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Low molecular weight alginate oligosaccharides have been shown to exhibit anti-microbial activity against a range of multi-drug resistant bacteria, including Pseudomonas aeruginosa . Previous studies suggested that the disruption of calcium (Ca 2+ )-DNA binding within bacterial biofilms and dysregulation of quorum sensing (QS) were key factors in these observed effects. To further investigate the contribution of Ca 2+ binding, G-block (OligoG) and M-block alginate oligosaccharides (OligoM) with comparable average size DPn 19 but contrasting Ca 2+ binding properties were prepared. Fourier-transform infrared spectroscopy demonstrated prolonged binding of alginate oligosaccharides to the pseudomonal cell membrane even after hydrodynamic shear treatment. Molecular dynamics simulations and isothermal titration calorimetry revealed that OligoG exhibited stronger interactions with bacterial LPS than OligoM, although this difference was not mirrored by differential reductions in bacterial growth. While confocal laser scanning microscopy showed that both agents demonstrated similar dose-dependent reductions in biofilm formation, OligoG exhibited a stronger QS inhibitory effect and increased potentiation of the antibiotic azithromycin in minimum inhibitory concentration and biofilm assays. This study demonstrates that the anti-microbial effects of alginate oligosaccharides are not purely influenced by Ca 2+ -dependent processes but also by electrostatic interactions that are common to both G-block and M-block structures.

Background: Peri-implantitis has become an inexorable clinical challenge in implantology. Topical immunomodulatory epoxy-tiglianes (EBCs), derived from the Queensland blushwood tree, which induce remodeling and resolve dermal infection via induction of the inflammasome and biofilm disruption, may offer a novel therapeutic approach., Design: In vitro antimicrobial activity of EBC structures (EBC-46, EBC-1013 and EBC-147) against Streptococcus mutans , Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis in minimum inhibitory concentration, growth curve and permeabilization assays were determined. Antibiofilm activity was assessed using minimum biofilm eradication concentration (MBEC) experiments. Biofilm formation and disruption assays were analyzed using confocal laser scanning microscopy, scanning electron microscopy and direct plate counting., Results: The observed antimicrobial efficacy of the tested compounds (EBC-1013 > EBC-46 > EBC-147) was directly related to significant membrane permeabilization and growth inhibition ( p  < 0.05) against planktonic S. mutans and P. gingivalis . Antibiofilm activity was evident in MBEC assays, with S. mutans biofilm formation assays revealing significantly lower biomass volume and increased DEAD:LIVE cell ratio observed for EBC-1013 ( p  < 0.05). Furthermore, biofilm disruption assays on titanium discs induced significant biofilm disruption in S. mutans and P. gingivalis ( p  < 0.05)., Conclusions: EBC-1013 is a safe, semi-synthetic, compound, demonstrating clear antimicrobial biofilm disruption potential in peri-implantitis., Competing Interests: This work was supported by QBiotics Group Limited, Yungaburra, Australia, who also supplied the EBC compounds used in this study. DWT. KEH, MFP and LCP have received research funding from QBiotics Group. JS is an employee of QBiotics Group. PR is the Executive Director and Chief Scientific Officer of QBiotics Group. The other authors have no conflicts of interest to disclose., (© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Background: The increasing prevalence of invasive fungal infections in immuno-compromised patients is a considerable cause of morbidity and mortality. With the rapid emergence of antifungal resistance and an inadequate pipeline of new therapies, novel treatment strategies are now urgently required., Methods: The antifungal activity of the alginate oligosaccharide OligoG in conjunction with nystatin was tested against a range of Candida spp. ( C. albicans , C. glabrata , C. parapsilosis , C. auris , C. tropicalis and C. dubliniensis ), in both planktonic and biofilm assays, to determine its potential clinical utility to enhance the treatment of candidal infections. The effect of OligoG (0-6%) ± nystatin on Candida spp. was examined in minimum inhibitory concentration (MIC) and growth curve assays. Antifungal effects of OligoG and nystatin treatment on biofilm formation and disruption were characterized using confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and ATP cellular viability assays. Effects on the cell membrane were determined using permeability assays and transmission electron microscopy (TEM)., Results: MIC and growth curve assays demonstrated the synergistic effects of OligoG (0-6%) with nystatin, resulting in an up to 32-fold reduction in MIC, and a significant reduction in the growth of C. parapsilosis and C. auris (minimum significant difference = 0.2 and 0.12 respectively). CLSM and SEM imaging demonstrated that the combination treatment of OligoG (4%) with nystatin (1 µg/ml) resulted in significant inhibition of candidal biofilm formation on glass and clinical grade silicone surfaces ( p < 0.001), with increased cell death ( p < 0.0001). The ATP biofilm disruption assay demonstrated a significant reduction in cell viability with OligoG (4%) alone and the combined OligoG/nystatin (MIC value) treatment ( p < 0.04) for all Candida strains tested. TEM studies revealed the combined OligoG/nystatin treatment induced structural reorganization of the Candida cell membrane, with increased permeability when compared to the untreated control ( p < 0.001)., Conclusions: Antimicrobial synergy between OligoG and nystatin against Candida spp. highlights the potential utility of this combination therapy in the prevention and topical treatment of candidal biofilm infections, to overcome the inherent tolerance of biofilm structures to antifungal agents., Competing Interests: DT and KH have received research funding from AlgiPharma AS. PR is the Chief Scientific Officer at AlgiPharma AS. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Powell, Adams, Quoraishi, Py, Oger, Gazze, Francis, von Ruhland, Owens, Rye, Hill, Pritchard and Thomas.)

