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Although infrequent in our laboratory, growth of bacterial colonies has been observed on top of the purified cultures of yeasts. In this study, the likelihood of bacterial excision from yeast under aging and starvation stresses was assessed using 10 gastric and 10 food-borne yeasts. Yeasts were identified as members of Candida or Saccharomyces genus by amplification and sequencing of D1/D2 region of 26S rDNA. For aging stress, yeasts were cultured on brain heart infusion agar supplemented with sheep blood and incubated at 30 °C for 3-4 weeks. For starvation stress, yeasts were inoculated into distilled water and incubated similarly. After seven days, starved yeasts were cultured on yeast extract glucose agar, incubated similarly and examined daily for appearance of bacterial colonies on top of the yeast's growth. Outgrowth of excised bacteria was observed on top of the cultures of 4 yeasts (Y1, Y3, Y13 and Y18) after 3-7 days. The excised bacteria (B1, B3, B13 and B18) were isolated and identified at the genus level according to their biochemical characteristics as well as amplification and sequencing of 16S rDNA. B1 (Arthrobacter) were excised from Y1 (Candida albicans) upon aging and B3 (Staphylococcus), B13 (Cellulomonas) and B18 (Staphylococcus) were excised from their respective yeasts; Y3 (Candida tropicalis), Y13 (Saccharomyces cerevisiae) and Y18 (Candida glabrata) upon starvation. DNA from yeasts was used for detection of 16S rDNA of their intracellular bacteria and sequencing. Amplified products from yeasts showed sequence similarity to those of excised bacteria. Under normal conditions, yeast exerts tight control on multiplication of its intracellular bacteria. However, upon aging and starvation the control is no longer effective and bacterial outgrowth occurs. Unlimited multiplication of excised bacteria might provide yeast with plenty of food in close vicinity. This could be an evolutionary dialogue between yeast and bacteria that ensures the survival of both partners., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier B.V. All rights reserved.) more...

Objective: Implant-related infection is a devastating complication in orthopedic surgery. Aiming to minimize this problem, many material modifications have been developed. Here we report a study of a surface modification of Ti-6 Al-4 V alloy using a methodology that enables the study of interactions between bacteria and the material in the presence of eukaryotic cells., Methods: We mixed different concentrations of collection or clinical strains of staphylococci isolated from implant-related infections with preosteoblastic cells using a previously published methodology, analyzing the minimal concentration of bacteria able to colonize the surface of the material through image analysis. Ti-6 Al-4 V alloy was modified by anodization to obtain two F-doped nanostructured surfaces that have been previously described to have antibacterial properties., Results: Our results show similar bacterial adhesion results to nanoporous and nanotubular F-doped surfaces. The presence of preosteoblastic cells increases the adherence of all bacterial strains to both structures. No effect of the surface on eukaryotic cells adherence was detected., Conclusion: To our knowledge, this is the first time that anin vitro study emulating the race for the surface evaluates and compares the osseointegration and antibacterial properties between two nanostructured- modified titanium alloy surfaces. Clinical strains show different behavior from collection ones in bacterial adherence. The presence of cells increased bacterial adherence. NP and NT surface modifications didn´t show significant differences in bacterial adhesion and preosteoblastic cells integration., (Copyright © 2018 Elsevier B.V. All rights reserved.) more...

The ability of bacteria to move is critical for their survival in diverse environments and multiple ways have evolved to achieve this. Two forms of motility have recently been described for Staphylococcus aureus, an organism previously considered to be non-motile. One form is called spreading, which is a type of sliding motility and the second form involves comet formation, which has many observable characteristics associated with gliding motility. Darting motility has also been observed in Staphylococcus epidermidis. This review describes how motility is defined and how we distinguish between passive and active motility. We discuss the characteristics of the various forms of Staphylococci motility, the molecular mechanisms involved and the potential future research directions. more...

Fish produce mucus substances as a defensive outer barrier against several bacterial infections. We have recently identified an antibacterial L-amino acid oxidase (psLAAO1) in the mucus layer of the flounder Platichthys stellate. In this study, the antibacterial protein psLAAO1 was expressed as a secretory bioactive recombinant protein in the methylotrophic yeast Pichia pastoris. The recombinant psLAAO1 inhibited the growth of bacteria to the same levels as native psLAAO1 present in mucus. In particular, Staphylococci and Yersinia were strongly suppressed, showing the highest growth retardation of the 21 species and strains tested. Moreover, Staphylococcus epidermidis was most sensitive to psLAAO1 with a minimum inhibitory concentration (MIC) of 0.078 μg/mL, whereas Escherichia coli was essentially resistant to psLAAO1 with a MIC of >10 μg/mL. Interestingly, psLAAO1-treated E. coli were found to upregulate the expression of the btuE gene, which encodes glutathione peroxidase (GPx). The biochemical function of GPx is to reduce free hydrogen peroxide and is induced under response to reactive oxygen species (ROS). Thus, E. coli confers resistance to the reduced free hydrogen peroxide produced by psLAAO1 by increasing GPx levels. Furthermore, the growth of Staphylococcus aureus was completely inhibited in the presence of recombinant psLAAO1. The morphology of psLAAO1-treated S. aureus showed cell surface damage, the formation of large aggregates and the cells showed severe deformations. Western blot analysis showed that psLAAO1 binds to the surface of S. aureus. Therefore, psLAAO1 binds to the surface of LAAO-sensitive S. aureus and directs peroxidative activity at the surface of the bacterial membrane. more...

