Convergent behavior of extended stalk regions from staphylococcal surface proteins with widely divergent sequence patterns.

Staphylococcus epidermidis and Staphylococcus aureus are highly problematic bacteria in hospital settings. A major challenge is their ability to form biofilms on abiotic or biotic surfaces. Biofilms are well-organized, multicellular bacterial aggregates that resist antibiotic treatment and often lead to recurrent infections. Bacterial cell wall-anchored (CWA) proteins are important players in biofilm formation and infection. Many have putative stalk-like regions or regions of low complexity near the cell wall-anchoring motif. Recent work demonstrated the strong propensity of the stalk region of S. epidermidis accumulation-associated protein (Aap) to remain highly extended under solution conditions that typically induce compaction. This behavior is consistent with the expected function of a stalk-like region that is covalently attached to the cell wall peptidoglycan and projects the adhesive domains of Aap away from the cell surface. In this study, we evaluate whether the ability to resist compaction is a common theme among stalk regions from various staphylococcal CWA proteins. Circular dichroism spectroscopy was used to examine secondary structure changes as a function of temperature and cosolvents along with sedimentation velocity analytical ultracentrifugation, size-exclusion chromatography, and SAXS to characterize structural characteristics in solution. All stalk regions tested are intrinsically disordered, lacking secondary structure beyond random coil and polyproline type II helix, and they all sample highly extended conformations. Remarkably, the Ser-Asp dipeptide repeat region of SdrC exhibited nearly identical behavior in solution when compared to the Aap Pro/Gly-rich region, despite highly divergent sequence patterns, indicating conservation of function by various distinct staphylococcal CWA protein stalk regions.

Staphylococcal Periscope proteins Aap, SasG, and Pls project noncanonical legume-like lectin adhesin domains from the bacterial surface.

Staphylococcus aureus and Staphylococcus epidermidis are frequently associated with medical device infections that involve establishment of a bacterial biofilm on the device surface. Staphylococcal surface proteins Aap, SasG, and Pls are members of the Periscope Protein class and have been implicated in biofilm formation and host colonization; they comprise a repetitive region ("B region") and an N-terminal host colonization domain within the "A region," predicted to be a lectin domain. Repetitive E-G5 domains (as found in Aap, SasG, and Pls) form elongated "stalks" that would vary in length with repeat number, resulting in projection of the N-terminal A domain variable distances from the bacterial cell surface. Here, we present the structures of the lectin domains within A regions of SasG, Aap, and Pls and a structure of the Aap lectin domain attached to contiguous E-G5 repeats, suggesting the lectin domains will sit at the tip of the variable length rod. We demonstrate that these isolated domains (Aap, SasG) are sufficient to bind to human host desquamated nasal epithelial cells. Previously, proteolytic cleavage or a deletion within the A domain had been reported to induce biofilm formation; the structures suggest a potential link between these observations. Intriguingly, while the Aap, SasG, and Pls lectin domains bind a metal ion, they lack the nonproline cis peptide bond thought to be key for carbohydrate binding by the lectin fold. This suggestion of noncanonical ligand binding should be a key consideration when investigating the host cell interactions of these bacterial surface proteins.

GraS Sensory Activity in Staphylococcus epidermidis Is Modulated by the "Guard Loop" of VraG and the ATPase Activity of VraF.

Antimicrobial peptides (AMPs) are one of the key immune responses that can eliminate pathogenic bacteria through membrane perturbation. As a successful skin commensal, Staphylococcus epidermidis can sense and respond to AMPs through the GraXRS two-component system and an efflux system comprising the VraG permease and VraF ATPase. GraS is a membrane sensor known to function in AMP resistance through a negatively charged, 9-residue extracellular loop, which is predicted to be linear without any secondary structure. An important question is how GraS can impart effective sensing of AMPs through such a small unstructured sequence. In this study, we verified the role of graS and vraG in AMP sensing in S. epidermidis, as demonstrated by the failure of the ΔgraS or ΔvraG mutants to sense. Deletion of the extracellular loop of VraG did not affect sensing but reduced survival with polymyxin B. Importantly, a specific region within the extracellular loop, termed the guard loop (GL), has inhibitory activity since sensing of polymyxin B was enhanced in the ΔGL mutant, indicating that the GL may act as a gatekeeper for sensing. Bacterial two-hybrid analysis demonstrated that the extracellular regions of GraS and VraG interact, but interaction appears dispensable to sensing activity. Mutation of the extracellular loop of VraG, the GL, and the active site of VraF suggested that an active detoxification function of VraG is necessary for AMP resistance. Altogether, we provide evidence for a unique sensory scheme that relies on the function of a permease to impart effective information processing. IMPORTANCE Staphylococcus epidermidis has become an important opportunistic pathogen that is responsible for nosocomial and device-related infections that account for considerable morbidity worldwide. A thorough understanding of the mechanisms that enable S. epidermidis to colonize human skin successfully is essential for the development of alternative treatment strategies and prophylaxis. Here, we demonstrate the importance of an AMP response system in a clinically relevant S. epidermidis strain. Furthermore, we provide evidence for a unique sensory scheme that would rely on the detoxification function of a permease to effect information processing.

