Listeria monocytogenes wall teichoic acid decoration in virulence and cell-to-cell spread.

Wall teichoic acid (WTA) comprises a class of glycopolymers covalently attached to the peptidoglycan of gram positive bacteria. In Listeria monocytogenes, mutations that prevent addition of certain WTA decorating sugars are attenuating. However, the steps required for decoration and the pathogenic process interrupted are not well described. We systematically examined the requirement for WTA galactosylation in a mouse oral-virulent strain by first creating mutations in four genes whose products conferred resistance to a WTA-binding bacteriophage. WTA biochemical and structural studies indicated that galactosylated WTA was directly required for bacteriophage adsorption and that mutant WTA lacked appreciable galactose in all except one mutant - which retained a level ca. 7% of the parent. All mutants were profoundly attenuated in orally infected mice and were impaired in cell-to-cell spread in vitro. Confocal microscopy of cytosolic mutants revealed that all expressed ActA on their cell surface and formed actin tails with a frequency similar to the parent. However, the mutant tails were significantly shorter - suggesting a defect in actin based motility. Roles for the gene products in WTA galactosylation are proposed. Identification and interruption of WTA decoration pathways may provide a general strategy to discover non-antibiotic therapeutics for gram positive infections. © 2016 John Wiley & Sons Ltd.

Antibacterial efficacy and cytotoxicity of low intensity direct current activated silver-titanium implant system prototype.

Silver-based devices activated by electric current are of interest in biomedicine because of their broad-spectrum antimicrobial activity. This study investigates the in vitro antibacterial efficacy and cytotoxicity of a low intensity direct current (LIDC)-activated silver-titanium implant system prototype designed for localized generation and delivery of silver ions at the implantation site. First, the antibacterial efficacy of the system was assessed against methicillin-resistant Staphylococcus aureus (MRSA) over 48 h at current levels of 3 and 6 µA in Mueller-Hinton broth. The cytotoxicity of the system was then evaluated over 48 h in two phases using an in vitro model with in which the activated electrodes were suspended in growth medium in a cell-seeded tissue culture plate. In phase-1, the system was tested on human osteosarcoma (MG-63) cell line and compared to titanium controls. In phase-2, the cytotoxicity characteristics were validated with normal human diploid osteoblast cells. The LIDC-activated system demonstrated high antimicrobial efficacy against MRSA, but was also toxic to human cells immediately surrounding the electrodes. The statistical analysis showed that the cytotoxicity was a result of the presence of silver, and the electric activation did not make it worse.

Use of bacteriophage to target bacterial surface structures required for virulence: a systematic search for antibiotic alternatives.

Bacteriophages (phage) that infect pathogenic bacteria often attach to surface receptors that are coincidentally required for virulence. Receptor loss or modification through mutation renders mutants both attenuated and phage resistant. Such attenuated mutants frequently have no apparent laboratory growth defects, but in the host, they fail to exhibit properties needed to produce disease such as mucosal colonization or survival within professional phagocytic cells. The connection between attenuation and phage resistance has been exploited in experimental demonstrations of phage therapy. In such experiments, phage resistant mutants that arise naturally during therapy are inconsequential because of their attenuated status. A more contemporary approach to exploiting this connection involves identifying small effector molecules, identified in high-throughput screens, that inhibit one or more of the steps needed to produce a functioning phage receptor. Since such biosynthetic steps are unique to bacteria, inhibitors can be utilized therapeutically, in lieu of antibiotics. Also, since the inhibitor is specific to a particular bacterium or group of bacteria, no off-target resistance is generated in the host's commensal bacterial population. This brief review covers examples of how mutations that confer phage resistance produce attenuation, and how this coincidental relationship can be exploited in the search for the next generation of therapeutic agents for bacterial diseases.

Effects of cathode design parameters on in vitro antimicrobial efficacy of electrically-activated silver-based iontophoretic system.

