Secreted Proteases Control Autolysin-mediated Biofilm Growth of Staphylococcus aureus

Staphylococcus epidermidis, a commensal of humans, secretes Esp protease to prevent Staphylococcus aureus biofilm formation and colonization. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases; however, the mechanism whereby Esp disrupts biofilms is unknown. We show here that Esp cleaves autolysin (Atl)-derived murein hydrolases and prevents staphylococcal release of DNA, which serves as extracellular matrix in biofilms. The three-dimensional structure of Esp was revealed by x-ray crystallography and shown to be highly similar to that of S. aureus V8 (SspA). Both atl and sspA are necessary for biofilm formation, and purified SspA cleaves Atl-derived murein hydrolases. Thus, S. aureus biofilms are formed via the controlled secretion and proteolysis of autolysin, and this developmental program appears to be perturbed by the Esp protease of S. epidermidis.     

Prevention of Staphylococcus aureus biofilm formation and reduction in established biofilm density using a combination of phage K and modified derivatives

Aims: To investigate the ability of a mixture of phage K and six of its modified derivatives to prevent biofilm formation by Staphylococcus aureus and also to reduce the established biofilm density. Methods and Results: The bioluminescence‐producing Staph. aureus Xen29 strain was used in the study, and incubation of this strain in static microtitre plates at 37°C for 48 h confirmed its strong biofilm‐forming capacity. Subsequently, removal of established biofilms of Staph. aureus Xen29 with the high‐titre phage combination was investigated over time periods of 24 h, 48 h and 72 h. Results suggested that these biofilms were eliminated in a time‐dependant manner, with biofilm biomass reduction significantly greater after 72 h than after 24–48 h. In addition, initial challenge of Staph. aureus Xen29 with the phage cocktail resulted in the complete inhibition of biofilm formation over a 48‐h period with no appearance of phage resistance. Conclusions: In general, our findings demonstrate the potential use of a modified phage combination for the prevention and successful treatment of Staph. aureus biofilms, which are implicated in several antibiotic‐resistant infections. Significance and Impact of the Study: This study highlights the first use of phage K for the successful removal and prevention of biofilms of Staph. aureus.     

Antimicrobial efficacy of the alkaloid harmaline alone and in combination with chlorhexidine digluconate against clinical isolates of Staphylococcus aureus grown in planktonic and biofilm cultures

Aims: To investigate the antimicrobial efficacy of an alkaloid, harmaline alone and in combination with chlorhexidine digluconate (CHG) against clinical isolates of Staphylococcus aureus (S. aureus) grown in planktonic and biofilm cultures. Methods: Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined for each micro‐organism grown in suspension and in biofilm using microbroth dilution method. Chequerboard assays were used to determine synergistic, indifferent or antagonistic interactions between harmaline and CHG, and the some of results were verified by confocal laser scanning microscopy. Results: Harmaline and CHG showed effective antimicrobial activity against suspensions and biofilm cultures of S. aureus, respectively. As determined by fractional inhibitory concentration index (FICI), synergistic antimicrobial effects between harmaline and CHG were observed in nine and 11 of the 13 S. aureus strains when in suspension and in biofilm, respectively. FICI values were from 0·375 to 1·25 when in suspension and from 0·25 to 1·25 when in biofilm. Conclusions: Synergistic activity of harmaline and CHG against clinical isolates of S. aureus (in suspension and in biofilm) was observed in vitro. Significance and Impact of the Study: This study might provide alternative methods to overcome the problem of drug‐resistance of S. aureus both in suspension and in biofilm.     

Anti-biofilm activity of Quercus infectoria G. Olivier against methicillin-resistant Staphylococcus aureus