Asp22 drives the protonation state of the Staphylococcus epidermidis glucose/H+ symporter.

The Staphylococcus epidermidis glucose/H+ symporter (GlcPSe) is a membrane transporter highly specific for glucose and a homolog of the human glucose transporters (GLUT, SLC2 family). Most GLUTs and their bacterial counterparts differ in the transport mechanism, adopting uniport and sugar/H+ symport, respectively. Unlike other bacterial GLUT homologs (for example, XylE), GlcPSe has a loose H+/sugar coupling. Asp22 is part of the proton-binding site of GlcPSe and crucial for the glucose/H+ co-transport mechanism. To determine how pH variations affect the proton site and the transporter, we performed surface-enhanced IR absorption spectroscopy on the immobilized GlcPSe We found that Asp22 has a pKa of 8.5 ± 0.1, a value consistent with that determined previously for glucose transport, confirming the central role of this residue for the transport mechanism of GlcPSe A neutral replacement of the negatively charged Asp22 led to positive charge displacements over the entire pH range, suggesting that the polarity change of the WT reflects the protonation state of Asp22 We expected that the substitution of the residue Ile105 for a serine, located within hydrogen-bonding distance to Asp22, would change the microenvironment, but the pKa of Asp22 corresponded to that of the WT. A167E mutation, selected in analogy to the XylE, introduced an additional protonatable site and perturbed the protonation state of Asp22, with the latter now exhibiting a pKa of 6.4. These studies confirm that Asp22 is the proton-binding residue in GlcPSe and show that charged residues in its vicinity affect the pKa of glucose/H+ symport.

Phenylpiperazine 5,5-Dimethylhydantoin Derivatives as First Synthetic Inhibitors of Msr(A) Efflux Pump in Staphylococcus epidermidis.

Herein, 15 phenylpiperazine 3-benzyl-5,5-dimethylhydantoin derivatives (1-15) were screened for modulatory activity towards Msr(A) efflux pump present in S. epidermidis bacteria. Synthesis, crystallographic analysis, biological studies in vitro and structure-activity relationship (SAR) analysis were performed. The efflux pump inhibitory (EPI) potency was determined by employing ethidium bromide accumulation assay in both Msr(A) efflux pump overexpressed (K/14/1345) and deficient (ATCC 12228) S. epidermidis strains. The series of compounds was also evaluated for the capacity to reduce the resistance of K/14/1345 strain to erythromycin, a known substrate of Msr(A). The study identified five strong modulators for Msr(A) in S. epidermidis. The 2,4-dichlorobenzyl-hydantoin derivative 9 was found as the most potent EPI, inhibiting the efflux activity in K/14/1345 at a concentration as low as 15.63 µM. Crystallography-supported SAR analysis indicated structural properties that may be responsible for the activity found. This study identified the first synthetic compounds able to inhibit Msr(A) efflux pump transporter in S. epidermidis. Thus, the hydantoin-derived molecules found can be an attractive group in search for antibiotic adjuvants acting via Msr(A) transporter.

The staphylococcal biofilm protein Aap forms a tetrameric species as a necessary intermediate before amyloidogenesis.