The management of antibiotic-resistant, bacterial biofilm infections in chronic skin wounds is an increasing clinical challenge. Despite advances in diagnosis, many patients do not derive benefit from current anti-infective/antibiotic therapies. Here, we report a novel class of naturally occurring and semisynthetic epoxy-tiglianes, derived from the Queensland blushwood tree ( Fontainea picrosperma) , and demonstrate their antimicrobial activity (modifying bacterial growth and inducing biofilm disruption), with structure/activity relationships established against important human pathogens. In vitro, the lead candidate EBC-1013 stimulated protein kinase C (PKC)-dependent neutrophil reactive oxygen species (ROS) induction and NETosis and increased expression of wound healing-associated cytokines, chemokines, and antimicrobial peptides in keratinocytes and fibroblasts. In vivo, topical EBC-1013 induced rapid resolution of infection with increased matrix remodeling in acute thermal injuries in calves. In chronically infected diabetic mouse wounds, treatment induced cytokine/chemokine production, inflammatory cell recruitment, and complete healing (in six of seven wounds) with ordered keratinocyte differentiation. These results highlight a nonantibiotic approach involving contrasting, orthogonal mechanisms of action combining targeted biofilm disruption and innate immune induction in the treatment of chronic wounds.

Chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) evolve to generate environmentally adapted biofilm communities, leading to increased patient morbidity and mortality. OligoG CF-5/20, a low-molecular-weight inhaled alginate oligomer therapy, is currently in phase IIb/III clinical trials in CF patients. Experimental evolution of P. aeruginosa in response to OligoG CF-5/20 was assessed using a bead biofilm model allowing continuous passage (45 days; ∼245 generations). Mutants isolated after OligoG CF-5/20 treatment typically had a reduced biofilm-forming ability and altered motility profile. Genotypically, OligoG CF-5/20 provided no selective pressure on genomic mutations within morphotypes. Chronic exposure to azithromycin, a commonly prescribed antibiotic in CF patients, with or without OligoG CF-5/20 in the biofilm evolution model also had no effect on rates of resistance acquisition. Interestingly, however, cross-resistance to other antibiotics (e.g., aztreonam) was reduced in the presence of OligoG CF-5/20. Collectively, these findings show no apparent adverse effects from long-term exposure to OligoG CF-5/20, instead resulting in both fewer colonies with multidrug resistance (MDR)-associated phenotypes and improved antibiotic susceptibility of P. aeruginosa IMPORTANCE The emergence of multidrug-resistant (MDR) pathogens within biofilms in the cystic fibrosis lung results in increased morbidity. An inhalation therapy derived from alginate, OligoG CF-5/20, is currently in clinical trials for cystic fibrosis patients. OligoG CF-5/20 has been shown to alter sputum viscoelasticity, disrupt mucin polymer networks, and disrupt MDR pseudomonal biofilms. Long-term exposure to inhaled therapeutics may induce selective evolutionary pressures on bacteria within the lung biofilm. Here, a bead biofilm model with repeated exposure of P. aeruginosa to OligoG CF-5/20 (alone and in combination with azithromycin) was conducted to study these long-term effects and characterize the phenotypic and genotypic adaptations which result. These findings, over 6 weeks, show that long-term use of OligoG CF-5/20 does not lead to extensive mutational changes and may potentially decrease the pathogenicity of the bacterial biofilm and improve the susceptibility of P. aeruginosa to other classes of antibiotics., (Copyright © 2021 Oakley et al.)