Aims: To establish the effect of Quercus infectoria G. Olivier extract and its main constituent, tannic acid, on staphylococcal biofilm and their anti‐biofilm mechanisms. Methods and Results: Anti‐biofilm activity of the plant materials on clinical isolated of methicillin‐resistant Staphylococcus aureus and methicillin‐susceptible Staph. aureus was employed using a crystal violet‐stained microtiter plate method. The extract at minimum inhibitory concentration (MIC; 0·25 mg ml^?1) was significantly reduced the biofilm formation of the isolates (P < 0·05). The effect on staphylococcal cell surface hydrophobicity (CSH) of the test compounds was investigated as a possible mode of action of the anti‐biofilm activity. The hydrophobicity index of all the bacterial isolates increased following treatment with supra‐MIC, MIC and sub‐MIC of the extract and tannic acid. Observation of the treated bacterial cells by electron microscopy revealed that the test compounds caused clumps of partly divided cocci with thickened and slightly rough cell wall. Conclusions: The results indicated that Q. infectoria extract and tannic acid affected staphylococcal biofilm formation and their effect on bacterial CSH and cell wall may involve in the anti‐biofilm activity. Significance and Impact of the Study: This evidence highlighted the anti‐biofilm potency of the natural products and clarified their anti‐biofilm mechanisms.     

sarA‐mediated repression of protease production plays a key role in the pathogenesis of Staphylococcus aureus USA300 isolates

Mutation of staphylococcal accessory regulator (sarA) results in increased production of extracellular proteases in Staphylococcus aureus, which has been correlated with decreased biofilm formation and decreased accumulation of extracellular toxins. We used murine models of implant‐associated biofilm infection and S. aureus bacteraemia (SAB) to compare virulence of USA300 strain LAC, its isogenic sarA mutant, and derivatives of each of these strains with mutations in all 10 of the genes encoding recognized extracellular proteases. The sarA mutant was attenuated in both models, and this was reversed by eliminating production of extracellular proteases. To examine the mechanistic basis, we identified proteins impacted by sarA in a protease‐dependent manner. We identified 253 proteins where accumulation was reduced in the sarA mutant compared with the parent strain, and was restored in the sarA/protease mutant. Additionally, in SAB, the LAC protease mutant exhibited a hypervirulent phenotype by comparison with the isogenic parent strain, demonstrating that sarA also positively regulates production of virulence factors, some of which are subject to protease‐mediated degradation. We propose a model in which attenuation of sarA mutants is defined by their inability to produce critical factors and simultaneously repress production of extracellular proteases that would otherwise limit accumulation of virulence factors.     

Clonal distribution of enterotoxigenic Staphylococcus aureus on handles of handheld shopping baskets in supermarkets

Aims: Shopping carts and handheld shopping baskets in supermarkets are subject to accidental bacterial contamination through contacts with a variety of food. We investigated the prevalence of Staphylococcus aureus on the handles of handheld shopping baskets in four supermarkets distantly located in Osaka district, Japan. Methods and Results: Fifty two strains of Staph. aureus were isolated from 760 basket handles. Among these, six strains were positive for staphylococcal enterotoxin B (SEB) production, representing 12% of total. This SEB producer ratio is considerably higher than among Staph. aureus isolated from nasal swabs of the supermarket workers (2%) and from independently collected clinical specimens (4%). These SEB‐producing Staph. aureus strains from the basket handles are clonal and belong to ST12. Coagulase typing showed that they are in group VII, which is the most common cause of food poisoning in Japan. Biofilm assays indicated that SEB gene (seb)‐positive strains including this clone produced a significantly higher amount of biofilm than seb‐negative strains. Conclusions: The frequent isolation of seb‐positive Staph. aureus on shopping basket handles raises the possibility that they could be a hidden reservoir for Staph. aureus with a potential to cause food poisoning and draws attention to the importance of shopping basket sanitation.     

