a leg cast over a 16 months observation time were recruited. Risk factors were identified by the nurse that placed the cast and skin lesions classified with the NPUAP scale when the cast was removed. RESULTS: In the 216 enrolled patients 17.6% (38) developed a pressure sore: 16/124 in orthopedic wards; 22/92 in oncology wards. The multivariate analysis identified the following risk factors: administration of cytotoxic drugs (p = 0.033; OR = 2.61; having a cancer did not increase the risk); skin redness before cast application (p = 0.001; OR = 4.44) and having reported symptoms after the application (p = 0.000; OR = 7.86). Pressure sores were mainly stage 1 and only 6/216 (2.4%) > or = stage II. The type of plaster cast, the material, the number of days it was worn and having had a surgery are not significant risk factors. CONCLUSIONS: Pressure sores related to leg plaster casts are a frequent complication in at risk sub-groups. The acknowledgement and identification of specific risk factors may allow to identify and evaluate preventive interventions to improve the care of these patients.

Molecular epidemiology of Staphylococcus aureus from implant orthopaedic infections: ribotypes, agr polymorphism, leukocidal toxins and antibiotic resistance.

Staphylococcus aureus is a leading pathogen of implant-related infections. In the field of biomaterials a variety of alternative approaches are currently proposed for prophylaxis and treatment of implant infections, but little is known on the role of the different pathogenetic mechanisms and spreading strategies that lead selected S. aureus clones to prevail and become epidemic. This study aimed at identifying and characterizing the major clones in a collection of 200 S. aureus isolates from implant orthopaedic infections. Strain typing by automated ribotyping identified 98 distinct ribogroups. Ribogroups corresponded to specific accessory gene regulatory (agr) polymorphisms and possessed peculiar arrangements of toxins. The agr type II allele was more represented in epidemic clones, while agr type I in sporadic clones. A clear trend was observed, where epidemic clones resisted antibiotics more than sporadic ones. Conversely, the gene for lukD/lukE leukotoxin, found in 68% of the isolates, was unrelated to the level of clonal spreading. Surprisingly, the isolates of the most prevalent ribogroup were susceptible to almost all antibiotics and never possessed the lukD/lukE gene, thus suggesting the role of factors other than antibiotic resistance and the here investigated toxins in driving the major epidemic clone to the larger success.

Strong biofilm production, antibiotic multi-resistance and high gelE expression in epidemic clones of Enterococcus faecalis from orthopaedic implant infections.

Enterococcus faecalis is an opportunistic pathogen, which today represents one of the leading aetiologic agents of nosocomial infections and, increasingly, of implant infections. Here, in a collection of 43 E. faecalis isolated from implant orthopaedic infections, virulence-related phenotypes (biofilm and gelatinase production) and genotypes (gelE and esp) were studied to characterize epidemic clones identified and grouped by ribotyping. The presence of the esp gene and a marked and steady biofilm formation ability appeared to be the features associated with the clonal spreading, as well as a conspicuous gelatinase production, whereas the simple presence of gelE appeared non-specific of the epidemic clones. Antibiotic multi-resistance and strong biofilm production abilities together with a high phenotypic expression of gelatinase are an important equipment of E. faecalis to colonize peri-prosthesis tissues and to spread out as causative agents of implant orthopaedic infections.

The role of Enterococcus faecalis in orthopaedic peri-implant infections demonstrated by automated ribotyping and cluster analysis.

Enterococcus faecalis is an emerging etiologic agent of hospital infections, exhibiting high rates of antibiotic resistances. Here, 43 isolates of E. faecalis, taken from patients with implant orthopaedic infections come at the Rizzoli Orthopaedic Institute from 13 different Italian regions, were genotyped by an automated RiboPrinter and analyzed for antimicrobial susceptibility. The three most represented ribogroups were the iris-ribogroup, with its nine strains, the daisy-ribogroup, containing eight isolates, and the violet-ribogroup, with five isolates. The isolates belonging to the iris-ribogroup interestingly share a basal antibiotic resistance pattern, all being resistant to tetracycline, gentamicin and erythromycin. Among the isolates belonging to the daisy-ribogroup, 3 out of 8 were multi-resistant, 2 of which with the same pattern. More varied appeared the resistance profiles of the violet-ribogroup, in which 2 out of the five isolates were multi-resistant, the other being only bi- or mono-resistant. Noteworthy was also the variety of geographic origins and of implant infection sites for all the isolates. Cluster analysis demonstrated that ribogroups had a high internal similarity and that the three largest ones belonged to well-defined clusters, highlighting the tendency of E. faecalis to give rise to resistant clones in orthopaedic peri-implant infections.