The recent worldwide spread of methicillin-resistant Staphylococcus pseudintermedius (MRSP) in dogs is a reason for concern due to the typical multidrug resistance patterns displayed by some MRSP lineages such as sequence type (ST) 71. The objective of this study was to compare the in vitro adherence properties between MRSP and methicillin-susceptible (MSSP) strains. Four MRSP, including a human and a canine strain belonging to ST71 and two canine non-ST71 strains, and three genetically unrelated MSSP were tested on corneocytes collected from five dogs and six humans. All strains were fully characterized with respect to genetic background and cell wall-anchored protein (CWAP) gene content. Seventy-seven strain-corneocyte combinations were tested using both exponential- and stationary-phase cultures. Negative binomial regression analysis of counts of bacterial cells adhering to corneocytes revealed that adherence was significantly influenced by host and strain genotype regardless of bacterial growth phase. The two MRSP ST71 strains showed greater adherence than MRSP non-ST71 (p < 0.0001) and MSSP (p < 0.0001). This phenotypic trait was not associated to any specific CWAP gene. In general, S. pseudintermedius adherence to canine corneocytes was significantly higher compared to human corneocytes (p < 0.0001), but the MRSP ST71 strain of human origin adhered equally well to canine and human corneocytes, suggesting that MRSP ST71 may be able to adapt to human skin. The genetic basis of the enhanced in vitro adherence of ST71 needs to be elucidated as this phenotypic trait may be associated to the epidemiological success and zoonotic potential of this epidemic MRSP clone. more...

Silver nanoparticles form promising template for designing antimicrobial agents against drug resistant pathogenic microorganisms. Thus, the development of a reliable green approach for the synthesis of nanoparticles is an important aspect of current nanotechnology research. In the present investigation, silver nanoparticles synthesized by a soil Bacillus sp. were characterized using UV-vis spectroscopy, FTIR, SEM, and EDS. The antibacterial potential of biosynthesized silver nanoparticles, standard antibiotics, and their conjugates were evaluated against multidrug-resistant biofilm-forming coagulase-negative S. epidermidis strains, S. aureus, Salmonella Typhi, Salmonella Paratyphi, and V. cholerae. Interestingly, silver nanoparticles (AgNPs) showed remarkable antibacterial activity against all the test strains with the highest activity against S. epidermidis strains 145 and 152. In addition, the highest synergistic effect of AgNPs was observed with chloramphenicol against Salmonella typhi. The results of the study clearly indicate the promising biomedical applications of biosynthesized AgNPs. more...

Bringing the study of bacterial adhesion down to a single-cell level is critical for understanding the molecular mechanisms involved in initial bacterial attachment. We have developed a simple and versatile method for making single-cell bacterial probes to study the adhesion of single bacterial cells by atomic force microscopy (AFM). A single-cell probe was made by picking up a bacterial cell from a glass surface using a tipless AFM cantilever coated with a commercial cell adhesive Cell-Tak. The method was applied to four different bacterial strains, and single-cell adhesion was measured on three surfaces (fresh glass, hydrophilic glass, and mica). Attachment to the cantilever was stable during the AFM force measurements that were conducted for 2 h, and viability was confirmed by Live/Dead fluorescence staining at the end of each experiment. The adhesion force and final rupture length were dependent on bacterial strains, surfaces properties, and contact time. The single-cell probe offers control of cell immobilization and thus holds advantages over the commonly used multicell probes with which random immobilization is obtained by submerging the cantilever in a bacterial suspension. The reported method provides a general platform for investigating single-cell interactions of bacteria with different surfaces and other cells by AFM force spectroscopy, thus improving our understanding of the mechanisms of bacterial attachment. more...

The current knowledge of in vitro adherence of Staphylococcus pseudintermedius to canine corneocytes is limited to comparative analyses between strains, staphylococcal species or corneocytes collected from different breeds, body sites and hosts. However, the role played by colonization status of corneocyte donors remains unknown. The aim of this study was to evaluate the adherence properties of commensal S. pseudintermedius strains to corneocytes collected from dogs with different colonization status. For this purpose, corneocytes were collected from five dogs that were classified as persistently colonized (D1 and D2), intermittently colonized (D3 and D4) or non-colonized (D5) on the basis of the results of a previous longitudinal study. Adherence to corneocytes originating from each of the five dogs was assessed by an in vitro adhesion assay using four genetically unrelated strains isolated from the colonized dogs (S1 to S4). Irrespective of their host of origin, all strains adhered significantly better to corneocytes from D1 and D2 than to corneocytes from D3, D4 and D5 (P<0.0001). The mean count of cells adhering to corneocytes from persistently colonized dogs was on average three times higher than the mean count using corneocytes from the other dogs. A significant difference between strains was only observed for one strain-corneocyte combination (S2-D4), indicating that S. pseudintermedius adherence to corneocytes is driven by host factors and only marginally influenced by strain factors. This finding has important implications for understanding and preventing S. pseudintermedius skin colonization and infection. more...

We have previously generated an affibody molecule for the disease-associated amyloid beta (Aβ) peptide, which has been shown to inhibit the formation of various Aβ aggregates and revert the neurotoxicity of Aβ in a fruit fly model of Alzheimer's disease. In this study, we have investigated a new bacterial display system for combinatorial protein engineering of the Aβ-binder as a head-to-tail dimeric construct for future optimization efforts, e.g. affinity maturation. Using the bacterial display platform, we have: (i) demonstrated functional expression of the dimeric binder on the cell surface, (ii) determined the affinity and investigated the pH sensitivity of the interaction, (iii) demonstrated the importance of an intramolecular disulfide bond through selections from a cell-displayed combinatorial library, as well as (iv) investigated the effects from rational truncation of the N-terminal part of the affibody molecule on surface expression level and Aβ binding. Overall, the detailed engineering and characterization of this promising Aβ-specific affibody molecule have yielded valuable insights concerning its unusual binding mechanism. The results also demonstrated that our bacterial display system is a suitable technology for future protein engineering and characterization efforts of homo- or heterodimeric affinity proteins., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.) more...

