Antimicrobial resistance and presence of <Emphasis Type="Italic">ileS-2</Emphasis> gene encoding mupirocin resistance in clinical isolates of methicillin-resistant <Emphasis Type="Italic">Staphylococcus</Emphasis> sp.

Seventy-eight staphylococcal strains were isolated from surgical-site, blood-stream and other hospital-acquired infections. Eighteen isolates were determined as methicillin (MET)-resistant S. aureus (MRSA), while the remaining were MET-resistant coagulase-negative staphylococci (CoNS). Fifty percent of CoNS strains were multiresistant, while 10 % of isolates were resistant only to β-lactams. Clinical isolates of CoNS were generally more resistant to antimicrobial agents than S. aureus strains. Thirty-nine % of S. aureus strains were resistant only to β-lactams. None of the MRSA strains carried ileS-2 gene; this gene was found in two strains of S. epidermidis.     

Adherence of Lactobacillus to Intestinal 407 Cells in Culture Correlates with Fibronectin Binding

Lactobacilli are members of the normal mucosal microflora of most animals. Many isolates of Lactobacillus spp. are adherent to epithelial cells. In this study, using Lactobacillus acidophilus and L. agilis, we detected adherence in a pattern that suggested that the bacteria were binding to extracellular matrix proteins. Fluorescent microscopy, by using anti-fibronectin antibody, demonstrated that the isolates localize in those areas where fibronectin was detected. In addition, fibronectin pretreatment of the bacterial cells decreased adherence to Intestinal 407 epithelial cell monolayers. Cellular binding to fibronectin was detected by enzyme-linked immunosorbent assay and affinity binding to radio-labeled fibronectin. Fibronectin may be one of the eukaryotic receptors mediating attachment of Lactobacillus to mucosal surfaces. Received: 19 January 2000 / Accepted: 2 March 2000     

LysGH15B,the SH3b Domain of Staphylococcal Phage Endolysin LysGH15, Retains High Affinity to Staphylococci

LysGH15, a phage endolysin, exhibits a particularly broad lytic spectrum against Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA). Sequence analysis reveals that this endolysin contains a C-terminal cell wall binding domain (SH3b), which causes the endolysin to bind to host strains. In this study, the substrate binding affinity of the SH3b domain (LysGH15B) was evaluated. A fusion protein of LysGH15B and green fluorescent protein (LysGH15B–GFP) were cloned and expressed in Escherichia coli. Laser scanning confocal microscopy was used to detect the fluorescence of the treated cells irradiated at different excitation wavelengths and to determine the binding activity of LysGH15B–GFP and GFP. We found that LysGH15B–GFP not only generated green fluorescence, but, more importantly, also displayed specific affinity to staphylococcal isolates, especially MRSA. In contrast, the single GFP did not display any binding activity. The high affinity was attributed to the portion of LysGH15B and the binding activity of the fusion protein was specific to staphylococci. This study provides an insight into the SH3b domain of LysGH15. The specific binding activity may cause LysGH15B to serve as an anchoring device, and offer an alternative approach for cell surface attachment onto staphylococci.     

Characterisation of Faecal Staphylococci from Roe Deer (Capreolus capreolus) and Red Deer (Cervus elaphus) and Their Susceptibility to Gallidermin

Our current knowledge of microbiota in wild ruminants is limited. The goal of this study was to evaluate staphylococcal species in red and roe deer for various attributes (haemolysis, DNase, and urease activities; lactic acid and biofilm production; and antibiotic profile) and their susceptibility to gallidermin. Sixteen staphylococcal strains were identified from faeces of 21 free-living animals (9 adult female Cervus elaphus—red deer and 12 young female Capreolus capreolus—roe deer) sampled by the Polish colleagues in the Strzałowo Forest District, Piska Primaeval Forest. The variability in the species of staphylococci was determined. Seven species (Staphylococcus capitis, S. epidermidis, S. haemolyticus, S. hominis, S. pseudintermedius, S. vitulinus and S. warneri) and five clusters/groups of coagulase-negative staphylococci (CoNS) were identified. The strains were generally not haemolytic and Dnase negative; did not form biofilms or only produced low-grade biofilms; exhibited high levels of lactic acid; were urease positive; and were generally susceptible to antibiotics (only two strains were resistant to multiple antibiotics). However, all of the strains were susceptible to the lantibiotic bacteriocin gallidermin, with a minimal inhibitory concentration of 0.0156 μg (up to 6400 AU/ml in arbitrary units). This is the first study to perform a detailed study of the properties of CoNS from roe and red deer.