The accumulation-associated protein (Aap) from Staphylococcus epidermidis is a biofilm-related protein that was found to be a critical factor for infection using a rat catheter model. The B-repeat superdomain of Aap, composed of 5-17 B-repeats, each containing a Zn2+-binding G5 and a spacer subdomain, is responsible for Zn2+-dependent assembly leading to accumulation of bacteria during biofilm formation. We previously demonstrated that a minimal B-repeat construct (Brpt1.5) forms an antiparallel dimer in the presence of 2-3 Zn2+ ions. More recently, we have reported the presence of functional amyloid-like fibrils composed of Aap within S. epidermidis biofilms and demonstrated that a biologically relevant construct containing five and a half B-repeats (Brpt5.5) forms amyloid-like fibrils similar to those observed in the biofilm. In this study, we analyze the initial assembly events of the Brpt5.5 construct. Analytical ultracentrifugation was utilized to determine hydrodynamic parameters of reversibly associating species and to perform linked equilibrium studies. Linkage studies indicated a mechanism of Zn2+-induced dimerization similar to smaller constructs; however, Brpt5.5 dimers could then undergo further Zn2+-induced assembly into a previously uncharacterized tetramer. This led us to search for potential Zn2+-binding sites outside of the dimer interface. We developed a Brpt5.5 mutant that was unable to form the tetramer and was concordantly incapable of amyloidogenesis. CD and dynamic light scattering indicate that a conformational transition in the tetramer species is a critical step preceding amyloidogenesis. This mechanistic model for B-repeat assembly and amyloidogenesis provides new avenues for potential therapeutic targeting of staphylococcal biofilms.

Trace amines produced by skin bacteria accelerate wound healing in mice.

Certain skin bacteria are able to convert aromatic amino acids (AAA) into trace amines (TA) that act as neuromodulators. Since the human skin and sweat contain a comparatively high content of AAA one can expect that such bacteria are able to produce TA on our skin. Here we show that TA-producing Staphylococcus epidermidis strains expressing SadA are predominant on human skin and that TA accelerate wound healing. In wounded skin, keratinocytes produce epinephrine (EPI) that leads to cell motility inhibition by ß2-adrenergic receptor (ß2-AR) activation thus delay wound healing. As ß2-AR antagonists, TA and dopamine (DOP) abrogate the effect of EPI thus accelerating wound healing both in vitro and in a mouse model. In the mouse model, the S. epidermidis wild type strain accelerates wound healing compared to its ΔsadA mutant. Our study demonstrates that TA-producing S. epidermidis strains present on our skin might be beneficial for wound healing.

Optomechanical detection of vibration modes of a single bacterium.

Low-frequency vibration modes of biological particles, such as proteins, viruses and bacteria, involve coherent collective vibrations at frequencies in the terahertz and gigahertz domains. These vibration modes carry information on their structure and mechanical properties, which are good indicators of their biological state. In this work, we harnessed a particular regime in the physics of coupled mechanical resonators to directly measure these low-frequency mechanical resonances of a single bacterium. We deposit the bacterium on the surface of an ultrahigh frequency optomechanical disk resonator in ambient conditions. The vibration modes of the disk and bacterium hybridize when their associated frequencies are similar. We developed a general theoretical framework to describe this coupling, which allows us to retrieve the eigenfrequencies and mechanical loss of the bacterium low-frequency vibration modes (quality factor). Additionally, we analysed the effect of hydration on these vibrational modes. This work demonstrates that ultrahigh frequency optomechanical resonators can be used for vibrational spectrometry with the unique capability to obtain information on single biological entities.

Use of bacterial cellulose film modified by polypyrrole/TiO2-Ag nanocomposite for detecting and measuring the growth of pathogenic bacteria.

The aim of this study was to use of bacterial cellulose/polypyrrole/TiO2-Ag (BC/PPy/TiO2-Ag) nanocomposite film to detect and measure the growth of 5 pathogenic bacteria. For this purpose, at first, 13 BC/PPy/TiO2-Ag films were fabricated, then bacterial suspensions were prepared according to McFarland standard. The results showed that by increasing the bacterial concentration, the electrical resistance of sensors was decreased and there was a relation between bacterial concentration and bacterial type with electrical resistance change of sensors. The obtained data showed that the sensitivity of the sensors was increased with increasing the concentration of polypyrrole and TiO2-Ag. FT-IR and SEM tests were performed to investigate the interaction between nanoparticles and determine the size of nanoparticles. The BC/PPy/TiO2-Ag biosensors are portable and the response time of these sensors is very short for target analysis. Therefore, these sensors have the potential to improve biological safety as diagnostic tools.

Overview of Staphylococcus epidermidis cell wall-anchored proteins: potential targets to inhibit biofilm formation.