A set of Cu-Mn-O and Ag-Cu-Mn-O films were sputter-deposited onto polished Ti-6Al-4V coupons and the microbiological adherence of Staphylococcus sp. was studied in these biomedical surfaces modified by using advanced ternary and quaternary oxides, these latter incorporated micrometric silver islands. Silver is known to have a natural biocidal character and its presence in the surface of Ti-6Al-4V forming large micrometric islands. In principle, predicted to enhance the antimicrobial properties of biomedical surfaces. Microbial adhesion tests were performed using collection strains and six clinical Staphylococcus aureus and Staphylococcus epidermidis strains. The adherence study was performed using a previously published protocol by Kinnari et al. Collection strains and clinical strains showed decreased adherence to modified materials; however, only on the clinical strains were there statistically significant differences between Cu-Mn-O and Ag-Cu-Mn-O containing silver islands. Nanocrystalline silver dissolves and releases both Ag(+) and Ag(0) whereas other silver sources release only Ag+. We can conclude that nanocrystalline silver coating, confirmed by XRD, appears to alter the biological properties of the solution, particularly antimicrobial activity., (Copyright © 2012 Wiley Periodicals, Inc.) more...

The Gram-positive bacterium Staphylococcus pseudintermedius is regarded as the major cause of canine bacterial pyoderma. Despite its clinical importance, there is only very limited knowledge about the pathogenesis of S. pseudintermedius infection and the specific bacterial virulence factors involved in causing disease. Using a whole-genome approach, we have previously identified 18 predicted cell-wall-anchored surface proteins representing possible virulence factors in a clinical isolate of S. pseudintermedius (strain ED99). They were designated S. pseudintermedius surface proteins A-R (SpsA-SpsR). The present study tested three of the putative Sps proteins (SpsD, SpsL and SpsO) for their ability to mediate adherence of bacteria to canine corneocytes. The three proteins were expressed on the surface of the nonpathogenic surrogate host Lactococcus lactis, a Gram-positive bacterium that does not adhere to canine corneocytes. Adherence assays were performed using corneocytes from different healthy canine donors (n = 5), and bacterial cells were quantified using computerized image analysis. Two of the proteins, SpsD and SpsO, mediated adherence of L. lactis to canine corneocytes, suggesting that they contribute to S. pseudintermedius pathogenesis and may represent novel therapeutic targets to combat infection., (© 2011 The Authors. Veterinary Dermatology. © 2011 ESVD and ACVD.) more...

The aim of this study was the determination of bacteria present in maxillary and ethmoid cavities in patients with chronic sinusitis and to correlate these findings with bacteria simultaneously present in their nasopharynx. The purpose of this correlation was to establish the role of bacteria found in chronically inflamed sinuses and to evaluate if the bacteria present colonized or infected sinus mucosa. Nasopharyngeal and sinus swabs of 65 patients that underwent functional endoscopic sinus surgery were cultivated and at the same time the presence of leukocytes were determined in each swab. The most frequently found bacteria in nasopharynx were Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus spp., Streptococcus viridans and Streptococcus pneumoniae. Maxillary or ethmoidal sinus swabs yielded bacterial growth in 47 (72.31%) patients. The most frequently found bacteria in sinuses were Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella spp. and Streptococci (pneumoniae, viridans and spp.). The insignificant number of leukocytes was present in each sinus and nasopharyngeal swab. Every published microbiology study of chronic sinusitis proved that sinus mucosa were colonized with bacteria and not infected, yet antibiotic therapy was discussed making no difference between infection and colonization. Chronic sinusitis should be considered a chronic inflammatory condition rather than bacterial infection, so routine antibiotic therapy should be avoided. Empiric antibiotic therapy should be prescribed only in cases when the acute exacerbation of chronic sinusitis occurs and the antibiotics prescribed should aim the usual bacteria causing acute sinusitis. In case of therapy failure, antibiotics should be changed having in mind that under certain circumstances any bacteria colonizing sinus mucosa can cause acute exacerbation of chronic sinusitis. more...

Biomaterial-associated infections (BAI) remain a serious clinical complication, often arising from an inability of host tissue-implant integration to out-compete bacterial adhesion and growth. A commercial polymer coating based on polyethylene glycol (PEG), available in both chemically inert and NHS-activated forms (OptiChem(®)), was compared for simultaneous growth of staphylococci and osteoblasts. In the absence of staphylococci, osteoblasts adhered and proliferated well on glass controls and on the NHS-reactive PEG-based coating over 48 h, but not on the inert PEG coating. Staphylococcal growth was low on both PEG-based coatings. When staphylococci were pre-adhered on surfaces for 1.5 h to mimic peri-operative contamination, osteoblast growth and spreading was reduced on glass but virtually absent on both reactive and inert PEG-based coatings. Thus although NHS-reactive, PEG-based coatings stimulated tissue-cell interactions in the absence of contaminating staphylococci, the presence of adhering staphylococci eliminated osteoblast adhesion advantages on the PEG surface. This study demonstrates the importance of using bacterial and cellular co-cultures compared to monocultures when assessing functionalized biomaterials coatings for infectious potential., (Copyright © 2010 Elsevier Ltd. All rights reserved.) more...