Extracellular DNA in Helicobacter pylori biofilm: a backstairs rumour

Aims: This study detected and characterized the extracellular DNA (eDNA) in the biofilm extracellular polymeric substance (EPS) matrix of Helicobacter pylori and investigated the role of such component in the biofilm development. Methods and Results: Extracellular DNA was purified and characterized in a 2‐day‐old mature biofilm developed by the reference strain H. pylori ATCC 43629, the clinical isolate H. pylori SDB60 and the environmental strain H. pylori MDC1. Subsequently, the role of eDNA in the H. pylori biofilm was evaluated by adding DNase I during biofilm formation and on mature biofilms. Extracellular DNA was detected in the 2‐day‐old EPS biofilm matrix of all analysed H. pylori strains. The DNA fingerprintings, performed by RAPD analysis, on eDNA and intracellular DNA (iDNA), showed some remarkable differences. The data obtained by microtitre biofilm assay as well as colony forming unit count and CLSM (confocal laser scanning microscopy) qualitative analysis did not show any significant differences between the DNase I‐treated biofilms and the corresponding not treated controls both in formation and on mature biofilms. Conclusions: In this study, we provide evidence that eDNA is a component of the EPS matrix of H. pylori biofilm. The different profiles of eDNA and iDNA indicate that lysed cells are not the primary source of eDNA release, suggesting that other active mechanisms might be involved in this process. Moreover, the biomass assay suggests that eDNA may not be the main component of biofilm matrix, suggesting that it could be primarily involved in other mechanisms such as recombination processes, via transformation, contributing to the wide genomic variability of this micro‐organism defined as a ‘quasi‐species’. Significance and Impact of the Study: The presence of eDNA in H. pylori biofilm can contribute to the active dynamic exchange of information aimed to reach the best condition for the bacterial survival in the host and in the environment.     

Inhibitory effects of antibiofilm compound 1 against Staphylococcus aureus biofilms

A novel benzimidazole molecule that was identified in a small‐molecule screen and is known as antibiofilm compound 1 (ABC‐1) has been found to prevent bacterial biofilm formation by multiple bacterial pathogens, including Staphylococcus aureus, without affecting bacterial growth. Here, the biofilm inhibiting ability of 156 μM ABC‐1 was tested in various biofilm‐forming strains of S. aureus. It was demonstrated that ABC‐1 inhibits biofilm formation by these strains at micromolar concentrations regardless of the strains' dependence on Polysaccharide Intercellular Adhesin (PIA), cell wall‐associated protein dependent or cell wall‐ associated extracellular DNA (eDNA). Of note, ABC‐1 treatment primarily inhibited Protein A (SpA) expression in all strains tested. spa gene disruption showed decreased biofilm formation; however, the mutants still produced more biofilm than ABC‐1 treated strains, implying that ABC‐1 affects not only SpA but also other factors. Indeed, ABC‐1 also attenuated the accumulation of PIA and eDNA on cell surface. Our results suggest that ABC‐1 has pleotropic effects on several biofilm components and thus inhibits biofilm formation by S. aureus.     

Chrysin protects mice from Staphylococcus aureus pneumonia

Aim: To elucidate the effect of chrysin on α‐haemolysin production by Staphylococcus aureus and protection against pneumonia in a murine model. Methods and Results: Haemolysis, Western blot and real‐time RT‐PCR assays were performed to evaluate the effect of chrysin on α‐haemolysin secretion by Staph. aureus. The efficacy of chrysin against human alveolar epithelial cell injury by α‐haemolysin was tested using live/dead staining or by measuring lactate dehydrogenase activity. Furthermore, we determined the protective effect of chrysin against Staph. aureus pneumonia through histopathology experiments in a mouse model. The production of α‐haemolysin by Staph. aureus was inhibited when presented with an increasing subinhibitory concentration of chrysin in vitro. Consistent with this result, chrysin prevented α‐haemolysin‐mediated cell injury and protected mice from Staph. aureus pneumonia. Conclusions: Chrysin is a potent inhibitor of α‐haemolysin expression by Staph. aureus, and it conferred a significant degree of protection against Staph. aureus pneumonia. Significance and Impact of Study: The chrysin‐mediated inhibition of α‐haemolysin production and protection against Staph. aureus pneumonia may offer a new strategy in combating pathogen infections.     

Molecular detection of enterotoxins E, G, H and I in Staphylococcus aureus and coagulase-negative staphylococci isolated from clinical samples of newborns in Brazil