Pseudomonas aeruginosa causes problematic chronic lung infections in those suffering from cystic fibrosis. This is due to its antimicrobial resistance mechanisms and its ability to form robust biofilm communities with increased antimicrobial tolerances. Using novel antimicrobials or repurposing current ones is required in order to overcome these problems. Manuka honey is a natural antimicrobial agent that has been used for many decades in the treatment of chronic surface wounds with great success, particularly those infected with P. aeruginosa . Here we aim to determine whether the antimicrobial activity of manuka honey could potentially be repurposed to inhibit pulmonary P. aeruginosa infections using two ex vivo models. P. aeruginosa isolates ( n = 28) from an international panel were tested for their susceptibility to manuka honey and clinically relevant antibiotics (ciprofloxacin, ceftazidime, and tobramycin), alone and in combination, using conventional antimicrobial susceptibility testing (AST). To increase clinical applicability, two ex vivo porcine lung (EVPL) models (using alveolar and bronchiolar tissue) were used to determine the anti-biofilm effects of manuka honey alone and in combination with antibiotics. All P. aeruginosa isolates were susceptible to manuka honey, however, varying incidences of resistance were seen against antibiotics. The combination of sub-inhibitory manuka honey and antibiotics using conventional AST had no effect on activity against the majority of isolates tested. Using the two ex vivo models, 64% (w/v) manuka honey inhibited many of the isolates where abnormally high concentrations of antibiotics could not. Typically, combinations of both manuka honey and antibiotics had increased antimicrobial activity. These results highlight the potential of manuka honey as a future antimicrobial for the treatment of pulmonary P. aeruginosa isolates, clearing potential infection reservoirs within the upper airway.

Acquisition of a mucoid phenotype by Pseudomonas sp. in the lungs of cystic fibrosis (CF) patients, with subsequent over-production of extracellular polymeric substance (EPS), plays an important role in mediating the persistence of multi-drug resistant (MDR) infections. The ability of a low molecular weight (Mn = 3200 g mol -1 ) alginate oligomer (OligoG CF-5/20) to modify biofilm structure of mucoid Pseudomonas aeruginosa (NH57388A) was studied in vitro using scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) with Texas Red (TxRd®)-labelled OligoG and EPS histochemical staining. Structural changes in treated biofilms were quantified using COMSTAT image-analysis software of CLSM z-stack images, and nanoparticle diffusion. Interactions between the oligomers, Ca 2+ and DNA were studied using molecular dynamics (MD) simulations, Fourier transform infrared spectroscopy (FTIR) and isothermal titration calorimetry (ITC). Imaging demonstrated that OligoG treatment (≥0.5%) inhibited biofilm formation, revealing a significant reduction in both biomass and biofilm height ( P  < 0.05). TxRd®-labelled oligomers readily diffused into established (24 h) biofilms. OligoG treatment (≥2%) induced alterations in the EPS of established biofilms; significantly reducing the structural quantities of EPS polysaccharides, and extracellular (e)DNA ( P  < 0.05) with a corresponding increase in nanoparticle diffusion ( P  < 0.05) and antibiotic efficacy against established biofilms. ITC demonstrated an absence of rapid complex formation between DNA and OligoG and confirmed the interactions of OligoG with Ca 2+ evident in FTIR and MD modelling. The ability of OligoG to diffuse into biofilms, potentiate antibiotic activity, disrupt DNA-Ca 2+ -DNA bridges and biofilm EPS matrix highlights its potential for the treatment of biofilm-related infections., Competing Interests: D.W.T. has a consultancy relationship and has, with K.E.H., received research funding from AlgiPharma AS. P.D.R. is a director/owner of AlgiPharma AS. The remaining authors declare no competing interests.