Post-operative infection is a major risk associated with implantable devices. Prior studies have demonstrated the effectiveness of ionic silver as an alternative to antibiotic-based infection prophylaxis and treatment. The focus of this study is on an electrically activated implant system engineered for active release of antimicrobial silver ions. The objective was to evaluate the effects of the cathode design, especially the cathode material, on the in vitro antimicrobial efficacy of the system. A modified Kirby-Bauer diffusion technique was used for the antimicrobial efficacy evaluations (24 h testing interval). In phase-1 of the study, a three-way ANOVA (n = 6, α = 0.05) was performed to determine the effects of cathode material (silver, titanium, and stainless steel), cathode surface area and electrode separation distance on the efficacy of the system against Staphylococcus aureus. The results show that within the design space tested, none of these parameters had a statistically significant effect on the antimicrobiality of the system (P > 0.15). Subsequently, one-way ANOVA (n = 6, α = 0.05) was conducted in phase-2 to validate the inference regarding the non-significance of the cathode material to the system efficacy using a broader spectrum of pathogens (methicillin-resistant S. aureus, Escherichia coli, Streptococcus agalactiae and Aspergillus flavus) responsible for osteomyelitis. The results confirmed the lack of statistical difference between efficacies of the three cathode material configurations against all pathogens tested (P > 0.58). Overall, the results demonstrate the ability to alter the cathode material and related design parameters in order to minimize the silver usage in the system without adversely affecting its antimicrobial efficacy.

Biocompatibility analysis of an electrically-activated silver-based antibacterial surface system for medical device applications.

The costs associated with the treatment of medical device and surgical site infections are a major cause of concern in the global healthcare system. To prevent transmission of such infections, a prophylactic surface system that provides protracted release of antibacterial silver ions using low intensity direct electric current (LIDC; 28 µA system current at 6 V) activation has been recently developed. To ensure the safety for future in vivo studies and potential clinical applications, this study assessed the biocompatibility of the LIDC-activated interdigitated silver electrodes-based surface system; in vitro toxicity to human epidermal keratinocytes, human dermal fibroblasts, and normal human osteoblasts, and antibacterial efficacy against Staphylococcus aureus and Escherichia coli was evaluated. The study concluded that the technological applications of the surface system for medical devices and surgical tools, which contact human tissues for less than 1.5 h, are expected to be self-sterilizing without causing toxicity in vivo.

In vitro properties of a Listeria monocytogenes bacteriophage-resistant mutant predict its efficacy as a live oral vaccine strain.

A Listeria monocytogenes glcV mutation precludes the binding of certain listerial phages and produces a profound attenuation characterized by the absence of detectable mutants in the livers and spleens of orally inoculated mice. In vitro, we found that the mutant formed plaques on mouse enterocyte monolayers as efficiently as the parent but the plaques formed were smaller. Intracellular growth rate determinations and examination of infected enterocytes by light and fluorescence microscopy established that the mutant was impaired not in intracellular growth rate but in cell-to-cell spreading. Because this property is shared by other immunogenic mutants (e.g., actA mutants), our glcV mutant was tested for vaccine efficacy. Oral immunization with the mutant and subsequent oral challenge (22 days postvaccination) with the parent revealed a ca. 10,000-fold increase in protection afforded by the mutant compared to sham-vaccinated controls. The glcV mutant did not stimulate innate immunity under the dose and route employed for vaccination, and an infectivity index time course experiment revealed pronounced mutant persistence in Peyer's patches. The immunogenicity of the glcV mutant compared to an isogenic actA mutant reference strain was next tested in an experiment with a challenge given 52 days postvaccination. Both mutant strains showed scant vital organ infectivity and high levels of protection similar to those seen using the glcV mutant in the 22-day postvaccination challenge. Our results indicate that oral administration of a profoundly attenuated listerial mutant can safely elicit solid protective immunity.

Passive immunization with a polyclonal antiserum to the hemoglobin receptor of Haemophilus ducreyi confers protection against a homologous challenge in the experimental swine model of chancroid.

Haemophilus ducreyi, the etiologic agent of chancroid, has an obligate requirement for heme. Heme is acquired by H. ducreyi from its human host via TonB-dependent transporters expressed at its bacterial surface. Of 3 TonB-dependent transporters encoded in the genome of H. ducreyi, only the hemoglobin receptor, HgbA, is required to establish infection during the early stages of the experimental human model of chancroid. Active immunization with a native preparation of HgbA (nHgbA) confers complete protection in the experimental swine model of chancroid, using either Freund's or monophosphoryl lipid A as adjuvants. To determine if transfer of anti-nHgbA serum is sufficient to confer protection, a passive immunization experiment using pooled nHgbA antiserum was conducted in the experimental swine model of chancroid. Pigs receiving this pooled nHgbA antiserum were protected from a homologous, but not a heterologous, challenge. Passively transferred polyclonal antibodies elicited to nHgbA bound the surface of H. ducreyi and partially blocked hemoglobin binding by nHgbA, but were not bactericidal. Taken together, these data suggest that the humoral immune response to the HgbA vaccine is protective against an H. ducreyi infection, possibly by preventing acquisition of the essential nutrient heme.

