A rapid PCR method for the detection of slime-producing strains of Staphylococcus epidermidis and S. aureus in periprosthesis infections.

In periprosthesis tissues, Staphylococcus epidermidis produces extracellular polysaccharide slime. Recently it has been shown that S. aureus also produces slime and that both S. epidermidis and S. aureus contain the ica operon responsible for slime production. In the operon, icaA encodes for N-acetylglutaminyltransferase, the enzyme for polysaccharide synthesis. However, co-expression of icaA and icaD is required for full slime synthesis. The slime-producing strains of both S. epidermidis and S. aureus are more virulent and are responsible for severe postsurgical or periprosthesis infections. The authors describe a simple, rapid, and reliable polymerase chain reaction method to detect icaA and icaD. The method was applied to the detection of ica genes on two reference strains, 15 strains each of S. epidermidis and S. aureus from periprosthesis infections and 10 strains from the skin and mucosa of healthy volunteers. icaA and icaD were detectable only in slime-producing strains (tested for slime production on Congo Red agar), and never in nonslime-producing ones. This method is a straightforward way of detecting the slime-producing ability by S. epidermidis and S. aureus. In clinical specimens this polymerase chain reaction method enables rapid diagnosis of virulent slime-producing strains with respect to the traditional culture method on Congo Red agar, which requires much more time. Rapid identification of the virulent properties of the bacterial strain responsible for a staphylococcal infection is crucial for deciding treatment.     

In catheter infections by Staphylococcus epidermidis the intercellular adhesion (ica) locus is a molecular marker of the virulent slime-producing strains.

Recently, it has been shown that S. epidermidis includes the ica operon responsible for slime production. In the operon, coexpression of icaA and icaD genes is required for full slime synthesis. In this study, the presence of icaA and icaD genes was searched for in a collection of 100 Staphylococcus epidermidis strains from catheter-associated infections by an original PCR method. Another 51 strains of S. epidermidis isolated from the skin or mucosa of healthy volunteers (26 of which derived from the hospital staff) were also investigated. Slime-forming ability was phenotypically tested on Congo red agar plates. Sixty-one percent of the strains isolated from catheters were icaA- icaD-positive and produced slime. The results indicate that detection of ica genes by a PCR method is a useful tool for prompt identification of S. epidermidis slime-forming strains isolated from catheter-related infections. Also, three saprophytic strains from the hospital staff were positive for slime synthesis and presence of ica genes, suggesting a potential diffusion of slime-forming strains in hospital personnel.     

A multiplex PCR method for the detection of all five individual genes of ica locus in Staphylococcus epidermidis. A survey on 400 clinical isolates from prosthesis-associated infections

In Staphylococcus epidermidis, ica locus encodes for the synthesis of a polysaccharide intercellular adhesin (slime or biofilm). A multiplex polymerase chain reaction (PCR) for the detection of the five individual genes of ica locus was developed, with the aim to probe the set of genes in a large collection of Staphylococcus epidermidis clinical isolates. Single representative fragments for icaR, icaA, icaD, icaB, and icaC genes were selected. Multiplex PCR was applied to two reference Staphylococcus epidermidis strains [the non-biofilm-forming ATCC 12228 and the biofilm-forming ATCC 35984 (RP62A)] and to 400 clinical isolates of Staphylococcus epidermidis from orthopedic prosthesis associated infections. The gene profile was compared with the phenotypic biofilm-forming ability, evaluated by means of an optimized Congo red agar (CRA) plate test. Among the clinical isolates, 228 (57%) turned out completely ica positive and were biofilm producing. Among the 172 non-biofilm-forming strains (43%), 164 (41%) were completely ica negative and 8 strains (2%) harbored all five ica genes. The ica locus thus proves to be a cluster of strictly linked genes, without any evidence of single gene deletion.     

Detection of ica genes and slime production in a collection of Staphylococcus epidermidis strains from catheter-related infections in neutropenic patients

Slime production, principal virulence factor of Staphylococcus epidermidis associated with catheter-related infections is mediated by icaADBC operon wich expression is subject to phase variation. Reversible transposition of IS256 element into this operon is one of the most important mechanisms of biofilm phenotypic variation. Our study compared 28 S. epidermidis strains from catheter-related infection to 28 strains from nasal carriage concerning slime production on Congo red agar plate and ica genes and IS256 presence by PCR. ica operon was present among all slime-producing strains, and was absent among slime-negative strains. Only 79% of ica-positive strains were slime producers and no insertion of IS256 element was detected inside ica genes. A significative difference was found between catheter-related infections strains and commensal ones in terms of oxacillin (67,8 versus 35,7%) and ofloxacin resistance (75 versus 35,7%), slime production (64,2 versus 28,5%), phase variability (46,4 versus 7,1%) and ica genes presence (82,1 versus 35,7%). Our study demonstrates the role of ica genes, of phenotypic variability of slime production and antibiotic multiresistance as virulence factors of S. epidermidis associated with catheter-related infections; it confirms also the complexity and the diversity of regulation mechanisms implicated in biofilm formation.     