     

Quantitation of bacterial adherence by image analysis.

In studies of the adherence of pathogenic bacteria to host eukaryotic cells in vitro, the counting of the bacteria is often challenging, especially if many experiments are involved. We developed a method to use digital imaging and computer-aided recognition for the quantitation of bacteria attached to cultured cells. We employed an immunocytochemical method to stain the bacteria and leave the hosts cells relatively unstained. We describe this method for use with five species of bacteria, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, and Chlamydia pneumoniae. To demonstrate an application of this method, we studied the attachment of H. influenzae and S. pneumoniae to target epithelial cell lines derived from the respiratory tract.     

Staphylococci Bind Heparin-Binding Host Growth Factors

Staphylococcus aureus, which mediated binding to heparan sulfate, and also strains of coagulase-negative staphylococci (CNS) adhered in high numbers to polymers with end-point attached heparin. A characteristic feature of several cell growth factors is strong affinity for heparin. In the present study, binding of the ¹²⁵I-labeled heparin-binding growth factors (HBGF), acidic and basic fibroblast growth factor (aFGF, bFGF), and platelet-derived growth factor (PDGF) by S. aureus and CNS strains was examined. Staphylococcal strains used in this study bind bFGF and PDGF, but not aFGF. The binding of bFGF and PDGF was time dependent, influenced by pH and ionic strength for S. aureus Cowan 1. Preincubation of staphylococcal cells with unlabeled bFGF enhanced bFGF binding, but heparin, protamine sulfate, poly-L-lysine, and suramin were potent inhibitors of ¹²⁵I-bFGF binding to cells of S. aureus Cowan 1. Glycosaminoglycans of comparable size (chondroitin sulfate), other polysulfated polymers (λ-carrageenan, fucoidan), and some polysulfated polysaccharides (dextran sulfate, pentosan polysulfate) inhibited binding of both GFs to various extents. The partial inhibition of binding of both GFs after protease and periodate treatments indicates that both proteinaceous and other carbohydrate moieties participate in the binding. A lysozyme cell surface extract and bacterial lysates of S. aureus Cowan 1 competitively inhibited binding of ¹²⁵I-bFGF and ¹²⁵I-PDGF. These results suggest that staphylococci have the ability to bind two of the HBGFs, bFGF and PDGF, but not aFGF, via more than one cell structure. These binding structures seem to be exposed on the cell surface and deeply anchored in the cytoplasmic membrane as well.     

Pilin independent binding of Neisseria gonorrhoeae to immobilized glycolipids

The adherence process in pathogenesis involves the attachment of bacteria to structures present on eukaryotic cell surfaces. To investigate components necessary for this interaction, we have characterized the binding of N. gonorrhoeae to eukaryotic glycolipids immobilized on thin layer chromatograms. The gonococci specifically bind to a subset of glycolipids consisting of lactosylceramide, gangliotriosylceramide, and gangliotetraosylceramide. This binding was identified in both piliated and nonpiliated cells, and is postulated to be mediated by a nonpilin lectin-like adhesin protein.     

Staphylococcus aureus fibronectin binding protein-A induces motile attachment sites and complex actin remodeling in living endothelial cells

Staphylococcus aureus fibronectin binding protein-A (FnBPA) stimulates alpha5beta1-integrin signaling and actin rearrangements in host cells. This eventually leads to invasion of the staphylococci and their targeting to lysosomes. Using live cell imaging, we found that FnBPA-expressing staphylococci induce formation of fibrillar adhesion-like attachment sites and translocate together with them on the surface of human endothelial cells (velocity approximately 50 microm/h). The translocating bacteria recruited cellular actin and Rab5 in a cyclic and alternating manner, suggesting unsuccessful attempts of phagocytosis by the endothelial cells. Translocation, actin recruitment, and eventual invasion of the staphylococci was regulated by the fibrillar adhesion protein tensin. The staphylococci also regularly produced Neural Wiskott-Aldrich syndrome protein-controlled actin comet tails that further propelled them on the cell surface (velocity up to 1000 microm/h). Thus, S. aureus FnBPA produces attachment sites that promote bacterial movements but subvert actin- and Rab5 reorganization during invasion. This may constitute a novel strategy of S. aureus to postpone invasion until its toxins become effective.     