Antibiotic multiresistance strictly associated with IS256 and ica genes in Staphylococcus epidermidis strains from implant orthopedic infections.

In this study the presence both of the ica genes, encoding for biofilm exopolysaccharide production, and the insertion sequence IS256, a mobile element frequently associated to transposons, was investigated in relationship with the prevalence of antibiotic resistance among Staphylococcus epidermidis strains. The investigation was conducted on 70 clinical isolates derived from orthopedic implant infections. Among the clinical isolates investigated a dramatic high level of association was found between the presence of ica genes as well as of IS256 and multiple-resistance to all the antibiotics tested (oxacillin, penicillin, gentamicin, erythromycin, clindamycin, chloramphenicol, sulfamethoxazole + trimethoprim, ciprofloxacin, vancomycin). Noteworthy, a striking full association between the presence of IS256 and resistance to gentamicin was found, being none of the IS256-negative strain resistant to this antibiotic. This association is probably because of the link of the corresponding aminoglycoside-resistance genes, and IS256, often co-existing within the same staphylococcal transposon. In conclusion, in orthopedics, the presence of ica genes and that of IS256 in S. epidermidis genome should both be considered as informative markers of clinically relevant strains equipped with greatest and broadest resistance potential to survive to medical treatments.

Detection of biofilm formation in Staphylococcus epidermidis from implant infections. Comparison of a PCR-method that recognizes the presence of ica genes with two classic phenotypic methods.

Biofilm-forming ability is increasingly being recognized as an important virulence factor in Staphylococcus epidermidis. This study compares three different techniques for the detection of biofilm-positive strains. The presence of icaA and icaD genes responsible for biofilm synthesis was investigated by a PCR method in a collection of 80 S. epidermidis strains isolated from orthopedic implant infections. The results from molecular analysis were compared with those obtained by two classic phenotypic methods, the Congo red agar (CRA) plate test and the microtiter plate test (MtP). Fifty-seven percent of all the examined strains were found icaA/icaD-positive, of which only three were not positive for CRA test. Differently, by the MtP method, 66% of the strains were found to be biofilm-producers but only a limited agreement with the PCR-method was noticeable because of the observation of (icaA/icaD+)/MtP- strains (8%) and of a surprising ambiguous result of (icaA/icaD-)/MtP+ strains (16%). The category of the weak biofilm-producers provided the highest contribution to these mismatching results (10%). The better agreement between the CRA plate test with the molecular detection of ica genes indicates the former as a reliable test for the phenotypic characterization of virulence of clinical isolates. However, MtP method remains a precious tool for the in vitro screening of different biomaterials for the adhesive properties using a reference strain.

Antibiotic resistance in exopolysaccharide-forming Staphylococcus epidermidis clinical isolates from orthopaedic implant infections.

The opportunistic pathogen Staphylococcus epidermidis is able to produce biofilm and to frequently cause implant infections. In recent years, it has also exhibited an increasing antimicrobial drug resistance. Here, the resistance to a panel of 16 different antibiotics in 342 clinical strains of S. epidermidis from orthopaedic implant infections has been investigated. The isolates were pheno- and genotyped for extracellular polysaccharide production, relevant to staphylococcal biofilm formation, in order to ascertain possible associations with antibiotic resistance. Approximately 10% of the isolates were found to be sensitive to all screened antibiotics. In all, 37-38% were resistant to beta-lactams such as oxacillin and imipenem, while the resistance to penicillin, ampicillin, cefazolin, cefamandole, was consistently observed in over 80% of the strains. Erythromycin- and clindamycin- resistant strains were approximately 41% and 16%, respectively. Of the isolates, 10% was resistant to chloramphenicol, 23% to sulfamethoxazole and 26% to ciprofloxacin. Resistance to vancomycin was never observed. Interestingly, exopolysaccharide-producing strains exhibited a significantly higher prevalence in the resistance to the four aminoglycosides (gentamicin, amikacin, netilmicin, tobramycin), to sulfamethoxazole and to ciprofloxacin with respect to non-producing isolates. Moreover, multiple resistance to antibiotics was more frequent among exopolysaccharide-forming strains.

Staphylococcus lugdunensis, an aggressive coagulase-negative pathogen not to be underestimated.