Incorporation of secretory immunoglobulin A into biofilms can decrease their resistance to ciprofloxacin.

Extracellular matrices utilized by biofilms growing on inert surfaces are generally produced entirely by the bacteria growing within those biofilms, whereas symbiotic (mutualistic) biofilms growing in or on a wide range of plants and animals utilize host-derived macromolecules, such as mucoid substances, as components of their extracellular matrix. Incorporation of host-derived molecules may have a profound effect on the resistance to antibiotics of symbiotic biofilms, which may have important implications for medicine and biology. As an initial probe of the potential effects of host-derived molecules in the extracellular matrix on the sensitivity of biofilms to antibiotics, an in vitro model was used to evaluate the effects of ciprofloxacin on biofilms grown in the presence and absence of SIgA, a host-derived glycoprotein associated with biofilms in the mammalian gut. In five out of six strains of Escherichia coli tested, the incorporation of SIgA into the biofilms apparently reduced the resistance of the bacteria to ciprofloxacin. On the other hand, SIgA generally increased the resistance of planktonic bacteria to ciprofloxacin, perhaps due in part to the SIgA-mediated aggregation of the bacteria. These findings suggest that incorporation of host-derived molecules into the extracellular matrix of symbiotic biofilms might profoundly alter the properties of those biofilms, including the resistance of those biofilms to antibiotics.

Identification and characterization of two Bordetella avium gene products required for hemagglutination.

Bordetella avium causes bordetellosis in birds, a disease similar to whooping cough caused by Bordetella pertussis in children. B. avium agglutinates guinea pig erythrocytes via an unknown mechanism. Loss of hemagglutination ability results in attenuation. We report the use of transposon mutagenesis to identify two genes required for hemagglutination. The genes (hagA and hagB) were adjacent and divergently oriented and had no orthologs in the genomes of other Bordetella species. Construction of in-frame, unmarked mutations in each gene allowed examination of the role of each in conferring erythrocyte agglutination, explanted tracheal cell adherence, and turkey poult tracheal colonization. In all of the in vitro and in vivo assays, the requirement for the trans-acting products of hagA and hagB (HagA and HagB) was readily shown. Western blotting, using antibodies to purified HagA and HagB, revealed proteins of the predicted sizes of HagA and HagB in an outer membrane-enriched fraction. Antiserum to HagB, but not HagA, blocked B. avium erythrocyte agglutination and explanted turkey tracheal ring binding. Bioinformatic analysis indicated the similarity of HagA and HagB to several two-component secretory apparatuses in which one product facilitates the exposition of the other. HagB has the potential to serve as a useful immunogen to protect turkeys against colonization and subsequent disease.

Immunization with the Haemophilus ducreyi hemoglobin receptor HgbA with adjuvant monophosphoryl lipid A protects swine from a homologous but not a heterologous challenge.

Haemophilus ducreyi, the etiological agent of chancroid, has a strict requirement for heme, which it acquires from its only natural host, humans. Previously, we showed that a vaccine preparation containing the native hemoglobin receptor HgbA purified from H. ducreyi class I strain 35000HP (nHgbAI) and administered with Freund's adjuvant provided complete protection against a homologous challenge. In the current study, we investigated whether nHgbAI dispensed with monophosphoryl lipid A (MPL), an adjuvant approved for use in humans, offered protection against a challenge with H. ducreyi strain 35000HP expressing either class I or class II HgbA (35000HPhgbAI and 35000HPhgbAII, respectively). Pigs immunized with the nHgbAI/MPL vaccine were protected against a challenge from homologous H. ducreyi strain 35000HPhgbAI but not heterologous strain 35000HPhgbAII, as evidenced by the isolation of only strain 35000HPhgbAII from nHgbAI-immunized pigs. Furthermore, histological analysis of the lesions showed striking differences between mock-immunized and nHgbAI-immunized animals challenged with strains 35000HPhgbAI but not those challenged with strain 35000HPhgbAII. Mock-immunized pigs were not protected from a challenge by either strain. The enzyme-linked immunosorbent assay (ELISA) activity of the nHgbAI/MPL antiserum was lower than the activity of antiserum from animals immunized with the nHgbAI/Freund's vaccine; however, anti-nHgbAI from both studies bound whole cells of 35000HPhgbAI better than 35000HPhgbAII and partially blocked hemoglobin binding to nHgbAI. In conclusion, despite eliciting lower antibody ELISA activity than the nHgbAI/Freund's, the nHgbAI/MPL vaccine provided protection against a challenge with homologous but not heterologous H. ducreyi, suggesting that a bivalent HgbA vaccine may be needed.