Staphylococcus is the most prevalent pathogen causing bacteremia and many of its isolates possess the ability to form biofilm. In this study Staphylococcus isolates from the blood of patients with bacteremia were analyzed by two biofilm detection phenotypic methods: Congo red agar (CRA) and microtiter-plate adherence (MPA) in relation to the presence of ica genes, detected by PCR. Their oxacillin susceptibility was also evaluated. Among 127 isolates evaluated, 47 were S. aureus and 80 were coagulase negative staphylococci (CNS). Seventy-four (58.3%) isolates were mecA gene positive (27.7%S. aureus and 76.3% CNS isolates). Among the 40 S. aureus isolates which were positive for the ica genes, 25 (62.5%) were positive in MPA and 27 (67.5%) in CRA, whereas both methods combined detected 34 (85%) isolates as biofilm producers. Among 12 S. epidermidis isolates carrying ica genes, 8 were positive in MPA and 5 in CRA. The combination of CRA and MPA methods provided a better prediction of the presence of ica genes in S. aureus isolates than did either method alone., (© 2010 The Societies and Blackwell Publishing Asia Pty Ltd.) more...

TiO(2)-coated biomaterials that have been excited with UV irradiation have demonstrated biocidal properties in environmental applications, including drinking water decontamination. However, this procedure has not been successfully applied towards the killing of pathogens on medical titanium-based implants, mainly because of practical concerns related to irradiating the inserted biomaterial in situ. Previous researchers assumed that the photocatalysis on the TiO(2) surface during UV application causes the bactericidal effects. However, we show that a residual post-irradiation bactericidal effect exists on the surface of Ti6Al4V, not related with photocatalysis. Using a combination of staining, serial dilutions, and a biofilm assay, we show a significant and time-dependent loss in viability of different bacterial strains of Staphylococcus epidermidis and Staphylococcus aureus on the post-irradiated surface. Although the duration of this antimicrobial effect depends on the strains selected, our experiments suggest that the effect lasts at least 60 min after surface irradiation. The origin of such phenomena is discussed in terms of the physical properties of the irradiated surfaces, which include the emission of energy and changes in surfaces charge occurring during electron-hole recombination processes. The method here proposed for the preparation of antimicrobial titanium surfaces could become especially useful in total implant surgery for which the antimicrobial challenge is mainly during or shortly after surgery., (Copyright 2010 Elsevier Ltd. All rights reserved.) more...

Aims: To produce surfaces of defined linear topographical features which reflect those found on worn and new stainless steel, to monitor the effect of feature dimensions on the retention of Listeria monocytogenes and Staphylococcus sciuri., Methods and Results: Surfaces were fabricated with parallel linear features of 30 microns or of microbial dimensions (1.02 and 0.59 microm width) and used in microbial retention assays with Staph. sciuri and L. monocytogenes. Retained cells were distributed uniformly across the smooth 30 micron featured surfaces but were retained in high numbers on microtopographies at the 'peaks' between the wide grooves. On smaller features, retention was attributed to the maximum area of contact between cells and substratum being attained, with cocci being embedded in 1.02-microm-width grooves, and rods aligned along (and across) the densely packed parallel 0.59-microm grooves., Conclusions: The dimensions of surface features may enhance or impede cell retention. This phenomenon is also related to the size and shape of the microbial cell., Significance and Impact of Study: Findings may help describe and evaluate properties of hygienic and easily cleanable surfaces. more...

Staphylococcus sp. strain BP/SU1, capable of degrading the biopolymer and utilize it as a source of carbon and energy, was isolated from activated sludge using METABOLIX (MBX D411G). It was found that this strain was capable of accumulating poly(3-hydroxybutyric acid) P(3-HB), as granule poly (3-hydroxybutyric acid), p(3-HB), inclusion bodies when grown under suitable nutrient conditions. These strains could sustain cell growth up to a dry mass of 9.24 g/l with a doubling time of 8 to 10 hr and could accumulate P(3-HB) as granular inclusion bodies to a cell dry weight of more than 12%. P(3-HB) accumulated by this organism was isolated and characterized through NMR, FT-IR spectroscopy, UV Spectroscopy, Mass spectroscopy and Differential Scanning Calorimetry. P(3-HB) granules so isolated showed physical and chemical properties that should be possessed by a superior quality thermoplastic biopolymer. more...

Staphylococcus epidermidis adheres to hydrophilic glass and hydrophobic dimethyldichlorosilane (DDS)-coated glass in similar numbers, but in different modes. Real-time observation of staphylococcal adhesion under a shear rate of 15 s(-1) revealed different adhesion dynamics on both substrata. The number of adsorption and desorption events to achieve a similar number of adhering bacteria was twofold higher on hydrophilic than on hydrophobic DDS-coated glass. Moreover, 22% of all staphylococci on glass slid over the surface prior to adhering on a fixed site ("mobile adhesion mode"), but mobile adhesion was virtually absent (1%) on DDS-coated glass. Sliding preceded desorption on hydrophilic glass in about 20% of all desorption events, while on hydrophobic DDS-coated glass 2% of all staphylococci desorbed straight from their adhesion site. Since acid-base interactions between the staphylococci and a hydrophobic DDS-coating are attractive, it is suggested that these interactions facilitate a closer approach of the bacteria and therewith enhance immobile adhesion at local, high affinity sites. Alternatively, if the local site is low affinity, this may lead to desorption. In the absence of attractive acid-base interactions, as on hydrophilic glass, bacteria can be captured in the minimum of the DLVO-interaction energy curve, but this does not prevent them from sliding under flow at a fixed distance from a substratum surface until immobilization or desorption at or from a local high or low affinity site, respectively. more...

The aim of this work was to identify the initial binding sites to the bacterial membranes of the antimicrobial peptide alphas2-casein f(183-207) and also to acquire further insight into membrane permeabilization of this peptide. Furthermore, cell morphology was studied by transmission electron microscopy. In all the experiments, bovine LFcin was employed as a comparison. Results showed that initial binding sites of alphas2-casein f(183-207) peptide were lipoteichoic acid in Gram-positive bacteria and lipopolysaccharide in Gram-negative. The peptide was able to permeabilize the outer and inner membranes. Moreover, the alphas2-casein peptide f(183-207) generated pores in the outer membrane of Gram-negative bacteria and in the cell wall of Gram-positive bacteria. In the Gram-negative bacteria, f(183-207) originated cytoplasm condensation, and in the Gram-positive bacteria the cytoplasmic content leaked into the extracellular medium. Furthermore, the experiments of inner and outer membrane permeabilization performed with LFcin-B showed that this peptide also has the ability to permeabilize both the inner and outer membranes. more...