Pseudomonas aeruginosa plays a major role in many chronic infections. Its ability to readily form biofilms contributes to its success as an opportunistic pathogen and its resistance/tolerance to antimicrobial/antibiotic therapy. A low-molecular-weight alginate oligomer (OligoG CF-5/20) derived from marine algae has previously been shown to impair motility in P. aeruginosa biofilms and disrupt pseudomonal biofilm assembly. As these bacterial phenotypes are regulated by quorum sensing (QS), we hypothesized that OligoG CF-5/20 may induce alterations in QS signaling in P. aeruginosa QS regulation was studied by using Chromobacterium violaceum CV026 biosensor assays that showed a significant reduction in acyl homoserine lactone (AHL) production following OligoG CF-5/20 treatment (≥2%; P < 0.05). This effect was confirmed by liquid chromatography-mass spectrometry analysis of C 4 -AHL and 3-oxo-C 12 -AHL production (≥2%; P < 0.05). Moreover, quantitative PCR showed that reduced expression of both the las and rhl systems was induced following 24 h of treatment with OligoG CF-5/20 (≥0.2%; P < 0.05). Circular dichroism spectroscopy indicated that these alterations were not due to steric interaction between the AHL and OligoG CF-5/20. Confocal laser scanning microscopy (CLSM) and COMSTAT image analysis demonstrated that OligoG CF-5/20-treated biofilms had a dose-dependent decrease in biomass that was associated with inhibition of extracellular DNA synthesis (≥0.5%; P < 0.05). These changes correlated with alterations in the extracellular production of the pseudomonal virulence factors pyocyanin, rhamnolipids, elastase, and total protease ( P < 0.05). The ability of OligoG CF-5/20 to modify QS signaling in P. aeruginosa PAO1 may influence critical downstream functions such as virulence factor production and biofilm formation., (Copyright © 2018 American Society for Microbiology.)

Aims: A novel alginate oligomer (OligoG CF-5/20) has been shown to potentiate antifungal therapy against a range of fungal pathogens. The current study assessed the effect of this oligomer on in vitro virulence factor expression and epithelial invasion by Candida species., Methods and Results: Plate substrate assays and epithelial models were used to assess Candida albicans (CCUG 39343 and ATCC 90028) invasion, in conjunction with confocal laser scanning microscopy and histochemistry. Expression of candidal virulence factors was determined biochemically and by quantitative PCR (qPCR). Changes in surface charge of C. albicans following OligoG treatment were analysed using electrophoretic light scattering. OligoG induced marked alterations in hyphal formation in the substrate assays and reduced invasion in the epithelial model (P < 0·001). Significant dose-dependent inhibition of phospholipase activity in C. albicans was evident following OligoG treatment (P < 0·05). While OligoG binding failed to affect alterations in surface charge (P > 0·05), qPCR demonstrated a reduction in phospholipase B (PLB2) and SAPs (SAP4 and SAP6) expression., Conclusion: OligoG CF-5/20 reduced in vitro virulence factor expression and invasion by C. albicans., Significance and Impact of the Study: These results, and the previously described potentiation of antifungal activity, define a potential therapeutic opportunity in the treatment of invasive candidal infections., (© 2017 The Society for Applied Microbiology.)

In chronic respiratory disease, the formation of dense, 3-dimensional "microcolonies" by Pseudomonas aeruginosa within the airway plays an important role in contributing to resistance to treatment. An in vitro biofilm model of pseudomonal microcolony formation using artificial-sputum (AS) medium was established to study the effects of low-molecular-weight alginate oligomers (OligoG CF-5/20) on pseudomonal growth, microcolony formation, and the efficacy of colistin. The studies employed clinical cystic fibrosis (CF) isolates ( n = 3) and reference nonmucoid and mucoid multidrug-resistant (MDR) CF isolates ( n = 7). Bacterial growth and biofilm development and disruption were studied using cell viability assays and image analysis with scanning electron and confocal laser scanning microscopy. Pseudomonal growth in AS medium was associated with increased ATP production ( P < 0.05) and the formation (at 48 h) of discrete (>10-μm) microcolonies. In conventional growth medium, colistin retained an ability to inhibit growth of planktonic bacteria, although the MIC was increased (0.1 to 0.4 μg/ml) in AS medium compared to Mueller-Hinton (MH) medium. In contrast, in an established-biofilm model in AS medium, the efficacy of colistin was decreased. OligoG CF-5/20 (≥2%) treatment, however, induced dose-dependent biofilm disruption ( P < 0.05) and led to colistin retaining its antimicrobial activity ( P < 0.05). While circular dichroism indicated that OligoG CF-5/20 did not change the orientation of the alginate carboxyl groups, mass spectrometry demonstrated that the oligomers induced dose-dependent (>0.2%; P < 0.05) reductions in pseudomonal quorum-sensing signaling. These findings reinforce the potential clinical significance of microcolony formation in the CF lung and highlight a novel approach to treat MDR pseudomonal infections., (Copyright © 2017 American Society for Microbiology.)