Adaptation in a mouse colony monoassociated with Escherichia coli K-12 for more than 1,000 days.

Although mice associated with a single bacterial species have been used to provide a simple model for analysis of host-bacteria relationships, bacteria have been shown to display adaptability when grown in a variety of novel environments. In this study, changes associated with the host-bacterium relationship in mice monoassociated with Escherichia coli K-12 over a period of 1,031 days were evaluated. After 80 days, phenotypic diversification of E. coli was observed, with the colonizing bacteria having a broader distribution of growth rates in the laboratory than the parent E. coli. After 1,031 days, which included three generations of mice and an estimated 20,000 generations of E. coli, the initially homogeneous bacteria colonizing the mice had evolved to have widely different growth rates on agar, a potential decrease in tendency for spontaneous lysis in vivo, and an increased tendency for spontaneous lysis in vitro. Importantly, mice at the end of the experiment were colonized at an average density of bacteria that was more than 3-fold greater than mice colonized on day 80. Evaluation of selected isolates on day 1,031 revealed unique restriction endonuclease patterns and differences between isolates in expression of more than 10% of the proteins identified by two-dimensional electrophoresis, suggesting complex changes underlying the evolution of diversity during the experiment. These results suggest that monoassociated mice might be used as a tool for characterizing niches occupied by the intestinal flora and potentially as a method of targeting the evolution of bacteria for applications in biotechnology.

Quantitative glycomics from fluidic glycan microarrays.

A hallmark of cell-surface processes involving glycans is their multivalent interaction with glycan binding proteins (GBPs). Such a multivalent interaction depends critically on the mobility and density of signaling molecules on the membrane surface. While glycan microarrays have been used in exploring multivalent interactions, the lack of mobility and the difficulty in controlling surface density both limit their quantitative applications. Here we apply a fluidic glycan microarray, with glycan density varying for orders of magnitude, to profile cell surface interaction using a model system, the adhesion of Escherichia coli to mannose. We show the quantitative determination of monovalent and multivalent adhesion channels; the latter can be inhibited by nanopartices presenting a high density of mannosyl groups. These results reveal a new E. coli adhesion mechanism: the switching in the FimH adhesion protein avidity from monovalent to multivalent as the density of mobile mannosyl groups increases; such avidity switching enhances binding affinity and triggers multiple fimbriae anchoring. Affinity enhancement toward FimH has only been observed before for oligo-mannose due to the turn on of secondary interactions outside the mannose binding pocket. We suggest that the new mechanism revealed by the fluidic microarray is of general significance to cell surface interactions: the dynamic clustering of simple sugar groups (homogeneous or heterogeneous) on the fluidic membrane surface may simulate the functions of complex glycan molecules.

A Listeria monocytogenes mutant defective in bacteriophage attachment is attenuated in orally inoculated mice and impaired in enterocyte intracellular growth.

A Listeria monocytogenes bacteriophage was used to identify a phage-resistant Tn917 insertion mutant of the mouse-virulent listerial strain F6214-1. The mutant was attenuated when it was inoculated orally into female A/J mice and failed to replicate efficiently in cultured mouse enterocytes. Phage binding studies indicated that the mutant had a cell surface alteration that precluded phage attachment. All phenotypes associated with the mutation could be complemented in trans by a single open reading frame (ORF) that corresponded to the ORF interrupted by the Tn917 insertion. The complementation effected was, in all cases, at a level indistinguishable from that of the parent. The Tn917 insertion interrupted a gene that is predicted to encode a group 2 glycosyl transferase (provisionally designated glcV). A similar glcV gene is present in Listeria welshimeri and Listeria innocua and in some serotypes of L. monocytogenes. We speculate that the loss of the glcV product results in a defective phage receptor and that this alteration coincidentally influences a feature of the normal host-pathogen interaction required for virulence. Interestingly, the glcV lesion, while preventing phage attachment, did not alter the mutant's ability to bind to cultured mouse enterocyte monolayers. Rather, the mutation appeared to alter a subsequent step in intracellular replication measured by a reduction in plaque-forming efficiency and plaque size. In vivo, the mutant was undetectable in the liver and spleen 48 h after oral inoculation. The mutation is significant in part because it is one of the few that produce attenuation when the mutant is delivered orally.