The new emerging coagulase-negative pathogen Staphylococcus lugdunensis is responsible for severe cardiac and joint infections. Since the biochemical phenotypic systems designed for the identification of CoNS do not appear to be species specific and are hardly reliable for the discrimination of S. lugdunensis from other staphylococci, its precise identification requires fine molecular methods. The pathogenic mechanisms by which S. lugdunensis causes severe infections are not yet completely elucidated and in this review its virulence and toxic determinants are surveyed as well as its adhesins and biofilm production.

Exopolysaccharide production by Staphylococcus epidermidis and its relationship with biofilm extracellular DNA.

Implant-related infections are difficult to treat because they are very often associated with biofilm-forming micro-organisms capable of resisting host immune defenses and surviving conventional antibiotic treatments. In Staphylococcus epidermidis biofilm-forming strains, the polysaccharide intercellular adhesin (PIA), whose expression is encoded by the icaADBC operon, is recognized as a main staphylococcal accumulation mechanism. Nevertheless, various observations have shown that PIA expression is dispensable and a variety of additional/alternative accumulation mechanisms, including extracellular DNA (eDNA) and several other factors of proteic nature, can compensate for icaADBC low expression or even for its absence. A suggestive hypothesis points to the possibility that changes in biofilm extracellular matrix composition can be induced in different environmental niches. In this study we aimed at investigating the relationship between the exopolysaccharide and eDNA biofilm components, screening 55 S. epidermidis clinical isolates by means of a simple fluorescence-based microtiter-plate assay. Our findings indicate the existence of a certain degree of correlation, although not a strict one, between eDNA and the exopolysaccharide component. The presence of exopolysaccharide greatly varied even in strains belonging to the same strain type determined by automated riboprinting.

Biofilm extracellular-DNA in 55 Staphylococcus epidermidis clinical isolates from implant infections.

Biofilm formation is broadly recognized as an important virulence factor in many bacterial species implicated in implant-related opportunistic infections. In spite of a long history of research and many investigative efforts aimed at elucidating their chemical composition, structure, and function, the nature of bacterial biofilms still remains only partly revealed. Over the years, different extracellular polymeric substances (EPS) have been described that contribute functionally and structurally to the organization of biofilms. Recently extracellular DNA (eDNA) has emerged as a quantitatively conspicuous and potentially relevant structural component of microbial biofilms of many microbial species, Staphylococcus aureus and S. epidermidis among them. The present study aims at comparatively investigating the amount of eDNA present in the biofilm of 55 clinical isolates of S. epidermidis from postsurgical and biomaterial-related orthopedic infections. Quantification of eDNA was performed by a non-destructive method directly on bacterial biofilms formed under static conditions on the plastic surface of 96-well plates.

Polymorphisms of agr locus correspond to distinct genetic patterns of virulence in Staphylococcus aureus clinical isolates from orthopedic implant infections.

Staphylococcus aureus is the leading etiologic agent of orthopedic implant infections. It is endowed with the accessory gene regulator (agr) locus that modulates expression of many virulence genes. Four allelic groups of agr have been recognized within this bacterial species. Here, 200 S. aureus isolates from orthopedic implant infections, typed at the start depending on their agr group, were screened for the presence of adhesin and leukotoxin genes. Interestingly, specific virulence gene patterns emerged in association with agr groups. The most frequently observed agr groups, agr I and agr II, were associated with the presence of sdrE, fib (agr II more than agr I), fnbB (agr I more than agr II), and lukE/lukD (agr II more than agr I). The third more frequent agr group, agr III, differed clearly from agr I and II, exhibiting high prevalence of bbp, generally not harbored by agr I and II, and copresence of bbp with cna, whereas high prevalence of the tandem sdrE/fib marked definitely agr II (91% of agr II isolates), and, though less strictly, agr I, in which prevailed the peculiar fib/fnbB pattern. The only four isolates belonging to agr IV showed full copresence of bbp with fib. Results point out distinct patterns of virulence genes, which underlie distinct evolutive strategies associated to agr groups in S. aureus causing orthopedic implant infections.

Characterization of 26 Staphylococcus warneri isolates from orthopedic infections.