Detection of slime production by means of an optimised Congo red agar plate test based on a colourimetric scale in Staphylococcus epidermidis clinical isolates genotyped for ica locus

This investigation was conduced on a collection of 113 S. epidermidis strains isolated from biomaterial-associated infections. All strains were examined both for the presence of icaA and icaD genes responsible for slime synthesis by a PCR method and for the in vitro slime production ability by the Congo red agar (CRA) plate test. In the present study, the original CRA test was optimised adopting a six-colour reference scale for a fine classification of colonies colours. The six-colour tones of the scale were as follows: very black (vb), black (b), almost black (ab), which were considered as positive results, and bordeaux (brd), red (r), and very red (vr), interpreted as negative. 57.5% of all the strains were found to be icaA icaD-positive as well as slime-forming onto CRA, exhibiting the following colonies colours: vb (35.4%); b (15.9%); ab (6.2%). The percentage of icaA icaD-negative strains was 42.5% and all of them were negative onto CRA: brd (19.5%), r (14.2%), vr (8.8%). The comparison of colour classification with the information on ica genes confirmed the validity of the scale adopted, providing support to the criteria used for a correct interpretation of the colonies colour during the execution of the CRA test. Overall these results indicate a fine consistency between these two experimental methods and a good reliability of CRA plate test, especially when this is supported by a colourimetric scale.     

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.     

Staphylococcus caprae strains carry determinants known to be involved in pathogenicity: a gene encoding an autolysin-binding fibronectin and the ica operon involved in biofilm formation

The atlC gene (1,485 bp), encoding an autolysin which binds fibronectin, and the ica operon, involved in biofilm formation, were isolated from the chromosome of an infectious isolate of Staphylococcus caprae and sequenced. AtlC (155 kDa) is similar to the staphylococcal autolysins Atl, AtlE, Aas (48 to 72% amino acid identity) and contains a putative signal peptide of 29 amino acids and two enzymatic centers (N-acetylmuramoyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase) interconnected by three imperfect fibronectin-binding repeats. The glycine-tryptophan (GW) motif found in the central and end part of each repeat may serve for cell surface anchoring of AtlC as they do in Listeria monocytogenes. The S. caprae ica operon contains four genes closely related to S. epidermidis and S. aureus icaA, icaB, icaC, and icaD genes (> or = 68% similarity) and is preceded by a gene similar to icaR (> or =70% similarity). The polypeptides deduced from the S. caprae ica genes exhibit 67 to 88% amino acid identity to those of S. epidermidis and S. aureus ica genes. The ica operon and icaR gene were analyzed in 14 S. caprae strains from human specimens or goats' milk. Some of the strains produced biofilm, and others did not. All strains carry the ica operon and icaR of the same sizes and in the same relative positions, suggesting that the absence of biofilm formation is not related to the insertion of a mobile element such as an insertion sequence or a transposon.     

The ica operon and biofilm production in coagulase-negative Staphylococci associated with carriage and disease in a neonatal intensive care unit

Coagulase-negative staphylococci (CoNS) are a major cause of sepsis in the neonatal intensive care unit (NICU). We evaluated the hypothesis that the ica operon and biofilm production are associated with CoNS disease in this setting. CoNS associated with bacteremia or blood culture contamination and from the skin of infants with CoNS bacteremia or healthy controls were obtained during a prospective case-control study on a busy NICU. A total of 180 strains were identified, of which 122 (68%) were Staphylococcus epidermidis and the remainder were S. capitis (n = 29), S. haemolyticus (n = 11), S. hominis (n = 9), S. warneri (n = 8), and S. auricularis (n = 1). The presence of the genes icaA, icaB, icaC, and icaD was determined by PCR, and biofilm production was examined using qualitative (Congo red agar [CRA]) and quantitative (microtiter plate) techniques. There were no significant differences in the presence of the ica operon or CRA positivity among the four groups of strains. However, quantitative biofilm production was significantly greater in strains isolated from either the blood or the skin of neonates with S. epidermidis bacteremia. We conclude that the quantity of biofilm produced may be associated with the ability to cause CoNS infection. This conclusion suggests that the regulation of biofilm expression may play a central role in the disease process.     