In silico Predicted Glucose-1-phosphate Uridylyltransferase (GalU) Inhibitors Block a Key Pathway Required for Listeria Virulence.

Peptidoglycan walls of gram positive bacteria are functionalized by glycopolymers called wall teichoic acid (WTA). In Listeria monocytogenes, multiple enzymes including the glucose-1-phosphate uridylyltransferase (GalU) were identified as mandatory for WTA galactosylation, so that the inhibition of GalU is associated with a significant attenuation of Listeria virulence. Herein, we report on a series of in silico predicted GalU inhibitors identified using structure-based virtual screening and experimentally validated to be effective in blocking the WTA galactosylation pathway in vitro. Several hits such as C04, a pyrimidinyl benzamide, afforded promising experimental potencies. This proof-of-concept study opens new perspectives for the development of potent and selective GalU inhibitors of high interest to attenuate Listeria virulence. It also underscores the high relevance of using molecular modeling for facilitating the identification of bacterial virulence attenuators and more generally antibacterials.

Secretory IgA and mucin-mediated biofilm formation by environmental strains of Escherichia coli: role of type 1 pili.

Recent studies suggest the importance of secretory IgA (SIgA) and mucin in the mediation of biofilm formation by commensal bacteria within the mammalian gut. Studies using a variety of strains of Escherichia coli have indicated that the interaction between E. coli and SIgA is dependent on the type 1 pilus. In this study, the importance of the pilus in SIgA-mediated biofilm formation by a laboratory strain (MG1655) and environmental (fecal) strains of E. coli was evaluated. Transient expression of the type 1 pilus by the laboratory strain of E. coli failed to facilitate SIgA-mediated biofilm formation, whereas constitutive expression of the type 1 pilus by the laboratory strain was sufficient. In contrast, transient expression of the type 1 pilus was sufficient to facilitate SIgA-mediated biofilm formation by environmental isolates. The "threshold" for mucin-mediated biofilm formation appeared to be lower than that for SIgA-mediated biofilm formation, perhaps reflecting disparate roles of mucin and SIgA in mediating biofilm formation in the gut. These studies also provide the first procedures for the growth of bacterial biofilms on live epithelial cells in vitro, an important development that may facilitate future studies on the effects of bacterial biofilms on epithelial cells.

Host and bacterial factors in listeriosis pathogenesis.

Members of the Genus Listeria are ubiquitous environmental saprophytic microorganisms. If ingested they can cause a severe disseminated disease (listeriosis) that has a high mortality rate, the highest of any food-borne pathogen, even with antibiotic therapy. Central to the high mortality rate is the hallmark characteristic of the microorganism to grow intracellularly. The presence of listeriae in food processing plants has resulted in many outbreaks of human disease and large scale recalls of processed foods. Despite the ubiquity of the microorganism, the actual disease rate (those animals showing disease signs over those exposed) is quite low and disease is almost always associated with an underlying predisposition (pregnancy being the most common in otherwise normal individuals). There are many features of the pathogenesis of listeriosis that have remained mysterious despite the extensive use of the microorganism in the study of cell-mediated immunity and intracellular growth. Informational advances such as the sequence of the mouse and listerial genomes, and technical advances such as the discovery of listeria-susceptible mouse strains, may renew interest in the study of the natural pathogenesis of the disease. This may be further facilitated by studies that employ the natural inoculation route and mimic common predisposing conditions witnessed in victims of natural outbreaks.

Evaluation of silver-titanium implants activated by low intensity direct current for orthopedic infection control: An in vitro and in vivo study.

Silver is an alternative antimicrobial of interest for the prophylaxis of prosthetic infections and electrical activation is known to augment its oligodynamic efficacy. In this study, we evaluated the in vitro and in vivo efficacy of a silver (Ag)-titanium (Ti) implant activated by 30 µA direct current compared with three controls - passive Ag-Ti, active Ti-Ti, and passive Ti-Ti. We hypothesized that the experimental group would provide better resistance to pathogenic colonization on the implant. Modified Kirby-Bauer technique was used to evaluate in vitro efficacy of the four groups against five bacteria and one fungus. For in vivo evaluation, forty-eight rats were divided into four groups. The implant was secured in a wound cavity along the posterior margin of the femur. The wound was inoculated with 7.5 × 10(5) CFU of Staphylococcus aureus. Rats were euthanized 14 days postsurgery and quantitative cultures were performed on the implant segments and the wound cavity tissue. In vitro tests showed that the growth of all six pathogens was inhibited around the active Ag anodes of the experimental group. In vivo, none of the four groups were able to prevent wound infection, but the experimental group resulted in reduced colonization. The mean bacterial loads on Ti segments were significantly lower in the implants which also had an Ag segment (p = 0.0007), and this effect was more pronounced with electrical activation (p = 0.0377). The results demonstrate the antimicrobial potential of LIDC-activated Ag-Ti implants. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1023-1031, 2016.