The lichen species namely Usnea ghattensis, Heterodermia podocarpa, Arthothelium awasthii and Parmotrema tinctorum have been cultured in vitro and were screened for their antioxidant and antibacterial potential using different assay systems. The methanol extract of lichens showed antioxidant and antibacterial activities according to the order U. ghattensis>A. awasthii>H. podocarpa>P. tinctorum. The IC(50) values for the antioxidant activities of U. ghattensis and A. awasthii are less or equivalent to that of standard antioxidants. The methanolic extracts of the mycobiont and photobiont cultures of lichenU. ghattensis and A. awasthii were effective against Bacillus licheniformis, Bacillus megaterium, Bacillus subtilis and Staphylococcus aureus. The minimum inhibitory concentration (MIC) of the extract was found between 5 and 10microg extract/ml. The results suggested that the extract of mycobiont and photobiont cultures of lichen U. ghattensis and A. awasthii could be of use as an easily accessible source of natural antioxidants and antibacterial properties for the possible food supplement or in the pharmaceutical industry. more...

Background: Our aim was to determine the antibiotic susceptibility of the preoperative conjunctival bacterial flora against 25 commonly used antibiotics, especially the new fluoroquinolones levofloxacin, gatifloxacin, and moxifloxacin., Patients and Methods: The Kirby-Bauer disk-diffusion technique was used to test for the in vitro antibiotic susceptibility of conjunctival bacterial strains isolated from 160 patients (median=74 years, mean=71 years) undergoing cataract surgery at the Department of Ophthalmology, Stanford University, CA, USA., Results: Among the 256 bacteria isolated, 201 (79%) were coagulase-negative staphylococci (CNS), 26 Staphylococcus aureus, 15 Streptococcus group D and 14 gram-negative rods. A total of 100 of these 256 strains (39%) were classified as multiresitant (resistant to>or=five antibiotics). The resistance rate (RR) of commonly used antibiotics for all CNS was: gatifloxacin=moxifloxacin more...

Antimicrobial peptides and proteins are being studied with increasing interest because of their broad range antimicrobial activity. Among plant antimicrobial proteins, the wheat seed polypeptides, puroindoline a and puroindoline b, are particularly interesting because of their established antibacterial activity. In this paper we describe different strategies used to clone His tagged and GST tagged puroindolines obtaining 1.5 mg recombinant protein from 1 l culture. The antimicrobial activity of recombinant and native puroindolines was comparable. more...

Biodegradable polyurethanes have potential for use as implantable devices (orthopedic, maxillofacial, cardiovascular, wound dressing and plastic surgery) because of their controllable elasticity, and the possibility of changing their chemistry and structure. Studying bacterial and cell adhesion to polyurethanes helps to determine surface cytocompatibility and suitability for in vivo trials. Staphylococcus aureus, Staphylococcus epidermidis and hTERT human fibroblast cells were used to determine the cytocompatibility of experimental biodegradable polyurethanes (PUs) with different hydrophobic-to-hydrophilic (pho:phi) content ratios (100% pho, 70% pho, and 30% pho). Poly(L/DL-lactide) 70/30% (PLDL) and Thermanox were used as control surfaces. Surface characterization using noncontact profilometry, contact angles, and scanning electron microscopy (SEM) showed that the three PU surfaces, PLDL, and Thermanox have different properties. On the 100% PU and 30% PU surfaces, hTERT cells spread less in comparison to the 70% PU, PLDL, and Thermanox surfaces. The adsorption of fibronectin to the surfaces had no effect on the adhesion and spreading of hTERT cells when compared to the uncoated surfaces. The trend for S. aureus was the most adhered on the 70% PU and 30% PU, then Thermanox, followed by 100% PU and PLDL, respectively. The amount of S. epidermidis adhesion followed the trend of the most on 70% PU, then 100% PU, then 30% PU and PLDL, and the least on Thermanox. These results suggest that the 70% PU surface is cytocompatible to hTERT fibroblasts, while the 100% PU and 30% PU were not. All surfaces encouraged S. aureus and S. epidermidis colonization, particularly the 70% PU., (Copyright (c) 2006 Wiley Periodicals, Inc.) more...

Infections with multiresistant bacteria have become a serious problem in joint arthroplasty. This study reports about in vitro antibacterial activity against multiresistant bacteria and in vitro cytotoxicity of polymethylmetacrylate bone cement loaded with metallic silver particles with a size of 5-50 nm called NanoSilver. In vitro antibacterial activity against S. epidermidis, methicillin-resistant S. epidermidis (MRSE), and methicillin-resistant S. aureus (MRSA) was studied by microplate proliferation tests. Quantitative elution testing and qualitative ongrowth of human osteoblasts was done to study in vitro cytotoxicity. Only NanoSilver cement showed high-antibacterial activity against all strains, including MRSE and MRSA. Gentamicin cement was not effective against MRSA and MRSE due to the high-level gentamicin resistance of the tested strains. Plain cement did not inhibit proliferation of any strains. There was no significant difference regarding in vitro cytotoxicity between NanoSilver and the non-toxic control. Cytotoxicity of cement loaded with silver salts made this kind of silver unsuitable for all day clinical use in the past. This new form of silver called NanoSilver was free of in vitro cytotoxicity and showed high effectiveness against multiresistant bacteria. If the results can be confirmed in vivo NanoSilver may have a high interest in joint arthroplasty. more...