Concerns about acquisition of antibiotic resistance have led to increasing demand for new antimicrobial therapies. OligoG CF-5/20 is an alginate oligosaccharide previously shown to have antimicrobial and antibiotic potentiating activity. We investigated the structural modification of the bacterial cell wall by OligoG CF-5/20 and its effect on membrane permeability. Binding of OligoG CF-5/20 to the bacterial cell surface was demonstrated in Gram-negative bacteria. Permeability assays revealed that OligoG CF-5/20 had virtually no membrane-perturbing effects. Lipopolysaccharide (LPS) surface charge and aggregation were unaltered in the presence of OligoG CF-5/20. Small angle neutron scattering and circular dichroism spectroscopy showed no substantial change to the structure of LPS in the presence of OligoG CF-5/20, however, isothermal titration calorimetry demonstrated a weak calcium-mediated interaction. Metabolomic analysis confirmed no change in cellular metabolic response to a range of osmolytes when treated with OligoG CF-5/20. This data shows that, although weak interactions occur between LPS and OligoG CF-5/20 in the presence of calcium, the antimicrobial effects of OligoG CF-5/20 are not related to the induction of structural alterations in the LPS or cell permeability. These results suggest a novel mechanism of action that may avoid the common route in acquisition of resistance via LPS structural modification.

The host- and bacteria-derived extracellular polysaccharide coating of the lung is a considerable challenge in chronic respiratory disease and is a powerful barrier to effective drug delivery. A low molecular weight 12-15-mer alginate oligosaccharide (OligoG CF-5/20), derived from plant biopolymers, was shown to modulate the polyanionic components of this coating. Molecular modeling and Fourier transform infrared spectroscopy demonstrated binding between OligoG CF-5/20 and respiratory mucins. Ex vivo studies showed binding induced alterations in mucin surface charge and porosity of the three-dimensional mucin networks in cystic fibrosis (CF) sputum. Human studies showed that OligoG CF-5/20 is safe for inhalation in CF patients with effective lung deposition and modifies the viscoelasticity of CF-sputum. OligoG CF-5/20 is the first inhaled polymer therapy, represents a novel mechanism of action and therapeutic approach for the treatment of chronic respiratory disease, and is currently in Phase IIb clinical trials for the treatment of CF.

The oligosaccharide OligoG, an alginate derived from seaweed, has been shown to have anti-bacterial and anti-biofilm properties and potentiates the activity of selected antibiotics against multi-drug resistant bacteria. The ability of OligoG to perturb fungal growth and potentiate conventional antifungal agents was evaluated using a range of pathogenic fungal strains. Candida (n = 11) and Aspergillus (n = 3) spp. were tested using germ tube assays, LIVE/DEAD staining, scanning electron microscopy (SEM), atomic force microscopy (AFM) and high-throughput minimum inhibition concentration assays (MICs). In general, the strains tested showed a significant dose-dependent reduction in cell growth at ≥6% OligoG as measured by optical density (OD600; P<0.05). OligoG (>0.5%) also showed a significant inhibitory effect on hyphal growth in germ tube assays, although strain-dependent variations in efficacy were observed (P<0.05). SEM and AFM both showed that OligoG (≥2%) markedly disrupted fungal biofilm formation, both alone, and in combination with fluconazole. Cell surface roughness was also significantly increased by the combination treatment (P<0.001). High-throughput robotic MIC screening demonstrated the potentiating effects of OligoG (2, 6, 10%) with nystatin, amphotericin B, fluconazole, miconazole, voriconazole or terbinafine with the test strains. Potentiating effects were observed for the Aspergillus strains with all six antifungal agents, with an up to 16-fold (nystatin) reduction in MIC. Similarly, all the Candida spp. showed potentiation with nystatin (up to 16-fold) and fluconazole (up to 8-fold). These findings demonstrate the antifungal properties of OligoG and suggest a potential role in the management of fungal infections and possible reduction of antifungal toxicity.