Relative Susceptibilities to Vancomycin and Quinupristin-Dalfopristin of Adhered and Planktonic Vancomycin-Resistant and Vancomycin-Susceptible Coagulase-Negative Staphylococci

Quinupristin-dalfopristin, a novel streptogramin antibiotic, has proven efficacious against multi-drug-resistant, Gram-positive bacteria, particularly glycopeptide-resistant coagulase-negative staphylococci (CoNS), and CoNS within biofilms. We examined its activity, along with the glycopeptide antibiotic vancomycin, against laboratory-derived, vancomycin-resistant (van R) CoNS and their vancomycin-susceptible (van S) parent strains, both in the planktonic state and after their adhesion to silicone urinary catheters. The laboratory-derived van R CoNS displayed lower adhesion and biofilm formation capabilities than did their van S parent strains. Compared with silicone, the adhesion to hydrogel-silver urinary catheters was approximately one log lower for both van R and van S CoNS. Adhesion of van R and van S CoNS to silicone catheters increased their tolerance to vancomycin. However, adhered van R CoNS succumbed to concentrations of quinupristin-dalfopristin markedly (16- to 32-fold) lower than adhered van S CoNS. This anomaly may be due to the presence of vancomycin sequestered in the cell wall of van R CoNS. Quinupristin-dalfopristin in combination with vancomycin may provide enhanced inhibitory effects against van R CoNS in the adhered state.     

Diversity of staphylococcal cassette chromosome in coagulase-negative staphylococci from animal sources

Aims:  To type the staphylococcal cassette chromosome (SCC) in coagulase-negative staphylococci (CoNS) from animal sources.
Methods and Results:  A total of 92 CoNS isolates recovered from farm animals was analysed. The top three staphylococcal species were Staphylococcus lentus (34), S. sciuri (31), and S. xylosus (13). The presence of the cassette chromosome recombinase (ccr) genes ccrA1 , ccrB1 , ccrA2 , ccrB2 , ccrA3 , ccrB3 and ccrC , the mec regulatory genes mecI and mecR1 , and Tn 554 was used to differentiate the SCC. A total of 60 of the 92 isolates were methicillin resistant. Among the 60 methicillin-resistant Staphylococcus spp. isolates, SCC mec ( mecA -carrying SCC) types I, III, IV and V were identified in 24 isolates based on the combinations of the ccr genes and the mec regulatory genes, with type III being predominant. The single S. epidermidis carried SCC mec type IV. SCC type III was also identified in two of 32 methicillin-susceptible isolates. Identical SCC mec types were present in different species of CoNS. Pulsed-field gel electrophoresis (PFGE) generated 64 patterns out of 81 PFGE typeable isolates. Indistinguishable clones were detected in animals from different farms.
Conclusions:  Heterogeneous SCC existed in CoNS of diverse genetic background. Both clonal transmission of methicillin-resistant CoNS and horizontal transfer of SCC mec occurred in the animal production environment.
Significance and Impact of the Study:  This study adds to our knowledge of SCC mec type and the diversity of SCC in CoNS.     