Attachment of staphylococci to silicone catheters in vitro

The adherence of radiolabeled staphylococci to silicone catheters was investigated in vitro. Staphylococcus aureus and Staphylococcus epidermidis strains bound to the same extent to the catheters. Also, S. epidermidis strains isolated from patients with plastic-related infections showed binding similar to that of other S. epidermidis strains. By preincubation of catheters the influence of purified staphylococcal cell surface components on the binding was evaluated. The most potent inhibitors of the binding of S. aureus were the two surface proteins, clumping factor and protein A, and the cytoplasmic membrane. Surface proteins and the cell membrane of S. epidermidis also blocked the binding. Only protein-containing surface proteins inhibited the binding. The production of slime correlated with the degree of S. epidermidis binding. Human plasma and serum, as well as purified albumin and IgG, inhibited the binding of both staphylococcal species. Fibrinogen, and to a certain extent fibronectin, inhibited the binding of S. epidermidis, while both these purified plasma proteins enhanced the binding of S. aureus.     

肺癌患者中心静脉导管相关表皮葡萄球菌icaA、icaD表达及转化生长因子β1与 生物膜的关系**☆

背景：研究证实,以生物材料为中心的感染细菌临床株致病力与其在中心静脉导管材料表面形成细菌生物膜的能力呈正相关。 目的：分析肺癌患者中心静脉导管相关表皮葡萄球菌icaA、icaD mRNA表达及外周血转化生长因子β1水平与细菌生物膜形成的关系。 方法：种属鉴定相关性血流感染肺癌患者表皮葡萄球菌类型后行细菌基因组 DNA 抽提,PCR法检测生物膜形成相关基因icaA、icaD mRNA表达及生物膜表型。酶联免疫吸附试验检测相关性血流感染与未感染肺癌患者血清转化生长因子β1水平。 结果与结论：相关性血流感染肺癌患者表皮葡萄球菌操纵子icaA、icaD基因表达与生物膜形成呈正相关(P < 0.01),且表皮葡萄球菌生物膜阳性患者外周血转化生长因子β1水平较无相关性血流感染肺癌患者高(P < 0.05)。表明置入中心静脉插管引起表皮葡萄球菌感染icaA、icaD基因表达阳性肺癌患者较易形成细菌生物膜,外周血高水平转化生长因子β1对细菌生物膜形成有积极作用。     

Phase variation of biofilm formation in Staphylococcus aureus by IS 256 insertion and its impact on the capacity adhering to polyurethane surface

While ica gene of Staphylococcus epidermidis is known to undergo phase variation by insertion of IS256, the phenomenon in Staphylococcus aureus has not been evaluated. Six biofilm-positive strains were tested for the presence of biofilm-negative phase-variant strains by Congo red agar test. For potential phase-variant strains, pulsed-field gel electrophoresis was done to exclude the possibility of contamination. To investigate the mechanism of the biofilm-negative phase variation, PCR for each ica genes were done. Changes of ica genes detected by PCR were confirmed by southern hybridization, and their nucleotides were analyzed by DNA sequencing. Influence of ica genes and biofilm formation on capacity for adherence to biomedical material was evaluated by comparing the ability of adhering to polyurethane surface among a biofilm-negative phase-variant strain and its parent strain. A biofilm-negative phase-variant S. aureus strain was detected from 6 strains tested. icaC gene of the phase-variant strain was found to be inactivated by insertion of additional gene segment, IS256. The biofilm-negative phase-variant strain showed lower adhering capacity to polyurethane than its parent strain. This study shows that phase variation of ica gene occurs in S. aureus by insertion of IS256 also, and this biofilm-negative phase variation reduces adhering capacity of the bacteria.     

Detection of the intercellular adhesion gene cluster (ica) and phase variation in Staphylococcus epidermidis blood culture strains and mucosal isolates.