Aims: The objective of this study was to investigate the detection of SEE, SEG, SEH and SEI in strains of Staphylococcus aureus and coagulase‐negative staphylococci (CNS) using RT‐PCR. Methods and Results: In this study, 90 Staph. aureus strains and 90 CNS strains were analysed by PCR for the detection of genes encoding staphylococcal enterotoxins (SE) E, G, H and I. One or more genes were detected in 54 (60%) Staph. aureus isolates and in 29 (32·2%) CNS isolates. Staphylococcus epidermidis was the most frequently isolated CNS species (n = 64, 71·1%), followed by Staphylococcus warneri (n = 8, 8·9%) and other species (Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus simulans, Staphylococcus saprophyticus and Staphylococcus xylosus: n = 18, 20%). The genes studied were detected in Staph. epidermidis, Staph. warneri, Staph. haemolyticus, Staph. hominis, Staph. simulans and Staph. lugdunensis. The highest frequency of genes was observed in Staph. epidermidis and Staph. warneri, a finding indicating differences in the pathogenic potential between CNS species and highlighting the importance of the correct identification of these micro‐organisms. RT‐PCR used for the detection of mRNA revealed the expression of SEG, SEH and/or SEI in 32 (59·3%) of the 90 Staph. aureus isolates, whereas expression of some of these genes was observed in 10 (34·5%) of the 90 CNS isolates. Conclusions: Staphylococcus epidermidis was the most toxigenic CNS species. Among the other species, only Staph. warneri and Staph. lugdunensis presented a positive RT‐PCR result. PCR was efficient in confirming the toxigenic capacity of Staph. aureus and CNS. Significance and Impact of the Study: This study permitted to confirm the toxigenic capacity of CNS to better characterize the pathogenic potential of this group of micro‐organisms. In addition, it permitted the detection of SEG, SEH and SEI, enterotoxins that cannot be detected by commercially available immunological methods.     

Safety-related properties of staphylococci isolated from food and food environments

Aims: To test some safety‐related properties within 321 staphylococci strains isolated from food and food environments. Methods and Results: The isolates were identified as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus pasteuri, Staphylococcus sciuri, Staphylococcus warneri and Staphylococcus xylosus. Decarboxylase activity was quite common for the various Staphylococcus spp., and tyrosine was the most frequently decarboxylated amino acid. The frequency of antibiotic resistance was highest in Staph. pasteuri and Staph. xylosus. Several of the isolates were tolerant to QAC compounds, and in some cases, QAC tolerance was present in antibiotic‐resistant strains. Most of the strains displayed moderate to high adhesion rates to stainless steel and Teflon^®. The strains that readily formed biofilms belonged to the species Staph. aureus, Staph. epidermidis and Staph. pasteuri. Conclusions: An high incidence of some safety hazards was found within the staphylococcal strains of food origin tested in this study. In particular, amino acid decarboxylase activity and biofilm‐forming ability were common within strains, and antibiotic resistance and tolerance to QAC‐based compounds occurred frequently as well. These characteristics are an important safety concern for food industry. Significance and Impact of the Study: This work gives a first picture of safety hazards within staphylococcal species isolated from food environments. The presence of disinfectant‐resistant staphylococci is a concern because resistance can be genetically transferred between the various Staphylococcus species. This could lead an increase and spread of resistant enterotoxic staphylococci and/or pathogenic staphylococci.     

Enhanced extracellular α-amylase production in Brevibacillus choshinensis by optimizing extracellular degradation and folding environment

A strategy for optimizing the extracellular degradation and folding environment of Brevibacillus choshinensis has been used to enhance the extracellular production of recombinant α-amylase. First, a gene (bcp) encoding an extracellular protease and another encoding an extracellular chaperone (prsC) were identified in the genome of B. choshinensis HPD31-SP3. Then, the effect of extracellular protein degradation on recombinant α-amylase production was investigated by establishing a CRISPR/Cas9n system to knock out bcp. The effect of extracellular folding capacity was investigated separately by coexpressing extracellular chaperones genes from different sources (prsA, prsC, prsL, prsQ) in B. choshinensis. The final recombinant strain (BCPPSQ), which coexpressed prsQ in a genetic background lacking bcp, produced an extracellular α-amylase activity of 6940.9 U/ml during shake-flask cultivation. This was 2.1-fold greater than that of the original strain BCWPS (3367.9 U/ml). Cultivation of BCPPSQ in a 3-l fermenter produced an extracellular α-amylase activity of 17925.6 U/ml at 72 h, which was 7.6-fold greater than that of BCWPS (2358.1 U/ml). This strategy demonstrates its great potential in enhancing extracellular α-amylase production in B. choshinensis. What''s more, this study provides a strategic reference for improving the extracellular production of other recombinant proteins in B. choshinensis.     