Staphylococcus warneri is a coagulase negative Staphylococcus (CNS) commonly present in the flora of human epithelia and mucosal membranes. Over the last two decades, similarly to other CNS species, S. warneri has been reported as a new emerging pathogen, capable of causing serious infections usually in association with the presence of implant materials, but, at times, even in the absence of a foreign body and in patients considered immunocompetent. At present, there is still a lack of scientific data on the pathogenesis and epidemiology of this species. The present study investigated a collection of 26 clinical isolates derived from orthopedic infections, some associated with implant materials and others not. Automated ribotyping showed the existence of 5 distinct ribogroups. Except for the least numerous ribogroup consisting of a single isolate, all other ribogroups included at least one strain obtained from an infection not associated with implant materials, suggesting that putative virulence factors necessary for infections even in the absence of a foreign body could be transversal to most ribogroup categories. Orthopedic infections were found to involve S. warneri strains with low antibiotic resistance potential, differing in this respect from the strains isolated at neonatal intensive care units, where this species has been described to figure among the principal causative agents and exhibit an alarming profile of antibiotic resistance.

Prevalence of genes for aminoglycoside-modifying enzymes in Staphylococcus epidermidis isolates from orthopedic postsurgical and implant-related infections.

Staphylococcus epidermidis, a main etiologic agent of implant-related infections, is showing increasing resistance to several antibiotic substances, among them the aminoglycosides, a class of drugs playing a relevant role in current medical protocols to prevent and treat clinical infections. Here we investigated the prevalence of aac(6')-Ie-aph(2''), aph(3')-IIIa, and ant(4') genes, encoding for the three forms of aminoglycoside-modifying enzymes (AME), responsible for resistance to aminoglycoside antibiotics, in 70 clinical isolates of S. epidermidis from orthopedic postsurgical and implant-related infections. In addition, ermA and ermC, the two most common staphylococcal genes conferring antibiotic resistance to macrolides, lincosamides, and streptogramin B (MLS(B)) were included in this investigation. All isolates were characterized by automated ribotyping, so that the presence of antibiotic resistance determinants was investigated in strains exhibiting different ribopatterns. Interestingly, combinations of coexisting AME genes appeared to be typical of specific ribopatterns. The aac(6')-Ie-aph(2'') gene was the most prevalent AME gene, being observed in 44% of the isolates. As far as the determinants for MLS(B) antibiotics are concerned, the ermC gene was observed in 33% of the isolates, while ermA was detected in a single isolate. These results provide a detailed characterization in terms of antibiotic resistance determinants of clones of S. epidermidis frequently isolated from implant orthopedic infections, providing useful indications for more effectual future strategies of infection prevention/eradication based on the incorporation of antibiotic drugs in biomaterials.

Cluster analysis of ribotyping profiles of Staphylococcus epidermidis isolates recovered from foreign body-associated orthopedic infections.

Staphylococcus epidermidis is an opportunistic pathogen of major clinical interest for its high prevalence in implant-associated infections. However, only little information is available on the phylogeny of its major clonal entities and their virulence and resistance markers. Therefore, purpose of this study was to characterize four clusters identified by automated ribotyping of 70 isolates derived from orthopedic, mostly foreign body-related infections. The ica locus, encoding the polysaccharide intercellular adhesin, the IS256 insertion element as well as the resistance to gentamicin, clindamycin, chloramphenicol, and ciprofloxacin were all traits uniquely observed in two of these clusters. The phylogenetic analysis of the S. epidermidis clusters offered a detailed insight into the clonal origin of exopolysaccharide- producing and multiresistant strains with transposons appearing to be actively involved in genetic exchanges.

The presence of both bone sialoprotein-binding protein gene and collagen adhesin gene as a typical virulence trait of the major epidemic cluster in isolates from orthopedic implant infections.

Staphylococcus aureus is a major, highly clonal, pathogen causing implant infections. This study aimed at investigating the diverse distribution of bacterial adhesins in most prevalent S. aureus strain types causing orthopaedic implant infections. 200 S. aureus isolates, categorized into ribogroups by automated ribotyping, i.e. rDNA restriction fragment length polymorphism analysis, were screened for the presence of a panel of adhesins genes. Within the collection of isolates, automated ribotyping detected 98 distinct ribogroups. For many ribogroups, characteristic tandem genes arrangements could be identified. In the predominant S. aureus cluster, enlisting 27 isolates, the bbp gene encoding bone sialoprotein-binding protein appeared a typical virulence trait, found in 93% of the isolates. Conversely, the bbp gene was identified in just 10% of the remaining isolates of the collection. In this cluster, co-presence of bbp with the cna gene encoding collagen adhesin was a pattern consistently observed. These findings indicate a crucial role of both these adhesins, able to bind the most abundant bone proteins, in the pathogenesis of orthopaedic implant infections, there where biomaterials interface bone tissues. This study suggests that specific adhesins may synergistically act in the onset of implant infections and that anti-adhesin strategies should be targeted to adhesins conjointly present.