Application of percoll density gradients in studies of the adhesion ofStreptococcus pyogenes to human epithelial cells

A new assay was used to study the adhesion ofStreptococcus pyogenes strains to epithelial cells. [³H]thymidine-labeled bacteria were incubated with standardized preparations of epithelial cells collected from oral-pharyngeal surfaces of human volunteers. The mixtures were then centrifuged in 50% Percoll to form a density gradient. Epithelial cells with attached bacteria formed a band near the top of the tube, whereas unattached bacteria were located near the bottom. The epithelial cells were collected on membrane filters, and the number of adherent bacteria was then determined by scintillation counting.The abilities of M-protein-positive (M⁺) and M-protein-negative (M^–) strains ofS. pyogenes to attach to human pharyngeal, buccal, and tongue epithelial cells were compared. The results obtained confirmed the significant difference previously shown to exist between the attachment of M⁺ and M^– strains to human epithelial cells. M⁺ strains ofS. pyogenes exhibited a much greater ability to bind to pharyngeal epithelial cells than did M^– variants. Also, M⁺ strains were bound in higher numbers to pharyngeal epithelial cells than to buccal or tongue epithelial cells. The adhesion ofS. pyogenes strains to epithelial cells was time dependent, and a significant increase in the adhesion of M⁺ strains occurred after 3–4 h of exposure of the bacteria to epithelial cells.The adsorption ofS. pyogenes strains to epithelial cells was described by a Langmuir isotherm. With this model, the number of binding sites and the affinities of the streptococci for epithelial cells were estimated. Significantly higher numbers of binding sites were calculated to be present on pharyngeal epithelial cells for M⁺ strains ofS. pyogenes than on buccal cells. However, the affinity of the organisms was similar for both types of cells.Adsorption of M⁺ strains to human pharyngeal epithelial cells was inhibited by certain galactosides and fucose, but not by glucose or xylose. This suggests that saccharide moities play a role in the binding of M⁺ strains ofS. pyogenes to human pharyngeal epithelial cells.     

The amino-terminal domain of the P-pilus adhesin determines receptor specificity

Pyelonephritic isolates of Escherichia coli commonly express P-pili, which mediate bacterial attachment to glycolipids on epithelial cell surfaces. Three classes of P-pili have been defined, based on varying specificity for galabiose-containing glycolipids. Variation in adhesive capacity is correlated with a shift in preferred host, suggesting that host tropism depends largely on detailed specificity for the globoseries glycolipids. In this study we examined the importance of the PapG adhesin in determining receptor specificity. Translational fusions were constructed between the ammo-terminus of the PapG adhesin from each of the three pilus classes and a reporter protein. The binding specificity of the purified fusion proteins in vitro was identical to that seen with whole bacteria. Adherence of intact bacteria to cultured kidney cells was markedly reduced by a monoclonal antibody specific for the Class III adhesin (previously denoted PrsG), confirming the importance of the ammo-terminus of PapG in mediating attachment to a receptor when presented on the eukaryotic cell surface. These results suggest that the detailed receptor specificity resides solely within the amino-terminus of the PapG adhesin and is independent of the complex pilus architecture.     

Glycosaminoglycan binding by Borrelia burgdorferi adhesin BBK32 specifically and uniquely promotes joint colonization

Microbial pathogens that colonize multiple tissues commonly produce adhesive surface proteins that mediate attachment to cells and/or extracellular matrix in target organs. Many of these ‘adhesins’ bind to multiple ligands, complicating efforts to understand the role of each ligand‐binding activity. Borrelia burgdorferi, the causative agent of Lyme disease, produces BBK32, first identified as a fibronectin‐binding adhesin that promotes skin and joint colonization. BBK32 also binds to glycosaminoglycan (GAG), which, like fibronectin is ubiquitously present on cell surfaces. To determine which binding activity is relevant for BBK32‐promoted infectivity, we generated a panel of BBK32 truncation and internal deletion mutants, and identified variants specifically defective for binding to either fibronectin or GAG. These variants promoted bacterial attachment to different mammalian cell types in vitro, suggesting that fibronectin and GAG binding may play distinct roles during infection. Intravenous inoculation of mice with a high‐passage non‐infectious B. burgdorferi strain that produced wild‐type BBK32 or BBK32 mutants defective for GAG or fibronectin binding, revealed that only GAG‐binding activity was required for significant localization to joints at 60 min post‐infection. An otherwise infectious B. burgdorferi strain producing BBK32 specifically deficient in fibronectin binding was fully capable of both skin and joint colonization in the murine model, whereas a strain producing BBK32 selectively attenuated for GAG binding colonized the inoculation site but not knee or tibiotarsus joints. Thus, the BBK32 fibronectin‐ and GAG‐binding activities are separable in vivo, and BBK32‐mediated GAG binding, but not fibronectin binding, contributes to joint colonization.     