Simple production of resilin-like protein hydrogels using the Brevibacillus secretory expression system and column-free purification

Resilin, an insect structural protein, has excellent flexibility, photocrosslinking properties, and temperature responsiveness. Recombinant resilin-like proteins (RLPs) can be fabricated into three-dimensional (3D) structures for use as cell culture substrates and highly elastic materials. A simplified, high-yielding production process for RLPs is required for their widespread application. This study proposes a simple production process combining extracellular expression using Brevibacillus choshinensis (B. choshinensis) and rapid column-free purification. Extracellular production was tested using four representative signal peptides; B. choshinensis was found to efficiently secrete Rec1, an RLP derived from Drosophila melanogaster, regardless of the type of signal peptide. However, it was suggested that Rec1 is altered by an increase in the pH of the culture medium associated with prolonged incubation. Production in a jar fermentor with controllable pH yielded 530 mg Rec1 per liter of culture medium, which is superior to productivity using other hosts. The secreted Rec1 was purified from the culture supernatant via (NH₄)₂SO₄ and ethanol precipitations, and the purified Rec1 was applied to ring-shaped 3D hydrogels. These results indicate that the combination of secretory production using B. choshinensis and column-free purification can accelerate the further application of RLPs.     

Generation and characterization of an inter-generic bivalent alpha domain fusion protein αCS from Clostridium perfringens and Staphylococcus aureus for concurrent diagnosis and therapeutic applications

Aim: To evaluate an inter‐generic recombinant alpha domain fusion protein for simultaneous detection and neutralization of Clostridium perfringens and Staphylococcus aureus alpha toxins. Methods and Results: Truncated portions of clostridial and staphylococcal alpha haemolysin genes were PCR amplified and linked to each other through a hydrophilic flexible Glycine linker sequence using overlap‐extension PCR to form a chimeric gene αCS. The recombinant αCS fusion protein was expressed and characterized for its toxicity, cell binding capacity and haemolysis inhibition properties. The fusion protein was nontoxic and effectively retarded staphylococcal alpha haemolysis, probably by competitively interacting with putative staphylococcal alpha haemolysin receptors on erythrocytes. Murine hyperimmune polysera raised against r‐αCS specifically detected 42‐kDa and 33‐kDa proteins when culture supernatants of Cl. perfringens (clostridial alpha toxin) and Staph. aureus (staphylococcal alpha toxin), respectively, were analysed in Western blot. The polyclonal antisera effectively diminished the haemolytic action of both the wild‐type toxins in vitro. Conclusions: The r‐αCS fusion protein was nontoxic competitive inhibitor of staphylococcal alpha haemolysin. The protein elicited specific immune response against Cl. perfringens and Staph. aureus alpha toxins. The antisera also neutralized the toxicities of both the native wild‐type toxins in vitro. Significance of the Study: The bivalent recombinant αCS protein could be a novel intervention in the field of diagnostics and therapeutics against Cl. perfringens and Staph. aureus infections, particularly, in case of co‐infections like gangrenous ischaemia, gangrenous mastitis, etc.     

Development and application of a polymicrobial, in vitro, wound biofilm model

Aims: The goal of this investigation was to develop an in vitro, polymicrobial, wound biofilm capable of supporting the growth of bacteria with variable oxygen requirements. Methods and Results: The strict anaerobe Clostridium perfringens was isolated by cultivating wound homogenates using the drip‐flow reactor (DFR), and a three‐species biofilm model was established using methicillin‐resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and Cl. perfringens in the colony‐drip‐flow reactor model. Plate counts revealed that MRSA, Ps. aeruginosa and Cl. perfringens grew to 7·39 ± 0·45, 10·22 ± 0·22 and 7·13 ± 0·77 log CFU per membrane, respectively. The three‐species model was employed to evaluate the efficacy of two antimicrobial dressings, Curity? AMD and Acticoat?, compared to sterile gauze controls. Microbial growth on Curity? AMD and gauze was not significantly different, for any species, whereas Acticoat? was found to significantly reduce growth for all three species. Conclusions: Using the colony‐DFR, a three‐species biofilm was successfully grown, and the biofilms displayed a unique structure consisting of distinct layers that appeared to be inhabited exclusively or predominantly by a single species. Significance and Impact of the Study: The primary accomplishment of this study was the isolation and growth of an obligate anaerobe in an in vitro model without establishing an artificially anaerobic environment.     