Currently, the treatment of infections by Staphylococcus epidermidis (S. epidermidis) represents a challenge because some strains have multidrug-resistance to antimicrobial products (antibiotic and biocides) and can produce biofilms. These biofilms protect bacterial cells from both antimicrobials and the host immune response. Therefore, it is crucial to encourage research on the development of new treatments. One method is immunotherapy, targeting components of S. epidermidis, such as S. epidermidis surface (Ses) proteins. Ses is expressed constitutively in most strains, and they participate in biofilm formation. This review is an update on Ses, regarding their structure, biological function, their relationship with S. epidermidis biofilm formation, and its possible role as therapeutic targets to develop immunotherapeutic treatments to prevent infections by S. epidermidis.

Small Molecules Produced by Commensal Staphylococcus epidermidis Disrupt Formation of Biofilms by Staphylococcus aureus.

The microbiota influences host health through several mechanisms, including protecting it from pathogen colonization. Staphylococcus epidermidis is one of the most frequently found species in the skin microbiota, and its presence can limit the development of pathogens such as Staphylococcus aureusS. aureus causes diverse types of infections ranging from skin abscesses to bloodstream infections. Given the increasing prevalence of S. aureus drug-resistant strains, it is imperative to search for new strategies for treatment and prevention. Thus, we investigated the activity of molecules produced by a commensal S. epidermidis isolate against S. aureus biofilms. We showed that molecules present in S. epidermidis cell-free conditioned media (CFCM) caused a significant reduction in biofilm formation in most S. aureus clinical isolates, including all 4 agr types and agr-defective strains, without any impact on growth. S. epidermidis molecules also disrupted established S. aureus biofilms and reduced the antibiotic concentration required to eliminate them. Preliminary characterization of the active compound showed that its activity is resistant to heat, protease inhibitors, trypsin, proteinase K, and sodium periodate treatments, suggesting that it is not proteinaceous. RNA sequencing revealed that S. epidermidis-secreted molecules modulate the expression of hundreds of S. aureus genes, some of which are associated with biofilm production. Biofilm formation is one of the main virulence factors of S. aureus and has been associated with chronic infections and antimicrobial resistance. Therefore, molecules that can counteract this virulence factor may be promising alternatives as novel therapeutic agents to control S. aureus infections.IMPORTANCES. aureus is a leading agent of infections worldwide, and its main virulence characteristic is the ability to produce biofilms on surfaces such as medical devices. Biofilms are known to confer increased resistance to antimicrobials and to the host immune responses, requiring aggressive antibiotic treatment and removal of the infected surface. Here, we investigated a new source of antibiofilm compounds, the skin microbiome. Specifically, we found that a commensal strain of S. epidermidis produces molecules with antibiofilm activity, leading to a significant decrease of S. aureus biofilm formation and to a reduction of previously established biofilms. The molecules potentiated the activity of antibiotics and affected the expression of hundreds of S. aureus genes, including those associated with biofilm formation. Our research highlights the search for compounds that can aid us in the fight against S. aureus infections.

Butyric Acid from Probiotic Staphylococcus epidermidis in the Skin Microbiome Down-Regulates the Ultraviolet-Induced Pro-Inflammatory IL-6 Cytokine via Short-Chain Fatty Acid Receptor.

The glycerol fermentation of probiotic Staphylococcus epidermidis (S. epidermidis) in the skin microbiome produced butyric acid in vitro at concentrations in the millimolar range. The exposure of dorsal skin of mice to ultraviolet B (UVB) light provoked a significant increased production of pro-inflammatory interleukin (IL)-6 cytokine. Topical application of butyric acid alone or S. epidermidis with glycerol remarkably ameliorated the UVB-induced IL-6 production. In vivo knockdown of short-chain fatty acid receptor 2 (FFAR2) in mouse skin considerably blocked the probiotic effect of S. epidermidis on suppression of UVB-induced IL-6 production. These results demonstrate that butyric acid in the metabolites of fermenting skin probiotic bacteria mediates FFAR2 to modulate the production of pro-inflammatory cytokines induced by UVB.

Antagonistic Interaction of Staphylococcus aureus and Staphylococcus epidermidis with Rhizopus arrhizus Mediated by Phenol Soluble Modulins and Organic Acids.