The rate of lysostaphin-catalyzed lysis of staphylococci follows the Michaelis-Menten equation at [E](0) << [S](0), i.e., the activity of the enzyme is proportional to its concentration. This equation is proposed for determining the specific activity of lysostaphin. The apparent activation energy of hydrolysis of pentaglycine bridges in Staphylococcus peptidoglycan is 77.9 kJ/mol. more...

Staphylococcus aureus is a major pathogen, associated with medical-device related infections. Converting biomaterial surfaces into non-interactive surfaces requires a specific surface/interface design. One approach is to polish the surface, and a second is to coat the surface with an antimicrobial or protein resistant coating. This study showed that polishing a titanium surface or coating titanium with various treatments that decreased the surface's coefficient of friction, had no significant effect on minimising S. aureus adhesion to these surfaces under static conditions in comparison to standard medical grade titanium. The cell promoting coating, TAST, was found to increase the S. aureus density on its surface as expected. The only coating that significantly decreased the density of adhering S. aureus was the titanium surface coated with sodium hyaluronate. Thus such a coating could have potential use as a coating for ostoesynthesis, orthopaedic or dental implants. more...

The application of NIR in-line to monitor and control fermentation processes was investigated. Determination of biomass, glucose, and lactic and acetic acids during fermentations of Staphylococcus xylosus ES13 was performed by an interactance fiber optic probe immersed into the culture broth and connected to a NIR instrument. Partial least squares regression (PLSR) calibration models of second derivative NIR spectra in the 700-1800 nm region gave satisfactory predictive models for all parameters of interest: biomass, glucose, and lactic and acetic acids. Batch, repeated batch, and continuous fermentations were monitored and automatically controlled by interfacing the NIR to the bioreactor control unit. The high frequency of data collection permitted an accurate study of the kinetics, supplying lots of data that describe the cultural broth composition and strengthen statistical analysis. Comparison of spectra collected throughout fermentation runs of S. xylosus ES13, Lactobacillus fermentum ES15, and Streptococcus thermophylus ES17 demonstrated the successful extension of a unique calibration model, developed for S. xylosus ES13, to other strains that were differently shaped but growing in the same medium and fermentation conditions. NIR in-line was so versatile as to measure several biochemical parameters of different bacteria by means of slightly adapted models, avoiding a separate calibration for each strain. more...

We investigated the role of blood components in the adhesion of staphylococci to biological and artificial surfaces under well-defined flow conditions by using the Cone and Plate(let) Analyzer. An enzyme-linked immunosorbent assay-like binding assay with biotinylated bacteria determined the extent of bacterial adhesion to subendothelial extracellular matrix (ECM), polystyrene (PS) and adult bovine aortic endothelial (ABAE) cell monolayer. The presence of adsorbed plasma proteins on PS and ECM did not increase and in some cases reduced staphylococcal adhesion under flow conditions (200s(-1)). However, their presence on ABAE cells increased bacterial adhesion but to a level still lower than the adhesion to PS and ECM. In contrast, adhered platelets significantly increased staphylococcal adhesion to both PS and ECM, but did not affect the adhesion to ABAE cells. Furthermore, bacterial adhesion to the platelets coated ECM and PS under flow conditions (200s(-1)) was increased by 1.4 to 2.6-fold compare to static conditions. The platelet-enhanced bacterial adhesion was markedly inhibited by blockade of the platelet GPIb receptor. In conclusion, staphylococcal extensive adhesion to ECM and PS surfaces is increased by venous flow and mediated by surface adhered activated platelets via a GPIb dependent mechanism. On the other hand, ABAE cells demonstrated limited bacterial adhesion that is mediated by adsorbed plasma proteins. Our results suggest that under physiological venous flow conditions the intact vessel wall is less prone for bacterial adhesion than damaged vessel wall. more...

Background: Bacterial infections occur frequently on the skin of atopic dermatitis (AD) patients. The objectives of this study were to evaluate the microbiology of the skin of AD patients for staphylococci, the frequency and density of each species, and their susceptibility to antimicrobial drugs., Methods: To study the staphylococci present on the skin of 21 AD outpatients and of 12 healthy subjects (HS), cutaneous organisms were obtained using the contact-plate method., Results: Staphylococcus aureus was isolated in 85.7% of AD patients (mild type, 77.8%; moderate type, 87.8%; and severe type, 100%) and in 25% of HS, while Staphylococcus epidermidis was isolated in 83.3% of HS and in 38.1% of AD patients. Among the coagulase-negative staphylococci (CNS) identified, S. epidermidis was the common type and several other CNS were detected in both AD patients and HS. As the eruption grade of dermatitic skin became more severe, the average density of S. aureus increased (severe, 2.68 +/- 0.86; moderate, 2.49 +/- 0.48; mild, 2.28 +/- 0.44). A reversed tendency was seen in S. epidermidis (severe, 1.80; moderate, 1.90; mild, 2.10). Among nine antimicrobial drugs tested against S. aureus, S. epidermidis, and some other types of CNS isolates, vancomycin (VCM) and minocycline (MINO) were the most active, gentamycin (GM) was the less active, and ampicillin (ABPC) was the least active., Conclusions: The skin of AD patients was more frequently colonized with S. aureus than that of normal controls. As the severity of the AD lesions increased, the numbers of S. aureus isolated increased. The skin of HS was more colonized with S. epidermidis. Other species of CNS were isolated from several cases of AD patients and HS. In addition, S. aureus, S. epidermidis, and the other CNS showed poor susceptibility to some of the tested antimicrobial drugs. more...