The effects of subinhibitory concentrations of costus oil on virulence factor production in Staphylococcus aureus

Aim: To determine the antimicrobial activity of costus (Saussurea lappa) oil against Staphylococcus aureus, and to evaluate the influence of subinhibitory concentrations of costus oil on virulence‐related exoprotein production in staph. aureus. Methods and Results: Minimal inhibitory concentrations (MICs) were determined using a broth microdilution method, and the MICs of costus oil against 32 Staph. aureus strains ranged from 0.15 to 0.6 μl ml^?1. The MIC₅₀ and MIC₉₀ were 0.3 and 0.6 μl ml^?1, respectively. Western blot, haemolytic, tumour necrosis factor (TNF) release and real‐time RT‐PCR assays were performed to evaluate the effects of subinhibitory concentrations of costus oil on virulence‐associated exoprotein production in Staph. aureus. The data presented here show that costus oil dose dependently decreased the production of α‐toxin, toxic shock syndrome toxin 1 (TSST‐1) and enterotoxins A and B in both methicillin‐sensitive Staph. aureus (MSSA) and methicillin‐resistant Staph. aureus (MRSA). Conclusion: Costus oil has potent antimicrobial activity against Staph. aureus, and the production of α‐toxin, TSST‐1 and enterotoxins A and B in Staph. aureus was decreased by costus oil. Significance and Impact of the Study: The data suggest that costus oil may deserve further investigation for its potential therapeutic value in treating Staph. aureus infections. Furthermore, costus oil could be rationally applied in food products as a novel food preservative both to inhibit the growth of Staph. aureus and to repress the production of exotoxins, particularly staphylococcal enterotoxins.     

Dimethyl formamide-free, urea-NaCl fluorescence in situ hybridization assay for Staphylococcus aureus

Aims: To test the feasibility of identifying Staphylococcus aureus with a fluorescence in situ hybridization (FISH) assay that uses a single hot‐plate and urea‐NaCl reagents. Methods and Results: Slides spotted with S. aureus and treated with methanol and lysozyme were incubated with urea‐NaCl reagents on a hot‐plate with a precise temperature control and identified with specific DNA probes. Conclusions: Staphylococcus aureus was detected and differentiated from Staphylococcus epidermidis in 1 h with a novel FISH method that used a single hot‐plate and in the absence of dimethyl formamide. Significance and Impact of Study: A rapid hot‐plate FISH assay with urea‐NaCl and without toxic dimethyl formamide might be useful if FISH is run infrequently or where resources are limited.     

The Enterococcal Surface Protein, Esp, Is Involved in Enterococcus faecalis Biofilm Formation

The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.     

Staphylococcus aureus biofilm formation and antibiotic susceptibility tests on polystyrene and metal surfaces

Aim: We compared the MBEC?‐HTP assay plates made of polystyrene with metal discs composed of TMZF^® and CrCo as substrates for biofilm formation. Methods and Results: Staphylococcus aureus was grown on polystyrene and on metal discs made of titanium and chrome–cobalt. Antibiotic susceptibility was assessed by examining the recovery of cells after antibiotic exposure and by measuring the biofilm inhibitory concentration (BIC). The minimal inhibitory concentration (MIC) was assessed with planktonic cells. Bacterial growth was examined by scanning electron microscopy. The antibiotic concentration for biofilm inhibition (BIC) was higher than the MIC for all antibiotics. Microscopic images showed the biofilm structure characterized by groups of cells covered by a film. Conclusions: All models allowed biofilm formation and testing with several antibiotics in vitro. Gentamicin and rifampicin are the most effective inhibitors of Staph. aureus biofilm‐related infections. We recommend MBEC?‐HTP assay for rapid testing of multiple substances and TMZF^® and CrCo discs for low‐throughput testing of antibiotic susceptibility and for microscopic analysis. Significance and Impact of the Study: In vitro assays can improve the understanding of biofilms and help developing methods to eliminate biofilms from implant surfaces. One advantage of the TMZF^® and CrCo discs as biofilm in vitro assay is that these metals are commonly used for orthopaedic implants. These models are usable for future periprosthetic joint infection studies.