The Haemophilus ducreyi trimeric autotransporter adhesin DsrA protects against an experimental infection in the swine model of chancroid.

Adherence of pathogens to cellular targets is required to initiate most infections. Defining strategies that interfere with adhesion is therefore important for the development of preventative measures against infectious diseases. As an adhesin to host extracellular matrix proteins and human keratinocytes, the trimeric autotransporter adhesin DsrA, a proven virulence factor of the Gram-negative bacterium Haemophilus ducreyi, is a potential target for vaccine development. A recombinant form of the N-terminal passenger domain of DsrA from H. ducreyi class I strain 35000HP, termed rNT-DsrAI, was tested as a vaccine immunogen in the experimental swine model of H. ducreyi infection. Viable homologous H. ducreyi was not recovered from any animal receiving four doses of rNT-DsrAI administered with Freund's adjuvant at two-week intervals. Control pigs receiving adjuvant only were all infected. All animals receiving the rNT-DsrAI vaccine developed antibody endpoint titers between 3.5 and 5 logs. All rNT-DsrAI antisera bound the surface of the two H. ducreyi strains used to challenge immunized pigs. Purified anti-rNT-DsrAI IgG partially blocked binding of fibrinogen at the surface of viable H. ducreyi. Overall, immunization with the passenger domain of the trimeric autotransporter adhesin DsrA accelerated clearance of H. ducreyi in experimental lesions, possibly by interfering with fibrinogen binding.

Extrauterine listeriosis in the gravid mouse influences embryonic growth and development.

Gravid mice and other rodents inoculated with Listeria monocytogenes typically fail to clear an intrauterine infection and either succumb or expel their intrauterine contents. We took advantage of this property to investigate the effects of an extrauterine infection on parameters of pregnancy success. Pregnant mice were selected for our study if they showed no clinical signs of listeriosis following oral inoculation at 7.5 gestational days (gd), and had no detectable intrauterine colony forming units (cfu) at near term (18.5 gd). The range of oral doses employed was 106-108 cfu per mouse for two listerial serotype strains (4nonb and 1/2a). At all doses, inoculation resulted in a decrease in average near-term (18.5 gd) fetal weight per litter compared to sham inoculated controls. Additionally, embryonic death (indicated by intrauterine resorptions) was exhibited by some inoculated mice but was absent in all sham inoculated animals. In parallel experiments designed to detect possible loss of placental function, gravid uteruses were examined histopathologically and microbiologically 96 h after oral inoculation. Placental lesions were associated with high (> 106), but not low (< 10²) or absent intrauterine cfu. In vitro, mouse embryonic trophoblasts were indistinguishable from mouse enterocytes in terms of their sensitivity to listerial exposure. A model consistent with our observations is one in which products (host or bacterial) generated during an acute infection enter embryos transplacentally and influences embryonic survival and slows normal growth in utero.

Antibacterial efficacy of silver nanoparticles of different sizes, surface conditions and synthesis methods.

Silver nanoparticles (Ag-nps) are used as a natural biocide to prevent undesired bacterial growth in clothing and cosmetics. The objective of this study was to assess the antibacterial efficacy of Ag-nps of different sizes, surface conditions, and synthesis methods against Escherichia coli, Ag-resistant E. coli, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and Salmonella sp. Ag-nps samples were synthesized by: Base reduction with unmodified surfaces and used as synthesized ('unwashed'; 20, 50 and 80 nm) or after 20 phosphate buffer washes ('washed'; 20, 50 and 80 nm), or synthesized by laser ablation with carbon-stabilized surfaces ('carbon-coated'; 25 and 35 nm). Unwashed Ag-nps were toxic to all bacterial strains at concentrations between 3.0-8.0 µg/ml. The washed Ag-nps and carbon-coated Ag-nps were toxic to all bacterial strains except Ag-resistant E. coli at concentrations between 64.0-1024.0 µg/ml. Ag-resistant E. coli died only when treated with unwashed Ag-nps or its supernatant, both of which contained formaldehyde.