In accordance with Recommendation 30b of the International Code of Nomenclature of Bacteria, minimal standards are proposed for the genus Staphylococcus and the description of newly recognized species in this genus. Assignment of a strain to the genus Staphylococcus requires that it is a Gram-positive coccus that forms clusters, produces catalase, has an appropriate cell wall structure (including peptidoglycan type and teichoic acid presence) and G + C content of DNA in a range of 30-40 mol%. The recommended minimal standards for describing a new Staphylococcus species are based on the results of phenotypic and genomic studies of at least five independently isolated strains. They include colony morphology and the results of the following conventional tests: pigment production, growth requirements, fermentative and oxidative activity on carbohydrates, novobiocin susceptibility, enzymic activities (nitrate reductase, alkaline phosphatase, arginine dihydrolase, ornithine decarboxylase, urease, cytochrome oxidase, staphylocoagulase in rabbit plasma, heat-stable nuclease, amidases, oxidases, clumping factor, and haemolytic activity on sheep or bovine blood agar). DNA-DNA hybridization experiments may distinguish species when the difference between the binding in the homologous reaction and the binding in the heterologous reaction expressed as a percentage is less than 70%. In addition, rRNA signature sequence criteria, ribotyping characterization of the nomenclature type strain and other strains of the species, and reference strains of other species is recommended to describe the strains of the new species with sets of genetic attributes and reveal possible grouping errors. This proposal has been endorsed by the members of the Subcommittee on the taxonomy of staphylococci and streptococci of the international Committee on Systematic Bacteriology. more...

Of 41 methicillin-resistant coagulase-negative staphylococcal clinical isolates collected during a 5-month period between late 1995 and early 1996, 28 showed tube dilution teicoplanin MICs of 4 to 8 microg/ml which increased to 16 to 32 microg/ml upon prolonged incubation. Cultures of such bacteria were heterogeneous; they contained subpopulations with frequencies of 10(-5) to 10(-4) that could grow on up to 50 microg of teicoplanin per ml. The same cultures were also heterogeneous with respect to susceptibility to vancomycin; while the MICs for the majority of cells were 2 to 4 microg/ml, subpopulations that could grow on 6 to 12 microg of vancomycin per ml were also present at frequencies of 10(-5) to 10(-7). Selective enrichment of such cultures for the resistant subpopulation occurred with relative ease under laboratory conditions. Heterogeneous phenotypes for teicoplanin (but not for vancomycin) susceptibility were also identified in several Staphylococcus epidermidis isolates collected during the preantibiotic era. The addition of half the MIC of teicoplanin inhibited autolysis and caused formation of cellular aggregates which disintegrated to individual bacteria in the stationary phase when the titer of teicoplanin in the medium fell to undetectable levels, indicating removal of the antibiotic from the culture medium by the bacteria. more...

Infections of cerebrospinal fluid (CSF) shunts constitute a serious clinical problem. The role of adhesion by coagulase negative staphylococci, the most common etiological agent, was examined in vitro to polyvinyl chloride (PVC), silicone, and to PVC and silicone with end-point attached (EPA) heparin. These are flexible materials commonly used in neurosurgical implants. Bacterial adhesion was quantitated by bioluminescence. The bacterial adhesion to biomaterial surfaces increased with increasing concentrations of bacterial cells. Scatchard plot analysis showed continuous negative (concave) slopes, indicating multiple interactions between biomaterial and bacteria. The thermodynamic studies showed a positive value of the standard entropy change at 37 degrees C, which indicates that hydrophobic interactions are important in bacterial adhesion to polymers. Incubation with CSF for 1 h decreased bacterial adhesion in 75% of the samples compared to incubation in buffer. Thus, the contribution of CSF proteins, like fibronectin, for the initial bacterial adhesion might be small. Heparinization of silicone and PVC decreased the numbers of adhered bacteria by 23 to 54% and 0 to 43% compared to unheparinized surfaces. Among putative inhibitors tested, suramin, chondroitin sulfate, and fucoidan inhibited adhesion to 81 +/- 19, 78 +/- 22, and 64 +/- 7%, respectively. These findings indicate that hydrophobic interactions play an important role, and heparinization rendering the biomaterial surface hydrophilic is therefore effective to reduce bacterial adhesion. Heparinized polymers incubated with putative inhibitors may be the optimal way to prevent shunt infections. more...

Multicellular cells were efficiently induced in Staphylococcus haemolyticus by the addition of protease to exponentially growing cultures at 30 C. Electron microscopy revealed the formation of tetrad-shaped multicells that were septated but not separated from each other. Incubation of the multicells with extract from the cells grown without protease resulted in a fourfold increase in the number of colony-forming units as compared with the untreated control. An electrophoretic analysis showed that protease caused a loss of cell wall-lytic activity of the cell, which possibly led to the formation of multicells through cessation of cross wall separation. more...

Staphylococcus saprophyticus isolates (n = 10) from urinary tract infections (UTIs) could be reproducibly distinguished from S. saprophyticus strains from respiratory tract infections (n = 5) by distinct electrophoretic mobility. Thus, the method of "bacteriopheresis" may offer a new approach to discriminate pathogenic microbial strains of different origin with respect to defined surface structures (hemagglutinins, lectins), generating distinct surface charges. more...

A new subspecies, Staphylococcus capitis subsp. ureolyticus, was isolated from human skin and is described on the basis of studies of 15 to 26 strains. DNA-DNA reassociation reactions demonstrated that these strains were closely related to Staphylococcus capitis but were significantly divergent. The strains of S. capitis subsp. ureolyticus can be distinguished from S. capitis by their positive urease activity, their ability to produce acid from maltose under aerobic conditions, their fatty acid profile, and their colony morphology. The type strain of the new subspecies is strain ATCC 49326. more...