Rhizopus arrhizus (R. arrhizus) is a common causative agent of mucormycosis that usually enters the human body through the respiratory tract and skin. Both these sites harbor staphylococci as a part of the normal microflora, indicating the possibility of interspecies interactions. We aimed to elucidate this interaction and identify the molecular mechanisms involved. Both Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) substantially hindered R. arrhizus radial growth, spore germination, and liquid culture biomass. Secreted components in the stationary-phase supernatant were responsible for this activity. The active components, based on molecular weight-based fractionation, mass spectrometry, and ion exclusion chromatography, were identified as a truncated version of phenol soluble modulin α2 (Δ1Δ2PSMα2) and PSMα3 in S. aureus, PSMδ in S. epidermidis, and organic acids in both the species. Exposure to the phenol soluble modulins (PSMs) extensively damaged the fungal spores and pre-existing hyphae, leading to bleb formation, shriveling, hyphal shrinkage, and cell distortion.

Design, synthesis, and discovery of novel oxindoles bearing 3-heterocycles as species-specific and combinatorial agents in eradicating Staphylococcus species.

A series of new functionalized 3-indolylindolin-2-ones, 3-(1-methylpyrrol-2-yl)indolin-2-ones, and 3-(thiophen-2-yl)indolin-2-ones were synthesized by using novel indium (III)-catalysed reaction of various 3-diazoindolin-2-ones with indoles, 1-methylpyrrole, or thiophene via one-pot procedure. The newly synthesized compounds were characterized and screened for their in vitro antibacterial activity against various Staphylococcus species, including methicillin-resistant Staphylococcus aureus. results revealed that five compounds KS15, KS16, KS17, KS19, and KS20 exhibited potent and specific antibacterial activity against Staphylococcus species albeit inactive against Gram-negative bacteria. Especially, compounds exhibited superior antibacterial potency against Staphylococcus epidermidis compared to the reference drug streptomycin. The most potential compound KS16 also increased the susceptibility of Staphylococcus aureus to ciprofloxacin, gentamicin, kanamycin, and streptomycin. Among them, KS16 was found to be a synergistic compound with gentamicin and kanamycin. Furthermore, the cellular level of autolysin protein was increased from the KS16-treated Staphylococcus aureus cells. Finally, in vitro CCK-8 assays showed that KS16 exhibited no cytotoxicity at the minimum inhibitory concentrations used for killing Staphylococcus species. From all our results, novel oxindole compounds directly have lethal action or boost existing antibiotic power with the reduction of doses and toxicity in the treatment of multidrug-resistant Staphylococcus species.

Whole genome sequencing revealed new molecular characteristics in multidrug resistant staphylococci recovered from high frequency touched surfaces in London.

The rise of antibiotic resistance (AMR) is one of the most important public health threats worldwide.Today, increasing attention is being paid to multidrug resistant staphylococci isolated from healthcare and non-healthcare environments as the treatment of these bacteria has become increasingly difficult. In this study, we compared staphylococci isolates recovered from high frequency touched surfaces from public areas in the community and hospitals in East and West London. 281 out of 600 (46.83%) staphylococci isolates recovered were multidrug resistant, of which 49 (8.17%) were mecA positive. There was significantly higher proportion of multidrug resistant staphylococci (P = 0.0002) in East London (56.7%) compared to West London (49.96%). The most common species identified as multidrug resistant were S. epidermidis, S. haemolyticus and S. hominis, whereas penicillin, fusidic acid and erythromycin were the most frequent antibiotics the isolates were resistant to. Whole genome sequenced of mecA positive isolates revealed that S. sciuri isolates carried the mecA1 gene, which has only 84.43% homology with mecA. In addition, other frequently identified resistance genes included blaZ, qacA/B and dfrC. We have also identified a diverse range of SCCmec types, many of which were untypable due to carrying a novel combination of ccr genes or multiple ccr complexes.

Acquisition of time-frequency localized mechanical properties of biofilms and single cells with high spatial resolution.