Transient appearance of lectin receptors onRhizobium trifolii

The appearance on the surface ofRhizobium trifolii 0403 of determinants important to both clover lectin (trifoliin) binding and adherence of the bacteria to clover root epithelial surfaces was studied by quantitative agglutination, immunofluorescence, and direct microscopic techniques. These unique determinants were found for only transient periods of time—as cells left lag phase and as they entered stationary phase of growth in broth. When present, these receptors were associated with a fibrillar polyanionic capsule surrounding the cells when grown on solid medium. These studies support earlier proposals that the architecture of the rhizobial cell surface is not constant in composition, but changes with the phase of growth.     

Identification of a novel lectin inStreptococcus pyogenes and its possible role in bacterial adherence to pharyngeal cells

During investigation of the interaction of human lactoferrin (HLf) with variou bacteria, it was found that inStreptococcus pyogenes, HLf binding occurred to agar-rather than broth-grown cells irrespective of the nutrients used. Furthermore, binding of HLf to broth-grown, heat-killed bacteria was induced by overnight incubation on agar media or short-time exposure of the cells to water-soluble agar extract. The binding pattern was revealed in most of 92S. pyogenes strains representing various M-or T-types with no apparent type variation. The component thus bridging the attachment of HLf to the streptococcal cell surface was recovered in extracts of agar-grown cells and isolated by affinity chromatography on HLf-sepharose. By gel filtration in the presence of radiolabeled HLf, this component exhibited similar elution position as crude water-soluble agar extract. Chemical analysis identified the active HLf-binding agar component to be a galactose-rich polysaccharide (GRP). Further binding tests showed that the interaction between streptococci and GRP was stable in the presence of high molar NaCl, KSCN, or urea and was unaffected by various serum or matrix proteins or by streptococcal lipoteichoic acid; however, a moderate inhibition by heparin or bovine mucin was observed. Studies on isogenic mutants ofS. pyogenes did not support the involvement of M-protein or the hyaluronate capsule in the binding of GRP. SDS-PAGE and Western blot analyses revealed a GRP-binding protein of approximately 70 kDa in the cell-wall extracts of two strains ofS. pyogenes, types M19 and M55. Finally, the adherence of (broth-grown)³H-thymidine-labeledS. pyogenes, type M19, to the pharyngeal epithelial cell line DT-562 or to normal tonsillar epithelial cells was inhibited by GRP in a dose-related manner. We thus propose that the streptococcal GRP-binding component may represent a novel surface lectin acting as a mucosal adhesin forS. pyogenes, in accordance with previous data indicating that galactosecontaining sugar moieties may serve as ligands for the adherence of streptococci to pharyngeal cells. Our results also indicate that GRP-like components such as mucin or heparin might act to block epithelial adherence ofS. pyogenes at the mucosal level.     

A method for the enumeration of bacterial adhesion to epithelial cells using image analysis

Abstract Computerised image analysis was utilised to enumerate the attachment of Staphylococcus epidermidis to HEp2 cell monolayers. A differential staining technique was employed such that individual staphylococcal cells stood out in sharp contrast against the uneven cell surface and granular contents of the epithelial cells. The primary image analysis operation involved subtracting an out-of-focus image from an in-focus image of the bacteria on the monolayer, thereby accentuating the bacterial image. Enumeration, using a particle counting routine, was rapid and reproducible, facilitating counting in excess of 700 bacteria per field at ×500 magnification. The computerised programme compared favourably with manual counting and would provide a rapid, objective and morphologically discriminatory method for evaluating bacterial attachment to various tissues.     

Time-resolved fluorimetry in the study of attachment of staphylococci and streptococci on substrate-bound fibronectin

Abstract The application of time-resolved fluorimetry was evaluated in the study of staphylococcal and streptococcal attachment to fibronectin-coated coverslips. The test system allowed the use of low bacterial concentrations (2 × 10⁵−10⁷ bacteria per ml), in contrast to the much higher concentrations of bacteria used in earlier assays. The bacteria attached much better to fibronectin-coated plastic surfaces than to albumin-coated ones, but there were differences between the individual strains. Soluble fibronectin inhibited the adsorption of staphylococci but enhanced streptococcal attachment to fibronectin-coated surfaces. Purified antibodies to fibronectin inhibited both staphylococcal and streptococcal adhesion in a dose-dependent way. Our results show that time-resolved fluorimetry is a very sensitive method for quantitating bacterial attachment.     