The effect of highly purified staphylococcal alpha-toxin on HEp-2 neoplastic cells growing in culture was studied by electron microscopy. In addition to some nuclear modifications, the most significant morphological changes were in mitochondrial structure, alpha-toxin seems to induce a defined sequence of changes in mitochondrial configuration which can be summarized as an earlier phase of 'condensation' of mitochondrial structure and a later phase of mitochondrial swelling with an intermediate 'transitional' configuration. The significance of these findings is discussed in the light of the present state of knowledge of the biological characteristics of staphylococcal alpha-toxin. more...

The hydrophobicity-hydrophilicity of various strains of Staphylococcus has been studied by a technique involving partitioning of the cells between aqueous and hydrocarbon phases. S. aureus was typically hydrophobic, and to a greater degree in stationary- than in exponential-phase cultures. Mutants that lacked teichoic acid, protein A or coagulase production were hydrophobic, indicating that none of these factors was responsible for hydrophobicity. The presence of a capsule rendered strains hydrophilic. Thus, determination of hydrophobicity may be a useful additional test for capsulation. However, a non-capsulate S. aureus strain was hydrophilic. Trypsin treatment converted strains from hydrophobic to hydrophilic. Isolated bacterial cell wall preparation, either crude or purified, and peptidoglycan were hydrophilic. These results indicate that the determinant of hydrophobicity is a protein or protein-associated molecule localised at the cell surface of the organism, i.e., a component of either the cell wall, cell membrane, or both. Twenty-five strains of twelve coagulase-negative species were examined and most (18) were hydrophobic, again indicating that protein A is not a major determinant of hydrophobicity in these staphylococci. Four of seven hydrophilic strains were capsulate; three strains of S. sciuri were hydrophilic but non-capsulate. more...

The laboratory conditions for reproducible routine determination of staphylococcal cell-surface hydrophobicity by the salt aggregation test were standardized. Fresh bacterial suspensions standardized to 5 x 10(9) cfu/ml gave the most reproducible results with both Staphylococcus aureus and coagulase-negative staphylococci. For relatively hydrophobic strains a 5-min reading time was necessary to detect bacterial aggregation in ammonium sulphate solutions ranging from 0.1 M to 1.5 M, pH 6.8. A x 10 hand lens facilitated reading aggregations. Overnight storage of bacterial suspensions at 20 degrees C reduced cell-surface hydrophobicity of all species, while storage at 4 degrees C reduced the hydrophobic nature of Staph. aureus strains. The hydrophobicity of coagulase-negative staphylococci rarely changed at 4 degrees C. A 10-fold dilution of fresh, standardized bacterial suspensions made it impossible to detect bacterial aggregation in ammonium sulphate solutions even with a hand lens. Under standardized conditions three types of staphylococcal cell aggregations were observed. The first looked like the slide agglutination for O antigens of Enterobacteriaceae, the second resembled H-agglutination, while the third had a filamentous appearance. These patterns indicated that more than one component might contribute to cell-surface hydrophobicity of both Staph. aureus and coagulase-negative staphylococci, or the same component might have different position on the cell surface. more...

The process of cell separation and arrangement of Staphylococcus was investigated using a scanning electron microscope. After two cycles of cell division, the Staphylococcal cells cultured on an agar medium were generally observed to be arranged in three morphological types: linear, square, and crooked arrangements. Results of the examination of cell surface structure revealed that separations had occurred in these clustered cells following two patterns. One type of second separation occurred parallel to the transversal axis of the preceding pair of the parental cells (X-type) and the other occurred tangential to it (Y-type). In the former type, the four daughter cells were usually arranged tetragonally after the separations, and in the latter type they were arranged either linearly or crookedly depending on the direction of the second separation. The final pattern of the cell arrangement was thus determined by the type of septal wall formation and the direction of cell separation. After several cycles of cell divisions, the cells were finally arranged in an irregular grape-like cluster, even though the cross walls were formed regularly at the rectangular face of the preceding cross walls. more...

The adherence of coagulase-negative staphylococci to smooth surfaces was assayed by measuring the optical densities of stained bacterial films adherent to the floors of plastic tissue culture plates. The optical densities correlated with the weight of the adherent bacterial film (r = 0.906; P less than 0.01). The measurements also agreed with visual assessments of bacterial adherence to culture tubes, microtiter plates, and tissue culture plates. Selected clinical strains were passed through a mouse model for foreign body infections and a rat model for catheter-induced endocarditis. The adherence measurements of animal passed strains remained the same as those of the laboratory-maintained parent strain. Spectrophotometric classification of coagulase-negative staphylococci into nonadherent and adherent categories according to these measurements had a sensitivity, specificity, and accuracy of 90.6, 80.8, and 88.4%, respectively. We examined a previously described collection of 127 strains of coagulase-negative staphylococci isolated from an outbreak of intravascular catheter-associated sepsis; strains associated with sepsis were more adherent than blood culture contaminants and cutaneous strains (P less than 0.001). We also examined a collection of 84 strains isolated from pediatric patients with cerebrospinal fluid (CSF) shunts; once again, pathogenic strains were more adherent than were CSF contaminants (P less than 0.01). Finally, we measured the adherence of seven endocarditis strains. As opposed to strains associated with intravascular catheters and CSF shunts, endocarditis strains were less adherent than were saprophytic strains of coagulase-negative staphylococci. The optical densities of bacterial films adherent to plastic tissue culture plates serve as a quantitative model for the study of the adherence of coagulase-negative staphylococci to medical devices, a process which may be important in the pathogenesis of foreign body infections. more...

Certain antibiotic compounds including penicillin, cloxacillin and 6-APA caused a prolonged survival of Staph. aureus cells undergoing autolysis in physiologic saline. The retardation of the death rate was dependent upon the type of compound and its concentration. The results suggest that the compounds inhibit the autolytic function of the bacteria. more...

Small-cell formation of Staphylococcus aureus by subinhibitory concentration of nitrofuran derivatives was examined by scanning and transparent electron microscopy.