Biofilms are a cluster of bacteria embedded in extracellular polymeric substances (EPS) that contain a complex composition of polysaccharides, proteins, and extracellular DNA (eDNA). Desirable mechanical properties of the biofilms are critical for their survival, propagation, and dispersal, and the response of mechanical properties to different treatment conditions also sheds light on biofilm control and eradication in vivo and on engineering surfaces. However, it is challenging yet important to investigate mechanical behaviors of biofilms with a high spatial resolution because biofilms are very heterogeneous. Moreover, biofilms are viscoelastic, and their time-dependent mechanical behavior is difficult to capture. Herein, we develop a powerful technique that combines the high spatial resolution of an atomic force microscope (AFM) with a rigorous history-dependent viscoelastic analysis to deliver highly spatial-localized biofilm properties within a wide time-frequency window. By exploiting the use of static force spectroscopy in combination with an appropriate viscoelastic framework, we highlight the intensive amount of time-dependent information experimentally available that has been largely overlooked. It is shown that this technique provides a detailed nanorheological signature of the biofilms even at the single-cell level. We share the computational routines that would allow any user to perform the analysis from experimental raw data. The detailed localization of mechanical properties in space and in time-frequency domain provides insights into the understanding of biofilm stability, cohesiveness, dispersal, and control.

Propidium iodide staining underestimates viability of adherent bacterial cells.

Combining membrane impermeable DNA-binding stain propidium iodide (PI) with membrane-permeable DNA-binding counterstains is a widely used approach for bacterial viability staining. In this paper we show that PI staining of adherent cells in biofilms may significantly underestimate bacterial viability due to the presence of extracellular nucleic acids (eNA). We demonstrate that gram-positive Staphylococcus epidermidis and gram-negative Escherichia coli 24-hour initial biofilms on glass consist of 76 and 96% PI-positive red cells in situ, respectively, even though 68% the cells of either species in these aggregates are metabolically active. Furthermore, 82% of E. coli and 89% S. epidermidis are cultivable after harvesting. Confocal laser scanning microscopy (CLSM) revealed that this false dead layer of red cells is due to a subpopulation of double-stained cells that have green interiors under red coating layer which hints at eNA being stained outside intact membranes. Therefore, viability staining results of adherent cells should always be validated by an alternative method for estimating viability, preferably by cultivation.

Structural organization of a Type III-A CRISPR effector subcomplex determined by X-ray crystallography and cryo-EM.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins provide an immune-like response in many prokaryotes against extraneous nucleic acids. CRISPR-Cas systems are classified into different classes and types. Class 1 CRISPR-Cas systems form multi-protein effector complexes that includes a guide RNA (crRNA) used to identify the target for destruction. Here we present crystal structures of Staphylococcus epidermidis Type III-A CRISPR subunits Csm2 and Csm3 and a 5.2 Å resolution single-particle cryo-electron microscopy (cryo-EM) reconstruction of an in vivo assembled effector subcomplex including the crRNA. The structures help to clarify the quaternary architecture of Type III-A effector complexes, and provide details on crRNA binding, target RNA binding and cleavage, and intermolecular interactions essential for effector complex assembly. The structures allow a better understanding of the organization of Type III-A CRISPR effector complexes as well as highlighting the overall similarities and differences with other Class 1 effector complexes.

Antibacterial investigation of titanium-copper alloys using luminescent Staphylococcus epidermidis in a direct contact test.

Commercially pure titanium (CP-Ti), used as oral implants, is often populated by various bacterial colonies in the oral cavity. These bacteria can cause Peri-implantitis, leading to loss of bone tissue and failure of implants. With the increased awareness of antibiotic resistance, research has been directed towards alternative solutions and recent findings have indicated titanium­copper (Ti-Cu) alloys as a promising antibacterial material. The aim of this study was to produce homogeneous Ti-Cu alloys, with various concentrations of copper, and to characterise their antibacterial properties through direct contact tests, using luminescent bacteria, in addition to traditional materials characterisation techniques. Samples of CP-Ti and four different Ti-Cu alloys (1, 2.5, 3 and 10 wt%Cu) were produced in an arc-furnace, heated treated and rapidly quenched. X-ray diffraction revealed that Ti2Cu, was present only in the 10 wt%Cu alloy, however, scanning electron microscopy (SEM) indicated precipitates at the grain boundaries of the 3 wt%Cu alloy, which were confirmed to be of a copper rich phase by energy dispersive x-ray spectroscopy (EDS) analysis. EDS line scans confirmed that the alloys were homogenous. After 6 h, a trend between copper content and antibacterial rate could be observed, with the 10 wt%Cu alloy having the highest rate. SEM confirmed fewer bacteria on the 3 wt%Cu and especially the 10 wt%Cu samples. Although the 10 wt%Cu alloy gave the best antibacterial results, it is desired that the Cu concentration is below ~3 wt%Cu to maintain similar mechanical and corrosive performance as CP-Ti. Therefore, it is proposed that future work focuses on the 3 wt%Cu alloy.