Host cell binding of the flagellar tip protein of Campylobacter jejuni

Flagella are nanofibers that drive bacterial movement. The filaments are generally composed of thousands of tightly packed flagellin subunits with a terminal cap protein, named FliD. Here, we report that the FliD protein of the bacterial pathogen Campylobacter jejuni binds to host cells. Live‐cell imaging and confocal microscopy showed initial contact of the bacteria with epithelial cells via the flagella tip. Recombinant FliD protein bound to the surface of intestinal epithelial cells in a dose‐dependent fashion. Search for the FliD binding site on the host cell using cells with defined glycosylation defects indicated glycosaminoglycans as a putative target. Heparinase treatment of wild type cells and an excess of soluble heparin abolished FliD binding. Binding assays showed direct and specific binding of FliD to heparin. Addition of an excess of purified FliD or heparin reduced the attachment of viable C. jejuni to the host cells. The host cell binding domain of FliD was mapped to the central region of the protein. Overall, our results indicate that the C. jejuni flagellar tip protein FliD acts as an attachment factor that interacts with cell surface heparan sulfate glycosaminoglycan receptors.     

Fibronectin binding to a Streptococcus pyogenes strain.

In previous studies, Staphylococcus aureus has been shown to bind fibronectin (P. Kuusela, Nature (London) 276:718-720, 1978), an interaction that may be important in bacterial attachment and opsonization. Recently some strains of streptococci of serological groups A, C, and G were also found to bind fibronectin. The binding to one selected strain of Streptococcus pyogenes has been characterized here. The binding of [125I]fibronectin to streptococcal cells resembles that to staphylococcal cells and was found to be time dependent, functionally irreversible, and specific in the sense that unlabeled proteins other than fibronectin did not block binding. Bacteria incubated with proteases largely lost their ability to bind fibronectin, and material released from the streptococci by a brief trypsin digestion contained active fibronectin receptors. This material inhibited the binding of [125I]fibronectin to the streptococci. The inhibitory activity was adsorbed on a column of fibronectin-Sepharose but not on a column of unsubstituted Sepharose 4B or egg albumin Sepharose. The receptor appeared to be a protein nature since the inhibitory activity of the trypsinate was destroyed by papain and was not absorbed on a column containing monoclonal antibodies directed against lipoteichoic acid bound to protein A-Sepharose. Binding sites in fibronectin for streptococci and staphylococci, respectively, were localized by analyzing the ability of isolated fragments to inhibit [125I]fibronectin binding to bacteria and by adsorbing 125I-labeled tryptic fragments with staphylococcal and streptococcal cells. Both species of bacteria appeared to preferentially bind a fragment (Mr = approximately 25,000) originating from the N-terminal region of the protein. In addition, streptococci also bound a slightly smaller fragment (Mr = approximately 23,000). Fibronectin receptors solubilized from either streptococci or staphylococci inhibited the binding of fibronectin to both species of bacteria.     

Inhibition of wall autolysis of staphylococci by sodium polyanethole sulfonate “liquoid”

Summary Liquoid (polyanethole sulfonate) was neither capable of influencing the growth nor the viability of staphylococci. But liquoid induced a suppression of the activity of different autolytic wall systems of normally growing staphylococci, i.e., autolysins which participate in cross wall separation as well as autolysins which are responsible for cell wall turnover. Additionally, the lysostaphin-induced wall disintegration of staphylococci was inhibited by liquoid.However, no indication could be found for a direct inhibition of lytic wall enzymes by liquoid; rather an interaction of liquoid with the target structure for the autolytic wall enzymes, the cell wall itself, was postulated. On the basis of the experimental data with the teichoic acid^- mutant S. aureus 52A5 the sites of wall teichoic acid were supposed to be an important target for the binding of liquoid to the staphylococcal cell wall.