Staphylococcus epidermidis is a common cause of catheter-associated infections and septicemia in immunocompromised patients. To answer the question whether S. epidermidis skin isolates differ from isolates causing septicemic diseases, 51 strains obtained from blood cultures, 1 strain from shunt-associated meningitis, and 36 saprophytic isolates were characterized. The study demonstrates that most of the blood culture strains formed a multilayered biofilm on plastic material, whereas skin and mucosal isolates did not. Moreover, biofilm-producing strains were found to generate large bacterial autoaggregates in liquid culture. Autoaggregation and biofilm formation on polymer surfaces was associated with the presence of a DNA sequence encoding an intercellular adhesion gene cluster (ica) that mediates the production of a polysaccharide intercellular adhesin. The presence of the intercellular adhesion genes in blood culture isolates was also found to be correlated with the exhibition of black colonies on Congo red agar, whereas the adhesin-negative strains formed red colonies. Upon subcultivation on Congo red agar, the black colony forms of the blood culture strains exhibited red colony variants which were biofilm and autoaggregation negative and occurred at a frequency of 10(-5). The DNA analysis of these S. epidermidis variants by pulsed-field gel electrophoresis and Southern hybridization with an ica-specific gene probe revealed no detectable difference between the black and red colony types. Moreover, after repeated passage, the phenotype of the parent strain could be restored. Therefore, these colony forms were regarded as phase variants. This phenotypic change was observed exclusively in adhesin-positive clinical isolates and not in adhesin-negative saprophytic strains of S. epidermidis.     

表皮葡萄球菌生物膜形成与ica操纵子的相关性研究

目的　研究表皮葡萄球菌 (表葡 )生物膜形成与ica操纵子存在之间的相关性并比较 3种检测表皮葡萄球菌生物膜形成的方法。方法　通过PCR法扩增icaA基因以检测ica操纵子的存在。用半定量粘附实验、扫描电子显微镜法和光学显微镜法检测自临床分离到的 19株表葡的粘附能力。结果　在 19株表葡中 ,通过PCR法测得 7株为icaA阳性 ,通过半定量粘附实验测得 5株icaA(+)菌株为粘附株 ;大多数菌株在含糖培养基中所测得的吸光度 (A)值大于不含糖培养基中所测得的A值。结论　ica操纵子的存在与表葡生物膜形成在统计学上显著相关 (P <0 .0 2 5 )。培养基中补充葡萄糖有利于大多数表葡的粘附。半定量粘附实验和光镜检测法适用于细菌粘附的初步测定和筛选 ,而扫描电镜检测法有更高的灵敏度以区分粘附菌株和非粘附菌株。     

Detection by PCR of adhesins genes and slime production in clinical Staphylococcus aureus

The presence of the ica loci and adhesins genes in clinical Staphylococcus aureus strains were considered important factors of virulence. In this study, 46 strains of Staphylococcus aureus were isolated from auricular infection, and were investigated for slime production using Congo Red Agar method (CRA). In order to detect the adhesins genes (ica A, ica D, fnb A, cna, Clf A) Polymerase Chain Reaction was used. Qualitative biofilm production of S. aureus using CRA plates revealed that 56.5% of strains were slime producers. In addition 78.26% of strains were ica A and ica D positive. While the fnbA gene was present in 76.1% of isolated strains. Furthermore, 56.5% of strains have the cna gene and 30.4% were clfA positives. Overall this study confirms the presence of fnb A and ica A/ica D genes in the majority of studies S. aureus strains isolated from Staphylococcal sepsis. ((c) 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).     

Presence and expression of collagen adhesin gene (cna) and slime production in Staphylococcus aureus strains from orthopaedic prosthesis infections.

Prosthesis-associated infections still represent one of the most serious complications in the clinical use of biomaterials. The most frequent causes are Staphylococcus aureus and Staphylococcus epidermidis. Several studies have been devoted to identify adhesion mechanisms for these bacteria. Slime in particular has been extensively investigated. Recently, in Staphylococcus aureus species, considerable attention has been given to the host protein receptors that have been shown in in vitro assays to serve as substrates for bacterial adhesion. Collagen-rich tissues, as bone and cartilage, that are the preferential sites of staphylococcal infections, are also the tissues that harbour orthopaedic implants. These can be easily coated in vivo by collagen and thus become prone to adhesion of Staphylococci strains which carry the collagen adhesin gene (cna). In this study the frequency of cna was determined within a collection of 35 Staphylococcus aureus strains from orthopaedic prosthesis infections by a PCR method. Also the collagen-binding ability and slime forming capacity was evaluated. 29% of the strains were cna-positive and also able to bind collagen in vitro. 83% of the strains were slime forming. The results indicate that in the examined bacterial population slime-positive strains predominate over the cna-positive strains, with a striking association of the two adhesion mechanisms in cna-positive strains.     