Pseudomonas aeruginosa (PA) biofilm-associated infections are a common cause of morbidity in chronic respiratory disease and represent a therapeutic challenge. Recently, the ability of a novel alginate oligomer (OligoG) to potentiate the effect of antibiotics against gram-negative, multi-drug-resistant bacteria and inhibit biofilm formation in vitro has been described. Interaction of OligoG with the cell surface of PA was characterized at the nanoscale using atomic force microscopy (AFM), zeta potential measurement (surface charge), and sizing measurements (dynamic light scattering). The ability of OligoG to modify motility was studied in motility assays. AFM demonstrated binding of OligoG to the bacterial cell surface, which was irreversible after exposure to hydrodynamic shear (5,500 × g). Zeta potential analysis (pH 5-9; 0.1-0.001 M NaCl) demonstrated that binding was associated with marked changes in the bacterial surface charge (-30.9 ± 0.8 to -47.0 ± 2.3 mV; 0.01 M NaCl [pH 5]; P < 0.001). Sizing analysis demonstrated that alteration of surface charge was associated with cell aggregation with a 2- to 3-fold increase in mean particle size at OligoG concentrations greater than 2% (914 ± 284 to 2599 ± 472 nm; 0.01 M NaCl [pH 5]; P < 0.001). These changes were associated with marked dose-dependent inhibition in bacterial swarming motility in PA and Burkholderia spp. The ability of OligoG to bind to a bacterial surface, modulate surface charge, induce microbial aggregation, and inhibit motility represents important direct mechanisms by which antibiotic potentiation and biofilm disruption is affected. These results highlight the value of combining multiple nanoscale technologies to further our understanding of the mechanisms of action of novel antibacterial therapies.

Objectives: The in vitro effect of a novel, oligosaccharide nanomedicine OligoG against oral pathogen-related biofilms, both alone and in the presence of the conventional anti-bacterial agent triclosan, was evaluated., Methods: The effect of OligoG±triclosan was assessed against established Streptococcus mutans and Porphyromonas gingivalis biofilms by bacterial counts and image analysis using LIVE/DEAD(®) staining and atomic force microscopy (AFM). The effect of triclosan and OligoG surface pre-treatments on bacterial attachment to titanium and polymethylmethacrylate was also studied., Results: OligoG potentiated the antimicrobial effect of triclosan, particularly when used in combination at 0.3% against S. mutans grown in artificial saliva. OligoG was less effective against established P. gingivalis biofilms. However, attachment of P. gingivalis, to titanium in particular, was significantly reduced after surface pre-treatment with OligoG and triclosan at 0.01% when compared to controls. Light microscopy and AFM showed that OligoG was biocidal to P. gingivalis, but not S. mutans., Conclusions: OligoG and triclosan when used in combination produced an enhanced antimicrobial effect against two important oral pathogens and reduced bacterial attachment to dental materials such as titanium, even at reduced triclosan concentrations. Whilst the use of triclosan against oral bacteria has been widely documented, its synergistic use with OligoG described here, has not previously been reported. The use of lower concentrations of triclosan, if used in combination therapy with OligoG, could have environmental benefits., Clinical Importance: The potentiation of antimicrobial agents by naturally occurring oligomers such as OligoG may represent a novel, safe adjunct to conventional oral hygiene and periodontal therapy. The ability of OligoG to inhibit the growth and impair bacterial adherence highlights its potential in the management of peri-implantitis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Outbreaks of Staphylococcus aureus infections in neonatal units require prompt investigation and implementation of control measures. From January to March 1990, a marked increase in the number of S. aureus infections was observed in a neonatal nursery. Twenty-seven S. aureus isolates from 23 patients were analyzed by phage typing and restriction endonuclease analysis (REA). Only nine strains were differentiated by phage type. However, REA with HindIII, CfoI, and ClaI differentiated 20 strains. The REA results indicated that the outbreak was due to several different S. aureus strains and did not represent transmission of a single epidemic strain. REA may enable more accurate determination of the presence or absence of an epidemic strain during an outbreak than would traditional methods such as phage typing.