表皮葡萄球菌临床株生物被膜形成及其icaA基因的分析

目的 检测表皮葡萄球菌临床株生物被膜的形成能力,了解icaA基因及其表达与生物被膜形成的关系.方法 收集205株临床分离表皮葡萄球菌,刚果红平板试验检测其黏附性,半定量黏附试验检测其生物被膜的形成能力,扫描电镜观察生物被膜形态,PCR方法 扩增icaA基因片段,RT-PCR方法 分析icaA基因表达情况.结果 205株表皮葡萄球菌中刚果红平板试验阳性24株,半定量黏附试验阳性22株,28株枪测到icaA基因.半定量黏附试验阳性菌株的icaA基因表达水平呈现高于半定量黏附试验阴性菌株的趋势.结论 表皮葡萄球菌临床株具有一定的形成生物被膜的能力,icaA基因的存在及其正常表达是表皮葡萄球菌形成生物被膜的重要分子生物学基础,icaA基因表达尚有其他因索调控.     

Anaerobic conditions induce expression of polysaccharide intercellular adhesin in Staphylococcus aureus and Staphylococcus epidermidis

Products of the intercellular adhesion (ica) operon in Staphylococcus aureus and Staphylococcus epidermidis synthesize a linear beta-1,6-linked glucosaminylglycan. This extracellular polysaccharide mediates bacterial cell-cell adhesion and is required for biofilm formation, which is thought to increase the virulence of both pathogens in association with prosthetic biomedical implants. The environmental signal(s) that triggers ica gene product and polysaccharide expression is unknown. Here we demonstrate that anaerobic in vitro growth conditions lead to increased polysaccharide expression in both S. aureus and S. epidermidis, although the regulation is less stringent in S. epidermidis. Anaerobiosis also dramatically stimulates ica-specific mRNA expression in ica- and polysaccharide-positive strains of both S. aureus and S. epidermidis. These data suggest a mechanism whereby ica gene expression and polysaccharide production may act as a virulence factor in an anaerobic environment in vivo.     

The Intercellular Adhesion (ica) Locus Is Present in Staphylococcus aureus and Is Required for Biofilm Formation

Nosocomial infections that result in the formation of biofilms on the surfaces of biomedical implants are a leading cause of sepsis and are often associated with colonization of the implants by Staphylococcus epidermidis. Biofilm formation is thought to require two sequential steps: adhesion of cells to a solid substrate followed by cell-cell adhesion, creating multiple layers of cells. Intercellular adhesion requires the polysaccharide intercellular adhesin (PIA), which is composed of linear beta-1,6-linked glucosaminylglycans and can be synthesized in vitro from UDP-N-acetylglucosamine by products of the intercellular adhesion (ica) locus. We have investigated a variety of Staphylococcus aureus strains and find that all strains tested contain the ica locus and that several can form biofilms in vitro. Sequence comparison with the S. epidermidis ica genes revealed 59 to 78% amino acid identity. Deletion of the ica locus results in a loss of the ability to form biofilms, produce PIA, or mediate N-acetylglucosaminyltransferase activity in vitro. Cross-species hybridization experiments revealed the presence of icaA in several other Staphylococcus species, suggesting that cell-cell adhesion and the potential to form biofilms is conserved within this genus.     

Phase variation of slime production in Staphylococcus aureus: implications in colonization and virulence.

Two methods commonly used for slime detection in coagulase-negative staphylococci (tube biofilm formation and colony morphology in Congo red agar) were used to study 144 ruminant mastitis Staphylococcus aureus strains. Slime production was detected in 21 strains. A majority of cells (85%) in slime-producing (SP) strains and a minority of cells (5%) in non-slime-producing (NSP) strains showed a condensed exopolysaccharide matrix (slime) surrounding the bacterial cell wall, as revealed by electron microscopy and immunofluorescence. In vivo slime production was also detected immunohistochemically after experimental infection of the mammary gland in sheep. Upon repeated subcultures in Congo red agar, NSP variants were obtained from four ovine and four bovine SP strains at a frequency ranging from 0.5 x 10(-4) to 10(-4). Because SP variants could not be obtained from NSP strains within this range or at a higher frequency, they were obtained by the tube biofilm formation (requiring repeated subculturing of NSP strains in tryptic soy broth containing 2% glucose for subsequent recovery of colonies adherent to the walls of the culture tubes). In experimental challenge, the SP variant showed a significantly higher colonization capacity than did the NSP variant of the same strain used (P < 0.001). However, the NSP variant had a higher virulence than did the SP variant (P < 0.001). These results may help to explain the different roles of S. aureus slime production cell types (SP and NSP) coexisting in disease.